Laser en Autismo

Advances in Experimental Medicine and Biology 1116
Clinical and Experimental Biomedicine
Mieczyslaw Pokorski Editor
Clinical
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Advances in Experimental Medicine
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Volume 1116
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Mieczyslaw Pokorski
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Mieczyslaw Pokorski
Opole Medical School
Opole, Poland
ISSN 0065-2598 ISSN 2214-8019 (electronic)

Advances in Experimental Medicine and Biology
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Contents
Bioprogressive Paradigm in Physiotherapeutic and Antiaging

Strategies: A Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Mieczyslaw Pokorski, Giovanni Barassi, Rosa G. Bellomo,
Loris Prosperi, Matteo Crudeli, and Raoul Saggini
Influence of Proprioceptive Neuromuscular Facilitation
on Lung Function in Patients After Coronary Artery Bypass
Graft Surgery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Małgorzata Bujar-Misztal and Andrzej Chciałowski
Remote Ischemic Preconditioning in Renal Protection During
Elective Percutaneous Coronary Intervention . . . . . . . . . . . . . . . . 19
Małgorzata Wojciechowska, Maciej Zarębiński, Piotr Pawluczuk,
Dagmara Gralak-Łachowska, Leszek Pawłowski, Wioletta Loska,
Marzena Goszczyńska, Krzysztof Flis, and Agnieszka
Cudnoch-Jędrzejewska
Prognostic Impact of Extracapsular Lymph Node Invasion
on Survival in Non-small-Cell Lung Cancer: A Systematic
Review and Meta-analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Seyed Vahid Tabatabaei, Christoph Nitche, Maximilian Michel,
Kurt Rasche, and Khosro Hekmat
Influence of Transurethral Resection of Bladder Cancer
on Sexual Function, Anxiety, and Depression . . . . . . . . . . . . . . . . 37
Wojciech Krajewski, Urszula Halska, Sławomir Poletajew,
Radosław Piszczek, Bartosz Bieżyński, Mateusz Matyjasek,
Andrzej Tukiendorf, Fryderyk Menzel, Małgorzata Mazur,
Joanna Rymaszewska, and Romuald Zdrojowy
Cognitive Predictors of Cortical Thickness in Healthy Aging . . . . 51
Patrycja Naumczyk, Angelika K. Sawicka, Beata Brzeska,
Agnieszka Sabisz, Krzysztof Jodzio, Marek Radkowski,
Karolina Czachowska, Paweł J. Winklewski, Karolina Finc,
Edyta Szurowska, Urszula Demkow, and Arkadiusz Szarmach
v
vi Contents
Osteoprotegerin, Receptor Activator of Nuclear Factor

Kappa B Ligand, and Growth Hormone/Insulin-Like Growth
Factor-1 Axis in Children with Growth Hormone Deficiency . . . . . 63
Ewelina Witkowska-Sędek, Małgorzata Rumińska,
Anna Stelmaszczyk-Emmel, Maria Sobol, Urszula Demkow,
and Beata Pyrżak
Inhibition of Cross-Reactive Carbohydrate Determinants
in Allergy Diagnostics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Maciej Grzywnowicz, Emilia Majsiak, Józef Gaweł,
Karolina Miśkiewicz, Zbigniew Doniec, and Ryszard Kurzawa
Effects of Glutathione on Hydrolytic Enzyme Activity
in the Mouse Hepatocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Iwona Stanisławska, Bożena Witek, Marek Łyp,
Danuta Rochon-Szmejchel, Adam Wróbel, Wojciech Fronczyk,
Agnieszka Kamińska, Adam Kołątaj, and Daniel Załuski
Adaptation to Occupational Exposure to Moderate
Endotoxin Concentrations: A Study in Sewage Treatment
Plants in Germany . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
M. A. Rieger, V. Liebers, M. Nübling, T. Brüning, B. Brendel,
F. Hoffmeyer, and M. Raulf
Effects of Low-Level Laser Therapy in Autism Spectrum
Disorder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Gerry Leisman, Calixto Machado, Yanin Machado,
and Mauricio Chinchilla-Acosta
Epidemiology of Granulomatosis with Polyangiitis in Poland,
2011–2015 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
Krzysztof Kanecki, Aneta Nitsch-Osuch, Paweł Gorynski,
Patryk Tarka, Magdalena Bogdan, and Piotr Tyszko
Adv Exp Med Biol - Clinical and Experimental Biomedicine (2018) 3: 1–9
https://doi.org/10.1007/5584_2018_281
Published online: 16 October 2018
Bioprogressive Paradigm
in Physiotherapeutic and Antiaging
Strategies: A Review
Mieczyslaw Pokorski, Giovanni Barassi, Rosa G. Bellomo,

Loris Prosperi, Matteo Crudeli, and Raoul Saggini
Abstract the proprio-neuromuscular facilitation, a

This review updates the knowledge on the use combination of stretch technique that steps
of bioprogressive philosophy in current reha- into the bioprogressive approach, as currently
bilitation paradigms, focusing on age-related the best method in the world of physical
ailments and antiaging strategies. It is a holis- rehabilitation.
tic approach that combines aspects of biology
and function into the realm of rehabilitation Keywords
therapy. The bioprogressive philosophy, with Aging · Antiaging strategy · Bioprogressive
assistance of modern technological paradigm · Physical dysfunction ·
developments, such as microgravity- Physiotherapy · Proprioceptive neuromuscular
producing devices and techniques, enables facilitation · Rehabilitation
personalized and targeted therapeutic
approach that seems the most effective in reha-
bilitation and prevention of neuro-myo-sen- 1 Bioprogressive Rehabilitation
sory disorders that compromise the
homeostatic body harmony, particularly in Aging is usually defined as a progressive loss of
old age. The review defines the aging, function accompanied by increasing morbidity
discusses the most common physical and mortality. Causes of the aging process are
dysfunctions, linked to posture, balance, or multifactorial and can be classified into two
gait, and gives cues to modern antiaging reha- groups: intrinsic (notably genetic modifications)
bilitative approaches. The emphasis is put on and extrinsic (environmental) factors. Several
studies convincingly demonstrate the fast pace
of aging of the general population worldwide
M. Pokorski (*)
and how this process is going to advance in the
Opole Medical School, Opole, Poland
e-mail: [email protected] next few years. As the elderly population grows
in terms of age and size, so do the requests for
G. Barassi, L. Prosperi, M. Crudeli, and R. Saggini
Department of Medical and Oral Sciences and assistance for senior persons (Christensen et al.
Biotechnologies, “G.d’Annunzio” University Chieti, 2009). The main feature of this phenomenon is
Chieti, Italy the elderly need for care which is required to
R. G. Bellomo maintain their healthy condition, for instance,
Department of Biomolecular Sciences, “Carlo Bo” performing some kind of exercise aimed to
University Urbino, Urbino, Italy
1
2 M. Pokorski et al.
avoid the risk of falling or to begin a rehabilitation frameworks often occur in adulthood, and
protocol (Israely et al. 2018; Zur et al. 2018; behaviors are generally modifiable through a
Aalen et al. 2014; Arbeev et al. 2009). series of rehabilitative interventions (Atkins
A bioprogressive multisensory rehabilitation et al. 2014; Barzilai et al. 2012; Bergman et al.
approach has proven the most suitable to face a 2007). Thus, nutrition, frequency of physical
physiological reduction in stability. The activity, and other crucial factors need to be
approach, supported by modern technology, examined to develop the appropriated lifestyle,
enables the creation of a changing training envi- which would also provide succor to metabolic
ronment, with the aim to increase proprioceptive activity, cerebral functionality, and postural con-
perception and balance reaction (Bellomo et al. trol (Si and Liu 2014; Bosma-den Boer et al.
2009). Improvements can be monitored in a mod- 2012; Misirli et al. 2012; Agnoli et al. 2011; Hu
ern gait analysis laboratory to describe variations 2002).
in balance reactions, which enables the therapist Studying the inter-system correlations and the
to periodically adapt the training program to rules that underlie the perceptive-motor pattern
changes in the patient condition to reach increas- holds a central role in the understanding of
ingly important therapeutic goals (Basile et al. relationships between movement, function, envi-
2016). Parkinson’s patients treated with a ronment context, and psycho-behavioral
bioprogressive rehabilitation paradigm, consisting characteristics of subjects. The human being
of a combination of dynamic antigravity postural forms a unique biosystem whose management
system (SPAD), auditory cue system (metro- requires a holistic approach, personalized for spe-
nome), high-intensity vibratory system (VISS), cific patient’s characteristics (Saggini et al. 2012).
and active stretching based on virtual reality reha- Posture is identified as the morphological, senso-
bilitation system (VRRS) for several months, rial, and motor expression of the evolved identity.
experience a distinct improvement in balance sta- The somatosensory system, visual and auditory
bility and gait pattern, with a reduction in falls structures, and vestibular receptors are essential
and overall enhancement of quality of life for maintaining the brain-regulated body motion
(Bellomo et al. 2014). Modern assistance and balance, as well as a feel of one’s comfort
methods enable the elderly to access the training (Zimmermann et al. 2013). A potential alteration
facilities at any time and to use them in a safe of sensory afferentation can be compensated
environment while continuing to provide through the redundancy characteristics of a sys-
instructions for rehabilitation and monitoring of tem, which also respond to appropriate
progress (Akushevich et al. 2013). Some innova- myofascial adaptations (Reghem et al. 2014).
tive examples for the preventive and rehabilitative The aging process underlies a progressive deteri-
intervention, which illustrate this increasingly oration of the delicate mechanisms of neurologi-
diversified scope and ever-changing future per- cal osteomuscular-fascial optimization,
spective of the intervention program, have been manifesting as an altered “afferent stimulation”
described by Young (1983). that determines “postural dysfunction” (Barassi
Aging concerns physical, cognitive, and social et al. 2018a, b, c, d).
spheres of individuals. The cardiovascular sys- Already at gestational age week 5, a trilaminar
tem, muscle-skeletal mass, osteoarthritic struc- embryo may be recognized, with the interposition
ture, immuno-humoral metabolism, and central of mesoderm between ectoderm and endoderm.
nervous system disorders predominate in the The epithelial to mesenchymal cell transition
aged, altering the organism’s functionality emerges, during which the structure assumes its
(Israely et al. 2018; Carmeli et al. 2016). Diabetes function and form. In this mechanism, muscular
and cardiovascular diseases, closely linked to chains and fascial continuity appear to be the
inflammation and oxidative stress, belong to at evolutionary expressions of the spinal cord
least partly modifiable risk factors, with a proper (Barassi et al. 2018b). Paraxial mesodermal
diet and correct lifestyle. Such pathologic elements of the skeletal muscular system are
Bioprogressive Paradigm in Physiotherapeutic and Antiaging Strategies: A Review 3
formed as the splanchnic mesodermal derivations, 2018). There appears a plausibility that such
in particular smooth muscles, derived from the structures play a central role in the body’s self-
occipital-to-sacral region somites and head-region management of edema to recreate homeostatic
somitomeres (Saggini et al. 1996). harmony (Johnson 1977). Thus, contrarily to the
It is known that fascial connective tissue is old physiological tenet of functional neutrality of
able to pick up nociceptive and proprioceptive connective tissue, purportedly serving the crea-
information thanks to the presence of mechano- tion of a parenchymal scaffold for cell arrange-
and nociceptors and its spatial organization (Van ment, connective cells appear to host a multitude
der Wal 2009). Studies also demonstrate that of active functions, inter alia, inter-cell communi-
connective tissue creates a network between the cation, proprioceptive signal transmission, regu-
musculoskeletal, neural, endocrine, circulatory, lation of extracellular space fluid, and others
respiratory, and immune systems, creating a (Bordoni and Zanier 2015). Nowadays, these
milieu for inter-tissue cooperation (Vecchiet cells appear to underlie the effectiveness of the
et al. 1999). In particular, fascia is composed of bioprogressive rehabilitation paradigms that are
cells that have the ability to contract and commu- set to affect their function to restore a proper body
nicate with one another, ensuring the supervision neuro-myo-sensory integration, as schematically
of body activity, such as the transmission of ten- depicted in Fig. 1.
sion created by muscles or management of inter-
stitial fluids (Bordoni and Zanier 2015; Schleip
2003). One of the latest studies performed on 2 Stretch Exercise
fascia tissue demonstrates, using a confocal laser
endomicroscopy, the presence of “reticular One of the most common exercise techniques is
structures” in visceral submucosa and dermal muscular-fascial stretch. Stretching technique
periarterial stroma, which are hypothesized to be modifies the Golgi apparatus activity which, via
an extension of the interstitial space (Benias et al. the spinal cord, decreases the firing rate of alpha
BODY DYNAMICS
PROPRIOCEPTIVE RECEPTORS SENSORIAL NEURAL STIMULATION
MESODERM
(Connective Tissue)
Microgravity Environment
Tissue Remodelling Rehabilitation Rehabilitation
Action
GOLGI OSTEO-MYO-FASCIAL
APPARATUS SYSTEM
STRETCHING MAV
Mechano-Acoustic Vibration Terrestrial Acquatic
ISOMETRIC PNF ISOTONIC

Proprioceptive
Neuromuscular
Facilitation
BODY DYNAMICS
OPTIMIZATION
Fig. 1 Schematic diagram of the bioprogressive philosophy of physiotherapeutic interventions, pointing to the central
role of connective tissue in maintaining the neuro-myo-sensory integration
motor neurons, and, in turn, muscle tone, for held in that position, while rehabilitant contracts
instance, as occurs in Yoga posture (Barassi the muscle isometrically to its maximum for a
et al. 2018c; Schleip 2003). There exist various certain amount of time, followed by a brief
types of stretching technique. The unresolved phase of passive stretching. CRAC is an exten-
issue with physical stretch is what it should be sion of the former technique in which after the
like in the setting of health-care practitioners, maximum isometric contraction of the agonistic
particularly those engaged in sports training or muscle, rehabilitant contracts the corresponding
rehabilitative framework in the elderly (Caplan antagonistic muscle for a certain amount of time.
et al. 2009). There are three types of stretch exer- Ultimately, it seems that a mix of stretch modes,
cise: static stretching, dynamic stretching, and in particular static stretch followed by dynamic
proprioceptive neuromuscular facilitation (PNF) stretch, optimizes the benefits gained from physi-
(Fig. 1). A combination of these approaches cal rehabilitation, simultaneously reducing the
guarantees autogenic inhibition, mutual inhibi- number of exercise-induced muscle impairments
tion, stress relaxation, and a reduction of pain or injuries (Rowlands et al. 2003). To this end,
consequently to the gate control theory, all of meditation techniques consisting of a variable
which leads to improvements in the range of motion ingredient and resembling the CRAC
motion of joints (Nelson et al. 2005; Wicke sequence of stretches, such as is Tai Chi and the
et al. 2014; Hindle et al. 2012). Static stretching like, advance in popularity of late (Hindle et al.
consists of holding muscles in an elongated posi- 2012). An additional benefit of meditation is the
tion for an extended period. Contrarily, dynamic improvement in mood, emotional stability, and
stretch consists of moving joints through their cognition (Pokorski and Suchorzynska 2018;
range of motion. There are pros and cons of Lam et al. 2014).
both types of stretch. Static isometric exercise,
although being physically motionless, is a strong
respiratory stimulant that provides succor to arte- 3 Aging and Rehabilitation
rial blood and, consequently, tissue oxygenation.
It also increases heart rate and blood pressure Old age should not be seen as a disease or pathol-
(Pokorski et al. 1990). These effects, thought of ogy but as a stage of life that can be fully explored
as typical and beneficial from the standpoint of to pursue personal and social goals. Human func-
physical rehabilitation or training, may jeopardize tionality has an outstanding plasticity and is capa-
the frail elderly due to abruptly increased meta- ble of compensating for the lost structural
bolic rate. Static stretch may also lead to muscle elements caused by the age-related dampening
weakness, which hampers subsequent physical of cellular metabolism. That may be exemplified,
performance. Dynamic stretch, on the other for instance, by the adaptive hyperventilatory
hand, gives proneness to injuries, which may responses to strenuous stimuli, such as exercise,
actually hamper the range of motion that already in old age, despite a severe structural deterioration
declines with age but is important for daily life of the lungs and airways, limiting the alveolar gas
activities (Konrad and Tilp 2014). Dynamic exchange area and thus oxygen diffusion
stretch, however, increases the nerve conduction (Pokorski and Marczak 2003). Likewise, cogni-
velocity, muscle compliance, enzymatic turnover, tive brain functions are maintained due to a
and the central control running down from the greater neuronal synchronization and a flexible
brain to muscles (Wicke et al. 2014). The PNF use of cognitive reserve or recruitment of alterna-
combines the stretching and contraction of tive neuronal pathways, normally constituting a
reciprocally related muscle groups in a cyclical dormant reserve. The antiaging strategy is a pro-
pattern. “Contract-relax” (CR) and “contract- cess that not only involves combating the disease
relax-antagonist-contract” (CRAC) methods are and its effects but also a large psychological com-
the representative techniques of PNF. In the CR ponent that is closely linked to behavior, person-
technique, the target muscle is lengthened and ality traits, and other factors (Angel and Hogan
1992). Exercise is an antiaging strategy linked to regulation of genes that are responsible for
physical and cognitive rehabilitation, develop- expression of sarcomeric proteins and for recruit-
ment of positive stress, and lifestyle changes ment of satellite cells. Integrated treatment with
(Rebelo-Marques et al. 2018). The mind-to-body focused mechano-acoustic vibrations has a bene-
connection is indispensable for a motivated ficial effect on bone mineral density (BMD) and
engagement in antiaging behaviors, such as exer- T-score and on muscle strength and quality of life
cise, and in physical rehabilitation in case of a of osteoporotic subjects (Kwak et al. 2017;
disease-induced handicap (Wrycza and Baudisch Saggini et al. 2017).
2012; Depp and Jeste 2009). Exercise has a pro- The body’s agility to react to external forces
tective effect on cognitive functions and helps plays a central role in rehabilitation. Acting on
combat the deleterious effects of stress and this mechanism enables the most individual adap-
aging. The practice of proper physical exercise tation of a therapeutic protocol and thus the use of
can improve physiological and functional the entirety of the rehabilitation power and also
responses, notably cardiovascular fitness and the exposition of compensative reactions and
respiratory function, and thus physical health rebalancing of human body network (Fig. 1). To
and quality of life of the elderly (Bellomo et al. this end, microgravity environment is particularly
2017). Exercise also plays a key role in the assess- effective in reversing functional detriments and
ment and prevention of frailty in the elderly limits concerning daily activities in patients with
(Singh 2002). In adulthood, aerobic skills and muscle and balance disorders (Kaneda et al.
general physical activity are associated with 2008). The exemplary is the aquatic microgravity
lower mortality and morbidity from cardiovascu- environment, produced by immersion in water,
lar diseases. Benefits of physical activity also which improves body stability due to a decrease
include reductions in a risk for stroke, diabetes in neural system activation caused by a central
mellitus, cancer, and osteoporosis (Ruiz et al. redistribution of the blood volume (Barassi et al.
2008; Lee and Skerrett 2001; Bijnen et al. 1999; 2018a). A decrease in sympathetic activity,
Hakim et al. 1998; Paffenbarger et al. 1993). accompanied by increased parasympathetic activ-
Aging is inherently involved with a loss of ity, promoting vasodilation and blood circulation,
muscle mass and function leading to sarcopenia increases blood flow, accelerates cellular metabo-
and, consequently, to postural dysfunction lism and removal of unwanted materials, and
(Carmeli 2017; Lin and Bhattacharyya 2012). decreases pain sensitivity (Forestier and Françon
Sarcopenia is underlain by a reduction in protein 2008). The autonomic nervous system
synthesis and an increase in muscle protein deg- remodeling positively affects the perception pain
radation (Delli Pizzi et al. 2017). Further, the and fatigue associated with musculoskeletal
ability of muscle regeneration is severely disorders due mostly to decreasing muscle ten-
compromised by aging, which can lead to disabil- sion (Barassi et al. 2018d; Bellomo et al. 2012;
ity, particularly in patients with concomitant Yasui et al. 2010). The rebalancing of gait pattern
diseases or organ failures. Skeletal muscle dys- involves the development of relatively stable
function can be ameliorated by well-suited exer- changes in spinal and supraspinal networks that
cise that improves the function of muscle are modeled by proprioceptive and somatosen-
mitochondria (Barbieri et al. 2015). Beneficial sory sensory information specific for the task.
effects of focal vibrations on the well-being of The development of specialized training
ailing elderly patients are beyond doubt when protocols for assisted exercise in the microgravity
analyzing the results of studies on proprioception, environment hinges on the realization of the
muscle tone, and quality of gait (Toosizadeh et al. importance of locomotor learning and how the
2018; Saggini and Bellomo 2015). Vibrations of use of specific devices alters the demand stem-
300 Hz effectively increase muscle strength, act- ming from motor tasks (Wolpert et al. 2001). The
ing on neuromuscular receptors, Golgi tendon literature unambiguously shows that a specific
organs, Pacini corpuscles, as well as through the reeducation protocol in relieving body weight
can improve the static and dynamic balance, lead- of biology and function into the realm of rehabil-
ing to a better overall motor performance itation therapy (Fig. 1). It enables the optimiza-
(Moreno et al. 2013; Threlkeld et al. 2003). In tion of redundant biological circuits and
our daily practice, we implement the single- arrangements through personalized therapeutic
photon avalanche detector (SPAD), a photon interventions rather than application of rigidly
detecting system for optical communications. fixed therapy schemes (Trieschmann 1987). Fur-
SPAD is a device able to compose a microgravity ther research should scrutinize the physiological
environment during gait motion, which optimizes underpinnings of the bioprogressive approach to
the body tridimensional alignment in the body better understand its function and potential
weight support modality. It has a dual action: benefits to be gained in physiotherapy.
mechanical, which allows for a cortical-
subcortical neuromotor retraining aimed at the Ethical Approval This review article has been written in
reacquisition of a body scheme in three planes compliance with the ethical standards of the institutional
and/or national research committees and with the 1964
of the space, and functional, which minimizes the
Helsinki declaration and its later amendments. The article
energy consumption and increases the proprio- does not contain any studies with human participants or
ceptive signal inflow to cortical areas during gait animals performed by any of the authors.
(Delli Pizzi et al. 2017; Saggini et al. 2004). Thus,
SPAD improves postural stability through an
increase in exteroceptive and proprioceptive sen-
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# Springer International Publishing AG, part of Springer Nature 2018
Published online: 21 August 2018
Influence of Proprioceptive Neuromuscular

Facilitation on Lung Function in Patients
After Coronary Artery Bypass Graft Surgery
Małgorzata Bujar-Misztal and Andrzej Chciałowski
Abstract surgery of restrictive character, accompanied

Coronary heart disease is the most common by air trapping. We found a gradual improve-
cardiovascular disease. Primary prevention, ment after 2 months of home rehabilitation,
medication, and invasive approach, along with a significant trend for a better outcome
with the coronary artery bypass graft using PNF. We conclude that PNF holds a
(CABG), are used in cardiovascular prophy- potential for optimizing the outcome of pul-
laxis. Pulmonary rehabilitation, whose main monary rehabilitation after coronary bypass
task is to restore the patient’s optimum effi- surgery.
ciency and prevent complications of the respi-
ratory system, particularly resulting from Keywords
limited physical activity and immobilization, Cardiac rehabilitation · Coronary artery
plays an important role in the postsurgical bypass · Physiotherapy · Proprioceptive
treatment. The aim of this study was to evalu- neuromuscular facilitation · Pulmonary
ate the influence on lung function of proprio- function · Rehabilitation
ceptive neuromuscular facilitation
(PNF) vs. standard physiotherapy in patients
undergoing CABG surgery. The study 1 Introduction
included 42 patients scheduled for surgery,
divided into PNF (n ¼ 22; aged 62.3 8.4) Coronary heart disease is the most common car-
and standard rehabilitation (n ¼ 20; aged diovascular disease (Nichols et al. 2014). An
62.0 7.4) groups. The findings demonstrate important treatment element, aside from pharma-
a decrease in post-surgery lung function in cotherapy, is to counteract risk factors, adjust
both groups. The increased ratios of FEV1/ physical activity, balance the conditions of work
FVC and RV%TLC speak for a transient post- and rest, and treat comorbid conditions such as
operative disturbance in lung function after hypertension, diabetes, or obesity (Cassar et al.
2009). In some cases, invasive treatment is
required such as balloon angioplasty, stenting,
M. Bujar-Misztal (*)
Department of Cardiac Surgery, Military Institute of or coronary artery bypass graft (CABG) (Bravata
Medicine, Warsaw, Poland et al. 2007).
e-mail: [email protected] CABG surgery is classically performed
A. Chciałowski through median sternotomy. The violation of the
Department of Infectious Diseases and Allergology, chest wall and internal structures contributes to
Military Institute of Medicine, Warsaw, Poland
11
12 M. Bujar-Misztal and A. Chciałowski
pain when breathing, chest mobility impairments, groups: PNF (aged 62.3 8.4; n ¼ 22) and
cough reflex weakness, retention of secretions in standard rehabilitation (aged 62.0 7.4;
the bronchial tree, and sometimes lower lung n ¼ 20). The surgery was performed on a beating
lobes collapse, all of which may manifest in heart, without the use of extracorporeal circula-
lung dysfunction and arterial blood gas disorders tion. The principles of the rehabilitation methods
(El-Kader 2011; Wynne and Botti 2004; are presented in Table 1.
Westerdahl et al. 2001). Pulmonary rehabilita- During the 2-month period after surgery,
tion, whose task is to restore the patient’s opti- patients in both groups continued cardiac rehabil-
mum breathing efficiency, prevents itation according to the rules established during
complications mostly arising from physical inac- the hospital stay. Spirometry and body
tivity or immobilization (Pack et al. 2013; Leon plethysmography examinations were performed
et al. 2005), particularly after surgery. The using BodyBox 5500 (Medisoft, Limassol,
existing methods of physiotherapy take into Cyprus), according to the guidelines the
account the scope of hospital and posthospital European Respiratory Society (ERS) and Ameri-
treatment and the possibility of home-based reha- can Thoracic Society (ATS) (Curtis et al. 1994;
bilitation (Perk et al. 2012; Dylewicz et al. 2004). Efthimiou et al. 1991; Berrizbeitia et al. 1989).
Proprioceptive neuromuscular facilitation Table 2 shows the schedule of pulmonary func-
(PNF), known as Kabat-Kaiser rehabilitation, is tion examinations conducted in the study.
currently used in daily physiotherapy practice. In Data of respiratory variables are as the percent-
the method “P” stands for proprioception, i.e., age means SD of predicted values. The Shapiro-
sensory receptor-mediated collection of informa- Wilk test was used to assess normality of data
tion about the executed motion and spatial body distribution. A paired t-test or Wilcoxon’s test
positon, “N” for activation of neuromuscular was used to assess differences in each variable
structures, and “F” for facilitation. PNF between the time points of measurement in either
represents a holistic approach taking into account group patients of post-surgery rehabilitation. A
the therapeutic influence on the whole person, p-value <0.05 defined the statistically significant
including mental and social spheres, rather than differences. Calculations were carried out with R
just disease symptoms. The method is 2.13.0 software for Windows.
underpinned by a belief that bodily parts are inti-
mately interconnected and the treatment efforts
ought to be addressed to the whole (Adler et al. 3 Results
2008). Therefore, the aim of this study was to
determine the influence on lung function of 3.1 Spirometry
PNF vs. standard physiotherapy in patients after
CABG surgery. We found no signs of obstruction in any of the
patients subjected to either PNF or standard type
of cardiological rehabilitation after CABG sur-
2 Methods gery. The FEV1%FVC index was around 80%
and 90% in the PNF and standard rehabilitation
The study was approved by the Ethics Committee groups, respectively, and changed irrelevantly
for Scientific Research of the Academy of Physi- from the baseline level after 2-month-long post-
cal Education in Warsaw and by the Ethics Com- surgery rehabilitation (Table 3). In general, all the
mittee of the Military Institute of Medicine in variables were modestly reduced after 2-month-
Warsaw, Poland, and the patients gave informed long rehabilitation with either method; the reduc-
consent for study procedures. Forty-two patients, tion was more distinct in the PNF group in terms
aged 48–85, diagnosed with stable coronary of statistical significance, albeit the variables
artery disease, scheduled for elective CABG, remained in the normal level with the use of
were randomly divided into two physiotherapy both rehabilitative methods.
Influence of Proprioceptive Neuromuscular Facilitation on Lung Function in. . . 13
Table 1 Cardiological rehabilitation

Proprioceptive neuromuscular facilitation (PNF) Standard rehabilitation
Respiratory exercises Respiratory muscle training
Using proprioceptive stimulation of respiration Learning abdominal breathing
Learning of conscious and controlled breathing Intensified breath
Learning correct posture with the relaxation of shoulder girdle Teaching effective cough with chest stabilization
Learning effective cough with chest stabilization Light exercise tailored to the patient’s condition
Learning intensified breath
Relaxation
Physiotherapy sessions lasting 45 min; 2–3 times a day; 5 days a week
Anticoagulation was an additional pharmacotherapy in both groups
Table 2 Pulmonary function tests

Timeline of examinations
Pre-
surgery Post-surgery
Examination Pulmonary variables Day 3 Day 7 2 months
Spirometry FVC, FEV1, FEV1%FVC, PEF, FEF25, FEF50, FEF75 X – – X
Plethysmography VC, TGV, RV, TLC, RV%TLC X X X X
X marks the time of examination, FVC forced vital capacity, FEV1 forced expiratory volume in 1 s, FEV1/%FVC
Tiffeneau-Pinelli index, FEF25, FEF50, and FEF75 forced expiratory flow at 25%, 50%, and 75% of FVC, VC vital
capacity, TGV thoracic gas volume, RV residual volume, TLC total lung capacity
Table 3 Changes in spirometry variables from the base- standard method in patients with coronary artery bypass
line level after 2-month-long post-surgery rehabilitation graft (CABG)
using proprioceptive neuromuscular facilitation (PNF) or
PNF method Standard method
Variable Pre-surgery baseline 2 months post-surgery Pre-surgery baseline 2 months post-surgery
FVC 99.11 3.99 95.91 3.97 *** 104.98 3.77 96.98 3.65 **
FEV1 96.73 4.01 89.45 3.80 *** 99.54 4.05 90.73 3.66
FEV1%FVC 79.51 8.25 80.81 8.81 90.55 9.33 89.51 7.45
PEF 100.16 4.96 97.26 5.27 *** 104.96 4.02 98.69 4.84
FEF25 89.67 5.68 71.87 6.71* 71.75 8.00 58.33 7.19
FEF50 89.23 7.53 72.74 5.49 *** 90.34 8.73 76.92 6.22 *
FEF75 66.11 5.67 68.56 5.83* 98.45 6.90 93.59 5.74
Data are presented as % predicted value; *p < 0.05; **p < 0.01; ***p < 0.001
3.2 Plethysmography changed to 130.3 23.4% and 134.2 21.3%

on Day 7 post-surgery in the respective groups.
In the pre-surgery baseline examination, the RV% The tests conducted 2 months post-surgery,
TLC index, i.e., the proportion of residual capac- after a period of home-based rehabilitation,
ity to total lung capacity, amounted to showed a decline the RV%TLC index to
113.2 19.1% and 114.0 26.7% predicted in 109.3 30.1% and 108.2 19.5% in the PNF
the patients subjected to PNF and standard reha- and standard rehabilitation groups, respectively,
bilitation, respectively. This index increased compared with Day 3 post-surgery as well as with
significantly to 133.8 26.5% and the baseline pre-surgery examination, albeit the
133.8 24.5% on Day 3 post-surgery and latter decline failed to reach statistical signifi-
cance in both groups.
Table 4 Plethysmography variables in patients treated with proprioceptive neuromuscular facilitation (PNF) and
standard rehabilitation method at baseline pre-surgery and on the third day post-surgery
Variable Pre-surgery baseline Day 3 post-surgery Pre-surgery baseline Day 3 post-surgery
VC 88.38 20.27 49.35 13.53 100.54 20.44 60.75 15.06 *
TGV 112.36 20.69 96.09 20.29 *** 126.82 31.36 101.72 28.34 **
RV 111.39 23.35 90.91 34.44 120.74 40.91 99.47 39.50 *
TLC 93.89 16.79 63.01 16.12 ** 99.89 20.26 69.21 18.76
RV%TLC 113.21 19.09 133.75 26.51 114.04 26.68 133.80 24.49
standard rehabilitation method on the third and the seventh day post-surgery
Variable Day 3 post-surgery Day 7 post-surgery Day 3 post-surgery Day 3 post-surgery
VC 49.35 13.53 64.31 20.94 * 60.75 15.06 71.68 16.54 ***
TGV 96.09 20.29 101.00 27.70 *** 101.72 28.34 115.56 17.17 **
RV 90.91 34.44 107.12 34.75 *** 99.47 39.50 120.00 35.33 **
TLC 63.01 16.12 77.94 20.77 *** 69.21 18.76 84.57 17.74 ***
RV%TLC 133.75 26.51 130.32 23.41 133.80 24.49 134.20 21.28 **
standard rehabilitation method on the seventh day and then 2 months post-surgery
Variable Day 3 post-surgery 2 months post-surgery Day 3 post-surgery 2 months post-surgery
VC 64.31 20.94 88.51 21.66 *** 71.68 16.54 97.15 16.61
TGV 101.00 27.70 113.23 29.07 *** 115.56 17.17 118.47 23.49 ***
RV 107.12 34.75 109.02 39.53 *** 120.00 35.33 110.75 29.06
TLC 77.94 20.77 92.33 20.77 *** 84.57 17.74 97.17 16.59 *
RV%TLC 130.32 23.41 109.30 30.12 *** 134.20 21.28 108.17 19.50
Data are presented as % predicted value; *p < 0.05; ***p < 0.001
The results of body plethysmography are post-surgery in both groups. The examination
presented in detail in Tables 4, 5, 6, and 7. conducted 2 months post-surgery showed a sub-
There were a number of significant differences stantial increase in the variables, with an advan-
in specific variables, beyond those in the RV% tage for the PNF treatment.
TLC index above outlined. However, RV%TLC
is a reflection of lung air trapping after CABG
surgery, and thus it is of key importance for lung
4 Discussion
health.
It is worth noticing that the plethysmography
Rehabilitation is an important part of the treat-
variables were generally lower in patients treated
ment for patients after cardiovascular and thoracic
with PNF than those with standard rehabilitation.
surgeries, as it helps improve physical and respi-
The CABG caused a significant decrease in the
ratory capacity. Breathing exercises lead to a
value of individual variables, particularly on Day
reduction or complete elimination of potential
3 post-surgery, with a gradual rebound on Day 7
pulmonary complications resulting from impaired
standard rehabilitation method at baseline pre-surgery and 2 months post-surgery
Variable Pre-surgery baseline 2 months post-surgery Pre-surgery baseline 2 months post-surgery
VC 88.38 20.27 88.51 21.66 *** 100.54 20.44 97.15 16.61
TGV 112.36 20.69 113.23 29.07 *** 126.82 31.36 118.47 23.49 **
RV 111.39 23.35 109.02 39.53 120.74 40.91 110.75 29.06
TLC 93.89 16.79 92.33 20.77 ** 99.89 20.26 97.17 16.59
RV%TLC 113.21 19.09 109.30 30.12 114.04 26.68 108.17 19.50
Data are presented as % predicted value; **p < 0.01; ***p < 0.001
respiratory mechanics, increasing pain, cough, changes in the lungs. In a study of Shenkman
phlegm retention, or diaphragm impairment et al. (1997), impaired lung function, expressed
(Efthimiou et al. 1991). as the 25% reductions in FVC, FEV1, and PEF,
The present study enabled to relate functional was observed 3–4 months after surgery, while
breathing variables in patients after coronary reductions in FEF50 and FEF75 were sustained
artery bypass graft surgery to the pre-surgery for a longer time. A decline in lung function is
baseline and to compare the effects on lung func- due likely to a modified breathing pattern after
tion of PNF and standard rehabilitation methods chest opening. Damage to ribs limits the chest
in the extended post-surgery period of up to wall motion, and respiratory muscles weakness
2 months. We, generally, noticed reductions in usually persist for several months. In addition,
spirometry indices 2 months after revasculariza- atelectatic changes in the basal segments of
tion, pointing to impairment of airflow in large lower lung lobes and pleural damage contribute
and small bronchi, irrespective of rehabilitation. to respiratory dysfunction (Montes et al. 2004).
The reduction was somehow less in the The diaphragm dysfunction caused by a transient
PNF-rehabilitated patients. The FEV1/FVC phrenic nerve damage as a result of heart hypo-
index did not change significantly at the post- thermia also is a serious adverse effect that occurs
surgery time marks studied, which was due likely in about 22–31% of patients, even as late as up to
to the proportional reduction of both components. 1 year after surgery (Westerdahl et al. 2003, 2005;
Body plethysmography also revealed a decline in Curtis et al. 1994; Efthimiou et al. 1991). A
individual variables on the third and the seventh weaker cardiac mechanical function with low
day post-surgery. The decline was associated with ejection fraction may predispose to the develop-
increased value of RV%TLC that became ment of foci of pulmonary edema and an abnor-
normalized 2 months later but only in patients mal ventilation/perfusion ratio, followed by
undergoing PNF rehabilitation. hypoxia (Staton et al. 2005). Vaidya et al.
The PNF method apparently has never before (1996) have shown that spirometry variables
been used for rehabilitation after CABG surgery, decrease after mitral valve surgery for up to
so there is no comparative reference to the present 3 months, despite improved hemodynamic heart
findings. However, the general impairment of function.
lung function after CABG operations is in line Staton et al. (2005) have shown significant
with the results of other studies. A decline, some- reductions in FEV1 and FVC, with unchanged
times reaching 40–50%, in FVC and FEV1 has FEV1/FVC ratio, compared with the pre-surgery
been observed in the first 3 days post-surgery, baseline. However, the adoption of FEV1/FVC as
gradually normalizing after a few months the exponent of a possible restriction has been due
(Wynne and Botti 2004; Dubach et al. 1995; largely to the lack of TLC measurement.
Berrizbeitia et al. 1989). Rachwalik et al. (2006) Rachwalik et al. (2006, 2007), after a triple exam-
have observed a slight increase in FEV1/FVC, ination of patients, on the fifth day and 2.5 and
which points to the trend toward restrictive 6 months after cardiopulmonary bypass surgery,
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Published online: 29 September 2018
Remote Ischemic Preconditioning

in Renal Protection During Elective
Percutaneous Coronary Intervention
Małgorzata Wojciechowska, Maciej Zarębiński,

Piotr Pawluczuk, Dagmara Gralak-Łachowska,
Leszek Pawłowski, Wioletta Loska, Marzena Goszczyńska,
Krzysztof Flis, and Agnieszka Cudnoch-Jędrzejewska
Abstract criteria of the serum creatinine (SC) and

Remote ischemic preconditioning (RIPC) cystatin C (CC) levels. The rates of major
exerts protection in remote organs. The pur- cardiac and cerebral adverse events (MACCE)
pose of this study was to investigate the poten- during 1 year follow-up were evaluated. We
tial of RIPC to prevent contrast induced found that contrast-induced nephropathy
nephropathy. One hundred and twenty four assessed by SC occurred in 4.9% (3/61) patients
patients were randomized to elective percuta- with RIPC and in 12.1% (7/58) patients without
neous coronary intervention with or without it (p ¼ 0.20). Nephropathy assessed by CC
RIPC. RIPC was performed using three cycles occurred in 1.7% (1/58) patients with RIPC
of 5-min inflation to 200 mmHg of a standard and 3.5% (2/57) patients without it (p ¼ 0.62).
upper arm blood pressure cuff. The time There was no coincidence between the diagno-
between the last inflation cycle and the coro- sis of contrast-induced nephropathy based on
nary intervention was less than 2 h. The pri- SC and CC (McNemar test 0.012, κ ¼ 0.28);
mary endpoint was the incidence of contrast- SC was a more sensitive marker of nephropathy
induced nephropathy based on the standard than CC (ten and three cases, respectively). The
MACCE rate during the year of follow-up
M. Wojciechowska (*) tended to be lower with the ischemic
Department of Experimental and Clinical Physiology, preconditioning than without it, four vs. six
Center for Preclinical Research, Warsaw Medical
University, Warsaw, Poland
cases, respectively. We conclude that RIPC
prior to percutaneous coronary intervention
Invasive Cardiology Unit, Western Hospital, Grodzisk
Mazowiecki, Poland
has no major influence on the development of
e-mail: [email protected] contrast-induced nephropathy and does not
M. Zarębiński, P. Pawluczuk, D. Gralak-Łachowska, L.
improve the one-year outcome.
Pawłowski, W. Loska, M. Goszczyńska, and K. Flis
Invasive Cardiology Unit, Western Hospital, Grodzisk Keywords
Mazowiecki, Poland Coronary intervention · Kidney insufficiency ·
A. Cudnoch-Jędrzejewska Nephropathy · Percutaneous coronary
Department of Experimental and Clinical Physiology, intervention · Remote ischemic
Center for Preclinical Research, Warsaw Medical
University, Warsaw, Poland
preconditioning · Renal protection
19
20 M. Wojciechowska et al.
1 Introduction 2 Methods
Contrast-induced nephropathy is defined as an 2.1 Study Population

increase in the serum creatinine
(SC) concentration of >0.5 mg/dL or 25% above This is a prospective, single-center trial that was
the baseline level within 48–72 h after parenteral conducted from October 2012 to September 2013
administration of a radiocontrast dye. The inci- in the Invasive Cardiology Unit of Western
dence of nephropathy, despite the use of low– Hospital in Grodzisk Mazowiecki, Poland. A
osmolar contrast media and appropriate hydra- hundred and twenty-four patients aged 18 years
tion, remains significant (Bartorelli and Marenzi or older, with stable coronary angina and no his-
2008; Wong and Irwin 2007; McCullough et al. tory of end-stage renal disease, admitted to the
2006; Rihal et al. 2002). The Mehran risk score is hospital for elective PCI, were enrolled into the
a simple and well-tested scale that has been devel- study. Sixty-two patients were allocated to
oped to provide the risk of contrast-induced receive standard therapy (control group) and
nephropathy after percutaneous coronary inter- another 62 to receive standard therapy plus
vention (PCI) (Raingruber et al. 2011; Mehran RIPC (RIPC group). One patient from the control
et al. 2004). group was excluded due to the acute stent throm-
Serum cystatin C (CC) is an early predictive bosis and the need of urgent angioplasty 2 h after
biomarker for acute kidney injury. Various stud- primary PCI.
ies demonstrate the superiority of CC compared
to SC, especially to detect a minor reduction in
glomerular filtration rate (GFR). In addition, SC
2.2 Study Protocol
concentration typically goes up 48–72 h after the
contrast use, whereas CC shows to highest value
Patients were numbered depending on the order
after 12–24 h (Herget-Rosenthal et al. 2004;
of admission to the hospital. They were then
Dharnidharka et al. 2002; Mussap et al. 2002).
assigned to receive elective PCI (control group)
Remote ischemic preconditioning (RIPC) is a
or PCI with RIPC (RIPC group). All the patients
method consisting of multiple brief episodes of
were given 500 ml of saline and 600 mg of
ischemia applied to one organ, which carries pro-
N-acetylcysteine intravenously twice: before and
tection against a subsequent prolonged ischemia
after PCI. They were also asked to drink 1.5 L of
applied to remote organs (Zimmerman et al.
still water during the day of the procedure. Con-
2011; Hausenloy and Yellon 2008). Reversed
comitant drugs, such as diuretics, angiotensin-
distribution of blood in the kidneys and ischemia
converting enzyme inhibitors (ACEI), angioten-
of the outer kidney medulla are two of the
sin II receptor antagonists (AT1 blockers), and
pathomechanisms for developing contrast-
metformin, were permitted.
induced nephropathy (Sendeski 2011;
Goldenberg and Matetzky 2005; Heyman et al.
1994). Suitability of RIPC to prevent kidney
injury after contrast administration has been 2.3 Procedures
evaluated in a few studies. The results are incon-
clusive (Gholoobi et al. 2015; Menting et al. RIPC was performed by three cycles of 5-min
2015; Yamanaka et al. 2015; Igarashi et al. inflation to 200 mmHg and 5-min deflation of a
2013; Er et al. 2012). Therefore, the purpose of standard upper arm blood pressure cuff. The time
this trial was to investigate the potential of RIPC between the last inflation cycle and PCI was <2 h.
for renal protection during elective PCI. PCI was performed according to the standard
clinical practice. In all patients, a nonionic
Remote Ischemic Preconditioning in Renal Protection During Elective. . . 21
iodinated, water-soluble, radiographic contrast 3 Results

medium (iopromide; iodine concentration
370 mg/ml, osmolality 0.77 Osm/kg H2O at Demographic, clinical, basic biochemical, and
37 C) was used. Patients from the control angiographic data of the patients are shown in
group had blood pressure measured only 2 h Table 1.
before PCI. The clinicians performing PCI were
not involved in the recruitment of patients into the
study, numbering the patients, or in the
3.1 Contrast-Induced Nephropathy
preconditioning procedure.
Assessed by Serum Creatinine
(SC)
2.4 Outcomes The assessment of contrast-induced nephropathy

was possible in 119 patients (61 RIPC, 58 control)
The primary outcome was the incidence of as 1 patient from RIPC and 3 patients from the
contrast-induced nephropathy defined as SC ele- control group had no complete biochemical data.
vation 25% or 0.5 mg/dL over the baseline The mean increase in SC after 24 and 72 h did not
level in a period of 24–72 h after contrast admin- differ between the two groups (24 h: RIPC
istration. Additionally, the incidence of contrast- 0.01 0.10 vs. control 0.04 0.10; p ¼ 0.087;
induced nephropathy, defined as serum CC eleva- 72 h: 0.03 0.09 vs. 0.04 0.08; p ¼ 0.623).
tion 25% 24 h after contrast administration, was Nephropathy measured by SC occurred in 4.9%
evaluated, and the coincidence between CC and patients (3/61) in RIPC group and in 12.1%
SC was assessed. Standard assays were used to patients (7/58) in the control group, p ¼ 0.20
measure the serum concentration of creatinine (Table 2).
and cystatin C. The composite endpoint of
major adverse cardiac and cerebrovascular events
(MACCE) consisted of acute coronary syndrome, 3.2 Contrast-Induced Nephropathy
transient ischemic attack, or stroke during a Assessed by Serum Cystatin C
1-year follow-up. (CC)
The assessment of contrast-induced nephropathy

2.5 Statistical Analysis was possible in 115 patients (58 RIPC and 57 con-
trol) as 4 patients from RIPC and 4 patients from
Continuous variables are presented as arithmetic control group had no data on CC after 24 h. The
means SD for normally distributed variables or mean increase in CC after 24 h did not differ
as medians and interquartile ranges in case of a between the RIPC and control groups
skewed distribution. Differences between groups (0.01 0.13 vs. 0.01 0.11; p ¼ 0.827).
for continuous data were tested by Student’s t-test Nephropathy measured by CC occurred in 1.7%
or Mann-Whitney rank test, as appropriate. Cate- patients in RIPC and 3.5% patients in the control
gorical variables are reported as counts and group; p ¼ 0.62 (Table 2).
percentages. Differences between categorical
data were assessed by Chi-squared test or Fisher’s
exact test. A two-sided p-value <0.05 was consid- 3.3 Coincidence Between Serum
ered statistically significant. All analyses were Cystatin C (CC) and Serum
performed with SAS software v9.2 (SAS Insti- Creatinine (SC)
tute; Cary, NC).
Data on CC were available in all patients with
contrast-induced nephropathy identified on the
Table 1 Patients’ demographic, clinical, basic biochemical, and angiographic indices

RICP (n ¼ 62) Control (n ¼ 61) p
Dermographics
Age, yr 64.4 10.5 62.9 8.6 0.398
BMI, kg/m2 29.6 4.6 29.3 4.4 0.709
Female, n (%) 20 (32.3%) 19 (31.2%) 0.895
Biochemical indices
Serum creatinine, mg/dL 0.86 0.27 0.86 0.26 0.949
GFR, mL/min 105.6 36.3 107.3 36.5 0.801
Urea, mg/dL 36 (28–45) 37 (30–44) 0.422
Urea-to-creatinine ratio 43.5 9.8 46.3 11.8 0.159
Serum cystatin C, mg/L 0.88 0.23 0.88 0.24 0.855
Troponin Ths, ng/mL 0.01 (0.01–0.02) 0.01 (0.01–0.02) 0.931
Hemoglobin, g/dL 14.2 1.2 13.8 1.3 0.066
Hematocrit, % 41.2 3.0 40.1 3.3 0.052
Clinical data (examination, medical history, and medications)
SBP, mmHg 137.2 19.4 136.4 21.9 0.827
DBP, mmHg 82.3 13.8 80.6 12.6 0.472
LVEF, % 52.5 7.6 53.2 8.6 0.605
CKD, n (%) 3 (4.8%) 2 (3.3%) 1.000
Diabetes, n (%) 13 (21.0%) 16 (26.2%) 0.492
Arterial hypertension, n (%) 45 (72.6%) 47 (77.1%) 0.568
MVD, n (%) 33 (53.2%) 33 (54.1%) 0.923
ACEI/AT1 blockers, n (%) 54 (87.1%) 58 (95.1%) 0.121
Statins, n (%) 56 (90.3%) 55 (90.2%) 0.976
Diuretics, n (%) 29 (46.8%) 31 (50.8%) 0.654
Percutaneous coronary intervention
Contrast volume, mL 155.5 76.7 143.7 55.7 0.331
Duration of PCI, min 45 (30–60) 43 (30–60) 0.978
Mehran risk score 3.2 2.6 2.9 2.3 0.555
Mehran risk score > 2.0 30 (48.4%) 30 (49.2%) 1.000
RIPC remote ischemic preconditioning group, BMI body mass index, GFR glomerular filtration rate, Troponin Ths high-
sensitivity troponin T, SBP systolic blood pressure, DBP diastolic blood pressure, LVEF left ventricular ejection fraction,
CKD chronic kidney disease, MVD multivessel coronary disease, ACEI angiotensin-converting enzyme inhibitors, AT1-
blockers angiotensin II receptor antagonists, TIA transient ischemic attack, PCI percutaneous coronary intervention
Table 2 Remote ischemic preconditioning (RICP) and incidence of contrast-induced nephropathy (CIN)
RICP (n ¼ 62) Control (n ¼ 61) p
Serum creatinine 24-B, mg/dL 0.01 0.09 0.04 0.10 0.087
Serum creatinine 72-B, mg/dL 0.03 0.09 0.04 0.08 0.623
Serum cystatin C 24-B, mg/dL 0.00 0.13 0.01 0.11 0.827
CIN (SC) 3/61 (4.9%) 7/58 (12.1%) 0.197
CIN (CC) 1/58 (1.7%) 2/57 (3.5%) 0.618
CIN (CC) contrast-induced nephropathy assessed by serum cystatin C criteria, CIN (SC) contrast-induced nephropathy
assessed by serum creatinine criteria, Serum creatinine 24-B difference between serum creatinine concentration
measured after 24 h and the baseline (B) level, Serum creatinine 72-B difference between serum creatinine concentration
measured after 72 h and the baseline level, Serum cystatin C 24-B difference between serum cystatin C concentration
measured after 24 h and the baseline level
basis of SC. There was no coincidence between been demonstrated to prevented contrast-induced
the diagnosis of contrast-induced nephropathy nephropathy in patients with ST-elevation
based on SC and CC (McNemar test: 0.02, myocardial infarction (Yamanaka et al. 2015) or
κ ¼ 0.28). Serum creatinine measured twice in coexisting chronic kidney disease (Igarashi
(24 and 72 h after PCI) appeared to more accu- et al. 2013; Er et al. 2012), with a high Mehran
rately identify all patients with contrast-induced risk score (Mehran et al. 2004). After a thorough
nephropathy than CC measured once (from analysis of all the studies, along with the present
10 cases of contrast-induced nephropathy based results, we conclude that RIPC may be of less
on SC, only 3 met criteria to recognize contrast- importance in case of elective PCI and low-risk
induced nephropathy based on CC). patients, but it may be particularly useful in unsta-
ble patients with a high to very high risk of
contrast-induced nephropathy.
3.4 One-Year Follow-Up The mechanisms underlying contrast-induced
nephropathy are multifactorial, and it is possible
One-year follow-up was possible in case of that they vary depending on comorbidities and
95 patients: 9 (90%) with contrast-induced indications for PCI (elective vs. urgent). In
nephropathy (CIN+) and 86 (79%) without it lower-risk patients, the plausible mechanism of
(CIN–). The RIPC had no appreciable influence contrast-induced nephropathy may be a direct
on the occurrence of major adverse cardiac and toxicity of contrast on tubular cells, caused or
cerebrovascular events during the 1-year follow- aggravated by patient’s dehydration (Sendeski
up, nor did it significantly change the MACCE 2011). In case of higher-risk patients, the
index (Table 3). pathomechanism may be more complicated as
hypoxic injury may be a predominant factor.
RIPC may prevent hypoxic injury and thus may
4 Discussion have a renoprotective effect in higher-risk
patients, but not in the general population.
In this study, general risk for developing contrast- Urea-to-creatinine (U-to-SC) ratio is used to
induced nephropathy was 8.4% (10/119 patients), determine the patient hydration. An abnormal
and it was compatible with the predicted proba- value that could indicate hypovolemia and a
bility based on the Mehran risk score (Mehran reduced renal perfusion is more than 40 (Higgins
et al. 2004). The study demonstrates that RIPC 2016). In the study, baseline mean U-to-SC ratio
applied prior to elective PCI failed to provide an in the patients of both groups was 45.1 1.3, and
added benefit to hydration and N-acetylcysteine it was over 40 in 63.4% of patients. That indicates
in decreasing the incidence of contrast-induced that more than 60% of patients could be
nephropathy. There was a trend toward lower hypovolemic on admission. We demonstrate a
numerical incidence of contrast-induced nephrop- positive association between higher baseline U-
athy, assessed on the basis of both SC and CC, to-SC ratio and contrast-induced nephropathy
and also lower increase in SC and CC levels after (55.1 14.8 in CIN+ vs. 43.9 10.0 in CIN–;
PCI in the RIPC group; the differences were p < 0.002), which means that more adequate fluid
however insignificant. As the risk for developing therapy should be implemented in selected
contrast-induced nephropathy was generally low patients for preventing nephropathy. In this
in our patients (Mehran risk score about 3 points; study, patients did not interrupt treatment with
Table 1), these results are in line with similar potentially nephrotoxic drugs, such as ACEI and
studies in the literature, where RIPC failed to AT1 blockers, so we indirectly demonstrate that
improve kidney function after contrast adminis- such drugs did not increase the risk of
tration in patients with low-to-moderate risk of nephropathy.
contrast-induced nephropathy (Gholoobi et al. CC is considered an early marker of kidney
2015; Menting et al. 2015). However, RIPC has injury. SC typically goes up between 48–72 h
Table 3 Effects on the occurrence of major adverse cardiac and cerebrovascular events of remote ischemic
preconditioning (RIPC) during 1-year follow-up
RICP (n ¼ 62) Control (n ¼ 61)
n ¼ 47 (75.8%) n ¼ 48 (78.7%) p
TIA/stroke n (%) 1 (2.1%) 1 (2.1%) 1.000
ACS (UA/MI) n (%) 4 (8.5%) 5 (10.4%) 1.000
MACCE n (%) 4 (8.5%) 6 (12.5%) 0.740
TIA Transient ischemic attack, ACS (UA/MI) acute coronary syndrome (unstable angina/myocardial infarct), MACCE
composite endpoint defined as acute coronary syndrome, transient ischemic attack, or stroke
after the use of contrast, whereas CC reaches the after percutaneous coronary intervention does not
highest value 12–24 h after PCI (Herget- detect all cases of contrast-induced nephropathy.
Rosenthal et al. 2004; Dharnidharka et al. 2002; Remote ischemic preconditioning before percuta-
Mussap et al. 2002). As patients are usually neous coronary intervention does not influence
discharged from the hospital 24 h after PCI, CC 1-year follow-up outcome.
might be a better option to identify contrast-
induced nephropathy. The present results indi- Conflicts of Interest The author declares no conflicts of
cate, however, that SC measured twice, 24 and interest in relation to this article.
72 h after PCI, may be more accurate to detect
nephropathy as we found ten CIN+ cases based Ethical Approval All procedures in this study were
performed in accordance with the ethical standards of the
on SC and three on CC.
institutional research committee and with the 1964
In previous studies, RIPC performed before Helsinki Declaration for Human Research and its later
PCI has been shown to improve clinical outcomes amendments.
during the following 4 (Yamanaka et al. 2015) or
6 weeks (Er et al. 2012). In this study, RIPC, Informed Consent Written informed consent was
performed before elective PCI, failed to influence obtained from all individual participants included in the
study.
the incidence of contrast-induced nephropathy or
the MACCE index during in 1-year follow-up.
Nonetheless, we cannot rule out the appearance
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Published online: 29 June 2018
Prognostic Impact of Extracapsular Lymph

Node Invasion on Survival in Non-small-Cell
Lung Cancer: A Systematic Review
and Meta-analysis
Seyed Vahid Tabatabaei, Christoph Nitche, Maximilian Michel,

Kurt Rasche, and Khosro Hekmat
Abstract with publication date since 1990. Prospective

The extracapsular tumor extension (ECE) of or retrospective studies reporting data on prog-
nodal metastasis is an important prognostic nostic parameters in subjects with NSCLC
factor in different types of malignancies. How- with positive ECE or with only intracapsular
ever, there is a lack of recent data in patients lymph node metastasis were retrieved. Data
with non-small-cell lung cancer (NSCLC). In were summarized using risk ratios (RR) for
addition, the TNM staging system does not the survival with 95% confidence intervals
include ECE status as a prognostic factor. (CI). The data was analyzed using Mix 2 (ref:
This systematic review and meta-analysis has Bax L: MIX 2.0 – Professional software for
been conducted to summarize and pool meta-analysis in Excel. Version 2.015.
existing data to determine the prognostic role BiostatXL, 2016. https://www.meta-analysis-
of ECE in patients with lymph node-positive made-easy.com). There 2,105 studies were
NSCLC. Two authors performed an indepen- reviewed. Five studies covering a total of
dent search in PubMed using a predefined 828 subjects met the inclusion criteria and
keyword list, without language restrictions were included in the meta-analysis. Two hun-
dred and ninety-eight (35.9%) patients were
categorized as ECE+, of whom 54 (18.1%)
S. V. Tabatabaei and K. Rasche survived at the end of follow-up. In the
Department of Internal Medicine, Division of Pulmonary,
Allergy, and Sleep Medicine, HELIOS Clinic Wuppertal,
ECE-negative group, 257 patients (48.4%)
Witten/Herdecke University, Wuppertal, Germany survived by the end of follow-up. Thus, ECE
C. Nitche
status is associated with a significantly
Department of Internal Medicine, Marien Hospital decreased survival rate: pooled RR 0.45
Euskirchen, Teaching Hospital of Uniklinik Bonn, (95% CI 0.35–0.59), Q(4) ¼ 4.06, Pvalue ¼ 0.39,
Euskirchen, Germany and I2 ¼ 68.00% (95 CI 0.00–79.55%). In
M. Michel conclusion, ECE has a significant impact on
Institute of Zoology, University of Cologne, Cologne, survival in NSCLC patients and should be con-
Germany
sidered in diagnostic and therapeutic decisions
K. Hekmat (*) in addition to the current TNM staging. Post-
Department of Cardiothoracic Surgery, University Clinic
of Cologne, Cologne, Germany
operative radiotherapy may be an option in
e-mail: [email protected] ECE-positive pN1 NSCLC patients.
27
28 S. V. Tabatabaei et al.
Keywords better prognosis than patients with N2 status

Extracapsular lymph node invasion · Meta- (hilar lymph nodes on the same side of the
analysis · Metastases · Non-small-cell lung tumor) (Dai et al. 2016). However, even within
cancer · Prognostic factor · Survival rate the same lymph node stage, prognosis for patients
differs according to the number and the ratio of
the positive mediastinal lymph nodes
(Jonnalagadda et al. 2011; Wisnivesky et al.
1 Introduction 2011), single-station or multiple-station involve-
ment (Misthos et al. 2008), and the presence of
Lung cancer is the most common cancer due to skip metastasis or micrometastasis (Zhang et al.
the increasing number of cigarette smokers and 2016; Kerr and Nicolson 2013; Wang et al. 2001).
air pollution exposure (Eckel et al. 2016). Cur- Importantly, extracapsular lymph node extension
rently, it is the leading cause of malignancy- (ECE) has been shown to have a prognostic sig-
related death in male patients and runs in second nificance in various types of cancer such as
among female cancer patients worldwide (Torre breast (Fujii et al. 2016), head and neck (Kelder
et al. 2016). Approximately 75–85% of all lung et al. 2012), colorectum (Veronese et al. 2016),
cancer patients are diagnosed with non-small-cell oral cavity (Taghavi and Yazdi 2015), gastric
lung cancer (NSCLC) (Le Péchoux 2011). (Alakus et al. 2010), cervix cancer (Metindir and
According to the current guidelines, curative Bilir Dilek 2008), melanoma (Khosrotehrani et al.
resection by tumor surgery is the first-choice 2014), and vulva cancers (Luchini et al. 2016).
treatment for stage IA-IIIA NSCLC (Ettinger The present study describes the results of a meta-
et al. 2014; Van Schil et al. 2013). Postoperative analysis we conducted to evaluate the prognostic
adjuvant chemotherapy and thoracic radiotherapy significance of ECE in NSCLC. The data suggest
have been shown to improve the outcome in that ECE status should be taken into consider-
resected stage II and IIIA NSCLC and minimize ation as a prognostic factor in NSCLC.
the risk of locoregional recurrence (Hui et al.
2015; Gomez and Komaki 2012; Bradley et al.
2005). Bronchovascular reconstructive 2 Methods
procedures are evolving in order to avoid pneu-
monectomy in functionally unsuitable patients 2.1 Inclusion and Exclusion Criteria
(Maurizi et al. 2016).
Several clinical factors such as tumor differen- This systematic review adheres to the MOOSE
tiation (Kuo et al. 2014), lymphatic (Al-Alao et al. guidelines (Stroup et al. 2000) and PRISMA
2014) or blood vessel invasion (Naito et al. 2010), statement (Liberati et al. 2009), using predefined
serum carcinoembryonic antigen elevation, and protocols. The following criteria were employed
tumor size (Igai et al. 2011) are considered to for the studies to be eligible for inclusion: (1) pro-
have a significant influence on survival and spective or retrospective; (2) use of a curative
local recurrence rate after a curative tumor surgical approach such as lobectomy or pneumo-
resection. In contrast, tumor histology for nectomy; (3) presence of a statistical comparison
adenocarcinoma vs. squamous cell carcinoma of survival between ECE+ and ECE– patients;
does not seem to play an important role in prog- (4) ECE+ was defined as cancer cells invading
nosis (Kerr and Nicolson 2013). Intriguingly, the beyond the capsule of a lymph node; (5) non-
impact of lymph node staging on patient progno- small-cell lung cancer needed to be histologically
sis is highly complex. As such, staging of the diagnosed; (6) published in a peer-reviewed jour-
mediastinal lymph nodes has been reported to nal; (7) publication year between 1990 and 2017
result in a very heterogeneous picture (Goldstraw in any language; (8) patients with nodal status
et al. 2007). In NSCLC stage IV, patients with N1-N2; and (9) NSCLC staging IIa–IIIa. Exclu-
lymph nodes in the lung (status of N1) have a sion criteria were the following: (1) copresence of
Prognostic Impact of Extracapsular Lymph Node Invasion on Survival in. . . 29
other malignancies; (2) lack of data about prog- Scores 5 out of the 9 possible were judged to
nostic parameters in the title/abstract; (3) insuffi- indicate a high risk of bias.
cient data to enable a statistical comparison of The pooled risk ratios (RR) and 95% confi-
survival between ECE+ and ECE– patients; dence intervals (CI) of all-cause mortality
(4) NSCLC stage IV; (5) palliative therapy of a between ECE+ and ECE patients were calcu-
subgroup of patients without sufficient data to lated using DerSimonian–Laird random-effect
exclude them in a secondary analysis; models (DerSimonian and Laird 1986). Hetero-
(6) in vitro or animal studies; (7) neo-adjuvant geneity across studies was assessed by the I2
chemoradiotherapy; and (8) nodal status N3. metric and χ2 statistics (Higgins and Thompson
2002).
2.2 Data Sources and Literature

Search Strategies 3 Results
Two investigators independently conducted a We identified 2105 non-duplicated articles, out of

PubMed search in June 2017 with a time limit of which 2096, after reviewing the title and abstract,
publication date since 1990 using the following were judged as inadequate for further elaboration
combination of mesh terms: (1) ((“Carcinoma, to meet the goal of this meta-analysis. The
Bronchogenic”[Mesh]) AND “Neoplasm remaining nine full-text articles were retrieved
Metastasis”[Mesh]) AND “Lymph Nodes”[Mesh])); for more detailed analysis. Out of the nine
(2) ((“Prognosis”[Mesh]) AND “Carcinoma, articles, four were further excluded for the follow-
Bronchogenic”[Mesh]) AND “Lymphatic ing reasons: one was an editorial (Riquet 1996),
Metastasis”[Mesh])). References of previously two lacked a comparison of outcomes between
published related review articles were also consid- ECE+ and ECE patients (Riquet et al. 1995;
ered. The reviewers screened the titles and abstracts Bollen et al. 1994), and one provided only a
of all retrieved articles. If extracapsular or p-value (Borghetti et al. 2016). As a result, five
intracapsular tumor extension was mentioned, the articles were considered for this meta-analysis
full text was included into the review. The final list (Liu et al. 2015; Moretti et al. 2009; Lee et al.
of chosen studies was reached through a consensus. 2007; Vansteenkiste et al. 1997; Duran-Cantolla
One investigator extracted the data from the et al. 1994).
included articles. Data were validated by another
investigator and inserted into an Excel database.
For each study, the authors’ information, year and 3.1 Study and Patient Characteristics
country of publication, number and gender of
patients, age, tumor stage, smoking, vascular Data from 828 patients were analyzed. The
invasion, and duration of follow-up were patients’ clinical and demographic characteristics
extracted. are provided in Table 1. Two studies were
conducted in Europe, two in Asia, and one in
the USA. While four of the articles contained
2.3 Outcomes the patients’ characteristics, Duran–Cantolla
et al.’s (1994) did not. There was a greater pro-
The primary outcome was survival after treatment portion of men than women patients reported,
at the end of the follow-up period. We applied the with a mean or median age in a range
Newcastle–Ottawa Scale to evaluate the method- 59–62 years. Two articles investigated a head-
ological quality of non-randomized trials. The to-head survival of ECE+ and ECE patients
following three areas of the studies were (Liu et al. 2015; Lee et al. 2007). In the remaining
evaluated: selection of participants, comparability articles, data were extracted from a subgroup
of participants, and comparability of outcomes. analysis (Moretti et al. 2009; Vansteenkiste et al.
Table 1 Patients’ characteristics
Poor Vascular
Mean/Median differentiation invasion Squamous cell carcinoma Pneumonectomy R0–Resection
age (year) Male (%) T4 (%) N2 (%) (%) (%) (%) Smoking (%) Stage IIIA (%) (%) (%)
Study ECE ECE ECE ECE ECE ECE ECE ECE ECE
Author country NOS + ECE + ECE + ECE + ECE + ECE ECE+ ECE + ECE + ECE + ECE ECE+ ECE + ECE
Lee et al. Taiwan 8 62 (32–-82) 58 42 NR NR NR 67 33 86 14 51 49 57 43 75 25 NR 100

(2007)
Liu et al. China 8 59 (27–79) 21 79 0 24 76 22 78 NR 15 85 21 79 0 28 72 100

(2015)
Duran Spain 7 NR NR NR 33 NR NR NR NR 37 NR NR
Cantolla et al.
(1994)
Vansteenkiste Belgium 7 61 (40–76) 93 9 14 35 21 61 NR NR 80 87

et al. (1997)
Moretti et al. USA NR 61 (35–85) 58 NR 100 NR NR 38 NR 100 NR 85

(2009)
NOS Newcastle–Ottawa Scale score, ECE+ with extracapsular tumor extension, ECE without extracapsular tumor extension, T4 locally advanced lung cancer, N2 cancer is in lymph nodes,
Stage IIIA cancer spread to lymph nodes on the same side of the chest as the tumor, R0-Resection a microscopically margin-negative resection, NR not reported
1997; Duran-Cantolla et al. 1994). Only Liu 4 Discussion

et al. (2015) and Moretti et al. (2009) report a
tumor-free survival as the main outcome. In the The extracapsular tumor extension (ECE) of
former article, there was neither T4 nor stage IIIa nodal metastasis has been known as a prognostic
reported, although these stages were not set as the factor for multiple cancer malignancies, such as
exclusion criteria. The median score on the oral cavity, colon, gastric, cervix cancer, and
Newcastle–Ottawa Scale was 7 (range 7–8 melanoma. In each case, ECE-positive status is
points), with no single score pointing to the high associated with a higher rate of all-cause mortal-
risk of quality bias, i.e., 5 points. ity (Chen et al. 2016; Taghavi and Yazdi 2015;
Khosrotehrani et al. 2014; Alakus et al. 2010;
Metzger et al. 2009; Metindir and Bilir Dilek
3.2 Risk Ratio for Overall Survival 2008; Wind et al. 2007). The ECE has also been
included in the last versions of staging for squa-
Pooling data from the five studies reporting mous cell carcinoma of the vulva (Luchini et al.
survival, it turned out that only 18.1% of the 2016). Our results emphasize the importance of
ECE+ patients survived, while the survival rate ECE status in the prognosis of NSCLC. In this
amounted to 48.4% in the ECE– patients. This meta-analysis covering 828 patients, we show
difference in the survival rate was highly signifi- that ECE+ status is associated with poorer out-
cant, showing that ECE-positive status leads to a come in NSCLC patients. ECE+ status has also
distinct decrease in patient survival. The pooled been associated with a higher rate of high-grade
RR was 0.45 (95% CI 0.35–0.59); Q(4) ¼ 4.06, tumors, with vascular and lymph vessel invasion,
Pvalue ¼ 0.39, and I2 ¼ 68.00% (95 CI 0.00–79.55%) and with tumor protein P53 overexpression (Lee
(Fig. 1). The contour-enhanced funnel plot et al. 2007). These could be the physiopathologic
(Peters et al. 2008) of the studies included in the causes behind a worse outcome in ECE+ status.
meta-analysis indicated no publication bias, as Postoperative radiotherapy (PORT) has
confirmed by the Begg–Mazumdar test become the standard therapy in NSCLC patients
(Kendall’s τ ¼ 0.00, Pvalue ¼ 1.0) (Fig. 2). with stage II to IIIA in the last decade. The
According to classic fail-safe N, 33 nonsignificant locoregional relapse of tumor is reported to be
studies are needed to make the pooled Pvalue around 40% in these patients, and PORT plays
nonsignificant. an important role in patient survival (Jing et al.
2016). In contrast, for patients with pN0 and pN1
nodal disease, PORT is associated with a signifi-
cant decrease in survival. Consequently, PORT is
Fig. 1 Summary of survival risk ratios (RR) in original studies and the pooled estimate; RR risk ratio
Fig. 2 Publication bias

analysis using a contour-
enhanced funnel plot; RR
risk ratio
reserved only for pN2 and pN3 status. Since the This result, in conjunction with our finding,
findings of the present review suggest a decreased suggests that ECE plays an important role in the
survival in the ECE+ group, we hypothesize that prognosis of solid tumors as well as NSCLC, and
PORT could benefit the subgroup of pN1 patients consequently it should be incorporated into the
with extra-nodal lymph node invasion. It would TNM staging criteria.
be interesting whether this approach increases It is important to know that the penetration of
survival compared to no pN1-PORT in NSCLC the lymph node capsule can be investigated after
(Borghetti et al. 2016; Zaric et al. 2013), similar surgical lymph node dissection (Gu et al. 2017).
to pN1 breast cancer where PORT has been Since ECE is very focal, the surrounding fatty
shown to benefit ECE+ patients (Belkacemi tissue has to be completely included in surgical
et al. 2015). Lynch et al. (2014) have shown that resection of lymph nodes, in order to make a valid
in patients with squamous cell carcinoma of the statement about tumor extension (Veronese et al.
tonsil, ECE status is predictive of contralateral 2016). In lung cancer patients, endobronchial
nodal recurrence. Consequently, prophylactic ultrasound-guided transbronchial needle aspira-
contralateral radiotherapy has been suggested to tion (EBUS-TBNA) has recently been widely
improve overall survival in these patients. The used for mediastinal lymph nodes staging and as
same could also be true for NSCLC patients a minimal invasive staging tool has replaced
with lymph node status N1 (Borghetti et al. video-mediastinoscopy. EBUS-TBNA provides
2016). More data is required to discuss this issue both cytologic and histologic materials, but it
in NSCLC, particularly with lymph node status cannot provide reliable information regarding
N2 and N3. ECE status.
Furthermore, Fig. 3 represents a graphical In the present systematic review and meta-
reconstruction of summarized all-cause mortality analysis, we examined three prospective and two
data from studies included in three systematic retrospective observational studies involving
reviews published in 2016 and 2007 (Luchini 828 patients affected by NSCLC with metastatic
et al. 2016; Veronese et al. 2016; Wind et al. lymph nodes. Of these, 298 patients had ECE+ of
2007). As seen, ECE+ is associated with a signif- lymph node metastasis, while 530 showed only
icantly higher all-cause mortality in solid tumors. intra-nodal metastasis. While the results of this
Fig. 3 Pooled estimate of all-cause mortality in different types of cancer. The data is summarized using a random-effect
model; RR risk ratio
meta-analysis are statistically robust, it is impor-

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Influence of Transurethral Resection

of Bladder Cancer on Sexual Function,
Anxiety, and Depression
Wojciech Krajewski, Urszula Halska, Sławomir Poletajew,

Radosław Piszczek, Bartosz Bieżyński, Mateusz Matyjasek,
Andrzej Tukiendorf, Fryderyk Menzel, Małgorzata Mazur,
Joanna Rymaszewska, and Romuald Zdrojowy
Abstract TURB was prospectively evaluated. The Hos-

The standard of care in non-muscle invasive pital Anxiety and Depression Scale (HADS),
bladder cancer consists of transurethral tumor simplified International Index of Erectile
resection. The aim of this study was to evalu- Function (IIEF-5), and Sexual Satisfaction
ate the influence of transurethral resection of Questionnaires (SSQ) were administered to
bladder tumor (TURB) on the patients’ self- patients before and 10 days after tumor resec-
reported depression, anxiety, sexual satisfaction. We found that primary anxiety, depres-
tion, and erectile dysfunction. Psychological sion, and erectile dysfunction were all worse in
condition of 252 male patients who underwent cancer patients than those in the general popu-
lation, and all further worsened after tumor
W. Krajewski (*), B. Bieżyński, F. Menzel, M. Mazur,
resection. The post-resection worsening was
and R. Zdrojowy influenced by the operation and catheterization
Department of Urology and Oncological Urology, time, complications, and the tumor
Wrocław Medical University, Wrocław, Poland characteristics. Taxonomical analysis shows
e-mail: [email protected]
that the greatest risk of depression aggravation
U. Halska concerned patients who were younger, had a
Department of Physiotherapy, Opole Medical School,
Opole, Poland
higher body mass index, and a medium-sized
tumor. We conclude that transurethral resec-
S. Poletajew and M. Matyjasek
Department of General, Oncological and Functional
tion of non-muscle invasive bladder cancer
Oncology, Medical University of Warsaw, Warsaw, may adversely affect sexual function, anxiety,
Poland and depression. Patients should be informed
R. Piszczek about potential complications to prevent the
Department of Urology and Oncologic Urology, Lower abandonment of a follow-up. The findings of
Silesian Specialistic Hospital, Wrocław, Poland this study stress the role of personalized medi-
A. Tukiendorf cine pursued by a multidisciplinary
Department of Social Medicine, Wrocław Medical medical team.
University, Wrocław, Poland
J. Rymaszewska
Department of Psychiatry, Wrocław Medical University,
Wrocław, Poland
37
38 W. Krajewski et al.
Keywords outcomes of operation. Therefore, in the present

Anxiety · Bladder cancer · Depression · study, we set out to evaluate the influence on the
Erectile dysfunction · Sexual satisfaction · self-reported symptoms of anxiety and depres-
Transurethral tumor resection sion, and on sexual satisfaction and erectile func-
tion, of TURB surgery in male patients.
1 Introduction
2 Methods
The majority of urinary bladder cancers are
non-muscle invasive cancer at the time of diagno- The study was approved by the Ethics Committee
sis. This type of cancer in which tumor resides in of Wroclaw Medical University in Wroclaw,
the mucosa or submucosal layer of the bladder Poland, and was performed in accordance with
contrasts with muscle invasive cancers that infil- the recommendation of the Declaration of
trate or pass through the muscular layer (Babjuk Helsinki for Human Research. Informed consent
et al. 2013). The standard of care in non-muscle was obtained from all participants enrolled into
invasive bladder cancer consists of transurethral the study. Further, the investigating urologist
tumor resection (TURB) with or without addi- explained the purpose of the study to each patient,
tional intravesical therapy. TURB surgery is a the protection of participant’s confidentiality, and
procedure that allows the visualization of the the participant’s freedom to drop out of the study
urethra and the bladder walls, and the removal at any time.
of any suspicious foci. It is commonly performed
under spinal anesthesia with the use of a rigid,
hollow, steel tube (resectoscope) equipped with 2.1 Patients and General Procedures
an optic system and cutting tools, which are
introduced through the urethra. We conducted a prospective observational analy-
Treatment results of a surgical procedure are sis of bladder cancer patients who underwent
evaluated by both clinical outcomes and patient’s TURB surgery. The patients were recruited dur-
reported outcomes; the latter described by the ing 12 months, from October 2016 to October
patient himself and devoid of any interpretation 2017 in the Department of Urology and Urologic
by a third party. The vast majority of available Oncology of Wroclaw Medical University;
studies concerning the patient’s reported Department of General, Oncological and Func-
outcomes in bladder cancer relate to the muscle tional Oncology of Warsaw Medical University,
invasive cancer and its extensive mutilating Warsaw; and the Department of Urology and
effects (Feuerstein and Goenka 2015). Despite Oncologic Urology of Lower Silesian Specialty
that TURB is one of the most commonly Hospital in Wrocław, Poland. Three hundred
performed surgical procedures in urology and sixty-seven male patients scheduled for TURB
the belief of the operation safety, reports were initially considered for the study. Patients
concerning the self-reported outcomes in those with a clinical suspicion of a bladder tumor in
who survive the malignancy are lacking. It is whom resection with cutting current was
noteworthy that patients after TURB surgery performed were included into the study. Those
have a significantly higher readmission rate com- in whom only removal of a tumor with cold
pared with other urological procedures due to a forceps or cold biopsy with fulguration was
relatively high occurrence of postoperative lower performed were excluded. Other exclusion
urinary tract symptoms, bleeding, and bladder criteria were the following: age under 18 years
perforation risk (Rambachan et al. 2014). These old, inability to understand the information about
factors raise the possibility that TURB might have the study or to cooperate with psychological
a considerable impact on the patient’s reported evaluations, and the lack of contact after
Influence of Transurethral Resection of Bladder Cancer on Sexual Function. . . 39
operation. Patients taking medications that could 2.2 Survey Questionnaires

affect their mental status were also disqualified.
Psychometric tools used in the study consisted Hospital Anxiety and Depression Scale (HADS)
of the Hospital Anxiety and Depression Scale The HADS, presented by Zigmond and Snaith
(HADS), the Sexual Satisfaction Questionnaire (1983), was used in a Polish adaptation by
(SSQ), and the simplified International Index of Watrowski and Rohde (2014). This is a tool suit-
Erectile Function (IIEF-5). From the initially con- able for different kinds of patients, including
sidered population of 367 patients, 68 were those with both cancerous and noncancerous
unable to fill out the HADS questionnaire and diseases. As the items of the questionnaire are
were discarded from further evaluation. Forty- not concentrated on severe symptomatology, the
seven patients did not resent the second set of scale is applicable to the general population
questionnaires and also were discarded. Those samples (Hinz et al. 2010).
who stated that they were not sexually active The scale consists of two subscales with seven
(56 patients), but filled out the HADS question- items, scored 0–3 each, rating anxiety (HADS-A)
naire, were included into the study. In the end, and depression (HADS-D); the higher the score
252 patients, scheduled for TURB due to con- the worse anxiety and depressive symptoms.
firmed bladder cancer, were included in the anal- Based on the individual sums of scores, patients
ysis (56 with the HADS questionnaire only and can be assigned to one of the three categories:
196 with the HADS, SSQ, and IIEF-5 non-case/normal (0–7), borderline (8–10), and
questionnaires). The patients’ mean age was definite (11–21) depression and anxiety.
66 12 years (range 18–98 years). The mean
duration of surgery was 34 21 min (range Sexual Satisfaction Questionnaire (SSQ) The
10–140 min). The questionnaires were completed SSQ was designed by Nomejko and Dolińska-
after admission, before surgery. After surgery, Zygmunt (2014). This tool is dedicated for adults
patients were discharged with the addressed enve- of all ages and it allows to measure a patient’s
lope containing a second copy of the attitude (cognitive and emotional) toward their
questionnaires, which were to be completed in sexual activity (Zak–Łykus and Nawrat 2013).
10–14 days and resent to the departments. The questionnaire consists of 10 items and the
Demographic, clinical, and psychometric data answers are provided using a 4-point Likert scale:
were collected. Clinical details such as the Amer- 1 – absolutely not, 2 – rather not, 3 – rather yes,
ican Society of Anesthesiologists (ASA) Score – and 4 – definitely yes. A result of the question-
a score that assesses the physical status of patients naire is the sum of points. Questions 1, 3, 5, 8, and
before surgery, body mass index (BMI), nicotine 9 are encoded inversely. The theoretical distribu-
abuse, history of previous transurethral surgeries, tion of the results fits within the 10–40 point
recurrence/year index, previous intravesical che- range; the lower the score, the worse perception
motherapy or Bacillus Calmette-Guérin (BCG) of sexual satisfaction. The method does not have
immunotherapy for bladder cancer were recorded. a reference level; the result is interpreted in rela-
Blood and urine biochemical indices were tion to the examined population (Nomejko et al.
checked, along with urological data such as 2012).
tumor size and focality, surgeon’s experience
(resident/specialist), anesthesia type, obturator Simplified International Index of Erectile Func-
nerve block, and the use of postoperative cyto- tion (IIEF –5) The IIEF-5 is a condensed version
static agent installation. Additionally, catheteriza- of the 15-item International Index of Erectile
tion and hospitalization times were recorded. Function (IIEF) and is composed of five
Clinical and pathological stages were evaluated questions, designed to diagnose the presence
for all the patients according to the TNM staging and severity of erectile dysfunction. Each IIEF-5
system (Sobin and Gospodarowicz 2010). item is scored on a 5-point scale Likert scale,
where the higher the value, the better the erectile logistic regression for binary dependent variables.
function and intercourse satisfaction. The IIEF-5 Finally, non-regression taxonomy was used for
score above 21 is considered a normal erectile selected variables to detect the patient types
function; the lower the score, the worse erectile which were compared to psychological and sex-
function. Erectile dysfunction is classified into ual performance outcomes. The computation was
four categories: severe (1–7), moderate (8–11), performed in the R platform (Team 2017).
moderate (12–16), and mild (17–21) (Rhoden
et al. 2002). The tool has been validated in vari-
ous settings (Tang et al. 2014; Deveci et al. 2008). 2.5 Taxonomy
We also constructed dendrograms of taxonomic

distances between the patients, based on selected
2.3 Surgical Procedures
variables Marczewski and Steinhaus (1958),
using a complete linkage method of Maechler
All surgical steps were performed in accordance
(2017). Taxonomy relies on the symmetric differ-
with the good quality white-light transurethral
ence between objects (A, B); the distance
resection of bladder tumors (GQ-WLTURBT)
(D) between objects is defined as D ¼ (A–B)/
and European Association of Urology (EAU)
max (A, B). The method enables the defining of
guidelines. Perioperative antibiotics were
the types of patients grouped on the basis of
administered in all cases. All but two resections
shared characteristics.
were performed under spinal anesthesia. Obtura-
tor nerve blockade was not performed routinely;
in six cases the blockade was performed due to an
3 Results
extensive obturator reflex. Rigid resectoscopes
with a monopolar energy source were used.
Patients’ clinical characteristics are displayed in
Each patient had a catheter placed after the resec-
Table 1, and the questionnaire quantitative and
tion, which was removed as soon as reasonably
qualitative baseline (before) and post-surgical
possible. Immediate administration of cytostatic
results are in Table 2. The mean differences in
drug was given in accordance with EAU
the patients’ ailments before and post-surgery
guidelines. Intraoperative and postoperative
were compared with a t-test for dependent
complications were categorized according to
samples; all of the differences in the
Clavien and Dindo (2007) classification.
questionnaires were significant.
Linear Regression Linear regression was

2.4 Statistical Analysis conducted to show the associations between clin-
ical variables. Significant (p < 0.05) and border-
Regression and non-regression data analysis was line (p < 0.10) relations are displayed in Table 3.
performed. At first, linear modeling of continuous As an example of data interpretation in Table 3,
outcome variables was conducted. In this age was a factor reducing primary sexual satisfac-
approach, means of intercepts and slopes with tion nearly by 0.2 points per year of advancing
standard errors and p-values were estimated. age in the SSQ survey. The other results are to be
Then, univariate and multivariate logistic interpreted analogously.
regressions were used. The outcomes were
expressed by a classical odds ratio (OR) with a Logistic Regression To model the probability of
confidence 95% interval (CI95%) and a p-value. a worsening in the SSQ, HADS, and IIEF-5
Moreover, for variables with more than two scales after surgery, we used logistic regression
response categories, an ordered logistic regres- analysis. Significant (p < 0.05) and borderline
sion was implemented which is an extension of results (p < 0.10) are reported in Table 4.
Table 1 Patients’ characteristics and surgical data

Clinical feature Stratification n (%) Clinical feature Stratification n (%)
Age (year) <65 95 (37.7) Level of training of Specialist 83 (32.9)
65–70 68 (27.0) surgeon Resident 169 (67.1)
70–75 36 (14.3) Number of tumors 1 162 (64.3)
>75 53 (21.0) 2 39 (15.5)
ASA class 1 60 (23.8) 3 51 (20.2)
2 141 (55.9) Tumor size (cm) <1 91 (36.1)
3 51 (20.3) 1–3 108 (42.9)
BMI (kg/m2) 25.0 72 (28.6) >3 53 (21.0)
25.1–30.0 112 (44.4) Hospitalization time 1 202 (80.1)
30.1–35.0 40 (15.9) (days) 2 21 (8.3)
>35.1 17 (6.7) >2 29 (11.5)
Missing 11 (4.4) Catheterization time (h) 24 203 (80.5)
Nicotine abuse Yes 115 (45.6) 24–48 14 (5.6)
No 126 (50.0) >48 35 (14.0)
Missing 11 (4.4) Tumor stage (T) 0 35 (14.0)
Prior TURB 0 101 (40.0) A 111 (44.0)
1 72 (28.6) 1 56 (22.0)
2 37 (14.7) 2 32 (12.1)
3 42 (16.7) Other than 6 (2.4)
UCC
Recurrences/year 0 101 (40.0) Missing 4 (1.6)
<1 81 (32.2) CIS 8 (3.2)
>1 70 (27.8) High grade tumor Yes 87 (34.5)
Preoperative chemotherapy Yes 7 (2.8) No 161 (63.9)
No 242 (96.0) Missing 4 (1.6)
Missing 3 (1.2) Concomitant CIS Yes 35 (13.9)
Preoperative BCG Yes 34 (13.5) No 213 (84.5)
immunotherapy No 216 (85.7) Missing 4 (1.6)
Missing 2 (0.8) Concomitant CD 1 114 (45.0)
Preoperative hematuria Yes 91 (36.1) 2 33 (13.1)
No 150 (59.5) Chemotherapeutic Yes 132 (52.4)
Missing 11 (4.4) instillation No 110 (43.6)
Preoperative pyuria Yes 101 (40.1) Missing 10 (4.0)
No 140 (55.5)
Missing 11 (4.4)
ASA American Society of Anesthesiologists Score, BMI body mass index, TURB transurethral resection of bladder tumor,
BCG Bacillus Calmette-Guérin, CIS carcinoma in situ, CD cancer disease
Taxonomic Analysis Dendrograms of taxonomic related to changes in the level IIEF-5 and depres-
distances grouped patients of a given type into sion (one-way ANOVA, Table 5). A rough inter-
aggregates, forming hierarchical super-groups pretation of the clinical patterns of patients is as
(Figs. 1 and 2). The groups were defined follow- follows: a population with a relatively average
ing the increment of the joining heights between BMI 30 kg/m2, aged >45, with nonsmoking
objects. After pruning the dendrogram along the status generates the most negative IIEF-5 changes
observed cut-off value (red line), six groups of after surgery, compared to the remaining types of
patients have been formed. These collections patients (type 1 in the right-hand part of Table 5).
were used for further statistical comparisons. Further, all the mean differences between the
The assigned taxonomic types of patients were patient groups are significant.
Table 2 Questionnaire results at baseline (pre-TURB) and after transurethral resection of bladder (post-TURB) tumor
Psychometric tool/level Pre-TURB Post-TURB
HADS – anxiety (points) 7.9 3.7 (1–19) 8.8 4.4 (1–21)
HADS – anxiety qualitative borderline (n; %) 80 (31.7) 58 (23.0)
HADS – anxiety qualitative definitive (n; %) 56 (22.2) 83 (32.9)
HADS – depression (points) 5.7 4.1 (0–18) 7.3 4.9 (0–20)
HADS – depression qualitative borderline (n; %) 36 (14.3) 45 (17.9)
HADS – depression qualitative definitive (n; %) 35 (13.9) 61 (24.2)
SSQ sexual satisfaction (points) 29.1 5.3 (13–40) 27.3 5.5 (11–40)
IIEF-5 (points) 16.2 5.0 (2–25) 13.4 5.4 (5–25)
IIEF-5 without ED (n; %) 28 (14.3) 13 (6.7)
IIEF-5 mild ED (n; %) 72 (36.8) 44 (22.4)
IIEF-5 mild to moderate ED (n; %) 61 (31.1) 65 (33.2)
IIEF-5 moderate ED (n; %) 24 (12.2) 44 (22.4)
IIEF-5 severe ED (n; %) 11 (5.6) 30 (15.3)
Data are mean point scores SD (range) or number (%); HADS Hospital Anxiety and Depression Scale, SSQ Sexual
Satisfaction Questionnaire, IIEF-5 simplified International Index of Erectile Function, ED erectile dysfunction. All of the
pre-TURB/post-TURB differences were significant at p < 0.05
interrelationships. We collected a variety of data

4 Discussion
types such as continuous, binary, and categorical
data and adopted both classical regression and
To ensure optimal disease-free survival in
non-regression methods of evaluation, such as
patients suffering from non-muscle-invasive
taxonomy statistics, to find the best possible
bladder cancer (NMIBC), lifelong surveillance
data fit.
with repeated invasive procedures such as
cystoscopies, resections, and in some cases radi-
cal operations are required. That necessitates a
close cooperation between physician and patient 4.1 Hospital Anxiety and Depression
and a good compliance with diagnostic and ther- Scale (HADS)
apeutic schedules. However, manipulation on
genitourinary tract often causes or aggravates It is known that anxiety and depressive symptoms
psychological problems such as anxiety about are enhanced in oncological patients compared
losing masculinity, inferior self-esteem and with healthy individuals (Hinz et al. 2010). Fur-
sense of sexuality, anxiety about the value of ther, employment of therapeutic procedures may
intimate relationships, and a lack of body accep- additionally increase the patient’s psychological
tance (Gerharz et al. 2005). Those factors may distress (Palapattu et al. 2004). The present
lead to the avoidance or total abandonment of the finding confirmed the presence of elevated anxi-
diagnostic and therapeutic proceedings and lead ety and depressive symptoms in NMIBC patients
to uncontrolled disease development. Thus, it is on par with other kinds of malignancy (Kulpa
crucial to value both clinical and patient-reported et al. 2016; Kulpa et al. 2014; Hinz and Brahler
outcomes of therapeutic methods. 2011). We also demonstrated that some cancer
In this study, we seek to address changes in characteristics, such as the number of tumors and
anxiety, depression, erectile function, and sexual disease aggressiveness, aggravated the
satisfaction in a homogenous group of male symptoms. It seems that not only the knowledge
patients who underwent TURB procedure due to of having a tumor but also the awareness of its
NMIBC. Clinical, peri-, and postoperative data advancement reduces the patient’s psychological
were also evaluated to find possible wellbeing. We further demonstrated that anxiety
Table 3 Linear regression elaboration
Clinical Clinical Clinical
outcome Regression outcome Regression outcome Regression
Before
tumor After tumor
resection Parameter Mean SE p Resection Parameter Mean SE p Δ Difference Parameter Mean SE p
SSQ Intercept 40.7 2.2 <0.0001 SSQ Intercept 40.8 2.3 <0.0001 SSQ Intercept 0.16 0.41 0.6920
Age 0.2 0.0 <0.0001 Age 0.2 0.0 <0.0001 Surgery time 0.05 0.01 <0.0001
Intercept 33.7 0.8 <0.0001 Intercept 29.1 0.7 <0.0001 Intercept 1.33 0.29 <0.0001
Prim. 0.6 0.1 <0.0001 Surgery 0.1 0.0 0.0046 Cath. Time 0.01 0.01 0.0107
Anxiety time
Intercept 32.0 0.6 <0.0001 Intercept 31.2 0.8 <0.0001 Intercept 3.19 0.69 <0.0001
Prim. 0.6 0.1 <0.0001 Anxiety 0.5 0.1 <0.0001 ASA 0.74 0.34 0.0316
Depression
Intercept 17.7 1.0 <0.0001 Intercept 31.3 0.6 <0.0001 Anxiety Intercept 0.09 0.35 0.7980
Prim. IIEF-5 0.7 0.1 <0.0001 Depression 0.6 0.1 <0.0001 Surgery time 0.03 0.01 0.0016
Anxiety Intercept 8.3 0.3 <0.0001 Intercept 17.6 0.8 <0.0001 Intercept 0.27 0.22 0.2310
Tumor 0.2 0.1 0.0832 IIEF-5 0.7 0.1 <0.0001 Cath. Time 0.01 0.00 <0.0001
focality
Influence of Transurethral Resection of Bladder Cancer on Sexual Function. . .
Intercept 4.9 0.3 <0.0001 Anxiety Intercept 9.2 0.3 <0.0001 Intercept 0.41 0.23 0.0688
Depression 0.5 0.1 <0.0001 TURB 0.3 0.2 0.0426 SSQ change 0.19 0.06 0.0021
Intercept 11.9 0.9 <0.0001 Intercept 8.1 0.4 <0.0001 Depression Intercept 0.65 0.30 0.0307
IIEF-5 0.3 0.1 <0.0001 Tumor size 0.4 0.1 0.0070 SSQ change 0.03 0.01 0.0002
Depression Intercept 9.1 1.0 <0.0001 Intercept 4.5 0.4 <0.0001 Intercept 1.13 0.19 <0.0001
IIEF-5 0.2 0.1 <0.0001 Depression 0.6 0.04 <0.0001 Cath. Time 0.01 0.00 <0.0001
Intercept 1.4 1.4 0.3340 Intercept 12.3 0.8 <0.0001 Intercept 1.22 0.18 <0.0001
Age 0.1 0.0 <0.0001 Sec. IIEF-5 0.3 0.1 <0.0001 SSQ change 0.16 0.05 0.0016
IIEF-5 Intercept 28.9 2.0 <0.0001 Depression Intercept 1.2 1.7 0.4990 Intercept 1.27 0.14 <0.0001
Age 0.2 0.0 <0.0001 Age 0.1 0.0 <0.0001 Anxiety 0.41 0.05 <0.0001
change
43
(continued)
Table 3 (continued)
44
Clinical Clinical Clinical

outcome Regression outcome Regression outcome Regression
Before
tumor After tumor
resection Parameter Mean SE p Resection Parameter Mean SE p Δ Difference Parameter Mean SE p
Intercept 11.9 0.8 <0.0001 IIEF-5 Intercept 0.70 0.48 0.1500
Sec. IIEF-5 0.4 0.1 <0.0001 Surgery time 0.06 0.01 <0.0001
IIEF-5 Intercept 15.1 0.7 <0.0001 Intercept 2.03 0.38 <0.0001
Surgery 0.1 0.0 0.0039 Tumor size 0.40 0.14 0.0061
time
Intercept 2.07 0.34 <0.0001
Cath. Time 0.02 0.01 0.0010
Intercept 1.51 0.25 <0.0001
SSQ change 0.71 0.07 <0.0001
Intercept 2.60 0.28 <0.0001
Anxiety 0.22 0.10 0.0243
change
Intercept 2.03 0.32 <0.0001
Depression 0.49 0.11 <0.0001
change
SSQ Sexual Satisfaction Questionnaire, IIEF-5 simplified International Index of Erectile Function, Cath Catheterization, TURB transurethral resection of the bladder tumor
W. Krajewski et al.
Table 4 Logistic regression elaboration

Sign of change Risk factor OR CI 95% p-value
SSQ BMI 1.05 (1.00–1.10) 0.072
Age 0.96 (0.93–0.98) <0.001
Weight 1.01 (1.00–1.03) 0.092
Anxiety Catheterization time 0.991 (0.98–1.00) 0.059
Depression Height 1.07 (1.01–1.13) 0.014
Tumor stage (T) 0.79 (0.62–1.01) 0.056
Postoperative chemotherapeutic instillation 2.19 (0.92–5.24) 0.078
IIEF-5 BMI 1.04 (0.99–1.10) 0.088
Age 0.95 (0.93–0.98) <0.001
Preoperative chemotherapy 0.15 (0.02–1.26) 0.081
Operation time 1.02 (1.01–1.03) 0.015
SSQ Sexual Satisfaction Questionnaire, IIEF-5 simplified International Index of Erectile Function, BMI body mass index,
OR odds ratio, CI confidence intervals
Fig. 1 Dendrogram of taxonomic analysis of depressive symptoms. Red line presents the cut-off level for statistically
different patients groups
and depressive symptoms were positively results, was clearly influenced by surgery, cathe-
associated with each other and depressive terization, and the occurrence of complication.
symptoms were influenced by the patient’s age That may be related to psychological stress stem-
and presurgery urine abnormalities. That lends ming from the hospitalization and confirmation of
support to previous studies that have a neoplasm, bothersome and demeaning catheter-
demonstrated that urinary infection and lower ization, and physical complaints caused by
urinary tract symptoms (LUTS) might influence LUTS, bleeding, or a post-anesthesia syndrome,
quality of life (Jeong et al. 2015). which often occurs after surgery (Ghoneim and
We demonstrated that TURB procedure O’Hara 2016). However, in some cases, particu-
caused an increase in the level of anxiety and larly in TURB naïve patients, we noticed a sub-
depressive symptoms. The second post-surgery stantial decrease in post-surgery anxiety; the
of HADS survey, which showed worsened reason could be a “falsely” elevated anxiety
Fig. 2 Dendrogram of taxonomic analysis of erectile function based on the International Index of Erectile Function
(IIEF-5). Red line presents the cut-off level for statistically different patients groups
level before unknown circumstances of the first- 4.2 Sexual Satisfaction

time TURB procedure. On the other hand, Questionnaires (SSQ)
patients who experienced multiple TURBs may and International Index
accept this situation as a part of their lives and of Erectile Function (IIEF)
might not be suffering as they did before
(Yoshimura et al. 2005; Ficarra et al. 2000). Despite the fact that sexual well-being diminishes
In this study, it was possible to find various with age (Laumann and Waite 2008;
“taxonomical” types of patients that were Hatzimouratidis 2007) and that the average age
associated with changes in depressive symptoms. of a patient diagnosed with bladder cancer is
Taxonomy enables the extraction of strongly nearly 70 years (Babjuk et al. 2013), many older
homogenous subpopulations from the general adults still participate in some form of sexual
patient population, which are characterized by actions including intercourse, oral sex, and mas-
one or more predefined features and demonstrate turbation (Lindau et al. 2007). The patients of this
comparable outcomes. Contrarily, the method can study displayed erectile dysfunction akin to that
also identify subgroups that are at a significant reported in other studies (Tang et al. 2014;
distance in terms of the features from other Mazzola et al. 2012; Deveci et al. 2008). We
groups of patients. We demonstrated that the demonstrated that the basic sexual satisfaction
greatest risk of aggravation of HADS depression and erectile function inversely associated with
concerned patients who were relatively young, age and with anxiety and depression. In addition,
had greater BMI, and had a medium-sized patients who stated that they were not sexually
tumor. On the opposite side, the youngest age, active were older and had a greater score of
the lowest BMI, and the smallest tumor size were depressive symptoms; these findings are in line
associated with the smallest change in depression with those in other studies (Corona et al. 2010;
level. We failed to unravel similar statistical Wylie and Kenney 2010). The TURB surgery
correlates in the taxonomic evaluation of changes adversely affected sexual life. Notably, post-
in anxiety. surgery survey scores decreased in patients with
an extended time of surgery or urethral catheteri-
zation, and with the occurrence of complication
Table 5 Patterns of patients in taxonomic analysis (one-way ANOVA analysis)
Depression (Fig. 1) IIEF-5 (Fig. 2)
Type BMI (kg/m2) Age (year) Tumor size (cm) Depression change Type BMI Age Smoking IIEF-5 change
1 28.3 3.9 60.8 5.1 2.0 0.8 0.6 2.2 1 30.3 3.9 47.7 4.7 No 5.8 6.0
2 27.3 3.2 55.2 9.5 4.5 0.7 2.0 3.6 2 26.2 2.4 63.5 5.1 No 3.2 4.1
3 26.7 2.9 54.8 6.0 0.5 0.4 0.7 1.5 3 24.4 3.2 77.6 7.6 No 1.8 3.3
4 44.9 10.5 61.7 8.1 1.3 1.5 2.3 2.2 4 36.9 9.7 68.5 6.2 No 4.1 3.3
5 26.4 3.5 72.3 7.2 0.7 0.5 1.9 2.5 5 27.8 4.8 67.6 9.7 Yes 2.1 3.4
6 27.1 4.3 73.0 6.3 4.5 1.7 2.0 2.5 p-value <0.0001 <0.0001 – 0.0264
p-value <0.0001 <0.0001 <0.0001 0.0295
Values means SD
Influence of Transurethral Resection of Bladder Cancer on Sexual Function. . .
47
or administration of cytostatic agents. A number sexuality, still, true influence of TURB is

of patients reported not engaging in any sexual unknown (Bowen et al. 2015; Placer et al.
activity for a time after surgery (data not shown), 2015). Either type of surgery may lead to erectile
which is consistent with other reports (van der Aa dysfunction due to thermal or chemical tissue
et al. 2009). As in case of depressive symptoms, injury, or due to psychological issues relating to
we identified taxonomical groups related to surgery and hospitalization (Ibrahim et al. 2002;
changes in erectile function. We demonstrate Schmidt et al. 2015; Jaidane et al. 2010). In con-
that patients with a greater BMI ( 30 kg/m2), trast to majority of studies analyzing PROs after
lower age, and nonsmokers were subject to the TURP, we assessed ailments shortly after
most negative IIEF changes after surgery com- procedure. It is possible that during longer fol-
pared to the other patients. low-up the TURB influence on sexual function
The male psychological condition is substan- will blur. However, TURP is often performed
tially influenced by sexuality, including erectile once in a lifetime, and TURB (and similarly
function, sexual desire, ability to have an orgasm, cystoscopies) is in general performed repeatedly.
and the ejaculatory capacity (Gilbert et al. 2013; For that reason, it is important to inform the
Wittmann et al. 2009). On the other side, sexual patient about potential consequences of surgery
function closely associates with the psychological in order to prevent abandonment of follow-up
well-being, also in older age, which helps main- procedures.
tain satisfaction from a relationship and provides
a considerable emotional and physical pleasure
(Thomas et al. 2015; Nomejko and Dolińska-
5 Conclusions
Zygmunt 2014). In turn, quality of sexual life
associates with the health condition (Lindau and
This study was conducted in the high-volume
Gavrilova 2010). Any disorder of the lower abdo-
oncology-orientated medical centers, which
men and pelvis may lead to sexual dysfunction
certifies the quality and repeatability of surgery
due to psychogenic or organic causes. That is
procedures. Nonetheless, TURB, like every sur-
particularly apparent in patients with urological
gical procedure, may be performed a slightly
or gynecological malignancies (Guercio and
different way among urologists. There was a pos-
Mehta 2018). In addition, any medical
sible qualification bias, as we included only
intervention may lead to a worsening of
patients that were able to fill out surveys without
symptoms and to the fear of harming a partner
a help of a third party. We thus lost the
during sexual interaction (Kowalkowski et al.
participants who were unfit to fill out the initial
2014; van der Aa et al. 2009).
questionnaires due to poor health condition or
Literature data concerning sexual function
excessive presurgery stress. Some of the enrolled
refer, almost solely, to patients treated for muscle
patients failed to return the questionnaire or its
invasive blood cancer (Mohamed et al. 2012).
second post-surgery version and were discarded
Radical proceedings in majority of such patients
from the final evaluation. Therefore, this study
cause irreversible erectile dysfunction, signifi-
may not include a complete cross section of
cantly disturbing sexual satisfaction (Asgari
NMIBC patients. We believe, however, we have
et al. 2013; Selvin et al. 2007). Scarce data avail-
conclusively shown that the TURB procedure
able in NMIBC patients demonstrate a high prev-
caused untoward changes in anxiety, depressive
alence of sexual dysfunction compared with an
symptoms, and sexual function. The findings
age- and gender-matched healthy population and
underscore the role of personalized medicine
show that therapy may also adversely influence
based on the effort of a multidisciplinary team
sexual function (van der Aa et al. 2009; Sighinolfi
which includes professionals engaged in the
et al. 2007). When discussing endoscopic
assessment and therapy of psychological health
procedures, only transurethral resection of pros-
apart from somatic ailments.
tate (TURP) was analyzed in terms of changes in
Acknowledgments This research did not receive any material discursive intra–psychic approach. Cult
specific grants from public, commercial, or nonprofit Health Sex 15:881–895
agencies. Guercio C, Mehta A (2018) Predictors of patient and
partner satisfaction following radical prostatectomy.
Sex Med Rev 6(2):295–301
Conflicts of Interest The authors declare no conflicts of
Hatzimouratidis K (2007) Epidemiology of male sexual
interest in relation to this article.
dysfunction. Am J Mens Health 1:103–125
Hinz A, Brahler E (2011) Normative values for the hospi-
tal anxiety and depression scale (HADS) in the general
German population. J Psychosom Res 71:74–78
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Cognitive Predictors of Cortical Thickness

in Healthy Aging
Patrycja Naumczyk, Angelika K. Sawicka, Beata Brzeska,

Agnieszka Sabisz, Krzysztof Jodzio, Marek Radkowski,
Karolina Czachowska, Paweł J. Winklewski, Karolina Finc,
Edyta Szurowska, Urszula Demkow, and Arkadiusz Szarmach
Abstract were examined on 3.0 T MRI scanners. The

This study seeks to define the role of predictive protocol included standard anatomical
values of the motor speed, inhibition control, sequences, to exclude brain pathology, and a
and fluid and crystallized intelligence in high-resolution T1-weighted sequence used to
estimating the cortical thickness in healthy estimate the cortical thickness. The neuropsy-
elderly. Forty-six older healthy subjects chological protocol included fluid intelligence
(37 women, 9 men) over 60 years of age assessment (Raven Progressive Matrices),
were included in the study. The participants crystalized intelligence assessment
P. Naumczyk (*) and K. Jodzio

Institute of Psychology, University of Gdansk, Gdansk,
Poland
A. K. Sawicka
Department of Bioenergetics and Nutrition, Gdansk K. Czachowska
University of Physical Education and Sport, Gdansk, Department of Human Physiology, Faculty of Health
Poland Sciences, Gdansk Medical University, Gdansk, Poland
B. Brzeska P. J. Winklewski
Department of Biology and Pharmaceutical Botany, Department of Human Physiology, Faculty of Health
Faculty of Pharmacy, Gdansk Medical University, Sciences, Gdansk Medical University, Gdansk, Poland
Gdansk, Poland
Second Department of Radiology, Faculty of Health
Department of Human Physiology, Faculty of Health Sciences, Gdansk Medical University, Gdansk, Poland
Sciences, Gdansk Medical University, Gdansk, Poland
Department of Clinical Anatomy and Physiology, Faculty
Second Department of Radiology, Faculty of Health of Health Sciences, Pomeranian University in Slupsk,
Sciences, Gdansk Medical University, Gdansk, Poland Slupsk, Poland
A. Sabisz, E. Szurowska, and A. Szarmach K. Finc
Second Department of Radiology, Faculty of Health Center for Modern Interdisciplinary Technologies,
Sciences, Gdansk Medical University, Gdansk, Poland Nicolaus Copernicus University, Torun, Poland
M. Radkowski U. Demkow
Department of Immunopathology of Infectious and Department of Laboratory Diagnostics and Clinical
Parasitic Diseases, Warsaw Medical University, Warsaw, Immunology of Developmental Age, Warsaw Medical
Poland University, Warsaw, Poland
51
52 P. Naumczyk et al.
(information or vocabulary subtest of the The structural brain aging theories seek the
Wechsler Adult Intelligence Scale-Revised origins of pathological aging in noxious processes
(WAIS-R)), and executive functioning (Color in different areas of the brain. Namely, the frontal
Traits Test). The findings unraveled several hypothesis focuses on the damage in the anterior
interdependencies. The higher the intelligence, cortical and subcortical regions (West 1996),
the thicker was the grey matter in nine regions whereas the neurodegenerative hypothesis
of both hemispheres, but also some paradoxi- assumes that aging follows roughly the
cal reversed associations were found in four Alzheimer’s disease pattern of brain damage,
areas; all of them were localized along differ- but at a slower rate (Swerdlow 2007), which
ent sections of the cingulate gyrus in both points to the possible brain pathology in the tem-
hemispheres. An inverse association was poral lobes. The expansion of in vivo neuroimag-
found between crystallized intelligence and ing techniques, such as magnetic resonance
the thickness of the pars opecularis of the imaging (MRI), sheds some light on this theoreti-
right hemisphere. The better the executive cal debate. In the studies on normal healthy aging,
functioning, the thicker was the grey matter brain volume shrinkage has been repeatedly
of a given region. The better the motor perfor- reported (Fjell et al. 2013; Fjell et al. 2009a; Raz
mance, the thicker was the grey matter of the et al. 2010; Raz et al. 2005), with the annual
rostral middle frontal area of the left hemi- percentage change of cortical volume up to
sphere and the lingual gyrus of both 0.8% (Fjell et al. 2014). Yet, current data give
hemispheres. In conclusion, the associations more support to the frontal hypothesis. Although
unraveled demonstrate that the neural cortical atrophy is widespread and present both in
mechanisms underlying healthy aging are frontal and temporal areas (Tamnes et al. 2013;
complex and heterogenic across different cog- Enzinger et al. 2005), it seems that cortical thin-
nitive domains and neuroanatomical regions. ning is more prevalent in the anterior regions
No brain aging theory seems to provide a (Fjell et al. 2009b; Resnick et al. 2003). Nonethe-
suitable interpretative framework for all the less, the frontal lobes dysfunction does not
results. A novel, more integrative approach to explain the diversity of functional changes
the brain aging should be considered. accompanying normal aging. Healthy old people
increasingly seek clinical advice on cognitive
Keywords problems. It has been shown that a range of cog-
Aging process · Cortical thickness · Grey nitive functions deteriorate with age (Josefsson
matter · Healthy aging · Inhibition control · et al. 2012; Finkel et al. 2007). The deterioration
Intelligence · Motor speed primarily concerns working memory (Mattay
et al. 2006; Balota et al. 2000), inhibitory control
(Park and Reuter-Lorenz 2009), the pace of infor-
mation processing (Salthouse 2000), and a long-
1 Introduction
term memory – specifically in the episodic mem-
ory retrieval domain (Reuter-Lorenz and Park
There are four major hypotheses describing the
2010; Grön et al. 2003).
neuronal underpinnings of aging: the frontal
In the research focused on these four areas,
hypothesis, the neurodegenerative hypothesis, the
elderly participants perform worse than the
acceleration in right hemisphere’s aging hypothe-
young ones. Unfortunately, those results are not
sis (the right-hemi theory), and the hemispheric
easily explained in the framework of the frontal
asymmetry reduction in older adults model
hypothesis. Although patients with the anterior
(HAROLD). The first two are grounded in struc-
brain damage experience deficits in the
tural studies, whereas the latter two are rooted in
information processing, inhibition control, and
neuropsychological data on healthy aging in which
working memory, their dysfunction usually far
the subject functions well in everyday life.
Cognitive Predictors of Cortical Thickness in Healthy Aging 53
exceeds what we attribute to the typical pattern of thickness with motor speed, inhibition control,
cognitive aging. To account for the variability in and fluid and crystallized intelligence. Whereas
elderly individuals’ outcomes, the hypothesis of crystallized intelligence is, roughly speaking,
accelerated aging in the right hemisphere’s, a unaffected by normal aging, the other three
right-hemi model has been proposed (Dolcos factors decline with age. Our goal was to compare
et al. 2002). This model suggests an asymmetry the predictive value of all these factors in the
in the age-related vulnerability between the estimation of the grey matter thickness and its
hemispheres. The right-hemi theory was rooted variations across different brain regions in healthy
in the neuropsychological findings of a corre- elderly. In the study, we remained attentive to the
spondence between cognitive performance of patterns supporting or undermining the
healthy aging individuals and patients suffering hypotheses of the brain aging outlined above.
from lateralized brain damage (Taconnat et al.
2007; Uekermann et al. 2006; Royall et al.
2005; Joy et al. 2001). Yet, the studies of struc- 2 Methods
tural brain do not support a notion of asymmetry
in cortical atrophy associated with aging. In the 2.1 Participants
light of such findings, the HAROLD model was
put forward (Cabeza 2002). It postulates that The study protocol was approved by the Ethics
aging is reflected mostly in the functional archi- Committees of the Universities involved in the
tecture of the brain in the form of a reduction of project (permissions NKBBN/296/2017 and
prior specialization of cerebral regions. The 7/2014), and all participants gave written
HAROLD model is, to an extent, consistent with informed consent for the study procedures. The
previous structural findings (see Tamnes et al. participants consisted of 46 pensioners
2013) suggesting that higher-order cortical (37 women and 9 men) over 60 years of age.
systems are especially prone to atrophy in elderly. Individuals with any psychoneurological
Nonetheless, the current understanding of the incidents, brain pathology diagnosed in magnetic
mechanisms underlying age-related brain resonance scan images, dementia symptoms
changes and healthy cognitive aging remains based on the Mini Mental State Examination
inadequate. There are many factors, unaccounted (MMSE), metal bodily implants, or overt neuro-
for, which have a protective or harmful effect on degenerative condition excluded from the study.
the brain’s structure and function during aging. Detailed demographic data of participants are
On the other hand, there are no data providing a depicted in Table 1.
direct contrast between the hypotheses mentioned
above, which would require a difficult task of
collecting and matching both anatomical and neu- 2.2 MRI Acquisition and Processing
ropsychological data. Rather, the two structural or
the two functional models are compared with The participants were examined in two equivalent
each other (Fjell et al. 2009a; Dolcos et al. 3.0 T MRI scanners (Philips Achieva TX;
2002). Further, some previous studies that try to Andover, MA and GE 750 Discovery; Chicago,
link cortical thickness and cognitive functioning IL). The protocol included standard anatomical
in the elderly have failed to refer to the sequences, as brain pathology reference, and a
hypotheses described (Lee et al. 2016; Persson high-resolution T1-weighted sequence, later
et al. 2016; Gautam et al. 2015; Fjell et al. 2006). used for the estimation of cortical thickness. The
The present study seeks to contribute to the T1 sequence parameters for the Philips were:
debate on the theories of aging. We focused on repetition time (TR) ¼ 8.10 ms, echo time
defining neuropsychological correlates of grey (TE) ¼ 3.7 ms, voxel size 111 mm, flip
matter thickness in the elderly. Specifically, we angle 8 ; and for the GE were: TR ¼ 8.19 ms,
examined the relationships between cortical TE ¼ 3.18 ms, voxel size 1x1x1 mm, flip
Table 1 Demographic and neuropsychological characteristics of the study group

Demographic variable Mean SD Min–Max
Age (year) 69.0 4.6 63–86
Education (years) 14.4 3.4 7–26
Retirement (years) 10.3 7.6 0–31
Gender n (%) –
Women 37 (80) –
Men 9 (20) –
Cognitive function tests
Fluid intelligence 75.3 25.5 3–99
(RPM-S percentile score)
Fluid intelligence 7.4 1.8 2–10
(RPM-S sten score)
Crystallized intelligence 13.1 2.7 8–18
(WAIS-R calculated normative score)
Motor speed and visual attention 55.7 15.6 32–97
(time of CTT part 1 completion (s))
Executive control and visual attention 112.0 33.0 57–194
(time of CTT part 2 completion (s))
Inhibition control 1.1 0.6 0.02–2.70
(CTT interference score)
RPM-S Raven Progressive Matrices-standard version, WAIS-R Wechsler Adult Intelligence Scale-Revised, CTT Color
Traits Test
angle ¼ 12 . All anatomical scans were processed thickness of each region was measured by aver-
with FreeSurfer v.6.0 software (Athinoula aging all individual metrics of vertices for a given
A. Martinos Center for Biomedical Imaging at region. The evaluation resulted in 68 cortical
Massachusetts General Hospital, Boston, MA) thickness values for every subject (66 regions
following the recommended processing stream and 2 mean cortical thickness scores).
(http://surfer.nmc.mgh.harvard.edu/), previously
described in detail works (Dale et al. 1999; Fischl
et al. 2004a, b; Fischl et al. 2002; Fischl et al.
2.3 Neuropsychological Assessment
1999). In short, the processing includes transla-
tion into Talairach space and removal of
All of the participants underwent a thorough neu-
non-brain tissue from the images, followed by a
ropsychological assessment that took place about
conversion of the voxel-based images into the
10 days after the MRI session. The neuropsycho-
surface-based 3D representations of triangles
logical protocol included fluid intelligence assess-
(vertices), basing on white and gray matter
ment with the Raven Progressive Matrices
boundaries. The procedure allows for a precise
(RPM-S) standard version, crystallized intelli-
subject-to-subject and subject-to-template
gence assessment with the Wechsler Adult Intel-
registrations upon individual gyral and sulcal
ligence Scale-Revised (WAIS-R) or vocabulary
topology. Anatomical landmarks derived from
subtest of the WAIS-R, and the executive func-
the Desikan-Killiany brain atlas (Desikan et al.
tioning assessment with the Color Traits Test
2006) were fitted into the data resulting in
(CTT).
66 regions of interest, 33 per hemisphere, for
As normative fluid intelligence scores, both
each participant. The atlas is presented in Fig. 1.
percentile and sten scores, were non-normally
The closest distance from the grey matter bound-
distributed (Shapiro-Wilk (46) ¼ 0.787,
ary to the pial boundary at each vertex represents
p < 0.001 and Shapiro-Wilk (46) ¼ 0.901,
cortical thickness at a given vertex. The cortical
Fig. 1 The Desikan-Killiany brain atlas used for automated cortical parcellations. (Reproduced from Klein and
Tourville (2012) under the terms of the Creative Commons Attribution License)
p ¼ 0.001, respectively), the normally distributed 1 completion, divided by the time needed for
raw scores of the RPM-S were used for further part 1 completion. The interference score is 0, if
evaluation. For crystallized intelligence, a calcu- there is no difference in the performance between
lated normative score of a given subtest was cho- the test parts. The higher the interference score,
sen to account for differences in the subtests used. the worse the executive functioning, as more time
As for the CTT, three metrics were derived from is needed to account for the increased
the test: the time taken to complete part 1 – inhibition load.
marker of visual attention and motor speed, the
time taken to complete part 2 – marker of execu-
tive control and visual attention, and the interfer-
2.4 Statistical Evaluation
ence index – marker of the flexibility of voluntary
attention, especially over the inhibition processes.
The evaluation focused on the relationship
The last marker was computed as a difference
between the neuropsychological metrics
between the time needed for part 2 and part
obtained, i.e., fluid IQ raw score, crystallized IQ
calculated normative score, time for part 1 of the 3 Results

CTT, time for part 2 of the CTT, and the interfer-
ence index of CTT, age, and the cortical thickness 3.1 Neuropsychological Data
score derived from the MRI examination. The
evaluation was conducted in three steps. First, The study participants’ both fluid intelligence
the normality of variable distribution was (mean percentile score on RPM-S of
verified, based on the Shapiro-Wilk test and 75.3 25.5; range 3–99) and crystallized intelli-
Z-skewness/Z-kurtosis estimations. For gence (normative score on WAIS-R of
non-normally distributed variables, a log transfor- 13.1 2.7, range 8–18) are grossly in line with
mation was performed and the normality test was the average level present in the Polish elderly
repeated. For eight variables (time of CTT – both population at large. Likewise, participants
part 1 and part 2 – and grey matter thickness of showed normal executive functioning. The mean
lateral occipital area of left hemisphere (LH), LH interference index of 1, achieved in our sample,
posterior cingulate cortex, LH insula, caudal ante- indicates that it took the subjects twice as long to
rior cingulate of right hemisphere (RH), and RH perform part 2 of the test, compared with part
superior frontal area), the transformation resulted 1. The neuropsychological results are
in normal distribution, so that the log-version of summarized in Table 1.
scores was further compared. For seven variables
(participants’ age, and grey matter thickness of
LH isthmus cingulate area, LH lateral
orbitofrontal area, LH pars opecularis, RH pars 3.2 Cognitive Predictors of Cortical
triangularis, RH posterior cingulate cortex, and Thickness
RH precentral gyrus) the log-transformation was
not sufficient, so that they were further evaluated To account for multiple comparisons, a Bonferroni
with nonparametric tests. The second step correction was applied, so that p < 0.05 was
included paired correlations between all of the corrected to p < 0.007. As this is a highly conser-
variables with Pearson’s and Spearman’s tests, vative approach, the uncorrected results also are
depending on whether a given variable was reported to verify the trends in data. After account-
distributed normally. The third step, for all of ing for the shared variance (see Methods for
the anatomical regions of interest (ROIs), for details), cortical thickness of 30 anatomical regions
which more than one of the neuropsychological/ in both hemispheres and the global metric of grey
demographical variables correlated with cortical matter thickness of the right hemisphere were, to
thickness, a hierarchical stepwise regression (for different extents, explained by neuropsychological
normally distributed variables) or a partial or demographical data. Out of those, five measures
Spearman’s correlation (for non-normally of thickness remained significant after the rigorous
distributed variables) was performed to determine multiple comparisons correction.
which of the factors examined explained most of Only six of the uncorrected relationships were
the variance of grey matter thickness. explained by participants’ age. The age-related
Additional measures of the effect size were effects were found only in the right hemisphere,
computed for all significant associations to deter- mostly in the temporal lobe, with a fraction of
mine the percentage of variance in the cortical variance explained varying from 9% (middle tem-
thickness scores explained by the variables of poral area, RH) to 26% (inferior temporal area,
interest. Those measures were the adjusted r2 RH; this link remained significant after
metric for regression models and Pearson’s/ Bonferroni correction). All of them were a
Spearman’s r2 coefficient for correlation models. reversed linkage, meaning the higher the
Statistical analyses were performed using a com- participants’ age, the thinner the grey matter.
mercial statistical package for social sciences – The neuropsychological variables explained
SPSS v24 (IBM; New York, NY). the rest of the associations found (24 uncorrected
significant linkages in both hemispheres, 4 signif- Age-related effects were notable only in the
icant corrected linkages). The factor that right hemisphere, mostly in the temporal lobe.
accounted for most of them (13 uncorrected and This result, particularly the mean thickness score
2 Bonferroni corrected) was fluid intelligence. prediction, is in line with the right-hemi model
Higher fluid intelligence was associated with described in the introduction. Despite the tempo-
thicker grey matter in nine regions of both ral location, it does not support the neurodegener-
hemispheres, but also some paradoxical reversed ative hypothesis, as the Alzheimer’s pathologies
associations were found in four areas; all of them affect primarily parahippocampal and entorhinal
were localized along different sections of the gyri (Braak and Braak 1991). Still, this is a
cingulate gyrus in both hemispheres. The same finding contradictory to previous studies, where
reversed direction was significant for crystallized no profound asymmetry of age-related effects has
intelligence and the thickness of the pars been found (Lee et al. 2016; Zhou et al. 2013;
opecularis of the right hemisphere. Fjell et al. 2009b; Raz et al. 2005). There are a
There was an inverse relationship between the few explanations possible. The first refers to the
interference index and cortical thickness; the gender inequality present in this study. Our group
higher the interference index, the worse the exec- had 37 women and only 9 men, and there is
utive performance. This relationship indicates research suggesting that age-related brain atrophy
that the better participants’ executive functioning, is less pronounced or even statistically insignifi-
i.e., the smaller the interference index, the thicker cant in females, when compared to peer males
the grey matter of a given region. Thus, executive (Thambisetty et al. 2010; Hutton et al. 2009).
functioning explained underlying differences in Another distinguishing feature is the demo-
variance in five regions of both hemispheres graphic structure. Our participants were all
(one region after Bonferroni correction). Also, elderly, with age ranging from 62 to 86 years,
the better was the participants’ motor perfor- which was a rather low age disparity compared to
mance, the thicker the grey matter of the rostral other studies in which the spread was as wide as
middle frontal area of the left hemisphere and the 18–93 years (Fjell et al. 2009a), 20–79 years (Lee
lingual gyrus of both hemispheres (the left et al. 2016), 22–60 years (Hutton et al. 2009), or
hemisphere’s association was significant after 5–59 years (Zhou et al. 2013). It might be that in
Bonferroni correction). The results, with the our group the difference in brain atrophy was not
corresponding measures of the effect size, are so much apparent within a 24-year span between
provided in Table 2. the eldest and youngest participants. In the main,
our group also was older than some of the others,
which could affect regional variability in the cor-
4 Discussion tical damage observed. An interesting possibility,
however, cannot be ruled out that the method we
This study defined the predictive value of age and employed in this study enabled a direct compari-
cognitive variables for cortical thickness in differ- son of the predictive value of age with variables
ent anatomical regions. Specifically, fluid and associated with cognitive functioning; the latter is
crystallized intelligence, motor speed, and inhibi- evincibly a better predictor of brain atrophy in
tion control were chosen as factors with declining multiple regions.
versus roughly invariable developmental The relationship between fluid and crystallized
trajectories. After correcting for multiple intelligence, and grey matter thickness, on the
comparisons, significant “predictor-cortical thick- other side, we unraveled was twofold. For some
ness” pairs were found in five regions. It is worth associative regions throughout the frontal, parie-
noting that the uncorrected results were in line tal, and temporal lobes of both hemispheres, the
with the ones that passed the rigorous threshold; linkage was positive, meaning that higher intelli-
therefore, those trends will be briefly discussed gence accompanied thicker grey matter, whereas
as well. for others (localized in different sections of
Table 2 Cognitive and demographic predictors of the grey matter thickness

Variable (indicator)
Brain region % variance explained Beta/r p (uncorr) p (corr)
Age
Mean thickness, RH 9 0.292 0.049 ns.
Middle temporal, RH 9 0.296 0.046 ns.
Supramarginal, RH 10 0.318 0.031 ns.
Precentral RH 11 0.325 0.040 ns.
Superior temporal, RH 12 0.352 0.016 ns.
Inferior temporal, RH 26 0.521 <0.001 0.01
Intelligence (fluid – RPM-S)
Caudal anterior cingulate, RH 22 0.466 0.001 0.05
Isthmus cingulate, RH 19 0.459 0.001 0.05
Rostral anterior cingulate, LH 13 0.387 0.008 ns.
Isthmus cingulate, LH 10 0.316 0.032 ns.
Temporal pole, RH 10 0.311 0.036 ns.
Inferior parietal, LH 10 0.312 0.035 ns.
Fusiform, LH 11 0.326 0.027 ns.
Superior parietal, LH 9 0.332 0.024 ns.
Inferior temporal, LH 12 0.352 0.016 ns.
Banks STS, RH 11 0.354 0.016 ns.
Temporal pole, LH 13 0.36 0.014 ns.
Lateral orbitofrontal, RH 13 0.363 0.013 ns.
Pars orbitalis, LH 15 0.389 0.008 ns.
Intelligence (crystallized – WAIS-R)
Pars opecularis, RH 10 0.314 0.034 ns.
Motor speed and visual attention (time of CTT – part 1 completion)
Lingual, RH 12 0.346 0.018 ns.
Rostral middle frontal, LH 12 0.369 0.012 ns.
Lingual, LH 16 0.402 0.006 0.05
Inhibition control (interference index of CTT)
Lateral occipital, LH 9 0.297 0.045 ns.
Pars triangularis, RH 10 0.311 0.036 ns.
Inferior parietal, RH 9 0.326 0.027 ns.
Superior parietal, RH 11 0.326 0.027 ns.
Precuneus, LH 21 0.453 0.002 0.05
Significance of results after Bonferroni correction for multiple comparisons is marked in the last right-hand column; %
variance explained, the percentage of variance in cortical thickness score of a given region explained by a variable; Beta
standardized regression coefficient, r Pearson’s/Spearman’s correlation coefficient, p(uncorr) significance threshold
unaccounted for by multiple comparisons, p(corr) significance threshold accounted for by multiple comparisons, ns
not significant
RPM-S Raven Progressive Matrices-standard version, WAIS-R Wechsler Adult Intelligence Scale-Revised, CTT Color
Traits Test, LH left hemisphere, RH right hemisphere, uncorr significance threshold unaccounted for by multiple
comparisons, corr significance threshold accounted for by multiple comparisons, ns not significant
cingulate cortex for the fluid intelligence and in 2013), the negative ones raise more interest. It is
the pars opecularis of the right hemisphere for intriguing that such paradoxical associations
crystallized intelligence), it was negative, mean- between the global and/or crystallized intelli-
ing that better intelligence related to thinner grey gence and grey matter thickness, although previ-
matter. As the positive linkages are in line with ously reported, have been left out of discussions
previous studies (Lee et al. 2016; Menary et al. by the authors (Fjell et al. 2006). Although some
of our results should be taken with caution, as whereas crystallized intelligence is based on
they were not corrected in the multiple compari- declarative knowledge and the ability to utilize
son analysis, those previous reports of the para- facts (Reber and Reber 2001). Therefore, the for-
doxical associations lend support for interpreting mer has a strong hereditary component (Gray and
them as non-incidental findings. Thompson 2004), while the latter is acquired
There are a variety of possible mechanisms through learning and then shaped by experience
underlying this reversed (paradoxical) relation- (Cattell 1963). Our results suggest that a stronger
ship (higher intelligence – thinner cortical grey prognostic is congenital traits.
matter) found in this and previous studies. One The present results also identified five regions
possible explanation comes from research on the in which the core predictor of grey matter thick-
developing brain. During childhood and adoles- ness was the performance of inhibitory control in
cence, cortical thickness metrics follow a reversed the elderly. Those were localized mostly in asso-
U-shape trajectory. Starting at some age (different ciative regions of the frontal, parietal, and occipi-
between regions and individuals), the thickness of tal lobes. Worse inhibitory control was related to
grey matter continuously decreases greater cerebral atrophy in all the linkages found.
(Khundrakpam et al. 2016). That is interpreted This is a novel finding, as in previous studies on
as a healthy process of synaptic pruning, which neuroanatomy-cognition correspondence in the
plays a key role in regional functional specializa- elderly, the executive domain has either been
tion of the brain (Haartsen et al. 2016). Menary left out of investigation (Raz and Rodrigue
et al. (2013) has provided support to this hypoth- 2006; Persson et al. 2016) or few significant
esis, demonstrating a reversed relationship results have been found (Fjell et al. 2006). Also,
between lexicon scores and grey matter thickness an additional finding in the present study was the
of frontal areas, meaning that the youth with predictive value of the motor speed and visual
extensive vocabulary had thinner cortices. Thus, attention outcome for thickness of three areas of
it may be that thinner grey matter of the cingulate the cerebral cortex. This factor also was not of
cortex and pars opecularis we found, rather than primary interest in previous studies. Lee et al.
atrophy, represents a greater higher degree of (2016) have identified a principal component
specialization of those regions with increased they attributed to motor performance, which is
participants’ intelligence. associated with cortical thickness of multiple
The mechanism above outlined lends support regions, such as inferior temporal, olfactory,
for the HAROLD model. This theory puts empha- parahippocampal, and inferior frontal orbital
sis on the dedifferentiation of previously areas. In contrast, our findings were few, which
specialized brain regions in triggering the cogni- may be a result of using univariate scores of only
tive performance changes observed in the elderly. one test as an indicator in the study.
As fluid and crystallized intelligence are known Nonetheless, the predictive power of the
preservative cognitive factors, their predictive elderly’s executive and motor performance is
value for a greater specialization of certain brain hardly interpretable under any of the theoretical
regions would suggest that these variables play a contexts provided. The results do not support the
key role in restraining the asymmetry reduction frontal hypothesis (as only two linkages in ante-
with aging. From this perspective, it is interesting rior regions were found), nor do they confirm a
that it was fluid, not crystallized, intelligence that higher effect in the temporal areas (as the neuro-
accounted for most of the associations unraveled degenerative model postulates). Also, no obvious
in this study. There are noteworthy differences lateralization was present or absent in the data;
between the two constructs that may contribute thus, it cannot be interpreted as enhanced deterio-
to this result. Fluid intelligence, often synony- ration of the right hemisphere, nor as a reduction
mous with abstract reasoning, is based on the in asymmetry between the hemispheres
ability to solve novel problems creatively, (as HAROLD model assumes). It seems rather
that the structure-function relation is References

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Osteoprotegerin, Receptor Activator

of Nuclear Factor Kappa B Ligand,
and Growth Hormone/Insulin-Like
Growth Factor-1 Axis in Children
with Growth Hormone Deficiency
Ewelina Witkowska-Sędek, Małgorzata Rumińska,

Anna Stelmaszczyk-Emmel, Maria Sobol, Urszula Demkow,
and Beata Pyrżak
Abstract the GH-deficient and GH-sufficient children.

The growth hormone/insulin-like growth The OPG content and total alkaline phospha-
factor-1 (GH/IGF-1) axis is involved in the tase (ALP) activity increased significantly
regulation of the receptor activator of nuclear after the initiation of GH treatment, while
factor kappa B ligand (RANKL)/RANK/ total sRANKL remained unchanged. The
osteoprotegerin (OPG) system, but the exact variables baseline BMI SDS for height-age
mechanism of the associations is not fully (β ¼ 0.42; p < 0.05), baseline ALP activity
explained. In this study we investigated the (β ¼ 0.36; p < 0.05), weight SDS for height-
serum OPG and total sRANKL concentrations age at 6 months of GH treatment (β ¼ 1.86;
in short children who had differences in GH p < 0.01), and total ALP activity at 6 months of
secretory status. We also investigated the GH treatment (β ¼ 0.48, p < 0.01) were
associations between the GH/IGF-1 and identified as independent predictors of
OPG/RANKL systems in GH-deficient ΔOPG6-month-baseline. We conclude that OPG
children during GH treatment. There were no and total sRANKL concentrations are inde-
significant differences in any anthropometric pendent from GH secretory status in short
or biochemical parameters evaluated between children. OPG elevation during GH treatment
is independently associated with total ALP
activity and nutritional status in GH-deficient
E. Witkowska-Sędek (*), M. Rumińska, and B. Pyrżak children.
Department of Paediatrics and Endocrinology, Medical
University of Warsaw, Warsaw, Poland
e-mail: [email protected] Keywords
Bone turnover · Children · Growth hormone ·
A. Stelmaszczyk-Emmel and U. Demkow
Department of Laboratory Diagnostics and Clinical Hormone replacement therapy ·
Immunology of Developmental Age, Medical University Osteoprotegerin · RANKL/RANK/OPG
of Warsaw, Warsaw, Poland pathway
M. Sobol
Department of Biophysics and Human Physiology,
Medical University of Warsaw, Warsaw, Poland
63
64 E. Witkowska-Sędek et al.
1 Introduction increasing the RANKL/OPG ratio and thereby

favoring osteoclast recruitment, which result in
The receptor activator of nuclear factor kappa B stimulation of bone resorption, include parathor-
ligand (RANKL)/receptor activator of nuclear mone (PTH), 1,25-dihydroxyvitamin D, and
factor kappa B (RANK)/osteoprotegerin (OPG) glucocorticoids (Kohli and Kohli 2011; Ma
system is the main trimolecular complex which et al. 2001; Hofbauer and Schoppet 2004). On
mediates osteoblast-controlled osteoclastogenesis the other hand, factors that decrease the RANKL/
and bone degradation and maintains physiologic OPG ratio, such as estrogens, androgens, and
bone remodeling (Kohli and Kohli 2011; Yates leptin, inhibit bone resorption and consequently
and Górecki 2006; Theoleyre et al. 2004; Khosla decelerate bone turnover (Rubin et al. 2002;
2001; Yasuda et al. 1998; Simonet et al. 1997). Burguera et al. 2001; Szulc et al. 2001; Lee and
The balance between OPG and RANKL/RANK Lorenzo 1999).
is crucial for normal osteoclastic bone resorption. In healthy children and adolescents, bone mass
RANKL is expressed as a surface-bound mole- depends on the balance between bone formation
cule on cells such as osteoblasts, bone marrow and bone resorption, which are regulated in grow-
stromal cells, chondrocytes, activated T ing bone by several systemic and local factors.
lymphocytes and CD4+/CD8+ thymocytes or as The growth hormone/insulin-like growth factor-1
a soluble molecule released by tumor necrosis (GH/IGF-1) and RANKL/RANK/OPG pathways
factor-alpha convertase. RANK receptors are seem to be the major determinants of those pro-
expressed on preosteoclasts and on other cells of cesses (Guerra–Menéndez et al. 2013; Kohli and
this lineage. OPG is produced and secreted by Kohli 2011; Boyce and Xing 2008; Hofbauer and
osteoblasts and by many other cell types, such Schoppet 2004), but the clinical application of the
as the heart, kidney, liver, spleen, and B cells RANKL/RANK/OPG pathway may be difficult
(Boyce and Xing 2008; Kearns et al. 2008; Li due to a lack of well-established reference data.
et al. 2007; Takayanagi 2007; Vega et al. 2007; Growth hormone deficiency and IGF-1 defi-
Wada et al. 2006; Ikeda et al. 2001). The interac- ciency are some of the main risk factors for mark-
tion between RANKL and RANK stimulates edly reduced bone mineral density, increased
osteoclast formation, differentiation, and survival fracture risk, and decreased bone remodeling
in bone by activation of a number of transcription (Giustina et al. 2008). GH replacement therapy
factors and signaling pathways, whereas OPG increases bone turnover as evaluated by changes
acts as a soluble decoy receptor by binding in biochemical markers of bone formation and
RANKL and consequently inhibits RANKL- bone resorption, which peak at 3–6 months after
induced osteoclastic bone resorption (Rubin the start of GH treatment, suggesting the activa-
et al. 2002; Mundy et al. 2001). Both local and tion of both osteoblasts and osteoclasts
systemic factors such as various cytokines and (Witkowska–Sędek et al. 2016, 2017, 2018;
calciotropic hormones, which are crucial in the Andersson et al. 2015; Giustina et al. 2008;
regulation of osteoclastogenesis and bone Nielsen et al. 1991). Experimental studies have
remodeling, are associated with changes in the shown that the GH/IGF-1 axis is involved in the
stromal RANKL/OPG ratio. The discovery of regulation of the RANKL/RANK/OPG pathway
those interactions has helped to understand the and subsequently modulates bone formation and
pathophysiology of several disorders which affect bone resorption, but the exact mechanism of those
bone metabolism, such as glucocorticoid-induced associations is not fully explained (Guerra–
osteoporosis, postmenopausal osteoporosis, rheu- Menéndez et al. 2013; Mrak et al. 2007; Wang
matoid arthritis, multiple myeloma, vascular cal- et al. 2006; Rubin et al. 2002; Ueland et al. 2002).
cification, chronic periodontitis, and rare genetic Moreover, it is emphasized that OPG and
disorders of mineral metabolism (Friedmann et al. RANKL serum levels do not fully reflect their
2014; Vega et al. 2007; Kruk et al. 2002). Factors concentrations in bone microenvironment, which
Osteoprotegerin, Receptor Activator of Nuclear Factor Kappa B Ligand, and. . . 65
is a further difficulty in the exact evaluation of the children. In the 41 strong group of GH-deficient
relationships between the GH/IGF-1 axis and the children, 18 of them were treated with GH for
RANKL/RANK/OPG pathway. 12 months, 14 were treated for 6 months, and in
This study seeks to define serum OPG and 9 children only baseline data were available. All
total sRANKL concentrations in short children, GH-deficient children received recombinant
with different GH secretory status, and to evalu- human GH subcutaneously once daily at bedtime.
ate the associations between the GH/IGF-1 axis Mean GH doses are reported in Table 2. Height,
and the OPG/RANKL pathway in GH-deficient weight, and body mass index (BMI) were
children during GH replacement therapy. expressed as standard deviation scores (SDS)
according to standards of the Institute for Mother
and Child in Warsaw, Poland. BMI was calcu-
2 Methods lated as weight in kilograms divided by height in
square meters (kg/m2). Height was normalized for
2.1 Subjects and General Protocol chronological age, while weight and BMI were
normalized for height-age. Height-age was
This study was conducted at the Department of defined as the age that corresponds to the patient’s
Pediatrics and Endocrinology of the Medical Uni- height when plotted at the 50th percentile on a
versity of Warsaw, Poland, and included 41 growth chart. Baseline height velocity (HV) was
GH-deficient and 20 GH-sufficient short children calculated in all children using data from the
and adolescents recruited prospectively. All the period of 6–18 months before the initiation of
children underwent GH testing in order to evalu- GH treatment or taking baseline anthropometric
ate the GH secretory status. GH deficiency was and biochemical measurements in GH-sufficient
diagnosed according to the following criteria: children. Bone age was evaluated using the
height below the 3rd percentile for sex and age standards of Greulich and Pyle (1969). Pubertal
according to Polish growth charts, height velocity status was assessed according to staging system
less than –1 SD below mean for sex- and of Tanner (1962).
age-matched Polish population, bone age delay,
exclusion of causes of short stature other than GH
deficiency, and the GH level below 10 ng/mL in a 2.2 Biochemical Analysis
test of spontaneous nocturnal growth hormone
secretion (five measurements of GH Serum OPG, total sRANKL, IGF-1, PTH,
concentrations made every 30 min during the 25-hydroxyvitamin D (25(OH)D), calcium and
first 2 h after falling asleep) and in two pharma- phosphorus content, total alkaline phosphatase
cological tests with different stimuli such as clo- (ALP) activity, and 24-h urine calcium and phos-
nidine, insulin, glucagon, or arginine. Children phorus excretion were measured in all children at
with GH release equalling or exceeding 10 ng/ baseline and at 6 and 12 months of GH replace-
mL were considered as GH-sufficient. The maxi- ment therapy in the GH-deficient group. Serum
mum release of GH (GHmax) in a particular content of OPG and total sRANKL were
patient was defined as the highest GH concentra- measured with ELISA (DRG Instruments
tion in any measurement in three tests in GmbH, Marburg, Germany) and those of GH,
GH-deficient children and in any measurement IGF-1, and PTH with an immunoassay using the
in the test or tests performed in GH-sufficient IMMULITE 2000 Xpi analyzer (Siemens,
children. Erlangen, Germany). The serum content of 25
The protocol included baseline anthropometric (OH)D was measured by immunoassay using
and biochemical evaluation of both GH-deficient the ARCHITECT analyzer (Abbott Diagnostics,
and GH-sufficient children and anthropometric Abbott Park, IL). The serum ALP activity, cal-
and biochemical evaluation at 6 and 12 months cium and phosphorus content, and urine calcium
of GH replacement therapy in GH-deficient and phosphorus excretion in 24-h urine samples
were measured by dry chemistry system using the the Spearman correlation analysis for nonpara-
VITROS 5600 chemistry analyzer (Ortho Clinical metric data. The independent predictors of OPG
Diagnostics, Raritan, NJ). IGF-1 concentrations elevation during the first 6 months of GH treat-
were normalized for sex and bone age and were ment were analyzed using backward stepwise
expressed as SDS according to the normative data regression beginning with the three variable
provided by the manufacturer (Siemens regression models. A p-value <0.05 defined sta-
Healthcare Diagnostics Inc., Deerfield, IL). tistically significant differences. The analysis was
performed using a commercial Statistica v13.1
package (StatSoft, Tulsa, OK).
2.3 Statistical Evaluation
Data were reported as means SD or medians 3 Results

with interquartile ranges. Data normality was
checked with the Shapiro-Wilk test. Comparisons 3.1 Anthropometric and Biochemical
between the GH-deficient and GH-sufficient chil- Parameters at Baseline
dren were conducted using a t-test or Mann and During Growth Hormone
Whitney U test. Comparisons between baseline Therapy
and treatment values for the corresponding
parameter were conducted using the repeated At baseline there were no significant differences
measures ANOVA with Bonferroni post hoc test in any evaluated anthropometric or biochemical
for parametric data and the Friedman test with parameters between the GH-deficient and
post hoc comparisons for nonparametric data. GH-sufficient children (Table 1).
Correlation analyses were performed using the After the initiation of GH treatment, as
Pearson correlation test for parametric data and expected, HV increased significantly
Table 1 Anthropometric and biochemical parameters in GH-deficient and GH-sufficient children at baseline
Parameter GH-deficient children GH-sufficient children p
Number of patients 41 20 –
Prepubertal/pubertal status) 31/10 13/7 –
Age (years) 10.3 3.5 10.8 3.4 ns
Bone age (years) 8.2 3.5 9.7 3.7 ns
Height SDS 2.59 0.58 2.60 0.57 ns
Weight SDS for height-age 0.09 0.72 0.36 0.57 ns
BMI SDS for height-age 0.08 1.00 0.45 0.75 ns
HV (cm/year) 5.3 1.4 5.2 1.4 ns
IGF-1 SDS 0.37 1.04 0.20 1.41 ns
OPG (pmol/L) 5.6 (3.6–10.1) 9.4 (4.8–11.5) ns
Total sRANKL (pmol/L) 226.3 (164.5–317.3) 243.8 (179.9–337.2) ns
Total ALP (U/L) 202.0 (171.0–231.0) 193.5 (160.0–236.5) ns
PTH (pg/mL) 20.7 (12.2–24.1) 17.6 (11.1–22.9) ns
25(OH)D (ng/mL) 25.8 (22.2–34.0) 27.6 (24.3–34.6) ns
Serum Ca (mg/dL) 10.0 (9.8–10.3) 10.1 (10.0–10.2) ns
Serum P (mg/dL) 5.0 0.46 5.1 0.53 ns
Urine Ca excretion (mg/kg/24 h) 2.9 (1.5–4.0) 2.5 (1.0–4.2) ns
Urine P excretion (mg/kg/24 h) 16.8 (14.0–20.1) 19.5 (16.1–23.1) ns
Data are presented as means SD or medians with interquartile ranges (IR) as appropriate; ns not significant, GH growth
hormone, SDS standard deviation score, BMI body mass index, HV height velocity, IGF-1 insulin-like growth factor-1,
OPG osteoprotegerin, sRANKL receptor activator of nuclear factor kappa B ligand, ALP alkaline phosphatase, PTH
parathormone, 25(OH)D 25-hydroxyvitamin D, Ca calcium, P phosphorus
(p < 0.001), which led to an increase in height significantly higher than that at baseline after
SDS observed after the first 6 months of treatment 12 months of GH treatment (p < 0.05), but its
(p < 0.001). Weight SDS and BMI SDS for 24-h urine excretion did not change significantly
height-age did not change significantly during during therapy. Nor did serum phosphorus con-
the first 12 months of GH treatment. The mean tent change during GH treatment. The anthropo-
IGF-1 SDS increased significantly after 6 months metric and biochemical parameters at baseline
of GH treatment compared to the baseline value and at 6 and 12 months of GH replacement ther-
(p < 0.001) but then declined back toward the apy are presented in Table 2.
baseline value after another 6 months of treat-
ment, remaining insignificantly higher than that
at baseline. The OPG content and total ALP
3.2 Associations Between OPG or
activity increased significantly after the first
Total sRANKL
6 months of GH treatment (p < 0.01 and
and Anthropometric
p < 0.05, respectively) and then reached a plateau,
and Biochemical Parameters
remaining significantly higher than that at base-
line after 12 months of treatment (p < 0.001 and
Baseline associations between OPG or total
p < 0.05, respectively). Total sRANKL, PTH, and
sRANKL and the anthropometric and biochemi-
25(OH)D did not change significantly during GH
cal parameters investigated, taken as independent
treatment. Serum calcium content was
variables, in GH-deficient and GH-sufficient
Table 2 Anthropometric and biochemical parameters at baseline and during GH treatment in GH-deficient children
Parameter Baseline 6 months 12 months pa pb pc
Number of patients 41 32 18 – – –
Age (years) 10.3 3.5 11.3 3.4 11.7 3.6 – – –
Bone age (years) 8.2 3.5 – 9.9 3.5 – <0.001 –
Height SDS 2.59 0.58 2.23 0.59 2.01 0.64 <0.001 <0.001 <0.01
Weight SDS for height-age 0.09 0.72 0.17 0.72 0.06 0.72 ns ns ns
BMI SDS for height-age 0.08 1.00 0.18 0.98 0.03 1.01 ns ns ns
HV (cm/year) 5.3 1.4 – 9.8 1.03 – <0.001 –
GH dose (mg/kg/week) – 0.19 0.01 0.19 0.01 – – –
IGF-1 SDS 0.37 1.04 2.02 2.51 0.94 1.06 <0.001 ns <0.05
OPG (pmol/L) 5.55 7.57 9.70 <0.01 <0.001 ns
(3.56–10.05) (4.31–12.16) (6.36–12.42)
Total sRANKL (pmol/L) 226.3 260.9 316.4 ns ns ns
(164.5–317.3) (155.4–354.0) (129.3–511.4)
Total ALP (U/L) 202.0 244.0 264.0 <0.05 <0.05 ns
(171.0–231.0) (212.0–291.0) (225.0–282.0)
PTH (pg/mL) 20.7 (12.2–24.1) 19.2 (11.2–30.3) 16.6 (12.6–22.5) ns ns ns
25(OH)D (ng/mL) 25.8 (22.2–34.0) 27.3 (23.1–31.2) 28.0 (23.1–31.8) ns ns ns
Serum Ca (mg/dL) 10.0 (9.8–10.3) 10.2 (9.9–10.3) 10.2 (10.0–10.5) ns <0.05 ns
Serum P (mg/dL) 5.00 0.46 5.30 0.36 5.40 0.80 ns ns ns
Urine Ca excretion (mg/kg/ 2.9 (1.5–4.0) 2.3 (2.0–3.2) 2.0 (1.2–4.4) ns ns ns
24 h)
Urine P excretion (mg/kg/ 16.8 (14.0–20.1) 20.1 (14.5–26.1) 17.8 (14.3–22.3) ns ns ns
24 h)
Data are presented as means SD or medians with interquartile ranges (IR) as appropriate; pa p-value baseline vs. 6 months,
pb p-value baseline vs. 12 months, pc p-value 6 vs. 12 months, ns not significant, GH growth hormone, SDS standard
deviation score, BMI body mass index, HV height velocity, IGF-1 insulin-like growth factor-1, OPG osteoprotegerin,
sRANKL receptor activator of nuclear factor kappa B ligand, ALP alkaline phosphatase, PTH parathormone, 25(OH)D
25-hydroxyvitamin D, Ca calcium, P phosphorus
Table 3 Spearman’s correlation coefficients (r) between baseline serum OPG (pmol/L) or total sRANKL (pmol/L)
concentrations and baseline anthropometric and biochemical parameters in GH-deficient and GH-sufficient children
GH-deficient children GH-sufficient children
Parameter OPG Total sRANKL OPG Total sRANKL
Age (years) ns ns ns 0.45 (<0.05)
Bone age (years) ns ns ns 0.46 (<0.05)
Height SDS ns ns ns ns
Weight SDS for height-age 0.33 (<0.05) 0.46 (<0.01) ns ns
BMI SDS for height-age ns 0.41 (<0.01) ns 0.47 (<0.05)
HV (cm/year) ns ns ns ns
GHmax (ng/mL) ns ns ns ns
IGF-1 SDS ns ns 0.55 (< 0.05) ns
Total ALP (U/L) ns ns ns ns
PTH (pg/mL) ns ns ns ns
25(OH)D (ng/mL) ns 0.34 (<0.05) ns ns
Serum Ca (mg/dL) ns ns 0.46 (<0.05) ns
Serum P (mg/dL) ns ns ns ns
Urine Ca excretion (mg/kg/24 h) ns ns ns ns
Urine P excretion (mg/kg/24 h) ns ns ns ns
GHmax maximum growth hormone release, SDS standard deviation score, BMI body mass index, HV height velocity,
IGF-1 insulin-like growth factor-1, OPG osteoprotegerin, sRANKL receptor activator of nuclear factor kappa B ligand,
ALP total alkaline phosphatase, PTH parathormone, 25(OH)D 25-hydroxyvitamin D, Ca calcium, P phosphorus, ns not
significant
children, are presented in Table 3. There were no with baseline height SDS (r ¼ 0.49; p < 0.01),
significant associations between either maximum weight SDS and BMI SDS for height-age
GH release, baseline height SDS or HV and either (r ¼ 0.54; p < 0.01 and r ¼ 0.58; p < 0.001,
baseline OPG or total sRANKL levels in GH- respectively), and with baseline IGF-1 SDS
deficient and GH-sufficient children. In (r ¼ 0.46; p < 0.01). Total sRANKL content did
GH-deficient children, the strongest associations not change significantly during GH treatment.
were found between the total sRANKL content Likewise, we did not find any associations of
and the nutritional parameters such as weight significance between OPG concentrations after
SDS for height-age and BMI SDS for height-age the first 6 months of GH treatment and any of
(r ¼ 0.46; p < 0.01 and r ¼ 0.41; p < 0.01, the anthropometric or biochemical parameters
respectively). In GH-sufficient children, OPG measured after the first 6 months of GH treatment.
content was adversely associated with IGF-1 The increment in IGF-1 during the first 6 months
SDS (r ¼ 0.55, p < 0.05), while total sRANKL of therapy (ΔIGF-16-month-baseline) and GH doses
was adversely associated with age (r ¼ 0.45; administered in that period were not associated
p < 0.05), bone age (r ¼ 0.46; p < 0.05), and with OPG concentrations after 6 months of ther-
BMI SDS for height-age (r ¼ 0.47; p < 0.05). apy either. ΔOPG6-month-baseline was associated
There were no statistically significant with height SDS (r ¼ 0.46; p < 0.01), weight
associations between OPG or total sRANKL SDS, and BMI SDS for height-age (r ¼ 0.61;
levels at 6 months of GH treatment and baseline p < 0.001 and r ¼ 0.50; p < 0.01, respectively)
anthropometric and biochemical parametres. Fur- and total ALP activity (r ¼ 0.39; p < 0.05) and
ther analysis revealed that the increment in OPG was adversely associated with 25(OH)D
concentration in the first 6 months of treatment concentrations (r ¼ 0.45; p < 0.05) evaluated
(ΔOPG6-month-baseline) was adversely associated after the first 6 months of GH treatment. Total
with GHmax (r ¼ 0.41; p < 0.05) and positively sRANKL concentration after the first 6 months of
GH treatment was associated only with total ALP months of therapy. Backward stepwise regression
activity at 6 months of therapy (r ¼ 0.42; analysis was not performed at 12 months of GH
p < 0.05). treatment because there were too few patients
To evaluate the relationship between the who could be included in such analysis.
ΔOPG6-month-baseline, considered as a dependent
variable, and the independent study variables,
measured at baseline (models 1, 2, and 3) and at 4 Discussion
6 months of GH treatment (models 4, 5, and 6),
backward stepwise regression analysis was used. Several authors have investigated the relationships
Regression models included the following inde- between the GH/IGF-1 axis and the RANKL/
pendent variables: model 1, baseline height SDS, RANK/OPG pathway in experimental and clinical
weight SDS, and BMI SDS for height-age; model studies (Xia et al. 2015; Guerra–Menéndez et al.
2, GH max, baseline bone age, and IGF-1 SDS for 2013; Flint et al. 2009; Wang et al. 2006; Lanzi
bone age; model 3, baseline total ALP, PTH, and et al. 2003; Ueland et al. 2003), but such studies in
25(OH)D; model 4, height SDS, weight SDS, and GH-deficient patients, especially in children and
BMI SDS for height-age, all at 6 months of GH adolescents, are scarce. IGF-1 influences OPG and
treatment; model 5, IGF-1 SDS for bone age, RANKL concentrations in a dose- and time-
ΔIGF-16-month-baseline, and GH dose administered dependent manner in vitro and in vivo, which
in the first 6 months of treatment; model 6, total confirms its role as a coupling agent in direct
ALP, PTH, and 25(OH)D, all at 6 months of GH activation of bone formation and indirect, through
treatment. The variables, baseline BMI SDS for OPG/RANKL, activation of bone resorption
height-age (β ¼0.42; p < 0.05), baseline ALP (Rubin et al. 2002). It is hypothesized that GH
activity (β ¼0.36; p < 0.05), weight SDS for induces OPG production and is able to modulate
height-age at 6 months of GH treatment (β bone remodeling by directly influencing
¼1.86; p < 0.01), and total ALP activity at osteoblast-osteoclast cross talk (Mrak et al.
6 months of GH treatment (β ¼ 0.48; p < 0.01), 2007). The results of studies evaluating OPG and
were identified as independent predictors of RANKL concentrations in children and
ΔOPG6-month-baseline in models 1, 3, 4, and adolescents are divergent. Further multicenter
6, respectively. Neither GH max, nor IGF-1 studies, including large cohorts of children and
SDS or increase in IGF-1 SDS after the initiation adolescents, are needed to establish valuable refer-
of GH treatment, nor GH dose, nor bone age was ence data for pediatric population and to foster the
found to be significant independent predictors of clinical use of the RANKL/RANK/OPG pathway.
OPG elevation after the initiation of therapy. Buzi et al. (2004) have found that the OPG level in
After the first 12 months of GH treatment, we healthy children is gender independent but changes
failed to find any significant associations between with age and is significantly higher in the first
the OPG content or ΔOPG12-month-baseline and any 4 years of life than at 4–14 years of age, while
of the anthropometric and biochemical RANKL content slightly declines with age. Those
parameters. GH doses administered in the first authors have also reported that serum OPG content
12 months of replacement therapy were adversely in children affected by Turner’s syndrome
associated with OPG at 12 months (r ¼ 0.60; (TS) and early or precocious puberty (PP) is com-
p < 0.05). We also noticed that there was a ten- parable to those in healthy peers, while that in
dency for greater elevation in OPG in the first children with rheumatoid arthritis (RA) is signifi-
12 months of therapy in children treated with cantly higher than in controls. Simultaneously,
lower GH doses, but that result was statistically they have also found that serum RANKL does
insignificant (r ¼ 0.49; p ¼ 0.06). Total not differ significantly between TS-, PP-, and
sRANKL content at 12 months of GH treatment RA-afflicted children and age-matched healthy
was not associated with any of the anthropometric population. On the other hand, Wasilewska et al.
and biochemical parameters evaluated at 12 (2009) have suggested that in healthy children and
adolescents, OPG concentration is independent of response to the missing anabolic actions of GH

gender, age, and pubertal status, while RANKL is and IGF-1 in GH-deficient children, but the lack
higher in boys than girls and increases with age of significant associations between OPG, IGF-1,
and weight. Meazza et al. (2014), in turn, have and the maximum GH release in diagnostic tests
failed to find any significant differences in the would weigh against such relationships. The pres-
OPG concentration between healthy controls and ent study revealed that in both GH-deficient and
GH-deficient children before the initiation of GH GH-sufficient children, neither OPG nor total
treatment. Studies, also those in the adult popula- sRANKL content was associated with maximum
tion, show a positive association between serum GH release, but there was an inverse association
OPG and age, which is considered a compensatory between OPG concentration and IGF-1 SDS for
response aimed at counteracting bone loss in the bone age in GH-sufficient children. We found that
aging skeleton, while serum RANKL seems to OPG concentration was inversely associated with
decrease with age (Abrahamsen et al. 2005; weight SDS for height-age, while total sRANKL
Kudlacek et al. 2003). In different stages of patho- concentration was positively associated with
logical bone metabolism with elevated bone nutritional status in GH-deficient children. In con-
resorption, OPG content usually increases, which trast, in GH-sufficient children, there was an
could be interpreted as a compensatory bone inverse association of sRANKL content with
response to increased osteoclastic activity (Siomou age and bone age. In our opinion, the main limi-
et al. 2011; Kearns et al. 2008; Buzi et al. 2004; tation of this study, and that of Flint et al. (2009),
Ueland et al. 2001). Both untreated GH deficiency is a small number of short children investigated.
and IGF-1 deficiency, which are characterized by A larger sample size of short children and
decreased bone turnover and increased fracture adolescents should be evaluated to better explore
risk, and GH replacement therapy, which leads to the relationships between the RANKL/RANL/
an increase in bone turnover, could be reflected in OPG system and GH secretory status.
changes in serum RANKL and OPG In the present study, we also set out to evaluate
concentrations (Ueland and Bollerslev 2005; the effects of GH replacement therapy on OPG
Lanzi et al. 2003). and total sRANKL content and the relationship
In the present study, we set out to evaluate the between OPG or RANKL and other biochemical
differences in serum OPG and total sRANKL parameters, including total ALP activity that is
content between GH-deficient and GH-sufficient considered a bone formation marker, and
short children. We hypothesized that the OPG calcium-phosphorus balance in GH-deficient
and total sRANKL content could be associated children. We found that after the initiation of
with the GH secretory status and IGF-1 concen- GH treatment, both OPG content and ALP activ-
tration. We found that although GH-deficient ity significantly increased, while total sRANKL,
children showed a tendency for higher BMI and PTH, and 25(OH)D remained unchanged. The
lower IGF-1 SDS for bone age, accompanied by a content of serum calcium also increased during
greater bone age delay, their OPG and sRANKL GH treatment, but its urine excretion did not
were not different than those detected in change significantly. Nor were there changes in
GH-sufficient children. Similar results have been the content of serum phosphorus and its urine
previously reported by Flint et al. (2009), who excretion. We also noticed that the increase in
further conclude that that neither OPG nor OPG in the first 6 months of GH treatment was
RANKL correlates with BMD z-score in the adversely associated with the maximum GH
short children. In addition, those authors have release and positively associated with height
revealed that IGFBP-3, which is closely related SDS and nutritional status at baseline and at
to GH secretory status, is the only significant 6 months of therapy, with baseline IGF-1 SDS
negative predictor of serum OPG concentration. for bone age, and with total ALP activity at
The authors put forward a notion that increased 6 months of therapy. The backward stepwise
OPG content might represent a compensatory regression models revealed that only ALP activity
and nutritional status independently predicted the disorders in GH-deficient adults. They demon-
elevation of OPG after the initiation of GH treat- strate that plasma OPG content is higher in
ment. We also found that GH doses were GH-deficient adults than in healthy controls and
adversely associated with the increase in OPG in is associated with age, BMI, waist and hip circum-
the first year of replacement therapy. The plausi- ference, waist-hip ratio, and the content of fasting
ble association between GH administration and insulin, total cholesterol, triglycerides, high sensi-
OPG content ought to be confirmed in a larger tivity C-reactive protein, and interleukin-6. The
group of patients. Meazza et al. (2014) have also authors suggest that OPG could be a potential
investigated the effects of 12-month GH treat- therapeutic target in cardiovascular disorders.
ment on OPG content and on various metabolic Those results suggest that the RANKL/RANK/
parameters, including adipokine profile, in OPG pathway is associated not only with bone
GH-deficient children, and report, in opposition turnover but also with metabolic status and could
to the present study, a lack of significant changes be used as a marker of impaired metabolic status
after the initiation of GH treatment. Those authors leading to increased cardiovascular risk. Although
have found that height SDS, both at baseline and those observations have been made in
after 12 months of GH treatment, was adversely GH-deficient adults, it is possible that the same
associated with the OPG content in GH-deficient associations could also apply for GH-deficient
children. They also found an adverse association children and adolescents. Erol et al. (2016) have
between the baseline OPG and IGF-1 content found that OPG content is lower in obese than
before treatment. The discrepancy between the nonobese children, but they do not find any
present findings and those above outlined points associations between OPG and insulin resistance.
to the need of further exploration of OPG content In conclusion, we found that serum OPG or
changes due to GH treatment. sRANKL was independent from GH secretory
The results of studies in adult populations par- status and IGF-1 content in short children. After
tially correspond to those reported in children. the initiation of GH replacement treatment, serum
Ueland et al. (2001) have reported that serum OPG content increased significantly, while total
OPG content does not depend on GH secretory sRANKL remained unchanged. The increase in
status and does not differ between GH-deficient OPG during GH treatment was positively
and healthy adults. Lanzi et al. (2003), who associated with total ALP activity and nutritional
investigated the effects of 6-month-long GH treat- status in GH-deficient children. The exploration of
ment in both childhood and adult-onset GH defi- mutual relationships between the RANKL/RANK/
ciency, have reported a significant increase in OPG pathway and both bone turnover and
plasma OPG content after the first 6 months of glucolipid metabolism could lead to new avenues
GH treatment, which is accompanied by signifi- of clinically applicable research concerning GH
cant increases in total serum calcium and urinary deficiency and GH replacement treatment.
calcium excretion with unchanged PTH and 25
(OH)D levels. In the same study, the authors also Conflicts of Interest The authors declare no conflicts of
evaluate the associations between the OPG level interest in relation to this article.
during GH treatment and two bone turnover
markers: osteocalcin, considered a bone formation Ethical Approval All procedures performed in this study
were in accordance with the ethical standards of the insti-
marker, and urine deoxypyridinoline excretion,
tutional research committee and with the 1964 Helsinki
considered a bone resorption marker. They have Declaration and its later amendments. The study protocol
reported an inverse association of OPG increase was approved by the Bioethics Committee of the Medical
during GH treatment with the increase in University of Warsaw (permit: KB/93/2016).
osteocalcin and with deoxypyridinoline excretion.
Xia et al. (2015) have revealed that OPG, despite Informed Consent Informed written consent was
obtained from all individual participants included in the
its associations with bone metabolism, might also
study or from their parents and legal guardians.
play a role in the development of cardiovascular
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Inhibition of Cross-Reactive
Carbohydrate Determinants in Allergy
Diagnostics
Maciej Grzywnowicz, Emilia Majsiak, Józef Gaweł,

Karolina Miśkiewicz, Zbigniew Doniec, and Ryszard Kurzawa
Abstract inhibitor was applied, the anti-CCD antibody

Despite being clinically largely irrelevant, content decreased in 16, remained unchanged
antibodies against cross-reactive carbohydrate in 3, and increased in 5 samples. In samples
determinants (CCD) are an important issue in positive for plant allergens, the asIgE content
the in vitro diagnostics, as they may produce dropped by an average of 72% when the CCD
false positive or falsely elevated results of the inhibitor was used in the assay, except the
immunoglobulin E class (asIgE) in relation to antibodies to tree and grass pollen allergens,
the actually present level of asIgE. The present for which the asIgE content remained above
chapter demonstrates an effective resolution of 100 kU/L. We conclude that the use of a CCD
this diagnostic issue by the use of a CCD inhibitor in in vitro assays is a viable option to
inhibitor in in vitro tests. A synthetic CCD mitigate the influence of anti-CCD antibodies
inhibitor, Polycheck® CCD inhibitor, was on the measured level of asIgE immunoglobu-
used in the laboratory diagnostics of 24 chil- lin, which increase the reliability of testing
dren diagnosed with allergic diseases. The particularly in cases displaying multiple
anti-CCD antibody content was measured in allergies.
the serum using a Polycheck® Atopic 30-I
panel (Biocheck GmbH; Münster, Germany), Keywords
a screening assay for the quantitative determi- Allergy · Anti-CCD antibodies · CCD
nation of multiple allergen-specific IgE. We inhibitor · Cross-reactive carbohydrate
found that the baseline anti-CCD antibody determinants (CCD) · IgE immunoglobulin
content, without the CCD inhibitor, ranged
from 0.7 to 3.5 kU/L in the sera of the majority
of 16 out of 24 children. When the CCD 1 Introduction
Cross-reactive carbohydrate determinants (CCD)

M. Grzywnowicz and E. Majsiak
are structurally simple carbohydrate residues
Polish-Ukrainian Foundation of Medicine Development,
Lublin, Poland which become attached to amino acid protein
chains during glycosylation as part of post-
J. Gaweł, K. Miśkiewicz, Z. Doniec, and R. Kurzawa (*)
Allergology and Pulmonology Clinic, Institute of translational processing in eukaryotic cells. This
Tuberculosis and Lung Diseases, Regional Branch in process, which leads to glycoprotein production,
Rabka-Zdrój, Rabka-Zdrój, Poland is common in both plants and animals. What
75
76 M. Grzywnowicz et al.
makes CCD different from other carbohydrate positive levels of anti-CCD antibodies. To this
residues is their antigenicity and ability to stimu- end, we compared the asIgE content in relation
late the production of allergen specific immuno- to 30 allergens using multiparametric
globulin E (asIgE). The term “cross-reactive” Polycheck® tests with and without a CCD
determinants was first used more than 30 years inhibitor.
ago (Aalberse et al. 1981). Subsequent research
has demonstrated that anti-CCD antibodies are of
no clinical relevance, since their presence is not
2 Methods
associated with any symptoms (Mertens et al.
2010; Muller et al. 2009; Mari et al. 2008; Ebo
The study was approved by the institutional
et al. 2004; Mari 2002). While basophil activation
Ethics Committee and was performed according
tests have demonstrated bioactivity of anti-CCD
to the guidelines of the Helsinki Declaration for
asIgE, no connection has been substantiated to
Human Research. There were 24 children of
clinical symptoms in patients (Mertens et al.
either genders (12 boys and 12 girls) aged 3–10
2010; Foetisch et al. 2003).
(average 5 years) enrolled into the study. They
Despite the seeming clinical irrelevance, anti-
were hospitalized with chronic upper and lower
CCD antibodies pose a serious problem in case of
respiratory tract symptoms and skin symptoms in
the in vitro asIgE diagnostics with respect to the
the Regional Branch of Warsaw Institute of
allergens in which they appear, making it harder to
Tuberculosis and Lung Diseases in the mountain-
correctly diagnose the allergy source. That is
ous city of Rabka-Zdrój from July to August
caused by the structural heterogeneity of CDD
2017. The goal of hospitalization was to conduct
and their resemblance in various allergen sources.
the diagnostics procedures and to determine ther-
The most important type of CCD, having a
apy. Seventeen children were diagnosed with
sequence of N-glycan with alpha(1,3)-fructose
asthma, associated with allergic rhinitis in 8 of
attached to the polypeptide chain through nitrogen
them, and 7 children were diagnosed with moder-
atom in the asparagine radical, is present in both
ately severe atopic dermatitis. Anti-CCD
plant allergens, including inhalable allergens and
antibodies were found in the sera of all children
various food allergens, hymenoptera venom, and
in a range of 0.43–25 kU/L (median 1.5 kU/L).
latex (Altmann 2016). Therefore, when an exam-
The Polycheck® Atopic 30-I (Biocheck
ined person produces anti-CCD antibodies, they
GmbH, Münster, Germany), an allergy test rou-
can bind with allergens which contain CDDs dur-
tinely used in allergy screening, was the diagnos-
ing the test, giving a false positive or falsely ele-
tic method applied. This test is a multiparametric,
vated result in relation to the actual asIgE content.
quantitative test with a lower limit of detection of
Taking into account that the prevalence of anti-
0.15 kU/L. As recommended by the producer, it
CCD antibodies in people subjected to the in vitro
was assumed that positive results start from the
asIgE testing can reach 25%, they pose a serious
level of 0.35 kU/L. The allergens included in the
diagnostic problem in the allergy testing (Muller
assay were the following: cow’s milk, alpha-
et al. 2009; Ebo et al. 2004; Mari et al. 2008). The
lactalbumin, beta-lactoglobulin, casein, egg
prevalence of anti-CCD antibodies is even higher
white, egg yolk, codfish, peanut, cacao, soybean,
among young people, amounting to 35%.
apple, carrot, tomato, flour mix, chicken meat,
One way to circumvent or eliminate the prob-
citrus mix, rice, grass mix, rye, dog epithelia, cat
lem posed by anti-CCD antibodies in the in vitro
epithelia, Cladosporium herbarum, Alternaria
asIgE diagnostics is to perform the component-
alternata, Aspergillus fumigatus,
resolved diagnostics, based on the antigens free
Dermatophagoides pteronyssinus, D. farinae,
from CCD or utilizing a CCD inhibitor. The
hazel pollen, birch pollen, and mugwort pollen.
premise behind the present study was to evaluate
The test also includes a CCD marker, pineapple
the influence of a CCD inhibitor on the asIgE
bromelain.
content in individuals previously diagnosed with
Inhibition of Cross-Reactive Carbohydrate Determinants in Allergy Diagnostics 77
A Polycheck® CCD inhibitor was used Table 1 Anti-CCD antibody content assayed without and
according to the producer’s recommendations, with the CCD inhibitor (kU/L)
added at a ratio of 1:50 to each tested serum. CCD inhibitor Difference
This synthetic inhibitor is composed of polyva- Sample Without With %
lent CCD structures of bromelain, with up to four 1 25.00 6.30 75
amino acids left after the proteolysis and purifica- 2 0.47 0.22 53
tion processes, which guarantees the lack of pep- 3 0.78 0.41 47
tide epitopes. The glycopeptide obtained in such a 4 1.00 0.56 44
way is attached to a protein carrier, which is 5 2.40 1.40 42
6 1.90 1.20 37
highly purified human serum albumin.
7 8.90 6.10 31
Data were presented in the tabular form as two
8 0.89 0.62 30
nominal variables (sample number and the 9 0.80 0.59 26
absence or presence of CDD inhibitor) and one 10 0.82 0.63 23
measurement variable (content of anti-CCD anti- 11 0.76 0.60 21
body). Differences in the content of anti-CCD 12 2.70 2.30 15
antibody assayed without and with the addition 13 0.56 0.49 13
of a CCD inhibitor, expressed as the percentage 14 2.80 2.50 11
of the former, were statistically analyzed using 15 0.87 0.80 8
the Wilcoxon signed-rank test. A p-value <0.05 16 3.40 3.30 3
defined a statistically significant difference. Sta- 17 1.50 1.50 0
tistical analysis was performed using a commer- 18 2.00 2.00 0
19 2.20 2.20 0
cial GraphPad Prism v7 package (San Diego,
20 1.50 1.60 +7
CA).
21 1.00 1.10 +10
22 0.43 0.48 +12
23 2.40 2.90 +21
3 Results 24 6.30 7.90 +25
We found that the anti-CCD antibody content was

significantly lower when the samples were Table 2 Anti-CCD antibody content assayed without and
incubated in the serum containing the CCD inhib- with the CCD inhibitor in a patient positive for plant-based
allergens (kU/L)
itor than in those without the inhibitor (p < 0.05).
The results for individual samples are presented Polycheck®
in Table 1. The anti-CCD content decreased in Atopic 30-I CCD inhibitor %
16, remained unchanged in 3, and increased in Without With
5 samples. In most of the samples (16 out of the Peanut 3.50 2.00 43
24), the initial anti-CCD antibody content, with- Cacao 0.34 0.25 26
Soybean 0.78 0.38 51
out the inhibitor, was in a range 0.7–3.5 kU/L,
Apple 6.20 0.42 93
i.e., in radioallergosorbent test (RAST) class
Carrot 56.00 16.00 71
2, which is in line with the studies presented in
Tomato 4.20 0.15 96
the literature (Holzweber et al. 2013). Flour mix 4.00 0.58 86
From among the samples analyzed, special Citrus mix 9.20 0.72 92
attention should be paid to those displaying a Rice 9.00 0.65 93
positive result in relation to plant allergens. Grass mix >100 >100 –
Table 2 presents an example of the presence of a Rye >100 >100 –
multitude of plant allergens in a patient’s serum Hazel pollen >100 >100 –
and their changes after the addition of the CDD Birch pollen >100 >100 –
inhibitor. After application of the inhibitor, there Mugwort pollen >100 >100 –
was an across-the-board decrease in the anti-CCD CCD 0.82 0.63 23
78 M. Grzywnowicz et al.
antibodies to plant allergens, except for the the limit of detection, completely mitigating the
antibodies related to tree and grass pollens false positive. On average, the level of the entire
which remained at a level exceeding 100 kU/L. anti-CCD antibody content was lowered by 72%,
despite a 23% decrease in the CCD marker. That
clearly illustrates an advantage of the use of a
CCD inhibitor, instead of testing different levels
4 Discussion
of a CCD marker. Unfortunately, in the patients
investigated in this study, despite positive values
A CCD inhibitor is used in the asIgE detection
for CCD, only one sample displayed numerous
test to limit false positive reactions, by competi-
positive results for asIgE. It is worth emphasizing
tively binding anti-CCD antibodies in the sample
that the use of multiparametric tests makes it
assayed before they become bound with allergens
much easier for a physician to identify the pres-
containing a CCD residue. In general, CCD inhib-
ence of anti-CCD IgE antibodies than when
itor, as opposed to allergen-extract molecules, is
interpreting individual results (Altmann 2016;
composed of a mix of molecules that are unasso-
Holzweber et al. 2013).
ciated with the solid phase of the test, which is
The CCD inhibitor is a tool for in vitro asIgE
why the CCD inhibitor-anti-CCD IgE complexes
testing, which helps reduce the effect of anti-CCD
can be washed out along with the other unbound
antibodies on the test results, and thereby
elements during the procedure, and thus do not
increases the specificity of the method. The appli-
influence the test result. The CCD inhibitor used
cation of this reagent can contribute to the better
in this study has been tested before in a group of
and faster diagnostics in cases of multiple
more than 1,300 CCD-positive patients, effec-
allergies tested in vitro. The present findings
tively reducing the number of false positives
lead to a conclusion that the use of CCD inhibi-
(Holzweber et al. 2013).
tion is a viable option in a clinical setting.
The present study shows that the use of a CCD
Multiparametric tests in the diagnostics of
inhibitor lowers the content of anti-CCD
IgE-dependent allergies facilitate the identifica-
antibodies directly analyzed in the test, which
tion of an influence of anti-CCD antibodies on
complies with the previous observations
the results for other allergens. However, due to
(Altmann 2016). The absence of an inhibitory
additional costs stemming from the need to retest
effect in some samples we analyzed remains
with CCD inhibitor, the use of a CCD inhibitor
unexplained. That could stem from the lack of
should be considered on a case-by-case basis.
duplicate measurements. Also, a small quantity
of the material tested or unique patient’s antibody
Conflicts of Interest The authors declare no conflicts of
characteristics may constitute some limitation. A interest in relation to this article.
low content of asIgE found in the present sample
of patients (median 1.5 kU/L, with no inhibitor)
can be yet another factor making the statistical
References
evaluation difficult, based on the CCD marker
alone. Be it as it may, other authors have also Aalberse RC, Koshte V, Clemens JG (1981) Immunoglob-
noticed the lack of a uniformly inhibitory effect ulin E antibodies that crossreact with vegetable foods,
on the anti-CCD antibody content of a CCD pollen, and Hymenoptera venom. J Allergy Clin
Immunol 68:356–364
inhibitor, although there was a marked reduction
Altmann F (2016) Coping with cross-reactive carbohy-
in false positives (Holzweber et al. 2013). drate determinants in allergy diagnosis. Allergo J Int
As exemplified by a sample showing, in 25:98–105
in vitro testing, different plant-based allergies, Ebo DG, Hagendorens MM, Bridts CH, De Clerck LS,
Stevens WJ (2004) Sensitization to cross-reactive car-
the action of the CCD inhibitor was clearly visible
bohydrate determinants and the ubiquitous protein
in case of reduced specific asIgE (Table 2). In profilin: mimickers of allergy. Clin Exp Allergy
case of the tomato allergen, asIgE dropped below 34:137–144
Inhibition of Cross-Reactive Carbohydrate Determinants in Allergy Diagnostics 79
Foetisch K, Westphal S, Lauer I, Retzek M, Altmann F, by double-blind placebo-controlled oral challenge of

Kolarich D, Scheurer S, Vieths S (2003) Biological the clinical relevance of IgE antibodies against plant
activity of IgE specific for cross-reactive carbohydrate glycans. Allergy 63:891–896
determinants. J Allergy Clin Immunol 111:889–896 Mertens M, Amler S, Moerschbacher BM, Brehler R
Holzweber F, Svehla E, Fellner W, Dalik T, Stubler S, (2010) Cross-reactive carbohydrate determinants
Hemmer W, Altmann F (2013) Inhibition of IgE bind- strongly affect the results of the basophil activation
ing to cross-reactive carbohydrate determinants test in hymenoptera-venom allergy. Clin Exp Allergy
enhances diagnostic selectivity. Allergy 68:1269–1277 40:1333–1345
Mari A (2002) IgE to cross-reactive carbohydrate Muller UR, Johansen N, Petersen AB, Fromberg-Nielsen J,
determinants: an analysis of the distribution and Haeberli G (2009) Hymenoptera venom allergy: anal-
appraisal of the in vivo and in vitro reactivity. Int ysis of double positivity to honey bee and Vespula
Arch Allergy Immunol 129:286–295 venom by estimation of IgE antibodies to species-
Mari A, Ooievaar-de Heer P, Scala E, Giani M, Pirrotta L, specific major allergens Api m1 and Ves v5. Allergy
Zuidmeer L, Bethell D, van Ree R (2008) Evaluation 64:543–548
Effects of Glutathione on Hydrolytic

Enzyme Activity in the Mouse
Hepatocytes
Iwona Stanisławska, Bożena Witek, Marek Łyp,

Danuta Rochon-Szmejchel, Adam Wróbel, Wojciech Fronczyk,
Agnieszka Kamińska, Adam Kołątaj, and Daniel Załuski
Abstract weight, whereas the latter received 12 μL/g of

In this study, the effect of glutathione (GSH) on physiological saline, all given intraperitoneally
the activity of hydrolytic enzymes of lysosomal, daily for 7 days. Then, fragments of liver tissue
microsomal, and cytosolic fractions was studied were collected from the euthanized animals and
in the mouse hepatocytes. The experiments processed to obtain lysosomal, microsomal, and
involved 30 Swiss male mice, divided into the cytosolic fractions of hepatocytes. The activity of
experimental and control groups of 15 mice each. the following enzymes was investigated in vitro:
The former group received injections of 12 μL/g β-glucuronidase, β-galactosidase, β-glucosidase,
of GSH solution at a dose of 100 μg/g body N-acetyl-hexosaminidase, lysosomal esterase and
lipase, acid phosphatase, cathepsin D and L, leu-
I. Stanisławska, M. Łyp (*), and A. Kołątaj cine aminopeptidase, and alanine aminopepti-
Department of Dietetics, College of Rehabilitation, dase. We found that GSH, administered in vivo
Warsaw, Poland in the mouse, in the main (73% cases), increased
e-mail: [email protected] the in vitro activity of the majority enzymes
B. Witek abovementioned, although the effect was some-
Department of Animal Physiology, The Jan Kochanowski how variable, depending on the fraction of
University, Kielce, Poland
hepatocytes and the type of enzyme. The findings
D. Rochon-Szmejchel imply that GSH supplementation may intensify
“Dandiete” Diet Outpatient Clinic, Nowe Miasto
Lubawskie, Poland the rate of cellular hydrolytic degradation, i.e., the
rate of disposal by the cell of unwanted materials.
A. Wróbel
“Expertdent”, Kielce, Poland
Keywords
W. Fronczyk
Institute of Genetics and Animal Breeding, Polish Cellular fraction · Glutathione · Hepatocytes ·
Academy of Sciences, Magdalenka, Poland Hydrolytic enzymes · Mouse liver
A. Kamińska
The Cardinal Stefan Wyszyński University, Warsaw,
Poland 1 Introduction
D. Załuski
Department of Pharmacognosy, The Ludwik Rydygier The liver plays a key role in the metabolism of
Collegium Medicum, Nicolaus Copernicus University, glutathione (GSH), a peptide involved in the
Bydgoszcz, Poland
81
82 I. Stanisławska et al.
regulation of cellular redox state (Kalinina et al. After weaning at 4–5 weeks, animals obtained
2015; Hultberg and Hultberg 2006). GSH also feed consisting of 16% protein for another 7 days.
plays a protective role against oxidative stress The composition and nutritional value of the feed
and damage in the hepatocytes, scavenging a are presented in Table 1. Then, the animals were
variety of free radicals (Wang et al. 2015). GSH divided into the glutathione- and 0.9% NaCl-
is notably present in the hepatocyte cytosol treated (control) groups, consisting of 15 animals
(approx. 90%), where it is synthesized in a each. Over the next 7 days, the mice received
two-step reaction, and in the mitochondrial matrix reduced glutathione (Sigma-Aldrich; St. Louis,
(10%) to which it passes from the cytosol MS) in a dose of 100 μg/g dissolved in 12 μL/g
(Koehler et al. 2006). body mass of 0.9% NaCl or saline alone. All
Lysosomes are the membrane-bound cellular injections were made i.p., twice daily, between
entities that contain hydrolytic enzymes whose 8.15–8.45 a.m. and 4.15–4.45 p.m. 6 h after the
main role, although not explicitly limited to, is last injection, under brief diethyl ether anesthesia,
to degrade and dispose of unwanted by the cell the mice were euthanized by cervical dislocation
material and its debris (Settembre et al. 2013). and were decapitated. Fragments of liver tissue
Oxidative stress contributes to the destabilization were immediately isolated, perfused with 0.9%
of lysosomal membranes causing a release of NaCl solution at 5 C, and placed in a 0.25 M
hydrolases into the cytosol, with a subsequent saccharose solution containing 2 mM EDTA, at a
induction of cellular apoptosis or necrosis (Bidere concentration of 1 g tissue per 6 ml solution. The
et al. 2003; Jóźwik et al. 2003). fragments were homogenized at 200 rpm in a
Recently, GSH supplementation is a heady Potter-Elvehjem tissue grinder with a Teflon
trend in the general public. The peptide is consid- pestle (Omni International; Kennesaw, GA),
ered a food additive with the presumed enhancing according to Beaufay’s (1972) method.
role in the cellular clearance and energy pro- Homogenates were subjected to sequentially
cesses, which also makes it of use in the sports increasing centrifugations: 8 min at 5,500 rpm in
field to increase physical efficacy. However, the a K26D centrifuge, 20 min at 14,000 rpm (Sorvall
influence of GSH on the activity of specific lyso- RC-5C), and 60 min at 10,000 rpm (Beckman
somal processes is largely unknown. The present L7–66; GMI, Ramsey, MN), according to the
study seeks to examine the effect of exogenous method of Marzella and Glaumann (1980).
GSH, applied in vivo, on the in vitro activity of The lysosomal and microsomal sediments
hydrolytic enzymes in the lysosomal, micro- were suspended in 100 mM phosphate-buffered
somal, and cytosolic fractions of the mouse liver. saline (PBS), pH 6.0, with the addition of 0.1%
Table 1 Compound and energy content of the mouse

2 Methods feed used in the experiment
Ingredient Content (%)
2.1 Animals and Study Protocol
Premix LSM 1.0
Salt 0.39
The experiment was performed in 30 Swiss male Chalk 1.6
mice, aged 8 weeks, weighing 25.0 1.2 g. The Phosphates 1.0
animals were kept in standard cages at a room Milk powder 5.8
temperature of 20–22 C, relative humidity of Soybean meal 10.1
50–60%, with water ad libitum, and a 12 h light/ Corn meal 64.7
dark cycle (lights on at 8:00 a.m. and off at 8:00 p. Cereal germs 15.4
m.). All animals were maintained in a good phys- Total energy (MJ/kg) 14.0
ical condition and were under a professional Leucine (% in protein) 8.9
veterinary care. Lysine (% in protein) 4.7
Effects of Glutathione on Hydrolytic Enzyme Activity in the Mouse Hepatocytes 83
Triton X100. They were then frozen and thawed was determined using a modified method of
twice and centrifuged for 10 min at 700 rpm in a Kirschke et al. (1972). All substrates were pur-
K-26D centrifuge. After another double freezing chased from SERVA Feinbiochemica GmbH &
and thawing, the cytosolic fractions were then Co (Heidelberg, Germany).
assayed. Data were presented as means SD.
Differences between group means of the glutathi-
one and control groups were compared using
analysis of variance ANOVA. A p-value <0.05
2.2 Cellular Enzymes
defined statistically significant differences. A
commercial statistical package of SAS/STAT
Activity of cellular enzymes was determined in
software was used (SAS Institute Inc., Cary, NC).
each of the subcellular fraction obtained during
the preparation procedures described in the sub-
section above, i.e., lysosomal, microsomal, and
cytosolic fractions, using the appropriate 3 Results
substrates and methods depicted in Table 2. A
Nicolet Evolution 300 BB spectrophotometer In the glutathione group, activity of β-GlcUr sig-
setup (Thermo Electron Corporation, Rugby, nificantly decreased, on average, in the lysosomal
Warwickshire, UK) was used for the enzyme fraction of hepatocytes (to 64.4% of that in the
activity determination. control group) and increased in microsomal
Activities of β-GlcUr, β-Gal, β-Glu, Hex, AcP, (to 174.9% of control) and cytosolic (to 119.9%
EL, and LL were determined using 4-nitrophenyl of control) fractions. The activity of β-Gal and
substrates at 420 nm wavelength according to β-Glu increased in all the fractions studied, while
Barrett and Heath’s (1977) micromethod. Cath the activity of Hex markedly increased in lyso-
D and Cath L were determined using 6% somal and cytosolic fractions only (to 130.5%
azocasein as a substrate at 366 nm wavelength and 136.2% of control, respectively) (Table 3).
according to Langner et al.’s (1973) method. GSH-fortified animal feed distinctly enhanced the
LeuAP and AlaAP were determined using Fast activity of all remaining hydrolytic enzymes in
Blue BB salt (4-benzoylamino-2,5-diethoxyben- the subcellular fractions of mouse liver, with the
zenediazonium chloride) substrates at 540 nm exception of esterase, leucine and alanine
wavelength according to McDonald and Barrett’s aminopeptidases, and cathepsins in the cytosol
(1986) method. The enzyme activity was and lipase in the microsomal fraction.
expressed in nmol/mg protein/h. The protein
Table 2 Lysosomal enzymes analyzed in the study

Enzyme Enzyme code Assay method
β-glucuronidase (β-GlcUr) EC 3.2.1.31 Barrett and Heath (1977)
β-galactosidase (β-Gal) EC 3.2.1.23 ”
β-glucosidase (β-Glu) EC 3.2.1.21 ”
N-acetyl-hexosaminidase (Hex) EC 3.2.1.30 ”
Acid phosphatase (AcP) EC 3.1.3.2 ”
Esterase (E) EC 3.1.1.2 ”
Lipase (L) EC 3.1.1.13 ”
Cathepsin D (Cath D) EC 3.4.23.5 Langner et al. (1973)
Cathepsin L (Cath L) EC 3.4.22.15 ”
Leucine aminopeptidase (LeuAP) EC 3.4.11.1 McDonald and Barrett (1986)
Alanine aminopeptidase (AlaAP) EC 3.4.11.2 ”
(”) – same as above
Table 3 Activity of glycosidases in lysosomal (L), microsomal (M), and cytosolic (C) fractions of liver tissue in the
in vivo glutathione (GSH)-treated and control untreated mice
Enzyme Fraction Control GSH % of Control
L 303.1 30.0 195.1 17.0** 64.4
β-GlcUr M 94.0 9.2 164.4 16.0** 174.9
C 62.4 5.0 74.8 6.1* 119.9
L 347.8 35.0 450.5 40.0** 129.5
β-Gal M 59.5 5.8 112.9 11.0** 189.7
C 325.5 27.0 394.6 32.0** 121.2
L 117.1 12.0 215.3 19.0** 183.9
β-Glu M 23.9 2.2 34.2 3.3** 143.1
C 134.0 11.0 157.2 12.0* 117.3
L 842.1 84.0 1098.7 99.0** 130.5
Hex M 287.1 29.0 245.5 24.0 85.5
C 225.0 19.0 306.5 25.0** 136.2
L 1979.4 198.0 2571.4 233.0*** 129.9
AcP M 795.7 79.0 1226.9 122.0*** 154.2
C 1520.0 127.0 2444.5 203.0*** 160.8
L 1434.1 143.0 1932.0 175.0*** 134.7
E M 864.9 86.0 1017.7 101.0* 117.78
C 320.5 27.0 302.2 25.0 94.3
L 574.8 58.0 728.8 66.0*** 126.8
L M 592.6 59.0 576.4 57.0 97.3
C 134.2 11.0 157.6 12.0 117.4
L 95.6 9.7 143.7 12.0*** 150.3
AlaAP M 208.2 21.0 328.7 32.0*** 157.9
C 134.1 11.0 132.3 10.0 98.7
L 146.8 15.0 236.3 21.0*** 161.0
LeuAP M 232.5 23.0 359.8 35.0*** 154.8
C 75.1 6.1 80.7 6.6 107.5
L 22.7 2.4 33.7 2.9*** 148.5
Cath. D & L M 3.0 0.1 3.9 0.2*** 130.0
C 0.89 0.2 0.55 0.1*** 61.8
β-GlcUr β-glucuronidase, β-Gal β-galactosidase, β-Glu β-glucosidase, Hex N-acetyl-hexosaminidase, AcP acid phos-
phatase, E esterase, L lipase, AlaAP alanine aminopeptidase, LeuAP leucine aminopeptidase, Cath A & L cathepsins D
and L. Enzyme activity was expressed in nmol/mg protein/h; *p 0.05; **p 0.01, and ***p 0.001 for the intergroup
difference
rate, so that they are exposed to large amounts of

4 Discussion
reactive oxygen species (ROS) and permanent
oxidative stress. It is known that oxidative stress
The mechanisms responsible for maintenance of
contributes to cellular injury through a reduction
cellular glutathione homeostasis are not fully
in the content of cellular GSH, shifting the bal-
well-understood. GSH is synthesized in hepatic
ance toward the oxidative side of glutathione
cytosol, where it is transported into the
disulfide (GSSG). Oxidation of the sulfhydryl
mitochondria (Fernandez-Checa and Kaplowitz
groups in cysteine residues of proteins forms
2005), with the resulting high content of GSH in
disulfides (Mosharov et al. 2000). Cysteine is
liver cells. The rate of GSH synthesis in
the rate-limiting element in cellular glutathione
hepatocytes is balanced by its secretion into the
biosynthesis (Radtke et al. 2012) and this
blood and bile (Zhu et al. 2014). Hepatocytes are
proteinogenic amino acid is hardly replenishable
rich in mitochondria and have a high respiratory
Table 4 Synopsis of in vivo GSH supplementation on the in vitro activity of all hydrolases investigated in the cellular
fractions of mouse hepatocytes
Activity of hydrolases
Hepatocyte fraction Increase (n) No change (n) Decrease (n)
Lysosomal 9 0 1
Microsomal 8 2 0
Cytosolic 5 4 1
Total; n (%) 22 (73.4) 6 (20.0) 2 (6.6)
with foods. We reasoned that exogenously in effect intensifying hydrolytic degradation by

supplemented glutathione could protect the cell of unwanted materials.
hepatocytes from oxidative stress by inhibiting The content of GSH in the liver increases
mitochondrial lipid and protein peroxidation or along with protein intake (Hum et al. 1992;
by chelating nonheme iron that easily Morini et al. 1991), providing succor to protective
accumulates in liver mitochondria, the processes antioxidative membrane processes. McCarty and
that lead to mitochondrial and lysosomal dysfunc- DiNicolantonio (2015) have shown that protein
tion and damage (Seo et al. 2008). Oxidative intake increases the pool of amino acids available
stress-induced injury also is mediated through to glutathione synthesis. In this study we used a
lysosomal damage (Ollinger and Brunk 1995), high-protein diet along with GSH supplementa-
thereby decreasing the ability of the cell to dis- tion, which may contribute to enhanced activity
pose of unwanted materials. Thus, we deemed it of endogenous peptidases noticed. The exception
warranted to examine whether and how the was cathepsins whose content decreased in the
administration of exogenous glutathione would cytosolic fraction of hepatocytes. Cathepsins are
influence the process of hydrolytic degradation released from lysosomes into the cytosol in
mediated by hydrolases in the in vitro response to apoptogenic factors, including oxida-
preparations of lysosomal, microsomal, and cyto- tive stress. Their release induces changes in the
solic fractions of mouse hepatocytes. This kind of outer mitochondrial membranes (Bidere et al.
study design finds support in the knowledge that 2003). Thus, decrease in cathepsins content
GSH-fortified diet can appreciably influence GSH speaks for the possibility of less oxidative stress
cellular content (Valencia et al. 2001). The and less propensity for apoptosis, as an effect of
findings of this study, in the main, are in line GSH supplementation noticed in this study.
with the rationale above outlined. We found that The enzyme family of lipases and esterases
in vitro activity of all hydrolases investigated, shows a great degree of complex functional diver-
with a single exception of β-GlcUr, increased in sity (Anthonsen et al. 1995). These proteins are
the lysosomal fraction. The enzymes activity, usually activated on contact with the aggregated
with a few exceptions, also increased in the substrate. Thus, increase in their activity noticed
other cellular fractions, the least so in the cytosol in this study also is in line with the augmentation
(Table 4). The predominant effect on the activity of cellular degradative processes, induced by
of lysosomal hydrolases of exogenous GSH has GSH given in vivo.
also been described in renal tissue by Śliwa- In conclusion, the findings of this study sug-
Jóźwik et al. (2004), which is suggestive of a gest that GSH-supplemented diet activates degra-
high demand of liver and renal cells for the anti- dation of unwanted cellular material in
oxidant. There is a biological plausibility that hepatocytes, particularly in the most hydrolyti-
improvement in structural functionality of cally active lysosomal fraction. That points to
lysosomes, due to the antioxidative action of the potential role of exogenous GSH as an agent
GSH, leads to enhanced activity of hydrolases, modulating the reactivity of the degradative lyso-
somal enzymes in the liver. GSH-enriched diet
might thus play a protective role in liver dysfunc- Hum S, Koski KG, Hoffer LJ (1992) Varied protein intake
tional states by developing adaptive responses alters glutathione metabolism in rats. J Nutr 122
(10):2010–2018
aimed at rebalancing of a distorted “redox state” Jóźwik A, Śliwa-Jóźwik A, Fronczyk W, Kołątaj A (2003)
through the enhancement of cellular antioxidant The level of in brain young and old mice. J Physiol
capacity. The findings of this study imply a poten- Pharmacol 2:64–65
tial benefit of GSH supplementation for decreas- Kalinina EV, Chernov NN, Novichkova MD (2015) Role
of glutathione, glutathione transferase, and
ing oxidative damage, particularly in tissues of glutaredoxin in regulation of redox-dependent pro-
high metabolic demand, at the cellular level. cesses. Biochem Mosc 79(13):1562–1583
There is an urgent need to explore the safety of Kirschke H, Langner J, Wiederanders B, Ansorge S,
GSH-fortified diet, which requires alternative Bohley P (1972) Intracellular protein degradation.
IV. Isolation and characterization of peptidases from
study designs. rat liver lysosomes. Acta Biol Med Ger 28(2):305–322
Article in German
Conflicts of Interest The authors declare no conflict of Koehler CM, Beverly KN, Leverich EP (2006) Redox
interest in relation to this article. pathways of the mitochondrion. Antioxid Redox Sig-
nal 8(5–6):813–822
Langner J, Wakil A, Zimmermann M, Ansorge S,
Ethical Approval All procedures performed in this study Bohley P, Kirschke H, Wiederanders B (1973)
were in accordance with the ethical standards of the insti- Aktivitätsbestiung proteolytischer Enzym mit
tution at which the studies were conducted and with the Azokasein als substrat. Acta Biol Med Ger 31:1–18
1964 Helsinki Declaration and its later amendments. All (Article in German)
applicable international, national, and/or institutional Marzella L, Glaumann H (1980) Increased degradation in
guidelines for the care and use of animals were followed. rat liver induced by vinblastine. I. Biochemical charac-
The experiments were carried out with the approval of the terization. Lab Investig 42:8–17
Bioethics Committee of the Świętokrzyska Chamber of McCarty MF, DiNicolantonio JJ (2015) An increased need
Physicians in the city of Kielce in Poland (permit for dietary cysteine in support of glutathione synthesis
no. 49/2016). may underlie the increased risk for mortality associated
with low protein intake in the elderly. Age (Dordr) 37
(5):96
McDonald JK, Barrett AJ (1986) Exopeptidases. In:
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nutritional and pharmacologic viewpoints: part C3A cells exposed to excessive energy substrates.
IV. Nutrition 17:783–784 Nutrients 6:2552–2571
Wang SL, Zhu XY, Zhang DW, Zhang ZJ, Gao HJ, Yang
CQ (2015) Relevance of plasma malondialdehyde
Adaptation to Occupational Exposure

to Moderate Endotoxin Concentrations:
A Study in Sewage Treatment Plants
in Germany
M. A. Rieger, V. Liebers, M. Nübling, T. Brüning, B. Brendel,

F. Hoffmeyer, and M. Raulf
Abstract plants. Subjects were matched to pairs

Acute or chronic inhalation of endotoxin may (8 workers escorted by 10 students).
lead to changes of lung function and inflam- Inflammatory markers were investigated
matory markers in the airways. Adaptation to before, directly after, and 16 h after the shift
workplace exposure may be possible. In this end. Additional NALF samples were collected
study, we investigated the possible difference in students without any specific exposure after
in inflammatory markers assessed in nasal 3 days. In NALF, total cell count, and interleu-
lavage fluid (NALF) in chronical exposure kin (IL)-8 and IL-1β concentrations were sig-
compared to voluntary subjects exposed nificantly higher in workers than in students at
acutely to endotoxin. We sought to define the all times pointing to workplace-related long-
variability of inflammatory markers in NALF lasting exposure resulting in adaptation. How-
and the dose-related changes after moderate ever, concentration of inflammatory markers
exposure in naïve subjects. Endotoxin expo- without specific exposure in students showed
sure (4–1039 EU/m3) resulted from routine a great variability, covering the whole range of
work during one shift in sewage treatment values recorded in the workers. The findings of
this study make us to recommend a repeated
assessment of inflammatory markers in
M. A. Rieger (*) healthy volunteers before the investigation of
Institute of Occupational and Social Medicine and Health
Services Research, University Hospital of Tübingen,
exposure-related changes and a sample size
Tübingen, Germany adequate for statistical analysis.
V. Liebers, T. Brüning, F. Hoffmeyer, and M. Raulf Keywords
Institute for Prevention and Occupational Medicine of the Cumulative effect · Dose-response
German Social Accident Insurance (IPA), Ruhr University relationship · Endotoxin exposure ·
Bochum, Bochum, Germany
Inflammation · Nasal lavage fluid ·
M. Nübling Occupational exposure · Sewage
Research Center for Occupational and Social Medicine
(FFAS), Freiburg, Germany
B. Brendel
Institute for Clinical Epidemiology and Applied Biometry,
Tübingen University Hospital, Tübingen, Germany
89
90 M. A. Rieger et al.
1 Introduction syndrome (ODTS). It has been shown in farmers

that ODTS is a major predictor of chronic bron-
Occupational exposure to endotoxins is typical chitis (Radon et al. 2003). Yet, especially when
for working conditions in the waste or wastewater exposed to moderate endotoxin concentrations,
industries and agriculture. Exposure to endotoxin many workers seem to develop no symptoms,
may induce three different kinds of reactions: but they adapt to exposure (von Essen and
acute effects due to a single exposure or chronic Romberger 2003) or develop endotoxin tolerance
effects due to chronic or repeated exposure during (Bakirci et al. 2007). Therefore, there might be a
a longer period (Rylander 1995). Depending on difference in the reaction pattern to chronic expo-
the dose, location, route, and time of release in the sure to endotoxin compared with voluntary
blood, the response to endotoxin varies. Several subjects exposed acutely.
studies have shown adverse health effects In this study, we focused on inflammatory
induced by acute or chronic inhalation of endo- markers in the nasal lavage fluid (NALF) such
toxin. Changes in lung function and inflammatory as the total cell count, proportion of neutrophils,
markers such as ECP, interleukin (IL)-8, IL-1β, total protein, IL-8, IL-6, IL-1β, TNF-α, and
tumor necrosis factor (TNF)-α, and C-reactive sCD14, and we wanted to answer the following
protein (CRP) in the airways or blood are well two questions:
recognized (Liebers et al. 2008; Liebers et al.
2006; Rylander 2002; Thorn 2001). As it is 1. Is there a difference in the content of inflam-
known from different studies, no-effect levels of matory markers in NALF between chronically
endotoxin range from 9 to 170 ng/m3. Castellan exposed (workers of sewage treatment plants)
et al. (1987) have described a threshold of and just acutely exposed subjects (students)?
90 endotoxin units (EU)/m3 for acute mucous 2. Are there any differences in the patterns of
membrane irritation and pulmonary change inflammatory markers in NALF of healthy
among cotton workers. Against this backdrop, volunteers after exposure to a moderate endo-
the Dutch Expert Committee on Occupational toxin concentration (work in a sewage treat-
Standards of the National Health Council has ment plant) compared with nonspecific
proposed a health-based recommended occupa- exposure? Are there any dose-related or
tional exposure limit (HBROEL) for endotoxin cumulative changes?
in the workplace of 90 EU/m3 in 2010 (8-hour
time-weighted average) (Health Council of the
Netherlands 2010).
Several studies have been undertaken to 2 Methods
describe a dose-response relationship concerning
the clinical symptoms (Castellan et al. 1987) or 2.1 Study Design
inflammatory markers (Danuser et al. 2000;
Michel et al. 1997), but data still seem to be Study protocol was created in accordance with
insufficient in work-related (Thorn 2001) or envi- the Declaration of Helsinki and was approved
ronmental exposure (Walser et al. 2015). Clinical by the Ethics Committee of the University of
and epidemiological findings have linked the Wuppertal. All participants gave written informed
endotoxin exposure at the work place to fever, consent to the study protocol. As the exposure
cough, shortness of breath, wheezing, headache, scenario we chose the areas of the chamber filter
nose and throat irritation, chest tightness, acute press or the sand trap in sewage treatment plants.
airway flow restriction, and inflammation Our previous measurements in these areas have
(Liebers et al. 2006). The effects of acute expo- shown rather high concentrations of endotoxin
sure to higher levels of endotoxin have been (up to 942 EU/m3 during flushing of the chamber
summarized in the so-called organic dust toxic filter press after treatment of sewage sludge with
Adaptation to Occupational Exposure to Moderate Endotoxin Concentrations: A. . . 91
iron (III) chloride and lime) combined with low 2.2.1 Inclusion Criteria
concentrations of other biological hazards (e.g., and Sociodemographic Data
total bacteria: 105–106 CFU/m3 and total molds: Students The following inclusion criteria were
<103 CFU/m3), and no relevant exposures to applied for students: healthy male non-smokers,
ammonia (Rieger and Diefenbach 2007; without any clinically manifested allergy and not
Steinberg 2001). Based on those results and fur- living or working in a nearby farm, sewage treat-
ther exploratory measurements, we selected four ment plant, or waste industry. The students were
sewage treatment plants of a regional association aged 24.7 (range 22–29) years and had a body
for sewage treatment in North Rhine-Westphalia mass index (BMI) of 23.7 (range 20.7–26.6) kg/m2
(Germany) for the present study. Data were (Table 1). After inclusion in the study, a screening
assessed in 2002–2003. test was performed at least 1 month prior to expo-
To get insight into the adaptation to endotoxin sure. The test consisted of a skin prick test with
exposure, we compared chronically exposed common aero-allergens. In addition, specific
workers on the sewage treatment plants to just immunoglobulin E (sIgE) in response to a variety
acutely exposed voluntary subjects (students) of ubiquitous aero-allergens was assessed in the
regarding the possible changes in various serum (atopy screen sx1 Phadiatop®, CAP-FEIA;
outcomes during one working shift. The workers ThermoFisher Scientific, Waltham, MA). Allergen
and students were matched in pairs, i.e., the sIgE 0.35 kU/L was considered positive. Based
workers were escorted by students during their on the screening results and medical history, 4 out
ordinary work on the plant. The students of the 10 students were classified as having atopy.
documented the workers’ activities in a log. To On the screening day, students also were trained by
investigate a possible dose-response relationship, a laboratory’s specialist to gather NALF.
students were asked to participate in the study
several times with intervals of several months On the day of examination, sociodemographic
between the repeated exposures. Four students data, general symptoms, and the judgment of the
were purposefully exposed to high concentrations stay on the sewage treatment plant were assessed
of endotoxin on two following days to assess a by means of a standardized questionnaire. None of
possible cumulative effect on inflammatory the students indicated any concurrent possible
markers. Lastly, specimens were obtained from exposure to biological hazards due to occupation
five students not only during their stay on sewage or leisure time activities. Concerning the occupa-
treatment plants but also after 3 days free of any tional history, only two students indicated former
specific exposure in offices and lecture rooms at occupational exposure to airborne biological
the university. Thus, variability of parameters hazards (agricultural work years before the study)
within 1 day could be compared with changes and none had any other previous inhalation expo-
evoked by specific exposure. sure. Concerning the judgment of stress due to the
working environment of the sewage treatment
plant, most of the inquired aspects were rated as
2.2 Study Sample little-to-moderate (heat, dust, chemicals, humidity,
cold, and time pressure) and four aspects as
As described above, a major goal of the study was moderate-to-high (noise, humidity, infectious
a comparison of chronically exposed workers in agents, and odor), which was assessed on a
sewage treatment plants (n ¼ 8) to just acutely 3-point Likert scale: 1 ¼ little stress and
exposed voluntary subjects, represented by 3 ¼ high stress. The self-reported students’ health
students (n ¼ 10). In addition, a possible dose- status was rated to be very good (mean 1.4), based
response relationship of the immunological on the following scale: 1 ¼ very good, 2 ¼ good,
reactions and a cumulative effect of exposure to and 6 ¼ insufficient. The evaluation of general
endotoxin on two following days were complaints and medical history showed no specific
investigated.
Table 1 Sociodemographic data of students

Agea BMIa Atopyb SHSc (1 ¼ very good Subjective evaluation of work environment on
Students (years) (kg/m2) (yes/no) 6 ¼ insufficient) sewage plantd (range 9–27 points)
s1 29 25.3 Yes ni 14
s2 25 22.7 Yes 2 16
s3 29 26.3 No 2 14
s4 23 20.7 No 1 18
s5 24 22.8 No 1 17
s6 23 26.6 No 1 17
s7 23 22.8 Yes 1 16
s8 22 22.7 Yes 2 15
s9 25 22.8 No 2 15
s10 24 24.2 No 1 ni
Mean 24.7 23.7 – 1.4 15.8
a
In case of a repeated participation, the age, body mass index (BMI), and indications in the questionnaire of the first day
of examination are given
b
Assessed by skin prick test using common aero-allergens, confirmed by specific immunoglobulin E in response to a
variety of ubiquitous aero-allergens (atopy screen sx1 Phadiatop) and medical history
c
SHS Subjective health status
d
Working environment was judged by the students regarding nine aspects: heat, noise, dust, chemicals, infectious agents,
odor, humidity, cold, and time pressure (1 ¼ little stress, 2 ¼ moderate stress, 3 ¼ high stress)
ni no indication
findings. Spirometry revealed no abnormalities in chemicals, humidity, cold, and time pressure and
the morning of the examination day. as moderate-to-high due to infectious agents and
odor. The majority of workers (7/8) indicated a
Workers The workers were recruited among the high work satisfaction. The evaluation of general
staff of selected sewage treatment plants working complaints and medical history showed no spe-
at the chamber filter press or sand trap. Only cific findings. The subjective ability to work was
workers fit for work on the day of examination rated slightly better (mean 2.3, range 1–4) than
were asked to participate in the study. All workers the own health status (mean 2.8, range 2–4)
were men, 3/8 workers were current and 2/8 were (1 ¼ very good, 2 ¼ good, and 6 ¼ insufficient).
former smokers. The mean age was 40.9 (range In the morning of the examination day, spirome-
24–56) years. The BMI was 28.4 (range try resulted in regular findings in all but one
23.6–37.6) kg/m2, with one worker being obese worker; the latter presenting with expiratory
according to the definitions of WHO. The mean flow limitation. Based on sIgE (in all but one
working duration on the sewage treatment plant worker) and the medical history, one worker
was 13.4 (range 4.0–33.8) years and the workers was described as being atopic.
indicated to work >6 h/day in the chamber filter As the recruitment of sewage treatment plants
press hall (Table 2). Beyond the work on sewage turned out difficult, the time interval between the
treatment plants, only two workers reported a measurements in single plants was between 1 and
previous occupational exposure to airborne 9 months. That made it possible that four students
biological hazards (agricultural work or sewage (s4, s7, s8, and s9) could be examined on two
sludge treatment of about 7 years’ duration) and different plants and one student (s5) on three
one worker reported a former inhalation exposure plants. According to the usual size of the sewage
from mining of 6 years’ duration. In a treatment plant, only two or three workers per
standardized questionnaire, the workers judged plant could be recruited for participation in the
the stress as little-to-moderate by such aspects of study.
the work environment as heat, noise, dust,
Table 2 Sociodemographic data of workers
Daily work in Work on Subjective evaluation of Work satisfaction Subjective ability to
SHSb (1 ¼ very chamber filter the sewage work environment on (single item; 1 ¼ little, work (school grades,
Age BMI Atopya good press hall plant sewage plantc (range 9–27 2 ¼ medium, 1 ¼ very good
Workers (years) (kg/2) (yes/no) 6 ¼ insufficient) (hours) (years) points) 3 ¼ high) 6 ¼ insufficient)
w1 38 28.1 No 4 >6 8.3 15 3 3
w2 43 27.8 No 4 >6 6.0 17 3 2
w3 38 26.2 No 2 >6 8.3 12 3 2
w4 24 23.6 No 2 >6 4.0 17 3 2
w5 49 29.1 No 3 >6 19.8 15 3 4
w6 44 26.3 Yes 3 >6 17.7 15 2 2
w7 56 37.6 No 2 <3 33.8 10 3 1
w8 35 28.7 No 2 Highly variable 9.5 16 3 2
All Mean Mean Yes: 2.8 > 6 h: 6/8 Mean 13.4 Mean 14.6 Mean 3 Mean 2.3
workers 40.9 28.4 1/8
a
Assessed by antibodies against inhalative allergens (IgE, sx1-test) and medical history
b
SHS Subjective health status
Adaptation to Occupational Exposure to Moderate Endotoxin Concentrations: A. . .
c
Working environment was judged by the workers regarding nine aspects: heat, noise, dust, chemicals, infectious agents, odor, humidity, cold, and time pressure (1 ¼ little stress,
2 ¼ moderate stress, 3 ¼ high stress)
93
2.3 Assessment of Exposure 2.4.1 Time Points for Data Assessment

During Exposure
Exposure to endotoxin was assessed by personal According to the cross-shift approach, the
sampling of aerosols from the inhalable dust frac- parameters were assessed at three time points
tion using the PGP-GSP system (GSA and intervals were chosen according to similar
Messgerätebau GmbH; Ratingen, Germany). studies (e.g., Doreswamy et al. 2011; Danuser
Usually the sampling equipment was carried by et al. 2000; Sigsgaard et al. 2000; Wang et al.
workers during their routine work at the sewage 1997):
treatment plant. Endotoxin was assessed using a
commercially available chromogen-kinetic Limu- Time point A: morning, before exposure (i.e.,
lus amoebocyte lysate (LAL) assay (COAMATIC 6:30–9:30 am, depending on the single plant)
Chromo-LAL®; Chromogenix, Hemochrom) as Time point B: afternoon, 8 h after start of expo-
described elsewhere (Liebers et al. 2006; Rieger sure (i.e., 2:45–4:00 pm, depending on the
et al. 2005). Endotoxin exposure was measured single plant)
twice per day for a period of 112–265 min (mean Time point C: next morning (usually about 16 h
166 min, median 178 min) and reached after time point B, i.e., 6:45–10:00 am)
concentrations between 4 and 121 EU/m3
(median 24.5 EU/m3) during routine work and
between 162 and 1039 EU/m3 (median 589.5
2.4.2 Nasal Lavage Fluid (NALF): Cellular
EU/m3) in areas with high exposure (Table 3). A
and Soluble Inflammatory Markers
great variability of exposure on single working
Before the collection of NALF, the subjects were
days and some rather low endotoxin
asked to blow their nose. Then a syringe with a
concentrations during the day resulted from the
suitable adapter containing 7 mL of sterile physi-
different tasks the workers performed during their
ological saline solution was introduced into a
daily work, including periods without or with
nostril, followed by its aspiration; the procedure
only low specific exposure, such as administra-
was repeated five times (Raulf et al. 2015; Raulf-
tive or monitoring work in an office near the
Heimsoth et al. 2011; Raulf-Heimsoth et al.
chamber filter press hall (mean 131 min/day,
2000). The residual volume of NALF was
range 70–215 min/working day) or legal breaks.
recorded (3 mL being considered as good result),
There was no relevant exposure to airborne bac-
sent overnight under standardized conditions to
teria (mean 2.4 104 CFU/m3) or molds (mean
the laboratory and centrifuged (5 min at
3.8 102 CFU/m3) during the working day. All
1300 rpm). The cell-free supernatant was divided
analyses were performed by the same laboratory
into aliquots and frozen at 80 C until further
(Mikrobiologisches Labor Dr. Balfanz –
analysis of soluble inflammatory markers. The
Dr. Lohmeyer GbR, Münster, Germany).
cell pellets were resuspended, and the total cell
number was determined using the Neubauer
counting chamber. For differential cell counts,
2.4 Assessment of Effects slides were prepared by cytospin (Cytospin 2;
Shandon Corp., Pittsburgh, PA) and stained with
To assess a broad spectrum of possible effects due May-Grünwald-Giemsa. Two independent
to the exposure to endotoxin, subjective observers counted 200 cells on each slide under
(symptoms – standardized questionnaire) and light microscopy. The results were expressed as
objective (cellular and soluble inflammatory the percentage and calculated as absolute cell
markers in blood and NALF; lung function) count based on the total cell number.
parameters were repeatedly gathered. The present The inflammatory markers were determined in
evaluation focuses on NALF with regard to the thawed cell-free supernatants. All samples
inflammatory markers. underwent only one freeze-thaw cycle. The
Table 3 Endotoxin exposure on sewage treatment plants during the days of examination assigned to study participants
EU/m3
Plant no. (date) Students Workers Two measurements/daya; average
1 s1 w1 90/46; 68.0
(23/4/2002) s2 w2 17/16; 16.5
s3 w3 12/7; 9.5
2 s4 w4 32/41; 36.5
(22/5/2002) s5 w4 32/41; 36.5
s6 w5, (w6)b 99/91; 95.0
s7 – 121/47; 84.0
3c s7 w7 10/7; 8.5
(12/2/2003) s8 w7 10/7; 8.5
s9 w8 4/5; 4.5
s5 w8 4/5; 4.5
(13/2/2003) s10 w7 58/13; 35.5
s5 w8 12/33; 22.5
s9 – 51/8; 29.5
4c,d s4 318/234; 276.0
(3/11/2003) s5 798/885; 841.5
s8 – (No personal sampling); 465.0e
s9 394/162; 278
(4/11/2003) s4 536/643; 589.5
s5 231/1039; 635.0
s8 – (No personal sampling); 659.7e
s9 866/643; 754.5
a
Exposure was assessed by personal sampling during routine work on sewage treatment plants (especially in chamber
filter press hall and sand trap) before and after lunch break, sampling equipment usually carried by the worker(s), duration
of measurements: 112–265 min (mean 166 min, median 178 min)
b
W6 no full exposure due to part-time work on the day of examination
c
Plant 3 and plant 4: measurements on two following days
s student, w worker, EU endotoxin unit
d
Plant 4: students only, no routine work but high exposure near the sand trap
e
Student s8 no personal sampling but exposure assigned as mean value of exposure of students s4, s5, and s9 (each
personal sampling)
following soluble markers were measured: inter- PeliKine™-Tool sets (CLB, Amsterdam,
leukin (IL)-1β, IL-5, IL-6, IL-8, soluble (s)CD14, Netherlands) in a standard range of 0.4–300 pg/
tumor necrosis factor (TNF)-α, eosinophilic cat- mL for IL-1β and a standard range of
ionic protein (ECP), NO, total protein content, 1.4–1000 pg/mL for TNF-α. IL-8 was measured
and albumin. The present evaluation of cellular with the OptEIA™ ELISAs (BDBiosciences
and soluble inflammatory markers in NALF Pharmingen, Heidelberg, Germany) in a standard
focuses on total cell count, proportion of range of 3–200 pg/mL and most of the samples
neutrophils, and the concentrations of total pro- must be diluted to reach the standard range. IL-6
tein, IL-8, IL-6, TNF-α, IL-1β, and sCD14. and sCD14 were determined with the DuoSet™
Therefore the methods of these selected soluble ELISA (R&D Systems, Wiesbaden, Germany) in
markers are described in more detail. a standard range of 3–600 pg/mL for IL-6 and of
Interleukins, sCD14, and TNF-α were 250–16,000 pg/mL for sCD14. Total protein con-
measured using purchased monoclonal “sand- tent was determined according to the method of
wich” enzyme immunoassay (EIA) kits. IL-1β Bradford (1976) with bovine serum albumin as a
and TNF-α were measured with the respective standard solution (range 10–100 μg/mL). For all
soluble markers, concentrations below the limit of For this major goal of the study, 12 pairs of
quantification (LOQ) were set at 2/3 of LOQ. workers and students were combined and
investigated on three sewage treatment plants
2.4.3 Spirometry (see Table 4)
Vital capacity (VC), forced vital capacity (FVC),
and forced expiratory volume in 1 s (FEV1) as 2. Variability of inflammatory markers without
well as peak expiratory flow (PEF), peak inspira- specific exposure, possible dose-response rela-
tory flow (PIF), and maximal instantaneous tionship, and cumulative effects of the endo-
forced expiratory flow when x% of the FVC toxin exposure:
remains to be expired (MEFx%; 25%, 50%, and
75%) were measured pre- and post-shift, as well Five students (s4, s5, s7, s8, and s9) were
as in the next morning with a portable spirometer investigated without any specific exposure with
(Jaeger SpiroPro®; Viasys Würzburg, Germany) a time interval of at least 3 weeks after the last
using the software V1.0. Measurements were exposure to endotoxin (s4, s5, s8, and s9:
performed according to the guidelines of the 3 weeks, s7: 9 months). This examination was
American Thoracic Society (ATS 1995), but repeated twice each with a 1-week interval.
repeated measurements were not possible at all Dose-related changes of inflammatory markers
time points due to organizational constraints on concentrations were investigated based on
the sewage treatment plants. Reference values 20 values as some students participated repeat-
were chosen according to Quanjer et al. (1993). edly in the study. The students’ values were
grouped according to three categories of endo-
2.4.4 Questionnaires toxin concentration: <50 EU/m3 – eight students;
The occupational and medical history (including 50–200 EU/m3 – three students; >200 EU/m3 –
smoking), the current occupation, potential other four students. Possible cumulative effects could
sources of exposure (e.g., leisure time activities, be investigated in four students (s4, s5, s8, and s9)
or place of living), and the subjective stress who were exposed to high endotoxin
caused by the working conditions on the sewage concentrations on plant 4 on two following days
treatment plant were assessed by means of a (see Table 5).
standardized questionnaire and by the subjective
evaluation of health and workability. In addition,
the study participants were asked to indicate the 2.6 Statistical Analysis
extent of symptoms in line with a mucous mem-
brane irritation syndrome (e.g., symptoms from Data were expressed as median with min–max
the eyes, nose, and throat), general effects of range (tables) or stem and leaf plots (box plots)
exposure to endotoxin conforming to an organic illustrating the interquartile range (75th percentile
dust toxic syndrome (e.g., limb pain, fever, and – 25th percentile) and minimum and maximum
feeling like getting a flu), or other symptoms (e.g., (figures). As the concentration of inflammatory
nausea and diarrhea) at time point A, B, and C, markers in NALF showed no normal distribution,
using a Likert scale (0 ¼ not at all, 4 ¼ very logarithmic (total cells, total protein, IL-8, IL-1β,
strong). and sCD14) or dichotomous values (neutrophils
categorized below/equal or above 40%, IL-6 and
TNF-α categorized as below/equal or above the
2.5 Sample Size for Single Research respective limit of quantification) were used for
Issues statistical analysis comparing workers and
students (research issue 1). In this context, after
1. Comparison of chronically and only acute univariate analysis (logarithmic values: regres-
exposed subjects: sion analysis and analysis of variance, with a
UNIANOVA procedure; dichotomous values:
Table 4 Cellular and soluble inflammatory markers in nasal lavage fluid (NALF) before exposure (time point A) in chronically exposed (workers) and so far not exposed
subjects (students). These data were assessed on plant 1–3 (only Day 1)
Students (n ¼ 10; n ¼ 12 values) Workers (n ¼ 8; n ¼ 8 values) Students vs. workersa
Number of values >LOQ (%; Number of values >LOQ (%; Univariate
NALF 100% ¼ 12) Median (range) 100% ¼ 8) Median (range) analysis GEE
Total cell count 12 (100%) 6.0 (4.0–12.0) 8 (100%) 10.0 (4.0–24.0) p ¼ 0.090 p ¼ 0.079
(104)
Neutrophils (%) 12 (100%) 78.8 (27.5–93.3) 8 (100%) 35.8 (0.0–94.0) ns/nt ns/nt
Total protein 12 (100%) 58.5 (18.1–160.3) 8 (100%) 61.3 (10.0–370.3) ns/nt ns/nt
IL-8 (pg/mL) 12 (100%) 147.1 (38.3–802.3) 8 (100%) 231.9 (59.1–1162.1) p ¼ 0.024 p ¼ 0.128
IL-6 (pg/mL) 1 (8%) 2.0 (2.0–43.2) 2 (25%) 2.0 (2.0–18.4) ns/nt ns/nt
TNF-α (pg/mL) 8 (67%) 1.6 (0.9–90.9) 5 (63%) 1.6 (0.9–20.2) ns/nt ns/nt
IL-1β (pg/mL) 10 (83%) 1.5 (0.3–6.3) 8 (100%) 2.1 (0.8–36.6) p ¼ 0.019 p ¼ 0.085
sCD14 (pg/mL) 12 (100%) 3461.0 (1657.8–12083.8) 8 (100%) 3907.6 (1266.3–12876.2) ns/nt ns/nt
a
This statistical analysis is based on the values of all three time points; at time point A, a marked difference was found for the total cell count only
ns no statistically significant difference between students and workers (i.e., p > 0.05)
Adaptation to Occupational Exposure to Moderate Endotoxin Concentrations: A. . .
nt no tendency regarding significant differences between students and workers (i.e., p > 0.100)
LOQ limit of quantification, GEE general estimating equation
97
98
Table 5 Cellular and soluble inflammatory markers in nasal lavage fluid (NALF) in students at time point A (morning) and B (afternoon) according to endotoxin exposure during
the day
Time point A
(morning) Time point B (afternoon after endotoxin exposure)b
(n ¼ 10 students/ No specific exposurea < 50 EU/m3 (n ¼ 8 50–200 EU/m3 (n ¼ 3 > 200 EU/m3 (n ¼ 4/n ¼ 8 values); median (range)
n ¼ 31 values); (n ¼ 5 students/n ¼ 14 students/n ¼ 9 values)c; students/n ¼ 3 values)c;
NALF median (range) valuesd); median (range) median (range) median (range) Day 1c Day 2c
Total cellsa 8.0 (4.0–16.0) 4.0 (4.0–12.0) 8.0 (4.0–12.0) 8.0 (8.0–8.0) 4.0 (4.0–8.0) 4.0 (4.0–4.0)
104
Neutrophils 67.7 (0.0–93.3) 38.7 (0.0–84.7) 56.1 (9.0–94.0) 27.0 (16.0–81.3) 12.2 (1.0–55.0) 60.3 (7.0–66.0)
(%)
Total 73.6 (6.7–359.1) 74.7 (21.6–372.7) 37.5 (21.3–150.9) 42.9 (21.5–43.3) 49.6 (44.2–164.7) 47.9 (33.6–133.5)
protein (μg/
mL)
IL-8 246.7 373.6 (96.2–2982.0) 137.1 (38.8–572.2) 105.5 (89.0–414.2) 379.9 (169.2–636.9) 558.2 (51.7–1324.2)
(pg/mL) (38.3–1590.9)
IL-6 2.0 (2.0–43.2) 2.0 (2.0–33.6) 2.0 (2.0–26.2) 2.0 (2.0–2.0) 2.0 (2.0–2.0) 2.0 (2.0–5.7)
(pg mL)
TNF-α 0.9 (0.9–90.9) 1.7 (0.9–3.2) 1.5 (0.9–9.5) 6.6 (0.9–7.6) 0.9 (0.9–7.7) 0.9 (0.9–1.6)
(pg/mL)
IL-1β 2.8 (0.3–17.7) 1.2 (0.3–15.7) 0.9 (0.3–4.2) 0.6 (0.5–1.7) 1.8 (1.4–5.7) 1.4 (0.9–3.4)
(pg/mL)
sCD14 3548.8 3337.4 (1563.8–18374.0) 2968.0 (943.2–7415.5) 2495.9 (2046.5–3473.3) 3108.2 (1937.3–6620.7) 2426.8 (1943.2–7741.1)
(pg/mL) (275.3–13574.1
The number of values is based on repeated participation of students in the study (see Table 3)
a
No specific exposure: offices and lecture rooms of the university
b
Endotoxin exposure derived from the students’ stay on a sewage treatment plant (plant 1–3: escorting workers during their routine working day)
c
Concentrations per category: <50 EU/m3: median 17 EU/m3 (4–36 EU/m3), 50–200 EU/m3: median 84 EU/m3 (70–95 EU/m3); >200 EU/m3: Day 1 median 356 EU/m3
(162–885 EU/m3; mean: 465 EU/m3), Day 2 median 643 EU/m3 (231–1039 EU/m3; mean 659.7 EU/m3)
d
Student 8: did not participate on the third day without specific exposure
M. A. Rieger et al.
logistic regression) regarding the possible effects work shift. In addition, workers w3 and w4
either due to group (chronic or only acute expo- showed a tendency of exposure-induced changes
sure), time points (A, B, and C), or the in lung function, whose significance remains
interactions of group and time points, generalized unclear due to methodological reasons i.e. no
linear models (GLM) with generalized estimating possibility of repeated measurements due to orga-
equations (GEE) were calculated, as some nizational constraints on the sewage treatment
students participated several times in the study. plants. All students had normal spirometry results
In addition, the possible effect of group and plant at all time points and showed no signs of
on the values gathered at time point A was exposure-induced changes in lung function.
analyzed by univariate analysis (logarithmic
values) or logistic regression (dichotomous
values). Due to a limited number of subjects, we 3.2 Research Issue 1: Effects
additionally reported results ignoring the cluster- of Chronic Endotoxin Exposure
ing caused by repeated measurements of the same
subjects. A two-sided significance level of 0.05 The emphasis of the present study was on the
was chosen for all tests. According to small num- possible effects of the endotoxin exposure on
bers, no statistical analysis was possible for cellular and soluble inflammatory markers in
research issue 2. Data were analyzed using a NALF. For the comparison of chronically
commercial SPSS software v22 and v23 (IBM exposed workers with acutely exposed students,
Co, Armonk, NY). 12 pairs of workers and students based on data of
8 workers and 10 students could be examined on
3 sewage treatment plants. There were significant
3 Results differences between both groups in IL-8 (univari-
ate analysis: p ¼ 0.024, GEE: p > 0.128) and
3.1 General Findings IL-1β (univariate analysis: p ¼ 0.019, GEE:
p ¼ 0.085), each with a lower concentration in
None of the healthy volunteers cancelled the students than workers. Similarly, total cell count
exposure on the sewage treatment plant. Only tended to be lower in students than workers (uni-
after the exposure to a high endotoxin concentra- variate analysis: p ¼ 0.090, GEE: p ¼ 0.079).
tion on plant 4, some students indicated TNF-α was the only inflammatory marker with a
symptoms in line with a mucous membrane irrita- significant concentration difference at the three
tion in the standardized questionnaire (increase time points (univariate analysis: p ¼ 0.079,
from “no symptoms” to “little” or “clear” irrita- GEE: p ¼ 0.016) with fewer values below LOQ
tion especially of the nose) which were no longer at time point A, compared to time point C. For
prevalent on the next morning. One student TNF-α, no significant effect or interaction with
reported stomach ache, diarrhea, and nausea on the variable “group” could be detected. For the
the morning of the second day of exposure on other inflammatory markers, no effect or interac-
sewage plant 4, but the symptoms eased during tion regarding “group” or “time point” could be
the day. The sewage workers did not report any seen in the statistical analysis.
changes of symptoms during the working day in Concerning the possible effect of
the standardized questionnaire. chronic vs. acute exposure (variable “group”) or
the effect of plant on the concentration resulting
3.1.1 Lung Function at time point A, a tendency toward a significant
All but one worker (w8) had normal values in the difference was found for the variable “group”
spirometric examination before the exposure and only in the total cell count in NALF (p ¼ 0.064)
showed no clinically relevant changes after the (lower concentration in students) and the variable
work shift. Worker w8 had a peripheral flow “plant” turned out to be significantly associated
limitation which seemed to increase during a with a higher concentration of this marker on
plant 3 than on plant 1 (p ¼ 0.034). Likewise, endotoxin concentration than that without spe-
logistic regression showed a significant effect of cific exposure (median: 12.2% (exposure >200
the variable “plant” on TNF-α (p ¼ 0.027), with EU/m3) vs. 53.3% (no specific exposure)). Gen-
more results below the limit of detection on plant erally, concentrations of inflammatory markers
1 (five samples) than on plant 2 or 3 (only one without specific exposure included most other
sample each). Concerning both markers, there values gathered after endotoxin exposure in the
was no significant interaction between the samples investigated in the present study (see
variables “group” and “plant”. For the other Table 5).
inflammatory markers gathered at time point A, Concerning the pattern of inflammatory
no such effects of the two variables could be seen marker concentrations, a higher endotoxin con-
in this univariate analysis. The concentrations of centration seemed associated with an increased
inflammatory markers in students and workers at concentration of total protein, IL-8, IL-1β, and
time point A are given in Table 4. sCD14, and with a decrease in the percentage of
neutrophils and in the concentration of IL-6 in
descriptive analysis. Differently, comparing the
inflammatory marker concentrations in the after-
3.3 Research Issue 2: Different
noon of the first and second days with high endo-
Patterns of Inflammatory
toxin exposure on plant 4 (mean endotoxin
Markers in NALF After Exposure
concentration 465 EU/m3 (Day 1) and 659.7
to a Moderate Endotoxin
EU/m3 (Day 2) (for details regarding the exposure
Concentration as Compared
see Table 3 and legend to Table 5)), marked
to Nonspecific Exposure. Dose-
differences between the values gathered at time
Response Relationship of Acute
point B on the first and second days seemed
Exposure, Including
detectable regarding the total cells (lower on
a Cumulative Exposure
Day 2), neutrophils (higher on Day 2), IL-8
(higher), IL-6 (higher), and sCD14 (lower) (see
The inflammatory markers in NALF at time point
last columns in Table 5 and Fig. 1). Compared to
B for all students participating in the study were
the initial findings on the morning of Day 1 (time
analyzed according to the respective endotoxin
point A1), the concentrations found on the morn-
exposure during the day (plant 3: values of the
ing of Day 2 (C1/A2) and Day 3 (C2) during the
first day of exposure only) and compared to time
cross-shift examination showed almost complete
point B values without specific exposure and to
recovery regarding the neutrophils (C1 and C2)
all values gathered before the exposure (i.e., time
but slight changes regarding IL-6 and especially
point A) (Table 5). The values gathered from five
IL-1β (C1 and C2), and an increase in variability
students at time point B, on 3 days without any
especially at time point C1 regarding the total
specific exposure (see Table 5 – column “no
cells, total protein, TNF-α, and sCD14 (Fig. 1).
specific exposure”), and the respective time
Due to a small sample size, combined with a
point A- and C-values (data not shown) showed
great variability in the concentration of all
a great variability and there was no marked dif-
markers investigated, no statistical analysis was
ference between A-, B-, and C-values. Due to this
possible to compare the values at time point B,
great variability, comparison of NALF markers
according to different kinds of exposure or to
gathered on sewage plant 4 with those gathered in
compare between all of the time points (A1 to
the same four students during 3 days without
C2), or between the three time points of each day
specific exposure revealed no significant differ-
(A1 to C1 and A2 to C2), with or without specific
ence between both conditions (see Table 5). Only
exposure. The study’s main finding was the great
the percentage of neutrophils at time point B1
variability of inflammatory marker concentrations
tended to be lower after exposure to a high
in naïve subjects without specific exposure.
12 100
NALF total cells (10e4)
NALF neutrophils (%)

80
10
60
8
40
6
20
4 0
A1 B1 C1/A2 B2 C2 A1 B1 C1/A2 B2 C2
15 14
NALF TNF-a (pg/mL)

12
NALF IL-6 (mg/mL)
10
10
8
6
4
5
2
0
A1 B1 C1/A2 B2 C2 A1 B1 C1/A2 B2 C2
Time of collection (Day 1 & Day 2) Time of collection (Day 1 & Day 2)
350
NALF total protein (mg/mL)
1000
300
NALF IL-8 (pg/mL)
250
200
100
150
100
50
0 10
A1 B1 C1/A2 B2 C2 A1 B1 C1/A2 B2 C2
6 12000
10000
NALF sCD14 (pg/mL)
NALF IL-1b (pg/mL)
5 8000
6000
4
4000
3
2
2000
1
0
A1 B1 C1/A2 B2 C2 A1 B1 C1/A2 B2 C2
Time of collection (Day 1 & Day 2) Time of collection (Day 1 & Day 2)
Fig. 1 Cellular and soluble inflammatory markers in nasal lavage fluid (NALF) in four students at time point A (before
exposure), time point B (afternoon of Day 1 and Day 2), and time point C (the morning after exposure during Day 1 and
Day 2); mean endotoxin exposure of 465 EU/m3 (Day 1) and 659.7 EU/m3 (Day 2)
4 Discussion recruitment of sewage treatment plants and

workers proved to be difficult, the time gap
4.1 Study Design and Study Sample between the single study days was rather long.
In addition, the endotoxin exposure on a single
In the present study, we chose a quasi- sewage plant was very heterogeneous and, nota-
experimental design to investigate the effects of bly, it was much lower than expected as the
an endotoxin exposure lasting several hours on workers performed a variety of tasks during the
inflammatory markers in NALF and to compare working day. Therefore, a fourth sewage treat-
chronically and acutely exposed subjects. We ment plant was chosen, where only students
chose not to expose the study participants in a were exposed to high endotoxin concentrations
laboratory setting, but rather to assess the expo- in a specific plant area on two following days.
sure during routine work in selected areas of
sewage treatment plants in Germany (plant 1–3: Exposure Assessment We used personal sam-
chamber filter press, plant 4: sand trap) where our pling to assess the amount of endotoxin inhaled
previous studies have shown high endotoxin by the workers and the escorting students. The
concentrations (>900 EU/m3) and at the same assessment of endotoxin concentrations followed
time a rather low exposure to bacteria, molds, the German specifications for standardized
and chemical hazards (Rieger and Diefenbach measurements (Rieger et al. 2005; BGIA-
2007; Steinberg 2001). Likewise, high Arbeitsmappe 1989) using a commercially avail-
concentrations of endotoxin in certain areas of able chromogen-kinetic Limulus amoebocyte
sewage treatment plants (e.g., 729–887 EU/m3 lysate (LAL) test as described elsewhere (Rieger
during cleaning of the filter press) have been et al. 2005). The LAL test has turned out suitable
reported in a Dutch study (Visser et al. 2006). for the comparison of exposure in different sew-
This particular exposure to endotoxin, combined age treatment plants (Reynolds et al. 2005) and
with a much lower concentration of other the assessment of exposure during working
biological hazards and without dust, is different activities (Liebers et al. 2008).
from the situation, e.g., in swine confinement Due to a surprisingly high proportion of
buildings used for similar investigations by administrative and monitoring tasks and duties
other authors (Wang et al. 1997). related to the work in the chamber filter press
The exposure to a “natural mixture” of endo- hall, the exposure due to routine work on plants
toxin was different from laboratory settings where 1–3 was lower than expected based on previous
usually lipopolysaccharide derived from one spe- studies (Rieger and Diefenbach 2007; Visser et al.
cific bacterium have been used for standardized 2006; Steinberg 2001), but it reflected a well-
exposure (Danuser et al. 2000; Sigsgaard et al. known variability of exposure typical for
2000; Rylander et al. 1989). Generally, the use of biological hazards (Liebers et al. 2008). The
a working environment for an investigation of concentrations we found clearly exceeded the
possible effects of endotoxin exposure has HBROEL recommended in 2010 in the
facilitated the recruitment of workers and a simul- Netherlands (Health Council of the Netherlands
taneous examination of several study participants, 2010) only on plant 4 (median 356 EU/m3 (Day
such as pairs of chronically and acutely exposed 1) and 643 EU/m3 (Day 2)) and ranged from
subjects. Yet it lacks the possibility of a 4 EU/m3 up to 95 EU/m3 (i.e., 0.4 ng/m3 up to
standardized exposure, a feature of a laboratory 9.5 ng/m3) for the routine work on plants 1–3,
setting. where pairs of workers and students were
The advantage of the present quasi- investigated. Thus, in only 6/22 measurements
experimental design was a very realistic exposure on plant 1–3, the endotoxin concentrations
scenario and a rather good possibility to address exceeded the HBROEL recommendations of
chronically exposed subjects. Yet, as the 50 EU/m3 at the time of the study (DECOS 1998).
Concerning condition “no specific exposure”, the latter question has not yet been addressed in
we decided not to perform any exposure assess- the occupational settings, where endotoxin is the
ment, as all office and lecture rooms, where the leading biological hazard, as opposed to studies
students stayed during the 3 days of the investi- examining workers and naïve controls in occupa-
gation, were in a good condition and had a good tional settings with more mixed biological
indoor climate with no apparent risk of elevated exposures, e.g., waste collection (Wouters et al.
concentrations of endotoxin, as reported to occur 2002) or pig farming (Sahlander et al. 2012; von
due to dampness in schools (Jacobs et al. 2014; Essen and Romberger 2003). As the workers on
Jacobs et al. 2013). the sewage treatment plants were only men, we
recruited no female students. Thus, we could not
Inflammatory Markers in NALF The noninva- examine any gender effect despite this might have
sive collection of NALF turned out well applica- been interesting according to the findings of a
ble for this field study, and it has been applied in Taiwanese study addressing the recovery in lung
many studies addressing the reaction to endotoxin function after cessation of exposure to organic
exposure in both field and laboratory settings dust (Lai et al. 2015).
(Raulf et al. 2017; Sigsgaard et al. 2015; Wouters There were some differences in the
et al. 2002; Douwes et al. 2000). According to the sociodemographic data of workers and students.
concept of “united airway disease” in the occupa- The workers were older, some were smokers, and
tional setting, inflammatory markers assessed in some showed signs of peripheral flow limitation
NALF may not only point to the inflammatory before (one worker) or after (three workers) the
processes in the upper but also lower airways work shift. This is in line with cross-sectional
(Quirce et al. 2010). As the students were trained studies in wastewater industries (Steinberg
in collecting their NALF at the study onset, the 2001), waste collectors (Raulf et al. 2017;
recovery rate and quality of specimens were gen- Schantora et al. 2015), compost workers
erally high. The time points for NALF collection (Hoffmeyer et al. 2014), or agricultural workers
(B about 8 h and C about 24 h after time point A) (Spierenburg et al. 2015) presenting with respira-
were chosen according to other studies, where tory symptoms during their employment. How-
NALF was gathered at 1 h (Danuser et al. ever, with respect to the ATS recommendations,
2000), 3 h (Sigsgaard et al. 2000), 6/7 h methodological limitations of our spirometry
(Doreswamy et al. 2011; Wang et al. 1997), or assessment have to be quoted. In general, there
23/24 h or later after endotoxin exposure was no possibility of repeated measurements due
(Danuser et al. 2000). In most of those studies, to organizational constraints on the sewage treat-
the exposure-related effect in inflammatory ment plants. On the other hand, students were
markers has occurred 3 or 6 h after exposure, only non-smokers and had normal spirometry
with a recovery approximately 24 h after a single findings.
exposure. Thus, time points B and C seemed to be Atopy was determined in 1/8 workers and 4/10
chosen properly in the present study. students by objective parameters. Students rated
their general health better than workers
Study Sample The constitution of pairs of (1.4 vs. 2.8; 1 ¼ very good, 6 ¼ insufficient)
workers (i.e., chronically exposed subjects) and and indicated a higher stress than workers due to
acutely exposed healthy volunteers (students), some aspects of the working environment on the
escorting the workers, was designed to compare: sewage treatment plants, e.g., noise 2.7
(a) the baseline concentration of inflammatory (students) vs. 1.8 (workers) or humidity 2.4
markers in NALF in the morning and (b) the (students) vs. 1.4 (workers) (1 ¼ little, 3 ¼ high
patterns of inflammatory markers in NALF after stress), whereas both groups indicated a similarly
exposure to endotoxin in the afternoon and on the high stress due to odor and infectious hazards.
next morning in both groups. To our knowledge,
The fact that the workers, despite partly was not reflected in statistical analysis (n ¼ 12
longstanding work on sewage treatment plants, values from students, n ¼ 8 values from workers).
indicated a rather good health status and even The difference between workers and only
better ability to work in conjunction with a high acutely exposed students is similar to the findings
work satisfaction might point to a healthy worker comparing current and former compost workers
survival effect. Yet, one out of eight workers to white collar workers. There, current workers
showed pathologic findings in spirometry before show a higher concentration of inflammatory
exposure, and he and two additional workers markers in induced sputum than former compost
showed a tendency of exposure-induced changes workers or nonexposed subjects (Raulf et al.
in lung function at time point B. According to the 2015). Similarly, before exposure, workers from
questionnaire, some students experienced slight biofuel plants have a higher IL-8 concentration in
symptoms of mucous membrane irritation, but no NALF compared to controls from conventional
severe complaints. To contrast the findings fuel plants (Zheng et al. 2014). Pig farmers com-
derived from the high exposure, some students pared to naïve controls show signs of local and
were also asked to participate in the assessment systemic inflammation associated with altered
during 3 days without specific exposure. Yet, no innate immunity, which has been ascribed to
data of chronically exposed subjects were avail- adaptation to chronic endotoxin exposure
able in either condition. (Sahlander et al. 2012; von Essen and Romberger
2003). So far, this effect has not been described
for workers in wastewater industries.
Other than in studies comparing farm workers
4.2 Research Issue 1: Effects
to naïve subjects in swine confinement (Sahlander
of Chronic Endotoxin Exposure
et al. 2012; von Essen and Romberger 2003), in
the present study there was no definable higher
We aimed to answer the question of whether there
concentration of inflammatory markers in
was a difference between chronically exposed
students than in workers at time point B or C, as
(workers of sewage treatment plants) and acute
a result of exposure to endotoxin on plant 1, 2, or
exposed subjects (students) regarding the markers
3. This might be due to a rather low endotoxin
in NALF before and after exposure to endotoxin
concentration associated with routine work on the
during a routine work on a plant. The results of
days of examination and the fact that the sample
univariate analysis and GEE pointed to a certain
size was too limited to detect differences between
difference between students and workers, which
workers and students.
can be interpreted as the effect of chronic expo-
sure: workers showed higher concentrations of
some inflammatory markers in NALF (i.e., total
4.3 Research Issue 2: Different
cell count, IL-8, and IL-1β) across all three time
Patterns of Inflammatory
points but without significant clinical symptoms
Markers in NALF After Exposure
or findings due to the exposure. Focusing only on
to a Moderate Endotoxin
the values gathered at time point A, this differ-
Concentration as Compared
ence occurred in the total cell count. To summa-
to Nonspecific Exposure. Dose-
rize, the effect of chronic exposure may result in
Response Relationship of Acute
higher concentrations of inflammatory markers in
Exposure, Including
NALF. Yet, these findings are based on only a
a Cumulative Exposure
small sample size (n ¼ 10 students, n ¼ 8
workers). In addition, regarding the comparison
We found a great intra- and interindividual
of concentrations at time point A, the uneven
variability concerning the concentrations of
distribution due to repeated participation of
inflammatory markers in NALF gathered in five
some students has to be accounted for, as this
healthy students in the morning and afternoon of
3 days without any specific exposure. Due to a comparing time point B values of the students
small sample size we could not perform any sta- exposed to different endotoxin concentrations,
tistical analysis, but descriptive analysis suggests higher endotoxin concentrations seemed
that there were no circadian differences in the associated with increased concentration of total
inflammatory markers investigated. protein, IL-8, IL-1β, and sCD14, and with
In the literature, we could not find any study decreased percentage of neutrophils and IL-6
focusing on the variability of inflammatory concentration. A cumulative exposure to the
markers in NALF (or other specimens) in naïve endotoxin concentration > 200 EU/m3 (Day 1:
subjects without a specific exposure. Yet, hints median 356 EU/m3 (162–885 EU/m3; mean:
for a great variability can be driven from studies 465 EU/m3), Day 2: median 643 EU/m3
where the results of (sometimes repeated) (231–1039 EU/m3; mean 660 EU/m3)), for
measurements before exposure are reported. A about 8 h each, seemed associated with a differ-
broad concentration range of inflammatory ence in the concentration of time point B values
markers in NALF has been reported, e.g., in for- collected on the first and second day. The differ-
mer waste collectors, especially in never smokers ence, particularly, were present in total cell count
(Raulf et al. 2017), in former compost or white (lower on Day 2), percentage of neutrophils
collar workers in induced sputum (Raulf et al. (higher on Day 2), and concentrations of IL-8
2015), or in NALF and exhaled breath condensate (higher), IL-6 (higher), and sCD14 (lower). Yet
in workers from conventional fuel plants (Zheng these effects should be considered cautiously
et al. 2014). A repeated collection of NALF with- against the background of a great intra- and inter-
out exposure (0 min, 30 min, and 2, 6, and 24 h) individual variability of inflammatory markers in
in six healthy volunteers or pre-exposure in NALF specimens collected at time point B in five
16 healthy volunteers (Raulf-Heimsoth et al. students on 3 days without any specific exposure,
2010) has shown a broad range of inflammatory as well as compared to a broad range of values
markers comparable to the present findings. collected at time point A in the students.
Unfortunately, other authors have not reported Beside a small sample size combined with
the range of repeated measurements of inflamma- great variability of concentrations, a reason for
tory markers in healthy subjects, but just the the lack of a distinguished dose-dependent reac-
means or medians, e.g., in 20 healthy volunteers tion in NALF inflammatory markers in the pres-
with repeated measurements (Kitz et al. 2008) or ent study could be that the endotoxin exposure
in 9 healthy volunteers with rather “similar” during routine work (plant 1–3) or at specific
concentrations (Michel et al. 1997). Therefore, places known to result in a high endotoxin expo-
no comparison is possible between the present sure (plant 4) was too small to induce a detectable
results and those reported previously. Interest- response. So far, there have been several studies
ingly, the median concentration of inflammatory where the dose-dependent changes of inflamma-
markers at different time points after saline instil- tory markers are investigated, mainly with the
lation reported by Danuser et al. (2000) do not goal to describe the effect of the lowest possible
show such high variability as found in the present level of endotoxin exposure in healthy volunteers
study. Thus, our findings seem rather new as a or chronically exposed workers (Danuser et al.
great variability of inflammatory markers has so 2000; Michel et al. 1997).
far been described explicitly only in response to Soluble inflammatory markers in NALF are
organic dusts (Sigsgaard et al. 2005). the main outcomes in an experimental study of
In the present study, the concentration of Danuser et al. (2000), where seven subjects were
inflammatory markers without a specific endo- exposed to 0, 10, and 40 μg lipopolysaccharide
toxin exposure in the students entailed the major- (LPS) by nasal instillation for 10 s. The instilla-
ity of values collected after the exposure (see tion of 10 μg LPS increased IL-6 over 2-fold and
Table 5). When possible dose-related changes in that of 40 μg caused an increase in nasal
the marker concentration were investigated by symptoms and a 13-fold increase in IL-6
compared to saline treatment, with a peak at 6 h. are akin to the present findings concerning
As there were no repeated measurements in single neutrophils and sCD14, at variance with IL-8.
individuals, the variability of the effects is To wrap it up, there still seems to be a lack of
unknown. Yet, comparing the present findings replicated inhalation studies in healthy volunteers
to those of that study suggests that the exposure describing a consistent dose-response relation-
on plant 4 was high enough to produce a distinct ship or cumulative effects due to endotoxin expo-
reaction in the NALF inflammatory markers. sure. That holds true for both work-related (Thorn
Michel et al. (1997) published the results of a 2001) and environmental exposures (Walser et al.
study on the dose-response relationship to inhaled 2015). Concerning the present study, it remains
endotoxin, focusing on clinical symptoms, blood unclear whether exposure to a far higher endo-
and induced sputum indices, and lung function in toxin concentration than found on plant 4 would
nine healthy volunteers after a weekly bronchial have induced changes in inflammatory markers
challenge with increasing doses of 0.5, 5, and distinct from the greatly variable concentrations
50 μg LPS. They noticed significant increases in found without any specific exposure in the
the number of neutrophils and monocytes in the students. Inversely, it could be that the apparent
sputum after inhalation of 5 μg LPS, which was changes described in the experimental studies
significantly greater after 50 μg LPS, compared to cited would not come to sight if those studies
saline. No repeated measurements were reported. accounted for the variability of findings without
Thus, variability of the effects in the nine specific exposure.
volunteers is unknown. The authors have set a
threshold value for no response in sputum inflam-
matory cells at 0.5 μg inhaled LPS, which 5 Conclusions
corresponds to the exposition for 10 h a day and
lung ventilation of 1 m3 per hour of air containing We replicated previous findings of a great
50 ng LPS. This concentration corresponds to variability of the concentration of inflammatory
about 500 EU/m3, which was exceeded by the markers in NALF in healthy subjects without
mean concentration the majority of students specific exposure at different time points, i.e.,
were exposed to for about 8 h on plant 4 in the within 1 day and between several days. The con-
present study. Yet, in the cited study, significant centration range was even broader than the appar-
effects in sputum inflammatory cells were ent reaction after exposure to a rather high
observed only at a concentration of 5 μg LPS endotoxin concentration (mean: 465 EU/m3
and higher, corresponding to a rather high expo- (Day 1)/660 EU/m3 (Day 2)). Therefore, in our
sure of about 5000 EU/m3. Thus, exposure even study we could not replicate findings on a dose-
on sewage treatment plant 4 possibly was too low response relationship or cumulative effects
to evoke distinct effects. obtained by other authors.
Concerning the effect of repeated exposure Based on our findings, it seems necessary to
(Day 1 and Day 2 on plant 4) at time point B on carefully assess individual ranges of concentra-
the total cell count (lower on Day 2), neutrophils tion for single inflammatory markers without any
(higher on Day 2), IL-8 (higher), IL-6 (higher), specific exposure before investigating a reaction
and sCD14 (lower on Day 2), our present results to a specific exposure such as endotoxin or other
can be compared with a study of Doreswamy hazardous substances. In addition, a great
et al. (2011). Those authors have reported a sig- variability of findings in single individuals
nificant increase in neutrophils and a decrease in underscores the importance of a sample size,
sCD14 and IL-8-concentration in NALF after a which should be large enough for the use of
6-hour experimental exposure (instillation of non-parametric tests (i.e., 20 subjects) or corre-
2 μg LPS in the nostril) on the third day com- spond to the sample size indicated in the test-
pared to pre-exposure or 6 h after just a single specific table accounting for the predictable
exposure in 15 healthy volunteers. Those results great variability of findings (Wilcoxon 1945).
Concerning the adaptation to chronic endo- Bradford MM (1976) A rapid and sensitive method for the
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concentration changes of inflammatory markers. Lipopolysaccharide-induced nasal cytokine response:
a dose-response evaluation. Eur Arch Otorhino-
Acknowledgments The study was financed by a grant of laryngol 257:527–532
the Lieselotte und Dr. Karl Otto Winkler Stiftung für DECOS (Dutch Expert Committee on Occupational
Arbeitsmedizin and own research funds of the institutions Standards) (1998) Endotoxins: health based
involved (Department of Applied Physiology, Occupa- recommended exposure limit. In: A report of the
tional Medicine and Infectiology, University of Health Council of the Netherlands, publication no
Wuppertal, Germany, and Department of Allergology/ 1998/03WGD. Health Council of the Netherlands,
Immunology, Institute for Prevention and Occupational Rijswijk, pp 1–82
Medicine, Institute of the German Social Accident Insur- Doreswamy V, Alexis NE, Zhou H, Peden DB (2011)
ance, Ruhr University Bochum, Germany). The work of Nasal PMN response to repeated challenge with endo-
the Institute of Occupational and Social Medicine and toxin in healthy volunteers. Inhal Toxicol 23:142–147
Health Services Research (IASV) is supported by an unre- Douwes J, Wouters I, Dubbeld H, van Zwieten L,
stricted grant of the employers’ association of the metal Steerenberg P, Doekes G, Heederik D (2000) Upper
and electric industry Baden-Württemberg airway inflammation assessed by nasal lavage in com-
(Südwestmetall). post workers: a relation with bio-aerosol exposure. Am
The authors thank the Emscher-Genossenschaft/ J Ind Med 37:459–468
Lippeverband (EGLV) for giving access to their sewage Health Council of the Netherlands (2010) Endotoxins.
treatment plants, all volunteers for study participation, and Health-based recommended occupational exposure
the involved staff of IPA, IASV, and University limit. Health Council of the Netherlands, The Hague
Wuppertal, especially Monika Lahr, Heiko Diefenbach, Hoffmeyer F, van Kampen V, Taeger D, Deckert A,
and Nicole Blomberg for technical assistance in collection Rosenkranz N, Kaßen M, Schantora AL, Brüning T,
of specimens and on the sewage treatment plants Raulf M, Bünger J (2014) Prevalence of and relation-
and Gerda Borowitzki, Susanne Freundt, and Sabine ship between rhinoconjunctivitis and lower airway
Bernard for technical assistance in analysis of the NALF diseases in compost workers with current or former
samples. exposure to organic dust. Ann Agric Environ Med
21:705–711
Jacobs JH, Krop EJ, de Wind S, Spithoven J, Heederik DJ
Conflicts of Interest The authors declare no conflicts of (2013) Endotoxin levels in homes and classrooms of
interest in relation to this article. Dutch school children and respiratory health. Eur
Respir J 42:314–322
Jacobs JH, Krop EJ, Borras-Santos A, Zock J, Taubel M,
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Published online: 29 June 2018
Effects of Low-Level Laser Therapy

in Autism Spectrum Disorder
Gerry Leisman, Calixto Machado, Yanin Machado,

and Mauricio Chinchilla-Acosta
Abstract difference in the baseline to study endpoint

The study examined the efficacy of low-level change in the ABC irritability subscale score
laser therapy, a form of photobiomodulation, between test and placebo participants was
for the treatment of irritability associated with 15.17 in favor of the test procedure group.
autistic spectrum disorder in children and ANCOVA analysis found this difference to be
adolescents aged 5–17 years. Twenty-one of statistically significant (F ¼ 99.34, p < 0.0001)
the 40 participants received eight 5-min compared to the baseline ABC irritability sub-
procedures administered to the base of the scale score. The study found that low-level
skull and temporal areas across a 4-week laser therapy could be an effective tool for
period (test, i.e., active treatment participants). reducing irritability and other symptoms and
All the participants were evaluated with the behaviors associated with the autistic spectrum
Aberrant Behavior Checklist (ABC), with the disorder in children and adolescents, with pos-
global scale and five subscales (irritability/agi- itive changes maintained and augmented
tation, lethargy/social withdrawal, stereotypic over time.
behavior, hyperactivity/noncompliance, and
inappropriate speech), and the Clinical Global Keywords
Impressions (CGI) Scale including a severity- Autism spectrum disorder · Brain · Clinical
of-illness scale (CGI-S) and a global improve- trial · Low-level laser therapy · Neuronal
ment/change scale (CGI-C). The evaluation networks · Photobiomodulation
took place at baseline, week 2 (interim),
week 4 (endpoint), and week 8 (post-
procedure) of the study. The adjusted mean 1 Introduction
G. Leisman (*) Autism spectrum disorder (ASD) is a complicated

Faculty of Social Welfare and Health Sciences, University syndrome characterized clinically by language
of Haifa, Haifa, Israel impairment, dysfunction in social engagement,
National Institute for Brain and Rehabilitation Sciences, stereotypical movements and behaviors, and var-
Nazareth, Israel ious cognitive deficits (McPartland and Volkmar
Faculty ‘Manuel Fajrdo’, University of Medical Sciences, 2012; South et al. 2012; Zappella 2012; Melillo
Havana, Cuba and Leisman 2010). In earlier work, we have also
noted an excess of high-frequency EEG,
C. Machado, Y. Machado, and M. Chinchilla-Acosta suggesting an imbalance in the excitation-
Institute for Neurology and Neurosurgery, Havana, Cuba
111
112 G. Leisman et al.
inhibition homeostasis in the neocortex patterns of synchronization and information flow

(Machado et al. 2015). (Brovelli et al. 2004) between connected
Recent theories have proposed that deficits in elements.
the integration of transient activity patterns in A significant literature exists on the ability of
diverse brain regions suggest a possible temporal low-level light therapy (LLLT) to penetrate the
binding deficit in ASD (Leisman et al. 2012; skull. Low-energy light passes the skull and a
Testa–Silva et al. 2012; Vissers et al. 2012; therapeutic effect likely exists. LLLT systems
Melillo and Leisman 2009). Binding denotes the employ the so-called quantum optically induced
effective incorporation of dissimilar neural infor- transparency effect (Scherman et al. 2012; Weis
mation in shaping an individual’s unified experi- et al. 2010). This effect controls optical properties
ence of the world and self, reportedly impaired in of dense media enhancing transparency contrast
ASD (Machado et al. 2015; McPartland and by a factor of five. Therefore, the skull, spine, or
Volkmar 2012; South et al. 2012; Zappella joints can be penetrated even with moderate
2012; Melillo and Leisman 2010). We have intensity light reaching deep layers in muscles,
suggested that synchronous activity in the neo- connective tissue, and even bone, enabling
cortex may be critical for sensory integration and transcranial effects of LLLT (Hamblin 2018;
is associated with integrated perceptual Hiwaki and Miyaguchi 2018; Grover Jr et al.
experiences (Ding et al. 2017), selective attention 2017; deTaboada et al. 2006).
(Salo et al. 2017), and working memory (Griffiths LLLT achieves a therapeutic effect by
and Kumar 2017), all of which are part of the employing non-ionizing light, including lasers,
myriad deficits reported in ASD. The evidence light-emitting diodes, or broadband light in the
from both animal and human studies visible red (600–700 nm) and near-infrared
demonstrates that changes in neuronal synchrony (780–1100 nm) spectra (Shanks and Leisman
occur during all of these processes. 2018; deFreitas and Hamblin 2016). LLLT is a
Augmented coherence values, reflecting nonthermal process occurring when a chromo-
inefficiencies in brain networking noted in ASD, phore is exposed to a suitable wavelength of
likely reflect rigid neuronal networks that could light. Chromophores are responsible for the
explain the typical manifestation of repetitive color associated with biological compounds
behaviors, deficiencies in social interaction, and such as hemoglobin and cytochromes (Cotler
communication and imagination, characteristics et al. 2015). With chromophore absorption of a
of ASD (Machado et al. 2015; Gadow and photon of light, an electron transits to an excited
Drabick 2012; Zappella 2012). state, with a physiologic effect occurring when
Brain specialization does not fully explain photons dissociate the inhibitory signaling mole-
most aspects of brain function. Confirmatory cule nitric oxide (NO) from cytochrome-C-oxi-
studies indicate that integrative functions over dase, increasing the electron transport,
multiple distributed systems and areas support mitochondrial membrane potentials, and produc-
diverse cognitive processes such as visual recog- tion of mitochondrial products such as ATP and
nition (Behrmann and Plaut 2013), language NADH (deFreitas and Hamblin 2016; Wang et al.
(Friederici and Gierhan 2013), cognitive control 2016, 2015). Other effects include the production
(Power and Petersen 2013), emotion (Pessoa of reactive oxygen species (ROS) which activate
2012), and social cognition (Barrett and Satpute transcription factors, leading to the cellular pro-
2013). The neural substrate enabling integration liferation and migration (Farivar et al. 2014).
of distributed neural information and the emer- Based on these complex characteristics, LLLT
gence of a coherent cognitive state depends partly possesses physiologically modifying properties
on neural communication among specialized associated with light characteristics such as wave-
brain regions existing within a network of interre- length and irradiance, varied by exposure
gional projections (Fuster 1997; Goldman-Rakic parameters, such as energy density, irradiation
1988). The result is associated with large-scale duration, and treatment frequency. On the basis
Effects of Low-Level Laser Therapy in Autism Spectrum Disorder 113
of the above, we investigated behavioral and cog- their parents seeking treatment for the symptoms
nitive changes in ASD as a consequence of the of ASD and from among the individuals who
delivery of red LLLT. responded to recruitment flyers and print ads.
Participants did not receive any financial compen-
sation for participation.
2 Methods
2.2 Inclusion and Exclusion Criteria
2.1 Participants
Each eligible participant satisfied each of the fol-
The study received an approval from the Helsinki
lowing inclusion criteria and none of the exclu-
Committee of the Institute for Neurology and
sion criteria. Males or females aged 5–17 years
Neurosurgery in Havana, Cuba, and was
meeting the criteria of the Diagnostic and Statis-
registered with the FDA (Identifier:
tical Manual of Mental Disorders, 4th Edition, for
NCT03379662). Informed written consent was
ASD, are diagnosed by a qualified medical pro-
obtained from the parent or guardian of each
fessional. Diagnosis was confirmed by the
participant after a full explanation of the
Autism Diagnostic Interview (ADI-R)
procedures to be undertaken. The informed con-
(Constantino et al. 2003). Each participant
sent forms, research protocol, and approvals are
demonstrated “irritable” behaviors such as
available for inspection in the Office of Research
tantrums, aggression, self-injurious behavior, or
Integrity at the Institute of Neurology and Neuro-
a combination of thereof. The participant’s Aber-
surgery in Havana, Cuba.
rant Behavior Checklist (ABC) irritability sub-
The participants consisted of 40 individuals
scale (Rojahn et al. 2003) score was >18; the
distributed at baseline in the fashion described in
Clinical Global Impressions Severity Scale
Tables 1, 2, and 3. Participants in both groups
(CGI-S) (Berk et al. 2008) score was 4
spanned 5–16 years, with the mean participant
(4 ¼ moderately ill). The participants’ therapeu-
age of approx. 8 years. A t-test for independent
tic/intervention plan had been consistent/stable
samples revealed no statistically significant dif-
for 3 months. They abstained from undertaking
ference in age between test and placebo group
new treatments during the study time.
participants (μa-μb ¼ 0.13; t ¼ 0.13;
Exclusion criteria were the following: history
df ¼ 38; p(two-tailed) ¼ 0.90 ( p > 0.05)).
of a primary or concurrent diagnosis of another
All study participants were recruited from
disorder, including neurological, use of a psycho-
among the investigator’s normal pool of new
tropic drug, or any participation in a research
and existing patients who voluntarily came with
study within 30 days prior to the commencement
of the current study.
Table 1 Age of study participants by the procedure group
Age (years) Testa (n ¼ 21) Placebo (n ¼ 19)
Mean 8.2 8.4 2.3 Baseline Concomitant
Standard deviation 3.0 3.2 Medications and Low-Level Light
Range 5–16 5–16 Therapy (LLLT)
a
Test group was the active treatment group
Table 4 presents the OTC and prescription
Table 2 Participant gender breakdown for the test and medications reported by participants at the base-
placebo group participants line evaluation used in the past by both test and
Gender Testa (n ¼ 21) Placebo (n ¼ 19) placebo group participants. No participant took
Male 16 14 any OTC medication during the study time.
Female 5 5 Table 5 lists the non-drug therapies routinely
a
Test group was the active treatment group used by both test and placebo group participants
Table 3 Participant ethnicity by the procedure group

Ethnicity Testa (n ¼ 21) Placebo (n ¼ 19)
Caucasian 3 4
Hispanic 9 1
African 8 13
Hispanic and African 1 0
Asian 0 1
a
Table 4 Prior medication used to treat the symptoms of per week, 3–4 days apart at the investigator’s test
autistic disorder by the procedure group site. A pulsed laser of 635 nm with a power output
Medicationa Testb (n ¼ 21) Placebo (n ¼ 19) of 15 mW and a red 635 nm LED were used as
Carbamazepine 10 7 treatment and placebo, respectively.
Risperdal 9 8 Participants were required to maintain their
Valproate 2 3 regular medication schedule and treatment
Methylphenidate 3 1 regimens, as reported at the baseline evaluation,
Haloperidol 1 3 to treat symptoms related to autistic disorder
Chlorpromazine 1 2 throughout the study time. All of them complied
Risperidone 1 1 with this requirement.
Conductosa 1 1
Thioridazine 0 2
Levomepromazine 0 1
a 2.5 Outcome Measures
Some participants previously used several medications;
therefore, medication use by participant group adds up to
greater than the participant sample size per group; btest Pre-post treatment outcome measures included
group was the active treatment group the Aberrant Behavior Checklist (ABC). The
global score and the five subscale scores
to treat the symptoms of ASD at the time of entry consisted of (a) irritability and agitation,
to the study. Therapy used to treat symptoms of (b) lethargy and social withdrawal,
ASD was minimal and equal between test and (c) stereotypic behavior, (d) hyperactivity and
placebo group participants. noncompliance, and (e) inappropriate speech.
Medications routinely used by both test and The ABC is an informant rating instrument
placebo group participants at the time of study empirically derived by principal component anal-
enrollment for indications other than to treat the ysis. The global score for the ABC has not been
symptoms of autistic disorder are listed in psychometrically derived and is not statistically
Table 6. valid (Farmer and Aman 2012).
The second outcome measure consisted of the
Clinical Global Impressions (CGI) scale includ-
2.4 Procedure ing a severity-of-illness scale (CGI-S) and a
global improvement/change scale (CGI-C).
All the 40 participants completed the course Leucht and Engel (2006) have noted that both
according to the protocol. Twenty-one of them the CGI and the Brief Psychiatric Rating Scale
were randomized to the test (active treatment) (BPRS) are often employed in drug trials. Those
procedure group, and 19 were randomized to the authors have found that by calculating the effect
placebo procedure group. size and its 95% confidence interval for both
Participants received eight 5-min laser light continuous (standardized mean differences) and
applications to the base of the skull and temporal dichotomous (odds ratio) outcomes, there was no
areas with the Erchonia® EAL Laser (active or significant differences between tests, which
sham) across a 4-week period: two applications indicates a good inter-test reliability.
Table 5 Therapies used to treat the symptoms of autistic disorder by the procedure group
Therapy Testa (n ¼ 21) Placebo (n ¼ 19)
Logopedic therapy for language 2 2
Hydrotherapy for hyperactivity 1 0
a
Table 6 Baseline non-autism-related concomitant medication use by the procedure group

Medication (Indication) Testa (n ¼ 21) Placebo (n ¼ 19)
Ketotifen (allergies) 3 1
Benadryl (allergies) 0 1
Meclizine (allergies) 0 1
Fluticasone (Asthma) 1 0
Vitamins A, B6, and C 0 1
a
The ABC and CGI-S and CGI-C were 3.2 Primary Efficacy Outcome
administered prior to treatment and then 2 (mid- Analysis
point) and 4 weeks (endpoint) of treatment and
finally 8 weeks after treatment (post-procedure The evaluation time point at which study success
evaluation). was analyzed (study endpoint) was predetermined
as 4 weeks following baseline (pretreatment)
evaluation. The study was predetermined to be
considered successful if, using the intent-to-treat
3 Results
(ITT) last observation carried forward (LOCF)
analysis, the primary endpoint was statistically
3.1 Pre-procedure Measures
significant at the 0.05 level. The primary efficacy
outcome measure was predefined as the mean
change from baseline to endpoint (end of the
Aberrant Behavior Checklist (ABC) Mean and
4-week treatment period) in the ABC irritability
standard deviation baseline (pre-procedure) of
and agitation subscale score, with a minimum
the ABC global and subscale scores for test and
mean difference of 8.5 points between the test
placebo group participants are provided in
and placebo groups.
Table 7.
The primary outcome measure was evaluated
for the following two populations:
T-tests for independent samples revealed no sta-
tistically significant differences in the baseline
1. Intent-to-Treat (ITT). ITT analysis included all
ABC global score or in any of the five ABC
participants with valid measurements at base-
subscale scores between test and placebo group
line, randomized to a procedure group.
participants ( p > 0.05) as shown in Table 8.
Dropouts and terminated participants were
handled by carrying forward the last observa-
Clinical Global Impressions Severity (CGI-S)
tion for all time points following the dropout
Score Table 9 shows the CGI-S baseline score
(LOCF). If a participant was not a dropout but
for the test and placebo group participants. The
had no data in a relative day range, the last
majority of participants in both groups had a
observation prior to the time point being
baseline CGI-S score of 6, corresponding to
analyzed was employed.
“severely ill”.
2. Per-Protocol Population. Per-protocol analy-
sis of results intended to corroborate
Table 7 ABC global and subscale scores by the procedure group

Testa (n ¼ 21) Placebo (n ¼ 19)
Mean SD Mean SD
Global score 30.5 6.7 29.6 6.8
Irritability and agitation 23.1 9.3 24.7 5.1
Lethargy and social withdrawal 13.7 4.1 12.3 5.6
Stereotypic behavior 32.8 7.8 36.9 7.9
Hyperactivity and noncompliance 7.2 3.1 6.4 4.0
Inappropriate speech 107.3 20.3 104.7 28.7
a
Table 8 T-test results for differences in ABC global and subscale scores between the two procedure group
(test vs. placebo)
μa-μb t df p
Global score 0.95 +0.44 38 0.66
Irritability and agitation 1.64 0.68 38 0.50
Lethargy and social withdrawal 1.40 +0.91 38 0.37
Stereotypic behavior 4.09 1.64 38 0.11
Hyperactivity and noncompliance 0.77 +0.69 38 0.49
Inappropriate speech 2.60 +0.33 38 0.74
Table 9 CGI-S baseline scores by the procedure group

CGI-S Score Testa (n ¼ 21) Placebo (n ¼ 19)
7: Among the most extremely ill patients 3 0
6: Severely ill 14 15
5: Markedly ill 4 4
a
conclusions drawn from the ITT analysis. This adjusted means for the absolute change in ABC
analysis excluded the participants with major irritability subscale score from baseline to end-
protocol deviations and incompletes point, adjusting for the covariate of baseline ABC
(dropouts, non-compliant participants, irritability subscale scores.
disqualified participants, etc.). All participants The adjusted mean difference in the baseline to
in this study completed all study visits and endpoint change in the ABC between the test and
procedures and had all study measurements placebo groups was 15.2, almost double the
recorded through to the study endpoint evalu- pre-established study success criteria of 8.5
ation. Therefore LOCF was not applied. points between the procedure groups, in favor of
the test group (F ¼ 99.3; p < 0.0001). Table 11
shows the observed mean and standard deviation
at baseline and endpoint for the ABC score and
change between the evaluations by the procedure
3.3 General Statistical Evaluation
group.
The ABC score decreased 14.8 points for
For the ABC irritability subscale score, changes
participants in the test group, while it increased
were evaluated by analysis of covariance
by 0.32 points for participants in the placebo
(ANCOVA), with baseline measures as a covari-
group, resulting in an observed difference
ate and a procedure group (active or placebo) as
between the two groups in the ABC irritability
the main effect. Table 10 shows the observed and
Table 10 Change in ABC irritability subscale score from baseline to endpoint, adjusting for baseline ABC irritability
subscale score
ABC irritability subscale score Testa (n ¼ 21) Placebo (n ¼ 19)
Observed mean 14.81 0.32
Adjusted mean 14.83 0.34
a
Table 11 ABC irritability subscale score from baseline to endpoint by the procedure group
ABC irritability subscale score Mean SD Mean SD
Baseline 30.5 6.7 29.6 6.8
Endpoint 15.7 9.9 29.9 6.6
Change 14.8 6.4 0.3 1.4
a
subscale score from baseline to endpoint evalua- on the participant’s primary caregiver’s rating and
tion of 15.1. (b) CGI-I rating of 1 (very much improved) or
2 (much improved) at study endpoint as deter-
T-Test for Two Correlated Samples A correlated mined by the clinician’s evaluation.
sample t-test was used to compare the difference Participants attaining a 25% reduction
in the mean change in ABC score from study from baseline to endpoint in the ABC irritability
baseline to endpoint. The evaluation for partici- subscale score. Eighteen (86%) of the 21 test
pant groups separately found the mean change to group participants attained a minimum of 25%
be significant for the test group (t ¼ +10.6; reduction in the ABC score from baseline to end-
df ¼ 20; p < 0.001), but not for the placebo point, while none of the 19 placebo participants
group (t ¼ 1.0; df ¼ 18, p ¼ 0.33). did. Table 12 shows the mean and standard devi-
ation of the percent (%) change in ABC scores
T-Test for Independent Samples A t-test for two from baseline to endpoint for the test and placebo
independent samples, used to compare the mean groups.
ABC score change from baseline to endpoint
between the test and placebo groups, found that ABC Irritability Subscale A t-test for two inde-
the mean difference of 15.1 was significant in pendent samples was performed to compare the
favor of the participants in the test group mean difference in percent change in ABC scores
(t ¼ 10.1; df ¼ 38; p < 0.0001). from baseline to endpoint between the test group
and placebo groups. The mean difference of
-52.5% was significant in favor of the test group
participants (t ¼- 9.79; df ¼ 38; p < 0.0001).
3.4 Secondary Efficacy Outcome
Analysis
CGI-I Ratings Seventeen (81%) of the 21 test
group participants received a CGI-I rating of
3.4.1 Positive Responder Rate (PRR)
1 or 2 at the study endpoint evaluation, while
The evaluation of the difference in PRR between
none of the 19 placebo participants did.
the procedure groups was performed as a
predetermined secondary efficacy outcome mea-
Positive Responder Rate Successes Table 13
sure to provide support for the primary efficacy
shows the number and percentage of test and
analysis. The PRR was defined as satisfaction
placebo group participants meeting the dual
with both of the following: (a) 25% reduction
criteria for PRR. There was a difference of 81%
from baseline to endpoint in the ABC score based
Table 12 Percentage change in ABC irritability subscale score by the procedure group
% change in ABC irritability score Testa (n ¼ 21) Placebo (n ¼ 19)
Mean SD 51.0 22.4 1.5 6.6
a
Table 13 Positive responder rate by the procedure group

Participants Testa (n ¼ 21) Placebo (n ¼ 19)
Meeting success criteria (n, %) 17 (81) 0 (0)
a
Table 14 Comparison of the proportion of positive responder rates between the procedure groups
2 2 table Positive responder rate (PRR) Negative responder rate (NRR)
Testa group 17 4
Placebo group 0 19
p < 0.00001 17 23
a
in the proportion of participants who met the PRR score as a covariate and the group as a main effect
between procedure groups: 81% of test group (Tables 16 and 17). The F values were statistically
participants met the PRR criteria compared with greater for the mean change for each of the ABC
none in the placebo group. Fisher’s exact test for scores between groups ( p < 0.0001). With
two independent proportions was conducted to removal of individual differences in baseline
compare the proportion of PRR between the scores, the two adjusted means significantly dif-
groups (Table 14). The difference in this pro- fered ( p < 0.0001), and the means were signifi-
portion was significant ( p < 0.00001). The cant for the test but not for placebo group
greater PRR from baseline to endpoint for the participants (Tables 18 and 19).
test group relative to the placebo group also was A t-test analysis for two independent samples
significant. comparing differences in the mean change in the
ABC scores from baseline to endpoint between
3.4.2 Aberrant Behavior Checklist (ABC) the two groups is provided in Table 20; the dif-
Global and Subscale Scores ference was significantly greater for the test than
The evaluation of the mean change from baseline placebo group participants.
to endpoint for each of the ABC global score and The evaluation of the mean change in each of
the remaining four ABC subscale scores (leth- the ABC global score and the five subscale sores
argy/social withdrawal, stereotypic behavior, (irritability/agitation, lethargy/social withdrawal,
hyperactivity/noncompliance, and inappropriate stereotypic behavior, hyperactivity/noncompli-
speech) between the two procedure groups was ance, and inappropriate speech) across the study
performed as a predetermined secondary efficacy duration between the procedure groups was
outcome measure to provide support for the pri- performed as a predetermined supportive measure
mary efficacy analysis outcome. Table 15 shows providing support for the primary efficacy analy-
the mean and standard deviation of baseline and sis. The ABC scores were recorded at the follow-
endpoint ABC global and subscale scores and the ing four evaluation points: baseline
changes for the test and placebo participants (pre-procedure), week 2 (interim procedure
(Aman et al. 1985a, 1985b). The ABC global administration), week 4 (study endpoint), and
and subscale scores were evaluated with week 8 (i.e., 4 weeks post-procedure). Table 21
ANCOVA, with the baseline measure for each shows these data for the test and placebo groups.
Table 15 ABC global and subscale scores from baseline to endpoint by the procedure group
Mean SD Mean SD
Global score Baseline 107.3 20.3 104.7 28.7
Endpoint 63.8 30.5 105.4 28.4
Change 43.5 19.1 0.7 2.6
Lethargy and social withdrawal Baseline 23.1 9.3 24.7 5.1
Endpoint 13.8 8.8 24.7 5.1
Change 9.3 5.8 0.1 0.2
Stereotypic behavior Baseline 13.7 4.1 12.3 5.6
Endpoint 8.2 5.1 12.3 5.6
Change 5.5 4.0 0.0 0.0
Hyperactivity and noncompliance Baseline 32.8 7.8 36.9 7.9
Endpoint 21.1 9.5 37.3 7.4
Change 11.7 7.5 0.4 1.1
Inappropriate speech Baseline 7.2 3.1 6.4 4.0
Endpoint 4.9 2.4 6.4 3.9
Change 2.3 2.3 0.0 0.3
a
Table 16 Change in ABC scores from baseline to endpoint, adjusted for baseline score
ABC global and subscales Mean scores Testa (n ¼ 21) Placebo (n ¼ 19)
Global score Observed 43.53 0.74
Adjusted 43.60 0.82
Lethargy and social withdrawal Observed 9.29 0.05
Adjusted 9.44 0.22
Stereotypic behavior Observed 5.47 0
Adjusted 5.43 0.05
Hyperactivity and noncompliance Observed 11.67 0.37
Adjusted 11.95 0.68
Inappropriate speech Observed 2.29 0
Adjusted 2.21 0.08
a
Table 17 Significance of the mean baseline to endpoint change in the ABC global and subscale scores between the test
and placebo groups
Observed difference Adjusted difference F p
Global score 44.3 44.4 99.0 <0.0001
Lethargy and social withdrawal 9.7 9.3 58.1 <0.001
Stereotypic behavior 5.5 5.4 33.8 0.000001
<0.00005
Hyperactivity and noncompliance 12.0 12.6 50.3 <0.0001
Inappropriate speech 2.3 2.1 19.3 0.000091
<0.0001
Figures 1, 2, 3, 4, 5, and 6 show the mean indicate that each ABC score in the test (active
changes in each of the ABC global and five sub- treatment group) decreased significantly from the
scale scores across the study duration by the pro- baseline level across the three evaluation points;
cedure group. The figures as well as Table 21 the decrease was progressively augmented over
Table 18 t-test analysis for the mean change in the ABC global and subscale scores for the test group participants
t df p
Global score +10.45 20 <0.0001
Lethargy and social withdrawal +7.34 20 <0.0001
Stereotypic behavior +6.34 20 <0.0001
Hyperactivity and noncompliance +7.14 20 <0.0005
Inappropriate speech +4.59 20 <0.0001
Table 19 t-test analysis for the mean change in the ABC placebo groups was performed as a
global and subscale scores for the placebo group predetermined secondary efficacy outcome mea-
participants
sure providing support for the primary efficacy
t df p analysis outcome. Table 24 shows the number of
Global score 1.25 18 0.23 participants by the CGI-S rating at baseline and
Lethargy and social withdrawal 1.00 18 0.33 endpoint for both groups of participants. All
Stereotypic behavior –0.0 18 –1.00
21 test (active treatment) participants showed a
Hyperactivity and noncompliance 1.44 18 0.17
one-category or greater improvement in CGI-S
Inappropriate speech 0.0 18 1.00
ratings from baseline to endpoint. In contradis-
tinction, the majority of placebo participants
Table 20 t-test analysis for the mean change in the ABC (17) showed no change in CGI-S rating, one
scores between the test and placebo groups demonstrated a worsening of one category, and
t df p another demonstrated an improvement of
Global score 10.01 38 <0.0001 one-category rating from baseline to endpoint. A
Lethargy and social 7.01 38 <0.0001 2 3 Fisher’s exact test for matched categorical
withdrawal data was conducted to compare the proportion of
Stereotypic behavior 6.02 38 <0.0001 participants whose CGI-S rating improved,
Hyperactivity and 6.92 38 <0.0005 showed no change, or worsened from baseline
noncompliance
to endpoint in the test and placebo groups
Inappropriate speech 4.32 38 <0.0001
(Table 25).
A difference in the proportion of participants
time, including a 4-week follow-up period during whose CGI-S rating changed one or more
which no further treatment occurred. Conversely, categories from baseline to endpoint between the
there was no such change in the placebo group. test and placebo groups was significant
One-way ANOVA and Tukey’s HSD post hoc ( p < 0.00001), with a greater proportion of test
analyses were performed to evaluate the mean group participants who had improved CGI-S
change in each ABC global and subscale scores ratings from baseline to endpoint relative to the
across the four study evaluation points in the test placebo group (Table 26). In both test and pla-
and placebo groups. Table 22 shows that the score cebo groups, all participants (100%) were rated at
improvement in each of the ABC subscales was baseline in the top three severity-of-condition
significant across almost all the comparative ratings, i.e., marked, severe, and most extreme.
evaluations in the test group. Conversely, score All placebo group participants (100%) retained
changes in the placebo participants were not sta- these top CGI-S ratings (categories 5–7 in
tistically significant (Table 23). Table 26) by week 8, while only 3 of the 21 of
active test treatment (14%) received the same top
3.4.3 Clinical Global Impressions ratings. This continuously progressive and sub-
Severity-of-Illness (CGI-S) Ratings stantial improvement for the test over placebo
The evaluation of the change in CGI-S ratings group participants was illustrated in Fig. 7 which
from baseline to endpoint between the test and indicates the percentage of participants who
Table 21 ABC global and subscale scores across the study duration by the test and placebo groups
Mean SD Mean SD
Global score Baseline 107.3 20.3 104.7 28.7
Week 2 89.3 24.1 110.0 19.4
Week 4 63.8 30.5 105.4 28.4
Week 8 48.8 25.1 105.3 31.6
Irritability Baseline 30.5 6.7 29.6 6.8
Week 2 23.4 7.4 29.9 7.5
Week 4 15.7 9.9 29.9 6.5
Week 8 11.8 10.0 29.9 6.6
Lethargy and social withdrawal Baseline 23.1 9.3 24.7 5.1
Week 2 18.2 9.3 24.7 5.1
Week 4 13.8 8.8 24.7 5.1
Week 8 10.7 9.8 24.7 5.1
Stereotypic behavior Baseline 13.7 4.1 12.3 5.6
Week 2 11.6 4.4 12.3 5.6
Week 4 8.2 5.1 12.3 5.6
Week 8 6.9 5.1 12.6 5.7
Hyperactivity and noncompliance Baseline 32.8 7.8 36.9 7.9
Week 2 30.3 8.2 36.7 7.8
Week 4 21.1 9.5 37.3 7.4
Week 8 16.0 10.5 37.3 7.4
Inappropriate speech Baseline 7.2 3.1 6.4 4.0
Week 2 5.8 2.6 6.4 4.0
Week 4 4.9 2.4 6.4 3.9
Week 8 3.5 2.2 6.5 3.9
a
received the 5–7 categories of CGI-S ranking relative to baseline, with the majority (13) receiv-
across the study duration by the procedure group. ing a rating of “much improved”. Conversely, no
placebo group participant was rated as
3.4.4 Clinical Global Impressions demonstrating symptom improvement at end-
Improvement/Change (CGI-C) point relative to baseline, with the majority
Ratings (17) rated as “no change” and the remaining
The evaluation of CGI-C ratings at study end- 2 as “minimally worse”. A proportion of
point between the procedure groups was participants in both test and placebo groups
performed as a predetermined secondary efficacy whose CGI-C rating at study endpoint, relative
outcome measure to provide support for the pri- to baseline, showed “improvement” (CGS-C
mary efficacy analysis outcome. The number of rating of 1, 2, and 3), “no change” (CGI-C rating
participants by the CGI-C rating at endpoint for of 4), or “worsening” (CGI-C rating of 5, 6, and
the test and placebo groups is shown in Table 27. 7) in symptoms, evaluated with 2 x 3 Fisher’s
Twenty out of the 21 test group participants exact test, is shown in Table 28. A difference in
showed some degree of improvement in the this proportion between the two groups was sig-
autism-related symptoms at study endpoint nificant ( p < 0.00001). There was a greater
Fig. 1 Aberrant Behavior 120

Checklist (ABC) global
score in the test (active 100
treatment) and placebo
Points
group participants 80
60
40
Baseline Week 2 Week 4 Week 8
Test Placebo

Checklist (ABC) irritability 30
subscale score in the test
Points
(active treatment) and 25
placebo group participants 20
15
10
Test Placebo

Checklist (ABC) lethargy/
social withdrawal subscale 25
Points
score in the test (active

treatment) and placebo 20
group participants
15
10
Test Placebo

Checklist (ABC)
stereotypic behavior
12
Points
subscale score in the test 10

(active treatment) and
placebo group participants 8
6
Test Placebo

Checklist (ABC) 35
hyperactivity/
Points
noncompliance subscale 30
score in the test (active
25
treatment) and placebo
group participants 20
15
Test Placebo

Checklist (ABC) 7
inappropriate speech Points
subscale score in the test 6
(active treatment) and
5
placebo group participants
4
3
Test Placebo
Table 22 One-way ANOVA and Tukey’s HSD post hoc follow-up results for the change in the Aberrant Behavior
Checklist (ABC) score across the study duration in the test (active treatment) group participants
ABC scale/subscale F p Tukey’s HSD
Global score 101.6 <0.0001 Changes between each possible evaluation
Combination was significant at p < 0.01
Irritability 114.5 <0.0001 Changes between each possible evaluation
Combination was significant at p < 0.01
Lethargy and social 49.6 <0.0001 Changes between each possible evaluation
withdrawal Combination was significant at p < 0.01, except week 4–8 which was
significant at p < 0.05
Stereotypic behavior 38.2 <0.0001 Baseline to week 2 significant at p < 0.05
All others significant at p < 0.01, except week 4–8 which was not
significant
Hyperactivity and 59.4 <0.0001 All significant at p < 0.01, except baseline to week 2 which was not
noncompliance significant
Inappropriate speech 26.9 <0.0001 All significant at p < 0.01, except week 2–4 which was not significant
Table 23 One-way ANOVA results for the change in Aberrant Behavior Checklist (ABC) score across the study
duration in the placebo group participants
ABC scale/subscale F p
Global score 0.4 0.789
Irritability 0.4 0.739
Lethargy and social withdrawal 1.0 0.399
Stereotypic behavior 1.0 0.399
Hyperactivity and noncompliance 2.4 0.081
Inappropriate speech 0.4 0.739
Table 24 Clinical Global Impressions severity-of-illness (CGI-S) ratings at baseline and endpoint by the procedure
group
CGI-S category Baseline Endpoint Baseline Endpoint
7: Most extreme of all autism patients 3 – – –
6: Severe 14 2 15 16
5: Marked 4 1 4 3
4: Moderate – 13 – –
3: Mild – 4 – –
2: Borderline – 1 – –
1: Normal – – – –
a
Table 25 Fisher’s exact test for matched categorical data to compare the proportion of participants whose Clinical
Global Impressions severity-of-illness (CGI-S) ratings improved
2 3 table Improved Unchanged Worsened n
Testa group 21 0 0 21
Placebo group 1 17 1 19
Total 22 17 1 40
a
Test group was the active treatment group; p < 0.00001
Table 26 Clinical Global Impressions severity-of-illness (CGI-S) ratings across the study duration by the procedure
groups
CGI-S Baseline Week 2 Week 4 Week 8 Baseline Week 2 Week 4 Week 8
7: Most extreme 3 2 – 1 – – – –
6: Severe 14 1 2 2 15 15 16 15
5: Marked 4 15 1 – 4 4 3 4
4: Moderate – 3 13 9 – – – –
3: Mild – – 4 6 – – – –
2: Borderline – – 1 3 – – – –
1: Normal – – – – – – – –
a
%
100
80
60
40
20
0
Test Placebo
Fig. 7 Percentage of participants receiving a 5–7 rating in the severity-of-illness subscale of the Clinical Global
Impressions Scale (CGI-S) across the study duration
Table 27 Clinical Global Impressions improvement/change subscale (CGI-C) ratings at the study endpoint by the
procedure group
CGI-C category Testa (n ¼ 21) Placebo (n ¼ 19)
1: Very much improved 4 –
2: Much improved 13 –
3: Minimally improved 3 –
4: No change 1 17
5: Minimally worse – 2
6: Much worse – –
7: Very much worse – –
a
Table 28 Fisher’s exact test comparing the proportion of participants with Clinical Global Impressions improvement/
change subscale (CGI-C) ratings of ASD symptoms at the study endpoint
2 3 table Improved Unchanged Worsened n
Test groupa 20 1 0 21
Placebo group 0 17 2 19
Total 20 18 2 40
a
proportion of participants demonstrating primary efficacy analysis outcome. CGI-C ratings

“improvement” at study endpoint in the test than were recorded across the following evaluation
placebo group, which is viably attributed to the points: after 2 weeks (interim procedure adminis-
application of LLLT. tration), 4 weeks (study endpoint), and 8 weeks
The evaluation of change in the CGI-C ratings (4 weeks post-procedure). Table 29 shows the
across the study duration by the procedure group number of participants by CGI-C rating category
was performed as a predetermined supportive for the test and placebo groups. The CGI-C
analysis to provide additional support for the ratings for the test (active treatment) participants
Table 29 Clinical Global Impressions improvement/change subscale (CGI-C) ratings across the study duration by the
procedure group
CGI-C rating Week 2 Week 4 Week 8 Week 2 Week 4 Week 8
1: Very much improved – 5 10 – – –
2: Much improved 9 12 8 – – –
3: Minimally improved 11 3 3 – – –
4: No change 1 1 – 18 17 17
5: Minimally worse – – – 1 2 2
6: Much worse – – – – – –
7: Very much worse – – – – – –
a
indicated a continuous progressive improvement LLLT is more effective than placebo in effecting
in symptoms. As early as 2 weeks into the 4-week a positive change in the ASD symptoms.
procedure, 20 out of the 21 participants received All participants of the active treatment showed
CGI-C ratings of improvement, relative to base- a one-category or greater improvement in CGI-S
line, with 11 showing “minimal improvement” from baseline to endpoint, while 17 of the placebo
and 9 “much improvement”. By week 8, the pre- participants showed no CGI-S rating change. The
sentation of autistic symptoms and behaviors was difference in the proportion of participants whose
reported as “very much improved” for almost half CGI-S rating changed one or more categories
(48%) of active treatment participants. Con- from baseline to endpoint between the two groups
versely, there was essentially no change in the was significant ( p < 0.00001). This difference is
CGI-C ratings across the study duration for the attributable to the efficacy of LLLT compared
placebo participants, indicating no observable with placebo. All 21 active treatment participants
symptomatic improvement. showed a one-category or greater improvement in
CGI-S from baseline to endpoint.
Twenty out of the 21 active treatment
participants showed some degree of improvement
4 Discussion
in autism-related symptoms at endpoint relative to
baseline. The majority (13) received ratings of
Baseline ABC irritability subscale scores
“much improved”. No placebo group participant
indicated that LLLT was significantly more effec-
demonstrated improvement in symptoms at end-
tive than placebo in treating symptoms of ASD in
point relative to baseline. The majority
children and adolescents independent of baseline
(17) demonstrated “no change”, and the
ABC scores, which accounted for 6% of the vari-
remaining 2 placebo participants rated “mini-
ance in the ABC score change from baseline to
mally worse”. We found that the ABC global
endpoint. The difference in the proportion of PPR
and five subscale scores decreased progressively
between the LLLT and placebo groups was sta-
and significantly from baseline across each of the
tistically significant ( p < 0.00001).
three successive evaluation points; the decrease
Considering the baseline score as a covariate,
progressed over time, including a 4-week follow-
F values were statistically significant
up during which no further LLLT occurred. Con-
( p < 0.0001) for the mean change from baseline
versely, the placebo group demonstrated no sig-
to endpoint between the LLLT and placebo
nificant change across the study duration,
groups and within participant groups for both
demonstrating the effectiveness of LLLT in
ABC global and subscale scores. Removing indi-
reducing ASD-associated symptoms.
vidual differences in the baseline score, the
For both test and placebo group, all
adjusted means significantly differed
participants were rated at baseline in the top
( p < 0.0001), supporting the conclusion that
three severity conditions. By week 8, all placebo 2013). Abnormal anatomical connectivity and
participants retained a 5–7 CGI-S score, while performance of hub regions have been
only 3 of the 21 active treatment (14%) received hypothesized to be associated with cognitive and
this rating, which speaks for a progressive behavioral dysfunction in numerous neurological
improvement in the symptom presentation across and psychiatric disorders (Bullmore and Sporns
the study time. As early as 2 weeks into the 2012; Seeley et al. 2009). For example, analyses
4-week active treatment, 20 out of the 21 test of structural and functional connectivity in
group participants received improved CGI-C schizophrenia have shown reduced frontal hub
ratings, with 11 participants showing “minimal” connectivity (Fornito et al. 2012).
and 9 demonstrating “much improvement”. By We have recently noted that altered functional
week 8, symptom presentation of ASD was connectivity, i.e., synchronous brain activity,
reported as being “very much improved” for might be associated with the deficits characteris-
almost half (48%) of the test group participants, tically found in ASD (Machado et al. 2015). Of
whereas no significant change in the CGI-C rating specific importance is the integrity of functional
was noted for the placebo group participants. connectivity in the default mode network (DMN),
There is a well-established literature on a network active during inactive resting states,
photobiomodulation, supporting improvement in and in cognitive functions linked to the
dysfunctional neuronal activity with the use of ASD-related social dysfunction. Assaf et al.
low intensity red and near-infrared (NIR) light (2010) have found a decreased functional connec-
(Hiwaki and Miyaguchi 2018; Naeser et al. tivity between the precuneus/medial/prefrontal/
2014). Ten-year experience in animal studies anterior/cingulate cortices and default mode net-
has indicated that transcranial photobiomo- work core areas, with the degree of functional
dulation has a positive effect in animal models connectivity in these regions inversely correlated
of traumatic brain injury (Wu et al. 2012a; Oron with the ASD communication and social deficits.
et al. 2007), Alzheimer’s (Wu et al. 2012b), These results support the hypothesis that default
depression (Purushothuman et al. 2015), and mode networks’ under-connectivity contributes
stroke (Hiwaki and Miyaguchi 2018; Lapchak to the core deficits seen in ASD.
and deTaboada 2010; Oron et al. 2006), while Of particular interest is the association of
human studies have included traumatic brain default mode networks with ASD (Buckner
injury (Naeser et al. 2011), depression (Schiffer et al. 2008). Numerous studies have found vari-
et al. 2009), and stroke (Zivin et al. 2009). Fur- ous “hubs” with the effectiveness of the entire
ther, low-level light energy has been found safe network highly dependent on the hub status. The
for humans in the stroke studies, providing a high most common ASD-related hubs are those
benefit-to-risk ratio, with no reported side effects reported by Raichle et al. (2001) and Greicius
of LLLT. et al. (2003) being associated with the ventrome-
Almost all aspects of cognitive function dial and dorsomedial prefrontal, posterior cingu-
require integration of widely distributed neural late, precuneus, lateral parietal, and entorhinal
activity. Network analysis of human brain con- cortices (Raichle 2015). Assaf et al. (2010) have
nectivity has reliably classified sets of neocortical examined three default mode sub-networks
areas essential for supporting optimized neuronal obtained from the resting fMRI scans of
signaling and communication. Optimal “brain 16 ASD individuals and 16 matched controls,
hubs” exist in the networks of effective connec- using independent component analysis. The
tivity, supporting the notion of the hub’s function ASD individuals demonstrate a reduced func-
in a wide range of cognitive tasks and a dynamic tional connectivity between the medial-prefron-
coupling in and across effective functional tal/anterior-cingulate/precuneus cortices and
networks. A high level of brain hub centrality other default mode sub-networks areas. The
renders these networks vulnerable to dysfunction degree of functional connectivity in these regions
and disconnection (van den Heuvel and Sporns
is inversely associated with the severity of social employed in this study. The authors would like to thank
and communication deficits. Ms. Elvira Walls for her assistance with the statistical
analysis and research methodology.
LLLT promotes cell and neuronal repair
(Dawood and Salman 2013) and brain network
Conflicts of Interest None of the authors have any fidu-
rearrangement (Erlicher et al. 2002) in many neu- ciary interests in the research reported herein or with the
rologic disorders identified with lesions in the source of funding.
hubs of default mode networks (Buckner et al.
2008). LLLT facilitates a fast-track wound-
healing (Dawood and Salman 2013) as
mitochondria respond to light in the red and References
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Published online: 4 July 2018
Epidemiology of Granulomatosis
with Polyangiitis in Poland, 2011–2015
Krzysztof Kanecki, Aneta Nitsch-Osuch, Paweł Gorynski,

Patryk Tarka, Magdalena Bogdan, and Piotr Tyszko
Abstract rate for GPA was 94%. In conclusion, the

Granulomatosis with polyangiitis (GPA) is a incidence and prevalence rates of GPA in
form of vasculitis that affects small- and Poland are similar to that reported in other
medium-sized vessels in many organs. The European countries. The study provides recent
aim of the study was to describe the epidemi- epidemiological data on GPA in Poland,
ology of GPA in Poland in 2011–2015, includ- which may be useful for comparisons with
ing the incidence and prevalence rates. The other geographical regions.
authors conducted a retrospective,
population-based study, using hospital dis- Keywords
charge records with GPA diagnosis. GPA inci- Autoimmune disease · Disease prevalence ·
dence was estimated on the basis of the data Epidemiological data · Granulomatosis with
from the Polish hospital morbidity study car- polyangiitis · Systemic vasculitis
ried out by the National Institute of Public
Health. The final study group consisted of
1491 patients (749 females, 742 males) who 1 Introduction
were first time hospitalized with the diagnosis
of GPA. The average annual incidence of GPA Granulomatosis with polyangiitis (GPA), previ-
in Poland was 7.7/1,000,000 (95% CI, ously known as Wegener’s granulomatosis, is a
4.1–11.4), and the point prevalence at the end systemic necrotizing vasculitis which affects
of 2015 was 36/1,000,000. A statistically small- to medium-sized vessels and is related to
significant decrease in the GPA incidence anti-neutrophil cytoplasmic antibodies.
was noticed in this study. A 1-year survival Clinically, it can involve multiple systems,
commonly pulmonary system in 78%, upper
respiratory tract in 86%, and kidneys in 61% of
K. Kanecki (*), A. Nitsch-Osuch, P. Tarka,
cases (Langford 2012). Elucidation of the
and M. Bogdan
Department of Social Medicine and Public Health, mechanisms underlying this type of vasculitis,
Medical University of Warsaw, Warsaw, Poland frequently accompanying lung diseases, is com-
e-mail: [email protected] plicated because of its rarity, which hampers the
P. Gorynski undertaking of large-scale studies, difficulties in
National Institute of Public Health–National Institute of the classification, and multifaceted clinical
Hygiene, Warsaw, Poland
presentations, which suggest the existence of sev-
P. Tyszko eral etiologic pathways (Gibelin et al. 2011).
Institute of Rural Health in Lublin, Lublin, Poland
131
132 K. Kanecki et al.
GPA can occur at all ages, and it has been respectively. The incidence was stable throughout
reported from infancy to old age. the study period. There was an increase in the
Results from the population-based surveys annual prevalence from 28.8 per million in 1990
support the existence of differential international to 64.8 per million in 2005 (Watts et al. 2009).
variations in the occurrence of GPA. It has Another British study has reported 462 GPA
become a well-accepted notion that the geo- cases diagnosed in 1997–2013 (Pearce et al.
graphic distribution of GPA decreases with the 2017). In that study, the overall estimate of GPA
decreasing latitude, both north and south of the incidence was 11.8 (95% CI, 10.7–12.9) per mil-
equator. In the Nordic countries, the incidence or lion person-years. The incidence was 0.88 (95%
prevalence of GPA is roughly two- to sevenfold CI, 0.40–1.96) in children (aged <16 years) and
higher than that in the southern Europe. A similar 14.0 (95% CI, 12.8–15.4) in adults. The preva-
discovery was made in the southern hemisphere, lence in 2013 was 134.9 (95% CI, 121.3–149.6)
with a threefold higher incidence of GPA, con- per million population.
trolled for age, sex, and ethnicity, in the southern- In a study from northern Germany in
most part of New Zealand compared with the 1998–2005, GPA incidence ranged between
northernmost part (O’Donnell et al. 2007). Ethnic 6 and 12 new cases per million per year (median
origin is also thought to play a part in the devel- 8.5) (Herlyn et al. 2008). In another German
opment of GPA. In a multiethnic population of study in 1994–2006, the prevalence of GPA rose
France, GPA is twice as prevalent among popula- from 58 to 98 per million population (Herlyn
tion of the European versus non-European et al. 2014). In a Swedish study conducted until
descent; and non-European cases of GPA are 30 June 2008, using hospital records and serology
rather rare (Mahr et al. 2004). Relatives of database, the annual incidence for GPA was
patients with GPA are at increased risk of being estimated at 9.8 (95% CI, 7.4–12.2) per million
diagnosed with other autoimmune and inflamma- (Mohammad et al. 2009). In a retrospective Nor-
tory diseases, indicating a shared susceptibility to wegian cohort study, using hospital discharge
those diseases (Knight et al. 2010a). records, the annual incidence increased from 5.2
HLA-DPB1*0401 is a GPA susceptibility allele, (95% CI, 2.7–9.0) during 1984–1988 to 12.0
and the population frequency of the allele may (95% CI, 8.0–17.3) during 1994–1998 per mil-
help explain variations in the GPA incidence lion. The point prevalence increased from 30.4
described in the literature (Watts et al. 2015). (95% CI, 16.6–51.0) to 95.1 (95% CI,
69.1–129.0) per million population (Koldingsnes
and Nossent 2000). A Finish study has shown an
1.1 Incidence and Prevalence annual increase in the incidence from 1.9 (95%
of Granulomatosis CI, 1.4–2.6) during 1981–1985 to 9.3 (95% CI,
with Polyangiitis (GPA) 8.1–10.6) during 1996–2000 per million (Takala
et al. 2008). In the region of Lugo of northwest
GPA is a rare disease. Therefore, collection of a Spain, a study based on the data from 1988–2001,
large population of patients to determine the inci- coming from a single reference hospital and
dence and prevalence takes time, which makes regarding all patients aged 15 or older, has
studies rather scarce. In a study conducted in reported the annual GPA incidence of 2.95
1990–2005, using of the General Practice (95% CI, 1.44–6.05) per million population
Research Database in the UK, 295 patients with (Gonzalez-Gay et al. 2003). In the Costa del Sol
newly diagnosed GPA have been identified. region of southern Spain in 2010, the annual
The overall annual incidence of GPA was 8.4 incidence and prevalence were 2.1 and 15.8 per
(95% confidence interval (95% CI), 7.5–9.4) million, respectively (Romero-Gómez et al.
per million. In that study, the annual rate was 2015). In northern Italy, the mean annual GPA
8.1 (95% CI, 6.8–9.6) per million and 8.8 (95% incidence increased from 1.7 during 1995–1999
CI, 7.4–10.3) per million in women and men, to 3.4 per million per year during 2005–2009.
Epidemiology of Granulomatosis with Polyangiitis in Poland, 2011–2015 133
The point prevalence increased from 17.8 (95% was determined on the basis of the International
CI, 7.7–35.1) in 1999 to 34.3 (95% CI, Classification of Diseases (M31.3) for the dis-
20.3–54.2) per million in 2009 (Catanoso et al. charge diagnosis using the Integrated South
2014). In a French urban multiethnic population, Australian Activity Collection (Hissaria et al.
GPA prevalence was estimated at 23.7 (95% CI, 2008). A different study also shows that discharge
16–31) per million adults (Mahr et al. 2004). In records might be a useful element in the epidemi-
Lithuania, the annual GPA incidence is reported ological evaluation of eosinophilic
at 2.1 (95% CI, 1.1–4.1) (Dadoniene et al. 2005), granulomatosis with polyangiitis, a necrotizing
whereas in Turkey at 4.8 per million population vasculitis (Kanecki et al. 2017).
(Pamuk et al. 2016). Between 2000 and 2004,
GPA prevalence in the Australian Capital Terri-
tory and the surrounding rural region was 2 Methods
148.0 (95% CI, 125.1–173.9) per million, and
the disease-specific incidence was 8.4 per million This study was approved by the institutional
per year (Ormerod and Cook 2008). A 5-year inci- board for clinical studies. It is a retrospective,
dence of GPA in South Australia was 56.0 (95% population-based study based on a review of hos-
CI, 44.1–68.4) per million over the period pital discharge files reporting the diagnosis of
2001–2005 (Hissaria et al. 2008). A 5-year preva- GPA in Poland in 2011–2015. The incidence of
lence of GPA varies in New Zealand between GPA was estimated on the basis of data obtained
29–75 cases per million population (de Zoysa from the Polish hospital morbidity study carried
2013). Using the National Inpatient Sample, the out by the National Institute of Public Health.
largest all-payer inpatient database in the USA, Data on all inpatients, discharged and those who
the annual hospitalization rate for patients with passed away, excluding psychiatric and military
the principal diagnosis of GPA increased by 24%, hospitals, were sent to the National Institute of
from 5.1 to 6.3 per 1 million US population, in the Public Health, on a monthly basis. The
period 1993–2011 (p < 0.0001 for trend) (Wallace anonymized data included the information on
et al. 2017). The annual incidence of GPA in hospitalization with ICD10-coded diagnoses,
Japan is 2.1 per million adult population (95% date of admittance and discharge, birth date, gen-
CI, 0.6–3.7 per million) (Fujimoto et al. 2011). der, and the place of residence. We evaluated the
Another study performed in a southern hemisphere patients with the first-time hospitalization for
region shows a 5-year GPA prevalence of GPA. The incidence rate was calculated using
152 (95% CI, 117–186) per million population the number of GPA patients and the
(Gibson et al. 2006). corresponding census data. In addition, demo-
In Poland, the average annual GPA incidence graphic data for the general Polish population
has been estimated at 4.9 per million in the gen- were obtained from the Central Statistical Office
eral population, 5.8 per million in the adult popu- in Poland (CSO 2016).
lation, and 1 per million in the population below
18 years of age (Kanecki et al. 2014). In view of
the paucity of epidemiological data, the present 2.1 Statistical Analysis
study seeks to define a current burden of GPA in
Poland. The updated information would also help Categorical data were expressed as percentages.
compare recent trends in GPA morbidity in Assuming the Poisson distribution of the cases
Poland with those in other geographical regions. analyzed, 95% confidence intervals (CI) were cal-
There is a growing interest in the development culated. Nonparametric tests were used when the
of surveillance ways for GPA, using the existing normality assumptions were unmet. A p-value of
resources such as hospital morbidity databases. In <0.05 defined statistically significant differences.
a New Zealand study, a 5-year incidence of GPA The incidence rate was calculated using the num-
in South Australia over the period 2001–2005 ber of disease cases divided by the population
figure. Linear regression was used for trend anal- The age distribution of patients at their first
ysis. All analyses were performed using Statistica hospitalization with GP diagnosis is presented in
software v2013 and WINPEPI software Fig. 3. The median and mean age of all patients
(Abramson 2011). were 55 and 52 years, respectively. The average
annual incidence of GPA in Poland was
estimated at 7.7 per million population (95%
CI, 4.1–11.4), and the point prevalence was
3 Results
38.4 per million at the end of 2015. GPA inci-
dence decreased significantly over time in
There were 6995 cases of hospitalization with
Poland between 2011 and 2015 (12.9 vs. 6.7
GPA diagnosis in 2011–2015, out of which 1491
per million, respectively; p < 0.001). Of note,
cases represented patients with the first-time GPA
there was a significant seasonal decreasing trend
diagnosis. The majority consisted of adult patients,
in the first-time GPA incidence from January to
1455 (97.5%; 18 years of age). The whole
December, presented in Fig. 4 (p < 0.01). There
patient group comprised 742 (49.8%) males and
were no significant differences in age according
749 (50.2%) females. The number of GPA patients
to gender at the time of GPA diagnosis. In all
by gender per year is presented in Fig. 1. The
6,995 hospitalized cases of GPA, there were
number of GPA patients in rural and urban regions
108 deaths during the study period (54 females
per year is presented in Fig. 2. There were signifi-
and 54 males; mean age 60 years, range
cant differences in the incidence rate between peo-
19–86 years). Fifty-one out of the 108 deaths
ple in urban (995 patients) and rural areas
were observed in patients hospitalized with
(496 patients); 67% vs. 33%, p < 0.001.
newly diagnosed GPA (27 females and
Fig. 1 Incidence of 300 Female

granulomatosis with
Number of Patients
250 Male
polyangiitis (GPA) by
gender in 2011–2015 200
150
100
50
0
2011 2012 2013 2014 2015
Calendar Year
Fig. 2 Incidence of 350 Urban

Number of Patients
granulomatosis with 300 Rural

polyangiitis (GPA) by place 250
of living in 2011–2015
200
150
100
50
0
2011 2012 2013 2014 2015
Calendar Year
Fig. 3 Age distribution of patients diagnosed with granulomatosis with polyangiitis (GPA) in 2011–2015
Fig. 4 Seasonal monthly incidence of granulomatosis with polyangiitis (GPA) in 2011–2015
24 males). During the study period, A 1-year Mohammad et al. 2009; Watts et al. 2009; Herlyn
survival rate for GPA was 94%. et al. 2008; Takala et al. 2008; Dadoniene et al.
2005; Gonzalez-Gay et al. 2003; Koldingsnes and
Nossent 2000). Moreover, the average annual
4 Discussion incidence in 2011–2015 was higher than that
reported in a previous epidemiological study
On the basis of the inpatient discharge files, we from Poland (Kanecki et al. 2014). The GPA
estimated that the average annual incidence of incidence was also higher than that previously
GPA in Poland was 7.7 per million population. reported in a recent British study (Pearce et al.
This finding is comparable with data from other 2017). The GPA prevalence in northern Germany
European countries (Pearce et al. 2017; Romero- rose from 58 to 98 per million population in
Gómez et al. 2015; Catanoso et al. 2014; 1994–2006 (Herlyn et al. 2014). However in
another study, it amounted to 6–12 new cases per distributed across age groups. Population-based
million per year in the period of 1998–2005 studies indicate that GPA may occur in a wide
(Herlyn et al. 2008). A high GPA incidence in range of age (Lane et al. 2003; Koldingsnes and
2011 in Poland and a significant drop in succes- Nossent 2000). A wide range of age at the first-
sive years may be related to environmental, time GPA hospitalization was also noticed in the
infectious, or other unknown factors. present study, the median and mean age of 55 and
Additionally, we noticed a significantly 52 years, respectively. The wide age ranges may
decreasing trend in GPA incidence month-to- imply that stochastic events are at work in the
month, with the highest rate of newly diagnosed disease pathogenesis (Gibelin et al. 2011). In the
GPA in January. It is a long-held belief that the UK, median age of patients with newly diagnosed
etiology of systemic vasculitides could be infec- GPA was 59 years (interquartile range
tious. For vasculitides with a frequent pulmonary 47–70 years), with a mean of 52 years (Watts
involvement, this hypothesis is all the more rele- et al. 2009). In other studies, mean age at the
vant because lung diseases are notably initiated diagnosis was 64 years (Herlyn et al. 2014) and
by airborne infections. The most compelling 55–65 years (Herlyn et al. 2008). In Finland,
observation to support the infectious cause mean age at the diagnosis was 53 years (Takala
comes from the circumstantial evidence showing et al. 2008), and in Norway it was 50 (range
that the onset of these diseases is preceded by 10–84 years). The highest incidence rate occurred
infectious events and from the studies showing in Norwegian men aged 65–74 (Koldingsnes and
that GPA first becomes evident during the fall and Nossent 2000).
winter months, the seasons with frequent In the present study, male to female ratio was
respiratory infection epidemics. However, there 0.99. Likewise, 51–52% of GPA patients in the
is a controversy regarding the concept of season- British studies were male (Wallace et al. 2016;
ality concerning the onset of disease, with other Watts et al. 2009). Yet another British study has
studies suggesting either no GPA seasonality also reported that GPA incidence is lower in
(Mahr et al. 2006a) or its increased occurrence females (adjusted incidence rate ratio ¼ 0.68;
in the summer (Mahr et al. 2006b). 95% CI, 0.56–0.81), with the greatest incidence
We found that there were significantly more in those aged 55–69 years (adjusted incidence
patients who lived in urban rather than rural rate ratio ¼ 6.8, 95% CI, 4.9–9.6; reference
areas. Likewise, a greater GPA prevalence was group 16–39 years of age) (Pearce et al. 2017).
observed in urban areas in northern Germany In Norway, 62% of GPA patients are male
(Herlyn et al. 2014). An Australian study has (Koldingsnes and Nossent 2000), whereas only
also reported a trend toward a higher incidence 49% of male patients have been reported in
of GPA in rural areas (Ormerod and Cook 2008). Finland (Takala et al. 2008).
In a multicultural population of New Zealand, During the present study period, 108 deaths in
GPA predominates in Europeans (de Zoysa all GPA patients were observed, with a 1-year
2013). A long-standing exposure to silica may survival rate for GPA was 94%. All the deaths
be associated with GPA onset (Hogan et al. were observed in adults. Among the dead, there
2007). Other positive relationships were were 51 patients with newly diagnosed GPA. In a
suggested for GPA and farming, particularly Swedish study, 5-year survival rate for GPA was
due to a contact with livestock and organic 83% (Mohammad et al. 2009). In the UK, mortal-
solvents (Lane et al. 2003). The role of occupa- ity was reported at 13.6% for the 1-year rate
tional exposures as determinants of GPA risk is (Pearce et al. 2017). In New Zealand, 1- and
at variance with a recent study that demonstrates 10-year probability of survival in GPA patients
no such relationship between (Knight et al. were 91% and 62%, respectively (Khan et al.
2010b). 2012). A meta-analysis of GPA cases indicates a
GPA occurs primarily during adulthood, but similar mortality risk, with a standardized mortal-
its age-specific incidence rates are not uniformly ity ratio of 2.63 (95% CI 2.02–3.43) (Tan et al.
2017). A population-based British study suggests prevalence at 36 per million at the end of 2015.
that the survival rate of GPA patients has The incidence rate of GPA in Poland is similar to
improved considerably over the past two decades, the data reported by other European countries. A
affirming the benefits of recent trends in the man- higher incidence of GPA was reported in urban
agement of GPA and its complications (Wallace than rural regions of Poland. During the study
et al. 2016). Using the National Inpatient Sample, period, a 1-year survival rate for GPA was 94%.
the largest all-payer inpatient database in the Trends in GPA incidence observed in this study
USA, it has been reported that inhospital deaths may be related to the environmental, infectious,
in patients with the principal diagnosis of GPA or other unknown factors.
declined by 73%, from 9.1% to 2.5% (p < 0.0001
for trend) from 1993 to 2011 (Wallace et al. Conflicts of Interest Authors declare no conflict of inter-
2017). Patients with GPA have a ninefold est in relation to this article.
increased risk of death in the first year of the
disease, attributed to infection, active vasculitis,
and renal failure. The risk is at its lowest between References
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Advances in Experimental Medicine and Biology 1116

Clinical and Experimental Biomedicine

Mieczyslaw Pokorski Editor

ISSN 0065-2598 ISSN 2214-8019 (electronic)

Library of Congress Control Number: 2018964103

# Springer Nature Switzerland AG 2018

Bioprogressive Paradigm in Physiotherapeutic and Antiaging

Osteoprotegerin, Receptor Activator of Nuclear Factor

Mieczyslaw Pokorski, Giovanni Barassi, Rosa G. Bellomo,

Abstract the proprio-neuromuscular facilitation, a

PROPRIOCEPTIVE RECEPTORS SENSORIAL NEURAL STIMULATION

ISOMETRIC PNF ISOTONIC

Influence of Proprioceptive Neuromuscular

Małgorzata Bujar-Misztal and Andrzej Chciałowski

Abstract surgery of restrictive character, accompanied

Table 1 Cardiological rehabilitation

Table 2 Pulmonary function tests

3.2 Plethysmography changed to 130.3 23.4% and 134.2 21.3%

both off-pump and on-pump, have shown the References

Remote Ischemic Preconditioning

Małgorzata Wojciechowska, Maciej Zarębiński,

Abstract criteria of the serum creatinine (SC) and

Contrast-induced nephropathy is deﬁned as an 2.1 Study Population

iodinated, water-soluble, radiographic contrast 3 Results

2.4 Outcomes The assessment of contrast-induced nephropathy

The assessment of contrast-induced nephropathy

Table 1 Patients’ demographic, clinical, basic biochemical, and angiographic indices

Prognostic Impact of Extracapsular Lymph

Seyed Vahid Tabatabaei, Christoph Nitche, Maximilian Michel,

Abstract with publication date since 1990. Prospective

Keywords better prognosis than patients with N2 status

2.2 Data Sources and Literature

Two investigators independently conducted a We identiﬁed 2105 non-duplicated articles, out of

Lee et al. Taiwan 8 62 (32–-82) 58 42 NR NR NR 67 33 86 14 51 49 57 43 75 25 NR 100

Liu et al. China 8 59 (27–79) 21 79 0 24 76 22 78 NR 15 85 21 79 0 28 72 100

Vansteenkiste Belgium 7 61 (40–76) 93 9 14 35 21 61 NR NR 80 87

Moretti et al. USA NR 61 (35–85) 58 NR 100 NR NR 38 NR 100 NR 85

1997; Duran-Cantolla et al. 1994). Only Liu 4 Discussion

Fig. 2 Publication bias

meta-analysis are statistically robust, it is impor-

Influence of Transurethral Resection

Wojciech Krajewski, Urszula Halska, Sławomir Poletajew,

Abstract TURB was prospectively evaluated. The Hos-

Keywords outcomes of operation. Therefore, in the present

operation. Patients taking medications that could 2.2 Survey Questionnaires

We also constructed dendrograms of taxonomic

Linear Regression Linear regression was

Table 1 Patients’ characteristics and surgical data

interrelationships. We collected a variety of data

Clinical Clinical Clinical

Table 4 Logistic regression elaboration

level before unknown circumstances of the ﬁrst- 4.2 Sexual Satisfaction

or administration of cytostatic agents. A number sexuality, still, true inﬂuence of TURB is

Cognitive Predictors of Cortical Thickness

Patrycja Naumczyk, Angelika K. Sawicka, Beata Brzeska,

Abstract were examined on 3.0 T MRI scanners. The

P. Naumczyk (*) and K. Jodzio

Table 1 Demographic and neuropsychological characteristics of the study group

calculated normative score, time for part 1 of the 3 Results