(uv) light on tear film and pollen ingredients
TRANSCRIPT
DISSERTATION
The effect of ultraviolet (UV) light on tear film and pollen
ingredients – an approach for better understanding allergic
and non-allergic reactions on the ocular surface.
submitted by
Andrea HEIDINGER, BSc MSc
for the Academic Degree of
Doctor of Medical Science (Dr. scient. med.)
at the
Medical University of Graz
Department of Ophthalmology
under the supervision of
a.o. Univ.-Prof. Mag. Dr. Otto SCHMUT
Dr. Dieter RABENSTEINER
PD DDr. Jasmin RABENSTEINER
2017
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Declaration
I hereby declare that this thesis is my own original work and that I have fully
acknowledged by name all of those individuals and organisations that have
contributed to the research for this thesis. Due acknowledgement has been made
in the text to all other material used.
Throughout this thesis and in all related publications I followed the “Standards of
Good Scientific Practice and Ombuds Committee at the Medical University of
Graz“.
October 16th, 2017
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Disclosures
Part of this thesis has been published in: Heidinger A, Rabensteiner DF,
Rabensteiner J, Kieslinger P, Horwath-Winter J, Stabentheiner E, Riedl R,
Wedrich A, Schmut O. Decreased viability and proliferation of Chang conjunctival
epithelial cells after contact with ultraviolet light-irradiated pollen. Cutaneous and
Ocular Toxicology. DOI: 10.1080/15569527.2017.1414226.
Co-Authors:
Dr. Dieter Franz Rabensteiner
PD Dr. Jutta Horwath-Winter
Univ.-Prof. Dr. Andreas Wedrich
Univ.-Prof. Mag. Dr. Otto Schmut
Department of Ophthalmology, Medical University of Graz, Auenbruggerplatz 4,
8036 Graz, Austria
PD DDr. Jasmin Rabensteiner
Petra Kieslinger, MSc
Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical
University of Graz, Auenbruggerplatz 15, 8036 Graz, Austria
Ass.Prof. Dr.phil. Edith Stabentheiner
Institute of Plant Sciences, University of Graz, Schubertstrasse 51, 8010 Graz,
Austria
Dipl.Ing. Dr. Regina Riedl
Institute for Medical Informatics, Statistics and Documentation, Medical University
of Graz, Auenbruggerplatz 2, 8036 Graz, Austria
Doctoral student Andrea Heidinger received funding from the
Medical University of Graz through the Doctoral School Sustainable Health.
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Acknowledgements
I would like to thank my supervisors a.o. Univ.-Prof. Mag. Dr. Otto Schmut, Dr.
Dieter Rabensteiner and PD DDr. Jasmin Rabensteiner for their continuous
support from planning the thesis, during conducting the experiments until
finalisation of the thesis.
Special thanks to Gabriele Trummer, Sieglinde Kirchengast, Manuela Fischl and
Christine Wachswender for their great technical assistance.
A great thanks to my family who has always inspired and supported me.
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Table of Contents
Declaration ............................................................................................................ 1
Disclosures ........................................................................................................... 2
Acknowledgements .............................................................................................. 3
1. Abbreviations and Definitions ..................................................................... 8
2. Figures ......................................................................................................... 10
3. List of Tables ............................................................................................... 13
4. Abstract in German ..................................................................................... 14
5. Abstract in English...................................................................................... 15
6. Introduction ................................................................................................. 16
6.1. Allergy - Definition .................................................................................. 16
6.2. Ocular allergies ...................................................................................... 18
6.2.1 Hay fever ..................................................................................................18
6.2.2 Therapy ....................................................................................................19
6.3. The ocular surface ................................................................................. 19
6.3.1 Tear film ingredients .................................................................................20
6.4. Non-allergic reactions ............................................................................ 20
6.5. Pollen and their ingredients .................................................................... 21
6.6. Pollen allergens ..................................................................................... 22
6.7. Further pollen ingredients ...................................................................... 23
6.8. ROS (reactive oxygen species) .............................................................. 23
6.9. Inflammatory cytokines .......................................................................... 24
6.10. Environmental pollutants .................................................................... 25
6.11. Classification of air pollutants ............................................................. 26
6.11.1 Particulate matter ......................................................................................26
6.11.2 Nitrogen dioxide ........................................................................................27
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6.11.3 Ozone .......................................................................................................27
6.12. Effects of air pollutants on human health ............................................ 28
6.13. Interaction between allergens and pollutants ..................................... 29
6.14. Global warming................................................................................... 30
6.15. Ultraviolet light .................................................................................... 30
6.16. Aim of the study .................................................................................. 34
7. Material and Methods .................................................................................. 35
7.1. Materials ................................................................................................ 35
7.2. Methods ................................................................................................. 39
7.2.1 Irradiation .................................................................................................39
7.2.2 Steaming with ozone ................................................................................39
7.2.3 Determination of histamine in histidine solutions .......................................40
7.2.3.1 Histamine ELISA ...................................................................................40
7.2.4 Determination of histamine and cytokines in human tears before and after irradiation .................................................................................................................41
7.2.5 Pilot study: The impact of ultraviolet light and ozone on tear film components. ............................................................................................................43
7.2.5.1 Inclusion and exclusion criteria .............................................................43
7.2.5.2 Study participant recruitment ................................................................44
7.2.5.3 Tear collection ......................................................................................44
7.2.5.4 Blood sampling .....................................................................................44
7.2.5.5 Cytological examination ........................................................................45
7.2.5.6 Ophthalmological examination ..............................................................45
7.2.5.7 Analysis ................................................................................................46
7.2.5.8 LC-MS/MS analysis ..............................................................................46
7.2.6 Determination of histamine in pollen before and after irradiation ...............48
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7.2.6.1 Collection of pollen................................................................................48
7.2.6.2 Irradiation of pollen ...............................................................................49
7.2.6.3 Determination of histamine content .......................................................50
7.2.6.4 Polyacrylamide gel electrophoresis .......................................................50
7.2.6.5 Pollen morphology ................................................................................51
7.2.6.6 GRAM and PAS staining .......................................................................51
7.2.7 Cell culture................................................................................................52
7.2.7.1 Determination of cell viability .................................................................53
7.2.7.2 Determination of cell proliferation ..........................................................54
7.2.7.3 Statistical analysis.................................................................................54
8. Results ......................................................................................................... 56
8.1. Irradiance measurements ...................................................................... 56
8.1.1 Irradiance measurements with UV-A and UV-B lamp ................................56
8.1.2 Irradiance measurements of natural sunlight ............................................57
8.2. Determination of histamine in histidine solutions before and after irradiation .......................................................................................................... 58
8.3. Determination of histamine in human tears ............................................ 62
8.4. Determination of cytokines in human tears ............................................ 63
8.4.1 ProcartaPlex kit ........................................................................................63
8.5. Pilot study .............................................................................................. 65
8.5.1 Histamine analysis ....................................................................................67
8.6. Histamine in alder and hazel pollen ....................................................... 69
8.7. Polyacrylamide gel electrophoresis ........................................................ 71
8.8. Pollen morphology ................................................................................. 73
8.9. Pollen, bacteria and fungi ....................................................................... 77
8.10. Cell culture ......................................................................................... 78
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8.10.1 Alder .........................................................................................................78
8.10.2 Hazel ........................................................................................................79
8.11. Cell Imaging ....................................................................................... 80
8.12. xCELLigence analysis ........................................................................ 82
9. Discussion ................................................................................................... 86
9.1. UV light measurements .......................................................................... 86
9.2. UV light induced histamine formation ..................................................... 86
9.3. Histamine content in human tears before and after irradiation ............... 88
9.4. Cytokines in tears................................................................................... 90
9.5. Ozone-induced histamine formation....................................................... 92
9.6. Pilot study .............................................................................................. 92
9.6.1 Ophthalmological examinations ................................................................92
9.7. Histamine content of pollen before and after irradiation ......................... 93
9.8. Protein content ....................................................................................... 94
9.9. Pollen morphology ................................................................................. 94
9.10. Pollen, bacteria and fungi ................................................................... 95
9.11. Cell Culture ......................................................................................... 96
10. Conclusion ............................................................................................... 99
10.1. Answers of the main study questions ................................................. 99
11. References ............................................................................................. 101
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1. Abbreviations and Definitions
ACN Acetonitrile
Aqua dest. Aqua destillata
ATD Aqueous tear deficient dry eye
BCC Basal cell carcinoma
CO Carbon monoxide
CO2 Carbon dioxide
DMEM Dulbecco´s Modified Eagle Medium
DNA Deoxyribonucleic acid
DPBS Dulbecco´s phosphate buffered saline
EDE Evaporative dry eye
ELISA Enzyme-linked immunosorbent assay
ESI-Q-TOF Electrospray ionization – quadrupol - time of flight
FEIA Fluorescent enzyme immunoassay
ICNIRP International Commission for Non-Ionizing Radiation Protection
IgE Immunoglobulin E
IgG Immunoglobulin G
IL Interleukin
MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-
sulfophenyl)-2H-tetrazolium)
NaCl Sodium chloride (physiological saline)
NADPH Nicotinamide adenine dinucleotide phosphate
NMSC Non-melanoma skin cancer
NOAA National Oceanic and Atmospheric Administration
NO2 Nitrogen dioxide
NOx Nitrous gases
O Oxygen
O2 Di-oxygen
O3 Ozone
OD Oculus dexter (right eye)
OS Oculus sinister (left eye)
PAS Periodic acid–Schiff staining
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PM Particulate matter
PS Penicilline / Streptomycine
RAST Radio-allergo-sorbent-test
ROS Reactive oxygen species
SAC Seasonal allergic conjunctivitis
SCC Squamous cell carcinoma
SD Standard deviation
SEM Standard error of the mean
SNAC Seasonal non-allergic conjunctivitis
SNAR Seasonal non-allergic rhinitis
SO2 Sulfur dioxide
SO3 Sulfur trioxide
TNF Tumor necrosis factor
TGF Transforming growth factor
UNEP United Nations Environment Programme
UV Ultraviolet light
UV-A Ultraviolet light, type A
UV-B Ultraviolet light, type B
UV-C Ultraviolet light, type C
UVI Ultraviolet light index
WHO World Health Organization
WMO World Meteorological Organization
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2. Figures
Figure 1. Possible formation of histamine in human tear fluid.
Figure 2. Optometer for irradiance measurements.
Figure 3. Ozone generator.
Figure 4. Ophthalmological examinations: (A) papillae on the upper eyelid; (B) Schirmer´s test; (C) corneal staining; (D) lissamine green staining.
Figure 5: UltiMate 3000 HPLC system (left) and TSQ Quantum Ultra (right) from Thermo Fisher Scientific, USA.
Figure 6. Male inflorescences of hazel pollen in the flowering period in March 2017.
Figure 7. UV-A lamp; white numbers on the lamp surface display different irradiances.
Figure 8. UV-B lamp; white numbers on the lamp surface display different irradiances.
Figure 9. Histamine formation of histidine solutions after UV light irradiation; error bars display ± 1 SD.
Figure 10. UV-A irradiation of histidine solutions (solved in sodium chloride) for different time periods; error bars represent minimum and maximum values; error bars display ± 1 SD.
Figure 11. UV-B irradiation of histidine solutions solved in sodium chloride for different time periods; error bars represent minimum and maximum values; error bars display ± 1 SD.
Figure 12. Comparison between solvents aqua dest. and sodium chloride on histamine formation after three hours UV-B irradiation.
Figure 13. Steaming of histidine solutions with different ozone concentrations and solvents.
Figure 14. Histamine in human tears before and after UV light irradiation and steaming with ozone; error bars display ± 1 SD.
Figure 15. External calibration curve of histamine, red circles with error bars show standard deviation; black circles show measured values (n = 3) for each concentration.
Figure 16. External calibration curve of L-histidine, red circles with error bars show standard deviation; black circles show measured values (n = 3) for each concentration.
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Figure 17. Histidine and histamine levels in tears measured by LC-MS.
Figure 18. Histamine content of alder pollen after UV light and sunlight irradiation. Reproduced from Heidinger et al. with permission of publisher (Taylor and Francis).
Figure 19. Histamine content of hazel pollen after UV light irradiation and sunlight irradiation. Reproduced from Heidinger et al. with permission of publisher (Taylor and Francis).
Figure 20. PAGE of alder pollen: Lane A = without irradiation; lane B = with UV-A light irradiation; lane C = with UV-B light irradiation; lane D = with sunlight irradiation; lane E = with ozone (100 µg/ml). Arrows highlight proteins that partly disappeared after irradiation.
Figure 21. PAGE of hazel pollen: Lane A = without irradiation; lane B = with UV-A light irradiation; lane C = with UV-B light irradiation; lane D = with sunlight irradiation; lane E = with ozone (100 µg/ml). Arrows highlight proteins that partly disappeared after irradiation.
Figure 22. Alder pollen in physiological saline: A= without irradiation, 400x magnification; B= without irradiation, 1000x magnification; C= irradiation with UV-A light for 3 days, 400x magnification; D= irradiation with UV-A light for 3 days, 1000x magnification; E= irradiation with UV-B light for 3 days, 400x magnification; F= irradiation with UV-B light for 3 days, 1000x magnification.
Figure 23. Hazel pollen in physiological saline: A= without irradiation, 400x magnification; B= without irradiation, 1000x magnification; C= irradiation with UV-A light for 3 days, 400x magnification; D= irradiation with UV-A light for 3 days, 1000x magnification; E= irradiation with UV-B light for 3 days, 400x magnification; F= irradiation with UV-B light for 3 days, 1000x magnification.
Figure 24. Non-irradiated alder pollen with SEM in normal vacuum.
Figure 25. UV-A irradiated alder pollen with SEM in normal vacuum.
Figure 26. UV-B irradiated alder pollen with SEM in normal vacuum.
Figure 27. Non-Irradiated pollen (A) vs. irradiated pollen (B).
Figure 28. Pollen without irradiation; pictures coloured with Pixelmator image editing program.
Figure 29. Pollen after UV-A irradiation; pictures coloured with Pixelmator image editing program.
Figure 30. Pollen after UV-B irradiation, pictures coloured with Pixelmator image editing program
Figure 31. Alder pollen with fungi after PAS staining.
Figure 32. UV-A light irradiated alder pollen and fungi after PAS staining.
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Figure 33. UV-B light irradiated alder pollen and fungi after PAS staining.
Figure 34. Cells with non-irradiated pollen after washing steps.
Figure 35. Cells with UV-A irradiated pollen after washing steps.
Figure 36. Cells with UV-B irradiated pollen after washing steps.
Figure 37. Cells in DMEM (control).
Figure 38. xCELLigence analysis of non-irradiated and irradiated alder pollen suspensions.
Figure 39. xCelligence growth curve of CHANG cells with DMEM and diluted DMEM with NaCl (ratio 1+1).
Figure 40. xCELLigence growth curve of CHANG cells and alder pollen. Reproduced from Heidinger et al. with permission of publisher (Taylor and Francis).
Figure 41. xCELLigence growth curve of CHANG cells and hazel pollen. Reproduced from Heidinger et al. with permission of publisher (Taylor and Francis).
All figures were provided by Andrea Heidinger, Department of Ophthalmology,
Medical University of Graz, Austria. A part of it is reproduced from “Heidinger A. et
al. Decreased viability and proliferation of Chang conjunctival epithelial cells after
contact with ultraviolet light-irradiated pollen. Cutaneous and Ocular Toxicology –
in press” with permission of publisher (Taylor & Francis).
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3. List of Tables
Table 1: Materials for steaming with ozone and irradiation with UV light
Table 2: Materials for histamine determination
Table 3: Materials for PAS staining
Table 4: Materials for electrophoresis
Table 5: Materials for cell culture
Table 6: Further reagents/materials
Table 7: Protocol synopsis.
Table 8. Sunlight irradiance in W/m2 on a sunny day.
Table 9. Sunlight irradiance in W/m2 under different weather conditions.
Table 10. Cytokine determination (ProcartaPlex Kit).
Table 11. Cytokine determination (BioPlex Kit).
Table 12. Characterization of study participants.
Table 13. Subjective symptoms.
Table 14. Fluorescein-break-up time (in seconds), and corneal and conjunctival staining; OD= oculus dexter (right eye), OS= oculus sinister (left eye).
Table 15. Quantitative estimation of histamine and histidine in human tears before and after UV light irradiation.
Table 16. MTS-test results of alder and hazel pollen.
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4. Abstract in German
Hintergrund
Die Zahl der Menschen, die an Allergien leiden, ist in den letzten Jahren deutlich
angestiegen. Man vermutet, dass die erhöhte Umweltbelastung durch Ozon und
Abgase das Allergiepotential vieler Pollen erhöht. Auch die globale Erwärmung,
die zu länger andauernden Blütezeiten vieler Gräser und Bäume führt, wird dafür
mitverantwortlich gemacht. Wir untersuchten, ob UV-Licht einen Effekt auf
Tränenfilminhaltsstoffe, Polleninhaltsstoffe und auf die Pollenmorphologie hat und
ob bestrahlte Pollen die Vitalität und das Wachstum menschlicher Bindehautzellen
beeinflussen.
Material und Methoden
Erle (Alnus glutinosa) und Hasel (Corylus avellana) Pollen wurden mit
Sonnenlicht, UV-A und UV-B Licht bestrahlt und der Histamingehalt vor und nach
Bestrahlung gemessen. Veränderungen des Proteinspektrums wurden mittels
Polyacrylamid-Gelelektrophorese analysiert. Rasterelektronenmikroskopie und
Lichtmikroskopie dienten dazu, Effekte der Bestrahlung auf die Morphologie der
Pollen darzustellen. Bindehautzellen (CHANG Zellen) wurden kultiviert und der
Einfluss bestrahlter und nicht-bestrahlter Pollen auf die Zellvitalität und
Proliferation untersucht. In einer Pilotstudie wurden Tränen von fünf freiwilligen
Probanden abgenommen, mit UV-Licht bestrahlt und anschließend der Histidin-
und Histamingehalt bestimmt.
Ergebnisse
UV-Licht Bestrahlung von Pollen führte zu einem Anstieg des Histamingehalts, zu
einem veränderten Proteinspektrum und einer veränderten Pollenmorphologie. Die
Inkubation der Bindehautzellen mit den Pollen zeigte einen signifikant stärkeren
Abfall der Zellvitalität mit bestrahlten Pollen gegenüber nicht-bestrahlten Pollen.
Eine Bestrahlung von Tränen mit UV-Licht führte zu keinem Histaminanstieg.
Schlussfolgerungen
Unsere Versuche zeigen, dass UV-A und UV-B Licht in der Lage sind, Pollen und
deren Inhaltstoffe zu verändern. Dies könnte unter anderem für die Zunahme an
Beschwerden während der Pollensaison mit verantwortlich sein.
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5. Abstract in English
Purpose
The number of patients suffering from allergic diseases increases from year to
year. It is suspected, that environmental factors such as ozone and exhaust gases
could increase the allergenic potential of pollen. Also climate change which leads
to extended flowering periods of tree and grass pollen is in suspicion to increase
the allergenic potential. We investigated the effect of ultraviolet (UV) light on tear
film ingredients, pollen ingredients, pollen morphology and the impact of irradiated
and non-irradiated pollen on the viability and proliferation of human conjunctival
cells.
Material und Methods
Alder (Alnus glutinosa) and hazelnut (Corylus avellana) pollen were irradiated with
sunlight or UV-A and UV-B light, respectively and the histamine content was
analysed and compared with non-irradiated pollen. Changes in the protein
spectrum of pollen were investigated with polyacrylamide gel electrophoresis
(PAGE). Scanning electron microscopy (SEM) and light microscopy were used to
investigate effects of UV light on pollen morphology. A conjunctival cell line
(CHANG cells) was used to study the effects of irradiated pollen on cell viability
and proliferation. In a pilot study tears were obtained from five voluntary subjects,
irradiated with UV light and analysed for their histidine and histamine content.
Results
UV light irradiation increased the histamine level of alder and hazelnut pollen in a
dose dependent manner and caused changes in the pollen protein spectrum and
pollen morphology. Treatment of CHANG cells with irradiated pollen induced a
statistically significant higher decrease of cell viability than treatment with non-
irradiated pollen.
Conclusion
Our results indicate, that UV-A and UV-B light cause pathological alterations of
pollen, which could be a contributory cause for the worldwide increase of
symptoms during the pollen season.
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6. Introduction
The prevalence of people suffering from allergic diseases increases from year to
year making it a major public-health concern. About 40-50 % of the world´s
population suffer from one or more allergies (1). Approximately 400 million people
suffer from allergic rhinitis, about 200 to 250 million people from food allergies and
about 300 million people from asthma. There are two types of asthma: allergic and
non-allergic asthma. Most of the children and about 50 % of adults suffer from
allergic asthma. While the prevalence of non-allergic asthma remains stable the
prevalence of allergic asthma constantly increases. Until 2025 it is expected that
asthma would affect up to 400 million people. Sensitization rates to one or more
common allergens are suspected to rise in the future further increasing the
number of patients suffering from allergies (1–5).
Some of the reasons for this increase are thought to be the increasing amount of
pollutants in the environment and the global warming with longer periods of sun
exposure on the earth´s surface (6–8). Indoor and outdoor pollution like tobacco
smoke, exhaust gases, particulate matter (PM) and longer sun irradiation periods
cause biological and chemical changes to pollen thus making them more harmful
(9). It is known that there is often a genetic predisposition in allergy sufferers. A
family history is a strong risk factor for development of hay fever, asthma or atopic
dermatitis. Nevertheless, since genetic changes occur within thousands of years
this might not be the explanation for the recent rapid increase of allergies. This
leads us to the assumption that environmental factors play a major role in the
pathogenesis of allergic diseases (10,11).
6.1. Allergy - Definition
An allergy is a hypersensitivity reaction of the bodies’ immune system against
normal harmless substances. These substances can include pollen grains, dust
mites, mildew, animal dander, drugs or certain foods. Getting in contact with the
substance leads to the formation of specific antibodies (immunoglobulins), which
induce an immunological reaction in the body. Allergic diseases can affect people
of all ages, from new-borns to the elderly. The most common allergies include
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allergic rhinitis, allergic asthma, ocular allergy and food allergy (12).
Our immune system protects us from invading organisms by producing specific
immunglobulins that defend these foreign substances. They are divided in several
subclasses. Immunoglobulins of type E (IgE) e.g. are responsible for the induction
of allergic reactions. After being built up by plasma cells these antibodies are
mainly located on mast cells, on circulating basophil and eosinophil granulocytes
and in different tissues (13). In small amounts IgE is important for defending
parasitic infections, in patients with allergies IgE is overproduced (the
concentration may increase several hundred-fold) which leads to the development
of allergic reactions and the induction of a cascade of immunological reactions.
Every substance that is able to induce an allergic reaction consists of several
allergens (also known as antigens). Allergens are ubiquitously distributed in the
environment. In healthy subjects the contact with the allergen induces no or only a
harmless immune response. In patients suffering from allergies very little amounts
of allergens are enough to induce an IgE production followed by a hypersensitivity
reaction and exaggerated immune response (14–16).
When the allergen encounters the human body for the first time it is recognised by
antigen-presenting cells, which bind the allergen and then adhere to specific type
of T-cells (Th2-cells). Through mediation of specific cytokines, the Th2-cells bind
to antibody-producing B-cells. These B-cells in further case produce great
amounts of IgE antibodies, which are then released in the blood where they
adhere to the surface of mast cells and basophil granulocytes. Every time the body
gets in contact with the same allergen, the allergen will bind to the antibodies on
the immune cells, activate them and initiate the first stage of the allergic reaction -
the early-phase reaction: mast cells begin to degranulate within a few minutes,
simultaneously histamine and a variety of other inflammatory mediators like
cytokines, interleukins, prostaglandins, etc., which are stored in granules inside
the cells are released. Depending on the strength of the allergy, this causes
several mild to severe symptoms like vasodilation, redness, itchiness, dyspnoea,
anaphylaxis. Simultaneously this liberation initiates the late-phase reaction, further
mediators and cells like mast cells, eosinophil, basophil and neutrophil
granulocytes and macrophages will be recruited within the following hours
promoting the immunological reaction (17–19).
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Allergic symptoms may be seasonal as for airborne allergies like pollen, or year-
round as for food-, mildew- or drug allergy.
Approximately 40–60 % of all patients with allergies suffer from ocular symptoms.
Allergic conjunctivitis might affect more than one billion people worldwide. It is one
of the most common anterior eye problems ophthalmologists have to deal with
(15). Allergic conjunctivitis is distinguished into several forms: seasonal allergic
conjunctivitis (SAC), persistent or perennial allergic conjunctivitis (PAC), vernal
keratoconjunctivitis (VKC) or atopic keratoconjunctivitis (AKC). The most common
form is SAC, better known as hay fever or pollinosis (15).
6.2. Ocular allergies
6.2.1 Hay fever
Hay fever is caused by airborne grass or tree pollen and affects 25 % – 50 % of all
patients with ocular allergy (20). Getting into contact with the allergen causes
symptoms as red, watery, itchy and tearing eyes. Patients with ocular allergies
usually also suffer from runny or itchy nose as the eye and the nasal mucosa will
react in the same way to allergens (21). Often hay fever is also accompanied by
asthma, and atopic dermatitis with symptoms as bronchial obstruction, coughing
and rash (16,22). Hay fever is mainly diagnosed through the above-mentioned
clinical symptoms and the patients’ medical history. Often the formation of papillae
or follicle, an accumulation of immune cells in the conjunctiva, can be observed,
especially for highly developed allergies. A skin prick test or blood sampling can
additionally confirm the diagnosis. For the skin prick test, several different antigens
will be applied on different locations on the inside forearm or on the back.
Afterwards a lancet is used to make a slight incision (prick) on the skin to allow the
allergen to penetrate. If an allergy is present a visible inflammatory reaction with
development of a wheal and reddening of the area will be observed. The diameter
of the wheal and intensity of reddening will be measured to estimate the severity of
the allergy. A wheal ≥3 mm in diameter is classified as positive result (23).
In the blood IgE can be detected by using radio-allergo-sorbent-test (RAST),
fluorescent-enzyme immunoassay (FEIA), enzyme-linked immunosorbent assay
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(ELISA) or others. Often also conjunctival scraping is performed if an allergy is
suspected: eosinophil granulocytes in the conjunctiva are inflammatory cells
known to be a hint for the presence of an allergy (24).
PAC is similar to SAC with exception that signs and symptoms may be perennial.
PAC is mainly caused by house dust mites and animal dander. Usually symptoms
are less intensive than for SAC. VKC is a form of allergy predominantly occurring
in adolescent males. VKC is similar to PAC as it is persistent during the whole
year, but symptoms are more serious. Ophthalmological signs are papillary
hypertrophy or in severe cases giant papillae, looking like cobblestones, punctate
epithelial erosions in the superior and central cornea, and trantas dots containing
eosinophil granulocytes. VKC patients may suffer from photophobia and blurred
vision additionally to the already mentioned allergic symptoms. AKC is similar to
VKC with exception that it is usually present in adults only. As AKC and VKC are
more severe forms of allergies they require a specific medical treatment (24,25).
6.2.2 Therapy
Whenever possible avoidance of the allergen should be achieved. Patients with
hay fever are free of complaints when they are not exposed to pollen. Eye rubbing
should also be avoided as it could lead to high tryptase levels associated with
increased allergic symptoms (26). The most common form of topical therapy is the
usage of lubricants. They are recommended as they could minimize patients’
symptoms and by using them a wash out of remaining allergens is possible.
Antihistaminic drops may be used as they prevent the initiation of histamine-
induced symptoms as itching, vasodilation or chemosis. Drops containing mast
cell stabilisers prevent the release of histamines and other mediators from the
mast cells (15).
6.3. The ocular surface
The ocular surface is constantly exposed to environmental influences, infectious
agents and pollen through its direct contact with the environment. Pollen interact
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with the conjunctiva or cornea and initiate a cascade of immunological reactions,
especially for subjects suffering from allergies. Pollen can also affect the tear film
where they can interact with tear film ingredients (24).
6.3.1 Tear film ingredients
The tear film can be categorised into three distinct layers: the outer lipid layer, the
middle aqueous layer, and the inner mucous layer. Together the layers consist of
several hundreds of proteins, lipids, mucins, antioxidants, electrolytes, etc. (27).
To maintain the physiological function, a sufficient tear volume and a balanced
composition of tears are important. A lack of tears or altered tear composition may
lead to intermittent dehydration of the ocular surface which is known as dry eye
disease or keratoconjunctivitis sicca. If the tear volume is not sufficient enough it is
called aqueous tear deficient dry eye (ATD). If there is a lack or reduced amount of
tear film ingredients the tear film evaporation increases, which is called
evaporative dry eye (EDE). Subjects with dry eye syndrome may suffer from
burning and reddened eyes, itching, stinging, foreign body sensation, light
sensitivity, visual disturbance etc. (28). Some of the symptoms are similar to those
from allergy sufferers, thus an allergic disease is often difficult to diagnose,
especially if laboratory tests do not exhibit any abnormalities (29).
The tear film naturally consists of a variety of antioxidants, such as superoxide
dismutase or glutathione peroxidase which act as free radical scavengers and
prevent tear film ingredients from oxidative damage (30,31). If the amount of
antioxidants is not sufficient this may also result in damage of tear film ingredients
evoking different eye complaints. Pollen may not only induce IgE-dependent
allergic reactions but also non-allergic reactions mainly mediated through further
ingredients as reactive oxygen species (ROS), lipids or enzymes (32).
6.4. Non-allergic reactions
People often experience allergy-like symptoms although they do not suffer from
any allergy, as diagnosed by allergy testing with skin prick test or blood test. This
could be due to two major causes: first, common allergy tests use only use certain
specific allergens, which are known to induce the majority of allergic reactions. But
21
there are also several other antigens present in pollen, so if in some cases the
allergy is directed against any other allergen it cannot be detected with these tests
(33).
The second reason might be the ability of pollen to induce also non-IgE mediated
reactions that cause allergy-alike symptoms. It is known that pollen ingredients like
enzymes (proteases, lipases) or lipids in pollen induce immunological and
chemical reactions, e.g. the so called seasonal non-allergic conjunctivitis (SNAC)
syndrome which was first described by Schmut et al. (32). Pollen enzymes,
especially proteases lead to the destruction or alteration of tear film ingredients
which causes ocular symptoms and may lead to non-classical allergic
inflammatory reactions (34,35). Pollen ingredients may also be able to induce
instability of the tear film, reduced tear film break-up time thus leading to the
development of dry eye (36).
Similar reactions have been described by Eriksson et al. and Wedbäck et al.
whose patients suffered from rhino-conjunctival symptoms during pollen seasons,
despite tests did not show any evidence for an allergy. They named this disease
seasonal non-allergic rhinitis (SNAR). The underlying mechanisms of SNAC and
SNAR are not fully understood today. It is known that pollen hydrate when getting
in contact with body fluids or mucosal tissues. The hydration is accompanied by a
release of proteins, enzymes and other mediators, which interact with cells and
epithelial barriers and may initiate the non-allergic reaction (37,38).
6.5. Pollen and their ingredients
Pollen is the collective term for multiple pollen grains discharged from the male
parts of a tree, grass or flower and is a fine powdery, typically yellow, substance.
Pollen grains have great varieties of shapes and sizes from 12 m to 300 m
diameter (39). They are very important for the fertilizing process and will be
transported by wind, insects or other animals to the female ovule where the
fertilization takes place. Similar to plant cells pollen consist of a cell wall,
cytoplasm and cytoplasmic organelles like golgi apparatus, mitochondria and an
endoplasmic reticulum with exception of chloroplasts (40).
22
The cell wall is composed of an intine and exine, the intine surrounds the pollen
cytoplasm, the exine is responsible for protecting the pollen grain from physical,
chemical or environmental factors (39). Typical pollen ingredients are sugars (30
%), proteins (20 %), water (< 10 %), free amino acids (< 10 %), lipids (< 5 %),
enzymes, vitamins, minerals, aromatics, dye stuffs and secondary plant
substances (41).
In central Europe the flowering period of tree pollen starts in early April. Alder and
hazel pollen are the first to release their pollen grains in the environment.
6.6. Pollen allergens
Pollen allergens are water-soluble proteins or glycoproteins with a molecular
weight usually higher than 10 kDa. Pollen allergens are resistant to pH changes,
high temperature (up to 100°C) and remain stable for centuries in a dry
atmosphere. They are capable of eliciting an allergic reaction within a few
seconds. Pollen allergen release may be influenced by environmental factors such
as high relative humidity, heavy rain and pollutants. (39,42).
There is a wide range of pollen-specific allergenic proteins that have been
identified today and can be found in an international Allergom database (43). It is
known that the amount of protein in pollen depends on the pollen species and also
on ambient factors. This may be among others a reason for the different
allergenicity of various pollen species. Allergens will be released when the pollen
grain is getting into contact with the ocular surface, the upper airway or nasal
mucosa. A release in ambient air, external to the organism is also common and
mainly caused by relative humidity, heavy rain and pollutants, especially diesel
engine exhaust particles (44,45).
The major allergen of alder pollen is Aln g 1 with a molecular mass of 18.5 kDa.
The major allergen of hazel pollen is Cor a 1 with a the molecular mass of 17 kDa,
about 95 % of all european citizens with hazel allergy are sensitized to it (43).
23
6.7. Further pollen ingredients
Lipids are the major component of the pollen coat. They are required for pollen
hydration, tube growth and the initial steps of fertilization but are also located
inside pollen (46). It is assumed that lipids can modify the antigenic properties of
proteins and that they can lead to an activation of human eosinophils and
neutrophils which plays an important role in the allergic or inflammatory response
(47).
Pollen grains contain a variety of enzymes, the most important ones are
proteases. These proteases lead to the destruction of tear film components and
cause damages to epithelial cells. Epithelial junctions get ruptured and thus
harmful substances can get into the cells (34,35). Another important enzyme in
pollen is NADPH (nicotinamide adenine dinucleotide phosphate) oxidase. Its
function is to transfer electrons and form superoxide to promote microbial killing.
NADPH could also lead to reactive oxygen species (ROS) formation when getting
into contact with the human body.
An important component of pollen grains are starch granules. They contain
specific pollen allergens, which are released into the air in case of pollen grain
rupture and might be responsible for the development of inflammation of the lower
airway. Pollen grains itself are too big in size to reach the lower airways, only
particles smaller than 10 µm could get into the trachea, bronchi, bronchioles, lungs
and alveoli. Starch granules are small in sizes (0.5 µm - 5 µm), it is assumed that
they are responsible for inducing lower airway inflammation (44,48).
6.8. ROS (reactive oxygen species)
ROS are natural by-products of the normal metabolism and include for example
singlet oxygen, peroxides, super oxides, hydroxyl radicals and hydrogen peroxide.
They are important for cell signalling, for pollen germination and the growth
process. If the level of ROS increases this can lead to destruction or damage of
cells which is known as oxidative stress (49). Results from former studies indicate
that ROS play an important role in the pathogenesis of allergic diseases (50,51).
24
They are able to activate mast cells and therefore might be co-responsible for the
induction of immune responses and the onset of allergic symptoms.
Environmental air pollutants such as ozone, diesel exhaust and cigarette smoke
are known to increase the production of ROS which could lead to worsening of
disease symptoms and oxidative stress-induced airway inflammation (52,53).
6.9. Inflammatory cytokines
Pollen are known to induce the release of inflammatory cytokines when getting
into contact with the ocular surface or mucosal tissues (54,55). Cytokines are
secreted by inflammatory cells and regulate a large number of biological effects in
the human body. They have both, detrimental effects and beneficial effects.
Beneficial effects include antimicrobial defence on the ocular surface or effects on
wound healing and axon regeneration after nerve injury. Detrimental effects
include their contributory role in onset of different eye complaints such as dry eye
or ocular allergy. Elevated cytokine levels might induce different eye complaints
and lead to the induction of oxidation and the production of ROS thus activating
the inflammatory cascade. Cytokines are present in the whole body, also in the
tear film. The levels in tears seem to be very stable throughout the day suggesting
cytokines as potential biomarkers for estimating the severity of eye diseases (56–
58). Common cytokines include interleukins such as IL-1α, IL-1β, IL-2, IL-4, IL-6
IL-7, IL-8, IL-13, IL-15, interferons such as IFN-γ, tumor necrosis factors (TNF-α),
transforming growth factors, etc. (59). IL-1β, IL-6, INF-γ and TNF-α are thought to
play a key role in pathogenesis of DED (60).
IL-1 is a proinflammatory cytokine and important mediator for pathogenesis of
inflammatory diseases. It is divided into IL-1α and IL-1β, which have similar
biological effects, but their impacts differ between different cell types. IL-6 is also a
proinflammatory cytokine and mediator of the acute phase response. It is reported
to be one of the key molecules in DED.
IFN-γ is a cytokine important for activation of macrophages and has antiviral and
immunoregulatory properties. TNF-α is also a proinflammatory cytokine and
mediator of the acute phase response and induces inflammation and apoptotic cell
death (59,61).
25
Patients suffering from allergies are known to have higher levels of inflammatory
cytokines in tears (62). Pollutants may have adjuvant effects on the release of
inflammatory cytokines and the onset of allergic inflammations (63,64). In a study
with ragweed-allergic subjects a nasal challenge with diesel exhaust particles
(DEP) and ragweed caused higher inflammatory cytokine release than challenge
with ragweed only (65).
A literature search revealed great differences between cytokine standard values in
tears: in a study where the authors used a Luminex high-sensitivity multiplex
cytokine kit the cytokine concentrations in tears of healthy subjects were the
following: 0.11 ± 0.03 pg/ml for IL-1β, 0.56 ± 0.14 pg/ml for IL-6, 4.49 ± 0.74 pg/ml
for IFN-γ, and 0.58 ± 0.07 pg/ml TNF-α (60).
In the paper of Wei et al. the authors summed up data from five published reports
where tears of healthy subjects were investigated with the same method: they
reported the cytokine levels the following: 39.0 ± 23.6 pg/ml for IL-1β, 42.2 ± 23.6
pg/ml for IL-6, 24.0 ± 18.0 pg/ml for IFN-γ, and 58.3 ± 36.9 pg/ml for TNF-α
(58,66–69). Comparing the data reveals that cytokine levels may strongly vary
even when using the same analysis method.
6.10. Environmental pollutants
Environmental pollutants are substances, which may cause long- or short-term
damage to humans, animals or vegetation if present in high enough concentration.
These substances include gases, particulate matter and volatile organic
chemicals. The environmental pollution is defined as “contamination of the
physical and biological components of the earth/atmosphere system to such an
extent that normal environmental processes are adversely affected” (70). There
are many types of environmental pollution, beyond water and soil pollution air
pollution is the most important one when talking about allergic diseases. The main
sources for air pollutants are manufacturing facilities and the production and
combustion of fossil fuels (70,71).
26
6.11. Classification of air pollutants
Pollutants can be categorized into particulate matter (PM), gaseous directly
emitted pollutants (= primary pollutants as CO, CO2, SO2, NO, NO2, hydrocarbons,
etc.) or pollutants, which are formed out of primary pollutants via physical and
chemical processes (= secondary pollutants as ozone, SO3, NOx, etc.). The former
are mainly produced by factories and road traffic whereas the second are
produced by photochemical reactions in the atmosphere (72,73).
6.11.1 Particulate matter
Particulate matter (PM) is a mixture of solid and liquid particles found in the air.
PM is categorized into particles with a diameter of less than 10 μm (PM10) and
particles with a diameter of less than 2.5 μm (PM2.5 or fine PM). PM can consist of
hundreds of different chemicals like inorganic ions, metals, polycyclic aromatic
hydrocarbons, sulfates, nitrates, ammonium and even allergens and microbial
compounds. PM can be formed by combustion engines, factories and agriculture
or due to chemical reactions of gaseous pollutants. A great variety of health effects
are known to be induced by PM, mainly affecting the cardiovascular system and
the respiratory system. As PM consists of inhalable particles which reach the
lower airways, they lead to respiratory symptoms, such as irritation or inflammation
of the airways, coughing or difficulty in breathing and enhanced airway
responsiveness (74,75). The risk might be increased for people with pre-existing
pulmonary or cardiovascular diseases. The existence of PM2.5 and PM10 strongly
depend on the geographical location and the weather. In cities or locations with
several factories the appearance is higher than in rural areas.
The average annual concentration of PM2.5 should not exceed 10 g/m3, the
concentration of PM10 should not exceed 20 g/m3 according to the WHO Air
quality guidelines for Europe (WHO AQG) (74).
27
6.11.2 Nitrogen dioxide
Nitrogen dioxide (NO2) is a toxic and irritant gas leading to significant health
effects. It is mainly derived from combustion by motor vehicles. Elevated levels of
NO2 are associated with reduced lung function and an increase of bronchial
symptoms in asthmatic patients and are assumed to potentiate the response to
allergens (76,77). A small number of studies suggest a potential effect of elevated
NO2 exposure on respiratory and cardiovascular mortality. It is difficult to estimate
the overall health risk of NO2 since the concentrations used in most studies
substantially exceeded those we encounter in daily life (78,79).
The WHO AQG suggest a threshold for NO2 at concentrations of 40 g/m3 for
annual mean and 200 g/m3 for one-hour mean (74). NO2 is the main source of
ozone formation on the earth´s atmosphere, which also has several negative
impacts for human health.
6.11.3 Ozone
Ozone is an irritant gas and a potent respiratory hazard on earth. It is formed by
complex photochemical reactions in the atmosphere or also near ground mainly
due to NO2 from vehicle emissions. Nitrogen dioxide (NO2) is cleaved into nitrogen
monoxide and oxygen. Oxygen (O) and di-oxygen (O2) than react to ozone (O3).
On sunny days the ozone formation is higher than on cloudy days (6). Near-
ground ozone negatively effects human health, it has irritating effects on mucosal
tissues as airways, nose or conjunctiva and triggers inflammation, causes cell
injury or cell death. Symptoms caused by ozone can include headache, respiratory
ailments or limitations of physical performance (80,81). In the lungs ozone induces
a reduction of lung function, leads to bronchoconstriction of the airways and
triggers inflammation (82). The daily level of ozone in our region lies between 70-
80 µg/m3. The threshold value for the daily ozone dose is 120 µg/m3 for an eight-
hour daily average. As recent studies have shown, health effects are also
occurring below these level, thus the WHO AQG recommended a threshold of 100
g/m3 for an 8-hour daily average. Levels higher than 240 µg/m3 are considered to
induce significant health effects. For every 10 µg/m3 increase in ozone an increase
of 0.3 % to 0.5 % in mortality due to ozone is expected (74).
28
Low concentrations of ozone lead to recruitment of leukocytes accompanied with
airway inflammation, high concentrations can lead to lung injury (83,84). In studies
researchers often use different units, µg/m3 or ppb (parts per billion). This often
makes it difficult to compare studies and their outcomes. 1 ppb ozone corresponds
to 2.15 µg/m3 ozone.
Ozone in the stratosphere (at an altitude of 10 km to 50 km) has positive effects
for the earth; it filters harmful solar UV-B radiation. Since the 1980s ozone levels
constantly decreased mainly due to hydrochlorofluorocarbons (HCFCs). Thus, an
ozone hole developed with higher levels of UV-B radiation reaching the earth´s
atmosphere. An increasing ozone layer depletion would have enormous health
effects: as calculated with computational models a 10 % decrease in stratospheric
ozone may lead to approximately 1.75 million more cases of cataracts worldwide
per year. A 1 % decrease in stratospheric ozone will lead to a 2 % increase of non-
melanoma skin cancer (NMSC) (85,86). Although HCFCs and other ozone-
depleting substances were reduced since the invention of the Montreal protocol
most of these substances are long-lasting and therefore still causing depletion of
the ozone layer. Additionally changes in global weather lead to longer periods of
sun exposure so it still remains interesting to investigate the effects of UV-B for
human health and related pathologies (61).
6.12. Effects of air pollutants on human health
Excessive research has been done in the last years to elucidate the risks of air
pollutants on human health. There might by a broad spectrum of effects caused by
pollutants, from eye irritation, nausea, skin irritation, difficulties in breathing to
cancer and reduced activity of the immune system leading to several mild to
severe diseases (87). Researchers agree that the rising amount of air pollutants
contributes to increased mortality and hospital admissions, primarily affecting the
cardiovascular and the respiratory system (88,89). An effect of pollutants on the
increasing prevalence of respiratory and allergic diseases is also described (90–
92).
29
In urban areas, there are higher levels of vehicle emissions, studies indicate that
the amount of people suffering from allergic diseases or asthma is higher in
polluted urban areas than in rural areas. Therefore, an enhanced susceptibility to
inhaled allergens caused by exposure to air pollution may be one of several
reasons (93–95). PM and ozone are known to induce an increased expression of
pro-inflammatory cytokines and lipid peroxidation which leads to pulmonary
inflammation. Air pollutants not only affect the human body but also pollen grains:
they can interact with the pollen surface and pollen ingredients thus making pollen
more allergenic (94,96).
6.13. Interaction between allergens and pollutants
Pollutants are able to modify or alter the allergenic potential of pollen and could
thus reinforce allergic symptoms (97). Through the influence of pollutants there
might emerge alterations of the physicochemical characteristics of the pollen grain
surface, which directly effects pollen-mediated allergic and non-allergic reactions.
They might also act as adjuvants amplifying the allergic reactions. Researchers
detected cracks in the pollen surface and increased fragility of the exine after
exposure to ambient air pollution with light, scanning- and transmission electronic
microscopy. They found PM accumulating on the pollen grain leading to changes
in the shape of pollen (98).
Exposure of pollen to ozone significantly induced the activity of NADPH in
ragweed pollen (49). Ozone and other pollutants are in discussion to make allergic
subjects more susceptible to the antigen they are sensitized to (99,100). A study
that evaluated the effect of outdoor pollutants on the risk of allergic diseases in
children revealed that simultaneous exposure to PM and mite allergens had a
synergistic effect on the development of asthma (101).
High temperature and enriched CO2-levels during plant growth lead to earlier
flowering periods, faster plant growth, an increased pollen production as well as
increased allergen content. CO2 is suspected to potentiate the severity of allergic
symptoms and is also the major reason for global warming (102–105).
30
6.14. Global warming
Global warming, also referred to as climate change, is a collective term for the
annually observed rise in the average earth temperature and its related
consequences. According to the National Oceanic and Atmospheric Administration
(NOAA) between 1880 and 2016 the average surface temperature increased by
0.95°C. This results in environmental and economic consequences such as longer
and hotter heat waves, heavier rainfall, more powerful hurricanes and also
consequences for human health. The warming of the oceans and melting of
glaciers are also directly related to the global warming (1).
The incidence of allergies and asthma is rising due to longer and more intense
growth-periods of pollen producing plants (106). Hydration or fragmentation of
pollen may be induced through thunderstorms or rainfall thus generating biological
aerosols carrying allergens (107,44).
Due to the warmer weather people are prone to spend more time outside, which
increases their exposure to UV light radiation. The negative effects of prolonged
exposure to UV light are well known: it may lead to the formation of free radicals,
which are highly reactive and in further consequence induce cell damage and
other pathological cell alterations. This may lead to development of several
diseases especially on the eyes and the skin as these organs are directly exposed
to the environment (108).
6.15. Ultraviolet light
UV light is categorized into UV-A (380-315 nm), UV-B (315-280 nm) and UV-C
(280-215 nm) radiation. UV-C is the most harmful radiation but is completely
filtered by the ozone layer. Only UV-A and approximately 10 % of UV-B radiation
reach the earth´s surface.
In small amounts UV light has beneficial effects for the body: it is important for the
formation of vitamin D in the skin and is used for treatment of diseases like
psoriasis or eczema. Larger amounts may lead to acute or chronic health effects
on the human body.
31
UV-C and UV-B light are absorbed in the cornea and conjunctiva of the eye. UV-A
light is able to reach deeper layers of the eye where it induces light damage. UV
light induced eye damages can include photokeratitis, pterygium, cataract, dry eye
or malignancies as basal cell carcinoma (BCC) and squamous cell carcinoma
(SCC). Excessive sun exposure especially in regions near the equator may be
responsible for up to 20 % of all cataracts according to the WHO (85).
Important for a potential development of diseases are the strength of solar
radiation and the duration of irradiation. The strength of UV light radiation varies
geographically; it is the highest near the equator where sunlight strikes the earth
most directly. It can also be influenced by factors as absorption, scattering by
molecules in the atmosphere and the appearance of clouds (109).
Although UV-A is less energetic and about 1,000-fold less efficiently absorbed by
DNA (desoxyribonucleic acid) than UV-B the interest in investigating the effects on
the human body increased, mainly due to the enhanced appearance of UV-A in
natural sunlight, compared to UV-B. UV-A is known to induce oxidative damage to
lipids, proteins and DNA and was classified as carcinogenic by the International
Agency for Research on Cancer (IARC) in 2012 (109,110).
As it is difficult to conceptualize the daily amount of harmful radiation the World
Health Organization (WHO), the United Nations Environment Programme (UNEP),
the World Meteorological Organization (WMO), and the International Commission
for Non-Ionizing Radiation Protection (ICNIRP) created the so called solar
ultraviolet light index (UVI). It is a unit less numeric value that describes the
intensity of solar radiation that reaches the earth´s surface per day. It serves as an
indicator for getting sunlight-induced erythema, which is an acute side effect of
prolonged UV light exposure and could lead to pathological alterations of the skin
(skin cancer) or premature skin aging. It should alert people to pay attention for
sunburns and encourage them to use sun protection. The value starts from zero
upwards – the higher the index, the greater the risk for getting skin and eye
damage (85).
UV light may also have other impacts, which have not been entirely identified
today, for example the formation of histamine from histidine. Histamine is a
32
hormone and neurotransmitter known to trigger lots of different symptoms in the
human body, like headache, indigestion, tachycardia and eczema (111,112). Also
different ophthalmic symptoms like itching, scratching, burning and redness can be
triggered due to histamine (113). Histamine is also known to stimulate the cytokine
secretion from epithelial cells, cell culture experiments with human bronchial
epithelial cells revealed an increasing release of IL-6 and IL-8 after stimulation with
histamine (114).
The amino acid histidine is the early stage in the formation of histamine. For
conversion into histamine the enzyme histidine-decarboxylase is necessary (115).
By in-vitro experiments with aqueous histidine solutions in our laboratory it was
found that instead of the histidine-decarboxylase, UV light and ozone were also
capable of converting histidine to histamine. Two aqueous 0.05 % histidine
solutions were prepared; one was irradiated with UV light and steamed with
ozone, the other one was left untreated. The histamine content was measured with
ESI-Q-TOF-MS (electrospray ionization - quadruple - time of flight) and was higher
in the treated than in the untreated sample. This phenomenon was also described
in the early years of the 20th century with UV light, cathode rays and x-rays, but
not with ozone. The irradiation of histidine (pulverized and in aqueous solutions)
solutions led to the formation of a substance with histamine-alike properties, later
identified as histamine. Results revealed that wavelengths shorter than 290 nm
are effective in formation of histamine from histidine whereas higher wavelengths
are nearly ineffective. Also experiments with sunlight were done, which showed a
really slow rate of histamine formation. It is presumed that histamine is degraded
short time after building, this was recognized through lower histamine levels after
longer irradiations periods then after shorter periods (116–121).
Histidine is also present in human tear fluid in concentrations of about 1.9 ± 0.7
μM in basal tears and 3.2 ± 1.9 μM in reflex tears, therefore it might be interesting
to investigate if UV light and ozone are able to promote the formation of histidine
to histamine in human tear fluid and thereby cause discomfort similar to symptoms
caused by allergic reactions (122).
33
Figure 1. Possible formation of histamine in human tear fluid.
34
6.16. Aim of the study
We hypothesize that environmental factors such as UV-A light, UV-B light and
ozone are able to convert histidine to histamine. We assume that they have an
influence on tear film ingredients - there might be a conversion from histidine to
histamine and an elevation of proinflammatory cytokines mediated by UV light.
This might be among others responsible for the strengthening of allergic
symptoms.
Further we assume that UV light is able to change the ingredients and the
morphology of pollen thus increasing their allergenic potential.
In this study, the following questions were investigated:
1. Is UV light capable of converting histidine to histamine?
2. Does UV light influence the histamine and histidine content of human tears?
3. Does UV light influence the cytokine content of human tears?
4. Is UV light capable of altering pollen ingredients?
5. Does UV light influence the protein content of pollen?
6. Is UV light capable of altering the morphology of pollen?
7. Does UV light influence the allergenic potential of pollen?
8. Does an UV light-irradiation of pollen influence the viability and proliferation
of human conjunctival cells?
35
7. Material and Methods
7.1. Materials
Materials needed for the experiments, have been categorised for every experiment
and are listed in the tables below in alphabetical order.
Table 1: Materials for steaming with ozone and irradiation with UV light
UV light irradiation/ ozone steaming
Reagents Additional information/
order number Manufacturer
Humazon® Unit Ozone generator Technomed GmbH,
Germany
Optometer P 9710 Gigahertz Optik GmbH,
Germany
UV-A lamp VL-115 L, 365 nm Tube,
Power: 30 W
Vilber Lourmat GmbH,
France
UV-B lamp VL-115 M, 312 nm Tube,
Power: 30 W
Vilber Lourmat GmbH,
France
Table 2: Materials for histamine determination
Histamine determination
Reagents Additional information/
order number Manufacturer
BEH-Amide ACQUITY UPLC
1.7µm (150mm x 2.1 mm) Column for HPLC Waters, Belgium
Histamine # US13779-5GM Merck KgaA, Germany
Histamine detection kit # BA-E 5800 ImmuSmol, France
Histamine research ELISA
kit # DEIA207 Creative Diagnostics, USA
Histidine # 1043510025 Merck KgaA, Germany
Multifuge 3 L-R - Thermo Fisher, Scientific,
Austria
TSQ Quantum Ultra Mass spectrometer Thermo Fisher Scientific,
MA, USA
UltiMate 3000 HPLC system Thermo Fisher Scientific,
USA
36
Table 3: Materials for PAS staining
GRAM/GIEMSA/PAS - staining
Reagents Additional information/
order number Manufacturer
Alcohol (70 %, 80 %, 90 %) - Pharmacy, State hospital
Graz, Austria
Butylacetate # 101974 Merck KgaA, Germany
Cover glasses # 631-1571 VWR, Austria
Ethanol absolute # 102428 Merck KgaA, Germany
Fireboy # 14400 Integra, Germany
Giemsa solution # 1.09204.1000 Merck KgaA, Germany
Giemsa puffer tablets (pH
7.2) # 1094680100 Merck KgaA, Germany
Gram (Color 2 kit) # 55542 bioMèrieux, France
Hämatoxylin (acidic) # 606070517 Gatt-Koller, Austria
HCl-Alcohol (0.6 %) - Pharmacy, State hospital
Graz, Austria
May-Grünwald solution 1014241000 Merck KgaA, Germany
M-Fix, spray fixative # 103981 Merck KgaA, Germany
Microscope slides # 631-0098 VWR, Austria
Pertex # 41-4010-00 Medite, Germany
Schiff´s reagent # 109033 Merck KgaA, Germany
0.5 % periodic acid # 403154017 Gatt-Koller, Austria
37
Table 4: Materials for electrophoresis
Electrophoresis
Reagents Additional information/
order number Manufacturer
Colloidal Blue Staining Kit # LC6025 Thermo Fisher Scientific,
Austria
Novex Sharp unstained
Protein Standard # LC501
Thermo Fisher Scientific,
Austria
NuPAGE™ 4-12 % Bis-Tris Protein Gels, 1.0 mm, 10-
well
# NP0321BOX Thermo Fisher Scientific,
Austria
NuPAGE™ MES SDS
Running Buffer (20X) # NP0002
Thermo Fisher Scientific,
Austria
NuPAGE™ LDS Sample
Buffer (4x) # NP007
Thermo Fisher Scientific,
Austria
XCell SureLock™ Mini-Cell
Electrophoresis System #EI0001
Thermo Fisher Scientific,
Austria
Table 5: Materials for cell culture
Cell culture
Reagents Additional information/
order number Manufacturer
Cell Titer 96® Aqueous One
Solution Cell Proliferation
Assay (MTS solution)
# G358C Promega, USA
CO2 incubator (Heracell 240) - Heraeus, Germany
DMEM (Dulbecco's Modified
Eagle Medium) # 31885049
Thermo Fisher Scientific,
Austria
DMSO (Dimethyl Sulfoxide) # 102952 Merck KgaA, Germany
E-Plates # 05469830001 ACEA Biosciences, USA
Fetal bovine serum # 10437036 Thermo Fisher Scientific,
Austria
Penicilline/ streptomycine # A 2212 Biochrom AG, Germany
Trypsine # P10-023100 PAN Biotech, Germany
25-cm2 culture flask # 83.3910.302 Sarstedt, Germany
96-well plates # 83.3924.005 Sarstedt, Germany
12-well plates # 83.3921.005 Sarstedt, Germany
38
Table 6: Further reagents/materials
Further reagents/materials
Reagents Additional information/
order number Manufacturer
Aqua dest. # B230673 Fresenius Kabi, Austria
Bio-Plex® 200 Multiplex
Immunoassay System # 171000201 BioRad, USA
Bio-Plex Pro™ Human
Chemokine Assays #171304090M BioRad, USA
Heraeus™ Labofuge™ 400 - Thermo Fisher, Scientific,
Austria
China mint oil - Bio Diät GmbH, Germany
Countess 2FL Cell Counter # AMQAF1000 Thermo Fisher Scientific,
Austria
Countess™ Cell Counting
Chamber Slides # C10228
Thermo Fisher Scientific,
Austria
DPBS # P04-36500 PAN Biotech, Germany
ELISA-Reader (1) Anthos 2010 Anthos Labtec Instruments
GmbH, Austria
ELISA-Reader (2) Flex Station 3 Multi-Mode
Microplate Reader from Molecular Devices, Austria
Eppendorf tubes # 0030125150 Eppendorf, Germany
HydroFlex™ microplate
washer -
Tecan Trading AG,
Switzerland
Glass capillaries # 708744 Brand GmbH + Co Kg,
Germany
Light microscope Axioskop HBO 50 Carl Zeiss Microscopy
GmbH, Germany
Micropipettes and
Multichannel pipettes 10-100 μl; 100-1000 μl Eppendorf, Austria GmbH
ProcartaPlex Human Basic
Kit # EPX010-10420-901
Thermo Fisher Scientific,
Austria
Physiological saline # 1313121 Fresenius Kabi, Austria
Scale AS R2
10 mg - 220 g Rauch, Austria
Shaker PROMAX 1020 Heidolph Instruments,
Germany
SEM (scanning electron
microscope) XL 30 ESEM FEI, The Netherlands
Sterile working bench BioWizard Silver SL-130 Kojair, Finland
xCELLigence RTCA DP # 05469759001 ACEA Biosciences, USA
39
7.2. Methods
7.2.1 Irradiation
Irradiation was done with an UV-A lamp (VL-115 L, 365 nm Tube, Power: 30 W)
and UV-B lamp (VL-115 M, 312 nm Tube, Power: 30 W) from Vilber-Lourmat
GmbH, France. For irradiation of histidine solutions, sealable, UV light-permeable
fused silica vessels were used. For irradiation of tear samples solutions were
pipetted into microtiter plates and irradiated by reversing the UV lamp, placing it
about 3 cm above the plates. We also used natural sunlight for irradiation;
therefore, the fused silica vessels were placed on the roof terrace of our
department. An Optometer P 9710 from Gigahertz Optik GmbH, Germany, was
used to measure the irradiance of the UV lamps and that of natural sunlight (see
Figure 2).
Figure 2. Optometer for irradiance measurements.
7.2.2 Steaming with ozone
An ozone generator (Humazon® Unit) from Technomed, Germany was used for
steaming samples with ozone (see Figure 3). Ozone was extracted with a syringe
and added to the test substances. Histidine solutions were steamed in glass tubes:
1 ml of the test solutions were filled into a 5 ml glass tube, the ozone generator
settings were adjusted to 100 g/ml, 1 ml ozone was extracted with a syringe,
40
added to the test substance, the tube was sealed and immediately shaken well for
10 seconds. For steaming with 300 l the ozone generator settings were adjusted
to 100 g/ml, 3 ml ozone were extracted with a syringe and added to the test
substance. Human tears were irradiated in plastic tubes: 30 l tears were filled into
a 1 ml plastic tube. Irradiation was done as described above.
Figure 3. Ozone generator.
7.2.3 Determination of histamine in histidine solutions
Histidine powder was dissolved in sodium chloride (NaCl) solution or aqua
destillata (Aqua dest.) in concentrations from 0.2 % - 1 %. Solutions were then
irradiated with UV-A or UV-B light for different time periods from 1 minute to 3
hours in sealable fused silica vessels. Additionally, the solutions were steamed
with 100 μg/ml and 300 μg/ml ozone. Histamine was then analysed with a
histamine enzyme-linked immunosorbent assay (ELISA) Kit.
7.2.3.1 Histamine ELISA
For histamine determination, we used two different competitive ELISA kits from
Creative Diagnostics, USA and ImmuSmol, France. The antigen is bound to the
solid phase of the microtiter plate, histamine in the standards, controls and
samples compete with a histamine anti-serum for free binding sites. After washing
steps histamine can be detected by using an anti-rabbit IgG-peroxidase conjugate.
All experiments were performed in a sterile workbench following the protocols
41
recommended by the manufacturer. 25 μl of the control, standard or sample were
pipetted into a microtiter plate, 25 μl acylation buffer and 25 μl acylation reagent
were added. The solutions were incubated for 1 hour at room temperature on a
shaker. Afterwards 200 μl aqua dest. were added to all tubes and incubated for 30
minutes at room temperature on a shaker.
Twenty μl of the acylated controls, standards and samples were then pipetted in
the wells of histamine microtiter strips and 100 μl antiserum were added to all
wells. The plate was shaken briefly, covered with adhesive foil and incubated for
20 hours at 2-8°C. On the following day the foil was removed, the contents
aspirated and washed 4 times with 300 μl wash buffer. The plate was blotted dry
by tapping the inverted plate on absorbent material. 100 μl of enzyme conjugate
were pipetted into all wells, covered with adhesive foil and incubated for 1 hour at
room temperature on a shaker. The foil was removed, the contents aspirated and
washed 4 times with 300 μl wash buffer. The plate was again blotted dry by
tapping the inverted plate on absorbent material. 100 μl substrate were pipetted
into all wells and incubated for 30 min. at room temperature on a shaker while
covered with foil. 100 μl stop solution were added to all wells and the absorbance
of the solutions in the wells were read with the Flex Station 3 Multi-Mode
Microplate Reader from Molecular Devices, Austria at a wave length of 450 nm
and a reference wave length of 620 nm within 10 minutes. The calibrator curve
was obtained by plotting the absorbance readings (mean absorbance) of the
standards linear on the y-axis against the corresponding standard concentrations
logarithmic on the x-axis using a 4-parameter non-linear regression for curve
fitting.
We used a competitive ELISA, the colour, which was measured photometrical at
the end was conversely related to the histamine content: the darker the colour
reaction, the less histamine was in the sample, the lighter the colour reaction, the
more histamine was in the sample.
7.2.4 Determination of histamine and cytokines in human tears before and
after irradiation
First experiments were carried out with tears from the principal investigator. Tears
were collected from the lower lateral tear meniscus with a glass capillary after
42
stimulation with China mint oil. Two to three drops of the oil were placed on a
swab and positioned under the eye without skin contact. Tears were transferred
into plastic microtubes (Eppendorf, Germany) stored on ice and immediately
analysed. Histamine was measured with the histamine ELISA kits from Creative
Diagnostics and ImmuSmol as described in section 7.2.3.1. Cytokine
measurements were done with the ProcartaPlex Human Basic Kit from
ThermoFisher Scientific using the Luminex xMAP (multianalyte profiling)
technology and the Bio-Plex Pro™ Human Chemokine Assay from BioRad on the
center for medical research, Core Facility Imaging/ Flow Cytometry. Three μl of
tear sample were diluted with 17 μl of sample diluent and then analysed following
the protocols recommended by the manufacturers. For cytokine measurements,
the Bio-Plex® 200 Multiplex Immunoassay System from BioRad was used.
All samples and standards were analysed in duplicates. The concentrations of the
samples were calculated by plotting the concentration of the standards against the
mean fluorescence intensity (MFI) generated by each standard. A 5PL algorithm
was used for curve fit. Final sample concentrations were multiplied with 6.67 to
correct the dilution factor.
As there is a great variety of different cytokines present in tears we decided to
analyse the 4 key inflammatory cytokines only: IL-1β, IL-6, INF-γ, and TNF-α.
Bead regions for the ProcartaPlex Assay were: 18 (IL-1β), 25 (IL-6), 43 (INF-γ)
and 45 (TNF-α).
Bead regions for the Bio-Plex Pro™ Assay were: 39 (IL-1β), 19 (IL-6), 21 (INF-γ),
36 (TNF-α).
Before analysis tears were irradiated with UV-A or UV-B light for different time
periods:
For histamine determination: 30, 60, 90 and 120 seconds.
For cytokine determination: 30 and 60 seconds and 3, 5 and 10 minutes.
An irradiation of tears in sealable vessels was not convenient due to the small
sample volumes that were available. Thus, we pipetted tear samples into 96-well
plates and irradiated them by reversing the UV lamp placing it directly above the
plate.
43
We also tested, whether ozone has an effect on the histamine and cytokine
content of human tears and steamed tears with 10 μg/ml and 100 μg/ml ozone
respectively.
Afterwards tears from five healthy subjects with normal tear function were obtained
in a pilot study. Simultaneosly additional parameters were assessed as described
in further detail below.
7.2.5 Pilot study: The impact of ultraviolet light and ozone on tear film
components.
Table 7: Protocol synopsis.
Title The impact of ultraviolet light and ozone on tear film
components, a pilot study.
Study design Open, mono-centric pilot study
Objective Does UV light influence the histamine and histidine content of
human tears?
Primary target values Histamine content of tears (ng/ml)
Histidine content of tears (ng/ml)
Planned number of study
participants 20
7.2.5.1 Inclusion and exclusion criteria
Inclusion criteria
- Males and females between 18 and 90 years
Exclusion criteria
- Patients suffering from allergic conjunctivitis
44
- Patients with eye diseases that require special tretament, e.g. viral or
bacterial conjunctivitis or keratitis, glaucoma
- Usage of cyclosporine A- or cortisone eye drops 30 days prior to study start
- Oral intake or topical use of antihistaminics
7.2.5.2 Study participant recruitment
Study participants were recruited during routine examinations in the dry eye unit at
the Department of Ophthalmology in Graz, Medical University of Graz, Austria.
After explaining the study procedures patients were asked for a study participation.
The study protocol was approved by the institutional review board (ethics vote
number: 29-129 ex 16/17)and written informed consent was obtained from all
subjects. Patients were asked for different eye complaints as itching, redness,
tearing, burning, foreign body sensation and light sensitivity. The following
answers were possible: 0= None of the time; 1= Some of the time; 2= Half of the
time; 3= Most of the time; 4= All of the time.
7.2.5.3 Tear collection
One hundred thirty μl tears were obtained from the right eye of all subjects using
glass capillaries and china mint oil for tear stimulation as described in section
7.2.4. Samples were transferred into plastic microtubes (Brand GmbH, Germany)
on ice and immediately frozen at -70°C until analysed with LC-MS (liquid
chromatography-mass spectrometry).
7.2.5.4 Blood sampling
To measure the amount of IgE 3 ml blood were withdrawn from all study
participants. IgE was analysed in the serum at the Institute for Clincial and
Chemical Laboratory Diagnostics, Graz, Austria. The normal range of IgE is 0-100
IU/ml, levels higher than 100 IU/ml indicate the presence of an allergy.
45
7.2.5.5 Cytological examination
A conjunctival scraping was done to investigate conjunctival smears for eosinophil
granulocytes. After application of one drop of a local anaesthetic, a plastic spatula
was used to wipe off a conjunctival specimen of the lower lid of both eyes, placed
on a slide and then stained with May-Grünwald-Giemsa technique. The slide with
material from conjunctival scraping was airdried and fixed with heat. Afterwards it
was stained with May-Grünwald solution for 5 minutes. Solution was decanted and
the slide dyed in Giemsa-colouring solution for 10 minutes. Solution was decanted
and the slide shortly rinsed with Giemsa puffer solution and aqua dest. Evaluation
of eosinophil granulocytes was done with a light microscope (Zeiss Axioskop HBO
50) from Zeiss, Austria.
7.2.5.6 Ophthalmological examination
All study participants were examind for signs of ocular allergy at the slitlamp:
conjunctival injection, conjunctival edema, lid edema and formation of papillae or
follicle on both eyes. The ability to produce tears was measured with the
Schirmer´s test: a filter paper was placed into the lower lid of both eyes for 5
minutes. After lapse of time the produced amount of tears were determined by
measuring the wettened area of the paper with a ruler. A Schirmer´s test shorter
than 10 mm is an indication for dry eyes (123). With the dye fluorescein the tear
film break-up time (T-BUT) and corneal staining were assessed. T-BUT is
measured in seconds after application of 1 μl fluoresceine from the time of a blink
until the first appearance of dry spots. A T-BUT lower than 10 seconds is
considered abnormal. Corneal staining is the assessment of punctate epithelial
erosions, a sign for ocular surface dryness. It is graded in all four quadrants and
the central area of the cornea, each with a score from 0 to 3. Lissamine green was
used for assessment of corneal and conjunctival staining. A Lissamine green strip
was wettened with 0.9 % physiological saline and then applied to the inferior
fornix. The nasal and temporal area, the upper and lower area of the conjunctiva
and the cornea were graded (see Figure 4) (124–126).
46
Figure 4. Ophthalmological examinations: (A) papillae on the upper eyelid; (B)
Schirmer´s test; (C) corneal staining; (D) lissamine green staining.
7.2.5.7 Analysis
Histamine and histidine in tears were measured with LC-MS at the center for
medical research, Core facility for Mass Spectrometry, Graz. Frozen tears were
thawed and splitted into three parts, two parts were irradiated with UV-A and UV-B
light.
1) without irradiation
2) irradiation with UV-A light for 20 seconds
3) irradiation with UV-B light for 20 seconds
7.2.5.8 LC-MS/MS analysis
Before sample analysis proteins were precipitated with three parts of -20°C cold
acetonitrile (ACN) with one part of tear liquid sample. The solution was mixed,
centrifuged at 2700 rpm (Multifuge 3 L-R from ThermoFisher Scientific, Austria) for
10 minutes at 4-8°C, and transferred to a 0.2 ml vial. 5 µL of the sample was used
for LC-MS/MS analysis. If concentration of L-histidine was out of the validated
linear range samples were further diluted with 25/75 (v/v) H2O/acetonitrile.
Chromatographic separation was carried out with UltiMate 3000 HPLC system
(Thermo Fisher Scientific, USA). The column was an BEH-Amide ACQUITY UPLC
1.7µm (150mm x 2.1 mm) (Waters, Belgium) operated at 45°C. Mobile phase A
was 99.9/0.1 (v/v) H2O/formic acid with 5 mM ammonium formate and mobile
phase B was 9.9/90/0.1 (v/v/v) H2O/acetonitril/formic acid with 5 mM ammonium
formate. A linear gradient from 90 to 35 % was run for 5 minutes, stabilized at 35
% for 2 minutes, and returned to 90 % B. The column was re-equilibrated for
further 10 minutes, the separation was performed at a flow rate of 150 µL min-1
and total run was 17 minutes.
A B C D
47
Mass spectrometric detection was performed with a TSQ Quantum Ultra (Thermo
Fisher Scientific, USA) triple quadrupole with electrospray ionization (ESI) source
in positive mode and with multiple reaction monitoring. ESI spray was set up as
followed: spray voltage 4000 V, capillary offset 35 V, skimmer offset 15 V,
nebulizer gas 30 arbitrary units, heated probe gas 10 arbitrary units, and capillary
temperature of 275°C. The transition of 112 to 95 m/z was used to quantify
histamine and 156 to 110 m/z for histidine, collision energy of 15 V was used for
both transitions.
48
Figure 5: UltiMate 3000 HPLC system (left) and TSQ Quantum Ultra (right) from Thermo Fisher Scientific, USA.
7.2.6 Determination of histamine in pollen before and after irradiation
7.2.6.1 Collection of pollen
Pollen of the common tree species alder (Alnus glutinosa (L.) Gaertn.) and
hazelnut (Corylus avellana L.) were harvested in rural areas (south and south-east
of Styria) in 2017 (see Figure 6 showing male inflorescences of hazel pollen in the
flowering period). Trees were monitored for beginning of the flowering period in
49
March, male inflorescences were harvested, sieved in the laboratory and stored at
room temperature in paper bags protected from light. Shortly before use pollen
were suspended in physiological saline in different concentrations from 10 mg/ml -
100 mg/ml.
Figure 6. Male inflorescences of hazel pollen in the flowering period in March
2017.
7.2.6.2 Irradiation of pollen
Alder and hazel pollen were used in a concentration of 100 mg/ml suspended in
physiological saline. For irradiation, the pollen suspensions were pipetted in
sealable, UV light-permeable fused silica vessels to avoid evaporation due to
irradiation and thus false positive results. Solutions were irradiated with UV-A or
UV-B light for the following time periods: 2, 4 or 6 hours. The irradiance used
corresponded to natural occurring irradiance for UV-A light, for UV-B light the
irradiance was ~40 times higher.
We also used natural sunlight for irradiation: the fused silica vessels were placed
on the roof terrace of our department for one day. Considering sunrise and sunset
the average sunshine duration amounted to 10 hours per day. After irradiation
pollen suspensions were centrifuged for five minutes at 1300 rpm and the
supernatant was collected for use and stored at -20°C until analysis.
50
7.2.6.3 Determination of histamine content
For histamine determination, the ELISA kits from Creative Diagnostics and
ImmuSmol were used, as described in section 7.2.3.1.
7.2.6.4 Polyacrylamide gel electrophoresis
Electrophoresis of pollen was performed using the XCell SureLock™ Mini-Cell
Electrophoresis System from Invitrogen, Life technologies. 100 mg alder and 100
mg hazel pollen were suspended in physiological saline, gently mixed and then
divided into five parts:
a) Solution was left untreated
b) Irradiation with UV-A light (45 W/m2, for one or two days)
c) Irradiation with UV-B light (43 W/m2, for one or two days)
d) Irradiation with sunlight (for one or two days)
e) Steaming with ozone (10 g/ml and 100 g/ml)
After irradiation, the samples were centrifuged at 1300 rpm for 5 minutes and the
supernatant was used for further analysis. First a running buffer was prepared
using 40 ml MES buffer diluted with aqua dest. to a final volume of 800 ml. Ten μl
of the pollen supernatants were diluted with 30 μl NuPAGE LDS 4x sample buffer
and gently mixed avoiding the formation of air bubbles. Twelve μl of the samples
were carefully pipetted into the wells of a NuPAGE 4–12 % Bis-Tris mini-gel
(Thermo Fisher Scientific, Austria). The gel was placed into the chamber of the
XCell Sure Lock™ Electrophoresis system from Thermo Fisher Scientific, Austria
and filled with the prepared running buffer. Subsequently gel electrophoresis was
performed at 200 V and 78 mA for 35 minutes. After lapse of time the gel was
taken out of the plastic chamber using a knife and incubated in a fixing solution
with aqua dest -methanol - acetic acid in a concentration of 5:6:1 for 10 minutes.
Staining was done with the colloidal blue stain kit using distilled water, methanol
and stainer A in a concentration of 6:3:1 for 10 minutes and then overnight after
adding 5 ml stainer B. On the following day, the gel was destained in aqua dest.
for several hours and subsequently photographed on a lamp with a Nikon D-500
reflex-camera.
51
For size determination of proteins, we used a Novex® Sharp Unstained Protein
standard (Thermo Fisher Scientific, Austria) with 12 protein bands in the range of
3.5 – 260 kDa.
7.2.6.5 Pollen morphology
A change in the morphology of pollen before and after irradiation was monitored
by using a light microscope (Axioskop HBO 50, Zeiss, Austria) and with scanning
electron microscopy (XL 30 ESEM from FEI; The Netherlands). Pollen were
investigated in dry condition and after being suspended in physiological saline for
one or two days. Non-irradiated pollen were also suspended in physiological saline
and served as a control. SEM pictures were coloured using image-editing
application Pixelmator (Version 3.6).
7.2.6.6 GRAM and PAS staining
We were interested, if alder or hazel pollen were contaminated with bacteria or
fungi and if UV light influences the vitality of these microorganisms. Irradiated and
non-irradiated pollen were placed on microscope slides (VWR, Austria) and
stained with periodic acid-schiff (PAS) colouring and gram staining. PAS is a
staining method to detect fungi or polysaccharides and mucous substances in
several tissues. For bacterial identification, we used the gram staining, which
detects peptidoglycan in the cell wall.
For Gram staining the slides with pollen material were air-dried and fixed with
heat, using the Fireboy from Integra, Germany. Slides were then coloured with the
gram colour 2 kit from bioMérieux, France applying reagent 1 for 1 minute followed
by rinsing with aqua dest. Afterwards staining with reagent 2 for 1 minute was
done, followed by rinsing with aqua dest. Slides were destained with reagent 3 and
rinsed with aqua dest. before colouring with reagent 4 for 1 minute.
For PAS staining slides were air-dried and fixed with a fixation spray from Merck
KgaA, Germany. Slides were inlayed into 0.5 % periodic acid solution (Gatt-Koller,
Austria) for five minutes followed by rinsing with piped water for 5 minutes and
briefly rinsing with aqua dest. Then slides were applied to Schiff´s reagent (Merck
KgaA) for 15 minutes, rinsed with piped water for five minutes and then rinsed with
aqua dest. briefly. Colouring in Hämalaun-solution (Gatt-Koller, Austria) was done
52
for 5 minutes, followed by rinsing with piped water for 10 minutes and briefly
rinsing with aqua dest. Then slides were applied to an ascending alcohol series,
beginning with 70 % ethanol. Slides were tiled with Pertex mounting medium
(Medite, Germany) before evaluating the samples under the light microscope.
7.2.7 Cell culture
We cultivated a human conjunctival cell line (CHANG cells, CCL-20.2, clone 1-5c-
4, Wong-Kilbourne derivative of CHANG conjunctiva) acquired from the American
Type Culture Collection (ATCC, Manassas, Va., USA). Until use the cells were
frozen in DMSO/DMEM (Dimethyl sulfoxide from Dulbecco´s modified eagle
medium) at –196°C in liquid nitrogen. Cells were then defrosted and resuspended
with 10 ml of DMEM containing 1 % P/S and 10 % fetal bovine serum
(ThermoFisher Scientific, Austria). They were then centrifuged at 1300 rpm for 5
minutes and resuspended in 1 ml fresh culture media.
A 25 cm2 culture flask (Sarstedt GmbH, Wiener Neudorf, Austria) was prefilled with
4 ml culture media and 1 ml cell suspension was added and then incubated in a
CO2 incubator (Heracell 240, Kendro Heraeus, Germany) at 37°C, 5 % CO2. Every
second or third day cell culture media was changed or cells were split and seeded
in two new flasks.
Cells were used for experiments when at least 90% confluent, assessed by an
inverse microscope (Axio Observer Z.1, Zeiss, Germany). Culture media was
removed and the cells were rinsed twice with DPBS (PAN Biotech, Germany).
After removal of DPBS 1 ml trypsin was added to the flask and incubated for 2 - 3
minutes in the incubator. When cells were fully detached from the bottom of the
flask, assessed via microscope, 5 ml culture media were added to inactivate
trypsin and the solution was transferred into a centrifuge tube and centrifuged at
1300 rpm for 5 minutes. Finally, the medium was decanted and the cells were
resuspended in 2 ml medium. The number of cells was determined with the
Countess 2FL cell counter (Thermo Fisher Scientific, Austria). For the experiments
cells were seeded in 96-well plates (Sarstedt, Austria) in a concentration of
100.000 cells per ml (10.000 cells per well) and incubated until the following day.
On the next day culture media was removed and the cells were rinsed twice with
DPBS. Wells were then filled with 100 μl of test solutions as described below:
53
Pollen were suspended in physiological saline in different concentrations.
Preliminary experiments revealed that alder pollen are much more harmful than
hazel pollen, thus they were used in lower concentrations:
Alder pollen (10 mg/ml) suspended in physiological saline
Hazel pollen (25 mg/ml) suspended in physiological saline
Solutions were divided into four parts:
1) solution was left untreated
2) irradiation with UV-A light (45 W/m2, for one, two or three days)
3) irradiation with UV-B light (43 W/m2, for one, two or three days)
4) irradiation with sunlight (for one, two or three days)
We tested whole pollen suspensions as well as pollen supernatants, therefore
pollen suspensions were centrifuged at 1300 rpm for five minutes and
supernatants were collected. Untreated cells served as control, DMEM without
cells served as blank.
All test solutions were incubated on the cells for 30 minutes at 37°C in a CO2
incubator. Afterwards solutions were removed and all wells were carefully washed
two times with DPBS to remove all pollen. Finally, all wells were filled with 100 l
DMEM.
7.2.7.1 Determination of cell viability
For assessment of cell viability, the Cell Titer 96® Aqueous One Solution Cell
Proliferation Assay (MTS) from Promega, USA was used. 10 μl of the reagent
were added to all wells and incubated for 2 hours in an incubator. The absorbance
of the MTS reaction product was measured using an ELISA reader (Anthos 2010,
ADAP software from Anthos Labtec Instruments GmbH, Germany) at wavelengths
of 492 nm and 620 nm. For calculation of cell viability, the mean of at least eight
wells per assay was used. Each experiment was repeated three times.
54
7.2.7.2 Determination of cell proliferation
Proliferation of CHANG cells after contact with pollen was measured using the
xCELLigence Real-Time Cell Analysis (RTCA) DP system from ACEA
Biosciences, USA. This device allows for monitoring of cell proliferation in real
time. It consists of an analyser, which is placed in the incubator at 37°C and 5 %
CO2 and a control unit (laptop with pre-installed software). The device uses non-
invasive electrical impedance measurements for monitoring of cell proliferation.
First, the experimental setup and a time schedule had to be programmed with the
software: Three steps were necessary, the first for the background reading, the
second for seeding of the cells (followed by 24 hours of incubation) and the third
for the experiment itself.
For preparation of test solutions alder and hazel pollen were solved in
physiological saline in concentrations of 20 mg/ml and 50 mg/ml. Solutions were
centrifuged for 5 minutes at 1300 rpm and supernatants were diluted with DMEM
in a ratio of 1+1 to reach final concentrations of 10 mg/ml for alder and 25 mg/ml
for hazel. 100 μl of cell culture media were added to each of the 16 wells of an E-
Plate (ACEA Biosciences, USA). First a background reading was performed (cell
index < 0.063 was required), then 100 μl cell suspension were added (100.000
cells/ml). The cells were incubated for 24 hours on the xCELLigence station inside
the incubator. Changes in impedance reflecting cell adhesion and proliferation
were measured every 20 minutes. On the following day culture media was
removed and 100 μl test solutions were added to the wells of the E-plate. Cells
with DMEM and physiological saline (in the same ratio 1+1) served as control. Cell
proliferation was measured every 20 minutes for 3 consecutive days. Beyond
pollen supernatants we also tested solutions with pollen grains, the incubation time
was 30 minutes, afterwards solutions were removed by washing the wells with
DPBS and 100 μl cell culture media was added. The cell proliferation was again
measured for 3 consecutive days. All samples were analysed in duplicates; tests
were repeated twice.
7.2.7.3 Statistical analysis
Data were analysed using SPSS version 24 (SPSS Inc. Chicago, Ill., USA). For
cell culture experiments groups were built for every experiment and compared
55
using ANOVA. For post-hoc analysis Bonferroni correction was used. In case of a
violating of the equal variances assumption the Games-Howell post hoc tests were
used instead. A significance level of p=0.05 was defined for all statistical analyses.
56
8. Results
8.1. Irradiance measurements
8.1.1 Irradiance measurements with UV-A and UV-B lamp
Before carrying out all experiments we measured the irradiance of the UV-A and
UV-B lamp with an optometer at different positions on the lamp. Measurements
revealed that the irradiances of the lamps were not the same over the whole lamp
surface: in the middle, it was higher than at the left or right end (see Figure 7 and
8). To ensure same conditions for all experiments, vessels were always placed in
the middle of the lamp, where irradiations where the highest.
Figure 7. UV-A lamp; white numbers on the lamp surface display different
irradiances.
Figure 8. UV-B lamp; white numbers on the lamp surface display different
irradiances.
57
For irradiation of tear samples, the UV lamps had to be reversed as samples were
irradiated in microtiter plates. Although we tried to minimize the space between the
lamp surface and the well plate while irradiating we had a gap of 5 centimetres
between them. Thus, irradiance of tear samples was diminished: a distance of five
centimetres reduced the irradiance from 46.8 W/m2 for UV-A to 10.1 W/m2 (-78.4
%) and 42.7 W/m2 for UV-B to 9.5 W/m2 (-77.8 %).
8.1.2 Irradiance measurements of natural sunlight
We measured the natural occurring irradiance of UV light at the roof terrace of our
department in April 2017 at three time points: 10 a.m., 12 a.m. and 2 p.m. Results
were 23.1 W/m2, 43.2 W/m2 and 30.0 W/m2 for UV-A and 1.3 W/m2, 2.1 W/m2 and
1.7 W/m2 respectively for UV-B. Beyond these time-dependent differences there
were also day-dependent differences in the irradiance. The sunshine duration was
very variable during the experiments in April 2017. This was observed by own
measurements and was also confirmed by measurements of the ZAMG (Central
Institute for Meteorology and Geodynamics in Vienna, Austria) which monitors the
sunshine duration and other meteorological factors such as air temperature,
rainfall, storms, etc. (127). There were lots of cloudy days with lower levels of UV
light irradiation. We measured the UV light irradiance on both, sunny and cloudy
days and detected great differences in the irradiances. Cloudy weather conditions
reduced the irradiance from three-fold to approximately six-fold. There are also
time-dependent differences, in the morning and afternoon the irradiance is lower
than at midday (see Table 8 and 9).
Table 8. Sunlight irradiance in W/m2 on a sunny day.
Time (sunny day) UV-A UV-B
10:00 a.m. 23.1 1.3
12:00 a.m. 43.2 2.1
2:00 p.m. 30.0 1.7
58
Table 9. Sunlight irradiance in W/m2 under different weather conditions.
Different weather conditions UV-A UV-B
12:00 a.m. - sunny day 43.0 0.9
12:00 a.m. - sunny day, little
clouds 31.3 0.7
12:00 a.m. - partly cloudy 11.8 0.5
12:00 a.m. - mostly cloudy 5.5 0.2
8.2. Determination of histamine in histidine solutions before and
after irradiation
We detected littlest amounts of histamine in histidine solutions. After irradiating the
histidine solutions with UV-A and UV-B light the histamine content raised
obviously. The higher the start concentration of histidine, the higher the histamine
concentration (see Figure 9). For UV-B light the histamine increase was much
higher than for UV-A light. The highest histamine formation was determined with
0.8 % histidine.
For 0.2 % histidine the baseline value was 0.7 ± 0.3 ng/ml, after three hours UV-A
light irradiation it increased to 1.8 ± 0,2 ng/ml, after three hours UV-B light
irradiation it increased to 26.1 ± 3.7 ng/ml. For 0.4 % histidine the baseline value
was 1.3 ± 0.1 ng/ml, after three hours UV-A light irradiation it increased to 2.9 ±
0.1 ng/ml, after three hours UV-B light irradiation it increased to 26.3 ± 7.6 ng/ml.
For 0.6 % histidine the baseline value was 1.4 ± 0.1 ng/ml, after three hours UV-A
light irradiation it increased to 3.8 ± 0.1 ng/ml, after three hours UV-B light
irradiation it increased to 41.1 ± 8.8 ng/ml. For 0.8 % histidine the baseline value
was 2.1 ± 0.2 ng/ml, after three hours UV-A light irradiation it increased to 4.9 ±
0.1 ng/ml, after three hours UV-B light irradiation it increased to 60.2 ± 3.7 ng/ml.
For 1 % histidine the baseline value was 2.4 ± 0.4 ng/ml, after three hours UV-A
light irradiation it increased to 4.6 ± 0.2 ng/ml, after three hours UV-B light
irradiation it increased to 33.9 ± 0.8 ng/ml.
59
Figure 9. Histamine formation of histidine solutions after UV light irradiation; error
bars display ± 1 SD.
Histamine formation did also depend on the duration of irradiation, longer
irradiation periods led to higher histamine formation (see Figure 10 and 11). The
highest histamine formation for UV-A light was determined after two hours of
irradiation; the highest histamine formation for UV-B light was determined after
three hours of irradiation.
The baseline histamine content (without irradiation) was 2.3 ± 0.1 ng/ml.
With UV-A light irradiation it raised to 2.6 ± 0.1 ng/ml after one minute, 2.6 ± 0.1
ng/ml after 30 minutes, 3.5 ± 0.2 ng/ml after one hour, 5.5 ± 0.3 ng/ml after two
hours and 5.4 ± 0.8 ng/ml after three hours.
With UV-B light irradiation the histamine content raised to 2.5 ± 0.1 ng/ml after one
minute, 3.3 ± 0.4 ng/ml after 30 minutes, 7.8 ± 0.8 ng/ml after one hour, 12.2 ± 0.6
ng/ml after two hours and 50.3 ± 2.9 ng/ml after three hours.
60
Figure 10. UV-A irradiation of histidine solutions (solved in sodium chloride) for
different time periods; error bars represent minimum and maximum values; error
bars display ± 1 SD.
Figure 11. UV-B irradiation of histidine solutions solved in sodium chloride for
different time periods; error bars represent minimum and maximum values; error
bars display ± 1 SD.
The histamine formation did also depend on the solvent used: We detected a
higher histamine formation when using sodium chloride instead of aqua dest. for
61
all histidine solutions (see Figure 12). The baseline histamine content (without
irradiation) of a 1 % histidine solution was 2.4 ng/ml when using aqua dest. and
2.6 ng/ml when using sodium chloride. After three hours of UV light irradiation the
histamine content increased to 27.2 ng/ml when using aqua dest. and 50.3 ng/ml
when using sodium chloride. Sodium chloride was therefore used as solvent for all
further experiments.
Figure 12. Comparison between solvents aqua dest. and sodium chloride on
histamine formation after three hours UV-B irradiation.
Steaming of histidine solutions with different ozone concentration did only lead to
small amounts of histamine. With high histidine (1 %) and high ozone (300 g/ml)
concentrations we detected a small histamine formation: the histamine content
rose from 2.6 ng/ml (without ozone) to 5.7 ng/ml after steaming with ozone (see
Figure 13). As observed for UV light too, the amounts of histamine were higher
when using sodium chloride compared to aqua dest.
62
Figure 13. Steaming of histidine solutions with different ozone concentrations and
solvents.
8.3. Determination of histamine in human tears
We irradiated human tears with UV-A and UV-B light for different time periods and
steamed them with different ozone concentrations. The aim was to find out, which
UV light and ozone concentrations induce the highest histamine formation and
could be further used for the planned pilot study.
Irradiation of human tears led to a slight increase in the histamine content after
irradiation with UV-A light and a strong decrease after irradiation with UV-B light
(see Figure 14). The highest histamine formation was detected for 30 seconds
irradiation with UV-A light.
The baseline histamine content was 5.7 ± 0.3 ng/ml, after 30 seconds UV-A light
irradiation the histamine content was 6.9 ± 0.5 ng/ml, after 60 seconds 6.1 ± 0.4
ng/ml, after 90 seconds 6.6 ± 0.3 ng/ml and after 120 seconds 6.2 ± 0.1 ng/ml.
After 30 seconds UV-B light irradiation the histamine content was 2.6 ± 0.7 ng/ml,
after 60 seconds 3 ± 0.2 ng/ml, after 90 seconds 4.3 ± 0.8 ng/ml and after 120
seconds 2.7 ± 0.5 ng/ml.
A strong decrease in histamine was also measured after steaming with both ozone
concentrations, 10 μg/ml and 100 μg/ml: histamine levels decreased from 5.7 ± 0.3
ng/ml (baseline) to 2.3 ± 0.1 for 10 μg/ml ozone and 2.8 ± 0.6 for 100 g/ml ozone,
63
respectively. There was no time-depending trend for and increase or decrease of
histamine recognizable, whether for UV-A light nor for UV-B light.
Figure 14. Histamine in human tears before and after UV light irradiation and
steaming with ozone; error bars display ± 1 SD.
For the pilot study we decided to use an irradiation of 20 seconds. We assumed
that shorter irradiation periods would not have lead to any effects and longer
irradiation periods would have lead to an evaporation of tears and thus false
positive results.
As results from ozone measurements provided no reliable results we decided not
to perform further experiments in the pilot study.
8.4. Determination of cytokines in human tears
8.4.1 ProcartaPlex kit
First experiments were made with the ProcartaPlex kit from ThermoFisher
Scientific, Austria. The basal cytokine levels in tears were 28.65 pg/ml for IL-1
beta, 200.66 pg/ml for IL-6 and 49.71 pg/ml for TNF-alpha. For IFN-gamma the
level was below the detection limit. Irradiation of tears with UV light for 10 minutes
64
led to an increase for UV-A and a decrease for UV-B. After steaming with 10 g/ml
and 100 g/ml ozone cytokine levels strongly decreased (see Table 10).
Table 10. Cytokine determination (ProcartaPlex Kit).
IL-1 beta IL-6 IFN-gamma TNF-alpha
Tears 28.65 200.66 OOR < 49.71
Tears + UV-A 10 min. 66.97 369.34 OOR < 142.86
Tears + UV-B 10 min. 24.84 78.79 OOR < *30.29
Tears + Ozone 10 μg/ml *7.72 OOR < OOR < *11.51
Tears + Ozone 100 μg/ml *5.40 OOR < OOR < *4.34
OOR< = out of range below; *Value extrapolated beyond standard range. Values are displayed in pg/ml.
The second cytokine kit from BioRad provided completely different results.
Cytokine levels were 2.47 pg/ml for IL-1 beta and 20.75 pg/ml for IFN-gamma
(both values extrapolated). For IL-6 and TNF-alpha levels were below the standard
range. Irradiation with UV-A light led to a slight increase of IFN-gamma after 30
seconds, 1, 3 and 5 minutes. For IL-1 beta no differences could be detected. IL-6
and TNF-alpha were still not detectable (see Table 11).
Table 11. Cytokine determination (BioPlex Kit). IL-1 beta IL-6 IFN-gamma TNF-alpha
Tears *2.47 OOR < *20.75 OOR <
Tears + UV-A 30 sec. *2.31 OOR < *34.67 OOR <
Tears + UV-A 1 min. *3.28 OOR < 59,92 OOR <
Tears + UV-A 3 min. *2.47 OOR < 83,30 OOR <
Tears + UV-A 5 min. *2.79 OOR < 83,30 OOR <
Tears + UV-B 30 sec. *2.87 OOR < 83,30 *0.65
Tears + UV-B 1 min. *2.63 OOR < 53,83 OOR <
Tears + UV-B 3 min. *2.47 OOR < OOR < OOR <
Tears + UV-B 5 min. *2.47 OOR < *4.49 OOR <
OOR< = out of range below; *Value extrapolated beyond standard range. Values are displayed in pg/ml.
65
As results from cytokine measurements provided no reliable results we decided
not to perform further cytokine experiments in the pilot study.
8.5. Pilot study
It was planned to include 20 patients in the pilot study, the main target was the
histamine content before and after UV light irradiation. After performing the first
histidine/histamine measurements we decided to stop patient recruitment: In four
out of five patients’ histamine was not found in human tears, neither before nor
after irradiation with UV light. Answering of the main question was thus not
possible. The results of the ophthalmological examination, blood test, conjunctival
scraping and histamine/histidine measurements of the first five patients are
displayed below. A statistical evaluation of the results was not reasonable due to
small sample size.
Table 12. Characterization of study participants.
Patient Number Sex Age
1 female 26
2 female 55
3 female 58
4 female 73
5 male 74
First, ophthalmological symptoms were assessed with a questionnaire: the
symptom scores were defined as follows: 0= none of the time; 1= some of the
time; 2= half of the time; 3= most of the time; 4= all of the time. All patients
suffered from itching, tearing and light sensitivity at least some of the time (see full
results in Table 13).
At the slit lamp, corneal and conjunctival parameters were assessed. For all
ophthalmological examinations, the following scores were used: 0= none; 1= mild;
2= moderate; 3= severe. All patients had mild to moderate conjunctival injection,
66
conjunctival edema was detected in one only patient and lid edema had not been
detected in any of the patients.
Table 13. Subjective symptoms.
Patient
number Itching Redness Tearing Burning
Foreign
body
sensation
Light
sensitivity
1 1 2 1 3 1 1
2 2 0 1 2 1 1
3 1 0 2 0 0 1
4 2 2 1 2 0 2
5 2 1 3 2 1 1
Papillae were detected in two patients only, for both in the upper eyelid on the left
and right eye: a mild grade for patient number one and a moderate grade for
patient number five. Follicles were detected in the lower lids of both eyes of patient
number one and on the upper and lower lids from the left and right eye of patient
number two. Patient number two had moderate follicles in the upper lid of the right
eye and mild follicles in the lower lid of the right eye and on both lids of the left
eye.
The results of fluorescein-break up time (F-BUT) measurements and results of
corneal and conjunctival staining are displayed in Table 14. All patients had a
shortened break-up time, which indicates an instability of the tear film. Patient one,
four and five have a documented medical history of dry eye, which is known to be
associated with an instability of the tear film.
When analysing conjunctival scrapings, we could not detect eosinophil
granulocytes in any of the samples. IgE levels were below the detection limit
(<18.10 IU/l) for patients one, two and three. For patient number four the levels
were 26.6 IU/ml and for patient number five 24.1 IU/ml. All values were within the
normal range of IgE in blood.
67
Table 14. Fluorescein-break-up time (in seconds), and corneal and conjunctival
staining; OD= oculus dexter (right eye), OS= oculus sinister (left eye).
8.5.1 Histamine analysis
The external calibration for L-histidine (12.5, 25, 100, 125, 300, 500, 1000 nM) and
histamine (1.25, 2.5, 10, 12.5, 30, 50, 100 nM) is shown in Figure 15 and 16.
Linear calibration ranges were 1.4 to 100 nM for histamine (n=3) and 11 nM to
1 µM for L-histidine (n=3) with a coefficient of determination of 99.2% and 97.7%
respectively. Lower limit of detection (LLOD) and lower limit of quantification
(LLOQ) were calculated according to equation 2 and 3 with pseudo blanks
24.1/75/0.9 (v/v/v) H2O/ACN/NaCl, which should imitate the tear liquid sample
after purification. The LLOD for histamine was 0.4 nM and the LLOQ 1.4 nM. The
limit of detection and quantification for histidine was 3.4 nM and 11 nM.
Histamine could be detected in the sample of subject number five only, after
irradiation with UV-A and UV-B light the histamine level slightly increased.
Histidine was detected in all five samples, in sample number one the histidine
content sank after irradiation, in samples two, three and four the histidine content
increased, higher for UV-A than for UV-B light. In sample number five, histidine
content increased, higher for UV-B than for UV-A light (see Table 15 and Figure
17). There was no trend for an increase or decrease of histidine and histamine
recognizable.
Patient
number
F-BUT
OD
(in sec.)
F-BUT
OS
(in sec.)
Corneal
staining
OD
Corneal
staining
OS
Lissamine
green
staining
OD
Lissamine
green
staining
OS
1 6.7 5.2 0 0 0 1
2 12.3 9.0 0 0 0 0
3 7.3 8.0 0 0 1 0.75
4 3.3 2.7 0 0 0.75 2
5 4.7 5.3 0.75 0.5 2 0.75
68
Figure 15. External calibration curve of histamine, red circles with error bars show standard deviation; black circles show measured values (n = 3) for each concentration.
Figure 16. External calibration curve of L-histidine, red circles with error bars show standard deviation; black circles show measured values (n = 3) for each concentration.
69
Table 15. Quantitative estimation of histamine and histidine in human tears before and after UV light irradiation.
BDL= below lower detection limit (0.0025 µM).
Figure 17. Histidine and histamine levels in tears measured by LC-MS.
8.6. Histamine in alder and hazel pollen
Histamine was detected in alder as well as in hazel pollen. The mean histamine
level of alder pollen was 5.9 ng/ml, for hazel it was 9.9 ng/ml. Irradiation of pollen
Quantitative estimation (µM)
Patient
number Histamine
Histamine
+ UV-A
20 sec.
Histamine
+ UV-B
20 sec.
Histidine
Histidine
+ UV-A
20 sec.
Histidine
+ UV-B
20 sec.
1 BDL BDL BDL 0.423 0.362 0.179
2 BDL BDL BDL 2.604 5.061 4.792
3 BDL BDL BDL 0.859 1.354 1.012
4 BDL BDL BDL 1.233 1.405 1.233
5 0.00727 0.00862 0.00852 1.854 2.018 2.225
70
solutions with UV light led to an increase of histamine, the longer the irradiation
period the higher the histamine content (128).
Alder pollen
After 2 hours of irradiation with UV-A light the histamine content of alder pollen
increased to 8.8 ng/ml, after 4 hours to 9.6 ng/ml and after 6 hours to 10.1 ng/ml.
For UV-B irradiation the histamine content was 11.4 ng/ml after 2 hours, 13.1
ng/ml after 4 hours and 24.6 ng/ml after 6 hours. Irradiation with sunlight for 1 day
(corresponds to approximately 10 hours’ irradiation) led to an increase of
histamine to 15.7 ng/ml (see Figure 18) (128).
Figure 18. Histamine content of alder pollen after UV light and sunlight irradiation.
Reproduced from Heidinger et al. with permission of publisher (Taylor and
Francis).
Hazel pollen
After 2 hours of irradiation with UV-B light the mean histamine level of hazel pollen
was 12.6 ng/ml, after 4 hours it was 16.6 ng/ml and after 6 hours it was 22.8 ng/ml.
For UV-B irradiation the histamine content was 18.1 ng/ml after 2 hours, 23.4
ng/ml after 4 hours and 39.5 ng/ml after 6 hours. Irradiation with sunlight for 1 day
(corresponds to approximately 10 hours’ irradiation) led to an increase of
histamine to 32.5 ng/ml (see Figure 19) (128).
71
Figure 19. Histamine content of hazel pollen after UV light irradiation and sunlight
irradiation. Reproduced from Heidinger et al. with permission of publisher (Taylor
and Francis).
8.7. Polyacrylamide gel electrophoresis
Separation of pollen proteins with polyacrylamide gel electrophoresis revealed that
both UV light and ozone, respectively had an influence on the pollen protein
spectrum: Irradiation with UV light and steaming with ozone led to an alteration
and partly destruction of pollen proteins.
Figure 20 and 21 illustrate protein bands disappearing after two days of irradiation
with UV-A, UV-B and sunlight (one day of irradiation provided similar results but
not that obvious; results not shown).
For alder pollen effects were stronger for UV-B than for UV-A, with sunlight
irradiation effects seemed to be the same as for UV-B. Steaming with ozone
resulted in nearly the same effect as irradiation with UV-A light. Four protein bands
between 3.5 and 30 kDa partly disappeared after irradiation.
72
For hazel pollen effects were the strongest for sunlight, effects with UV-A seemed
to be the same as for UV-B. Steaming with ozone resulted in nearly the same
effect as irradiation with UV-A light.
Figure 20. PAGE of alder pollen: Lane A = without irradiation; lane B = with UV-A
light irradiation; lane C = with UV-B light irradiation; lane D = with sunlight
irradiation; lane E = with ozone (100 µg/ml). Arrows highlight proteins that partly
disappeared after irradiation.
Figure 21. PAGE of hazel pollen: Lane A = without irradiation; lane B = with UV-A
light irradiation; lane C = with UV-B light irradiation; lane D = with sunlight
73
irradiation; lane E = with ozone (100 µg/ml). Arrows highlight proteins that partly
disappeared after irradiation.
8.8. Pollen morphology
After irradiating pollen suspensions with UV light, we were able to detect
morphological changes of alder and hazel pollen with light- and SEM microscopy.
The cell wall of irradiated pollen grains seemed to be deformed and pollen seemed
to be more polymorphic after irradiation, compared to non-irradiated pollen. The
longer the irradiation period, the more obvious were the changes. Figure 22 and
23 show non-irradiated pollen and pollen that have been irradiated for two days.
After one day of irradiation pollen also revealed morphological changes but not
that obvious. Alterations could be observed for both UV-A and UV-B light whereas
the difference was much better recognizable for UV-B light. We also investigated
the morphology of pollen in dry condition during irradiation, where we could not
detect any changes.
Figure 22. Alder pollen in physiological saline: A= without irradiation, 400x
magnification; B= without irradiation, 1000x magnification; C= irradiation with UV-A
light for 3 days, 400x magnification; D= irradiation with UV-A light for 3 days,
1000x magnification; E= irradiation with UV-B light for 3 days, 400x magnification;
F= irradiation with UV-B light for 3 days, 1000x magnification.
74
Figure 23. Hazel pollen in physiological saline: A= without irradiation, 400x
magnification; B= without irradiation, 1000x magnification; C= irradiation with UV-A
light for 3 days, 400x magnification; D= irradiation with UV-A light for 3 days,
1000x magnification; E= irradiation with UV-B light for 3 days, 400x magnification;
F= irradiation with UV-B light for 3 days, 1000x magnification.
First images of pollen were made with SEM under normal vacuum. The vacuum
causes a dehydration of pollen and thus a deformation (see Figures 23-25). This
means we were not able to see if UV light induces morphological changes as the
vacuum itself induces changes.
Figure 24. Non-irradiated alder pollen with SEM in normal vacuum.
75
Figure 25. UV-A irradiated alder pollen with SEM in normal vacuum.
Figure 26. UV-B irradiated alder pollen with SEM in normal vacuum.
Using a low-vacuum SEM made it able to detect morphological changes caused
by UV light. Pollen clumped after irradiation (see Figure 26) cell walls of irradiated
pollen grains seemed to be deformed and pollen seemed to be more polymorphic
after irradiation (see Figures 27-29).
Figure 27. Non-Irradiated pollen (A) vs. irradiated pollen (B).
76
Figure 28. Pollen without irradiation; pictures coloured with Pixelmator image
editing program.
Figure 29. Pollen after UV-A irradiation; pictures coloured with Pixelmator image
editing program.
Figure 30. Pollen after UV-B irradiation, pictures coloured with Pixelmator image
editing program
77
8.9. Pollen, bacteria and fungi
PAS and GRAM colouring of alder and hazel pollen revealed that both pollen
species were naturally contaminated with bacteria and fungi. Microbiological
analysis revealed that these were aerobe spore-forming bacteria and mildew (see
Figure 30). We could observe that mildew were mortified due to UV light,
especially with UV-B. This could be seen on the loss of typical purple colour in the
PAS colouring. The mortification is time-dependent; a reduced dyeing could be
observed upon the second day of irradiation. No mortification was detected with
UV-A light (see Figures 31 and 32).
Figure 31. Alder pollen with fungi after PAS staining.
Figure 32. UV-A light irradiated alder pollen and fungi after PAS staining.
78
Figure 33. UV-B light irradiated alder pollen and fungi after PAS staining.
8.10. Cell culture
Cell culture experiments revealed that the viability of cells decreased after
incubation with pollen. The decrease in cell viability was higher when using
irradiated pollen compared to non-irradiated pollen. The following results are
shown in percentage of cell viability ± 2 SE (128).
8.10.1 Alder
Cell viability of control cells (incubated with DMEM) was 100 % (± 15.08).
Incubation with non-irradiated alder pollen (10 mg/ml) led to a decrease of cell
viability to 86.55 (± 15.51), compared to the control the difference was not
significant (p>0.05; see Table 16).
When using pollen that have been preliminary irradiated for one day the cell
viability significantly decreased to 31.97 (± 5.58) for UV-A and 61.33 (± 5.02) for
UV-B (p<0.001) compared to the control. The decrease in cell viability between
irradiated and non-irradiated pollen was also significant for UV-A (p<0.001) and
UV-B (p=0.039).
When testing supernatants of pollen extracts experiments results revealed that
supernatants were less harmful than whole pollen suspensions. After incubation
with alder pollen supernatants the cell viability decreased to 93.97 (± 9.95),
compared to the control the difference was not significant (p>0.05). When using
79
pollen supernatants that have been preliminary irradiated for one day the cell
viability decreased to 90.70 (± 10.39) for UV-A and 61.64 (± 8.55) for UV-B.
Compared to the control the difference was statistically significant for UV-B
(p<0.001) but not for UV-A (p>0.05).
When comparing pollen supernatants with and without irradiation we observed
statistically significant results for UV-B (p=0.003) but not for UV-A (p>0.05; full
results shown in Table 16) (128).
8.10.2 Hazel
Cell viability of control cells incubated with DMEM was 100 % (± 12.53).
Incubation with non-irradiated hazel pollen (25 mg/ml) led to a decrease of cell
viability to 59.37 (± 10.99), compared to the control the difference was significant
(p=0.012).
When using pollen that have been preliminary irradiated for one day the cell
viability significantly decreased to 12.50 (± 6.48) for UV-A and 4.97 (± 2.38) for
UV-B (p<0.001) compared to the control. The decrease in cell viability between
irradiated and non-irradiated pollen was also significant for UV-A and UV-B
(p<0.001).
As observed for alder pollen supernatants of hazel pollen were also less harmful
than whole pollen suspensions. After incubation with hazel pollen supernatant the
cell viability decreased to 88.59 (± 6.37), compared to the control the difference
was not significant (p>0.678).
When using pollen supernatants that have been preliminary irradiated for one day
the cell viability decreased to 62.43 (± 13.04) for UV-A and 38.20 (± 13.76) for UV-
B. Compared to the control the difference was statistically significant for UV-B
(p=0.002) but not for UV-A (p=0.073; full results shown in Table 16) (128).
80
Table 16. MTS-test results of alder and hazel pollen.
Alder Cell viability in %
(± SE) Hazel
Cell viability in %
(± SE)
Cells (control) 100 (± 15.08) Cells (control) 100 (± 12.53)
Pollen suspension
Cells + Alder 10 mg/ml 86.55 (± 15.51) Cells + Hazel 25 mg/ml 59.37 (± 10.99) *
Cells + Alder 10 mg/ml
+ UV-A 31.97 (± 5.58) * †
Cells + Hazel 25 mg/ml +
UV-A 12.50 (± 6.48) * †
Cells + Alder 10 mg/ml
+ UV-B 61.33 (± 5.02) * †
Cells + Hazel 25 mg/ml +
UV-B 4.97 (± 2.38) * †
Pollen supernatant
Cells + Alder 10 mg/ml 93.79 (± 9.95) Cells + Hazel 25 mg/ml 88.59 (± 6.37)
Cells + Alder 10 mg/ml
+ UV-A 90.70 (± 10.39)
Cells + Hazel 25 mg/ml +
UV-A 62.43 (± 13.04) *
Cells + Alder 10 mg/ml
+ UV-B 61.64 (± 8.55) * †
Cells + Hazel 25 mg/ml
+ UV-B 38.20 ± 13.76) * †
* Value indicates statistically significant difference (p<0.05) between pollen and control
† Value indicates statistically significant difference (p<0.05) between non-irradiated pollen and
irradiated pollen
8.11. Cell Imaging
During the experiments, all wells were repeatedly evaluated visually with a phase-
contrast microscope to see, whether pollen influence the morphology or
adherence of the cells. The evaluation revealed two substantial phenomena:
(1) Despite several washing steps after incubation, pollen could not be fully
removed from the wells (see Figure 33).
(2) A marked loss of cells could be detected for all the wells where irradiated
pollen solutions had been used (see Figure 34 and 35). This was not the case for
control cells (see Figure 36). For non-irradiated pollen solutions, a minimal loss of
cells could be detected (see Figure 33).
81
Figure 34. Cells with non-irradiated pollen after washing steps.
Figure 35. Cells with UV-A irradiated pollen after washing steps.
Figure 36. Cells with UV-B irradiated pollen after washing steps.
82
Figure 37. Cells in DMEM (control).
8.12. xCELLigence analysis
We assessed the proliferation of conjunctival cells after incubation with irradiated
and non-irradiated pollen with the xCELLigence real time analysis system. We
compared pollen suspensions as well as pollen supernatants.
First experiments were carried out with pollen suspensions: alder pollen (10
mg/ml) were irradiated with UV-A and UV-B light for four days and analysed with
the xCELLigence system.
Results immediately revealed that pollen suspensions were not suitable for
analysing with xCELLigence. The principle of the measurement is a change in
impedance reflecting cell adherence and proliferation. Pollen itself change the
impedance of the wells of the E-Plate. This was recognized through the increasing
cell index of the blank (red line in Figure 37). The blank is the negative control,
which proofs that the test substance itself does not interfere with the electrodes on
the plate. As the wells with the blank do not contain any cells the cell index has to
be zero during the whole experiment.
83
Figure 38. xCELLigence analysis of non-irradiated and irradiated alder pollen
suspensions.
Therefore, we were compelled to use pollen supernatants for further use. All pollen
solutions were prepared in half medium and half physiological saline, thus we also
prepared the control. When comparing the diluted medium with the undiluted one
we could observe that the proliferation rate of cells in half physiological saline (half
medium is somewhat lower, but cells still proliferate normal (see Figure 38).
Figure 39. xCelligence growth curve of CHANG cells with DMEM and diluted DMEM with NaCl (ratio 1+1). Testing pollen supernatants revealed a strong influence of pollen on the ability of
cells to grow and proliferate. In comparison to a normal proliferation rate of control
cells, proliferation rate of cells incubated with pollen decreased after contact with
alder or hazel pollen solutions.
84
Cells treated with non-irradiated pollen stopped to proliferate after pollen contact,
which could be seen on the decrease of the growth curve. This indicates that
pollen had a cytostatic effect on conjunctival cells (see green lines in Figure 39
and 40).
Irradiated pollen exhibited a strong cytotoxic effect on conjunctival cells. Within a
few minutes after test solutions were added, there was a sharp decline of the
growth curve and the cell index decreased. This indicates that cells detached from
the surface of the wells. These results are consistent with the results of the MTS-
tests, where we also detected a loss of cells after pollen incubation.
For alder pollen, effects were stronger for UV-A (blue line in Figure 39) and UV-B
light (pink line in Figure 39), for sunlight effects were more or less the same as for
non-irradiated pollen (turquoise line in Figure 39) (128).
Figure 40. xCELLigence growth curve of CHANG cells and alder pollen.
Reproduced from Heidinger et al. with permission of publisher (Taylor and
Francis).
For hazel pollen, effects were also stronger for UV-A (blue line in Figure 40) and
UV-B light (pink line in Figure 40), for sunlight effects were nearly the same as for
non-irradiated pollen (turquoise line in Figure 40). For non-irradiated hazel pollen,
there was a sharp decline recognizable approximately four hours after adding the
pollen supernatant to the cells. This indicates that pollen supernatant itself has a
85
negative impact on the cells but in comparison to irradiated pollen the effect is
delayed (128).
Figure 41. xCELLigence growth curve of CHANG cells and hazel pollen.
Reproduced from Heidinger et al. with permission of publisher (Taylor and
Francis).
86
9. Discussion
Our experiments revealed that UV light is able to induce alterations of tear film
ingredients as well as alterations of pollen ingredients and pollen morphology.
9.1. UV light measurements
Before carrying out all experiments we made irradiance measurements of the UV
lamps and also of natural sunlight under different conditions. We detected
alterations in the irradiation of the UV lamps: in the middle of the lamp the
irradiation was the highest, to the sides it declined. To ensure same conditions for
all further experiments attention was paid to always put the vessels to be irradiated
in the middle of the lamp. Also differences in the sunshine duration and differences
in the irradiance from day to day were measured. As the irradiance was higher on
sunny days all samples were irradiated without exception on these days while
continuously monitoring the irradiance.
9.2. UV light induced histamine formation
In our study, we prepared histidine solutions in different concentrations and could
demonstrate that UV-A light and UV-B light are capable of inducing the formation
of histamine from histidine. Histamine concentrations increased with duration of
irradiation. There were also higher histamine levels when using higher
concentrated histidine start solutions. Interestingly we detected a higher histamine
formation when using sodium chloride instead of aqua dest. as solvent for
histidine. We cannot explain these discrepancies at the moment. As we detected
higher histamine concentrations with sodium chloride it was used for all further
experiments.
The amino acid histidine is the early stage in the formation of histamine. For
conversion into histamine the enzyme histidine decarboxylase is necessary. The
fact that UV light is able to convert histidine to histamine was published in 1928 by
Ellinger et al. and later analysed by Bourdillon et al. again (116–119).
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Unfortunately, the published results were inconclusive. Ellinger was able to detect
histamine after irradiating histidine (dissolved or in powder form) with UV light for
several time periods. Histamine was detected pharmacologically by injecting the
irradiated solutions into the small intestine of guinea pigs followed by monitoring
the intestine excitation. He also found out that UV light is not just able to convert
histidine into histamine but also to destroy histamine especially when longer
irradiation periods were used (116). In 1930 Bourdillon et al. repeated Ellingers
experiments and interestingly they were not able to detect a histamine formation
when using wavelengths >290 nm (belongs to irradiation with UV-A und UV-B).
When using wavelengths > 260 nm (belongs to irradiation with UV-A, -B and -C)
low histamine formation was detectable. When using wavelengths <280 nm or
>550 nm (which belongs to UV-C and visible light) they could detect a high
histamine formation (118). One of the reasons for this discrepancy might have
been the lack of suitable detection methods at that time. As late as in 1934 Peter
Holtz was able to detect histamine with chemical methods which are far more
accurate than the pharmacological analyses (120).
In our experiments, we were able to detect a histamine formation with UV-A und
UV-B light. We did not test UV-C as it almost does not appear at the earth´s
surface and therefore has no real consequence for human health. As expected
UV-B had stronger effects in histamine formation as it is known to be more harmful
than UV-A.
In the last years the amount of UV light we are exposed to is dramatically
increasing. This is mainly due to the warmer weather and thus increasing and
prolonged outdoor activities or changed sunbathing habits for cosmetically
reasons (tanned skin as ideal of beauty). The number of skin and eye diseases
also increases as these two organs are constantly exposed to the environment.
Main reasons might be the UV light-induced formation of free radicals, which are
highly reactive and induce cell damage and other pathological cell alterations
(129).
The eye is protected from the environment by the eyebrows and the eyelashes
and due to its location in the orbit. When the sun shines bright it is a normal
reaction to squint and screw up the eyes. The pupil gets constricted which
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minimizes the amount of sun rays getting into the eye. Due to strong ground
reflection from snow, water or sand the irradiation is increased leading to
pathological effects on the eyes, e.g. photokeratitis or photoconjunctivitis. As the
amount of UV radiation increases we assumed that UV light might also have an
influence on ingredients of the tear film leading to pathological alterations and the
development of different diseases. Thus, we investigated the effect of UV light on
histidine and histamine in human tears before and after irradiation.
9.3. Histamine content in human tears before and after irradiation
Histidine is present in human tears in concentrations of about 1.9 ± 0.7 μM in
basal tears and 3.2 ± 1.9 μM in reflex tears. It seemed interesting to investigate if
UV light and ozone are able to promote the formation of histidine to histamine in
human tear fluid (122). This might cause discomfort similar to symptoms caused
by allergic reactions. Histamine is a major mediator of allergic reactions, high
levels lead to itching, redness or burning on the ocular surface (20,130).
The standard values of histamine in tears are reported to be vary variable. Kari et
al. report normal levels of histamine in tears with 3.5 nmol/L (= 31.5 ng/ml),
Abelson et al. reported values from from 2.2 to 36 ng/ml with a mean of 10.3
ng/ml, in another study they reported values of 0.86 ± 0.23 ng/ml. In allergic
patients histamine levels in tears are reported to be higher than in control patients
(131–133).
In our study, the histamine content in tears from a non-allergic patient was 5.7 ±
0.3 ng/ml. We detected a slight increase in histamine when irradiating human tears
with 30, 60, 90 or 120 seconds UV-A light. The histamine formation was the
highest with 30 seconds irradiation. But 30 seconds irradiation do not reflect
physiological conditions, therefore we had to consider the optimal irradiation time.
Too short irradiation periods would not have led to any effects in histidine
depletion/ histamine formation. In average humans blink about 17 times per
minute when at rest, which means approximately every three to four seconds.
During conversation the levels are higher (~ 26 times per minute) and while
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reading blink rates are lower (~ 5 times per minute) (134). Finally we decided to
take a mean value and irradiated all tear samples with UV light for 20 seconds.
First intentions were to analyse all samples with an ELISA assay. Since the
required amount of sample volume is too high for tear samples (20 μl) we decided
to use a mass spectrometric based method, as this method normally requires a
reduced sample volume. It is also a good alternative for immunoassays as it has a
great specificity and short analysis time (135).
Other than supposed, the LC-MS/MS method could not be developed with a
smaller sample volume than 30 μl. This means the amount of tear fluid we
required for determination of histidine and histamine before and after irradiation
was 100 µl at minimum.
This was only possible by stimulating of tear secretion with a volatile oil. The
average amount of tears that are physiologically present in the conjunctival sac
are 7.0 ± 2.0 µl (136). With stimulation of tear secretion, we induced the production
of reflex tears. The normal tear fluid consists of several hundreds of proteins,
lipids, mucins, metabolites, hormones, etc. Reflex tears are known to differ in their
composition in comparison to natural tears, which has to be concerned when
interpreting the results. There are different methods for tear collection available:
the use of cellulose sponges, Schirmer´s strips or the capillary method we used. It
is known that the collection method could influence the composition of tears too
which makes it difficult to compare studies with different tear collection methods
(137,138). In our study, we used the capillary method for tear sampling as it is a
long approved technique in our department and used by several other well-known
researchers. After evaluating the results of the first patients it emphasized that the
method for histamine measurement is not appropriate to answer the study
question. In five out of six patients histamine was found in human tears neither
before nor after irradiation with UV light.
The major reasons therefore might have been the great amounts of tear fluid we
withdrew from the subjects. Producing reflex tears might have led to an
inadvertently dilution effect.
As histamine was the main target value for this study the study was terminated
early.
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Histidine was detected in all five samples in varying concentrations.
After irradiation, there was just a slight decrease for histidine in sample number
one but no increase of histamine. In the other samples histidine was higher after
irradiation than before. If our theory had been right, there should have been an
increase of histamine and a decrease of histidine after irradiation.
Our results indicated that there might be no effect of UV light on histamine
formation in human tears. The LC-MS/MS method we developed was not suitable
for detecting histamine and histidine in tears. A method that requires less sample
volume should be preferred to further address this issue.
We are aware that concluding from in-vitro tests to situations in-vivo might also be
difficult in this experimental setting. With every blink, the tear film is renewed, the
tear glands continuously produce new tears and old tears flow off the lacrimal
duct. Additionally the daily contact of the eyes, particularly the contact of the tear
film with UV light is different, for outdoor-workers doses may be higher than for
indoor-workers, for sunny days the irradiance is higher than for cloudy days, etc.
So, it is difficult to say which dose of UV light we are daily exposed to and if these
doses really have an effect on the tear film or not. Better experimental set-ups
have to be found to further investigate the question of UV light and its
consequence for tear film ingredients, especially histidine and histamine.
9.4. Cytokines in tears
Cytokines are released from different cell types in immunological or allergic
reactions. We hypothesized that UV light irradiation is also able to increase the
release of cytokines. It is really difficult to investigate this hypothesis: an irradiation
of human eyes is not allowed due to ethical reasons; irradiation of animals eyes
might be possible but it is not clear if reactions are the same in human eyes. The
next point is that it is difficult to do an irradiation in animals because it is a normal
reflex to close the eyes when there is excessive light. Thus, we withdrew tear fluid
from humans and tried to investigate the effect of UV light in vitro. When
withdrawing tear fluid there are always some conjunctival cells within the sample
(contact with the conjunctiva is difficult to avoid), so there might be cells present,
where cytokine can be released when irradiating them with UV light.
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For cytokine measurement in tears we used fluorescence based multiplex assays.
The advantage of multiplex assays in comparison to the traditional ELISA method
is the ability to measure more than one protein in each sample at the same time
(139). It also requires less sample volume than the ELISA method and is not that
expensive. Unfortunately, cytokine measurements with two different multiplex kits
were inconclusive; with the ProcartaPlex Kit we detected an increase of cytokines
after UV light irradiation, with the BioPlex Kit a decrease. The measured cytokine
levels from the ProcartaPlex Kit were approximately in the same range as reported
by other researchers, the cytokine levels from the BioPlex Kit where too low when
comparing it with previous studies. Other researchers also report troubles with
cytokine measurements: according to literature cytokine levels in tears are highly
depending on the analysis technique used. Even when using the same analysis
technique there are great differences. In the paper of Wei et al. results of five
papers measuring IL-1β level with the same technique are described: the mean
level of IL-1β of all five studies together was 39.0 ± 23.6 pg/ml. When looking at
the individual values of each study it exposes that the lowest measured value for
IL-1β in one study was 2.0 ± 1.7 pg/ml, the highest in another study was 101.4 ±
2.8 pg/ml (60), meaning there is great variability though using the same analysis
technique. This makes it very difficult to interpret data and to compare it to data
available in the literature (60).
As already described in section 9.4 tear film ingredients are somewhat different in
basal tears and reflex tears. A study with 270 healthy humans analysed
differences in cytokine levels between basal and reflex tears with an ELISA assay:
as an example in basal tears the concentration for IL-1β were 12.9 ± 2.3 pg/ml, in
reflex tears levels were below the lower detection limit (140). This might also be a
confounding factor as it is often not easy to avoid production of reflex tears when
withdrawing tears.
We assume that the number of conjunctival cells that were present in our tear
samples were too low to investigate the effect of UV light on cytokine release.
Thus, it is difficult to draw a conclusion from the results.
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9.5. Ozone-induced histamine formation
When using ozone for steaming of histidine solutions, we could detect a histamine
formation just when using high ozone concentrations (300 μg/ml). Histamine might
be destroyed as fast as it is formed, because ozone is a strong oxidant with
destructive properties (141). Using such high concentrations does not reflect
physiological conditions wherefore we used lower doses for steaming of tears. We
used ozone in a concentration of 100 μg/ml (the daily levels of ozone are around
80 g/ml) and observed a strong decrease of histamine, the same when using low
ozone concentrations (10 μg/ml). Ozone will lead to immediate destruction of
several tear ingredients. As we hypothesised that ozone will increase the
histamine content we decided not to further test the effect of ozone on histamine
formation in the pilot study.
9.6. Pilot study
As described in section 9.43 the LC-MS method for histamine and histidine
detection was not appropriate. Results from ophthalmological examinations,
conjunctival scraping or blood test did not show any significant differences
between the study participants.
9.6.1 Ophthalmological examinations
Ophthalmological examinations were done to examine if study participants exhibit
any ocular signs of an allergy: At the time of investigtion there were no objective
signs of an allergy present. All subjects for the pilot study were recruited from the
dry eye unit at the Department of Ophthalmology, Medical University of Graz.
Subject one, three and five had a documented medical history of dry eyes.
Papillae and follicles were detected in some of the patients. They are signs of an
ocular allergy but also signs for an active inflammation. Follicles are a response to
chronic mechanical, chemical or microbial irritation, papillae are signs of
conjunctival inflammation.
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Conjunctival scrapings on both eyes and IgE blood test were done, to categorize
the patients as allergic or non allergic too. All patients had IgE in the normal range
of 0-100 IU/ml. The presence of eosinophil granulocytes in conjunctival scrapings
is known to be a hint for an allergy. There were no eosinophil granulocytes present
in any of the conjunctival scrapings. The eosinophil granulocytes contain lots of
granules with toxic proteins that could harm the conjunctiva, cornea or tear film
ingredients. Eosinophils are therefore thought to play a contributory role in the
pathophysiology of other ocular non-allergic diseases (25). Although conjunctival
scraping is often used for allergy diagnostic tool, it is not a really reliable method
for allergy detection. Eosinophil granulocytes might also be present in the
conjunctiva of non-allergy sufferers. Vice-versa not all allergy-sufferers do have
eosinophil granulocytes present in their conjunctiva (142). Another difficulty is that
eosinophils are present not only in the superficial layer but also in deeper layers of
the conjunctiva which could not be reached by scraping. Thus a negative
conjunctival scraping does not mean that there are no eosinophil granulocytes
present in the conjunctiva (143). We recommend that conjunctial scrapings should
never be used as single diagnostic tool but together with a blood test, skin test or
an ophthalmological examination.
9.7. Histamine content of pollen before and after irradiation
Histamine is naturally inherited in living organisms such as plants, microbes and
also pollen (144–147). We could prove that alder and hazel pollen naturally
comprise histamine. When irradiating both pollen species, the histamine content
increased, higher for hazel than for alder pollen. The increase was also higher
when using UV-B light compared to UV-A light. With natural sunlight, an increase
in the histamine content could be detected too.
When the pollen grain gets in contact with a moist surface, for example the tear
film or the nasal mucosa, the pollen grains hydrate and release histamine and a
variety of other substances. These are capable of destroying proteins of the tear
fluid and could thus provoke different eye complaints and non-IgE-mediated
reactions. The residence time of pollen on the ocular surface might be several
hours, meaning there is enough time to trigger different early- and late-phase
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allergic symptoms as well as non-allergic symptoms (148). We assume that the
increase of histamine in irradiated pollen may contribute to higher allergenicity of
pollen and strengthens allergic as well as non-allergic reactions mediated by
pollen.
We carried out our experiments in spring when the irradiance of sunlight was lower
and also the sunshine duration was shorter than in the summer months. Probably
the observed effects will be stronger when investigating pollen species with
flowering periods in summer months. The effects might be also different when
doing these experiments in other countries where the levels of UV radiation are
higher.
9.8. Protein content
Electrophoretic analysis revealed that UV light is able to degrade pollen proteins.
Similar results were obtained by other researchers: Majd et al. found out that the
pollen spectrum from pollen collected in polluted areas differs from those of non-
polluted areas. In their study protein bands between 22 and 45 kDa disappeared,
in our study several protein bands between 3.5 and 60 kDa partly disappeared or
weakend (98). The capability of UV light to degrade proteins is known from earlier
studies (149). The degradation is accompanied by a release of amino acids, which
build up the proteins. The amino acid histidine is the early stage in the formation of
histamine and is beyond other amino acids a naturally inherited component in
pollen grains (115,150). As histidine may also be released through this UV light-
induced degradation process it could – in theory - serve as basis for new
histamine formation and may be an explanation for the increasing histamine
content after irradiation. This theory is supported by the fact that histamine
formation from histidine is also possible due to ultraviolet light
(119,118,117,151,152).
9.9. Pollen morphology
UV light irradiation induced alterations of the pollen surface as detected by SEM
and light microscopy. We were able to detect morphological changes of pollen
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after incubation with UV light, pollen seemed to be more polymorphic and looked
sticky and agglutinated. The agglutination of pollen could be a hint for changes of
the pollen surface. Pollen are composed of an intine and an exine, the intine
encompasses the cell and is mainly composed of cellulose and pectin and is not
that resistant. The main component of the exine is the very resistant sporopollenin,
which is responsible for protecting the pollen grain from numerous physical,
chemical or environmental factors (153,154). It is suspected that wind pollinated
pollen, such as alder and hazel have an increased pollen wall thickness (155).
Thus, it seems very unlikely that the exine of pollen alters the shape due to UV
light irradiation. On the other hand, it is known that UV-B light induces changes of
the cuticular wax composition position of pollen and causes membrane changes. It
triggers the production of free radicals which leads to decreased activities of
antioxidant enzymes and increased lipid peroxidation on the pollen surface
(101,156,157). Previous studies also detected altered shapes of pollen grains from
polluted areas compared to pollen from non-polluted areas. Airborne particles and
atmospheric fine dust accumulated on the surface of the pollen grains, detected by
SEM (98). Due to pollutants, a release of pollen material out of the pollen is
triggered which than agglomerate on the surface of pollen grains. In our light
microscopic images, we were able to detect alterations of the pollen shape and of
the surface (looking like an evagination of the pollen content). We assume that
these alterations might be agglomerations of pollen material as it has been
described previously (98,158).
The impact of these UV light-induced changes of the pollen surface is not clear at
that time. We assume that these changes, together with all the other observed
alterations are responsible for the strengthening of pathological effects of pollen.
9.10. Pollen, bacteria and fungi
As a microbiological analysis revealed, pollen grains are naturally contaminated
with bacteria and fungi. Viruses or bacteria are known to induce the histamine
production and the histamine release (151,152). All pollen can harbour several
species of bacteria and fungi which are suspected to be a part of the allergenic
96
effect of pollen, for example gram-negative bacteria produce endotoxins which can
act as an adjuvant in promoting the initial sensitization to pollen allergens (42,159).
If the increase of histamine may be among others due to involvement of these
microorganisms could not be answered in this study and has to be further
examined. One possibility might have been to sterile filter all pollen solutions, but
beyond microorganisms also all pollen grains would have been filtered out. Thus
we would not have been able to answer the main study question.
In previous experiments, we compared pollen supernatants with and without sterile
filtration to find out, whether the contamination with microorganisms also influence
the cell viability. As we could not find any significant differences between sterile
filtered and non-sterile filtered pollen supernatants we decided to use non-sterile
filtered pollen supernatants for the cell culture experiments to reproduce the
effects of pollen as natural as possible.
9.11. Cell Culture
Allergic reactions typically occur in the lung, nose or eyes where they evoke
different symptoms. In our study we focused on the effects on the eyes, especially
on the conjunctiva as this tissue is permanently exposed to the environment. We
used the MTS test and the xCELLigence real time analysis system to study the
effects of pollen on cell viability and proliferation. The MTS test is an easy to use,
accurate and rapid test to measure the cell viability. Its principle is the ability of
viable cells to reduce the MTS reagent to a coloured formazan product due to
mitochondrial activity. The colorimetric reaction can be measured photometrical
and is directly related to the number of viable cells in the well (160). MTS-test
results revealed a decrease of cell viability when incubating human conjunctival
cells with alder and hazel pollen. The decrease of cell viability was stronger when
using pollen that have been preliminary irradiated with UV light which indicates
that UV light changes pollen components thus making them more harmful for
conjunctival cells.
As previously described we could show that UV light induces several alterations of
pollen, from change in ingredients to change in morphology. If the decrease of cell
97
viability is due to alterations of pollen ingredients or due to alterations of the pollen
surface cannot be answered at the moment. Further studies are needed to
address this question.
We compared, whether pollen suspensions (with pollen grains) and pollen
supernatants (without pollen grains) had the same effect on conjunctival cells.
Experiments revealed that pollen suspensions were much more harmful than
supernatants. This indicates that irradiation might lead to alterations of pollen that
strengthen the negative effects on cell viability and cell adherence. It is known that
peptidases in pollen can disrupt epithelial tight junctions by degrading the
extracellular domains of these proteins. This causes an impairment of the
epithelial barrier or cell membrane and thus an influx of harmful substances into
the cell which in further case might influence the cell viability (161). Further we
assume that pollen grains have an influence on the cells, not only in a chemical
way but also in a mechanical way. Due to contact with the pollen grains cells might
be irritated which influences the viability, cell-to-cell interaction and the adherence.
These facts have to be considered when interpreting the results of the MTS test:
the MTS test measures the mitochondrial activity, which is directly correlated with
the number of cells. In our study, cells were partially washed away which led to a
lower cell viability even if pollen had an effect or not. We suggest that the
outcomes of the cell culture experiments thus should not be seen as definite
values. But all in all, we can conclude that there is an effect of pollen on cell
viability and this effect is greater, when using pollen that have been irradiated with
UV-A or UV-B light before.
Additionally, we detected a great influence of pollen on cell proliferation. Cells
stopped to proliferate after contact with pollen as detected with the xCELLigence
real time analysis system. The xCELLigence system uses special microtiter plates
equipped with gold microelectrodes at the bottom, which non-invasively monitor
the viability and proliferation of cultured cells using electrical impedance as
readout. The measurement-intervals can be individually chosen by the operator,
which makes the system appropriate for every kind of cell line (from slow to fast
growing) and every kind of experimental setting.
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The xCELLigence real time analysis system has some advantages compared to
standard laboratory test as MTS, MTT, XTT or LDH. Standard assays only provide
endpoint data, they are also more time consuming and often more expensive.
Through the continuous monitoring throughout the entire course of the experiment
it is possible to distinguish between several effects: one can distinguish between
cytotoxic effects (cell death or decreased cell viability) or cytostatic effects
(reduced cell proliferation or disability to grow) (162).
Cytotoxicity can be caused by environmental or physical factors (radiation, heat) or
chemical factors (noxious substances). This can result in a variety of cell fates
such as necrosis or apoptosis including cell lysis, loss of membrane integrity, rapid
swelling or shut down of the metabolism.
Cytostatic effects are a special category of cytotoxicity. Cells remain alive but fail
to grow and divide.
First experiments with the xCELLigence system were done with pollen
suspensions without cells: as expected pollen itself caused a change in
impedance thus influencing the whole measurement. Therefore, for further
experiments we used pollen supernatants. We could detect a marked effect of
pollen supernatants on cell viability and proliferation. When using irradiated pollen
supernatants, the effects were much greater.
Our study results indicate that non-irradiated alder and hazel pollen supernatants
have a cytostatic effect on cells and irradiated pollen supernatants have a
cytotoxic effect on pollen.
It is known that ingredients of pollen can cause several allergic and non-allergic
reactions at the ocular surface. Pollen grains contain many proteolytic enzymes
and oxidases, which produce reactive oxygen species (ROS) and lead to oxidative
stress within minutes. Through pollen contact the release of pro-inflammatory
cytokines such as interleukin-6 (IL-6), interleukin-8 (IL-8) or tumour necrosis
factor–α (TNF-α) and thus local immune responses in airway or conjunctiva can be
triggered (163,164). We hypothesize that an irradiation of pollen may strengthen
these effects thus provoking more symptoms on the eyes, in the nose or in the
lung.
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10. Conclusion
With our experiments, we could show that UV light is capable of changing pollen
components thus making them more harmful for conjunctival cells. The increasing
amounts of histamine after irradiation and the alteration of pollen ingredients and
morphology may contribute to allergic and non-allergic complaints (SNAC
syndrome) and support the theory that environmental factors contribute to the
increased number of patients suffering from allergic diseases.
It would be interesting to investigate whether these effects could be also observed
for other pollen species. Further experiments will be needed to answer this
question and to reveal possible other alterations of pollen ingredients due to UV
light.
10.1. Answers of the main study questions
1. Are UV light and ozone capable of converting histidine to histamine?
Yes, UV light and ozone are capable of converting histidine to histamine.
2. Does UV light influence the histamine and histidine content of human tears?
We´re not sure - results were inconclusive.
3. Does UV light influence the cytokine content of human tears?
We´re not sure - a more suitable detection method and better experimental setting
should be selected to further investigate this question.
4. Is UV light capable of altering pollen ingredients?
Yes, UV light is able to increase the histamine content of pollen.
5. Is UV light capable of altering the protein content of pollen?
Yes, UV light is able to alter and destroy proteins of pollen.
6. Is UV light capable of altering the morphology of pollen?
Yes, the pollen wall seems altered after UV light irradiation.
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6. Does UV light influence the allergenic potential of pollen?
Yes, UV light-irradiated pollen were more harmful for conjunctival cells.
8. Does an UV light-irradiation of pollen influence the viability and proliferation
of human conjunctival cells?
Yes, viability and proliferation of human conjunctival cells decreased after
incubation with pollen. The decrease was greater when using pollen that were
irradiated with UV light before.
101
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