Department of Oral Pathology and Medicine, Institute of Dentistry, University Of

Helsinki, Helsinki, Finland.

Department of Medicine, Institute of Clinical Medicine, University Of Helsinki,

Helsinki, Finland.

Department of Anatomy, Institute of Biomedicine, University Of Helsinki,

Helsinki, Helsinki, Finland.

ORAL IMMUNE DEFENSE

against

CHRONIC HYPERPLASTIC CANDIDOSIS

Ahmed S. Ali Musrati

Academic dissertation

2

Supervised by

1. Professor Yrjö T. Konttinen, M.D, Ph.D., Head of Biomaterial and Inflammation Research Center, Institute of Clinical Medicine, Department of Medicine, Biomedicum Helsinki, P.O. Box 700, 00029 HUS, Finland.

2. Professor Jarkko Hietanen, M.D., Ph.D., D.D.S., M.Sc., Department of Oral

Pathology, Institute of Dentistry, PL 41, 00014 University of Helsinki, Finland.

Reviewed by

1. Docent Ilmo Leivo, MD, PhD. Department of Pathology. Haartman Institute. University of Helsinki. Helsinki, Finland.

2. Professor Stephen Porter, BSc MD PhD FDS RCSE FDS RCS Professor of

Oral Medicine, Chairman of the Division of Maxillofacial Diagnostic Medical and Surgical Sciences, UCL Eastman Dental Institute 256 Grays Inn Road London WC1X 8LD, UK.

Opponent

Docent Aaro Miettinen, MD, PhD. Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, Helsinki, Finland.

ISBN 978-952-92-3870-5 (Paperback) ISBN 978-952-10-4702-2 (PDF) Helsinki 2008 Yliopistopaino

3

This thesis is dedicated to..... ... the memory of my late mother, Zakia Mohammed... (1948-1998)

4

CONTENTS

LIST OF ORIGINAL PUBLICATIONS ……………………………………...........7

ABBREVIATIONS …………………………………………......................................8

ABSTRACT…………………………………………………………………..............10

REVIEW OF THE LITERATURE………………………………………………...12

Anatomical & histological review of the oral cavity……………………....12

Fungi: Taxonomy and characteristics...…...…………………………….....17

Candida albicans……………………………………………………………..18

Taxonomy……………………………………………………....................18

Morphology……………………………………………………………….19

Biofilm formation………………………………………………………...20

Biological behavior……………………………………………………….21

Culture media……………………………………………………………..22

Identification……………………………………………………………...22

1) Pathophysiology of Candida albicans infections.…………………………24

Relationship between C. albicans and the host…………………………..24

C. albicans as a component of the host microflora…………….......24

When does C. albicans turn pathogenic?..........................................25

Role of C. albicans in nosocomial infections & candidaemia……..25

Oral candidosis…………………………………………………………….25

Definition and epidemiology……………………………………...25

Etiology and predisposing factors……………………………..…..26

Clinical variants................………………………………………....29

Chronic hyperplastic candidosis…………………………………...31

Clinical manifestation………………………………………31

Histopathology……………………………………………..31

Diagnosis…………………………………………………………..32

Management……………...………………………………………..32

Leukoplakia …………………………………………………………….....35

2) Host defense against oral candidosis………………………………………...37

Nonspecific host defense………………………………………………….37

5

1) Toll-like receptors………………………………………………..38

2) Proteins…………………………………………………………..38

i) Natural antimicrobial peptides…………………………...38

a) Defensins……………………………………………39

b) Histatins……………………………………………40

c) Protegrins…………………………………………….40

ii) Cytokines and Chemokines………………………………40

a) Interleukin-8………………………………………42

b) RANKL…………………………………………….42

3) Cells……………………………………………….…………….43

i) Neutrophils………………………………………………….44

ii) Dendritic cells………… ……………………………………46

iii) Mast cells…………………………………………………..47

Specific host defense…………………………………………………..48

1. Cell-mediated….…………………….………………………….. 48

2. Humoral immunity.....................................................................…50

AIMS OF THE STUDY...........………………………............…………………….52

MATERIALS AND METHODS.............................................................................53

Criteria for patient selection…………………………………….53

Biopsies: processing and storage…………………………………53

Histological staining……………………………………………55

a) Periodic Acid Schiff Staining.............................................55

b) Immunohistochemistry …………………………………55

Antigen retrieval methods…………………………..55

a) Heat-induced antigen retrieval……………...….55

b) Pepsin treatment……………………………….56

Primary antibodies used in the study………………....….56

Programmable Tech Mate staining robot...........................57

ABC staining.……………………………………….........57

Specificities & sensitivities of the primary antibodies......58

Western blotting…………………..…………………………....58

Cell culture..................................................................................59

Statistical analysis.......................................................................60

6

RESULTS & DISCUSSION……………………….…….................….......................61

Study I: Alpha-defensin-1 in CHC………………………..............61

Study II: RANKL in mast cells .............................…………..……65

Study III: Expression of RANKL in CHC…………………...……67

Study IV: Interleukin 8 and its receptor IL-8 RA in CHC…...……73

Study V: Toll-like receptors in CHC…….....….……………….…77

SUMMARY& CONCLUSION....................................................................................82

AKHNOWLEDGEMENT...........................................................................................87

REFERENCES..............................................................................................................90

7

*LIST OF ORIGINAL PUBLICATIONS

This thesis is based on the following original publications, referred to in the text by

their Roman numerals (I-V). The impact factor (IF) was according to rating up to 2006.

I. Ali A, Niissalo S, Hietanen J, Laine M, Rautemaa R, Konttinen YT. Expression of

alpha-defensin-10 in chronic hyperplastic candidosis. J Oral Pathol Med 34:347–51,

2005

II Ali A, Lax AS, Liljeström M, Ashammakhi N, Kovanen P, Konttinen YT. Mast cell

in atherosclerosis as a source of the cytokine RANK. Clinic Chem Lab Med 44:672–74,

2006

III. Ali A, Rautemaa R, Hietanen J, Beklen A, Konttinen YT. A possible CD1a

Langerhans cell-mast cell interaction in chronic hyperplastic candidosis. J Oral Pathol

Med 36:329-36, 2007

IV. Ali A, Rautemaa R, Hietanen J, Järvensivu A, Richardson M, Konttinen YT.

Expression of IL-8 and its receptor IL-8RA in chronic hyperplastic candidosis.

Oral Microbiol Immun 21:223-230, 2006

V. Ali A, Natah S, Konttinen YT. Differential expression of Toll-like receptors (TLRs)

in chronic hyperplastic candidosis. Oral Microbiol Immun In press

* The publishers have granted me their kind permission to use any material in all the

papers of this thesis.

8

LIST OF ABBREVIATIONS

ABC avidin-biotin-peroxidase complex

AIDS acquired immune deficiency syndrome

APC antigen presenting cell

ATP adenosine triphosphate

BSA bovine serum albumin

C. albicans Candida albicans

CHC chronic hyperplastic candidosis

DAB diaminobenzidine

DC dendritic cell

FBS fetal bovine serum

GCP granulocyte chemotactic protein

HCl hydrochloric acid

HIV human immunodefeciency virus

HNP human neutrophil peptide

Ig immunoglobulin

IL-8 interleukin-8

IL-8 RA interleukin-8 receptor A

LC Langerhans cell

LP Leukoplakia

LPS lipopolysaccharide

MCT mast cell tryptase MCTC mast cell tryptase and chymase

MDNCF monocyte-derived neutrophil chemotactic factor

MHC major histocompatibility complex

NADPH nicotinamide adenin dinucleotide phosphate

NAF neutrophil activating factor

NAP neutrophil activating protein

NCF neutrophil chemotactic factor

ODF osteoclast differentiation factor

OPG osteoprotegrin

OPGL osteoprotegrin ligand

9

PAS periodic acid Schiff

PG prostaglandin

PML polymorphonuclear leukocyte

RANK receptor activator of nuclear factor κB

RANKL receptor activator of nuclear factor κB ligand

SCID severe combined immunodeficiency syndrome

TCF T cell chemotactic factor

TLR toll-like receptor

TNF tumor necrosis factor

TTC triphenyltetrazolium chloride

VSMC vascular smooth muscle cell

10

Abstract Candida yeast species are widespread opportunistic microbes, which are usually

innocent opportunists unless the systemic or local defense system of the host becomes

compromised. When they adhere on a fertile substrate such as moist and warm, protein-

rich human mucosal membrane or biomaterial surface, they become activated and start

to grow pseudo and real hyphae. Their growth is intricately guided by their ability to

detect surface defects (providing secure “hiding”, thigmotropism) and nutrients (source

of energy, chemotropism).

The hypothesis of this work was that body mobilizes both non-specific and specific

host defense against invading candidal cells and that these interactions involve resident

epithelial cells, rapidly responding non-specific protector neutrophils and mast cells as

well as the antigen presenting and responding dendritic cell – lymphocyte – plasma cell

system. It is supposed that Candida albicans, as a result of darwinistic pressure, has

developed or is utilizing strategies to evade these host defense reactions by e.g.

adhering to biomaterial surfaces and biofilms.

The aim of the study was to assess the host defense by taking such key molecules of the

anti-candidal defense into focus, which are also more or less characteristic for the main

cellular players in candida-host cell interactions.

As a model for candidal-host interaction, sections of chronic hyperplastic candidosis

were used and compared with sections of non-infected leukoplakia and healthy tissue.

In this thesis work, neutrophil-derived anti-candidal α-defensin was found in the

epithelium, not only diffusely all over, but as α-defensin-rich front. Once they reach the

epithelium, neutrophils, which form the major immigrant host defense cell, organize

themselves into microabscess structures (study I). Mast cells, in addition to tumour

necrosis factor-α, were found to contain preformed receptor activator of nuclear factor

kappa B ligand (study II). This is important for the recruitment and maturation of

antigen presenting dendritic cells and T lymphocyte activation (study III).The presence

and effects of the chemokine interleukin-8 on the chemotaxis and transmigration of

neutrophils was studied (study IV). For the immune system to operate, it has to be

invoked first by a set of innate receptors known as Toll-like receptors (TLRs). Only

three classes of TLRs seem to be engaged in recognizing C. albicans, i.e. TLR2, TLR4

and TLR6. Hypha-rich candidal infection appears to try to elude the host response

through stimulating TLR2 rather than TLR4 (study V).

11

Chronic hyperplastic candidosis provides a system that is very useful to study local and

systemic host factors, which under normal circumstances restrain C. albicans to a

harmless commensal state, but failure of which may lead to chronic infection.

12

Anatomical & histological review of the oral cavity:

The oral cavity can be divided into two parts: the vestibulum oris (vestibule) and the

cavum oris proprium (oral cavity proper). The vestibular part is bordered by the lips

and cheeks on the outer side and by the teeth and alveolar ridges on its inner side. The

oral cavity proper part lies within the dental arches and bones of the jaw, being limited

posteriorly toward the pharynx by the anterior pillars of the fauces which is the passage

between the back of the mouth and the pharynx (Fig.1, A&B). The oral cavity is mostly

lined with mucous membrane and its underlying musculature and connective tissue.

The morphologic structure of the oral mucous membrane varies according to the

functional requirements in different areas of the oral cavity and the mechanical

influences which affect them. In case of considerable mechanical influences, e.g.

around the teeth, on the surface of tongue and on the hard palate where the epithelium

comes in contact with the rough surface of masticated food, the

mucosa is keratinized

and attached densely to

the underlying tissue and

/ or bone. In other areas

of the mouth where

chewing is not primary

concern, the mucosa is

nonkeratinized, loose and

unsupported by bone e.g.

labial and buccal regions. The same applies in those areas which are well-protected

with other tissues e.g. the floor of the mouth which is covered with the tongue. Due to

its unique function in chewing and tasting, the dorsum of the tongue is covered with a

mosaic of keratinized and nonkeratinized epithelium and specialized foliate, fungiform

and circumvallate papillae which contain the taste buds (Squier and Kremer 2001).

The oral mucous membrane is composed of two layers, 1) surface epithelium which

covers 2) underlying connective tissue. The lamina propria is the name used for the

connective tissue component of oral mucosa.

Figure 1 showing a schematic illustration A, and extraoral photograph B, of the oral cavity (modified from a website)

13

Epithelium

The oral mucosa, which is covered by stratified squamous epithelium, is traditionally

classified into three main types, keratinized, nonkeratinized and specialized. The

epithelial covering of the oral mucosa consists of several layers of cells which flatten as

they reach the surface. The deepest or the innermost layer which rests on the basement

membrane is called basal layer (stratum basale) and consists of cuboidal cells. Next to

the basal layer is a number of layers of polyhedral cells which form the prickle cell

layer (stratum spinosum), the name of which was derived from the prickly appearance

of the connected cells by their intercellular bridges. The cells of the prickle-cell layer

flatten out when they pass into the next two layers; the granular and keratinous. The

granular layer (stratum granulosum) is called so because its cells contain keratohyaline

granules. The surface of oral epithelium is covered with the last keratinous layer which

contains dead cells filled with keratin (only in the keratinized areas of the oral cavity).

The keratinized layer may take up two morphological forms, orthokeratinized or

parakeratinized, with the main difference between the two being the retainment of

pyknotic nuclei in the cells of the latter.

The epithelium of the oral cavity does not contain blood vessels although some of the

nerves actually pass into it. Papillae of underlying connective tissue protrude toward the

epithelium.

The epithelium forms reciprocal ridges that protrude toward the lamina propria. These

ridges interdigitate with the papillae and are called epithelial ridges or rete pegs. The

epithelium is separated from its underlying lamina propria by means of a basement

membrane.

Lamina propria

The lamina propria of the oral mucosa is a dense connective tissue layer of variable

thickness. It lies below and supports the overlying stratified epithelium. Its papillae

contain both blood vessels and nerves important also for the epithelium. The papillae

are arranged in such a way that the surface area of contact between the lamina propria

and epithelium is increased to facilitate exchange of material e.g. nutrients. The lamina

propria can be divided into two parts; the papillary superficial part located just beneath

the epithelium containing papillae, and the reticular deeper part. Besides blood vessels

and nerves, the lamina propria consists of other components of the dense and loose

connective tissue e.g. lymphatics, ducts of glands, and sense organs.

14

The lamina propria is further attached to an underlying connective tissue called

submucosa.

Submucosa

Submucosa is the connective tissue which lies below the lamina propria and which

attaches the oral mucous membrane to the underlying bony or muscular tissues. This

tissue contains blood vessels which divide into smaller branches, nerves and adipose

tissue. The blood vessel system in this tissue divides into subepithelial capillary

network in the papillae, and is accompanied by venous and lymphatic vessels. The

sensory nerves, which traverse the submucosa, are myelinated but just before they form

their terminal parts lose their myelin sheath and turn unmyelinated. This portion of the

oral cavity may contain minor salivary glands.

Salivary glands & saliva

The oral mucosa is bathed continually with a clear fluid, saliva, which is mostly

secreted by three major paired glands, the parotid, the submandibular and the

sublingual. In addition, there are numerous minor salivary glands, perhaps about 500 in

number, scattered over most of the oral surfaces with the exception of the gingivae and

the anterior third of the hard palate. All salivary glands, which are of merocrine type,

empty their secretions into the oral cavity through excretory ducts (e.g. Stensen’s and

Wharton’s ducts).

Apart from water, the other major constituents of saliva are mucus, α-amylase, lipase,

electrolytes and growth factors (Kagami, Hiramatsu et al. 2000). Besides, saliva, whose

electrolyte composition is different from plasma, contains shed epithelial cells, food

debris and oral microbiota (Edgar 1992). The basic secretory units of salivary glands

are clusters of cells called acinus (plural: acini). The basic morphology of acini differs

according to the type of secretion so that the serous acini tend to be circular in shape

while those producing mucinous secretions have a rather tubular morphology. These

cells secrete a fluid that contains water, electrolytes, mucus and enzymes, all of which

flow out of the acinus into the salivary ducts. Ducts of the salivary glands root out from

acini as intercalated ducts, which unite to form striated ducts which further coalesce to

form the excretory duct of the gland.

Within the ducts the composition of the secretion is altered. The intercalated ducts add

bicarbonate to the glandular secretions and reabsorb chloride ion from the primary-

produced saliva. The basal parts of striated duct cells are rich in mitochondriae whose

alignment together with the basal folds forming the cellular compartments inhabitating

15

the mitochondria give the ducts a striated appearance (hence its name). The

mitochondria of the striated ducts allow pumping of ions across the membrane, thus

regulating the ion concentration of saliva, e.g. much of the sodium is actively

reabsorbed, and potassium is secreted. These relatively small collecting ducts within

salivary glands lead further into a larger terminal (excretory) duct whose main function

is to eventually empty the saliva into the oral cavity. Both serous and mucous acini and

terminal parts of the secretory intercalated duct system are surrounded by spindle-

shaped smooth muscle cells called myoepithelial cells which, by their contraction, help

move the saliva from acini to the duct system and thus participate in the secretory

process.

Each major salivary gland is characterized by its own type of acini, which corresponds

to the type of saliva it produces:

• Parotid glands produce a serous, watery secretion

• Submandibular glands produce a mixed serous and mucous secretion;

however serous cells significantly outnumber the mucous cells.

• Sublingual glands are mixed as well but mucous cells predominate.

Secretion of saliva is under control of the autonomic nervous system, which in part

controls both the volume and type of saliva secreted. Stimulation of the sympathetic

division leads to a rather viscous secretion rich in proteins, while parasympathetic

stimulation produces more watery saliva.

Functions of saliva

The most important functions of saliva are summarized below:

1. Lubrication and binding: the mucin component of saliva lubricates and

protects the oral structures by acting as a barrier against irritants. Lubrication

aids in speech, mastication and swallowing (Tabak 1990). Lubrication of dry

food solubilizes it so that it can be tasted. A salivary protein, gustin, seems to be

essential for taste bud growth and development (de Almeida Pdel, Gregio et al.

2008). Salivary mucins are extremely effective in binding masticated food into a

slippery bolus that slides easily through the esophagus without inflicting

damage to mucosa.

2. Oral hygiene: saliva enhances oral health by its almost constant flushing oral

tissues, which floats away food debris and microorganisms thereby keeping the

mouth relatively clean and hindering microbial colonization. Saliva contains

16

antibacterial substances e.g. lysozyme, peroxidase, and lactoferrin, which can

lyse many bacteria or prevent overgrowth of oral microbiota (Rudney 1995).

Saliva participates in the immune response through IgA which it contains and

which is known to inhibit bacterial attachment to the oral mucosa by means of

clumping the bacterial cells together (Dowd 1999).

3. Initiation of food digestion: saliva starts digestion of food. Serous acinar cells

secrete an alpha-amylase which digests dietary starch into maltose. However,

this function remains minor compared to the pancreatic amylase which breaks

down the yet undigested starch in the intestine.

4. Buffering action: the salivary content of bicarbonate and urea is of great

importance in neutralizing the acidic environment of dental plaques, which

protects against dental decay.

5. Antisolubility: in the initial stages of dental caries, the hard tissue substance of

the tooth (enamel) is dissolved most probably due to the acid produced by

acidogenic bacteria. Thus, the mineralized constituents of enamel, calcium and

phosphate, are liberated. Instead of losing theses essential minerals for good,

saliva, already containing considerable calcium and phosphate, when it gets

saturated with them partly with the aid of certain salivary proteins, tends to

precipitate them again in the enamel (remineralization).

17

Fungi: Definition, taxonomy and characteristics

Fungi are eukaryotic, plant-like microorganisms which are ubiquitously spread in

nature. There are about 80,000 species of fungi which range from the simplest

unicellular yeasts to the more complicated multicellular mushrooms and mildews.

There are many ways to classify fungi, but the most common criterion upon which

fungi can be classified is according to their manner of reproduction and formation of

spores. With respect to reproduction, fungi are classified into five major groups:

• Ascomycetes- characterized by production of microscopic spores inside

elongated cells or sacs, known as asci.

• Basidiomycetes- the spores in this group are produced on the end of specialised

cells called basidia.

• Deuteromycetes-this class includes all fungi which reproduce only by asexual

spores without any known sexual reproduction.

• Oomycetes-the life cycle of these fungi include two phases: asexual (through

zoospore), and sexual (oospore)

• Zygomycetes-the hyphae of these fungi form coencytic mycelia. They

reproduce both asexually (chlamydospores) and sexually (formation of zygote).

Fungi grow well in dark and moist conditions; hence they thrive most often in soil and

aquatic environments. One of the important characteristics of fungi is that they lack

chlorophyll and therefore draw their nutrition from decaying organic matter of living or

dead plants and animals which they use as sources of energy. For that reason, fungi are

considered heterotrophic and said to be saprophytes. Due to their lack of chlorophyll,

modern biologists tend to place fungi in their own kingdom rather than in the plant

kingdom as used to be previously.

Many fungi play an important role in the natural cycle as they decompose organic

matters and return their end products to the soil. Fungi are even used for medical

purposes, such as species within the penicillium genus which provide antibiotics, e.g.

penicillin.

There are two basic morphological varieties of fungi, yeasts and molds. Fungi in the

yeast phase tend to form moist and shining colonies reproduce asexually and their cell

walls are sometimes surrounded with a capsule, e.g. Cryptococcus neoformans.

In contrast, fungi in the mold form consist of masses from which filamentous

projections, known as hyphae, branch out. The hyphae may be septate (where the

18

multicellular hyphae are separated by crosswalls) or nonseptate (called coenocytes,

multinucleate without partitions). Hyphae usually grow along a surface and branch

together forming tufts, collectively called mycelium. Common septate filamentous

fungi are Aspergillus, Fusarium, Cephalosporium, Paecilomyces, and Penicillium

species. The nonseptate filamentous fungi include the Mucor species.

Candida The genus Candida belongs to yeasts. It is also the most common cause of

opportunistic mycoses worldwide. It is a frequent

colonizer of human skin and mucous membranes.

Candida is a member of normal flora of skin, mouth,

vagina, and bowel. In addition to being a colonizer and a

pathogen, it is found in the environment, particularly on

leaves, flowers, water and soil. The genus Candida

includes around 154 species. Among these, six are most

frequently isolated in human infections. While Candida

albicans is the most abundant and significant species, Candida tropicalis, Candida

glabrata, Candida parapsilosis, Candida krusei, and Candida lusitaniae are also

isolated as causative agents of Candida infections. Importantly, there has been a recent

increase of infections due to non-albicans Candida spp., such as Candida glabrata and

Candida krusei (Abi-Said, Anaissie et al. 1997). Patients receiving fluconazole

prophylaxis are particularly at risk of developing infections due to fluconazole-resistant

Candida krusei and Candida glabrata strains (Barchiesi, Morbiducci et al. 1993). The

diversity of Candida spp. that are encountered in infections is expanding and the

emergence of species that were rarely described in infections in the past is now likely

(Blinkhorn, Adelstein et al. 1989).

Candida albicans C. albicans belongs to the Ascomycota class of fungi and is the most commonly studied

species because it causes a variety of mycotic infections in humans (Siqueira and Sen

2004).

Taxonomy

Kingdom: fungi

phylum: ascomycota

Subphylum: ascomycotina

Figure 2 showing hyphae of C. albicans, CH.(modified from a website)

19

Class: ascomycetes

Order: saccharomycetales

Family: saccharomycetaceae

Genus: Candida

Species: albicans

Morphology

The fungal pathogen C. albicans can be found in three morphological states as yeasts,

hyphae or intermediate forms i.e. pseudohyphae (Sudbery, Gow et al. 2004) (Fig. 2, 3).

Some mycologists prefer to consider the pseudohyphal and hyphal forms as one entity;

therefore C. albicans is usually said to be dimorphic in nature as it has two distinct

shapes. Yeast cells are unicellular and

spherical or oval in shape, and normally

form smooth, white dome-shaped

colonies. Yeasts multiply by a specific

process of mitotic division known as

budding, in which daughter cells exude

from the mother cells. Several classes of

fungi, including C. albicans, are

featured with the ability to form spores

i.e. blastospores and chlamydospores

(which are spherical, smooth surfaced

and highly refractile (Nobile, Bruno et al.

2003), thus more favorable for candidal survival). Pseudohyphae are considered

modified yeasts which continued in polarized growth without separation from the

mother cell at the end of each cell cycle (Sudbery, Gow et al. 2004). Pseudohyphae are

also characterized by unequal width of their cellular projections, being wider at the

center than at ends (Merson-Davies and Odds 1989). Hyphae are microscopic tubes

which contain compartmentalized cell units separated by septa, these units arise

initially from blastospores or also from already existing hyphae (Webb, Thomas et al.

1998). When it takes up the hyphal form, it forms filamentous projections with parallel-

sided walls, so keeping the width of their compartments the same throughout the

branched portion. Germ tube is a term applied to the projecting hyphae in the first cell

cycle just before septation (Calderone, Suzuki et al. 2000).

Figure 3 illustrating a drawing of the basic morphologies of C. albicans. a) yeast form, b) pseudohyphae, c) hyphae and d) Opaque

CD

BA

CD

BA

20

C. albicans is well known for its morphological plasticity, i.e. its ability to transform

from one morphological pattern to another known as switching (Whiteway and

Oberholzer 2004), which is thought to promote the pathogenicity of the organism (Lo,

Kohler et al. 1997). There are other minor morphological changes which take place

during switching. For example, opaque phase is a variety where the cell becomes

oblong instead of the usual oval form of the yeast. Cell signal transduction pathways

and various transcriptional effects have been linked to the different array of

morphological forms and switching of C. albicans (Liu 2002; Dhillon, Sharma et al.

2003).

Biofilm formation

Microorganisms can exist either in a floating planktonic state or attached to an external

surface. A biofilm is an assembly of surface-coating microbial cells that is attached to

the surface and enclosed in a matrix of polysaccharide material such as alginate, Psl and

Pel-encoded polysaccahride (Donlan 2002; Ryder, Byrd et al. 2007).

In addition to its association with many hospital acquired nosocomial infections (Potera

1999) biofilm formation is of clinical significance since it confers the associated

microorganisms an ability to resist external threats, e.g. antimicrobial drugs (Baillie and

Douglas 2000; Mah and O'Toole 2001; Mukherjee, Chandra et al. 2003). A biofilm

formed of C. albicans renders it a hundred times more resistant to the antifungal

fluconazole and 20-30 times more resistant to amphotericin B than planktonic cells

(Kumamoto 2002). Both yeast and hyphal forms of C. albicans can participate in

biofilm formation. Due to biofilm formation, C. albicans yeast is considered as one of

the most common microorganisms found in the bloodstream in hospitalized patients, in

whom it originates from the biofilm composed of yeast cells embedded in a protective

matrix of extracellular protein (Chaffin, Lopez-Ribot et al. 1998; Crump and Collignon

2000; Soustre, Rodier et al. 2004). Biofilm of C. albicans has been noticed in dentures

as well as other biomaterials, e.g. stents, shunts, endotracheal tubes and catheters

(Andes, Nett et al. 2004; Bulad, Taylor et al. 2004). In order to form a biofilm C.

albicans has to adhere first to a medical device, colonize it and then establish a biofilm.

Biofilm formation by C. albicans depends on many factors, e.g. nature of the device

surface, whether host-derived conditioning film is present and liquid flow (Chandra,

Kuhn et al. 2001; Kuhn, Chandra et al. 2002). Colonization of different biomaterials by

C. albicans has been accepted as an important major cause of medical device failure

(Jones, McGovern et al. 2001). In the biofilm composed of C. albicans a layer of yeast

21

cells is located lowermost attached to the device, follows a layer above the yeast layer

composed of filamentous cells in the hyphal form surrounded by extensive exoplasmic

matrix (Baillie and Douglas 1999).

Biological behavior

Generally speaking fungi have the ability to adapt themselves to different

environmental conditions. Two important biological features seem to contribute to such

adaptation, chemotropism and thigmotropism.

Chemotropism is the growth of an organism along a concentration gradient toward a

particular chemical in its environment. Despite some doubts, it is argued that C.

albicans hyphal tips might excrete enzymes e.g. SAPs (secreted aspartyl proteinases),

phospholipases, which break cellular components of the host, which are then sensed

back by receptors located on the hyphal apices (Davies, Stacey et al. 1999). This might

be used as a directional guidance for the candidal hyphae so they decrease or even stop

growing in the surface plane and extend instead down to the underlying cellular tissue.

Chemotropic response has been suggested to play a role when C. albicans infects the

vascular endothelium from where the candidal hyphae grow down to the underlying

cellular tissue due to chemical signals released

from the endothelial cells and detected by the

newly formed germ tubes (Rotrosen, Edwards et

al. 1985).

Thigmotropism is the ability of the organism to

sense and respond to changes in surface

topography upon which it rests. There are two

types of thigmotropism. Positive thigmotropism

causes an organism to grow toward an object and

negative thigmotropism away from it.

Thigmotropism has been demonstrated in hyphae of C. albicans (Sherwood, Gow et al.

1992). This contact sensing phenomenon seems of importance in candidaemia where

invasion of both epithelium and endothelium ensues. Although invasion of endothelium

involves direct penetration of this layer without any requirement of surface exploration,

epithelium is grown over by blastospore-derived hyphae prior to penetration (Filler,

Swerdloff et al. 1995). This might indicate that thigmotropism can be of importance in

both surface growth and invasion (Davies, Stacey et al. 1999). The molecular events

involved in the regualtion of thigmotropism are not yet known.

Figure 4 showing the creamy colored appearance of C. albicans in SDA. Permitted from Dr. David Ellis

22

Culture media

In general, fungi are cultivated in three basic types of culture media, natural,

dehydrated and synthetic.

The most frequently used medium for culturing C. albicans is SDA (Sabouraud

Dextrose Agar) which belongs to the dehydrated media. Basically it contains dextrose

and beef extract and is useful in clinical applications since this culture medium allows

growth of Candida while inhibiting growth of other microorganisms, e.g. bacteria. SDA

is seldom used alone since it is estimated that more than one type of Candida species

occurs in about 10% of oral samples (Williams and Lewis 2000). Examples of media

used in combination with SDA include Pagano-Levin agar, or commercially available

chromogenic agars (discussed in the next section). In SDA candidal colonies are white

to cream colored with smooth contours and glabrous shape (Fig. 4).

Identification

There are many species of the genus Candida e.g. C. glabrata, C. krusei, and C.

tropicalis. To differentiate C. albicans from the other species, certain identification

tests must be employed. C. albicans can be distinguished from other species by means

of three main criteria, chromogenic, morphological and physiological criteria. Only the

chromogenic and morphological criteria will be clarified.

23

Table 1. Chromogenic criteria in agar culture to differentiate C. albicans from

other candida species.

Agar medium Basis of differentiation Candida sp. identified (color)

Pagano-Levin Reduction of TTC

(triphenyltetrazolium chloride)

C. albicans (cream),

other species (red or pink)

CHROMagar

Candida

Chromogenic substrate for

hexosaminidase

C. albicans (green)

C. tropicalis (blue),

C. krusei (pale rose)

Albicans ID Chromogenic substrate for

hexosaminidase

C. albicans (blue),

other species (cream)

Table 2. Morphological criteria to differentiate C. albicans from other Candida species

(Williams and Lewis 2000).

Morphology

Candida species Germ tube Pseudohyphae Chlamydospores

C. albicans + + +

C. tropicalis some + some

C. stellatoida + + some

C. parapsilosis - + -

C. krusei - + -

C. guilliermondii - + -

C. glabrata - - -

C. kefyr - + -

24

Pathophysiology of Candida albicans infection

Relationship between C. albicans and the host

• C. albicans as a component of the host microflora

Candidal species are found in about 40-70% of healthy individuals, the main species

found being C. albicans (Lo, Kohler et al. 1997; Watts, Very et al. 1998; Liu 2001;

McCullough, Jaber et al. 2002). C. albicans comprises a component of the benign

commensals living in a variety of body locations (Lo, Kohler et al. 1997; Lockhart,

Pujol et al. 2002; Soll 2002). It belongs to the normal microbial flora which colonizes

mucocutaneous surfaces asymptomatically e.g. oral cavity, gastrointestinal tract,

genitourinary tract of healthy human host (Elahi, Pang et al. 2001; Newman, Bhugra et

al. 2005). In the vagina C. albicans, which colonizes the epithelial surfaces of 5 – 25%

of healthy women (Sobel 1988; Hauman, Thompson et al. 1993), is the causative agent

of vulvovaginal candidosis which affects 50 – 75% according to estimates of those

women at least once before their menopause (Kent 1991; Sobel 1992), while 5-10%

have a recurrent type of vulvovaginal candidosis (Fidel 2005).

• When does C. albicans cause infection?

As mentioned above, candidal yeasts can form a part of the normal microflora of the

body. Candidal infection ensues when the number of yeasts increases so it exceeds the

tolerance of the host tissue and causes inflammation, e.g. mucositis. In other words, it is

the immune host defense status of the host which is to be blamed rather than the

microorganism itself since as long as the host defense is intact with proper functional

integrity, the latter remains innocent. When the local or systemic host defense becomes

compromised, the delicate balance between C. albicans and the host is broken and the

fungus becomes pathogenic in which case it causes an infection called candidosis or

candidiasis. Factors which alter the status of the local and / or general host defense and

hence favor the growth of the candidal yeasts in the oral cavity will be discussed shortly

below as predisposing factors. Therefore, candidal infections have received the

designation “disease of the diseased”. Recently, the incidence of candidosis has

increased which is mainly due to the increasing number of immunocompromised

patients (McCullough, Jaber et al. 2002; Wellington, Bliss et al. 2003). Candidosis

ranges from simple superficial oral and vaginal infections to life-threatening systemic

candidosis in severely immunocompromised patients (Naglik, Newport et al. 1999;

25

Whiteway 2000). Biomaterials, when implanted to a host, form a good habitat for C.

albicans to organize a biofilm community which in turn can contribute to disease

(Kumamoto 2002).

• Role of C. albicans in nosocomial infections & candidaemia

C. albicans has been claimed to be the fourth most common nosocomial infectious

agent (Schaberg, Culver et al. 1991; Ashman, Farah et al. 2004). In addition to the role

of C. albicans in mucosal and mucocutaneous infections, it has an ability to invade also

other vital systems in the body. In patients whose immunity is suppressed, these forms

of invasive candidal infections, including blood-disseminated candidosis (candidal

sepsis) are usually associated with high morbidity and mortality despite the use of

appropriate antifungal agents (Fridkin and Jarvis 1996; Andriole 1999; Kao, Brandt et

al. 1999). In susceptible hosts C. albicans enters the blood compartment and causes

deep-seated infections in target organs (Ibrahim, Filler et al. 1998). Once in blood C.

albicans may disclose its virulence factors and so the condition transits from

candidaemia (mere presence of candida in the blood) to lethal septicemia (presence of

symptoms caused by candidal toxins) the morbidity of which has been estimated to be

around 50% (Edmond, Wallace et al. 1999; Barelle, Bohula et al. 2003).

Oral candidosis

• Definition and epidemiology

Oral candidosis is an opportunistic infection of the oral cavity caused by the

overgrowth of Candida species, usually of C. albicans (Fotos and Hellstein 1992;

Muzyka and Glick 1995). Candida species are present as commensal organisms of the

oral microbiota in about 20-60% of normal human population (MacFarlane TW 1989).

In the mouth, the primary site where C. albicans is located is the dorsum of tongue,

while other places such as tooth surfaces covered with plaque are less commonly

colonized (Arendorf and Walker 1980). There are certain factors which affect the

pattern of distribution of C. albicans in the mouth (Webb, Thomas et al. 1998). They

may include:

1. Saliva: One study showed that saliva has the ability to reduce candidal attachment to

the acrylic surface of oral biomaterials (Samaranayake, McCourtie et al. 1980),

although mannoprotein on the surface of C. albicans may also selectively absorb

26

salivary mucins, which can enhance candidal attachment to acrylic (Edgerton,

Scannapieco et al. 1993).

2. pH: The exact pH at which C. albicans adheres best to oral cells cannot be defined

precisely since the effect of pH varies considerably depending on the candidal strain

and type of mucosal cell (Mehentee and Hay 1989) but generally speaking, low pH has

been suggested to favor the growth and colonization of C. albicans.

3. Adhesion: The interaction between oral cells and C. albicans is thought to be

mediated through ligand-receptor interactions. Mannoprotein, for example, represents a

ligand on the candidal surface while there are many mammalian cell proteins acting as

receptors, e.g. iC3b, fibrinogen and laminin (Calderone and Braun 1991).

4. Cell surface hydrophobicity: Candidal cells can be hydrophilic or hydrophobic

depending on the composition of cell wall in the protein structure of the cell wall

(Hazen, Lay et al. 1990). When candidal cells are hydrophobic, they can bind diffusely

to hydrophobic surfaces of host cells without ligand-receptor interactions to areas

which are free of macrophages (Hazen, Brawner et al. 1991).

5. Oral bacteria: The growth and colonization of C. albicans may be augmented by the

presence of some bacteria e.g. Streptococcus sanguis, Streptococcus gordonii (Hsu,

Minah et al. 1990).

6. Hyphae: C. albicans is a biphasic fungus. The hyphal form is associated with more

invasive potential than the yeast form (Kimura and Pearsall 1980). This is thought to be

due to the release of some proteases during switching of C. albicans from the

unicellular yeast to the hyphal form (Borg and Ruchel 1988; Cutler 1991).

• Etiology and predisposing factors

Among the 150 Candida species known today, the most common etiological

microorganism of oral candidosis is C. albicans (Cannon, Holmes et al. 1995). This,

however, does not preclude the other human pathogenic species from being direct

causative agents in some cases of oral candidosis, namely C. parapsilosis, C. tropicalis,

C. glabrata, C. krusei, C. pseudotropicalis, or even C. dubliniensis, which has first

more recently been associated with oral candidosis in HIV-infected patients (Odds

1988; Sullivan, Westerneng et al. 1995). There are many factors which predispose to

the development of oral candidosis. These can act either by enhancing the growth and

colonization of candidal yeast or suppressing the immune system of the host or both.

27

They are summarized in table 3. Some factors, which are considered common, are

detailed further below.

1) Prostheses

Dental prosthesis, especially if they are ill-fitting and accompanied with poor oral

hygiene, may become a substrate for candidal growth. Constant physical irritation can

cause local microscopic breaches in the oral mucosa, which provide an entrance route

to the fungus. It has been noticed that salivary yeast counts are much higher in patients

wearing full dentures than in dentate subjects (Parvinen 1984). Since dental prostheses

may restrict the diffusion of oxygen and flow of saliva to the underlying tissue, a local

stagnant environment with a low pH and oxygen content may be produced. Such an

environment favors fungal overgrowth and ingrowth into the porous acrylic resin

matrix (Shay, Truhlar et al. 1997). C. albicans adheres avidly to denture-base materials

in vitro and this has been attributed to its hydrophobicity (Radford, Challacombe et al.

1999).

2) Epithelial alterations

Intact oral mucous membrane provides an effective physical barrier against ingress of

fungal or bacterial cells. When the turnover rate of epithelial cells alters, e.g. due to

radiation therapy or anticancer medication, the integrity of oral epithelial seal is

impaired, which predisposes the mouth to candidal infection (Bunetel and Bonnaure-

Mallet 1996).

3) Endocrine disorders

Deficiencies of certain hormones predispose to the emergence of oral candidosis e.g.

diabetes mellitus, hypothyroidism, hypoparathyroidism, hypoadenalism and Addison’s

disease (Fotos and Hellstein 1992). More commonly, the mucocutaneous form of

candidosis has been associated with multiple endocrine disorders (Firth, O'Grady et al.

1997) where the triad of chronic mucocutaneous candidosis, hypoparathyroidism and

adrenocortical failure comprise a compex condition known as APECED (Autoimmune

Polyendocrinopathy-Candidosis-Ectodermal Dystrophy) (Perheentupa 2006). Studies

have found that Candida species also in asymptomatic patients are more common in the

oral cavity of diabetic patients than in healthy people (Dourov and Coremans-Pelseneer

1987). Although it is still obscure, the mechanism of this overgrowth of Candida is

thought to be xerostomia, elevated glucose levels and impaired neutrophil function

(Rossie and Guggenheimer 1997).

28

4) Infectious and immunologic disorders

The cell-mediated and humoral immunity are of paramount importance in protecting

the oral mucosa against candidosis (Hedderwick and Kauffman 1997). Since Candida

species are opportunistic pathogens, fungal infections are common in patients whose

immune system is compromised, e.g. acquired immune deficiency syndrome (AIDS) in

which more than 90% of affected patients at some stage of the disease develop oral

candidosis (Ellepola and Samaranayake 2000). Human immune deficiency virus (HIV)

alters cell-mediated immunity so that T-cell function becomes impaired. Oral

candidosis in HIV +ve patients can take up a variety of expressions ranging from

pseudomembranous, atrophic and hyperplastic, all of which resemble clinically the

lesions seen in noninfected individuals. HIV-related oral candidosis can also extend

beyond the frontiers of the oral cavity to involve other organs such as the esophagus

and the trachea, which will cause dysphagia and restrosternal discomfort. Severe

combined immunodeficiency syndrome (SCID) is another condition characterized by

defects in the cell-mediated and humoral immune reactions. Chronic mucocutaneous

candidosis is often noticed in patients with SCID. Patients on immunosuppressive

drugs, e.g. following organ transplantation, are also susceptible to oral candidosis.

Table 3. Summary of factors predisposing to oral candidosis

Promote the growth of yeasts Suppress the host defense

Intrinsic Extrinsic Local Systemic

Imbalance of

oral microflora

High carbohydrate

diet

Hyposalivation Extremity of age, i.e.

infancy/senility

Low pH Dental prostheses Epithelial changes Immunosuppressive

infections e.g. AIDS

Poor oral

hygiene

Broad spectrum

antibiotics

Immunosuppressive

therapy e.g. cortisone

Already existing

lesions e.g.

leukoplakia

Malnutrition or

malabsorption, e.g.

iron deficiency

Endocrinpathies e.g.

diabetes

Radiation therapy Pregnancy

Heavy smoking

29

Classification of oral candidosis

Group 1: Candidosis confined to the oral mucosa

1. Acute

• Acute pseudomembranous candidosis (thrush)

• Acute atrophic (erythematous) candidosis

2. Chronic

• Chronic atrophic candidosis (denture stomatitis)

• Candida-associated angular cheilitis

• Median rhomboid glossitis

• Chronic hyperplastic candidosis (candidal leukoplakia)

Group 2: Oral manifestations of generalized candidosis

• Chronic mucocutaneous candidosis

Clinical variants of oral candidosis:

1) Acute pseudomembranous candidosis (thrush)

This type is characterized by the presence of extensive white superficial patches. These

curd-like patches are nothing but pseudomembranes consisting of desquamated

epithelial cells, fibrin and fungal hyphae (Akpan and Morgan 2002). The

pseudomembranes can be wiped off leaving an erythematous base. This lesion can be

found anywhere in the oral cavity, most often on the dorsum of tongue or on labial and

buccal mucosae. Young infants and old people are frequently affected.

2) Acute atrophic candidosis

This form of candidosis is also called acute erythematous candidosis. It is not as

frequent as thrush except perhaps after the use of broad spectrum antibiotic, but when it

occurs it causes painful burning sensation of the mouth (Lehner 1967). These lesions

are characterized by red, atrophic changes occurring most commonly on the palate and

tongue. Loss of tongue papillae (depapillation) leads to a bright red appearance and

soreness (Palmer, Robinson et al. 1996). This clinical type of candidosis is commonly

seen in HIV infections and after use of corticosteroids or broad-spectrum antibiotics.

3) Chronic atrophic candidosis (denture stomatitis)

This particular form is characterized by the presence of chronic erythematous and

edematous lesions, which range from small to large. It affects at some stage about half

of all complete denture wearers (Budtz-Jorgensen 1990). As the synonym suggests, it is

30

associated with ill-fitting and/or poorly cleaned dentures. Therefore, it tends to be

found exclusively in the denture-wearing areas, especially the palate. Clinically, it

manifests as bright red, somewhat velvety to pebbly surface.

4) Candida-associated angular cheilitis

Since angular cheilitis (perleche) is commonly associated with denture stomatitis, it

belongs to the same category as chronic atrophic candidosis but has a different location

(RA 1966; Budtz-Jorgensen 1974). As the name points, it affects the angles of the oral

cavity (commissures) and it starts as fissures which allow pooling of saliva and

incubation of candidal cells leading to erythematous lesions which may encrust or

erode and hence turn painful. Other predisposing factors include deep nasolabial folds

(Shay, Truhlar et al. 1997) and long-term use of dentures (Penhall 1980).

5) Median rhomboid glossitis

Median rhomboid glossitis is a chronic symmetrical depapillated area of the tongue

anterior to the circumvallate papillae. The region of papillary atrophy is usually

elliptical or rhomboid in shape and situated at the midline of the tongue (Scully, el-

Kabir et al. 1994).

6) Chronic hyperplastic candidosis

This clinical manifestation was the pivot of my study and is considered separately

below in more detail.

7) Chronic mucocutaneous candidosis

This term, which comprises an array of clinical manifestations, is applied when the

candidal infection involves organs other than the mouth e.g. skin, nail beds, vagina etc.

Four subtypes have been recognized, associated with familial susceptibility;

endocrinopathy; both familial susceptibility and endocrinopathy; and onset later than 10

years of age (Higgs and Wells 1974).

31

Chronic hyperplastic candidosis (CHC)

Lehner first reported the presence of candidal infection in an oral leukoplakia and

called it candidal leukoplakia (Lehner 1964; Lehner 1967). Despite the confusion it

may cause, most histopathologists prefer to use

the term “chronic hyperplastic candidosis”

instead of candidal leukoplakia. CHC is

considered the most important clinical form of

oral candidosis due to its association with the

development of malignancy at the lesion site

(Cawson and Lehner 1968; Bartie, Williams et al.

2001). This tendency should prompt the oral

diagnostician to consider an already-forming real cancer as a differential diagnosis.

Clinical manifestations

Chronic hyperplastic candidosis appears as discrete, raised lesions. These hyperplastic,

sometimes nodular lesions may be speckled or homogenous and they range in size from

small, palpable lesions to large dense plaques. They are most often white or cream-

colored (Fig. 5) but occasionally red (Fig. 6) (Appleton 2000). The red and speckled

lesions have the clinical appearance of numerous

white nodules on an erythematous base (Cawson

and Lehner 1968). They occur on the buccal

mucosa, palate, or dorsum of the tongue (Daniels,

Schwartz et al. 1985). These lesions cannot be

wiped off.

Histopathology

The clinical presentation of CHC (whether it is

homogeneous or speckled) may reflect the histopathological picture of the lesion so the

more nodular the lesion is, the more dysplasia the epithelium tends to show.

Histopthaologically candidal cells, yeasts and hyphae (Fig. 7) , are seen on the

uppermost tissue surface, and when they invade the epithelium (hyphal and

pseudohyphal invasion), they rarely penetrate beyond the spinous layer (Reichart,

Philipsen et al. 1995). The lesions show hyperparakeratotic or hyperorthokeratotic

epithelium with irregular separation and epithelial hyperplasia. There is a higher mitotic

activity, but it is restricted to the basal and suprabasal layers of the epithelium. A

Figure 5 showing white, cream colored presentation of CHC (Webb, Thomas et al. 1998)

Figure 6 showing CHC in its red lesion form (Akpan and Morgan 2002)

32

typical feature is the presence of microabscesses,

which are collections of polymorphonuclear

leukocytes in the epithelium. Lamina propria contains

an inflammatory cell infiltrate composed mostly of

lymphocytes, macrophages and plasma cells.

Diagnosis

To reach a precise diagnosis of oral candidosis, as

with any other oral lesion, a thorough medical and

dental history has to be obtained from the patient. The oral diagnostician has to conduct

a careful clinical examination where they first inspect the lesion to see if its appearance

matches any of the known clinical presentations of the disease. Therefore, a wide

knowledge of oral medicine is mandatory. The next step in clinical examination is

palpation which can indicate an important diagnostic feature i.e. whether the lesion can

be rubbed off or not.

If the clinical diagnosis is still provisional or doubtful, other tests are also available

such as exfoliative cytology, culture or tissue biopsy (Epstein and Polsky 1998;

Sherman, Prusinski et al. 2002). Exfoliative cytology is performed by scraping

superficial cells to samples. A swab culture should be taken on the undersurface of a

denture when denture stomatitis is suspected. If the candidal infection is thought to

have already invaded the tissue, sampling some tissue in form of a biopsy should be

considered (Fotos and Hellstein 1992). Hematological screening might be useful since

up to some 40 % of patients with oral candidosis may have some hematological

abnormalities (Challacombe 1986).

Management

a) Elimination of the predisposing factors

Once a diagnosis of oral candidosis is assured, the first line of treatment should aim to

eliminate or alleviate any dental and/or medical factors which contribute to the

occurrence of candidal infection in the oral cavity. The oral diagnostician may find it

necessary to refer his/her patient, in case there is suspicion of any pertinent medical

condition, to their physicians to seek for advice. Patient cooperation is sometimes

crucial especially when the predisposing factors are social rather than medical, e.g.

smoking. One of the most important factors contributing to chronic hyperplastic

candidosis is smoking; therefore, cessation of smoking is mandatory to relieve the

disease. Chronic atrophic candidosis is mostly associated with edentulous patients who

Figure 7 showing histopathology of CHC (Sitheeque and Samaranayake 2003)

33

wear dentures; therefore patient education and motivation to cleanse their dentures

regularly and apply the oral hygiene measures is necessary to restore the infected

edentulous mucosa to normalcy. If patients with acute atrophic candidosis are on broad-

spectrum antibiotics or corticosteroid, discussion with their physicians about the

possibility of withdrawal or substitution of the medicine could resolve the problem.

b) Antifungal treatment

Antifungal therapy has been used successfully in the management of oral candidosis.

Prior to prescription of any antifungal agents, advising the patient to gargle with a

physiological saline solution helps to decrease the oral fungal counts and thus soothe

the associated symptoms (Appleton 2000). Pharmacological treatment of oral

candidosis should be tailored to the individual patients according to their current

medical status and severity of infection (Sherman, Prusinski et al. 2002). Antifungal

agents are available in different forms (i.e. gels, ointment, creams, suspension,

lozenges, and tablets) and the dentist should manage to select the proper form upon

writing a prescription. Threre are many drug categories available to treat the different

clinical presentations of candidosis, summarized as follows:

• Polyenes: The candidal cell wall is composed of many layers, the innermost of

which is the plasma membrane. Polyenes are potent agents which act by binding to the

sterol part of the candidal cell membrane and disrupt its osmotic integrity leading to

leakage of essential ions e.g. potassium and magnesium. This category includes

nystatin and amphotericin B. Nystatin, which is of bacterial origin, is the drug of choice

for treatment of oral candidosis. It should be prescribed cautiously in patients with

uncontrolled diabetes or xerostomia. Recently, liposomal nystatin (Nyotran) has been

designed where nystatin was incorporated into liposomes and it is now in late phase

clinical trial (DiDomenico 1999). The preferred topical application of nystatin is oral

suspension (100,000 U/ml) or pastilles (100,000 IU) for 7-14 days (Farah, Ashman et

al. 2000). Amphotericin B can be used to treat superficial candidosis i.e. Fungizone

(oral), or more wide-spread systemic involvement i.e. Ambisome (intravenous).

• Antimetabolites: This group contains only one agent: flucytosine (5-

fluorocytosine). It is candidastatic since it inhibits the candidal protein synthesis by

replacing uracil with 5-flurouracil in fungal RNA. Its use has declined due to the

emergence of candidal resistance (Appleton 2000).

34

• Azoles: This class includes two major categories: imidazole (e.g. clotrimazole,

miconazole, econazole, ketoconazole) and triazole (e.g. fluconazole, itraconazole).

Although their effect targets the plasma membrane (same as polyene), they are

fungistatic rather than fungicidal (Wynn, Jabra-Rizk et al. 1999). Fluconazole is

considered as the first line of treatment in many cases of candidosis but its main

drawback is the emergence of candidal resistance.

• Glucan synthesis inhibitors: these (e.g. caspofungin) act by inhibiting the essential

component of the fungal cell wall, glucan.

• Miscellaneous agents: there is only one drug available of this class of cuurent use

i.e. griseofulvin which exerts its effect through disrupting the mitotic spindle.

Figure 8 showing a schematic diagram of C. albicans cell constituents as targets of antifuingal agents

35

Table 4 showing a summary of the most commonly prescribed antifungal agents for

treating oral candidosis

Agent name Administration (route and dose) Treatment course

Nystatin (Mycostatin®) Oral- lozenge (200.000 U): 1X4

-suspension (100.000 U/ml):

1tsp (for 2 mins) X4

10-14 days

Amphotericin B (Fungizone®)

Oral- tablet (10 mg): 1X4 4-6 weeks

Clotrimazole (Mycelex®) Oral-troche (10 mg): 1X5

-cream (10mg/g): X4

Troche: 14 days

Cream: 1-8 weeks

Miconazole (Daktarin®) Oral- gel (2%): 2.5 mlX4 Treatment continues

1 week after resolution of the infection

Leukoplakia (LP) The old official definition of leukoplakia (LP) was formulated in 1978 by the World

Health Organization (WHO) as “A white patch or plaque that cannot be rubbed off or

characterized clinically or pathologically as any other disease” (Sciubba 1995). WHO

stated in a newer defintion in 1997 as a predominantly white lesion of the oral mucosa

that cannot be characterized as any other definable lesion.

LP can appear at any time in life, but it occurs most common in the elderly people.

Clinically LP is a white or gray patch that develops on the tongue, inside of the cheek

or anywhere in the mouth. The lesion may have developed slowly over weeks to

months and be thick and slightly raised, eventually with a hardened and rough texture.

It is usually painless, but may be sensitive to touch, heat, spicy foods, or other irritation.

The transformation liability of LP to oral cancer is well-documented in the literature

and is considered to have the most precancerous potential among oral lesions

(Warnakulasuriya 2000). The highest malignant potential is seen in the nodular,

exophytic as well as speckled forms of LP and erythroplakia (Axell, Pindborg et al.

1996). Hansen et al., in 1985, suggested the term proliferative verrucous leukoplakia

(PVL) for leukoplakias which tend to recur and may change to precancerous lesions

(Hansen, Olson et al. 1985). Many studies screened the frequency of PVL and its

progression to carcinoma in variable samples in the western countries and the results

36

ranged from 60 to 100% (Fettig, Pogrel et al. 2000; Bagan, Jimenez et al. 2003;

Morton, Cabay et al. 2007)

LP is thought to be a reaction of the oral mucosal membrane to chronic irritation of the

mucous membranes. Hairy LP of the mouth is an unusual form of LP that is seen

mostly in HIV patients (but can be found in some other immunodepressed patients). It

appears as fuzzy (hence the name hairy) white patches on the tongue and less

frequently elsewhere in the mouth.

Etiology

• Irritation from rough teeth, fillings, or crowns, or ill-fitting dentures

• Chronic smoking, pipe smoking, or other tobacco use

• Alcohol abuse

• Sun exposure to the lips

Diagnosis

An experienced clinician may suspect LP upon examination; however, a biopsy is

usually necessary to rule out other causes, such as oral cancer. During the biopsy, a

small piece of tissue from the lesion is removed to be examined histopathologically in a

laboratory.

Treatment

Treatment, if needed, involves removal of the source of irritation. For example, if LP is

caused by a rough tooth or an irregular surface on a denture or filling, the offending

tooth should be smoothened and dental appliances repaired. If LP is caused by

smoking, quitting of smoking is critical in relieving the lesion.

LP is usually harmless, and lesions usually clear in a few weeks or months after the

source of irritation has been removed. If elimination of the source of irritation is

ineffective in reducing LP, the lesion may need to be surgically removed.

37

Host defense against oral candidosis Under normal conditions, the host tolerates the existence of C. albicans and other

eventual candidal components of the oral microflora. The host safeguards itself by

means of natural defense measures, e.g. the physical integrity of the intact epithelium,

mucosal membrane, which serves as a natural barrier against foreign intruders

(Marodi 1997). Thereby it acts as a selective gatekeeper allowing the passage of certain

substances but prevents ingrowth of Candida and other microbes (Dale 2002).This

relationship between the host and microflora is maintained and enables commensalism

as long as the innate and adaptive immunity of the host remain competent. Once the

host defense is compromised, opportunistic species of the microflora take benefit of it

breaking the mutual balance by invading the host. The host tries to defend itself against

such saprophytic opportunistic microbes by two main ways, utilizing both nonspecific

and specific host defense.

Nonspecific host defense (innate immunity)

This branch of the host defense system is the first line to become engaged when a host

encounters a pathogen. As the name indicates, it does not base its recognition

mechanisms on learning (adaptive behavior) and immunological memory; rather, it

senses and identifies any foreign substance, which imposes a threat to the host right

away. Any host is naturally armed with such a protective system; therefore, it is also

called innate immunity. Although it may utilize ligand specific receptors, these

receptors are not drawn from a huge number of different specificities but represent

rather broad recognition ranges. When a pathogen invades a host, the innate immunity

serves two major tasks. It recognizes the invading organism and tries to clear it in a

rapid but robust way. Second, it triggers a subsequent cascade whereby a more

sophisticated machinery of the immune system (specific or adaptive) receives a danger

signal (secondary stimulus) necessary for its activation by specific antigens (primary

stimulus). Understandably, the innate immunity is phylogenetically ancient unlike

adaptive immunity which is only found in vertebrates (Raj and Dentino 2002). Innate

immune defense utilizes many mechanisms involving various molecules. There are

some agents which have a shared task and form a bridge between innate and adaptive

immune response e.g. defensins. To avoid repetition, the key players of the innate host

defense will be considered under only one heading.

38

1. Toll-like receptors (TLRs)

Toll was first discovered in Drosophila melanogaster in 1996 (Lemaitre, Nicolas et al.

1996), its human homologues Toll-like receptors (TLRs) comprise an important family

of receptors for the detection of microbial components of a broad variety of pathogens,

which triggers an inflammatory host defense response and alert the adaptive arm of the

host defense (Netea, Van der Meer et al. 2004). In mammals, Toll has not only been

maintained but has developed to comprise 11 identified receptors which belong to the

family of TLRs (Takeda and Akira 2005). Structurally, TLRs are composed of an

extracellular portion, which harbors leucine-rich repeats and an intracellular domain

having a high degree of similarity to interleukin-1 receptor (Takeda and Akira 2004;

Takeda and Akira 2005). Zymosan is a generic name for the polysaccharide component

of the fungal cell wall (including yeasts). Zymosan stimulates many host cells via

TLR2 (Underhill, Ozinsky et al. 1999). The immunostimulatory properties of zymosan

are most probably conferred by β-glucan (Kataoka, Muta et al. 2002). In addition,

TLR2 and TLR4 can sense C. albicans through its cell wall constituents

phospholipomannan and mannan, respectively (Roeder, Kirschning et al. 2004). It is

still debatable whether the morphological form of C. albicans is a determinant factor in

excitation of or escapes from the host defense mechanisms through TLR2 and TLR4

which could perhaps explain some apparently conflicting results (Netea, Sutmuller et

al. 2004; Netea, Van der Meer et al. 2004; Villamon, Gozalbo et al. 2004; Villamon,

Gozalbo et al. 2004). While epidermal keratinocytes have been found to express TLR2

and TLR4 in response to microbial pathogens including C. albicans (Pivarcsi, Bodai et

al. 2003), very few studies have investigated, so far, the eventual participation of such

recently discovered structures in the immunity of the oral mucous membrane against

candidosis.

2. Proteins

a) Natural antimicrobial peptides

One of the mechanisms used by the host when it is invaded by a pathogen is production

and / or release of substances having antimicrobial effects in response to the microbe-

host cell contact (Yang, Biragyn et al. 2002). These substances are called antimicrobial

peptides. As the name denotes, these are small molecular-weight peptides, which are

engaged in killing or inactivating a peptide typical spectrum of pathogenic invaders, i.e.

viruses, bacteria or fungi (Bastian and Schafer 2001). The list of such natural defense

39

agents is already long but only the best known antifungal peptides will be presented in

this thesis.

Defensins

The discovery of defensins goes back to the time when a class of peptides, found to

have antimicrobial properties, was isolated from macrophages of rabbit lung (Selsted,

Brown et al. 1983). A couple of years later, similar peptides were found in human

neutrophils and therein the term defensin was coined (Ganz, Selsted et al. 1985).

Defensins are cysteine-rich antimicrobial peptides containing three pairs of disulfide

bridges (Lehrer and Ganz 1999). They have a molecular-weight of 2000-6000 Da and

their amphipathic properties allow hydrophilic, hydrophobic and cationic clustering

(Marshall 2004). There are three classes of defensins: alpha (α), beta (β) and theta (θ),

of which only the first two exist in humans (Izadpanah and Gallo 2005). Alpha-

defensins are composed of 29-35 amino acid residues and they are shorter than β-

defensins, whose amino acid number ranges from 38 to 42 (Raj and Dentino 2002).

These defensins have different locations of cysteine residues and disulfide motifs.

Alpha-defensins are subcategorized into six types designated from 1 to 6. The first four

subclasses (1 to 4) are produced by polymorphonuclear leukocytes (therefore, they are

referred to as Human Neutrophil Peptide or HNP) while the last two are produced by

Paneth cells of the intestine and the female genitourinary tract (Quayle, Porter et al.

1998). The four types of β-defensins are produced by epidermal keratinocytes of the

skin and epithelial cells of the mucous membranes of the mouth, gastrointestinal and

genitourinary tracts (Raj and Dentino 2002).

Microbial membranes are the main targets of the microbicidal effects of defensins.

Because of their positive charge, defensin peptides interact with the negatively charged

components or pathogen-associated molecular structures of the microbial membranes.

This antimicrobial protein-microbial membrane interaction results in disruption of the

latter and leakage of the microbial cell membrane leading to osmotic rupture and death

of the microbial cell (Weinberg, Krisanaprakornkit et al. 1998).

Along with their obvious role in innate immunity, α- and β-defensins are also linked to

the adaptive immune response. They augment the adaptive immune response of the host

through recruitment of inflammatory cells to the site of the microbial invasion. Alpha-

defensins act directly as chemotactic agents to recruit CD8 and CD4 T cells as well as

immature dendritic cells. They also stimulate release of the chemokine interleukin 8 by

40

epithelial cells which attracts neutrophils to the site of infection (Van Wetering,

Mannesse-Lazeroms et al. 1997; Zasloff 2002).

Histatins

Histatins, which are small histidine-rich cationic peptides, comprise an important group

of the antimicrobial peptides of saliva. They are secreted by the parotid and

submandibular salivary glands and their molecular size ranges from 7-38 amino acids

(Kavanagh and Dowd 2004). The proteolytic degradation of the products of the two

histatin genes (HIS1 and HIS2) results in the formation of at least 12 histatin fragments

in human saliva, of which histatin 5 is the most active form against C. albicans (Xu,

Levitz et al. 1991; Ruissen, Groenink et al. 2002). Histatin 5 tends to retard the

transition of C. albicans from the unicellular to the fungal form, a step necessary for the

tissue invasion and penetration (Cannon, Holmes et al. 1995). It has been postulated

that histatin 5, as all other cationic antimicrobial peptides, disrupts the osmotic integrity

of C. albicans by creating permanent pores in its cell membrane. Consequently, the

membrane loses its ability to control the passage of ions in and out of the

microorganism leading to its demise. Also other antifungal mechanisms have been

reported to histatin 5, e.g. binding to the fungal mitochondria which cause release of

cellular ATP out of the cell so that the candidal cell becomes depleted of its energy

supply (Helmerhorst, Breeuwer et al. 1999), and generation of lethal reactive oxygen

species (Helmerhorst, Troxler et al. 2001). The ability of histatin 5 to kill azole-

resistant fungi has made it a topic of intensive research as a potential synthetic

chemotherapeutic antifungal agent (Tsai and Bobek 1997).

Protegrins

Protegrins belong to a larger group of cathelin-containing antimicrobial peptides called

cathelicidins (Zanetti, Gennaro et al. 1995). Protegrins are small (2 kDa) as they

contain only 16 to18 amino acids particularly cysteine and arginine. They were first

isolated from porcine leukocytes (Fahrner, Dieckmann et al. 1996) and they occur in

five sequential types (PG-1 to PG-5).

b) Chemokines and cytokines

Chemokines are small secreted proteins whose main function is to attract leukocytes to

move in a specific direction, along the gradient of the chemokine concentration. In

other words, chemokines are chemotactic cytokines (hence their name, chemo for

chemotaxis and kine for cytokine) (Baggiolini 2001). Cytokines are regulatory proteins,

such as the interleukins and lymphokines that are released by cells of the immune

41

system and act as intercellular mediators in the generation of an immune response.

Thus, cytokine is a more general term than chemokine, which also participate in

intercellular communications in a well defined and narrow area. Structurally,

chemokines form a family of structurally related glycoproteins with potent leukocyte

chemotactic and activating properties. The molecular weight of chemokines ranges

from 8 to10 kD and they are 70 to 90 amino acids in length, having 20 to 70%

homologies in their amino acid sequences (Luster 1998). Most of them fit into two

subfamilies with four cysteine residues. These subfamilies segregate based on whether

the two amino terminal cysteine residues are immediately adjacent to each other (cc

chemokines) or separated by one amino acid (cxc or alpha chemokines). The CXC

group includes molecules such as interleukin-8 and platelet factor 4 most of them being

chemoattractants for neutrophils. Examples of the CC group (beta chemokines) include

eotaxin and monocyte chemoattractant protein-3, and such chemokines generally attract

monocytes, lymphocytes, basophils, and eosinophils (Luster 1998). There are also two

other small subgroups. The C group has one member (lymphotactin) which lacks one of

the cysteines in the four-cysteine motif, but shares homology at its carboxyl terminus

with the C-C chemokines. The C chemokine seems to be specific for lymphocyte. The

fourth subgroup is the C-X3-C subgroup. The C-X3-C chemokine (e.g. fractalkine /

neurotactin) has three amino acid residues between the first two cysteine amino acids. It

is tethered directly to the cell membrane via a long mucin stalk and induces both

adhesion and migration of leukocytes.

Chemokines can also be classified to either inflammatory or homing (homeostatic):

inflammatory chemokines are widely distributed in most tissues and their expression is

triggered by inflammatory stimuli, while homing chemokines are constitutively

expressed within lymphoid tissues (Baggiolini 2000). Chemokines exert their effects, as

any protein ligand, through receptors. This chemokine-receptor binding may, in the

future, be employed to provide a successful chemotherapy of certain drastic diseases,

e.g. AIDS. This is exemplified by the finding that the HIV antigen gp120 recognizes

overlapping receptors epitopes to which chemokines can bind (Baggiolini 1998).

Chemokines act via many seven-transmembrane-domain receptors, two of which,

CXCR1 (IL-8RA) and CXCR2 (IL-8RB), are for interleukin-8.

In this thesis, only two cytokines, IL-8 and RANKL, will be discussed

42

a) Interleukin-8 (IL-8)

Interleukin-8 (IL-8) is a chemotactic cytokine which attracts leukocytes and stimulates

monocyte adherence (Lane, Lore et al. 2001). The main cell target for the chemotactic

effect of IL-8 is neutrophil. The chemotactic effect of IL-8 results in recruitment and

transmigration of neutrophils across the microvasculature to the site of inflammation

(Schall and Bacon 1994). IL-8 is also a chemoattractant for T lymphocytes (Larsen,

Anderson et al. 1989) and basophils. IL-8 was also referred to as neutrophil

chemotactic factor (NCF), neutrophil activating factor (NAF) (Walz, Peveri et al.

1987), neutrophil activating protein (NAP) , monocyte-derived neutrophil chemotactic

factor (MDNCF) (Yoshimura, Matsushima et al. 1987), T cell chemotactic factor

(TCF), granulocyte chemotactic protein (GCP) and leukocyte adhesion inhibitor (Yang,

Huang et al.). It is released by several cell types including monocytes, macrophages,

neutrophils, T-lymphocytes, fibroblasts, endothelial cells, and epithelial cells after an

inflammatory stimulus, e.g. IL-1, TNF, LPS, or viruses (Yoshimura, Matsushima et al.

1987; Peveri, Walz et al. 1988; Strieter, Kunkel et al. 1988; Strieter, Phan et al. 1989;

Palter, Mulayim et al. 2001). IL-8 belongs to the CXC subfamily of chemokines, is a

member of the beta-thromboglobulin superfamily and structurally related to platelet

factor 4. IL-8 may play an important role in the progression of many cancerous

diseases, e.g. melanoma, breast cancer and lung cancer (Reed and Purohit 1997;

Poppenborg, Knupfer et al. 1999; Penson, Kronish et al. 2000). It was found that IL-8 is

produced by fibroblasts in the dental pulp when it is stimulated with substance P (Park,

Hsiao et al. 2004) or black-pigmented Bacteroids (Yang, Huang et al. 2003). IL-8

induces its chemotactic effect via two receptors: CXCR1 and CXCR2, also known as

IL-8RA and IL-8RB, respectively (Murphy and Tiffany 1991). Both receptors are

found plentifully on neutrophils while they are expressed less on monocytes and

myeloid cell lines (Sprenger, Lloyd et al. 1994). Beside its chemotactic effect and

recruitment of neutrophils to the site of inflammation, IL-8 has been claimed to trigger

the oxidative burst response in those cells (Baldwin, Weber et al. 1991), thereby aiding

their microbicidal efficacy.

In the oral mucosa, IL-8 plays a major role in combating pathogens such as C. albicans

through recruitment of neutrophils and lymphocytes to the site of infection.

b) RANKL

The cytokine receptor activator of nuclear factor κB ligand (RANKL) is a membrane-

bound ligand that belongs to the tumor necrosis factor family (Hofbauer and Heufelder

43

2001), which bestows RANKL an other synonym: TRANCE (TNF-related activation-

induced cytokine). It is a protein consisting of 316 amino acids with a molecular mass

of 35 kDa (Yasuda, Shima et al. 1998). RANKL is produced by osteoblastic lineage

cells (Gori, Hofbauer et al. 2000), fibroblasts (Mandelin, Li et al. 2003), immune cells

(Anderson, Maraskovsky et al. 1997) and some cancer cells (Nagai, Kyakumoto et al.

2000). The main function of RANKL is to orchestrate formation, maturation and

activation of osteoclast (Jeziorska, McCollum et al. 1998; Liu, Xu et al. 2003);

therefore, RANKL was also known as osteoclast differentiation factor (ODF). The

inductive action of RANKL on osteoclast progenitors is mediated through binding to its

receptor RANK. Binding of RANKL to RANK is counteracted by a decoy receptor

called osteoprotegerin (OPG), which is the reason why RANKL was also called

osteoprotegerin ligand (OPGL). The neutralizing effect of OPG on RANKL-RANK

interaction was based on the observation that OPG (-/-) knockout mice develop

osteoporosis and arterial calcification of aorta and renal arteries (Schoppet, Preissner et

al. 2002). RANKL can occur in three forms: either as a cell-bound, truncated or

secreted (Hofbauer, Shui et al. 2001). The role of RANKL in bone metabolism is

manifested in many pathological conditions, e.g. rheumatic disease (Haynes, Barg et al.

2003), atherosclerosis (Collin-Osdoby 2004), cancers such as multiple myeloma

(Farrugia, Atkins et al. 2003) and prostate cancer (Lynch, Hikosaka et al. 2005),

periodontitis (Crotti, Smith et al. 2003; Liu, Xu et al. 2003) and aseptic bone loss in

total hip replacement (Gehrke, Sers et al. 2003). The expression of RANKL is

upregulated by some factors which have been associated with bone resorption, e.g.

glucocorticoids, interleukins (IL-1, IL-6, IL-11 and IL-17), TNFα and PTH (Kong and

Penninger 2000). Another important role of RANKL, beside its osteoclastogenetic

effect, is immunity. The fact that activated CD4+ and CD8+ T lymphocytes display

RANKL in lymphoid structures, such as lymph nodes, Peyer’s intestinal patches and

thymus (Lacey, Timms et al. 1998), and that RANK is expressed by dendritic cells

evoked the concept of RANKL contributing to immunological processes. So far, no

analytical study has been conducted to assess the role of RANKL and RANK

interaction in an infectious disease, let alone candidosis.

3. Cells

Along with the cytokines and the natural antimicrobial peptides, there are certain cells

which belong to the innate immune system and aid in extinguishing the infectivity of

pathogens.

44

Such cells include phagocytic cells (macrophages and neutrophils) that can engulf

(phagocytose) foreign substances. Macrophages are thought to mature continuously

from circulating monocytes.

Phagocytic cells respond to the chemotactic stimuli by carrying out three main steps

which guide their migration towards the chemotactic stimulus. These steps are

margination and rolling, adhesion and transmigration, and then the final migration

towards the site of inflammation. The journey of the phagocytic cells to their destiny is

guided by chemotaxis. Chemotaxis is the process by which phagocytes are attracted to

where the microorganisms are located by means of chemotactic stimuli such as

microbial products, complement, damaged cells and white blood cell fragments.

Chemotaxis is followed by a direct contact of the phagocyte with the microorganisms.

This intimate adhesion is enhanced by opsonization, where protein components (called

opsonins) coat the surface of the pathogen. This is followed by ingestion, in which the

phagocyte extends projections, or pseudopods that engulf the foreign organism. Finally,

the pathogenic microbe in the phagosome fuses with a lysosome and is digested by

enzymes in the phagolysosome, a process involving also reactive oxygen species. Many

important cells are involved in the host immune defense against infection such as

neutrophils, dendritic cell and mast cells.

a) Neutrophil

The first phagocyte a pathogen is likely to encounter is a neutrophil. A neutrophil is a

type of white blood cell which plays major early role in the cellular innate immune

response against foreign invaders. Neutrophils are produced in the bone marrow at a

rate of 1011 cells per day, and then circulate around the blood stream as mature cells for

approximately ten hours before migrating into the peripheral tissue pools, where they

reside for their 1 to 2 day long living period (Smith 1994). After that neutrophils

undergo apoptosis (Savill, Fadok et al. 1993). It is thought that the short life span of

neutrophils might be due to their involvement in the maintenance of body homeostasis

which renders them susceptible to cellular stress (Smith 1994), to minimize propagation

of those pathogens that parasitize phagocytes, and to diminish the damaging effects of

the antimicrobial products on the host tissues.

Neutrophils constitute 50 to 60% of the total population of leukocytes (Smith 1994).

Like other phagocytes, they are recruited to the tissue site where the noxious substance

is located. Once there, they approach the invading stimulus and display a variety of

antimicrobial processes, e.g. elaboration of natural antimicrobial peptides, enzymes

45

(e.g. peroxidase, hydrolytic enzymes), and eventually engulf it. Because of their

peculiar nuclear morphology (the nucleus is termed segmented or polymorphonuclear),

neutrophils belong to the polymorphonuclear leukocytes (PMLs) (Huizinga, Roos et al.

1990). They contain granules and are also known as granulocytes. These cytoplasmic

granules are tiny vesicles containing enzymes that help the cell to efficiently perform its

microbicidal function. Neutrophils utilize two major microbicidal mechanisms, namely

oxidative and enzymatic processes. In this way, it kills and digests microorganisms that

it has already engulfed by phagocytosis. Degranulation process of the cytoplasmic

granules of neutrophils also causes an extracellular release of its destructive mediators.

Neutrophils are active phagocytes, capable of only one phagocytic event, expending all

of their glucose reserves in an extremely vigorous respiratory burst. The respiratory

burst involves activation of an NADPH oxidase enzyme, which produces large

quantities of superoxide converted to other reactive oxygen species. Beside their role as

direct scavengers of toxic substances in the body, neutrophils can also aid in the

regulation of the inflammatory process by secreting of humoral mediators such as

cytokines, e.g. TNF-α (Lloyd and Oppenheim 1992), IL-1β (Palma, Cassone et al.

1992), IL-6 (Melani, Mattia et al. 1993) and IL-8 (Strieter, Kasahara et al. 1990). Being

highly motile, neutrophils quickly congregate at the site of infection, dragged by

cytokines expressed by cells at the site of action, e.g. activated endothelium, mast cells

and macrophages. Neutrophils are also under normal circumstances produced in huge

numbers, but this may increase as much as 10-fold in response to an infection

(Cannistra and Griffin 1988).

In candidosis, neutrophils play a major role in combating the pathogen C. albicans by

expressing either candidastatic and/or candidacidal mechanisms. Neutrophils contain

calprotectin in their cytoplasm which inhibits the growth of the fungus without killing it

(Sohnle and Collins-Lech 1990). Neutrophils can eliminate the fungal pathogen from

the host by producing candidacidal proteins, e.g. HNP 1-4. The hyphal and

pseudohyphal forms of the organism can be engulfed and damaged by neutrophils

through oxidative mechanisms (Diamond, Clark et al. 1980). The importance of

neutrophils in defending the host against C. albicans is supported by the fact that

candidal infection predominates in patients with chronic granulomatous disease in

which leukocytes suffer from NADPH oxidase deficiency (Kim, Rodey et al. 1969).

46

b) Dendritic cells

Dendritic cells (DCs) belong to a class of cells known as antigen presenting cells

(APCs). They are derived from haematopoietic stem cells in the bone marrow and were

first identified in 1973 (Steinman and Cohn 1973; Scimone, Lutzky et al. 2005).

Possibly excluding the brain, testes and cartilage, DCs patrol almost all other tissues of

the body (Cutler, Jotwani et al. 2001). They are found most commonly in places with

direct contact with the environment, in skin and mucous membranes where they serve

as sentinels against ingress of pathogens. In humans, DCs are classified into three

subsets. Two are myeloid cells, namely Langerhans cells and dermal DCs (both of

which can be generated in vitro from human cord blood CD34+ hemopoietic

progenitors) (de Saint-Vis, Fugier-Vivier et al. 1998) and the third one is lymphoid DCs

(Cutler, Jotwani et al. 2001). When they circulate in the blood, DCs are

indistinguishable from monocytes but when they reside in peripheral tissues, e.g. skin

and mucosa, they develop long spiky projections called dendrites, from which their

name is derived (Cutler and Jotwani 2004). DCs are considered to represent an

important link between innate and adaptive immune responses (Palucka and

Banchereau 1999). In the mucous membranes and epidermis, DCs are present as

Langerhans cells (LCs), while in the dermis they are known as dermal DCs. Despite

their old discovery in 1868 by Paul Langerhans (after whom LCs are named), LCs were

categorized as a class of DCs very recently, in 1979 (Palucka and Banchereau 2006).

LCs are found above the basal layer in skin and mucosal surfaces of the mouth, nose,

gastrointestinal tract and genitourinary tract (Jotwani and Cutler 2003). The primary

function of LCs (and other DCs) is to control the potential pathogen entry sites and

hence they are considered as immunosurveillant cells. Having not exposed to an antigen

yet, LCs remain in a resting and immature state but characterized by high endocytotic

activity. When an antigen is encountered, e.g. C. albicans, LCs capture it and actively

internalize it by phagocytosis (Ramachandra, Chu et al. 1999). The manner of

phagocytosis depends on the morphological form of the fungal part being engulfed.

LCs engulf yeasts through coiling phagocytosis while zipper-like mechanism is the

prevalent phagocytosis type for hyphae (Montagnoli, Bacci et al. 2001). In the

cytoplasmic vacuolar compartments, LCs process the fungal cell, degrade it into

fragments and then associate it with major histocompatibility complex-antigen (MHC I

or II, depending on the antigen) (Romagnoli, Nisini et al. 2004). After the LCs have

packaged the antigen with MHC, they extrude the whole complex to the cell surface for

47

recognition by T cells (Adams, O'Neill et al. 2005). If antigen-specific T cells are

encountered, a response ensues and LCs transform to the active, mature form, they

migrate via blood or lymphatic vessels to the regional lymph nodes where they interact

with naïve T cells (Banchereau and Steinman 1998). This interaction is mediated

through upregulation of certain cell surface receptors, e.g. CD80 and CD86, which aid

in priming and enhancing the T lymphocytes.

c) Mast Cells

Paul Ehrlich, when still a medical student in 1878, was the first to describe mast cells

whose large size and metachromatic granular appearance led him to the mistaken belief

that they were either well-fed or feeding the surrounding tissues, and therefore gave

them the name ‘mastzellen’ which means “feeding cells”. Mast cells develop in the

bone marrow from CD34+ pluripotent stem cells. Being still immature, mast cells

circulate in the blood and lymphatics to home to tissues where they undergo maturation

by a locally-produced growth factor (Prussin and Metcalfe 2003). Throughout the

human body mast cells (infrequently known as mastocytes) are found in connective

tissues and submucosal areas (Kelley, Chi et al. 2000), preferably around the blood

vessels and beneath the epithelium (Church and Levi-Schaffer 1997). The role of mast

cells in the pathogenesis of many human diseases e.g. atherosclerosis, has become

strongly evident (Kovanen 1995). Although some researchers prefer to classify mast

cells according to their location into mucosal and connective tissue mast cells

(Gemmell, Carter et al. 2004), the granular content of mast cells remains the prominent

criterion for categorizing them into mast cell tryptase (MCT) and mast cell tryptase and

chymase (MCTC). As the name denotes, MCT contains tryptase in their granules and are

found most abundantly in the respiratory and intestinal mucosa and conjunctiva (Irani,

Schechter et al. 1986). On the other hand, MCTC contains both tryptase and chymase

and predominate in the skin and submucosa of the small intestine and subepithelial area

of the conjunctiva (Irani, Bradford et al. 1989). Mast cells are known best for their

participation in allergy through their expression of high affinity receptor (FcεRI) for

immunoglobulin E. The prominent location of mast cells in the vicinity of blood vessels

most probably dictates their primary function in the regulation of the vascular muscle

tone in the inflammatory process. Beside tryptase and chymase, mast cell granules

contain and / or rapidly produce an array of active mediators and cytokines, e.g.

histamine, heparin, carboxypeptidase, prostaglandins (PGD2, PGE2), thromoxanes, IL-

4, TNF-α, RANKL and growth factors (Kelley, Chi et al. 2000). When mast cells are

48

stimulated, they elaborate their granular contents to the extracellular environment by

exocytosis, a process known as degranulation. Mast cells are considered an integral part

of the immune system. In tissues, mast cells remain in close proximity to T cells

(Mekori and Metcalfe 1999) since the former has the ability to engulf and process

antigens, and present them to the latter, thereby aiding in the initiation of an adaptive

immune response (Malaviya, Twesten et al. 1996). Release of IL-16 by mast cells is

thought to be an important explanation for the presence of CD4+ T cells at sites of mast

cell activation (Rumsaeng, Cruikshank et al. 1997), since IL-16 is a preferential

chemoattractant for CD4+ T cells (Center, Kornfeld et al. 1996). Although mast cells

have been shown to be involved in the immune defense against many bacteria, e.g. K.

pneumoniae, there is no study; so far, attempting to clarify whether mast cells play a

role in the host defense against candidal infection.

Specific host defense (acquired immunity)

The second category of the immune system is called specific, acquired or adaptive

immunity. As the name suggests, this branch of the host defense is directed against the

particular and specific pathogens which have succeeded partially or totally in

overwhelming the shields of the innate immune armory system. It takes days for the

specific immunity to recruit, activate and expand the antigen-specific lymphocytes,

which is much longer than it takes to mount the immediate innate defense response.

Therefore, the specific host response is not effective in eliminating infections in their

early stages and its major role comes to play in cases of long lasting infections. The

specific host defense is divided into two major sections through which it exerts its

mechanisms: cell-mediated T cells and humoral (antibodies) immunity.

1. Cell-mediated immunity

The cellular arm of the specific immune defense is formed of T lymphocytes, which is

abbreviated as T cells. This subclass of white blood cells originates in the bone marrow

from pluripotent haematopoietic stem cells, and the newly-formed cells then circulate

through the thymus gland (the letter T which represents this class of lymphocytes is

derived from thymus) and differentiate to thymocytes (Vallejo, Davila et al. 2004).

They are usually divided into two major subsets that are functionally and

phenotypically different, cytotoxic T cells (CD8+) and helper T cells (CD4+) (Germain

2003). Cytotoxic T cells (sometimes called killer/suppressor, CD8+) are important in

the direct destruction of certain tumor cells, virally infected cells and sometimes

parasites. Most CD8+ T cells recognize antigens only when they are bound to the MHC

49

class I (MHC-I) molecules. The other category of T cells is CD4+ (helper) T cells which

constitute a pertinent coordinator of immune regulation. Their main function is to

augment or potentiate the immune responses by secretion of specialized factors or

cytokines, which activate other white blood cells to fight the infection. Most CD4+ T

cells recognize antigens associated with MHC II (Siu 2001). There are two subsets of

T-helper cells: Th1 cells which, through their secretion of IL-2 and INF-γ, enhance cell-

mediated immune response and in the same time inhibit the other subset of T-helper

cells, the Th2 cells. Th2 cells are engaged in minimizing cell-mediated immune

response through secretion of certain cytokines such as IL-4 and IL-10. Both types of T

cells can be found throughout the body. They often depend on the secondary lymphoid

organs, the lymph nodes and spleen, as sites where activation occurs, but they are also

found in other tissues of the body, most conspicuously in the mucosal-associated

lymphatic tissue in intestinal and reproductive tracts, but also in liver, lung and blood.

T cells are responsible for cell-mediated immunity which is of paramount importance in

the defense against viral, bacterial and fungal infections. T cells also aid B lymphocytes

to produce antibodies, recognize and reject foreign tissues due to their histocompatible

tissue type (or actually MHC molecules). T lymphocytes first have to find their way to

the site of infection. Once there, they are exposed to an antigen so that the antigen-

specific T cells divide rapidly and produce large numbers of new T cells sensitized to

the various epitopes of the pathogen specific set of antigens. T lymphocytes play a

central role in control of the acquired immune response and, furthermore, serve as

crucial effectors through antigen specific cytotoxic activity

As to candidal infections, cell-mediated immunity is necessary in the successful

defense of the host against candidosis, especially on mucosal membranes (Fidel 2002).

This is based on the experimental observation that T cell-deficient mice fail to resist

gastrointestinal candidosis whereas those mice with deficient phagocytic capability and

normal T cell function readily cleared the infection (Balish, Filutowicz et al. 1990). Th1

response in particular is the branch of cell-mediated immunity which provides

protection against candidosis, while Th2 pathway would lead to attenuation of the Th1

response (Romani, Mocci et al. 1991).

50

2. Humoral Immunity

Humoral immunity is the second branch of the adaptive immune response. It pertains to

antibody production and all the accessory processes involved, e.g. Th2 activation and

cytokine production, germinal center formation and isotype switching, affinity

maturation and memory cell generation. The main functions of antibodies are pathogen

and toxin neutralization, classical complement activation, opsonin promotion of

phagocytosis, and pathogen elimination (Cooper 1985; Edelman 1991).

The process of antibody production, which is T-cell dependent, is the responsibility of a

class of lymphocytes called B cells (B refers to Bursa of Fabricius in birds, necessary

for the maturation of B cells), which need two signals to initiate the humoral immune

response (Gulbranson-Judge and MacLennan 1996). With a T-dependent antigen, the

first signal comes from antigen-mediated cross-linking of B cell receptors and the

second signal derives from the Th2 cells. First, cross-linking of the T-dependent

antigen on B cell ensues, followed by presentation of that antigen associated with MHC

II to Th2 cells, which then provide co-stimulation to trigger B cell proliferation and

differentiation into plasma cells (Parker 1993; Baker, Gagnon et al. 2000). Cytokines,

predominantly IL-4, IL-5, and IL-6, stimulate the B cells to divide and differentiate into

antibody-secreting plasma cells. A few activated B cells differentiate into short-lived

plasma cells that migrate directly to the medullary cords and begin secreting IgM and

later IgG. Isotype switching to IgG, IgA, and IgE and memory cell generation occur in

response to T-dependent antigens and cytokines (Jumper, Splawski et al. 1994). In a

primary immune response, IgM is secreted before isotype switching occurs and is the

predominant isotype produced. Each antibody isotype has its specific effector functions

in humoral immunity.

Plasma cells are antibody-producing cells that no longer divide in response to antigen.

They are larger than B cells and have more ribosomes, endoplasmic reticulum, and

Golgi complexes. Some plasma cells survive only a few weeks, while others continue

to produce antibody for longer periods, providing rapid protection against an eventual

re-infection. Other B cells become memory cells with high avidity antigen-specific B-

cell receptors, which can be rapidly re-stimulated by antigen and are present in higher

frequency than the naïve resting B cells were before the infection and antigen-driven

oligoclonal expansion of the antigen-specific B cell clones.

51

There are five different antibody isotypes, IgG, IgM, IgA, IgE, and IgD. All theses

isotypes seem to have unique features and different implications in the humoral

immune response (Han, Mou et al. 1995). When microbes enter the body through the

epithelia of the oral mucosa, for example, they may begin replicating locally or get into

the circulation and move throughout the body. The Fc regions of the pentavalent IgM,

which is secreted first in a primary immune response, allow them to aggregate antigens

present at relatively high concentration and subsequently bind to Fc receptors of

professional phagocytes to be phagocytozed in e.g. splenic and liver sinusoids. Because

IgM is so large, it cannot enter the tissues very efficiently; but it is effective in

controlling pathogens in the circulation. Once isotype switching occurs, IgG with the

same antigen specificity starts to predominate in serum and in tissues. IgG both

neutralizes pathogens and their toxins and opsonizes them for phagocytosis by

neutrophils and macrophages (Robbins and Robbins 1986). Interestingly, only IgG

seems to be able to cross the placenta and impart immunity to the developing fetus. IgG

can also activate complement on the pathogen surface once concentrations are high

enough for two IgG molecules to bind to nearby epitopes. IgA is the predominant

antibody that is secreted across epithelial cells of the respiratory, digestive and genital

tracts to block pathogen entry into the body (Robbins and Robbins 1986). IgE binds to

FcεRI on mast cells surrounding the blood vessels throughout the body. When a

pathogen binds and cross-links the mast cell surface IgE, the mast cell immediately

releases inflammatory mediators that trigger coughing, sneezing or vomiting to expel

pathogens from the body.

Antibodies against candidal antigens are found in the sera of most people (Lehner,

Buckley et al. 1972), being higher in patients with chronic mucocutaneous candidosis

in whom the assessed antibodies seemed to be directed against the cell wall component

mannan (Lehner, Wilton et al. 1972). Nevertheless, the role of humoral immunity in the

defense process against mucosal or systemic candidosis is still questionable (Fidel

2002). There are reports about children having defective B cell functions, either

congenital or acquired, who did not show any susceptibility to candidal infection

(Rogers and Balish 1980). It is obvious that antibodies per se cannot exert any

candidastatic or candidacidal activities; rather they contribute by acting as opsonins for

neutrophils and macrophages at the site of infection.

52

AIMS OF THE STUDY

1. To assess and clarify the presence of α-defensin-1, its localization and eventual

immunohistopathological signs of its participation in the host response against

chronic candidal stimulus in CHC compared with candida-negative leukoplakia

lesions.

2. When performing immunohistochemical staining of RANKL in CHC samples,

we found accidentally that mast cells form a source of RANKL; therefore we

aimed to confirm this pioneering discovery in atherosclerotic samples. We

chose atherosclerosis because mast cells have a well-established role in the

pathogenesis of the disease.

3. To evaluate the histopathological distribution of CD1a Langerhans cells in CHC

compared with leukoplakia and healthy controls, and whether the number of

these RANK-positive antigen-specific cells correlates with that of RANKL-

producing mast cells.

4. To investigate the contribution of the chemokine IL-8 and its receptor IL-8 RA

in the host defense against C. albicans and check if there is any

immunohistopathological signs of their involvement in CHC compared with

healthy controls.

5. To study what TLRs (TLR1-9) plays roles in the immune response of the host

against C. albicans by comparison of the CHC lesions with leukoplakia and

healthy mucosa and by studying the effect of those candidal PAMPs on mucosal

epithelial cells which differed between CHC and comparators.

53

MATERIALS AND METHODS

Criteria for patient selection

a) For patients of CHC & LP

The local Ethical committee approved the study protocol. Ten biopsy samples of oral

mucosal lesions were obtained from patients with chronic hyperplastic candidosis

(n=10) and the same number from patients with leukoplakia (n=10) undergoing

examination of mucosal lesions (Table 5). Histopathology of both disease entities

disclosed their corresponding typical features. Periodic Acid Schiff (PAS) staining of

the biopsy samples and/or fungal culture of saliva samples on Dentocult CA® culture

medium (Orion Diagnostica, Espoo, Finland) were used to confirm or exclude the

fungal infection. Five biopsy samples were recruited from healthy individuals

undergoing dental procedure to serve as controls.

b) For patients of atherosclerosis

Coronary artery samples were collected from left anterior descending coronary arteries

from five unselected patients autopsied for medicolegal reasons. All coronary segments

contained a large confluent core of extracellular lipid. The local Ethical committee

approved the study protocol.

Biopsies: processing and storage

All biopsies (CHC, LP, healthy controls and atherosclerosis) were fixed in formalin

(10% phosphate buffered formalin, pH 7) and stored at 4+°C. After fixation, the tissue

biopsies were dehydrated by first using graded ethanol solutions, then graded xylene

solutions followed by liquid paraffin. The wax was allowed to solidify, forming blocks

into which the tissues were embedded in cassettes. Out of these paraffin blocks, 6 µm

and 4 µm (for study II) thick sections were cut and mounted on objective glass slides,

which were then stored till immunostaining was performed. The modern paraffin is

typically a mixture of paraffin wax and resin and it provides excellent morphological

detail and resolution.

54

Table 5. Clinical and demographic data of the patients with chronic hyperplastic candidosis (CHC), leukoplakia (LP) and healthy controls (HC).

M = Male, F =Female

Number Gender Age Location of the lesion

Clinical presentation

Additional Information

1 CHC M 59 Tongue Diffuse keratinization

Heavy smoker

2 CHC F 45 Tongue Red/white lesion Painful 3 CHC F 59 Tongue Homogenous 4 CHC F 67 Palate Verrucous Prosthesis 5 CHC F 55 Cheek/Commissures Hyperplastic Heavy smoker 6 CHC F 53 Tongue Nodular Sharp tooth edge 7 CHC M 44 Tongue Ulcerative 8 CHC M 53 Palate Verrucous 9 CHC F 81 Cheek Papular

leukoplakia Carcinoma of tongue

10CHC F 85 Tongue Hyperplastic 1 LP F 51 Alveolar ridge White patch 2 LP F 55 Tongue White patch 3 LP F 52 Cheek Striated patch 4 LP F 73 Floor of the mouth Exophytic Prosthesis, heavy

smoker 5 LP F 66 Palate Striated 6 LP F 64 Floor of the mouth Homogenous 7 LP F 50 Alveolar ridge Exophytic 8 LP F 48 Cheek Striated 9 LP F 84 Alveolar ridge Exophytic,

pigmented Prosthesis

10 LP M 49 Alveolar ridge White patch Number Gender Age Location of the sample Reason presenting patient to

the clinic (no inflammation) 1 HC F 56 Upper left sulcular mucosa Resection of left upper incisor 2 HC F 57 Upper left sulcular mucosa Wisdom tooth operation 3 HC F 44 Lower right sulcular mucosa Orthodontic treatment

4 HC M 32 Upper right sulcular mucosa Check-up

5 HC F 25 Upper posterior vestibular mucosa

Check up

55

Histological Staining

a) Periodic acid Schiff staining

Periodic acid-Schiff (PAS) staining of sections from lesions of both chronic

hyperplastic candidosis (CHC) and leukoplakia was used to confirm or exclude the

presence of candidal hyphae. It is also used for the demonstration of glycogen and

neutral mucins. PAS stain consists of 1% Periodic acid and Schiff's reagent (Basic

Fuchsin, Potassium metabisulphite, Hydrochloric acid, Deactivated Charcoal and

Distilled water).

Procedure

Following deparaffinization sections were 1) immersed in distilled water, 2) treated

with 0.5% periodic acid for 5 minutes at room temperature, 3) washed well in distilled

water, 4) treated with Schiff's reagent for 20 minutes, 5) washed in running tap water

for 10 minutes, 6) counterstained with Mayer's Haematoxylin for 5 minutes and 7)

dehydrated in alcohol, cleared in xylene and mounted in Diatex.

b) Immunohistochemistry

Immunohistochemistry is a method used to detect antigens in tissues using antibodies,

and it was the main method used in all projects of this study.

Antigen retrieval methods

Antigen retrieval is a process which helps to retrieve the immunoreactivity of a

particular antigen after it has been lost due to tissue fixation in formalin. In this study,

two methods were used, heat-induced treatment using microwave (for studies I-V), and

pepsin treatment (for study III). A special microwave was used in study V.

a) Heat-induced antigen retrieval

This method depends on application of heat treatment to the tissue being stained using

microwave. After deparaffinizing the sections, they were immersed in 10% Antigen

Retrieval Buffer (ChemMate Detection Kit) (for study I), in Tris EDTA buffer (10 mM

Tris and 1 mM EDTA, pH 9.0) (for study III) and in 10 mM citrate buffer (pH 6.0) (for

studies II, IV,V), followed by a period of 10 minutes of heating in a microwave at 600

W, with checking of the plastic box after the first five minutes to ascertain that it had

enough fluid to evaporate and to avoid drying-up of the slides. In study V, the

microwave used was programmed so that the heating cycle would run for 24 minutes at

94 ºC. Slides were kept 30 minutes at room temperature to cool them down.

56

b) Pepsin treatment antigen retrieval

This technique, which was applied only for study III, is based on the fact that pepsin is

a proteolytic enzyme and thus may digest (etch) the masking proteins, thereby exposing

the covered (hidden) antigen. In this technique, sections were immersed in a solution

containing pepsin and distilled water (1:250) followed by addition of 1-N HCl (1:100,

0.1 ml). All sections were kept in +37 ˚C incubator for 30 minutes and profusely

washed with running water, and finally washed in PBS for 5 minutes.

Primary antibodies used in the study

The primary anti-human antibodies which were used in the studies I-V are summarized

below in the following table.

Table 6. Primary anti-human antibodies used in this study

Antigen Antibody Source Subtype Concentration

μg/ml

Manufacturer

α-defensin-1 Polyclonal Goat IgG 20 Chemicon, Canada

RANKL Monoclonal Mouse IgG2b 5 R&D, USA

Tryptase Monoclonal Mouse IgG1 1.05 Dako, Denmark

Chymase Monoclonal Mouse IgG1 0.01 Neomarkers, USA

CD1a Monoclonal Mouse IgG1 0.012 Dako, USA

IL-8 Polyclonal Goat IgG 0.2 Santa Cruz, USA

IL-8 RA Polyclonal Rabbit IgG 2 Novus Biologicals, USA

TLR1 Polyclonal Rabbit IgG 0.8 Santa Cruz, USA

TLR2 Polyclonal Rabbit IgG 2.6l Santa Cruz, USA

TLR3 Polyclonal Rabbit IgG 2 Santa Cruz, USA

TLR4 Polyclonal Rabbit IgG 1.3 Santa Cruz, USA

TLR5 Polyclonal Rabbit IgG 1.3 Santa Cruz, USA

TLR6 Polyclonal Goat IgG 1 Santa Cruz, USA

TLR7 Polyclonal Goat IgG 0.8 Santa Cruz, USA

TLR8 Polyclonal Rabbit IgG 1 Santa Cruz, USA

TLR9 Polyclonal Rabbit IgG 0.5 Santa Cruz, USA

57

Programmable Tech Mate staining robot

In the first study (study I), tissue sections of both CHC and LP were stained using the

ChemMate staining system. In this automatic immunostaining protocol, sections were

run through three washes in buffer containing carrier protein, detergent and

preservative and contributing to blocking of non-specific tissue binding of the

immunological reagents used in the protocol. Then the sections were incubated in 20

µg/ml goat anti-human α-defensin-1 IgG (Chemicon, Temecula, CA) in antibody

diluent for 30 minutes at room temperature, 2 µg/ml biotinylated rabbit anti-goat IgG

for 25 minutes, endogenous peroxidase block solution for 8 minutes and finally with

streptavidin conjugated highly purified horse radish peroxidase and 0.03% H2O2 with

concentrated diaminobenzidine (DAB) as chromogen (160 μl of DAB) in horse radish

peroxidase substrate buffer. Sections were counterstained for 30 seconds with Mayer’s

haematoxylin solution before mounting.

ABC staining

Avidin-biotin-peroxidase complex (ABC) staining was applied for studies II-V. In this

protocol, the endogenous peroxidase enzyme activity of the tissues was quenched by

keeping the sections in a solution containing 1.5 ml of 30 % H2O2 diluted with 150 ml

methanol, for 30 minutes at room temperature. To avoid nonspecific staining, the

sections were incubated with normal serum (the normal serum of the species in which

the secondary antibody was produced) (1:50 in 0.1% BSA, Vector Laboratory,

Burlingame, UK) for 1 hour at room temperature. The sections were then incubated in

the primary antibody (please refer to table 2 for details about their types and

concentrations) and diluted according to the requirements of the experiment with 0.1%

BSA and incubated overnight at 4ºC. Next day, all sections were incubated in

biotinylated secondary antibody (dilution 1: 100, Vector) for 1 hour at room

temperature, followed by incubation in avidin-biotin-peroxidase complex (ABC)

(dilution 1:100; Vector) for 1 hour at room temperature. Finally, the sites of peroxidase

binding were revealed with a combination of 300 µl of 3% H2O2 and 0.023% 3, 3′-

diaminobenzidine tetra-hydrochloride solution (Sigma Chemical Co., St. Louis, MO,

USA). All sections were counterstained with Mayer’s haematoxylin solution for 30

seconds, dehydrated in graded ethanol, cleared in xylene and mounted in Diatex

(Becker Industrifärg AB, Märsta, Sweden).

58

Specificity & sensitivity of the primary antibody

During immunohistochemical staining of all the studies (I-V) the specificity of the

immunoreaction was asserted by using some sections for negative staining control

experiments. These control sections were stained with control antibodies of the same

concentration (and isotype when pertinent) as the primary antibody used in this study

(if a monoclonal antibody was used for control staining an antibody directed against

Aspergillus niger glucose oxidase (Dakopatts, Glostrup, Denmark), an enzyme which is

not present or inducible in mammalian tissues, was used).

To assess the specificity of the primary antibody in study I, antigen absorption test was

done by incubating the primary polyclonal goat anti-human α-defensin-1 IgG antibody

with recombinant human α-defensin-1 peptide (GF 099 Chemicon, Temecula, CA,

USA) in phosphate buffered saline containing 1% bovine serum albumin, at 1:10 on a

molar basis, for 1 hour at room temperature.

In study IV, to preclude any nonspecific binding of the ABC to biotin of the yeast, new

sections of CHC were incubated in avidin-biotin-peroxidase complex (ABC) (dilution

1:100; Vector) for one hour at room temperature. Then all steps were followed as

mentioned above. No positive staining could be seen in the control sections.

Western blotting

Western blotting technique was used in study II in which mast cells were investigated

to assess if they contain RANKL.

a) Cell lysis

The cell pellet of 105 human umbilical cord blood-derived mast cells sensitized with

IgE and activated with anti-IgE IgG was lysed with 25µl Passive Lysis Buffer

(Promega Corporation, Madison, WI, USA) with gentle shaking at room temperature

for 30 minutes. The cell debris was removed by centrifugation at 13 000 g for 10 min at

+4˚C and supernatant was stored at -70˚C.

b) Gel electrophoresis and immunoblot analysis

Cell lysis and culture media (10µl) were mixed with sodium dodecyl sulphate

containing sample buffer (5µl) (New England Biolabs, Beverly, MA, USA). The

samples were boiled for 5 minutes, and applied to the gels. Sodium dodecyl sulphate

polyacrylamide gel electrophoresis was performed with a 12% polyacrylamide slab gel.

After electrophoresis; the gels were blotted onto polyvinylidene fluoride (Millipore

Corporation, Bedford, MA, USA).

59

After blocking overnight with a 3% bovine serum albumin in Tris buffered saline

blocking solution and a ten minute incubation in washing solution (0.1% Tween 20, 50

mM Tris-HCl, 0.5 M NaCl, pH 7.5), blotted membranes were incubated with affinity

purified polyclonal rabbit anti-human sRANK ligand antibody (0.4µg/ml dilution with

washing buffer containing 2% bovine serum albumin) (PeproTech, Rocky Hill, NJ,

USA) for two hours at room temperature. The membranes were then washed with

washing buffer for 30 minutes with at least 3 changes of buffer. The membranes were

further incubated with alkaline phosphatase conjugated goat anti-rabbit Ig (1:5000

dilution with washing buffer containing 2% bovine serum albumin) (Jackson

Immunoresearch Laboratories, West Grove, PA, USA) for one hour at room

temperature. The membranes were then washed with washing buffer for 30 minutes

with at least 3 changes of buffer and a final wash in Tris buffered saline (50mM Tris-

HCl, 0.9% NaCl pH7.5). The alkaline phosphatase-binding sites were revealed in ten

millilitres of colour development solution (Alkaline Phosphatase Conjugate Substrate

Kit, Bio-Rad Laboratories, Richmond, CA, USA) based on the colorimetric detection of

alkaline phosphatase with a mixture of 5-bromo-4-chloro-3-indolyl phosphate (BCIP)

and nitroblue tetrazolium (NBT). After 30 minutes the color reaction was stopped by

washing the membranes in distilled water for ten minutes.

Cell culture

Cord blood-derived clusters of differentiation (CD) 34+ hematopoietic cells (Cambrex,

East Rutherford, NJ) were cultured in StemSpan SF expansion medium (StemCell

Technologies, Vancouver, Canada), supplemented with penicillin (100 U/ml),

streptomycin (100 μg/ml) and gentamicin (10 μg/ml; Life Technologies, Carlsbad, CA),

in the presence of 10 ng/ml of SCF (R & D Systems Inc. Minneapolis, MN) in an

atmosphere containing 5% CO2 and 5% O2 for 10 weeks, after which the cells were

cultured in the presence of 100 ng/ml of SCF, 10 ng/ml of IL-4 and 50 ng/ml of IL-6 (R

& D Systems Inc.) in an atmosphere of 5% CO2 and atmospheric oxygen for at least 4

weeks. The effect of serum-supplementation was investigated by adding 10% fetal

bovine serum (FBS; Life Technologies) under different oxygen tensions to cells

cultured for 10 weeks in serum-free and hypoxic conditions, in the presence of

cytokines as above. The number of viable cells was counted with Trypan blue (Sigma,

St. Louis, MO) and is expressed as mean ±SEM.

60

Statistical analysis

In study V, the differences in expression of the TLRs in the three epithelial layers

(upper layer, middle layer and basal layer) for the three groups (CHC, leukoplakia and

healthy mucosa biopsy samples) were analyzed separately with Pearson’s chi-squared

test in which the levels of the TLR expression of the epithelial cells were coded (0 = no

expression at all, 1 = 1-24% expression, 2 = 25-49% expression, 3 = 50-74%

expression, 4 = 75-100% expression) using the non-parametric Kruskal-Wallis test.

Differences between the two groups (candidosis versus control, candidosis versus

leukoplakia, leukoplakia versus control) were assessed by the non parametric Mann-

Whitney U test. All statistical analyses were performed using the SPSS version 14.0. p-

values less than 0.05 (two tailed test) was considered to be statistically significant.

61

RESULTS & DISCUSSION

Routine histopathology (for studies I, III, IV,

V)

Candidal hyphae were revealed in CHC confined

to the uppermost layers of the epithelia with

varying degree of penetration, while in

leukoplakia no hyphae were observed although

some yeast cells were occasionally found on the

surface of the mucosa in some of the samples. In

CHC, candidal hyphae were usually growing in a

perpendicular direction into the epithelium and

were randomly distributed along the epithelial

surface colonizing some areas while absent in others (Fig. 9, arrows). Numerical

grading was used to estimate the number of yeast cells and hyphae in individual cases

(included with the result tables of each study below). Staining of CHC sections with

hematoxylin and eosin showed parakeratinized epithelium with clear, broad and

bulbous rete ridges. In some areas keratin layer had eroded and the underlying

epithelium was exposed. Individual neutrophils and microabscesses were often seen in

the epithelia of CHC samples, whereas inflammatory cell infiltrates, composed of

lymphocytes and plasma cells, were seen in lamina propria in all sections.

Immunohistochemical staining

1. Study I: α-defensin-1 in CHC

The antimicrobial peptide α-defensin-1 was mainly located in neutrophils. Both CHC

and LP showed intravascular α-defensin-1 containing neutrophils, which was intense

and uniform. In the lamina propria of CHC α-defensin-1 containing neutrophils were

also found. There was no peri- or extracellular α-defensin-1 staining in the lamina

propria. In the epithelium, an apparent gradient or stratification of α-defensin-1 positive

neutrophils was obvious. This accumulation was particularly intense in heavily-

Candida-infected and neutrophil-infiltrated samples, which also contained

intraepithelial neutrophil microabscesses (Fig. 10). The cytoplasmic staining of many

individual neutrophils in CHC epithelia was relatively weak, but associated with peri-

and extracellular staining. This was particularly prominent in areas of microabscesses.

In addition, CHC epithelia contained an intense superficial band or rim of α-defensin-1

Figure 9. Candidal hyphae (arrows) attacking oral epithelium

62

deposition in the uppermost epithelial cell layers (Fig. 10, 11). However, in CHC the

number of candidal hyphae did not correlate with the number of α-defensin-1 positive

neutrophils in connective tissue (P=0.58) or in

the epithelium (P=0.35) (Table 7).

In contrast, the Candida-negative, control LPs

contained few neutrophils and very little

immunoreactive α-defensin-1, mostly in

intravascular neutrophils. LP epithelium was

not infiltrated by neutrophils and no epithelial

microabscesses or extracellular α-defensin-1

staining was observed.

In this work, we were interested in the eventual protective role of the α-defensin-1

against the opportunistic pathogen C. albicans in CHC, and based on our achieved

results we can narrate a possible step-by-step story of both the infectious and the

defense scenes, focusing on the role of the main spot of our main study, i.e. α-defensin-

1. The first event which follows invasion of the oral mucosa by C. albicans is the

activation of the host innate immunity that, subsequently, triggers a local inflammatory

reaction. Neutrophils, which are capable of killing and digesting smaller invading

microorganisms such as planktonized yeast cells in intracellular phagolysosomes

(Sohnle and Collins-Lech 1990), are considered the

first line of defense against such intruding pathogens,

are alarmed and thus recruited from the intravascular

compartment to the site of infection. In the

extravascular tissue and with the help of some

chemokines, e.g. IL-8, neutrophils traverse through

the lamina propria towards the location of the

pathogen. When neutrophils get into the epithelium

they accumulate and form microabscesses (localized

collection of PMNs in the epithelium). Due to their

activation, neutrophils degranulate their contents extracellularly, and among those

substances released is α-defensin-1. Although α-defensin-1 is released by neutrophils

(irrespective of their location at the time of α-defensin-1 release), most of our results

show that α-defensin-1 was concentrated in the upper most part of the epithelium. We

Figure 11 showing the strong α-defensin-1 rim concentration at the uppermost epithelium

Figure 10. Microabscess (arrow) and a strong α-defensin-1 band in the uppermost epithelium

63

hypothesize an explanation of this observation. Whenever α-defensin-1 is released by

the neutrophils, it may move along with the advancing epithelial cells as a result of the

normal epithelial cell growth, migration and shedding cycle (epithelial flow). This will

inevitably lead to the heavy accumulation of this antimicrobial peptide and thus to the

formation of the obvious immunoreactive rim at the most superficial part of the

epithelium. The presence of this α-defensin-1-rich zone might serve as a shield against

the ingress of C. albicans in the mucosa. Formation of this neutrophil-derived and

epithelially-deposited α-defensin-1 barrier may lead to an effective anti-candidal shield

against new ingrowths.

When a neutrophil faces a candidal pseudohyphen, it degranulates letting its granular

content with α-defensins come in contact with the candidal yeast membrane and this

can result in inhibition of the growth or even killing of the microorganism. Although

we conducted our study with the first class of α-defensin-1, we believe that it is more or

less similar to the behavior of the other neutrophil granule-derived α-defensin

subclasses as far as mucosal infections are concerned.

Neutrophils can produce, via activation of the cell surface NADPH oxidase, other

antimicrobial substances, e.g. superoxide anion (O¯2) and hydrogen peroxide (H2O2).

The problem with these “chemical weapons” is that their action is immediate; therefore

they seem not to be effective enough against chronic, long standing infections such as

CHC (Dahlgren and Karlsson 1999). Besides, even if these respiratory burst products

succeed in disrupting the pathogen(s), they will probably damage some “innocent

bystanders”, which can trigger an inflammatory reaction. This, however, does not

refrain neutrophils from using other options, e.g. α-defensins. The cooperation between

the immigrant neutrophils, which release α-defensins, and resident epithelial cells

bestows the host an important support in terms of candidal defense (Sawaki, Mizukawa

et al. 2002). As might seem paradoxical, we did not find a significant correlation

between prevalence of candidal hyphae and number of α-defensin-1 expressing

neutrophils. To justify this correlation discrepancy, we suggest that the effects of

epithelial β-defensins may supplement the neutrophil-derived α-defensin-1. This

synergistic action may account for the relatively low content of α-defensin-1 (produced

by the host) compared to the amount of the existing pathogens in some cases.

64

Table 7. Alpha-defensin-1 in chronic hyperplastic candidosis (CHC) and leukoplakia

(LP).

− = Negative, +/− = only occasional, + = some, ++ = moderate numbers of and +++ =

high numbers of hyphae, microabscesses, neutrophil infiltrates and α-defensin-1

positive cells. E = epithelium, CT = connective tissue.

Sample Hyphae Microabscesses Neutrophil Dysplasia α-defensin-1

1 CHC +++ - +++ Dysplasia moderata E+, CT +/-2 CHC + + + No dysplasia E-, CT+/- 3 CHC +++ + + No dysplasia E+, CT+/- 4 CHC + + +++ No dysplasia E+, CT+/- 5 CHC + + + No dysplasia E+, CT+/- 6 CHC +++ + + No dysplasia E+, CT- 7 CHC + + +++ Dysplasia moderata E+, CT- 8 CHC + + + No dysplasia E+, CT- 9 CHC +++ + + No dysplasia E+, CT- 10 CHC +++ +++ +++ No dysplasia E+, CT- 1 LP - - - Dysplasia levis - 2 LP - - - No dysplasia - 3 LP - - - No dysplasia - 4 LP - - - No dysplasia - 5 LP - - - No dysplasia - 6 LP - - - No dysplasia - 7 LP - - - Dysplasia levis - 8 LP - - - No dysplasia - 9 LP - - - No dysplasia - 10 LP - - +/- No dysplasia -

65

2. Study II: RANKL in mast cell (in atherosclerosis)

In sections of atherosclerotic left anterior descending arteries taken from patients with

coronary artery disease, we found mast cells in

the adventitia layer, which displayed strong

granular RANKL staining apparently confined to

the cellular cytoplasm, without any visible signs

of its release into the extracellular space. In

contrast, mast cells in atherosclerotic plaques

displayed granular cytoplasmic RANKL staining,

which was faint and associated with pericellular

RANKL-positive granules, apparently as a result

of mast cell activation and partial degranulation.

Immunocytochemistry of sections of mature human mast cells (developed from in vitro

culture of umbilical cord blood-derived CD34+ haematopoietic stem cells) showed a

strong and granular cytoplasmic RANKL staining (Fig. 12). Negative control did not

show any immunostaining. In Western blotting,

lysed mast cells displayed a band which, by its

size and immunoreactivity, was shown to

represent the soluble form of RANKL (Fig. 13)

In fact, the idea of this study emerged when we

were doing a series of immunohistochemical

staining of some CHC sections with different

cytokines using different antigen retrieval

protocols. This screening and use of pepsin

pretreatment for antigen retrieval culminated in our discovery that mast cells express

RANKL, a finding which had not been reported before. Therefore, we decided to assess

this observation in a well-known disease called atherosclerosis. As the name suggests

atherosclerosis is a general term applied to any obliterating blood vessel atheroma

formation (literally it comes from the Greek words atheroma, meaning “porridge”, and

Skleros, meaning “hard”). We investigated the role of mast cells in terms of RANKL

expression and whether it is involved in any mechanism behind the process of coronary

calcification. There are many processes that are thought to contribute to the

Figure 12. Mast cells cultured in vitro show positive RANKL granules

Figure 13. Western blot of lysed mast cells show a band of soluble RANKL

66

development of atherosclerosis, e.g. oxidant stress (Schulze and Lee 2005). Since the

ossified structure of the atherosclerotic plaque was found to have the same histological

features of normal bone including trabeculae, lacunae and marrow like spaces, Maria et

al. suggested that this structure be called “osteosome” (Jeziorska, McCollum et al.

1998). By our discovery that mast cells form a source of RANKL, we provided new

insight for the quantitative assessment of the cytokine RANKL in the pathogenesis of

atherosclerosis. RANKL, which is also called osteoprotegerin ligand (OPGL), is a

membrane-bound ligand regulating osteoclast maturation and activation (McClung

2006). The interaction of RANKL with its receptor (RANK) or decoy receptor

osteoprotegerin (OPG) comprises an essential step in bone physiology and

pathophysiology. RANKL and RANK have recently been found both in the endothelial

cells as well as the vascular smooth muscle cells (VSMCs) of tunica media of arteries,

suggesting that RANKL-RANK interaction is important for blood vessel

(patho)physiology (Collin-Osdoby 2004). Our study seems to expand this field by

incorporating RANKL-positive perivascular mast cells as new actor into this system.

Accordingly, OPG deficiency leads to osteoporosis and arterial calcification.

Due to the uncertainty of the exact role of RANKL in the pathophysiology of

atherosclerosis, we cannot form an opinion whether expression of RANKL has

beneficial or harmful effects in this particular condition. It might be that RANKL plays

a protective role in atherosclerosis by means of stimulating osteoclastic progenitors to

mature to osteoclasts which can resorb the already formed bone in the calcified plaques,

but this cannot be predicted based on only immunohistochemical experiments.

The present observations demonstrate that mast cells form a potent cellular source of

soluble RANKL, which may contribute to vascular wall inflammation and plaque

calcification. Molecular mechanisms responsible for the role of mast cells in the

regulation of pathological, dystrophic calcification in atherosclerotic plaques and bone

metabolism are only now emerging. It is noteworthy, that mast cells do respond to

mechanical stimulation. In this respect it was interesting to note that mast cells in the

adventitia were resting mast cells, whereas mast cells in the atherosclerotic plaque were

activated, i.e. partially degranulated. This raises the possibility that the shear stress and

the lever effect of the hard and calcified plaque may lead to mechanical mast cell

activation and degranulation. Degranulation leads to release of biologically active

mediators like RANKL and also to release of mast cell proteinases and collagenase

activators, which contribute to plaque rupture.

67

This is the first study to show that mast cells express RANKL, thereby suggesting

another critical role of mast cells in atherosclerosis and adding another cellular source

of RANKL to the list of RANKL producing cells already reported in the literature.

However, whether this affects osteoclast biology in the vicinity of the calcified lesion

or some other aspects of endothelial-smooth muscle cell interactions and vascular

pathology remains to be elucidated.

Therefore, mast cells in general and RANKL in mast cells in particular, together with

its co-actors, may deserve more attention in atherosclerosis and calcified tissue

research.

3. Study III: Role of RANKL in CHC

Langerhans cells, which are CD1a positive, were found in the epithelia of CHC, LP and

healthy controls. In the epithelium of LP and healthy tissue, these CD1a positive cells

were mostly found in the middle layer, while in CHC they formed a network which was

seen throughout the whole epithelium: in its upper, middle and lower thirds. In CHC,

there was an obvious match between the content of Langerhans cells, whose grading

values ranged from high (+++) to low (+), and the estimated quantity of candidal

hyphae in each case (Table 8). In the

epithelium, the morphology of CD1a positive

cells was typical of dendritic cells, whereas in

the lamina propria, they had the more regular

shape of inflammatory mononuclear cells. In

some sections of CHC, Langerhans cells were

in a very close proximity to, or even traversing

the basement membrane (Fig. 14).

In CHC and LP, the epithelia showed some

RANKL immunoreaction particularly at the region of the epithelial ridges with stronger

staining of the former than the latter. Epithelium of healthy control sections was

completely devoid of any immunoreaction to RANKL.

RANKL immunostaining was noticed to be dependent upon the method of

pretreatment. In case of pepsin pretreatment, the only RANKL-positive cells in lamina

propria were typical mast cells which in CHC displayed only faint granular cytoplasmic

staining, with pericellular RANKL positive granules and matrix. In contrast, the

number of mast cells was relatively low in LP and healthy control sections and RANKL

Figure 14. CD1a LCs traverse the basement membrane towards the lamina propria

68

staining was in these cells confined to the cytoplasm without any signs of extracellular

release of RANKL.

If instead of pepsin, Tris EDTA heat pre-treatment was used for antigen retrieval, also

other cells, e.g. fibroblasts and lymphocytes, were found to be RANKL positive. These

other RANKL positive cells were more intensely stained than mast cells and they were

distributed mainly in the subepithelial areas. Side by side comparison of pepsin pre-

treated and Tris EDTA heat pre-treated sections from the same location demonstrates

nicely this difference.

Neither CD1a nor RANKL positive cells were observed in negative staining controls.

This third work supports the previous study showing that also mast cells in oral mucosa

contain RANKL, which was in the second work described in mast cells residing in

connective tissue. In this study, CD1a positive Langerhans cells (LCs) were found in

mucosal sections from CHC, LP and healthy control tissues. LCs along with

neutrophils comprise a very essential nonspecific armory which plays a pivotal role in

extinguishing the infectious microbe C. albicans from the host. Besides their direct

antimicrobial effects i.e. phagocytosis, dendritic cells (immature and mature) can also

produce chemotactic factors that activate and recruit neutrophils (Scimone, Lutzky et

al. 2005). They are considered the most potent antigen catching, processing and

presenting cells of the immune defense system (Steinman 1991). The preferred place of

residence for LCs is mucosa where they act as sentinels against any foreign substances.

Our results suggest a role for CD1a LCs in the hindrance of the entry of such

pathogens. When an antigen source, e.g. candidal cell, succeeds in invading the

epithelial natural barrier and intrudes into epithelium, LCs which have already been

waiting for such a situation will stand in the way of the invading yeasts and hyphae and

will eventually capture and subsequently engulf them. Following endocytosis, LCs

process the engulfed candidal cell and associate its degraded components (epitopes)

with major histocompatibility complex molecule (MHC II). Dendritic cells undergo

maturation and leave the peripheral tissue to migrate to the regional lymph nodes where

they present the antigen to naïve T-cells (Cutler and Jotwani 2004). In the present work,

we demonstrated and compared the presence of CD1a positive Langerhans cells along

with RANKL positive cells in CHC with LP and healthy controls. We also assessed the

difference in the immunological response between CHC and LP since the former is

considered as LP superimposed by C. albicans infection. In CHC, the number of LCs

and their pattern of distribution, however, were variable, probably due to recruitment

69

and migration. The dendritic cell numbers are debatable in other oral infections and

inflammations, e.g. chronic periodontitis. In three separate studies, it was found that the

number of dendritic cells increased (Saglie, Pertuiset et al. 1987), decreased (Seguier,

Godeau et al. 2000), or did not change (Gemmell, Carter et al. 2002) in chronic

periodontitis compared to healthy controls. We found a lot of inter- and intraindividual

(intrasample) variation in the number and localization of CD1a positive Langerhans

cells in CHC, which probably reflects the dynamic nature of their involvement in local

disease mechanisms.

Our results showed a close match between the numerical quantities of LCs in the

epithelium of CHC sections with the numbers of candidal yeasts and hyphae. However,

we still think that the number of LCs per se in such mucosal infectious lesions does not

form a reliable parameter to assess the severity of the infection, since otherwise all

CHC sections should have shown higher numbers of LCs than their Candida negative

counterparts and comparators, i.e. LP and healthy controls, some of which did contain

nearly equal or even higher numbers of LCs than CHC. As has been stated, LCs were

usually found above the basal layer of mucosal epithelium in oral, nasal,

gastrointestinal and other areas (Girolomoni, Caux et al. 2002), which was the case also

in our LP and healthy control sections. But in CHC, however, Langerhans cells were

seen in all epithelial regions including the basement membrane area at the epithelial-

lamina propria junction. The presence of LCs in the upper part of the epithelium can

reflect the role of Langerhans cells in capturing and engulfing C. albicans. Once C.

albicans (yeast or hypha) is engulfed, LCs start to migrate away from the epithelium

towards the lamina propria where they have to traverse the basement membrane, as

shown in the Figure 14. The trafficking capability of LCs offers a convincing

explanation for the highly variable location of Langerhans cells throughout the

epithelium in CHC. The second theme of this work deals with studying the expression

of the cytokine Receptor activator of nuclear factor kappa-B ligand (RANKL) and

comparing it in CHC with LP and healthy control. RANKL is a membrane-bound

ligand which belongs to tumor necrosis factor superfamily. It is produced by

osteoblastic lineage cells, mesenchymal cells, VSMCs, chondrocytes, and various

immune cells, such as activated T cells (Hofbauer, Shui et al. 2001).

70

Table 8. Grading* of CD1a and RANKL staining in chronic hyperplastic candidosis

(CHC), leukoplakia (LP) and healthy control (H).

* (0) The structure itself is not present, (-) negative, (±) only occasional, (+) some, (++) moderate and (+++) high numbers of positive cell. DC= dendritic cell CT= connective tissue

CD1a positive DCs RANKL positive cells Cases

Yeasts and hyphae

Keratin DCs in epithelium DCs in

CT Epithelial cells

CT cells e.g. mast cells, other cells

CHC-1 +++ - ++ ++ - ++ CHC-2 + 0 + ++ ++ +++ CHC-3 +++ - +++ ++ ± + CHC-4 + - + ++ ± + CHC-5 + - ++ + ± ++ CHC-6 +++ - +++ ++ ± ++ CHC-7 + - + + ++ ++ CHC-8 ++ - +++ + + ++ CHC-9 +++ - +++ ++ + +++ CHC10 +++ - +++ ++ ++ +++ Neg. - - - - - - LP-1 - - ++ ++ ± ++

LP-2 - - ++ + ± ++ LP-3 - - +/- + ± + LP-4 - - + ++ ± ++ LP-5 - - ++ ++ ± + LP-6 - - + ++ ± ++ LP-7 - - + ++ + ++ LP-8 - - ++ ++ ± ++ LP-9 - - ++ - ± ++ LP-10 - - ++ + ± - H-1 - 0 ++ ++ - -

H-2 - - ++ ++ - + H-3 - - + ++ - ++

71

It binds to its receptor RANK on the surface of monocyte/macrophages, osteoclast

progenitors, endothelial cells and antigen presenting cells leading to their differentiation

(Matsuzaki, Udagawa et al. 1998; Burgess, Qian et al. 1999). T cells can modulate the

function of dendritic cells (Josien, Wong et al. 1999) through release of RANKL. This

augments the capacity of dendritic cells to stimulate naïve T-cell proliferation in a

mixed lymphocyte reaction (Anderson, Maraskovsky et al. 1997). As we found in the

previous study that mast cells produce RANKL, we proceeded with this observation to

assess whether it holds true for candidal mucosal infections involving antigen

presenting cell-T lymphocyte interactions. The antigen retrieval methods seemed to

affect the results of RANKL staining. When we treated the samples, which were

formalin-fixed and paraffin embedded, with pepsin digestion, mast cells could be

specifically studied as probably all cells containing cell surface bound RANKL become

negative. In contrast, when Tris EDTA heat treatment was used for antigen retrieval,

other RANKL-producing cells were also visible and stained positively, including

lymphocytes and fibroblasts. We suggest that the different location of the antigen

(RANKL) in different cells as well as to the mechanism of action of the antigen

retrieving agent, may account for the disparity of RANKL staining. Pepsin is a

proteolytic enzyme so its use entails the risk of cleaving some molecules exposed on

the cell surface. In this way, pepsin appears to destroy most of RANKL proteins which

were expressed by cells containing RANKL on their surfaces. In mast cells, however,

RANKL seems to be stored in cytoplasmic granules. Since pepsin cannot penetrate

plasma membranes (either cellular or in particular granular) composed mostly of lipid,

RANKL proteins located in mast cells avoids the splitting action of pepsin. The Tris-

EDTA based solution, on the other hand, is designed to break the protein cross-links,

without endangering the antigen proper itself. Thus, all RANKL positive cells were

seen after this method of antigen retrieval. This type of fixation artefact can be used if

in particular the eventual role of mast cell RANKL is in focus of interest.

In CHC, RANKL-positive mast cells had weak staining apparently due to release of

their RANKL laden-granules extracellularly, while in LP and healthy controls RANKL

staining of the mast cell cytoplasm was stronger and completely intracellular. This

observation might signify an active role of mast cell RANKL in CHC, which is not

taken into use in noninfectious lesion, e.g. LP, or in normal mucosa. We suggest that

this mast cell activation and RANKL release perhaps strengthens and speeds up the

72

maturation of the RANK positive Langerhans cells, when they communicate with the

RANKL positive T-cells. In this respect, our results may add a new aspect of the

participation of mast cells in the immunological response against pathogens.

4. Study IV: Role of IL-8 and its receptor in CHC

Immunohistochemical staining revealed IL-8 and IL-8 RA in all CHC samples and to a

lesser extent in healthy control sections. Deep microvasculature showed IL-8 positive

endothelial cells, which was more intense than in the superficial vasculature. In contrast

to IL-8, the intensity of staining of both deep and superficial endothelial cells for IL-8

receptor A was much weaker and more variable. Some areas were IL-8 RA negative,

although also strongly staining endothelia were found (Table 8). The microvascular

endothelia in the healthy control sections were

relatively weak in IL-8 and IL-8 RA staining

compared to CHC (Table 9, 10).

The basal (lower) cell layer of the CHC

epithelium showed stronger staining of IL-8 and

IL-8 RA than the spinous (middle) cell layer,

whereas the granular (upper) layer was

completely devoid of any immunoreactions

except for very scarce staining for IL-8 RA in some of the sections. Staining of the

healthy control epithelia was generally less intensive than their counterparts of CHC.

In the lamina propria and among inflammatory cells, IL-8 was found in many cell

types, including plasma cells, mast cells, macrophages and neutrophils. Heavily

infected CHC sections, in which there was a heavy neutrophil infiltration, showed

intense IL-8 staining of the inflammatory cells. While

polymorphonuclear cells had migrated on to the

epithelium where they formed intraepithelial

microabscesses containing IL-8 RA positive

neutrophils, mononuclear inflammatory cells were

retained in the submucosa. Negative staining controls

confirmed the specificity of the staining.

Figure 15 showing the positive reaction of the mother cell of C. albicans to IL-8

Figure 16. The tip of C. albicans hyphae seems to be positive to IL-8RA

73

IL-8 and IL-8 receptor A in candidal cells in tissue and in agar sections

The germs of C. albicans, which were found lying on the uppermost layers of the CHC

epithelia, stained positively for IL-8 and IL-8 RA. Candidal cells seemed to be

polarized so that the mature blastoconidia (candidal cell bodies) only expressed IL-8

(Table 9, Fig. 15), whereas the hyphal tips were IL-8 RA positive (Table 10, Fig. 16).

A set of agar sections with cultured C. albicans cells were similarly stained. These

experiments confirmed that only the mother cells of C. albicans express IL-8, while IL-

8 RA is expressed by the outermost parts or the tips of candidal hyphae. The negative

staining controls showed neither IL-8 nor IL-8 RA staining.

Table 9. IL-8 in chronic hyperplastic candidosis (CHC) and healthy controls

0 the structure itself is not present, – negative, ± only occasional, + some, ++ moderate and +++ high numbers of positive cells.

Epithelium Lamina propria Cases

Candidal mother cell

Candidal hyphae

Keratin layer

Medium layer

Basal layer

Superficial vascular endothelium

Deep vascular endothelium

1 CHC +++ + – ++ +++ + +++ 2 CHC ++ ++ – ++ +++ ++ +++ 3 CHC +++ ++ 0 ++ +++ ++ +++ 4 CHC 0 0 0 ++ +++ ++ +++ 5 CHC 0 0 – ++ ++ + +++ 6 CHC 0 0 – + +++ ++ +++ 7 CHC ++ ++ – ++ +++ + ++ 8 CHC +++ ++ – ++ +++ + +++ 9 CHC +++ ++ – + +++ + +++ 10 CHC 0 0 – ++ ++ ++ +++ 1 Control 0 0 0 + + + + 2 Control 0 0 0 ++ ++ +++ +++ 3 Control 0 0 0 + + ++ + Negative control staining

0 0 – – – – –

74

Table 10. IL-8 receptor A staining in chronic hyperplastic candidosis (CHC) and

healthy controls

0 the structure itself is not present, – negative, ± only occasional, + some, ++ moderate and +++ high numbers of positive cells.

As we have previously mentioned, the first line of cells which are called to the site of

candidal infection are neutrophils. But how neutrophils are called and recruited to the

place of tissue insult was the objective of this fourth work in which we were interested

in the eventual role of IL-8 (a cytokine known for its chemoattractive ability for

neutrophils) and its receptor IL-8 RA in neutrophil-mediated defense in CHC. The role

of cytokines, when synthesized, is to augment the defensive role of phagocytes

(Marodi 1997). In the present study, we were able to show that especially the vascular

endothelium and oral epithelial cells seem to react to candidosis by secretion of IL-8.

Interleukin-8 is a chemokine which belongs to the CXC subfamily and has potent

chemotactic effects on neutrophils both in vivo and in vitro (Baggiolini, Dewald et al.

Epithelium Lamina propria Cases

Candidal mother cell

Candidal hyphae

Keratin layer

Medium layer

Basal layer

Superficial vascular endothelium

Deep vascular endothelium

1 CHC ++ ++ ± ++ +++ +++ +++ 2 CHC 0 0 – ++ ++ + ± 3 CHC ++ ++ – ++ +++ +++ +++ 4 CHC 0 0 – ++ +++ + ± 5 CHC ++ +++ – + +++ – ± 6 CHC ++ ++ ± + +++ ++ ++ 7 CHC + ++ ± +++ +++ ++ ++ 8 CHC ++ ++ – ++ +++ ± ++ 9 CHC + ++ – ++ ++ ± – 10 CHC 0 0 – + ++ ± + 1 Control 0 0 0 + +++ – – 2 Control 0 0 0 ++ ++ ++ ++ 3 Control 0 0 0 + ++ ++ ++ Negative control staining

– – – – – – –

75

1994). Neutrophils apparently respond to IL-8 by migrating through the vascular wall

and lamina propria to the epithelium where the actual candidal hyphae are located.

Once they infiltrate the epithelium, neutrophils form microabscesses and release the

antimicrobial contents of their granules extracellularly. Probably due to the strong

innate defense shield, candidal hyphae (despite their penetrative capacity) seldom reach

beyond the spinous cell layer of the epithelium. There has long been a debate, due to

conflicting data, whether C. albicans stimulates epithelial cells to synthesize IL-8 in

vivo and in vitro (Hebert and Baker 1993). Whatever the mechanism, in this work we

demonstrate that the growth of C. albicans in CHC is associated with relatively strong

IL-8 and IL-8 RA staining. IL-8 is a strong chemotactic stimulus to neutrophils, but it

may also play a role for candidal cell biology.

The surprising observation was that mother cells of C. albicans express IL-8 (or IL-8 –

like protein), while IL-8 RA (or IL-8 RA-like protein) was localized mainly in the

hyphal tips. Although we did not find candidal homologues of IL-8 or IL-8 RA when

we used BLAST to assess whether C. albicans has DNA sequences coding these

proteins, we still believe there is a possibility that C. albicans encodes such proteins

since genomic sequencing of C. albicans has not been completed yet. These findings,

which were first done using CHC tissue sections, were later confirmed by pure candidal

cultures in agar. The peculiar location of the positive staining of IL-8 and IL-8 RA by

the mother cell and hyphal tips, respectively, seems to exclude the chance of a

haphazard occurrence. Based on our findings we assume that the continuous contact of

C. albicans with oral epithelium in health and disease (since C. albicans comprises the

major fungal oral microflora in about 25-75% of population) might have driven the

fungus to evolve mechanisms counteracting host immune defenses.

C. albicans has been demonstrated to have a property of contact sensing or

thigmotropism (Sherwood, Gow et al. 1992). This property is important in candidal

biofilm formation on intraoral devices such as acrylic dentures (Nikawa, Nishimura et

al. 1998). A rather new concept referred to as chemotropism has emerged in candidal

biology. Davies et al. argued that Candida hyphal tips could elaborate exoenzymes into

the underlying host structure. The hyphal tips, maybe through plasma membrane

receptors, would be able to detect if this produces any cellular breakdown products

(Davies, Stacey et al. 1999). Based on the present work, it might be that C. albicans

expresses IL-8 RA on its hyphal tips to be able to sense the presence of IL-8, which

might indicate potential danger. We tentatively name this phenomenon chemophobia. It

76

first seemed somewhat paradoxical that candidal cell body itself produces IL-8 or an

analogue. However, if the endogenous candidal IL-8 (or analogue) produced by the

mother cell communicates with the IL-8 RA (or analogue) at the tip of the hypha, its

repulsive effect might help to guide the growth of the hypha in a centrifugal direction,

away from the mother cell. When the tip of the hypha has grown to a distance from the

mother cell so that the communication between the cell body and hyphal tip ceases, this

very same ability might help to keep the sensitive hyphal tip away from IL-8-rich areas,

which might be or become heavily infiltrated by neutrophils. Likewise, if the hyphal

tip-located IL-8 receptor does not sense any IL-8, C. albicans might advance until it is

faced by some defense barrier, danger signal or nutrient. This implies that C. albicans

might have a strategy to sense the most suitable path for epithelial penetration.

In conclusion, IL-8 and IL-8 RA seem to be at work in host defense and may contribute

to the recruitment and migration of neutrophils from the vascular compartment through

lamina propria to epithelia, where they accumulate and get engaged in anti-candidal

defense. At the same time, the evolutionary pressure may have conferred C. albicans

the ability to utilize IL-8 system in its own survival strategy. The most straightforward

explanation for such an arrangement would be that the candidal cell utilizes this system

first for internal communication to direct the growth of the hyphae away from the cell

body, followed by its use in external intelligence with an aim to keep the vulnerable

hyphal tip away from danger

5. Study V: Differential expression of TLRs in CHC

The pattern of TLR immunostaining in the three categories of samples (i.e. CHC, LP,

and healthy controls) was different in terms of intensity and distribution of positive

cells. The epithelium of all sections was divided into the three classical layers: lower,

middle and upper. In most of the samples the strongest immunostaining was noticed in

the basal layer. In CHC the number of positive epithelial cells ranged from none (-) to

high (+++) for different TLRs. In hyphae-rich samples of CHC all the epithelium was

uniquely positive to TLR 4 while it showed very faint staining for TLR 2, except for

very few cells at the very bottom (Fig. 17). The three layers of the epithelia of the CHC

sections revealed varying degrees of immunostaining in such a way that the uppermost

layers tended to be devoid of staining while the underlying middle layers varied in

staining intensity from always weak (in case of TLR 2) to moderately immunopositive

(in case of TLR 4) to heavy staining (rest of TLRs), while the lower layer was always

77

positive. The picture was different in leukoplakia where TLRs staining of the middle

and lower epithelial layers was always positive except, in some sections for TLR 9 to

which the tissue showed weak staining or was even negative. In contrast, the epithelial

layers of the healthy control tissue

were always positive except

interestingly a few sections where

the lower layers were very weakly

stained or negative.

The specificity of the TLRs

epithelial staining was confirmed by

the lack of immunoreactive cells in

the negative staining controls.

From a statistical point of view, there

was consistently statistically

significant lower expression of the

TLR2 (P<0.05) among the three

epithelial layers either for candidosis

versus control or candidosis versus

leukoplakia comparisons. In addition

to that, among the upper layer in the candidosis versus normal status there was also

significantly lower expression of the TLR3-6 (P<0.05), and in the candidosis versus

leukoplakia there was a significantly lower expression of the TLR 6 and 7 (P<0.05).

Interestingly, there was a statistically significant higher expression of the TLR 4 and 6

(P< 0.05) in the leukoplakia lower layer versus controls. On the other hand, in the upper

and middle layers these two TLRs expression were observed to be significantly lowered

for candidosis versus leukoplakia or candidosis versus controls.

Lower or higher expression (P>0.05 but <0.066) was also observed for different TLR

among the three different epithelial layers.

In this work, the immunohistochemical expression of nine classes of TLRs (TLR1-9)

was assessed in a series of sections from CHC, LP and healthy tissue. In oral

candidosis, TLRs serve to recognize C. albicans (usually through its cell wall

component zymosan), thereby igniting a set of intracellular signaling cascades, which

end in production of proinflammatory cytokines and chemokines. In this study, we

Figure 17 showing the numerous candidal hyphae in chronic hyperplastic candidosis (A), in which TLR2 staining was very faint except few cells at the very basal layer (B) while the same section showed strong staining of TLR4 (C). The same finding was noticed in another hyphae-rich section (D, E).

78

compared the immunochemical staining of all known TLRs (except TLR10) in sections

from CHC, LP and healthy tissues. It has been shown that candidal cell wall

components constitute pathogen associated molecular patterns (PAMPs) for certain

TLRs, e.g. zymosan is recognized by TLR2/TLR6 heterodimers while mannan is a

ligand for TLR4 (Netea, Van Der Graaf et al. 2002; Roeder, Kirschning et al. 2004).

For practical analysis, the epithelium was divided into three layers namely; upper,

middle and lower. All TLRs, except those which are claimed to recognize C. albicans,

i.e. TLR2, TLR4 and TLR6, were found to be strongly positive especially by the

middle and lower layers. This may indicate that when epithelial TLRs (i.e. TLR1,

TLR3, TLR5-9) are not stimulated by a particular PAMP (in this context, zymosan or

phospholipomannan), they tend (continue) to be constitutively expressed. This

explanation is enhanced further by the staining pattern of the healthy tissue to the TLRs

(TLR1-9) and by a previous report which has documented the expression of TLRs by a

variety of oral mucosal cells (Mahanonda and Pichyangkul 2007). For TLR2 and

TLR4, it was another interesting story. In two samples out of five there were high

numbers of candidal hyphae compared to the unicellular yeasts and when stained with

TLR antibodies, they showed very little staining of TLR2 while TLR4 was comparably

strong, but in the other sections in which hyphae were scarce TLR4 was weaker. If we

take the dogma of negative regulation of TLRs into account (Han and Ulevitch 2005),

then we can suggest that TLR2 is more actively engaged in recognizing and mediating

the effects of candidal ligand (and thus more down-regulated) in the sections which

contained high hyphae: yeast ratio. Such findings drive us to suggest that candidal

hyphae could direct the immune response towards stimulating TLR2 rather than TLR4

which, otherwise, would also be weakly expressed. It has been intensely debated

whether C. albicans could stimulate TLR 2 and 4 in a manner which is dependent on

the fungus morphological form. Our results seem to accord the previous work of Netea

et al. who have shown that the opportunistic pathogen can exploit TLR2 to evade the

immune system (Netea, Van der Meer et al. 2004). The same group was the first to

work on this the idea and demonstrated that TLR2 knockout mice had gained more

resistance against systemic candidosis compared with wild type due to the less release

of anti-inflammatory cytokines, e.g. IL-4 and IL-10 which are elaborated by TLR2

activation (Netea, Sutmuller et al. 2004).

79

In this work we present a new aspect of C.

albicans virulence in mucosal candidosis in

the sense that candidal hyphae bring down the

anticandidal Th1 cytokines (by evading

TLR4), and simultaneously enhances the anti-

inflammatory Th2 process (through

stimulation of TLR2).

The lower layers of all epithelia of CHC

showed wider TLRs staining, comparatively,

than the superficial layers and this is likely

due to the synthesis of TLRs first in the lower

layer and then starts to decrease in quantity as

it travels upwards along the epithelial flow for

a variety of reasons, e.g. limited half-life and

down-regulation.

In leukoplakia, the strong epithelial staining

(for all TLRs except TLR9, which was quite

weak in some sections) extended to the

overlying keratinous layer (Fig. 18). If the

classical definition of leukoplakia was revised

(please refer to the leukoplakia section in this thesis), an explanation can be stated for

the less staining of TLR9 noticed in some sections. The definition of leukoplakia

denotes that it is a lesion and as a result; the host defense may be elicited and activated.

Activation of the defense system may be manifested by an exaggerated expression of

epithelial TLRs in order to protect the already diseased underlying tissue. Another

important clue which can be drawn from the definition is that the etiology of

leukoplakia is not yet known and its pathogenesis has not so far been attributed to any

microorganisms. Therefore, we consider it a logical finding that no particular TLR

(except TLR9) was down-regulated and, therefore, probably not involved in any long-

term ligand binding. The endosomal location of TLR9 parallels its ability to recognize

the unmethylated CpG DNA the origin of which can be bacterial, fungal or

mitochondrial (Bellocchio, Moretti et al. 2004; Takeda and Akira 2005). Based on that,

we suggest that, in leukoplakia, the mucosal host defense system may degrade the

invading microbes, whether bacteria or fungi, by phagocytosis, e.g. by PMNs or direct

Figure 18 showing the TLRs (1-9) immunostaining in leukoplakia.

80

lysis (by antimicrobial peptides resulting in leakage of the microbial cell contents

including its genetic material), which would keep on stimulating TLR9 leading to its

decreased expression. We, however, cannot generalize this assumption since it was

found only in some sections but this again could be due to involvement of other factors,

e.g. degree of the lesion chronicity and local immune status.

Sections of the healthy tissue, in contrast, showed strong expression of all TLRs

especially by the middle and superficial layers. The lower layers of some sections

showed faint or even absent TLRs staining which may broaden the phenomenon of

negative regulation in healthy tissue with unoccupied TLRs.

81

SUMMARY & CONCLUSION

Candida, which is normally present on the skin and mucous membranes, such as the

mouth, gastrointestinal tract, genitourinary tract, is the most frequently isolated fungal

pathogen of humans. Among its many species, Candida albicans is responsible for

most human candidal infections affecting immunocompromised patients ranging from

premature infants to AIDS (acquired immunodeficiency syndrome) sufferers. The

immune system of the host, under normal conditions, can maintain a strict control upon

C. albicans should it attempt to express its pathogenic potential. It is not a necessity

that patients be suffering from immunocompromising pathological conditions to

become infected by such opportunistic pathogens, since age extreme individuals, i.e.

infants and senile people, as well as denture wearers also remain suitable targets for C.

albicans infection. In mucosal niche, C. albicans can thrive in a fertile ground with a

moist and warm, protein-rich human mucosal membrane or biomaterial surface where

they become activated and start to grow pseudo and real hyphae resulting in

colonization. C. albicans can also travel through the blood stream and affect distant

sites, e.g. brain, kidney and heart valves, and cause what is known as systemic

candidosis.

Infection of the oral mucosa with the opportunistic candidal infection may result in

several clinical and histopathological forms ranging from atrophic, pseudomembranous

to hyperplastic candidosis. We have adopted the last category (chronic hyperplastic

candidosis) to present a model for studying the oral host defense against candidal

invasion.

We supposed that the host has to recognize the invading candidal microbe first through

binding of certain components of the candidal cell wall to germ-line encoded receptors

(the subject of study V). Once the host identifies the identity of the attacking stimulus,

it reacts by playing many tactics, e.g. elaboration of natural antimicrobial peptides (e.g.

defensins) and recruitment of immune cells (e.g. phagocytes). The style of α-defensin-1

release and its manner of contribution in the defense process against C. albicans was

investigated in study I, while the cellular scene of the host defense and the cytokines

involved in its regulation were the focus of the other studies (II, III, and IV). Generally

speaking, the aim of this thesis work was to draw a scenario of how the host responds

to candidal infection of the oral mucosa in terms of natural antimicrobial peptides,

cytokines, and cells and try to combine their tasks into a unified theme.

82

In the first work, we studied the neutrophil-derived anti-candida α-defensin-1 which

was found in the epithelium; not only diffusely all over, but as an α-defensin-1 rich

shield, while it was hardly seen in the lamina propria. But since the cellular source of α-

defensin-1 is neutrophil, which is recruited from the lamina propria one can ask how

this antimicrobial peptide reaches the uppermost part of the epithelium without being

expressed in the lamina propria? This question drove us to shape a probable step-by-

step process which went in accordance with our results and observations and some

basic biological facts. We suggested that the neutrophils transmigrate through the

microvasculature in the lamina propria and migrate towards the epithelium. On their

way, they would maintain their granules intact and advance further till they reach the

epithelium where they-upon contact with candidal germs-start to release their granular

contents, including α-defensin-1. While neutrophils gather and organize themselves

into microabscesses, α-defensin-1 will continue, maybe as a result of epithelial cell

flow, advancing towards the uppermost layer of the epithelium and accumulate there, a

process which

may be

responsible for the

α-defensin-1 rich

frontier shield.

The second work

(study II) does not

need much to

discuss because its

main purpose was

to confirm the

accidental finding

that mast cells can

express RANKL.

The pattern of

mast cell staining

was different in

calcified plaque

compared with RANKL-positive mast cells in the tunica adventitia. It seems that

Figure 19 showing a schematic summary of the interplay of the different components of the host defense (nonspecific and specific) which were studied in this thesis

83

RANKL has a particular role in the calcification process on the plaque since it was

partially released from the mast cells, while it appeared completely intracellular in areas

not affected by plaques.

The third work (study III) was done to check if mast cells-releasing-RANKL behave in

the same way in CHC as in the previous work of atherosclerosis. Mast cells seem to

react to the opportunistic C. albicans infection by local mast cell hyperplasia and

secretion of RANKL. This may be important for the recruitment and maturation of

antigen presenting dendritic cells and lymphocyte activation. Some sections showed

strong mast cell staining confined to the cell cytoplasm, without any signs of

extracellular release of RANKL. Others displayed granular cytoplasmic RANKL

staining, which was faint and associated with pericellular RANKL positive granules,

apparently as a result of mast cell activation and partial degranulation. Expression of

RANKL augments the ability of dendritic cells to enhance naïve T cell proliferation in

a mixed lymphocyte reaction. Dendritic cells and T lymphocytes comprise an important

category of immune system against fungal infections. Therefore, mast cells seem to

participate in the immunological processes conducted by the host against such a

pathological insult.

The fourth work (study IV) was conducted to examine the presence and effects of the

neutrophil chemokine IL-8 and its receptor IL-8 RA in CHC. We found that most of the

host tissue produces IL-8 at high concentrations, e.g. epithelium, endothelium and

inflammatory cells, which would activate neutrophils and lead to their degranulation.

The most important and novel observation was that the candidal cell body showed IL-8-

like immunoreaction whereas the tips of the candidal hyphae expressed IL-8 RA-like

immunoreactivity. This is very interesting as we speculate that endogenous candidal-

derived IL-8 at low concentrations could lead to centrifugal growth of the hyphae, away

from the candidal cell body, via interactions of IL-8 with its receptors at the hyphal tips.

At the same time, hyphal IL-8RA could prevent the tips from intruding into dangerous

areas where neutrophils will soon migrate or where they are already located.

In the fifth and last piece of work, a comparative analysis of TLRs (TLR1-9)

immunostaining in CHC, LP and healthy control was undertaken. From this

experiment, we concluded that the resultant immune response following C. albicans-

TLR binding depends largely on the morphological form of the fungus. In other words,

C. albicans seems to take advantage of its projecting hyphal form in eluding the host

84

defense by means of directing the immune defense towards TLR2 rather than TLR4.

When oral epithelium is infected with yeast-dominated candidosis, TLR4 seems to be

more involved than TLR2. In leukoplakia, the weak status of the diseased tissue makes

it vulnerable to further infection and this may explain the strongly positive TLRs

staining (except TLR9 which, in some sections, showed weak staining possibly due to

their engagement with the nuclear material of any invading microbe).

To summarize, the above long story can be shortened and sectioned into one complete

scene (omitting some unnecessary details): The mouth encompasses many candidal

cells which are floating around, some are attached. Some such candidal cells attempt to

establish adherence to the oral epithelia and invade the underlying tissue. We have two

scenarios here. If the host is healthy, such attempts will be doomed to failure due to the

strong physical integrity of the epithelium and the strict host defense. If the immune

status of the host is compromised, unpleasant consequences may follow. Once C.

albicans attaches to the oral epithelium, it will be recognized by TLR2, 4 and 6. Based

on literature analysis and the pattern of TLR expression, the most important of these in

recognizing C. albicans seem to be TLR2 and 4. If the invading cells have more

unicellular yeast form, TLR4 responds more extensively and the subsequent immune

response against candidosis will be promoted through elaboration of pro-inflammatory

cytokines and chemokines. IL-8 is important as it recruits the neutrophils to the

uppermost layers where the candidal germs are located. During their upward

movement, they release some of their α-defensin-1 extracellularly till they reach the

superficial layers where they group themselves into collections known as

microabscesses (Fig. 19). When the host senses that the infection is going to persist

longer (become chronic), antigen-presenting cells are engaged into the host defense in

order to pave the way for the more sophisticated specific immune defense. These

epithelial dendritic cells are Langerhans cells, which under such circumstances are

recruited to the epithelium where they engulf candidal cells (either by zipping or

coiling), leave the mucosa and in the regional lymph node present the antigen to naive

T cells. During their journey, they get augmented by RANKL secreted by resident mast

cells, so that they become more effective in communicating with T cells. On the other

hand, C. albicans seems to have developed some means to avoid the host defense. One

aspect is that when more hyphae are encountered in the infection, then the receptor

stimulation may shift towards TLR2 which results in production of anti-inflammatory

85

cytokines, thereby propagating the potency of the infection. Another smart technique is

the ability of the fungus to produce IL-8 or an analogue by the mother cell and express

its receptor IL-8RA at the hyphal tip so that it can stimulate itself in an autocrine

manner to drive the projecting hyphae away from the cell body. This apparent

chemophobic phenomenon may also serve to save the sensitive and more important part

of the candidal body (i.e. the hyphal tip- used in thigmotropism and chemotropism)

should it sense any risk for approaching neutrophils.

In the end, although the study of the pathogenesis of the opportunistic pathogen C.

albicans and the host defense against it has advanced progressively, there are still many

interesting puzzles which remain to be resolved. First, why does C. albicans thrive only

in some people? Second, when the host is healthy, why does not C. albicans cause any

infection? Is it because it stays merely commensal or it actually expresses some

pathogenic features which are immediately abrogated by the strong host defense? In

case it exerts its pathogenicity only when the host immunity is compromised, how does

it sense the change in the defense status? Third, out of study 5, how do candidal

hyphae, but not yeast, elude the host response by directing its stimulation towards

TLR2? Fourth, is there any purpose behind C. albicans chemophobia other than

avoiding the source of danger i.e. neutrophils? Fifth, why is it very rare, if ever, that C.

albicans could invade the mucosa beyond the spinous layer? Is it because of the strong

shield of the epithelium, or are some other factors involved? Sixth, what is the nature of

epithelial anti-Candida activity? Seventh, I wish if researchers, in the future, could

reach a clear and satisfactory defintion of LP because the one in use today is a bit

confusing and ambiguous. Eighth, regarding atherosclerosis, what is exactly the role of

RANKL in the pathogensis of the disease? Is it a causative factor or an effect?

Therefore, further efforts are needed to reveal other anti-Candida host defense

mechanisms. This might aid in paving the road towards lowering the incidence of

candidosis (whether mucosal or systemic) by improving the current medications and

developing new therapeutic modalities.

86

AKHNOWLEDGEMENTS

This study was conducted at the Department of Medicine, Institute of Clinical

Medicine, Departments of Oral Pathology and Oral Medicine, Institute of Dentistry and

Department of Anatomy, Institute of Biomedicine, all part of the Faculty of Medicine at

the University of Helsinki, Helsinki, Finland, during the period 2003-2008 (I joined the

university in 2002 but I started this thesis project in 2003).

First of all, I thank my Lord “Allah” for His utmost support, not just for this thesis, but

rather for the events in all of my life.

I also wish to express my sincere thanks to my dear supervisor Professor of Medicine

(and former Professor of Oral Medicine 1999-2003) Yrjö T. Konttinen, who was of

great help and support during the whole period of my study. He has helped me get the

keys for many scientific research windows thereby giving me the chance to come face

to face with the fascinating world of biomedical science. The efforts of his faithful

advice and encouragement were so valuable.

Along with Professor Konttinen, my thanks go to my second supervisor Professor

Jarkko Hietanen for his supply of the samples and for his kind help and advice,

Professor Malcolm Richardson and Dr. Riina Rautemaa for their helpful contribution to

our collaborative work, Professor Stephen Porter and Docent Ilmo Leivo for reviewing

my thesis and stating their valuable comments.

Moreover, I cannot forget the aid of Dr. Timo Sorsa who made my coming to Finland

possible through his certificate of admission which was mandatory for my visa to be

issued.

I would like to say to my old colleague Dr. Jian Ma, who is back in China now, thank

you for teaching me the principles of staining and lab techniques. I owe you for your

help and patience. I wish you a happy and an enjoyable life.

Among Finns whom I know, I have never seen such a kind person with an always

smiling face as Marjatta Kivekäs who, beside her nice character, prepared all of my

paraffin-embedded sections when needed without even a bit of hesitation.

My many thanks go to all of TULES group members for their good rapport and

friendship.

I present my deepest gratitude to my dear brother Professor Mohammed Elmusrati and

his family who, upon my first coming to Finland on the 10th of November 2001, offered

87

me respectful and generous hospitality. Besides, his help in showing me the principles

of computer science was really of great support in making my research easygoing and

pleasurable.

Coming to my dearest friend Dr. Nabil Nattah, I thank you so much for your

explanation and advice about some rules and regulations which I needed to know

during my first study days in Biomedicum. Our companionship when we used to and

still go out and eat pizzas will never vanish from my memory.

Although I have been living in a completely new culture with different standards for

more than five years, I have never had the feeling of being lonely or desolate. Thanks

for this go to my Libyan and Arab friends living in Helsinki, Turku and Tampere. The

nice moments that we spent together, which were full of useful discussions and

amusements, have inspired me with hope and certainty to drill my way ahead towards

accomplishing my thesis, and here it is, so thank you very much.

Finally, I am deeply thankful to my wife Dr. Dareen Fteita for her caring and support

and since we share the same specialty, her scientific views and comments were of great

aid in finalizing this thesis. Our loving son Salem has bestowed more beauty to the

picture of my life by letting me taste the meaning of fatherhood.

My last words are devoted for the two persons who together were the reason for my

existence before education: my father, Salem Elmusrati to whom I say: Cheer up dad!

The very moment that you have grown me for, since I was a kid, has come. Your

efforts did not go with the wind. The time is here to harvest what you have sown.

I say to the other most precious person: my mother, Zakia Mohammed who filled my

life with kindness and mercy before her passing away. I say to you: Oh mom! How a

model mother you were! You suffered a lot to make me stand on my feet. Even if days

have parted us farther and farther, you will remain closer and closer to my heart. I will

never forget the most beautiful days when you were with us. Being satisfied with the

will of Our Lord, I wish you had attended this day even though I am completely sure

that you share my happiness in your grave.

I finalize this acknowledgment with thanking the following institutes and foundations

for financing me and my work: Libyan Secretary of Higher Education, Finska

Läkaresällskapet, The National Center of Excellence in Biomaterials and Tissue

Engineering of the Academy of Finland, the National Graduate School of Biomaterials

88

BGS, and its successor the National Graduate School of Musculoskeletal Disorders and

Biomaterials TBGS (2007-), The Sigrid Jusélius Foundation, The Finnish Funding

Agency for Technology and Innovation (Microrobotic diagnostics and Therapy), MNT

ERA Net (A new Generation of Titanium Biomaterials), MATERA (BioNanoCoRe),

Suomalais-Norjalainen Lääketieteen Säätiö, Avohoidon Tutkimssäätiö, Väinö ja Laina

Kiven säätiö, Finnish Dental Society (Apollonia), The Invalid Foundation, Viikki

Graduate School (for supporting a practical course in Sweden), The University of

Helsinki, FEMS (Federation of European Microbiological Societies), ESCMID

(European Society of Clinical Microbiology and Infectious Diseases).

Helsinki, 12/05/2008

Ahmed S. Musrati

89

REFERENCES

Abi-Said, D., E. Anaissie, et al. (1997). "The epidemiology of hematogenous candidiasis caused by different Candida species." Clin Infect Dis 24(6): 1122-8.

Adams, S., D. W. O'Neill, et al. (2005). "Recent advances in dendritic cell biology." J Clin Immunol 25(3): 177-88.

Akpan, A. and R. Morgan (2002). "Oral candidiasis." Postgrad Med J 78(922): 455-9. Anderson, D. M., E. Maraskovsky, et al. (1997). "A homologue of the TNF receptor

and its ligand enhance T-cell growth and dendritic-cell function." Nature 390(6656): 175-9.

Andes, D., J. Nett, et al. (2004). "Development and characterization of an in vivo central venous catheter Candida albicans biofilm model." Infect Immun 72(10): 6023-31.

Andriole, V. T. (1999). "The 1998 Garrod lecture. Current and future antifungal therapy: new targets for antifungal agents." J Antimicrob Chemother 44(2): 151-62.

Appleton, S. S. (2000). "Candidiasis: pathogenesis, clinical characteristics, and treatment." J Calif Dent Assoc 28(12): 942-8.

Arendorf, T. M. and D. M. Walker (1980). "The prevalence and intra-oral distribution of Candida albicans in man." Arch Oral Biol 25(1): 1-10.

Ashman, R. B., C. S. Farah, et al. (2004). "Innate versus adaptive immunity in Candida albicans infection." Immunol Cell Biol 82(2): 196-204.

Axell, T., J. J. Pindborg, et al. (1996). "Oral white lesions with special reference to precancerous and tobacco- related lesions: conclusions of an international symposium held in Uppsala, Sweden, May 18-21 1994. International Collaborative Group on Oral White Lesions." J Oral Pathol Med 25(2): 49-54.

Bagan, J. V., Y. Jimenez, et al. (2003). "Proliferative verrucous leukoplakia: high incidence of gingival squamous cell carcinoma." J Oral Pathol Med 32(7): 379-82.

Baggiolini, M. (1998). "Chemokines and leukocyte traffic." Nature 392(6676): 565-8. Baggiolini, M. (2000). "Reflections on chemokines." Immunol Rev 177: 5-7. Baggiolini, M. (2001). "Chemokines in pathology and medicine." J Intern Med 250(2):

91-104. Baggiolini, M., B. Dewald, et al. (1994). "Interleukin-8 and related chemotactic

cytokines--CXC and CC chemokines." Adv Immunol 55: 97-179. Baillie, G. S. and L. J. Douglas (1999). "Role of dimorphism in the development of

Candida albicans biofilms." J Med Microbiol 48(7): 671-9. Baillie, G. S. and L. J. Douglas (2000). "Matrix polymers of Candida biofilms and their

possible role in biofilm resistance to antifungal agents." J Antimicrob Chemother 46(3): 397-403.

Baker, B. M., S. J. Gagnon, et al. (2000). "Conversion of a T cell antagonist into an agonist by repairing a defect in the TCR/peptide/MHC interface: implications for TCR signaling." Immunity 13(4): 475-84.

Baldwin, E. T., I. T. Weber, et al. (1991). "Crystal structure of interleukin 8: symbiosis of NMR and crystallography." Proc Natl Acad Sci U S A 88(2): 502-6.

90

Balish, E., H. Filutowicz, et al. (1990). "Correlates of cell-mediated immunity in Candida albicans-colonized gnotobiotic mice." Infect Immun 58(1): 107-13.

Banchereau, J. and R. M. Steinman (1998). "Dendritic cells and the control of immunity." Nature 392(6673): 245-52.

Barchiesi, F., V. Morbiducci, et al. (1993). "Emergence of oropharyngeal candidiasis caused by non-albicans species of Candida in HIV-infected patients." Eur J Epidemiol 9(4): 455-6.

Barelle, C. J., E. A. Bohula, et al. (2003). "Asynchronous cell cycle and asymmetric vacuolar inheritance in true hyphae of Candida albicans." Eukaryot Cell 2(3): 398-410.

Bartie, K. L., D. W. Williams, et al. (2001). "PCR fingerprinting of Candida albicans associated with chronic hyperplastic candidosis and other oral conditions." J Clin Microbiol 39(11): 4066-75.

Bastian, A. and H. Schafer (2001). "Human alpha-defensin 1 (HNP-1) inhibits adenoviral infection in vitro." Regul Pept 101(1-3): 157-61.

Bellocchio, S., S. Moretti, et al. (2004). "TLRs govern neutrophil activity in aspergillosis." J Immunol 173(12): 7406-15.

Blinkhorn, R. J., D. Adelstein, et al. (1989). "Emergence of a new opportunistic pathogen, Candida lusitaniae." J Clin Microbiol 27(2): 236-40.

Borg, M. and R. Ruchel (1988). "Expression of extracellular acid proteinase by proteolytic Candida spp. during experimental infection of oral mucosa." Infect Immun 56(3): 626-31.

Budtz-Jorgensen (1990). Candida-associated denture stomatitis and angular cheilitis. Oral candidosis. M. T. Samaranayake LP. London, Wright.

Budtz-Jorgensen, E. (1974). "The significance of Candida albicans in denture stomatitis." Scand J Dent Res 82(2): 151-90.

Bulad, K., R. L. Taylor, et al. (2004). "Colonization and penetration of denture soft lining materials by Candida albicans." Dent Mater 20(2): 167-75.

Bunetel, L. and M. Bonnaure-Mallet (1996). "Oral pathoses caused by Candida albicans during chemotherapy: update on development mechanisms." Oral Surg Oral Med Oral Pathol Oral Radiol Endod 82(2): 161-5.

Burgess, T. L., Y. Qian, et al. (1999). "The ligand for osteoprotegerin (OPGL) directly activates mature osteoclasts." J Cell Biol 145(3): 527-38.

Calderone, R., S. Suzuki, et al. (2000). "Candida albicans: adherence, signaling and virulence." Med Mycol 38 Suppl 1: 125-37.

Calderone, R. A. and P. C. Braun (1991). "Adherence and receptor relationships of Candida albicans." Microbiol Rev 55(1): 1-20.

Cannistra, S. A. and J. D. Griffin (1988). "Regulation of the production and function of granulocytes and monocytes." Semin Hematol 25(3): 173-88.

Cannon, R. D., A. R. Holmes, et al. (1995). "Oral Candida: clearance, colonization, or candidiasis?" J Dent Res 74(5): 1152-61.

Cawson, R. A. and T. Lehner (1968). "Chronic hyperplastic candidiasis--candidal leukoplakia." Br J Dermatol 80(1): 9-16.

Center, D. M., H. Kornfeld, et al. (1996). "Interleukin 16 and its function as a CD4 ligand." Immunol Today 17(10): 476-81.

Chaffin, W. L., J. L. Lopez-Ribot, et al. (1998). "Cell wall and secreted proteins of Candida albicans: identification, function, and expression." Microbiol Mol Biol Rev 62(1): 130-80.

91

Challacombe, S. J. (1986). "Haematological abnormalities in oral lichen planus, candidiasis, leukoplakia and non-specific stomatitis." Int J Oral Maxillofac Surg 15(1): 72-80.

Chandra, J., D. M. Kuhn, et al. (2001). "Biofilm formation by the fungal pathogen Candida albicans: development, architecture, and drug resistance." J Bacteriol 183(18): 5385-94.

Church, M. K. and F. Levi-Schaffer (1997). "The human mast cell." J Allergy Clin Immunol 99(2): 155-60.

Collin-Osdoby, P. (2004). "Regulation of vascular calcification by osteoclast regulatory factors RANKL and osteoprotegerin." Circ Res 95(11): 1046-57.

Cooper, N. R. (1985). "The classical complement pathway: activation and regulation of the first complement component." Adv Immunol 37: 151-216.

Crotti, T., M. D. Smith, et al. (2003). "Receptor activator NF kappaB ligand (RANKL) and osteoprotegerin (OPG) protein expression in periodontitis." J Periodontal Res 38(4): 380-7.

Crump, J. A. and P. J. Collignon (2000). "Intravascular catheter-associated infections." Eur J Clin Microbiol Infect Dis 19(1): 1-8.

Cutler, C. W. and R. Jotwani (2004). "Antigen-presentation and the role of dendritic cells in periodontitis." Periodontol 2000 35: 135-57.

Cutler, C. W., R. Jotwani, et al. (2001). "Dendritic cells: immune saviors or Achilles' heel?" Infect Immun 69(8): 4703-8.

Cutler, J. E. (1991). "Putative virulence factors of Candida albicans." Annu Rev Microbiol 45: 187-218.

Dahlgren, C. and A. Karlsson (1999). "Respiratory burst in human neutrophils." J Immunol Methods 232(1-2): 3-14.

Dale, B. A. (2002). "Periodontal epithelium: a newly recognized role in health and disease." Periodontol 2000 30: 70-8.

Daniels, T. E., O. Schwartz, et al. (1985). "Langerhans cells in candidal leukoplakia." J Oral Pathol 14(9): 733-9.

Davies, J. M., A. J. Stacey, et al. (1999). "Candida albicans hyphal invasion: thigmotropism or chemotropism?" FEMS Microbiol Lett 171(2): 245-9.

de Almeida Pdel, V., A. M. Gregio, et al. (2008). "Saliva composition and functions: a comprehensive review." J Contemp Dent Pract 9(3): 72-80.

de Saint-Vis, B., I. Fugier-Vivier, et al. (1998). "The cytokine profile expressed by human dendritic cells is dependent on cell subtype and mode of activation." J Immunol 160(4): 1666-76.

Dhillon, N. K., S. Sharma, et al. (2003). "Signaling through protein kinases and transcriptional regulators in Candida albicans." Crit Rev Microbiol 29(3): 259-75.

Diamond, R. D., R. A. Clark, et al. (1980). "Damage to Candida albicans hyphae and pseudohyphae by the myeloperoxidase system and oxidative products of neutrophil metabolism in vitro." J Clin Invest 66(5): 908-17.

DiDomenico, B. (1999). "Novel antifungal drugs." Curr Opin Microbiol 2(5): 509-15. Donlan, R. M. (2002). "Biofilms: microbial life on surfaces." Emerg Infect Dis 8(9):

881-90. Dourov, N. and J. Coremans-Pelseneer (1987). "Experimental chronic lingual

candidosis induced in streptozotocin diabetic rats." Mykosen 30(4): 175-83. Dowd, F. J. (1999). "Saliva and dental caries." Dent Clin North Am 43(4): 579-97. Edelman, G. M. (1991). "Antibody structure and molecular immunology." Scand J

Immunol 34(1): 1-22.

92

Edgar, W. M. (1992). "Saliva: its secretion, composition and functions." Br Dent J 172(8): 305-12.

Edgerton, M., F. A. Scannapieco, et al. (1993). "Human submandibular-sublingual saliva promotes adhesion of Candida albicans to polymethylmethacrylate." Infect Immun 61(6): 2644-52.

Edmond, M. B., S. E. Wallace, et al. (1999). "Nosocomial bloodstream infections in United States hospitals: a three-year analysis." Clin Infect Dis 29(2): 239-44.

Elahi, S., G. Pang, et al. (2001). "Nitric oxide-enhanced resistance to oral candidiasis." Immunology 104(4): 447-54.

Ellepola, A. N. and L. P. Samaranayake (2000). "Oral candidal infections and antimycotics." Crit Rev Oral Biol Med 11(2): 172-98.

Epstein, J. B. and B. Polsky (1998). "Oropharyngeal candidiasis: a review of its clinical spectrum and current therapies." Clin Ther 20(1): 40-57.

Fahrner, R. L., T. Dieckmann, et al. (1996). "Solution structure of protegrin-1, a broad-spectrum antimicrobial peptide from porcine leukocytes." Chem Biol 3(7): 543-50.

Farah, C. S., R. B. Ashman, et al. (2000). "Oral candidosis." Clin Dermatol 18(5): 553-62.

Farrugia, A. N., G. J. Atkins, et al. (2003). "Receptor activator of nuclear factor-kappaB ligand expression by human myeloma cells mediates osteoclast formation in vitro and correlates with bone destruction in vivo." Cancer Res 63(17): 5438-45.

Fettig, A., M. A. Pogrel, et al. (2000). "Proliferative verrucous leukoplakia of the gingiva." Oral Surg Oral Med Oral Pathol Oral Radiol Endod 90(6): 723-30.

Fidel, P. L., Jr. (2002). "Immunity to Candida." Oral Dis 8 Suppl 2: 69-75. Fidel, P. L., Jr. (2005). "Immunity in vaginal candidiasis." Curr Opin Infect Dis 18(2):

107-11. Filler, S. G., J. N. Swerdloff, et al. (1995). "Penetration and damage of endothelial cells

by Candida albicans." Infect Immun 63(3): 976-83. Firth, N. A., J. F. O'Grady, et al. (1997). "Oral squamous cell carcinoma in a young

person with candidosis endocrinopathy syndrome: a case report." Int J Oral Maxillofac Surg 26(1): 42-4.

Fotos, P. G. and J. W. Hellstein (1992). "Candida and candidosis. Epidemiology, diagnosis and therapeutic management." Dent Clin North Am 36(4): 857-78.

Fridkin, S. K. and W. R. Jarvis (1996). "Epidemiology of nosocomial fungal infections." Clin Microbiol Rev 9(4): 499-511.

Ganz, T., M. E. Selsted, et al. (1985). "Defensins. Natural peptide antibiotics of human neutrophils." J Clin Invest 76(4): 1427-35.

Gehrke, T., C. Sers, et al. (2003). "Receptor activator of nuclear factor kappaB ligand is expressed in resident and inflammatory cells in aseptic and septic prosthesis loosening." Scand J Rheumatol 32(5): 287-94.

Gemmell, E., C. L. Carter, et al. (2002). "Antigen-presenting cells in human periodontal disease tissues." Oral Microbiol Immunol 17(6): 388-93.

Gemmell, E., C. L. Carter, et al. (2004). "Mast cells in human periodontal disease." J Dent Res 83(5): 384-7.

Germain, R. N. (2003). "Ligand-dependent regulation of T cell development and activation." Immunol Res 27(2-3): 277-86.

Girolomoni, G., C. Caux, et al. (2002). "Langerhans cells: still a fundamental paradigm for studying the immunobiology of dendritic cells." Trends Immunol 23(1): 6-8.

93

Gori, F., L. C. Hofbauer, et al. (2000). "The expression of osteoprotegerin and RANK ligand and the support of osteoclast formation by stromal-osteoblast lineage cells is developmentally regulated." Endocrinology 141(12): 4768-76.

Gulbranson-Judge, A. and I. MacLennan (1996). "Sequential antigen-specific growth of T cells in the T zones and follicles in response to pigeon cytochrome c." Eur J Immunol 26(8): 1830-7.

Han, J. and R. J. Ulevitch (2005). "Limiting inflammatory responses during activation of innate immunity." Nat Immunol 6(12): 1198-205.

Han, W., J. Mou, et al. (1995). "Cryo atomic force microscopy: a new approach for biological imaging at high resolution." Biochemistry 34(26): 8215-20.

Hansen, L. S., J. A. Olson, et al. (1985). "Proliferative verrucous leukoplakia. A long-term study of thirty patients." Oral Surg Oral Med Oral Pathol 60(3): 285-98.

Hauman, C. H., I. O. Thompson, et al. (1993). "Oral carriage of Candida in healthy and HIV-seropositive persons." Oral Surg Oral Med Oral Pathol 76(5): 570-2.

Haynes, D. R., E. Barg, et al. (2003). "Osteoprotegerin expression in synovial tissue from patients with rheumatoid arthritis, spondyloarthropathies and osteoarthritis and normal controls." Rheumatology (Oxford) 42(1): 123-34.

Hazen, K. C., D. L. Brawner, et al. (1991). "Differential adherence of hydrophobic and hydrophilic Candida albicans yeast cells to mouse tissues." Infect Immun 59(3): 907-12.

Hazen, K. C., J. G. Lay, et al. (1990). "Partial biochemical characterization of cell surface hydrophobicity and hydrophilicity of Candida albicans." Infect Immun 58(11): 3469-76.

Hebert, C. A. and J. B. Baker (1993). "Interleukin-8: a review." Cancer Invest 11(6): 743-50.

Hedderwick, S. and C. A. Kauffman (1997). "Opportunistic fungal infections: superficial and systemic candidiasis." Geriatrics 52(10): 50-4, 59.

Helmerhorst, E. J., P. Breeuwer, et al. (1999). "The cellular target of histatin 5 on Candida albicans is the energized mitochondrion." J Biol Chem 274(11): 7286-91.

Helmerhorst, E. J., R. F. Troxler, et al. (2001). "The human salivary peptide histatin 5 exerts its antifungal activity through the formation of reactive oxygen species." Proc Natl Acad Sci U S A 98(25): 14637-42.

Higgs, J. M. and R. S. Wells (1974). "[Classification of chronic mucocutaneous candidiasis with observations on the clinical picture and its therapy]." Hautarzt 25(4): 159-65.

Hofbauer, L. C. and A. E. Heufelder (2001). "Role of receptor activator of nuclear factor-kappaB ligand and osteoprotegerin in bone cell biology." J Mol Med 79(5-6): 243-53.

Hofbauer, L. C., C. Shui, et al. (2001). "Effects of immunosuppressants on receptor activator of NF-kappaB ligand and osteoprotegerin production by human osteoblastic and coronary artery smooth muscle cells." Biochem Biophys Res Commun 280(1): 334-9.

Hsu, L. Y., G. E. Minah, et al. (1990). "Coaggregation of oral Candida isolates with bacteria from bone marrow transplant recipients." J Clin Microbiol 28(12): 2621-6.

Huizinga, T. W., D. Roos, et al. (1990). "Neutrophil Fc-gamma receptors: a two-way bridge in the immune system." Blood 75(6): 1211-4.

Ibrahim, A. S., S. G. Filler, et al. (1998). "Secreted aspartyl proteinases and interactions of Candida albicans with human endothelial cells." Infect Immun 66(6): 3003-5.

94

Irani, A. A., N. M. Schechter, et al. (1986). "Two types of human mast cells that have distinct neutral protease compositions." Proc Natl Acad Sci U S A 83(12): 4464-8.

Irani, A. M., T. R. Bradford, et al. (1989). "Detection of MCT and MCTC types of human mast cells by immunohistochemistry using new monoclonal anti-tryptase and anti-chymase antibodies." J Histochem Cytochem 37(10): 1509-15.

Izadpanah, A. and R. L. Gallo (2005). "Antimicrobial peptides." J Am Acad Dermatol 52(3 Pt 1): 381-90; quiz 391-2.

Jeziorska, M., C. McCollum, et al. (1998). "Observations on bone formation and remodelling in advanced atherosclerotic lesions of human carotid arteries." Virchows Arch 433(6): 559-65.

Jones, D. S., J. G. McGovern, et al. (2001). "Conditioning film and environmental effects on the adherence of Candida spp. to silicone and poly(vinylchloride) biomaterials." J Mater Sci Mater Med 12(5): 399-405.

Josien, R., B. R. Wong, et al. (1999). "TRANCE, a TNF family member, is differentially expressed on T cell subsets and induces cytokine production in dendritic cells." J Immunol 162(5): 2562-8.

Jotwani, R. and C. W. Cutler (2003). "Multiple dendritic cell (DC) subpopulations in human gingiva and association of mature DCs with CD4+ T-cells in situ." J Dent Res 82(9): 736-41.

Jumper, M. D., J. B. Splawski, et al. (1994). "Ligation of CD40 induces sterile transcripts of multiple Ig H chain isotypes in human B cells." J Immunol 152(2): 438-45.

Kagami, H., Y. Hiramatsu, et al. (2000). "Salivary growth factors in health and disease." Adv Dent Res 14: 99-102.

Kao, A. S., M. E. Brandt, et al. (1999). "The epidemiology of candidemia in two United States cities: results of a population-based active surveillance." Clin Infect Dis 29(5): 1164-70.

Kataoka, K., T. Muta, et al. (2002). "Activation of macrophages by linear (1right-arrow3)-beta-D-glucans. Impliations for the recognition of fungi by innate immunity." J Biol Chem 277(39): 36825-31.

Kavanagh, K. and S. Dowd (2004). "Histatins: antimicrobial peptides with therapeutic potential." J Pharm Pharmacol 56(3): 285-9.

Kelley, J. L., D. S. Chi, et al. (2000). "The molecular role of mast cells in atherosclerotic cardiovascular disease." Mol Med Today 6(8): 304-8.

Kent, H. L. (1991). "Epidemiology of vaginitis." Am J Obstet Gynecol 165(4 Pt 2): 1168-76.

Kim, M. H., G. E. Rodey, et al. (1969). "Defective candidacidal capacity of polymorphonuclear leukocytes in chronic granulomatous disease of childhood." J Pediatr 75(2): 300-3.

Kimura, L. H. and N. N. Pearsall (1980). "Relationship between germination of Candida albicans and increased adherence to human buccal epithelial cells." Infect Immun 28(2): 464-8.

Kong, Y. Y. and J. M. Penninger (2000). "Molecular control of bone remodeling and osteoporosis." Exp Gerontol 35(8): 947-56.

Kovanen, P. T. (1995). "Role of mast cells in atherosclerosis." Chem Immunol 62: 132-70.

Kuhn, D. M., J. Chandra, et al. (2002). "Comparison of biofilms formed by Candida albicans and Candida parapsilosis on bioprosthetic surfaces." Infect Immun 70(2): 878-88.

95

Kumamoto, C. A. (2002). "Candida biofilms." Curr Opin Microbiol 5(6): 608-11. Lacey, D. L., E. Timms, et al. (1998). "Osteoprotegerin ligand is a cytokine that

regulates osteoclast differentiation and activation." Cell 93(2): 165-76. Lane, B. R., K. Lore, et al. (2001). "Interleukin-8 stimulates human immunodeficiency

virus type 1 replication and is a potential new target for antiretroviral therapy." J Virol 75(17): 8195-202.

Larsen, C. G., A. O. Anderson, et al. (1989). "The neutrophil-activating protein (NAP-1) is also chemotactic for T lymphocytes." Science 243(4897): 1464-6.

Lehner, T. (1964). "Chronic Candidiasis." Trans St Johns Hosp Dermatol Soc 50: 8-21. Lehner, T. (1967). "Oral candidosis." Dent Pract Dent Rec 17(6): 209-16. Lehner, T., H. R. Buckley, et al. (1972). "The relationship between fluorescent,

agglutinating, and precipitating antibodies to Candida albicans and their immunoglobulin classes." J Clin Pathol 25(4): 344-8.

Lehner, T., J. M. Wilton, et al. (1972). "Immunodeficiencies in chronic muco-cutaneous candidosis." Immunology 22(5): 775-87.

Lehrer, R. I. and T. Ganz (1999). "Antimicrobial peptides in mammalian and insect host defence." Curr Opin Immunol 11(1): 23-7.

Lemaitre, B., E. Nicolas, et al. (1996). "The dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent antifungal response in Drosophila adults." Cell 86(6): 973-83.

Liu, D., J. K. Xu, et al. (2003). "Expression of RANKL and OPG mRNA in periodontal disease: possible involvement in bone destruction." Int J Mol Med 11(1): 17-21.

Liu, H. (2001). "Transcriptional control of dimorphism in Candida albicans." Curr Opin Microbiol 4(6): 728-35.

Liu, H. (2002). "Co-regulation of pathogenesis with dimorphism and phenotypic switching in Candida albicans, a commensal and a pathogen." Int J Med Microbiol 292(5-6): 299-311.

Lloyd, A. R. and J. J. Oppenheim (1992). "Poly's lament: the neglected role of the polymorphonuclear neutrophil in the afferent limb of the immune response." Immunol Today 13(5): 169-72.

Lo, H. J., J. R. Kohler, et al. (1997). "Nonfilamentous C. albicans mutants are avirulent." Cell 90(5): 939-49.

Lockhart, S. R., C. Pujol, et al. (2002). "In Candida albicans, white-opaque switchers are homozygous for mating type." Genetics 162(2): 737-45.

Luster, A. D. (1998). "Chemokines--chemotactic cytokines that mediate inflammation." N Engl J Med 338(7): 436-45.

Lynch, C. C., A. Hikosaka, et al. (2005). "MMP-7 promotes prostate cancer-induced osteolysis via the solubilization of RANKL." Cancer Cell 7(5): 485-96.

MacFarlane TW, S. L. (1989). Fungal infections. London, Wright. Mah, T. F. and G. A. O'Toole (2001). "Mechanisms of biofilm resistance to

antimicrobial agents." Trends Microbiol 9(1): 34-9. Mahanonda, R. and S. Pichyangkul (2007). "Toll-like receptors and their role in

periodontal health and disease." Periodontol 2000 43: 41-55. Malaviya, R., N. J. Twesten, et al. (1996). "Mast cells process bacterial Ags through a

phagocytic route for class I MHC presentation to T cells." J Immunol 156(4): 1490-6.

Mandelin, J., T. F. Li, et al. (2003). "Imbalance of RANKL/RANK/OPG system in interface tissue in loosening of total hip replacement." J Bone Joint Surg Br 85(8): 1196-201.

96

Marodi, L. (1997). "Local and systemic host defense mechanisms against Candida: immunopathology of candidal infections." Pediatr Infect Dis J 16(8): 795-801.

Marshall, R. I. (2004). "Gingival defensins: linking the innate and adaptive immune responses to dental plaque." Periodontol 2000 35: 14-20.

Matsuzaki, K., N. Udagawa, et al. (1998). "Osteoclast differentiation factor (ODF) induces osteoclast-like cell formation in human peripheral blood mononuclear cell cultures." Biochem Biophys Res Commun 246(1): 199-204.

McClung, M. R. (2006). "Inhibition of RANKL as a Treatment for Osteoporosis: Preclinical and Early Clinical Studies." Curr Osteoporos Rep 4(1): 28-33.

McCullough, M., M. Jaber, et al. (2002). "Oral yeast carriage correlates with presence of oral epithelial dysplasia." Oral Oncol 38(4): 391-3.

Mehentee, J. F. and R. J. Hay (1989). "In vitro adherence of Candida albicans strains to murine gastrointestinal mucosal cells and explants and the role of environmental pH." J Gen Microbiol 135(8): 2181-8.

Mekori, Y. A. and D. D. Metcalfe (1999). "Mast cell-T cell interactions." J Allergy Clin Immunol 104(3 Pt 1): 517-23.

Melani, C., G. F. Mattia, et al. (1993). "Interleukin-6 expression in human neutrophil and eosinophil peripheral blood granulocytes." Blood 81(10): 2744-9.

Merson-Davies, L. A. and F. C. Odds (1989). "A morphology index for characterization of cell shape in Candida albicans." J Gen Microbiol 135(11): 3143-52.

Montagnoli, C., A. Bacci, et al. (2001). "The plasticity of dendritic cells at the host/fungal interface." Immunobiology 204(5): 582-9.

Morton, T. H., R. J. Cabay, et al. (2007). "Proliferative verrucous leukoplakia and its progression to oral carcinoma: report of three cases." J Oral Pathol Med 36(5): 315-8.

Mukherjee, P. K., J. Chandra, et al. (2003). "Mechanism of fluconazole resistance in Candida albicans biofilms: phase-specific role of efflux pumps and membrane sterols." Infect Immun 71(8): 4333-40.

Murphy, P. M. and H. L. Tiffany (1991). "Cloning of complementary DNA encoding a functional human interleukin-8 receptor." Science 253(5025): 1280-3.

Muzyka, B. C. and M. Glick (1995). "A review of oral fungal infections and appropriate therapy." J Am Dent Assoc 126(1): 63-72.

Nagai, M., S. Kyakumoto, et al. (2000). "Cancer cells responsible for humoral hypercalcemia express mRNA encoding a secreted form of ODF/TRANCE that induces osteoclast formation." Biochem Biophys Res Commun 269(2): 532-6.

Naglik, J. R., G. Newport, et al. (1999). "In vivo analysis of secreted aspartyl proteinase expression in human oral candidiasis." Infect Immun 67(5): 2482-90.

Netea, M. G., R. Sutmuller, et al. (2004). "Toll-like receptor 2 suppresses immunity against Candida albicans through induction of IL-10 and regulatory T cells." J Immunol 172(6): 3712-8.

Netea, M. G., C. A. Van Der Graaf, et al. (2002). "The role of toll-like receptor (TLR) 2 and TLR4 in the host defense against disseminated candidiasis." J Infect Dis 185(10): 1483-9.

Netea, M. G., J. W. Van der Meer, et al. (2004). "Toll-like receptors as an escape mechanism from the host defense." Trends Microbiol 12(11): 484-8.

Newman, S. L., B. Bhugra, et al. (2005). "Enhanced killing of Candida albicans by human macrophages adherent to type 1 collagen matrices via induction of phagolysosomal fusion." Infect Immun 73(2): 770-7.

Nikawa, H., H. Nishimura, et al. (1998). "Relationship between thigmotropism and Candida biofilm formation in vitro." Mycopathologia 144(3): 125-9.

97

Nobile, C. J., V. M. Bruno, et al. (2003). "Genetic control of chlamydospore formation in Candida albicans." Microbiology 149(Pt 12): 3629-37.

Odds, F. C. (1988). Candida and candidosis. London, Balliere Tindall. Palma, C., A. Cassone, et al. (1992). "Lactoferrin release and interleukin-1, interleukin-

6, and tumor necrosis factor production by human polymorphonuclear cells stimulated by various lipopolysaccharides: relationship to growth inhibition of Candida albicans." Infect Immun 60(11): 4604-11.

Palmer, G. D., P. G. Robinson, et al. (1996). "Aetiological factors for oral manifestations of HIV." Oral Dis 2(3): 193-7.

Palter, S. F., N. Mulayim, et al. (2001). "Interleukin-8 in the human fallopian tube." J Clin Endocrinol Metab 86(6): 2660-7.

Palucka, A. K. and J. Banchereau (2006). "Langerhans cells: daughters of monocytes." Nat Immunol 7(3): 223-4.

Palucka, K. and J. Banchereau (1999). "Dendritic cells: a link between innate and adaptive immunity." J Clin Immunol 19(1): 12-25.

Park, S. H., G. Y. Hsiao, et al. (2004). "Role of substance P and calcitonin gene-related peptide in the regulation of interleukin-8 and monocyte chemotactic protein-1 expression in human dental pulp." Int Endod J 37(3): 185-92.

Parker, D. C. (1993). "T cell-dependent B cell activation." Annu Rev Immunol 11: 331-60.

Parvinen, T. (1984). "Stimulated salivary flow rate, pH and lactobacillus and yeast concentrations in persons with different types of dentition." Scand J Dent Res 92(5): 412-8.

Penhall, B. (1980). "Preventive measures to control further bone loss and soft tissue damage in denture wearing." Aust Dent J 25(6): 319-24.

Penson, R. T., K. Kronish, et al. (2000). "Cytokines IL-1beta, IL-2, IL-6, IL-8, MCP-1, GM-CSF and TNFalpha in patients with epithelial ovarian cancer and their relationship to treatment with paclitaxel." Int J Gynecol Cancer 10(1): 33-41.

Perheentupa, J. (2006). "Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy." J Clin Endocrinol Metab 91(8): 2843-50.

Peveri, P., A. Walz, et al. (1988). "A novel neutrophil-activating factor produced by human mononuclear phagocytes." J Exp Med 167(5): 1547-59.

Pivarcsi, A., L. Bodai, et al. (2003). "Expression and function of Toll-like receptors 2 and 4 in human keratinocytes." Int Immunol 15(6): 721-30.

Poppenborg, H., M. M. Knupfer, et al. (1999). "In vitro modulation of cisplatin resistance by cytokines." Cytokine 11(9): 689-95.

Potera, C. (1999). "Forging a link between biofilms and disease." Science 283(5409): 1837, 1839.

Prussin, C. and D. D. Metcalfe (2003). "4. IgE, mast cells, basophils, and eosinophils." J Allergy Clin Immunol 111(2 Suppl): S486-94.

Quayle, A. J., E. M. Porter, et al. (1998). "Gene expression, immunolocalization, and secretion of human defensin-5 in human female reproductive tract." Am J Pathol 152(5): 1247-58.

RA, C. (1966). Chronic oral candidosis, denture stomatitis and chronic hyperplastic candidosis. Symposium on Candida infections. H. R. Winner HI. Edinburgh, Livingstone.

Radford, D. R., S. J. Challacombe, et al. (1999). "Denture plaque and adherence of Candida albicans to denture-base materials in vivo and in vitro." Crit Rev Oral Biol Med 10(1): 99-116.

98

Raj, P. A. and A. R. Dentino (2002). "Current status of defensins and their role in innate and adaptive immunity." FEMS Microbiol Lett 206(1): 9-18.

Ramachandra, L., R. S. Chu, et al. (1999). "Phagocytic antigen processing and effects of microbial products on antigen processing and T-cell responses." Immunol Rev 168: 217-39.

Reed, M. J. and A. Purohit (1997). "Breast cancer and the role of cytokines in regulating estrogen synthesis: an emerging hypothesis." Endocr Rev 18(5): 701-15.

Reichart, P. A., H. P. Philipsen, et al. (1995). "Pseudomembranous oral candidiasis in HIV infection: ultrastructural findings." J Oral Pathol Med 24(6): 276-81.

Robbins, F. C. and J. B. Robbins (1986). "Current status and prospects for some improved and new bacterial vaccines." Annu Rev Public Health 7: 105-25.

Roeder, A., C. J. Kirschning, et al. (2004). "Toll-like receptors as key mediators in innate antifungal immunity." Med Mycol 42(6): 485-98.

Rogers, T. J. and E. Balish (1980). "Immunity to Candida albicans." Microbiol Rev 44(4): 660-82.

Romagnoli, G., R. Nisini, et al. (2004). "The interaction of human dendritic cells with yeast and germ-tube forms of Candida albicans leads to efficient fungal processing, dendritic cell maturation, and acquisition of a Th1 response-promoting function." J Leukoc Biol 75(1): 117-26.

Romani, L., S. Mocci, et al. (1991). "Th1 and Th2 cytokine secretion patterns in murine candidiasis: association of Th1 responses with acquired resistance." Infect Immun 59(12): 4647-54.

Rossie, K. and J. Guggenheimer (1997). "Oral candidiasis: clinical manifestations, diagnosis, and treatment." Pract Periodontics Aesthet Dent 9(6): 635-41; quiz 642.

Rotrosen, D., J. E. Edwards, Jr., et al. (1985). "Adherence of Candida to cultured vascular endothelial cells: mechanisms of attachment and endothelial cell penetration." J Infect Dis 152(6): 1264-74.

Rudney, J. D. (1995). "Does variability in salivary protein concentrations influence oral microbial ecology and oral health?" Crit Rev Oral Biol Med 6(4): 343-67.

Ruissen, A. L., J. Groenink, et al. (2002). "Histatin 5 and derivatives. Their localization and effects on the ultra-structural level." Peptides 23(8): 1391-9.

Rumsaeng, V., W. W. Cruikshank, et al. (1997). "Human mast cells produce the CD4+ T lymphocyte chemoattractant factor, IL-16." J Immunol 159(6): 2904-10.

Ryder, C., M. Byrd, et al. (2007). "Role of polysaccharides in Pseudomonas aeruginosa biofilm development." Curr Opin Microbiol 10(6): 644-8.

Saglie, F. R., J. H. Pertuiset, et al. (1987). "The presence of bacteria in the oral epithelium in periodontal disease. III. Correlation with Langerhans cells." J Periodontol 58(6): 417-22.

Samaranayake, L. P., J. McCourtie, et al. (1980). "Factors affecting th in-vitro adherence of Candida albicans to acrylic surfaces." Arch Oral Biol 25(8-9): 611-5.

Sawaki, K., N. Mizukawa, et al. (2002). "Immunohistochemical study on expression of alpha-defensin and beta-defensin-2 in human buccal epithelia with candidiasis." Oral Dis 8(1): 37-41.

Savill, J., V. Fadok, et al. (1993). "Phagocyte recognition of cells undergoing apoptosis." Immunol Today 14(3): 131-6.

Schaberg, D. R., D. H. Culver, et al. (1991). "Major trends in the microbial etiology of nosocomial infection." Am J Med 91(3B): 72S-75S.

99

Schall, T. J. and K. B. Bacon (1994). "Chemokines, leukocyte trafficking, and inflammation." Curr Opin Immunol 6(6): 865-73.

Schoppet, M., K. T. Preissner, et al. (2002). "RANK ligand and osteoprotegerin: paracrine regulators of bone metabolism and vascular function." Arterioscler Thromb Vasc Biol 22(4): 549-53.

Schulze, P. C. and R. T. Lee (2005). "Oxidative stress and atherosclerosis." Curr Atheroscler Rep 7(3): 242-8.

Scimone, M. L., V. P. Lutzky, et al. (2005). "Migration of polymorphonuclear leucocytes is influenced by dendritic cells." Immunology 114(3): 375-85.

Sciubba, J. J. (1995). "Oral leukoplakia." Crit Rev Oral Biol Med 6(2): 147-60. Scully, C., M. el-Kabir, et al. (1994). "Candida and oral candidosis: a review." Crit Rev

Oral Biol Med 5(2): 125-57. Seguier, S., G. Godeau, et al. (2000). "Immunohistological and morphometric analysis

of intra-epithelial lymphocytes and Langerhans cells in healthy and diseased human gingival tissues." Arch Oral Biol 45(6): 441-52.

Selsted, M. E., D. M. Brown, et al. (1983). "Primary structures of MCP-1 and MCP-2, natural peptide antibiotics of rabbit lung macrophages." J Biol Chem 258(23): 14485-9.

Shay, K., M. R. Truhlar, et al. (1997). "Oropharyngeal candidosis in the older patient." J Am Geriatr Soc 45(7): 863-70.

Sherman, R. G., L. Prusinski, et al. (2002). "Oral candidosis." Quintessence Int 33(7): 521-32.

Sherwood, J., N. A. Gow, et al. (1992). "Contact sensing in Candida albicans: a possible aid to epithelial penetration." J Med Vet Mycol 30(6): 461-9.

Siqueira, J. F., Jr. and B. H. Sen (2004). "Fungi in endodontic infections." Oral Surg Oral Med Oral Pathol Oral Radiol Endod 97(5): 632-41.

Sitheeque, M. A. and L. P. Samaranayake (2003). "Chronic hyperplastic candidosis/candidiasis (candidal leukoplakia)." Crit Rev Oral Biol Med 14(4): 253-67.

Siu, G. (2001). "Linking CD4 gene expression and T cell development." Curr Mol Med 1(5): 523-32.

Smith, J. A. (1994). "Neutrophils, host defense, and inflammation: a double-edged sword." J Leukoc Biol 56(6): 672-86.

Sobel, J. D. (1988). "Pathogenesis and epidemiology of vulvovaginal candidiasis." Ann N Y Acad Sci 544: 547-57.

Sobel, J. D. (1992). "Pathogenesis and treatment of recurrent vulvovaginal candidiasis." Clin Infect Dis 14 Suppl 1: S148-53.

Sohnle, P. G. and C. Collins-Lech (1990). "Comparison of candidacidal and candidastatic activities of human neutrophils." Infect Immun 58(8): 2696-8.

Soll, D. R. (2002). "Candida commensalism and virulence: the evolution of phenotypic plasticity." Acta Trop 81(2): 101-10.

Soustre, J., M. H. Rodier, et al. (2004). "Caspofungin modulates in vitro adherence of Candida albicans to plastic coated with extracellular matrix proteins." J Antimicrob Chemother 53(3): 522-5.

Sprenger, H., A. R. Lloyd, et al. (1994). "Genomic structure, characterization, and identification of the promoter of the human IL-8 receptor A gene." J Immunol 153(6): 2524-32.

Squier, C. A. and M. J. Kremer (2001). "Biology of oral mucosa and esophagus." J Natl Cancer Inst Monogr(29): 7-15.

100

Steinman, R. M. (1991). "The dendritic cell system and its role in immunogenicity." Annu Rev Immunol 9: 271-96.

Steinman, R. M. and Z. A. Cohn (1973). "Identification of a novel cell type in peripheral lymphoid organs of mice. I. Morphology, quantitation, tissue distribution." J Exp Med 137(5): 1142-62.

Strieter, R. M., K. Kasahara, et al. (1990). "Human neutrophils exhibit disparate chemotactic factor gene expression." Biochem Biophys Res Commun 173(2): 725-30.

Strieter, R. M., S. L. Kunkel, et al. (1988). "Monokine-induced gene expression of a human endothelial cell-derived neutrophil chemotactic factor." Biochem Biophys Res Commun 156(3): 1340-5.

Strieter, R. M., S. H. Phan, et al. (1989). "Monokine-induced neutrophil chemotactic factor gene expression in human fibroblasts." J Biol Chem 264(18): 10621-6.

Sudbery, P., N. Gow, et al. (2004). "The distinct morphogenic states of Candida albicans." Trends Microbiol 12(7): 317-24.

Sullivan, D. J., T. J. Westerneng, et al. (1995). "Candida dubliniensis sp. nov.: phenotypic and molecular characterization of a novel species associated with oral candidosis in HIV-infected individuals." Microbiology 141 ( Pt 7): 1507-21.

Tabak, L. A. (1990). "Structure and function of human salivary mucins." Crit Rev Oral Biol Med 1(4): 229-34.

Takeda, K. and S. Akira (2004). "Microbial recognition by Toll-like receptors." J Dermatol Sci 34(2): 73-82.

Takeda, K. and S. Akira (2005). "Toll-like receptors in innate immunity." Int Immunol 17(1): 1-14.

Tsai, H. and L. A. Bobek (1997). "Studies of the mechanism of human salivary histatin-5 candidacidal activity with histatin-5 variants and azole-sensitive and -resistant Candida species." Antimicrob Agents Chemother 41(10): 2224-8.

Underhill, D. M., A. Ozinsky, et al. (1999). "The Toll-like receptor 2 is recruited to macrophage phagosomes and discriminates between pathogens." Nature 401(6755): 811-5.

Vallejo, A. N., E. Davila, et al. (2004). "Biology of T lymphocytes." Rheum Dis Clin North Am 30(1): 135-57.

Walz, A., P. Peveri, et al. (1987). "Purification and amino acid sequencing of NAF, a novel neutrophil-activating factor produced by monocytes." Biochem Biophys Res Commun 149(2): 755-61.

Van Wetering, S., S. P. Mannesse-Lazeroms, et al. (1997). "Effect of defensins on interleukin-8 synthesis in airway epithelial cells." Am J Physiol 272(5 Pt 1): L888-96.

Warnakulasuriya, S. (2000). "Lack of molecular markers to predict malignant potential of oral precancer." J Pathol 190(4): 407-9.

Watts, H. J., A. A. Very, et al. (1998). "Thigmotropism and stretch-activated channels in the pathogenic fungus Candida albicans." Microbiology 144 ( Pt 3): 689-95.

Webb, B. C., C. J. Thomas, et al. (1998). "Candida-associated denture stomatitis. Aetiology and management: a review. Part 1. Factors influencing distribution of Candida species in the oral cavity." Aust Dent J 43(1): 45-50.

Webb, B. C., C. J. Thomas, et al. (1998). "Candida-associated denture stomatitis. Aetiology and management: a review. Part 2. Oral diseases caused by Candida species." Aust Dent J 43(3): 160-6.

101

Weinberg, A., S. Krisanaprakornkit, et al. (1998). "Epithelial antimicrobial peptides: review and significance for oral applications." Crit Rev Oral Biol Med 9(4): 399-414.

Wellington, M., J. M. Bliss, et al. (2003). "Enhanced phagocytosis of Candida species mediated by opsonization with a recombinant human antibody single-chain variable fragment." Infect Immun 71(12): 7228-31.

Whiteway, M. (2000). "Transcriptional control of cell type and morphogenesis in Candida albicans." Curr Opin Microbiol 3(6): 582-8.

Whiteway, M. and U. Oberholzer (2004). "Candida morphogenesis and host-pathogen interactions." Curr Opin Microbiol 7(4): 350-7.

Villamon, E., D. Gozalbo, et al. (2004). "Toll-like receptor 2 is dispensable for acquired host immune resistance to Candida albicans in a murine model of disseminated candidiasis." Microbes Infect 6(6): 542-8.

Villamon, E., D. Gozalbo, et al. (2004). "Toll-like receptor-2 is essential in murine defenses against Candida albicans infections." Microbes Infect 6(1): 1-7.

Williams, D. W. and M. A. Lewis (2000). "Isolation and identification of Candida from the oral cavity." Oral Dis 6(1): 3-11.

Wynn, R. L., M. A. Jabra-Rizk, et al. (1999). "Antifungal drugs and fungal resistance: the need for a new generation of drugs." Gen Dent 47(4): 352-5.

Xu, T., S. M. Levitz, et al. (1991). "Anticandidal activity of major human salivary histatins." Infect Immun 59(8): 2549-54.

Yang, D., A. Biragyn, et al. (2002). "Mammalian defensins in immunity: more than just microbicidal." Trends Immunol 23(6): 291-6.

Yang, L. C., F. M. Huang, et al. (2003). "Induction of interleukin-8 gene expression by black-pigmented Bacteroides in human pulp fibroblasts and osteoblasts." Int Endod J 36(11): 774-9.

Yasuda, H., N. Shima, et al. (1998). "Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL." Proc Natl Acad Sci U S A 95(7): 3597-602.

Yoshimura, T., K. Matsushima, et al. (1987). "Neutrophil chemotactic factor produced by lipopolysaccharide (LPS)-stimulated human blood mononuclear leukocytes: partial characterization and separation from interleukin 1 (IL 1)." J Immunol 139(3): 788-93.

Yoshimura, T., K. Matsushima, et al. (1987). "Purification of a human monocyte-derived neutrophil chemotactic factor that has peptide sequence similarity to other host defense cytokines." Proc Natl Acad Sci U S A 84(24): 9233-7.

Zanetti, M., R. Gennaro, et al. (1995). "Cathelicidins: a novel protein family with a common proregion and a variable C-terminal antimicrobial domain." FEBS Lett 374(1): 1-5.

Zasloff, M. (2002). "Antimicrobial peptides of multicellular organisms." Nature 415(6870): 389-95.