Pathological Changes at the Target Tissue Level in Sjögren's Syndrome

Pathological Changes at the Target Tissue Level in Sjögren’s Syndrome and Their Effect on the Exocrine Function of the Salivary Glands

Mikael Laine

Institute of Clinical Medicine / Invärtes medicin, University of Helsinki, Finland Department of Medicine, Helsinki University Central Hospital, Helsinki, Finland Institute of Biomedicine / Anatomy, University of Helsinki, Finland And ORTON Orthopaedic Hospital of the ORTON Foundation, Helsinki, Finland

ACADEMIC DISSERTATION

To be presented, with the permission of the Faculty of Medicine of the University of Helsinki, for public examination in Lecture Hall 3 at Biomedicum Helsinki, Haartmaninkatu 8, Helsinki on June 11th, 2010, at 12 o’clock noon.

Helsinki 2010 SUPERVISED BY

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CONTENTS

1. LIST OF ORIGINAL PUBLICATIONS <<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<< 0

2. ABSTRACT <<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<:;

3. ABBREVIATIONS <<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<:/

4. INTRODUCTION<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<:9

5. REVIEW OF THE LITERATURE <<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<:5 4<:< '-# R 7''8 <<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<:5 4<:<:< <<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<< :1 4<:

4<9< # '' <<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<< 66 4<9<:< ( # # '' <<<<<<<<<<<<<<<<<<<<<<<<<< 66 4<9

6. AIMS OF THE STUDY <<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<< 60

7. MATERIALS AND METHODS <<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<< 9; 1<:< % <<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<< 9; 1<:<:< D " <<<<<<<<<<<<<<<<<<<<<<<<<< 9; 1<:

8. RESULTS <<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<< 90 2<:< @ 3" ) @'( "" )- *" '' 78 <<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<< 90 2<:<:< @ : / " <<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<< 90 2<:

9. DISCUSSION <<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<41 0<:< % <<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<41 0

0<6< @ " <<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<< 5; 0<6<:< $" ) <<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<< 5; 0<9< ) " " ) # <<<<<<<<<<<<<<<< 5; 0<4< ) # 3 # ) # <<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<< 59 0<5< C !'%36 " ) ) # <<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<51 0<1< # " # ) # '-# R <<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<< 50

10. SUMMARY AND CONCLUSIONS <<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<< 1:

11. ACKNOWLEDGEMENTS <<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<16

12. REFERENCES <<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<14

ORIGINAL PUBLICATIONS I–IV <<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<< 21

1. LIST OF ORIGINAL PUBLICATIONS

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2. ABSTRACT

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3. ABBREVIATIONS

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4. INTRODUCTION

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5. REVIEW OF THE LITERATURE

5.1. SJÖGREN’S SYNDROME (SS)

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5.1.1. CLINICAL FEATURES

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5.1.2. DIAGNOSIS

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Table 1. The American-European consensus about six inclusion criteria, seven exclusion criteria and classification rules for primary and secondary Sjögren’s syndrome (modified from Vitali et al., 2002).

Symptoms / Inclusion criteria Clinical findings Ocular symptoms (I) Dry eyes over 3 months or sensation of sand or gravel in the eyes or usage of tear substitutes more than 3 times a day Oral symptoms (II) Dry mouth over 3 months or recurrently or permanently swollen parotid glands or need to drink liquids in order to aid swallowing Ocular signs (III) Schirmer’s I test, performed without anesthesia, ≤ 5 mm in 5 minutes or Rose Bengal score ≥ 4 Histopathological Focal lymphocytic sialoadenitis in labial salivary glands, with a focus findings (IV) score ≥ 1, defined as number of lymphocytic foci containing over 50 lymphocytes per 4 mm2 of glandular tissue Salivary gland Unstimulated whole saliva flow ≤ 1.5 ml in 15 minutes or parotid involvement (V) sialography indicating presence of diffuse sialectasia or salivary scintigraphy showing delayed uptake, reduced concentration and/or delayed excretion of tracer Auto-antibodies (VI) Presence of SS-A/Ro and/or SS-B/La auto-antibodies in the serum

Exclusion criteria Past head and neck radiation therapy Hepatitis C infection Acquired immunodeficiency syndrome (AIDS) Pre-existing lymphoma Sarcoidosis Graft versus host disease Use of anti-cholinergic drugs

Classification rules for primary Sjögren’s syndrome a) The presence of any 4 of the 6 items is indicative of primary SS, as long as either item IV (histopathology) or VI (serology) is positive b) The presence of any 3 of the 4 objective criteria items (III, IV, V, VI)

Classification rules for secondary Sjögren’s syndrome In patients with potentially associated disease (e.g. another connective tissue disease), the presence of item I or item II plus any 2 from among the item III, IV and V may be considered as indicative of secondary Sjögren’s syndrome

5.1.3. TREATMENT

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5.2. SALIVARY GLANDS

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Figure 1. Schematic view of the acinus and its ducts as discussed in the text. Figure does not show the distribution of the basal cells, which form a basal cell layer in excretory ducts but are also scattered around the salivon (Redrawn and modified from Rosset al., 2006; original diagram based on Johnson LR (ed) Gastrointestinal Physiology, 2nd ed., CV Mosby, 1981, p.47).

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5.2.1. DEVELOPMENTAL BIOLOGY

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Figure 2. A schematic view of the salivary gland development stages: 1. Prebud stage, 2. Initial bud stage, 3. Pseudoglandular stage, 4. Canalicular stage, 5. Terminal bud stage (Modified from the original figure published by Tucker, 2007).

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5.2.2. FUNCTION

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Table 2. Composition of stimulated adult saliva compared to adult plasma (modified from Roth and Calmes, 1981 and from the HUSLAB analysis instruction manual, Hospital District of Helsinki and Uusimaa [http:// huslab.fi/ohjekirja/index.html]).

Saliva Plasma Sodium (Na+) * 2–21 mmol/L * 137–145 mmol/L Potassium (K+) ** 10–36 mmol/L ** 3.3–4.9 mmol/L Calcium (Ca2+) 1.2–2.8 mmol/L 2.15–2.51 mmol/L Magnesium (Mg2+) 0.08–0.5 mmol/L * 0.71–0.94 mmol/L Chloride (Cl-) * 5–40 mmol/L * 96–107 mmol/L - Bicarbonate (HCO3 ) 25 mmol/L 21–28 mmol/L 2- Phosphate (HPO4 ) 1.4–39 mmol/L 0.76–1.53 mmol/L

Uric acid 2–3 mmol/L 0.155–0.48 mmol/L Urea 3–5mmol/L 2.6–8.1 mmol/L Glucose <1.0 mmol/L 4.0–6.1 mmol/L Total proteins (mg/ml) 1.5–2.5 60–90 * lower than plasma ** higher than plasma Y " #" " )3 "#" ) *3 " H 7 G%8< G%: G%4 G%2 # *" " " G% < G%4 " - " # 7 ' /;;58< " # # " \ * * < + # ) ) " $% 3 # " # " "

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5.3. THE BASEMENT MEMBRANE

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5.3.1 GENERAL STRUCTURE AND FUNCTION

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5.3.2. LAMININS (LM)

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Table 3. Nomenclature of 15 currently known laminins and some of their known integrin receptors (modified from Patarroyo et al., 2002 and from Aumailley et al., 2005).

Chains Abbreviated Previous names Integrin receptor α1β1γ1 111 Laminin-1, EHS-laminin α1β1,α2β1,α7β1,α6β4 α2β1γ1 211 Laminin-2, merosin α1β1,α2β1,α3β1,α6β1,α7β1,α6β4 α1β2γ1 121 Laminin-3, s-laminin ND α2β2γ1 221 Laminin-4, s-merosin Presumably similar to laminin-211 α3Aβ3γ2 332, or 3A32 Laminin-5 (-5A), kalinin, nicein, α3β1,α6β1,α6β4 epiligrin α3Bβ3γ2 3B32 Laminin-5B α3β1,α6β1,α6β4 α3Aβ1γ1 311, or 3A11 Laminin-6 (-6A), k-laminin ND α3Aβ2γ1 321, or 3A21 Laminin-7 (-7A), ND ks-laminin α4β1γ1 411 Laminin-8 α3β1,α6β1,α7β4 α4β2γ1 421 Laminin-9 ND α5β1γ1 511 Laminin-10 α3β1,α6β1,α7β1,α6β4,αVβ3a α5β2γ1 521 Laminin-11 α3β1, α6β1 α2β1γ3 213 Laminin-12 ND α4β2γ3 423 Laminin-14 ND α5β2γ3 523 Laminin-15 ND ND = not specified aTo recombinant laminin α5 IVa domain

5.3.2.1 General structure of laminins

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5.3.2.2 In vivo functions of different laminins

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5.3.3. LAMININ RECEPTORS

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5.3.3.1. Integrins (Int)

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# # *"" *" " F ) # *" 7( ! " :000M K /;;/8< # # " #" " ) " # 3 7 # " " 8 3 # # 7 # " C " 8< $" # " \ # *"" # < $" # 7( ! " :0008<

! " # /9 " 3 # " ) [ " " ) :2 3 2 33) 7( $ /;;68< $" ) ) " ) " ) 7 /;;/8< $" # # 6 *" " 3 33) " * < $" # ) ) ) " " # [ D *" ) 73/ 31 ) 8 7 /;;/8< $" # [ " #" ) " 3) ) " ) # 3 " " # [ # [ D " " * # 6< ' " * # [ " " # 6< $" F " D # " ) # " * " H " # " " " 7 /;;/8<

Figure 3. The integrin receptor family (modified from Hynes, 2002). *Also other integrins such as Int α1β1 and α2β1 function as laminin receptors (Patarroyo et al., 2002; Tulla et al., 2008). RGD refers to arginine-glycine-aspartic acid cell adhesion sequence in molecules such as fibronectin and vitronectin in vertebrates (Hynes, 2002).

! $ # # " # # # " # F < # < $" ) D ) < # H " " *"" ) < H " " ) 7 /;;/8< $" # 3 # "* " " # < 539 " " ) 7( :005M /;;:8< # " # # # F * " " " # < D 539 ) @ 3::: /:: 366/ 34:: *"" D @'( 7 '" :0028< )" " * " '' " ) 539 " * 3 " " " # F *" " 7E F /;;08< $" [ # " )* " H) )* 3 3 " < C " " F " 3: 36 39 ) # " # " " 7@ K /;;48< " # 3 " # D # [ # * " <

5.3.3.2. Non-integrin receptors for laminins

# " ) ) " #< " @" ) # # * 3 33 # * 7)- :002M % /;;:8<

!$ % ## $" @" ) # # " @" ) # *"" * # ) @" ) " 7@ 3 8 7C $ /;;58< $" @" ) # D # " D * # ) # 24 12 < @ 3 * D # " ) #

7C $ /;;58< @" ) # # [ @ 43" 7@ 34::4/:8 7% /;;:8< @ 34:: " ) " ) * *"" ## " 7% :001M % /;;:8< * " " @" ) # # " @'( * <

!$$ 3 # # # *"" * [ [ D " 3 " # D 7(8 # # " # " ) < ( * " * ) # " " 7Y /;;:8< # * 33 # < 3 # " ) " 33 # ) # < # 3) # *" 33 # ) " " 7)- :0028< # " ) ) " )H D " # " # )* " C " " # 7)- :002M Y /;;:8<

5.4. ANDROGENS IN SS

" " # " '' " 9;M4; < $" " # # # 3 < " '' # # < $" ## " # #" ) " * # " <

5.4.1. GENERAL ASPECTS CONSIDERING ANDROGENS IN SS

C # " ) *" # " ) ## ) # 7 Y /;;;8< " " # # # * D " " " " " * # ''< # # #" ) " " [ # '' " " '' * < % *" '' " ) " * C 3" 7E /;;:8<

C " ) C 3' ) " " # " *"" # # # 7 /;;:8< $" C C 3' 793 8 " " F " D ) " # 4; * < $" " " # " # #" ) " # *" ''< $" " C " " # # <

5.4.2. DEHYDROEPIANDROSTERONE (DHEA) AND ITS NORMAL FUNCTION IN VIVO

C " C 3' * 93 * # " " ) " F " D # " H " " "#" < ' C " # * ) < C [ " " D ". C 93 ) # # " 7@) :002M , F /;;;M ' /;;08 7# 98< $" C C 3' " " " "33 7% 83 # D< #" # " ") " 3 # " 7!8 ) " " " < $" ! " " \ ) 7 8 " * < ! " # 3 " 7 $8 *"" " # # " " C " < + # C C 3' *" # " #" " < $" C C 3' "#" ) " D )* /;M6; # # # # #< $" " " C C 3' " " # << " 3 " # [ # # 7@) :002M ' /;;0M # 98< C 3 # " # # ) # " ' ) *" # ) 7 /;;08< C " ) ) ") ) ) )

7@) /;;68< % 3D " ! '' '@C< " " ) *" " " " % 3D 7C-) /;;0M E /;;:M /;;68<

5.4.2.1. Intracellular processing of DHEA

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

#3 D < ) " " H "#" < $" *" ) " ) " 3 " < * C 3' *"" # " )* " # /; 6; *"" " C C 3' ) 7@) /;;68< $" ) " D ) " " # #3 *"" '' ) # F<

Figure 4. Schematic representation of the human steroid enzymes in peripheral intracrine tissues. Abbreviations: DHEA (dehydroepiandrosterone), DHEA-S (dehydroepiandrosterone-sulphate), 3β-hydroxysteroid dehydrogenase (3β-HSD), androstenedione (4-dione), androstenediol (5-diol), 17β-hydroxysteroid dehydrogenase (17β-HSD), dihydrotestosterone (DHT) (Modified from Labrieet al., 2003 and from Spaan et al., 2009).

5.4.2.2. Cysteine-rich secretory protein -3 (CRISP-3)

!'%36 7 * [ # /2 '(%/28 " " " 7K- :0058< Y" " " , " ) !'%36 " ) ) # H # < H " " " # 7KF" :0058< ) \ " * ) !'%36 7) /;;/8< !'%36 * # ) # 3 # 7 :0068< " !'%36 # ) " # " # 7 !C8 " # # " !'%36 # # 7 :0068< $" [ # '' " ) * 7" # # ''8 *" '' " ) " * C 7 - /;;:M E /;;:8< " !'%36 ) C 7 # 83# < $" " " " " D " " # " " D # <

5.4.2.3. Effect of sex steroids on laminin and integrin production

% " " * * ) " D " # # < $" " * C " " # <

5.4.3. SS AND SALIVARY GLANDS

# " # # C " " < ' # 3 # F< # C C 3' # 93 " ) ) F $ 7' /;;08< "" # $ ) 433: *" *" '' " " 7, F /;;;M % /;;28<

5.5. SS IN MALE PATIENTS

0 :; *" '' * 7 " :0248< # " :;U *" '' < $" '' ) ) " D 7E /;;/M K /;;08< * # D " *" '' # < " " 3 '' 9;M4; # 7$ :021M E /;;/M /;;48 " *" '' < $" " 3 '' * < Y " * # " # " # " " * # " C " D " < " " " ) " " C C 3' ) " D " 1;M2; # " C 3' " :;M/;U " # 7( FF /;;18< * C 3' # [ " # # ) ) " # < ) *" "< " * ) " C < # D ) / ) 7K < /;;18 ) # ) " " # < $" " D *" ) *" " "#" # 7! 3 /;;28<

6. AIMS OF THE STUDY

:< $ " ) " ) # "" )- *" '-# R <

/< $ " ) # 3 # " ) # "" )- *" '-# R <

6< $ " C C 3' " D !'%36 # # "" )- *" '-# R <

9< $ " # # # # D "" )- R *" '-# R <

7. MATERIALS AND METHODS

7.1. PATIENTS AND SAMPLES

$" " " O " " 7' 8 " - " " 73E8< $" # '' * ) " 3C '' 7E /;;/8< " )- * " " D " '' " * [ ) " # " * " " < + " "3 * " " # 7L@C8 *" " * " D *"" ) * < @'( "" " Z : *" " '' * * 1 : 73E8< )- * [ " < " D ) * " ) " D " " "<

7.1.1. INCLUSION AND EXCLUSION CRITERIA FOR THE STUDY

$" " * '' # ) " 3C < " " * ) " * F " C ) # '' 7E /;:;8< $" " *. :8 % '' # " 3C /8 ( # 1:9 3/; 7 # 3/; H RC /;;28M " * ) /60 ) " " '' % 68 ) C 3' 7" # # - * ) 2: "" * 41 "" 8< $" D * :8 # Z:2 \2; /8 68 ) # " 98 " ) 48 " 58 " 18 " " ) # 28 # # * 08 *" H :;8 [ ::8 # [

:/8 *" " # " '' # " " " :68 # " :; # <

7.1.2. COLLECTION OF THE LABIAL SALIVARY GLAND (LSG) SAMPLES (I-III)

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

7.1.3. COLLECTION OF THE RESTING AND STIMULATED SALIVA (III)

! # * /0 *" '' 7/1 / # 46 8 6/ "" )- 7:6 :0 # 94 8< $" # * )* 0

<< / << ')- " " " #" *" * " < * ) " )- "* [ 3*D 6; D "* [ " 4 D " *" * < *" '' " " " * D :4 ) " # < ' D < # " " * # :;;; # 4 " * F 31; F<

7.2. ANTIBODIES (I-IV)

$" ) " $) 9<

Table 4. Primary antibodies of this study and some of their characteristics

Antibody Antigen Source Reference Articles MAb 161EB7 LM α1 chain Mouse Virtanen et al., 2000 I MAb 2E8 LM α2 chain Mouse Leivo et al., 1988 I MAb BM2 LM α3 chain Mouse Rousselle et al., 1991 I MAb FC10 LM α4 chain Mouse Petäjäniemi et al., 2002 I MAb 4C7 LM α5 chain Mouse Engvall et al., 1986 I MAb 114DG10 LM β1 chain Mouse Virtanen et al., 1997 I MAb S-Laminin LM β2 chain Mouse Chemicon, Temecula, CA I MAb 113BC7 LM γ1 chain Mouse Määttä et al., 2001 I MAb D4B5 LM γ2 chain Mouse Mizushima et al., 1998 I MAb TS2/7 Int α1 subunit Mouse Hemler et al., 1984 II, IV MAb 10G11 Int α2 subunit Mouse Giltay et al., 1989 II, IV MAb J143 Int α3 subunit Mouse Fradet et al., 1984 II MAb GoH3 Int α6 subunit Rat Sonnenberg et al., 1987 II MAb 9.1 Int α7 subunit Mouse Vizirianakis et al., 2001 II MAb LM 142.69 Int αV subunit Mouse Cheresh and Spiro, 1987 II MAb 102DF5 Int β1 subunit Mouse Ylänne and Virtanen, 1989 II, IV MAb 90BB10 Int β3 subunit Mouse Ylänne et al., 1990 II MAb AA3 Int β4 subunit Mouse Tamura et al., 1990 II MAb IIH6 (IgM) α-DG Mouse Ervasti and Campbell, 1993 II MAb BRIC221 Lutheran ag Mouse Parsons et al., 1997 II MAb CRISP-3 CRISP-3 Rabbit Udby et al., 2005 III

7.3. INDIRECT IMMUNOFLUORESCENCE LABELING (I-II, IV)

'D ; " * [D *" 3/; :; 738< [D * # " * # . :8 *" :;

" " ) ;<:4 7% ' 1<98 # ;<: U $ V /8 *" % ' 68 ) # 98 ) " ) < " ) 7 ) $) 98 * #( ) D ( 6 *"" 3" #( ) 5 *"" 3" # ) 48 " " ) 58 ) " 3 -# ) # 3 #( ) 7 D 922 % ) C# +!8 # 3 #( 7O !" @) Y ( % 8 3" 5 #( # 3 # 7O 8 3" 3( # 5; b9 < 18 % :.:;;; % ' * F " < # " * *" * % ' + *"" " * ) \ # / 7 K+ ( 8 D + V1; 7+ )# ( 8 < ' # # * # % d # b d * bb d bbb d #< ! " # # F ) # " < % * " " # 7 # 8< # * # ) D #( 7 K+ V;06: :;; #8 " " " [ ) # -# ) < ' # * # # " [ " # ) # " ) " <

7.4. ANALYSIS OF CRISP-3 (III)

H " !'%36 # !, 7!, 8 !, * " ) ) # 7'(8 *" *" # D # 7 ' O 8< $" * )- H 3 " 7H!$3%!8 # @#" $

%! " 7! " " " ( 8 \ 'L ! ( ) [ # !'%36 7/9; ) 8 " ) # 7/61 ) 8 *"" * " # # " F " 7$) 48<

\ # " '( * F " # < D *" 6 U " * [ " " " * )F ;<4 U $ 3V % '< \ * # ;<53#@ )) 3" !'%36 # ) D 4523 -# # 3)) # ) < ) *" , 7, 8 ) !'%36D 7 /;M 04 " # # 8 * [ :3"# " ) * , ) 7 " @) % 8 $ 7 # ' # 8 D *" " # 75 D 8 " )) 3" !'%36 ) " D < $ @+,& [ ! 7 " @) % 8 * [ " *"" * F< $" F )) * # [ < ) # " # * ) 7/1 /0 ) # # # !'%36 8 * " [ " ) < $" ) " ) # F !'%36 " [D [ * " [ ) " " # 7) /;;/8<

Table 5. Primers used for cysteine-rich secretory protein-3 (CRISP-3) and porphobilinogen deaminase (PBGD) mRNA.

mRNA Sense Antisense CRISP-3 5’-TTCACCCTTCTTCCCAATCA-3’ 5’-CCTCCTTGAGTGGAGGGTATT-3’ PBGD 5’-ACATGCCCTGGAGAAGAATG-3’ 5’-AGATGCGGGAACTTTCTCTG-3’

7.5. SEX STEROID MEASUREMENTS (III)

$" H C 3' 7$" Y" 8 * # < C 3' * 42 *" '' 7 # 9:M5; 8 // # D3" "" )-< C F 7' ' # % 8 * " C < C * # 94 *" '' 7 # 9:M5; 8 :; #3 D3" "" )-<

7.6. HUMAN SUBMANDIBULAR GLAND CELL LINE (HSG) STIMULATIONS (III, IV)

'( )" " " ) ) # 7'" :02:8 * # !< K " ," < $" *" " # 3 3 *" 3 ) # *" 3 # ) 7! :0068< " D !$3%! * " '( # < 2;;;; '( C 3:/ , D 7() !@ ( ,L8 *" :; U ' / # :;; @ :;; ;#@ * #" # *" 3 # 7 ' O 8 /93* * ) /9 " *" *" :;; ; C C 3:/ , D *" :; U " D " '<

7.7. IMMUNOHISTOCHEMICAL AVIDIN-BIOTIN-PEROXIDASE COMPLEX (ABC) STAINING (III)

" " # 9 3 " [ * < ' * [ F *" D " # " < " * *" :; $ :4; , ) 7$ ' 1<48< # ! 7$"$ K+ ( 8 * * / 4 1;;Y " # < ')H " K+ $"$ F ) * " // # " * # . :8 " )) 3" !'%36 # ) 7;<5 3#@M $) 98 K+ " $ ) /4 M /8 ) # 3)) # ) $ ' # F /4 M 68 % D # ' 6 D 6 M 98 D3 -# /4 48 ') Y # ' # + 663 ) F " " 7" $

/ / 8 4 < # " # " * *" # " K+ " $ *" # ) 6 D 4 ) ) )* " < , 3 )) # ) # # " " # ) < " # " * " ) *" " D *" "

# " D " # 7D # '* 8<

7.8. IN SITU HYBRIDIZATION (III)

!'%36 , # ::10M:450 7! 'H , g;;5;5:8 7) /;;48 * K' 7'# @ O 8< F *" V" * 64'3) ) *" ' 64'3) )< ")F " ) 7!, 8 ) * [ 7 :;;M 8< [ F " 4 3 " * ")F *" /4M4; D :;5 64'3) !, ) 44 #"< $" * *" # # ) *" 4; 3#@ !, 3 7'# ' @ +8 6; b61 " ")F < " * #" 6M9 * 7' "3 " :0098<

7.9. SANDWICH ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) (III)

05 * \3) * #" *" )) 3" !'%36 ) # ) 7) /;;48< , 3[ ) # * ) *" ) 7;<4 , 6 K 2 , %+ K %+ : U ) / 9 / 9 ) : U $ V3:;; 1

4<; ;<;;5 U + # ;<;9 U 3" / / 7) < /;;/8< ) ) * 90/ < * <

7.10. SEARCH FOR ANDROGEN RESPONSIVE ELEMENTS (AREs) (III)

# ! 'H , g;;5;5: " , @ '$, *) 7".***< )< < "<# FH< #h d'"i d 8 * " #" " H " # < $" ".)< "#<- D<" *) * ) " ) # H :;<;;; ) /;;; ) * " # " " # D< $" " " * 53) " 3 " !C H 74R3(( $ $($$$36R8 7! " :00/8< " * *" " FF # " )* * H * D *" , " # @/'CG #<

7.11. EXPLANT TISSUE CULTURE STIMULATIONS (III-IV)

" @'( * ) * " E "" ) < $" * # 7 D / 68 53*3 C 3:/ 7() !@ ( ,L8 # :; U ' *" :;;; / @3# : # 7:;D8 #F 7/<4 3#M () !@ ( ,L8 #"< # " * # " * " # ) C 3:/ *" :; U ' / @3# :;; ;<: # 7:D 8 /<4 3# " < $ D * *" *" :;; ; C 1/ " ) )H H!$3%! !'%36 !, <

7.12. RNA ISOLATION AND cDNA SYNTHESIS (IV)

# * # * " " # *" 7 ' O 8< $ !, * # " $F 7 # ' # 8 !, * [ !, # " ) !, %[ K 7 + , *8< , * "F # '' ' , ' " ' !$3%! 7 # ) 8< H !, * , "<

7.13. QUANTITATIVE REAL TIME-PCR (qRT-PCR) (IV)

G 3 %! * # @#" $ %! " 7! " " " ( 8 'L ! ( ) # 7 : * 4R3($$($( $( 36R 4R3$$$$((($( (($ $$36RM # : * 4R3 $ ( ( (($ $$($$( 36R 4R3 ( $( ($$($( 36RM # / * 4R3 (($( (( $(( $$$( $36R 4R3 (($$(( $( ( ( (36R8< % ( " # # * F " ) # 4R3 $($(( ( ( $(36R * 4R3 ( $(((( $$$$$( 36R <

7.14. STATISTICAL ANALYSIS (I-IV)

" '%'' * :6<; 7'%'' "# @8 * < D %3% 1<;: * 7@ # 8 * " " !'%36 < $" " ) " ) * *" '" 3Y < ! D / " 7'C 8< ' R 3 " 3Y" * 3* " 3 ) ) <

8. RESULTS

8.1. LAMININ-CHAIN DISTRIBUTIONS IN LSGs IN HEALTHY CONTROL SUBJECTS COMPARED TO PATIENTS WITH SS (I)

" * )* " # @'( "" )- *" '' * < * " )* "" )- # 7 Z /8 \ @'(< $" * # @'( *"" " " " [< $" " * " " " \ @'( '' * * ) * # F *" " D @ 39 " 7 ) *8 " [ # " D ) *<

8.1.1. LAMININ α1 AND α2 CHAIN

@ 3: @ 3/ " * ) "" " @'( " # " *" " @ 3 " * " < *" '' " " # @ 3: @ 3/ " * * "" )-< $" # @ 3: '' * :

8.1.2. LAMININ α3 CHAIN

" @ 36 " # * # ) < $" " # * # "" )- *" '' 7 " " \ @'( 8 *" " # '' "" ) # /<4 /<1<

8.1.3. LAMININ α4 CHAIN

@ 39 " # * " *" # " < $" # @ 39 " * ) 3 " ""F< ' # " " * < @ 39 # * "" *" *" '' " # * #< " [ ) \ @ 39< @ 39 " D # # " # # # " " 78 * ) # # " @ 39 #< " [ # ) @ 39 # * * " \ @'( '' "" ) # * ) " # \ <

8.1.4. LAMININ α5 CHAIN

@ 34 " * ) " - " ) " < # * # *" '' "" )- # ) # /

8.1.5. LAMININ β- AND γ CHAINS

@ 33: " * D " " @'( # ) " < ' # * *" @ 33/ " # * < @ 34: 34/ " * " # " @'( # < @ 33: 34: 34/ " * " @'( " "" )- " @'( " *" '' *" \ ) * " \ @'( 7 ) 8<

8.2. DISTRIBUTION OF INTEGRIN AND NON-INTEGRIN RECEPTORS OF LAMININS IN HEALTHY CONTROL SUBJECTS AND PATIENTS WITH SS IN THE LSGs (II)

8.2.1. DISTRIBUTION OF INTEGRIN α SUBUNITS

" @'( " "" )- 6 5 ) " " " # " < " : / ) * " ) < '( 7 ) *8 : / ) * ) " 78 " * " [ " # [ *" " \ # " < $" # " : / ) * # " " < C" ) # " @'( " # ) < 1 E ) * " @'(<

8.2.2. DISTRIBUTION OF INTEGRIN β SUBUNITS

# 3: 39 ) * " " ) # " ) " '' "" < 36 ) * " " @'(<

8.2.3. DISTRIBUTION OF NON-INTEGRIN RECEPTORS

3 # * * " ) " ) ) " ) < @" ) # # # # " ) # " ) " '' "" <

8.2.4. COMPARISON BETWEEN THE HEALTHY CONTROL SUBJECTS AND PATIENTS WITH SS

6 5 3: 39 ) " " ) # " " @('<

, " # " ) * )* " "" )- *" ''< : / ) * " # # " ) " ) " @'( " "" )-< $" # # * * *" ''< 1 E 36 * " @'( *" 3 # * " ) " " " @'( "" )- " @'( *" ''< @" ) # # * # " " ) " " @'( "" )- " @'( *" ''<

8.3. DHEA CONCENTRATIONS AND ANDROGEN-REGULATED CRISP-3 IN SS AND HEALTHY CONTROL SUBJECTS (III)

8.3.1. AREs IN THE HUMAN CRISP-3 GENE COMPLEX

$" " # # :;;;; ) /;;; ) * " " 7b8 7M8 :; 6 H *"" / 726<6U " #8 " !C 4)3(( $ $($$$36)<

8.3.2. DHEA AND DHEA-S CONCENTRATIONS

42 *" '' 7# 9:M5; 8 " * C 3' *" // "" )- 7 / 'C /<; / ;

C * # [ * '' 7 d 948 " #3 D3 " "" 7 d :;8 7//9 /66<6 9:0 / 02<: M d ;<;;48<

8.3.3. ANDROGEN RESPONSIVENESS OF HSG CELLS AND LSG EXPLANTS

# [ " !'%36 !, ) * '( # *" 3 # *" :;; ; C /9 " 7 d ;<;:2 ) *8< $" !'%36 !, '( *" # * 00 / 45 :;4 % ( < $ " " # *" 3 # " !'%36 !, /45 / /;1 :;4 % ( < !'%36 !, * " 3# ) C " " # " :;; ; C " :/19 / :;24 !'%36 !, :;4 % ( < $" # D " * " " # * * " " !'%36 !, * # [ 7 d ;<;:2 ) Y D # 8< $" " !, " # * ## ) #" * " \ # !'%36 " < D " @'( * *" :;; ; C 1/ " " !'%36 !, * '( D * "" * D <

8.3.4. CRISP-3 STAINING

!'%36 * D " 3 " " @'( "" )- *" *< + " " @'( *" '' !'%36 # * * " F # F " < $" # " " * * " @'( " "" )- *" !'%36 * " " *" ''< @" " "" # " '' * ) !'%36 # [ < $" !'%36 # * ) " # @'( '' " " " [< C \ * # F !'%36 # *" ''< , )) # ) " 7 8 " [ ) * " # #

[ " [ # "" )- " *" ''< C" * # *"" ) # " # D< " H ) !'%363 " * <

8.3.5. IN SITU HYBRIDIZATION OF CRISP-3

" @'( " "" )- !'%36 !, * ) " *" " ) " )# < " @'( " *" '' ) # * * # F < " ) # D " " )# < $" [ " ")F * ) " # ) # # !, ) " !'%36 [ !, )< [) ) " )<

8.3.6. CRISP-3 PROTEIN IN SALIVA

$" !'%36 7 ;## 8 # D *" * * *" '' *" " "" )- 7 Z ;<;;48< " *" " \ * * " " !'%36 )* '' "" ) D # # [ 7 Z ;<;;;:8< !'%36 * * " D *" 7 Z ;<;;;: $) 58<

Table 6. CRISP-3 levels in resting and stimulated saliva, values are mean ± SEM.

Healthy controls Patients with SS P n = 32 n = 29 Resting CRISP-3, μg/mg salivary protein 14.0 ± 1.4 8.4 ± 1.4 <0.005 CRISP-3 output, μg/15 minutes 97.6 ± 12.0 21.1 ± 2.7 <0.0001 Stimulated CRISP-3, μg/mg salivary protein 13.1 ± 0.9 3.7 ± 0.4 <0.0001 CRISP-3 output, μg/15 minutes 240.1 ± 26.2 53.8 ± 7.1 <0.0001

8.4. EFFECT OF ANDROGEN STIMULATION ON THE SALIVARY GLAND CELL LAMININ AND INTEGRIN EXPRESSION (IV)

8.4.1. EFFECT OF MATRIGEL ON EXPRESSION OF LM α1 CHAIN AND INT α1 AND α2 SUBUNITS IN SALIVARY GLAND CELLS

# " '( " < $" " !, : 1/ " Q ) :;4 % ( 12; / 552 7 d68 :/5/ / 4;4 7 d68 6/66 / 64:/ 7 d98 /9 92 1/ " R< # : 7d;<;:68 / 7d;<6668 ) !, " # #< $" # " * #" " # # : / ) <

8.4.2. EFFECT OF SEX STEROIDS ON EXPRESSION OF LM α1 CHAIN AND INT α1 AND α2 SUBUNITS IN SALIVARY GLAND CELLS

'D @ :3" !, '( @'( D < CD " !, : / ) * 7 *" #8 7 #8 '( # [ 1/3" ) C < : :; :;; ; C 1/ " : ) !, ) 61 U 7d;<6:/8 95 U 7d;<;:58 990 U 7d;<;;98 < $" # / ) !, * 49 U 7d;<:468 59; U 7d;<;;68 ::;; U 7d;<;;18< : :; :;; ; C 1/ " : ) !, ) 0; U 7;<;468 :;/ U 7d;<;;08 ::6 U 7d;<5448 / ) !, ) 60 U 7d;<44:8 916 U 7d;<;958 6 U 7d;<4098 < ' *" :3 : / ) !, ) 10 U 7d;<;;08 :/; U 7d;<;/;8 < : / ) !, ) 1/ U 7d;<;/58 05 U 7d;<;258 < ' *" : :; : ; $ # [ " D : ) !, :;4 % ( ) " ) " # * . 66 U 6: U 44 U 7555/; / 64:14

d::M *"225:0 / /;2:2 d6M 21:1/ / :0;:4 d6M :;/01: / 1556 d6 8< $" # " # / ) !, * 9: U 6 U 5/ U 74;;2 / /951 d2M *" 1;59 / :/6: d6M 4:1; / 91; d6M 2:61 / :/02 d68< " : ) !, * :5 U 09 U 1; U 7:/;0/5 / 405/1 d2M *" :9;505 / 00651 d/M /69169 / ://:0: d6M /;4/29 / 049:: d68 / ) !, :64 U 20 U 0; U 709// / 1:51 d:4 *" //:64 / :0/11 d/M :1264 / ::;4: d6M :106/ / 550/ d68< # " * # [ < C " # [ ) 7 " * 8< ) " '( 92 1/ " " C #" : ) " " ) C < *" " H!$3%! / ) # * " * " " " : ) ) ) " " * # $ < ' # * # [ # " [ " # *" " ) " <

8.4.3. EFFECT OF DHEA STIMULATION ON LM α1 CHAIN AND INT α1 AND α2 SUBUNITS IN SALIVARY GLAND BIOPSIES

CD # " ) ) " D @

:3" @'( "" )- *" '' * ) *" # < $" # *" " < $" D : / ) * ) C @'( "" )- ) @'( *" '' " * < $" C " D : / ) " * #" " *" # # ) " "" '' @'(<

9. DISCUSSION

9.1. PATIENTS AND SAMPLE SELECTION

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

) " * " D " " " <

9.2. THE SPECIFICITY AND SENSITIVITY OF THE METHODS

" ) # " # # < $" # " * # " # ) *"" ) # "< $" ) * " -# *" ) *"" " # F F # D "< $" ) [ " " " < " " " # # \D) < " # " " ) \ " " " " <#< " 3) 3 D D7 8 " < " [ " " ) -# *" \ " 7$8 " " D #" " \ *"" " ) *" D [< \ " " ) " # ) # " * " " *" # # ) [ " << # # 3 )# < \ " ) " " # <#< ) "# 7 $" /;;28< " < $" " " " ) " * " " *"" ) *" # 3 ) " [ \ #< * " *" # * " " # " )* *" '' "" )- * " " ) " ) "#" ) [D [ ) *" # " # < Y" ) " " " )# # * F " ) # # * D # " H [ " " # # # < ) 7) $) 98 * " 3 3 [ )# #< ! "#" " # D

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

!, H!$3%! ) ) # " " # # !'%36 * D " @'(< $" " * E " C !%'36 @ " ) <

9.3. LIMITATIONS OF THE STUDY

$" " ) # # "#" ) [ " < !# " " " " # # " " " < $" ) *<

9.3.1. THE RESTRICTED PALETTE OF ANTIBODIES

$" ) " * " ) *# # F " 7% /;;/M E /;;;M %- /;;98< ) *" % E R " # ) " H * 3" # 3 ) ) * " " 3 # 3 ) < $) 9 " * " ) " < @ D * * " ) " " ) < $" " " #" ) @'(< * * " ) *"" * # @'( * * # < ) )* " # ) # D * * " " # " 7% /;;/8 *"" ) ) " " # " @'( " < ) " * * ) " " *"" " D " [ #<

9.4. DISTRIBUTION OF THE LAMININ CHAINS IN LABIAL SALIVARY GLANDS

@ :3" * D " " < @ :3" * " < " [ # " @ :3" ) "

7')# :0028< " " " D * # " 3" 91 ) 7C # < :0258< * " # F :3" ) " ) " * # F @ 43" @ :3 " 7C # < :025M $# :001M $) 98< $" " " ) 3 ) " " * " @ :3" ) @ 43" ) " " # < $" *" " [ # " *"" " 3" 91 ) * * * ) [ " [ # 7')# :0028< " " F :3" * F # " ) @ :3" [ 3" :5:C 1 ) " [ *"" " ) *" 7E /;;;8< $" # *" :5:C 1 ) " * " @ :3" # " # < ' # @

:3" " * # " # * " *"" * ) \ # " " # < " *" " # " @ : ) # * * '' @'( # " *" " ) # ) ''< ' # *" @ 3/3" # F # ) * " * " @'( " " < # " # " * ) " 3 4 " * " @ :3" @ 3::: " " " @'( 7% /;;/M $) 6 98< $" * " " @ :3" " # @ 3::: # " " *"" " # @'( *" ''< # " # " @ /3" ) # * ) " " < * " " " " " # ) " ) ) *" " " "" - # 7')# :0028< $* " " *" # D *" " * " )3 *"" #" D " # < $" ## " @ /3" " # @ 3/:: " " "" @'(< ' " ' [ " ) " " # " " " < " )) # " " " " HF " 3" "

7K 8< $" # " @ /3" * * *" '' " "" )-< $" ## " " # 3 " " # '' *" " " *"" " ''< % # * " " " ) # *" :; ) [ # F # " @ 93" 7%- /;;/8< % # # " ""F ) # @ 93" < $" *" [ # " * # " " [) ) " @ 93" 7 # L" /;;;8 *"" # ) )* # 7% /;:;8< @ 93" @'( @ 39:: ) @ 3/3" * " @ 39/: ) < $" # @ 93" * '' @'( *"" @ 39:: * " < " @ :3 /3" ) @ 93" * " " [ < $" [ # * \ 3 " < ! " ) " * " *" 3 \ $,3 @3:3 " @ 39 !, 7% /;:;8< $" @ 9 # " # ) \ ''< " D " ) " # ## ) # " D '' 7 " :0068< + " * " " #" " # 7 8 @

:3 /3" D * @'( *" '' " " ) @ 93" D * ''< " " ) * 3" ) 7@ 3208 " D [ *"" * < #" ) " " D " " D @ 3 9:: *"" ) [) ) " < $" ) " * " ) " " @ 39:: " " ) " # ) " )3 #< " ) " K* 3* " " " )" *" # @ 3: 39 D 7K* /;;58< $" " @ :3" * # [ D @'( *" '' " # * [) ) *" "#" [) < K* * )) 3" @ :3" ) 736;;8

# # :245M/;00 @ : " " # Y ) #< Y 3" @

: " ) 7 ):5:C 18 # ) @ : # " ) D # ) (93(4 C6 # " @ : " \ # 7E /;;;8< " '*" % 36;; * 14 U " # 7 #8 *" " ):5:C 1 " 7) ) #8< $" [ ):5:C 1 * [ ) # ) ) O ! " " 7E /;;;8< ) " " " " @ : " ) " * D " < " @ 93" * *" # " < 3%! " * @ 93" )* '' 7K* /;;58< * @ 93" "F ) [) ) " *"" # %! < ' *" " %! " " @ 9 " ) * '' *" * [) 7 d ;<;:8 ) '' *" "#" [) 7 d ;<;68 7K* /;;58< ) " @ 9 Y ) # "#" [) # \ " \ " < " " * " @ " ) # * " @'( @ 36:: 366/ 34:: ) ) " < " " @ [ # # " " " ) @'( *" @ 3 ::: 3/:: 39:: " [ 3 ) ) < #" " " ) @'( "#" D< " " " " " D @ 3::: 3/:: D @ 3 9:: '' " #< @ \ " # # " \ #< @ D # " " " [ " D @ 39:: *"" ) # " < $" ) " '' " " \ *<

9.5. DISTRIBUTION OF POTENTIAL INTEGRIN AND NON-INTEGRIN RECEPTORS OF LAMININS IN LABIAL SALIVARY GLANDS

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d ;<;/8< *" '' " # " ) 39 ) " " * ) *

" " 3 539 *"" * " # F )< Y"

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

* ) " " "" " @'( ''<

9.6. DHEA AND CRISP-3 CONCENTRATION IN SALIVA AND THEIR DISTRIBUTION IN LABIAL SALIVARY GLANDS

" !'%36 * ) # < " # # 7 !C8 " # # < C 3 # D * ) !C 7! " :00/M '"*F < :0048< # " ) " * ) # # " # 7 :0068< # ) " " !C " " # D ## # " " " # # < " " C " " ) ) # * < '' # D # *"" # [ < '( *" C " !'%36 !, ) " * # " # " # " '( < $" [ # * " D ) D @'( "" * D *"" 3 " !'%36 !, ) # " " " !'%36 ) # # ) C < !'%36 * ) # " # "" '' < @'( " # " 3 7 8 * # *" " " " # " < *" '' " ## # * ) ## # " " ) @'( ''< $" [ # * [ ) ")F *"" " * ) # *" #" # "" ) # *" *" '' !'%36 ) # * * < $" " !'%36 * "#" "" )- " *" '' 78< # " [ # " " " " # ) ) * # F " " @'(< " " " # " # ) '' # " ) # " \ [< $" *"" \ " 7K :0018< * " # ## " "

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

) " " # [ # " #" # *" <#< #<

9.7. ANDROGEN EFFECT ON LAMININ CHAINS AND INTEGRIN SUBUNITS IN SALIVARY GLANDS IN SJÖGREN’S SYNDROME

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

" # " # " # < " @'( *" '' C ) " D < " " " " " ''<

10. SUMMARY AND CONCLUSIONS

'-# R *"" * < $" )" " " ) < $" ## " " ) ) " " # " # < $" " ) # < " # # " # # " # ) '-# R <

MAJOR CONCLUSIONS:

:< @ " D "#" # ) @'(< @ " [ " # < @ 3::: " D " ''< @ 3/:: 39:: " [ ) *" @'(< +" " @ 36:: 366/ 34:: " # # # @'( * ) ''<

/< " ) # 3 # ) "#" D @'(< 63: 53: 539 * " @" ) # # " " # " @'( # ) " [ " " " < $" D :3: /3: " " " " " * D " )) ## # 3[ M < CD " # * * '' "" ## # " " @ 3::: 3/::3 3 :3: /3: '' " " @'(< * ) " # " # *"" ) * " ) # # <

6< @ * C C 3' *" '' " " # < $" # * [ # " # 3 !'%36 "

*"" * " ''< $" #" ) " " C " ) F # " *"" C ) <

9< " " # " " " ) # # " " # " " # # < $" # )H " " # ) # ) " @ 3:::3 3 :3: /3: < @ @ 3:::3 3 :3: /3: ) # " # < # :3: /3: D " # < C " " " # " # ) [ " @'( *" '' " " # ) F # " <

11. ACKNOWLEDGEMENTS

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

/;:;

12. REFERENCES

" % " Y $ ,. 'D " < " D " < O %" :024M :/:. 46:M4:< ( O . !D) '-# R < ,L ' /;;0M ::16. 1;:M4< ' '!. " C # . " " < CD C ) /;;:M :;0. 14M2/< . # " "# "< O :0/1M :. 4;9M5< '- $" ! K EC , ) $ '# ( # ! . CD # 3 ) # # " : ) : " / ) : 3 $ < O /;;6M :1:. /2;9M::< O$. C ) . ) * h C /;;0M /4. 26M2< '" ,. $" " ) ) < O :002M :06. :M/:< 3$ @ Y( F ! C# C) % . [ < D /;;4M /9. 6/5M6/< ' . # < ! $" /;;;M 4:. /2;M6;:< O O ! Y ' ( $%. ') ) H 4 # '-# R < @ /;;:M 642. :214M5< - K % O. % '-# R . )- " < CD !" :00;M 2. /26M2< #" C. CD D " \ " " " " " ) ) # < O :04;M 29. 6;6M/:< "" ' !. " #3(3 3 " " [) Y) < O " :021M /5/. :11;6M::< # L" %. . $" " < /;;;M /:2. /:6M69<

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