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Role of IgG1 M3R Autoantibodies in Secretory Dysfunction of Sjogren's Syndrome

Permanent Link: http://ufdc.ufl.edu/UFE0024871/00001

Material Information

Title: Role of IgG1 M3R Autoantibodies in Secretory Dysfunction of Sjogren's Syndrome
Physical Description: 1 online resource (55 p.)
Language: english
Creator: Huynh, Huy
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: anti, autoantibodies, autoantibody, b6, igg, igg1, m3r, nod, purification, secretion, secretory, sjogren, syndrome
Food Science and Human Nutrition -- Dissertations, Academic -- UF
Genre: Food Science and Human Nutrition thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Sjo umlautgren?s syndrome (SS), first described in 1933 by the Swedish ophthalmologist Henrik Sjo umlautgren, is an autoimmune disorder that targets the exocrine glands, especially the lacrimal and salivary glands. Two main symptoms that characterize this disorder are xerostomia (dry mouth) and xerophthalmia (dry eyes). Studies have suggested that autoantibodies from SS patients reactive to type-3 muscarinic acetylcholine receptors (M3R) may account for defective secretion in these patients. Thus, the objective of this study was to investigate the effects of IgG1 anti-mouse M3R autoantibodies on saliva secretion and calcium release by utilizing in vivo and in vitro systems, respectively. First, sera from C57BL/6J (control) mice and SS-prone NOD/ShiLtJ (experimental) mice were collected and purified into different isotypes of IgG (IgG1, IgG2b, IgG2c, and IgG3). ELISA analysis was performed for their quantification. Fractions of each isotype were injected intraperitoneally (IP) into immune-deficient B6.129S7-Rag1tm1Mom/J recipient mice. After 3 hours, saliva was collected for 10 minutes following an injection of a secretagogue mixture (pilocarpine and isoproterenol) to measure saliva flow. This procedure was repeated each day over a 5-day period. In addition, to investigate in vitro if IgG1 anti-M3R autoantibodies inhibit M3R-mediated intracellular calcium release, human salivary gland (HSG) cells were incubated with the purified fractions for 24 hours prior to agonist carbachol stimulation and subject to a calcium release assay. In the in vivo experiment, both NOD and C57BL/6J IgG1 and IgG2c fractions had a stimulatory effect on saliva secretion on Day 2 and Day 3, respectively, but a decrease in saliva secretion was observed on Day 5 in the NOD fractions compared to B6 fractions. In the in vitro experiments, significant reductions (p < 0.05) in calcium release were observed for both C57BL/6J IgG1 and IgG2b at 15 minutes of incubation, as well as for all C57BL/6J and NOD fractions (especially NOD IgG1) at 24 hours with an addition of 2 ?L Ig for both time treatments, compared to carbachol-stimulated HSG cells alone. The in vivo preliminary findings suggest that both NOD IgG1 and NOD IgG2c fractions can cause secretory dysfunction. This may be due to the binding of these fractions to M3R, thereby blocking the signaling pathway that normally controls saliva secretion. Conversely, C57BL/6J IgG1 and IgG2c fractions seemed to cause a stimulatory effect to increase saliva secretion. NOD IgG2b fractions did not appear to inhibit salivary flow. The in vitro experiments demonstrate that both C57BL/6J and NOD purified fractions cause a decrease in calcium release in HSG cells stimulated with carbachol, suggesting intracellular calcium release can be inhibited by chronic incubation (24 hours) with both C57BL/6J and NOD purified IgG isotypes. Also, it can be observed that HSG cell incubation with the same amount (0.2 ?g) of Ig fractions can cause a similarly decreasing trend compared to using the same volume (2 ?L). Overall, these results suggest that NOD IgG1 and IgG2c might have inhibitory effects on secretory function in vivo. Experimental optimization is needed to investigate if IgG1 is responsible for a decrease in carbachol-induced intracellular calcium release, in vitro, after 24-hour chronic incubation.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Huy Huynh.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Rodrick, Gary E.
Local: Co-adviser: Cha, Seunghee.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024871:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024871/00001

Material Information

Title: Role of IgG1 M3R Autoantibodies in Secretory Dysfunction of Sjogren's Syndrome
Physical Description: 1 online resource (55 p.)
Language: english
Creator: Huynh, Huy
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: anti, autoantibodies, autoantibody, b6, igg, igg1, m3r, nod, purification, secretion, secretory, sjogren, syndrome
Food Science and Human Nutrition -- Dissertations, Academic -- UF
Genre: Food Science and Human Nutrition thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Sjo umlautgren?s syndrome (SS), first described in 1933 by the Swedish ophthalmologist Henrik Sjo umlautgren, is an autoimmune disorder that targets the exocrine glands, especially the lacrimal and salivary glands. Two main symptoms that characterize this disorder are xerostomia (dry mouth) and xerophthalmia (dry eyes). Studies have suggested that autoantibodies from SS patients reactive to type-3 muscarinic acetylcholine receptors (M3R) may account for defective secretion in these patients. Thus, the objective of this study was to investigate the effects of IgG1 anti-mouse M3R autoantibodies on saliva secretion and calcium release by utilizing in vivo and in vitro systems, respectively. First, sera from C57BL/6J (control) mice and SS-prone NOD/ShiLtJ (experimental) mice were collected and purified into different isotypes of IgG (IgG1, IgG2b, IgG2c, and IgG3). ELISA analysis was performed for their quantification. Fractions of each isotype were injected intraperitoneally (IP) into immune-deficient B6.129S7-Rag1tm1Mom/J recipient mice. After 3 hours, saliva was collected for 10 minutes following an injection of a secretagogue mixture (pilocarpine and isoproterenol) to measure saliva flow. This procedure was repeated each day over a 5-day period. In addition, to investigate in vitro if IgG1 anti-M3R autoantibodies inhibit M3R-mediated intracellular calcium release, human salivary gland (HSG) cells were incubated with the purified fractions for 24 hours prior to agonist carbachol stimulation and subject to a calcium release assay. In the in vivo experiment, both NOD and C57BL/6J IgG1 and IgG2c fractions had a stimulatory effect on saliva secretion on Day 2 and Day 3, respectively, but a decrease in saliva secretion was observed on Day 5 in the NOD fractions compared to B6 fractions. In the in vitro experiments, significant reductions (p < 0.05) in calcium release were observed for both C57BL/6J IgG1 and IgG2b at 15 minutes of incubation, as well as for all C57BL/6J and NOD fractions (especially NOD IgG1) at 24 hours with an addition of 2 ?L Ig for both time treatments, compared to carbachol-stimulated HSG cells alone. The in vivo preliminary findings suggest that both NOD IgG1 and NOD IgG2c fractions can cause secretory dysfunction. This may be due to the binding of these fractions to M3R, thereby blocking the signaling pathway that normally controls saliva secretion. Conversely, C57BL/6J IgG1 and IgG2c fractions seemed to cause a stimulatory effect to increase saliva secretion. NOD IgG2b fractions did not appear to inhibit salivary flow. The in vitro experiments demonstrate that both C57BL/6J and NOD purified fractions cause a decrease in calcium release in HSG cells stimulated with carbachol, suggesting intracellular calcium release can be inhibited by chronic incubation (24 hours) with both C57BL/6J and NOD purified IgG isotypes. Also, it can be observed that HSG cell incubation with the same amount (0.2 ?g) of Ig fractions can cause a similarly decreasing trend compared to using the same volume (2 ?L). Overall, these results suggest that NOD IgG1 and IgG2c might have inhibitory effects on secretory function in vivo. Experimental optimization is needed to investigate if IgG1 is responsible for a decrease in carbachol-induced intracellular calcium release, in vitro, after 24-hour chronic incubation.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Huy Huynh.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Rodrick, Gary E.
Local: Co-adviser: Cha, Seunghee.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024871:00001


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1 ROLE OF IGG1 M3R AUTOANTIBODIES IN SECRETORY DYSF U NCTION OF SJGRENS SYNDROME By HUY HUYNH A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR TH E DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2009

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2 2009 Huy Huynh

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3 To everyone who supported my work.

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4 ACKNOWLEDGMENTS It is with sincere gratitude, humility, and thankfulness that I acknowledge the following individuals for their help and support during the last two years. Without their help, I would not have grown both professionally or personally to the potential that they saw within me to succeed. I would first like to thank my committee co -chai rs, Dr. Gary Rodrick and Dr. Seunghee Cha, for accepting me as a student and guiding me through my efforts. I would like to thank Dr. Rodrick, for his patience and honest advice that helped guide me in the right direction. Also I thank Dr. Cha for her unwavering support and invaluable advice that not only helped me further my career and knowledge as a student, but also gave me the faith to push myself through the hardest of times. Next, I would like to recognize my committee members for their support. I wish to thank Dr. Keith Schneider for his motivational talks about timeliness and his statistical expertise. Next, I wish to thank Dr. Ammon B. Peck for sharing his knowledge and passion for the field of autoimmunity. In addition, I would like to tha nk Dr. Minoru Satoh for his unforgettable humor and his never ending patience, while teaching me the intricacies of experimental technique, implementation, and understanding the reasoning behind our methods. Lastly, I would like to thank my laboratory a ssociates for always lending a helping hand and being such wonderful role models for me. I wish to thank Jason Weinstein, Dina Nacionales, and Tolga Barker in Dr. Satohs lab for always taking time out of their schedules to teach me the nuances of researc h, as well as making my research experience enjoyable. Also, to my associates in Dr. Chas lab oratory: Kaleb Pauley, Marievic Bulosan, Kyumee Yo, and Christopher Chuong. I would like to t hank them for being my support beam and a constant positive energy for me in the las t two years. Without everyone I would not have had the memorable experiences and knowledge th at helped motivate me to succeed.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................................... 4 LIST OF TABLES ................................................................................................................................ 7 LIST OF FIGURES .............................................................................................................................. 8 ABSTRACT .......................................................................................................................................... 9 CHAPTER 1 INTRODUCTION ....................................................................................................................... 11 Primary and Secondary Sjgren Syndrome ............................................................................... 11 Nutritional Aspects ...................................................................................................................... 12 Autoantibodies and Muscarinic Receptors Detected in SS ...................................................... 12 Anti -Ro/SSA and Anti La/SSB Autoantibodies ................................................................ 12 Role of Autoantibodies in the Pathogenesis of Sjgrens Syndrome ............................... 13 Anti -Muscarinic 3 Receptor Autoantibodies with Emphasis on IgG1 Isotype ................ 14 Mouse Models ............................................................................................................................. 16 Defective Secretory Function in SS ........................................................................................... 17 Specific Aims .............................................................................................................................. 18 Specific Aim #1 ................................................................................................................... 18 Objective ....................................................................................................................... 18 Hypothesis .................................................................................................................... 18 Rationale ....................................................................................................................... 18 Specific Aim #2 ................................................................................................................... 18 Objective ....................................................................................................................... 18 Hypothesis .................................................................................................................... 18 Rationale ....................................................................................................................... 18 2 MATERIALS AND METHODS ............................................................................................... 20 Specific Aim #1 ........................................................................................................................... 20 Objective (In vivo) ............................................................................................................... 20 Hypothesis ............................................................................................................................ 20 Rationale ............................................................................................................................... 20 Mouse Model ....................................................................................................................... 20 Serum Collection ................................................................................................................. 21 Dialysis and Antibody Fraction Column Preparation for Purification ............................. 21 Purification of Whole IgG ................................................................................................... 23 Purification of Isotypic Immunoglobulins: IgG1, IgG2b, IgG2c, and IgG3 .................... 23 Measurement of Concentration of Purified Ig Isotypes by ELISA .................................. 24

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6 Injection Strategy ................................................................................................................. 25 Saliva Collection .................................................................................................................. 26 Specific Aim #2 ........................................................................................................................... 26 Objective (In vitro) .............................................................................................................. 26 Hypothesis ............................................................................................................................ 26 Rationale ............................................................................................................................... 27 Cell Culture .......................................................................................................................... 27 Calcium Assay ..................................................................................................................... 27 3 RESULTS .................................................................................................................................... 31 Specific Aim #1 ........................................................................................................................... 31 A nalysis of Purified Fractions Measured Concentrations of Ig Isotypes with IgG1 Being the Highest ............................................................................................................. 31 NOD IgG1 and IgG2c Inhibit Secretory Dysfunction in Immunodefecient Mice In Vivo ................................................................................................................................... 31 Specific Aim #2: Altered Intracellular Calcium Release in HSG Cells in the Presence of Ig Isotypes ................................................................................................................................ 33 4 DISCUSSION .............................................................................................................................. 43 Specific Aim #1 ........................................................................................................................... 43 Specific Aim #2 ........................................................................................................................... 47 Future Directions ......................................................................................................................... 50 Conclusion ................................................................................................................................... 51 LIST OF REFERENCES ................................................................................................................... 52 BIOGRAPHICAL SKETCH ............................................................................................................. 55

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7 LIST OF TABLES Table page 2 1 B6 Ig fractions table that displays ratios used to calculate volume injected into each Rag1KO mouse in vivo .......................................................................................................... 29 2 2 NOD Ig fractions table that displays ratios used to calculate volume injected into each Rag1KO mouse in vivo ................................................................................................. 29 2 3 B6 Ig fractions table that displays injection amounts and volumes that were used in each 15 minute and 24 hour incubation experiment in vitro ............................................... 29 2 4 NOD Ig fractions table that displays injection amounts and volumes that were used in each 15 minute and 24 hour incubation experiment in vitro ........................................... 29 3 1 B6 Purified Ig Fraction ELISA results .................................................................................. 35 3 2 NOD Purified Fraction ELISA results .................................................................................. 35

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8 LIST OF FIGURES Figure page 1 1 Description of the three phases of SS -like d isease development in the NOD Aec1Aec2 mouse model (Modified from Lee et al. (25)). .................................................. 19 2 1 In vivo experiment with Rag1KO mice under four different treatment conditions:. ......... 30 2 2 In vivo experimental timeline over a 5 -day period. .............................................................. 30 3 1 In vivo results of saliva secretion after 3 hour incubation (on Day 1) with IP injected B6 (control) and NOD (experimental) purified IgG1 into Rag1KO mice. ...................... 36 3 2 In vivo results of saliva secretion after 3 hour incubation (on Day 1) with IP injected B6 (control) and NOD (experimental) purified IgG2b into Rag1KO mice. .................... 37 3 3 In vivo results of saliva secretion after 3 hour incubation (on Day 1) with IP injected B6 (control) and NOD (experimental) purified IgG2c into Rag1KO mice. ..................... 38 3 4 In vitro (control) and NOD (experimental) Ig fraction. .................................................................... 39 3 5 In vitro each respective B6 (control) and NOD (experimental) Ig fraction ..................................................................... 40 3 6 In vitro (control) and NOD (experiment al) Ig fraction ..................................................................... 41 3 7 In vitro (control) and NOD (experimental) Ig frac tion ..................................................................... 42

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9 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science ROLE OF IGG1 M3R AUTOANTIBODIES IN SECRETORY DYSF U NCTION OF SJGRENS SYNDR OME By Huy Huynh August 2009 Chair: Gary Rodrick Cochair: Seunghee Cha Major: Food Science and Human Nutrition Sjgrens s yndrome (SS), first described in 1933 by the Swedish ophthalmologist Henrik Sjgren, is an autoimmune disorder that targets the ex ocrine glands, especially the lacrimal and salivary glands. Two mai n symptoms that characterize this disorder are xerostomia (dry mouth) and xerophthalmia (dry eyes). Studies have suggested that autoantibodies from SS patients reactive to type 3 muscarin ic acetylcholine receptors (M3R) may account for defective s ecretion in these patients. Thus, the objective of this study was to investigate the effects of IgG1 anti mouse M3R autoantibodies on saliva secretion and calcium release by utilizing in vivo and in vitro systems, respectively. First, sera from C57BL/6J (control) mice and SS -prone NOD/ShiLtJ (experimental) mice were collected and purified into different isoty pes of IgG (IgG1, IgG2b, IgG2c, and IgG3). ELISA analysis was performed for their quantif ication Fractions of each isotype were injected intraperitoneally (IP) into immune -deficient B6.129S7 Rag1tm1Mom/J recipient mice After 3 hours, saliva was collected for 10 minutes following an injection of a secretagogue mixture (pilocarpine and isopr otere nol) to measure saliva flow. T his procedure was repeated each day over a 5 -day period. In addition, to investigate in vitro if IgG1 anti -M3R autoantibodies inhibit

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10 M3R -mediated intracellular calcium release, human salivary gland (HSG) cells were inc ubated with the purified fractions for 24 hours prior to agonist carbachol stimulation and subject to a calcium release assay. In the in vivo experiment, both NOD and C57 BL/6 J IgG1 and IgG2c fractions had a stimulatory effect on saliva secretion on Day 2 and Day 3, respectively, but a decrease in saliva secretion was observed on Day 5 in the NOD fractions compared to B6 fractions. In the in vitro experiments, significant reductions (p<0.05) in calcium release were observed for both C57 BL/6 J IgG1 and IgG2 b at 15 minute s of incubation, as well as for all C57 BL/6 J and NOD fractions (especially NOD IgG1) at 24 hours with an addition of 2 Ig for both time treatments, compared to carbachol -stimulated HSG cells alone. The in vivo preliminary findings suggest that both NOD IgG1 and NOD IgG2c fractions can cause secretory dysfunction This may be due to the bindi ng of these fractions to M 3R, thereby blocking the signaling pathway that normally controls saliva secretion. Conversely C57 BL/6 J IgG1 and IgG2c fractions seemed to cause a stimulatory effect to increase saliva secretion. NOD IgG2b fractions did not appear to inhibit sali vary flo w The in vitro experiments demonstrate that both C57 BL/ 6 J and NOD purified fractions cause a decrease in calcium release in HSG cells stimulated with carbachol, suggesting intracellular calcium release can be inhibited by chronic incubation (24 hours) wi th both C57 BL/6 J and NOD purified IgG isotypes. Also, it cause a similarly decreasing trend compared to using the same volume (2 ). Overall, these results suggest that NOD IgG1 and IgG2c might have inhibitory effects on secretory function in vivo Experimental optimization is needed to investigate if IgG1 is responsible for a decrease in carbachol induced intracellular calcium release, in vitro after 24 -hour chr onic incubation.

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11 CHAPTER 1 INTRODUCTION Sjgrens s yndrome (SS) was first described in 1933 by the Swedish ophthalmologist Henrik Sjgren as an autoimmune disorder that targets the exocrine tissues of the body, especially the lacrimal and salivary glands Other targets of the disease include symptoms localized in the cardiovascular, central nervous system, renal systems, lungs, and gastrointestinal tract (1). Two main symptoms that characterize the disorder are xerostomia (dry mouth) and xerophthalmia (dry eyes) Saliva secretion i s an important process within the body bec ause it is induced during food consumption and is vital in normal dietary function and oral health Saliva is first produced by both ductal and acinar cells. Acinar cells are divided into two types: mucus and ser ous cells (2) Salivary secretion is mainly controlled by the parasympathetic and sympathetic nervous systems. The sympathetic nerve is mainly responsible for protein secretion includ ing exocytosis found in acinar cells. Conversely the parasympathetic nerve is held responsible for water and electrolyte secretion (2). It has been shown that alterations in parasymp athetic neurotransmission can lead to problems within these secretory glands (3). With that understanding, it ca n be suggested that modifications of these cellular pathways can lead to dysfunction in saliva secretion. Primary and Secondary Sjgren Syndrome Sjgrens s yndrome is an autoimmune disease that displays both characteristics of kertoconjunctivitis sicca and xerostomia classified as primary and secondary. Primary SS (pSS) exhibits aforementioned traits without underlying diseases while secondary SS (sSS) di splays them in conjunction with another autoi mmune disease such as type 1 diabetes (T1D), systemic lup us erythematosus (SLE), rheumatoid arthritis (RA) and scleroderma (3).

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12 Nutritional Aspects Being that SS is an oral disorder, nutrient intake before and after disease onset has been observed. Normally, with decreased salivation, many oral problems arise such as candidiasis, dental caries, as well as difficulty in swallowing and speaking In women with primary SS, it has been reported that increased ingestion of lactose, thiamin, and riboflavin occurs due to higher milk intake (4). It has been found that milk is a good substi tute to help balance out low saliva secretion in patients with xerostomia (4). Along with nutrient intake studies, other b iological therapy methods have been applie d to dampen autoimmune responses in SS. Such methods include investigating the effects of B -cell targeted therapies such as infleximab, rituximab, epratuzumab, or belimumab (5). Autoantibodies and Muscarinic Receptors Detected in SS Anti -Ro/SSA and Anti -La/SSB Autoantibodies Not only are SS autoantibodies directed against IgG (rheumatoid factor), they are also directed agai nst Ro/SSA and La/SSB antigens. As part of the classification criteria proposed by the American -European consensus group, anti -Ro and anti -La autoantibodies are important disease markers for SS (6). These autoantibodies were first discovered in 1975 where 3 different types were found within sera from SS patients. Since their discovery, they have been so named anti Ro/SSA and anti -La/SSB (found only in patients with primary SS), as well as SS C which was later found to be RA nuclear antigen, a nuc lear antigen that was detected in cells associated with the Epstein Barr virus. Anti Ro and Anti -La autoantibodies are associated with numerous clinical symptoms such as h ypergammaglobulinemia, parotid swelling, severe salivary gland dysfunction, rheumatoid factor, as well as lymphopenia (7). These autoantigens are composed of a number of antigenic prot eins that are coupled with small RNA molecules. The protein makeup of these particular antibodies is composed of four

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13 small RNAs (hY1, hY2, hY3, and hY5). These RNAs are associa ted with 60kD Ro/SSA or 48kD La SSB (7) Antibodies against Ro 60 kD occur in 5090% of patients with SS, where their function is to bind to defective, small RNAs which eventually lead to their degradation (7), (8). Even though anti Ro antibodies are composed of two subunits, Ro52 kD is a structurally distinct protein whose p hysical interaction with Ro 60 kD has still not been shown (9) It is known that patients with SS and SLE have a surprisingly increased a mount of 52 kD Ro/SSA in PBMCs (10) The La/SSB protein is found at a much higher concentration than both Ro/SSA proteins about 50 fold higher in the cell (11) It is these RNA -protein molecules that can be found within all human cells as well as many other species of animals (12) Even though anti Ro/SSA antibodies are not specific markers for SS, they are found in 60% patients. Anti -La/S SB antibodies, conversely, are found in about 40% of patients with SS, where the only other disease that they are shown to be present in SLE (12) Role of Autoantibodies in the Pathogenesis of Sjgrens Syndrome In a study by Bacman et al. (13) IgG in patient sera was shown to bind and affect muscarinic receptors (MR) of rat parotid glands. Non-competitively, the antibodies were shown to have an inhibitory effect on the MR through decreasing the binding capabilities of 3H quinuclidinyl benzilate (MR agonist), which also led to other alterations, such as the decrease in cAMP. This study suggested that patient IgG binds to MR and competes for the MR agonist. The role of Ig in SS was further investigated by using t null mouse, a B cell deficient mous e model. P u rified IgG obtained from SS patients or SS -prone NOD/ShiLtJ mice has been null mice following intravenous infusions. Such changes do not occur if i nfused with serum IgG from healthy controls.

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14 Anti -Muscarinic 3 Receptor Autoantibodies with Emphasis on IgG1 Isotype Muscarinic acetylcholine type 3 r eceptors (M3R) are responsible for the neurological transmission of messages from extracellular to intra cellular space s a process that plays an integral part in the normal saliva pathway found in salivary and lacrimal glands. These receptors are found in both exocrine and non-exocrine tissues especially in acinar cells. It is understood that autoantibodi es produced against M3R are responsible for disabling the normal saliva pathway and inhibits excitatory enteric neurotransmission (14 ), (15) In addition, m any studies have suggested that M3R is the subtype that is responsible for smooth musc le dysfunction, strongly suggesting that it is the antibodys interaction with the receptor itself that causes these problems (16) In a study by Cha et al (17) responses from smooth bladder muscles stimulated with carb achol ( a M 3 R agonist) were measured after incubation from anti -M3R positive and anti M3R negative autoantibody containing sera from NOD/ Shi LtJ mice or human patients that exhibited pSS. It was determined that a lower carbachol response was observed in bla dder smooth muscle strips that were in an environment containing anti -M3R autoantibodies, compared to smooth muscle strips taken from anti -M3R autoantibody negative NOD/ Shi LtJ and C57BL/6 J control mice. It was also observed that chronic stimulation to M3R from anti M3R autoantibodies resulted in receptor desensitization. S era of patients with SS have shown an increase of a variety of autoantibodies. To better justify that the M3R serves as a primary site for SS, a study was done by Nakamura et a l. (18) In their study, a group of type 1 (M1R) and type 3 (M3R) receptor knockout mice were given dry food to ingest. After being fed, it was evident that the M3 R receptor knockout mice showed signs of difficulty ingesting food, due to the decreased salivation in their mouths (18)

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15 Therefor e, autoantibodies functioning as an antagonist for M3R pathway would certainly play a key role in secretory function in autoimmunity SS (15) Several attempts have been used to define an autoimmune disease, one of which is by Dawson et al (16) in which 5 areas of interest are vital in defi ning a particular antibody as pathogenic i n SS In turn, these criteria can be use d in better defining our study with SS particularly focusin g on anti M3R autoantibodies (16) (19) The first, Autoantibodies are presen t in patients with the disease (16) suggests that there are serum IgG antibodies found in primary and secondary SS patients that are capable of influencing the functionality of muscarinic receptors on smooth muscle of bladder a nd salivary acinar cells. D ue to ineffective and inefficient screening methods for these autoantibodies, further investigations must examine anti M3R in SS. The second criteria, Antibo dy reacts with the target antigen (16) explains the interaction where neurotransmission takes place. For SS, certain epitopes must be carefully examined to determine the exact location where reaction takes place. Previous studies sugge st that the 2nd extracellular loop of M3R is the target ed epitope but due to incomplete data, further in vestigation must be continued. The third criteria, Passive transfer of antibody reproduces features of disease (16) is notably the most important of the 5 criteria because the effects of the antibody to bring an onset of disease characteristics is vital to understanding disease pathology. Collected data from SS IgG transfer to mice have shown promising re sults with up regulation of M3R in bladder smooth muscles. The fourth criteria, Immunization with an tigen produces a model disease (16), is an important aspect that is still being considered. Because the target antigen of interest is still being investigated, this data is crucial in our study. Lastly, the fifth criteria, Reduction in antibody

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16 levels ameliorates the disease (16) observes the decrease in pathogenicity of disease after antibo dy levels have been diminished. It was observed that anti -muscarinic receptor antibody activity was reduced in vitro by IgG anti idio typic antibodies pooled from healthy patients leading to an improvement of bladder symptoms. Recently, considerable att ention has focused on the IgG1 subclass of IgG autoantib odies. In a study by Gao et al (15) experime nts using serum from SS patients confirmed the presence of anti -M3R autoantibodies, with results suggesting the presence of IgG1 autoantibodies These antibodies are thought to identify a tertiary epitope detected on the extracellular domains of the recep tor protein. High serum levels of IgG1 are usually found within SS patients, whereas IgG levels of other types such as IgG2, are found to be in lower concentrations (20) But this may only reflect the normal serum levels of these IgG isotypes in humans. Besides from looking at the effects of IgG1 antibodies, increased levels of IL 18 were found to be within acinar cells (20) This study proposes to address these criteria in hopes of gaining a better understanding of these autoantibodies and their effects. Mouse Models In order to monitor the effect s of auto antibodies in SS an in vivo study was performed in which t hree different mouse strains were utilized to observe and measure the effects of the proposed autoantibodies. C57BL/ 6J mice (B6), due to their non-SS disease phenotype, were used as the control mice. Their ability to resist autoimmune diabetes and exocrinopathy made them an ideal model for the control model (21) The NOD/ShiLtJ (NOD) mouse served as the experimental mouse exhibiting both T1D and SS. Besides from the aforementioned trait, the NOD mouse is prone to infiltration of mononuclear cells in the lacrimal and salivary gland, as well as symptoms such as infl ammation of the pancreatic islets of Langerhans (21) Lastly, the

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17 immunodeficient B6.129S7 Rag1tm1Mom/J (Rag1KO) mouse served as the recipient mouse due to its lack of B and T cells required for autoimmunity (22) The ages for the mouse models used were specific in range so that SS -like disease characteristics were developed and present in sera. For example, previous studies have shown the NOD mouse, which served as the experimental condition, displays disease phenotypes in its sera and saliva reduction betw een the ages of 17 20 weeks of age (23) This age range was used to help determine what age range was needed to gather sera from both B6 (control) and NOD (experimental) mo use models for immunogloblulin purification. Also, in a study by Gao et al (24) loss of sali vary secretion was still evident in mice up to 36 weeks of age. Lastly, it was suggested that there are 3 phase developments that were found in the NOD -Aec1Aec2 mouse model (Figure 1 1 (25) ) that we could correlate with our mice This presented an age limit as to the length of antibody effects still present in the Rag1KO recipient mice after 36 weeks of age. Defective Secretory Function in SS Ther e are many hypotheses as to how cellular functions are affected by SS. One of the main hypotheses is that autoantibodies that bind to M3R will cause an inhibition in the downstream control of fluid secretion. This can be invoked by an alteration in the normal calcium concentration release found within cells. As stated in Dawson et al (26) IgG antibodies extracted from SS patients reduced the effects of carbachol (muscarinic agonist and calcium inducing agent) within both mouse and human acinar cells In normal patients, when parasympathetic neurotransmitters bind to the M3R, a signaling cascade is stimulated that results in the re lease of intracellular calcium. This release provokes cellular functions to occur such as the activation of chloride channels in salivary glands for fluid secretion or smooth muscle contraction in the bladder (17) It can be assumed that the interactions between anti -M3R autoantibodies and the receptor will lead to a disruption in the

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18 normal intracellular signaling pathway that can alter calcium release, and thus disrupt normal salivary flow (17) Specific Aims In order to investigate the roles of IgG1 immu noglobulins in secretory function in SS, two specific aims were proposed and studied: Specific Aim #1 Objective : To determine, in vivo, if decreased fluid secretion i n Sjgrens s yndrome is dependent on isotypic autoantibodies, especially IgG1 anti -mouse M3 R autoantibodies. Hypothesis : I nfusion of the B and T -cell deficient Rag 1KO mice with purified NOD IgG1 targeting M3R will be sufficient to induce loss of secretion in the recipient mice. Rationale : To examine the effects of SS antibodies on Rag1KO mice, they were injected with varying isotypic mouse Ig fractions, where saliva secretion was collected and measured over a 5 -day period to determine their effect. Specific Aim #2 Objective : To investigate in vitro, if the inhibitory effect of IgG1 anti -mouse M3R autoantibodies on M 3 R can be observed in a cell culture system Hypothesis : Specific Aim #2 Hypothesis The binding of IgG1 anti -mouse M3R autoantibodies to the receptor is necessary to alter M3R mediated intracellular calcium release in vitro. Ratio nale : T o examine this, varying isotypic mouse Ig fractions were incubated with human salivary gland cells in order to induce inhibitory ef fects by monitoring intracellular calcium release in response to carbachol (muscarinic agonist).

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19 Figure 1 1. Descrip tion of the three phases of S S like disease development in the NOD Aec1Aec2 mouse model ( Modified from Lee et al. (25) ).

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20 CHAPTER 2 MATERIALS AND M ETHODS Specific Aim #1 Objective (In vivo) To determine if decrease d fluid secretion in Sjgrens s yndrome is dependent on isotypic autoantibodies, especially IgG1 anti -mouse M3R autoantibodies. Hypothesis Infusion of the B and T -cell deficient Rag 1KO m ice with purified NOD IgG1 targeting M3R will be sufficient to induce loss of secretion in the recipient mice. Rationale To examine the effects of SS antibodies on Rag1KO mice, they were injected with varying isotypic mouse Ig fractions, where saliva secretion was examined over a 5 day period to determine their effect. Mouse Model In order to carry out the in vivo experiment, three mouse strains were needed that were purchased from Jackson Laboratory. The first two mouse strains served as donor mice for sera to purify IgG subclass fractions. The first strain used as the control was the C57BL/6J (B6 Jackson Laboratory, Stock Number: 000664). This mouse served as a control mouse due to the fact that it is a widely used general purpose inbred strain. B6 m ice are used in many areas of research such as cardiovascular biology, diabetes, genetics, and immunology. Overall, B6 mice breed well, are long lived, and have a low susceptibility to tumors. The second mouse strain used as the experimental mouse was th e non -obese diabetic NOD/ShiLtJ (NODJackson Laboratory, Stock Number: 001976) m ouse. This is an appropriate mouse strain for our study due to its polyg enic model for T1D a nd a characteristic for SS. Because the NOD parental strain

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21 displays full disease p henotype characteristics around 17 weeks of age, sera were used from 1620 weeks of age from both NOD (experimental) and B6 (control) strains. The third strain, B6.129S7Rag1tm1Mom/J (Rag1KO Jackson Laboratory, Stock Number: 002216), served as the recipi ent mouse for the purified IgG fractions because of its immunodeficient background, where there is no production of mature B and T cells. The Rag1KO mice used in this study were obtained from the Cancer Genetics Research Complex (CGRC Gainesville, Florid a) and Dr. Minoru Satohs laboratory. Saliva was collected and measu red using mice between 1628 weeks of age. All studies were approved by the Institute of Animal Care and Use Committee (IACUC) at the University of Florida. Serum C ollection Before begin ning purification, blood was collected from both donor mouse strains (B6 control mice and NOD experimental mice) betwe en the ages of 16 20 weeks (approximately n=20 25/strain). Blood was collected from each mouse through orbital lobe excavation, where approximately 500 /mouse was collected T he blood was centrifuged for 10 minutes, at 4, 500 rpm and 4C to obtain sera in the supernatant. The serum from both strains was then pooled together to a volume of approximately 4.5 mL /strain that were then used for purification Dialysis and Antibody Fraction Column Preparation for Purification In order to obtain isotypic antibody fractions from the collected serum, a series of purification columns were assembled. To begin, dialysis was performed in order to create purification columns for the antibodies. Goat anti mouse IgG1 ( Southern Biotechnology, Birmingham, Alabama #107001) IgG2b ( #109001) IgG2c ( #107901) and IgG3 ( #110001) unlabel ed 1 mg antibodies were purchased and filled into dialysis tubes (Fisher Brand, No minal MWCO 12,00014,000 #2115216) Tubes were dialyzed in 0.1M NaHCO3 with 0.5M NaCl coupling buffer for a period of 3 4 days, where the b uffer was changed twice a day. Afterwards,

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22 the contents of the dialysis tubes were transferred to centrifuge tube s and centrifuged at 4C for 15 minutes at 12,000 rpm. The supernatants opt ical density (OD) was checked by spectrophotometer (Beckman DU 64 Spectrophotometer ) to verify the amount of IgG used and confirm the efficiency of conjugation. IgG concentration was estimated based on the conversion ratio of 1.42 OD280 = 1 mg/ mL IgG. About 1 g of CNBR activated Sepharose 4B beads (GE Healthcare, Label No. 71 118100ED, Lot. 311319) was mixed with 25mL of 103 M HCl and packed into 4, 10 mL Poly Prep Chromatography Columns (Bio Rad, Catalog 7311550). Once the beads began packing, HCl was continually added to prevent dehydration from occurring. Once 0.5 mL of the column was packed with beads and approximately 30 mL of HCl had run through, the column was washed o nce more but with 30 mL of coupling buffer. After the coupling buffer ran through, the supernatant of the antibody centrifuge tubes were added into each respective column, mixed gently with a pipette, and rotated overnight at 4C to mix/homogenize with t he beads. The next day, the coupling buffer in the c olumns was drained and the OD of the elu a te was measured by spectrophotometer to verify effective conjugation of IgG to the Sepharose beads If the OD was very low, that meant that the protein bound to the beads, which was desired. The columns were then washed again with 30 mL of coupling buffer and drained. Next, 5 mL of 1.0 M ethanolamine hydrochloride was used as a blocking buffer and mixed and rotated for 1 hour at room temperature with the column beads. Once completed, the column was drained and a series of three wash cycles occurred. To start, 10 mL of phosphate -buffered s aline, 1x, pH=7.4 (PBS Cellgro, Cat.No. 21 040CV) was washed through thoroughly to avoid any impurities found in the beads and columns. Next, the columns were washed with 10 mL of 0.5 M glycine elution (pH=2.5) buffer to detach any unwanted proteins from the beads. After the 3 wash cycles were

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23 completed using PBS and glycine, the columns were restored back to neutral conditi ons by running 5 mL of PBS through the beads with 2 mL left on top. This completed the column preparation and was ready for sera pass through. Purification of Whole IgG Before isotypic fractions were collected, total IgG was purified from the serum of bo th B6 and NOD mice In order to do so, a separate column was created for each mouse strain. Each column was packed with approximately 1 mL of Protein G Agarose (Sigma, Fast Flow from Streptococcus species P46915 mL ) beads Next, 10 mL of glycine buffer was washed through in order to eliminate any unwanted impurities. Lastly, neutral pH was restored by washing 10 mL of 1x PBS through the column. The serum was centrifuged for 15 minutes a t 12,000 rpm at 4C The supernatant was then run through the col umn for protein attachment. Once all sera was run through, approximately 10 mL of 1x PBS was passed through and collected as earlier flow through to clean the beads and return them back to their original white color. Columns were washed until the OD of the flow through returned back to the background level. IgG was eluted from the beads using 5 mL of glycine buffer To prevent the loss of antibody activity due to the acidic environment, 140 of 2 M Tris pH ~ 11 was added to every 1 mL of eluate immediately in order to neutralize the solution. The concentration of purified whole IgG was then measured using spectrophotometry as above. Purification of Isotypic Immunoglobulins: IgG1, IgG2b, I gG2c, and IgG3 After draining PBS from the antibody fraction purification columns, the total IgG from the previous step was ready to be passed though each of the 4 columns in the order of: IgG2c IgG1 IgG2b IgG3. The total IgG was run through one column at a time, collecting its pass through int o a tube, which was then transferred to the next column. After the contents of the transfer tube had run through each column, 2 mL of PBS was run through each purification

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24 column to wash out any residual from the whole IgG serum and again collected in the transfer tube. Doing so allowed the antibodies to bind to each respectiv e purification column. Glycine buffer pH 2.5 was then run through each column to elute bound antibodies from the beads so that approxi mately 4 mL of isotypic fractions were collected. Because of the acidic environment of glycine buffer pH 2.5 140 of 2 M Tris pH ~ 11 was also added per milliliter of elut e to neutralize This Glycine/Tris buffer became the environment that the Ig fractions were stored in, which would later on be used as one of the control treatments. Lastly, the purification col umns were neutralized and restored back to original conditions by washing and draining each column with 10 mL of PBS. Measurement of Concentration of Purified Ig Isotypes by ELISA In order to quantify and determine the concentration for each purified anti body fraction, a sand wich ELISA was performed. Wells of 96 well microtiter plates (Nunc, Immobillizer Amino) were incubated for 2 hours at 22C with 50 /well of a mixture co mposed of goat anti mouse kappa (Southern Biotechnology #105001, 1 mg/ mL ) and goat anti -mouse lambda (Southern Biotechnology #10601, 1 mg/ mL mL After incubation, both plates were washed once with TBS Tween 20, after which they were blocked with 150 /well of 0.5% BSA buffer (50 mM Tris HCl pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.3% Nonidet P 40 (NET/NP40)) for 3 0 minutes at room temperature. Total IgG was diluted 1:10,000 and the purified fractions were diluted to 1:1 000, both in 0.5% BSA NET/NP40. Serial d ilutions of the myeloma proteins (IgG1, IgG2b, IgG2c, IgG3, and IgM) were made from mL stock and served as a standard to create a standard curve to help calculate IgG concentration of samples. After th e incubation period passed, the 0.5% BSA solution was discarded. Lastly, the serial dilutions were added 100 /well and stored overnight at 4C.

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25 The next day, the 2 antibody mixtures were created using alkal ine phosphatase conjugated goat anti -mouse I gG1, IgG2b, IgG2c, and IgG3 ( Southern Biotechnology, Birmingham, Alabama) at a 1:1 000 dilution with 0.5% BSA NET/NP40 After the plates were washed three times with TBS Tween 20, the 2 antibody mixtures were added 100 /well and incubated for 2 hours i n room temperature, after which they were washed three times again with TBS Tween 20. The assay was developed using 100 /well diethanolamine/phosphatase substrate buffer (Sigma -Aldrich St. Louis, Missouri ) and absorbance was determined at 405 nm using a VERSAmax ELISA plate reader (Molecular Devices Sunnyvale, California ). Injection Strategy The most obvious characteristic of the disease that can be detected, measured, and confirmed is through the measurement of saliva secretion f rom the injected mice Rag1KO mice 1628 weeks old were used to infuse with different isotypic antibody fract ions In order to accomplish this, 4 different categories of Rag1KO mice were injected with respective solutions or no solution: B6 fractions, NOD fractions, Glycine/ Tris Buffer, and Non -Injected (Figure 2 1). The experimental groups used for this study were mice injected with fractions from B6 and NOD, while the control groups consisted of mice that were injected with a Glycine/Tris buffer and mice not injected with anything. In order to determine how much was injected into each mouse, the ELISA results (Table 3 1 and Table 3 2 ) were analyzed. Because we wanted to recreate a real life in vivo situation, the serum data from the ELISA s was used to calculate a ratio, which was then used to determine the appropriate volumes of antibody fraction to inject into each recipient mouse (Table 2 1 and Table 2 2 ). After determining the injection amounts, a 5 -day experimental plan was prepared (Figure 2 2). Only on the first day were the Rag1KO mice injected with purified Ig fractions. On this day, each Rag1KO mouse was injected through intraperitoneal injection (IP) with each

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26 purified fraction (i e ., mouse 1=B6 IgG1, mouse 2= B6 IgG2b, mouse 3=B6 IgG2c, mouse 4= NOD IgG1, etc ). A three hour period was observed so that each fraction had time to incubate within each mouse. After the incubation period had passed, saliva collection was performed over a 10 minute span. Saliva Collection Saliva secretion was stimulated by injec ting each mouse again through IP with a secretagogue mixture composed of 1 mg DL I soproteronol Hydrochloride (MP Biomedicals Cat. No. 151368, Lot No. 9192C) and 2 m g P ilocarpine Hydrochloride (MP Biomedicals, Cat. No. 151892, Lot No. 4218F) mixed in 1 mL of 1x PBS solution. One minute after secretagogue injection, saliva was collected for a period of 10 minutes through the oral cavity using a 200 pipette. The Ig injections and saliva collection were measured and observed over a 5 day period. Afterwa rds, each saliva volume was measured and normalized by dividing each saliva volume collected by the w eight of the respective mouse. S tatistical analysis was performed once all measurements had been taken using both a t test (nonparametric) and one -way AN OVA (non parametric) with Tukey Test to compare all paired values. Specific Aim #2 Objective (In vitro) To investigate if the inhibitory effect of IgG1 anti -mouse M3R autoantibodies on M3R can be observed in an in vitro cell culture system Hypothesis T he binding of IgG1 anti -mouse M3R autoantibodies to the receptor is necessary to alter M3R mediated intracellular calcium release in vitro.

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27 Rationale To examine this, varying isotypic mouse Ig fractions were incubated with human salivary gland cells in order to induce inhibitory effects by monitoring intracellular calcium release in response to carbachol (muscarinic agonist). Cell Culture In order to monitor intracellular calcium release within a cell, human salivary gland (HSG) cells were incubated with different IgG isotypes of SS and monitored through a fluorescence reader. HSG cells were grown in 12 mL culture plates at 37C with 1x DMEM (Dulbeccos Modification of Eagles Medium) as a medium. This medium consisted of 50 mL of heat inactivated 10% Fe tal Bovine Serum (Cell Gro, Catalog Number: 35 011CV) and 5 mL antibiotics Penicillin/Streptomycin Solution (Cell Gro, Catolog Number 3 0 -002CI). Calcium Assay After the culture was created and reached 80 90% confluency, it was re -seeded and transferred t o 3, 96 well plates (Nunc, Catalog Number: 161093), where 36 wells were utilized each plate. They were then incubated at 37C with both control and experimental isotypic autoantibodies for a period of 15 minutes (short term stimulation ) and 24 hours (chro nic stimulation ) as the experimental treatments Under each time point, 2 different treatments were applied: one plate using the same volume of 2 of each respective antibody for incubation, and one plate using the same amount of 0. (Table 2 3 and Table 2 4) Also, in order to ensure that any alterations in calcium release were due to the fractions t hemselves, other treatments, used as controls, were monitored for calcium changes that consisted of: blank, dye only, cells an d dye only (Negative Control), c arbachol (CCh) and cells (Experimental Positive Control), and i onomycin (I ono) and cells only well s (Assay Control). Once incubated with Ig fractions a Fluo 4 NW Calcium Assay Kit (Invitrogen, Lot Number:

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28 537564) was utilized in order to measure calcium release. The protocol provided by the manufacturer was followed and calcium release was measured using a fluorescence reader (Molecular Devices Spectramax M5) set at 494 nm for exc itation and 516 nm for emission. In order to stimulate calcium release, 10 of 100 M carbamoylcholine c hloride (CCh) (Sigma -Aldrich, Catalog Number: C43821G) an exper imental control and muscarinic receptor agonist, was injected into both control and experimental groups to begin calcium release. In order to ensure the assay was properly working, 1 of 5 M i onomycin (Iono) Calcium S alt (Sigma -Aldrich, Catalog Number: I3909) was used, acting as another calcium inducing agent and serving as an assay control. Once injected with both inducing agents calcium release was instantaneous; therefore fluorescence was read immediately after injection. Statistical analysis was utilized once all measurements had been taken using a one -way ANOVA (non -parametric) with Tukey Test to compare all paired values.

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29 Table 2 1. B6 Ig fractions table that displays ratios used to calculate volume injected into each Rag1KO mouse in vivo B6 Ig Fractions IgG1 IgG2b IgG2c B6 Serum Ratio 1.00 0 0.500 0 0.125 0 Amount Ratio 40.0 0 g 20.0 0 g 5.00 0 g Volume Injected into each Rag1KO mouse 266. 7 l 303. 9 l 337.6 l Table 2 2. NOD Ig fractions table that displays ratios used to calculate volume injected into each Rag1KO mouse in vivo NOD Ig Fractions IgG1 IgG2b IgG2c NOD Serum Ratio 1.0 00 0.92 00 0.15 00 Amount Ratio 40.0 0 g 36.8 0 g 6.00 0 g Volume Injected into each Rag1KO mouse 250.0 l 334.5 l 193.5 l Table 2 3. B6 Ig fractions table that displays injection amounts and volumes that were used in each 15 minute and 24 hour incubation experiment in vitro B6 Ig Fractions IgG1 IgG2b IgG2c Amount Injected in vitro per well (constant volume 2 l) 0.301 0 g 0.132 0 g 0.0296 0 g Volume Inject ed in vitro per well mouse (constant amount 0.2 g) 1.33 0 l 3.03 0 l 13.5 0 l Table 2 4. NOD Ig fractions table that displays injection amounts and volumes that were used in each 15 minute and 24 hour incubation experiment in vitro NOD Ig Fractions Ig G1 IgG2b IgG2c Amount Injected in vitro per well (constant volume 2 l) 0.317 0 g 0.224 0 g 0.0627 0 g Volume Injected in vitro per well mouse (constant amount 0.2 g) 1.26 0 l 1.79 0 l 6.38 0 l

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30 Figure 2 1. In vivo experiment with Rag1KO mice under four different treatment conditions: 1) Non -Injected Rag1KO mice (Control), 2) Rag1KO mice injected with Glycine/Tris buffer (Control), 3) Rag1KO mice injected with differ ent B6 purified Ig fractions (Control), 4) Rag1KO mice injected with different NOD purified Ig fractions (Experimental) Figure 2 2. In vivo experimental timeline over a 5 day period.

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31 CHAPTER 3 RESULTS Specific Aim #1 Analysis of Purified Fractions Measured Concentrations of Ig Isotypes with IgG1 Being the Highest In order to det ermine the amount of each IgG isotype needed to inject into each mouse in vivo, an ELISA was performed using the B6 and NOD purified Ig fractions. Analysis of each purified fraction revealed the concentration of each Ig fraction derived from B6 and NOD (T able 3 1 and Table 3 2 ). Using the concentration data the amount of each Ig fraction to be injected into Rag1KO mouse was calculated T here was high reactivity (marked by asterisks) found in each purified fraction. From the B6 ELISA, the results were a mL mL mL and IgG3 = <0.002. From the NOD ELISA, the results were as follows: IgG1 mL IgG2b mL ,, IgG2c mL and IgG3 = <0.002 mL The seru m data from Tabl es 3 1 and 3 2 were used to create a ratio that was used in conjunction with the above values to determine how much Ig would be injected per mouse (Table 2 1 and Table 2 2). For example, the serum values in Table 3 1 were used to determine a ratio of 1.00 : 0.500: 0.1250 for B6 Ig fractions. Next, this ratio was converted according ly using the starting amount of 40 This amount ratio was divided by the above concentrations (Table 3 1) to obtain the B6 Ig volumes that were injected into each Rag1KO mouse. NOD IgG1 and IgG2c Inhibit Secretory Dysfunction in Immunodefecient Mice In Vivo In order to gain a better un derstanding of the functional relationship of IgG1 and other autoantibody fractions t o SS an in vivo experiment was performed. To do so, 24 Rag1KO mice were injected, respectively, with purified fractions of mouse IgG1, IgG2b, and IgG2c (Table 2 1 and Ta ble 2 2 ) This was performed in order to gather data to test our hypothesis that NOD IgG1

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32 anti -mouse M3R autoantibodies causes saliva flow dysfunction. Note that IgG3 fractions from both B6 and NOD were not injected into mice due to their low concentrati ons defined by ELISA, probably due in part to the cryoglobul i n a emic characteristics of IgG3, which could have be en removed as precipitate during centrifugation at 4C After injection s of the appropriate isotypes saliva was collected through the oral cav ity and saliva volume was measured and data analyzed as presented in Figure 3 1 Figure 3 3 As shown i n Figure 3 1 NOD IgG1 purified fractions caused increased stimul ation on Day 2, but due to possible receptor desen si tization, there was a decrease of saliva secretion over time due to IgG1. Mice injected with B6 IgG1 pur ified fractions showed an initial increase in saliva secretion on Day 2, foll owed by a decrease until Day 4. By Day 5, signs of increase d saliva secretion occurred with mice treated w ith B6 IgG1 fractions. These data s upport the hypothesis that NOD IgG1 as well as NOD IgG2c autoantibodies are effective suppressors of saliva flow. In Figure 3 2 both B6 and NOD IgG2b fractions caused a stimulatory effect and showed max values on Day 2, but then B6 IgG2b fractions caused a rapid decrease in saliva volume though Day 5. NOD IgG2b fractions on the other hand, also caused a decrease, but at a much slower salivary flow rate (flat line) c ompared to B6. As presented i n Figure 3 3 B6 IgG2c fractions caused a stimulatory effect that peaked on Day 2, but slowly decreased through Day 5. NOD IgG2c fractions showed an increase peaking on Day 3 followed by a decrease in saliva secretion through Day 5. Lastly, both control groups, Glycine/Tris treated mice and non injected mice, showed very similar patterns and were used as a comparison in all three graphs over the course of the 5 days. Both groups showed decrease s in saliva flow rates from Day 2 Day 4, but an increase on Day 5 for the noninje cted mice, while Glycine/Tris -treated mice continued to show a decrease.

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33 Specific Aim #2 : Altered Intracellular Calcium Release in HSG Cells in the Presence of Ig Isotypes A Fluo 4 NW Calcium Assay Kit (Invitrogen Carlsbad, California ) was utilized in o rder to monitor the alterations in calcium release (stimulated by carbachol ( CCh) ) through fluorescence readings with the incubation of B6 (control) and NOD (experim ental) purified Ig fractions. This was performed to obtain data to test our hypothesis tha t IgG1 anti -mouse M3R autoantibodies would cause an alteration in the M3R mediated intracell ular calcium release in vitro In order to gain a better understanding of the effects of the a ntibodi es measurements were taken after two purified Ig incubation t imes of 15 minutes (short term stimulation) and 24 ho urs (chronic stimulation) Under each incubation time point, 2 different treatments were applied (as defined in Table 2 3 and Table 2 4) : one plate using the same volume of 2 of each respective anti body for incubation. Carbachol known to be a MR ago nist and calcium inducing agent, was added to HSG cells that contained purified Ig fractions (experimental conditio n), as wel l as our control set up of CChresponsive cells to stimulate calcium release. To ensure that our calc ium assay kit worked properly, i onomycin ( a calcium ionophore ) was utilized as an assay control to ensure that intracellular calcium was released In all of our t reatment studies in vitro, our i onomycin fluorescence readings ranged between 400 500 nm, which was a good indication that our assay properly worked and remained consistent throughout. Two of each respective B6 and NOD Ig fraction was incubated with HSG cells for 15 minutes to monitor the effects under a short -term stimulatory environment As shown in Figure 3 4, there was a significant difference (p<0.05) between both our negative and p ositive control s Next, it was observed that all fractions, both B6 and NOD caused an overall decrease in ca lcium

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34 release compared to CCh responsive cells It was interesting that both B6 IgG1 and B6 IgG2b fractions caused a significant decrease (p<0.05 ) in calcium release compared to this p ositive control response Lastly, it should be noted that NOD IgG1 caused a decrease in calcium release compared to the CCh responsive cells even though it was not significantly different. HSG cells were incubated with 2 of B6 and NOD Ig fractions for 24 hours in order to simulate a chronic environment. As shown in Figure 3 5, both positive and negative controls were significantly different (p<0.0 5) compared to each other. In addition the overall effects of ea ch B6 and NOD Ig fraction caused a decr e ase in calcium release that was significantly differ ent (p<0.05) compa red to the CCh responsive cells Using similar amount s of Ig fractions HSG cells were incubated with the IgG isotypic antibodies for 1 5 minutes as a short term stimulation. It was observed that both negative and positive controls were significantly different from each other (p<0.05). Also, even though they were not significantly different (p>0.05) from the CCh-responsive cell responses both B6 and NOD Ig fractions caused a n observable decrease in calcium release. HSG cells were then raction for a 24 hour period as a chronic stimulation. As shown in Figure 3 7, the data collected indicated a decrease in calcium release in all B6 and NOD Ig fractions (except NOD IgG1 ), even though the data w as not significantly different (p>0.05) compared to the responses CCh -responsive cells

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35 Table. 3 1. B6 Purified Ig Fraction ELISA results B6 IgG1 (g/ mL ) IgG2b (g/ mL ) IgG2c (g/ mL ) IgG3 (g/ mL ) Total volume ( mL ) Total Amount (g) IgG1 150.32 1.07 0. 51 <0.002 4.5 676.44 IgG2b <0.03 65.81 0.99 <0.002 4.5 296.145 IgG2c 1.52 3.68 14.81 <0.002 4.5 66.645 IgG3 <0.58 <0.039 <0.013 <0.002 4.5 ---Serum 1637.6 773 204.6 <0.4 1.5 Main r eactivity denoted by asterisks (*). The data marked by asterisks (*) multiplied by the total volume resulted in the total amount of each respective B6 Ig fraction. The serum data was utilized to determine an in vivo ratio that was used to calcula te Ig volumes injected into the Rag1KO mice (Table 2 1). Table. 3 2. NOD P urified Fraction ELISA results NOD IgG1 (g/ mL ) IgG2b (g/ mL ) IgG2c (g/ mL ) IgG3 (g/ mL ) Total volume (mL ) Total Amount (g) IgG1 158.36 1.61 0.53 <0.002 4.5 712.62 IgG2b 1.66 111.75 1.46 <0.002 4.5 502.875 IgG2c 2.03 12.46 31.34 <0.002 4.5 141.03 IgG3 0.03 0.01 0.01 <0.002 4.5 ---Serum 1628.8 1505.8 259.6 <0.002 1.5 Main r eactivity denoted by asterisks (*). The data marked by asterisks (*) multiplied by the total volume resulted in the total amount of each respective NOD Ig fraction. The ser um data was utilized to determine an in vivo ratio that was used to calcula te Ig volumes injected into the Rag1KO mice (Table 2 2).

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36 Combined IgG1 Normalized Day 1 Day 2 Day 3 Day 4 Day 5 0.0 2.5 5.0 7.5 10.0B6 (Control n=3) NOD (Experimental n=3) Non-Injected (n=3) Rag1KO recipient mice injected with purified fractions: Glycine/Tris (n=3) Salivary Flow Rate (ul/g) Figure 3 1 In vivo results of saliva secretion after 3 hour incubation (on Day 1) with IP injected B6 (control) and NOD (experimental) purified IgG1 into Rag1KO mice. Non injected condition and Glycine/Tris (purified Ig storage buffer) were both used as controls conditions. Saliva was collected over a 5 -day period. Statistical analysis was p erformed but data was not significant (p>0.05).

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37 Combined IgG2b Normalized Day 1 Day 2 Day 3 Day 4 Day 5 0.0 2.5 5.0 7.5 10.0Non-Injected (n=3) Glycine/Tris (n=3) NOD (Experimental n=3) Rag1KO recipient miceinjected with purified fractions: B6 (Control n=3) Salivary Flow Rate(ul/g) Figure 3 2 In vivo results of saliva secretion after 3 hour incubation (on Day 1) with IP injected B6 (control) and NOD (experimental) purified IgG2b into Rag1KO mice. Non in jected condition and Glycine/Tris (purified Ig storage buffer) were both used as controls conditions. Saliva was collected over a 5 -day period. Statistical analysis was performed but data was not significant (p>0.05).

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38 Combined IgG2c Normalized Day 1 Day 2 Day 3 Day 4 Day 5 0.0 2.5 5.0 7.5 10.0Non-Injected (n=3) Glycine/Tris (n=3) B6 (Control n=3) Rag1KO recipient mice injected with purified fractions: NOD (Experimental n=3) Salivary Flow Rate (ul/g) Figu re 3 3 In vivo results of saliva secretion after 3 hour incubation (on Day 1) with IP injected B6 (control) and NOD (experimental) purified IgG2c into Rag1KO mice. Non injected condition and Glycine/Tris (purified Ig storage buffer) were both used as con trols conditions. Saliva was collected over a 5 -day period. Statistical analysis was performed but data was not significant (p>0.05).

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39 15 Minute Incubation Cells Only (Neg StimNo stim) B6 IgG1 B6 IgG2b B6 IgG2c NOD IgG1 NOD IgG2b NOD IgG2c CCh + Cells (Exp Pos Control) Iono + Cells (Pos Control) 0 100 200 300 600 **p<0.05*Relative Fluorescence Figure 3 4 In vitro Calcium Assay with 15 minute incubation with 2 of each respective B6 (control) and NOD (experimental) Ig frac tion. Asterisks (*) indicate p< 0.05 compared to CCh + Cells (Positive Control).

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40 24 Hour Incubation Cells Only (Neg StimNo stim) B6 IgG1 B6 IgG2b B6 IgG2c NOD IgG1 NOD IgG2b NOD IgG2c CCh + Cells (Exp Pos Control) Iono + Cells (Assay Control) 0 100 200 300 500 *p<0.05* * *Relative Fluorescence Figure 3 5 In vitro Calcium Assay with 24 hour incubation with 2 of each respective B6 (control) and NOD (experimental) Ig fraction. Asterisks (*) indicate p< 0.05 compared to CCh + Cells (Positive Control).

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41 15 Minute Incubation Cells Only (Neg StimNo stim) B6 IgG1 B6 IgG2b B6 IgG2c NOD IgG1 NOD IgG2b NOD IgG2c CCh + Cells (Exp Pos Control) Iono + Cells (Assay Control) 0 100 200 300 500 *p<0.05*Relative Fluorescence Figure 3 6 In vitro (contr ol) and NOD (experimental) Ig frac tion. Asterisks (*) indicate p< 0.05 compared to CCh + Cells (Positive Control).

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42 24 Hour Incubation Cells Only (Neg StimNo stim) B6 IgG1 B6 IgG2b B6 IgG2c NOD IgG1 NOD IgG2b NOD IgG2c CCh + Cells (Exp Pos Control) Iono + Cells (Assay Control) 0 100 200 300 500 *p<0.05Relative Fluorescence Figure 3 7 In vitro (control) and NOD (experimental) Ig frac tion. Asterisks (*) indicate p< 0.05 compared to CCh + Cells (Positive Control).

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43 CHAPTER 4 DISCUSSION The purpose of this study was to measure and observe the effects of IgG1 sub class autoantibodies in secretory dysfunction in SS in both an in vivo and in vitro system. Different isotypic fractions of IgG1, IgG2b, IgG2c, and IgG3 were purified from sera from both B6 and NOD mice using affinity columns. Once completed, each fraction was analyzed through ELISA in order to de termine the amount and concentration of each fraction for injection or in vitro study For this project, two specific aims were addressed The objective for Specific Aim #1 was to determine if decreased fluid secretion in SS was dependent on isotypic aut oantibodies, especially IgG1 anti -mou se M3R autoantibodies. I t was hypothesized that infusion of the B and T cell deficient Rag1KO mice with purified NOD IgG1 fractions targeting M3R would be sufficient to induce loss of section in the recipient mice. Th e objective for Specific Aim #2 was to investigate and observe the inhibitory effects of IgG1 anti -mouse M3R autoantibodies on M3R in an i n vi tro cell culture system. I t was hypothesized that the binding of IgG1 anti -mouse M3R autoantibodies to the recept or was necessary to alter M3R mediated intracellular calcium release within this system. E fforts at understanding the effects of purified Ig fractions, especially IgG1, identified two important findings pertaining to loss of salivary function in SS. Firs t, it was observed that both purified NOD IgG1 and NOD IgG2c fractions caused secretory dysfunction in vivo Second, preliminary results suggested that both B6 and NOD purified fractions, especially NOD IgG1, caused a decrease in calcium release in HSG ce lls after 24 hour incubation in vitro. Specific Aim #1 For Specific A im # 1, it was proposed that injecting purified SS -prone NOD Ig fractions, not whole IgG, into Rag1KO mice in vivo, would be able to alter secretory flow, especially with

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44 the use of IgG1 fractions This hypothesis was based on the role of muscarinic receptors, more importantly M3R, in their functionality and relationship with IgG1 a utoantibodies. Studies have shown that autoantibodies produced against M 3R are responsible for inhibiting t he normal saliva secretion pathway and interfering excitatory enteric neurotransmission (14) (15) This concept is based on considerable work previously carried out in our laboratory. In a paper published by Robinson et al (27) null mice, with the infusion of human serum whole IgG purified from primary SS patients, resulted in either the loss or a gain in secretory function in the exocrine tissues most likely due to the state of immunity of the individual patient Moreover, the fractions obtained from SS sera disabled the proper binding of muscarini c receptor agonist to the salivary gland membrane. After exploring other studies and observing what they had found in regards to antibodies and S S, there was a general consensus that whole IgG from SS patients was able to produce an inhibitory effect on saliva secretion (13) (26) (27) (28) However it had never been investigated before if the purification of whole IgG into its s pecific constituent parts would also have the same effect or a differing one. In addition, Gao et al. (24) showed that secretory function is maintained when IL 4 is deficient, thus failing to generate IgG1 isotypic Ig, in the NOD genetic background mouse, indicating that IgG1anti -M3R antibody is essential in secretory dysfunction. Therefore, this project provided a new direction with the use of purified Ig fractions. In this study, whole IgG from both B6 (control mice) and NOD (SS like mice) were collected and purified into Ig fractions of IgG1, IgG 2b, IgG2c, and IgG3 and inject ed into recipient mice to monitor their effects. Due to null mouse because of its many SS like characteristic s and lacking functional B cells it was decided to find a similar mouse model in

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45 order to proceed with my study (27) The Rag1KO mice, which served as our recipient mice, provi ded a model for injection because of its immunodeficient ba c kground This was vital to this study because the introduction of a foreign agent into these recipient mice would avoid immunologic responses and generate antibodies. Therefore, this would ensure that the effects observed after injection of purified Ig fractions would originate not from antibodies or the function of T -cells but from injected Ig fractions After injection into the Ra g1KO mice with the differen t purified IgG fractions, it was expected to observe a decrease in salivary flow from the effects of IgG1 fractions But after analyzing the results ( Figure s 3 1 through 3 3 ), it was observed that each fraction appeared to cause varied results. These results were gathered i n order to gain a better understanding of the relationship of IgG1 and other au toantibody fractions t o normal cellular functions. The experimental project prompted for the use of 24 Rag1KO mice to be injected with respective purified mouse sera fractions of: IgG1, IgG2b, IgG2c. This was performed in order to gather data to support our hypothesis that IgG1 anti -mouse M3R autoantibodies c auses saliva flow dysfunction. However, IgG3 fractions from both B6 and NOD were not injected into mice due to the ir low concentrations based on the ELISA, which was resulted from their cryoglobul i n a emic characteristics d uring centrifugation. After injection, saliva was collected through the oral cavity and saliva volume was measured and analyzed Statistical analysis was performed once all measurements had been taken using both a t test (nonparametric) and one -way ANOV A (nonparametric) with Tukey Test to compare all paired values but the data was not significant (p>0.05). NOD IgG1 purified fractions caused increased stimulation on Day 2, but then due to possible receptor desensitization, there was a decrease of saliva secretion over time, thus

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46 supporting our hypothesis. One speculation for this observation is that cells may undergo a compensatory process by up regulating receptors where initially there is a possible overstimulation at M3R on Day 2, but ove r time, the cells downregulate receptors on the cell surface via endocytosis, thus there is a decrease in saliva secretion At this time, it was assume d that reduced saliva secretion may be the consequence of IgG1 affecting M3R The receptor, once blocked by IgG1, can lead to an alteration in the signaling pathway that leads to normal cellular functions. B oth B6 and NOD IgG2b fractions caused a stimulatory effect and showed max values on Day 2, but then B6 IgG2b fractions caused a rapid decrease in saliva volume th ough Day 5. NOD IgG2b fractions, on the other hand, also caused a decrease, but at a much slower salivary flow rate (flat line) compared to B6 on Day 5. This suggests that the effects of NOD IgG2b are less inhibitory than those of NOD IgG1. This can be observed as NOD IgG2b playing a very minimal role in the blocking of t he signaling pathway with M3R. In addition it was observed that B6 IgG2c fractions caused a stimulatory effect that peaked on Day 2, but slowly decreased through Day 5. NOD IgG2c frac tions showed an increase peaking on Day 3 followed by a decrease in saliva secretion through Day 5. This again, like NOD IgG1, displayed the same inhibitory effects that led to the decline in saliva secretion after Day 3. In addition to observing the ef fects of NOD IgG1 and NOD IgG2c, there were unexpected results that need to be further investigated. For instance, when observing the data from B6 (control) fractions, as well as the control conditions of non-i njected mice and Glycine/Tris treated mice, t hey also caused a decr ease in salivary flow. This may be explained by the amount of stress tha t the mice were under for the 5 -day period. The injections, handling, and saliva collection placed the mice in stressful and t iring conditions which can explain the decrease in saliva production in these mice In addition, considering that the Rag1KO mice were derived

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47 from both the CGRC and Dr. Satohs laboratory it is speculated that two different environmental conditions that the mice were housed in may have caused a discrepancy. This may have caused a n unanticipated fluctuation in saliva flow that must be observed in the future. The difference between these effects and those caused by NOD IgG1 and NOD IgG2c is that both NOD IgG1 and NOD IgG2c caused a notic eable stimulation in saliva secretion, either on Day 2 or Day 3, that eventually led to the decrease in saliva secretion, whereas the B6 (control) fractions and both control conditions caused very little initial stimulations or no stimulation at all. Thou gh the preliminary results support in part, our original hypothesis that there is an inhibitory factor present in both NOD IgG1 and NOD IgG2c, further inves tigation must be completed. Specific Aim #2 In addition to my in vivo study, this project also in vestigated the effects of the purified Ig frac tions, more importantly IgG1, on the inhibition of M3R mediated intracellular calcium release in an in vitro system. In a study by Dawson et al (26) it was found that whole IgG antibodies extracted from SS patients reduced the effects of carbachol (muscarinic agonist and calcium inducing agent) within both mouse and human acinar cells. W hen parasympathetic neurotransmitters bi nd to the M3R in normal individuals a sign aling cascade is stimulated and results in the release of intracellular calcium. This release provokes cellular functions to occur such as the activation of chloride chann els in salivary glands for fluid secretion or smooth muscle contraction in bladder (17) M any studies have shown the effects of whole IgG on intr acellular calcium release but similar to Specific Aim #1, little has been studied as to the effects of isotypi c Ig fractions on SS Therefore, this project provided new insight as to how these fractions can alter the normal signaling pathway involving M3R that leads to the release of intracellular calcium. In order to monitor the effects of both B6 (control) and NOD (experimental) Ig fractions in an in vitro

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48 setting, HSG cells were utilized as the target for incubation of each respective antibody This was performed in order to demonstrate the inh ibitory effects of NOD IgG1 on the binding of carbachol to M3R th us leading to a dysfunction in calcium signaling. Both B6 and NOD fractions were incubated under two different time points of 15 minutes (short term stimulation) and 24 hours (chronic stimulation), in which two Ig injection treatments of 2 or performed at each time point Due to the fact that HSG expresses M3R and a mouse cell line expressing M3R was not available, HSG cells were utilized. A NCBI Blast search showed the sequence homolo gy between human and mouse M3R is 86%. H igh sequence homology between both species suggests that the cross reactivity of antibody may occur, which allowed me to utilize HSG cells for calcium release studies. Therefore, HSG cells were incubated with each purified fraction. First, B6 IgG1 and B6 IgG2b caused a significant decrease in calcium release (p<0.05) compared to our CCh responsive cells in Figure 3 4 Also, the comparison between positive (CCh + cells) and negative (No stimulation) controls showed a significant diffe rence (p<0.05). This allowed me to assume that if the positive and negative controls were different, then any significant difference caused by the purified Ig fractions could be observed as acceptable and reliable There was a general decreasing trend in calcium release in al l B6 and NOD fract ions in comparison to the CCh -stimulated cells Second in Figure 3 5, the comparison between positive and negative controls displayed a significant decrease in calcium release (p<0.05 which meant any alterations caused by the Ig fract ions could be accepted as having an inhibitory or stimulatory effect. After analyzing the results for each B6 and NOD fraction, it was observed that each treatment caused a significant (p<0.05) decrease in calcium release. This can be explained by assumi ng that each fraction

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49 played an inhibitory role in blocking the M3R from the MR agonist (carbachol) thus affecting the signaling pathway downstream in the release of intracellular calcium Further comparisons revealed that the longer the incubation time with purified Ig fractions, the greater the inhibitory effect on calcium release This may be due to M3R desensitization because the longer the HSG cells are exposed to the purified fractions, the greater the inhibitory effect on calcium release. This wa s espe cially apparent for the 24 -hour incubation time with 2 injections of fractions. Third, the positive and negative controls were significantly different (p<0.05) in regards to a decrease in calcium release in Figure 3 6 The 15 minute incubation with the same amount of trend in calcium release for all B6 and NOD fractions, similar to both Figure 3 4 and Figure 3 5 where the same volume (2 ) of fractions were used. Even though the data for both B6 and NOD fractions ( Figure 3 6 ) were not significant, a decreasing trend of inhibitory effect s was noted. When comparing the 15 minute incubations there was a greater inhibition of calcium release (Figure 3 4 ). This may have been due to the greater amount of protein that was present in the fraction used to incubate, compared t o just using a constant amount but dif ferent volumes This suggests that there was a sufficient amount of anti -M3R autoantibodies present to affect the binding of carbachol to the receptor thus leading to a significant decrease in calcium release, whic h requires further verification Fourth, shown in Figure 3 7, both B6 and NOD purified Ig fractions (with the exception of NOD IgG1) caused an inhibitory effect on calcium release, even though their data was not significantly different (p>0.05). In this example the positive and negative controls were again not significantly different (p>0.05). This may have been due to not enough protein present in the fractions when incubated with the cells, thus causing a lesser effect compared to treatments The se d ata support the idea that because of a longer incubation time, each fraction was able to

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50 desensitize and block the binding site of M3R from carba c hol thus affecting neurotransmitters from a normal signaling pathway. The decreasing trend was observed in a ll fractions, again supporting the fact that there was some sort of inhibition factor in the fractions that caused this to happen. Lastly, NOD IgG1 was much different in Figure 3 5 when compared to Figures 3 4, 3 6, and 3 7 because of its inhibitory rath er than its stimulatory effect. This can be explained again by not enough protein present in the fraction to block the binding of carbachol to M3R, thus leading to a surge of intracellular calcium being released similar to the responses by CChresponsive cells (Positive control) W hen comparing the NOD IgG1 results of Figure s 3 4 through 3 7 the greater amount of Ig used to incubate with the cells caused a greater inhibitory effect. High concentration of IgG1 caused an inhibitory effect which suppor ts our original hypothesis that IgG1 leads to inhibition of calcium release by affecting M3R signaling pathway. Future Directions To acquire better results for anal ysis for future experiments, the experimental conditions further needs to be optimized. In r egards to the in vivo study, another step that should be performed is reconstituting the purified Ig fractions into whole IgG, and injecting that into the Rag1KO recipient mice. If the results mimic th ose of previous studies, then we could have a better i nterpretation of the effects of both whole IgG and purified Ig fractions on SS. In addition, a larger sample size of mice would greatly reduce statis tical errors, as well as give us a better depictio n of the population. As for the in vitro study, finding a suitable mouse cell line that expresses M3R may give a better representation of the effects o f the mouse Ig fractions. Since the results suggested that both B6 and NOD fractions inhibited calcium release in HSG cells, further studies need to be performed to determine whether this effect was due to specific binding of anti -M3R autoan tibodies or non-specific concentration dependent stimulation

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51 For example, knocking down M3R using small interfering RNA (siRNA), would allow us to determine if M3R express ion is required for this inhibition. I f inhibition is present in the absence of M3R, this would suggest that non -specific binding of antibody fractions can inhibit carbachol induced calcium release in HSG cells, as observed with B6 fractions. This could be due to the higher protein concentration in B6 fractions compared to NOD. In addition since using increased amounts of Ig fractions showed a decrease in calcium release, further investigation must performed to avoid discrepancies and errors. By doing so, dose effects of IgG1 can be analyzed while comparing it with the pathological inhibitory effects of anti -M3R autoantibodies Lastly, an effective and reliable assay to detect the presence of anti -M3R autoantibodies and the effect of each fraction on t he receptor should be perform ed to obtain a better understanding of its pathological effect on secretory dysfunction Conclusion In conclusion, the current data suggest that NOD IgG1 and NOD IgG2c both cause secretory dysfunction in vivo while NOD IgG1 causes an inhibition in calcium release in vitro The in vivo findings propose that both NOD IgG1 and IgG2c can cause secretory dysfunction, probably due to their ability to block the M3R binding site, thus inhibiting the signaling pathway for normal sali va secretion. The in vitro findings suggest that both B6 and NOD fractions can cause a decrease in calcium release in HSG cells after 24 ho urs of chronic stimulation, and NOD IgG1 s how inhibitory effects on the binding of carbachol to M3R which is suppor tive of my hypothesis By conducting further studies in vivo and in vitro using NOD purified fractions, it might be possible to gain a better understanding of functional implications of anti -M3R autoantibodies in autoimmune Sjgrens syndrome.

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52 LIST OF R EFERENCES 1. Cha, S., J. Brayer, J. Gao, V. Brown, S. Killedar, U. Yasunari, and A. B. Peck. 2004. A Dual Role for Interferon gren's Syndrome Like Autoimmune Exocrinopathy in the Nonobese Diabetic Mouse. Scandinavian Journal of Immunology 60:552565. 2. Mese, H., and R. Matsuo. 2007. Salivary secretion, taste and hyposalivation. Journal of Oral Rehabilitation 34:711723. 3. Waterman, S. A., T. Gordon, and M. Rischmueller. 2000. Inhibitory effects of muscarinic receptor autoantibodies on parasympathetic neurotransmission in Sjgren's syndrome. Arthritis & Rheumatism 43:16471654. 4. Cermak, J. M., A. S. Papas, R. M. Sullivan, M. R. Dana, and D. A. Sullivan. 2003. Nutrient intake in women with primary and seco ndary Sjgren's syndrome. European Journal of Clinical Nutrition 57:328. 5. Ramos Casals, M., and P. Brito -Zeron. 2007. Emerging biological therapies in primary Sjogren's syndrome. Rheumatology 46:13891396. 6. Vitali, C., S. Bombardieri, R. Jonsson, H. M. Moutsopoulos, E. L. Alexander, S. E. Carsons, T. E. Daniels, P. C. Fox, R. I. Fox, S. S. Kassan, S. R. Pillemer, N. Talal, and M. H. Weisman. 2002. Classification criteria for Sjogren's syndrome: a revised version of the European criteria proposed by the American -European Consensus Group. Annals of the Rheumatic Diseases 61:554558. 7. V on Mhlen, C. A., and E. M. Tan. 1995. Autoantibodies in the diagnosis of systemicrheumatic diseases. Seminars in Arthritis and Rheumatism 24:323358. 8. Wolin, S. L., and K. M. Reinisch. 2006. The Ro 60 kDa autoantigen comes into focus: Interpreting epitope mapping experiments on the basis of structure. Autoimmunity Reviews 5:367372. 9. Chen, X., and S. L. Wolin. 2004. The Ro 60 kDa autoantigen: insights into cellular function and role in autoimmunity. Journal of Molecular Medicine 82:232(238). 10. Espinosa, A., W. Zhou, M. Ek, M. Hedlund, S. Brauner, K. Popovic, L. Horvath, T. Wallerskog, M. Oukka, F. Nyberg, V. K. Kuchroo, and M. WahrenHerlenius. 2006. The Sjogren' s Syndrome -Associated Autoantigen Ro52 Is an E3 Ligase That Regulates Proliferation and Cell Death. Journal of Immunology 176:62776285. 11. Fabini, G., S. A. Rutjes, C. Zimmermann, G. J. M. Pruijn, and G. Steiner. 2000. Analysis of the molecular composit ion of Ro ribonucleoprotein complexes. European Journal of Biochemistry 267:27782789.

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5 3 12. JF, M., and S. RJ. 1992. Autoantibodies and their target antigens in Sjgren's syndrome. Netherland Journal of Medicine 40:140147. 13. Bacman, S., L. Sterin Borda J. J. Camusso, R. Arana, and O. H. E. Borda. 1996. Circulating antibodies against rat parotid gland M3 muscarinic receptors in primary Sjgren's syndrome. Clinical & Experimental Immunology 104:454459. 14. Nguyen, C., J. Cornelius, L. Cooper, J. Neff, J. Tao, B. Lee, and A. Peck. 2008. Identification of possible candidate genes regulating Sjogren's syndrome associated autoimmunity: a potential role for TNFSF4 in autoimmune exocrinopathy. Arthritis Research & Therapy 10:R137. 15. Gao, J., S. Cha, R. Jon sson, J. Opalko, and A. B. Peck. 2004. Detection of anti -type 3 muscarinic acetylcholine receptor autoantibodies in the sera of Sjgren's syndrome patients by use of a transfected cell line assay. Arthritis & Rheumatism 50:26152621. 16. Dawson, L., A. To bin, P. Smith, and T. Gordon. 2005. Antimuscarinic antibodies in Sjgren's syndrome: Where are we, and where are we going? Arthritis & Rheumatism 52:29842995. 17. Cha, S. H., E. Singson, J. Cornelius, J. P. Yagna, H. J. Knot, and A. B. Peck. 2006. Muscar inic acetylcholine type 3 receptor desensitization due to chronic exposure to Sjogren's syndrome associated autoantibodies. Journal of Rheumatology 33:296306. 18. Nakamura, T., M. Matsui, K. Uchida, A. Futatsugi, S. Kusakawa, N. Matsumoto, K. Nakamura, T Manabe, M. M. Taketo, and K. Mikoshiba. 2004. M3 muscarinic acetylcholine receptor plays a critical role in parasympathetic control of salivation in mice. Journal of Physiology 558:561575. 19. Drachman, D. 2003. Autonomic "myasthenia": The Case for an Autoimmune Pathogenesis Journal of Clinical Investigation 111:797799. 20. Eriksson, P., C. Andersson, C. Ekerfelt, J. Ernerudh, and T. Skogh. 2004. Relationship between serum levels of IL 18 and IgG1 in patients with primary Sjgren's syndrome, rheumatoi d arthritis and healthy controls. Clinical & Experimental Immunology 137:617620. 21. Andersson, A. 2009. Genetic control of disease in an experimental model for Sjogren's syndrome. Arthritis Research & Therapy 11:102. 22. Currie, M., A. M. Zaki, S. Neja t, G. M. Hirsch, and T. D. Lee. 2008. Immunologic targets in the etiology of allograft vasculopathy: Endothelium versus media. Transplant Immunology 19:120126.

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54 23. Jonsson, M. V., N. Delaleu, K. A. Brokstad, E. Berggreen, and K. Skarstein. 2006. Impaire d salivary gland function in NOD mice: Association with changes in cytokine profile but not with histopathologic changes in the salivary gland. Arthritis & Rheumatism 54:23002305. 24. Gao, J., S. Killedar, J. G. Cornelius, C. Nguyen, S. Cha, and A. B. Pe ck. 2006. Sjogren's syndrome in the NOD mouse model is an interleukin 4 time dependent, antibody isotype specific autoimmune disease. Journal of Autoimmunity 26:90103. 25. Lee, B., M. Tudares, and C. Nguyen. 2009. Sjgrens syndrome: An old tale with a new twist. Archivum Immunologiae et Therapiae Experimentalis 57:5766. 26. Dawson, L. J., J. Stanbury, N. Venn, B. Hasdimir, S. N. Rogers, and P. M. Smith. 2006. Antimuscarinic antibodies in primary Sjgren's syndrome reversibly inhibit the mechanism of fl uid secretion by human submandibular salivary acinar cells. Arthritis & Rheumatism 54:11651173. 27. Robinson, C. P., J. Brayer, S. Yamachika, T. R. Esch, A. B. Peck, C. A. Stewart, E. Peen, R. Jonsson, and M. G. Humphreys Beher. 1998. Transfer of human s erum IgG to function of exocrine tissues in Sjgren's syndrome. Proceedings of the National Academy of Sciences of the United States of America 95:75387543. 28. Wa terman, S., T. Gordon, and M. Rischmueller. 2000. Inhibitory effects of muscarinic receptor autoantibodies on parasympathetic neurotransmission in Sjgren's syndrome. Arthritis & Rheumatism 43:16471654.

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55 BIOGRAPHICAL SKETCH Huy Huynh grew up and attend ed school in Winter Haven, FL where he graduated high school in 2002. After high school, he began study at the University of Florida in Gainesville, where he obt ained a Bachelor of Science in human n utrition in May of 2007. In the fall of 2007, Huy was admitted into the masters studies program at the University of Florida where his degree was focused on food science. U nder the co-mentorship of Dr. Gar y Rodrick and Dr. Seunghee Cha, Huy was able to fulfill his requirements for his degree, as well as p erform his autoimmune research that investigated the role of IgG1 anti -M3R autoantibodies on secr etory dysfunction in Sjgrens s yndrome. In the fall of 2009, Huy will enter dental school at the University of Florida. His ultimate professional goal is to become a practicing general dentist with an interest in teaching and academia.