Sjogrens Syndrome (SS) is an autoimmune disease, characterized by widespread inflammation in the exocrine glands and other organs resulting in dryness of the lining surfaces of the body, most notably, dry mouth and dry eyes. In addition, it can involve numerous other organs such as the joints, skin and nervous system. The MPTB- SS Clinic conducts clinical investigations and clinical trials, and collaborates with laboratory investigators in MPTB, other NIH laboratories and Institutions outside the NIH in order to understand the mechanisms underlying this disease. The ultimate goal of our research is to find treatments for SS that are safe, effective and target specific steps in the pathogenesis of SS. Treatment study using efalizumab (Raptiva) an anti-LFA1 monoclonal antibody (5%) In this protocol we used Raptiva (efalizumab, Genentech), an anti-LFA1 monoclonal antibody, targeting the LFA1-ICAM pathway involved in several functions in Sjogrens Syndrome, including lymphocyte trafficking and antigen presentation. In stark contrast to our expectations, Raptiva has increased inflammation in the salivary gland, worsened salivary gland function in most patients and led to significant increase in immunoglobulin production and the emergence of new autoantibodies in two patients. Mechanistic studies to understand these changes are underway. Gene expression analysis in the peripheral blood showed activation of pathways of both T and B cell activation. MicroRNA microarrays showed a differential changes in microRNA expressions in the salivary glands between placebo (n=3) and Raptiva (n=6) recipients. The main targets of differentially expressed microRNAs are currently validated by RT-PCR. Salivary proteomics and metabolomics studies are underway. Autonomic nervous system (ANS) dysfunction in SS (5%) Compared to normal healthy individuals, SS patients have significantly more nervous system involvement including impaired ANS function (which regulates the homeostasis of basic physiologic functions via effects on the smooth muscle, glands and cardiovascular system). Despite the relatively high prevalance of ANS sbnormalities little is known about the underlying mechanisms of these. We hypothesize that ANS dysfunction is central to the pathogenesis of SS and, in collaboration with Dr. David Goldsteins group (NINDS), we are conducting a protocol to investigate ANS function in patients with SS and the association between ANS dysfunction and autoimmunity. This protocol consists of a comprehensive evaluation of autonomic function, using physiological, neuropharmacologic, neurochemical, and imaging approaches, to identify consistent distinctive patterns of ANS involvement in SS and thereby improve the diagnosis and understanding of pathophysiologic mechanisms of SS. We completed testing in 20 patients and 7 controls. Natural history of Sjogrens Syndrome protocol (70%) Biomarker studies 1. Salivary gland microRNA expression A major focus of this study was to evaluate the potential of microRNAs (miRNAs) as biomarkers. In a pilot study we used 24 Agilent microRNA microarrays to profile miRNAs isolated from healthy volunteers and Sjogrens patients'minor salivary glands (MSGs) with high grade (focus score of 12) or low grade inflammation (focus score of 1 or 2). We identified microRNA profiles that can serve as biomarkers for diagnosis (SS vs. controls), function (salivary flow) or degree of inflammation in SS. We identified two miRNAs the ratio of which correlated very well with the inflammatory status of the MSGs in a small set of samples. We have expanded these studies to a larger cohort of patients and formalin fixed paraffin embedded (FFPE) biopsies. Using a teaching cohort of 49 subjects, we first showed the deltaCt of hsa-mir-574-3p and hsa-mir-768-3p derived from FFPE samples can distinguish samples from Sjogrens from non-Sjogrens MSGs regardless of focus score (p 0.0001, one-way analysis of variance) as well as groups with various degree of inflammation.To validate the applicability of these miRNAs as biomarkers we determined the correlation between individual focus scores and the deltaCt in an independent set of 87 subjects. A Pearsons correlation analysis showed a significant relationship between deltaCt of hsa-mir-574-3p and hsa-mir-768-3p and focus scores (r=0.85, r squared=0.72, p= 0.0001) validating these miRNAs as biomarkers of MSG inflammation and suggesting that they may be used to reduce the subjectivity involved in the diagnosis of SS. We have further expanded these studies to serum with encouraging preliminary results. We are also analyzing more global microRNA expression profiles by microarrays in the teaching cohort to identify other biomarkers which will be tested in the validation cohort. 2. Functional target identification of individual differentially expressed miRNAs. The salivary gland inflammation in Sjogrens syndrome is associated with various cytokine abnormalities which impact salivary cell function. In this project we analyze microRNA responses of human SG cell lines to various cytokines to identify the microRNA signature of various cytokines and to identify microRNAs which may play a dominant role in SG dysfunction. We plan to use these in vitro microRNA to identify the effect of dominant cytokines in tissues and can be used as a tool to identify the most promising therapeutic targets or monitor response to therapy. The second objective of this project is to identify the cellular pathways targeted by the cytokine induced microRNAs which directly contribute to salivary gland dysfunction. 3. Exosomal microRNAs There is increasing evidence that exosomes represent a novel way of inter-cellular communication. Assuming that the release and content of exosomes is not random we hypothesized that exosomal microRNAs in various tissues and biologic fluids may reflects the functional state of an organ and maybe useful as biomarkers. In a previous collaboration we have shown that miRNA192 is found in higher levels in urinary exosomes of lupus patients with kidney disease than in patients disease without kidney disease and that the level of miRNA192 expression correlated with proteinuria. Based on these observations we are studying urinary exosomal microRNA in urine and sera in patients with lupus nephritis before and after treatment. 4. Genetic studies We have provided DNAs for the Sjogrens Genetics Network (SGENE) (PI: Dr. Kathy Moser, Oklahoma) for genome wide association studies SS. In addition to confirming the association of SS with some lupus associated genes and HLA, our initial studies identified a BLK, methyl-CpG binding protein 2, and musculin as novel genes associated with SS. MPTB translational research project on Sjogrens Syndrome (10%) To narrow the gap between basic science and clinical practice we continued our collaboration with several groups in NIDCR. We have successfully completed mRNA gene expression studies of normal volunteer and pSS samples in collaboration with the Chiorini group. One of the most interesting findings is the overexpression of several viral transcripts in the MSG in SS patients. The increased expression of viruses may indicate a direct etiologic role or an increase in overall viral load (due to decreased epithelial integrity) in the SG representing a target for a chronic inflammatory response. In another arm of this project we found that SG from males with SS have abnormal expression of XIST, a gene involved in silencing the second X chromosome in females, indicating a XXY karyotype in at least some cells. These findings were confirmed on DNA from peripheral blood using SNP analyses of genes on the X chromosome.
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