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). We addressed this question with two parallel projects. 1. Clinical and Laboratory Evaluation of the Autonomic Nervous System in Primary Sjogrens Syndrome In collaboration with Dr. David Goldsteins group (NINDS), we conducted a protocol of detailed investigation of ANS function in 21 pSS patients and 13 controls pSS. Subjects had a comprehensive evaluation of autonomic function to identify consistent distinctive patterns of ANS involvement in SS. Reduced gastric emptying was found in 45% and abnormally low sweat production w in 19% pSS patients. In response to the injection of an acetylcholine esterase inhibitor, unlike controls, pSS patients did not have an increase in saliva production but they did not differ in their cardiovascular responses, or adrenal hormone levels. Velocity index and acceleration index were significantly higher in pSS compared to controls before and during tilt table and glucagon tests suggesting an increased baseline cardiac contractility in pSS. Whether the observed higher cardiac contractility in pSS could be attributed to increased cardiac sympathetic outflow or decreased parasympathetic tonus remains unknown and warrants further study. 2. Ambulatory assessment of ANS function in SS We also evaluated cardiovascular (CV) ANS function in pSS by ambulatory real-time digital monitoring in over 109 consecutive pSS patients and 34 age- and sex-matched adult healthy volunteers (HV) using ANX 3.0 (ANSAR, Inc.). Small significant differences in heart rate variability between HV and pSS were seen in response to all three cardiovascular challenges indicating low cardiovagal tonus. The composite measures of both sympathetic and parasympathetic cardiovascular responses to all three challenges were significantly lower in pSS compared with HV while maintaining the sympathetic/parasympathetic balance. A logistic regression model identified two measures of CV responses which were predictive of the diagnosis of pSS. Both of these studies suggest that autonomic dysfunction is common in pSS but affects various ANS domains differently, with the GI domain being the most frequently involved. Abnormalities in the cardiovascular domain were more subtle but consistent across the two studies and suggest a relatively preserved sympathetic/parasympathetic balance but at a slightly different level of activation of the ANS. Treatment study using efalizumab (Raptiva) an anti-LFA1 monoclonal antibody Efalizumab is an anti-LFA1 monoclonal antibody targeting the LFA1-ICAM pathway involved in several functions in Sjogrens Syndrome, including lymphocyte trafficking and antigen presentation. As we reported earlier we treated nine patients in a study before the study was terminated. Immunehistochemistry of the biopsies showed an increase in macrophage numbers with treatment but there was no difference in the overall number of major lymphocyte subsets after efalizumab treatment despite a change in the inflammatory infiltrates from focal to diffuse all efalizumab treated subjects. Our findings suggest that efalizumab may disrupt the formation of inflammatory aggregates. Studies are ongoing to better understand the effect of LFA1-blockade on inflammation. microRNA studies Biomarker studies in tissue biopsies 1. Salivary gland microRNA expression In a pilot study we identified microRNA (miR) profiles that distinguished controls from patients and were associated with either inflammation or salivary dysfunction. In this study we identified two miRs the ratio of which correlated well with the inflammatory status of the MSGs in this small set of samples. We have expanded these studies to a larger cohort of patients and formalin fixed paraffin embedded (FFPE) biopsies and have shown that the deltaCt of hsa-mir-574-3p and hsa-mir-768-3p is different in pSS compared to both controls and subjects with sicca symptoms but is not statistically significantly different between HV and sicca patients. Pearsons correlation analysis showed a significant strong relationship between deltaCt of hsa-mir-574-3p and hsa-mir-768-3p and focus scores (r=0.68, p= 0.0001). Next, we expanded our analysis to extraglandular manifestations in Sjogrens patients. Mean values of deltaCt were not statistically different between SS patients with or without extraglandular manifestations or ANA positive or negative patients. 2. MicroRNA expression profiles in kidney biopsies of patients with lupus nephritis Lupus nephritis (LN) is the most common major organ manifestation in SLE. We have done a pilot study analyzing microRNA expression in FFPE biopsies of a small cohort of patients with lupus nephritis and different histological findings and renal outcomes. We found that miR-150 level was differentially expressed in kidney biopsies from patients with proliferative lupus nephritis with either low or high degree of chronicity. The positive correlation of miR-150 with chronicity index suggests that it may be a useful quantitative biomarker for evaluating kidney injury in lupus nephritis. MicroRNA biomarkers in biologic fluids In addition to studying miR biomarkers in tissue biopsies we are also interested in exploring them in more readily available samples such as serum, saliva and urine. 1. Source of circulating microRNAs in biologic fluids One of the unanswered questions in this area is whether miRs in the blood are circulating free or are within exosomes. To address this, we first optimized a protocol to isolate exosomes from a very small amount of fresh and frozen serum and saliva. The expression of selected microRNAs isolated either from exosomes or the exosome-free supernatant were compared by TaqMan RT-qPCR. The expression of all tested miRNAs in the pellet was at least 8 fold higher compared to the supernatant. The difference ranged from 8-128 folds (3-7 cycles). Most miRs were either undetectable (Ct >35) or detectable at a very low level in the exosome-free supernatant. These results show that the majority of miRs in the serum and saliva is packaged within microvesicles, primarily exosomes and not found free circulating in human samples. Although, the exosome isolation could mean an extra-step, it maybe necessary to reliably detect miRNAs which are present at low concentration in human fluids. MPTB translational research projects on Sjogrens Syndrome We continued our ongoing collaborations with other groups in MPTB. As part of the salivary gland initiative we have done gene expression studies on (MSG) we, in collaboration with Dr. Chiorini, have described differences in gene expression profiles between healthy male and female minor salivary glands (MSG) which may contribute to the increased risk of SS in females. We also identified an abnormal expression of XIST in male SGs. This may represent the presence of an extra X chromosome. Our collaboration with Dr. Ambudkar suggests an important role of STIM1 in Sjogrens in both mice and humans.

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