RESEARCH ACCOMPLISHMENTS Behcet's Disease CLINICAL Dr. Sibley traveled to Istanbul in December 2010 to collect patient disease questionnaire data, blood, DNA, RNA, and tissue specimens. Organ manifestations and patient questionnaires were compared between patient cohorts followed by Dr. Yusuf Yazici at New York University (NYU), Dr. Hasan Yazici at the University of Istanbul and NIAMS. While the mean age did not differ between sites, more women were seen in the United States (US) than in Turkey (p <0.0001). Prior organ manifestations were similar for oral ulcers, genital ulcers, skin disease, arthralgia, eye disease and thrombosis. However, percentages differed among NIH, NYU, and University of Istanbul patients with gastrointestinal disease (42.9, 37 and 0, respectively;p <0.0001) and neurologic disease (20, 17.3 and 4.5, respectively;p=0.02). American patients were treated with more prednisone and less colchicine (p <0.0001 and 0.0009, respectively). Other disease-modifying medication use did not differ. Disease activity scores were worse in the US compared to Turkey, with mean BSAS values of 42.5 at the NIH, 36.5 at NYU and 30.4 at the University of Istanbul (p=0.01) and mean BDCAF values of 5.9 at the NIH, 5.7 at NYU and 3.3 at the University of Istanbul (p <0.0001). No significant differences were seen between the NIH and NYU. Quality of life was also worse in the US, with mean BDQOL scores of 16.1 at the NIH compared to 12.5 in Istanbul (NS). Multivariable regression models accounting for age, sex, atypical ethnic background, and country of origin showed worse scores in ethnically atypical patients for BSAS and BDCAF (p=0.04 and p=0.01), patients from the US for BDCAF (p=0.001), older age for BDCAF (p=0.005) and women for BDQOL (p=0.01). (c) Enrollment has begun in an open label treatment study of mucocutaneous BD followed by a randomized withdrawal phase. One patient has been enrolled with several more planned over the next few months. Preliminarily, our first patient showed improvement in the number of oral ulcers and global disease activity. More definitive conclusions will be made after further patient enrollment. LABORATORY (a) The microbiome was compared between 6 Turkish paired patient samples of affected, ulcerated BD mucosa and unaffected, non-ulcerated BD mucosa and 4 healthy ethnically, age- and sex-matched control mucosa samples. 16S rDNA sequencing was performed. For most patients and controls, Streptococcus was the most common organism followed by a mix of anaerobes. No unique bacterial organisms were found in patients compared to controls. Microbial community membership did not differ between affected patient samples, unaffected patient samples, and healthy controls as analyzed by clustering analysis with Jaccard tree grouping. Based on this, we did not find a clear alteration in bacterial colonization in BD patients or controls and suspect that pathology may be due to an abnormal host response to bacteria in BD or other triggers rather than bacterial colonization itself. (b) RNA Sequencing of 17 paired Turkish oral mucosal affected and unaffected samples and 4 ethnically, age- and sex-matched controls has been performed. Principal Component Analysis (PCA) revealed that affected samples clustered together whereas unaffected samples clustered with controls. An Ingenuity canonical pathway analysis revealed that the most statistically significant and largest fold differences between affected and unaffected mucosa samples occurred in inflammatory pathways. Of the 34,799 reads, 4595 transcripts were two-fold or greater upregulated in ulcerated compared to unaffected BS samples, with 971 downregulated greater than two-fold. GSEA of NLR, TLR, IL-1 receptor and IL-10 pathways all showed enrichment in ulcerated compared to unaffected samples with normalized enrichment scores of 1.45, 1.48, 1.39 and 1.36 and nominal p-values of 0.02, 0.02, 0.11 and 0.05, respectively. These pathways did not differ between unaffected and control samples. (c) Serum samples have been obtained for all American patients with BD evaluated at the NIH under our protocols. It will be possible to evaluate cytokine profiles in the future and correlate these results with clinical parameters and results from RNA Sequencing and cell immunophenotyping studies. Adult Onset Still's Disease CLINICAL (a) The pilot study of rilonacept in AOSD has been completed. The data from this study have been monitored and the database is now locked. Preliminary analyses are now underway showing that three patients responded well to rilonacept. The two non-responders were switched to tocilizumab, an IL-6 receptor blocker, and are now showing clinical benefit. (b) Data from a preliminary study of the effect of treatment with IL-6 blockade are being analyzed for the two patients who were treated with this medication. So far, one patient showed clinical benefit whereas the other was limited by toxicity. We are considering expanding this study to more patients in order to study this more systematically. Other considerations for treatment include the use of JAK inhibitors for AOSD. LABORATORY (a) Serum samples at different time intervals and disease activity states have been obtained from patients with AOSD treated with rilonacept. These samples have been run and the analysis phase is beginning. Particular cytokines of interest are those that are implicated in the IL-1, IL-18 and IL-6 pathways, as well as other pathways known to be of importance in autoinflammation. We will correlate cytokine results with markers of clinical disease activity. CONCLUSIONS AND SIGNIFICANCE Methods similar methods to those used in the monogenic auto-inflammatory disorders NOMID and DIRA are employed to characterize clinical and inflammatory pathways in Behcets Disease and AOSD, polygenic autoinflammatory diseases in adults. 1. A clinical comparison has been made between patients with Behcet's Disease in the US and Turkey. American patients appear to report worse disease activity and worse quality of life when compared to those from Turkey. The etiology behind these findings remains to be determined. 2. A pilot treatment study of BD is underway with Anakinra, an IL-1 blocking medication. Preliminary results are encouraging. 3. Microbiome studies do not show differences in microbial colonization between BD patients and normal controls. 4. RNA sequencing results in BD show abnormalities in inflammatory pathways that are important in pathways of pathogen recognition 5. Cell immunophenotyping in BD is underway. 6. A pilot study of IL-1 inhibition with rilonacept has been completed in AOSD. Three of five patients showed good response to IL-1 blockade, whereas the remaining two patients responded well to a change of therapy to the IL-6 receptor blocker, tocilizumab. 7. Cytokine profiles will be analyzed from longitudinal serum samples collected from AOSD patients in an effort to better understand the underlying inflammatory pathophysiology.