RESEARCH ACCOMPLISHMENTS Behcet's Disease CLINICAL (a) and (b) 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 be compared between patient cohorts followed by Dr. Yusuf Yazici at NYU, Dr. Hasan Yazici at the University of Istanbul and our NIAMS cohort. The mean age did not differ between sites, however more women were seen in the US compared to Turkey (p<0.0001). Prior organ manifestations were similar for oral ulcers, genital ulcers, skin disease, arthralgia, eye disease and thrombosis. However, percentages differed at the NIH, NYU, and University of Istanbul for 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 that 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. A preliminary Ingenuity canonical pathway analysis revealed the most statistically significant and largest fold differences between affected and unaffected mucosa samples occurred in inflammatory pathways. We are in the process of further dissecting which inflammatory processes are most relevant. Future studies include a specific Ingenuity pathway analysis where factors known to be important in inflammation and pathogen recognition are pre-specified. Genes of interest will include TLRs, NLRs, cytokines, chemokines, complement components, JAKs, STATs and their downstream mediators. We also plan to correlate RNA Sequencing results with the microbial patterns we described to evaluate whether the host shows a different response to particular pathogens in BD patients compared to controls. (c) We have now optimized an antibody panel to immunophenotype peripheral blood cells with a particular interest in the monocyte and dendritic cell populations. This panel has been validated in control cells and will now be tested in Turkish and American BD samples. We have the ability to compare between the US and Turkey as well as to characterize immunophenotypes in different disease subsets and disease activity states. We anticipate that the samples will be characterized in the next 1-2 months with the analysis lasting a similar period of time. (d) 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 Stills Disease CLINICAL (a) The pilot study of rilonacept in AOSD has been completed. The data from this study has been monitored and the database is now locked. Preliminary analyses 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) A preliminary study of the effect of treatment with IL-6 blockade is being analyzed in 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 Similar methods to those used in the monogenic auto-inflammatory disorders NOMID and DIRA are underway 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 United States 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 in the process of being defined. 5. Cell immunophenotyping in BD is underway. 6. A pilot study of IL-1 inhibition with rilonacept has been completed in AOSD. 3/5 patients showed good response to IL-1 blockade whereas the remaining 2 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 with an effort to better understand the underlying inflammatory pathophysiology.
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