In the past year the Mucosal Immunity Section has been engaged in a number of research studies involving both inflammatory bowel disease and common variable immunodeficiency. In the area of IBD, we have conducted studies of IBD associated with LRRK2 risk polymorphisms, as described in detail in a previous annual report. In these studies we have established that the polymorphism is associated with increased levels of LRRK2 and this elevation is accompanied by increased gut inflammation in an induced colitis model driven by Dectin-1 induced NF-kappaB responses. Several observations derived from these studies have direct clinical significance. The first is that cells derived from patients who do not bear the LRRK2 risk polymorphism exhibit reduced pro-inflammatory cytokine responses in vitro when the cells are exposed to inhibitors of the kinase activity of LRRK2. The second is that induced colitis of normal mice or mice with elevated levels of LRRK2 is inhibited by administration of various LRRK2 inhibitors. These observations suggest that treatment of patients with inhibitors of LRRK2 can have a therapeutic effect in all patients with IBD regardless of their LRRK2 status. In this period, we have commenced a study of the safety and and immunologic effects of the administration of vorinostat, an HDAC inhibitor. The Clinical Protocol guiding the execution of this study (Protocol # 17-I-0101) has now obtained NIAID IRB and FDA approval. The study will ultimately enroll 20 patients who have failed other forms of Crohn's disease therapy; these patients will undergo a wide array of studies to evaluate the immunologic effects of HDAC inhibitor therapy including effects on regulatory T cells. So far one patient has been enrolled in the study and has completed its treatment phase (12 weeks of oral Vorinostat 100 mg BID); he will shortly return to complete the followup phase of the study. The patient has reported improvement in abdominal pain and cramping symptoms as well as some improvement in diarrhea; however, he still experiences intermittent watery stools. Importantly, his CDAI idex has decreased from > 300 to 180, a greater than 100 point drop. This represents a clinical response short of remission. No change in Treg cell number was observed in studies of cells present in peripheral blood. In the area of CVID we have continued to focus on gastrointestinal manifestations of this heterogeneous disease, i.e., patients with a CVID-entropathy characterized by diarrhea, malabsorption and villous atrophy. In prior studies of CVID we established that the CVID enteropathy is driven by cells producing IFN-gamma. In addition, we collaborated with Drs. Andriy Morgan and Natalia Stulzhenko in the Laboratory of Cellular and Molecular Immunology in studies of CVID that included microarray analyses of biopsy tissue of CVID patients with enteropathy showing that CVID enteropathy is characterized by up-regulation of genes related to various interferons. Based on this prior work, we have applied for and have obtained a U01 NIH cooperative research grant (PAR-13-029) with Drs. Morgan and Shulzhenko (now at Oregon State University) to expand on these findings. This Grant is entitled Anti-IL-12p40 Treatment of CVID Enteropathy: Gene Expression and Microbiota Analysis, and is centered around a clinical study of the safety and efficacy of a FDA-approved monoclonal anti-IL-12p40 in the treatment of CVID enteropathy. In addition, this Grant includes a study of the effect of this biologic agent on gene expression of inflammatory cytokines and on the intestinal micro biome of the patients. In an initial phase of these studies, we treated three patients with CVID enteropathy with a single dose of anti-IL-12p40 (270mg sc of Stelara)and found that all three patients experienced clinical improvement. In one of the two patients, there was long-term and dramatic cessation of diarrhea, whereas in the other two patients diarrhea was greatly reduced but rather transient. Despite this, improvement in objective measures of malabsorption, such as stool fat assessment, was not observed in any of the patients. As a result of this experience we concluded that a greater therapeutic effect might be obtained with a multiple dose regimen consisting of 270mg induction dose of Stelara followed by two addition 180mg doses over a six month period. Consequently, we applied for and received permission from the NIAID IRB to treat patients in this manner. In this second phase study (Protocol #14-I-0153) 4 subjects have been enrolled and two subjects have completed 40 weeks of treatment. These latter two patients underwent an endoscopy procedure at Week 48 for analysis of villous blunting (and evidence of intestinal inflammation). Furthermore, they had stool collections performed to evaluate changes in malabsorption including stool A1AT, fecal fat analysis, blood studies to evaluate D xylose absorption (carbohydrate malabsorption) and serum markers of nutrient absorption (ie total protein, albumin). Both patients experienced a greater than 8 kg weight gain and normalization of albumin and total protein levels; in addition, both exhibited improved duodenal histology (either marked improvement or disappearance of villous blunting). These patients, however, experienced intercurrent GI intestinal illnesses at the end of the study that precluded adequate assessment of malabsorption. They have continued taking Stelara (90 mg SC every 8 weeks). and are now without abdominal pain/bloating or diarrhea. The other two patients are almost at the midway point (24 weeks) of the study and have all shown significant weight gain and improvement in albumin/protein levels as well as cessation of diarrhea. No abdominal pain or bloating symptoms have been noted since starting on treatment. These patients will return to NIH at 24 weeks for stool malabsorption studies. Finally, two patients have been screened that are have proven to be candidates for the study; they will be enrolled in the study shortly. Meanwhile stool and tissue specimens of CVID patients with and without enteropathy have been sent to Drs. Morgun and Shulzhenko at Oregon State University for microbiome analysis before and after anti-IL-12p40 treatment as well as identification of a possible organism causing the enteropathy. A major outcome of this collaborative study has emerged from analysis of patient immunoglobulin levels in gut specimens. We have found that those patients with enteropathy have little or no expression of IgA in intestinal biopsies whereas those patients without enteropathy have only moderately reduced levels of IgA. Moreover, this deficiency does not extend to mucosal IgG levels. This finding indicates that enteropathy occurs in the subset of CVID patients with severe mucosal B cell immunodeficiency affecting the mucosal immunoglobulin, IgA. A second major outcome from these studies is the finding the duodenal specimens from patients with enteropathy contain Acinetobacter baumanii, an organism that, acting as an pathobiont in immunodeficient patients, induces an excessive IFN-gamma synthesis and resultant enteropathy. The findings in this study have been incorporated into a submitted paper that is now being considered for publication.
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