Michael Eberhardson

Profiling of CD4+ T Cells with Epigenetic Immune Lineage Analysis

Proper transcriptional control of pro- and anti-inflammatory responses of the immune system is important for a fine-tuned balance between protection and tolerance. Emerging evidence suggests a key role for epigenetic regulation in governing the Th cell differentiation, where effector cytokines direct the overall immune response. In this study, we describe a method to pinpoint the location of isolated human CD4+ T cells on any T cell effector axis based on specific CpG methylation of cytokine and transcription factor loci. We apply the method on CD4+ cells obtained from rheumatoid arthritis and multiple sclerosis patients and show that synovial fluid infiltrating CD4+ T cells are committed toward both Th1 and regulatory T cell phenotype, whereas the Th2 response is suppressed. Furthermore, we show that the IL-17A gene is regulated by promoter methylation and that Th17 commitment is not a common feature in the inflamed joints of rheumatoid arthritis patients. We conclude that the method described in this paper allows for accurate profiling of Th lineage commitment in ex vivo-isolated CD4+ T cells.

Trough s-infliximab and antibodies towards infliximab in a cohort of 79 IBD patients with maintenance infliximab treatment

BACKGROUND AND AIMS The anti-TNF antibody infliximab is effective in inducing remission in Crohns disease as well as in ulcerative colitis and many patients are treated for several years with sustained clinical remission. However, the role of monitoring s-infliximab and antibodies towards infliximab during maintenance treatment remains unclear. Our aim was to correlate serum drug levels and antibodies to clinical activity, CRP, albumin and concomitant immunosuppression in a cohort on maintenance infliximab treatment. METHODS We included 79 patients with Crohns disease or ulcerative colitis who had responded to infliximab and received maintenance treatment (4-69 infusions) in this retrospective study. Infliximab levels and antibodies towards the drug were analyzed with in-house-developed ELISA assays. RESULTS The mean s-infliximab was significantly higher in patients in remission (4.1μg/mL) as compared with disease flare (mean 1.8μg/mL); p<0.001. The s-infliximab showed a significant negative correlation with Harvey-Bradshaw index (r=-0.21; p<0.05). Serum-infliximab progressively decreased with the number of accumulated infusions (p<0.05). In patients with undetectable trough levels, 55% of the patients with concomitant immunosuppressive were positive for antibodies against infliximab, as compared with 94% of patients on monotherapy. Patients with undetectable serum-infliximab were in clinical remission at 25% of the visits. CONCLUSIONS The trough level 4.1μg/mL may serve as cut-off for clinical remission. Drug trough levels decreased during treatment and almost all patients with undetectable s-infliximab and monotherapy had developed antibodies against the drug.

Long-term effectiveness of vedolizumab in inflammatory bowel disease : a national study based on the Swedish National Quality Registry for Inflammatory Bowel Disease (SWIBREG)

Abstract Objectives: Clinical trials have demonstrated the efficacy of vedolizumab in inflammatory bowel disease (IBD). However, these findings may not reflect the clinical practice. Therefore, we aimed to describe a vedolizumab-treated patient population and assess long-term effectiveness. Materials and methods: Patients initiating vedolizumab between 1 June 2014 and 30 May 2015 were identified through the Swedish National Quality Registry for IBD. Prospectively collected data on treatment and disease activity were extracted. Clinical remission was defined as Patient Harvey Bradshaw index <5 in Crohn’s disease (CD) and Patient Simple Clinical Colitis Activity index <3 in ulcerative colitis (UC). Results: Two-hundred forty-six patients (147 CD, 92 UC and 7 IBD-Unclassified) were included. On study entry, 86% had failed TNF-antagonist and 48% of the CD patients had undergone ≥1 surgical resection. After a median follow-up of 17 (IQR: 14–20) months, 142 (58%) patients remained on vedolizumab. In total, 54% of the CD- and 64% of the UC patients were in clinical remission at the end of follow-up, with the clinical activity decreasing (p < .0001 in both groups). Faecal-calprotectin decreased in CD (p < .0001) and in UC (p = .001), whereas CRP decreased in CD (p = .002) but not in UC (p = .11). Previous anti-TNF exposure (adjusted HR: 4.03; 95% CI: 0.96–16.75) and elevated CRP at baseline (adjusted HR: 2.22; 95% CI: 1.10–4.35) seemed to be associated with discontinuation because of lack of response. Female sex was associated with termination because of intolerance (adjusted HR: 2.75; 95% CI: 1.16–6.48). Conclusion: Vedolizumab-treated patients represent a treatment-refractory group. A long-term effect can be achieved, even beyond 1 year of treatment.

HLA-DRhi and CCR9 Define a Pro-Inflammatory Monocyte Subset in IBD

OBJECTIVES:It has been demonstrated that circulating monocytes relocate to the intestinal mucosa during intestinal inflammation, but the phenotype and inflammatory mechanisms of these monocytes remain poorly understood. Here, we have investigated blood monocytes expressing high levels of HLA-DR and CCR9 in patients with inflammatory bowel disease (IBD).METHODS:Fifty-one patients with mild to severe ulcerative colitis (UC; n=31; UC-DAI 3–12) or Crohn’s disease (CD; n=20; Harvey–Bradshaw indices (HBI) 2–16) were included together with 14 controls, during IBD therapy for four consecutive weeks. The frequency of CD14+HLA-DRhi monocytes was monitored weekly in peripheral blood, using flow cytometry. The surface phenotype and cytokine profile of these monocytes were established using flow cytometry and real-time PCR. Clinical parameters were assessed weekly in all patients.RESULTS:The frequency of circulating CD14+HLA-DRhi monocytes was significantly higher in IBD patients with moderate to severe disease compared with healthy controls (P<0.001). During treatment with corticosteroids and granulocyte/monocyte apheresis, the proportion of circulating CD14+HLA-DRhi monocytes was significantly reduced. CD14+HLA-DRhi monocytes produced high levels of inflammatory mediators, such as tumor necrosis factor (TNF)-α, and expressed the gut-homing receptor CCR9. Furthermore, we found that the CCR9 ligand, CCL25/TECK, was expressed at high levels in the colonic mucosa in IBD patients with active disease.CONCLUSIONS:CD14+HLA-DRhi blood monocytes were increased in patients with active IBD. These monocytes exhibit a pro-inflammatory, gut-homing phenotype with regard to their TNF-α production and expression of CCR9. Our results suggest that these monocytes are important in mediating intestinal inflammation, and provide potential therapeutic targets in IBD.

Serum-Infliximab Trough Levels in 45 Children with Inflammatory Bowel Disease on Maintenance Treatment

The role of trough serum infliximab (s-IFX) and antibodies toward IFX (ATI) during maintenance treatment remains unclear in children. The aim of the present study was to investigate trough s-IFX and ATI to identify any correlation with inflammatory activity and clinical response in a pediatric inflammatory bowel disease (IBD) cohort. We investigated the s-IFX trough levels in pediatric IBD patients (n = 45) on maintenance IFX treatment. Ninety-three blood samples were collected and demographics, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and albumin were recorded. The mean s-IFX trough level was 5.2 µg/mL. The mean trough s-IFX level was significantly higher in the samples taken during remission (7.2 µg/mL) compared to active disease (4.5 µg/mL, p < 0.05). The trough s-IFX levels correlated with ESR, CRP, and albumin. S-IFX was undetectable in eight of the patients, all with positive ATI and active disease. Surprisingly, clinical and biochemical remission was observed at only 26 of the 93 visits. The correlation between dose variations and changes in trough s-IFX was not evident. In line with studies in adults, the s-IFX trough levels correlated with response to infliximab.

Safety and Immunogenicity of Inactivated Varicella-Zoster Virus Vaccine in Adults With Autoimmune Disease: A Phase 2, Randomized, Double-Blind, Placebo-Controlled Clinical Trial

Background Immunogenicity and safety of inactivated zoster vaccine (ZVIN) were evaluated in adults with autoimmune disease. Methods Adults with autoimmune disease treated with immunosuppressive therapy (biologic or nonbiologic) were randomized to receive 4 doses of ZVIN, ZVIN containing a higher quantity of antigen, or placebo. To measure varicella-zoster virus (VZV)-specific immune responses using glycoprotein enzyme-linked immunosorbent assay (gpELISA) and interferon-gamma enzyme-linked immunospot (IFN-γ ELISPOT), blood samples were collected at baseline, post-doses 2, 3, and 4. The primary hypothesis was that ZVIN would elicit significant VZV-specific immune responses, measured by gpELISA or ELISPOT, at approximately 28 days post-dose 4. Safety and tolerability was assessed through 28 days post-dose 4. Results ZVIN elicited a statistically significant VZV-specific immune response approximately 28 days post-dose 4, measured by gpELISA (estimated geometric mean fold rise from baseline [GMFR] = 1.6 [95% confidence interval [CI], 1.4,1.7], P value < .0001) and IFN-γ ELISPOT (estimated GMFR = 2.0 [95% CI, 1.6,2.6], P value < .0001); both results met the prespecified success criterion. Overall, 57% (164/289) of all ZVIN and 21% (13/62) of placebo recipients reported ≥1 injection-site adverse events (AEs), and 52% (149/289) and 47% (29/62) reported ≥1 systemic AEs, respectively. Eight ZVIN and 1 placebo recipients experienced serious AEs, including 2 events (ZVIN group) determined by the investigator to be vaccine related (keratitis; amnesia). Overall frequency of AEs decreased with subsequent doses of vaccine. Conclusions In adults with autoimmune disease, ZVIN was well tolerated and elicited statistically significant VZV-specific immune responses approximately 28 days post-dose 4, measured by gpELISA and IFN-γ ELISPOT. Clinical Trials Registration NCT01527383.

The sentinel node technique is useful for studies of intestinal immunology in inflammatory bowel disease patients

Objective Mesenteric lymph nodes may play a crucial role in the pathogenesis of human inflammatory bowel disease. We have used the sentinel node technique to analyze mesenteric lymph nodes draining inflammatory lesions sentinel nodes and corresponding site of inflamed bowel in patients with ulcerative colitis or Crohns disease. Methods Thirty-two patients undergoing surgical treatment of inflammatory bowel disease were included. Sentinel nodes were identified intraoperatively. The T cells were harvested from the mesenteric lymph nodes and characterized by flow cytometry. Results The distribution of CD4+CD62L+ (homing-marked) and CD4+CD69+ (activated) T cells was studied in mesenteric lymph nodes draining inflammation, nodes draining normal intestine, blood, and mucosa. The turnover of T cells was markedly increased in the lymph nodes connected to inflammatory segments. The immunologic activity of the sentinel lymph nodes correlated with the degree of intestinal inflammation. Conclusion Mesenteric sentinel nodes provide important information about locoregional immunology and pathogenesis in inflammatory bowel disease.

Randomised, Double-blind, Placebo-controlled Trial of CCR9-targeted Leukapheresis Treatment of Ulcerative Colitis Patients

Background and Aims Ulcerative colitis patients display increased numbers of circulating pro-inflammatory monocyte human leukocyte antigen-DR [HLA-DRhi] monocytes expressing high levels of the gut-homing C-C chemokine receptor 9 [CCR9] and tumour necrosis factor [TNF]-α. The aim of this first-in-human, double-blind, randomised, placebo-controlled trial was to evaluate selective removal of circulating CCR9-expressing monocytes by leukapheresis in patients with moderate to severe ulcerative colitis, with regards to safety, tolerability, and immunological response. Methods Patients with ulcerative colitis were treated every second day with leukapheresis during five sessions with a C-C chemokine ligand 25 [CCL25; CCR9 ligand] column or a placebo column. Results No major safety concerns were raised and the procedure was well tolerated. Pro-inflammatory HLA-DRhi cells decreased significantly in the active treatment group [p = 0.0391] whereas no statistically significant change was seen in the placebo group [p = 0.4688]. There was a significant decrease of HLA-DRhi monocytes in the active group compared with the placebo group when corrected for the imbalance in weight between the groups [p = 0.0105]. Mayo score decreased in the active group [p = 0.0156] whereas the change in the placebo group was not significant [p = 0.1250]. Mayo score ≤ 3 was observed in five out of 14 patients [35.7%] in the active group compared with one out of eight [12.5%] receiving placebo. The number of responders in the active treatment group was eight out of 14 patients [57.1%], whereas in the corresponding placebo group three out of eight patients [37.5%] responded to placebo. A dose-response correlation was observed between the blood volume processed and clinical outcome. Conclusion This clinical induction trial using CCL25-tailored leukapheresis demonstrates a safe and effective removal of activated monocytes with a clinical effect in patients with ulcerative colitis.

Peripheral blood leukocytes express differential chemokine receptor profiles in irritable bowel syndrome, Crohn’s disease and ulcerative colitis

Background Inflammatory bowel disease (IBD) is a collective term, generally referring to ulcerative colitis (UC) and Crohn’s disease (CD). Distinguishing between the sub-diagnoses of IBD as well as differential diagnoses such as irritable bowel syndrome (IBS) poses a challenge to the gastroenterologist because of the large overlap in clinical features. Making a correct diagnosis is important because treatment alternatives differ greatly between the two entities. Still, many patients are mis-diagnosed every year, and thus, there is need for developing improved diagnostic methods. In this study, we have used flow cytometry to investigate the expression of 20 chemokine receptors on the CD3+, CD14+ and CD19+ peripheral blood leukocyte populations in patients with UC, CD and IBS.