Mucosal α4ß7+ Lymphocytes and MAdCAM+ Venules Predict Response to Vedolizumab in Ulcerative Colitis.

BACKGROUND: Therapeutic strategies for patients with ulcerative colitis (UC) are based on patient- and disease-related factors in combination with drug characteristics but fail to predict success in individual patients. A considerable proportion of UC patients do not respond to the biological vedolizumab. Therefore, pretreatment biomarkers for therapeutic efficacy are urgently needed. Mucosal markers related to the integrin-dependent T lymphocyte homing could be potent predictors. METHODS: We prospectively included 21 biological- and steroid-naive UC patients with moderate-to-severe disease activity planned to escalate therapy to vedolizumab. At week 0, before initiating treatment, colonic biopsy specimens were obtained for immunophenotyping and immunohistochemistry. Clinical and endoscopic disease activity were determined at week 16 after 4 infusions of vedolizumab. In addition, we retrospectively included 5 UC patients who were first treated with anti-tumor necrosis factor α before receiving vedolizumab to compare with biological-naive patients. RESULTS: Abundance of α4ß7 on more than 8% of all CD3+ T lymphocytes in colonic biopsies at baseline was predictive for responsiveness to vedolizumab (sensitivity 100%, specificity 100%). The threshold for the proportion of MAdCAM-1+ and PNAd+ of all venules in the biopsies predictive for responsiveness to vedolizumab was ≥2.59% (sensitivity 89%, specificity 100%) and ≥2.41% (sensitivity 61%, specificity 50%), respectively. At week 16, a significant decrease of α4ß7+CD3+T lymphocytes was demonstrated in responders (18% [12%-24%] to 8% [3%-9%]; P = .002), while no difference was seen in nonresponders (4% [3%-6%] to 3%; P = .59). CONCLUSIONS: UC responders to vedolizumab have a higher percentage of α4ß7+CD3+ T lymphocytes and a higher proportion of MAdCAM-1+ venules in colonic biopsies than nonresponders before initiating therapy. Both analyses could be promising predictive biomarkers for therapeutic response and may lead to more patient tailored treatment in the future.

Dose-Escalating (50-500 mg) Gluten Administration Leads to Detectable Gluten-Immunogenic-Peptides in Urine of Patients with Coeliac Disease Which Is Unrelated to Symptoms, a Placebo Controlled Trial.

BACKGROUND: To determine the applicability and sensitivity of a urine self-test to detect gluten-immunogenic-peptides (GIP) in daily-life for patients with coeliac disease and correlate the test results with reported symptoms. METHODS: We performed a prospective double-blinded placebo-controlled study, including adults with coeliac disease adhering to a strictly gluten-free diet. Patients were administered gluten in test-cycles of ascending doses of 50, 100, 200, and 500 mg alternated with placebo. Urine portions from 2, 5-17 h after the ingestion were collected and analyzed for GIP using the iVYCHECK-GIP-Urine rapid lateral flow test. Patients completed a diary mapping symptoms (nausea, bloating, diarrhea, abdominal pain, and lower level of energy). RESULTS: We enrolled 15 patients and 7 received all 4 cycles with increasing gluten dosing. GIP was detected from urine in 47% of the patients receiving 50 mg gluten and in 86% with 500 mg gluten. We detected GIP in 20-50% of urine samples after placebo. There was no correlation between symptoms, gluten administration and/or GIP in urine. CONCLUSIONS: Gluten intake, even with a dose as low as 50 mg, leads to detectable urinary GIP concentrations. There is no correlation of coeliac disease ascribed symptoms with detection of urinary GIP.

Short Communication: Prolonged COVID-19 Infection in a Patient with Newly Diagnosed HIV/AIDS.

A 38-year-old male patient presented to the emergency department with fever and dyspnea. Hospitalization was warranted and soon coronavirus disease 2019 (COVID-19) was diagnosed based on a positive SARS-CoV-2-PCR. Over the following weeks his condition gradually worsened, leading to admission at the intensive care unit. Because of unexplained weight loss before admission, a HIV screening was performed. HIV was confirmed and additional tests showed an undetectable CD4+ T cell count, alongside a number of co-infections. Convalescent plasma therapy, which has been shown to be effective in severe humoral immunodeficiency was tried, but was not effective. One week after the HIV diagnosis, antiretroviral therapy was started and finally, 3 months after the initial positive test and after partial recovery of cellular immunity, the COVID-19 virus was cleared. In the end, the patient made a full recovery. Our case demonstrates a prolonged COVID-19 infection in a patient with undiagnosed HIV with severely impaired cellular immunity.

Homeostatic Function and Inflammatory Activation of Ileal CD8+ Tissue-Resident T Cells Is Dependent on Mucosal Location.

BACKGROUND & AIMS: Tissue-resident memory T (Trm) cells, both of the CD4 and CD8 lineage, have been implicated in disease flares in inflammatory bowel disease. However, data are conflicting regarding the profile of human CD8+ Trm cells, with studies suggesting both proinflammatory and regulatory functions. It is crucial to understand the functional profile of these cells in the context of (new) therapeutic strategies targeting (trafficking of) gut Trm cells. METHODS: Here, we performed imaging mass cytometry, flow cytometry, and RNA-sequencing to compare lamina propria and intraepithelial CD103+/-CD69+CD8+ Trm cells in healthy control subjects and patients with active ileal Crohn's disease. RESULTS: Our data revealed that lamina propria CD103+CD69+CD8+ T cells have a classical Trm cell profile with active pathways for regulating cell survival/death and cytokine signaling, whereas intraepithelial CD103+CD69+CD8+ T cells display tightly regulated innate-like cytotoxic profile. Furthermore, within lamina propria CD8+CD103- Trm cells, an Itgb2+GzmK+KLRG1+ population distinct from CD103+ CD8+ Trm cells is found. During chronic inflammation, especially intraepithelial CD103+CD69+CD8+ T cells displayed an innate proinflammatory profile with concurrent loss of homeostatic functions. CONCLUSIONS: Altogether, these compartmental and inflammation-induced differences indicate that therapeutic strategies could have a different impact on the same immune cells depending on the local compartment and presence of an inflammatory milieu, and should be taken into account when investigating short- and long-term effects of new gut T cell-targeting drugs.

Distribution of mucosal PD-1 expressing T cells in patients with colitis of different etiologies.

BACKGROUND: Immunotherapy, targeting programmed death-1 (PD-1) enhances antitumor T-cell activity in patients with malignancies. Blocking PD-1 or its ligand may lead to fulminant colitis as serious adverse event in these patients. Since little is known of the presence and role of PD-1+T cells in colitis of different etiologies, we determined PD-1+T cells in mucosal specimens of patients with inflammatory bowel disease, infectious colitis (InfC), immunotherapy-related colitis (ImC) and healthy controls (HC). METHODS: Newly diagnosed patients with ulcerative colitis (UC, n = 73), Crohn's disease (CD, n = 50), InfC (n = 5), ImC (n = 8) and HC (n = 8) were included. Baseline inflamed colonic biopsies were studied with immunohistochemistry and flowcytometry. RESULTS: Using immunohistochemistry, PD-1 was not present on lymphocytes in the epithelium of all patients, nor in HC. The percentage PD-1+ of all lymphocytes in the lamina propria was 40% in UC, 5% in InfC, 3% in ImC and 0% in HC. Flowcytometry showed significant higher percentages of PD-1+T cells in inflamed biopsy specimens of UC patients (22%) compared to all other groups: CD patients (13%), InfC (12%), ImC (5%) and HC (6%). CONCLUSION: There are relevant differences in distribution and frequencies of mucosal PD-1+ T-cell subsets in patients with UC, CD, InfC and ImC, supporting the hypothesis that these types of colitis are driven by different immunological pathways. The increased numbers of PD-1+ and PD-L1+ lymphocytes in the colonic mucosa of UC patients suggest that the PD-1/PD-L1 pathway might be more activated in UC than in CD.

Testing for IgA anti-tissue transglutaminase in routine clinical practice: Requesting behaviour in relation to prevalence of positive results.

OBJECTIVE: Due to the high awareness of coeliac disease and improvement of serological tests, the number of requested laboratory tests has increased substantially over the years. In the current study we have evaluated the requesting behaviour of distinct clinical disciplines in relation to the prevalence of positive results and in the context of existing guidelines. METHODS: Data were retrospectively extracted from the laboratory information system over a time-span of 5 years in a tertiary hospital and compared with the situation in a secondary hospital. RESULTS: Data reveal that for initial testing (n=18,183) the percentage positive results for IgA anti-TTG is <2%. Paediatricians have a slightly higher percentage of seropositive results (2.4-4.0%). Early confirmation (<2 months) of positive results by IgA anti-endomysium antibodies in an independent sample is only performed in a minority of paediatric patients. The majority of positive patients, however, have follow-up measurements (<14 months) in order to examine compliance to a gluten-free diet. Interestingly, initial requests for paediatric patients reveal an equal distribution between boys and girls, while in adult patients there is a two times preponderance of requests in female patients, similar to the female/male ratio in patients with positive results, being either paediatric or adult patients. CONCLUSION: Although laboratory testing for coeliac disease may be primarily used to exclude the disease, it is evident that the percentage positive results for IgA anti-TTG is extremely low. This may indicate that the clinical manifestations that warrant testing, should be further specified in order to increase the pre-test probability. As the specific serology is important to bypass a biopsy in the diagnosis of coeliac disease according to the paediatric guideline, the confirmation in an independent sample needs to get more attention.

Raising the Cut-Off Level of Anti-Tissue Transglutaminase Antibodies to Detect Celiac Disease Reduces the Number of Small Bowel Biopsies in Children with Type 1 Diabetes: A Retrospective Study.

OBJECTIVE: To study the optimal cut-off value for anti-tissue transglutaminase type 2 IgA antibodies (TG2A) in serum to select for diagnostic small bowel biopsies for celiac disease in children with type 1 diabetes mellitus. STUDY DESIGN: Children with type 1 diabetes mellitus with elevated TG2A titers and duodenal biopsies performed during the course of their diabetes treatment were included. Anti-endomysial antibodies were recorded if present. The optimal TG2A cut-off value, expressed as the ratio between obtained value and upper limit of normal (ULN), was determined using receiver operating characteristic curve analysis and compared with the cut-off value used in the European Society for Pediatric Gastroenterology, Hepatology and Nutrition guidelines in terms of sensitivity, specificity, positive and negative predictive value. RESULTS: We included 63 children. The optimal cut-off value for performing biopsies is demonstrated to be 11 times the ULN. Raising the cut-off value from 3 times the ULN to 11 times the ULN changed sensitivity from 96% to 87% and increased specificity from 36% to 73%, increased the positive predictive value from 88% to 94% and lowered negative predictive value from 67% to 53%. The percentage of normal histology was decreased from 12% to 6%. CONCLUSIONS: Increasing the TG2A cut-off value for performing duodenal biopsies in children with type 1 diabetes mellitus and suspected celiac disease leads to a substantial reduction of unnecessary biopsies. We advocate to adapt the European Society for Pediatric Gastroenterology, Hepatology and Nutrition 2012 guidelines for this group of children, including monitoring patients with TG2A levels of less than 11 times the ULN over time.

Intestinal CD103+CD4+ and CD103+CD8+ T-Cell Subsets in the Gut of Inflammatory Bowel Disease Patients at Diagnosis and During Follow-up.

BACKGROUND: The integrin CD103 is proposed to be a potential therapeutical target in inflammatory bowel disease (IBD), as it can form a heterodimeric integrin with ß7 (Etrolizumab, anti-ß7 integrin) on epithelial T cells. Therefore, we aimed to study the frequencies of different intestinal CD103+T-cell subsets, both CD4+ and CD8+, in newly diagnosed, untreated IBD patients at baseline and during follow-up, compared with healthy controls. METHODS: Intestinal biopsies from inflamed segments during colonoscopy and peripheral blood samples were prospectively taken from IBD patients at diagnosis and during follow-up. Blood and single cell suspensions from biopsies were analyzed for CD103+ T-cell subpopulations by flow cytometry and expressed as median percentages of the total T-cell population. RESULTS: In total, 75 Crohn's disease (CD) patients, 49 ulcerative colitis (UC) patients, and 16 healthy controls were included. At presentation, IBD patients displayed lower percentages of CD103+T-cell subsets in inflamed biopsies: 3% (1 to 5) CD103+CD4+ in IBD vs 5% (5 to 7) in healthy controls (P = 0.007) and 9% (4 to 15) CD103+CD8+ compared with 42% (23 to 57) in healthy controls (P = 0.001). The majority of intestinal T cells was composed of CD103-CD4+ T cells (65% [52 to 74]) in IBD compared with 30% (21 to 50) in healthy controls (P = 0.001). In patients with endoscopic remission during follow-up (n = 27), frequencies of CD103+ and CD103-T-cell subsets were comparable with healthy controls. CONCLUSION: At diagnosis, active inflammation in IBD was associated with decreased percentages of both CD103+CD4+ and CD103+CD8+T-cell subsets in colon and ileum biopsies. In active disease during follow-up, these T-cell populations remained low but increased in remission to values comparable with healthy controls. A shift toward more CD103-T cells was observed during active inflammation.

mTOR inhibition by metformin impacts monosodium urate crystal-induced inflammation and cell death in gout: a prelude to a new add-on therapy?

OBJECTIVE: Gout is the most common inflammatory arthritis worldwide, and patients experience a heavy burden of cardiovascular and metabolic diseases. The inflammation is caused by the deposition of monosodium urate (MSU) crystals in tissues, especially in the joints, triggering immune cells to mount an inflammatory reaction. Recently, it was shown that MSU crystals can induce mechanistic target of rapamycin (mTOR) signalling in monocytes encountering these crystals in vitro. The mTOR pathway is strongly implicated in cardiovascular and metabolic disease. We hypothesised that inhibiting this pathway in gout might be a novel avenue of treatment in these patients, targeting both inflammation and comorbidities. METHODS: We used a translational approach starting from ex vivo to in vitro and back to in vivo. RESULTS: We show that ex vivo immune cells from patients with gout exhibit higher expression of the mTOR pathway, which we can mimic in vitro by stimulating healthy immune cells (B lymphocytes, monocytes, T lymphocytes) with MSU crystals. Monocytes are the most prominent mTOR expressers. By using live imaging, we demonstrate that monocytes, on encountering MSU crystals, initiate cell death and release a wide array of proinflammatory cytokines. By inhibiting mTOR signalling with metformin or rapamycin, a reduction of cell death and release of inflammatory mediators was observed. Consistent with this, we show that patients with gout who are treated with the mTOR inhibitor metformin have a lower frequency of gout attacks. CONCLUSIONS: We propose mTOR inhibition as a novel therapeutic target of interest in gout treatment.

Frequencies and clinical associations of myositis-related antibodies in The Netherlands: A one-year survey of all Dutch patients.

Idiopathic inflammatory myopathies (IIM) are a heterogeneous group of connective tissue diseases, collectively known as myositis. Diagnosis of IIM is challenging while timely recognition of an IIM is of utter importance considering treatment options and otherwise irreversible (severe) long-term clinical complications. With the EULAR/ACR classification criteria (2017) considerable advancement has been made in the diagnostic workup of IIM. While these criteria take into account clinical parameters as well as presence of one autoantibody, anti-Jo-1, several autoantibodies are associated with IIM and are currently evaluated to be incorporated into classification criteria. As individual antibodies occur at low frequency, the development of line blots allowing multiplex antibody analysis has improved laboratory diagnostics for IIM. The Euroline myositis line-blot assay (Euroimmun) allows screening and semi-quantitative measurement for 15 autoantibodies, i.e. myositis specific antibodies (MSA) to SRP, EJ, OJ, Mi-2α, Mi-2ß, TIF1-γ, MDA5, NXP2, SAE1, PL-12, PL-7, Jo-1 and myositis associated antibodies (MAA) to Ku, PM/Scl-75 and PM/Scl-100. To evaluate the clinical significance of detection and levels of these autoantibodies in the Netherlands, a retrospective analysis of all Dutch requests for extended myositis screening within a 1 year period was performed. A total of 187 IIM patients and 632 non-IIM patients were included. We conclude that frequencies of MSA and MAA observed in IIM patients in a routine diagnostic setting are comparable to cohort-based studies. Weak positive antibody levels show less diagnostic accuracy compared to positive antibody levels, except for anti-NXP2. Known associations between antibodies and skin involvement (anti-MDA5, anti-TIF1-γ), lung involvement (anti-Jo-1), and malignancy (anti-TIF1-γ) were confirmed in our IIM study population. The availability of multiplex antibody analyses will facilitate inclusion of additional autoantibodies in clinical myositis guidelines and help to accelerate diagnosing IMM with rare but specific antibodies.

Intestinal T Cell Profiling in Inflammatory Bowel Disease: Linking T Cell Subsets to Disease Activity and Disease Course.

INTRODUCTION: A dysregulated intestinal T cell response is presumed in patients with inflammatory bowel disease [IBD]. In this longitudinal study, we investigated the changes in intestinal T lymphocyte subsets in IBD at first presentation and over time during endoscopic active or inactive disease, and relate them to disease activity and outcome. METHODS: We included 129 newly diagnosed patients (87 Crohn's disease [CD], 42 ulcerative colitis [UC]) and 19 healthy controls [HC]. Follow-up biopsy specimens were analysed from 70 IBD patients. Immunophenotyping of specimens was performed by flow cytometry identifying lymphocyte subpopulations. RESULTS: IBD patients at diagnosis displayed higher percentages of CD4 T+ cells, Tregs, and central memory T cells [TCM] and with lower percentages of CD8 and CD103 T lymphocytes than HC. Follow-up specimens of patients with endoscopic inactive disease showed T cell subset recovery comparable to HC. Endoscopic active disease at follow-up coincided with T cell subsets similar to those at diagnosis. In UC, lower baseline percentages of CD3 cells was associated with milder disease course without the need of an immunomodulator, whereas in CD, higher baseline percentages of CD4 and Tregs were associated with complicated disease course. CONCLUSIONS: The intestinal T cell infiltrate in IBD patients with active endoscopic disease is composed of increased percentages of CD4+ T cells, Tregs, and TCM, with lower percentages of CD8+ T cells and CD103+ T cells, compared with HC and endoscopic inactive IBD. Baseline percentages of CD3, CD4, and Tregs were associated with disease outcome. Further research is needed to demonstrate the predictive value of these lymphocyte subsets.

The value of serum antibodies in differentiating inflammatory bowel disease, predicting disease activity and disease course in the newly diagnosed patient.

BACKGROUND: Data on serum antibodies in untreated adult inflammatory bowel disease (IBD) patients at diagnosis are scarcely available, and results on the stability of antibody presence over time are inconsistent. Our aim was to investigate antibodies in newly diagnosed, untreated IBD patients in relation to disease phenotype and course. Furthermore, we analyzed antibody presence over time. METHODS: Baseline anti-Saccharomyces cerevisiae antibodies (ASCA), anti-chitobioside carbohydrate antibodies (ACCA), anti-laminaribioside carbohydrate antibodies (ALCA) and anti-mannobioside carbohydrate antibodies (AMCA) were measured with enzyme-linked immunosorbent assays and perinuclear anti-neutrophilic cytoplasmic antibodies (pANCA) was measured by indirect immunofluorescence in serum of 120 untreated IBD patients at diagnosis and 19 healthy controls. Antibodies were related to disease outcomes. Serial measurements were available in 71 patients. RESULTS: The combination of pANCA and ASCA enabled good discrimination between UC and CD (p = .004). Antibody presence was relatively stable over time, even though there were significant changes in concentrations. There was a trend towards larger fluctuations in concentration with immunosuppressive medication. Baseline pANCA in UC patients correlated with calprotectin values (rho = .545, p = .019) and change in pANCA status over time was associated with disease activity at that moment. No associations were found with antibodies at diagnosis and disease outcomes. CONCLUSION: Antibody profiles at diagnosis support the distinction between CD and UC. Anti-glycan antibodies are reasonably stable over time, but may fluctuate under the influence of immunosuppressive treatment which may explain the inconsistency in findings hitherto. The appearance or disappearance of pANCA antibodies during follow-up correlated with disease activity in UC and may be used in disease monitoring.

Candidate Serum Markers in Early Crohn's Disease: Predictors of Disease Course.

BACKGROUND AND AIMS: More than half of patients with Crohn's disease [CD] develop disease complications requiring aggressive medical therapy or surgery over time. However, predicting disease course and treatment response remains difficult. We therefore identified distinctive serum analytes associated with disease activity and course in newly diagnosed, untreated patients at presentation and during their follow-up. METHODS: In a pilot study, a multiplex immunoassay analysis on 36 markers was performed on serum from 20 CD patients at the time of primary diagnosis following endoscopic evaluation. The 12 most potent markers associated with disease activity, phenotype and course were analysed in a consecutive cohort of 66 CD patients at diagnosis and follow-up [n = 39]. A healthy control group [n = 20] was included as a reference. RESULTS: CD patients had higher baseline levels of sTNF-R2 [p = 0.001], sIL-2R [p = 0.0001], and MMP-1 [p = 0.001] compared with healthy controls. Serial measurements revealed that these three analytes dropped statistically significantly from baseline level during remission and were high during exacerbation. Great decline of sTNF-R1 levels was found during remission, with 6.7-fold lower levels than in healthy controls [p = 0.015]. Patients who did not respond to initial prednisone treatment had higher baseline levels of sTNF-R2 [p = 0.001]. Patients experiencing relapses during follow-up had lower baseline sTNF-R2 and VCAM levels compared with patients with long-lasting remission. CONCLUSIONS: In a large cohort of newly diagnosed untreated CD patients, we identified candidate serum markers [sTNF-R1, sTNF-R2, sIL-2R, and MMP-1] associated with disease activity. Furthermore, sTNF-R2 was associated with prednisone response and, together with VCAM, with long-lasting remission.

Effects of nongenetic factors on immune cell dynamics in early childhood: The Generation R Study.

BACKGROUND: Numbers of blood leukocyte subsets are highly dynamic in childhood and differ greatly between subjects. Interindividual variation is only partly accounted for by genetic factors. OBJECTIVE: We sought to determine which nongenetic factors affect the dynamics of innate leukocytes and naive and memory lymphocyte subsets. METHODS: We performed 6-color flow cytometry and linear mixed-effects modeling to define the dynamics of 62 leukocyte subsets from birth to 6 years of age in 1182 children, with 1 to 5 measurements per subject. Subsequently, we defined the effect of prenatal maternal lifestyle-related or immune-mediated determinants, birth characteristics, and bacterial/viral exposure-related determinants on leukocyte subset dynamics. RESULTS: Functionally similar leukocyte populations were grouped by using unbiased hierarchical clustering of patterns of age-related leukocyte dynamics. Innate leukocyte numbers were high at birth and predominantly affected by maternal low education level. Naive lymphocyte counts peaked around 1 year, whereas most memory lymphocyte subsets more gradually increased during the first 4 years of life. Dynamics of CD4+ T cells were predominantly associated with sex, birth characteristics, and persistent infections with cytomegalovirus (CMV) or EBV. CD8+ T cells were predominantly associated with CMV and EBV infections, and T-cell receptor γδ+ T cells were predominantly associated with premature rupture of membranes and CMV infection. B-cell subsets were predominantly associated with sex, breast-feeding, and Helicobacter pylori carriership. CONCLUSIONS: Our study identifies specific dynamic patterns of leukocyte subset numbers, as well as nongenetic determinants that affect these patterns, thereby providing new insights into the shaping of the childhood immune system.

The Complexity of alpha E beta 7 Blockade in Inflammatory Bowel Diseases.

Monoclonal antibodies targeting integrins are emerging as new treatment option in inflammatory bowel diseases. Integrins are molecules involved in cell adhesion and signalling. After the successful introduction of anti-α4ß7, currently anti-ß7 is under evaluation in a phase three trial. Anti-ß7 blocks both α4ß7/MAdCAM-1 and αEß7/E-cadherin interaction, targeting both the homing to and the retention in the gut of potential pathological T cells. Since the physiological and potential pathological roles of immune cells expressing αEß7 are less distinct than of those expressing α4ß7, an overview of the current state of knowledge on αEß7 in mice and humans in both health and inflammatory bowel diseases is presented here, also addressing the potential consequences of anti-ß7 treatment.