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Background: Mycobacterium chelonae is a rare cause of infective endocarditis that is difficult to diagnose and treat. After we found M chelonae in a series of patients, we aimed to investigate its role in cardiovascular prosthesis dysfunction and contamination of bioprostheses as a possible cause of infection., Methods: In this collaborative microbiological study, we report on nine patients treated in three cardiovascular surgical departments in Germany, who were found to have M chelonae infection after receiving BioIntegral bioprostheses. We performed fluorescence in-situ hybridisation (FISH) combined with broad-range 16S rRNA gene amplification and sequencing (FISHseq) on samples of native cardiovascular tissue and explanted bioprosthetic material, as well as on 12 unused BioIntegral prostheses. We confirmed FISHseq findings with histological examination by staining for acid-fast bacilli, and M chelonae was differentiated from M abscessus by molecular techniques., Findings: Between Dec 1, 2020, and Feb 28, 2022, we identified M chelonae in BioIntegral bioprostheses from three initial patients treated in Berlin that were explanted following dysfunction or suspected endocarditis, visualising morphologically intact FISH-positive mycobacteria. Despite negative mycobacterial culture, we also detected M chelonae in all 12 unused BioIntegral prostheses. The competent authorities in the EU prompted an alert, leading to the identification of six additional patients between March 1, 2022, and July 31, 2023. To find other cases of M chelonae endocarditis, we reviewed the FISHseq results of 1237 cardiovascular samples that were analysed between Jan 1, 2015, and Aug 31, 2022, including 295 samples from 228 bioprostheses supplied by other manufacturers. M chelonae was only detected in six of 41 patients who had received BioIntegral products., Interpretation: Bioprostheses manufactured by BioIntegral Surgical might be colonised by M chelonae, which can lead to implant dysfunction. These infections are likely to be missed by conventional routine diagnostics and should be considered in patients with BioIntegral implants and suspected infection or dysfunction. Cases should be reported to public health and regulatory authorities. Routine safety testing of bioprostheses during manufacture should be reconsidered., Funding: German Federal Ministry of Education and Research., Competing Interests: Declaration of interests JK and AM founded and run MoKi Analytics GmbH. AM founded and runs Moter Diagnostics. These two diagnostics laboratories investigated the samples with FISHseq originally and, after finding NTM, initiated and handed over this collaborative research to all other partners., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background and Aims: Fistula formation is a major complication in Crohn's disease [CD] and the role of the immune cell compartment remains to be elucidated. Thus, we compared the immune cell compartment of CD fistula to inflammatory CD colitis using imaging mass cytometry [IMC] and immunofluorescence., Methods: A 36-marker panel including structural, functional, and lineage markers for use in IMC was established. This panel was applied to analyse paraffin-embedded CD fistula tract [n = 11], CD colitis [n = 10], and colon samples from non-inflamed controls [n = 12]. Computational methods for cell segmentation, dimensionality reduction, and cell type clustering were used to define cell populations for cell frequency, marker distribution, and spatial neighbourhood analysis. Multiplex immunofluorescence was used for higher resolution spatial analysis., Results: Analysis of cell frequencies in CD fistulas compared to CD colitis and control colonic samples revealed a significant increase in neutrophils, effector cytotoxic T cells, and inflammatory macrophages in CD fistula samples, whereas regulatory T cells were decreased. Neutrophils in CD fistula expressed significantly more matrix metalloproteinase 9 [MMP9], correlating with extracellular matrix remodelling. Neighbourhood analysis revealed a strong association between MMP9+ neutrophils and effector cytotoxic T cells in both CD fistulas and colitis., Conclusions: This study presents the first highly multiplexed single cell analysis of the immune cell compartment of CD fistulas and their spatial context. It links immune cell dynamics, particularly MMP9+ neutrophils, to extracellular matrix remodelling in CD fistulas, offering insights into the complex network of cellular interactions and potential therapeutic targets for CD complications., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Crohn’s and Colitis Organisation. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: Flow cytometry of intestinal lymphocytes is discussed to be a stronger predictor of enteropathy-associated T-cell lymphoma development in refractory celiac disease than T-cell clonality analysis., Aims: To investigate possible associations between clinical characteristics of refractory celiac disease patients and aberrant intraepithelial lymphocytes and to evaluate the accuracy of immunophenotyping for the identification of high-risk refractory celiac disease., Methods: Flow cytometry of isolated lymphocytes from duodenal biopsies of controls and celiac disease patients was performed and results were compared to clinical data., Results: Flow cytometry analysis was performed on 42 controls, 37 non-complicated celiac disease and 30 refractory celiac disease cases with or without T-cell receptor clonality. Elevated aberrant intraepithelial lymphocyte counts were significantly associated with severe malabsorption. A 15% cut-off (aberrant lymphocytes among all lymphocytes) had the best discriminatory ability to identify high-risk patients. However, this technique failed to identify some high-risk cases (sensitivity 63%, specificity 100%). The severity of malabsorption was added to the criteria for high-risk refractory celiac disease, improving the correct patients' allocation (sensitivity 100%, specificity 96%)., Conclusion: Immunophenotyping of aberrant intraepithelial lymphocytes is a good predictor for high-risk refractory celiac disease. Furthermore, adding the evaluation of malabsorption to the diagnostic assessment of refractory celiac disease optimizes accuracy., (Copyright © 2022. Published by Elsevier Ltd.)

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in children and is associated with overweight and insulin resistance (IR). Almost nothing is known about in vivo alterations of liver metabolism in NAFLD, especially in the early stages of non-alcoholic steatohepatitis (NASH). Here, we used a complex mathematical model of liver metabolism to quantify the central hepatic metabolic functions of 71 children with biopsy-proven NAFLD. For each patient, a personalized model variant was generated based on enzyme abundances determined by mass spectroscopy. Our analysis revealed statistically significant alterations in the hepatic carbohydrate, lipid, and ammonia metabolism, which increased with the degree of obesity and severity of NAFLD. Histologic features of NASH and IR displayed opposing associations with changes in carbohydrate and lipid metabolism but synergistically decreased urea synthesis in favor of the increased release of glutamine, a driver of liver fibrosis. Taken together, our study reveals already significant alterations in the NASH liver of pediatric patients, which, however, are differently modulated by the simultaneous presence of IR.

Objective: A considerable proportion of patients with irritable bowel syndrome (IBS) may be wheat-sensitive and respond to a gluten-free diet (GFD) although they do not have coeliac disease. However, a diagnostic test for wheat sensitivity (WS) is missing. Our study evaluated the diagnostic accuracy (sensitivity and specificity) of confocal laser endomicroscopy (CLE) for the identification of WS as primary outcome., Design: In this prospective, double-blind diagnostic study 147 non-coeliac patients fulfilling the Rome III criteria for IBS were tested by CLE for duodenal changes after wheat (index test), soy, yeast or milk exposure. Patients with IBS responding to 2 months of GFD were classified as having WS (reference test) using response criteria recommended by regulatory bodies for pharmaceutical trials of patients with IBS. After 2 months, CLE results were unblinded and patients were advised to exclude those food components that had led to a positive CLE reaction. The clinical response was assessed at follow-up after 6 and 12 months., Results: Of 130 patients who completed the study per protocol, 74 (56.9%) responded to GFD and were classified as WS after 2 months, and 38 of these 74 patients were correctly identified by CLE (sensitivity 51.4%; 97.5% CI: 38.7% to 63.9%). A total of 38 of 56 patients without WS were correctly identified by CLE (specificity 67.9%; 97.5% CI: 52.9% to 79.9%). At 6 months follow-up, CLE correctly identified 49 of 59 food-sensitive patients (sensitivity 83.1%; 97.5% CI: 69.9% to 91.3%) but specificity was only 32% (97.5% CI: 15.7% to 54.3%)., Conclusion: In light of the high proportion of patients with IBS responding to GFD, the diagnostic accuracy of CLE is too low to recommend widespread use of this invasive procedure., Trail Registration Number: This study was registered as clinical trial in the German Registry for Clinical Studies (DRKS00010123)., Competing Interests: Competing interests: CBo, MS and RU received research grants from Dr. Schär AG. ME obtained consulting and lecture fees from Maunakeatech, Boston Scientific, Takeda, Abbvie, Janssen. CS received research grants and lecture fees from Olympus and Pentax. AS obtained consulting fees from Abbvie, Amgen, Astellas, Biogen, Celltrion, Consal, CSL Behring, Galapagos, Gilead, Institut Allergosan, Janssen, MSD, Norgine, Pfizer Pharma, Roche, Shire and Takeda, lecture fees and travel support from Abbvie, Astellas, Celltrion, Falk Foundation, Ferring, Janssen, MSD, Recordati Pharma and Takeda, and research support from Abbvie. SD obtained lecture fees from BMS, Recordati, Amgen and Falk. BS has served as consultant for Abbvie, Arena, BMS, Boehringer, Celgene, Falk, Janssen, Lilly, Pfizer, Prometheus and Takeda and received speaker’s fees from Abbvie, CED Service, Falk, Ferring, Janssen, Novartis, Pfizer, Takeda (served as representative of the Charité)., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The SARS-CoV-2 pandemic has so far claimed over three and a half million lives worldwide. Though the SARS-CoV-2 mediated disease COVID-19 has first been characterized by an infection of the upper airways and the lung, recent evidence suggests a complex disease including gastrointestinal symptoms. Even if a direct viral tropism of intestinal cells has recently been demonstrated, it remains unclear, whether gastrointestinal symptoms are caused by direct infection of the gastrointestinal tract by SARS-CoV-2 or whether they are a consequence of a systemic immune activation and subsequent modulation of the mucosal immune system. To better understand the cause of intestinal symptoms we analyzed biopsies of the small intestine from SARS-CoV-2 infected individuals. Applying qRT-PCR and immunohistochemistry, we detected SARS-CoV-2 RNA and nucleocapsid protein in duodenal mucosa. In addition, applying imaging mass cytometry and immunohistochemistry, we identified histomorphological changes of the epithelium, which were characterized by an accumulation of activated intraepithelial CD8 + T cells as well as epithelial apoptosis and subsequent regenerative proliferation in the small intestine of COVID-19 patients. In summary, our findings indicate that intraepithelial CD8 + T cells are activated upon infection of intestinal epithelial cells with SARS-CoV-2, providing one possible explanation for gastrointestinal symptoms associated with COVID-19., (© 2021. The Author(s).)

Disseminated nocardiosis is a rare, life-threatening disease. Particularly at risk are immunocompromised patients, highlighting the crucial role of host factors. Conventional intensive antibiotic treatment has improved survival rates, but the overall prognosis of patients with disseminated nocardiosis remains unsatisfactory. In this Grand Round, we present a case of severe nocardiosis that did not respond to standard therapy. The patient's condition deteriorated when antibiotic therapy was given alone and improved substantially only after coadministration of interferon gamma. We review the literature relevant to adjuvant interferon gamma therapy of nocardiosis and discuss its potential harms and benefits. Overall, we consider such treatment as beneficial and low risk if the patient is followed-up closely. We conclude that clinicians should consider this regimen in refractory cases of severe Nocardia infection., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Objectives: Access to full texts of randomised controlled clinical trials (RCTs) is often limited, so brief summaries of studies play a pivotal role. In 2008, a checklist was provided to ensure the transparency and completeness of abstracts. The aim of this investigation was to estimate adherence to the reporting guidelines of the Consolidated Standards of Reporting Trials (CONSORT) criteria for abstracts (CONSORT-A) in RCT publications., Primary Endpoint: Assessment according to the percentage of compliance with the 16 CONSORT-A criteria per study., Materials and Methods: This study is based on a full survey (212 RCT abstracts in dental implantology, PubMed search, publication period 2014-2016, 45 journals, median impact factor: 2.328). In addition to merely documenting 'adherence' to criteria, the authors also assessed the 'complete implementation' of the requested information where possible. The collection of data was performed independently by two dentists, and a final consensus was reached. The primary endpoint was evaluated by medians and quartiles. Additionally, a Poisson regression was conducted to detect influencing factors., Results: A median of 50% (Q1-Q3: 44%-63%) was documented for the 16 criteria listed in the CONSORT-A statement. Nine of the 16 criteria were considered in fewer than 50% of the abstracts. 'Correct implementation' was achieved for a median of 43% (Q1-Q3: 31%-50%) of the criteria. An additional application of Poisson regression revealed that the number of words used had a locally significant impact on the number of reported CONSORT criteria for abstracts (incidence rate ratio 1.001, 95% CI 1.001 to 1.002)., Conclusion: Transparent and complete reporting in abstracts appears problematic. A limited word count seems to result in a reduction in necessary information. As current scientific knowledge is often not readily available in the form of publications, abstracts constitute the primary basis for decision making in clinical practice and research. This is why journals should refrain from limiting the number of words too strictly in order to facilitate comprehensive reporting in abstracts., Competing Interests: Competing interests: This study is part of the doctoral thesis written by Jeremias Loddenkemper in pursuit of a doctoral degree in dental medicine (‘Dr. med. dent.’) at Witten/Herdecke University. Furthermore, the results contained in this article have already been presented as part of a poster presentation at the 33rd DGI Congress, Hamburg (28–30 November 2019). The authors declare that they have no competing financial, professional or personal interests that might have influenced the performance or presentation of the work described in this manuscript., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in children and adolescents. About 30% of patients with NAFLD progress to the more severe condition of nonalcoholic steatohepatitis (NASH), which is typically diagnosed using liver biopsy. Liver stiffness (LS) quantified by elastography is a promising imaging marker for the noninvasive assessment of NAFLD and NASH in pediatric patients. However, the link between LS and specific histopathologic features used for clinical staging of NAFLD is not well defined. Furthermore, LS data reported in the literature can vary greatly due to the use of different measurement techniques. Uniquely, time-harmonic elastography (THE) based on ultrasound and magnetic resonance elastography (MRE) use the same mechanical stimulation, allowing us to compare LS in biopsy-proven NAFLD previously determined by THE and MRE in 67 and 50 adolescents, respectively. In the present work, we analyzed the influence of seven distinct histopathologic features on LS, including septal infiltration, bridging fibrosis, pericellular fibrosis, hepatocellular ballooning, portal inflammation, lobular inflammation, and steatosis. LS was highly correlated with periportal and lobular fibrosis as well as hepatocellular ballooning while no independent association was found for inflammation and steatosis. Based on this analysis, we propose a composite elastography score (CES) which includes the four key histopathologic features identified as mechanically relevant. Interestingly, CES-relevant histopathologic features were associated with zonal distribution patterns of pediatric NAFLD. Mechano-structural changes associated with NAFLD progression can be histopathologically staged using the CES, which is easily determined noninvasively based on LS measured by time-harmonic elastography., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Non-alcoholic fatty liver disease (NAFLD) is a significant health burden in obese children for which there is currently no specific therapy. Preclinical studies indicate that epoxyeicosanoids, a class of bioactive lipid mediators that are generated by cytochrome P450 (CYP) epoxygenases and inactivated by the soluble epoxide hydrolase (sEH), play a protective role in NAFLD. We performed a comprehensive lipidomics analysis using liver tissue and blood samples of 40 children with NAFLD. Proteomics was performed to determine CYP epoxygenase and sEH expressions. Hepatic epoxyeicosanoids significantly increased with higher grades of steatosis, while their precursor PUFAs were unaltered. Concomitantly, total CYP epoxygenase activity increased while protein level and activity of sEH decreased. In contrast, hepatic epoxyeicosanoids showed a strong decreasing trend with higher stages of fibrosis, accompanied by a decrease of CYP epoxygenase activity and protein expression. These findings suggest that the CYP epoxygenase/sEH pathway represents a potential pharmacologic target for the treatment of NAFLD., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background & Aims: Point of care tests (POCTs) might be used to identify patients with undiagnosed celiac disease who require further evaluation. We performed a large multicenter study to determine the performance of a POCT for celiac disease and assessed celiac disease prevalence in endoscopy centers., Methods: We performed a prospective study of 1055 patients (888 adults; median age, 48 yrs and 167 children; median age, 10 yrs) referred to 8 endoscopy centers in Germany, for various indications, from January 2016 through June 2017. Patients were tested for celiac disease using Simtomax, which detects immunoglobulin (Ig)A and IgG antibodies against deamidated gliadin peptides (DGP). Results were compared with findings from histologic analyses of duodenal biopsies (reference standard). The primary aim was to determine the accuracy of this POCT for the detection of celiac disease, to identify candidates for duodenal biopsy. A secondary aim was to determine the prevalence of celiac disease in adult and pediatric populations referred for outpatient endoscopic evaluation., Results: The overall prevalence of celiac disease was 4.1%. The POCT identified individuals with celiac disease with 79% sensitivity (95% CI, 64%-89%) and 94% specificity (95% CI, 93%-96%). Positive and negative predictive values were 37% and 99%. When we analyzed the adult and pediatric populations separately, we found the test to identify adults with celiac disease (prevalence 1.2%) with 100% sensitivity and 95% specificity. In the pediatric population (celiac disease prevalence 19.6%), the test produced false-negative results for 9 cases; the test therefore identified children with celiac disease with 72% sensitivity (95% CI 53%-86%). Analyses of serologic data revealed significantly lower DGP titers in the false-negative vs the true-positive group., Conclusions: In a study of more than 1000 adults and children, we found the Simtomax POCT to detect celiac disease with lower overall levels of sensitivity than expected. Although the test identifies adults with celiac disease with high levels of sensitivity and specificity, the prevalence of celiac disease was as low as 1.2% among adults. The test's lack of sensitivity might be due to the low intensity of the POCT bands and was associated with low serum DGP titers. Study ID no: DRKS00012499., (Copyright © 2019 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Objectives: Today, nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in children and adults alike. Yet, the noninvasive evaluation of disease severity remains a diagnostic challenge. In this study, we apply multifrequency magnetic resonance elastography (mMRE) for the quantification of liver steatosis and fibrosis in adolescents with NAFLD., Methods: Fifty adolescents (age range, 10-17 years; mean BMI, 33.9 kg/m; range, 21.4-42.1 kg/m) with biopsy-proven NAFLD were included in this prospective study. Multifrequency magnetic resonance elastography was performed using external multifrequency vibrations of 30 to 60 Hz and tomoelastography postprocessing, resulting in penetration rate (a) and shear wave speed (c). Hepatic fat fraction was determined using Dixon method. The diagnostic accuracy of mMRE in grading liver steatosis and staging liver fibrosis was assessed by receiver operating characteristic curve analysis., Results: Multifrequency magnetic resonance elastography parameters c and a were independently sensitive to fibrosis and steatosis, respectively, providing area under the receiver operating characteristic values of 0.79 (95% confidence interval [CI], 0.66-0.92), 0.91 (95% CI, 0.83-0.99), and 0.90 (95% CI, 0.80-0.99) for the detection of any (≥F1), moderate (≥F2), and advanced (≥F3) fibrosis, and 0.87 (95% CI, 0.76-0.97) and 0.87 (95% CI, 0.77-0.96) for the detection of moderate (≥S2) and severe (S3) steatosis., Conclusions: One mMRE measurement provides 2 independent parameters with very good diagnostic accuracy in detecting moderate and advanced fibrosis as well as moderate and severe steatosis in pediatric NAFLD.

Background: Whipple's disease (WD) is a rare infection with Tropheryma whipplei that is fatal if untreated. Diagnosis is challenging and currently based on invasive sampling. In a case of WD diagnosed from a kidney biopsy, we observed morphologically-intact bacteria within the glomerular capsular space and tubular lumens. This raised the questions of whether renal filtration of bacteria is common in WD and whether polymerase chain reaction (PCR) testing of urine might serve as a diagnostic test for WD., Methods: We prospectively investigated urine samples of 12 newly-diagnosed and 31 treated WD patients by PCR. As controls, we investigated samples from 110 healthy volunteers and patients with excluded WD or acute gastroenteritis., Results: Out of 12 urine samples from independent, therapy-naive WD patients, 9 were positive for T. whipplei PCR. In 3 patients, fluorescence in situ hybridization visualized T. whipplei in urine. All control samples were negative, including those of 11 healthy carriers with T. whipplei-positive stool samples. In our study, the detection of T. whipplei in the urine of untreated patients correlated in all cases with WD., Conclusions: T. whipplei is detectable by PCR in the urine of the majority of therapy-naive WD patients. With a low prevalence but far-reaching consequences upon diagnosis, invasive sampling for WD is mandatory and must be based on a strong suspicion. Urine testing could prevent patients from being undiagnosed for years. Urine may serve as a novel, easy-to-obtain specimen for guiding the initial diagnosis of WD, in particular in patients with extra-intestinal WD., (© The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America.)

Background and Aims: Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in children and adolescents today. In comparison with adult disease, paediatric NAFLD may show a periportal localization, which is associated with advanced fibrosis. This study aimed to assess the role of genetic risk variants for histological disease pattern and severity in childhood NAFLD., Methods: We studied 14 single nucleotide polymorphisms (SNP) in a cohort of 70 adolescents with biopsy-proven NAFLD. Genotype was compared to an adult control cohort (n = 200) and analysed in relation to histological disease severity and liver tissue proteomics., Results: Three of the 14 SNPs were significantly associated with paediatric NAFLD after FDR adjustment, rs738409 (PNPLA3, P = 2.80 × 10 -06 ), rs1044498 (ENPP1, P = 0.0091) and rs780094 (GCKR, P = 0.0281). The severity of steatosis was critically associated with rs738409 (OR=3.25; 95% CI: 1.72-6.52, FDR-adjusted P = 0.0070). The strongest variants associated with severity of fibrosis were rs1260326, rs780094 (both GCKR) and rs659366 (UCP2). PNPLA3 was associated with a portal pattern of steatosis, inflammation and fibrosis. Proteome profiling revealed decreasing levels of GCKR protein with increasing carriage of the rs1260326/rs780094 minor alleles and downregulation of the retinol pathway in rs738409 G/G carriers. Computational metabolic modelling highlighted functional relevance of PNPLA3, GCKR and UCP2 for NAFLD development., Conclusions: This study provides evidence for the role of PNPLA3 as a determinant of portal NAFLD localization and severity of portal fibrosis in children and adolescents, the risk variant being associated with an impaired hepatic retinol metabolism., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Purpose: The optimal method for detecting CMV colitis in patients with inflammatory bowel disease (IBD) has not been established. We wanted to investigate which diagnostic test would be most accurate when defining CMV colitis rather by the further clinical course than by using another diagnostic modality., Methods: All consecutive patients with moderately or severely active IBD who had been tested for CMV by PCR, histology, or antigenemia assay at the two campuses CBF and CCM of the Charité - Universitätsmedizin Berlin between September 2006 and September 2009 were included in this retrospective study. During that time, in patients with a positive CMV test, immunosuppressive treatment of any kind was immediately reduced and antiviral treatment was started. This allowed identifying patients who responded to antiviral treatment and those who only responded to later escalation of immunosuppressive therapy., Results: One hundred and nine patients were identified, out of whom nine were considered to have clinically relevant CMV colitis. Sensitivity and specificity were 1 and 0.94 for CMV PCR and 0.5 and 1 for pp65 antigen immunofluorescence assay from peripheral blood, 0.67 and 0.98 for immunohistochemistry, and 0.17 and 0.98 for hematoxylin-eosin staining. When using absence of leukocytosis, splenomegaly, and steroid refractory disease as clinical parameters to test for CMV colitis, blood CMV PCR and immunohistochemistry were able to exclude CMV colitis in negative patients with a 75% likelihood of positive patients to have clinically relevant CMV colitis., Conclusions: Blood-based CMV PCR together with simple clinical parameters can exclude clinically relevant CMV colitis at a high specificity.

To date, little is known about the interaction between (pre-)malignant B cells and T cells. We generated transgenic mice that allow B cell-specific induction of the oncogene SV40 large T-antigen (TAg) to analyze the role of oncogene-specific T cells during sporadic B-cell lymphoma development. Constitutive TAg expression in CD19-Cre × LoxP-Tag mice resulted in TAg-tolerant CD8 + T cells and development of B-cell lymphomas. In contrast, CD19-CreER T2 × LoxP-Tag mice retained TAg-competent CD8 + T cells at time of oncogene induction and TAg expression in few B cells of adult mice resulted in exceptionally rare lymphoma formation late in life. Increased lymphoma incidence in the absence of TAg-specific T cells suggested T cell-mediated inhibition of lymphoma progression. However, TAg-initiated B cells were not eliminated by T cells and detected long term. Our results demonstrate a failure of the immune system to eradicate lymphoma-initiating B cells, retaining the risk of lymphoma development., (© 2018 by The American Society of Hematology.)

Purpose To measure in vivo liver stiffness by using US time-harmonic elastography in a cohort of pediatric patients who were overweight to extremely obese with nonalcoholic fatty liver disease (NAFLD) and to evaluate the diagnostic value of time-harmonic elastography for differentiating stages of fibrosis associated with progressive disease. Materials and Methods In this prospective study, 67 consecutive adolescents (age range, 10-17 years; mean body mass index, 34.7 kg/m 2 ; range, 21.4-50.4 kg/m 2 ) with biopsy-proven NAFLD were enrolled. Liver stiffness was measured by using time-harmonic elastography based on externally induced continuous vibrations of 30 Hz to 60 Hz frequency and real-time B-mode-guided wave profile analysis covering tissue depths of up to 14 cm. The diagnostic accuracy of time-harmonic elastography in staging liver fibrosis was assessed with area under the receiver operating characteristic curve (AUC) analysis. Liver stiffness cutoffs for the differentiation of fibrosis stages were identified based on the highest Youden index. Results Time-harmonic elastography was feasible in all patients (0% failure rate), including 70% (n = 47) of individuals with extreme obesity (body mass index above the 99.5th percentile). AUC analysis for the detection of any fibrosis (≥ stage F1), moderate fibrosis (≥ stage F2), and advanced fibrosis (≥ stage F3) was 0.88 (95% confidence interval [CI]: 0.80, 0.96), 0.99 (95% CI: 0.98, 1.00), and 0.88 (95% CI: 0.80, 0.96), respectively. The best liver stiffness cutoffs were 1.52 m/sec for at least stage F1, 1.62 m/sec for at least stage F2, and 1.64 m/sec for at least stage F3. Conclusion US time-harmonic elastography allows accurate detection of moderate fibrosis even in pediatric patients with extreme obesity. Larger clinical trials are warranted to confirm the accuracy of US time-harmonic elastography., (© RSNA, 2018 Online supplemental material is available for this article.)

Graft-versus-host disease (GvHD) is a major complication of allogeneic stem cell transplantation. High-resolution in vivo histology of the intestine by confocal endomicroscopy (CEM) detects acute GvHD (aGvHD) with high sensitivity. This pilot study aims to evaluate the diagnostic value of CEM for intestinal chronic GvHD (cGvHD). The study included 20 patients with gastrointestinal symptoms and confirmed cGvHD in other organs as well as 20 patients with clinically suspected acute GvHD for control. Confocal endomicroscopy was performed as gastroscopy followed by sigmoidoscopy after intravenous injection of fluorescein (10%) and topical application of acriflavine (0.05%). Histopathology from H&E-stained biopsy samples throughout the intestinal tract complemented the survey. All histological features of intestinal cGvHD were predominantly mild to moderate. Stroma fibrosis detected by standard histology (16/20 patients) was not seen by CEM. Apoptosis assessed by histology in 12/20 patients was concordant with CEM (8/12 patients). Confocal endomicroscopy revealed esophageal manifestation of cGvHD in 3 patients. For each biopsy site, CEM correlated with intestinal histology (r = 0.64). Classical histology from intestinal biopsy samples taken under CEM monitoring confirmed the final diagnosis of cGvHD. The sensitivity of CEM with 40% in cGvHD was significantly lower compared to 70% in patients with aGvHD. Confocal endomicroscopy detected acute features of cGvHD and contributed to the diagnosis of esophageal cGvHD but failed to display stroma fibrosis in vivo. Although CEM represents a useful noninvasive tool in routine diagnostic of intestinal aGvHD, the method is not sufficient to fully establish the diagnosis of cGvHD within the intestinal tract. Confocal endomicroscopy allowed acquisition of targeted biopsies in patients suspected of having cGvHD., (Copyright © 2017 John Wiley & Sons, Ltd.)

Background: Watery diarrhea is the cardinal symptom of lymphocytic colitis (LC). We have previously shown that colonic Na malabsorption is one of the major pathologic alterations of LC and found evidence for an epithelial barrier defect. On these grounds, this study aimed to identify the inherent mechanisms of this epithelial barrier dysfunction and its regulatory features., Methods: Epithelial resistance (R epi ) was determined by one-path impedance spectroscopy and 3 H-mannitol fluxes were performed on biopsies from sigmoid colon in miniaturized Ussing chambers. Tight junction proteins were analyzed by Western blot and confocal microscopy. Inflammatory signaling was characterized in HT-29/B6 cells. Apoptosis and mucosal surface parameters were quantified morphologically., Results: R epi was reduced to 53% and 3 H-mannitol fluxes increased 1.7-fold in LC due to lower expression of claudin-4, -5, and -8 and altered subcellular claudin-5 and -8 distributions off the tight junction. TNFα and IFNγ could mimic subcellular redistribution in HT-29/B6 cells, a process which was independent on MLCK activation. Epithelial apoptosis did not contribute to barrier dysfunction in LC and mucosal surface area was unchanged., Conclusions: Epithelial barrier dysfunction in LC occurs through downregulation of claudin-4, -5, and -8, and redistribution of claudin-5 and -8 off the tight junction, which contributes to diarrhea by a leak-flux mechanism. The key effector cytokines TNFα and IFNγ turned out to be the trigger for redistribution of claudin-5 and -8. Thus, alongside sodium malabsorption, leak-flux is yet another important diarrheal mechanism in LC.

During six years as German National Consultant Laboratory for Spirochetes we investigated 149 intestinal biopsies from 91 patients, which were histopathologically diagnosed with human intestinal spirochetosis (HIS), using fluorescence in situ hybridization (FISH) combined with 16S rRNA gene PCR and sequencing. Aim of this study was to complement histopathological findings with FISH and PCR for definite diagnosis and species identification of the causative pathogens. HIS is characterized by colonization of the colonic mucosa of the human distal intestinal tract by Brachyspira spp. Microbiological diagnosis of HIS is not performed, because of the fastidious nature and slow growth of Brachyspira spp. in culture. In clinical practice, diagnosis of HIS relies solely on histopathology without differentiation of the spirochetes. We used a previously described FISH probe to detect and identify Brachyspira spp. in histological gut biopsies. FISH allowed rapid visualization and identification of Brachyspira spp. in 77 patients. In most cases, the bright FISH signal already allowed rapid localization of Brachyspira spp. at 400× magnification. By sequencing, 53 cases could be assigned to the B. aalborgi lineage including "B. ibaraki" and "B. hominis", and 23 cases to B. pilosicoli. One case showed mixed colonization. The cases reported here reaffirm all major HIS Brachyspira spp. clusters already described. However, the phylogenetic diversity seems to be even greater than previously reported. In 14 cases, we could not confirm HIS by either FISH or PCR, but found colonization of the epithelium by rods and cocci, indicating misdiagnosis by histopathology. FISH in combination with molecular identification by 16S rRNA gene sequencing has proved to be a valuable addition to histopathology. It provides definite diagnosis of HIS and allows insights into phylogeny and distribution of Brachyspira spp. HIS should be considered as a differential diagnosis in diarrhea of unknown origin, particularly in patients from risk groups (e.g. patients with colonic adenomas, inflammatory polyps, inflammatory bowel disease or HIV infection and in men who have sex with men)., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: The pathology and diagnosis of acute and chronic rejection in intestinal transplantation (ITX) are far from being completely understood. We established models of acute and chronic intestinal graft rejection and analyzed peripheral and intragraft immune responses., Methods: We performed ITX from Dark Agouti into Lewis rats applying single-dose tacrolimus (TAC) at varying concentrations. Graft histology and immunohistology were assessed on postoperative day (POD)7, 14, and 45. Intragraft and peripheral gene expressions of inflammatory and anti-inflammatory markers and lipopolysaccharide binding protein (LBP) plasma as well as alloantibodies were measured simultaneously., Results: A 1-mg TAC resulted in acute rejection and recipient death; 3 mg and 5 mg prolonged survival and led to severe or moderate chronic rejection, respectively, with 50% of the 5-mg TAC recipients surviving the observation period. Consequently, we observed severe infiltration on POD7 in untreated and 1-mg TAC recipients compared with minor histological alterations in 3 and 5 mg TAC groups. Only 5-mg TAC treatment prevented accumulation of CD4+ T cells and ED1+ macrophages over the entire observation period. Peripheral and intragraft expressions of T cell activation inhibitor-mitochondrial were stable in long-term surviving 5-mg TAC recipients but declined before acute or chronic rejection in 1 and 3 mg TAC recipients. In contrast, LBP levels increased during acute and chronic rejections., Conclusions: We studied acute and chronic rejections in a preclinical model of ITX, which recapitulates clinical findings and highlights the importance of monitoring peripheral T cell activation inhibitor-mitochondrial expression, LBP levels, and antidonor antibodies for revealing rejection.

The mucosal immune system is relevant for homeostasis, immunity, and also pathological conditions in the gastrointestinal tract. Inducible NO synthase (iNOS)-dependent production of NO is one of the factors linked to both antimicrobial immunity and pathological conditions. Upregulation of iNOS has been observed in human Helicobacter pylori infection, but the cellular sources of iNOS are ill defined. Key differences in regulation of iNOS expression impair the translation from mouse models to human medicine. To characterize mucosal iNOS-producing leukocytes, biopsy specimens from H. pylori-infected patients, controls, and participants of a vaccination trial were analyzed by immunohistochemistry, along with flow cytometric analyses of lymphocytes for iNOS expression and activity. We newly identified mucosal IgA-producing plasma cells (PCs) as one major iNOS(+) cell population in H. pylori-infected patients and confirmed intracellular NO production. Because we did not detect iNOS(+) PCs in three distinct infectious diseases, this is not a general feature of mucosal PCs under conditions of infection. Furthermore, numbers of mucosal iNOS(+) PCs were elevated in individuals who had cleared experimental H. pylori infection compared with those who had not. Thus, IgA(+) PCs expressing iNOS are described for the first time, to our knowledge, in humans. iNOS(+) PCs are induced in the course of human H. pylori infection, and their abundance seems to correlate with the clinical course of the infection., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Purpose: Mantle-cell lymphoma (MCL) is a rather aggressive B-cell malignancy whose considerable variability of individual outcome is associated with clinical characteristics (Mantle Cell Lymphoma International Prognostic Index [MIPI]). The Ki-67 index is a strong independent prognostic factor; however, the biologic MIPI (MIPI-b) distinguishes only two groups, which does not appropriately depict the clinical heterogeneity. By using the cohort from the European MCL Younger and MCL Elderly trials, we aimed to evaluate the additional prognostic impact of cytology and growth pattern and to improve risk stratification with the Ki-67 index and MIPI., Patients and Methods: Diagnostic tumor biopsies were reviewed by the European Mantle Cell Lymphoma Pathology Panel to determine Ki-67 index by using published guidelines, cytology, and growth pattern. We evaluated prognostic effects for overall survival (OS) by Cox regression. For the cohort used for MIPI-b development (German Low-Grade Lymphoma Study Group [GLSG] 1996 and GLSG2000), we checked whether the equally weighted combination of Ki-67 index (dichotomized at the validated 30% cutoff) and MIPI risk groups was adequate and compared the prognostic power of this modified combination to MIPI and MIPI-b for the MCL Younger/MCL Elderly cohort., Results: The Ki-67 index was assessed in 508 of 832 patients (median age, 62 years). Blastoid cytology was associated with inferior OS independently of MIPI but not independently of the Ki-67 index. Growth pattern was not independently prognostic. The modified combination of the Ki-67 index and MIPI separated four groups with 5-year OS: 85%, 72%, 43%, and 17% (P < .001) and was more discriminative than MIPI and MIPI-b., Conclusion: Using the Ki-67 index is superior to using cytology and growth pattern as prognostic factors in MCL. The modified combination of the Ki-67 index and MIPI showed a refined risk stratification, reflecting their strong complementary prognostic effects while integrating the most relevant prognostic factors available in clinical routine., (© 2016 by American Society of Clinical Oncology.)

Background: Lymphocytic colitis (LC) causes watery diarrhea. We aimed to identify mechanisms of altered Na absorption and regulatory inputs in patients with LC by examining the epithelial Na channel (ENaC) function as the predominant Na transport system in human distal colon., Methods: Epithelial Na channel function and regulation was analyzed in biopsies from sigmoid colon of patients with LC and in rat distal colon in Ussing chambers. ENaC-subunit expression was measured by real-time PCR and RNA sequencing. Correction factors for subepithelial resistance contributions were determined by impedance spectroscopy. Upstream regulators in LC were determined by RNA sequencing., Results: Epithelial Na channel-mediated electrogenic Na transport was inhibited despite aldosterone stimulation in human sigmoid colon of patients with LC. The increase in γ-ENaC mRNA expression in response to aldosterone was MEK1/2-dependently reduced in LC, since it could be restored toward normal by MEK1/2 inhibition through U0126. Parallel experiments for identification of signaling in rat distal colon established MEK1/2 to be activated by a cytokine cocktail of TNFα, IFNγ, and IL-15, which were identified as the most important regulators in the upstream regulator analysis in LC., Conclusions: In the sigmoid colon of patients with LC, the key effector cytokines TNFα, IFNγ, and IL-15 inhibited γ-ENaC upregulation in response to aldosterone through a MEK1/2-mediated pathway. This prevents ENaC to reach its maximum transport capacity and results in Na malabsorption which contributes to diarrhea.

Clinical manifestations of leptospirosis are highly variable: from asymptomatic to severe and potentially fatal. The outcome of the disease is usually determined in the immunological phase, beginning in the second week of symptoms. The underlying mechanisms, predictive factors, and individual immune responses that contribute to clinical variations are not well understood. The aim of this study was to determine the specifics of CD4(+) T-cell reactivity and cytokine release after stimulation with leptospiral antigens in patients with leptospirosis of different disease severities (patients with mild and severe symptoms) and in control subjects (with and without proven exposure to Leptospira). Whole-blood specimens were stimulated with Leptospira antigens in vitro. Subsequently, intracellular staining of cytokines was performed, and flow cytometry was used to assess the expression of CD40 ligand (CD40L) and the production of gamma interferon (IFN-γ), interleukin-10 (IL-10), IL-2, and tumor necrosis factor alpha (TNF-α) by CD4(+) T cells. The production of inflammatory cytokines such as TNF-α by CD4(+) T cells after stimulation with leptospiral antigens was highest in patients with severe disease. In contrast, the ratio of IL-10 production to TNF-α production was higher in exposed subjects than in patients with mild and severe disease. Levels of proinflammatory cytokines such as TNF-α may be useful markers of the severity of the immunological phase of leptospirosis. IL-10 production by T cells after antigen-specific stimulation may indicate a more successful downregulation of the inflammatory response and may contribute to an asymptomatic course of the disease., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Syphilis is often called the great imitator because of its frequent atypical clinical manifestations that make the disease difficult to recognize. Because Treponema pallidum subsp. pallidum, the infectious agent of syphilis, is yet uncultivated in vitro, diagnosis is usually made using serology; however, in cases where serology is inconclusive or in patients with immunosuppression where these tests may be difficult to interpret, the availability of a molecular tool for direct diagnosis may be of pivotal importance. Here we present a fluorescence in situ hybridization (FISH) assay that simultaneously identifies and analyzes spatial distribution of T. pallidum in histological tissue sections. For this assay the species-specific FISH probe TPALL targeting the 16S rRNA of T. pallidum was designed in silico and evaluated using T. pallidum infected rabbit testicular tissue and a panel of non-syphilis spirochetes as positive and negative controls, respectively, before application to samples from four syphilis-patients. In a HIV positive patient, FISH showed the presence of T. pallidum in inguinal lymph node tissue. In a patient not suspected to suffer from syphilis but underwent surgery for phimosis, numerous T. pallidum cells were found in preputial tissue. In two cases with oral involvement, FISH was able to differentiate T. pallidum from oral treponemes and showed infection of the oral mucosa and tonsils, respectively. The TPALL FISH probe is now readily available for in situ identification of T. pallidum in selected clinical samples as well as T. pallidum research applications and animal models., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Classic Whipple disease (CWD) is a systemic infection caused by Tropheryma whipplei. Different diagnostic tools have been developed over the last decades: periodic acid-Schiff (PAS) staining, T whipplei-specific polymerase chain reaction (PCR), and T whipplei-specific immunohistochemistry (IHC). Despite all these advances, CWD is still difficult to diagnose because of a variety of clinical symptoms and possibly a long time span between first unspecific symptoms and the full-blown clinical picture of the disease. Herein, we report an observational cohort study summarizing epidemiologic data, clinical manifestations, and diagnostic parameters of 191 patients with CWD collected at our institution. Gastrointestinal manifestations are the most characteristic symptoms of CWD affecting 76% of the cohort. Although the small bowel was macroscopically conspicuous in only 27% of cases, 173 (91%) patients presented with characteristic histological changes in small bowel biopsies (in 2 patients, these changes were only seen within the ileum). However, 18 patients displayed normal small bowel histology without typical PAS staining. In 9 of these patients, alternative test were positive from their duodenal specimens (ie, T whipplei-specific PCR and/or IHC). Thus, in 182 patients (95%) a diagnostic hint toward CWD was obtained from small bowel biopsies. Only 9 patients (5%) were diagnosed solely based on positive T whipplei-specific PCR and/or IHC of extraintestinal fluids (eg, cerebrospinal fluid, synovial fluid) or extraintestinal tissue (eg, lymph node, synovial tissue), respectively. Thus, despite efforts to diagnose CWD from alternative specimens, gastroscopy with duodenal biopsy and subsequent histological and molecular-biological examination is the most reliable diagnostic tool for CWD.

The cell adhesion molecule CD2 facilitates antigen-independent T-cell activation and CD2 deficiency or blockade reduces intestinal inflammation in murine models. We here aimed to evaluate the therapeutic potential of monoclonal antibodies (mAb) specific for human CD2 in colitis treatment. Transfer colitis induced by naïve CD4(+) T cells expressing human CD2 was treated with anti-human CD2 mAb. The mAb CB.219 protected from severe colitis in a preventive treatment regimen, while therapeutic treatment ameliorated intestinal inflammation. Diminished intestinal tissue damage was paralleled by a profound suppression of lamina propria lymphocytes to produce pro-inflammatory cytokines and tumor necrosis factor α as well as the neutrophil chemoattractant CXC motif ligand 1 and the CC chemokine ligand 3. Furthermore, infiltration with macrophages and T cells was low. Thus, reduced intestinal inflammation in our humanized colitis model by targeting CD2 on T cells with the mAb CB.219 suggests a novel approach for colitis treatment., (Copyright © 2015. Published by Elsevier Inc.)

Accumulation of Tropheryma whipplei-stuffed macrophages in the duodenum, impaired T. whipplei-specific Th1 responses, and weak secretion of interleukin-12 (IL-12) are hallmarks of classical Whipple's disease (CWD). This study addresses dendritic cell (DC) functionality during CWD. We documented composition, distribution, and functionality of DC ex vivo or after in vitro maturation by fluorescence-activated cell sorting (FACS) and by immunohistochemistry in situ. A decrease in peripheral DC of untreated CWD patients compared to healthy donors was due to reduced CD11c(high) myeloid DC (M-DC). Decreased maturation markers CD83, CD86, and CCR7, as well as low IL-12 production in response to stimulation, disclosed an immature M-DC phenotype. In vitro-generated monocyte-derived DC from CWD patients showed normal maturation and T cell-stimulatory capacity under proinflammatory conditions but produced less IL-12 and failed to activate T. whipplei-specific Th1 cells. In duodenal and lymphoid tissues, T. whipplei was found within immature DC-SIGN(+) DC. DC and proliferating lymphocytes were reduced in lymph nodes of CWD patients compared to levels in controls. Our results indicate that dysfunctional IL-12 production by DC provides suboptimal conditions for priming of T. whipplei-specific T cells during CWD and that immature DC carrying T. whipplei contribute to the dissemination of the bacterium., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Two long and one truncated isoforms (termed LAP*, LAP, and LIP, respectively) of the transcription factor CCAAT enhancer binding protein beta (C/EBPβ) are expressed from a single intronless Cebpb gene by alternative translation initiation. Isoform expression is sensitive to mammalian target of rapamycin (mTOR)-mediated activation of the translation initiation machinery and relayed through an upstream open reading frame (uORF) on the C/EBPβ mRNA. The truncated C/EBPβ LIP, initiated by high mTOR activity, has been implied in neoplasia, but it was never shown whether endogenous C/EBPβ LIP may function as an oncogene. In this study, we examined spontaneous tumor formation in C/EBPβ knockin mice that constitutively express only the C/EBPβ LIP isoform from its own locus. Our data show that deregulated C/EBPβ LIP predisposes to oncogenesis in many tissues. Gene expression profiling suggests that C/EBPβ LIP supports a pro-tumorigenic microenvironment, resistance to apoptosis, and alteration of cytokine/chemokine expression. The results imply that enhanced translation reinitiation of C/EBPβ LIP promotes tumorigenesis. Accordingly, pharmacological restriction of mTOR function might be a therapeutic option in tumorigenesis that involves enhanced expression of the truncated C/EBPβ LIP isoform., Key Message: Elevated C/EBPβ LIP promotes cancer in mice. C/EBPβ LIP is upregulated in B-NHL. Deregulated C/EBPβ LIP alters apoptosis and cytokine/chemokine networks. Deregulated C/EBPβ LIP may support a pro-tumorigenic microenvironment.