Distinct immunological signatures discriminate severe COVID-19 from non-SARS-CoV-2-driven critical pneumonia.

Immune profiling of COVID-19 patients has identified numerous alterations in both innate and adaptive immunity. However, whether those changes are specific to SARS-CoV-2 or driven by a general inflammatory response shared across severely ill pneumonia patients remains unknown. Here, we compared the immune profile of severe COVID-19 with non-SARS-CoV-2 pneumonia ICU patients using longitudinal, high-dimensional single-cell spectral cytometry and algorithm-guided analysis. COVID-19 and non-SARS-CoV-2 pneumonia both showed increased emergency myelopoiesis and displayed features of adaptive immune paralysis. However, pathological immune signatures suggestive of T cell exhaustion were exclusive to COVID-19. The integration of single-cell profiling with a predicted binding capacity of SARS-CoV-2 peptides to the patients' HLA profile further linked the COVID-19 immunopathology to impaired virus recognition. Toward clinical translation, circulating NKT cell frequency was identified as a predictive biomarker for patient outcome. Our comparative immune map serves to delineate treatment strategies to interfere with the immunopathologic cascade exclusive to severe COVID-19.

A Direct in vivo RNAi screen identifies MKK4 as a key regulator of liver regeneration.

The liver harbors a distinct capacity for endogenous regeneration; however, liver regeneration is often impaired in disease and therefore insufficient to compensate for the loss of hepatocytes and organ function. Here we describe a functional genetic approach for the identification of gene targets that can be exploited to increase the regenerative capacity of hepatocytes. Pools of small hairpin RNAs (shRNAs) were directly and stably delivered into mouse livers to screen for genes modulating liver regeneration. Our studies identify the dual-specific kinase MKK4 as a master regulator of liver regeneration. MKK4 silencing robustly increased the regenerative capacity of hepatocytes in mouse models of liver regeneration and acute and chronic liver failure. Mechanistically, induction of MKK7 and a JNK1-dependent activation of the AP1 transcription factor ATF2 and the Ets factor ELK1 are crucial for increased regeneration of hepatocytes with MKK4 silencing.

Preexisting comorbidities shape the immune response associated with severe COVID-19.

BACKGROUND: Comorbidities are risk factors for development of severe coronavirus disease 2019 (COVID-19). However, the extent to which an underlying comorbidity influences the immune response to severe acute respiratory syndrome coronavirus 2 remains unknown. OBJECTIVE: Our aim was to investigate the complex interrelations of comorbidities, the immune response, and patient outcome in COVID-19. METHODS: We used high-throughput, high-dimensional, single-cell mapping of peripheral blood leukocytes and algorithm-guided analysis. RESULTS: We discovered characteristic immune signatures associated not only with severe COVID-19 but also with the underlying medical condition. Different factors of the metabolic syndrome (obesity, hypertension, and diabetes) affected distinct immune populations, thereby additively increasing the immunodysregulatory effect when present in a single patient. Patients with disorders affecting the lung or heart, together with factors of metabolic syndrome, were clustered together, whereas immune disorder and chronic kidney disease displayed a distinct immune profile in COVID-19. In particular, severe acute respiratory syndrome coronavirus 2-infected patients with preexisting chronic kidney disease were characterized by the highest number of altered immune signatures of both lymphoid and myeloid immune branches. This overall major immune dysregulation could be the underlying mechanism for the estimated odds ratio of 16.3 for development of severe COVID-19 in this burdened cohort. CONCLUSION: The combinatorial systematic analysis of the immune signatures, comorbidities, and outcomes of patients with COVID-19 has provided the mechanistic immunologic underpinnings of comorbidity-driven patient risk and uncovered comorbidity-driven immune signatures.

Comprehensive clinical and virological characterization of three cases of fulminant liver failure owing to HSV1 primary infection.

Herpes simplex virus 1 (HSV-1) is a frequently unrecognized, yet deadly cause of acute liver failure (ALF). We, therefore, analysed three cases of fatal HSV-1-induced ALF. All patients shared clinical (extremely elevated transaminases, LDH and AST/LDH ratio < 1) and virological characteristics (ratio of viral load in plasma versus throat swabs: 60-700-fold, lack of anti-HSV-1-IgG antibodies or low IgG-avidity during primary infection), which may help to identify patients at risk. Additionally, in vitro chemosusceptibility assays revealed high efficacy of the helicase-primase inhibitors (HPI), pritelivir and drug-candidate IM-250 compared to acyclovir (ACV) using HSV-1-isolates from two patients; hence, ACV/HPI-combinations might offer new therapeutic options for HSV-induced ALF.

Evaluation of bowel wall thickness by ultrasound as early diagnostic tool for therapeutic response in Crohn's disease patients treated with ustekinumab.

BACKGROUND: Ustekinumab was approved for the treatment of patients with moderate to severe CD 2. Development of predictors for selecting patients responding to ustekinumab has to be the next step. US offers a noninvasive method with great sensitivity in detecting CD activity 11. AIM: To evaluate BWT by BS as early diagnostic tool for treatment response in CD patients treated with ustekinumab at week 8. METHODS: This is a prospective monocentric study. Twenty-three CD patients had BS at the time of first and second application. BS was performed by one of 2 experienced DEGUM certificated sonographers, with evaluation by both independently and blindly. Primary endpoint was substantial sonographic response defined as decrease of BWT ≥ 1 mm. Secondary endpoint was concordance between sonographic and clinical response, defined as decrease of CDAI ≥ 70 points and sonographic and biochemical response defined as decrease of CRP ≥ 0.5 mg/dl. RESULTS: At week 8, BS detected in 10 of 23 patients a substantial decrease of BWT ≥ 1 mm; in 7, a decrease < 1 mm. Compared to baseline, all 17 patients showed generally improved blood data and 16/17 generally improved clinical data. Of those with a decrease of BWT ≥ 1 mm, we observed a substantial decrease of CDAI ≥ 70 points in 9/10 patients and a substantial decrease of CRP ≥ 0.5 mg/dl in 8/10 patients. CONCLUSION: Our study suggests that sonographic measurement of BWT can be a helpful parameter for selecting patients responding early to ustekinumab and for providing assistance in terms of further treatment interval at week 8.

Dynamics of glucose metabolism after liver transplantation: prediabetes as a window of opportunity for patient survival and long-term kidney function.

Post-transplantation diabetes mellitus (PTDM) is a relevant complication following liver transplantation with profound impact on morbidity and mortality. To date, little is known about the evolution and dynamics of glucose metabolism and the impact of prediabetes in long-term follow-up. To address this issue, all consecutive adult liver transplant recipients (n = 429) from a European university hospital transplant center between 2007 and 2017 were analyzed retrospectively. In patients without pre-existing diabetes (n = 327), we conducted a longitudinal characterization of glucose metabolism. Median follow-up was 37 [9-64, IQR] months. Median prevalence of prediabetes was 39 [37-39]% and of PTDM 21 [17-22]%. Throughout follow-up, intra-individual glucose regulation of patients was highly variable, continuously fluctuating between different states of glucose metabolism (normal glucose tolerance, prediabetes, PTDM). Whereas overall survival and long-term kidney function of patients with PTDM were significantly lower than that of patients with normal glucose metabolism, prediabetes was not associated with adverse outcome. This study provides new insight into the dynamics and impact of glucose metabolism after liver transplantation. Unlike PTDM, prediabetes is not associated with adverse outcome, providing a window of opportunity for targeted intervention. The results underline the need for constant screening and intervention in posttransplant care of liver allograft recipients.

Significance and clinical impact of routinely tested urinary ethyl glucuronide after liver transplantation - development of a risk score.

Alcohol abuse after liver transplantation can seriously impact graft and patient survival. However, to date, there is no defined standard procedure to identify patients consuming alcohol after liver transplantation. The aim of this study was to analyze the diagnostic value and clinical impact of routinely measured urinary ethyl glucuronide (uEtG) - a metabolite of ethanol - in patients after liver transplantation. Data of 362 consecutive patients after liver transplantation who visited the University Hospital of Tuebingen for outpatient follow-up were analyzed. Forty-eight patients (13%) displayed positive uEtG results. The uEtG positive group contained significantly more patients with pretransplant alcoholic liver disease. However, two thirds of the uEtG positive patients had no history of pretransplant alcoholic liver disease. Several clinical parameters were significantly associated with positive uEtG. In order to enable a more cost-effective application of uEtG in the future, a clinical risk score was developed (specificity 0.95). In conclusion, routine testing for uEtG reveals a considerable percentage of patients practicing alcohol intake after liver transplantation. Application of our proposed risk score could help focusing uEtG testing on patients at risk.

Impact of the COVID-19 pandemic on an interdisciplinary endoscopy unit in a German "hotspot" area: a single center experience.

BACKGROUND AND STUDY AIMS: Since December 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative pathogen of coronavirus disease 2019 (COVID-19), has posed a pandemic threat to global health and has challenged health care system in all affected countries. PATIENTS AND METHODS: This is a combined study including a descriptive part about the changes in the daily work routine of an Interdisciplinary Endoscopic Unit (IEU) and a prospective analysis of patients tested positive for SARS-CoV-2 who required endoscopic interventions. Conclusively, we present the finding of a point-prevalence analysis in the staff of the IEU. RESULTS: We present effects of the COVID-19-related restructuring of processes in our interdisciplinary endoscopy unit (IEU) with respect to cancelation of examinations, relocation of staff to other departments, impact of SARS-CoV-2 on medical staff of the IEU, and supply of protective clothing. Additionally, we analyzed the cohort of COVID-19 patients: Sixteen endoscopic interventions were done in ten patients. In all patients with confirmed infection with SARS-CoV-2, emergency endoscopies were required for relevant bleeding situations. Re-endoscopies were required only in critically ill COVID-19 patients. CONCLUSIONS: The restructuring of processes in the IEU was feasible in short time, effective, and can also be applied broadly at least in developed countries [Garbe et al. in Gastroenterology 159:778-780, 2020; Repici A, Pace F, Gabbiadini R, Colombo M, Hassan C, Dinelli M, Group IG-CW, Maselli R, Spadaccini M, Mutignani M, Gabbrielli A, Signorelli C, Spada C, Leoni P, Fabbri C, Segato S, Gaffuri N, Mangiavillano B, Radaelli F, Salerno R, Bargiggia S, Maroni L, Benedetti A, Occhipinti P, De Grazia F, Ferraris L, Cengia G, Greco S, Alvisi C, Scarcelli A, De Luca L, Cereatti F, Testoni PA, Mingotto R, Aragona G, Manes G, Beretta P, Amvrosiadis G, Cennamo V, Lella F, Missale G, Lagoussis P, Triossi O, Giovanardi M, De Roberto G, Cantu P, Buscarini E, Anderloni A, Carrara S, Fugazza A, Galtieri PA, Pellegatta G, Antonelli G, Rosch T, Sharma P (2020) Endoscopy units and the COVID-19 Outbreak: a Multi-Center Experience from Italy. Gastroenterology;]. The endoscopy-related rate of SARS-CoV-2 infection of staff is low, but supply of protective equipment is crucial for this. Endoscopic procedures in COVID-19 patients were not directly related to SARS-CoV-2 infection, but to other underlying diseases or typical complications of long-term ICU treatment.

Hypoxia induced Sonic Hedgehog signaling regulates cancer stemness, epithelial-to-mesenchymal transition and invasion in cholangiocarcinoma.

Aberrant activation of Sonic Hedgehog (SHH) pathway has been implicated in a variety of cancers including cholangiocarcinoma (CC); however, the influencing factors are still unknown. Additionally, intratumoral hypoxia is known to contribute towards therapeutic resistance through modulatory effects on various pathways. In this study, we investigated the relationship between hypoxia and SHH pathway activation and the effect of this interplay on cancer stemness and epithelial-to- mesenchymal transition (EMT) during cholangiocarcinogenesis. Hypoxia promoted SHH pathway activation, evidenced by upregulated SHH and SMO levels, and enhanced glioma-associated oncogene homolog 1 (GLI1) nuclear translocation; whereas silencing of HIF-1α impaired SHH upregulation. Hypoxia also enhanced the expression of cancer stem cell (CSC) transcription factors (NANOG, Oct4, SOX2), CD133 and EMT markers (N-cadherin, Vimentin), thereby supporting invasion. Cyclopamine treatment suppressed hypoxia induced SHH pathway activation, consequently reducing invasiveness by downregulating the expression of CSC transcription factors, CD133 and EMT. Cyclopamine induced apoptosis in CC cells under hypoxia, suggesting that hypoxia induced activation of SHH pathway has modulatory effects on CC progression. Therefore, SHH signaling is proposed as a target for CC treatment, which is refractory to standard chemotherapy.

Infodemiology of Crohn's disease and Ulcerative colitis using Google Trends - an approach to investigate patient needs.

INTRODUCTION: Inflammatory bowel diseases (IBD) highly affect quality of life. The course of disease and success of treatment are variable. These factors result in a high number of psychiatric comorbidities with patients requiring extensive medical consultation or additional psychotherapy. Unfortunately, time is often limited in daily clinical care, which leaves patients not feeling sufficiently informed about their disease. Patients often compensate by searching the internet, with possibly harmful information. We aim to identify information gaps to allow a more complete education of patients. METHODS: We analyzed the internet search behavior using the key words [Morbus Crohn] (CD) and [Colitis ulcerosa] (UC) using Google Trends. In addition, we investigated which websites are the first hits on Google, as those are most likely visited by patients. RESULTS: Symptoms, nutrition and therapy are central topics for persons interested in IBD. The searches concerning symptoms or therapies do not match the actual incidence or prevalence of comorbidities as well as the more commonly used therapies or established nutritional recommendations. We found a distinct impact of well-known personalities on disease related searches. The first suggested websites on google showed great heterogeneity of responsible publishers, often with possible conflict of interests. In line with those observations, quality of website content is highly heterogeneous. CONCLUSION: This study showed a need of information concerning symptoms, nutrition and therapy that should be considered during patient education. Since time in physician patient dialogue is short it may be helpful to further evaluate websites independently in order to give recommendations of websites offering reliable information.

Ubiquitously expressed Human Beta Defensin 1 (hBD1) forms bacteria-entrapping nets in a redox dependent mode of action.

Ever since the discovery of endogenous host defense antimicrobial peptides it has been discussed how these evolutionary conserved molecules avoid to induce resistance and to remain effective. Human ß-defensin 1 (hBD1) is an ubiquitously expressed endogenous antimicrobial peptide that exhibits qualitatively distinct activities between its oxidized and reduced forms. Here, we explore these antimicrobial mechanisms. Surprisingly, using electron microscopy we detected a so far unknown net-like structure surrounding bacteria, which were treated with the reduced but not the oxidized form of hBD1. A transmigration assay demonstrated that hBD1-derived nets capture bacteria and inhibit bacterial transmigration independent of bacterial killing. The presence of nets could completely prevent migration of hBD1 resistant pathogens and are stable in the presence of human duodenal secretion with a high amount of proteases. In contrast to HD6, cysteins are necessary for net formation. This redox-dependent function serves as an additional mechanism of action for hBD1 and differs from net formation by other defensins such as Paneth cell-derived human α-defensin 6 (HD6). While hBD1red and hBD1ox have distinct antimicrobial profiles and functions, only the reduced form provides additional host protection by entrapping bacteria in extracellular net structures preventing bacterial invasion. Better understanding of the modes of action of endogenous host peptides will help to find new antimicrobial strategies.

Silencing of Kangai 1 C-terminal interacting tetraspanin suppresses progression of cholangiocarcinoma.

Cholangiocarcinoma (CC) is the second most common primary hepatic malignancy. CC treatment options are very limited especially for patients with distant metastasis. Kangai 1 C-terminal interacting tetraspanin (KITENIN) is highly expressed in numerous cancers, but the role of KITENIN in CC remains unknown. Here, we have investigated for the first time the function of KITENIN in human CC cell lines (TFK-1, SZ-1), tissues and a CC mouse model (Alb-Cre/LSL-KRASG12D/p53L/L). KITENIN was expressed in 92.2% of human CC tissues, in murine CC samples and also in human CC cell lines. Knockdown of KITENIN by small interfering RNA (siRNA) effectively reduced proliferation, migration, invasion and colony formation in both intra- and extra-hepatic CC cells. The expression of epithelial-mesenchymal transition (EMT) markers like N-cadherin, Vimentin, Snail and Slug were suppressed in KITENIN knockdown CC cells. Our results indicate that KITENIN is crucial for cholangiocarcinogenesis and it might become a potential therapeutic target for human CC treatment.

Development and implementation of a comprehensive ultrasound curriculum for undergraduate medical students - a feasibility study.

BACKGROUND: Ultrasound is one of the most important imaging techniques in clinical medicine with unique advantages. Skills in ultrasound imaging are very usefull for physicians including novices and thus also mandated by the Task Force "National Competence-Based Learning Objectives for Undergraduate Medical Education" (NKLM) in Germany and as well as by the German Ultrasound Society (Deutsche Gesellschaft für Ultraschall in der Medizin, DEGUM). Since ultrasound is best learned hands-on in very small supervised groups, we developed and implemented a comprehensive ultrasound-curriculum for all undergraduate medical students of our faculty using a peer-teaching concept. METHODS: We used Kern's six-step model of curricular development comprising (1) problem identification and general needs assessment, (2) needs assessment of the targeted learners, (3) goals and objectives, (4) educational stategies, (5) implementation, and (6) evaluation and feedback. RESULTS: The developed curriculum covers basic ultrasound of the abdomen and the throat, eFAST (Extended Focused Assessment with Sonography for Trauma), lung-ultrasound, FEEL (Focused Echocardiography in Emergency Life Support) and compression duplex sonography of the thigh deep vein system. All 5th year medical students receive a 90 min lecture on ultrasound basics by a faculty member and then a 12.5 h hands-on course divided into three sessions with one student tutor for every 4 students. The students are provided with a script (PDF-File) that covers all the learning goals, including example images of pathologies. The student tutors are trained during a 1 week ultrasound course and a 21-day rotation through seven different ultrasound laboratories. In addition, they undergo a standardized 1.5 day didactical training. Prior to the implementation for all students, the overall course was tested on 27 volunteer students. These students rated (on a 6-point Likert scale from 1 = excellent to 6 = very poor) the satisfaction with the student tutors and the faculty members as 1.4 ± .9 (mean ± stddev) and 1.3 ± .5 respectively. CONCLUSION: A comprehensive ultrasound curriculum for all undergraduate medical students using a peer-teaching concept is feasible. Further studies are needed to evaluate in detail the learning outcomes for students and student tutors.

Sulphite oxidase (SO) - a mitochondrial autoantigen as target for humoral and cellular immune reactions in primary sclerosing cholangitis.

BACKGROUND: In a recent study we had evidence that sulphite oxidase (SO) may be a relevant autoantigen in primary sclerosing cholangitis (PSC). Aim of the present study was, therefore, to analyse humoral and cellular immune-reactivity towards SO in these patients in more detail. METHODS: Sera from 53 patients with PSC (30 untreated and 23 treated with ursodeoxycholic acid [UDCA] at time of analysis), from 422 patients with different hepatic and non-hepatic disorders, and from 50 healthy individuals were tested by ELISA for antibodies against full-length-SO (SO-fl) and its three major domains expressed in E.coli (SO-I, SO-II, SO-III). For epitope-mapping, 29 overlapping peptides were used. Peripheral blood mononuclear cells (PBMC) were obtained from 33 PSC-patients and analysed for SO-induced proliferation, production of cytokines, and expression of the activation marker cluster of differentiation (CD) 69. RESULTS: 43% of the 30 untreated and 26% of the 23 treated PSC-patients had IgG anti-SO-antibodies predominantly reacting with SO-fl, SO-I and SO-II. Antibody-reactivity decreased after UDCA-treatment. Prevalence and reactivity of anti-SO-antibodies were significantly higher in PSC than in patients with other hepatic and non-hepatic disorders. Epitope mapping revealed no distinct immuno-dominant regions within SO. Incubation of PBMC from PSC-patients (but not from controls) with SO-antigens revealed an activation of B-cells and a T-helper cell type-2 reaction pattern (production of interleukin [IL]-13, IL-10). CONCLUSIONS: PSC-patients show humoral and cellular immune response towards SO. Antibodies may be predominantly directed against conformational epitopes. SO enhances in vitro especially T-helper cell type-2 immune-reactions, which may be pro-fibrotic. SO is a detoxifying enzyme present also in bacteria; further studies analysing its role in the aetiology and pathogenesis in PSC may, therefore, be important.

Rendezvous endoscopic recanalization for complete esophageal obstruction.

BACKGROUND AND STUDY AIM: Complete esophageal obstruction after (chemo)radiation for head and neck cancers is rare. However, inability to swallow one's own saliva strongly inflicts upon quality of life. Techniques for endoscopic recanalization in complete obstruction are not well established. We assessed the efficacy and safety of rendezvous recanalization. PATIENTS AND METHODS: We performed a retrospective review of all patients who underwent endoscopic recanalization of complete proximal esophageal obstruction after radiotherapy between January 2009 and June 2016. Technical success was defined as an ability to pass an endoscope across the recanalized lumen, clinical success by changes in the dysphagia score. Adverse events were recorded prospectively. RESULTS: 19 patients with complete obstruction (dysphagia IV°), all of whom had failed at least one trial of conventional dilatation, underwent recanalization by endoscopic rendezvous, a combined approach through a gastrostomy and perorally under fluoroscopic control. Conscious sedation was used in all patients. In 18/19 patients (94.7%), recanalization was technically successful. In 14/18 patients (77.8%), the post-intervention dysphagia score changed to ≤ II. Three patients had their PEG removed. Factors negatively associated with success were obstruction length of 50 mm; and tumor recurrence for long-term success. No severe complications were recorded. CONCLUSIONS: Rendezvous recanalization for complete esophageal obstruction is a reliable and safe method to re-establish luminal patency. Differences between technical and clinical success rates highlight the importance of additional functional factors associated with dysphagia. Given the lack of therapeutic alternatives, rendezvous recanalization is a valid option to improve dysphagia.

Crohn's disease-derived monocytes fail to induce Paneth cell defensins.

Crohn's disease (CD) is associated with a multitude of genetic defects, many of which likely affect Paneth cell function. Paneth cells reside in the small intestine and produce antimicrobial peptides essential for the host barrier, principally human α-defensin 5 (HD5) and HD6. Patients with CD of the ileum are characterized by reduced constitutive expression of these peptides and, accordingly, compromised antimicrobial barrier function. Here, we present a previously unidentified regulatory mechanism of Paneth cell defensins. Using cultures of human ileal tissue, we showed that the secretome of peripheral blood mononuclear cells (PBMCs) from healthy controls restored the attenuated Paneth cell α-defensin expression characteristic of patients with ileal CD. Analysis of the Wnt pathway in both cultured biopsies and intestinal epithelial cells implicated Wnt ligands driving the PBMC effect, whereas various tested cytokines were ineffective. We further detected another defect in patients with ileal CD, because the PBMC secretomes derived from patients with CD were unable to restore the reduced HD5/HD6 expression. Accordingly, analysis of PBMC subtypes showed that monocytes of patients with CD express significantly lower levels of canonical Wnt ligands, including Wnt3, Wnt3a, Wnt1, and wntless Wnt ligand secretion mediator (Evi/Wls). These studies reveal an important cross-talk between bone marrow-derived cells and epithelial secretory Paneth cells. Defective Paneth cell-mediated innate immunity due to inadequate Wnt ligand stimulation by monocytes provides an additional mechanism in CD. Because defects of Paneth cell function stemming from various etiologies are overcome by Wnt ligands, this mechanism is a potential therapeutic target for this disease.