Paired guide RNA CRISPR-Cas9 screening for protein-coding genes and lncRNAs involved in transdifferentiation of human B-cells to macrophages.

CRISPR-Cas9 screening libraries have arisen as a powerful tool to identify protein-coding (pc) and non-coding genes playing a role along different processes. In particular, the usage of a nuclease active Cas9 coupled to a single gRNA has proven to efficiently impair the expression of pc-genes by generating deleterious frameshifts. Here, we first demonstrate that targeting the same gene simultaneously with two guide RNAs (paired guide RNAs, pgRNAs) synergistically enhances the capacity of the CRISPR-Cas9 system to knock out pc-genes. We next design a library to target, in parallel, pc-genes and lncRNAs known to change expression during the transdifferentiation from pre-B cells to macrophages. We show that this system is able to identify known players in this process, and also predicts 26 potential novel ones, of which we select four (two pc-genes and two lncRNAs) for deeper characterization. Our results suggest that in the case of the candidate lncRNAs, their impact in transdifferentiation may be actually mediated by enhancer regions at the targeted loci, rather than by the lncRNA transcripts themselves. The CRISPR-Cas9 coupled to a pgRNAs system is, therefore, a suitable tool to simultaneously target pc-genes and lncRNAs for genomic perturbation assays.

Dynamic changes in intron retention are tightly associated with regulation of splicing factors and proliferative activity during B-cell development.

Intron retention (IR) has been proposed to modulate the delay between transcription and translation. Here, we provide an exhaustive characterization of IR in differentiated white blood cells from both the myeloid and lymphoid lineage where we observed highest levels of IR in monocytes and B-cells, in addition to previously reported granulocytes. During B-cell differentiation, we found an increase in IR from the bone marrow precursors to cells residing in secondary lymphoid organs. B-cells that undergo affinity maturation to become antibody producing plasma cells steadily decrease retention. In general, we found an inverse relationship between global IR levels and both the proliferative state of cells, and the global levels of expression of splicing factors. IR dynamics during B-cell differentiation appear to be conserved between human and mouse, suggesting that IR plays an important biological role, evolutionary conserved, during blood cell differentiation. By correlating the expression of non-core splicing factors with global IR levels, and analyzing RNA binding protein knockdown and eCLIP data, we identify a few splicing factors likely playing an evolutionary conserved role in IR regulation. Our work provides new insights into the role of IR during hematopoiesis, and on the main factors involved in regulating IR.

Low incidence of colonic complications after severe Shiga toxin-producing E. coli O104:H4 infection.

BACKGROUND: In summer 2011, Shiga toxin producing Escherichia coli (EHEC) serotype O104:H4 caused the most severe EHEC outbreak in Germany to date. The case of a previously recovered patient with symptomatic postinflammatory colonic stenosis following EHEC- infection prompted us to conduct a prospective study to assess the macro- and microscopic intestinal long-term damage in a cohort of patients who had suffered from severe EHEC colitis. METHODS: Following EHEC infection in 2011, 182 patients were offered to participate in this study between January 2013 and October 2014 as part of the post-inpatient follow-up care at the University Medical Center Hamburg-Eppendorf and to undergo colonoscopy with stepwise biopsies. Prior to colonoscopy, medical history and persistent post-infectious complaints were assessed. RESULTS: Out of 182 patients, 22 (12%) participated in the study, 18 (82%) were female. All patients had been hospitalized due severe EHEC enterocolitis: 20 patients (90%) had subsequently developed hemolytic uremic syndrome (HUS), 16 patients (72%) had additionally required dialysis. On assessment prior to colonoscopy, all patients denied any abdominal complaints before EHEC-infection but 8 (36%) patients reported persistent post-infectious symptoms. According to the ROME IV criteria, 4 (18%) patients met the definition for post-infectious irritable bowel syndrome (PI-IBS). In all patients with persistent symptoms, colonoscopies and histological examination were unremarkable. Only in one symptom-free patient, biopsy revealed a locally limited cryptitis of the caecum, while all patients without complaints had inconspicuous histological and endoscopical findings. CONCLUSION: Following infection colonic stenosis is a serious but rare long-term complication in patients who had suffered from severe enterocolitis. However, a significant proportion of these patients develop PI-IBS.

Increased Seroprevalence of Campylobacter jejuni, but not HEV, in healthcare workers in gastroenterological endoscopy.

BACKGROUND: The exposure of healthcare workers (HCW) to fecal-orally transmitted pathogens like hepatitis E Virus (HEV), Campylobacter jejuni or Helicobacter pylori is still not known. The potential risk for employees or patients to acquire these infections through asymptomatic infected healthcare personnel has not yet been studied. Physicians and nurses in gastroenterology working in endoscopic workspaces were recruited. Employees from cardiology, presumed to possess a lower exposure, served as controls. The cytomegalovirus (CMV) seroprevalence was analyzed as a control pathogen without fecal-oral route of transmission. This study provides an objective view onto the potential exposure risk for HCW and patients in endoscopic workspaces. We hypothesize that HCW in gastroenterological endoscopy show a higher seroprevalence for fecal-oral pathogens like HEV, C. jejuni and H. pylori compared to HCW in cardiology. OBJECTIVE: Primary objective was the assessment of antibody titers against HEV, C. jejuni and H. pylori in serum of HCW from gastroenterological endoscopy as well as cardiology. As a secondary objective we analyzed the seroprevalence against CMV. METHODS: 65 HCW were from gastroenterological endoscopy (n=42) and cardiology (n=23) in three medical centers in the German federal states of Brandenburg, Hamburg and Schleswig-Holstein and were prospectively studied. Antibody titers were determined via ELISA in serum. RESULTS: HCW in gastroenterological endoscopy showed a significantly higher C. jejuni seroprevalence for IgG (19.1 %) compared to HCW from the field of cardiology (8.7 %; p=0.04). IgA titers against C. jejuni were negligible. HEV seroprevalence for IgG did not differ significantly between HCW in gastroenterological endoscopy (7.1 %) and cardiology (8.7 %), respectively. IgA and IgM titers against HEV were also negligible. All other antibody titers against CMV and H. pylori showed no significant difference. CONCLUSIONS: Only the C. jejuni seroprevalence was significantly increased in HCW from the field of gastroenterological endoscopy. HEV seroprevalence showed no differences. The results for CMV and H. pylori were without pathological findings. However, there is no elevated risk for HEV exposure in medical staff working at an endoscopy unit, but for C. jejuni the protective measures might need to be improved.

Diagnostic benefit of high b-value computed diffusion-weighted imaging in acute brainstem infarction.

BACKGROUND AND PURPOSE: Diffusion-weighted imaging (DWI) is a cornerstone in diagnostic of ischemic stroke. The aim of this study was to investigate the usefulness of high-b-value computed DWI (c-DWI) in comparison to standard DWI in patients with acute brainstem infarction. MATERIALS AND METHODS: 56 patients with acute brainstem infarction were retrospectively analysed by two readers. DWI was obtained with the b-values 0, 500 and 1000 s/mm² on either a 1.5 or 3 T magnetic resonance imaging (MRI) scanner. c-DWI was calculated with a monoexponential model with high b-values 2000, 3000, 4000 and 5000 s/mm². All c-DWI series with high-b-values were compared to the standard DWI sequence at b-value of 1000 s/mm² in terms of image artifacts, lesion extent and contrast. RESULTS: There was no statistically significant difference between 1.5 and 3 T MRI regarding the measured ischemic lesion size. There were no statistically significant differences between the ischemic lesion sizes on DWI at b-values of 1000 s/mm² and on c-DWI at higher b-values. Overall, the contrast between the lesion and the surrounding normal areas improved with increasing b-value on the isotropic DWIs: maximum at b = 5000, followed by that at b 2000 and b 1000 s/mm², in order. The best relation between artifacts and lesion contrast was identified for b 2000 s/mm². CONCLUSION: High b-value DWI derived from c-DWI has a higher visibility for ischemic brainstem lesions compared to standard DWI without additional time cost. The b-2000 image is recommended to use in clinical routine, higher b-value images lead to more imaging artifacts, which might result in misdiagnosis.

Endoscopic neo-anastomosis by Rendez-vous technique for the treatment of complete occlusion of bilioenteric anastomoses and distal bile ducts (case series).

BACKGROUND AND AIMS: The complete occlusion of bilioenteric anastomoses is a rare and challenging clinical condition. Repeated surgery is burdened with technical difficulties and significant morbidity. We report the first series of completely occluded bilioenteric anastomoses resp. distal bile duct successfully treated by simultaneous percutaneous and retrograde endoscopic interventions. PATIENTS AND METHODS: This case series includes 4 patients with obstructive jaundice and/or recurring cholangitis and pain due to complete fibrotic occlusion of a hepaticojejunostomy (3 patients) and the distal bile duct (1 patient). After performing PTCD and stepwise dilation of the biliocutaneous tract, we tried to approach the occluded anastomosis from 2 sides by simultaneous percutaneous cholangioscopy and peroral device-assisted enteroscopy/duodenoscopy. By cutting through the separating tissue layer with a needle knife under endoscopic and fluoroscopic control using diaphanoscopy, a new anastomosis should be established followed by dilation of the neoanastomosis with subsequent percutaneous transhepatic drainage for a minimum of 1 year to prevent re-occlusion. RESULTS: The Rendez-vous maneuver was successful in 3/4 cases. In one case, the retrograde access to the anastomosis failed, so the neoanastomosis was cut under cholangioscopic and fluoroscopic guidance only. The neoanastomosis could be established successfully in all 4 cases. Jaundice, cholangitis, and pain disappeared. Minor periinterventional adverse events were cholangitis (n = 1) and pneumonia (n = 1) due to aspiration, which could be managed conservatively. No serious adverse events were observed, and no re-occlusion of any neoanastomosis occurred during the follow-up before and after removal of the percutaneous drainage. CONCLUSION: Simultaneous percutaneous cholangioscopy and device-assisted enteroscopy/duodenoscopy with endoscopic creation of a neoanastomosis is a possible concept for the treatment of completely occluded bilioenteric anastomoses and distal bile ducts. This case series confirms the feasibility, safety, and long-term effectiveness of this treatment.

Granulomatous Inflammation in ANCA-Associated Vasculitis.

ANCA-associated vasculitis (AAV) comprises granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), and eosinophilic granulomatosis with polyangiitis (EGPA). While systemic vasculitis is a hallmark of all AAV, GPA is characterized by extravascular granulomatous inflammation, preferentially affecting the respiratory tract. The mechanisms underlying the emergence of neutrophilic microabscesses; the appearance of multinucleated giant cells; and subsequent granuloma formation, finally leading to scarred or destroyed tissue in GPA, are still incompletely understood. This review summarizes findings describing the presence and function of molecules and cells contributing to granulomatous inflammation in the respiratory tract and to renal inflammation observed in GPA. In addition, factors affecting or promoting the development of granulomatous inflammation such as microbial infections, the nasal microbiome, and the release of damage-associated molecular patterns (DAMP) are discussed. Further, on the basis of numerous results, we argue that, in situ, various ways of exposure linked with a high number of infiltrating proteinase 3 (PR3)- and myeloperoxidase (MPO)-expressing leukocytes lower the threshold for the presentation of an altered PR3 and possibly also of MPO, provoking the local development of ANCA autoimmune responses, aided by the formation of ectopic lymphoid structures. Although extravascular granulomatous inflammation is unique to GPA, similar molecular and cellular patterns can be found in both the respiratory tract and kidney tissue of GPA and MPA patients; for example, the antimicrobial peptide LL37, CD163+ macrophages, or regulatory T cells. Therefore, we postulate that granulomatous inflammation in GPA or PR3-AAV is intertwined with autoimmune and destructive mechanisms also seen at other sites.

[Clinical practicability of the cognitive screening battery BICAMS in patients with multiple sclerosis: results of the feasibility study in Germany]. / Klinische Umsetzbarkeit der kognitiven Screeningbatterie BICAMS bei Patienten mit Multipler Sklerose: Ergebnisse der Machbarkeitsstudie in Deutschland.

BACKGROUND: Patients with multiple sclerosis (MS) suffer from cognitive impairment in 40-70% of the cases. There is evidence that the cognitive status is predictive for working ability and early retirement. Regular assessment of cognitive functionality is therefore urgently needed. PURPOSE: The German validation of the Brief International Cognitive Assessment for Multiple Sclerosis (BICAMS) battery was evaluated in a multicentric way with respect to its feasibility in private neurological centers across Germany. METHODS: Physician assistants were trained with respect to application and scoring of BICAMS. All scored test materials were evaluated by independent neuropsychological experts. RESULTS: A total of 1606 BICAMS datasets were collected from 65 neurological centers. Of these 1573 datasets were analyzed of which 49.7% were correctly applied and scored while mistakes in application, scoring and transformation were found in 50.3%. Interrater reliability for each subtest was found to be ICC [Formula: see text] 0.953 when datasets containing mistakes were excluded. DISCUSSION: In general, BICAMS is highly recommended to be applied in standard clinical care; however, it should be emphasized that although the interrater reliability in the final sample was high, serious mistakes were found in 50.3% of cases. From these findings we conclude that nonpsychological staff have to be even more intensively trained and supervised by experts in the application and scoring of BICAMS.

On the validity of single tests, two-test combinations and the full Brief International Cognitive Assessment for Multiple Sclerosis (BICAMS) in detecting patients with cognitive impairment.

BACKGROUND: The international standard to screen for cognitive impairment in multiple sclerosis (MS) is BICAMS (Brief International Cognitive Assessment for MS). However, with an application time of approximately 20 minutes, the battery might be too time consuming from a pragmatic perspective of a routine examination. OBJECTIVES: To examine the relative sensitivity and specificity of a BICAMS short version and its validity compared to the total battery. METHODS: The German BICAMS version was applied comprising the Symbol Digit Modalities Test (SDMT), the Brief Visuospatial Memory Test-Revised (BVMT-R) and the Rey Auditory Verbal Learning Test (RAVLT; German VLMT). Single tests and two-test combinations were compared regarding conformity with the total battery. RESULTS: Examining 1320 MS patients, the two-test combination of SDMT-BVMT-R was the most sensitive (92.7%) to impairment and showed the strongest agreement with the total battery (κ = 0.95). Performing binary logistic regression analyses, this combination was also validated by its association with employment status. CONCLUSION: Application of the total BICAMS battery should be the goal to strive for. However, in time-restricted clinical settings, the combined application of SDMT and BVMT-R is a recommendable alternative with an application time of 10 minutes, while single tests alone are not sufficiently sensitive.

Fibrinolysis and the Influence of Tranexamic Acid Dosing in Cardiac Surgery.

OBJECTIVE: The present study aimed to determine whether underlying disease, performed surgery, and dose of tranexamic acid influence fibrinolysis measured with D-dimer levels. DESIGN: Retrospective analysis. SETTING: Single institution (Department of Cardiac Surgery and Section of Clinical Hemostaseology at the Düsseldorf University Hospital). PARTICIPANTS: The study comprised 3,152 adult patients undergoing elective cardiac surgery between February 2013 and October 2016. INTERVENTIONS: Two doses of tranexamic acid during surgery were administered. MEASUREMENTS AND MAIN RESULTS: D-dimer levels were analyzed at the start of surgery and before protamine administration. D-dimer levels at the start of surgery were compared according to disease. Intraoperative D-dimer development was analyzed according to the type of surgery and within 2 cohorts with different tranexamic acid doses. Interindividual variability was pronounced for D-dimer levels at the start of surgery, with significant differences among patients with coronary artery disease, valve disease, and aortic disease and patients undergoing heart transplantation compared with patients receiving a left ventricular assist device (p < 0.01). Aortic dissection, endocarditis, and extracorporeal life support were associated with higher D-dimer levels (p ≤ 0.01). With tranexamic acid at a fixed dose, intraoperative D-dimer levels decreased in on-pump and off-pump coronary bypass surgery, valve surgery, and left ventricular assist device surgery (p ≤ 0.02), but levels increased in aortic surgery and heart transplantations (p < 0.01). A decrease or increase in D-dimer levels during surgery was influenced significantly by a higher or lower tranexamic acid dose (p ≤ 0.01). CONCLUSIONS: D-dimer testing allows for the assessment of individual fibrinolytic activity in cardiac surgery, which is influenced by disease type, surgery type, and dose of tranexamic acid. The assessment of the fibrinolytic status may have the potential to facilitate dose-adjusted antifibrinolytic therapy in the future.

Interactions between rheumatoid arthritis synovial fibroblast migration and endothelial cells.

Leukocytes travel within the circulation and enter connective tissues by interactions with endothelium of postcapillary venules mediated by cell adhesion molecules, summarized as the leukocyte adhesion cascade. In the severe combined immunodeficient (SCID) mouse model, rheumatoid arthritis (RA) synovial fibroblasts (SF) migrated to distant cartilage through the vasculature. Therefore, RASF adhesion toward endothelial cells (EC) and E- and P-selectins were analyzed. Cell-to-cell binding assays between SF and EC were performed. Interactions of SF with tumor necrosis factor α (TNFα)-activated EC or selectins were analyzed in flow adhesion assays. Immunohistochemistry for E-selectin ligand CD15s was performed. CD15s induction in RASF by human serum or media was evaluated. Wild-type and E-/-/ P-/- Selectin-SCID mice were used for inverse-wrap surgery. After laser-mediated microdissection, real-time PCR for E-/P-selectin/vascular cell adhesion molecule 1 was performed. Adhesion between SF/EC under static conditions was highest in Roswell Park Memorial Institute-cultured RASF to TNFαα-activated human umbilical vein endothelial cells (2.25-fold) and RASF adhesion was higher toward venous than arterial EC (Dulbecco's modified eagle medium P = 0.0419, RPMI P = 0.0119). In flow chamber assays, RASF adhesion to E-selectin was higher than to P-selectin (e.g. 0.9 dyn cm-2 P = 0.0001). Osteoarthritis synovial fibroblasts showed lower rolling/adhesion properties (e.g. 0.5 dyn cm-2 , P = 0.0010). RASF adhesion to TNFαα-activated EC was increased (e.g. 0.9 dyn cm-2 , P = 0.0061). CD15s induction in RASF was strongest in RA serum. Vimentin/CD15s double-positive cells were detectable. In E-/P-selectin-deficient mice, contralateral invasion was reduced (P = 0.023). E- and P-selectin, and vascular cell adhesion molecule 1 expression in EC of implants was confirmed. Our data indicate that the milieu within vessels induces CD15s which enables RASF to interact with E-selectin/EC under flow. Therefore, RASF may migrate to distant sites and leave the vasculature similarly to leukocytes.

[The Impact of Immigrant Status on the Mode of Hospital Referral, Impression of Disease Severity and Length of Stay in Inpatient General Pediatric Care]. / Die Bedeutung des Migrationshintergrundes für Einweisungsmodus, Krankheitseindruck und Verweildauer in der stationären pädiatrischen Versorgung.

BACKGROUND: National as well as international research often shows differences in health and health behavior between children and youth with and without migration background. It is also noted that there are differences in the use of health services depending on the migration background. PATIENTS AND METHODS: Data from 266 pediatric patients, regarding their hospitalization, general impression of health status and length of stay in a hospital, were analyzed depending on the migration background. Information on migration background and treatment-related data were obtained from the parents of the patient or from the hospital information system (SAP). 20.7% of patients (n=55) had a migration background. RESULTS: Migrants were hospitalized more often under a participation of the emergency room than non-migrants; also migrants showed a more severe illness picture. Regarding the number of diagnoses and length of stay as well as the distribution of the main diagnoses, no differences were found. DISCUSSION: Language barriers, culture-specific ideas about illness and insufficient knowledge of the German health care system were discussed as possible reasons to for the differences between migrants and non-migrants. CONCLUSION: This study confirms already known differences in the use of health services by people with and without migration background. The results indicate worse health status in migrant patients compared to non-migrants by hospitalization. Future research with greater numbers of participants should specifically investigate on this point.

Phosphazenyl Phosphines: The Most Electron-Rich Uncharged Phosphorus Brønsted and Lewis Bases.

It was discovered that phosphazenyl phosphines (PAPs) can be stronger P-superbases than the corresponding Schwesinger type phosphazene N-superbases. A simple synthetic access to this class of PR3 derivatives including their homologization is described. XRD structures, proton affinities (PA), and gas-phase basicities (GB) as well as calculated and experimental pK BH + values in THF are presented. In contrast to their N-basic counterparts, PAPs are also privileged ligands in transition metal chemistry. In fact, they are currently the strongest uncharged P-donors known, exceeding classical and more recently discovered ligands such as PtBu3 and imidazolin-2-ylidenaminophosphines (IAPs) with respect to their low Tolman electronic parameters (TEPs) and large cone angles.

Anti-Shiga toxin 2 antibodies in enterohemorrhagic Escherichia coli O104:H4 infected patients may predict hemolytic uremic syndrome.

BACKGROUND AND AIM: An outbreak of Shiga toxin 2 (Stx2) producing enterohemorrhagic and enteroaggregative Escherichia coli O104:H4 infection in May 2011 in Germany caused enterocolitis and an unprecedented high 22% rate of hemolytic uremic syndrome (HUS). We hypothesized that anti-Stx2 IgM or IgG titers might predict HUS development. METHODS: Thirty-two patients infected with enterohemorrhagic Escherichia coli O104:H4 (HUS: n = 23; non-HUS: n = 9) were retrospectively screened for anti-Stx2 IgM/IgG and matched with clinical data regarding HUS development, fever, superinfection, dialysis, neurological symptoms, intensive care, antibiotic treatment, and plasmapheresis. RESULTS: Only HUS patients showed a prominent Stx2-specific humoral response in the early acute phase. Despite a strong trend towards prediction of HUS development, statistical analysis revealed no significant correlation between high IgM/IgG titers and further key clinical parameters such as fever, superinfection, neurological symptoms, antibiotic treatment, and plasmapheresis. CONCLUSIONS: Anti-Stx2 antibodies seem to accompany or even precede HUS development.

Headspace solid-phase microextraction and gas chromatographic analysis of low-molecular-weight sulfur volatiles with pulsed flame photometric detection and quantification by a stable isotope dilution assay.

Low-molecular-weight volatile sulfur compounds such as thiols, sulfides, disulfides as well as thioacetates cause a sulfidic off-flavor in wines even at low concentration levels. The proposed analytical method for quantification of these compounds in wine is based on headspace solid-phase microextraction, followed by gas chromatographic analysis with sulfur-specific detection using a pulsed flame photometric detector. Robust quantification was achieved via a stable isotope dilution assay using commercial and synthesized deuterated isotopic standards. The necessary chromatographic separation of analytes and isotopic standards benefits from the inverse isotope effect realized on an apolar polydimethylsiloxane stationary phase of increased film thickness. Interferences with sulfur-specific detection in wine caused by sulfur dioxide were minimized by addition of propanal. The method provides adequate validation data, with good repeatability and limits of detection and quantification. It suits the requirements of wine quality management, allowing the control of oenological treatments to counteract an eventual formation of excessively high concentration of such malodorous compounds.

Remodeling of extracellular matrix of the lamina propria in the uninvolved human rectal mucosa 10 and 20 cm away from the malignant tumor.

In recent years, it has been demonstrated that malignancy arises and advances through the molecular interplay between tumor cells and non-malignant elements of the tumor stroma, that is, fibroblasts and extracellular matrix. However, in contrast to the mounting evidence about the role of tumor stroma in the genesis and progression of the malignant disease, there are very few data regarding the uninvolved stromal tissue in the remote surrounding of the tumor. Using the objective morphometric approach in patients with adenocarcinoma, we demonstrate the remodeling of extracellular matrix of the lamina propria in the uninvolved rectal mucosa 10 and 20 cm away from the neoplasm. We show that the representation of basic extracellular matrix constituents (reticular and collagen fibers and ground substance) is decreased. Also, the diameter of empty spaces that appear within the extracellular matrix of the lamina propria is increased. These spaces do not represent the blood or lymphatic vessel elements. Very likely, they reflect the development of tissue edema in the remote, uninvolved lamina propria of the mucosa in patients with the malignant tumor of the rectum. We hypothesize that the remodeling of extracellular matrix in lamina propria of the rectal mucosa may increase its stiffness, modulating the mechano-signal transduction, and thus promote the progression of the malignant disease.

Chelating P2-Bis-phosphazenes with a (R,R)-1,2-Diaminocyclohexane Skeleton: Two New Chiral Superbases.

The linkage of two P2 -phosphazenyl groups through a C2 -symmetric (R,R)-1,2-diaminocyclohexane (DACH) backbone yielded the new chiral superbases DACH-P2 NMe2 and DACH-P2 Pyr (Pyr=pyrrolidinyl). These bases were prepared by a Kirsanov reaction and studied with respect to their spectroscopic and structural characteristics. Theoretical calculations concerning their basicity properties revealed remarkable pKBH+ values of 38.1 and 39.9 on the acetonitrile scale; this makes them the strongest nonionic chiral superbases known to date.

Orthotopic tracheal transplantation using human bronchus: an original xenotransplant model of obliterative airway disorder.

Bronchiolitis obliterans syndrome (BOS) is a main cause of allograft dysfunction and mortality after lung transplantation (LTx). A better understanding of BOS pathogenesis is needed to overcome this treatment-refractory complication. Orthotopic tracheal transplantation using human bronchus was performed in Brown Norway (BN) and nude (RNU) rats. Allografts were recovered in both strains at Day 7 (BN7 , n = 6; RNU7 , n = 7) or Day 28 (BN28 , n = 6; RNU28 , n = 6). Immune response of the host against the bronchial graft was assessed. Human samples from BOS patients were used to compare with the histological features of the animal model. Obstruction of the allograft lumen associated with significant infiltration of CD3+ and CD68+ cells was observed in the BN group on Day 28. Immune response from type 1 T-helper cells against the tracheal xenograft was higher in BN animals compared to nude animals on Days 7 and 28 (P < 0.001 and P = 0.035). Xenoreactive antibodies were significantly higher at Day 7 (IgM) and Day 28 (IgG) in the BN group compared to RNU (respectively, 37.6 ± 6.5 vs. 5.8 ± 0.7 mean fluorescence, P = 0.039; and 22.4 ± 3.8 vs. 6.9 ± 1.6 mean fluorescence, P = 0.011). Immunocompetent animals showed a higher infiltration of S100A4+ cells inside the bronchial wall after 28 days, associated with cartilage damage ranging from invasion to complete destruction. In vitro expression of S100A4 by human fibroblasts was higher when stimulated by mononuclear cells (MNCs) from BN rats than from RNU (2.9 ± 0.1 vs. 2.4 ± 0.1 mean fluorescence intensity, P = 0.005). Similarly, S100A4 was highly expressed in response to human MNCs compared to stimulation by T-cell-depleted human MNCs (4.3 ± 0.2 vs. 2.7 ± 0.1 mean fluorescence intensity, P < 0.001). Obliterative bronchiolitis has been induced in a new xenotransplant model in which chronic airway obstruction was associated with immune activation against the xenograft. Cartilage infiltration by S100A4+ cells might be stimulated by T cells.

EUS-guided choledochoduodenostomy for malignant distal biliary obstruction using a lumen-apposing fully covered metal stent after failed ERCP.

BACKGROUND: A novel lumen-apposing, self-expanding metal stent to perform EUS-guided drainage procedures has been recently developed. The aim of this study was to analyze the safety, technical and clinical effectiveness of this device for EUS-guided choledochoduodenostomy (EUS-CD) with palliative intent. METHODS: Retrospective analysis of all consecutive patients with unresectable malignant distal bile duct obstruction who, between March 2012 and September 2014, underwent EUS-CD using the study devices (AXIOS™ and Hot AXIOS™, Xlumena Inc., Mountain View, CA, USA) after unsuccessful ERCP in seven European centers was carried out. RESULTS: Fifty-seven patients (M/F 31/26; median age 73) underwent EUS-CD using the AXIOS™ stent or the Hot AXIOS™ delivery system. ERCP failure was due to duodenal obstruction in 41 patients (71.9 %) and to inability to cannulate the papilla in the remaining 16 patients (28.1 %). The procedure was technically successful in 56/57 patients (98.2 %), with a mean procedural time of 22.4 min (range 11-65). Clinical success was achieved in 54 of these 56 patients (96.4 %; 94.7 % of the entire cohort). Overall major procedural complication rate was 7 % (two duodenal perforations, one bleeding and one transient cholangitis). During follow-up, 5 out of 54 (9.3 %) patients with clinica success required re-intervention for stent migration in one case and a sump syndrome with transient increase in serum bilirubin concentrations with sludge in the distal duct reservoir in the remaining four patients. CONCLUSIONS: Our study shows that EUS-CD using the AXIOS™ and the Hot AXIOS™ devices is a safe procedure, with high technical and clinical success rates.

Development of a novel IGRA assay to test T cell responsiveness to HBV antigens in whole blood of chronic Hepatitis B patients.

BACKGROUND: Interferon gamma release assays (IGRA) have been developed to support easy and fast diagnosis of diseases like tuberculosis, and CMV in transplant patients. IGRAs focus on cellular immunity especially memory T cells and thus also allow rapid screening prior to complex flow cytometric testing. Here, we describe a novel, sensitive whole blood based cytokine release assay capable of assessing T cell responsiveness to HBV antigens in Hepatitis B patients and assessing hepatitis B vaccination status in healthy individuals. METHODS: Seventy two chronic Hepatitis B patients (CHB), 8 acute hepatitis B patients (AHB) and 80 healthy controls (HC) were tested by ELISA for IFNγ- and IL2-secretion in whole blood after challenge with synthetic peptide libraries of hepatitis B core antigen (HBcAg) or hepatitis B surface antigen (HBsAg). RESULTS: The developed IGRA test reliably differentiated between Hepatitis B patients, vaccinees and unvaccinated healthy controls. Treatment naïve and treated CHB patients showed a weaker IFNγ response to HBcAg (16 ± 5 and 35 ± 28 pg/ml, respectively) compared to the AHB group (82 ± 39 pg/ml), whereas HC remained unresponsive (6 ± 1 pg/ml). IL2 levels after HBcAg challenge were also higher in the AHB group compared to naive and treated CHB as well as HC (47 ± 21 vs. 12 ± 3, 15 ± 10 and 12 ± 9 pg/ml, respectively). HBsAg stimulation led to increased IFNγ and IL2 levels in the AHB group (33 ± 12 and 22 ± 12 pg/ml) and even higher levels in HC due to a high hepatitis B vaccination rate (41 ± 10 and 167 ± 58 pg/ml). Naive and treated CHB patients developed no or only weaker IFNγ or IL2 responses to HBsAg (5 ± 2 and 12 ± 7 pg/ml, for naive CHB, 12 ± 10 and 18 ± 15 pg/ml, for treated CHB). For HC, IL2 release after HBsAg stimulation depicted hepatitis B vaccination status with a diagnostic sensitivity and specificity of 85 % and 90 %. CONCLUSION: Our novel whole blood based cytokine release assay constitutes an easy and robust tool for screening HBV specific cellular immunity as alternative to flow cytometry or ELISPOT assays.