In Planta Production of the Receptor-Binding Domain From SARS-CoV-2 With Human Blood Group A Glycan Structures.

Glycosylation of viral envelope proteins is important for infectivity and immune evasion. The SARS-CoV-2 spike protein is heavily glycosylated and host-derived glycan modifications contribute to the formation of specific immunogenic epitopes, enhance the virus-cell interaction or affect virus transmission. On recombinant viral antigens used as subunit vaccines or for serological assays, distinct glycan structures may enhance the immunogenicity and are recognized by naturally occurring antibodies in human sera. Here, we performed an in vivo glycoengineering approach to produce recombinant variants of the SARS-CoV-2 receptor-binding domain (RBD) with blood group antigens in Nicotiana benthamiana plants. SARS-CoV-2 RBD and human glycosyltransferases for the blood group ABH antigen formation were transiently co-expressed in N. benthamiana leaves. Recombinant RBD was purified and the formation of complex N-glycans carrying blood group A antigens was shown by immunoblotting and MS analysis. Binding to the cellular ACE2 receptor and the conformation-dependent CR3022 antibody showed that the RBD glycosylation variants carrying blood group antigens were functional. Analysis of sera from RBD-positive and RBD-negative individuals revealed further that non-infected RBD-negative blood group O individuals have antibodies that strongly bind to RBD modified with blood group A antigen structures. The binding of IgGs derived from sera of non-infected RBD-negative blood group O individuals to blood group A antigens on SARS-CoV-2 RBD suggests that these antibodies could provide some degree of protection from virus infection.

The FXR agonist PX20606 ameliorates portal hypertension by targeting vascular remodelling and sinusoidal dysfunction.

BACKGROUND & AIMS: Steroidal farnesoid X receptor (FXR) agonists demonstrated potent anti-fibrotic activities and lowered portal hypertension in experimental models. The impact of the novel non-steroidal and selective FXR agonist PX20606 on portal hypertension and fibrosis was explored in this study. METHODS: In experimental models of non-cirrhotic (partial portal vein ligation, PPVL, 7days) and cirrhotic (carbon tetrachloride, CCl4, 14weeks) portal hypertension, PX20606 (PX,10mg/kg) or the steroidal FXR agonist obeticholic acid (OCA,10mg/kg) were gavaged. We then measured portal pressure, intrahepatic vascular resistance, liver fibrosis and bacterial translocation. RESULTS: PX decreased portal pressure in non-cirrhotic PPVL (12.6±1.7 vs. 10.4±1.1mmHg; p=0.020) and cirrhotic CCl4 (15.2±0.5 vs. 11.8±0.4mmHg; p=0.001) rats. In PPVL animals, we observed less bacterial translocation (-36%; p=0.041), a decrease in lipopolysaccharide binding protein (-30%; p=0.024) and splanchnic tumour necrosis factor α levels (-39%; p=0.044) after PX treatment. In CCl4 rats, PX decreased fibrotic Sirius Red area (-43%; p=0.005), hepatic hydroxyproline (-66%; p<0.001), and expression of profibrogenic proteins (Col1a1, α smooth muscle actin, transforming growth factor ß). CCl4-PX rats had significantly lower transaminase levels and reduced hepatic macrophage infiltration. Moreover, PX induced sinusoidal vasodilation (upregulation of cystathionase, dimethylaminohydrolase (DDAH)1, endothelial nitric oxide synthase (eNOS), GTP-cyclohydrolase1) and reduced intrahepatic vasoconstriction (downregulation of endothelin-1, p-Moesin). In cirrhosis, PX improved endothelial dysfunction (decreased von-Willebrand factor) and normalized overexpression of vascular endothelial growth factor, platelet-derived growth factor and angiopoietins. While short-term 3-day PX treatment reduced portal pressure (-14%; p=0.041) by restoring endothelial function, 14week PX therapy additionally inhibited sinusoidal remodelling and decreased portal pressure to a greater extent (-22%; p=0.001). In human liver sinusoidal endothelial cells, PX increased eNOS and DDAH expression. CONCLUSIONS: The non-steroidal FXR agonist PX20606 ameliorates portal hypertension by reducing liver fibrosis, vascular remodelling and sinusoidal dysfunction. LAY SUMMARY: The novel drug PX20606 activates the bile acid receptor FXR and shows beneficial effects in experimental liver cirrhosis: In the liver, it reduces scarring and inflammation, and also widens blood vessels. Thus, PX20606 leads to an improved blood flow through the liver and decreases hypertension of the portal vein. Additionally, PX20606 improves the altered intestinal barrier and decreases bacterial migration from the gut.

Non-linear significant relationship between use of glycopeptides and isolation of vancomycin-resistant Enterococcus species in a university hospital setting.

BACKGROUND: Emergence of colonization and infection with vancomycin-resistant enterococci (VRE) has become a worldwide challenge. To investigate whether the increasing incidence of VRE isolation can be correlated with use of glycopeptides in the hospital setting, we conducted a hospital-wide two-year study in the university hospital of Vienna. METHODS: Within the period from January 2011 through December 2012 all patients with isolation of invasive or non-invasive VRE were retrospectively included. Specialty-specific data concerning the consumption of vancomycin and teicoplanin, fluoroquinolones and third generation cephalosporins in defined daily doses (DDDs) from June 2010 through May 2012 were extracted from the hospital pharmacy computer system. To assess the relationship between the usage of those antibiotics and the incidence of VRE (VRE-rate per 10 000 patients) a Poisson regression was performed. FINDINGS: In the study period 266 patients were colonized or infected with VRE. Specialty-specific VRE isolation was as follows: general surgical units (44 patients), bone marrow transplant unit (35 patients), general medical units (33 patients), cardiothoracic surgery (27 patients), nephrology (26 patients), haematooncology (22 patients), gastroenterology (17 patients), urology (17 patients), and the infectious diseases unit (11 patients). Hospital-wide consumption of glycopeptides was higher for teicoplanin than for vancomycin (26 242 versus 8677 DDDs). Specialty-specific VRE incidence significantly increased with the use of glycopeptides, fluoroquinolones or third generation cephalosporins (p < 0.001). The results of the Poisson regression for vancomycin (p = 0.0018) and teicoplanin (p < 0.0001) separately were both highly significant. Spearman's correlation coefficient indicated a strong correlation between the two variables (rho = 0.8). CONCLUSION: Overall usage of glycopeptides, fluoroquinolones or third generation cephalosporins contributed to the emergence of VRE in the hospital setting.

Sepsis in standard care: patients' characteristics, effectiveness of antimicrobial therapy and patient outcome--a cohort study.

PURPOSE: Fast diagnosis and initiation of appropriate antibiotic therapy is pivotal for the survival of sepsis patients. However, most studies on suspected sepsis patients are conducted in the intensive care unit or in the emergency room setting, neglecting the standard care setting. This study evaluated sepsis risk factors, microbiological accurateness of the initial empiric antimicrobial therapy and its effect on hospital mortality in standard care patients. METHODS: In this prospective observational cohort study, patients with clinically suspected sepsis meeting two or more SIRS criteria were screened on standard care wards. After hospital discharge, occurrence of an infection was assessed according to standardized criteria, and empirical antibiotic therapy was evaluated using antibiograms of recognized pathogens by expert review. RESULTS: Of the 2384 screened patients, 298 fulfilled two or more SIRS criteria. Among these were 28.2 % SIRS patients without infection, 46.3 % non-bacteremic/fungemic sepsis patients and 25.5 % bacteremic/fungemic sepsis patients. Occurrence of a malignant disease and chills were associated with a higher risk of patients having bacteremic/fungemic sepsis, whereas other described risk factors remained insignificant. In total, 91.1 % of suspected sepsis patients received empirical antimicrobial therapy, but 41.1 % of bacteremic sepsis patients received inappropriate therapy. Non-surviving bacteremic sepsis patients received a higher proportion of inappropriate therapy than those who survived (p = 0.022). CONCLUSIONS: A significant proportion of bacteremic sepsis patients receive inappropriate empiric antimicrobial therapy. Our results indicate that rapid availability of microbiological results is vital, since inappropriate antimicrobial therapy tended to increase the hospital mortality of sepsis patients.

Successful implementation of infection control strategies prevents P. aeruginosa transmission among cystic fibrosis patients inside the hospital.

BACKGROUND: The aim of this study was to characterise the epidemiology of P. aeruginosa isolated from cystic fibrosis (CF) patients at the Vienna General Hospital (VGH) by molecular genetic fingerprinting in order to understand transmission ways and to evaluate the established infection control protocols. METHODS: The outpatient clinic for CF patients at the VGH cares for children and adolescents up to the age of 18 years. Among an average of 139 patients cared for at the clinic, 41 were tested positive for P. aeruginosa during the study period. Fifty P. aeruginosa isolates, obtained between August 2010 and March 2012 from routine examinations of CF patients, were subject to molecular characterization using the DiversiLab(®) method. RESULTS: 42 distinguishable molecular-biological patterns were identified, 7 of which were found multiple times. 40 out of 42 genotypes were retrieved from single patients only, while two patterns were present in two patients each. Nine patients presented with two or more phenotypically diverse P. aeruginosa isolates. In five of these cases the retrieved isolates belonged to the same genotype. CONCLUSION: The broad genetic heterogeneity of P. aeruginosa in the studied patient population suggests that the majority of CF patients cared for at the VGH acquire P. aeruginosa from environmental sources. It may be concluded that implemented infection control guidelines have been successful in preventing nosocomial transmission of P. aeruginosa among CF patients within the VGH and patient-to-patient transmission outside the hospital. Chronic polyclonal infection/colonization was rare in the study population.

Safety of Ommaya reservoirs in children with brain tumors: a 20-year experience with 5472 intraventricular drug administrations in 98 patients.

The Ommaya reservoir facilitates repetitive delivery of drugs into the CSF and is a pharmacologically rational system for intrathecal chemotherapy. Because previous studies have found a high rate of infection and other complications we herein studied our experience with this device. Between 1993 and 2013, 98 children with brain tumors aged 3 months to 21 years (38 ≤ 3 years) had an Ommaya reservoir placed. All patients received perioperative antibiotics. Only specially trained personnel that followed standardized guidelines were allowed to access the reservoir. As of April 2014, 5,472 chemotherapy instillations were performed amounting to a median of 36 deliveries (2-280) per reservoir. Ommaya reservoirs were present for 199,956 days and a median of 1,336 days per device. Median survival of the 52 patients still alive is 7.5 years. Only one patient developed an Ommaya reservoir infection (1 %) that could be temporarily sterilized but eventually required Ommaya reservoir explantation. Early complications related to Ommaya reservoir placement occurred in two patients, in one catheter malposition was corrected intraoperatively and in the other kinking of the catheter at the burr-hole required minor surgical correction. Two delayed complications requiring surgical revision included malpositioning of the catheter tip after rapid shrinkage of the ventricles and disconnection of the ventricular catheter after 24 accesses. No leucodystrophic changes occurred along the catheter track. In conclusion, Ommaya reservoirs are safe and complications infrequent providing that all personnel involved in implanting and subsequently accessing the device are specially trained and pay meticulous attention to strict aseptic conditions.

High detection rate of Trichomonas vaginalis in benign hyperplastic prostatic tissue.

While Trichomonas vaginalis, a protozoan parasite, is a well-investigated pathogen in the female population, there is little awareness of its significance in the male uro-genital tract. The presence of T. vaginalis in the prostate gland has only been scarcely investigated and has never been attested in conditions other than clinical prostatitis. Still, by some authors, this organ is regarded as ecologic niche for T. vaginalis. Since normal prostate tissue of sufficient quality is hard to come by, we investigated samples from 86 patients (mean age 68.7 ± 7.6 years) suffering from benign prostatic hyperplasia (BPH), a medical condition currently ranked as noninfectious, but characterized by chronic inflammatory tissue infiltrates of unknown etiology. Applying two different PCR protocols and sequence analysis of the respective amplicons, we detected T. vaginalis DNA in 29/86 (34%) BPH tissue samples, whereas in only 2/86 (2.3%) cases T. vaginalis grew in culture. Detection of T. vaginalis DNA correlated significantly (P < 0.01) with elevated peripheral blood monocytic cell counts, appearing along with protozoan infections. Given the unexpected high prevalence of T. vaginalis in BPH tissue of a nonselected, elderly study population from Austria, further epidemiological studies have to confirm this finding. Potential interactions of T. vaginalis in its prostatic habitat may be investigated with respect to their possible contribution to the inflammatory pathogenesis of BPH, since inflammatory cytokines have been shown to sustain prostatic hyperplastic growth.

NOTCH2 links protein kinase C delta to the expression of CD23 in chronic lymphocytic leukaemia (CLL) cells.

One characteristic of chronic lymphocytic leukaemia (CLL) lymphocytes is high expression of CD23, which has previously been identified as a downstream target for NOTCH2 signalling. The mechanisms regulating NOTCH2-dependent CD23 expression, however, are largely unknown. This study showed that peripheral CLL cells overexpressed transcriptionally active NOTCH2 (N2(IC)), irrespective of their prognostic marker profile. When placed in culture, NOTCH2 activity was spontaneously decreased in 25 out of 31 CLL cases (81%) within 24 h. DNA-bound N2(IC) complexes could be maintained by the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) or by gamma-interferon (IFN-gamma), two CLL characteristic inducers of CD23 expression. Inhibition of PKC-delta by RNA interference or by rottlerin antagonised PMA-induced NOTCH2 activation and also suppressed NOTCH2 activity in CLL cases with constitutively activated NOTCH2 signalling. In 23 out of 29 CLL cases tested (79%), DNA-bound N2(IC) complexes were found to be resistant to the gamma-secretase inhibitor (GSI) DAPT, suggesting that GSIs will be only effective in a subset of CLL cases. These data suggest that deregulation of NOTCH2 signalling is critically involved in maintaining the malignant phenotype of CLL lymphocytes and point to a link between PKC-delta and NOTCH2 signalling in the leukemic cells.

Myelin-mediated inhibition of oligodendrocyte precursor differentiation can be overcome by pharmacological modulation of Fyn-RhoA and protein kinase C signalling.

Failure of oligodendrocyte precursor cell (OPC) differentiation contributes significantly to failed myelin sheath regeneration (remyelination) in chronic demyelinating diseases. Although the reasons for this failure are not completely understood, several lines of evidence point to factors present following demyelination that specifically inhibit differentiation of cells capable of generating remyelinating oligodendrocytes. We have previously demonstrated that myelin debris generated by demyelination inhibits remyelination by inhibiting OPC differentiation and that the inhibitory effects are associated with myelin proteins. In the present study, we narrow down the spectrum of potential protein candidates by proteomic analysis of inhibitory protein fractions prepared by CM and HighQ column chromatography followed by BN/SDS/SDS-PAGE gel separation using Nano-HPLC-ESI-Q-TOF mass spectrometry. We show that the inhibitory effects on OPC differentiation mediated by myelin are regulated by Fyn-RhoA-ROCK signalling as well as by modulation of protein kinase C (PKC) signalling. We demonstrate that pharmacological or siRNA-mediated inhibition of RhoA-ROCK-II and/or PKC signalling can induce OPC differentiation in the presence of myelin. Our results, which provide a mechanistic link between myelin, a mediator of OPC differentiation inhibition associated with demyelinating pathologies and specific signalling pathways amenable to pharmacological manipulation, are therefore of significant potential value for future strategies aimed at enhancing CNS remyelination.

Is benign prostatic hyperplasia (BPH) an immune inflammatory disease?

OBJECTIVES: Chronic inflammation has been documented for years in benign prostatic hyperplasia (BPH), but only now has it become evident as a major factor in disease progression. This review highlights the immunologic key features of chronic inflammation in BPH and the present interpretation of these changes in the development and progression of BPH. RESULTS: Almost all BPH specimens show inflammatory infiltrates at histologic examination, but correlation to bacterial or other foreign antigens has not been established. Recognition of prostate secretion products by autoreactive T cells and animal models on experimental prostatitis demonstrate an autoimmune component to chronic inflammation. The infiltrate consists predominantly of chronically activated CD4(+) T lymphocytes, which are permanently recruited to prostate tissue via elevated expression of interleukin 15 (IL-15) and interferon gamma (IFN-gamma), proinflammatory cytokines produced by smooth muscle and T cells, respectively. With the appearance of infiltrates, T cell-derived cytokine production of IFN-gamma, IL-2, and transforming growth factor beta increases, the former two ultimately reaching 10-fold and the latter 2-fold higher levels in fully developed BPH than in normal prostates. As "mature" BPH nodules develop, IL-4 and IL-13 expression increases >2-fold, corresponding to a T-helper (Th)0/Th2 cytokine pattern. Dysregulation of the immune response in BPH may occur via elevated expression of proinflammatory IL-17, which stimulates a multifold production of IL-6 and IL-8, key executors of stromal growth in BPH. CONCLUSIONS: These data strongly suggest that BPH is an immune inflammatory disease. Unravelling the specific nature of immune dysregulation may help design novel drugs with these specific targets in mind.