Barriers and enablers of implementing bubble continuous positive airway pressure (CPAP) : perspectives of health professionals in Malawi

The study explored the factors that influence the implementation of Continuous Positive Airway Pressure (bubble CPAP) among health care professionals in secondary and tertiary hospitals in Malawi. Influencing factors occurred in an interconnected manner and included: inadequate healthcare provider training; rigid division of roles and responsibilities among providers; lack of effective communication between providers and newborn’s caregivers; human resources constraints; and inadequate equipment and infrastructure. Complications of prematurity are the leading cause of neonatal deaths, an important consideration in Malawi, which has the highest rate of preterm births in the world, at 18% of live-births.Global Affairs Canada (GAC)Canadian Institutes for Health Research (CIHR

Advantages and disadvantages of a Pro-Ribbon high-frequency driver in comparison to a highfrequency driver with a horn application in view of large PA systems

Diese Arbeit beschäftigt sich mit Vor- und Nachteilen von Hochtontreibern in Großbeschallungsanlagen. Untersucht werden hierbei die Pro Ribbon Hochtontreiber der Firma Alcons Audio und Kompressionstreiber der Firma d&b Audiotechnik. Die Lautsprecher werden anhand der Bauprinzipien, mittels akustischer Grundlagen sowie eigenen Messungen miteinander verglichen

One-helix protein 2 is not required for the synthesis of photosystem II subunit D1 in Chlamydomonas

International audienceIn land plants and cyanobacteria, co-translational association of chlorophyll (Chl) to the nascent D1 polypeptide, a reaction center protein of photosystem II (PSII), requires a Chl binding complex consisting of a short-chain dehydrogenase (high chlorophyll fluorescence 244 [HCF244]/uncharacterized protein 39 [Ycf39]) and one-helix proteins (OHP1 and OHP2 in chloroplasts) of the light-harvesting antenna complex superfamily. Here, we show that an ohp2 mutant of the green alga Chlamydomonas (Chlamydomonas reinhardtii) fails to accumulate core PSII subunits, in particular D1 (encoded by the psbA mRNA). Extragenic suppressors arose at high frequency, suggesting the existence of another route for Chl association to PSII. The ohp2 mutant was complemented by the Arabidopsis (Arabidopsis thaliana) ortholog. In contrast to land plants, where psbA translation is prevented in the absence of OHP2, ribosome profiling experiments showed that the Chlamydomonas mutant translates the psbA transcript over its full length. Pulse labeling suggested that D1 is degraded during or immediately after translation. The translation of other PSII subunits was affected by assembly-controlled translational regulation. Proteomics showed that HCF244, a translation factor which associates with and is stabilized by OHP2 in land plants, still partly accumulates in the Chlamydomonas ohp2 mutant, explaining the persistence of psbA translation. Several Chl biosynthesis enzymes overaccumulate in the mutant membranes. Partial inactivation of a D1-degrading protease restored a low level of PSII activity in an ohp2 background, but not photoautotrophy. Taken together, our data suggest that OHP2 is not required for psbA translation in Chlamydomonas, but is necessary for D1 stabilization

Construction of a hepatitis B virus neutralizing chimeric monoclonal antibody recognizing escape mutants of the viral surface antigen (HBsAg)

Hepatitis B virus (HBV) infection is a global burden on the health-care system and is considered as the tenth leading cause of death in the world. Over 248 million patients are currently suffering from chronic HBV infection worldwide and annual mortality rate of this infection is 686000. The “a” determinant is a hydrophilic region present in all antigenic subtypes of hepatitis B surface antigen (HBsAg), and antibodies against this region can neutralize the virus and are protective against all subtypes. We have recently generated a murine anti-HBs monoclonal antibody (4G4), which can neutralize HBV infection in HepaRG cells and recognize most of the escape mutant forms of HBsAg. Here, we describe the production and characterization of the chimeric human-murine antibody 4G4 (c-4G4). Variable region genes of heavy and light chains of the m-4G4 were cloned and fused to constant regions of human kappa and IgG1 by splice overlap extension (SOE) PCR. The chimeric antibody was expressed in Chinese Hamster Ovary (CHO)-K1 cells and purified from culture supernatant. Competition ELISA proved that both antibodies bind the same epitope within HBsAg. Antigen-binding studies using ELISA and Western blot showed that c-4G4 has retained the affinity and specificity of the parental murine antibody, and displayed a similar pattern of reactivity to 13 escape mutant forms of HBsAg. Both, the parental and c-4G4 showed a comparably high HBV neutralization capacity in cell culture even at the lowest concentration (0.6μg/ml). Due to the ability of c-4G4 to recognize most of the sub-genotypes and escape mutants of HBsAg, this antibody either alone or in combination with other anti-HBs antibodies could be considered as a potent alternative for Hepatitis B immune globulin (HBIG) as an HBV infection prophylactic or for passive immunotherapy against HBV infection

A concerted action of HNF4alpha and HNF1alpha links hepatitis B virus replication to hepatocyte differentiation.

International audienceHepatitis B virus (HBV) is an important human pathogen, which targets the liver extremely efficient, gaining access to hepatocytes by a so far unknown receptor and replicating in a hepatocyte-specific fashion. Cell differentiation seems to determine HBV replication. We here show that the level of hepatocyte differentiation, as indicated by hepatocyte polarization and metabolic activity, is closely correlated to the transcription of the HBV RNA pregenome. Pregenome transcription determined the level of HBV replication in various cell lines of hepatocellular origin and in primary human hepatocytes. A variety of hepatocyte-enriched nuclear factors have been described to regulate transcription of the pregenome, but it remained unknown which factors link HBV replication to hepatocyte differentiation. We determined that high expression levels of HNF4alpha but not its potential cofactors or other hepatocyte-enriched transcription factors were essential for efficient HBV replication, and link it to hepatocyte differentiation. HNF1alpha contributed to the control of HBV replication because it regulated the expression of HNF4alpha. Thus, a concerted action of HNF4alpha and HNF1alpha, which also determines morphological and functional differentiation of hepatocytes, links HBV replication to hepatocyte differentiation

T cells engineered to express a T-cell receptor specific for glypican-3 to recognize and kill hepatoma cells in vitro and in mice

BACKGROUND & AIMS: Cancer therapies are being developed based on our ability to direct T cells against tumor antigens. Glypican-3 (GPC3) is expressed by 75% of all hepatocellular carcinomas (HCC), but not in healthy liver tissue or other organs. We aimed to generate T cells with GPC3-specific receptors that recognize HCC and used them to eliminate GPC3-expressing xenograft tumors grown from human HCC cells in mice. METHODS: We used mass spectrometry to obtain a comprehensive peptidome from GPC3-expressing hepatoma cells after immune-affinity purification of human leukocyte antigen (HLA)-A2 and bioinformatics to identify immunodominant peptides. To circumvent GPC3 tolerance resulting from fetal expression, dendritic cells from HLA-A2-negative donors were cotrans-fected with GPC3 and HLA-A2 RNA to stimulate and expand antigen-specific T cells. RESULTS: Peptide GPC3(367) was identified as a predominant peptide on HLA-A2. We used A2-GPC3(367) multimers to detect, select for, and clone GPC3-specific T cells. These clones bound the A2-GPC3(367) multimer and secreted interferon-g when cultured with GPC3(367), but not with control peptide-loaded cells. By genomic sequencing of these T-cell clones, we identified a gene encoding a dominant T-cell receptor. The gene was cloned and the sequence was codon optimized and expressed from a retroviral vector. Primary CD8(+)T cells that expressed the transgenic T-cell receptor specifically bound GPC3(367) on HLA-A2. These T cells killed GPC3-expressing hepatoma cells in culture and slowed growth of HCC xenograft tumors in mice. CONCLUSIONS: We identified a GPC3(367)-specific T-cell receptor. Expression of this receptor by T cells allows them to recognize and kill GPC3-positive hepatoma cells. This finding could be used to advance development of adoptive T-cell therapy for HCC

Interferon-gamma and Tumor Necrosis Factor-alpha Produced by T Cells Reduce the HBV Persistence Form, cccDNA, Without Cytolysis

BACKGROUND & AIMS: Viral clearance involves immune cell cytolysis of infected cells. However, studies of hepatitis B virus (HBV) infection in chimpanzees have indicated that cytokines released by T cells also can promote viral clearance via non-cytolytic processes. We investigated the noncytolytic mechanisms by which T cells eliminate HBV from infected hepatocytes. METHODS: We performed a cytokine enzyme-linked immunosorbent assay of serum samples from patients with acute and chronic hepatitis B. Liver biopsy specimens were analyzed by in situ hybridization. HepG2-H1.3 cells, HBV-infected HepaRG cells, and primary human hepatocytes were incubated with interferon-gamma (IFN gamma) or tumor necrosis factor-a (TNF-alpha), or co-cultured with T cells. We measured markers of HBV replication, including the covalently closed circular DNA (cccDNA). RESULTS: Levels of IFN gamma and TNF-alpha were increased in serum samples from patients with acute vs chronic hepatitis B and controls. In human hepatocytes with stably replicating HBV, as well as in HBV-infected primary human hepatocytes or HepaRG cells, IFN gamma and TNF-alpha each induced deamination of cccDNA and interfered with its stability; their effects were additive. HBV-specific T cells, through secretion of IFN gamma and TNF-alpha, inhibited HBV replication and reduced cccDNA in infected cells without the direct contact required for cytolysis. Blocking IFN gamma and TNF-alpha after T-cell stimulation prevented the loss of cccDNA. Deprivation of cccDNA required activation of nuclear APOBEC3 deaminases by the cytokines. In liver biopsy specimens from patients with acute hepatitis B, but not chronic hepatitis B or controls, hepatocytes expressed APOBEC3A and APO-BEC3B. CONCLUSIONS: IFN gamma and TNF-alpha, produced by T cells, reduce levels of HBV cccDNA in hepatocytes by inducing deamination and subsequent cccDNA decay