Comparative purification and characterization of hepatitis B virus-like particles produced by recombinant vaccinia viruses in human hepatoma cells and human primary hepatocytes

This study describes the comparative expression and purification of hepatitis B surface antigen (HBsAg) particles produced upon infection of human primary hepatocytes and human hepatoma cell lines (HuH-7 and HepG2) with recombinant vaccinia viruses. The highest levels of HBsAg expression were found in HuH-7 hepatoma cells following infection with recombinant vaccinia viruses, which contain the S gene under control of a 7.5 k-promoter. Four different methods for purification of the HBsAg particles were examined: isopycnic ultracentrifugation, sucrose cushion sedimentation, isocratic column gel filtration, and binding to anti-HBs-coated microparticles. The highest degree of purity of HBsAg particles was reached by the method based on anti-HBs-coated microparticles. The resulting product was > 98% pure. Biochemical analysis and characterization of purified HBsAg particles were performed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), western blotting, and electron microscopy. The HBsAg, purified from human hepatoma cell lines and from human primary hepatocytes, consisted of both the non-glycosylated (p25) and the glycosylated (gp27) form and assembled into typical 22-nm particles, and thus may be of great interest and importance for research, diagnostics, and medical treatments

Cytokine profiles of umbilical cord blood mononuclear cells upon in vitro stimulation with lipopolysaccharides of different vaginal gram-negative bacteria

Inflammatory immune responses induced by lipopolysaccharides (LPS) of gram-negative bacteria play an important role in the pathogenesis of preterm labor and delivery, and in neonatal disorders. To better characterize LPS-induced inflammatory response, we determined the cytokine profile of umbilical cord blood mononuclear cells (UBMC) stimulated with LPS of seven vaginal gram-negative bacteria commonly found in pregnant women with preterm labor and preterm rupture of membrane. UBMC from ten newborns of healthy volunteer mothers were stimulated with purified LPS of Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, Proteus mirabilis, Acinetobacter calcoaceticus, Citrobacter freundii, and Pseudomonas aeruginosa. UBMC supernatants were tested for the presence of secreted pro-inflammatory cytokines (IL-6, IL-1β, TNF), anti-inflammatory cytokine (IL-10), TH1-type cytokines (IL-12, IFN-γ), and chemokines (IL-8, MIP-1α, MIP-1β, MCP-1) by Luminex technology. The ten cytokines were differentially induced by the LPS variants. LPS of E. coli and E. aerogenes showed the strongest stimulatory activity and P. aeruginosa the lowest. Interestingly, the ability of UBMC to respond to LPS varied greatly among donors, suggesting a strong individual heterogeneity in LPS-triggered inflammatory response

How Do We Treat Pregnancy-Related Venous Thromboembolism?

Venous thromboembolism (VTE) is a major cause of maternal morbidity and mortality during pregnancy and the postpartum period. Due to a lack of adequate study data, therapeutic strategies for pregnancy-related VTE are deduced from observational studies and extrapolated from recommendations for nonpregnant patients. Because heparins do not cross the placenta, weight-adjusted therapeutic-dose low-molecular-weight heparins (LMWHs) are the anticoagulant treatment of choice in cases of VTE during pregnancy. Once- and twice-daily dosing regimens are suitable. There is no evidence that measurement of factor Xa activities and consecutive LMWH dose adjustments improve clinical outcomes. There is no support for the routine use of vitamin K antagonists, direct oral thrombin or factor Xa inhibitors, fondaparinux, or danaparoid in uncomplicated pregnancy-related VTE. Management of delivery deserves special attention, and treatment strategies depend on the time interval between the diagnosis of acute VTE and the expected delivery date. In lactating women, an overlapping switch from LMWH to warfarin is possible. Anticoagulation should be continued for at least 6 weeks postpartum or for a minimum period of 3 months

A novel model to study neonatalEscherichia colisepsis and the effect of treatment on the human immune system using humanized mice

ProblemNeonatal sepsis is a serious threat especially for preterm infants. As existing in vitro and in vivo models have limitations, we generated a novel neonatal sepsis model using humanized mice and tested the effect of Betamethasone and Indomethacin which are used in the clinic in case of premature birth. Method of studyHumanized mice were infected with Escherichia coli (E. coli). Subsequently, the effect of the infection itself, and treatment with Betamethasone and Indomethacin on survival, recovery, bacterial burden, leukocyte populations, and cytokine production, was analyzed. ResultsThe human immune system in the animals responded with leukocyte trafficking to the site of infection and granulopoiesis in the bone marrow. Treatment with Indomethacin had no pronounced effect on the immune system or bacterial burden. Betamethasone induced a decline of splenocytes. ConclusionThe human immune system in humanized mice responds to the infection, making them a suitable model to study neonatal E. coli sepsis and the immune response of the neonatal immune system. Treatment with Betamethasone could have potential negative long-term effects for the immune system of the child

Perinatal Gram-Positive Bacteria Exposure Elicits Distinct Cytokine Responses In Vitro

During pregnancy, infections caused by the gram-positive bacteria Enterococcus faecalis (E. faecalis), Streptococcus agalacticae (S. agalacticae), and Staphylococcus aureus (S. aureus) are major reasons for preterm labor, neonatal prematurity, meningitis, or sepsis. Here, we propose cytokine responses to bacterial infections by the immature perinatal immune system as central players in the pathogenesis of preterm birth and neonatal sepsis. We aimed to close the gap in knowledge about such cytokine responses by stimulating freshly isolated umbilical blood mononuclear cells (UBMC) with lysates of E. faecalis, S. agalacticae, and S. aureus collected from pregnant women in preterm labor. Bacterial lysates and, principally, S. aureus and S. agalacticae distinctly triggered most of the eleven inflammatory, anti-inflammatory, TH1/TH2 cytokines, and chemokines quantified in UBMC culture media. Chemokines depicted the most robust induction. Among them, MIP-1 beta was further enhanced in UBMC from female compered to male newborn infants. Due to its stability and high levels, we investigated the diagnostic value of IL-8. IL-8 was critically upregulated in cord blood of preterm neonates suffering from infections compared to gestational age-matched controls. Our results provide novel clues about perinatal immunity, underscoring a potential value of IL-8 for the timely detection of infections and suggesting that MIP-1 beta constitutes an early determinant of sex-specific immunity, which may contribute, e.g., to male's vulnerability to preterm birth

Perinatal Gram-Positive Bacteria Exposure Elicits Distinct Cytokine Responses In Vitro

During pregnancy, infections caused by the gram-positive bacteria Enterococcus faecalis (E. faecalis), Streptococcus agalacticae (S. agalacticae), and Staphylococcus aureus (S. aureus) are major reasons for preterm labor, neonatal prematurity, meningitis, or sepsis. Here, we propose cytokine responses to bacterial infections by the immature perinatal immune system as central players in the pathogenesis of preterm birth and neonatal sepsis. We aimed to close the gap in knowledge about such cytokine responses by stimulating freshly isolated umbilical blood mononuclear cells (UBMC) with lysates of E. faecalis, S. agalacticae, and S. aureus collected from pregnant women in preterm labor. Bacterial lysates and, principally, S. aureus and S. agalacticae distinctly triggered most of the eleven inflammatory, anti-inflammatory, TH1/TH2 cytokines, and chemokines quantified in UBMC culture media. Chemokines depicted the most robust induction. Among them, MIP-1β was further enhanced in UBMC from female compered to male newborn infants. Due to its stability and high levels, we investigated the diagnostic value of IL-8. IL-8 was critically upregulated in cord blood of preterm neonates suffering from infections compared to gestational age-matched controls. Our results provide novel clues about perinatal immunity, underscoring a potential value of IL-8 for the timely detection of infections and suggesting that MIP-1β constitutes an early determinant of sex-specific immunity, which may contribute, e.g., to male’s vulnerability to preterm birth

Duty Rosters andWorkloads of Obstetricians in Germany: Results of a Germany-wide Survey

Background Compiling a daily hospital roster which complies with existing laws and tariff regulations and meets the requirements for ongoing professional training while also taking the legal regulations on the health of employees into account makes planning the duty roster a challenge. The aim of this study was to obtain a realistic picture of existing duty roster systems and of the current workloads of obstetricians in Germany. Method This online survey was sent to 2770 physicians training to become obstetricians or specializing in specific areas of obstetric care. The survey consisted of an anonymized 95item questionnaire which collected data on different types of duty roster systems and the workload of obstetricians in Germany for the period from 17.02.2015 to 16.05.2015. Results Out of a total of 2770 physicians who were contacted, 437 (16%) completed the questionnaire. Across all forms of care, the care provided outside normal working hours usually (75%) consisted of a combination of regular working times and on-call duty or even consisted entirely of standby duty. Level I perinatal centers were most likely 20% (n = 88) to have a shift system in place. Working a shift system was significantly more common in care facilities which had previously carried out a job analysis. The number of physicians in hospitals who are present during the night shift was higher in facilities with higher numbers of births and in facilities which offered higher levels of care. In addition to regularly working overtime and the fact that often not all the hours worked were recorded, it was notable that the systems used to compile duty rosters often did not comply with legal regulations or with collectively agreed working hours nor were they compatible with the staff planning requirements. Outlook The results of this study show that the conditions of work, the working times, and the organization of working times in obstetric departments are in need of improvement. Recording the actual times worked together with an analysis of the activities performed during working times and while on standby would increase the level of transparency for employers and employees