An in vitro study on the effect of bevacizumab on endothelial cell proliferation and VEGF concentration level in patients with hereditary hemorrhagic telangiectasia.

Previous studies have demonstrated that vascular endothelial growth factor (VEGF) is upregulated in patients with hereditary hemorrhagic telangiectasia (HHT). The use of Bevacizumab as an anti-angiogenic treatment agent seems promising. The purpose of the present in vitro study was to determine the efficacy and potential toxicity levels of bevacizumab on cell proliferation and VEGF concentrations in endothelial cells of HHT patients. In this in vitro study, endothelial cells from patients with HHT and HUVECs (control) were incubated with different concentration levels of bevacizumab (2, 4, 6, 8 or 10 mg/ml). After 24, 48 or 72 h, the cell proliferation was assessed by Alamar Blue® Assay and the VEGF levels in the cell culture supernatants were measured by VEGF-ELISA. All endothelial cells incubated with bevacizumab showed an initial decrease in cell proliferation. Cell proliferation recovered within 72 h in cell cultures incubated with concentration levels of up to 4 mg/ml bevacizumab, whereas those incubated with higher concentration levels showed a continuous decline in cell proliferation. VEGF expression decreased after 24 h in cell cultures incubated with bevacizumab concentration levels of 2 and 4 mg/ml but increased again after 48 h. Cell cultures incubated with bevacizumab concentration levels of 10 mg/ml showed a constant decline in VEGF expression without any tendency for recovery. Translating these results into daily clinical practice, the present study suggests that the intranasal submucosal injection of bevacizumab in HHT patients should not exceed a concentration level of 4 mg/ml. Overall, higher bevacizumab concentration levels not only reduce VEGF expression but pose a higher risk of toxic effects on endothelial cells as they jeopardize cell proliferation.

Sphingolipid Metabolism and Transport in Chlamydia trachomatis and Chlamydia psittaci Infections.

Chlamydia species infect a large range of vertebral hosts and have become of major economic and public health concern over the last decades. They are obligate intracellular bacteria that undergo a unique cycle of development characterized by the presence of two distinct bacterial forms. After infection of the host cell, Chlamydia are found inside a membrane-bound compartment, the inclusion. The surrounding membrane of the inclusion contributes to the host-Chlamydia interface and specific pathogen-derived Inc proteins shape this interface allowing interactions with distinct cellular proteins. In contrast to many other bacteria, Chlamydia species acquire sphingomyelin from the host cell. In recent years a clearer picture of how Chlamydia trachomatis acquires this lipid emerged showing that the bacteria interact with vesicular and non-vesicular transport pathways that involve the recruitment of specific RAB proteins and the lipid-transfer protein CERT. These interactions contribute to the development of a new sphingomyelin-producing compartment inside the host cell. Interestingly, recruitment of CERT is conserved among different Chlamydia species including Chlamydia psittaci. Here we discuss our current understanding on the molecular mechanisms used by C. trachomatis and C. psittaci to establish these interactions and to create a novel sphingomyelin-producing compartment inside the host cell important for the infection.

Molecular surveillance of carbapenemase-producing Pseudomonas aeruginosa at three medical centres in Cologne, Germany.

Background: Pseudomonas aeruginosa is a common pathogen causing hospital-acquired infections. Carbapenem resistance in P. aeruginosa is either mediated via a combination of efflux pumps, AmpC overexpression, and porin loss, or through an acquired carbapenemase. Carbapenemase-producing P. aeruginosa (CPPA) strains are known to cause outbreaks and harbour a reservoir of mobile antibiotic resistance genes, however, few molecular surveillance data is available. The aim of this study was to analyse the prevalence and epidemiology of CPPA in three German medical centres from 2015 to 2017. Methods: Identification and susceptibility testing were performed with VITEK 2 system. P. aeruginosa non-susceptible to piperacillin, ceftazidime, cefepime, imipenem, meropenem and ciprofloxacin (4MRGN according to the German classification guideline) isolated from 2015 to 2017 were analysed. A two-step algorithm to detect carbapenemases was performed: phenotypic tests (EDTA- and cloxacillin-combined disk tests) followed by PCR, Sanger sequencing, and eventually whole genome sequencing. CPPA isolates were further genotyped by RAPD and PFGE. In-hospital transmission was investigated using conventional epidemiology. Results: Sixty two P. aeruginosa isolates were available for further analysis, of which 21 were CPPA as follows: blaVIM-1 (n = 2), blaVIM-2 (n = 17), blaNDM-1/blaGES-5 (n = 1) and the newly described blaIMP-82 (n = 1). CPPA were mostly hospital-acquired (71.4%) and isolated on intensive care units (66.7%). All (except one) were from the tertiary care centre. PFGE typing revealed one large cluster of VIM-2-producing CPPA containing 13 isolates. However, using conventional epidemiology, we were only able to confirm three patient-to-patient transmissions, and one room-to-patient transmission, on several intensive care units. Conclusions: These data give insight into the epidemiology of CPPA in three centres in Germany over a period of 3 years. Carbapenemases are a relevant resistance mechanism in 4MRGN-P. aeruginosa, illustrated by genetically related VIM-2-producing strains that seem to be endemic in this region. Our data suggest that infection control measures should especially focus on controlling transmission on the ICU and support the need for a local molecular surveillance system.

Large-scale recoding of a bacterial genome by iterative recombineering of synthetic DNA.

The ability to rewrite large stretches of genomic DNA enables the creation of new organisms with customized functions. However, few methods currently exist for accumulating such widespread genomic changes in a single organism. In this study, we demonstrate a rapid approach for rewriting bacterial genomes with modified synthetic DNA. We recode 200 kb of the Salmonella typhimurium LT2 genome through a process we term SIRCAS (stepwise integration of rolling circle amplified segments), towards constructing an attenuated and genetically isolated bacterial chassis. The SIRCAS process involves direct iterative recombineering of 10-25 kb synthetic DNA constructs which are assembled in yeast and amplified by rolling circle amplification. Using SIRCAS, we create a Salmonella with 1557 synonymous leucine codon replacements across 176 genes, the largest number of cumulative recoding changes in a single bacterial strain to date. We demonstrate reproducibility over sixteen two-day cycles of integration and parallelization for hierarchical construction of a synthetic genome by conjugation. The resulting recoded strain grows at a similar rate to the wild-type strain and does not exhibit any major growth defects. This work is the first instance of synthetic bacterial recoding beyond the Escherichia coli genome, and reveals that Salmonella is remarkably amenable to genome-scale modification.

Shark Attack: high affinity binding proteins derived from shark vNAR domains by stepwise in vitro affinity maturation.

A novel method for stepwise in vitro affinity maturation of antigen-specific shark vNAR domains is described that exclusively relies on semi-synthetic repertoires derived from non-immunized sharks. Target-specific molecules were selected from a CDR3-randomized bamboo shark (Chiloscyllium plagiosum) vNAR library using yeast surface display as platform technology. Various antigen-binding vNAR domains were easily isolated by screening against several therapeutically relevant antigens, including the epithelial cell adhesion molecule (EpCAM), the Ephrin type-A receptor 2 (EphA2), and the human serine protease HTRA1. Affinity maturation was demonstrated for EpCAM and HTRA1 by diversifying CDR1 of target-enriched populations which allowed for the rapid selection of nanomolar binders. EpCAM-specific vNAR molecules were produced as soluble proteins and more extensively characterized via thermal shift assays and biolayer interferometry. Essentially, we demonstrate that high-affinity binders can be generated in vitro without largely compromising the desirable high thermostability of the vNAR scaffold.