Protection against Experimental Melioidosis with a Synthetic manno-Heptopyranose Hexasaccharide Glycoconjugate

This is the final version of the article. Available from the publisher via the DOI in this record.Melioidosis is an emerging infectious disease caused by Burkholderia pseudomallei and is associated with high morbidity and mortality rates in endemic areas. Antibiotic treatment is protracted and not always successful; even with appropriate therapy, up to 40% of individuals presenting with melioidosis in Thailand succumb to infection. In these circumstances, an effective vaccine has the potential to have a dramatic impact on both the scale and the severity of disease. Currently, no vaccines are licensed for human use. A leading vaccine candidate is the capsular polysaccharide consisting of a homopolymer of unbranched 1→3 linked 2-O-acetyl-6-deoxy-β-d-manno-heptopyranose. Here, we present the chemical synthesis of this challenging antigen using a novel modular disaccharide assembly approach. The resulting hexasaccharide was coupled to the nontoxic Hc domain of tetanus toxin as a carrier protein to promote recruitment of T-cell help and provide a scaffold for antigen display. Mice immunized with the glycoconjugate developed IgM and IgG responses capable of recognizing native capsule, and were protected against infection with over 120 × LD50 of B. pseudomallei strain K96243. This is the first report of the chemical synthesis of an immunologically relevant and protective hexasaccharide fragment of the capsular polysaccharide of B. pseudomallei and serves as the rational starting point for the development of an effective licensed vaccine for this emerging infectious disease.This work was funded by the United Kingdom Ministry of Defence. The mass spectral data described here were acquired on an Orbitrap Fusion mass spectrometer funded by National Institutes of Health grant 1S10OD010645-01A1

Coupling of alpha(1)-Adrenoceptors to ERK1/2 in the Human Prostate

Introduction: alpha(1)-Adrenoceptors are considered critical for the regulation of prostatic smooth muscle tone. However, previous studies suggested further alpha(1)-adrenoceptor functions besides contraction. Here, we investigated whether alpha(1)-adrenoceptors in the human prostate may activate extracellular signal-regulated kinases (ERK1/2). Methods: Prostate tissues from patients undergoing radical prostatectomy were stimulated in vitro. Activation of ERK1/2 was assessed by Western blot analysis. Expression of ERK1/2 was studied by immunohistochemistry. The effect of ERK1/2 inhibition by U0126 on phenylephrine-induced contraction was studied in organ-bath experiments. Results: Stimulation of human prostate tissue with noradrenaline (30 mu M) or phenylephrine (10 mu M) resulted in ERK activation. This was reflected by increased levels of phosphorylated ERK1/2. Expression of ERK1/2 in the prostate was observed in smooth muscle cells. Incubation of prostate tissue with U0126 (30 mu M) resulted in ERK1/2 inhibition. Dose-dependent phenylephrine-induced contraction of prostate tissue was not modulated by U0126. Conclusions: alpha(1)-Adrenoceptors in the human prostate are coupled to ERK1/2. This may partially explain previous observations suggesting a role of alpha(1)-adrenoceptors in the regulation of prostate growth. Copyright (C) 2011 S. Karger AG, Base

SARS-CoV-2 neutralizing antibodies: Longevity, breadth, and evasion by emerging viral variants.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) antibody neutralization response and its evasion by emerging viral variants and variant of concern (VOC) are unknown, but critical to understand reinfection risk and breakthrough infection following vaccination. Antibody immunoreactivity against SARS-CoV-2 antigens and Spike variants, inhibition of Spike-driven virus-cell fusion, and infectious SARS-CoV-2 neutralization were characterized in 807 serial samples from 233 reverse transcription polymerase chain reaction (RT-PCR)-confirmed Coronavirus Disease 2019 (COVID-19) individuals with detailed demographics and followed up to 7 months. A broad and sustained polyantigenic immunoreactivity against SARS-CoV-2 Spike, Membrane, and Nucleocapsid proteins, along with high viral neutralization, was associated with COVID-19 severity. A subgroup of "high responders" maintained high neutralizing responses over time, representing ideal convalescent plasma donors. Antibodies generated against SARS-CoV-2 during the first COVID-19 wave had reduced immunoreactivity and neutralization potency to emerging Spike variants and VOC. Accurate monitoring of SARS-CoV-2 antibody responses would be essential for selection of optimal responders and vaccine monitoring and design

Revolutionizing Clinical Microbiology Laboratory Organization in Hospitals with In Situ Point-of-Care

BACKGROUND: Clinical microbiology may direct decisions regarding hospitalization, isolation and anti-infective therapy, but it is not effective at the time of early care. Point-of-care (POC) tests have been developed for this purpose. METHODS AND FINDINGS: One pilot POC-lab was located close to the core laboratory and emergency ward to test the proof of concept. A second POC-lab was located inside the emergency ward of a distant hospital without a microbiology laboratory. Twenty-three molecular and immuno-detection tests, which were technically undemanding, were progressively implemented, with results obtained in less than four hours. From 2008 to 2010, 51,179 tests yielded 6,244 diagnoses. The second POC-lab detected contagious pathogens in 982 patients who benefited from targeted isolation measures, including those undertaken during the influenza outbreak. POC tests prevented unnecessary treatment of patients with non-streptococcal tonsillitis (n = 1,844) and pregnant women negative for Streptococcus agalactiae carriage (n = 763). The cerebrospinal fluid culture remained sterile in 50% of the 49 patients with bacterial meningitis, therefore antibiotic treatment was guided by the molecular tests performed in the POC-labs. With regard to enterovirus meningitis, the mean length-of-stay of infected patients over 15 years old significantly decreased from 2008 to 2010 compared with 2005 when the POC was not in place (1.43±1.09 versus 2.91±2.31 days; p = 0.0009). Altogether, patients who received POC tests were immediately discharged nearly thrice as often as patients who underwent a conventional diagnostic procedure. CONCLUSIONS: The on-site POC-lab met physicians' needs and influenced the management of 8% of the patients that presented to emergency wards. This strategy might represent a major evolution of decision-making regarding the management of infectious diseases and patient care

Synergy Between Intercellular Communication and Intracellular Ca2+ Handling in Arrhythmogenesis

Calcium is the primary signalling component of excitation-contraction coupling, the process linking electrical excitability of cardiac muscle cells to coordinated contraction of the heart. Understanding Ca2þ handling processes at the cellular level and the role of intercellular communication in the emergence of multicellular synchronization are key aspects in the study of arrhythmias. To probe these mechanisms, we have simulated cellular interactions on large scale arrays that mimic cardiac tissue, and where individual cells are represented by a mathematical model of intracellular Ca2þ dynamics. Theoretical predictions successfully reproduced experimental findings and provide novel insights on the action of two pharmacological agents (ionomycin and verapamil) that modulate Ca2þ signalling pathways via distinct mechanisms. Computational results have demonstrated how transitions between local synchronisation events and large scale wave formation are affected by these agents. Entrainment phenomena are shown to be linked to both ntracellular Ca2þ and coupling-specific dynamics in a synergistic manner. The intrinsic variability of the cellular matrix is also shown to affect emergent patterns of rhythmicity, providing insights into the origins of arrhythmogenic Ca2þ perturbations in cardiac tissue in situ

Engineered Single-Domain Antibodies with High Protease Resistance and Thermal Stability

The extreme pH and protease-rich environment of the upper gastrointestinal tract is a major obstacle facing orally-administered protein therapeutics, including antibodies. Through protein engineering, several Clostridium difficile toxin A-specific heavy chain antibody variable domains (VHHs) were expressed with an additional disulfide bond by introducing Ala/Gly54Cys and Ile78Cys mutations. Mutant antibodies were compared to their wild-type counterparts with respect to expression yield, non-aggregation status, affinity for toxin A, circular dichroism (CD) structural signatures, thermal stability, protease resistance, and toxin A-neutralizing capacity. The mutant VHHs were found to be well expressed, although with lower yields compared to wild-type counterparts, were non-aggregating monomers, retained low nM affinity for toxin A, albeit the majority showed somewhat reduced affinity compared to wild-type counterparts, and were capable of in vitro toxin A neutralization in cell-based assays. Far-UV and near-UV CD spectroscopy consistently showed shifts in peak intensity and selective peak minima for wild-type and mutant VHH pairs; however, the overall CD profile remained very similar. A significant increase in the thermal unfolding midpoint temperature was observed for all mutants at both neutral and acidic pH. Digestion of the VHHs with the major gastrointestinal proteases, at biologically relevant concentrations, revealed a significant increase in pepsin resistance for all mutants and an increase in chymotrypsin resistance for the majority of mutants. Mutant VHH trypsin resistance was similar to that of wild-type VHHs, although the trypsin resistance of one VHH mutant was significantly reduced. Therefore, the introduction of a second disulfide bond in the hydrophobic core not only increases VHH thermal stability at neutral pH, as previously shown, but also represents a generic strategy to increase VHH stability at low pH and impart protease resistance, with only minor perturbations in target binding affinities. These are all desirable characteristics for the design of protein-based oral therapeutics