Rare Occurrence of Inhibitors in Von Willebrand Disease: A Case Report

Introduction: Type 3 Von Willebrand Disease (VWD) is the least common but the most severe form of a disease, with a prevalence of about 0. 5 to 1 per million in Western countries. The prevalence of type 3 VWD in the developing countries, with a high degree of consanguinity, is about 6 per million. Moreover, due to underdiagnosis of the milder cases, the prevalence of type 3 VWD is about 50% of the cases. Rarely, some patients develop the Von Willebrand Factor (VWF) inhibitors, which may subsequently develop severe anaphylactic reactions on further exposure to the VWF containing factor replacement therapy. The prevalence of inhibitor development in patients with type 3 VWD has been shown to be in the range of 5.8 to 9.5%. In the absence of a gold standard assay for the quantitation of VWF inhibitors, a correct diagnosis and management of these patients are often challenging.Objectives: The objective of this study is to standardize the Bethesda assay for the VWF inhibitors and to estimate the VWD inhibitor titer in two cases of congenital type 3 VWD, which developed the VWF inhibitors.Results and Conclusions: We could successfully standardize the Bethesda assay for the quantitation of VWF inhibitors in two patients with congenital type 3 VWD with inhibitors.

Engineering well-expressed, V2-immunofocusing HIV-1 envelope glycoprotein membrane trimers for use in heterologous prime-boost vaccine regimens

HIV-1 vaccine immunofocusing strategies have the potential to induce broadly reactive nAbs. Here, we engineered a panel of diverse, membrane-resident native HIV-1 trimers vulnerable to two broad targets of neutralizing antibodies (NAbs), the V2 apex and fusion peptide (FP). Selection criteria included i) high expression and ii) infectious function, so that trimer neutralization sensitivity can be profiled in pseudovirus assays. Initially, we boosted the expression of 17 candidate trimers by truncating gp41 and introducing a gp120-gp41 SOS disulfide to prevent gp120 shedding. "Repairs" were made to fill glycan holes and other strain-specific aberrations. A new neutralization assay allowed PV infection when our standard assay was insufficient. Trimers with exposed V3 loops, a target of non-neutralizing antibodies, were discarded. To try to increase V2-sensitivity, we removed clashing glycans and modified the V2 loop's C-strand. Notably, a 167N mutation improved V2-sensitivity. Glycopeptide analysis of JR-FL trimers revealed near complete sequon occupation and that filling the N197 glycan hole was well-tolerated. In contrast, sequon optimization and inserting/removing other glycans in some cases had local and global "ripple" effects on glycan maturation and sequon occupation in the gp120 outer domain and gp41. V2 mAb CH01 selectively bound trimers with small high mannose glycans near the base of the V1 loop, thereby avoiding clashes. Knocking in a N49 glycan perturbs gp41 glycans via a distal glycan network effect, increasing FP NAb sensitivity - and sometimes improving expression. Finally, a biophysical analysis of VLPs revealed that i) ~25% of particles bear Env spikes, ii) spontaneous particle budding is high and only increases 4-fold upon Gag transfection, and iii) Env+ particles express ~30-40 spikes. Overall, we identified 7 diverse trimers with a range of sensitivities to two targets that should enable rigorous testing of immunofocusing vaccine concepts.

Direct Comparison of the Lowest Effect Concentrations of Mutagenic Reference Substances in Two Ames Test Formats

The Ames assay is the standard assay for identifying DNA-reactive genotoxic substances. Multiple formats are available and the correct choice of an assay protocol is essential for achieving optimal performance, including fit for purpose detection limits and required screening capacity. In the present study, a comparison of those parameters between two commonly used formats, the standard pre-incubation Ames test and the liquid-based Ames MPF™, was performed. For that purpose, twenty-one substances with various modes of action were chosen and tested for their lowest effect concentrations (LEC) with both tests. In addition, two sources of rat liver homogenate S9 fraction, Aroclor 1254-induced and phenobarbital/β-naphthoflavone induced, were compared in the Ames MPF™. Overall, the standard pre-incubation Ames and the Ames MPF™ assay showed high concordance (>90%) for mutagenic vs. non-mutagenic compound classification. The LEC values of the Ames MPF™ format were lower for 17 of the 21 of the selected test substances. The S9 source had no impact on the test results. This leads to the conclusion that the liquid-based Ames MPF™ assay format provides screening advantages when low concentrations are relevant, such as in the testing of complex mixtures.

A novel biparatopic antibody-ACE2 fusion that blocks SARS-CoV-2 infection: implications for therapy

In the absence of a proven effective vaccine preventing infection by SARS-CoV-2, or a proven drug to treat COVID-19, the positive results of passive immune therapy using convalescent serum provides a strong lead. We have developed a new class of tetravalent, biparatopic therapy, 89C8-ACE2. It combines the specificity of a monoclonal antibody (89C8) that recognizes the relatively conserved N-terminal domain (NTD) of the viral S glycoprotein, and the ectodomain of ACE2, which binds to the receptor-binding domain (RBD) of S. This molecule shows exceptional performance in vitro, inhibiting the interaction of recombinant S1 to ACE2 and transduction of ACE2-overexpressing cells by S-pseudotyped lentivirus with IC50s substantially below 100 pM, and with potency approximately 100-fold greater than ACE2-Fc itself. Moreover, 89C8-ACE2 was able to neutralize authentic virus infection in a standard assay at low nanomolar concentrations, making this class of molecule a promising lead for therapeutic applications.

Clinical meanings of rapid serological assay in patients tested for SARS-Co2 RT-PCR

BackgroundRT-PCR test for identification of viral nucleic acid is the current standard diagnostic method for the diagnosis of COVID-19 disease but technical reasons limit the utilization of this assay onlarge scalescreenings.MethodWe verified in a consecutive series of 191 symptomatic patients the clinical information that new rapid serological colorimetric test qualitatively analyzing IgM/IgG expression can provide with respect to standard assay and with respect to clinical outcome of patients.ResultsRapid serological test showed a sensitivity of 30% and a specificity of 89% with respect to the standard assay but, interestingly, these performances improve after 8 days of symptoms appearance. After 10 days of symptoms the predictive value of rapid serological test is higher than that of standardassay. When the behaviour of the two immunoglobulins was evaluated with respect to time length of symptoms appaerance, no significant difference in immunoglobulins behaviour was shown.ConclusionsThe rapid serological test analyzed in the present study is candidate to provide information on immunoreaction of the subject to COVID-19 exposure.

TiO2 Sol-gel Coating as a Transducer Substrate for Impedimetric Immunosensors

Given the importance of the transducer elements in the performance of sensors for various applications, as well as the growing search for devices that are capable of providing the response in shorter time, in this work, titanium dioxide was examined as a candidate for application in an electrochemical biosensor. A TiO2 coating deposited by sol-gel method on a silicon wafer was obtained with an anatase crystalline structure, as an n-type<br /> semiconductor with donor density equal to 2.954 · 1017 cm–3. Its surface was functionalized to be tested as a biosensor to detect snake venom of the Bothrops genera, and each step of the functionalization was investigated using Electrochemical Impedance Spectroscopy (EIS) and Cyclic voltammetry. Despite being less sensitive than the reference method ELISA, the TiO2-based biosensor was also capable of detecting the analyte of interest at 20 μg mL–1, revealing an increase in its leakage resistance and phase shift after incubation in this solution. Furthermore, the total time for carrying out the biodetection with the TiO2-coated device (41.24 ± 0.05 min) was estimated to be approximately 80 % shorter than that required by the labelled standard assay, which indicates that TiO2 is a promising electrochemical transducer for biosensing applications.

Method for identification of heme-binding proteins and quantification of their interactions

The standard assay for characterization of interaction of heme with proteins is absorbance spectroscopy. However, this approach demands relatively large quantities of proteins and it is difficult to perform in high-throughput manner. Here, we describe an immunosorbent assay based on the covalent in situ conjugation of heme to a pre-coated carrier. Advantage of this assay is that it allows both identification of heme-binding proteins and quantification of their binding avidity, using only minimal amounts of protein (1-10 μg). Importantly, the same approach can be used for covalent linkage of other natural or synthetic compounds and analyzing their interactions with proteins.

Markers of CNS Injury in Adults Living With HIV With CSF HIV Not Detected vs Detected <20 Copies/mL

The presence of quantifiable HIV RNA in cerebrospinal fluid (CSF) during antiretroviral therapy (ART) can associate with central nervous system (CNS) pathology, but the significance of RNA detected below the limit of quantification (LOQ) on a standard assay during ART remains unknown. We compared CNS parameters between individuals with CSF RNA detected below the LOQ (20 copies/mL) with those with HIV RNA not detected. Detection of CSF HIV RNA associated with decreased blood–brain barrier integrity and with decreased executive function, but not with CNS immune activation or poorer performance in overall neuropsychological testing.

Mutagenic properties of modified hydrothermal nanotitania extract

Backgrounds: The mutagenic properties of modified hydrothermal nanotitania extract were carried out using the Ames test (genotoxicity). Materials and methods: The Ames test was performed on Salmonella strains (TA98, TA100, TA1535, TA1537 and TA 102) which contain mutations in several genes with and without S9 metabolic activation from rat liver using the standard assay. The materials were extracted in distilled water and the serial dilutions of concentration ranging from 313 to 5000 μg/mLwere used after the incubation period of 24 h at 37° C. Results: These results suggested that all tested concentrations of the material extracts did not produce mutagenic effect in all the strains tested. Conclusions: Findings from this study showed that the modified hydrothermal nanotitania extract was non-mutagenic under present conditions. Bangladesh Journal of Medical Science Vol.19(1) 2020 p.159-162