Assessment of humoral responses in COVID-19 using various quantitative antibody tests.

BACKGROUND: Quantitative antibody tests are expected to be useful in diagnostics of COVID-19 and investigation of herd immunity against SARS-CoV-2. To make it proper to perform them, understanding of the immunological aspects is critically important. The present study aimed to assess humoral responses in COVID-19 using various quantitative antibody tests. METHODS: Four quantitative antibody tests that are different in targeted antigens, detectable immunoglobulin classes and avidity were used. Diagnosis was confirmed by RT-PCR for SARS-CoV-2 detection. Antibody titres of 117 samples collected from 24 COVID-19 patients and 23 non-COVID-19 patients were measured to evaluate correlations between different tests. For 24 COVID-19 patients, antibody titres measured at various time points after the onset or the RT-PCR diagnosis were subjected to assessment of humoral responses. RESULTS: Correlations between tests were observed to some degree, although there were discrepancies putatively due to differences in measurement principle. Seronegative COVID-19 was diagnosed for some patients, in whom antibody titres were less than the cut-off value in each test throughout the time courses. IgG seroconversion without prior IgM seroconversion most frequently occurred, while predominance of IgM responses over IgG responses was observed in some severe cases. Viral burdens estimated according to threshold cycle values at the RT-PCR seemed to impact antibody responses. CONCLUSIONS: The results provide insights into the nature of humoral responses to SARS-CoV-2 and diagnostic performance of antibody tests.

Application of clot-fibrinolysis waveform analysis to assessment of in vitro effects of direct oral anticoagulants on fibrinolysis.

INTRODUCTION: Acceleration of fibrinolysis by direct oral anticoagulants (DOACs) has been reported by several groups, suggesting contribution of not only anticoagulant but also fibrinolytic effects to the therapeutic efficacy. The present study aims to evaluate the usability of clot-fibrinolysis waveform analysis (CFWA) for assessment of in vitro effects of DOACs on fibrinolysis. METHODS: The experimental conditions were optimized according to how t-PA concentrations and a time length after t-PA adjustment affect parameters of CFWA. Addition of the activated partial thromboplastin time (APTT) reagent followed by that of calcium and t-PA was done to obtain clotting and fibrinolytic reaction curves which were mathematically differentiated for CFWA (APTT-CFWA). The positive and negative modes of waveforms were defined as the direction toward fibrin generation and that toward fibrin degradation, respectively. The maximum positive and negative values (Maxp 1 and Maxn 1) correspond to the maximum coagulation velocity and the maximum fibrinolysis velocity, respectively. Plasma spiked with each of DOACs (rivaroxaban, apixaban, edoxaban, and dabigatran) was subjected to APTT-CFWA. RESULTS: Optimization of t-PA use was based on Maxn 1. Roughly biphasic effects of rivaroxaban and dabigatran but not apixaban or edoxaban on fibrinolysis were observed through Maxn 1 and the fibrinolysis peak time, which was defined as a time length from the time when Maxp 1 (Maxp 1 time) to the time when Maxn 1 appears (Maxn 1 time). CONCLUSION: The results suggest the usability of CFWA for assessment of DOAC effects and provide insights into relevance of anticoagulation to therapeutic efficacy and bleeding risk from the perspective of fibrinolysis.

Intensive optimization and evaluation of global DNA methylation quantification using LC-MS/MS.

DNA methylation is a typical epigenetic phenomenon. Numerous methods for detecting global DNA methylation levels have been developed, among which LC-MS/MS has emerged as an excellent method from the viewpoint of sensitivity, reproducibility, and cost. However, LC-MS/MS methods have limitations due to a lack of the stability and the standardization required for a laboratory assay. The present study aimed to establish a robust assay that guarantees highly accurate measurements of global DNA methylation levels. There are at least three facets of the developed method. The first is discovery of the solvent conditions to minimize sodium adducts. The second is improvement of separation of nucleosides by LC using the columns that had not been used in previous similar studies. The third is success in reduction of the uncertainty of the measurement results, which was achieved by the calibration using the ratio of mdC but not the absolute amount in the presence of internal standards. These facets represent the advantage over methods reported previously. Our developed method enables quantification of DNA methylation with a short time length (8 min) for one analysis as well as with the high reproducibility of measurements that is represented by the inter-day CV% being less than 5%. In addition, data obtained from measuring global DNA methylation levels in cultured cell lines, with or without pharmacological demethylation, support its use for biomedical research. This assay is expected to allow us to conduct initial screening of epigenetic alterations or aberration in a variety of cells.

Assessment of in vitro effects of direct thrombin inhibitors and activated factor X inhibitors through clot waveform analysis.

AIMS: Clot waveform analysis (CWA) has been reported to extend the interpretation of clotting time measurement. The parameters obtained from successive derivatives of the clotting reaction curves reflect the rates of activation of individual coagulation factors, theoretically dissecting the cascade pathway. This study aims to assess the in vitro effects of direct thrombin inhibitors (DTIs) and activated factor X (FXa) inhibitors. METHODS: CWA was applied to the activated partial thromboplastin time (APTT) assay of plasma samples spiked with each drug. For CWA of APTT measurement curves (APTT-CWA), the positive mode of clotting reaction curves was defined as the direction towards fibrin generation. RESULTS: All the maximum positive values in the successive derivatives were decreased dependently on the concentrations of each drug. Moreover, the negative values in the second and third derivatives appeared putatively due to consumption of thrombin and factor FXa, respectively, to form complexes with plasma serine protease inhibitors. The decrease of the maximum negative values observed dependently on the concentrations of each drug appeared to be consistent with the decreased generation of thrombin and factor FXa. The analysis of Hill coefficients of each drug in the dose-response of changes in the APTT-CWA parameters revealed a difference in anticoagulant cooperativity between DTIs versus FXa inhibitors. CONCLUSIONS: The APTT-CWA demonstrated evidence for the blockade of thrombin-positive feedback by DTIs and FXa inhibitors and that for the differences in anticoagulant cooperativity between them. The results demonstrate the usability of CWA for assessment of anticoagulation and provide insights into direct anticoagulants.

Assessment of tailor-made prevention of atherosclerosis with folic acid supplementation: randomized, double-blind, placebo-controlled trials in each MTHFR C677T genotype.

This study aimed at assessing the effect of folic acid supplementation quantitatively in each MTHFR C677T genotype and considered the efficiency of tailor-made prevention of atherosclerosis. Study design was genotype-stratified, randomized, double-blind, placebo-controlled trials. The setting was a Japanese company in the chemical industry. Subjects were 203 healthy men after exclusion of those who took folic acid or drugs known to effect folic acid metabolism. Intervention was folic acid 1 mg/day p.o. for 3 months. The primary endpoint was plasma total homocysteine level (tHcy). In all three genotypes, there were significant tHcy decreases. The greatest decrease was in the TT homozygote [6.61 (3.47-9.76) micromol/l] compared with other genotypes [CC: 2.59 (1.81-3.36), CT: 2.64 (2.16-3.13)], and there was a significant trend between the mutated allele number and the decrease. The tHcy were significantly lowered in all the genotypes, but the amount of the decrease differed significantly in each genotype, which was observed at both 1 and 3 months. Using these time-series data, the largest benefit obtained by the TT homozygote was appraised as 2.4 times compared with the CC homozygote. Taking into account the high allele frequency of this SNP, this quantitative assessment should be useful when considering tailor-made prevention of atherosclerosis with folic acid.