Antibody-like proteins that capture and neutralize SARS-CoV-2.

To combat severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) and any unknown emerging pathogens in the future, the development of a rapid and effective method to generate high-affinity antibodies or antibody-like proteins is of critical importance. We here report high-speed in vitro selection of multiple high-affinity antibody-like proteins against various targets including the SARS-CoV-2 spike protein. The sequences of monobodies against the SARS-CoV-2 spike protein were successfully procured within only 4 days. Furthermore, the obtained monobody efficiently captured SARS-CoV-2 particles from the nasal swab samples of patients and exhibited a high neutralizing activity against SARS-CoV-2 infection (half-maximal inhibitory concentration, 0.5 nanomolar). High-speed in vitro selection of antibody-like proteins is a promising method for rapid development of a detection method for, and of a neutralizing protein against, a virus responsible for an ongoing, and possibly a future, pandemic.

Distribution of norovirus genotypes and subtypes in river water by ultra-deep sequencing-based analysis.

To determine the distribution of Norovirus (NoV) genotypes in natural river water in Thailand, we conducted a genome analysis using a next-generation sequencer. Twenty-five river water samples were collected at five different sites of the Khlong Klon River in the suburbs of Bangkok between August 2013 and December 2014. The partial genome of NoV was detected in 15 of the 25 samples (60·0%). Seven of these 15 samples (46·7%) contained multiple NoV GII genotypes: GII.4, GII.6, and GII.17. Our data showed that GII.17 had already emerged in August 2013 as a minor population, and it became a major genotype in December 2014. Our findings indicate that the virus was likely to have been circulating in the community before it appeared in the river water. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study was to investigate the frequencies of multiple genogroups and genotypes of norovirus in the river water near Bangkok, Thailand, by ultra-deep sequencing-based analysis. This study revealed that the epidemic strain was likely to have been circulating in the community before it appeared in the river water. Monitoring of the Norovirus (NoV) genomes in the natural environment may contribute to an understanding of the emergence of new epidemic NoV strains in human populations.

The IL15 +96522 A>T functional polymorphism is related to the differentiation of Th17 cells and the severity of Hashimoto's disease.

To clarify the association between the genetic producibility of IL-15, a pro-inflammatory cytokine, and the pathogenesis of autoimmune thyroid diseases (AITDs), we genotyped +96522 A>T and +82889 A>G polymorphisms in the IL15 gene using 127 patients with Hashimoto's disease (HD), including 55 patients with severe HD and 48 patients with mild HD; 130 patients with Graves' disease (GD), including 52 patients with intractable GD and 44 patients with GD in remission; and 79 healthy volunteers. Both the IL15 +96522 A allele and AA genotype were more frequent in patients with severe HD than in those with mild HD. The serum levels of IL-15 were higher in individuals with the IL15 +96522 AA genotype than in those with the T allele, and they were also higher in patients with severe HD than in those with mild HD. On the other hand, the mRNA levels of IL-15 were not significantly different among individuals with each genotype of both SNPs. After incubation with recombinant human IL-15, the proportions of Th17 cells in CD4+ cells were increased, and those of Treg cells in CD4+ cells were maintained. Our study indicates that the IL15 +96522A/C polymorphism correlates with the severity of HD, most likely by increasing Th17 cells.

Expression analysis of stemness genes in a rat thyroid cell line FRTL5.

Detection and analysis of a small subpopulation of cells such as stem cells or cancer stem cells are recognized to be a key technique in a recent regeneration and cancer science. However, in the thyroid, no marker that identifies stem cells has been established yet. We previously established a novel method to analyze cells collected by fluorescence-activated cell sorting (FACS), named mRNA quantification after FACS (FACS-mQ). By using this method, the biological characteristics of the sorted cells can be determined by analyzing their gene expression profile. In this study, we analyzed the expression of stemness genes in a rat thyroid cell lines FRTL5 using FACS-mQ. 3 stemness genes, NANOG, ABCG2 and GATA4, were expressed in FRTL5. In FRTL5 cells, varied expression of thyroglobulin (TG) among cells was observed by flow cytometry. Cell populations with high or low TG expression were analyzed by FACS-mQ. The cell population with low TG expression showed increased expression of the stemness genes. Furthermore, Ki67-positive cells showed increased expression of TG, which suggested that cells with high TG proliferated rapidly. These results indicated that FRTL5 contains a cell population with high stemness gene expression and less differentiated features, resembling stem cells. These cells might regulate proliferation in FRTL5.

Associations of single nucleotide polymorphisms in precursor-microRNA (miR)-125a and the expression of mature miR-125a with the development and prognosis of autoimmune thyroid diseases.

It is important to search the biomarker to predict the development and prognosis of autoimmune thyroid diseases (AITDs) such as Hashimoto's disease (HD) and Graves' disease (GD). MicroRNA (miR) bind directly to the 3' untranslated region of specific target mRNAs to suppress the expression of proteins, promote the degradation of target mRNAs and regulate immune response. miR-125a is known to be a negative regulator of regulated upon activation normal T cell expressed and secreted (RANTES), interleukin (IL)-6 and transforming growth factor (TGF)-ß; however, its association with AITDs remains unknown. To clarify the association between AITDs and miR-125a, we genotyped the rs12976445 C/T, rs10404453 A/G and rs12975333 G/T polymorphisms in the MIR125A gene, which encodes miR-125a, using direct sequencing and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods in 155 patients with GD, 151 patients with HD and 118 healthy volunteers. We also examined the expression of miR-125a in peripheral blood mononuclear cells (PBMCs) from 55 patients with GD, 79 patients with HD and 38 healthy volunteers using quantitative real-time PCR methods. We determined that the CC genotype and C allele of the rs12976445 C/T polymorphism were significantly more frequent in patients with HD compared with control subjects (P < 0·05) and in intractable GD compared with GD in remission (P < 0·05). The expression of miR-125a was correlated negatively with age (P = 0·0010) and down-regulated in patients with GD compared with control subjects (P = 0.0249). In conclusion, miR-125a expression in PBMCs and the rs12976445 C/T polymorphism were associated with AITD development and prognosis.

The functional polymorphisms of VDR, GC and CYP2R1 are involved in the pathogenesis of autoimmune thyroid diseases.

Vitamin D is a multi-functional immune regulator, and a low serum concentration of vitamin D promotes autoimmune inflammation. In this study, we evaluate the association between the prognosis of autoimmune thyroid disease (AITD) and the functional polymorphisms of genes that regulate vitamin D metabolism. For 139 Graves' disease (GD) patients, 116 Hashimoto's disease (HD) patients and 76 control subjects, we genotyped the following polymorphisms using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP): vitamin D receptor (VDR): rs731236, rs7975232, rs2228570 and rs1544410; group-specific component (GC): rs7041 and rs4588; and CYP2R1: rs10741657. The frequency of the TT genotype for the rs731236 polymorphism was higher in GD patients than in HD patients (P = 0·0147). The frequency of the C allele for the rs7975232 polymorphism was higher in GD patients than in control subjects (P = 0·0349). The proportion of GD patients whose anti-thyrotrophin receptor antibody (TRAb) level was >51% was higher in those with the CC genotype than in those with the CA+AA genotypes (P = 0·0065). The frequency of the CC genotype for the rs2228570 polymorphism was higher in HD patients than in control subjects (P = 0·0174) and GD patients (P = 0·0149). The frequency of the Gc1Gc1 genotype for the GC polymorphism and the AG genotype for the CYP2R1 polymorphism were lower in intractable GD than in GD in remission (P = 0·0093 and 0·0268, respectively). In conclusion, genetic differences in the VDR gene may be involved in the development of AITD and the activity of GD, whereas the genetic differences in the GC and CYP2R1 genes may be involved with the intractability of GD.

Association of polymorphisms in DNMT1, DNMT3A, DNMT3B, MTHFR and MTRR genes with global DNA methylation levels and prognosis of autoimmune thyroid disease.

To clarify the association between factors regulating DNA methylation and the prognosis of autoimmune thyroid diseases (AITDs), we genotyped single nucleotide polymorphisms in genes encoding DNA methyltransferase 1 (DNMT1), DNMT3A, DNMT3B, methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR), which are enzymes essential for DNA methylation. Subjects for this study included 125 patients with Hashimoto's disease (HD), including 48 patients with severe HD and 49 patients with mild HD; 176 patients with Graves' disease (GD), including 79 patients with intractable GD and 47 patients with GD in remission; and 83 healthy volunteers (control subjects). The DNMT1+32204GG genotype was more frequent in patients with intractable GD than in patients with GD in remission. Genomic DNA showed significantly lower levels of global methylation in individuals with the DNMT1+32204GG genotype than in those with the AA genotype. The MTRR+66AA genotype was observed to be more frequent in patients with severe HD than in those with mild HD. The DNMT1+14395A/G, DNMT3B-579G/T, MTHFR+677C/T and +1298A/C polymorphisms were not correlated with the development or prognosis of AITD. Our study indicates that the DNMT1+32204GG genotype correlates with DNA hypomethylation and with the intractability of GD, and that the MTRR+66AA genotype may correlate with the severity of HD.

Prolonged hybridization with a cRNA probe improves the signal to noise ratio for in-tube in situ hybridization for quantification of mRNA after fluorescence-activated cell sorting.

We developed an in-tube in situ hybridization method for mRNA quantification after fluorescence-activated cell sorting (FACS-mQ). A specific RNA in a particular cell type is stained with a cRNA probe and a fluorescent dye, which allows the stained cells to be selected by FACS without excessive RNA degradation. Our previous protocol required 4 h for hybridization with a cRNA probe, which might not produce enough fluorescence signal for sorting genes with low expressions. We determined the effect of prolonged hybridization for in-tube in situ hybridization on quantitative measurement of intracellular RNAs. During the hybridization step, the quantity of ACTB mRNA decreased gradually until 4 h, but remained constant from 4 to 16 h below 63.6° C. For flow cytometry, cells hybridization with cRNA probes for TG mRNA at 60° C for 16 h showed both increased signal and decreased background fluorescence compared to those hybridized for 4 h. These results indicate that when performing in-tube in situ hybridization, hybridization temperature can be raised to 63.6° C and the hybridization step can be extended up to 16 h without excessive intracellular RNA degradation.

Evaluation of amphotericin B and micafungin combination against clinical isolates of Aspergillus species.

We aimed to evaluate the efficacy of a combination of amphotericin B (AMB) and micafungin (MCFG) against 25 clinical isolates of Aspergillus species in vitro. We examined fungal growth in the presence of these drugs using a checkerboard method with the tetrazolium salt: 2,3-bis (2- methoxy-4-nitro-5-sulphophenyl)-2H-tetrazolium-5-carboxyanilide inner salt (XTT) to determine the efficacy of an AMB/MCFG combination in inhibition of filamentous fungal growth, evaluated based on 50% reduction of metabolic activity. The fractional inhibitory concentration index showed that the drugs synergistically inhibited 36% of the isolates. Activity was judged as indifferent for 64% isolates; antagonistic interaction was not detected. The AMB/MCFG combination was more effective than AMB alone when sub-inhibitory concentrations of AMB were used. This report demonstrates the efficacy of AMB/MCFG combination for inhibiting the growth of Aspergillus species in vitro, warranting the extension of such studies to animal models.

Association of functional GITR gene polymorphisms related to expression of glucocorticoid-induced tumour necrosis factor-receptor (GITR) molecules with prognosis of autoimmune thyroid disease.

The glucocorticoid-induced tumour necrosis factor (TNF)-receptor (GITR) affects the functions of regulatory T (T(reg)) and effector T (T(eff)) cells, but the significance of this phenomenon is still unclear. To examine the association of single nucleotide polymorphisms (SNPs) in the GITR gene with the expression of GITR molecules on T cells and with the pathological conditions in patients with autoimmune thyroid disease (AITD), we examined the frequencies of four candidate SNPs in AITD patients and healthy volunteers by restriction enzyme analysis and direct sequence analyses. We also analysed the GITR expression on peripheral T(reg) and T(eff) cells in AITD patients by three-colour flow cytometry. The CC genotype in the rs3753348 C/G SNP was significantly more frequent in patients with mild Hashimoto's disease (HD) than in those with severe HD [P = 0·0117, odds ratio (OR) = 3·13]. The AA genotype in the rs2298213 A/G SNP was significantly more frequent in patients with mild HD than in patients with severe HD (P = 0·010, OR = 4·43). All patients and healthy individuals had the GG genotype in rs60038293 A/G and rs11466696 A/G SNPs. The proportions of GITR(+) cells in T(reg) and T(eff) cells were significantly higher in AITD patients with the CC genotype of the rs3753348 SNP than in those with the GG genotype (P = 0·004 and P = 0·011, respectively). In conclusion, the rs3753348 C/G SNP in the GITR is associated with HD prognosis and expression on T(reg) and T(eff) cells.

Association of functional polymorphisms in promoter regions of IL5, IL6 and IL13 genes with development and prognosis of autoimmune thyroid diseases.

To clarify the association of genetic producibility of interleukin (IL)-5, IL-6 and IL-13, which are secreted by T helper type 2 (Th2), with the development and prognosis of autoimmune thyroid disease (AITD), we genotyped IL5-746C/T, IL6-572C/G and IL13-1112C/T polymorphisms, which are functional polymorphisms in the promoter regions of the genes regulating these cytokines. Fifty-seven patients with intractable Graves' disease (GD), 52 with GD in remission, 52 with severe Hashimoto's disease (HD), 56 with mild HD and 91 healthy controls were examined in this study. The IL13-1112T allele, which correlates with higher producibility of IL-13, was more frequent in patients with GD in remission than in those with intractable GD [P=0·009, odds ratio (OR)=3·52]. The IL5-746T allele, which may correlate with lower levels of IL-5, was more frequent in patients with GD in remission than controls (P=0·029, OR=2·00). The IL6-572G allele carriers (CG and GG genotypes), which have higher producibility of IL-6, were more frequent in AITD patients (P=0·033, OR=1·75), especially in GD in remission (P=0·031, OR=2·16) and severe HD (P=0·031, OR=2·16) than in controls. Interestingly, both allele and genotype frequencies of Th2 cytokine genes were similar between GD and HD patients. In conclusion, functional polymorphisms in the genes encoding Th2 cytokines are associated differently with the development and prognosis of AITD from each other.

Association of functional polymorphisms related to the transcriptional level of FOXP3 with prognosis of autoimmune thyroid diseases.

The severity of Hashimoto's disease (HD) and intractability (or inducibility to remission) of Graves' disease (GD) varies among patients. Forkhead box P3 (FoxP3) is a crucial regulatory factor for the development and function of regulatory T (T(reg) ) cells, and deficiency of the FoxP3 gene (FOXP3) suppresses the regulatory function of T(reg) cells. To clarify the association of the functional polymorphisms of the FOXP3 with the prognosis of GD and HD, we genotyped -3499A/G, -3279C/A and -2383C/T polymorphisms in FOXP3 gene obtained from 38 patients with severe HD, 40 patients with mild HD, 65 patients with intractable GD, in whom remission was difficult to induce, 44 patients with GD in remission and 71 healthy volunteers. The -3279CA genotype was more frequent in patients with GD in remission than in patients with intractable GD, and the -3279AA genotype, which correlates to defective transcription of FOXP3, was absent in patients with GD in remission. The -2383CC genotype was more frequent in patients with severe HD than in those with mild HD. In conclusion, the -3279A/C polymorphism is related to the development and intractability of GD and the -2383CC genotype to the severity of HD.

Association of the -31C/T functional polymorphism in the interleukin-1beta gene with the intractability of Graves' disease and the proportion of T helper type 17 cells.

Interleukin (IL)-1beta is a proinflammatory cytokine and has been implicated in the pathogenesis of several autoimmune diseases. To evaluate the hypothesis that the functional -31C/T polymorphism (rs1143627) in the gene encoding IL-1beta is associated with the intractability and the severity of autoimmune thyroid diseases, we genotyped this polymorphism in 64 patients with intractable Graves' disease (GD), 28 GD patients in remission, 49 patients with Hashimoto's disease (HD) who developed hypothyroidism (severe HD), 28 untreated euthyroid HD patients (mild HD) and 59 healthy volunteers. The -31T allele, which is related to the high producibility of IL-1beta, was significantly more frequent in patients with intractable GD than in those with GD in remission (P = 0.0017; odds ratio 2.8; 95% confidence interval 1.5-5.3), although there was no difference in this frequency between two groups of HD patients. We showed additionally that the proportion of IL-17-producing T helper type 17 (Th17) cells, whose differentiation and proliferation are promoted by IL-1beta, was higher in autoimmune thyroid disease patients with the T allele than in those with CC genotypes. In conclusion, our data indicated that the T allele of -31C/T polymorphism in the IL1B gene was involved in the intractability of GD, and this involvement may arise through the differentiation and proliferation of Th17 cells.

Involvement of functional polymorphisms in the TNFA gene in the pathogenesis of autoimmune thyroid diseases and production of anti-thyrotropin receptor antibody.

The severity of Hashimoto's disease (HD) and intractability of Graves' disease (GD) varies among patients. Severity of HD is associated with the functional +874A/T polymorphism for interferon-gamma, an inflammatory cytokine. To clarify the association between functional polymorphisms in two other inflammatory cytokine genes [tumour necrosis factor (TNF)-alpha and interleukin (IL)-2] and the severity of autoimmune thyroid disease (AITD), we examined the TNF-alpha-1031T/C, TNF-alpha-857C/T and IL-2 -330T/G polymorphisms in genomic DNA samples. We genotyped 41 patients with intractable GD, 34 patients with GD in remission, 41 patients with severe HD, 36 patients with mild HD and 70 healthy controls. The frequency of carriers of TNF-alpha-1031C (CT + CC), which correlates with higher TNF-alpha production, was significantly higher in HD and GD patients than in controls, but was not associated with the severity of HD. In GD patients, the levels of anti-thyrotropin receptor antibody (TRAb) at onset of the disease was higher in patients with the TNF-alpha-857T (CT + TT) genotype, which correlates with higher TNF-alpha production, than in those with the -857CC genotype. We found no differences in the IL-2 -330T/G polymorphism among groups of AITD patients. In conclusion, the functional -1031T/C polymorphism of the TNFA gene is associated with the development of AITD and the functional -857C/T polymorphism is associated with the levels of TRAb in active GD patients.

Arteriosclerosis obliterans associated with anti-cardiolipin antibody/beta2-glycoprotein I antibodies as a strong risk factor for ischaemic heart disease in patients with systemic lupus erythematosus.

OBJECTIVE: The main objective of this study was to clarify the role of aPLs in the pathogenesis of arteriosclerosis obliterans (ASO), ischaemic heart disease (IHD) and cerebral vascular disorder (CVD) in patients with SLE. METHODS: We evaluated 155 patients with SLE by using objective tests for diagnosing ASO, IHD and CVD and laboratory tests including ELISA for aCL/beta2-glycoprotein I antibodies (aCL/beta2-GPI) and anti-phosphatidylserine/prothrombin antibodies (anti-PS/PT). RESULTS: Twenty-five (16.1%) of the 155 SLE patients were diagnosed with ASO. Both aCL/beta2-GPI and anti-PS/PT levels were significantly higher in SLE patients with ASO (mean +/- S.E., 104.3 +/- 38.8 U/ml for aCL/beta2-GPI, P < 0.01; 72.6 +/- 48.9 U/ml for anti-PS/PT, P < 0.05) than in SLE patients without ASO (22.8 +/- 9.9 U/ml for aCL/beta2-GPI; 18.3 +/- 4.4 U/ml for anti-PS/PT). Multivariate logistic analysis including aCL/beta2-GPI, anti-PS/PT and traditional risk factors (hypercholesterolaemia, hypertension and diabetes mellitus) confirmed that the presence of aCL/beta2-GPI was the most significant risk factor for ASO in SLE patients [odds ratio (OR) 3.45; 95% CI 1.40, 8.56; P < 0.01]. Furthermore, the prevalence of ASO was associated strongly with IHD (OR 11.8; 95% CI 3.45, 40.1; P < 0.0001) but not CVD (OR 1.84; 95% CI 0.65, 5.21; P = 0.25). CONCLUSIONS: The presence of aCL/beta2-GPI contributes to the risk of development of ASO, which may represent an important mechanism for the pathogenesis of IHD in patients with SLE.

A new predictive indicator for development of pressure ulcers in bedridden patients based on common laboratory tests results.

BACKGROUND: Various scales have been devised to predict development of pressure ulcers on the basis of clinical and laboratory data, such as the Braden Scale (Braden score), which is used to monitor activity and skin conditions of bedridden patients. However, none of these scales facilitates clinically reliable prediction. AIMS: To develop a clinical laboratory data-based predictive equation for the development of pressure ulcers. METHODS: Subjects were 149 hospitalised patients with respiratory disorders who were monitored for the development of pressure ulcers over a 3-month period. The proportional hazards model (Cox regression) was used to analyse the results of 12 basic laboratory tests on the day of hospitalisation in comparison with Braden score. RESULTS: Pressure ulcers developed in 38 patients within the study period. A Cox regression model consisting solely of Braden scale items showed that none of these items contributed to significantly predicting pressure ulcers. Rather, a combination of haemoglobin (Hb), C-reactive protein (CRP), albumin (Alb), age, and gender produced the best model for prediction. Using the set of explanatory variables, we created a new indicator based on a multiple logistic regression equation. The new indicator showed high sensitivity (0.73) and specificity (0.70), and its diagnostic power was higher than that of Alb, Hb, CRP, or the Braden score alone. CONCLUSIONS: The new indicator may become a more useful clinical tool for predicting presser ulcers than Braden score. The new indicator warrants verification studies to facilitate its clinical implementation in the future.