No association of Borna disease virus with psychiatric disorders among patients in Northern Kyushu, Japan
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There is controversy over the prevalence of Borna disease virus (BDV) antibodies and its RNA in the peripheral blood mononuclear cells (PBMCs) of psychiatric patients, and the contribution of BDV to human psychiatric disorders. We examined 299 plasma and 229 PBMC samples. No plasma samples were positive for BDV-p40, p24 or gp18 antibodies by western blot analysis. The prevalence of BDV RNA in the psychiatric (schizophrenic) patients (1.8%) was not significantly different from that in the healthy volunteers (0.6%). The nucleotide sequences of BDV p40 and p24 were highly conserved with those of BDV He/80. Our results suggested that there is a lack of association between BDV infection and psychiatric disorders among the patients in Northern Kyushu, Japan. J. Med. Virol. 61:336–340, 2000. © 2000 Wiley-Liss, Inc.Cite
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An important concern for the use of antibodies in various applications, such as western blot (WB) or immunohistochemistry (IHC), is specificity. This calls for systematic validations using well-designed conditions. Here, we have analyzed 13 000 antibodies using western blot with lysates from human cell lines, tissues, and plasma. Standardized stratification showed that 45% of the antibodies yielded supportive staining, and the rest either no staining (12%) or protein bands of wrong size (43%). A comparative study of WB and IHC showed that the performance of antibodies is application-specific, although a correlation between no WB staining and weak IHC staining could be seen. To investigate the influence of protein abundance on the apparent specificity of the antibody, new WB analyses were performed for 1369 genes that gave unsupportive WBs in the initial screening using cell lysates with overexpressed full-length proteins. Then, more than 82% of the antibodies yielded a specific band corresponding to the full-length protein. Hence, the vast majority of the antibodies (90%) used in this study specifically recognize the target protein when present at sufficiently high levels. This demonstrates the context- and application-dependence of antibody validation and emphasizes that caution is needed when annotating binding reagents as specific or cross-reactive. WB is one of the most commonly used methods for validation of antibodies. Our data implicate that solely using one platform for antibody validation might give misleading information and therefore at least one additional method should be used to verify the achieved data.
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Objective To study the effect of staphylococcal enterotoxin B(SEB) on human peripheral blood mononuclear cells and to explore whether apoptosis is one of its immunosuppressive mechanism.Methods Human peripheral blood mononuclear cells in vitro were stimulated by different concentrations of SEB(100 μg/ml,50 μg/ml,25 μg/ml,10 μg/ml,5 μg/ml,1 μg/ml,0.1 μg/ml,0.01 μg/ml,0.001 μg/ml and 0.0001 μg/ml).PBMCs in negative control group were cultivated in RPMI1640,and PHA was added in positive control group.After cultivation for 48 h,MTT colorimetric assay was used to analyze proliferation of PBMC.Same process was also carried out in negative control group in RPMI1640 and positive control group of PHA.After 48 h,the repeated stimulation with 10 μg/ml SEB was done on PBMC for two times.Results Different concentrations of SEB could initially stimulate human peripheral blood mononuclear cells in vitro to proliferate,but the proliferation of PBMC activated by 10 μg/ml SEB was most dramatic.There was no significant difference between group of 10 μg/ml SEB and group of PHA.Their rates of proliferation were 23.1% and 24.3% respectively.After repeated stimulation at 48 hr later,PHA could continue to stimulate the proliferation of PBMC,but 10 μg/ml SEB could inhibit the proliferation of PBMC.Conclusion SEB may initially stimulate the proliferation of human PBMC in vitro.The repeated stimulation with SEB may inhibit the proliferation of PBMC.
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We have generated iPSCs from peripheral blood mononuclear cells (PBMCs) of a healthy man using heat sensitive and non-integrative Sendai virus containing Sox2, Oct3/4, c-Myc and Klf4. Human GRX-MCiPS4F-A2 cell line was established and characterized through this study.
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In a prospective study we tested the appearance of IgG and IgM positive viral protein bands in Western blots from six people who seroconverted for anti-HIV antibody. Quantification of the immunoblotted bands was performed by reading the Western blot stripes in Camag scanner and analysed on a 350 computer (digital equipment). In the first serum, all people were negative for anti-HIV antibodies. In the second serum, after 16 to 122 days, all people showed IgM HIV-antibodies to p24. IgG HIV-antibodies were detectable in all people after 18 to 114 days after the second collection. Our data clearly demonstrated that for early analysis of HIV infection only the detection of IgM antibodies to viral protein bands of the Western blot technique provides reliable results and that scanning and advanced integration analysis of the Western blot peaks offer the advantage of direct quantitative comparison of the results, not just qualitative description. Further, this direct quantitative comparison of antibodies to HIV virus protein bands can be used as a prognostic marker for disease states.
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Objective To explore the killing efficacy of cord blood mononuclear cells in HO9810 and the activation effect of rhIFNα-2b on it.Methods Mononuclear cells were isolated from fetal blood and adult peripheral blood respectively,whose killing efficacy in HO9810 at the presence or absence of rhIFNα-2b were measured with MTT.Results The natural killing efficacy of cord blood mononuclear cells was lower than that of the peripheral blood.When activated,however,it was enhanced markedly and could be equal to that of the latter.Conclusions The mononuclear cells in the cord blood can be activated into killing cells by rhIFNα-2b,which shows increased killing efficacy to HO9810 and have a promised future in antitumor immunotherapy.
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There was 100% agreement between the results of indirect immunofluorescence (IF) and Western blot testing when these methods were used to detect antibodies to the human immunodeficiency virus in sera from 25 patients with acquired immune deficiency syndrome (AIDS), 20 patients with AIDS-related complex, 186 subjects at high risk for AIDS, and 40 healthy heterosexuals. However, there was only an 88.7% correlation between IF and Western blot results for 728 sera from blood and plasma donor centers that were selected on the basis of screening enzyme immunoassay reactivity. IF tests yielded nine false-negatives and were equivocal, yielding a nonspecific pattern of reactivity for both infected and uninfected cells for 73 of these specimens. The IF and Western blot methods were equal in performance for the detection of anti-human immunodeficiency virus antibodies in the high-risk and unselected low-risk groups, proving to be a practical approach for testing specimens from these subjects. However, the Western blot was the most acceptable method for the validation of specimens from groups at low risk for AIDS that were selected based on enzyme immunoassay reactivity.
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Supplementary Figure 3 from Common Transcriptional Signature of Tumor-Infiltrating Mononuclear Inflammatory Cells and Peripheral Blood Mononuclear Cells in Hepatocellular Carcinoma Patients
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Uncontrolled preanalytical variables can reduce the accuracy and reproducibility of downstream analytical results from peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from EDTA and citrate-anticoagulated blood samples, obtained from healthy subjects and patients with inflammatory and infectious conditions. PBMC-derived RNA samples were examined for gene expression changes induced by extended blood pre-centrifugation delays at 4 °C and RT. We used Taqman RTqPCR to evaluate the combination of two target genes for their "diagnostic performance" in identifying EDTA and citrate-anticoagulated PBMC samples with extended pre-centrifugation times. We established the PBMC preanalytical score, a gene expression metric to asses the PBMC quality related to the pre-centrifugation delay at room temperature for different anticoagulants. The PBMC preanalytical score measurement can identify: EDTA PBMC samples or RNA extracted from these PBMCs with RT precentrifugation times >48 h with 98% sensitivity and 87% specificity at a cutoff of 57. citrate PBMC samples or RNA extracted from these PBMCs with RT precentrifugation times of >48 h with 92% sensitivity and 84% specificity at a cutoff of 348. The proposed PBMC preanalytical score may enable objective PBMC sample qualification for downstream applications, which may be influenced by blood precentrifugation delays.
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