Analysis of J beta gene segment usage by CD4+ and CD8+ human peripheral blood T lymphocytes.

Certain T cell antigen receptor V gene products in man have been shown by us and others to display a reproducible bias for preferential expression in CD4+ or CD8+ T cell subsets. In order to investigate whether such a skewed representation of V gene segments is also present at the J gene segment level, we tested the relative J beta gene usage by V beta 5.1 + T cells, as this V beta gene is biased towards CD4+ T cell expression in virtually all individuals. To analyze the usage of the 13 J beta gene segments, we developed a new approach using V beta 5.1 and C beta specific oligonucleotides as 5' and 3' primers respectively for polymerase chain reaction (PCR) amplification of cDNA derived from CD4+ or CD8+ peripheral blood lymphocyte (PBL) T cells. The PCR products were visualized for reactivity with individual J beta 1.1-1.6 and J beta 2.1-2.7 32P-labelled oligonucleotide probes using autoradiography and quantitative gel-scanning. Eleven normal blood donors provided the PBL T cells. The results showed that in every individual's V beta 5.1+ T cell populations (CD4 and CD8), all V beta/J beta combinations were used although at varying but reproducible levels for each J beta gene. Thus, no discernible disallowance of combinations existed. Moreover, we could show that six of 13 J beta genes were unequally expressed when compared in pairs with regard to expression in CD4+ and CD8+ T cell subsets.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evolutionary conservation of chymotrypsinogen gene: Genomic analysis and protein modeling

Summary Chymotrypsinogen is widely present in various animal pancreases. To study evolutionary relationship of chymotrypsinogen gene in species, we used a cDNA probe of human prechymotrypsinogen to investigate the species distribution of chymotrypsinogen gene, and designed oligodeoxynucleotide primers to investigate the genomic organization in the three domains of active sites. The genomic analyses showed that chymotrypsinogen gene is evolutionary conserved in species. On the basis of the deduced amino acid residues, a three-dimensional model for human chymotrypsinogen was further built by computer graphics. The model showed high similarity to the X-ray crystal structure of bovine chymotrypsinogen A, thus, demonstrated that the three-dimensional structure is more conserved in evolution than protein sequences.     

The polymerase chain reaction in the diagnosis of early mycosis fungoides

The earliest or patch stage of mycosis fungoides may present diagnostic difficulty both clinically and pathologically. The present study of the polymerase chain reaction (PCR) as a diagnostic tool in early mycosis fungoides was therefore undertaken, using a rapid PCR method for the detection of γ- and β-chain T-cell receptor (TCR) gene rearrangements in routine formalin-fixed, paraffin-embedded histological sections. Forty-two biopsies were studied from 26 patients with mycosis fungoides. Twenty-three skin biopsies with a clinicopathological diagnosis of early, or patch stage, mycosis fungoides were investigated. Of these, 18 (78 per cent) showed TCR-γ or both β- and γ-chain TCR gene rearrangements. TCR gene rearrangements were shown in seven of the 14 plaque stage lesions (50 per cent) and also in the single case of tumour stage disease. Where gene rearrangements were identified, these were identical in all biopsies from an individual patient, irrespective of the site of the lesion, the disease stage, or the time lapse between the biopsies. The PCR is therefore a highly sensitive technique, which can be performed on routine pathological material, in cases where the diagnosis of early mycosis fungoides cannot be made with certainty on conventional histopathological and immunohistochemical grounds. © 1997 John Wiley & Sons, Ltd.     

Evidence for selective in vivo expansion of synovial tissue-infiltrating CD4+ CD45RO+ T lymphocytes on the basis of CDR3 diversity

In this study we have analyzed the TCR V and V_ß regionsat the DNA level in the CD4⁺CD45RO⁺ memory T cell populationof synovial tissue infiltrating T lymphocytes of three rheumatoidarthritis (RA) patients and one patient with chronic arthritis.Cell lines of CD4⁺CD45RO⁺, CD4⁺CD45RO^–, CD8⁺CD45RO⁺ andCD8⁺CD45RO^– T lymphocyte populations were generated followingFACS cell sorting of freshly isolated synovial tissue mononuclearcell infiltrates (STMC) and of freshly isolated peripheral bloodmononuclear cells (PBMC) of these patients. The phenotyplc andmolecular analyses have revealed the following. (I) The TCRrepertoires of tissue infiltrating T lymphocytes in the varioussubsets were extensive on the basis of TCR V gene family usage.(II) Furthermore, each patient displayed individual specificTCR V gene expression patterns in the various STMC and PBMCderived T cell subsets. However, the majority of these arthritispatients manifested increased expression of multiple TCR V genefamilies in the synovial tissue derived CD4⁺CD45RO^– Tcell population when compared with the peripheral blood derivedCD4⁺CD45RO⁺ subset. Of these gene families, we found enhancedexpression of the TCR V7 and V_ß11 gene segments insynovial tissue to be shared by all four patients analyzed.OH) Nucleotlde sequence analysis of the CDR3 regions of a numberof TCR V regions in the CD4⁺CD45RO⁺ T cell subsets has revealedthat the CDR3 regions comprised within synovial tissue derivedTCR V regions differed from those found in peripheral bloodderived TCR V regions. These differences in CDR3 diversity mightbe the consequence of a specific interaction with particularMHC-peptlde complexes expressed at the site of inflammation.(Iv) The CDR3 region analysis also showed individual specificamlno acid motifs within the N-D-N regions of all analyzed TCRV_ß genes derived from PBMC as well as STMC.     

超抗原SEA和SEB对正常人TCRVβ基因的限制性取用

金黄色葡萄球菌肠毒素 (SE )作为一种超抗原 ,以MHC非限制性及TCRVβ特异性的方式激活T细胞。本文用定量PCR方法 ,分析SEA和SEB刺激的正常人外周血淋巴细胞T细胞抗原受体Vβ的取用格局。揭示在不同HLA遗传背景下 ,SEA刺激的淋巴细胞均选择性地取用Vβ3和Vβ6两种基因片段 ,而SEB刺激的淋巴细胞则优势表达Vβ2、Vβ8和Vβ9。通过比较分析 ,Sigma产的SEB刺激淋巴细胞后则优势表达Vβ3、Vβ4、Vβ16和Vβ2 0 ,提示外周血淋巴细胞在超抗原作用下发生寡克隆扩增。     

Accumulation of multiple T cell clonotypes in the synovial lesions of patients with rheumatoid arthritis revealed by a novel clonality analysis.

T cell activation in the characteristic synovial lesions of rheumatoid arthritis may play a major role in the pathogenesis of this autoimmune disease. Analysis of T cell clonal diversity in these sites remains equivocal. Using the PCR and subsequent single-strand conformation polymorphism analysis it is possible to assess the degree of junctional diversity in the TCR with minimal selection bias. Concentrating on the beta-chain of the TCR, a paucity of clonotypic T cell expansion is demonstrated in the peripheral blood of healthy individuals. After polyclonal stimulation in vitro (with concanavalin A or phytohemagglutinin) this pattern does not change. In contrast, some T cell clonotypes appear following in vitro stimulation with purified protein derivative. Analysis of the peripheral blood, synovial fluid, and synovial tissue of patients with rheumatoid arthritis indicated many dominant T cell clonotypes. These data argue for a clonally diverse T cell response in the affected tissues of rheumatoid arthritic subjects.     

Variant of hepatitis B virus isolated in zimbabwe

The polymerase chain reaction (PCR) was used to amplify an approximately 1.2 kb DNA fragment encompassing the pre-S/S gene region of HBV DNA from serum of patients with acute hepatitis B virus infection. Nucleotide sequence analysis revealed a number of interesting features in the S gene region. Two Bam HI sites were located at nucleotide positions 557 and 872, respectively, in the S gene. Guanine (G) was found at nucleotide position 903 as part of AGA, the codon for arginine (R) corresponding to amino acid position 122 of the S protein. Ade-nine (A) was found at nucleotide position 1017 as part of AXIA, the codon for lysine (K) corresponding to amino acid position 160 of the S protein. Nucleotide sequence alignment revealed a 97% homology to the corresponding domain of an HBVadw genome (clone pFDW294). Within the second loop of the “a” determinant, two mutations resulting in substitution of serine or threonine with the hydrophobic amino acids, methio-nine at position 143 and with alanine in place of glycine at position 145, are predicted from the consensus nucleotide sequence of the PCR-derived clones. Subtyping with monoclonal antibodies showed that the HBsAg was of the ayw subtype. © 1994 Wiiey-Liss, Inc.     

The human T cell receptor V beta repertoire of normal peripheral blood lymphocytes before and after mitogen stimulation.

Mitogen stimulation of T cells in vitro has been employed in the analysis of the T cell antigen receptor (TCR) repertoire and as a method of generating T cell lines and clones. It has been suspected for some time that mitogen stimulation may bias the repertoire. We have addressed this problem employing a semi-quantitative technique utilizing the polymerase chain reaction (PCR) and flow cytometry. Using this PCR method and a panel of primers to 22 V beta subgroups, the V beta repertoire of both unstimulated and phytohaemagglutinin (PHA)-stimulated peripheral T cells from eight healthy individuals was investigated. The samples were also analysed by flow cytometry using anti-V beta 2, V beta 5 and V beta 8 MoAbs. A significant increase in the expression of V beta 6, V beta 7.2 and V beta 10.1 was found in all eight samples of PHA-stimulated T cells compared with unstimulated T cells using the PCR method. In contrast, no differences were found between unstimulated and PHA-stimulated T cells by flow cytometry. These results question the validity of using mitogen-stimulated T cells to investigate TCR gene usage.     

Loop-mediated isothermal amplification method for detection of human papillomavirus type 6, 11, 16, and 18

A new method was developed for detection of human papillomavirus (HPV) by loop-mediated isothermal amplification (LAMP), which was compared with the polymerase chain reaction (PCR), and real-time PCR for specificity and sensitivity. All initial validation studies with the control DNA proved to be type-specific. In order to evaluate the reliability of HPV type-specific LAMP detecting HPV DNA from clinical samples, tissue specimens were obtained from 27 patients with external genital polypoid lesions. The histologic diagnoses included condyloma acuminatum (n = 21), bowenoid papulosis (n = 2), seborrheic keratosis (n = 2), epidermolytic acanthoma (n = 1), and hairy nymphae (n = 1). HPV-6 DNA and HPV-11 DNA were detected in 18 and 3 of 21 condylomata acuminata, respectively, and there was no simultaneous infection. HPV-16 DNA was detected in one of two bowenoid papuloses. HPV DNA was not detected in the seborrheic keratoses, epidermolytic acanthoma, and hairy nymphae. These results correlated perfectly with those from real-time PCR analysis. Most positive samples contained high copy numbers of HPV DNA. HPV-11 DNA was detected in one case that could not be detected by PCR. The average reaction time was about 59 min. There was a linear correlation between the genome quantity and reaction time to reach the threshold. The LAMP method has an additional advantage as a quantitative method, and is superior in terms of sensitivity, specificity, rapidity, and simplicity, and can potentially be a valuable tool for the detection of HPV DNA.     

多发性硬化症脑脊液中活化T细胞抗原受体V_β基因的表达

应用定量逆转录聚合酶链反应（ＲＴ－ＰＣＲ）对４例多发性硬化症（ＭＳ）脑脊髓液（ＣＳＦ）细胞及末梢血单个核细胞（ＰＢＭＣ）中Ｔ细胞抗原受体（ＴＣＲ）Ｖ_β基因（Ｖ_β１～Ｖ_β２０）表达进行了探讨。结果表明：未经刺激的ＣＳＦ细胞和ＰＢＭＣ表达广谱Ｖ_β基因，经ＴＣＧＦ／ＩＬ－２／ＩＬ－４短期培养的ＣＳＦ细胞则表达限制性Ｖ_β基因，而经同样培养的ＰＢＭＣ各Ｖ_β基因表达的百分率同培养前ＰＢＭＣ相同。说明在ＭＳ疾病的中枢神经系统中存在着对某种抗原的特异性Ｔ细胞应答，为ＭＳ的免疫治疗提供了新的启示。     

Genotype distribution of hepatitis C virus infection in Taiwan

To investigate the prevalence of genotype distribution of hepatitis C virus (HCV) infection in Taiwan, genotypes were identified in 122 (36 anti-HCV-positive blood donors, 44 anti-HCV-positive aborigines, 28 hemodialysis patients, and 14 patients with chronic liver diseases) of 280 subjects, using polymerase chain reaction by Okamoto's type-specific primer method. Type II was the dominant (66.7%) type among anti-HCV-positive blood donors, followed by type III and type IV with the same percentages (16.7%), while none of type I was detected. The prevalence of genotype distribution were 75.0%, 81.1%, and 64.3% for type 11,4.6%, 17.9%, and 21.4% for type III, 13.6%, 0%, and 7.1% for type IV, for the aborigines, hemodialysis, and chronic liver diseases groups, respectively. Four subjects revealed mixed infections by two different genotypes: two cases of II and III; and each one case of II and IV, and III and IV. Diverse genotype distributions in two hemodialysis groups disclose the existence of obvious regional differences even within a region. The results reveal the highest prevalence of type II as in Japan. However, there is a higher prevalence rate of type IV than in Japan. © 1994 Wiley-Liss, inc.     

Inter-laboratory comparison of HCV-RNA assay results: implications for multi-centre research

To investigate whether it is appropriate to assume comparability of hepatitis virus C (HCV)-RNA results across laboratories in multi-centre studies, nine laboratories of the European Paediatric HCV Network participated in an international proficiency study of HCV-RNA assays. A panel of 12 samples of different dilutions and genotypes was sent to each laboratory and tested with qualitative and/or quantitative HCV-RNA assays according to local procedures. Commercial assays were used in seven laboratories and in-house assays in two. All six laboratories in which a commercial qualitative assay was used were proficient, as were four of six runs (in five laboratories) in which a commercial quantitative assay was used. The proficiency of the laboratories where in-house assays were used could not be assessed according to the VQC definition because of differences in the methods used. Overall, there were several false-negative results, but only one false-positive result with a quantitative assay and none with a qualitative assay. The false-negative results may have implications for the diagnosis of infection, and highlight the need for an antibody test to be performed at 18 months to confirm the absence of infection. The results of qualitative assays were generally consistent across laboratories but it was difficult to evaluate and compare the results of quantitative assays. Multivariate analysis of data collected in multi-centre studies should therefore allow for centre and/or assay used.     

Systemic Mycobacterium kansasii infection mimicking peripheral T‐cell lymphoma

Nontuberculous mycobacteria are opportunistic pathogens which predominantly infect the immunocompromised host. The clinical and pathologic diagnosis of mycobacterial infection is generally not difficult. However, it may mimic malignancy on account of the clinical manifestations or the morphology of atypical lymphocytes with epithelioid histiocytes. The latter can be found in some types of lymphomas, especially T‐cell lymphoma. This report describes two immunocompetent patients with systemic Mycobacterium kansasii infection presenting with fever, systemic lymphadenopathy, and osteolytic bone lesions. The microscopic features of these two cases were similar and were characterized by effacement of the nodal architecture by lymphocytic infiltrates and small aggregates of epithelioid histiocytes throughout. These lymphocytes showed mild atypia and expressed predominantly CD3. Bone marrow was also involved in the same process in one case and T‐cell lymphoma with lymphoepithelioid features was the initial impression. However, further studies reported germline arrangements of T‐cell receptor genes, presence of acid‐fast bacilli, and recovery of M. kansasii in culture. At follow‐up, the lymphadenopathy was seen to have disappeared during antimycobacterial treatment. This report describes two infectious cases with small aggregates of epithelioid histiocytes and atypical lymphocytes mimicking peripheral T‐cell lymphoma; and such cases may become more common as the number of immunosuppressed hosts is increasing worldwide. We have reviewed the literature and summarized useful morphologic criteria for differentiation.     

Rapid preparation of diagnostic probes for the fragile X syndrome by direct PCR amplification of human chromosomal DNA

Summary The fragile X syndrome is a common familial form of mental retardation and is associated with a rare fragile site at Xq27.3 (FRAXA). This disorder has recently been reported to correlate with length variations of restriction genomic DNA fragments which may due to the amplification of (CCG)_n trinucleotide repeats located at the FRAXA locus. We described here a rapid preparation method of diagnostic DNA probes for the fragile X syndrome by direct enzymatic amplification of human chromosomal DNA. ThePstI-assay, which is Southern blot analysis of DNA samples probed by PCR products, was shown to be sensitive method for diagnostic purposes to detect the size variations specific in the fragile X syndrome.     

Polymerase chain reaction screening of immunoglobulin heavy chain and T cell receptor γ gene rearrangements: A practical approach to molecular DNA analysis of non-Hodgkin's lymphoma in a surgical pathology laboratory

Characterization of the clonality of non-Hodgkin's lymphoma (NHL) by the rearranged segments of immunoglobulin heavy chain (Ig(H)) or T cell receptor (TCR) genes is not only useful in the confirmation of the diagnosis but also for the future assessment of how a secondary lymphoma, such as a recurrence or another primary lymphoma, occurs. As a practical approach to obtaining and registering this information in a surgical pathology laboratory, FR3 and FR1 regions of Ig(H) gene and TCRgamma gene were concurrently amplified by polymerase chain reaction (PCR) using each pair of consensus primers and the same PCR protocol. Examined samples consisted of 134 primary NHL (phenotypically, 108 B cell and 26 T cell NHL), 19 reactive lymphadenopathies, as well as five secondary lymphomas whose primary lesions were included in this study. Among the primary NHL, the combined PCR analysis disclosed the clonality in 103 of 134 NHL (77%), by FR3 PCR in 77 B cell and two T cell NHL, by FR1 PCR in 59 B cell and one T cell NHL, and by TCRgamma PCR in 11 B cell and 17 of 26 T cell NHL, but in none of the reactive lymphadenopathies. Among the secondary lymphomas, the same pattern of PCR analysis was obtained in two cases (the durations between first and second lymphomas; 6 and 10 months), which suggested recurrence. In contrast, different results were obtained in three cases (17-37 months), which indicated another primary or emergence of the subclones. The results of Southern blot analysis were concordant with the PCR results of the first and the secondary lymphomas. Although the combined PCR analysis cannot replace Southern blot hybridization because of its lower detection rate, it can select those cases suitable for further Southern blot analysis thus reducing the number of unnecessary examinations by nearly 75%. This approach may also be useful in the comparative evaluation of primary and secondary lymphomas.