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PubMed2005

Rapid and accurate genotyping of YMDD motif variants in the hepatitis B virus genome by an improved reverse dot blot method.

By using the "flow-through hybridization" principle, we developed a new, rapid and accurate reverse dot blot (RDB) method to detect lamivudine resistance-associated YMDD motif variants in hepatitis B virus (HBV) genome. The improved RDB method was very fast at simultaneously detecting HBV YMDD wild-type and mutant motifs. In a blind analysis, 100 samples previously genotyped by DNA clonal sequencing analysis were used to evaluate the sensitivity and specificity of this assay. Conventional restriction fragment length polymorphism (RFLP) data were also used to test our method. In blind experiments, our improved RDB method had an accuracy and specificity of 100%, which was much higher than RFLP, which had an accuracy and specificity of only 83.0%. In clinical detection practice, 49 patients highly suspected of lamivudine resistance were successfully diagnosed by this method. Our improved RDB assay is a simple, rapid, cheap, semiautomatic, accurate, sensitive, and contamination-proof method of detecting lamivudine resistance-associated mutants in the human hepatitis B virus genome.

Amino Acid Motifs↗
PubMed1981

Clonal parasites in the analysis of resistance to reinfection with Fasciola hepatica.

A feature of fascioliasis in mice and rats is incomplete and generally poor resistance to reinfection. The possibility exists that population heterogeneity amongst Fasciola hepatica parasites (in characteristics such as antigenic variability and infectivity) contributes to the incomplete resistance expressed by already infected rodents to challenge infection. Using different exposure regimes, mice and rats were dosed with infective metacercariae of different single snail-derived clones and challenged with the same or different clonal parasites. The results clearly demonstrate that no better resistance to reinfection is seen with parasites of the homologous clone than with heterologous clone challenge. Thus the poor resistance to reinfection seen in fascioliasis cannot be ascribed readily to antigenic or infectivity differences between clonal metacercariae.

Animals↗
PubMed2000

Hypoxanthine-guanine phosphoribosyltransferase reporter gene mutation for analysis of in vivo clonal amplification in patients with HTLV type 1-associated Myelopathy/Tropical spastic paraparesis.

We tested a surrogate selection approach utilizing mutation at a reporter gene [hypoxanthine-guanine phosphoribosyltransferase (hprt)] as a probe for in vivo cell division, for detection of clonal T cell expansion in human T lymphotropic (HTLV-1) carriers. Peripheral blood samples from HTLV-1-infected individuals with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) were tested to determine the hprt mutant frequency (Mf). Wild-type and hprt mutant T cell clones were isolated, and clonal identity determined by multiplex PCR and DNA sequencing of T cell receptor (TCR) variable region beta-chain (TCR BV) and third complementarity determining regions (CDR3). Seven samples from HAM/TSP patients were tested, and Mfs were within the normal range for adults (mean 11.3 x 10(-6), max 22.4 x 10(-6), min 5.6 x 10(-6)). The frequency of HTLV-1 infection in wild-type and hprt mutant T cells from HAM/TSP patients was determined to identify enrichment in the mutant fraction of cells. This analysis was performed on 196 isolates from 6 individuals with HAM/TSP. In each case, there is enrichment for virally infected cells in the hprt mutant fraction of isolates. Ten mutant and eight wild-type isolates from sample LS42A (Mf 8.4 x 10(-6)) were tested for clonality by TCR BV PCR and sequencing. Of the 10 hprt mutants, there were two in vivo-expanded clones (four isolates with two identical TCRs, or 80% unique TCR sequences). These studies may provide new insights into the precise mechanism of HTLV-1 leukemogenesis, and aid in the study of mutator phenotypes generated by a combination of Tax-mediated in vivo expansion and mutagenesis.

Clone Cells↗
PubMed2005

Vaccination-induced cutaneous pseudolymphoma.

BACKGROUND: Although mild early cutaneous transient reactions to vaccinations are common, late-onset chronic lesions have been scarcely reported. We report herein a series of 9 patients presenting with cutaneous and subcutaneous pseudolymphoma. OBSERVATIONS: Nine patients presenting with late-onset, chronic skin lesions occurring at the site of antihepatitis B (8 cases) and antihepatitis A (one case) vaccination were reported. Histopathologic and immunohistochemic studies, and molecular analysis of clonality of skin biopsy specimens, were performed. Furthermore, the presence of vaccine products was investigated in skin lesions by using histochemical, microanalytic, and electronic microscopy techniques. RESULTS: Histopathologic studies showed dermal and hypodermal lymphocytic follicular infiltrates with germinal center formation. The center of follicles was mostly composed of B cells without atypia, whereas CD4+ T cells were predominant at the periphery. Molecular analysis of clonality revealed a polyclonal pattern of B-cell and T-cell subsets. Aluminium deposits were evidenced in all cases by using histochemical staining in all cases, and by microanalysis and ultrastructural studies in one case. Associated manifestations were vitiligo (one case) and chronic fatigue with myalgia (two cases). CONCLUSION: Cutaneous lymphoid hyperplasia is a potential adverse effect of vaccinations including aluminium hydroxide as an adjuvant. Further prospective studies are warranted to evaluate the incidence of this complication in the immunized population.

Adolescent↗
PubMed1990

Rapid, nonradioactive detection of clonal T-cell receptor gene rearrangements in lymphoid neoplasms.

Southern blot hybridization analysis of clonal antigen receptor gene rearrangements has proved to be a valuable adjunct to conventional methods for diagnosing lymphoid neoplasia. However, Southern blot analysis suffers from a number of technical disadvantages, including the time necessary to obtain results, the use of radioactivity, and the susceptibility of the method to various artifacts. We have investigated an alternative approach for assessing the clonality of antigen receptor gene rearrangements in lymphoid tissue biopsy specimens. This approach involves the amplification of rearranged gamma T-cell receptor genes by the polymerase chain reaction and analysis of the polymerase chain reaction products by denaturing gradient gel electrophoresis. By use of this approach, clonal rearrangements from neoplastic lymphocytes constituting as little as 0.1-1% of the total cells in the tissue are detected as discrete bands in the denaturing gel after the gel is stained with ethidium bromide and viewed under ultraviolet light. In contrast, polyclonal rearrangements from reactive lymphocytes appear as a diffuse smear along the length of the gel. Our findings suggest that polymerase chain reaction combined with denaturing gradient gel electrophoresis may offer a rapid, nonradioactive, and sensitive alternative to Southern blot analysis for the diagnostic evaluation of lymphoid tissue biopsy specimens.

Base Sequence↗
PubMed1985

Phenotypic and functional heterogeneity of human peripheral blood T lymphocytes producing colony stimulating factor. A clonal and precursor frequency analysis.

In this report we describe the precursor frequency and the subset distribution of peripheral blood human T cells producing lymphokine(s) acting on the proliferation and differentiation of bone marrow precursors of the granulocyte/macrophage series, as assessed in a liquid microculture assay. Because the sensitivity of this system was similar to that of the classic colony formation assay in semi-solid (methylcellulose) medium, it is likely that the lymphokine activity measured in this assay corresponds to the colony-stimulating factor (CSF) activity. Single human T lymphocytes, isolated from peripheral blood by E rosetting and Ficoll-Hypaque gradients, were seeded into microculture wells by limiting dilution or micromanipulation techniques and were incubated under culture conditions that allow clonal expansion of essentially all T cells. After 15 to 20 days, microcultures were stimulated with PHA and CSF activity was assayed in culture supernatants 24 hr thereafter. About 45% (1/2.3) of peripheral blood T cells were found to give rise to CSF-producing progenies. Moreover, when fluorescence-activated cell sorter-purified T4+ and T4- (or T8- and T8+) were analyzed, the frequency of the precursors of CSF-producing cells was 1/1.5 in the T4+ subset, whereas approximately one-third of the T8+ cells had this functional potential. To additionally characterize T cells responsible for CSF production, unfractionated T cells as well as T4+ and T4- cells were cloned by single cell micromanipulation. The resulting clones were analyzed simultaneously for the production of IL 2, production of CSF and for cytolytic activity in a lectin-dependent assay. It was found that 25/48 clones obtained from unfractionated T cells produced CSF, whereas 23/48 and 19/48 produced IL 2 or had cytolytic activity respectively. Six of the 25 CSF-producing clones had only this functional capability, whereas the remaining clones in addition displayed cytolytic activity (4/25), IL 2 production (10/25), or both (5/25). A similar functional heterogeneity was observed among T4+ and T4- clones, thus indicating that T cells producing CSF are functionally heterogeneous within both the T4+ and T8+ subsets.

Cell Differentiation↗
PubMed1994

Repertoire of rat MBP-reactive T cells: DNA sequencing analysis further demonstrates the clonal heterogeneity of rat T cells reactive against encephalitogenic epitopes.

Reports of the expression of very similar TCR structures by disparate rodent encephalitogenic T cells reactive with regions of MBP have aroused much interest for both theoretical and practical reasons. To ascertain the extent to which structural requirements of epitope recognition constrain TCR expression by MBP-reactive T cells, we set out to estimate the size of the overall repertoire of TCR beta-chain V beta-D beta-J beta (VDJ) assemblies in T cells of Lewis rats specific for MBP(68-88) as well as those specific for MBP(87-99). We previously reported that such T cells can express a diversity of V beta genes as revealed by PCR analysis. In this study, we have used direct sequencing of PCR products amplified from encephalitogenic T-cell clones and pauciclonal T-cell lines to demonstrate that VDJ structures of the rat T cells specific for either residues 68 to 88 or 87 to 99 of MBP are highly heterogeneous. Our results showed that (1) no pattern is evident in the utilization of germline J gene segments by individual T-cell clones; from a total of over 100 successfully sequenced clones displaying in-frame rearrangements, all the J beta segments have been demonstrated. (2) Even among the T-cell clones which share the V beta expression, J beta is variable. (3) Due to joining variations between the V beta, D beta, and J beta gene segments, no two of the T-cell clones examined share entire VDJ structures. Our study is the first report of nucleotide and amino acid sequences of TCR beta-chains from rat encephalitogenic T cells expressing V beta genes other than V beta 8.2. It demonstrates that the TCR repertoire of the MBP-reactive as well as encephalitogenic T cells is heterogeneous, even though a certain T-cell subset frequently dominated by the mechanism needs to be clarified.

Amino Acid Sequence↗
PubMed1996

Light chain-restricted autoantibodies in chronic idiopathic thrombocytopenic purpura, but no evidence for circulating clone B-lymphocytes.

In chronic idiopathic thrombocytopenic purpura (ITP) platelet destruction is caused by antibodies directed against platelet membrane glycoproteins (GP), and the predominant autoantigens are known to be GPIb/IX and GPIIb/IIIa. In a recent study we reported that these antibodies frequently had a restricted light chain phenotype, thereby supporting a clonal origin. Similar findings and the presence of clonal B-cell populations in immune thrombocytopenias have been reported by others. In the present study we further explored the hypothesis of clonal B-cell expansions in chronic ITP. Twenty patients with chronic ITP were investigated. Antibodies were detected with an ELISA (MAIPA) specific for GPIb/IX and GPIIb/IIIa; circulating clonal B lymphocytes were assessed by flow-cytometric (FACS) clonal-excess analysis and by analyzing Ig-gene rearrangements (CDR3) with the PCR technique. Nine patients displayed a GP-specific antibody restricted to either kappa or lambda phenotype. However, FACS analysis and Ig-gene rearrangement studies did not disclose any circulating clonal B-cell population. Considering the sensitivity of the FACs analysis and Ig-gene rearrangement for detection of clonal B-cell populations, the hypothesis of clonally derived autoantibodies in ITP is still valid. Most probably, the clonal B-cell expansion responsible for the production of autoantibodies in ITP, if present, is below the detection limit for the techniques employed.

Adolescent↗
PubMed2001

From immunoglobulin gene fingerprinting to motif-specific hybridization: advances in the analysis of B lymphoid clonality in rheumatic diseases.

In rheumatic diseases, autoantibody-producing cells of interest are often hidden in a polyclonal B-lymphocyte population. Immunoglobulin gene fingerprinting is a useful approach to screen for expanding clones and to detect recirculation between different locations. The gene fingerprinting approach and the Southern blot technique have been amalgamated, using electrophoretic transfer of a PCR product from an acrylamide gel onto a nylon membrane followed by hybridization with specific oligonucleotide probes. In contrast to conventional fingerprinting, the authenticity of immunoglobulin genes can be confirmed, individual genes can be detected and handling radionucleotides can be avoided. Also, the membrane may be reused for further investigations.

B-Lymphocytes↗
PubMed2006

Molecular-genetic biodiversity in a natural population of the yeast Saccharomyces cerevisiae from "Evolution Canyon": microsatellite polymorphism, ploidy and controversial sexual status.

The yeast S. cerevisiae is a central model organism in eukaryotic cell studies and a major component in many food and biotechnological industrial processes. However, the wide knowledge regarding genetics and molecular biology of S. cerevisiae is based on an extremely narrow range of strains. Studies of natural populations of S. cerevisiae, not associated with human activities or industrial fermentation environments, are very few. We isolated a panel of S. cerevisiae strains from a natural microsite, "Evolution Canyon" at Mount Carmel, Israel, and studied their genomic biodiversity. Analysis of 19 microsatellite loci revealed high allelic diversity and variation in ploidy level across the panel, from diploids to tetraploids, confirmed by flow cytometry. No significant differences were found in the level of microsatellite variation between strains derived from the major localities or microniches, whereas strains of different ploidy showed low similarity in allele content. Maximum genetic diversity was observed among diploids and minimum among triploids. Phylogenetic analysis revealed clonal, rather than sexual, structure of the triploid and tetraploid subpopulations. Viability tests in tetrad analysis also suggest that clonal reproduction may predominate in the polyploid subpopulations.

Alleles↗
PubMed1990

Analysis of inferred cytogenetic clonal evolution in a metastatic human ovarian carcinoma.

Cytogenetic analyses of solid tumors are hampered both by the difficulties in preparing high-quality banded metaphases and the high degree of karyotypic heterogeneity. Three specimens were obtained from a single patient with serous carcinoma for karyotyping and compared to determine whether the tumor was multifocal or metastatic. All three specimens were hypodiploid, sharing marker chromosomes, thus indicating metastasis. To simplify comparison between the specimens, a diagram of the inferred cytogenetic evolution of the cells analysed was developed. This method afforded a simple means of comparing the different tumor sites in a single patient and might also be applied to sequential samples from patients.

Chromosome Aberrations↗
PubMed1983

Clonal expansion and functional analysis of virus-specific T lymphocytes from cerebrospinal fluid in measles encephalitis.

T cells were directly cloned from the cerebrospinal fluid (CSF) of a patient with acute measles encephalitis by limiting dilution in the presence of irradiated feeder cells and T cell growth factor (TCGF). A total of 42 colonies was established. Functional analysis revealed 27 of them to be derived from a cytotoxic T lymphocyte as demonstrated by the ability to exert phytohaemagglutinin (PHA)-dependent cytotoxicity against uninfected allogeneic PHA blasts. Twenty-three of the cytotoxic colonies were specific for measles virus and restricted to self HLA-A or -B antigens. Three clones were also found to give measles virus-specific proliferative responses. The results show that the CSF in measles encephalitis contains a highly enriched population of in vivo sensitized antigen-specific T cells. We propose that the clinical symptoms in measles encephalitis are caused by a T cell-mediated reaction against virus-infected brain cells.

Child↗
PubMed1991

Immunoglobulin gene 'fingerprinting': an approach to analysis of B lymphoid clonality in lymphoproliferative disorders.

Rearrangement of the immunoglobulin heavy chain (IgH) gene is widely exploited as a marker of B cell lineage and clonality in the pathology of lymphoproliferative disorders. We have developed a simple, polymerase chain reaction (PCR) based method for detecting IgH gene rearrangement which relies on the observation that by using a panel of PCR amplimers specific for each of the six heavy chain variable region families in conjunction with a common joining region amplimer, clonal rearrangement can be detected in over 90% of cases of B lymphoid malignancy. By using radiolabelled amplimers and exploiting the size heterogeneity resulting from independent IgH rearrangement events, we show that high resolution gel electrophoresis can be used to generate a 'fingerprint' representing the spectrum of B cell clonality in complex populations of B lymphocytes. The method effectively scans the entire IgH gene rearrangement repertoire and is capable of detecting rare clonal or oligoclonal B lymphoid cell populations. In normal bone marrow mononuclear cells, clonal IgH rearrangement could be readily detected at a sensitivity of 10(-3). We illustrate the application of the method in assessing the spectrum of B cell clonality occurring in an autoimmune condition. Hashimoto's thyroiditis, and in a malignant B cell disorder, chronic lymphocytic leukaemia. In addition, we explore the potential application of the technique in tracking minimal residual disease and for monitoring clonal evolution in acute lymphoblastic leukaemia.

B-Lymphocytes↗
PubMed2000

Analysis of plasma cell clonality in localized AL amyloidosis.

AL amyloidosis is characterized by fibrillar tissue deposits composed of monoclonal immunoglobulin light chains(IgLs). It has been speculated that clonal expansion of plasma cells may occur locally and produce amyloidogenic IgLs. Both immunohistochemistry and molecular genetics are useful for examining plasma cell clonality from paraffin-embedded tissue sections, which are easy to obtain. We evaluated plasma cell clonality in 16 biopsy cases of localized AL amyloidosis using these two methods. A clonal excess of plasma cells was detected in 6 (37.5%) cases immunohistochemically, in 10 (62.5%) cases molecularly, and in 13 (81.3%) cases by at least one of the two methods. These results support local synthesis of the light chain proteins in localized AL amyloidosis.

Adult↗
PubMed2001

Molecular analysis of T-cell clonality with concomitant specific T-cell proliferation in vitro in nickel-allergic individuals.

BACKGROUND: The peripheral blood mononuclear cells (PBMC) of individuals with nickel contact allergy are reported to proliferate to a varying degree upon nickel stimulation in vitro. Different phenotypes of the T cells involved are described. With regard to preferential use of the T-cell receptor (TCR), analysis of the several families of the TCR-gamma gene allows rearrangement evaluation of all T cells regardless of predominant surface expression of TCR alpha/beta. METHODS: The PBMC of 10 nickel-allergic and five nonallergic individuals were cultured for 4 days in the presence of either medium, PHA, NiSO4, or tetanus toxoid (TT). Proliferation was measured by radioactive thymidine uptake and expressed as stimulation index (SI). T-cell clonality was assessed by analysis of the TCR-gamma chain gene, including the use of PCR with a primer combination covering the four main groups (Vgamma1-8, Vgamma9, Vgamma10, and Vgamma11) of the variable region of the TCR-beta chain gene. RESULTS: In the allergic individuals, proliferation to NiSO4 was significantly (P<0.05) higher than in nonallergics (mean SI: 18.05/17.87 vs 0.67/2.27). In unstimulated and PHA-stimulated cultures, there was a random TCR spectrum in both groups. In contrast, in nickel-allergic individuals or individuals with recent TT-booster, oligoclonality could be observed in the correspondingly stimulated cultures. CONCLUSION: In addition to proliferation assay, analysis of T-cell clonality may be a further means to characterize clinical hypersensitivity reactions on the basis of antigen-dependent oligoclonal T-cell expansion, as in the case of tissue-infiltrating lymphocytes.

Adult↗
PubMed2004

Plasmodium falciparum clonal population dynamics during malaria treatment.

We have developed a new fragment-analysis method to enumerate the clones and to quantify their proportions within Plasmodium falciparum isolates. We prospectively enrolled 20 adult patients with uncomplicated malaria who were returning to France from various sub-Saharan countries, from January 2000 through July 2001. The analysis of clonal populations was performed on blood samples obtained at 10 times: 1 before treatment with oral quinine and 9 during the first 96 h of the treatment. The resistance genotypes pfcrt and dhfr were determined for chloroquine and antifolinics. Multiple P. falciparum genotypes were detected in 19 (95%) of 20 patients: 2, 3, 4, and 5 genotypes were found in 4, 9, 4 and 2 patients, respectively. Disappearance and reappearance of some clones within a few hours was observed. Individual clones represented 0.4%-99.4% of total parasitemia. Surprisingly, in 10 of 15 subjects tested, resistance genotypes varied according to the time of blood collection. These findings may have important implications with regard to the interpretations of resistance studies.

Animals↗
PubMed1987

DNA clonal heterogeneity of hepatocellular carcinoma demonstrated by Feulgen-DNA analysis.

To demonstrate DNA clonal heterogeneity of hepatocellular carcinomas (HCC), the DNA histographic pattern of both primary HCCs and their recurrent or metastatic lesions were studied among 36 patients (33 men and 3 women). Thirty-six paired aspirations or imprints taken from primary, recurrent or metastatic lesions were stained, using the modified Feulgen method, and the DNA content was measured with a scanning microdensitometer at a wavelength of 550 nm. Paired aspirations or imprints taken from different parts of the same HCC were examined in 17 cases; the DNA distribution patterns were similar in 15 (88%) and differed in only two (12%). A similar DNA histogram was also shown among different tumors in 10 (71%) of 14 patients with multiple HCCs, with a DNA ploidy discrepancy in only four (29%). Two of two subcutaneous metastases and two of three recurrent tumors showed DNA distribution patterns similar to those in their primary HCCs. In summary, a DNA clonal heterogeneity of HCC was found in 19% (7/36). In contrast, the similar DNA histographic patterns found in most instances among different parts of the HCC and between the primary and recurrent or metastatic lesions suggest that HCC may derive from a single cell clone in the majority of cases.

Adult↗
PubMed1989

Molecular genetic analysis of lymphoid tumors arising after organ transplantation.

A variety of gene analyses were performed on lymphoid tumors from transplant patients who received cyclosporine A for immunosuppression. Epstein-Barr virus DNA was detected in the tumors, and the structure of circular episomal virus DNA was used as a measure of cell clonality. This analysis was correlated with clonality determined by study of immunoglobulin gene rearrangement. Some of the tumors had DNA rearrangements near the c-myc gene. Analysis suggested the pathogenesis of the tumors and indicated four categories of lymphoproliferation, three neoplastic and one reactive.

B-Lymphocytes↗