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PubMed1986

Analysis of the clonal origins of autoantibodies against thyroglobulin and DNA in autoimmune thyroiditis and systemic lupus erythematosus.

We have analysed the isoelectric focusing (IEF) spectra of antithyroglobulin autoantibodies in the sera of patients with autoimmune thyroiditis, and of anti-dsDNA autoantibodies in the sera of patients with systemic lupus erythematosus (SLE), NZB/NZW F1 hybrid, MRL-lpr/lpr and MRL/++ male and female mice. Ninety-two per cent of patients with anti-thyroglobulin autoantibodies had a polyclonal spectrotype compared with only 25% of SLE patients analysed for anti-dsDNA. Fifty-five per cent of the latter had monoclonal spectrotypes, the remainder being either biclonal or having a dominant clone on a polyclonal background. By contrast, only two out of 61 autoimmune thyroiditis patients expressed monoclonal anti-thyroglobulin autoantibodies. All of the lupus mice had highly restricted spectrotypes (monoclonal or biclonal) of anti-dsDNA autoantibodies. The implications of these results for the aetiology of autoimmunity are discussed.

Animals↗
PubMed1984

T cell regulation of myelopoiesis: analysis at a clonal level.

Colony-stimulating factor (CSF) production by a series of cloned human T lymphocyte cell lines was examined by substituting cloned T cells for peripheral blood mononuclear cells in the feeder layer of a double-layer agar CFU-C assay system. Of 12 T cell lines tested, all produced CSF when stimulated by specific antigen, whereas CSF production in the absence of stimulation was generally negligible. In the case of soluble antigen-specific (ragweed or tetanus toxoid) clones, this required both nominal antigen and the appropriate MHC gene product on autologous antigen-presenting cells, whereas in the case of clones specific for EBV-transformed B cell lines (allogeneic or autologous), surface-bound EBV-related antigen and MHC was necessary. When tested in this manner, CSF production by different cloned T cells was heterogeneous in both amount and subclass. Thus, although most clones stimulated growth of granulocyte, monocyte, and eosinophil colonies, certain clones were identified which preferentially stimulated some colony types but not others. This heterogeneity was particularly evident with respect to eosinophil colony production. In addition, a soluble inhibitor of granulocyte colony growth was produced by one clone. These findings provide further support for the notion that antigen-specific T cells may, on activation, regulate myelopoiesis in a precise way, and provide a possible cellular basis for selective eosinophilia, monocytosis, or neutrophilia seen in certain disease states.

Antigens, Surface↗
PubMed1997

Analysis of T-cell clonality in bone marrow of patients with acquired aplastic anaemia.

Acquired aplastic anaemia (AA) represents a state of bone marrow (BM) failure which is characterized by BM hypocellularity and pancytopenia. It has been hypothesized that in some AA patients, bone marrow failure is secondary to the targeted destruction of haemopoietic stem cells by autoreactive T cells. The response of T cells to antigenic stimulation has been shown, in a number of animal models and in autoimmune diseases, to result in the (oligo)clonal expansion of positively reacting T cells. For this reason, we studied the utilization of 24 T-cell receptor-variable gene segments (TCRBV) and the clonality in BM aspirates and peripheral blood (PB) of seven AA patients. BM from transplant donors served as controls. Determination of TCRBV gene segment usage revealed no significant differences between patients and controls. Clonality within each family was analysed by single-strand conformation polymorphism (SSCP) analysis. Clonal and clonally predominant bands were seen in BM of three AA patients in five to eight TCRBV families. Clonal rearrangements were encountered less often in BM of control subjects. In conclusion, our results suggest an antigen-driven T-cell response in the BM of predominantly AA patients resulting in oligoclonal T-cell outgrowth.

Adolescent↗
PubMed2003

Resolution of complex feline leukocyte antigen DRB loci by reference strand-mediated conformational analysis (RSCA).

The DRB genes of the domestic cat are highly polymorphic. Studies based on clonal sequence analysis have suggested the existence of two distinct loci within individual animals and good evidence for 24 distinct FLA-DRB alleles. This variability, the complexity of clonal sequence analysis and its susceptibility to PCR-induced artefacts has represented a bottleneck to further progress. In this study we have applied reference strand-mediated conformational analysis (RSCA) to FLA-DRB. This protocol has been shown to be highly reproducible. Using five reference strands including two derived from non-domestic felines, we could distinguish 23 FLA-DRB alleles. We used RSCA to explore genetic polymorphism of FLA-DRB in 71 cats including 31 for which clonal sequence analysis was also available. On average, RSCA identified 0.9 more alleles within cats than clonal sequence analysis. Reference strand-mediated conformational analysis was also able to identify animals containing new alleles that could be targeted for sequence analysis. Analysis of allele patterns showed clear evidence for different allele distributions between breeds of cats, and suggested the Burmese breed may have highly restricted FLA-DRB polymorphism. Results from two families provided clear evidence for variation in the number of DRB genes on different haplotypes, with some haplotypes carrying two genes and some containing three. This study highlights the utility of RSCA for the resolution of complex amplicons containing up to six distinct alleles. A simple, rapid method for characterizing FLA-DRB makes possible studies on vaccine response and susceptibility/resistance to viral infections, which are a significant clinical problem in cats.

Alleles↗
PubMed1997

Clonal X-inactivation analysis of human tumours using the human androgen receptor gene (HUMARA) polymorphism: a non-radioactive and semiquantitative strategy applicable to fresh and archival tissue.

Assessment of clonality of cellular proliferations is important in experimental and clinical cancer research. X-chromosome inactivation studies are widely used to assess clonality, but most assays require relatively large amounts of high molecular weight DNA. Two PCR-based strategies, the phosphoglycerate kinase (PGK) and the human androgen receptor (HUMARA) clonality assays allow studies of small tissue samples. The HUMARA assay was adapted to non-radioactive analysis taking advantage of an automated sequencer providing high resolution of alleles and immediate quantitation. This assay was validated by comparison with X-inactivation patterns obtained by Southern analysis with the probes M27 beta and PGK. Fifteen gastrointestinal carcinomas, 25 benign goiter nodules and normal peripheral leukocytes of 27 individuals (12 who were under 15 years and 15 over 80 years) were analysed. Furthermore, DNA extracted from formalin-fixed paraffin-embedded tissue (FPT) was analysed with the two PCR-based methods and compared with X-inactivation patterns determined by Southern analysis of high molecular weight (HMW) DNA. This modified HUMARA assay is reliable in most patients; as with other clonality assays, constitutive skewing in normal tissue precludes clonal analysis in some individuals. Extremely skewed X-inactivation patterns were found in normal peripheral leukocytes of 7 out of 15 old females (over 80 years) and in 1 of 12 of the young females tested (under 15 years). Comparison of results obtained with HMW and FPT DNA yielded consistent results for the HUMARA assay whereas the PGK PCR assay was much less reliable. The HUMARA assay thus permits studies of selected areas of tissue sections without significant stromal components, allowing correlation of histological and genotype findings in fresh and archival specimens.

Adolescent↗
PubMed2002

Simultaneous analysis of T cell clonality and cytokine production in rheumatoid arthritis using three-colour flow cytometry.

In this study we examined the cytokine production by T cells and TCRVbeta subsets in peripheral blood (PB) and synovial fluid (SF) from six RA patients and PB from 10 normal subjects, using three-colour flow cytometry. In two RA subjects we assessed T cell clonality by RT PCR using TCRBV family-specific primers and analysed the CDR3 (complementarity determining region 3) length by GeneScan analysis. A high percentage of IFN-gamma- and IL-2- producing cells was observed among the PB T cells in both the RA patients and normal controls and among the SF T cells in RA patients. In contrast, the percentage of T cells producing IL-4 and IL-5 was small among PB T cells in both RA patients and normal controls and among SF T cells in RA patients. There was no significant difference in the production of IFN-gamma, IL-2 and IL-5 between the two compartments (PB and SF); however, there were significantly more IL-4-producing cells in SF. Molecular analysis revealed clonal expansions of four TCRBV families in SF of two of the RA patients studied: TCRBV6.7, TCRBV13.1 and TCRBV22 in one and TCRBV6.7, TCRBV21.3 and TCRBV22 in the second. These expansions demonstrated cytokine expression profiles that differed from total CD3+ cells, implying that T cell subsets bearing various TCR-Vbeta families may have the potential to modulate the immune response in RA patients.

Adult↗
PubMed1998

Clonality Studies in the Analysis of Adrenal Medullary Proliferations: Application Principles and Limitations.

Clonality remains as the hallmark of neoplasms. A dual genetic approach using markers nonrelated (e.g., X-chromosome inactivation assays) and related to the malignant transformation (such as loss of heterozygosity analyses of tumor-suppressor genes) would provide useful clonality information from early and advanced tumor stages, respectively. Tumor progression and clonal selection would result in genetic instability and heterogeneous expression of those molecular markers related to the malignant pathway. Therefore, only the coexistence of multiple genetic abnormalities would support the clonal nature as an expression of convergent cell selection. Considering those facts, the currently available evidence on tumorigenesis and clonality in the adrenal medulla can be summarized as follows: 1. Multistep tumorigenesis defines the evolution of pheochromocytomas, as evidenced by the presence of several genetic alterations. 2. Both the significant association of nonrandom genetic alterations (specially 1p and 22q interstitial deletions) and the topographic accumulation of genetic deletions at the peripheral tumor compartment support a convergent clone selection for these neoplasms. 3. Although many genetic loci show nonrandom abnormalities, the most frequently involved locates on chromosome 1p regardless of genetic tumor background (sporadic or inherited predisposition). 4. Most pheochromocytomas should begin as monoclonal proliferations that do not always correlate with histopathologic features, particularly in inherited tumor syndromes. 5. Early histopathologic stages, described as adrenal medullary hyperplasias, are defined by hyperproliferative features in animal models and monoclonal patterns in the adrenal nodules from patients with MEN-2a.

Journal Article↗
PubMed1998

Age-related persistent clonal expansions of CD28(-) cells: phenotypic and molecular TCR analysis reveals both CD4(+) and CD4(+)CD8(+) cells with identical CDR3 sequences.

In a small group of subjects we had identified persistent expansions (range 6-72%) of CD4(+)CD8(+) double-positive (DP) peripheral blood (PB) cells which express the CD8 alpha/alpha homodimer. Here, DP cells present in a larger cohort were further investigated and found by FACS analysis to express a single or a dominant TCRBV family. In these subjects, with a mean age of about 64 years, expansions of CD4(+) cells with the same TCRBV family specificity as in the respective DP cells also were consistently detected. TCR heterogeneity of the dominant TCRBV family was specifically evaluated: The amplified CDR3 region was cloned and found to consist of one single or two largely dominant sequence patterns. Furthermore, cloning of the CDR3 region from FACS-sorted DP, CD4(+), or CD8(+) cells indicates that both DP and CD4(+), but not CD8(+) cells, isolated from the same individual possess a striking identity of the CDR3 regions. As indicated by FACS analysis, the clonally expanded cells occur in the CD4(+)CD28(-) cells. Taken together, these results suggest that expanded CD4(+)CD28(-) cells might also acquire CD8 alpha/alpha expression and become DP and imply that CD4 clonality is a more frequent phenomenon than previously suspected. In conclusion, the persistent expansions described in this report represent a novel group of age-related benign clonal expansions of still undefined significance of a rare CD28(-) T cell subset.

Adult↗
PubMed1993

Clonal predominance of T cell receptors within the CD8+ CD45RO+ subset in normal human subjects.

Structural models for the TCR alpha/beta predict that the CDR1, CDR2, and CDR3 loops of both the alpha- and beta-chains contribute to specific interactions with the Ag/MHC complex. The CDR3 loops are constructed by joining events involving the V-(D)-J segments, and thus may vary in both sequence and length. We have developed a polymerase chain reaction assay to assess the length variation of the CDR3 loop in TCR derived from seven V beta segment families (V beta 2, V beta 3, V beta 4, V beta 9, V beta 14, V beta 16, and V beta 17). Peripheral blood T cells from 10 normal adults as well as five cord blood samples were studied. CD4+ and CD8+ T cells were analyzed separately. We observed extreme predominance of particular CDR3 lengths in half of the normal adults. These TCR were shown to be clonal by direct sequence analysis. This clonal dominance was found in the CD8+, CD45RO+ T cell population, and was observed in various V segment families. These patterns of TCR clonality were persistent over many months of observation in some individuals. In one subject, the new appearance of a predominant clone was associated with a booster vaccination for hepatitis B. These studies reveal a surprising degree of oligoclonality in the CD8+ cells of normal subjects which may be due to both environmental and genetic factors; the functional significance of persistent clonal dominance in the CD8 compartment remains to be determined.

Adult↗
PubMed1996

Molecular and biologic characterization of a newly established Philadelphia-positive acute lymphoblastic leukemia cell line (Z-33) with an autocrine response to GM-CSF.

We have recently established a new Philadelphia chromosome (Ph1)-positive acute lymphoblastic leukemia (ALL) cell line, designated Z-33. This line has L2 morphology, ultrastructural characteristics of lymphoblasts and typical B lineage surface markers identical to those observed in the Ph1-positive ALL patient from whom the line was derived. In addition, a rearranged immunoglobulin heavy-chain gene (JH) band was found in Z-33 cells by Southern blot analysis, confirming B cell clonality. Cytogenetic analysis of the cell line revealed t(9;22)(q34;q11.2). Polymerase chain reaction (PCR)-amplified cDNA from Z-33 cells demonstrated an e1-az BCR-ABL junction, and the p190BCR-ABL protein was detected in them by the immune complex kinase assay. Z-33 cells produce interleukin (IL)-1 beta, IL-6, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), tumor necrosis factor (TNF)-alpha, and transforming growth factor (TGF)-beta, Neither IL-1 beta, G-CSF, TNF-alpha, nor their corresponding antibodies affected the cell line's growth. In contrast, anti-GM-CSF neutralizing antibodies suppressed Z-33 colony formation, and GM-CSF stimulated it in a dose-dependent fashion. In addition, receptor studies with biotinylated GM-CSF demonstrated specific binding to Z-33 cells, indicating that the cells express GM-CSF receptors. Taken together, our data suggest that the Ph1-positive Z-33 ALL cells produce GM-CSF, express GM-CSF receptors, and show an autocrine proliferative response to this cytokine.

Antibodies↗
PubMed2004

Use of quantitative antibiogram analysis to determine the clonality of coagulase-negative Staphylococcus species from blood culture.

Two phenotypic methods, quantitative antibiogram analysis and colony morphology, were compared to pulsed-field gel electrophoresis (PFGE) for distinguishing the clonality of coagulase-negative Staphylococcus (CNS) species. The results of these three methods were correlated with the patients' clinical findings for 23 episodes in which CNS species were isolated from two blood culture bottles within a 24-h period. Quantitative antibiogram and colony morphology at 24 h correlated with PFGE typing in 21 (91%) and 20 (87%) episodes, respectively. All episodes associated with CNS strains with identical PFGE patterns had quantitative antibiogram similarity coefficients < 10, whereas most episodes associated with strains with different PFGE patterns had quantitative antibiogram similarity coefficients >or= 17. The CNS isolate pairs were less likely to be associated with infection if the strains had different PFGE types or a quantitative antibiogram similarity coefficient >or= 17. Clinical microbiology laboratories should consider use of the quantitative antibiogram similarity coefficient to aid clinicians in distinguishing infection-associated CNS blood isolates from contaminants.

Adolescent↗
PubMed2002

Stochastic patterns in globin gene expression are established prior to transcriptional activation and are clonally inherited.

We have undertaken a detailed characterization of mouse globin gene expression patterns in the nucleus and cytoplasm of single erythroid cells. We demonstrate an imbalance of alpha- versus beta-globin expression in a significant proportion of cells both in nuclear transcription patterns and cytoplasmic mRNA levels. Clonal cell analysis showed these expression patterns to be clonally inherited, while analysis of a multicopy transgenic locus showed an all-or-none effect in the activation of all the genes in one locus. These data provide strong evidence for a stochastic basis of globin gene activation resulting in heritable all-or-none expression patterns.

Animals↗
PubMed2000

[Detection of minimal residual diseases in B-cell tumors using PCR specific for the immunoglobulin heavy chain gene].

In B-cell non-Hodgkin's lymphomas (NHL), clonal rearrangement of the immunoglobulin heavy chain (IgH) gene provides a useful marker for the detection of minimal residual disease (MRD) after treatment. To explore clinical usefulness of polymerase chain reaction (PCR) analysis of clonal IgH gene rearrangement in the detection of MRD a follow up study of 10 patients with B-cell NHL have been performed. At the time of diagnosis, tumor DNAs were PCR-amplified using sense primer specific for the heavy chain variable region (VH) and antisense primer specific for the heavy chain joining region (JH) of the IgH gene. The clonal rearrangement of IgH gene detected by PCR was used as clonal marker to determine MRD after treatment. In three cases, where clinical remission was not achieved, clonal IgH gene rearrangement was detected after the treatment. In seven cases, clinical remission was achieved after induction therapy but the PCR analysis revealed clonal IgH gene rearrangement in three of the cases. In all of the three cases, where MRD was detected by PCR, clinical relapse developed after 7-28 months of the therapy. In all cases that have relapsed, the IgH gene rearrangement was identical at the time of initial diagnosis and at the relapse. This study demonstrates that PCR analysis of clonal IgH gene rearrangement is a useful method to monitor and detect MRD before clinical relapse.

Biomarkers, Tumor↗
PubMed1993

A novel clonality assay based on transcriptional analysis of the active X chromosome.

The clonal origin of malignancy and hematopoiesis is a principal tenet of modern biology and medicine. This paper describes a highly specific and sensitive assay for the detection of clonality in cells and cell lineages suitable for studies in a large proportion of females. The specific ligase chain and/or ligase detection reactions (LCR/LDR) are utilized at a polymorphic glucose-6-phosphate dehydrogenase (G-6-PD) locus for discrimination of the mRNA transcripts of the active X chromosome. This combination approach circumvents problems encountered with other currently used assays of clonality based either on peptide G-6-PD polymorphism or on DNA methylation differences between the active and inactive X chromosomes. The veracity of this assay was verified by analysis of 19 random healthy females as well as by the study of hemopoietic and nonhemopoietic tissues from a patient with clonal hemopoiesis/polycythemia vera. Furthermore, we demonstrate that the G-6-PD locus used in our clonal assay does not display marked differences in methylation between the active and the inactive X chromosomes.

Alleles↗
PubMed1988

Clonality of preneoplastic liver lesions: histological analysis in chimeric rats.

The clonality of chemically induced altered hepatocellular foci was examined in rat liver. Chimeric rats composed of two histologically distinguishable cell lineages were placed on an initiation-promotion protocol for liver cancer induction. This resulted in multiple lesions of altered enzyme expression. These altered hepatocellular foci are generally considered to be initiated sites susceptible to cancer formation. The cellular origins of these lesions were determined by aligning sections demonstrating cell lineage with serial sections stained for altered enzyme expression. Analysis included 110 areas of deficient ATPase (EC 3.6.1.3) activity and 59 glucose-6-phosphatase (EC 3.1.3.9; G-6-Pase) deficient lesions, 744 foci of re-expression of gamma-glutamyl transpeptidase (EC 2.3.2.2; gamma-GT), and decreased glycogen mobilization (187 lesions). Of the 1100 focal enzyme alterations, 1054 were shown to be composed entirely of cells from a single lineage of the two lineages present in the mosaic tissue. Multiple alterations occurred within given lesions. Lesions with up to four phenotypic alterations were found to consist of cells of a single lineage. These results suggest that individual enzyme-altered foci are clonal in origin and that phenotypic heterogeneity within altered hepatocellular foci is due to lesion progression within a clonal population and not to a multicellular derivation.

Animals↗
PubMed2002

Retrospective analysis of the first clonal outbreak of nalidixic acid-resistant Shigella sonnei shigellosis in Israel.

Reported here is a retrospective molecular analysis of the isolates recovered from the first outbreak of nalidixic acid (NA)-resistant Shigella sonnei shigellosis to occur in Israel. The outbreak affected 94 children. In the retrospective analysis, a total of 13 NA-resistant isolates and five NA-susceptible isolates recovered during the outbreak period were examined. Restriction fragment length polymorphism profiles obtained by digestion with BamHI, PvuI, HinfI or SmaI yielded identical profiles for all 18 isolates. All NA-resistant strains had an identical plasmid profile, but this profile differed from that displayed by the susceptible strains. In all of the NA-resistant strains a 304 bp fragment in the gyrA gene coding for a region associated with NA resistance was sequenced and showed a single point mutation, Ser83-->Phe. In this outbreak, the isolates of NA-resistant Shigella sonnei belonged to a single clone and NA resistance was associated with a point mutation in the gyrA gene.

Adolescent↗