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PubMed1981

A clonal analysis of the IgE response and its implications with regard to isotope commitment.

In a clonal analysis of the IgE response, it was found that a small proportion of primary or nonimmune B cells in spleen and mesenteric lymph nodes can be stimulated by antigen to produce IgE-secreting clones. In addition, there appears to be no substantial difference in the frequency of such cells between the classical low and high IgE responder strains. An analysis of immune, or memory, B cells revealed substantial increases in the frequency of B cells secreting IgE as compared with primary B cells, although the actual proportion of B cells secreting IgE remained relatively low. When the IgE-secreting clones derived from either primary or secondary B cells were reanalyzed for the presence of other isotypes, it was found that all clones secreting IgE were secreting at least one other isotype, with the majority secreting two or three other isotypes in addition to IgE. This demonstrates that there is no distinct subpopulation of B cells committed to IgE expression per se.

Animals↗
PubMed1979

[Clonal analysis of a transplantable rat seminoma].

The study of rat testis seminoma by the method of clonal analysis and ectopic transplantations has shown that typical and anaplastic types are different stages of tumor progression in the course of which atypical and anaplastic features of the seminoma cell elements increase. It does not seem inconceivable that the spermacytic form, never encountered during our investigation of 400 tumors, may be an independent seminoma form incapable of transforming into a typical one. A spindle-cell form of seminoma has been described most frequently occurring during transplantation of a typical seminoma into testis and during cloning. The obtained evidence permits to consider the small dark cells of the seminoma as being cambial cells of the tumor which occur abundantly in growing tumors, and are absent in tumors with ceased proloferating. At later stages of the seminoma development, the small dark cells may be absent, their function being performed practically by all tumor cells. Acinar structures found in part of clones from lungs and in the majority of transplants from eye anterior chamber suggest a histogenetic relationship between the typical seminoma and embryocarcinoma, and thus enable us to regard the latter as the most differentiated variants of the seminoma.

Anaplasia↗
PubMed2001

[Clonal analysis of the heterogeneity of neoplastic hepatocytes].

The heterogeneity of hepatoma MH-22a cell line was studied by clonal analysis on the basis of the following factors: splenocyte-induced apoptosis of tumor hepatocytes, tumor hepatocyte-induced apoptosis of splenocytes and profile of B1-associated DNA fragments. Induction of apoptosis of tumor hepatocytes by splenocytes and of splenocytes--by tumor hepatocytes was carried out in the main population of hepatocytes and in five clonal lines of hepatoma MH-22a. Genetic heterogeneity was studied using the same material and PCR primed for murine B1-elements. It was shown that apoptosis of hepatocytes of the main population and two clonal lines of hepatoma MH-22a was induced by splenocytes. The ability to induce apoptosis of splenocytes was observed in the same clonal lines and population of tumor hepatocytes. The latter involved enhanced genetic heterogeneity which was identified by B1-PCR analysis. Thus, a correlation was established in tumor hepatocytes between apoptosis-related characteristics and frequency of genome rearrangements.

Animals↗
PubMed1983

Direct demonstration of the clonogenic potential of every human peripheral blood T cell. Clonal analysis of HLA-DR expression and cytolytic activity.

In an attempt to determine the clonogenic properties of human peripheral blood T cells, we have developed a limiting dilution microculture system using phytohemagglutinin (PHA) as T cell activator and supernatant from PHA-stimulated spleen cultures as a source of T cell growth factors. The frequencies of cells capable of extensive proliferation under these culture conditions were 0.52-0.73, 0.98-1.11, and less than 0.02 in peripheral blood mononuclear, E-rosette-positive, and E-rosette-negative cell populations, respectively. The clonogenic potential of virtually all T cells was confirmed in experiments using single cells isolated by micromanipulation. Clone size ranged between 5 and 30 X 10(4) cells on day 14 of culture. The same microculture system was used to determine the precursor frequency of all cytolytic T lymphocytes (CTL-P). As assessed by a lectin-dependent 51Cr release assay, the CTL-P frequency in purified T cell populations ranged between 0.30 and 0.34. In comparison, the precursor frequency of T cells capable of lysing K562 target cells was ranging between 0.14 and 0.16. Parallel analysis of individual clonal cultures for both lytic activities showed that 50% of the clones exhibiting lectin-dependent lysis were also active against K562 target cells. All of the proliferating clones expressed HLA-DR antigens, although to a varying degree as assessed by flow cytofluorometry. Given the high cloning efficiency of this culture system, it appears now possible to determine the precursor frequencies of the various classes of functional cells in T cell populations.

Cells, Cultured↗
PubMed1990

Clonal analysis of joint fluid T lymphocytes in patients with juvenile rheumatoid arthritis.

Synovial fluid (SF) lymphocytes from 4 patients with pauciarticular juvenile rheumatoid arthritis (JRA) and 4 patients with polyarticular JRA were examined for their phenotypic and functional characteristics. In all 8 patients there was a high proportion of activated SF T cells, together with an increased proportion of CD2+CD3- and the presence of CD3+CD4-CD8-WT31- lymphocytes. The functional analysis at the clonal level in 5 patients (427 clones) showed a relevant proportion of cytotoxic T cell clones, which were not confined to typically cytolytic phenotypes, but were also present among CD3+CD4+CD8- cultures. Compared to those with pauciarticular JRA, patients with polyarticular disease had a significantly higher proportion of T cell clones with cytotoxic activity. Although derived from a limited number of patients, our data suggest a direct involvement of T cells in the pathogenetic mechanisms that originate and maintain the articular damage, and the possibility of different or more pronounced T cell reactivities in the clinically more diffuse JRA types.

Adolescent↗
PubMed1997

Clonality analysis of hematopoiesis in essential thrombocythemia: advantages of studying T lymphocytes and platelets.

Essential thrombocythemia (ET) is a myeloproliferative disorder characterized by a sustained elevation of the platelet count in the absence of other causes of thrombocytosis. ET is difficult to diagnose, and the demonstration of clonal hematopoiesis may be of value. However, clonality analysis of hematopoietic cells based on the study of the X-chromosome inactivation pattern is complicated by the observation that some normal females present skewed lyonization. Moreover, DNA methylation of X-linked genes in hematopoietic cells may differ from that in other tissues. Appropriate controls for skewed lyonization are therefore critical for the study of clonality. We developed two techniques based on X-chromosome inactivation and polymerase chain reaction (PCR) analysis of polymorphisms, to study clonality in ET patients. Reverse transcriptase-PCR analysis of IDS, P55, and G6PD mRNAs was used to examine the different hematopoietic cell lineages including platelets in patients heterozygous for these polymorphisms and analysis of the HUMARA gene methylation pattern permitted us to study clonality in all nucleated cell fractions of the other patients. Using both types of assay and T lymphocytes as a control tissue for lyonization, clonal hematopoiesis was demonstrated in 28 patients. In 14 patients, the granulocytes were polyclonal; among these patients, platelets were monoclonal in 3 cases, polyclonal in 7 cases, and in the remaining 4 cases this fraction could not be studied because the patients were homozygotes for all RNA markers. No conclusion about clonality could be drawn in 6 cases. Polyclonal hematopoiesis was found in all the cases of reactive thrombocytosis. These findings confirm the high frequency of monoclonal hematopoiesis in ET, the utility of studying platelets, and the possibility of using T lymphocytes as a control tissues for X-chromosome inactivation patterns.

Adolescent↗
PubMed1995

Clonal analysis of haemopoietic cells in essential thrombocythaemia.

Essential thrombocythaemia (ET) is a myeloproliferative disease (MPD) predominantly involving the platelet lineage, with a diagnosis often difficult to establish in practice. The demonstration of a clonal haemopoiesis can contribute to diagnosis. The clonal origin of blood cells can be assessed in female patients using X-chromosome-linked polymorphisms, assuming a random inactivation of the X chromosome. The human androgen receptor gene HUMARA has been used for this purpose, taking advantage of a highly polymorphic trinucleotide repeat in the first exon. The close proximity of the polymorphic trinucleotide repeat to four methylation sites permits a clonal analysis based on the polymerase chain reaction. We have used this technique to study the clonality of haemopoietic cells in 32 patients with ET and 23 age-matched control heterozygotes for the HUMARA polymorphism. A monoclonal pattern of haemopoiesis was detected in unfractionated nucleated blood cells from 22 patients, suggesting that haemopoiesis is essentially monoclonal in a majority of cases in this disease. In some patients a monoclonality of granulocytes was documented, whereas the mononuclear fraction had a polyclonal pattern of X-inactivation. Finally, in two patients for whom a polyclonality had been found in unfractionated blood, analysis of G6PD transcripts in platelets revealed a clonal megakaryocytopoiesis. These findings confirm the heterogeneity of haematological abnormalities in ET patients and the potential utility of a multifaceted laboratory approach to investigate these patients.

Adult↗
PubMed1991

In vitro clonal analysis of quail cardiac neural crest development.

The developmental potentials of cardiac neural crest cells were investigated by in vitro clonal analysis. Five morphologically distinct types of clones were observed: (1) "pigmented" clones contained melanocytes only; (2) "mixed" clones consisted of pigmented and unpigmented cells; (3) "unpigmented dense" clones consisted of flattened, closely aligned unpigmented cells; (4) "unpigmented loose" clones consisted of a few loosely arranged, flattened cells; and (5) "unpigmented large" clones included a large number of small, stellate cells that were highly proliferative. The binding patterns of antibodies against lineage-specific markers showed that cells in the different clones expressed characteristic phenotypes. The following phenotypes were expressed in addition to pigment cells: smooth muscle cells, connective tissue cells, chondrocytes, and cells in the sensory neuron lineage. Mixed clones expressed all five phenotypes. Unpigmented dense clones contained smooth muscle cells, connective tissue cells, chondrocytes, and sensory neurons. Unpigmented loose clones exclusively consisted of smooth muscle cells, whereas unpigmented large clones contained chondrocytes and sensory neuron precursors. Based on these results, the following conclusions can be drawn: (1) Pigmented and unpigmented loose clones are most likely formed by precursors that are committed to the melanogenic and myogenic cell lineages, respectively. (2) Mixed and unpigmented dense clones are derived from pluripotent cells with the capacity to give rise to four or five phenotypes. (3) Unpigmented large clones originate from progenitor cells that appear to have a partially restricted developmental potential, that is, these cells are capable of generating two phenotypes in clonal cultures. Thus, the data indicate that the early migratory cardiac neural crest is a heterogeneous population of cells, consisting of pluripotent cells, cells with a partially restricted developmental potential, and cells committed to a particular cell lineage.

Animals↗
PubMed1998

Establishment of a novel B cell clonality analysis using single-strand conformation polymorphism of immunoglobulin light chain messenger signals.

The remarkable diversity of the complementarity determining region (CDR) 3 of the immunoglobulin (Ig) heavy (H) chain gene rearrangements has been exploited to identify the clonal populations of B cells in B cell malignancies. However, when B cell malignancies of different categories were examined, the overall detection rate was found to be approximately 70%. The development of a simple clonality analysis using Ig light (L) chain CDR3 diversity has been hampered due to the sparseness of knowledge regarding the sequence of Vkappa and Vlambda gene segments and the restriction of L chain CDR3 length. Based on the recently reported Vkappa and Vlambda gene sequences, we designed Vkappa and Vlambda framework 3 consensus primers. We combined the reverse transcriptase polymerase chain reaction (RT-PCR) of IgL chain transcripts with a single-strand conformation polymorphism (SSCP) analysis and then analyzed samples from patients with B cell malignancies. Clonal B cell populations were detected as discrete bands, and identical clones showed a similar mobility in a RT-PCR SSCP analysis. This method was thus found to be a useful supplement to the previously described approach of VH gene amplification for detecting clonal B cell populations. By using SSCP, we were able to determine the clonal identities of B cell expansion in different samples.

Arthritis, Rheumatoid↗
PubMed1997

Arbitrarily primed polymerase chain reaction fingerprinting and clonal analysis of oral Fusobacterium nucleatum isolates.

F. nucleatum is the most commonly isolated microorganism from subgingival plaque, but the role of this microorganism in periodontal diseases remains undefined. Arbitrarily primed polymerase chain reaction (AP-PCR) was evaluated as a method for fingerprinting F. nucleatum isolates and for use in clonal analysis. Pulsed field gel electrophoresis was used to further differentiate F. nucleatum isolates, with identical AP-PCR patterns. Extremely heterogeneous AP-PCR fingerprints were observed among the 98 F. nucleatum isolates, with 36 different genotypes observed with primer C1 and 30 different genotype detected with primer C2. Combining the results of the AP-PCR genotype analysis from C1 and C2 primer amplifications revealed that up to 7 different genotypes could be distinguished from isolates from the same oral cavity and that up to 4 different genotypes were observed within a single site. An intense amplicon at approximately 450 bp generated in AP-PCR amplification with primer C2 was associated with F. nucleatum subsp. nucleatum (ATCC 25586) and with 15 F. nucleatum isolates from diseased sites and 2 isolates from healthy sites. Pulsed field gel electrophoresis confirmed the AP-PCR genotypes and demonstrated increased discriminatory power over AP-PCR. The results indicated that AP-PCR and pulsed field gel electrophoresis provide a simple and sensitive means for differentiating oral F. nucleatum isolates and further demonstrate the heterogeneity of this species. These techniques may serve as useful tools in the clonal and epidemiological analysis of F. nucleatum isolates, which may help define the role of these microorganisms in periodontal diseases.

Bacterial Typing Techniques↗
PubMed1990

Clonal analysis in the ultrastructure of cell-to-cell interaction between a human glioma cell line and autologous tumor-specific cytotoxic T lymphocytes.

The clonal analysis in the ultrastructure of tumor-lymphocyte interaction was carried out in order to investigate the precise mechanism responsible for CTL-mediated cytolysis of tumor cells. A glioma-derived cell line (GI-1) and autologous tumor-specific cytotoxic T lymphocyte (CTL) clones were established. The CTL lines were composed of the morphologically homogeneous lymphocytes with intracytoplasmic electron-dense secretory granules. After the stimulation by GI-1, the size of the CTLs increased, and the intracytoplasmic organellas were developed. It was noted that the intracytoplasmic secretory granules markedly increased in number and size, and many of them exhibited an "immature" appearance. On the other hand, the tumor cells underwent a progressive degeneration. In contrast, the stimulation by other antigens caused only small morphological changes in the CTLs. It is suggested, therefore, that the secretory function of tumor-specific CTLs is activated by the stimulation of the specific antigen, and that soluble factors in the secretory granules in the CTLs may be closely associated with the mechanism of target cell lysis.

Cell Communication↗
PubMed1996

Clonal analysis of meningiomas.

Meningiomas are primary brain tumors arising from meningothelial cells. They usually grow slowly and are surgically easy to separate from the brain. A recent clonal analysis of meningiomas, using methylation-sensitive restriction fragment length polymorphisms, suggested a monoclonal origin. Using the same technique but with a highly informative X chromosome probe (M27 beta), we found that 17 (85%) of the 20 meningiomas analyzed were informative. Of the 17 informative tumors, 8 (47%) were monoclonal, 3 (18%) had loss of heterozygosity on the X chromosome, and, unexpectedly, 6 (35%) had a polyclonal pattern. Samples from two areas of one tumor showed a monoclonal pattern and loss of heterozygosity, respectively, on the X chromosome. A review of the histopathological and radiological features of the 17 informative tumors did not help to distinguish the clonal from the polyclonal tumors. We conclude that meningiomas are heterogeneous in clonal composition.

Adult↗
PubMed1987

Clonal analysis using recombinant DNA probes from the X-chromosome.

It has been demonstrated that restriction fragment length polymorphisms of X-chromosome genes can be used in conjunction with methylation patterns to determine the clonal composition of human tumors. In this report, we show that several X-chromosome probes can be used for such analyses. In particular, probes derived from the hypoxanthine phosphoribosyltransferase gene and the phosphoglycerate kinase gene could be used for clonal analysis in over 50% of American females. The X-inactivation patterns observed with these probes were found to accurately reflect clonality in more than 95% of 92 tumors tested.

Alleles↗
PubMed1996

Clonal analysis of focal nodular hyperplasia of the liver.

Recent evidence suggests that focal nodular hyperplasia of the liver (FNH) may represent a hyperplastic response to a vascular malformation, but the precise etiology remains unclear. We performed a clonal analysis of ten FNHs from nine patients by patterns of X chromosome inactivation. DNA isolated from paraffin-embedded specimens was subjected to polymerase chain reaction amplification for a highly polymorphic region of the human androgen receptor gene (HUMARA). Predigestion of tumor DNA with the methylation-sensitive, restriction enzyme HpaII allowed for selective amplification of the methylated (inactivated) allele. Of the nine patients analyzed, seven were heterozygous for the HUMARA polymorphism and informative for analysis. One informative patient had two lesions, for a total of eight FNHS. Amplification of lesional DNA after HpaII digestion demonstrated clonality in six of the eight informative cases. Paired tissue samples from different lesional areas were available in four of the six FNHs with evidence of clonality. In three of the four cases, DNA extracted from the two tissue samples showed both evidence of clonality and an identical pattern of X chromosome inactivation. In the remaining case, one sample showed evidence of clonality whereas the other was nonclonal. Three hepatic adenomas from two informative patients were also analyzed for comparative purposes, all of which showed evidence of clonality after HpaII digestion. The current study illustrates that most cases of FNH show a uniform pattern of X chromosome inactivation consistent with clonality.

Adenoma↗
PubMed1985

A clonal analysis of the requirement for the trithorax gene in the diversification of segments in Drosophila.

The requirement for the trithorax gene during imaginal cell proliferation has been analysed by clonal analysis. Clones carrying the allelic Regulator of bithorax and induced by mitotic recombination at different developmental stages. The results indicate that the trithorax gene is required at least until the beginning of the third larval instar to ensure the correct differentiation of head, thoracic, and abdominal structures, and are consistent with a role for the gene in maintaining the appropriate levels of Antennapedia and bithorax complex expression.

Abdomen↗
PubMed1993

Phenotypic and clonal analysis of T lymphocytes in childhood immune thrombocytopenic purpura.

In an attempt to identify and characterize T-lymphocyte immunoregulatory abnormalities in immune thrombocytopenic purpura (ITP), we have performed phenotypic and clonal analysis on peripheral T lymphocytes from 23 children with ITP. Quantitation of lymphocyte subpopulations showed that children with acute ITP had higher numbers of CD45RA+ and lower numbers of CD45RO+ T cells than children with chronic ITP or controls, but these differences may be age related. Analysis of T-cell receptor variable beta gene usage identified 2 boys with chronic ITP and elevated numbers of V beta 8+ T cells. Eight T-cell clones were established (6 CD4+, 4B4+ helper-inducer lines and 2 CD8+ lines) that showed in vitro proliferation against allogeneic platelets. The addition of autologous antigen-presenting cells enhanced the proliferation of six clones, but not for two clones that coexpressed natural killer (NK) markers. Four of seven positive clones also had measurable interleukin (IL)-2 secretion following platelet stimulation, providing further evidence for T-cell reactivity. Our results provide the first evidence that patients with ITP may have platelet-reactive T lymphocytes identifiable at the clonal level, supporting the hypothesis that autoreactive peripheral T lymphocytes may mediate or participate in the pathogenesis of this disorder.

Acute Disease↗
PubMed1999

Clonal analysis of adenosquamous carcinoma of the lung.

Adenosquamous carcinoma of the lung is a subset of pulmonary carcinomas, and comprises less than 4% of lung carcinomas. Its histogenesis remains unclear. The clonality of adenosquamous carcinoma from four female patients was analyzed to determine whether the clonality between the squamous cell and adenocarcinomatous components coincides. Each lesion was precisely microdissected from methanol-fixed sections. Adjacent normal lung tissue was collected as a normal control. DNA was extracted for clonal analysis based on an X-chromosome-linked polymorphic marker, the human androgen receptor gene (HUMARA). HUMARA was found to be amplified with or without previous digestion by the methylation-sensitive restriction endonuclease HpaII. All four cases were informative. Squamous cell and adenocarcinomatous components showed identical monoclonal patterns in all four patients. In one case, only the squamous cell carcinomatous component showed loss of heterozygosity of the HUMARA locus. The results suggest that the squamous cell and adenocarcinomatous components originate from the same cell.

Aged↗
PubMed1990

Clonal analysis of chronic myeloproliferative disorders using X-linked DNA polymorphisms.

Restriction fragment length polymorphisms of the X-chromosome genes phosphoglycerate kinase and hypoxanthine phosphoribosyl transferase were used to study clonality in peripheral blood leukocytes from 48 women with chronic myeloproliferative disorders (c-MPD). A total of 50% of patients were heterozygous for one or both of the polymorphic loci. These included 17 cases with polycythemia vera, four patients with essential thrombocythemia (ET), and three cases with idiopathic myelofibrosis (IMF). A clear-cut monoclonal X-inactivation pattern was observed in 17 of 24 cases including all IMF patients. Only one patient with PV exhibited a nonclonal composition of her leukocytes, while six cases demonstrated a predominantly clonal pattern in peripheral blood cells. Among the latter category reckoned three of four ET patients. Cell separation analyses were performed in one ET and three PV patients. In all four cases a monoclonal pattern of the granulocyte fraction could be established, while T lymphocytes of these patients were of nonclonal origin. These data suggest that the vast majority of c-MPDs arise from multipotent hematopoietic stem cells. Moreover, this type of clonal analysis might be of help in discriminating between primary MPD and reactive processes.

Adult↗