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PubMed1990

Clonal analysis of a human antibody response. Quantitation of precursors of antibody-producing cells and generation and characterization of monoclonal IgM, IgG, and IgA to rabies virus.

We quantitated and characterized the changes in the human B cell repertoire, at the clonal level, before and after immunization with rabies virus. Moreover, we generated 10 monoclonal cell lines producing IgM, IgG, and IgA antibodies to the virus. We found that in healthy subjects, not previously exposed to the virus, nearly 2% of the circulating B lymphocytes were committed to the production of antibodies that bound the virus. These B cells expressed the surface CD5 molecule. The antibodies they produced were polyreactive IgM that displayed a relatively low affinity for the virus components (Kd, 1.0-2.4 x 10(-6) g/microliters). After immunization, different anti-virus (IgG and IgA) antibody-producing cells consistently appeared in the circulation and increased from less than 0.005% to greater than 10% of the total B cells committed to the production of IgG and IgA, respectively. Most of such B cells do not express CD5 and produce monoreactive antibodies of high affinity for rabies virus (Kd, 6.5 x 10(-9) to 1.2 x 10(-10) g/microliters). One of these IgG mAbs efficiently neutralized rabies virus in vitro and in vivo, as detailed elsewhere (Dietzschold, B., P. Casali, Y. Ueki, M. Gore, C. E. Rupprecht, A. L. Notkins, and H. Koprowski, manuscript submitted for publication). Hybridization experiments using probes specific for the different human V gene segment families revealed that cell precursors producing low affinity IgM binding to rabies virus utilized a restricted number of VH gene segments (i.e., only members of the VHIIIb subfamily), whereas cell precursors producing high affinity IgG and IgA to rabies virus utilized an assortment of different VH gene segments (i.e., members of the VHI, VHIII, VHIV, and VHVI families and VHIIIb subfamily). In conclusion, our studies show that EBV transformation in conjunction with limiting dilution technology and somatic cell hybridization techniques are useful methods for quantitating, at the B cell clonal level, the human antibody response to foreign Ags and for generating human mAbs of predetermined specificity and high affinity.

Antibodies, Monoclonal↗
PubMed1990

A clonal analysis of glial lineages in neonatal forebrain development in vitro.

Retrovirus-mediated gene transfer combined with triple immunostaining for astro- and oligodendroglial markers (antibodies to glial fibrillary acidic protein, GD3 ganglioside, and galactocerebroside, and the O4 antibody) was used to study clonal aspects of glial lineage in primary cultures of the neonatal rat striatum. We found two major clonal populations: astrocyte clones containing GFAP+, but GD3-, O4-, and GC- cells, and oligodendrocyte clones containing cells expressing various combinations of GD3, O4, and GC, with rare GFAP+ cells. These results indicate that astrocytes and oligodendrocytes belong to separate lineages in forebrain postnatal development.

Animals↗
PubMed1999

Clonal analysis of a solitary follicular nodule of the thyroid with the polymerase chain reaction method.

Solitary follicular nodules of the thyroid occasionally create a diagnostic problem, especially in the differential diagnosis between adenoma and nodular hyperplasia To obtain confident histologic parameters of clonal lesions, we analyzed DNA samples prepared from paraffin-embedded archival tissue from 20 solitary follicular nodules of the thyroid for clonality with the polymerase chain reaction (PCR) method. On the base of X chromosome inactivation mosaicism, we tested restriction fragment-length polymorphism of the phosphoglycerate kinase (PGK) gene and a highly polymorphic short tandem repeat of the human androgen receptor (HUMARA) gene. Of 18 informative cases, 10 were monoclonal, 7 were polyclonal, and 1 showed microsatellite instability. All of the five completely encapsulated nodules were monoclonal. Four of the five unencapsulated nodules showed polyclonality. Of the seven partially encapsulated nodules, four were monoclonal, and the others were polyclonal. The former showed 50% or more of encapsulation degree, whereas the latter showed less than 50%. The capsule tended to be thicker in monoclonal nodules (mean, 0.33 mm) than in polyclonal nodules (mean, 0.13 mm). Other histologic features of the nodules and surrounding parenchymal changes had no significance with respect to predicting clonality. This study suggests that the degree of encapsulation and capsular thickness are morphologically important for predicting the clonality of the thyroid nodule.

Adult↗
PubMed1991

Clonal analysis of sucrase-isomaltase expression in the human colon adenocarcinoma Caco-2 cells.

To investigate the biosynthetic basis for the mosaic expression of brush border enzymes in confluent Caco-2 cells, a human colon carcinoma cell line exhibiting characteristics of adult small intestinal enterocytes, we have obtained a series of clones differing markedly in their growth rates, amounts of transforming growth factor-alpha/epidermal growth factor-like activity released into the culture medium, and sucrase-isomaltase (SI) activity. Other intestinal markers (aminopeptidase N, dipeptidylpeptidase IV, lactase, alkaline phosphatase and 'crypt cell antigen') displayed a much more limited variability in expression, suggesting that the Caco-2 cell clones we have obtained did not differ in their overall ability to differentiate. Immunofluorescence staining, metabolic labelling with radioactive methionine and hybridization analysis of SI mRNA abundance were used to investigate SI synthesis and its regulation in clones endowed with low, intermediate or high sucrase activity. The results obtained have demonstrated heterogeneous SI expression, even in clonal cell lines, and a negative correlation between SI expression and growth factor concentrations in the culture medium, suggesting an autocrine regulation of cell proliferation and differentiation in confluent Caco-2 cells. Pulse-chase experiments using the two clones endowed with the lowest and highest levels of SI activity, followed by immunoprecipitation of labelled SI with epitope-specific antibodies and SDS/PAGE analysis, suggested that both transcriptional and post-translational mechanisms play a role in the regulation of SI expression in intestinal cells.

Adenocarcinoma↗
PubMed1989

Temporal study of staphylococcal species on the skin of human subjects in isolation and clonal analysis of Staphylococcus capitis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

The staphylococcal skin floras of an isolated group of subjects were studied for 1 year. A wide variation in isolation patterns was found for different species. Staphylococcus intermedius, previously thought to be of veterinary origin, was found to be part of the resident flora of some subjects, and this may indicate a wider role for it in clinical infection. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of S. capitis isolates indicated persistent skin colonization at some sites; each region was colonized by only one clone of that species, although an adjacent area could be inhabited by a separate clone. Nine clonal groups were identified by SDS-PAGE; there was a degree of specialization between the groups with regard to the sites which they colonized. The interaction between species at a single site was less well defined. Noncolonizing isolates often exhibited phenotypic similarities that were lower than expected when compared with their presumed source. The results of SDS-PAGE analysis were compared with immunoblotting, antibiograms, and biotyping, and SDS-PAGE analysis was found to be a useful and practical tool for epidemiological work.

Adult↗
PubMed1998

Clonal analysis of superficial depressed-type gastric carcinoma in humans.

BACKGROUND: An important unanswered question concerning the histogenesis of superficial-type gastric carcinoma is whether it is monoclonal or multiclonal in origin. Therefore, the authors analyzed multiple areas of each cancer with a clonality assay based on trinucleotide repeat length polymorphism of the human androgen receptor gene (HUMARA) that was subject to random inactivation of X chromosomes. METHODS: The HUMARA assay was applied to 15 gastric carcinomas, early and advanced stage, manifested in superficial, depressed lesions of various sizes and at least some signet ring cells. DNA was extracted from fresh frozen and formalin fixed tumor tissues that were microdissected from the mucosal lesions, and the HUMARA locus was amplified by polymerase chain reaction with and without prior digestion of nonmethylated DNA with Hpa II. The amplified DNA samples were loaded on polyacrylamide gels, electrophoresed, and visualized by a silver-staining method. RESULTS: In the 15 cases examined, 9 cancers were informative (had features of the types sought in this study), and in these 9 cancers a total of 57 areas were analyzed. In 7 of the 9 cancers, the inactivated allele was common to all the informative areas of each tumor, irrespective of the macroscopic shape of the tumor or the degree of histologic heterogeneity within it. In one of the two remaining cancers, the inactivated allele of one of the areas examined was different from those in the other areas. CONCLUSIONS: Most of the superficial depressed-type gastric carcinomas in this study were demonstrated to be of monoclonal origin. This finding supports a notion expressed previously in the literature that superficial-type carcinoma has a long natural history, and it indicates that efforts to detect gastric carcinomas in early stages to improve patients' survival should be encouraged.

Carcinoma↗
PubMed1995

Clonal analysis of colorectal tumors using K-ras and p53 gene mutations as markers.

Mutations to the K-ras oncogene and p53 tumor suppressor gene are two of the most common genetic lesions in human cancers. In the present study we examined the clonality of colorectal tumors with respect to each of these genetic alterations. Screening for mutations was carried out using the polymerase chain reaction-based technique of single-strand conformation polymorphism. Eleven primary colorectal adenocarcinomas and two secondary adenocarcinomas were analyzed at four different sites within the tumor. Involved pericolic lymph nodes were collected from nine of these cases, a metastatic deposit in the liver was obtained in one case, and adjacent adenomatous lesions were collected in two cases. Seven tumors contained mutations in either the K-ras or p53 genes. In all cases, DNA derived from multiple sites within an individual tumor or metastatic deposits arising from that tumor showed the same pattern of gene mutation. Immunohistochemical staining for p53 protein overexpression also showed similar patterns of reactivity within individual tumors and their metastatic deposits. These results suggest that the major clonal expansion of colorectal carcinomas occurs after the acquisition of mutations in these genes. Our results also indicate that sampling errors are unlikely to occur in molecular studies aimed at defining the role of these genes in colorectal cancer progression.

Carcinoma↗
PubMed2001

Clonal analysis of esophageal squamous cell carcinoma with intraepithelial components.

OBJECTIVE: In tumors of the upper aerodigestive tract, field carcinogenesis is a prevailing concept which suggests that such tumors are commonly of multiclonal origin. METHODS: To test this possibility, we applied a PCR-based clonality assay utilizing the polymorphic locus of the human androgen receptor gene (HUMARA) in female patients with esophageal squamous cell carcinomas (SCCs). DNA was extracted from small pieces of tissues microdissected from multiple points of intraepithelial and invasive parts of each tumor and the adjacent epithelia in 12 cases. The HUMARA locus was PCR amplified with or without prior digestion with Hpa II. PCR products were analyzed by a genetic analyzer and polyacrylamide gel electrophoresis followed by silver staining. RESULTS: In each of 8 informative cases, the pattern of X chromosome inactivation of the major cell population in each sample was common among the samples from the invasive part and among those samples, if any, from the intraepithelial part, and was concordant between the intraepithelial and invasive parts in 5 cases and discordant in 1 case. Out of the samples of adjacent epithelia, a monoclonal pattern was demonstrated in 8 basal cell hyperplasias and 3 dysplasias, of which 2 and 1, respectively, showed inactivation patterns discordant with those of the concomitant cancers. CONCLUSION: Esophageal SCCs may often be preceded or accompanied by multiclonal precancerous lesions, and may develop through the outgrowth of single or less commonly multiple dominant clones.

Aged↗
PubMed1991

Clonal analysis of the cytotoxic T-lymphocyte response to reovirus.

Reovirus has previously been classified into three serotypes based on hemagglutination inhibition assays. In the present study, the specificity of cytotoxic T lymphocytes (CTL) generated in reovirus type 3-infected C3H/HeN mice was investigated at the population and clonal levels. Short-term CTL lines generated in response to reovirus type 3 preferentially lysed cells infected with reovirus type 1 or 3 and, in some instances, type 2-infected cells as well. Eleven CTL clones established from the lines demonstrated two unique patterns of recognition. A single clone was exquisitely specific for reovirus type 3-infected cells and did not cross-react on reovirus type 1- or 2-infected cells. Ten of the clones recognized reovirus type 1- and type 3-infected cells. These clones had low levels of cross-reactivity on reovirus type 2-infected cells that was revealed only at high effector:target cell ratios. Precursor frequency analysis further revealed that the majority of the CTL generated against reovirus type 3 could cross-react on both reovirus type 1- and type 2-infected cells. Some CTL could be detected that had a more restricted pattern of recognition and recognized reovirus type 3-infected cells exclusively or recognized reovirus type 3-infected cells and either reovirus type 1- or type 2-infected cells. These results indicate that a minimum of four epitopes are recognized by reovirus-specific CTL and that the response is dominated by CTL that recognize an epitope common to all three serotypes of reovirus.

Animals↗
PubMed1985

Clonal analysis of the involvement of T11 molecules in antigen receptor-mediated T lymphocyte functions.

In this study, we utilized two anti-T11 monoclonal antibodies (mAbs) that inhibited the specific cytolytic activity of mixed lymphocyte culture (MLC) activated T cells to analyze, at the clonal level, the involvement of T11 molecules in 1) antigen specific versus non-specific mechanisms of target cell lysis 2) antigen-driven T cell proliferation and IL-2 production versus IL-2-induced cell proliferation. In contrast to anti-T3 or anti-T8 mAbs, antibodies to T11 molecules inhibited the cytolytic activity of MLC-derived allospecific clones in a uniform manner. In addition, anti-T11 antibodies inhibited the specific activity of CTL clones resistant to anti-T3 antibodies, even after antibody-induced modulation of T3 molecules (while anti-T3 mAbs had no effect). Similarly, anti-T11 antibodies inhibited the alloantigen induced proliferation and IL-2 release of alloreactive clones independent on their T4+ or T8+ phenotype. The inhibitory activity of anti-T11 antibodies appears to be confined to antigen-specific T cell functions since neither natural killer-like activity of CTL clones nor the IL-2 induced clonal proliferation was affected. Thus, our results indicate that T11 molecules are functionally involved in antigen recognition by T cell regardless of their function and T4/T8 phenotype.

Antibodies, Monoclonal↗
PubMedDate not supplied

Clonal analysis of sacrococcygeal "teratomas".

Congenital masses of the sacrococcygeal region commonly contain multiple tissues and have variously been subclassified as neoplasms or congenital hamartomas based on clinicopathological and embryological observations. We have used a polymerase chain reaction-based assay for nonrandom X chromosome inactivation to infer the clonality of three cogenital sacrococcygeal tumors previously diagnosed as teratomas. One solid immature teratoma was monoclonal, and a predominantly cystic histologically mature mass was polyclonal. A third immature teratoma was noninformative because of baseline asymmetry of polyclonal tissue X inactivation. We confirm that immature teratomas at this site appear to be monoclonal neoplasms and suggest that at least some histologically mature "teratomas" are more appropriately classified as hamartomas.

Cloning, Molecular↗
PubMed1992

Clonal analysis of cardiac morphogenesis in the chicken embryo using a replication-defective retrovirus. III: Polyclonal origin of adjacent ventricular myocytes.

Replication-incompetent variants of the avian spleen necrosis virus (SNV) encoding cytoplasmic or nuclear-directed beta-galactosidase (beta-gal) have been used to trace the clonal growth of myocytes during left ventricular free-wall formation. Tubular-stage hearts were infected with a mixed suspension of both retroviruses and, after hatching, the progeny of marked cells in the ventricular wall were examined by X-gal histochemistry. When a small number of virions was introduced individual blue patches contained myocytes with only one label type (cytoplasmic or nuclear). These results confirmed our previous conclusion that each cluster or patch represents a single clone (Mikawa et al., 1992, Dev. Dynamics, 193:11-23). Each of these clones formed a clone-shaped patch which often extended through the entire thickness of the ventricular myocardium, but typically each patch was heterogeneous, containing a mixture of labeled and unlabeled cells. We then asked whether the two populations of myocytes in each patch were clonally related or generated from more than one progenitor. When hearts were infected with high titer viral suspensions many patches were observed in which cytoplasmic-tagged myocytes were intermingled with nuclear-tagged myocytes. Thus, the cone-shaped myocyte patches in the ventricular wall are polyclones derived from separate progenitors in the precardiac mesoderm. This finding led us to examine the separation of clonally related ventricular myocytes in the developing hearts. Embryos were infected with retroviral suspensions at varying stages of development and the resulting colonies examined after hatching.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Clonality analysis of defined B-cell populations in archival tissue sections using microdissection and the polymerase chain reaction.

A simple microdissection technique involving the use of a drawn-out glass pipette was developed for isolation of defined cell subsets from tissue sections. Using this technique and the polymerase chain reaction (PCR), clonally rearranged immunoglobulin (Ig) heavy chain genes were reliably amplified in single neoplastic follicles or few hundreds of tumour cells isolated from archival haematoxylin and eosin or immunostained sections of B-cell lymphomas. A polyclonal nature was consistently demonstrated in reactive lymphoid follicles or interfollicular reactive B-cells within the same lymphoma sections. Microdissection of lymphoma cells from within foci of chronic inflammation improved the resolution of tumour-specific PCR products by reducing amplification of background polyclonal B-cell sequences. The combination of microdissection and PCR techniques, therefore, provides an important tool for the investigation of B-cell lymphomas and also allows simple and specific access for other molecular genetic analyses of different cell subsets on tissue sections.

B-Lymphocytes↗
PubMed2006

Wotherspoon criteria combined with B cell clonality analysis by advanced polymerase chain reaction technology discriminates covert gastric marginal zone lymphoma from chronic gastritis.

BACKGROUND AND AIMS: Gastric mucosa associated lymphoid tissue lymphoma is a well defined B cell lymphoma yet often impossible to distinguish from severe chronic gastritis on morphological grounds alone. Therefore, it was suggested to use the clonality of the immunoglobulin (Ig) heavy chain (H) genes, as detected by polymerase chain reaction (PCR), as a decisive criterion. However, there is controversy as to whether B cell clonality also exists in chronic gastritis, hence rendering this approach futile at present. METHODS: An expert panel re-examined the histology and immunohistochemistry of a total of 97 cases of gastric biopsies, including clearcut marginal zone lymphoma, chronic gastritis, and ambiguous cases, applying the Wotherspoon criteria on the basis of haematoxylin-eosin and CD20 immunostainings. In addition, a new and advanced PCR system for detection of clonal IgH gene rearrangements was independently applied in two institutions in each case. RESULTS: The overall IgH clonality assessments of both institutions were in total agreement. Overt lymphoma (Wotherspoon score 5) was clonal in 24/26 cases. Chronic gastritis (Wotherspoon scores 1 and 2) was not clonal in 52/53 cases; the clonal case being Wotherspoon score 2. Of 18 cases with ambiguous histology (Wotherspoon scores 3 and 4) four were clonal. CONCLUSIONS: Using advanced PCR technology, clonal gastritis is extremely rare, if it exists at all. Thus B cell clonality in Wotherspoon 3 and 4 cases is regarded as suitable for definitively diagnosing gastric marginal zone lymphoma.

Algorithms↗
PubMed1998

The avian embryo as a model in developmental studies: chimeras and in vitro clonal analysis.

The avian embryo is a model in which techniques of experimental embryology and cellular and molecular biology can converge to address fundamental questions of development biology. The first part of the chapter describes two examples of transplantation and cell labeling experiments performed in ovo. Thanks to the distinctive histologic and immunocytochemical characteristics of quail and chick cells, the migration and development of definite cells are followed in suitably constructed chimeric quail-chick embryos. Isotopic transplantations of neural tube portions between quail and chick, combined with in situ hybridization with a nucleic probe specific for a quail oligodendrocyte marker, allowed study of the origin and migration of oligodendroblasts in the spinal cord. Heterotopic transplantations of rhombomeres were performed to establish the degree of plasticity of these segments of the hindbrain regarding Hox gene expression, which was revealed by labeling with chick-specific nucleic probes. The second part describes in vitro cell cloning experiments devised to investigate cell lineage segregation and diversification during development of the NC. An original cloning procedure and optimal culture conditions permitted analysis of the developmental potentials of individual NC cells taken at definite migration stages. The results revealed a striking heterogeneity of the crest cell population, which appeared to be composed of precursors at different states of determination. Clonal cultures also provide a means to identify subsets of cells that are the target of environmental factors and to understand how extrinsic signals influence the development of responsive cells.

Animals↗
PubMed1995

Clonal analysis in acute myeloid leukemia by polymerase chain reaction.

We have used PCR to amplify a polymorphic portion of the X-chromosome linked phosphoglycerate kinase gene (PGK) combined with a methylation-sensitive restriction enzyme digestion of the active X chromosome to examine the frequency of heterozygosity in Taiwanese females and analyze clonality in 18 female patients with acute myeloid leukemia (AML). We used hair follicles as normal tissue control. We found that the incidence of heterozygosity of the PGK gene in 102 hematological normal females and 18 patients tested was 35% (42/120). In five AML patients, a monoclonal X-inactivation pattern of leukemic blasts was found at presentation, which then returned to polyclonal at remission. In two of these five cases, a monoclonal pattern recurred at relapse. We also found that the hair follicles were readily accessible normal tissue for control, were easy to obtain, and were non-invasive.

Base Sequence↗
PubMed2003

Minimal residual disease detection in hairy cell leukemia. Comparison of flow cytometric immunophenotyping with clonal analysis using consensus primer polymerase chain reaction for the heavy chain gene.

We studied 86 specimens from 24 patients with hairy cell leukemia (HCL) to determine the sensitivity of routine flow cytometry (FC) and consensus primer PCR (cpPCR) in this disease. FC was more sensitive, detecting HCL in 48 (56%) of 86 specimens, while clonal B-cell populations were detected by cpPCR in only 23 (27%) of 86 specimens. FC and cpPCR were both more sensitive than morphologic examination. A positive cpPCR result is associated with higher tumor cell numbers than a negative cpPCR result, as determined by FC (P = .0017). We determined cutoff values for number of tumor cells at which cpPCR is consistently positive. At 6.8 tumor cells per microliter, cpPCR would be expected to be positive in at least 90% of the samples. FC was adequate in 86 cases (100%), while cpPCR was adequate in 74 cases (86%). FC is superior to cpPCR for detecting minimal residual HCL. It is more sensitive and more specific and permits quantitation of tumor cell number.

Cell Count↗
PubMed1986

Clonal analysis of T lymphocytes in spleens from patients with Hodgkin's disease. Frequent occurrence of unusual T4-positive cells which co-express cytolytic activity and production of interleukin-2.

T-lymphocyte populations isolated from spleens of untreated patients with Hodgkin's disease (HD) were grown by combining limiting dilution techniques and an assay system that allows clonal proliferation of virtually all human T cells. Under these conditions, high proportions of splenic T lymphocytes (50-80%) underwent clonal expansion, so that the set of clones obtained could be considered representative of the starting T-cell population. A total number of 229 clones from 6 HD spleens (3 uninvolved and 3 histologically involved by the disease) and 133 clones from 3 control spleens (obtained from otherwise healthy individuals, who underwent post-traumatic splenectomy) were examined for surface markers and tested in functional assays. One hundred and seventy-five clones from HD spleens and 75 clones from control spleens expressed the "helper/inducer" (T3+ T4+ T8-)phenotype, whereas as 54 clones from HD spleens and 58 control clones expressed the "cytotoxic/suppressor" (T3+T4-T8+) phenotype. As assessed by a non-specific lectin-dependent lytic assay, the proportion of HD clones displaying cytolytic activity was higher than that of cytolytic clones derived from control spleens. The majority of T4+ clones obtained from HD spleens (either uninvolved or histologically involved by the disease) were cytolytic, whereas only a small proportion (less than 10%) of T4+ clones derived from normal peripheral blood or spleens displayed cytolytic activity. The cytolytic potential of T4+ clones obtained from HD spleens did not reflect the activity of natural killer (NK) cells, since a minority of these clones exerted NK activity on K562 target cells. In addition, most of the T4+ cytolytic clones derived from HD spleens produced particularly high amounts of interleukin-2 (IL-2). These data indicate that T lymphocytes which concentrate in spleens of patients with HD consist at least in part of an infrequent T4+ cell subset co-expressing cytolytic activity and production of IL-2. These cells may reflect a cytotoxic reaction against unknown antigens associated with self class-II histocompatibility antigens.

Antigens, Differentiation, T-Lymphocyte↗