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PubMed1998

Monoclonality of both pale cells and cuboidal cells of sclerosing hemangioma of the lung.

Sclerosing hemangioma of the lung remains poorly understood, and it is still unclear whether this lesion is neoplastic or not. It consists of two major cell types, pale cells and cuboidal cells. We analyzed the clonality of each cell types from six female cases of surgically resected sclerosing hemangioma. The pale cells and cuboidal cells were separated by microdissection from methanol-fixed sections, and DNA was extracted for clonal analysis based on an X-chromosome-linked polymorphic marker, the human androgen receptor (HUMARA) gene or the phosphoglycerate kinase (PGK) gene. The HUMARA and PGK genes were found to be amplified with or without digestion by the methylation-sensitive restrictive endonuclease HpaII. Five of six cases were informative. Pale cells and cuboidal cells showed the same monoclonality in all of the informative cases, whereas the control cells showed a polyclonal pattern. Our results demonstrated that sclerosing hemangioma is caused by monoclonal expansion of cells, confirming that it is a neoplasia. Moreover, the present data indicate that both pale cells and cuboidal cells are derived from the same cell.

Adolescent↗
PubMed1998

The generation of fiber diversity during myogenesis.

Adult muscle is composed of different fiber types distinguished by their speed of contraction and metabolism. The generation of these differences is related both to the sequence in which muscle fibers form and to differences between the myogenic cells involved. Fibers form in two successive waves (primary and secondary) whose time of appearance can be correlated with the existence of successive populations of myogenic cells (embryonic and fetal). The differences between fibers arise through an interplay between heritable cellular commitment, where cells are preprogrammed to produce particular types of fiber and influences from the limb environment. The techniques of genetically marking cells and clonal analysis in vivo and in vitro are starting to reveal the relationship between these different influences. Although the process of myogenesis is similar in birds and mammals it is likely that cell autonomous behaviour plays a more important role during avian development as compared to mammals. The identification of muscle specific transcription factors has provided some clues to the mechanisms by which development is controlled but the expression of relatively few of these has been correlated with the sequence of events seen in myogenesis.

Adult↗
PubMed1996

Critical determinants of melanoma metastasis.

The process of metastasis consists of a series of linked, sequential steps. Although some of the steps in this process contain stochastic elements, metastasis as a whole favors the survival and growth of a few subpopulations of cells that preexist within the parent neoplasm. Metastases can have a clonal origin, and different metastases can originate from the proliferation of single cells. The outcome of metastasis depends on the interaction of metastatic cells with various host factors. Organ-specific metastases have been demonstrated in a variety of tumors and may even be specific to a particular site within one organ. Studies from our laboratory and from others have shown that the implantation of human cancer cells derived from surgical specimens into correct anatomic sites of nude mice provides a suitable model of metastasis of human tumors. Clonal analysis of human melanomas revealed that these tumors are heterogeneous for metastatic properties and that growth in the environment of specific organs is selective. These data suggest that systemic physiologic signals can be recognized by neoplastic cells, presumably by mechanisms similar to those shared by their normal cell counterparts. Elucidation of the mechanisms that regulate metastasis should lead to better therapeutic interventions.

Animals↗
PubMed1976

Patches in the livers of chimaeric mice.

Sections of adult chimaeric livers have been histochemically stained for beta-glucuronidase activity and patches of two cell populations visualized. A one-dimensional clonal analysis has been used to estimate the number of coherent clones in the adult liver. The data are consistent with a total of 9-22 million regular, coherent clones, comprising 10-34 nuclei, or a smaller number of irregular, branched coherent clones. Both of these alternatives suggest considerable cell mixing during liver morphogenesis.

Animals↗
PubMed1998

Differentiation of oligodendroglial progenitors derived from cortical multipotent cells requires extrinsic signals including activation of gp130/LIFbeta receptors.

We have previously isolated epidermal growth factor (EGF)-responsive multipotent progenitor cells from the early postnatal rodent cerebral cortex independent of generative zones. In this study we have examined the mechanisms regulating the generation of differentiated oligodendrocytes (OLs) from these multipotent cells. Although cultures of primary cortical OL progenitor cells propagated at clonal density spontaneously gave rise to differentiated OLs in defined medium, cultures of multipotent progenitors isolated from identical regions supported the elaboration of OL progenitors but not differentiated OLs. These observations indicate that the terminal maturation of OL progenitors derived from multipotent cells is dependent on signals present within the cellular environment. Application of cytokines such as basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), or neurotrophin 3 (NT3) to clonal density cultures of cortical multipotent progenitors increased the proportion of OL progenitors but failed to support the generation of differentiated OLs. By contrast, application of factors that activate gp130/leukemia inhibitory factor beta (LIFbeta) heterodimeric receptors, such as ciliary neurotrophic factor (CNTF), activated signal transducers and activators of transcription-3 in these OL progenitor cells and promoted the generation of differentiated OLs. Clonal analysis also demonstrated that CNTF directly targets OL progenitors derived from the multipotent cells. These observations suggest that two distinct progenitor cell pathways contribute to the generation of differentiated OLs during postnatal cortical gliogenesis. Although oligodendroglial maturation of classical OL progenitor cells is driven by cell autonomous mechanisms, our findings demonstrate that the generation of differentiated OLs from cortical multipotent progenitor cells is dependent on environmental cues, including activation of gp130/LIFbeta receptors.

Animals↗
PubMed1998

Characterization of the pathogenic autoreactive T cells in cyclosporine-induced syngeneic graft-versus-host disease.

Administration of the immunosuppressive drug cyclosporine after syngeneic bone marrow transplantation paradoxically elicits a systemic autoimmune syndrome resembling graft-vs-host disease (GVHD). This syndrome, termed syngeneic GVHD, is associated with the development of CD8+ cytolytic T lymphocytes that promiscuously recognize MHC class II molecules in association with a peptide from the invariant chain (CLIP). Clonal analysis reveals a major subset of cells that are pathogenic and require the N-terminal flanking region of CLIP for activation, while there is a minor subset of nonpathogenic T cells that require the C-terminal flanking region. The present studies show that pathogenic T cells produce type 1 cytokines (IL-2; IFN-gamma), while the nonpathogenic clones produce type 2 cytokines (IL-4; IL-10). Moreover, the repertoire of the pathogenic T cells is highly conserved with respect to V beta and V alpha TCR gene expression. The vast majority of clones express V beta8.5 (12/12) and V alpha11 (11/12). Although a limited number was evaluated, the nonpathogenic clones have only a V alpha restriction. Sequence analysis of the pathogenic T cell clones reveals a marked heterogeneity in the complementarity-determining region 3 domain and differential J region gene expression for both TCR alpha- and beta-chains. Evaluation of the specificity of these clones suggests that the functional interaction between the N-terminal flanking region of CLIP (defined by the amino acid sequence -KPVSP-) and the V region of the TCR is critical, allowing effective target cell recognition and tissue destruction in syngeneic GVHD.

Amino Acid Sequence↗
PubMed1986

Diffuse large cell lymphoma in a patient with hairy cell leukemia: immunoglobulin gene analysis reveals separate clonal origins.

A 75-year-old man with hairy cell leukemia (HCL) was found to have an immunoblastic lymphoma of the small bowel. Immunologic and genotypic characterization of these neoplasms revealed both the HCL and the immunoblastic lymphoma to be of the B cell lineage. The HCL expressed the HCL-associated antigens detected by the monoclonal antibodies HC-1 and HC-2, whereas the immunoblastic lymphoma failed to react with these antibodies. In addition, surface immunoglobulin light chains could not be accurately determined for the hairy cells, whereas the immunoblastic lymphoma was shown to express only kappa immunoglobulin light chains. These immunophenotype differences were compatible with either the clonal evolution of the HCL into the immunoblastic lymphoma or a separate clonal origin for these two neoplasms. An analysis of tumor DNA by Southern blot hybridization revealed different heavy-chain and kappa light-chain gene rearrangements in these two malignancies. Thus the occurrence of the large cell lymphoma most likely represents the emergence of a second clonally unrelated B cell malignancy.

Aged↗
PubMed1991

A sensitive and efficient induction system for murine IgE. Single cell analysis at the clonal level.

A culture system is described which permits the analysis of IgE expression by single murine cells within clones of B cells. The system is based on the use of a CB5.1 stroma cell line as a feeder which optimally supports the IL-4-induced switch to IgE of LPS-stimulated B cells in culture. In this system 100 U/ml IL-4 induces the switch to IgE, in 3-5% of B cells and the switch frequency to IgG1 was as high as 2%. Five ng IgE or 12 ng IgG1 were produced per clone containing on average 13-15 PFC. The detection of single IgE secreting B cells was possible due to two newly developed, highly specific rat anti-mouse IgE antibodies used in a sandwich-ELISA. The frequency of IgE-secreting B cells was enhanced 2.5 times when the fibroblastoid CB5.1 cells rather than thymocytes were used as feeder cells. CB5.1 cells supported the differentiation of B cells to IgM-PFC almost as well as rat thymocytes (which have, to date, been used as the standard feeder layer) whereas the amount of secreted IgG1 was about 3 times lower than in thymocyte cultures. Optimal switching to IgE occurred at concentrations of IL-4 which were 10-fold lower than that required for IgG1 expression, a situation quite opposite to that observed in the rat thymocyte-supported culture system. In confirmation of established data the switch of B cells to IgE or IgG1 occurred randomly. The advantages of CB5.1 cells as feeder cells are (1) the use of a homogeneous and defined cell line, (2) their limited release of defined lymphokines (IL-6 and GM-CSF), and (3) the low degradation and consumption of cytokine factors. The combination of the CB5.1 cell line with a highly specific IgE ELISA assay made it possible to analyse the appearance of IgE producing cells within a developing B cell clone.

Animals↗
PubMed2000

Cytogenetic and Southern blot analysis to demonstrate clonality and to estimate prognosis in patients with myelodysplastic syndromes.

We examined the bone marrow of 109 patients with myelodysplastic syndrome (MDS) at the time of diagnosis and during the course of the disease by means of Southern blot analysis and/or cytogenetic studies to detect and evaluate clonal markers, their implications for the prognosis of the disease, and the response to treatment. The patients either were enrolled in an EORTC study and received low-dose Ara-C with (n=31) or without (n=21) growth factors, according to the study protocol, or were treated supportively (only one patient received regular chemotherapy for concomitant lymphoma). Full or at least partial remission was achieved by 34% of the treated patients (n=54). In 57% (53 of 93) of all patients a clonal marker of either kind was detected by Southern blot analysis and/or cytogenetic examination. Clonal chromosomal aberrations were found in 45% (35 of 77) of the cases examined at diagnosis, with solitary del(5q) aberrations occurring in 10% of the cases and complex aberrations in 18%, trisomy 8 or monosomy 7 being a frequent finding. Of all patients, 49% (28 of 57) were characterized by one or more gene rearrangements (e.g., Ig-JH, TcR-beta, M-bcr, GM-CSF, G-CSF, or IL-3) at diagnosis. In five of 21 cases (24%) studied in hematological remission of the disease chromosomal aberrations were still detectable, and in seven of 23 (30%) a gene rearrangement persisted. We also found six cases with multiple clones exhibiting different susceptibilities to treatment and thereby indicating the oligoclonal character of this disease. Clinical evaluation revealed that the prognosis of the respective patients was directly related to the particular clonal markers detected at diagnosis: Risk groups were subdivided according to the karyotypes, with a solitary del(5q) aberration meaning a favorable, a normal karyotype an intermediate, solitary aberrations without del(5q) a poor, and complex karyotypes a very poor prognosis. We showed that densitometry helps to increase the sensitivity of Southern blot analysis by quantifying the amount of altered DNA, which often increases shortly before or at progression of MDS. Overall, there was a high level of concordance of both clonality examinations with the clinical course of the disease and the response rate. Therefore, we recommend cytogenetic studies and Southern blot analysis to detect clonal markers at diagnosis of MDS, to detect oligoclonality and clonal evolution, or to quantify the amount of clonal DNA, which appears to be a sensitive tool for evaluating the prognosis and response to therapy in MDS.

Aged↗
PubMed2005

BIOMED-2 multiplex immunoglobulin/T-cell receptor polymerase chain reaction protocols can reliably replace Southern blot analysis in routine clonality diagnostics.

To establish the most sensitive and efficient strategy of clonality diagnostics via immunoglobulin and T-cell receptor gene rearrangement studies in suspected lymphoproliferative disorders, we evaluated 300 samples (from 218 patients) submitted consecutively for routine diagnostics. All samples were studied using the BIOMED-2 multiplex polymerase chain reaction (PCR) protocol. In 176 samples, Southern blot (SB) data were also available, and the two types of molecular results were compared. Results of PCR and SB analysis of both T-cell receptor and immunoglobulin loci were concordant in 85% of samples. For discordant results, PCR results were more consistent with the final diagnosis in 73% of samples. No false-negative results were obtained by PCR analysis. In contrast, SB analysis failed to detect clonality in a relatively high number of samples, mainly in cases of low tumor burden. We conclude that the novel BIOMED-2 multiplex PCR strategy is of great value in diagnosing patients with suspected B- and T-cell proliferations. Because of its higher speed, efficiency, and sensitivity, it can reliably replace SB analysis in clonality diagnostics in a routine laboratory setting. Just as with SB results, PCR results should always be interpreted in the context of clinical, immunophenotypical, and histopathological data.

B-Lymphocytes↗
PubMed1987

Functional analysis of a clonal expansion of Leu 11 positive NK active lymphoid cells.

A female patient with an unusual lymphoproliferative disease associated with marked neutropenia has been observed for 36 months. The expanded cell population consists of large lymphocytes, many of which contain large azurophilic granules with acid phosphatase activity. These cells were T3, T8, T11 and Leu 11 positive but lacked the M1, T10, IL-2 receptor and HLA.DR antigens. The majority of these cells (60-70%) were also Leu 7 (HNK-1) positive. Strong natural killer (NK) activity was found in both the Leu 7 positive and negative cell populations. This cytotoxic activity was inhibited by monoclonal antibodies known to inhibit NK activity but was unaffected by antibodies which block T cell and T/NK cell cytotoxicity. Further functional analysis indicated that these cells suppressed normal T cell responses to mitogens, MLC responses and PWM induced B cell immunoglobulin synthesis. No effect on bone marrow progenitor cell growth was demonstrated. Antibody dependent cellular cytotoxic (ADCC) activity was barely detectable despite the presence of the Leu 11 antigen. Southern blot DNA analysis demonstrated clonal rearrangement of the T cell receptor beta gene thereby confirming that this variant of T gamma lymphoproliferative disease was a neoplastic condition.

Adult↗
PubMed1986

Proliferative and cytotoxic responses of human peripheral blood lymphocytes to autologous malignant effusions. An analysis at the clonal level.

Peripheral blood lymphocytes from five patients were stimulated initially in mixed lymphocyte:tumour culture (MLTC) with autologous malignant effusions and cloned by limiting dilution in interleukin-2 prior to phenotyping and assay for different functional capabilities namely proliferative responsiveness to autologous and allogeneic tumours in the primed lymphocyte test (PLT) and cytotoxicity (CTX) toward a range of fresh tumour and cell line targets. For individual clones the two functional activities tended to be mutually exclusive. Clones (or 'cloids' containing more than one PLT precursor) from three of four tumours analysed were responsive to autologous tumour cells in the PLT of which two shared antigens with allogeneic tumours of similar tissue provenance. All phenotyped PLT positive clones were T3+T4+T8-. Cytotoxic clones were generated from all MLTCs. Their target cell repertoire (based on an analysis of greater than 30) was generally broad including cell lines sensitive to natural killer (NK) cells, and less frequently and to a weaker extent, fresh autologous and allogeneic tumours. An ovarian carcinoma was exceptional, insofar as the CTX of 8/9 clones was apparently restricted to the autologous tumour. Phenotypically cytotoxic clones were T3+T4-T8+, less usually T3+T4+T8-, but invariably B73.1- (a monoclonal antibody reactive with the peripheral blood NK subset). Analysis at the clonal level emphasises the diversity of responses to putative human tumour-associated antigens, and the need to identify the critical functionally active molecules in the MLTC.

Aged↗
PubMed1993

Two-dimensional electrophoresis as an aid in the analysis of the clonality of immunoglobulins.

In this paper, we summarize our five-year observation of two-dimensional polyacrylamide gel electrophoretic (2-D PAGE) analysis of immunoglobulin (Ig) light (L) chain patterns on serum/plasma and/or purified human Ig, and compare this technique with agarose electrophoresis and/or immunofixation examination. Polyclonal Ig L chains were seen as large "fuzzy" areas with several zones of high density. The majority (71%) of the monoclonal Ig L chains of monoclonal gammopathy detected by conventional electrophoresis appeared as a single large and well-defined spot on 2-D PAGE analysis, with the remaining appearing as multiple spots. The presence of oligoclonal Ig, reflected by multiple spots in 2-D PAGE, and several bands in immunofixation, was observed in 5 of 26 patients after allogeneic bone marrow transplantation and in 5 patients with tumors. In the majority (77%) of hypergammaglobulinemia, L chains appeared as a wide spread of small and well-defined spots in 2-D PAGE analysis. This pattern suggested oligoclonal Ig-secreting B cell clone expansion, and corresponding abnormalities were not detected with immunofixation. 2-D PAGE analysis also detected oligoclonal Ig expansions whereas conventional electrophoretic examination was normal in 10 additional patients after bone marrow transplantation, and in 6 of 10 immunocompetent patients with acute severe infections. Analysis of the Ig L chain pattern of a severe combined immune-deficient mouse populated with human peripheral blood leukocytes confirmed the skewed human Ig production in the model. In summary, 2-D PAGE appears to be a sensitive tool for the analysis of Ig diversity in various clinical situations.

Animals↗
PubMedDate not supplied

Detection of clonality by heteroduplex analysis of amplified junctional region of T-cell receptor gamma in patients with T-cell acute lymphoblastic leukemias.

BACKGROUND AND OBJECTIVE: Traditional gel electrophoresis of PCR amplification products of regions from T-cell receptor genes often does not differentiate monoclonal from polyclonal rearrangements. We used polymerase chain reaction (PCR) and heteroduplex analysis on polyacrylamide gels to improve the detection of monoclonal rearrangements. METHODS: We investigated heteroduplex analysis of the amplified V gamma-J gamma junctions of the rearranged T-cell receptor gamma (TcR-gamma) gene by electrophoretic separation on non-denaturing polyacrylamide gel (PAGE) in 8 T-cell acute lymphoblastic leukemia (T-ALL) patients analyzed at diagnosis. RESULTS: Clonal homoduplex and heteroduplex bands were present only in the T-ALL samples and not in controls. We confirmed clonality by direct sequencing of the V gamma-J gamma junction. In 2 instances the analysis was performed on samples obtained from the same patient at diagnosis and at relapse, respectively; the presence of the same clonal TcR-gamma rearranged cell remained detectable during clinical progression of the disease. INTERPRETATION AND CONCLUSIONS: Our heteroduplex analysis showed that separation of the PCR product by electrophoresis on non-denaturing PAGE is a rapid and convenient method for the detection of clonal TcR-gamma rearrangements in T-ALL.

Gene Rearrangement↗
PubMedDate not supplied

NotI macrorestriction analysis suggests a clonal relationship of Salmonella enterica, ser. enteritidis phage lysotype 4 strains.

30 different strains of Salmonella enterica ser. enteritidis (S. enteritidis) isolated at the Institute for Medical Microbiology, Hannover, were subjected to a NotI macrorestriction analysis. The results were compared with the respective phage lysis patterns. 25 of 30 isolates belonged to phage lysotype 4; all these strains displayed an identical genomic NotI restriction fragment pattern. In contrast, five isolates of other phage lysotypes revealed more or less prominent differences in their NotI restriction fragment pattern. These results provide genetic evidence for a clonal expansion of a single phage lysotype 4 strain underlying the recent surge in salmonella infections and support phage lysotyping as a reliable epidemiological typing method for S. enteritidis.

Bacteriophage Typing↗
PubMed1988

Graves' disease: phenotypic and functional analysis at the clonal level of the T-cell repertoire in peripheral blood and in thyroid.

We have investigated at the clonal level the repertoire of intrathyroid and peripheral T lymphocytes in three patients with Graves' disease using a high efficiency cloning technique. Clonal efficiencies ranged from 10 to 31% for intrathyroid, and from 19 to 100% for peripheral T cells. In Graves' disease the phenotypic analysis showed similar percentages of CD3+ CD4+ CD8- and CD3+ CD4- CD8+ clones in thyroid infiltrates and peripheral blood. The functional evaluation showed similar or lower proportions of cytolytic clones in thyroid infiltrates with respect to peripheral blood. Furthermore, the proportions of intrathyroid and peripheral T-cell clones capable of releasing interleukin-2 and/or gamma-interferon in response to mitogen stimulation were similar. Finally, 44% of intrathyroid clones were neither cytolytic nor able to release IL-2 and gamma-interferon. These results are strikingly different from those obtained in Hashimoto's thyroiditis, where the large majority of intrathyroid T-cell clones are cytolytic and the proportions of clones able to release gamma-IFN are remarkably increased in thyroid infiltrates when compared to those obtained from peripheral blood. Taken together, these data suggest a different role for T lymphocytes in the pathogenesis of the two major human autoimmune thyroid diseases.

Adult↗
PubMed2003

Analysis of a clonally related mantle cell and Hodgkin lymphoma indicates Epstein-Barr virus infection of a Hodgkin/Reed-Sternberg cell precursor in a germinal center.

The simultaneous occurrence of a Hodgkin lymphoma (HL) and a non-Hodgkin lymphoma (NHL) is a rare event, and single cell analyses of such composite lymphomas revealed that NHL and Hodgkin/Reed-Sternberg (HRS) tumor cells are frequently descendants of the same tumor clone precursors. Here we present a composite lymphoma consisting of a mantle cell lymphoma (MCL) and an HL with EBV- and EBV+ HRS cells. Analysis of rearranged V genes of single cells revealed a clonal relationship between MCL and HL tumor cells. Although V gene rearrangements of the MCL were unmutated, mutations were observed in HRS cells. Besides mutations shared by all HRS cells, the EBV+ HRS cells carried identical additional mutations. These findings show that both lymphomas derive from a common precursor, most likely a pre germinal center (GC) B cell that already carried some transforming event(s). However, the presence of mutations in the V genes of the HRS cells further corroborates the importance of the GC reaction for the pathogenesis of HL. Importantly, the finding that only a subclone of the HRS clone, defined by a particular mutation pattern, was EBV infected represents a strong indication that EBV infection of the HRS cell precursor happened in the GC.

Adult↗
PubMed2003

Single-cell repertoire analysis demonstrates that clonal expansion is a prominent feature of the B cell response in multiple sclerosis cerebrospinal fluid.

Single-cell RT-PCR was used to sample CD19(+) B cell repertoires in cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS) or viral meningitis. Analysis of amplified Ab H and L chain products served to identify the rearranged germline segment and J segment, and to determine the degree of homology for the H and L chain sequence of individual B cells. The B cell repertoire of viral meningitis CSF was predominantly polyclonal, whereas B cell clonal expansion was a prominent feature of the IgG repertoire in three of four MS patients. Two dominant clonal populations in one MS CSF accounted for approximately 70% of the IgG H chain V regions sequenced, while the corresponding IgM repertoires were more heterogeneous. One clonal B cell population revealed multiple L chain rearrangements, raising the possibility of a role for receptor editing in shaping the B cell response in some MS patients. The most immediate implications of identifying rearranged Ig sequences in MS B cells is the potential to accurately recreate recombinant Abs from these overrepresented H and L chains that can be used to discover the relevant Ag(s) in MS.

Acute Disease↗