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D N Shapiro

Publications and source records attributed to D N Shapiro.

At least 19 recordsLinked to original sources
PubMed1992

Monoclonal antibodies to the myogenic regulatory protein MyoD1: epitope mapping and diagnostic utility.

Monoclonal antibodies (MoAbs) were developed against recombinant wild-type murine MyoD1 protein. Each of 4 MoAbs was immunologically reactive with recombinant MyoD1 protein by enzyme-linked immunosorbent assay, and each specifically stained the nuclei of myogenic cells. Epitopes were mapped using fusion protein constructs with specific deletions of defined regions of the MyoD1 molecule. MoAb 5.2F recognized an epitope in the amino terminal region between amino acid residues (AAR) 3 and 56, whereas epitopes for MoAbs 1.1A, 5.4G, and 5.8A were in the carboxyl terminus (AAR 167-318) of the MyoD1 protein. The epitope for MoAb 5.8A was further delineated to AAR 170-209 by Western analysis and immunoprecipitation of in vivo transcribed and translated MyoD1 protein having specific deletions in the carboxyl terminus. The 5.8A epitope was ultimately localized to the region between AAR 180 and 189 of the protein by enzyme-linked immunosorbent assay using 10-amino acid residue synthetic peptides. This sequence is apparently unique to MyoD1 and has little homology to other myogenic regulatory proteins (myogenin, Myf5, Myf6, and MRF4). Transfection of cDNA for murine MyoD1 into a nonmuscle cell line conferred 5.8A reactivity, confirming the specificity of this reagent. MoAb 5.8A was then used to examine the expression of MyoD1 in normal and malignant human tissues. MyoD1 was not detected in any normal adult tissue but was detected in 25 of 25 histologically confirmed rhabdomyosarcomas. Staining was localized to the nucleus and showed marked heterogeneity between cells as well as differential staining within nuclei. Specific subcellular localization of 5.8A was further determined by immunoelectron microscopy, where antibody was found to localize to electron-dense areas, more frequently associated with the nuclear submembranous region. In addition to rhabdomyosarcomas, MoAb 5.8A stained 2 of 5 Wilms' tumors and one ectomesenchymoma, neoplasms known to contain myogenic elements. The 5.8A reagent was also of value in the accurate histopathological classification of 2 of 4 tumors previously diagnosed as extraosseous Ewing's sarcoma and 2 of 3 tumors diagnosed as undifferentiated sarcomas.

Amino Acid Sequence↗
PubMed1992

Atypical Wiskott-Aldrich syndrome in a girl.

Wiskott-Aldrich syndrome (WAS) is a fully penetrant X-linked recessive disorder characterized by thrombocytopenia with small platelets, eczema, and defects of both T-cell and B-cell immunity. Obligate carriers of this disorder show no signs of the gene defect because in the cell lineages primarily affected by the disorder they demonstrate preferential use of the normal, nonmutant X as the active X. This can be explained by the selective disadvantage in proliferation and/or survival experienced by the cells with the mutant X as the active X. We have recently evaluated an 8-year-old girl with a disorder phenotypically identical to WAS. Cytogenetic studies did not show any structural abnormalities of the X chromosome and X chromosome inactivation analysis showed that both of her X chromosomes could function as the active X. These findings suggest that there is an autosomal recessive disorder that is very similar to classic WAS.

Antigens, CD↗
PubMed1992

Chromosomal sublocalization of the 2;13 translocation breakpoint in alveolar rhabdomyosarcoma.

A characteristic balanced reciprocal chromosomal translocation [t(2;13)(q35;q14)] has been identified in more than 50% of alveolar rhabdomyosarcomas. As the first step in characterization of the genes involved in this translocation, we constructed somatic cell hybrids that retained either the derivative chromosome 2 or the derivative chromosome 13 without a normal chromosome 13 homologue. Ten linked DNA probes known to be located within bands 13q13-q14 were mapped relative to the breakpoint on chromosome 13, allowing localization of the breakpoint region between two loci separated by 5.5 cM. A long-range restriction map extending approximately 2,300 kb around these loci failed to provide evidence of rearrangement. Additionally, we confirmed that the FMS-like tyrosine kinase gene (FLT), previously localized to 13q12 by in situ hybridization, is located proximal to the breakpoint, and we demonstrated that FLT is not a target for disruption by this tumor-specific translocation.

Blotting, Southern↗
PubMed1992

Sublocalization of the chromosome 5 breakpoint of the 3;5 translocation in myelodysplastic syndromes and acute myeloid leukemia.

A t(3;5)(q25.1;q34) reciprocal translocation identifies a subset of cases of myelodysplastic syndrome or acute myeloid leukemia (AML) that are characterized by increased numbers of megakaryocytes and severe trilineage dysplasia. As a first step in characterizing the t(3;5) breakpoints, we asked whether the translocation involves the CSFIR/PDGFRB locus at 5q33-q35. Pulsed-field gel electrophoretic analysis of a region extending 580 kb 5' to the PDGFRB gene and 120 kb 3' to the CSFIR gene did not reveal aberrant restriction fragments in leukemic cell DNA, confirming that the breakpoint does not occur in the vicinity of these genes. To sublocalize the breakpoint, we performed Southern blot hybridizations using DNA from human x hamster somatic cell hybrids containing the normal 3, the normal 5, the derivative 3, or the derivative 5 human chromosome. Using a series of polymorphic DNA probes from the long arm of chromosome 5, which have been linked by genetic recombination, we bracketed the breakpoint to within a region that spans approximately 13 centimorgans (sex average) and is flanked by the q34-qter markers cKK5.19 and L1200 (D5S62). This analysis places the chromosome 5 breakpoint of the t(3;5) considerably telomeric to the CSFIR/PDGFRB locus, confirming our studies with pulsed-field electrophoresis. Future efforts to identify the genes affected by the t(3;5) should focus on the 5q segment described in this study.

Animals↗
PubMed1992

Assignment of the genes encoding human interleukin-8 receptor types 1 and 2 and an interleukin-8 receptor pseudogene to chromosome 2q35.

Two human cDNA clones that encode different interleukin-8 (IL8) receptors have recently been isolated. The interleukin-8 receptor type 1 (IL8R1) binds IL8 only, whereas the interleukin-8 receptor type 2 (IL8R2) (previously designated IL8RA) also binds growth regulated gene (GRO), and neutrophil activating protein-2 (NAP-2) with high affinity. In the process of screening a genomic library with these cDNAs to obtain large clones for use in chromosomal localization studies, we isolated an interleukin-8 receptor pseudogene (IL8RP) that bears greatest similarity to IL8R2. Using Southern hybridization analysis of human x rodent somatic cell hybrid DNAs with cDNA probes for IL8R1 and IL8R2 and probes from the IL8RP locus, we assigned the three loci to chromosome 2; fluorescence in situ hybridization (FISH) to metaphase chromosome preparations using genomic clones from each locus refined this localization to chromosome 2, band q35, for all three. By virtue of their chromosomal location, IL8R1 and IL8R2 may be considered candidate genes for several human disorders in which the involved locus has been mapped to distal 2q or that are associated with structural abnormalities of this segment, including van der Woude syndrome and the neoplastic diseases rhabdomyosarcoma and uterine leiomyomata. In addition, because this region of chromosome 2q is homologous to proximal mouse chromosome 1 in the segment containing the Lsh-Ity-Bcg locus involved in mediating host resistance to infection with intracellular pathogens, examination for abnormalities of the murine homologues of the IL8R genes should be considered in mice affected by mutations of this locus.

Amino Acid Sequence↗
PubMed1991

Reassignment of the human CSF1 gene to chromosome 1p13-p21.

Human macrophage colony-stimulating factor (CSF-1 or M-CSF) is encoded by a single gene that was previously assigned to the long arm of chromosome 5, band q33.1, in a region adjacent to the gene encoding its receptor (Pettenati MJ, et al, Proc Natl Acad Sci USA 84:2970, 1987). Using fluorescence in situ hybridization with genomic probes to examine normal metaphase chromosomes, we reassigned the human CSF1 gene to the short arm of chromosome 1, bands p13-p21. We confirmed this result by hybridizing a CSF1 cDNA probe to filters containing flow-sorted chromosomes and by identifying CSF1 sequences in DNAs extracted from human x rodent somatic cell hybrids that contained human chromosome 1 but not human chromosome 5. Our findings are consistent with studies that have shown tight linkage between the murine CSF1 and amylase genes, as part of a conserved linkage group between mouse chromosome 3 and the short arm of human chromosome 1, which also includes the genes encoding the beta subunits of thyrotropin and nerve growth factor. Assignment of the CSF1 gene to chromosome 1 at bands p13-p21 raises the possibility that it may be altered by certain nonrandom chromosomal abnormalities arising in human hematopoietic malignancies and solid tumors.

Cell Line↗
PubMed1991

Rhabdomyosarcoma. From the laboratory to the clinic.

The prospect of identifying and developing new agents for treatment of rhabdomyosarcomas is discussed in the light of current prognosis for children with advanced stage disease. Preliminary attempts to identify tumor-specific agents using in vitro cell culture show potential promise, but as yet remain unproven. The more complex system of identifying therapeutically active agents using human tumor xenografts has demonstrated usefulness. The potential problems associated with this system are discussed.

Animals↗
PubMed1991

MyoD1 protein expression in alveolar soft part sarcoma as confirmatory evidence of its skeletal muscle nature.

A typical case of alveolar soft part sarcoma was found to express in a strong and widespread fashion the marker MyoD1 protein. This nuclear phosphoprotein is the product of a regulatory gene that controls the commitment of a cell to myogenic lineage; it therefore provides strong and perhaps definitive evidence in support of the skeletal muscle nature of this enigmatic neoplasm.

Cell Transformation, Neoplastic↗
PubMed1991

Relationship of tumor-cell ploidy to histologic subtype and treatment outcome in children and adolescents with unresectable rhabdomyosarcoma.

Clinical and histopathologic features are often inadequate for accurate prediction of relapse or survival of individual patients with rhabdomyosarcoma (RMS). We therefore studied the cellular DNA content (ploidy) of RMS cells in relation to histology and response to therapy in 37 patients with unresectable tumors. Using flow cytometric techniques, we found that about one third of patients had diploid tumor stem lines, regardless of the histologic subtype. In the group with abnormal ploidy, a hyperdiploid classification (1.10 to 1.80 times the DNA content of normal diploid cells) was exclusively associated with embryonal histology (P = .001). By contrast, near-tetraploidy (1.80 to 2.60 times the DNA content of normal cells) was strongly associated with alveolar histology (P = .001). Thus, in these histologic subtypes of RMS, abnormal ploidy appears to arise through different mechanisms. Tumor-cell ploidy had a significant impact on survival that was especially apparent in patients with unresectable, nonmetastatic (group III) tumors. In this subgroup, hyperdiploidy conferred the best prognosis and diploidy the worst (P less than .0001). None of the eight patients with diploid tumors survived for more than 18 months. Tumor-cell ploidy was the best predictor of treatment outcome for patients with either embryonal (P less than .001; relative risk, 25.5) or alveolar (P = .073; relative risk 7.1) RMS and contributed significantly after adjustment for disease stage and anatomic site. Patients with unresectable diploid RMS have an unacceptably high risk of treatment failure, justifying new therapeutic approaches for this distinct subgroup.

Adolescent↗
PubMed1990

Morphological and molecular characterization of spontaneous myogenic differentiation in a human rhabdomyosarcoma cell line.

Skeletal muscle differentiation consists of an ordered withdrawal of committed cells from the cell cycle and their fusion to form multinucleated myotubes. To determine if differentiation of malignant myoblasts parallels that of normal skeletal muscle, a cell line (Rh28) was established from an alveolar rhabdomyosarcoma. Rh28 displays a constant population doubling time of 45-55 h until passage 60, when the doubling time progressively increases until proliferation ceases. Loss of proliferative capacity is associated with morphological evidence of differentiation to multinucleated myotubes, fusion, and the expression of numerous muscle-specific genes. In contrast to normal myogenic differentiation, multinucleated cells continue to synthesize DNA and express abundant c-myc transcripts. These observations suggest synchronous replication and possible arrest in the G2-phase of the cell cycle, since there was no evidence of mitotic activity in differentiated cells. Terminal differentiation of early passage Rh28 cells was induced in the presence of 10% dialyzed fetal calf serum but not by medium containing 2% undialyzed serum, suggesting a role for low molecular weight growth factors in this process. Our data indicate that the Rh28 cell line may be of value in elucidating the relationship between oncogenic transformation and differentiation in rhabdomyosarcoma.

Cell Differentiation↗
PubMed1990

Superior vena cava syndrome associated with childhood malignancy: analysis of 24 cases.

Twenty-four children with superior vena cava obstruction at initial presentation or associated with disease recurrence were treated at St. Jude Children's Research Hospital from 1973 to 1988. Of the 16 patients with superior vena cava syndrome at presentation, eight had non-Hodgkin's lymphoma, four had acute lymphoblastic leukemia, two had Hodgkin's disease, one had neuroblastoma, and one had a yolk sac tumor. Their clinical condition at presentation was often critical and required rapid treatment. In all cases, histopathologic diagnosis was obtained without complication by either bone marrow aspiration, lymph node biopsy, thoracentesis, or thoracotomy prior to the initiation of definitive therapy. Eight children had superior vena cava syndrome as a late complication during the course of their therapy. None had an antecedent history of superior vena cava obstruction. In contrast to the patients with superior vena cava obstruction at presentation, this group was composed predominantly of patients with recurrent solid tumors. Other causes included disseminated candidiasis and superior vena cava thrombosis, thus underscoring the importance of recognizing the etiology of superior vena cava syndrome to facilitate proper treatment.

Adolescent↗
PubMed1990

Myogenic regulatory protein (MyoD1) expression in childhood solid tumors: diagnostic utility in rhabdomyosarcoma.

Transcripts for the muscle regulatory gene MyoD1 are expressed during normal skeletal muscle myogenesis and in rhabdomyosarcomas but not in other tissues or in soft-tissue sarcomas. Here we report the distribution of MyoD1 protein, determined by reactivity with anti-MyoD1 polyclonal sera in normal tissues, rhabdomyosarcoma cell lines, and in a variety of pediatric solid tumors. The distribution of MyoD1 protein was highly restricted in normal tissues and was detected only in fetal skeletal muscle and more faintly in adult skeletal muscle. All six human rhabdomyosarcoma cell lines analyzed expressed MyoD1 mRNA transcripts as well as immunoreactive protein. The immunohistochemical expression of MyoD1 protein was then examined in 49 surgical specimens from a variety of pediatric solid tumors. Each of 16 rhabdomyosarcoma specimens was positive for MyoD1, including four that did not express the intermediate filament protein desmin. Two of five specimens originally designated sarcoma type indeterminate (STI) and two of three specimens originally designated extraosseous Ewing's sarcoma (EOE) were positive for MyoD1, suggesting commitment to myogenic differentiation. Three of eight Wilms' tumors, which also expressed desmin and had clearly evident myogenic elements, also were positive for MyoD1. Tumors that failed to express MyoD1 protein included neuroblastoma, primitive neuroectodermal tumor, non-Hodgkins lymphoma, embryonal sarcoma of the liver, malignant fibrous histiocytoma, malignant rhabdoid tumor, and Ewing's sarcoma of the bone. These results indicate that expression of MyoD1 protein is highly restricted in normal human tissues and that expression of this gene product in malignant tissue may be diagnostic for rhabdomyosarcoma. Furthermore MyoD1 staining may be a valuable adjunct in the classification of pediatric soft-tissue sarcomas.

Child↗
PubMed1989

Activation of a murine T-cell hybridoma by cationized bacteria.

Cationic particles interact by electrostatic forces with membrane components of diverse cell types, including lymphocytes. Contact with cationized streptococci was shown to induce a murine T-cell hybridoma to transcribe lymphokine mRNA as well as secrete interleukin-2. This activation was accompanied by a rise in intracellular calcium. Cationized streptococci-induced activation of this T-cell hybridoma could be specifically inhibited by either chelating extracellular calcium or by treating with CD4 monoclonal antibody. These data indicate that the in vitro behaviour of T cells can be modulated by charged microbial particles; such interactions may have relevance for chronic inflammation associated with some bacterial infections.

Animals↗
PubMed1985

Immune regulation of in vitro murine megakaryocyte development. Role of T lymphocytes and Ia antigen expression.

Mitogen-activated murine T lymphocytes or T cell hybridomas produce an activity (megakaryocyte [Mk] potentiator activity) that enhances the in vitro growth and development of Mk colonies. This activity was found in optimal concentrations (2.5%) in T cell hybridoma-conditioned medium, and was also produced by feeder layers of concanavalin A-activated T cells. A subpopulation of murine Mk progenitor cells (colony-forming units; CFU-Mk) bears the Ia antigen. Separate experiments indicated that T cell products stimulate CFU-Mk by increasing their basal levels of Ia expression as well as the frequency of cells actively synthesizing DNA. The hypothesis that the expression of this antigen was related to the cell cycle status of these progenitor cells was confirmed in studies that indicated that ablation of actively cycling cells in vivo abrogated the cytotoxic effects of anti-Ia monoclonal antibodies. The interdependence of T cell lymphokine regulation of both Ia expression and cell cycle status was also seen in in vitro experiments in which Ia+ progenitor cells were eliminated by complement-dependent cytotoxicity. The removal of Ia+ cells prevented 5-hydroxyurea-mediated inhibition of cells in S phase. We hypothesize that immune modulation of megakaryocytopoiesis occurs via soluble T cell products that augment Mk differentiation. Further, the mechanism of immune recognition/modulation may occur via Ia antigens present on the surface of these progenitor cells.

Animals↗
PubMed1985

Antigen-specific T cell activation results in an increase in cytoplasmic free calcium.

Free intracellular calcium acts as a messenger in response to extracellular stimuli, including those that result in cellular proliferation. For example, mitogenic lectins have been shown to increase intracellular calcium concentration ([Ca+2]i) during proliferation of T lymphocytes. To determine if similar changes in [Ca+2]i occur when T cells are activated by nominal antigen, [Ca+2]i was measured in murine T cells from a bovine insulin-specific, major histocompatibility-restricted T hybridoma by using the calcium-sensitive fluor quin-2. Quin-2-loaded T hybridoma cells were activated by incubation with antigen-pulsed antigen-presenting cells (APC) and [Ca+2]i determined by measurement of quin-2 fluorescence. T cell [Ca+2]i rose sharply within 20 min after incubation with APC. Incubation of T cells with unpulsed APC resulted in [Ca+2]i not significantly different from resting levels. Further evidence that this activation was antigen specific was demonstrated at the level of both the APC and the T cell. Incubation of quin-2-loaded T cells with APC pulsed with the inappropriate antigen, porcine insulin, did not result in an increase in [Ca+2]i. Additionally, pretreatment of T cells with a monoclonal antibody against the T cell antigen receptor abrogated the [Ca+2]i increase. Finally, the antigen-induced rise in [Ca+2]i could be blocked by pretreatment of APC with appropriate but not inappropriate Ia monoclonal antibodies. These results suggest that a rapid rise in [Ca+2]i is an early event in the antigen-specific activation of the T cell and may be related to later steps, such as the secretion of lymphocyte monokines.

Animals↗
PubMed1984

Anti-idiotypic regulation of an insulin-reactive T cell clone.

A series of MHC-restricted, bovine-insulin-(BI) reactive T cell clones were generated. The specificity of one group was shown to be for an insulin A-chain loop determinant; the other group apparently demonstrated specificity of a B-chain determinant and/or amino acid residue A4. Guinea pig anti-idiotypic antisera were prepared against two idiotypically related BI monoclonal antibodies of similar A-chain loop specificity. These reagents were able to modulate the antigen-specific proliferation of an insulin-reactive, A-chain loop-specific T cell clone. Because the monoclonal antibodies and the T cell clone recognize a similar molecular domain of the insulin molecule, these data suggest that the anti-idiotypic sera mimic an insulin-like determinant, perhaps by bearing an "internal image" of the antigen and thereby interfering with T cell antigen recognition. Further, these results suggest that such reagents may be useful in characterization of T cell antigen receptor specificity and lend further credence to the concept of idiotypic-anti-idiotypic regulation of the immune response.

Animals↗
PubMed1978

Subcutaneous parotid effusion after mandibular osteotomy.

A case of subcutaneous parotid secretion after extraoral vertical mandibular osteotomy for prognathism has been described. The possible causes have been discussed and a rationale for conservative management has been suggested.

Humans↗