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H Yeger

Publications and source records attributed to H Yeger.

At least 55 records · Page 3Linked to original sources
PubMed1996

Expression of gastrin-releasing peptide receptor gene in developing lung.

Bombesin (BN) or its mammalian counterpart, Gastrin-releasing peptide (GRP) is produced by pulmonary neuroendocrine cells (PNEC). The function of GRP as a growth factor involves lung morphogenesis, but the precise mechanism and the target cells have not been defined. We used a nonradioactive in situ hybridization (NISH) and Northern blot analysis for both GRP receptor (GRP-R) and its ligand GRP on samples of fetal and newborn human and rabbit lung. For immunolocalization of BN/GRP and the proliferating lung cell population we used anti-BN/GRP or MIB-1 antibodies, respectively. NISH and Northern blot showed peak expression of GRP-R mRNA on day 24 gestation in the fetal rabbit lung. At the cellular level, GRP-R mRNA was localized mainly in the distal airway epithelial tubes and surrounding mesenchyme, whereas proximal airways showed decreased signal. Epithelium expressing GRP-R showed positive labeling for proliferation antigen in contrast to PNEC, which were negative. In human post-natal lung, strong signal for GRP-R mRNA was localized in the airway epithelial cells and submucosal glands. PNEC in both rabbit and human lung expressed mRNA for GRP, but only in human lung were they immunoreactive for the peptide. However BN/GRP immunoreactive cells were detected in rabbit fetal lung culture. The expression of GRP-R in mammalian lung is developmentally regulated, peaking both spatially and temporally during the phase of rapid airway growth and differentiation. Both epithelial and mesenchymal components express GRP-R consistent with paracrine mechanism for GRP activity during lung morphogenesis.

3T3 Cells↗
PubMed1996

Pulmonary neuroendocrine cells in cultures of human infant airway mucosa from postmortem tissues.

Airway mucosa was isolated by enzymatic dissociation from tracheas and bronchi obtained at autopsy from stillbirth, infants, and children. An epithelial cell fraction was recovered that could be cultured on a collagen-coated substratum for at least 7 days. Epithelial cells were identified immunocytochemically with anticytokeratin antibody; pulmonary neuroendocrine cells (PNEC) were identified with antibodies against serotonin, bombesin/GRP, and MOC-1 cell surface antigen. Transmission electron microscopy confirmed neuroendocrine ultrastructural features of PNEC including the presence of neurosecretory dense core vesicles. The ability to recover and maintain viable PNEC in human lung airway epithelial cell cultures from postmortem tissues should facilitate further investigations of PNEC function in normal human lung and in various disease states.

Autopsy↗
PubMed1996

Immunohistochemical detection of p53 in Wilms' tumors correlates with unfavorable outcome.

The role of p53 in the pathogenesis and progression of Wilms' tumors is only partly understood. Although p53 mutations were initially reported only in anaplastic Wilms' tumors, we had reported that, of two of twenty-one cases that had a p53 mutation, one tumor showed no evidence of anaplasia. To determine the significance of p53 expression in all clinical stages of Wilms' tumor, twenty-eight cases were analyzed for p53 immunoreactivity. Paraffin sections were immunolabeled with two different monoclonal antibodies, recognizing both mutant and wild-type p53. Fifteen of sixteen tumors in the recurrent/metastatic group and three of twelve tumors in the nonmetastatic/nonrecurrent group showed p53 immunopositivity. Only one of three positive tumors in the latter group showed moderate to strong positivity, whereas twelve of sixteen metastatic/recurrent tumors revealed a similar degree of p53 positivity. The positivity was stronger in the metastasis/recurrences as compared with the corresponding primary tumor. Western blot analysis revealed p53 expression in all of the Wilms' tumors tested, suggesting its involvement in the development of Wilms' tumors. Single-strand conformation polymorphism analysis performed on twenty-three of these tumors revealed p53 mutations in four of fourteen recurrent/metastatic tumors and none in the nonmetastatic/nonrecurrent group. Our results show that, whereas 60% of cases were immunopositive for p53 protein, mutations were detected in only 16% of tumors, indicating that wild-type p53 protein is retained in the other tumors. We conclude that p53 immunopositivity strongly correlates with recurrence/metastasis in Wilms' tumors. Furthermore, the accumulation of p53 in these tumors is not only due to mutations but may also involve stabilization of normal p53 with other proteins.

Blotting, Western↗
PubMed1995

Acellular matrix: a biomaterials approach for coronary artery bypass and heart valve replacement.

We have developed a multistep detergent-enzymatic extraction process (involving hypotonic and hypertonic solutions, the detergents octyl-phenoxy-polyethoxyethanol and sodium dodecyl sulfate, as well as DNAse and RNAse) which, while inhibiting autolysis, removes all cells from tissues and, with them, cellular antigens together with lipids and more soluble glycosaminoglycans. What remains is acellular matrix with the structural proteins well conserved and normally arranged. Canine arteries extracted to acellular matrix were implanted as coronary artery bypass allografts in a canine model, without the use of cardiopulmonary bypass, and compared with autogenous saphenous veins. Of nine pilot acellular matrix implants, four were patent, as compared with four of seven saphenous vein grafts. All occlusions in both graft types occurred acutely soon after implantation, with almost all patent grafts followed up for 6 months. The acellular matrix allografts showed no inflammation and only minimal cellular repopulation. This model needs further development, but appears promising for preclinical evaluation. Canine aortic and pulmonic valves extracted to acellular matrix using a modification of our extraction process, eliminating the detergent sodium dodecyl sulfate, were implanted heterotopically as allografts in the left main pulmonary artery in dogs, a location chosen to avoid the need for cardiopulmonary bypass. At 1 month, two-dimensional echocardiography of six implants showed leaflet motion and 3- to 5-mm Hg transvalvular gradients. Explant histology of four valves at 1 month showed no inflammation, cellular repopulation at the base of the valve, and partial endothelialization.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Mutations of the p53 tumor suppressor gene occur infrequently in Wilms' tumor.

Mutations of the p53 tumor suppressor gene occur frequently in a variety of adult-onset tumors, including colon, breast, lung, and brain, yet are infrequently identified in pediatric malignancies. Wilms' tumor, a common solid tumor of childhood, can be associated with mutations of the WT1 gene. Alterations of the p53 gene have been shown to modulate the ability of WT1 to transactivate its targets. Although positive p53 immunostaining has been demonstrated in Wilms' tumors, the correlation to p53 gene mutations is not clear. We examined Wilms' tumor samples for p53 mutations utilizing polymerase chain reaction-single-strand conformation polymorphism analysis and single-strand DNA sequencing. Mutations in the coding region of the p53 gene were demonstrated in 2 of 21 (9.5%) Wilms' tumors. Each mutation yielded a substitution of amino acid residues. One mutation was located in exon 6 and the other in exon 7. Both mutations were found in tumors from patients with advanced stage disease. Focal anaplasia was demonstrated in one of these tumors. Our data suggest that although p53 mutations occur infrequently in Wilms' tumor, they may be associated with advanced disease.

Adult↗
PubMed1994

NUB-7: a stable I-type human neuroblastoma cell line inducible along N- and S-type cell lineages.

Human NB cell lines express features of one or more of three recognizable phenotypes that include N-type (neuroblastic), S-type (Schwannian), and I-type (intermediate phenotype) cells. The I-type cell, which shares properties of both N- and S-type cells, is thought to represent the progenitor cell from which the other two cell types are derived. The MYCN amplified NB cell line NUB-7, established in our laboratory, is now shown to be composed principally of I-type cells. The observed phenotype was stable in culture and was representative of the original surgically resected tumor. The I-type cell designation was established based on morphological characteristics, the coexpression of various N-type (neurofilaments, peripherin, GAP-43, NCAM, MYCN) and S-type cell (vimentin, laminin, fibronectin) markers, and the relatively high level of expression of these markers in comparison to five predominantly N-type, one S-type, and one N/S mixed NB cell lines. Dibutyryl cyclic AMP and retinoic acid induced enhanced expression of N- and S-type phenotypes, respectively, in NUB-7 as supported by specific morphological changes, reduced growth, and changes in the levels of expression of both N- and S-type markers. Our studies with the NUB-7 cell line have now provided convincing evidence for the existence of a bipotential progenitor of N- and S-type cells in NB. As well, the NUB-7 cell line may also represent the tumor counterpart of a sympathetic ganglion progenitor cell.

Bucladesine↗
PubMed1993

Oxygen sensing in airway chemoreceptors.

Pulmonary neuroepithelial bodies, composed of innervated clusters of amine- and peptide-containing cells, are widely distributed throughout the airway mucosa of human and animal lungs. Structurally, neuroepithelial bodies resemble chemoreceptors (such as carotid body, taste buds) and are thought to function as hypoxia sensitive airway sensors. Evidence for this is indirect, however, and the mechanism of oxygen sensing by these cells is unknown. Here we culture neuroepithelial bodies isolated from rabbit fetal lungs and identify voltage-activated potassium, calcium and sodium currents using the whole-cell patch clamp technique. Upon exposure to hypoxia there is a reversible reduction (25-30%) in the outward potassium current, with no change in inward currents. In addition, we demonstrate the expression of an oxygen-binding protein (b-cytochrome, NADPH oxidase) on the plasma membrane of these cells. The identification of an oxygen-sensing mechanism (namely the presence of an O2-sensitive potassium channel coupled to an O2 sensor protein) in the cells of pulmonary neuroepithelial bodies indicates that they are transducers of the hypoxia stimulus and hence may function as airway chemoreceptors in the regulation of respiration.

Animals↗
PubMed1993

Homozygous somatic Wt1 point mutations in sporadic unilateral Wilms tumor.

Wilms tumor may be caused by loss of function of genes at different loci. A Wilms tumor suppressor gene, WT1, at chromosome 11 band p13, has recently been cloned and characterized. WT1 has been implicated in the development of Wilms tumor by virtue of mutations in patients with genitourinary anomalies and susceptibility to Wilms tumor. Homozygous intragenic mutations have been reported in Wilms tumors, but usually not in sporadic unilateral Wilms tumors, which constitute the majority of Wilms tumor cases. Using the single-strand conformational polymorphism assay, we have identified three sporadic unilateral Wilms tumors with homozygous point mutations: one with a de novo germ-line nonsense point mutation within WT1 exon 8, and two carrying a somatic mutation within WT1 exon 10. In all three cases loss of the wild-type allele was demonstrated by tumor loss of heterozygosity. This report provides an example of two somatic mutations in the same tumor expected to inactivate WT1 function.

Amino Acid Sequence↗
PubMed1993

Expression and distribution of peripherin protein in human neuroblastoma cell lines.

A series of human neuroblastoma (NB) cell lines was analyzed for expression of peripherin, a class-III intermediate filament protein expressed at high levels in ganglia of the peripheral nervous system. By Western blotting, peripherin protein was detected in all human NB cell lines examined. The highest level of peripherin was found in the NUB-7 cell line, previously characterized as homogeneously neuroblastic. By immunofluorescence labeling, peripherin was shown to be organized in a perinuclear filamentous pattern and, exemplified by IMR32 cells, was also shown to be localized to spontaneously formed neurites. Peripherin was expressed in neuroblastic but not substrate-adherent cells, and was found at low levels in I-type cells. There was a pronounced redistribution of peripherin to neurites formed in response to dibutyryl cyclic adenosine monophosphate (dbcAMP) and all-trans-retinoic acid (RA). In NUB-7 cells, which do not extend neurites in response to nerve growth factor, there was no change in the level of peripherin protein following treatment with this agent. Both dbcAMP and RA induced a redistribution of peripherin to neurite extensions, but only treatment with RA increased the level of the protein as demonstrated with NUB-6A4 and NUB-6C4 subclones. Peripherin was also variably expressed in peripheral neuroepithelioma (NE) cell lines tested, but was organized into a more basket-like filamentous pattern in these cells. The heterogeneous expression and distribution of peripherin in NB and NE cell lines indicate that this protein is associated with maturation of the neuronal phenotype and hence serves as a differentiation marker for tumors derived from the neural crest.

Blotting, Western↗
PubMed1993

Cell biology of pulmonary neuroepithelial bodies--validation of an in vitro model. I. Effects of hypoxia and Ca2+ ionophore on serotonin content and exocytosis of dense core vesicles.

Pulmonary neuroendocrine (NE) cells including the innervated clusters of NE cells--neuroepithelial bodies (NEB)--are difficult to study because of their small numbers and diffuse distribution within the airway mucosa of the lung. We have previously reported a method for isolation and culture of NE cells from rabbit fetal using a combination of mechanical and enzymatic dissociation followed by gradient centrifugation. This method provides single cell suspension of mixed lung cells enriched in NE cells, particularly those originating from NEB. This study further validates our in vitro model by detailed morphologic characterization of cultured NEB cells using high resolution light microscopy, transmission and scanning electron microscopy, HPLC for detection of serotonin (5-HT), and molecular (Northern blot) analysis of mRNA encoding for 5-HT synthesizing enzymes, tryptophane hydroxylase, and aromatic L-amino acid decarboxylase. In addition the effects of hypoxia on NEB cells in vitro were investigated to define the role of these cells as possible airway chemoreceptors. Exposure of NEB cultures to hypoxia resulted in decreased intracellular content of 5-HT accompanied by increased exocytosis of dense core vesicles (DCV). The amount of 5-HT release correlated with the degree of hypoxia, suggesting modulation by ambient pO2 levels. The role of Ca2+ ions in exocytosis of DCV and 5-HT release from NEB cells was tested in experiments with Ca2+ ionophore (A23187). Exposure of cultures to 5 micrograms/ml of ionophore resulted in up to 40% reduction in 5-HT content of NEB cultures as well as increased exocytosis of DCV. Our overall findings are consistent with a view that NEB cells are chemosensory in nature and that Ca2+ signaling pathway is involved in stimulus-secretion coupling. Further refinements in cell separation and culture methodology are required before more detailed investigation of NEB cell membrane properties, signal transduction mechanisms, and intracellular signaling pathways can be carried out.

Animals↗
PubMed1993

Variant translocations of chromosome 22 in Ewing's sarcoma.

Relatively few variant translocations have been reported in primary Ewing's sarcomas (ES). We report two new variant translocations, both of which involve chromosomal rearrangements of 22q12. Cytogenetic studies of tumor cells from a 12-year-old girl revealed a variant translocation, t(7;22)(p22;q12), the second example reported of a simple variant of the 22q12 reciprocal translocation in this type of sarcoma. The identity of this rearrangement was confirmed by in situ hybridization. In addition, a complex translocation was identified in a dysmorphic 15-year-old girl, t(4;11;22)(q21;q24;q12). No previous cases of variant translocations in ES have involved band 7p22 or 4q21, and there are no previous reports of an association between congenital abnormalities and unusual karyotype abnormalities in ES. Both variant translocations conserve the junction on the der (22), providing additional cytogenetic evidence that the sequences on chromosome 22 are critical.

Child↗
PubMed1993

Detection of MYCN gene amplification and deletions of chromosome 1p in neuroblastoma by in situ hybridization using routine histologic sections.

BACKGROUND: Amplification of the MYCN oncogene and partial deletion of chromosome 1p are genetic changes frequently seen in neuroblastoma that are indicators of poor prognosis. The identification techniques usually used--Southern blotting for MYCN gene amplification, and karyotypic analysis of viable tumor cells for large 1p deletions--are time-consuming and suited for specialized laboratories. EXPERIMENTAL DESIGN: We have developed an immunofluorescence in situ hybridization assay suitable for detection of MYCN amplification and 1p deletion in formalin-fixed, paraffin-embedded tissue sections. The technique is rapid, does not involve the use of radioactivity, and can be carried out in laboratories already familiar with conventional in situ hybridization and immunohistochemical detection methods. RESULTS: MYCN gene amplification appeared as multiple signals per nucleus, corresponding to double minute chromosomes. Deletion of 1p was detected by loss of one of the normal signals present within the nucleus of a neuroblastoma cell line. Use of different fluorophores enabled the simultaneous detection of MYCN gene amplification and 1p deletion at the individual cell level. Detection was found to be enhanced by RNase and pepsin treatment of tissue sections before hybridization and by a novel microwave denaturation method. CONCLUSIONS: Application of this methodology to formalin-fixed samples of neuroblastoma will permit comprehensive retrospective studies of these two genetic markers using archived tumors. In situ hybridization surveys will have widespread applications for studying known genetic aberrations in individual cells of a variety of solid tumors and for determining interrelationships and clinical significance in relation to tumor progression and patient outcome.

Chromosomes, Human, Pair 1↗
PubMed1992

Detection of amplified DNA sequences in human tumor cell lines by fluorescence in situ hybridization.

An unambiguous and rapid characterization of amplified DNA sequences in tumor cells is important for the understanding of neoplastic progression. This study was conducted to evaluate the potential of fluorescence in situ hybridization (FISH) to identify such amplified DNA sequences in human tumor cell lines. Applying this technique, we followed the metaphase location and interphase position of amplified DNA sequences corresponding to the SAMK, MYC, and MYCN genes in four cell lines derived from human tumors: two gastric carcinoma lines (KATO III and SNU-16), a neuroblastoma (NUB-7), and a neuroepithelioma (NUB-20) line. In metaphase cells of KATO III, NUB-7, and NUB-20 lines, the amplified regions were clearly visible and easily identified at an intrachromosomal location: in KATO III and NUB-7 at a terminal position and in NUB-20 at an interstitial position. In SNU-16, on the other hand, the amplified SAMK and MYC sequences were identified in extrachromosomal double minute chromosomes (DMs). In this line, the SAMK and MYC sequences were coamplified in the same cells and were colocated on the same DMs. FISH also allowed the identification of amplified DNA sequences in nondividing cells, enabling us to distinguish, at interphase, whether the amplification gave rise to intrachromosomal amplified regions (IARs) or to extrachromosomal DMs. The FISH technique also allowed us to determine at metaphase as well as at interphase the extent of amplification and the size of the IARs.

DNA Probes↗
PubMed1992

Loss of heterozygosity mapping in Wilms tumor indicates the involvement of three distinct regions and a limited role for nondisjunction or mitotic recombination.

Loss of heterozygosity (LOH) for polymorphic markers is a frequently occurring event in some tumors, reflecting the role of allele loss in the development of these tumors. We have determined LOH in 38 cases of Wilms tumor for the 2 known loci on chromosome arm 11p and for a newly detected locus on chromosome arm 16q. Only 7 of the 38 tumors studied showed reduction to homozygosity of 11p13 markers. In 4 of these tumors, reduced expression of WT1 and WIT1, genes located at 11p13 and implicated in Wilms tumorigenesis, was noted. However, this was also found in 2 of 7 tumors showing LOH exclusively of 11p15 markers and in 15 of the remaining 24 tumors in which there was no LOH for 11p markers. This suggests that events not involving mitotic recombination or chromosome nondisjunction are the most common mechanisms for mutations at the 11p Wilms tumor locus. We also noted that mitotic recombination involving 11p15 loci occurred in addition to reduced expression of the 11p13 locus genes in 2 tumors, suggesting a possible interaction between these 2 loci. In addition, LOH for 16q markers was observed in 6 tumors. In one case this was coincident with reduction of WT1 and WIT1 gene expression, and in 3 other cases it occurred in addition to 11p LOH. This indicates that an additional locus on 16q is likely to be involved in Wilms tumorigenesis.

Adolescent↗
PubMed1992

Increased globotriaosylceramide in familial dysautonomia.

Familial Dysautonomia (FD) is an autosomal recessive disease of unknown etiology, occurring primarily in Ashkenazi Jews. Patients are neurologically impaired, with deficits primarily in autonomic and sensory functions. The biochemical and genetic defects have remained elusive, precluding carrier detection and prenatal diagnosis. High-performance liquid chromatography data indicated up to a threefold increase in the neutral glycosphingolipid globotriaosylceramide in Dysautonomic fibroblasts and lymphoblasts. Total ganglioside values, measured by colorimetric, fluorometric or specific sodium borohydride incorporation, were decreased. Affected fibroblasts exhibited a range of pleomorphic phenotypes, such that the usual swirl-like confluent growth pattern of normal fibroblasts was distorted to varying degrees, suggesting abnormalities in the FD plasma membrane, possibly affecting cell-cell contacts. The glycosphingolipid increase could not be accounted for on the basis of markedly decreased alpha-galactosidase activity, as in Fabry's disease, where patients also display decreased autonomic function.

Cell Line↗
PubMed1992

Isolation and culture of neuroendocrine cells from fetal rabbit lung using immunomagnetic techniques.

We describe a novel method for the isolation and subsequent culture of pulmonary neuroendocrine cells (PNEC) from normal fetal rabbit lung using immunomagnetic techniques with a monoclonal antibody, MOC-1. This surface antigen has originally been identified on small cell carcinoma of the lung. Our immunohistochemical studies have shown that MOC-1 cross-reacts with PNEC of human and rabbit fetal lungs on frozen sections, and in fixed cultures of rabbit fetal lung. Using a combination of mechanical and enzymatic disaggregation, a single-cell suspension of fetal rabbit lung was obtained. These cells were incubated with MOC-1 conjugated to magnetic beads. PNEC were selectively removed from the heterogeneous mixture using a magnet, giving up to 2-fold enrichment compared with our previously reported method. These cells were maintained in culture in a functional state for up to 7 days. The ability to prepare PNEC from rabbit fetal lung offers an opportunity to develop in vitro models to investigate the physiologic and biochemical properties of these cells, and ultimately it may lead to a better understanding of their function in health and disease.

Animals↗
PubMed1992

Coordinate expression of Wilms' tumor genes correlates with Wilms' tumor phenotypes.

The cloning and molecular characterization of two putative tumor genes, WT1 and WIT1, from the chromosome 11p13 region has provided a means of evaluating their role in the generation of Wilms' tumor heterogeneity. A series of 29 tumors were analyzed for WT1 and WIT1 expression by Northern blot or RNase protection analyses, and results were compared with tumor histopathology. Tumors were scored for the percentage of mesenchymal and epithelial derived tissue components. Homotypic tumors comprised blastema, tubular epithelium, and a fibroblast-like mesenchyme. In addition to these tissue components, the group of tumors designated as heterotypic also contained ectopic cell phenotypes such as muscle and squamous epithelium. The analyses suggest that heterotypic differentiation patterns occur when WT1 and WIT1 expression is low relative to normal fetal kidney. In situ hybridization using antisense RNA probes showed that WT1 and WIT1 were concordantly expressed in normal fetal kidney and in the blastema of tumors. The ratio of WT1:WIT1 expression remained relatively constant in homotypic tumors but deviated significantly in heterotypic tumors. These results suggest that expression patterns of the WT1 and WIT1 genes can be closely correlated to Wilms' tumor histopathology.

Blotting, Northern↗