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Biomedical subjects

A H Ross

Publications and source records attributed to A H Ross.

At least 73 records · Page 4Linked to original sources
PubMed1990

Identical D region sequences expressed by murine monoclonal antibodies specific for a human tumor-associated antigen.

Sequence analysis has revealed significant structural similarities among murine mAb specific for the human tumor-associated Ag GA733. Antibodies generated in independent immunizations, either with Ag-positive tumor cells or with anti-idiotypic antibodies produced by immunization of goats with a GA733-specific antibody, all use members of the same gene families; remarkably, they also express identical amino acid sequences in their H chain CDR3. Inasmuch as this region normally exhibits considerable sequence variability, the identity displayed by the antibodies indicates a requirement for this particular sequence in the generation of their specificity for the GA733 antigen. Moreover, this homology suggests that the antibodies recognize a common determinant on the Ag, and that the polyclonal anti-idiotypic antibodies can functionally mimic the Ag at the level of the structure of the Ag-specific antibody that is induced.

Amino Acid Sequence↗
PubMed1990

Transient expression of full-length and truncated forms of the human nerve growth factor receptor.

To facilitate the characterization of nerve growth factor (NGF) receptor and mutated forms of the receptor, we have set up a rapid, efficient transient expression system utilizing COS cells. The human NGF receptor is a 427 amino acid protein with a hydrophobic signal sequence, a 222 amino acid extracellular domain, a single transmembrane domain and a 155 amino acid intracellular domain. The NGF receptor and a truncated form lacking the cytoplasmic domain were expressed in a COS cell expression system. Both recombinant proteins were detected on the cell surface and at a perinuclear site. Specific binding of 125I-NGF to the recombinant proteins was detected by chemical cross-linking. The extracellular domain of the NGF receptor was also expressed in the same system and detected in the COS cell endoplasmic reticulum and in the culture supernatant. This recombinant protein also specifically binds NGF.

Animals↗
PubMed1990

Introduction of nerve growth factor (NGF) receptors into a medulloblastoma cell line results in expression of high- and low-affinity NGF receptors but not NGF-mediated differentiation.

Expression of the cloned human nerve growth factor receptor (NGFR) cDNA in cell lines can generate both high- and low-affinity binding sites. Since the inability to respond appropriately to differentiation factors such as NGF may contribute to determining the malignant phenotype of neuroblastomas, we sought to determine whether the same is true of medulloblastomas. To generate a human central nervous system neuronal cell line that would respond to NGF, we infected the medulloblastoma cell line D283 MED with a defective retrovirus carrying the cDNA coding for the human NGFR. The resultant cells (MED-NGFR) expressed abundant low- and high-affinity NGFRs, and NGF treatment induced a rapid transient increase of c-fos mRNA in the NGFR-expressing cells but not in the parent line or in cells infected with virus lacking the cDNA insert. However, the MED-NGFR cells did not internalize the NGFR at high efficiency, nor did they differentiate in response to NGF. Three important conclusions emerge from this study: (i) internalization of NGFRs is not necessary for some early rapid transcriptional effects of NGF; (ii) an unknown factor(s) that cooperates with the cloned NGFR in allowing high-affinity NGF binding is found in a primitive central nervous system cell line; and (iii) NGFRs introduced into and expressed by D283 MED (i.e., MED-NGFR) cells are partially functional but are unable to induce differentiation in these primitive neuron-like tumor cells, implying that high-efficiency receptor-mediated endocytosis of NGF and its receptor may be a necessary step in the cascade of events leading to NGF-mediated differentiation.

Animals↗
PubMed1990

Lateral diffusion of nerve growth factor receptor: modulation by ligand-binding and cell-associated factors.

We compared the properties in human melanoma cell line A875 and rat pheochromocytoma cell line PC12 of nerve growth factor receptor (NGFr). We also analyzed NGFr and a truncated NGFR lacking the cytoplasmic domain, which were transiently expressed in COS cells. The full-length NGFR expressed in COS cells bound nerve growth factor (NGF) with positive cooperativity, but A875 NGFr and truncated NGFr in COS cells did not display positive cooperativity. The anti-human NGFr monoclonal antibody NGFR5 was characterized and found not to compete with NGF for binding to NGFr. Fabs were prepared from NGFR5 and 192, an anti-rat NGFR monoclonal antibody that was previously shown not to compete with NGF for binding. Fluorescein-labeled Fabs were used to measure the distribution and lateral diffusion of the NGFr. NGFr expressed on COS and A875 cells are diffusely distributed, but NGFr on the surface of PC12 cells appeared, for some cells, to be patched. In A875 cells, 51% of the NGFr was free to diffuse with diffusion coefficient (D) approximately 7 X 10(-10) cm2/s. In COS cells, 43% diffused with D approximately 5 X 10(-10) cm2/s. There was no significant difference in diffusibility between the full-length NGFr and the truncated NGFr. We compared NGFr diffusion on PC12 cells in suspension or adherent to collagen-coated coverslips. For suspension cells, we obtained 32% recovery with D approximately 2.5 X 10(-9) cm2/s. On adherent cells, we obtained 17% recovery with 6 X 10(-9) cm2/s. Binding of NGF enhanced lateral diffusion of NGFr in A875 cells and in PC12 cells in suspension but did not alter lateral diffusion of NGFr in COS cells or in adherent PC12 cells. NGF had no effect on the diffusing fraction or the distribution of NGFR for any cell line.

Animals↗
PubMed1990

Monoclonal antibody-dependent, cell-mediated cytotoxicity against human malignant gliomas.

Two monoclonal antibodies (MAbs), IgG2a MAb ASHG4 and IgG2b MAb ASHE2, were produced in mice immunized with cultured human malignant glioma cells. Both MAbs bound strongly to the surfaces of long-term cultured glioma cells, and MAb ASHE2 also bound strongly to short-term cultured glioma cells. Sections of frozen glioma tissues bound both MAbs strongly, whereas normal brain tissues showed weaker reactivities, and tissues derived from carcinomas of various histological types were completely unreactive. Furthermore, the MAbs did not bind to peripheral blood cells or bone marrow cells. Although both MAbs bound to the same Mr 27,000-29,000 protein, they may detect different or overlapping epitopes on this antigen. Because MAbs ASHE2 and ASHG4 lysed cultured glioma cells with human peripheral blood lymphocytes as effector cells, they are promising reagents for approaches to immunotherapy of human malignant gliomas.

Antibodies, Monoclonal↗
PubMed1990

Nerve growth factor-induced differentiation of human neuroblastoma and neuroepithelioma cell lines.

A series of neuroepithelioma and neuroblastoma cell lines were screened for nerve growth factor (NGF)-induced differentiation. All three neuroepithelioma cell lines and all nine neuroblastoma cell lines with amplified N-myc oncogene did not show any apparent NGF-induced differentiation. However, neurite extension was observed for three of six neuroblastoma cell lines with single-copy N-myc oncogene. The three responsive lines had a neuronal phenotype (short processes) which was enhanced by the addition of NGF. The three nonresponsive cell lines were flat without any processes. The addition of NGF to the responsive cell lines resulted in an up-regulation of neurofilament mRNA expression. Peripherin and synapsin, two markers of terminal neuronal differentiation, were not induced. There was little effect of NGF on the rate of cell growth or colony formation on soft agar. Binding of NGF to eight of the cell lines was analyzed by the method of Scatchard. Two responsive neuroblastoma cell lines and one nonresponsive neuroepithelioma cell line expressed both low- and high-affinity binding sites. Two nonresponsive neuroblastoma cell lines expressed only a small number of high-affinity binding sites, and two other nonresponsive neuroblastoma cell lines did not detectably bind NGF. Hence, NGF-induced differentiation is confined to a particular class of neural-related tumors, and, even for these cell lines, differentiation is incomplete.

Cell Differentiation↗
PubMed1990

Neural patterns in inflammatory bowel disease: an immunohistochemical survey.

The differential diagnosis of inflammatory bowel disease remains a significant diagnostic problem for surgical pathologists. Neural abnormalities, such as hypertrophy of nerve plexi, hyperplasia of ganglion cells, and ultrastructural axonal degeneration have been described in patients with regional enteritis. We performed an immunohistochemical survey of forty cases of regional enteritis, ulcerative colitis, nonspecific colitis, and normal colon. A panel of antibodies, directed against neuron-specific enolase, S-100 protein, synaptophysin, neurofilament protein, and nerve growth factor receptor, was utilized to evaluate the distribution of nerve fibers in paraffin-embedded tissue. Anti-synaptophysin and anti-nerve growth factor receptor highlighted small, arborizing nerve fibers in the mucosa, not apparent in the routinely stained sections. Intense staining of these fibers was observed in regional enteritis with antinerve growth factor receptor. This antibody may aid the discrimination of inflammatory bowel disease from other causes of colonic inflammation and facilitate the identification of regional enteritis in endoscopic biopsies.

Colitis, Ulcerative↗
PubMed1989

Analysis of nerve growth factor receptor expression in human neuroblastoma and neuroepithelioma cell lines.

A series of 22 neuroepithelioma and neuroblastoma cell lines were screened for expression of nerve growth factor receptor (NGFR) by flow cytometry, Western blotting, and Northern blotting. All 5 neuroepithelioma cell lines expressed cell surface NGFR, with 30-69% of cells NGFR positive, but the 17 neuroblastoma cell lines tested had a smaller percentage of cell surface NGFR-positive cells (0-21%) and 10 lines were completely lacking cell surface NGFR. SY5Y, a variant line with a neuronal phenotype derived from neuroblastoma line SKNSH, expressed much more NGFR than SHEP, a variant line with an epithelial-like phenotype also derived from SKNSH. By Western blotting, the Mr approximately 69,000 NGFR band was detected for all four neuroepithelioma cell lines tested but was visible for only 8 of 15 neuroblastoma cell lines tested. The band was most intense for neuroepithelioma cell lines SKNMC and TC32. For these two lines, a Mr approximately 56,000 and a Mr approximately 60,000 band were also detected. By Northern blotting, all three neuroepithelioma cell lines tested were positive for the 3.8 kilobase NGFR mRNA, but only 8 of 15 neuroblastoma cell lines were positive. Neuroepithelioma cell line TC32 and neuroblastoma cell line GICAN had the strongest expression of NGFR mRNA. These results demonstrate that NGFR is a biological marker for neuroepithelioma and that NGFR expression is heterogeneous for neuroblastoma cell lines. This series of neural cell lines differing in NGFR expression will be useful for future studies of regulation of NGFR expression and neuronal differentiation.

Antibodies, Monoclonal↗
PubMed1989

Management of infection after prosthetic abdominal rectopexy (Wells' procedure).

The management and results of treatment of eight cases of implant infection after a Wells' rectopexy for rectal prolapse are reported. Most infections presented within 3 months of the rectopexy. Fever, abdominal or pelvic pain, diarrhoea, and the passage of pus per rectum were common presenting features. Removal of the infected implant per rectum or per vaginum was successful in four of five attempts and is the recommended initial approach, particularly in cases occurring early after surgery. Despite removal of the implant early after rectopexy recurrent prolapse did not occur.

Abdomen↗
PubMed1989

Rapid quantitative immunohistochemical assessment of human peripheral neuropathies using a monoclonal antibody against nerve growth factor receptor.

An analysis of nerve growth factor (NGF) receptor expression and density in human sural nerve biopsies was performed by immunocytochemistry with a murine monoclonal antibody against the human NGF receptor. Quantitative assessment of immunostaining density was made by histospectrophotometry on frozen sections. Although there was enhanced expression of NGF receptor within endoneurium in all patients with clinical neuropathies, expression was highest in nerves with axonal disease, consistent with the proposal that disruption of axon-Schwann cell interactions triggers the re-expression of the NGF receptor. These results with human nerves, together with previous studies with animal models, suggest that NGF and NGF receptor play important roles in the general response to neuronal injury.

Adult↗
PubMed1989

Sequence investigation of the major gastrointestinal tumor-associated antigen gene family, GA733.

The monoclonal antibody-defined, tumor-associated antigen GA733 was purified from the SW948 human colorectal carcinoma cell line and its partial amino acid sequence was determined. By using a synthetic oligonucleotide probe, two recombinants were isolated from a total human genomic library. We prove the existence of a family of GA733 genes. One of the genomic isolates is demonstrated to be an intronless gene, which is transcribed in pancreatic carcinoma cell lines and in placenta. The GA733 proteins were observed to contain sequences homologous to a repeat unit occurring 10 times in thyroglobulin and once in the HLA-DR-associated invariant chain. A more evolutionarily distant relationship was found with the alpha chain of the interleukin 2 growth factor receptor.

Amino Acid Sequence↗
PubMed1989

Monoclonal antibodies as applied to tumor-associated antigens: the CO17-1A antigen.

The development of antitumor MAbs has led to the identification of many TAAs but no TSAs. It now seems unlikely that there are antigens exclusively associated with any tumor type. Nonetheless, there are numerous applications of the antitumor MAbs. They are used in serum assays to monitor tumor burden and in histologic assays to identify the tumor type. Initial attempts to use MAbs therapeutically have benefited some patients, but much work is needed to better understand the mechanism and to enhance this tumoricidal effect. Finally, the antitumor MAbs will allow biochemical and genetic analyses of the roles of TAAs in tumor progression.

Animals↗
PubMed1988

Molecular cloning and characterization of an antigen associated with early stages of melanoma tumor progression.

The melanoma-associated antigen ME491 is expressed strongly during the early stages of tumor progression. The ME491 gene was molecularly cloned by means of DNA-mediated gene transfer followed by screening a lambda genomic library with human repetitive Alu sequences as a probe. The cloned DNA, after transfection into mouse L-cells, generated a protein with characteristics that were indistinguishable in Western blot analysis from the ME491 antigen expressed by human melanoma cells. Repeat-free subfragments of the cloned DNA were used for further studies. By Northern blot analysis, the subfragments detected a single 1.2-kilobase mRNA in the transformants and various human melanoma cell lines. ME491 complementary DNA clones were then obtained by probing a melanoma complementary DNA library with the genomic subfragments. Nucleotide sequence analysis of the cloned complementary DNA indicated that the ME491 antigen consists of 237 amino acids (Mr 25,475) with four transmembrane regions and three putative N-glycosylation sites. No significant structural homology was observed with other proteins thus far reported. We observed that the amounts of mRNA varied greatly with different melanoma cell lines. Southern blot analysis revealed no amplification or rearrangement of the ME491 gene in the human melanoma cell lines tested, including both high and low expressors of this antigen. The ME491 gene has been mapped to chromosome region 12p12----12q13 by somatic cell hybrid analysis and more narrowly localized to 12q12----12q14 by in situ hybridization.

Amino Acid Sequence↗
PubMed1988

Cultures of human Schwann cells isolated from fetal nerves.

We describe a technique for the preparation of highly purified populations of Schwann cells (SC) from human fetal nerves. Cultures were prepared by chemical and mechanical dissociation of human fetal sciatic nerves by modification of the method of Kreider et al. developed for newborn rat nerve. A time course analysis of some SC-associated markers at different times in vitro was performed employing immunofluorescence (IF) and immunoperoxidase (IP) to determine the percentage of SC in culture and to evaluate the maintenance of specific SC characteristics. We compared this method with that of Askanas et al. which produces enriched SC cultures by utilizing successive re-explantation of the original nerve explant. After 48 h, approximately 90% of the cells were bipolar and S-100+ and over the next two weeks about 70-80% of cells were SC by cytologic and immunocytologic criteria. At 35 days, 35% were SC, whereas less than or equal to 2.5% of 35-day-old multi-explant cultures were SC. The SC obtained by this method displayed the typical morphological and immunological characteristics: they expressed surface laminin and nerve growth factor receptors, whereas fibronectin, which is localized on fibroblast surface, was absent.

Cell Separation↗
PubMed1988

Expression of nerve growth factor receptor in human peripheral neuropathies.

Nerve growth factor (NGF) receptors in human sural nerve biopsies were detected immunohistochemically using a monoclonal anti-human NGF receptor antibody. NGF receptors were not visualized within the endoneurium of normal adult nerves but were readily demonstrable within the endoneurium of nerves undergoing active axonal degeneration. Immunostaining of adjacent sections for S-100 beta protein, a specific Schwann cell marker, suggested that Schwann cells expressed the NGF receptors induced by axonal degeneration. Residual Schwann cells in nerves in which axons were completely depleted also expressed NGF receptors. NGF receptors were not detected in relation to thinly myelinated regenerating axons, nor were they detected in the endoneurium of nerves that had undergone segmental demyelination and remyelination. The increased expression of NGF receptors in human axonal neuropathies may contribute to regeneration by NGF-responsive neurons in these disorders.

Adult↗
PubMed1988

Expression of nerve growth factor receptor during human peripheral nerve development.

The expression of NGF receptors on human Schwann cells during development and myelination and in culture was analyzed using a murine monoclonal antibody to human NGF receptor. Nonmyelinated femoral nerves from 13- to 14-week fetuses stained strongly for NGF receptor, whereas tissues from later stages of development showed a decrease in the staining intensity. These changes correlated with the initiation of myelination (17-19 weeks), as observed by phase-contrast and electron microscopy, and the reactivity with monoclonal antibody 4C5, a marker of mature Schwann cells. In adult nerves, only the perineurium and few endoneurial cells were stained with anti-NGF receptor antibody. Cultured human fetal Schwann cells were positive for NGF receptor by immunofluorescence irregardless of donor age or length of time in culture. The decreased staining of NGF receptor with nerve maturation may reflect a dependence of antigen expression on Schwann cell differentiation and/or neuron-Schwann cell interaction.

Cells, Cultured↗