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Biomedical subjects

A C Morgan

Publications and source records attributed to A C Morgan.

At least 73 records · Page 4Linked to original sources
PubMed1984

Production of large quantities of human immunoglobulin in the ascites of athymic mice: implications for the development of anti-human idiotype monoclonal antibodies.

Human-murine hybridomas, generated by fusion of human CLL cells with non-Ig-secreting murine myeloma NS-1, and secreting the IgM kappa expressed on the CLL cell surface, were grown in the peritoneal cavity of doubly pristane-primed nude mice to produce large quantities of highly concentrated human IgM kappa for the development of anti-idiotype monoclonal antibodies. Although the hybridomas secreted only 0.8 to 1.5 micrograms/ml/10(6) cells in tissue culture, they produced 8 to 12 ml of ascites from two mice containing 1.7 mg/ml of human IgM. Partial purification was then easily accomplished by euglobulin precipitation. Two weekly injections of 100 micrograms of this material into BALB/c mice resulted in the development of both anti-idiotypic and anti-human IgM antibodies. This system overcomes the major hurdle of generating sufficient quantities of the human idiotypic Ig to immunize and screen for specific murine antiidiotypes, and provides the material to produce these therapeutically promising reagents rapidly for clinical trials. This is, to our knowledge, the first report of a human Ig produced in mouse ascites.

Animals↗
PubMed1983

Monoclonal antibody defined-human melanoma-associated antigens: molecular and phylogenetic studies in normal serum.

Normal human sera were analyzed for the presence and molecular form of two human melanoma-associated antigens (MAAs), the 250 "melanoma-specific" glycoprotein and the 100 K "common tumor antigen". The 250 K MAA was not synthesized by any cultures other than human melanoma and was not detectable in normal human serum. In contrast, the 100 K MAA, which is present in spent medium of cultured human melanoma, carcinoma and fetal melanocytes but not of adult normal cells, was found in normal human serum in nanogram quantities. This serum form of the 100 K MAA was also found in pooled sera of higher apes but not of lower species. The cell-derived form of the 100 K MAA, present in spent culture medium, had a similar phylogenetic distribution. The molecule was produced by cultured brain glia from gorilla, but not by melanoma cells from miniature swine or dog. The 100 K MAA from gorilla glia had a mol. wt identical to the molecule produced by human melanoma cells. Molecular characterization of this MAA in normal human serum showed that it was heterogeneous in size and was present in fractions greater than 100 kd after analytical HPLC gel sieving under non-denaturing conditions. In contrast, MAA from spent culture medium of melanoma cells was 100 kd or less in chemically defined medium (CDM) with no protein supplement, but had a higher mol. wt in CDM with BSA or fetal calf serum supplement, similar to the serum form of the molecule. An association of the 100 K MAA with albumin was demonstrated by analytical HPLC gel sieving and SDS-PAGE analysis of monoclonal antibody immunoprecipitates. The 100 K MAA was dissociated from albumin in normal human serum by treatment with SDS and fractionation by gel sieving. Under these conditions 100 K MAA from serum co-migrated with similarly treated 100 K from melanoma cells. These results indicate that the 100 K MAA is a normal serum constituent which forms a strong, non-covalent association with albumin and is evolutionarily restricted to higher apes or humans.

Animals↗
PubMed1983

Circulating immune complexes and tumor cell cytotoxins as prognostic indicators in malignant melanoma: a prospective study of 53 patients.

To evaluate the relationship between tumor burden and circulating immune complexes (IC) in malignant melanoma, we tested sera collected serially from 15 normal donors and 53 patients. Forty-eight of these had Stage III or IV disease at the outset of the study. The median survival time (MST) of ten patients with Stage IV disease whose sera contained C1q-binding IC at the outset of the study was 4.7 months; the MST of the 25 Stage IV patients whose sera were initially free of IC by this test was 8.65 months (p less than 0.02). C1q-binding IC were not found in the initial serum samples from 13 patients with Stage III or 5 patients with Stage I disease. Abnormal C1q binding tests were measured in 4 of 67 sera (6%) from 13 patients who remained free of evident tumor for up to 41 months. IC were detected in 13 of 39 sera (33%) from 19 patients with progressively growing tumors and in 21 of 68 sera (31%) from 21 patients who were initially free of disease but developed recurrences later, or who had significant remissions of variable duration during follow-up. The MST of 31 patients whose serial serum samples remained free of C1q-binding IC was 15.8 months. Twelve patients whose sera were initially free of circulating IC later developed abnormal serum C1q-binding levels. Their MST was 10.3 months. The MST of ten patients with persistently abnormal serum IC levels was 4.7 months. C1q-binding IC were reciprocally related to the presence of complement-dependent antibodies, cytotoxic for cultured allogeneic malignant melanoma cells in sera from 29 of these patients (r = -0.491;p = 0.003). These results suggest that the appearance of circulating C1q-binding IC is pathophysiologically important in malignant melanoma. Measurement of C1q-binding IC may be useful in assigning prognosis in this disease.

Antigen-Antibody Complex↗
PubMed1983

Monoclonal antibodies to human melanoma-associated antigens: an amplified enzyme-linked immunosorbent assay for the detection of antigen, antibody, and immune complexes.

An amplified, indirect biotin-avidin micro-enzyme-linked immunosorbent assay was developed for the measurement of human melanoma-associated antigens, either free or circulating with associated immunoglobulin in patient sera. Parameters and specificity of detection were assessed using monoclonal antibody to human melanoma-associated antigens. The main advantages of the assay are its flexibility, through the use of indirect detection and a variety of formats, and its sensitivity, with a lower limit of antibody detection at 100 pg/well and a lower limit of soluble antigen detection at 10 pg/well. The assay was applied to cell surface antigen detection with monoclonal antibody 9.2.27 to a melanoma-associated antigen against a panel of glutaraldehyde-fixed cells, and gave similar binding specificity as assessed by a previous 125I-Protein A assay. Utilizing a unique "sandwich" format, aMr 100,000 melanoma-carcinoma-associated antigen was quantitated in melanoma patient sera and found highly elevated in Stage IV disease. The same sandwich format was also used to detect and determine the class of human immunoglobulin associated with circulating Mr 100,000 human melanoma-associated antigens in normal donor sera. Thus, the sensitivity and flexibility of this enzyme-linked immunosorbent assay system make it particularly suitable for numerous applications in the study of monoclonal antibody-defined tumor-associated antigens.

Antibodies, Monoclonal↗
PubMed1982

"Hidden" cytotoxic antibodies that react with allogeneic cultured fetal and tumor cells contained in soluble immune complexes from normal human sera.

Hidden complement-dependent cytotoxins were demonstrated in normal donor sera after removing anti-immunoglobulins that normally block the ability of these immunoglobulin Gs to react with surface antigens on tumor cells. The blocking antibodies had certain properties of anti-idiotypes. Immunoglobulin G from Cohn Fraction II, after removing these anti-immunoglobulins, was cytotoxic for cultured fetal cells and for malignant melanoma, breast, lung, colon, and other tumor cells maintained either in tissue culture or by serial passage in nude mice. The cytotoxins were not adsorbed by extracts from normal lymphoid, liver, skin, or red blood cells. These results suggest that a heterogeneous group of natural antibodies reactive with antigens expressed on a variety of neoplastic and fetal cells circulate in normal donor sera as part of a soluble immune complex, together with a blocking anti-immunoglobulin.

Animals↗
PubMed1981

Immunochemical delineation of an oncofetal antigen on normal and simian virus 40-transformed human fetal melanocytes.

Human melanoma cells of uveal origin shed 94,000- and 240,000-dalton glycoproteins in common with most melanoma cell lines of dermal origin. Normal human melanocytes derived from fetal uvea shed a 90,000-dalton glycoprotein that was found to be immunologically identical with the 94,000-dalton glycoprotein of melanoma cells. Expression of this 90,000-dalton molecule was confined to fetal cells of ectodermal origin. After simian virus 40 (SV40) transformation of human fetal melanocytes, there was an apparent increase in molecular size of this component to 94,000 daltons. In contrast, the 240,000-dalton glycoprotein was not synthesized or shed by uninfected or SV40-transformed fetal melanocytes. These data suggest that the 94,000-dalton glycoprotein is an oncofetal antigen of ectodermal origin.

Antigens, Neoplasm↗
PubMed1981

Production and characterization of monoclonal antibody to a melanoma specific glycoprotein.

An immunogen consisting of a 4M urea extract derived from human melanoma cells (M14), that was devoid of HLA-A,B,C, HLA-DR antigens and fibronectin was adsorbed to lens culinaris lectin-Sepharose 4B and used to immunize mice for production of monoclonal antibody to a melanoma-specific glycoprotein. Screening for hybridomas secreting antibodies to melanoma associated antigens was facilitated by use of a solid phase target antigen of chemically defined medium of melanoma cells (CDM). Use of these procedures allowed us to select 40 hybridomas secreting antibody which recognized determinants on melanoma cells not found on lymphoid cells. Further characterization of one of these hybridomas, 9.2.27, indicated that the antibody it secreted recognized a 240K dalton glycoprotein found on all melanoma cell lines tested but not on carcinoma, lymphoid, or fibroblastoid cultures. These results demonstrate the utility of soluble antigen preparations devoid of strongly immunogenic non tumor-specific molecules in the elicitation of tumor specific antibody. Preliminary results suggest that immunogens of this kind are superior to intact melanoma cells for production of tumor specific hybridomas.

Animals↗
PubMed1981

Human melanoma-associated antigens: role of carbohydrate in shedding and cell surface expression.

The shedding of 2 tumor-specific glycoprotein antigens from human melanoma cells into spent culture medium was selectively inhibited by nontoxic doses (0.5 microgram/ml) of tunicamycin, an inhibitor of N-asparagine-linked glycosylation. The inhibition of shedding of these 2 antigens with m.w. of 240K and 94K is complete within 24 hr after addition of tunicamycin. During this time interval, our data suggest that these glycosylated cell surface antigens are replaced by their nonglycosylated forms. Removal of tunicamycin or addition of N-acetyl glucosamine restores shedding of these melanoma-associated antigens with initially reduced glycosylation. This same selective inhibition of shedding was observed with cultures adapted to grow in high doses (2.5 microgram/ml) of tunicamycin that otherwise killed > 98% of the cells upon first exposure. In contrast to other glycoproteins found in spent culture medium of melanoma cells, the shedding of melanoma-associated antigens is strictly dependent on glycosylation.

Animals↗
PubMed1980

Human melanoma associated antigens: a solid-phase assay for detection of specific antibody.

A solid-phase radioimmunometric binding assay is described utilizing 125I-labeled protein A for the detection of antibody to human melanoma associated antigens. The novel aspect of this assay is the use of chemically defined spent culture medium of melanoma cells at target antigens previously depleted of fibronectin by affinity chromatography. This makes it possible to screen for antibody in unabsorbed antiserum. Sensitivity, reproducibility and ease of performance of the assay are optimized by conjugating target antigens to a background of bovine serum albumin dried onto polyvinyl 96-well microtiter plates and cross-linked with glutaraldehyde. The use of an immobilized soluble antigen target derived from a large pool of spent culture medium facilitates direct interassay comparisons and permits extensive absorption analysis of antisera. The assay has considerable potential in screening for alloantibody and both poly- and monoclonal xeonoantibody to human melanoma associated antigens.

Animals↗
PubMed1978

Rheumatoid factor positive plasma and humoral cytotoxicity to malignant melanoma.

Complement dependent cytotoxicity to malignant melanoma tumor cells was demonstrated in incubations that included rheumatoid factor (RF) positive plasma and normal plasma. Cytotoxicity was not demonstrated to cells from a number of normal tissues. The phenomenon involved coating of tumor cells with immunoglobulins and complement fixation. The activity in RF positive plasma was present in high titer an may not be RF. The activity in normal plasma was present in low titer, was found in plasma from eight out of 11 healthy subjects and may be a naturally occurring antibody. This previously undescribed humoral cytotoxicity system may participate in tumor host interactions, especially after therapy, when the majority of patients become RF seropositive.

Animals↗
PubMed1978

Detection of immune complex-like materials in cancer patients' sera: a comparative study of results obtained with the C1q deviation and C1q binding tests.

In a collaborative study involving three laboratories, randomly coded sera from 47 patients and healthy donors were tested for soluble immune complexes by two versions of the C1q BT and by the C1q DT. Analysis of ranked data showed a close correlation between results obtained in all laboratories as well as good reproducibility on testing coded duplicate samples included in each panel of sera. However, with the use of values for normal donors established independently in each laboratory, the tests did not always agree in discriminating normal from abnormal sera, particularly with sera from cancer patients. Results reflected to some extent the methods used to inactivate the endogenous C1 complex in the test sera. Studies of 130 additional cancer patients revealed that 44 (34%) gave abnormal C1q BT values when inactivated with EDTA but only 35 (27%) were abnormal when heat inactivated (56 degrees for 30 min). Similarly 12 of 65 sera (18%) from patients with nonneoplastic diseases and 10 of 80 (13%) from healthy donors gave significantly abnormal results after addition of EDTA. Eight (12%) of the disease and six (8%) of the healthy controls' sera were similarly outside normal limits when heat inactivated. Repeated freezing and thawing of sera or changes in the concentration of PEG used to precipitate complex-bound 125I-C1q influenced C1q BT results more than duration of storage at -70 degrees C or changes ascribed to presence or absence of rheumatoid factors and CRP.

Antigen-Antibody Complex↗
PubMed1976

Human neoplastic and normal cells in tissue culture. I. Cell lines derived from malignant melanomas and normal melanocytes.

Forty-six cell lines derived from 31 human melanomas obtained from 28 patients were cultured. Fourteen of 16 lines have produced malignant tumors when injected into nude (thymus-deficient) mice. Tumors in 5 of the nude mice metastasized to distant lymph nodes and/or to the lungs of the mouse host. Extreme variability from line to line was observed for doubling time (34 to 106 hr), plating efficiency (0-86%), and melanin production. All tested lines had type B glucose-6-phosphate dehydrogenase, thereby excluding HeLa cell contamination. HeLa cells have been grown for some time in our laboratory. Our results clearly demonstrated that HeLa cell contamination does not occur invariably in heteroploid lines growing in a laboratory simultaneously with Hela cells, provided that proper care is taken to avoid such occurrence. Multiple cell lines derived from the same tumor had identical phosphoglucomutase enzyme phenotype, which suggested a lack of significant cross-contamination between the lines. Four long-term cultures of normal human uveal embryo melanocytes have also been established and characterized. Although all produced melanin after reaching saturation density, they differed from the melanoma cells morphologically; they were flat, not refringent, and lacked piling up and plating ability. When melanoma cells were exposed to bromodeoxyuridine (BUDR) for long periods, a phenotypic change toward non-neoplastic characteristics was observed. Cells became flat and not refringent and, when injected into nude mice, tumors appeared after a long latent period. These changes were completely reversible in vitro and in vivo. The BUDR-treated cultures were undistinguishable from the untreated mother cultures after 2 to 3 passages. Lines derived from tumors in nude mice (obtained by injection of BUDR-treated cells) were again indistinguishable from the untreated mother line. Normal melanocytes were mostly euploid; all the melanoma cells were aneuploid. All 29 cell lines derived from 14 patients had an average chromosome number higher than 46. Detailed group-by-group chromosome analysis always showed an excess of C chromosomes, which suggested that hyperreduplication of one or more C chromosomes is a specific characteristic of human melanomas.

Adult↗
PubMed1976

Selective lethal effect of supranormal temperatures on human neoplastic cells.

The effects of supranormal temperatures upon normal human cells (derived from normal adult and embryonal tissues) and neoplastic human cells (derived from biopsies of malignant tumors) have been quantitatively studied in tissue culture. Melanoma cells have been compared with melanocytes derived from fetal uveas. Colon carcinoma cells have been compared with embryonal intestinal epithelial cells. Malignant neuroepithelial cells derived from a teratocarcinoma of the ovary have been compared with neuroepithelial cells derived from fetal brain. Fibrosarcoma cells have been compared with normal adult fibroblasts. All cells defined as neoplastic have produced malignant tumors when injected into nude thymus-deficient mice at doses of 1 X 10(7) cells or less. Exposure to temperatures of 42.5-43.0 degrees for 4 to 8 hr has been shown to have significantly greater lethal effect on the tumor cells than on the nonneoplastic cells.

Carcinoma↗
PubMedDate not supplied

Enhanced in vitro tumor cell retention and internalization of antibody derivatized with synthetic peptides.

The Fab fragments of two antitumor monoclonal antibodies, NR-ML-05 and NR-LU-10, have been covalently derivatized with synthetic peptides designed to provide secondary sites of attachment to enhance their retention on tumor cells. Analogs of the peptide "GALA", an amphipathic peptide previously reported to interact with uncharged lipid bilayers, gave antibody conjugates of different molecular weight and bound peptide stoichiometry when attached to Fab fragments using the heterobifunctional cross-linker sulfo-SMCC. This attached peptide enhanced the retention and internalization of Fab fragments of NR-ML-05 on FEMX human melanoma cells, but not of NR-LU-10 on HT-29 human colon carcinoma cells, indicating that this effect might be specific for individual tumor antigen-antibody systems. This peptide appeared to increase nonspecific interactions of the conjugate with antigen-negative cells. Other membrane-active peptides were also tested. None were as effective as the "GALA" analogs. A synthetic ion channel peptide attached to NR-ML-05 Fab exhibited the greatest enhanced internalization of these tested peptides.

Amino Acid Sequence↗
PubMedDate not supplied

Synthesis of cobalamin-biotin conjugates that vary in the position of cobalamin coupling. Evaluation of cobalamin derivative binding to transcobalamin II.

Six cobalamin-biotin conjugates have been prepared. The cobalamin-biotin conjugates were prepared to evaluate the effect that the location of attachment had on the binding with transcobalamin II (TCII), the cobalamin binding protein in plasma, and to evaluate their potential use for in vitro and in vivo applications. This study focused only on the effect of binding with TCII. To decrease the possibility of steric problems in binding of the cobalamin conjugates with TCII, and biotin's binding with streptavidin or avidin, moieties of 11-18 atoms in length were used as linkers. Four biotin conjugates were prepared which were attached to the corrin ring of the cobalamin molecule (on b-, c-, d-, and e-side chains). One conjugate was attached to the 5'-OH of the ribose moiety, and another conjugate was attached at the cobalt metal (in place of the cyanide moiety of cyanocobalamin). Competitive binding studies were conducted where various amounts of the cobalamin-biotin conjugates and their precursor cobalamin derivatives competed with [57Co]cyanocobalamin for binding of recombinant human TCII (rhTCII). Evaluation of cobalamin derivatives which were conjugated at the 5'-OH of ribose or the cobalt metal center indicated that conjugation at either of these positions had little effect on binding with rhTCII. However, conjugates where the attachment was made on the corrin ring substituents had a large variation in binding with rhTCII. Conjugates on the e-propionamide side chain had little effect (relative affinity was equal to or decreased less than a factor of 3) on binding with rhTCII, conjugates of the b-isomer had decreased binding (relative affinity decreased less than a factor of 10), conjugates of the d-propionamide had further decreased binding (relative affinity decreased between 44 and 69 times), and conjugates on the c-acetamide group had poor binding to rhTCII (relative affinity decreased between 295 and 1160 times). The significance of the side chains on the corrin ring in providing specificity and high-affinity binding with rhTCII is discussed.

Binding, Competitive↗