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Biomedical subjects

M Liebert

Publications and source records attributed to M Liebert.

At least 73 records · Page 4Linked to original sources
PubMed1987

A semi-automated fluorescent (SAF) assay using viable, whole cells for screening hybridoma supernatants.

In the production of monoclonal antibodies, a rapid, sensitive, accurate assay is needed for the critical step of screening. We report the modification of an assay using viable whole cells for screening hybridoma supernatants. The modified assay uses fluorescent second antibodies for detection and has been adapted to an instrument capable of automating a number of assay steps. The modified assay is compared to a dot radioimmunoassay developed and used in our laboratory. The fluorescence assay is highly sensitive but shows more background effect, especially in samples with high protein content, such as ascites. The automated fluorescence assay is very rapid, capable of completing an assay in less than 90 min, and can be performed with minimal operator involvement. The assay was performed successfully with several different antibodies and cell types. This screening procedure should be especially useful for laboratories with large numbers of fusions to evaluate.

Antibodies, Monoclonal↗
PubMed1987

Detection of SSEA-1 on human renal tumors.

Stage-specific embryonic antigen-1 (SSEA-1) was localized on paraffin embedded, formalin fixed specimens of human renal tumors by immunoperoxidase staining using a monoclonal antibody. Of 19 renal cell carcinoma (RCC) samples tested, 12 were positive for SSEA-1; SSEA-1 was also found on distinct elements in two samples of Wilms' tumor. No correlation was found between expression of SSEA-1, and RCC morphology or pattern of growth. Because SSEA-1 is found on proximal tubules in the normal kidney, these results support the hypothesis that RCC arises from the cells of the proximal tubule. Furthermore, since greater than 60% of the RCCs examined expressed SSEA-1, this antigen may prove to be a useful target for immunolocation or therapy of metastatic RCC.

Antigens, Neoplasm↗
PubMed1987

High-dose, unlabeled, nonspecific antibody pretreatment: influence on specific antibody localization to human melanoma xenografts.

Nonspecific uptake of radiolabeled monoclonal antibodies in normal tissues is a significant problem for tumor imaging. A potential means of decreasing nonspecific antibody binding is to "blockade" nonspecific antibody binding sites by predosing with cold, nonspecific isotype-matched antibody, before injecting specific antibody. Nontumor-specific murine monoclonal antibody LK2H10 (IgG1) or Ab-1 (IgG2a) was given i.v. at doses of 0 to 3.5 mg to nude mice with xenografts of human melanoma. These mice were then given i.v. 4 micrograms of 131I anti-high molecular weight antigen of melanoma (HMWMAA) monoclonal antibody 763.24T (IgG1) or 225.28S (IgG2a), respectively. These mice were also given a tracer dose of 125I LK2H10 or Ab-1, respectively. Specific tumor uptake of anti-HMWMAA antibodies was see in all cases. No drop in tumor or nontumor uptake was demonstrated for either of the tumor-specific or nonspecific monoclonal antibodies due to nonspecific monoclonal antibody pretreatment. These data suggest that high doses of isotype-matched unlabeled nonspecific monoclonal antibody given before 131I tumor-specific monoclonal antibody, will not enhance tumor imaging.

Animals↗
PubMed1987

Monoclonal antibody (5G6.4) against ovarian carcinoma shows inhibition of in vitro colony formation.

Monoclonal antibodies (MAbs) have the potential for diagnosis and therapy of cancer, 5G6.4 is a MAb of the IgG2a class which was produced by immunization of BALB/c mice with human ovarian carcinoma (Ov Ca) cells. To further characterize 5G6.4, its effect on cell growth was tested using a human Ov Ca cell line established in our laboratory. A clonogenic assay was set up in 48-well plates in a double agar system. The cells were plated and 5G6.4 was added at different concentrations. Control plates consisted of cells with media without MAb. Negative control plates were also prepared using the same concentrations of an isotype-matched antimelanoma MAb, 225.28s. Colony formation (CF) was reduced to 50% or less of control with increasing amounts of 5G6.4 up to 50 micrograms/ml. Although CF was still depressed at concentrations above 50 micrograms/ml, the inhibition did not follow a directly proportional line; instead, it followed a bell-shaped curve. Plates with the control MAb, 225.28s, did not show this response. Similar results were obtained with cells from malignant Ov Ca ascites in the same clonogenic assay. Our study suggests that in the evaluation of the in vitro effect of MAb on growth, the concentration of MAb is crucial and may not show a linear response and that 5G6.4 may have a direct therapeutic effect by blocking the growth of Ov Ca cells. 5G6.4 is presently under study for therapy in an animal model.

Antibodies, Monoclonal↗
PubMed1987

Products of cells cultured from gliomas. V. Cytology and morphometry of two cell types cultured from glioma.

Explants of cells of a human glioma were evaluated with the nuclear fluorochrome 4',6-diamidino-2-phenylindole, by phase-contrast illumination, and by Giemsa staining correlated with double immunofluorescence for glial fibrillary acidic protein (GFAP) and fibronectin (FN). FN-positive (FN+) cells lacked GFAP detectable by immunofluorescence. Their mean nuclear-to-cytoplasmic ratio was large (0.192). Actual mean areas of nuclei (1,252 microns2) and cytoplasm (8,376 microns2) of FN+ cells compared with mean areas of fibroblasts suggested that the high nuclear-to-cytoplasmic ratio of FN+ cells was due to their microscopically evident reduced cytoplasmic spreading rather than to larger nuclei. Some FN+ cells showed marked variation in nuclear and nucleolar size and shape. Others had abnormal mitoses or hyperchromatic nuclei. GFAP-positive (GFAP+) cells lacked FN detectable by immunofluorescence. GFAP+ cells were smaller and less round than FN+ cells. Their usual location was growing on a layer of FN+ cells. The mean nuclear-to-cytoplasmic ratio (0.245) of GFAP+ cells was the highest in the study, surpassing the ratio of the continuous glioma line LM (0.176). Mean areas of nuclei (289 microns2) and of cytoplasm (1,350 microns2) of GFAP+ cells suggested that their high nuclear-to-cytoplasmic ratio was due to their microscopically evident reduced cytoplasmic spreading. Reduced spreading was associated with extension of long, thin cytoplasmic processes. The majority of GFAP+ cells showed marked cytoplasmic basophilia, nuclear hyperchromasia, and clumped chromatin. Features observed in both FN+ and GFAP+ cells from this high-grade astrocytoma are features associated with malignant transformation in more thoroughly studied tumor systems.

Antigens, Neoplasm↗
PubMed1987

Kinetics of interstitially administered monoclonal antibodies for purposes of lymphoscintigraphy.

The clearance rates of radiolabeled murine monoclonal intact IgG, F(ab')2 Fab and of an IgM following subcutaneous administration were evaluated in normal mice and rats using nuclear imaging and counting techniques. These studies suggest no significant difference in clearance rate exists between intact IgG and its F(ab')2 fragment, and little difference between these moieties and intact IgM. Fab is cleared considerably faster than the others, however. While significant differences in clearance rates exist, the magnitude of the differences are not as large as those following intravenous injection particularly when ambulation by the animal is allowed. When ambulation is allowed, clearance rates of all classes and fragments are accelerated and quite similar. Injection into the subcutaneous tissues of the footpad results in consistently faster clearance than an injection into the subcutaneous tissues of the abdomen. Ambulation considerably increased the clearance of antibodies, presumably by increasing lymph flow. These studies imply that the choice of intact antibody versus fragments for kinetic reasons may be less critical (particularly if ambulation is allowed) by the subcutaneous as compared with the intravenous delivery route. This kinetic information should be useful in designing imaging protocols with radiolabeled antibodies administered subcutaneously for purposes of imaging disease processes involving the lymphatics.

Animals↗
PubMed1986

Effect of herpesvirus infections on T-lymphocyte subpopulations and blastogenic responses in renal transplant recipients receiving cyclosporine.

The immunosuppressive effects of three herpesviruses--cytomegalovirus (CMV), Epstein-Barr virus (EBV), and herpes simplex virus (HSV)--were assessed in 29 renal transplant recipients treated with cyclosporine and prednisone. The ratios of Leu 3-positive ("helper-inducer") to Leu 2-positive ("suppressor-cytotoxic") T lymphocytes in peripheral blood were only moderately and transiently decreased during primary CMV infection, with or without concurrent reactivated EBV and HSV infections. This effect was due to an increase in absolute numbers of Leu 2-phenotypic and decrease in Leu 3-phenotypic T cells and was associated with symptomatic viral illness. Reactivated CMV infection alone or together with reactivated EBV and HSV infections resulted in less significant alterations in T-cell subsets than did primary CMV infection. Lymphocyte blastogenesis was not significantly altered during the herpesvirus infections. The data suggest that cyclosporine treatment inhibits the activation of suppressor cells and depression of cellular immune function that have been associated with herpesvirus infections in renal transplant recipients undergoing conventional immunotherapy.

Adolescent↗
PubMed1986

The influence of monoclonal antibody dose on tumor uptake of radiolabeled antibody.

The impact of antibody protein dose on tumor accumulation of radiolabeled monoclonal antibody was studied in nude mice with xenografts of human melanomas. 225.28S, a murine monoclonal antibody reactive with a high-molecular weight antigen of melanoma, was radiolabeled with I-125 and administered intraperitoneally to nude mice with human melanoma xenografts. Three days later, the animals were sacrificed, and tumor and normal tissue uptake of I-125 antibody was determined. At doses of 6.25, 62.5, 625 and 1875 ug of monoclonal antibody, there were no significant differences in percent of injected dose reaching the tumor/g of tumor or in the non-tumor uptakes achieved. These findings indicate that in the melanoma system, antibody dose is not a critical determinant of tumor uptake, and additionally indicate that low doses of antibody protein are appropriate for studies involving radioiodinated antibody localization.

Animals↗
PubMed1985

A method of membrane preparation for immunoassay.

Membranes prepared from a variety of solid tissues were used as solid-phase antigens for ELISA or RIA after fixation onto polylysine-primed 96-well plates. The preservation of antigens in these membrane preparations was tested by reactivity in ELISA using 2 monoclonal antibodies: W6/32, which recognizes an HLA framework antigen (a protein antigen) and anti-SSEA-1, directed to a carbohydrate antigen carried on glycoproteins. Levels of antigen deposition and usefulness as solid-phase antigens were assessed for ELISA as compared to RIA. Coated plates may be frozen for many months with preservation of antigenic activity. This method is relatively simple, rapid, and is useful for preparation of tissue antigens for immunoassay, especially for screening monoclonal antibodies.

Animals↗
PubMed1983

The relative roles of MHC and non-MHC genes in heart and skin allograft survival.

Although the role of the major histocompatibility complex (MHC) of the rat (RT1) in graft rejection has been established, the role of non-RT1 genes is not well understood. To investigate the influence of MHC and non-MHC genes in graft rejection, various combinations of congenic and inbred strains of rats were used as donors and recipients of skin grafts and perfused abdominal heart grafts. In addition, hemagglutinating and cytotoxic antibody responses were evaluated to assess loci that were serologically active in transplantation. Our results demonstrate that: (1) RT1 antigens are the most important determinant in heart and skin rejection; (2) antigens controlled by non-MHC genes also play a major role in rejection because they cause disparate heart grafts to be rejected by day 18 and skin grafts by day 26; (3) RT2 cell antigens alone do not cause graft rejection; and (4) allogeneic differences at the RT1, RT2, RT3, and RT6 loci elicit an antibody response in heart transplantation.

Animals↗
PubMed1983

Comparison of antigens in the rat MHC that act as CML determinants.

We have examined a variety of RT1 haplotypes using congenic strains of rats to investigate the comparative relationships among MHC antigens that have been reported to serve as cell-mediated lympholysis (CML) targets. These studies demonstrate that: (1) RT1.A antigens can act as CML determinants, and in some haplotypes they display identical serological and CML reaction patterns; (2) in some donor-recipient strain combinations RT1.A antigens may act as immunodominant determinants, preventing the generation of effectors capable of recognizing other antigens, and (3) a portion of the antigenic determinants that define the anti-CT CML response can be mapped to the same region of the rat MHC as the RT1.E locus.

Animals↗
PubMed1982

CML characterization of a product of a second class I locus in the rat MHC.

In the rat, genes that control the expression of target antigens detected by cell-mediated lympholysis (CML) are present in the major histocompatibility complex (MHC). The relationship of these loci, CT and Ag-L, to each other and to other loci within the MHC is unknown. In this report, we demonstrate the existence of a CML target antigen in the (DA X BN)F1 anti-DA.1I(BI) strain combination. The gene coding for this antigen is linked to the RT1 complex as indicated by the CML reactivity of targets from backcross and congenic animals. Inhibition studies demonstrated that this antigen has the widespread tissue distribution characteristic of class I antigens, and the gene coding for this CML antigen maps coincident with the RT1.E class I locus as indicated by the lysis of targets from the recombinant strains r10 and r11. The CML can be blocked by antisera directed against a product of the RT1.E locus. The locus controlling this CML reactivity, like CT and Ag-L, has been separated from RT1.A by recombination; unlike CT and Ag-L, the produce of this CML locus appears to be identical with an RT1.E allelic product that has been serologically identified and biochemically characterized.

Animals↗
PubMed1981

Gene order in the major histocompatibility complex of the rat.

The loci in the major histocompatibility complex (MHC) of the rat which code for class I and class II antigens--RT1.A and RT1.B, respectively--have previously been separated by laboratory-derived recombinants and by observations in inbred and wild rats. Closely linked to the MHC is the growth and reproduction complex (Grc) which contains genes influencing body size (dw3) and fertility (ft). These phenotypic markers were used in this study to orient the A and B loci of the MHC. Two recombinants were used for mapping. The BIL(R1) animal is a recombinant between the MHC and Grc, and it carries the haplotype RT1.AlBlGrc+. The r10 animal is an intra-MHC recombinant, and it has the haplotype RT1.AnB1Grc. These recombinants were characterized serologically, by mixed lymphocyte reactivity, by immune responsiveness to poly (Glu52Lys33Tyr15) and by the presence of the dw-3 gene. The data demonstrate that the gene order of the loci is: dw-3--RT1.B--RT1.A.

Animals↗
PubMed1977

Expression of HLA-B12 on platelets, on lymphocytes and in serum: a quantitative study.

Expression of the antigen HLA-B12 on platelets, on lymphocytes and in serum of normal subjects was studied using several quantitative techniques. HLA-B12 was found to be expressed on platelets from each of 25 heterozygous HLA-B12-positive donors in amounts varying about 35-fold from weakest to strongest. Expression of B12 on platelets was enhanced in the presence of the first locus antigen A11, but decreased in the presence of A2, A3, and Aw28. The antigen was expressed in equal strength on lymphocytes sampled from the same donor population. Serum content of B12 assayed in five subjects was found to be roughly proportional to the expression of that antigen on platelets. Expression of B12 on platelets is under genetic control but the precise mechanism of inheritance has not yet been defined. The applicability of these findings to transfusion of single donor platelets to alloimmunized patients is under investigation.

Blood Platelets↗
PubMedDate not supplied

Expression of the alpha6beta4 integrin provides prognostic information in bladder cancer.

Integrins are cell surface receptors for extracellular matrix components that may participate in metastatic processes. Normal urothelial tissues show a polarized expression of alpha6beta4 integrin on basal cells at their junction with the lamina propria. We have previously shown that bladder cancers frequently overexpress one member of the integrin family, the alpha6beta4 integrin. In this study, we evaluated the level of alpha6beta4 integrin expression in bladder cancer specimens from 57 patients and correlated the expression level with patient survival. Expression was evaluated by immunoperoxidase staining. Three patterns of alpha6beta4 expression were observed: negative (13 patients); strong overexpression throughout the tumor cells (21 patients); and weak expression that most closely resembled expression in normal urothelium (23 patients). Individuals with weak staining tumors had a statistically significantly better survival (p=0.041) than patients whose tumors exhibited either no expression or strong overexpression. These data indicate that evaluation of the expression of alpha6beta4 integrin may provide valuable prognostic information on clinical outcome in patients with bladder cancer.

Antigens, Surface↗