Biomedical subjects

A L Epstein

Publications and source records attributed to A L Epstein.

At least 91 records · Page 5Linked to original sources

PubMed1987

Monoclonal antibody DLC-48: an effective reagent for use in the depletion of malignant lymphoma from human bone marrow.

A combination of large-cell lymphoma cell lines was used as immunogen in the production of DLC-48, a mouse monoclonal antibody (MAb) to a B-cell-associated antigen. This reagent identifies a protein found on a majority of peripheral blood B-cells, B-cell derived tumor cell lines, and lymphoma biopsy specimens. In reactive lymph-node sections, DLC-48 stains both germinal center and mantle zone B-lymphocytes when the immunoperoxidase technique is used. DLC-48 is an IgM, and is highly cytotoxic in the presence of human serum complement. The conditions for complement-mediated cytotoxicity were first optimized by utilizing the chromium-release assay with 2 large-cell lymphoma cell lines, and were then applied to the treatment of the cell lines prior to cloning in agar. A single treatment with antibody and human serum eliminated 4 orders of magnitude of target cells. The cytolytic activity of this antibody was not inhibited by the presence of a 20-fold excess of normal human bone marrow. Treatment with antibody and autologous serum did not affect the growth of human hematopoietic progenitor cells (CFU-GEMM and CFU-GM). These results suggest that DLC-48, either alone or in combination with other MAb reagents, should be effective in eliminating malignant cells of B-lineage from human bone marrow for purposes of autologous bone-marrow transplantation.

Antibodies, Monoclonal↗

PubMed1987

Two new monoclonal antibodies, Lym-1 and Lym-2, reactive with human B-lymphocytes and derived tumors, with immunodiagnostic and immunotherapeutic potential.

Two new monoclonal antibodies (Lym-1 and Lym-2), reactive with the cell surface of B-lymphocytes and derived tumors, have been produced using tumor cell nuclei preparations as immunogens. Specificity screens using live cell radioimmunoassay techniques with 52 well-characterized human lymphoma and leukemia cell lines showed that both Lym-1 and Lym-2 bound to cell lines of B-cell lineage but were unreactive with those of T-cell, myeloid, or erythroid derivation. The B-cell specificity of these reagents was confirmed on 36 lymphoma and 15 leukemia biopsy specimens by using immunoperoxidase or immunofluorescence techniques. Additionally, flow cytometric analysis of 22 lymphoma biopsies showed that the majority of B-cell tumors were Lym-1 and/or Lym-2 positive and that within a given biopsy, a high percentage of the malignant cell population was stained. In both the immunoperoxidase and flow cytometric studies, reactive T-cells or T-cell lymphomas were consistently negative with the exception of Hodgkin's disease tissues which, in some instances, showed a higher than expected positivity with Lym-1 and Lym-2. Approximately 40% of B-cell chronic lymphocytic leukemias were found to be positive with Lym-1 while 80% were positive with Lym-2. Immunoperoxidase staining of frozen sections of human lymphoid tissues showed that both Lym-1 and Lym-2 stained germinal center and mantle zone B-lymphocytes as well as interfollicular histiocytes. Flow cytometric analysis of normal peripheral blood demonstrated specific staining of B-cells which comprised approximately 8% of circulating lymphocytes. Immunoperoxidase staining of nonlymphoid human organs and tissues revealed weak reactivity of Lym-1 with surface colonic epithelium only. Consistent with these findings, 35 solid tumor cell lines of diverse nature were found unreactive with both Lym-1 and Lym-2. Although standard techniques have thus far failed to identify the antigen recognized by Lym-2, the membrane antigen which binds Lym-1 has been shown by immunoprecipitation and competitive radioimmunoassay studies to be a polymorphic variant of the HLA-Dr antigen. Solid-phase radioimmunoassay techniques have shown that the antigens recognized by Lym-1 and Lym-2 are not significantly modulated after antibody exposure nor shed into the circulation of lymphoma patients. Finally, using iodine-125 labeled preparations of purified Lym-1 and Lym-2, we have determined that both reagents have a relatively large number of antibody binding sites per tumor cell and increased avidity for lymphoma cells when compared to normal and reactive lymph node B-cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibodies, Monoclonal↗

PubMed1987

Quantitative analysis of a nuclear antigen in interphase and mitotic cells.

The quantification of an interchromatin-associated antigen, designated p 105, during cellular passage through mitosis is described. Indirect immunofluorescence microscopy and immunogold electron microscopy demonstrated a qualitative increase in p 105 within the mitotic cytoplasm. Multiparameter flow cytometric analysis was performed on fixed cells sequentially stained with anti-p 105 immunofluorescence and/or propidium iodide. This analysis demonstrated approximately a tenfold increase in intracellular p 105 content as a function of progression from the G2 to the M phase. This increase was corroborated by the quantitative immunoblot analysis of colchicine-treated cell cultures and of cells sorted on the basis of anti-p 105 immunofluorescence. The data reveal that the increased levels of anti-p 105 immunofluorescence in conjunction with flow cytometry may be used effectively to quantitate mitotic index and isolate mitotic cells. The function and modulation of p 105 throughout the cell cycle is discussed.

Antigen-Antibody Complex↗

PubMed1987

Modulation of the nuclear antigen p105 as a function of cell-cycle progression.

The characterization of the proliferation-associated nuclear antigen designated p105 in quiescent and proliferating lymphocytes is described. Through the use of novel flow cytometric and cell-sorting strategies the intracellular content of p105 was assessed in situ on a per cell basis. These analyses demonstrated the presence of multiple cellular subpopulations within the cell cycle differing significantly in p105 content. The data revealed that the flow cytometric quantitation of p105 levels may effectively discriminate cycling from noncycling cells. Immunogold electron microscopy revealed that the modulation of this interchromatin-associated antigen was correlated with a significant degree of nuclear restructuring. In conjunction with cell sorting, immunogold electron microscopy and immunoblot controls demonstrated that the cell-cycle-related modulation in p105 cannot be accounted for by increased cellular mass or antigen sequestration. The significance of these controls and of the potential role of p105 in cellular proliferation is discussed.

Autoantigens↗

PubMed1987

Screening for anti-idiotypic monoclonal antibodies on paraformaldehyde-fixed lymphoma cells.

Paraformaldehyde-fixed, human lymphoma cells on glass slides were used to screen murine hybridoma supernatants for anti-idiotypic monoclonal antibodies by a rapid indirect immunofluorescence technique. The method is shown to require far fewer cells for screening than current techniques, and it provides results that correlate with the results of screening by flow cytometry or by capture ELISA. We conclude that paraformaldehyde fixation preserves the idiotypic determinants of lymphomas. The assay described in this report, therefore, has significant advantages over current methods for producing anti-idiotypic monoclonal antibodies.

Animals↗

PubMed1987

Genetic and environmental determination of herpes simplex virus type 1 penetration into non-permissive rat XC cells.

We have tested a number of herpes simplex virus type 1 (HSV-1) populations for their ability to enter and express virus polypeptides in non-permissive rat XC cells. The viruses tested included 40 intratypic F(MP) recombinants and different batches of virus, belonging to the same strains, that had been produced in two different permissive systems (HEp-2 or Vero cells). Our results indicated that the ability to infect XC cells was determined in part by genetic elements of the virus genome and in part by phenotypic characteristics conferred by the permissive cell that had produced the virus: a virus strain like HSV-1 MP which, when produced in HEp-2 cells, was able to infect XC cells, lost this ability when produced in Vero cells. Working only with viruses produced in HEp-2 cells we showed that the ability to enter XC cells could be transferred from the MP strain to the F strain (which does not normally infect XC cells efficiently) by transfer of the cloned BamHI B (map units 0.745 to 0.81) or BamHI L (map units 0.706 to 0.745) fragments isolated from MP DNA. The implicated locus or loci seemed to segregate, however, from loci controlling gC synthesis and cell fusion, which have been described as mapping in the same region.

Animals↗

PubMed1987

Immunohistochemical characterization of a 183 KD myeloid-specific-DNA-binding protein in B5 fixed, paraffin-embedded tissues, and bone marrow aspirates by monoclonal antibody BM-1.

A monoclonal antibody, designated BM-1, which is reactive in B5 formalin-fixed, paraffin-embedded tissues, has been generated against a cytoplasmic and nuclear antigen expressed in human myeloid precursor cells and derived leukemias. Using the avidin-biotin-complex immunoperoxidase procedure, BM-1 was found to stain selectively myeloid precursor cells in normal bone marrow and mature granulocytes in the blood. In a screen of 26 normal adult and fetal human organs fixed in B5 formalin, BM-1 was negative in all nonhematopoietic tissues with the exception of tissue granulocytes and scattered cells in the peripheral cortex of the thymus. Likewise a screen of 30 solid tumor cell lines including a spectrum of carcinomas, sarcomas, and neural-derived tumors was negative. BM-1 was also negative with 21 T and B cell lymphomas and 11 Hodgkin's disease tumors. A preliminary study of tumors of the hematopoietic system revealed that BM-1 was reactive with M2 and M3 acute myelogenous leukemias (AML), chronic myelogenous leukemias (CML) and myelomonocytic leukemias, and granulocytic sarcomas. M1, M4, M5, and M6 AML clot preparations were negative in this study, indicating that BM-1 may have a role in the histopathologic diagnosis of myelogenous leukemia. Myeloid leukemic cell lines HL-60, ML-2, KG1, and TPH-1-O showed BM-1 nuclear and/or cytoplasmic reactivity in a subpopulation of cells, but erythroid and lymphoid leukemias and all lymphoma cell lines were negative. Immunoperoxidase studies of a panel of fetal tissues showed BM-1 positive cells in the peripheral cortex of the thymus and portal myelopoietic regions of the liver at 18 weeks gestation. Finally, DNA-cellulose and solid phase radioimmunoassay (RIA) techniques developed in our laboratory demonstrate that the BM-1 antigenic domain is reactive only after binding to eukaryotic but not prokaryotic single- or double-stranded DNA. Immunoblot techniques using a DNA-cellulose purified protein sample revealed that BM-1 recognizes a 183 kD protein. These studies indicate that BM-1 is recognizing a myeloid-specific antigen that, because of its DNA binding characteristics, may have an important role in the differentiation of myeloid cells at the molecular level.

Animals↗

PubMed1987

Eosinophil peroxidase is detectable with a monoclonal antibody in collagen bands of nodular sclerosis Hodgkin's disease.

An IgG2a murine monoclonal antibody derived against human eosinophils was shown to immunoprecipitate the 78,000 dalton form of human eosinophil peroxidase (EPO). To confirm the specificity of the antibody, we used a glucose-oxidase avidin biotin procedure to immunostain 32 human cell lines and tissues. An eosinophilic subline of HL-60 promyelocytic leukemia was the only cell type other than eosinophils to be recognized by the antibody. Because previous reports have described occult eosinophilic degranulation in tissues with a variety of pathological conditions, we immunostained cryostat sections of four consecutive lymph node biopsies of nodular sclerosis Hodgkin's disease with the monoclonal antibody. Our objective was to characterize by immunohistology the eosinophilic infiltration in a lymphoma that frequently contains substantial numbers of eosinophils. In all four cases of nodular sclerosis Hodgkin's disease, there was striking and extensive deposition of EPO in a dendritic pattern throughout the connective tissue and collagen bands. The extent of deposition of EPO in the bands far exceeded the degree of infiltration by intact eosinophils, as determined by examination of routinely stained tissue sections. A similar dendritic pattern was not observed in any of six benign lymph nodes that were immunostained for EPO. We conclude that the monoclonal antibody described in this report is specific for EPO and that eosinophils extensively degranulate and release EPO in the bands of nodular sclerosis Hodgkin's disease. Moreover, the degree of eosinophilic infiltration in this disorder cannot be assessed solely on the basis of intact eosinophils.

Antibodies, Monoclonal↗

PubMed1987

Comparative serum stability of radiochelates for antibody radiopharmaceuticals.

Serum incubation of monoclonal antibodies chelate labeled by DTPA, benzyl-EDTA and benzyl-TETA with 111In, 57Co, and 67Cu demonstrated marked differences in their stability. In serum, 111In-benzyl-EDTA-antibody was more stable than 111In-DTPA-antibody. Cobalt-57 or 67Cu chelated antibody were less stable than either 111In chelated antibody; 67Cu was only firmly attached to the antibody as 67Cu-benzyl-TETA-antibody. The relative stability of the radiometal chelated antibodies was paralleled by the relative stability in serum of the radiometal chelates themselves. These in vitro studies suggest that in vivo behavior of metal chelates exposed to a complex protein environment cannot be predicted by classical equilibrium constants.

Antibodies, Monoclonal↗

PubMed1986

Immunochemical aspects of monoclonal antibodies important for radiopharmaceutical development.

Successful development of radiopharmaceuticals from monoclonal antibodies will require the control of several immunochemical aspects of the antibody molecules. A proposed set of methods is presented here for evaluating these immunochemical parameters. This approach consists of monitoring each monoclonal antibody harvest by selective affinity chromatography to determine the presence of detectable alterations in molecular homogeneity. The products are then evaluated by radioimmunoassay technique standardized for total immunoglobulin immunoreactivity. These assays are utilized to detect variation in the immunoreactivity secondary to changes in the hybridoma cell lines, and to measure any detectable denaturation secondary to purification, fragment production and radiolabeling. HPLC molecular sieving chromatography is presented as a practical and informative monitor of molecular stability of these radiopharmaceuticals in vitro and in vivo.

Antibodies, Monoclonal↗

PubMed1986

Serum stability of 67Cu chelates: comparison with 111In and 57Co.

Simple chelates and chelate conjugated monoclonal antibodies labeled with 111In, 57Co and 67Cu demonstrate marked differences in stability when exposed to a serum environment. Among these radiometals, on DTPA, the order of stability is 111In greater than 57Co much greater than 67Cu. On benzyl-EDTA, the order of stability is 111In congruent to 57Co much greater than 67Cu. Among those investigated, the only serum stable 67Cu chelate found was 67Cu-TETA. The order of stability observed for 57Co vs 67Cu is contrary to published equilibrium constants. These in vitro studies suggest that the in vivo behavior of metal chelates exposed to a complex molecular environment may not be predicted by classically determined equilibrium constants.

Antibodies, Monoclonal↗

PubMed1986

123I radiolabeling of monoclonal antibodies for in vivo procedures.

When labeled to monoclonal antibodies (MAbs) or their fragments, 123I can be used for imaging or for predicting the treatment potential and radiation dosimetry of 131I labeled to the same molecular species. Because 123I (p,5n) from the Crocker Nuclear Laboratory is in dilute solution, when compared with commercial 125I of labeling grade, we have evaluated labeling parameters using Chloramine-T as the oxidant and derived an optimum set of labeling conditions that provide a 60-80% radiochemical yield of highly immunoreactive antibody. When Lym-1, an IgG-2a murine antibody against human lymphoma, was used, yields of labeled immunoglobulin were decreased by protein or Chloramine-T concentrations less than 0.4 microgram/microliter and 0.8 microgram/microliter, respectively; denaturation of the immunoglobulin occurred when the Chloramine-T concentration was greater than 1.0 microgram/microliter. Optimum labeling occurred at pH 7-8 with deleterious effects when the pH was below 5 or above 10. An optimum method for labeling antibodies with multimillicurie amounts of 123I (less than one iodine atom per 100 antibody molecules) is described. Some of the observations derived from this study are also applicable to the preparation of treatment doses of 131I-labeled antibodies, wherein the amount of antibody can be a restrictive factor.

Antibodies, Monoclonal↗

PubMed1986

Phenotypic analysis in diffuse, large cell lymphoma. Clinical and histologic associations.

To investigate the possible relationships between immunologic phenotype, histologic subtype, and clinical features in diffuse, large cell lymphoma (DLCL), a computerized registry has been established for the prospective collection of immunologic, histologic, and clinical data. A combination of immunofluorescence and immunoperoxidase technics on single-cell suspensions, frozen tissues, and B5-fixed, paraffin-embedded specimens was used to study the first 33 biopsies. A definitive phenotype was established in all but two cases. Monoclonal antibody reagents reactive in B5-fixed, paraffin-embedded tissue sections helped assign a B-cell lineage in four cases lacking surface or cytoplasmic immunoglobulin, monoclonal light chains, and T-cell markers. There was no statistically significant association between the immunologic phenotype (whether mature B or not) and any clinical or histologic parameter, including response to therapy and survival. Bone marrow involvement was found to be associated significantly with both vague nodularity and a cleaved cell subtype. Through the use of a multifaceted approach to the immunophenotypic analysis of the DLCLs, a distinct lineage and stage of differentiation could be assigned to most biopsy specimens. That such analysis has significant clinical implications for patients with DLCL could not be demonstrated in this series.

Adult↗

PubMed1986

Sensitivity and specificity of immunostaining in the diagnosis of mantle zone lymphoma.

The diagnosis of mantle zone lymphoma is sometimes difficult to make solely on the basis of morphology because the mantle zone pattern may be present in other disorders such as benign mantle zone hyperplasia, follicular center cell (FCC) lymphomas, and Castleman disease. To distinguish mantle zone lymphoma from the other disorders mentioned previously, the authors performed immunoperoxidase studies on B-5-fixed, paraffin-embedded sections or cryostat sections of lymph nodes from nine patients with a diagnosis of mantle zone lymphoma. The results then were compared with the immunostaining pattern seen in FCC lymphomas and various benign lymphoid hyperplasias. A monoclonal proliferation of mantle zone cells, as shown by staining for immunoglobulin light chains, was noted in the mantle zones and interfollicular areas in all six cases from which cryostat sections were available. The cells in the residual follicular centers uniformly had a polyclonal light chain marking pattern. Two novel monoclonal antibodies (LN-1 and LN-2) that identify FCCs and B-cells in B-5-fixed, paraffin-embedded tissues also were used in this study. Of the six cases in which the monoclonal antibodies could be used, the cells in the residual follicles were uniformly LN-1 positive, LN-2 positive, while the mantle zone and interfollicular cells were almost completely LN-1 negative, LN-2 positive. The data suggest that mantle zone lymphomas are a distinctive neoplasm of monoclonal B-cells of non-FCC origin. The authors conclude that immunostaining is a sensitive technic for identifying a malignant neoplasm of B-cells in the mantle zone and interfollicular areas. In addition, the method is relatively specific and useful for distinguishing mantle zone lymphoma from similar-appearing disorders such as benign mantle zone hyperplasias and certain FCC lymphomas.

Aged↗

PubMed1986

Assessment of cell cycle-associated antigen expression using multiparameter flow cytometry and antibody-acridine orange sequential staining.

A novel approach which enables direct assessment of the differential expression of cellular antigens in noncycling (G0) and cycling cell subpopulations is presented. The method involves flow cytometric analysis and sorting of cells stained by use of indirect immunofluorescence, followed by restaining using acid acridine orange, to relate the immunofluorescence of sorted lymphoid subpopulation(s) to cell proliferation status (i.e., G0 vs. G1 vs. S vs. G2 and M). In the present study, this technique successfully identifies the proliferation-associated modulation of a heterochromatin-associated antigen in pokeweed mitogen-stimulated human lymphoid cultures. The potential utility of this method for documenting early antigenic changes associated with the G0-G1 transition is discussed.

Acridine Orange↗

PubMed1986

Immunoglobulin gene rearrangements and expression in diffuse histiocytic lymphomas reveal cellular lineage, molecular defects, and sites of chromosomal translocation.

We have examined the immunoglobulin gene configurations in cell lines from eight patients with diffuse histiocytic lymphoma in order to establish the cellular lineage and stage of differentiation of these lymphomas. The presence of heavy and light chain gene rearrangements as well as heavy chain class switching in seven cells placed these tumors within the B cell lineage. In contrast, one cell (SU-DHL-1), which lacks B cell-restricted surface antigens, retained germline heavy and light chain loci, indicating that it may represent a true histiocyte or uncommitted cell. Truncated RNAs for both the heavy and light chain immunoglobulins were responsible for the lack of surface immunoglobulin in the SU-DHL-2 cell line. Another cell line (SU-DHL-6), which possesses a t(14;18)(q32;q21) translocation, demonstrated an unexpected recombination within its heavy chain gene locus that may be the interchromosomal breakpoint.

Antigens, Surface↗

PubMed1986

Simultaneous nuclear antigen and DNA content quantitation using paraffin-embedded colonic tissue and multiparameter flow cytometry.

The simultaneous quantitation of nuclear antigens and DNA content is presented using monoclonal antibodies and flow cytometric analysis, with paraffin-embedded human colonic pathology specimens utilized as source material. The monoclonal antibodies evaluated were shown by immunogold electron microscopy to recognize nuclear proteins preferentially associated with interchromatin (p105) and heterochromatin (p34) regions. Indirect immunofluorescence analysis of p105 revealed two distinct G1-G0 cell subpopulations in cells from normal colonic epithelium and colonic adenocarcinomas. In addition, enhanced levels of both p105 and p34 were observed in aneuploid DNA content stemlines, relative to diploid cells. Cell-sorting experiments performed on cells sorted on the basis of p105 and DNA contents reveal the capability of this method for identifying morphologically heterogeneous cell subpopulations. Other data suggest that p105 is differentially expressed in well-differentiated versus poorly differentiated tumor regions. The potential utility of this approach for the retrospective study of proliferation-associated antigens and protooncogene protein products is discussed.

Adenocarcinoma↗

PubMed1985

Radioimmunotherapy of human lymphoma in athymic, nude mice as monitored by 31P nuclear magnetic resonance.

Human B cell lymphoma (Raji) growing in athymic, nude mice has been successfully treated with a single pulse dose of 131I-labeled monoclonal antibody (Lym-1) specific for this tumor. Sequential in vivo measurements of phosphate metabolites in the tumors by 31P surface coil nuclear magnetic resonance showed a significant initial decrease of phosphocreatine following radioimmunotherapy. Diminution of relative ATP to Pi peak area ratio suggesting tissue damage occurred within 3-4 days. The contribution from metabolites resonating at ca 3.8 ppm (putative sugar phosphate region) increased. There was no significant change in pH either as a function of tumor volume or treatment. The sequence of alterations of nuclear magnetic resonance spectra from tumors of treated mice were strikingly different from sequential nuclear magnetic resonance spectra obtained from tumors of control mice. These observations lead us to conclude that 31P surface coil nuclear magnetic resonance is a promising non-invasive method for assessing and predicting the efficacy of radioimmunotherapy. Further spatial discrimination of the region of tissue observed by the surface coil nuclear magnetic resonance experiment is under exploration in an effort to increase the utility of these methods.

Adenosine Triphosphate↗