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Biomedical subjects

A L Epstein

Publications and source records attributed to A L Epstein.

At least 55 records · Page 3Linked to original sources
PubMed1995

Behavioral disorders: an unrecognized epidemic with implications for providers.

It is reported that behavioral and mood-related problems are associated with more than 25% of medical visits. Many patients seeking help are misdiagnosed or not effectively treated by their physicians. We believe that medical education has not prepared primary care providers to evaluate and treat behavioral problems. The traditional medical or biological model needs to be replaced by a more integrated and comprehensive biopsychosocial approach. HMO delivery systems should take the lead in developing this new framework for care.

Health Maintenance Organizations↗
PubMed1995

A human-mouse chimeric Lym-1 monoclonal antibody with specificity for human lymphomas expressed in a baculovirus system.

A murine anti-human B-cell monoclonal antibody, Lym-1, has shown considerable promise for the treatment of human malignant lymphomas and has been utilized as a new radioimmunotherapy for refractory lymphoma. In order to enhance its clinical potential, a genetically engineered chimeric Lym-1 (chLym-1) with murine variable regions and human gamma 1 and kappa constant regions was constructed and expressed. The goal of this study was to generate a Lym-1 reagent with decreased immunogenicity and improved effector functions. Murine Lym-1 variable region cDNAs were isolated from the murine Lym-1 hybridoma cell line, fused to gamma 1 and kappa constant region cDNAs, and expressed in an insect cell expression system with the baculovirus transfer vector pAcUW31. The chLym-1 antibody expressed in this system was correctly processed and assembled into the expected immunoglobulin monomer. Chimeric Lym-1 bound to both target antigen-bearing Raji cells and a Lym-1 anti-idiotype antibody and had a similar binding affinity as murine Lym-1. The chimeric and murine versions of Lym-1 were assayed for their ability to mediate antibody-dependent cellular cytotoxicity (ADCC) and to induce complement-mediated cytotoxicity (CMC) against Raji targets. Chimeric Lym-1 mediated a two-fold higher level of ADCC than murine Lym-1 and slightly lower levels of CMC than murine Lym-1. In addition, in Raji lymphoma-bearing nude mice, chLym-1 localized to the tumor with approximately equal uptake at 24 and 72 hours. Chimeric Lym-1, however, cleared from the blood of nontumor-bearing mice approximately 5 times faster than murine Lym-1 (20 h vs. 5 days), as expected for a xenogeneic protein. The improved in vitro and in vivo activities of this genetically engineered monoclonal antibody render it a new potential immunotherapeutic reagent for the treatment of human malignant lymphomas.

Animals↗
PubMed1994

Herpes simplex virus 1 (HSV-1) helper co-infection affects the distribution of an amplicon encoded protein in glia.

HSV-1 derived amplicons expressing cytoplasmic beta-galactosidase (pA-SF1), or plasma membrane targeted TIMP-Thy1 (pA-TT1), were used to transduce glial cells in vitro. By monitoring the expression of reporter genes from both amplicons and helper virus, we determined that many cells were infected by both particles. In glial cells infected only by pA-SF1 beta-galactosidase immunoreactivity was restricted to the cytoplasm; co-infection with helper HSV-1 (wild type), resulted in additional nuclear beta-galactosidase immunoreactivity. Co-infection of cells with amplicon pA-TT1 and helper virus did not affect the plasma membrane localization of TIMP/Thy1. Thus, co-infection with wild type helper virus altered the localization of an amplicon encoded cytoplasmic, but not plasma membrane protein.

Cell Membrane↗
PubMed1994

Effect of seven new vasoactive immunoconjugates on the enhancement of monoclonal antibody uptake in tumors.

BACKGROUND: To enhance monoclonal antibody uptake in tumors, seven novel vasoactive immunoconjugates were developed that selectively alter the vascular permeability and/or blood volume of tumors in vivo. These immunoconjugates, composed of IL-1 beta, IL-2, TNF-alpha, physalaemin, leukotriene B4, histamine, and bradykinin chemically linked to TNT-1, a murine monoclonal antibody that binds necrotic regions in tumors, have been tested for their effects on antibody uptake in vivo. METHODS: Groups of four mice, each bearing the ME-180 human cervical carcinoma, were pretreated either 3 or 24 hours before the administration of I-125 labeled TNT-1 F(ab')2 fragment. Three-day biodistribution studies then were performed to determine the amount of radiolabeled antibody in the tumors and normal organs of the mice. In addition, mechanism of action studies were performed to determine if the vasoactive immunoconjugate affected the vascular permeability or blood volume of the tumor vessels. RESULTS: TNT-1/IL-2 gave the highest percent injected dose/g in tumor (4.80), compared with TNT-1/TNF (4.00), TNT-1/IL-1 (3.83), TNT-1/leukotriene-B4 (2.84), TNT-1/histamine (2.80), TNT-1/physalaemin (2.19), TNT-1/bradykinin (1.57), or TNT-1 alone (1.28). All of these immunoconjugates showed specific enhancement of monoclonal antibody uptake in tumor with no changes seen in normal tissues. Quantitative studies that demonstrated the mechanism of action of these immunoconjugates showed that TNT-1/IL-2 and TNT-1/histamine produced a marked change in the vasopermeability of tumor vessels but had no effect on tumor blood volume. In contrast, TNT-1/IL-1 and TNT-1/TNF produced a combination of effects, and TNT-1/leukotriene B4, TNT-1/bradykinin, and TNT-1/physalaemin affected only tumor blood volume. CONCLUSIONS: These studies indicate that pretreatment with vasoactive immunoconjugates may improve monoclonal antibody uptake in tumors significantly and thereby increase the therapeutic index of monoclonal antibody-directed immunotherapy.

Animals↗
PubMed1994

Technetium-99m-labeled monoclonal antibodies: influence of technetium-99m binding sites.

UNLABELLED: A number of 99mTc-labeled monoclonal antibodies (Mabs) are being evaluated for diagnostic applications. Preparations are carried out using both direct and indirect (bifunctional chelating agent, BFCA) labeling procedures in which nonspecific 99mTc binding has been postulated. METHODS: By using the ascorbic acid (AA) reduction mediated direct labeling procedure and diaminotetrathiol (N2S4) as a BFCA method, we examined the role of specific binding sites and aliphatic E amino groups of lysine as nonspecific binding sites for 99mTc. RESULTS: Labeling yields for the direct and N2S4 "regular" preparations averaged 73% +/- 2.8% and 91% +/- 2%, for the "specific" preparations, 60% +/- 3.5% and 75% +/- 2% and for the "nonspecific" preparations 13% +/- 1.0% and 16% +/- 1% respectively. All preparations were evaluated in tumor-bearing mice. The control and specific preparations permitted excellent scintigraphic visualization of tumors; the percentages of specific preparations in the tumors being 2.1% +/- 0.5% and 3.1% +/- 0.4%, respectively. With nonspecific preparations, tumors were not visualized and the percentages of administered radioactivity per gram of tumor were only 0.9% +/- 0.2% and 0.9% +/- 0.3%, respectively. CONCLUSIONS: In these 99mTc labeling procedures, the amino group-mediated nonspecific binding of 99mTc to Mabs can be as high as 16% and contributes to increased liver uptake and decreased tumor uptake of radioactivity.

Animals↗
PubMed1993

EWS and ATF-1 gene fusion induced by t(12;22) translocation in malignant melanoma of soft parts.

The genes involved in the t(12;22)(q13;q12) translocation found recurrently in malignant melanoma of soft parts have been characterized and shown to form, in four cases studied, hybrid transcripts. The deduced chimaeric protein encoded by the der(22) chromosome consists of the N-terminal domain of EWS linked to the bZIP domain of ATF-1, a transcription factor which may normally be regulated by cAMP. ATF-1 has not previously been implicated in oncogenesis. EWS was first identified as forming a hybrid transcript in Ewing's sarcoma, which links its N-terminal domain to the DNA binding domain of the FLI-1 gene. Thus the oncogenic conversion of EWS follows a common scheme of activation, exchanging its putative RNA binding domain with different DNA binding domains that appear to be tumour-specific.

Activating Transcription Factor 1↗
PubMed1993

Immunologic and biochemical analysis of TNT-1 and TNT-2 monoclonal antibody binding to histones.

Two novel murine monoclonal antibodies that bind to intracellular antigens, designated TNT-1 and TNT-2, have been generated by immunizing mice with nuclear extracts from human lymphoma cells. The monoclonal antibodies were initially identified by indirect immunofluorescence on lymphoma cell lines and subsequently were found to stain the nucleus of all cell types from several species including plants by indirect immunofluorescence techniques. Immunoblot analysis demonstrated that TNT-1 bound to a protein of 22 kD and TNT-2 bound to two proteins of 15 and 22 kD, consistent with the known molecular weight of histones. To characterize their immunoreactivity, competition radioimmunoassays were performed using purified histone fractions H1, H2a, H2b and H3. By these assays, TNT-1 was found to bind to histone fraction H1 and TNT-2 to an epitope common to histone fractions H1 and H3. Histones are found in abundance in the nucleus of the cell and are known to play a major role in chromosome structure and gene expression. Upon cell death, histones remain tightly bound to DNA and consequently provide an abundant intracellular antigen that can be exploited in targeting necrotic cells, such as those found in tumors.

Animals↗
PubMed1992

The evaluation of 186Re-labeled antibodies using N2S4 chelate in vitro and in vivo using tumor-bearing nude mice.

We have recently described a method for radiolabeling monoclonal antibodies, with metallic radionuclides using a new chelating agent N2S3. Using this chelate the monoclonal antibodies Lym-1 and B72.3 were labeled with 186Re and their biological integrity was evaluated in vitro and in vivo. 186Re-labeled antibodies using N2S4 methodology were found to be stable in human serum and retained their immunoreactivity. Intravenous administration of 0.5 mCi 186Re-labeled antibodies resulted in partial or complete regression of tumor tissue in mice.

Animals↗
PubMed1992

An improved method of direct labeling monoclonal antibodies with 99mTc.

An improved method of direct labeling MAbs with 99mTc is described. Two murine monoclonal antibodies, designated Lym-1 and B72.3, have been successfully labeled with 99mTc in 0.1 M borate buffer at pH 9.3. The choice of buffer and pH was essential for obtaining a radiolabeling yield greater than or equal to 98%. In vitro studies demonstrated that the radiolabeled antibodies were stable and retained their immunoreactivity. Imaging and biodistribution studies using Raji and LS174T human tumor-bearing nude mice demonstrated a significant tumor uptake at 24-h post-injection of 99mTc-labeled MAbs. This improved labeling method showed better stability than those of previously published methods and resulted in significant improvement in the uptake of antibody in tumor. External images at 24 h post-injection revealed clearly visible tumors demonstrating the benefit of this method for tumor immunoscintigraphy.

Animals↗
PubMed1991

Flow cytometric quantitation of the proliferation-associated nuclear antigen p105 and DNA content in advanced gastric cancers.

Flow cytometric quantitation of the proliferation-associated nuclear antigen p105 was done on cancer cell suspensions from 114 advanced gastric cancers and correlated with clinical behavior. DNA diploidy was observed in 45 (39.5%) and aneuploidy in 69 (60.5%) cases. By setting the cutoff line at the level used in a negative control study without primary antibody in the same sample, the p105-labeling rate was calculated by the p105-DNA dual fluorescence analysis. The mean p105-labeling rate was 37.7% (range, 9.3% to 79.0%). The p105-labeling rates were significantly higher (P less than 0.05) for aneuploid DNA, liver metastasis, vascular invasion, and histologically well-differentiated tumors. The 5-year survival rate of patients with high p105-labeling tumors (p105-labeling rate, greater than 30%) was significantly poorer (P less than 0.01) than that of patients with low-labeling tumors. When the p105-labeling rate and the clinicopathologic parameters were entered simultaneously into the Cox regression model, the stage of disease, DNA ploidy, p105-labeling rate, and vascular invasion emerged as independent prognostic parameters. These findings indicate that the measurement of p105 may provide useful information for predicting prognosis in advanced gastric cancers.

Adult↗
PubMed1991

Enhanced tumor uptake of macromolecules induced by a novel vasoactive interleukin 2 immunoconjugate.

Low uptake of monoclonal antibodies (MAbs) in cancer lesions is a significant problem in cancer therapy. Recent studies have shown that antibody uptake in tumor is controlled in large part by the tumor blood flow and the vascular permeability of the tumor endothelium. We have hypothesized that these physiological properties of tumor vessels may be altered by pretreatment with vasoactive drugs or peptides linked to tumor-specific MAbs. To test this hypothesis, two MAbs, Lym-1 directed against human malignant lymphomas and B72.3 reactive with the TAG-72 antigen expressed in solid tumors, were chemically conjugated with human recombinant interleukin 2 (IL-2). IL-2 has been used in humans to activate lymphokine-activated killer cells for the treatment of cancer but is also known to produce a generalized vascular permeability by an unknown mechanism when used systemically. Chemical conjugation of IL-2 to MAbs appears to destroy its cytokine function as shown by T-cell proliferation studies in vitro. Despite this finding, MAb/IL-2 immunoconjugates retain their ability to produce an enhanced vascular permeability when injected i.v. into nude mice bearing relevant tumor models only. Biodistribution studies using 125I-labeled tracer Lym-1 have demonstrated that the Lym-1/IL-2 immunoconjugate can increase antibody uptake in tumor by a factor of 4 in a time (2.5-h pretreatment)- and dose (30 micrograms/mouse)-dependent manner. In contrast, treatment of mice with free IL-2 and antibody showed this effect in all organs of the mouse including the tumor. Bidirectional crossover imaging studies in individual tumor-bearing nude mice showed improved uptake and decreased blood pool when the MAb/IL-2 immunoconjugates were used compared to controls. Finally, tumor blood flow and vascular permeability studies demonstrate that the physiological effect of the MAb/IL-2 is due to a reversible and specific vascular leakage at the tumor site. These studies indicate that pretreatment with this novel immunoconjugate may enhance the diagnostic and therapeutic potential of MAbs, drugs, and other macromolecules for the treatment of cancer.

Animals↗
PubMed1991

Chromatographic methods for large-scale preparation of immunoglobulin G2a monoclonal antibodies Lym-1 and TNT-1 F(ab')2 fragments.

Lym-1 and TNT-1 are two murine immunoglobulin G2a monoclonal antibodies (MAbs) which have been used for clinical trials in cancer patients. This paper describes methods for large-scale preparation of F(ab')2 fragments from 50 mg to 4 g of MAbs Lym-1 and TNT-1. Digestion of MAbs with pepsin was optimized and performed at pH 3.8, a pepsin/antibody ratio of 1:250, and 3-4 h of incubation at 37 degrees C. The F(ab')2 fragments were purified by tandem column procedures using fast protein liquid chromatography. Quality control analyses of the products included protein purity, isoelectric point, immunoreactivity, and endotoxin level. The results revealed that the chromatographic procedures are practical, simple, and effective, and can be used to produce gram quantities of clinical-grade F(ab')2 fragments for the diagnosis of cancer in patients.

Antibodies, Monoclonal↗
PubMed1991

Diffusion and binding of monoclonal antibody TNT-1 in multicellular tumor spheroids.

Tumor spheroids of HT-29 human colon adenocarcinoma and A375 melanoma were established to investigate the uptake and clearance kinetics of TNT-1, a monoclonal antibody that targets necrotic cells of tumors. Our data reveal that there was rapid uptake of TNT-1 and its F(ab')2 fragment in both spheroid models, whereas an antibody of irrelevant specificity, Lym-1, and its F(ab')2 fragment bound poorly to the spheroids. Unlike previously reported monoclonal antibodies to tumor cell-surface antigens, TNT-1 showed 1) a linear uptake that increased over time without saturation in tumor spheroids and 2) an unexpected uptake by a subpopulation of cells in the viable outer rim of the spheroids. These preclinical studies provide important information concerning the therapeutic potential of TNT monoclonal antibodies for the treatment of cancer and micrometastases.

Adenocarcinoma↗
PubMed1991

Effects of 131I-labeled TNT-1 radioimmunotherapy on HT-29 human colon adenocarcinoma spheroids.

Radiolabeled murine monoclonal antibody TNT-1, directed against the nuclear histones of degenerating cells, was used to treat human colon adenocarcinoma HT-29 spheroids in vitro. The therapeutic effects of 131I-TNT-1 were investigated as a function of the radioactive dose, treatment time, and number of treatments. Efficacy of treatment was assessed by TNT-1 antibody uptake, spheroid growth delay, and morphological examination using light microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). From these studies, it was determined that the therapeutic effect increased with the number of doses and the duration of treatment. Spheroids treated for 24 h showed approximately two to four times more cell death than those with a 2-h treatment. As previously shown in animal models, additional treatment with radiolabeled TNT-1 produced an expanding number of TNT-1 targets, and subsequent treatments were more effective as shown by antibody uptake studies. Microscopic examinations demonstrated that morphological changes consistent with spheroid destruction correlated well with antibody uptake data and increased gradually with dose, treatment time, and frequency of treatments. At the ultrastructural level, destruction of cells in the treated spheroids included the formation of porous cell membranes, crater-like holes (SEM), blebbing, and dissolution of cytoplasmic organelles (TEM). With continued culture, the injured spheroids were found to disaggregate after intensive 131I-TNT-1 therapy (e.g. 50 microCi/ml or 100 microCi/ml with two or three 24-h treatments). These findings suggest that tumor spheroids can be used as an in vitro model to evaluate monoclonal antibody therapy using TNT-1 and other candidate mAbs directed against intracellular antigens exposed in degenerating cells of tumors.

Adenocarcinoma↗
PubMed1991

Synthesis and preliminary evaluation of a new chelate N2S4 for use in labeling proteins with metallic radionuclides.

The successful attachment of various metallic radionuclides such as 99mTc to monoclonal antibodies for targeting tumor tissue in vivo depends upon the development and use of suitable bifunctional chelating agents. We have successfully synthesized a new N2S4 (compound 4) chelate. This chelate forms a stable complex with 99mTc and is capable of coupling to different proteins. The N2S4 compound is quite stable if kept as a hydrochloride salt and is coupled to antibody under neutral conditions with better than 95% efficiency without the loss of immunoreactivity.

Antibodies, Monoclonal↗
PubMed1991

Cell based radioimmunoassays to quantitate the immunoreactivity of TNT monoclonal antibodies directed against intracellular antigens.

Direct and indirect radioimmunoassay (RIA) procedures to determine the amount of binding of a mouse monoclonal antibody (MoAb) reactive with an intracellular antigen present in human cells are described. In these RIAs, mouse IgG2a MoAb, designated as Tumor Necrosis Treatment (TNT-1) antibody, paraformaldehyde/acetone fixed cells, and Sephadex beads were used to standardize the assay conditions. In the direct RIA, 83% of the 125I-labeled TNT-1 MoAb was bound to the target cells within 30 min after the addition of reagents. The amount of binding of the MoAb was directly proportional to the amount of antigen present in the assay. When the direct RIA was carried out using different types of target cells, 125I-labeled TNT-1 MoAb showed greater than 70% binding. In the indirect RIA, the amount of binding of secondary 125I-labeled goat anti-mouse IgG antibody to the target cells was linear. These results suggest that the indirect RIA can be used to estimate the immunoreactivity of the unlabeled TNT-1 MoAb present in crude protein preparations. Based on the results of RIAs the following two conclusions are drawn: 1) the direct RIA can be used to estimate rapidly the amount of immunoreactive TNT-1 MoAb present in 125I-labeled antibody preparations and 2) the indirect RIA which estimates the amount of immunoreactivity of unlabeled TNT-1 MoAb can be used to monitor the purification and study the characteristics of the MoAb present in crude protein preparations. These methods enable the quantitative measurement of MoAbs reactive against intracellular antigens using standard RIA procedures.

Animals↗
PubMed1990

Characterization of a cell surface molecule expressed on B-lymphocytes and Hodgkin's cells.

An antigen specifically expressed on the surface of plasma membrane of B-lymphocytes and Reed-Sternberg cells was identified using a newly developed monoclonal antibody produced by immunization by BALB/c mouse with a Hodgkin's cell line (HDLM-3). The antibody was termed anti-BLA.36 (B lymphocyte antigen.36) to indicate its predominant reactivity and the molecular weight of the corresponding antigen. By using immunoperoxidase techniques, expression of BLA.36 was detected on Hodgkin's, B-, and pre-B-cell lines, but not on other hematopoietic, melanoma, or carcinoma cell lines. In normal tissues, BLA.36 was detectable predominantly on cells in the germinal center and mantle zone of reactive follicles in lymph nodes and spleen. In hematopoietic malignancy, BLA.36 was detectable on the surface of Reed-Sternberg cells, mononuclear Hodgkin's cells, and also on malignant cells of B-cell lineage. Under these conditions, T-lymphocytes, histiocytes, granulocytes, macrophages, stromal cells in lymphoid tissue, and both normal and neoplastic epithelial cells were consistently negative for the expression of the antigen, with the single exception of a variable proportion of Kupffer cells in normal liver. Biochemical and immunological analyses indicate that BLA.36 is distinct from previously identified antigens of hematopoietic cell lineage, including CD20 and CD75 (LN2) which have similar molecular weights. When BLA.36-positive cell lines were cultured in the presence of the antibody, cell growth was adversely affected. Such an effect was eliminated by removal of the antibody from the culture, suggesting a possible growth-related function of the antigen. Anti-BLA.36 may serve as a probe to study growth-related functions of the corresponding antigen during normal growth of the B-lymphocyte, as well as in the neoplastic proliferations occurring in Hodgkin's disease and antigen-positive B-cell lymphomas. Finally, the antibody has already demonstrated its usefulness for the identification of Reed-Sternberg and Hodgkin's cells, and also normal and malignant B-lymphocytes in frozen as well as formalin- or B5-fixed/paraffin-embedded tissue sections.

Animals↗