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D M Gersten

Publications and source records attributed to D M Gersten.

At least 19 recordsLinked to original sources
PubMed1996

Immunostaining of human melanomas by a monoclonal antibody to B700 mouse melanoma antigen.

Previous studies have shown that B700, an albumin-like murine melanoma antigen, has a human homologue termed H700. Polyclonal antibodies to B700 also bind to all cultured human, swine and hamster melanoma cells, suggesting that B700 is a "pan-melanoma" antigen. The objects of this investigation were: (a) to determine if 2-3-3, a monoclonal antibody to B700, can be used to identify human melanomas in formalin-fixed, paraffin-embedded tissues, and (b) to determine the specificity and potential diagnostic value of 2-3-3. Forty-eight of the 49 human melanomas, including spindle melanoma cells, stained positively, as did five of the eight pigmented naevi including cellular spindle naevi. Twenty-six of the 32 human non-melanomatous lesions were negative for 2-3-3 staining (weakly positive on one breast carcinoma and positive on five neural tumours). These results indicate that 2-3-3, a monoclonal antibody to the mouse melanoma antigen B700, can be used to identify H700 in archival specimens. 2-3-3 may have an advantage over HMB45, which is the most commonly used antibody for melanoma diagnosis, because of its immunoreactivity with spindle melanocytic lesions. Antibodies to B700 may prove to be a useful adjunct in the diagnosis of human melanoma and related lesions.

Animals↗
PubMed1995

Studies on the expression and immunogenicity of the B50 melanoma antigen and its relationship with calreticulin.

B50 is a 50 kDa protein antigen originally identified and isolated from cultured B16 murine melanoma cells; it is found in close association with a melanoma-specific antigen termed B700. Using a specific rabbit antiserum, B50 (or B50 cross-reactive molecules) has been shown to be expressed by 35 out of 36 cell lines, including melanomas, sarcomas, fibrosarcomas, carcinomas, gliomas, immortalized and primary fibroblasts, melanocyte and keratinocyte cell lines obtained from murine, human, hamster, swine, and canine donors. B50 expression is localized on the cellular membrane and in the cytoplasm in varying amounts in seven of the nine cell lines tested. Mice immunized to B50 demonstrated a significant tumour rejection response when subsequently challenged with B16 F10 melanoma cells. Previous studies had indicated that B50 has significant N-terminal amino acid sequence homology with calreticulin. Calreticulin, a calcium-binding protein, is part of the Ro/SS-A complex. This complex is the primary autoantigenic determinant of the autoimmune diseases systemic lupus erythematosus and primary Sjogren's syndrome. We now show that sera from patients with those diseases contain antibodies which bind B50, although B50 itself does not bind calcium. Thus, B50 and calreticulin are closely related but distinct antigens.

Animals↗
PubMed1994

Further studies of the therapeutic effects of murine melanoma-specific monoclonal antibodies.

The results presented here further characterize four murine monoclonal antibodies (mAb) that recognize melanoma-specific antigens (9B6, T97, 2-3-1 and 2-3-3). These melanoma-specific mAbs are of the IgG2b isotype and are significantly therapeutic when administered systemically against established pulmonary melanoma metastases. Here we show a consistent and significant inhibition of the growth of melanoma lung metastases by all four mAbs and the existence of a time 'window' at days 5-8 after tumor inoculation for optimal therapy. Since these mAbs were found not to be cytotoxic or cytolytic in vitro, we looked for host immune response regulation as being responsible for the therapeutic effects. Natural killer (NK) cells were implicated as one arm of the host immune system involved in this response since depletion of NK cells in vivo by alpha asialoGM1 or alpha NK1.1 antibodies partially abrogated the inhibitory effect of the mAbs. The observed antimetastatic effects could also be partially abrogated using antibodies directed against the T-cell subset surface markers, CD4+ and CD8+. Intramuscular melanoma tumor growth was also found to be suppressed by mAb 2-3-1, but only if administered in the area of tumor growth and only if multiple inoculations are administered over a 13-day period. The beneficial effect of mAb antimetastatic therapy was found to be useful against several syngeneic melanomas, including JB/MS, B16 and several sublines of the B16 F10 melanoma.

Animals↗
PubMed1994

Novel experimental approaches to melanoma diagnosis and therapy.

We have investigated the potential use of immune therapies on the growth of melanoma metastases in a new animal model that more closely approximates the clinical situation. We have found that significant benefits towards decreased metastatic growth and subsequent animal survival can be achieved by treatment of tumor-bearing mice with melanoma-specific monoclonal antibodies or alternatively, with various types of monovalent or polyvalent vaccines. The beneficial effects of those vaccines can be significantly enhanced by concomitant interleukin-2 therapy.

Animals↗
PubMed1993

Cross-reactivity between murine melanoma antigen B700 and a human melanoma-associated antigen (M-66) recognized by autologous antibody: evidence suggesting shared epitopes.

We have previously reported the purification and partial characterization of a human melanoma-associated antigen (M-66) recognized by autologous antibody. This antigen was found to be an unusually acidic 66 kDa glycoprotein. In studies of murine melanoma, a 67-kDa albumin-like melanoma-associated antigen (MAA) isolated from B16 melanoma cells has also been reported by our laboratories. Because the murine MAA, B700, has a molecular weight that is nearly the same as M-66, we sought to determine what similarities and differences existed between these two antigens. Human sera S150, which is known to recognize M-66, was found to bind to murine melanoma cell line B16. The addition of purified M-66 inhibited binding of S150 to B16 cells. Binding by S150 was not noted against murine melanoma cell line S91, which is known not to express cell surface B700. Conversely, reactivity of S150 against Y-Mel 84:420, known to express M-66, could be inhibited by preincubation with B16 cells. Four monoclonal antibodies known to recognize B700 were evaluated for-binding against murine B16 and human melanoma cell line Y-Mel 84:420. Binding was noted against both B16 and Y-Mel 84:420 which could be inhibited by the addition of M-66. Binding of S150 was also noted against purified B700 as tested by ELISA. While a comparison of the amino acid composition of the two antigens revealed similarities, M-66 contained 2.8 times as much serine and 0.4 times as much proline as B700. B700 has been reported to be related to serum albumin, which is not the case for M-66.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Generation of cytotoxic antibodies to the B16 murine melanoma using a formalinized vaccine.

The goal of our experiments was to determine the extent to which the humoral response to a melanoma vaccine elicits the production of cytotoxic antibodies in tumor-challenged mice. Mice were immunized with a vaccine produced from formalinized extracellular antigens (FECA) derived from B16 F10 melanomas. The production of antibodies that recognized the vaccine preparation was determined by ELISA, as was their cross-reactivity with the B700 melanoma antigen. The antibodies were shown to be anti-proliferative by inhibition of tritiated thymidine incorporation into the DNA of cultured target cells and cytotoxic by assays for complement-mediated and antibody-dependent cellular cytotoxicity. Flow cytometric analyses indicated that approximately 60% of the target cells specifically bound antibody from the immune sera. These results confirm that B700 is a significant antigenic component of the FECA vaccine, and provide encouragement for this approach to developing useful melanoma vaccines.

Animals↗
PubMed1993

Towards stoichiometric silver staining of proteins resolved in complex two-dimensional electrophoresis gels: real-time analysis of pattern development.

Toward the ultimate goal of deriving quantitative protein data from silver-stained proteins resolved in complex two-dimensional electrophoresis gels we have performed computerized real-time analysis during pattern protein "silver stain" development. Three points emerged from this study: (i) Development time is a far more important variable than originally envisioned. (ii) Previous studies which, on the basis of a single development time, sought to relate silver stain slope and threshold properties to amino acid composition are non-informative since it will be shown that slope values change markedly with development time. (iii) The combination of real-time analysis and total protein load consideration allows, for the first time, protein "spots" to be grouped into categories, which in turn aid in quantification.

Animals↗
PubMed1993

Preimmunization of mice with formalinized extracellular antigens of melanoma in combination with IL-2 and surgical resection increased survival and tumor control in metastatic melanoma model.

Recently we found that immunization with formalized extracellular antigens (FECAs) could induce the production of specific antimelanoma antibodies and increase the defense mechanisms of antimelanoma cellular and humoral immunity. In experiments we used pathogen-free female mice C57BL/6 18-20 g. We injected FECA (0.02 mg of protein/per S.C.--subcutaneous injection) for 1 month, once per week. Concurrently we injected S.C. human recombinant IL-2: 100 U/g of weight (2,000 U/per mouse). Interleukin-2 (IL-2) was injected for 1 month, 5 days/week. On days 7, 14, 21, and 28 we took retroorbital blood from mice for the study of anti-FECA and anti-IL-2 antibody production with ELISA. Control and experimental mice were then given a subcutaneous injection with 0.5 x 10(6) cells B16-F10 melanoma in 25 microliters into the middle of the tail. By 18 days 100% developed local melanoma tumors. We resected tails of all control and experimental animals 5 mm distal the base of the tail under metaphan anesthesia. The production of antibodies to FECA and IL-2 started after the 21st day and was higher in the group of mice immunized with FECA and with IL-2 than in control animals. Combining preimmunization with FECA and IL-2 and resection of local melanoma tumors decreased the mortality and the number of mice with local recurrence and metastatic melanoma tumors to the lungs.

Animals↗
PubMed1992

Production of monoclonal antibodies against the B700 murine melanoma antigen and their antimetastatic properties.

Two unique murine melanoma antigens, termed B700 and B50, have been identified and isolated from several different murine melanoma cell lines. Both antigens can be detected on the cell surface, are actively shed in culture, and are often found in close association intracellularly. In previous studies, the antigen B700, which is related to serum albumin by biochemical and immunological criteria, was shown to function as a melanoma-specific tumor rejection antigen. We have also shown that animals sensitized to irradiated JB/RH melanoma cells produce antibodies which recognize B700 and/or B50, with B700 evoking the stronger humoral response. Animals testing positive by ELISA for antibody production to B700 or B50 were used for preparation of hybridomas and four different murine monoclonal antibodies have been produced whose specificities should facilitate epitope mapping. Clones have been used to generate ascites fluid in nude mice; the antibodies specifically recognize B700 and intact murine melanoma cells, but not B50. Two of these monoclonal antibodies have been administered systemically to C57Bl/6 mice bearing 5 day pulmonary metastases of the JB/MS melanoma, and significant inhibition of metastatic growth was observed for both antibodies.

Animals↗
PubMed1992

Polyacrylamide gel electrophoresis in vertical, inverse and double-crossing gradients of soluble polymers.

The presence of soluble dextrans, methylcellulose and polyethylene glycol polymers incorporated into vertical sodium dodecyl sulfate (SDS)-polyacrylamide slabs during electrophoresis can have a pronounced effect on protein separations. The effects of various standard and inverse gradients of polymers on the electrophoretic mobility of marker proteins in 10% T, 2.66% C Laemmli-style SDS gels, and the effects of simultaneous pore size and polymer gradients were investigated. These experiments demonstrate that the inclusion of polymers is a new, additional parameter that can be useful in resolving complex mixtures of proteins.

Dextrans↗
PubMed1992

Separation of proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of soluble, aqueous polymers: Ficoll and polyvinylpyrrolidone.

In previous studies we have demonstrated that water soluble polymers of dextran and methylcellulose, when incorporated into sodium dodecyl sulfate (SDS)-polyacrylamide gels, can be used to sharpen the protein bands. We have extended these studies to enhance the separations in the mid-molecular weight range. Enhanced separation of protein molecular weight markers between 30,000-67,000 was best achieved in SDS-gels containing 2.5% polyvinylpyrrolidone (average molecular weight 10,000).

Electrophoresis, Polyacrylamide Gel↗
PubMed1992

B700 antigen as a component of an antimelanoma vaccine: formalinized extracellular antigens.

Formalin fixation has enjoyed widespread use in the preparation of antibacterial and other vaccines, but rather less use in antitumor vaccines. Previous studies from our laboratories have demonstrated the efficacy of antimelanoma vaccines in mice, produced from formalinized antigens shed by cultured melanoma cells. In this study, we provide evidence that the immunodominant component of that vaccine is the well-characterized B700 melanoma antigen.

Animals↗
PubMed1992

Proteinuria of B700, a 67 kD albumin-like melanoma-specific antigen.

B700 is a murine melanoma antigen that is closely related to, but distinct from, serum albumin. The present study examined the metabolic fate and anatomic distribution of radioiodinated B700 and mouse serum albumin (MSA) administered s.c. to mice. In blood, both proteins were associated with the plasma fraction where the halflife of B700, a glycoprotein, was 0.5 days, compared to 2.7 days for MSA. Of particular interest was the observation that B700, a 67 kD anionic protein, was excreted primarily in urine. The selective B700-proteinuria did not alter urinary volumes or produce hematuria or edema. SDS-polyacrylamide gel electrophoresis and western blot analysis using the H-2-3-3 B700-specific monoclonal antibody revealed that B700 proteinuria occurred in B-16 murine melanoma bearing animals but not in control mice. These studies demonstrate that the tumor-bearing host readily distinguishes between very similar normal protein (MSA) and tumor-associated antigen (B700) molecules and processes them differently.

Animals↗
PubMed1992

Antigens of murine melanoma and their cross-species reactivity.

Many investigators have taken a two-stage approach to the study of tumor antigens. The first is the evaluation of antigens produced by animal tumors and the second is the determination of the extent to which animal antigens have relevant human homologs. Accordingly, we discuss the known protein antigens of murine melanoma with emphasis on those expressed by more than one species. These include six mouse-specific and five cross-species antigens, and 1 human antigen transfected into mouse cells. Of the five cross-species antigens, B700 and HMW have been demonstrated in four different species, making them candidate 'pan-melanoma' antigens.

Animals↗
PubMed1991

Gel electrophoresis in the presence of soluble, aqueous polymers: horizontal sodium dodecyl sulfate-polyacrylamide gels.

Because little is known about the use of aqueous polymers in polyacrylamide gel electrophoresis, we undertook a feasibility study that enables the discontinuous Laemmli-formulated system of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis to be performed in a horizontal format by the addition of large-sized aqueous polymers (i.e., dextrans and methylcelluloses). We studied four parameters: the cross-linking agent (bisacrylamide vs AcrylAide) and the polymer concentration, nature, and size. Three concentrations of each polymer were used. The best differentiation between the standard markers and the sharpest bands were obtained using concentrations of 2.5 and 0.06% (w/v) for Dextran T-500 and methylcellulose 4000, respectively. There was no predictable pattern to the variation in the plots of log Mr vs Rf caused by varying the concentration and length of the dextrans; however, the methylcellulose patterns suggest that gel viscosity is important. The results suggest that the combination of 0.06% methylcellulose 4000 polymers with bisacrylamide is a convenient and inexpensive means of performing flatbed Laemmli SDS-polyacrylamide gel electrophoresis.

Dextrans↗