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Biomedical subjects

E Stockert

Publications and source records attributed to E Stockert.

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

Immunophenotyping of melanomas for tyrosinase: implications for vaccine development.

Tyrosinase (EC 1.14.18.1), the key enzyme in melanin synthesis, has been shown to be one of the targets for cytotoxic T-cell recognition in melanoma patients. To develop serological reagents useful for immunophenotyping melanoma for tyrosinase, human tyrosinase cDNA was expressed in an Escherichia coli expression vector. The purified recombinant tyrosinase was used to generate mouse monoclonal and rabbit polyclonal antibodies. The prototype monoclonal antibody, T311, recognized a cluster of protein moieties ranging from 70 to 80 kDa in tyrosinase mRNA-positive melanoma cell lines and melanoma specimens as well as in L cells transfected with tyrosinase cDNA. Untransfected L cells and L cells transfected with tyrosinase-related protein 1, TRP-1(gp75), were nonreactive. Immunohistochemical analysis of melanomas with T311 showed tyrosinase in melanotic and amelanotic variants, and tyrosinase expression correlated with the presence of tyrosinase mRNA. Melanocytes in skin stained with T311, whereas other normal tissues tested were negative. The expression pattern of three melanosome-associated proteins--tyrosinase, TRP-1(gp75), and gp100--in melanoma was also compared. Tyrosinase and gp100 are expressed in a higher percentage of melanomas than TRP-1(gp75), and the expression of these three antigens was discordant. Tyrosinase expression within individual tumor specimen is usually homogenous, distinctly different from the commonly observed heterogeneous pattern of gp100 expression.

Amino Acid Sequence↗
PubMed1995

Serological response patterns of melanoma patients immunized with a GM2 ganglioside conjugate vaccine.

Gangliosides, such as GM2, GD2, GD3, and 9-O-acetyl GD3, are receiving attention as targets for antibody-based and vaccine-based therapies of melanoma. GM2 appears to be a particularly immunogenic ganglioside in humans, as indicated by the presence of naturally occurring IgM anti-GM2 antibodies in approximately 5% of humans and the fact that immunization with irradiated GM2-expressing melanoma cells or purified GM2 adherent to bacillus Calmette-Guérin elicits GM2 antibodies of low to moderate titers in a high proportion of vaccinated patients. To develop vaccines that consistently induce high titers of IgM as well as IgG anti-GM2 antibodies, vaccines containing GM2 conjugated to keyhole limpet hemocyanin as the carrier protein and QS-21 as the adjuvant have been constructed. The serological response of vaccinated patients was monitored by ELISA using purified GM2 ganglioside for IgM and IgG anti-GM2 antibodies and for GM2 cell surface-reactive antibodies by immune adherence assays and cytotoxic tests (IgM antibodies) and mixed hemadsorption assays (IgG antibodies). The majority of vaccinated patients developed IgM and IgG antibodies detectable by ELISA. In most cases, the results of IgM ELISA correlated with assays for cell surface-reactive IgM antibodies. This was not true for IgG anti-GM2 antibodies, where strong discrepancies were seen between high titers in ELISA and little or no reactivity in mixed hemadsorption tests for cell surface-reactive antibodies. These IgG antibodies (and the less frequent IgM antibodies that show similar discrepancies) may be directed against GM2 determinants that are buried, hidden, or not present on GM2-expressing target cells. With regard to a major objective of ganglioside vaccines--i.e., generation of cytotoxic antibodies--the GM2-keyhole limpet hemocyanin/QS-21 vaccine is clearly superior to the previously tested GM2/bacillus Calmette-Guérin vaccine. However, variability in patient response and lack of persistence of high-titered IgM cytotoxic antibodies in many patients are problems that remain to be solved.

Adjuvants, Immunologic↗
PubMed1994

Specificity analysis of blood group Lewis-y (Le(y)) antibodies generatedagainst synthetic and natural Le(y) determinants.

Le(y)-reactive monoclonal antibodies (mAbs) were generated in mice by immunization with synthetic Le(y) neoglycoproteins or with Le(y)-expressing cells. Serological analysis indicated that mAbs raised against synthetic Le(y) (i) reacted strongly with synthetic Le(y) but poorly with natural Le(y), (ii) cross-reacted with Le(x) or H-type 2 structures, and (iii) were IgG1, IgG2a, or IgG2b. mAbs raised against Le(y)-expressing cells (i) reacted with both synthetic Le(y) and natural Le(y), (ii) were of two types: cross-reactive with Le(x) or H-type 2 structures or specific for Le(y), and (iii) were IgM or IgG3. One of the mAbs raised against natural Le(y), mAb 3S193 (IgG3), showed high specificity for Le(y) in ELISA tests with synthetic Le(y) and Le(y) containing glycoproteins and glycolipids; it also reacted strongly in rosetting assays and cytotoxic tests with Le(y)-expressing cells. mAb 3S193 did not lyse O, A, AB, and B human erythrocytes in the presence of human complement. In flow cytometry, there was weak reactivity with granulocytes, a reactivity also observed with two previously described highly specific Le(y) mouse mAbs--BR55-2 (IgG3) and B3 (IgG1). A humanized version of mAb 3S193 has been constructed, and the specificity pattern and reactivity for Le(y) remain very similar to mouse mAb 3S193.

Animals↗
PubMed1994

The tumor protein MAGE-1 is located in the cytosol of human melanoma cells.

The MAGE-1 protein has been localized to the cytosol of human melanoma culture cells (MZ2-MEL.43) by subcellular fractionation. Its distribution between nuclear, mitochondrial, microsomal and cytosolic fractions was established by SDS-PAGE and immunoblotting with a rabbit antiserum and compared to that of markers for the main cell compartments. The immunoreactive material was recovered in the cytosolic fraction as a polypeptide migrating at 42 kDa, which has been further identified as the MAGE-1 protein because it comigrates with the product of cell-free transcription-translation of the MAGE-1 cDNA. Location to the cytosol was confirmed by immunofluorescence microscopy.

Antigens, Neoplasm↗
PubMed1994

Localization of a putative tumor suppressor gene by using homozygous deletions in melanomas.

The p21 region of human chromosome 9 is thought to contain a gene (MLM) involved in genetic susceptibility to melanoma and a gene or genes that influence progression of certain other tumors. Genomic clones that span a large region in 9p21 surrounding the presumptive tumor suppressor gene(s) have been isolated. A set of sequence-tagged sites in this region has been developed. By using these markers and others previously reported, the 9p21 region has been studied by physical mapping in 84 melanoma cell lines. A putative tumor suppressor gene, perhaps MLM itself, has been localized to a region of less than 40 kb that lies proximal (centromeric) to the alpha-interferon gene cluster.

Chromosome Mapping↗
PubMed1994

A cell cycle regulator potentially involved in genesis of many tumor types.

A putative tumor suppressor locus on the short arm of human chromosome 9 has been localized to a region of less than 40 kilobases by means of homozygous deletions in melanoma cell lines. This region contained a gene, Multiple Tumor Suppressor 1 (MTS1), that encodes a previously identified inhibitor (p16) of cyclin-dependent kinase 4. MTS1 was homozygously deleted at high frequency in cell lines derived from tumors of lung, breast, brain, bone, skin, bladder, kidney, ovary, and lymphocyte. Melanoma cell lines that carried at least one copy of MTS1 frequently carried nonsense, missense, or frameshift mutations in the gene. These findings suggest that MTS1 mutations are involved in tumor formation in a wide range of tissues.

Base Sequence↗
PubMed1994

TL antigen as a transplantation antigen recognized by TL-restricted cytotoxic T cells.

In contrast to broadly expressed classical class I antigens of the major histocompatibility complex, structurally closely related TL antigens are expressed in a highly restricted fashion. Unlike classical class I antigens, TL antigens are not known to be targets of cytotoxic T cells or to mediate graft rejection. Whereas classical class I antigens function as antigen-presenting molecules to T cell receptors (TCR), the role of TL is yet to be defined. To elucidate the function of TL, we have derived transgenic mice expressing TL in most tissues including skin by introducing a TL gene, T3b of C57BL/6 mouse origin, driven by the H-2Kb promoter. By grafting the skin of transgenic mice, we demonstrate that TL can serve as a transplantation antigen and mediate a TCR-alpha/beta+ CD8+ cytotoxic T cell response. This T cell recognition of TL does not require antigen presentation by H-2 molecules. Furthermore, we show that C57BL/6 F1 mice develop CD8+ T cells that are cytotoxic for C57BL/6 TL+ leukemia cells, providing further support for the concept that aberrantly expressed nonmutated proteins such as TL can be recognized as tumor antigens.

Animals↗
PubMed1994

Identification of the MAGE-1 gene product by monoclonal and polyclonal antibodies.

The human MAGE-1 gene encodes a melanoma peptide antigen recognized by autologous cytotoxic T lymphocytes. To produce antibodies against the MAGE-1 gene product, several approaches were taken. Three oligopeptides were synthesized based on predicted MAGE-1 amino acid sequences and were used to generate rabbit anti-peptide anti-sera. In addition, a truncated MAGE-1 cDNA was cloned into an Escherichia coli expression vector, and recombinant protein was produced and purified. All three rabbit anti-peptide antisera showed reactivity against the immunizing peptide, and one reacted with the recombinant MAGE-1 protein by immunoblotting, but none reacted with cell lysates from MAGE-1 mRNA-positive cells. The recombinant MAGE-1 protein was then used for the generation of mouse monoclonal and rabbit polyclonal antibodies. One IgG1 monoclonal antibody, MA454, as well as rabbit polyclonal antisera recognized a 46-kDa protein in extracts of MAGE-1 mRNA-positive melanoma cell lines. The antibodies showed no apparent cross-reactivity with products of the closely related MAGE-2 and MAGE-3 genes. Serological typing of normal and tumor cell lysates was in full agreement with mRNA analysis, showing expression of MAGE-1 protein in MAGE-1 mRNA-positive testis and a subset of melanomas but not in MAGE-1 mRNA-negative normal or tumor tissues. Transfection of the MAGE-1 gene into a MAGE-1 mRNA-negative melanoma cell line resulted in the expression of the 46-kDa protein, confirming the identity of this protein as the MAGE-1 gene product.

Amino Acid Sequence↗
PubMed1994

Identification of a high molecular weight endothelial cell surface glycoprotein, endoGlyx-1, in normal and tumor blood vessels.

BACKGROUND: The vascular endothelium participates in diverse physiologic and pathologic processes, ranging from blood clotting to leukocyte trafficking and tumor metastasis. Many of these functions are mediated by endothelial surface molecules, and monoclonal antibodies (mAb) have been used extensively to define endothelial cell surface structures in normal and lesional tissues. EXPERIMENTAL DESIGN: MAbs raised against human umbilical vein endothelial cells were tested on a panel of cultured cell types representing diverse cell lineages. In vivo antigen expression was examined through immunohistochemical tests with normal and lesional tissues. Immunochemical methods were employed to characterize mAb-defined antigens in endothelial cell extracts. RESULTS: The newly-derived mAbH572 reacts with a high molecular weight glycoprotein complex of cultured endothelial cells comprised of disulfide-bonded subunits of molecular weight 190,000, 140,000, 125,000, and 110,000. This antigen, designated endoGlyx-1, is expressed on the surface of cultured endothelium, with no changes in expression after cytokine-stimulation. Immunohistochemical assays detect endoGlyx-1 in endothelial cells of large, medium-sized, and small blood vessels, including capillaries, in all human organs tested; only hepatic and splenic sinusoids lack the molecule. EndoGlyx-1 is not found in any of the normal fetal and adult nonendothelial cell types tested. In a study of > 100 tumors, endoGlyx-1 immunostaining was consistently found in tumor capillaries, including "hot spots" of neoangiogenesis in certain tumors. EndoGlyx-1 is also expressed in hemangiomas and angiosarcomas. CONCLUSIONS: The highly restricted expression of endoGlyx-1 in normal and tumor blood vessels suggests a role for this molecule unique to endothelial cell physiology. In fact, endoGlyx-1 appears to be the only endothelial lineage-specific cell surface glycoprotein identified to date. The availability of endoGlyx-1 antibodies may aid in the characterization and quantification of the angiogenic phase of solid tumors and facilitate the search for endothelial precursors in bone marrow or peripheral blood.

Adult↗
PubMed1993

Comparison of antigen expression on fresh and cultured ascites cells and on solid tumors of patients with epithelial ovarian cancer.

The antigenic phenotype of malignant cells from ascites of patients with epithelial ovarian cancers was examined and compared to that of their primary and metastatic sites. Cell-surface antigens on frozen sections of primary and metastatic tumors and frozen cell pellets from ascites were analyzed with a panel of murine monoclonal antibodies using the indirect immunoperoxidase method. In addition, ascites cells cultured with and without autologous cell-free ascitic fluid were evaluated by immunofluorescence. The pattern of antigen expression detected on fresh and cultured ascitic epithelial cells was shown to be identical to the expression in autologous solid tumor tissues. When placed in culture, malignant epithelial cells generally persisted for a minimum of one, but no more than five, passages. Addition of autologous ascitic fluid to cultures of ascites cells did not alter the phenotype of the epithelial tumor cell population and did not enhance the growth of these cells. From one culture of ascites cells a permanent malignant epithelial ovarian cancer cell line (designated SK-OV-8) was established. The demonstration that epithelial tumor cells found in ascites of patients with epithelial ovarian cancer have the identical antigenic phenotype as their solid tumor counterpart, at least for the panel of antigens studied, may be useful in planning imaging and therapeutic trials with monoclonal antibodies.

Adult↗
PubMed1989

Highly restricted tumor-specific antigen of radiation leukemia virus-induced murine leukemias.

A tumor-specific Ag expressed by the radiation leukemia virus-induced BALB/c leukemia BALBRVC has been serologically and biochemically identified with mAb of rat origin--RH221 and RH16. These mAb recognize two distinct epitopes on a Mr 85,000 glycoprotein. Expression of the RH221/16 Ag was also detected on two radiation leukemia virus-induced C57BL/6 leukemias, B6RV1 and B6RV2. The Ag was not detected on 66 other leukemias, 16 sarcomas, or 19 normal tissues tested. The results of sequential immunoprecipitation and partial peptide analyses of the RH221/16 Ag indicate that this tumor-specific Ag is immunologically and structurally distinct from murine leukemia virus-related determinants expressed on the three RH221/16+ leukemias.

Animals↗
PubMed1989

Expression of TL, H-2, and chimeric H-2/TL genes in transgenic mice: abnormal thymic differentiation and T-cell lymphomas in a TL transgenic strain.

To investigate the genetic regulation of TL expression, 12 transgenic mouse strains on a C3H (TL-nonexpressing) background have been derived: two Tg.Tlaa-3 strains with Tlaa-3 isolated from A-strain TL+ thymocytes, four Tg.T3b strains with T3b from a TL+ leukemia arising in a C57BL/6 (TL-) mouse, three Tg.Con.3 strains with an H-2Kb/T3b chimeric gene (construct 3,5'flanking region and exon 1 of H-2Kb and exons 2-6 of T3b), one Tg.Con.4 strain with a T3b/H-2Kb chimeric gene (construct 4, 5' flanking region and exon 1 of T3b and exons 2-8 of H-2Kb), and two Tg.H-2Kb strains with H-2Kb. Expression of the transgenes was determined by the presence of TL or H-2Kb products or transcripts. Both Tg.Tlaa-3 strains expressed high levels of TL antigen in thymus, indicating that (i) the 9.6-kilobase Tlaa-3 DNA fragment contains sufficient information for correct tissue-specific expression in thymocytes and (ii) TL- thymocytes of C3H provide conditions for the transcriptional activation of Tlaa-3. In contrast, neither the four Tg.T3b strains nor the Tg.Con.4 strain expressed transgenes, indicating that (i) T3b lacks elements necessary for TL expression in normal thymocytes and (ii) the corresponding endogenous TL genes of C3H mice also lack these elements. The pattern of TL expression in two of the three Tg.Con.3 strains was similar to that of H-2Kb expression, indicating that transcription of this H-2Kb/T3b chimeric gene was driven by the regulatory sequences of H-2Kb. The thymuses of mice derived from the Tg.Tlaa-3-1 strain were smaller than C3H thymuses, and the surface phenotype of Tg.Tlaa-3-1 thymocytes resembled thymocyte precursors (TL+L3T4-Lyt-2-Thy-1+H-2+). These mice developed a high incidence of lymphomas with the same thymocyte precursor phenotype. The study of TL transgenic strains should prove useful in defining the role of TL in normal and abnormal T-cell differentiation.

Animals↗
PubMed1988

Influence of 5' flanking sequences on TL and H-2 expression in transfected L cells.

TL (thymus leukemia) antigens are encoded by genes in the major histocompatibility complex (MHC) of the mouse. Although similar in overall structure to other class I MHC antigens (H-2, Qa), TL expression is regulated in a highly distinctive fashion. In contrast to the broad distribution of H-2 and the intermediate distribution of Qa, TL expression is restricted to cells of T-cell derivation during development in the thymus and is lost when T cells migrate to the periphery. Some mouse strains do not express TL antigens on thymocytes (TL- strains), but leukemias occurring in these mice can have a TL+ phenotype, indicating activation of normally silent TL genes. In transfection studies with H-2 or TL genes in L cells (mouse fibroblasts), H-2 is expressed at high levels, whereas TL is poorly expressed. To identify genetic elements that regulate expression in transfected L cells, chimeric genes were constructed by transposing the 5' and 3' regions of TL and H-2 genes. Antigen expression was not influenced by transposing the cytoplasmic domain and 3' untranslated region. In contrast, interchanging the 5' flanking sequences and exon 1 had a marked influence on antigen expression, with 5' sequences from the H-2 gene increasing TL expression 10- to 50-fold, and 5' sequences from the TL gene markedly decreasing H-2 expression. With both the parental TL gene (p20-TL) and the highly expressed chimeric TL gene (construct 3), levels of TL mRNA and TL antigen correlated with the number of transfected gene copies. However, in cells transfected with equal copy numbers, much higher levels of TL mRNA and TL antigen were found in construct-3 transfectants than in p20-TL transfectants. In addition, there was marked heterogeneity in TL mRNA size in L cells transfected with p20-TL, in contrast to a more homogeneous transcript size in construct-3 transfectants. These results point to regulatory sequences in the 5' flanking region of class I genes that control proper initiation and processing of TL transcripts.

Animals↗
PubMed1987

Vaccines containing purified GM2 ganglioside elicit GM2 antibodies in melanoma patients.

GM2, GD2, and GD3 gangliosides are expressed on the surface of human melanoma cells and represent potential targets for immunological control of melanoma growth by monoclonal antibodies and active immunization. The immunogenicity of GM2 was investigated by analyzing the humoral immune response of melanoma patients to vaccination with cell lines selected for high GM2 expression and with vaccines containing purified GM2. The whole-cell vaccine and vaccines containing purified GM2 and bacillus Calmette-Guérin (BCG) elicited GM2 antibody in a high proportion of patients, particularly in GM2/BCG-vaccinated patients pretreated with cyclophosphamide and given a GM2/BCG booster immunization. Vaccines containing purified GM2 and Salmonella minnesota R595 as the adjuvant were also effective, but only in patients pretreated with cyclophosphamide. GM2 antibodies in vaccinated patients were of the IgM class and were cytotoxic for GM2-positive targets in the presence of human complement.

Animals↗
PubMed1986

Molecular basis of a unique tumor antigen of radiation leukemia virus-induced leukemia B6RV2: its relation to MuLV gp70 of xenotropic class.

Hybridomas secreting monoclonal antibodies that reacted with the B6 radiation leukemia virus (RadLV)-induced leukemia B6RV2 were produced by fusion of BALB/c NS-1 myeloma cells with spleen cells from (BALB/c X B6)F1 mice immunized with B6RV2. By direct and absorption analyses with 28 B6 and BALB/c leukemias, the monoclonal antibodies NU7-4 and NU7-99 were shown to react only with B6RV2, indicating that they recognized an individually distinct antigen on B6RV2 that was identified previously with conventional (BALB/c X B6)F1 anti-B6RV2 serum. Another monoclonal antibody, NU1-132, showed relatively restricted reactivity with B6 RadLV leukemias. These three monoclonal antibodies all precipitated material of approximately 80,000 daltons, which is the same size as that precipitated by anti-xenotropic MuLV gp70 serum. Sequential immunoprecipitation analysis revealed that the molecules precipitated by NU7-4 were not removed by pretreatment of NU7-99 or NU1-132 and that the molecules precipitated by NU7-99 were not removed by NU7-4 or NU1-132. The molecules precipitated by NU1-132 were partially removed by pretreatment with NU7-4, but not with NU7-99. The molecules precipitated by these three monoclonal antibodies were removed by pretreatment with anti-xenotropic gp70. These results suggested heterogeneity of the xenotropic MuLV gp70-related molecules expressed on B6RV2 and a possible relation between serologically defined unique tumor antigens and gp70-related molecules.

Animals↗
PubMed1986

Cell surface antigens of murine leukemias induced by radiation leukemia virus. Recognition of individually distinct cell surface antigens by cytotoxic T cells on leukemias expressing crossreactive transplantation antigens.

The specificity of transplantation immunity and T cell cytotoxicity against leukemias induced by RadLV was examined. Subcutaneous inoculation of two RadLV leukemias induced in BALB/c mice, BALBRVB and BALBRVD, resulted in initial tumor growth in CB6F1 mice, followed by complete tumor regression. Mice that had rejected leukemias BALBRVB or BALBRVD were subsequently challenged with various tumors of BALB/c origin. The growth of all five RadLV leukemias tested, and of one radiation-induced leukemia, was significantly inhibited. Another radiation-induced leukemia, a methylcholanthrene-induced sarcoma, and a leukemia induced by the Moloney leukemia virus, were not inhibited. The results indicate that RadLV leukemias share cell surface antigens that induce transplantation immunity in vivo. Cytotoxic lymphocytes were generated by coculturing spleen cells from mice that had rejected leukemia BALBRVB or BALBRVD with the corresponding leukemia cells. Direct tests and inhibition tests showed that such cytotoxic cells recognized individually specific antigens on leukemias BALBRVB and BALBRVD, distinct from the shared antigens detected in transplantation experiments. The effector cells in cytotoxicity assays were Thy-1+, Lyt-1+,-, Lyt-2+, and Lyt-3+ T cells.

Animals↗
PubMed1985

Thymus-leukemia (TL) antigens of the mouse. Analysis of TL mRNA and TL cDNA TL+ and TL- strains.

A thymus-leukemia (TL)-specific probe, pTL1, has been generated from a TL-coding gene of BALB/c mice. Multiple species of TL mRNA were detected in TL+ cells by Northern blot analysis with pTL1, and different Tla haplotypes could be distinguished on the basis of characteristic patterns of TL mRNA. No TL-related message was found in normal or leukemic TL- cells, including thymocytes from Tlab mice. However, TL mRNA could be detected in TL+ leukemias occurring in Tlab mice. A cDNA library has been made from ASL1 (a TL+ leukemia of A mice [Tlaa]), and pTL1+ clones have been sequenced. At least three structurally distinct TL genes are expressed in ASL1. Sequence comparison of TL genes from three Tla haplotypes indicates that TL genes are highly conserved (greater than 90% homology) and are more distantly related to H-2 genes. Several polyadenylation sites have been found in the 3' untranslated region of TL genes, and differential polyadenylation contributes to the size heterogeneity of TL transcripts. The predicted amino acid sequence of TL products indicates that TL and H-2 are similar in domain structure and disulfide bonds, but differ in glycosylation sites and in cytoplasmic domain sequences.

Amino Acid Sequence↗