Biomedical subjects

E Stockert

Publications and source records attributed to E Stockert.

At least 37 records · Page 2Linked to original sources

PubMed2000

Monoclonal antibody MA454 reveals a heterogeneous expression pattern of MAGE-1 antigen in formalin-fixed paraffin embedded lung tumours.

Cancer/testis (CT) antigens such as those encoded by the MAGE-gene family are expressed in a wide variety of malignant neoplasms. In normal tissues, expression is generally restricted to testis. Current knowledge of the expression pattern of CT antigens is mainly based on mRNA analysis. Little is known about actual protein expression. We previously developed MA454, a monoclonal antibody (mAb) to MAGE-1 recombinant protein. By employing antigen retrieval techniques, we show that MA454 is reactive on formalin-fixed paraffin-embedded tissues. Immunohistochemical (IHC) analysis of a normal tissue panel revealed staining solely in germ cells of testes. A series of 59 lung tumours was co-typed for MAGE-1 expression by RT-PCR and by immunohistochemistry with MA454. MA454 was positive in 19/59 cases (32%). MAGE-1 mRNA was found in 17 of the 54 cases (32%) available for RT-PCR. Of the 19 MA454-reactive tumours, 15 showed a highly heterogeneous pattern of expression. The other 4 MA454 positive cases revealed immunoreactivity in >25% of tumour areas. Of the 53 cases typed for both, mRNA and protein expression, 48 co-typed whereas 5 cases were discrepant, a likely consequence of heterogeneous MAGE-1 expression. The predominantly focal expression of MAGE-1 suggests that this antigen might not be sufficient as a sole target for immunotherapeutic approaches.

Antibodies, Monoclonal↗

PubMed1999

Cancer-testis antigens and ING1 tumor suppressor gene product are breast cancer antigens: characterization of tissue-specific ING1 transcripts and a homologue gene.

SEREX (serological analysis of recombinant tumor cDNA expression libraries) has been applied to several different tumor types and has led to the identification of a wide range of tumor antigens. In this study, a breast cancer library and a normal testicular library were analyzed using autologous and allogeneic breast cancer sera. Thirty genes were isolated, including 27 known genes and 3 previously unknown genes. Among the known genes, two cancer-testis (CT) antigens, NY-ESO-1 and SSX2, previously defined by SEREX analysis, were found. In addition, ING1, a candidate breast cancer suppressor gene, was isolated. This ING1 gene product was also recognized by 2 of 14 allogeneic sera from breast cancer patients but not 12 normal adult sera. Comparison of ING1 cDNA from normal and tumor tissues showed no mutation in the index breast cancer case and revealed the presence of at least three different mRNA transcripts with variable transcription initiation sites and exon usage. Tissue-specific expression of these transcripts was found in normal tissues and tumor cell line mRNAs. Furthermore, a novel gene, designated as ING2, sharing 76% nucleotide homology with ING1 was identified in the breast cancer cDNA library. The basis of the immunogenicity of ING1 and the biological role of ING1 and ING2 need further exploration.

Alternative Splicing↗

PubMed1999

Antigens recognized by autologous antibody in patients with renal-cell carcinoma.

The screening of cDNA expression libraries derived from human tumors with autologous antibody (SEREX) is a powerful method for defining the structure of tumor antigens recognized by the humoral immune system. Sixty-five distinct antigens (NY-REN-1 to NY-REN-65) reactive with autologous IgG were identified by SEREX analysis of 4 renal cancer patients and were characterized in terms of cDNA sequence, mRNA expression pattern, and reactivity with allogeneic sera. REN-9, -10, -19, and -26 have a known association with human cancer. REN-9 (LUCA-15) and REN-10 (gene 21) map to the small cell lung cancer tumor suppressor gene locus on chromosome 3p21.3. REN-19 is equivalent to LKB1/STK11, a gene that is defective in Peutz-Jeghers syndrome and cancer. REN-26 is encoded by the bcr gene involved in the [t(9:22)] bcr/abl translocation. Genes encoding 3 of the antigens in the series showed differential mRNA expression; REN-3 displays a pattern of tissue-specific isoforms, and REN-21 and REN-43 are expressed at a high level in testis in comparison to 15 other normal tissues. The other 62 antigens were broadly expressed in normal tissues. With regard to immunogenicity, 20 of the 65 antigens reacted only with autologous sera. Thirty-three antigens reacted with sera from normal donors, indicating that their immunogenicity is not restricted to cancer. The remaining 12 antigens reacted with sera from 5-25% of the cancer patients but not with sera from normal donors. Seventy percent of the renal cancer patients had antibodies directed against one or more of these 12 antigens. Our results demonstrate the potential of the SEREX approach for the analysis of the humoral immune response against human cancer.

Aged↗

PubMed1999

Humoral immune responses of cancer patients against "Cancer-Testis" antigen NY-ESO-1: correlation with clinical events.

Humoral immune responses against the "Cancer-Testis" (CT) antigen NY-ESO-1 are frequently observed in patients with NY-ESO-1 expressing tumors. This is in contrast to other known tumor antigens (TA) defined by antibody or cytotoxic T cell (CTL) reactivity, i.e., MAGE-1, MAGE-3, SSX2, Melan A, and tyrosinase. No NY-ESO-1 antibody has been detected in healthy controls and patients with NY-ESO-1 negative tumors. In this study, we have assessed the NY-ESO-1 serum antibody response in patients with NY-ESO-1 positive tumors of different histological types and stages using Western blotting and an ELISA. Of the 12 patients analyzed, 10 had demonstrable NY-ESO-1 antibodies at the start of the study. All patients were followed for changes in NY-ESO-1 antibody titers during the course of tumor treatment and clinical evolution. In 4 patients, an increase of NY-ESO-1 antibody titer was observed with progression of disease or extensive tumor necrosis under treatment. One patient showed a stable NY-ESO-1 antibody titer over 3 years along with gradual regression of a large tumor mass. In 5 patients, a decrease of NY-ESO-1 antibody was detected: in 1 patient after curative tumor resection, in 3 patients with partial regression of metastatic disease under chemo- and immunotherapy, and in another patient with a NY-ESO-1 negative tumor relapse. Our results indicate that the induction and maintenance of NY-ESO-1 antibody is dependent on the presence of NY-ESO-1 expressing tumors. Furthermore, changes in NY-ESO-1 antibody titers correlate with the evolution of NY-ESO-1 positive disease.

Adult↗

PubMed1999

Isoforms of the human PDZ-73 protein exhibit differential tissue expression.

Patients with renal and colon cancer frequently develop IgG autoantibodies toward the NY-CO-38/PDZ-73 antigen, a protein of 652 amino acids (73 kDa) which contains three copies of the PDZ protein-protein interaction domain. The gene encoding PDZ-73 mapped to chromosome 11p15.4-p15.1. Additional tissue-specific isoforms were identified: PDZ-45, which lacks the third PDZ domain and the putative PEST protein degradation motif, is expressed in kidney, colon, small intestine, brain and testis; PDZ-54 and PDZ-59, which also lack the third PDZ domains, have unique carboxyl terminal amino acids and are expressed in brain, kidney, bladder, colon cancer and renal cancer; and a putative PDZ-37 isoform, containing only the third PDZ domain, that is expressed in the central nervous system. Immunohistochemical staining with anti-PDZ 73 monoclonal antibodies showed strong cytoplasmic reactivity in epithelial cells of the small intestine, colon and kidney tubules, with a prominent apical staining pattern in cells of the small intestine. The reactivity pattern of the antibodies with various tissues correlated with the mRNA expression pattern of the PDZ-45 isoform. The existence of multiple PDZ-73 isoforms with variations in tissue distribution, PDZ domains, protein degradation sequences and carboxyl terminal structure indicate that these isoforms have distinct tissue-specific functions.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Expression of melanocyte-associated markers gp-100 and Melan-A/MART-1 in angiomyolipomas. An immunohistochemical and rt-PCR analysis.

Angiomyolipomas are tumours of uncertain histogenesis, most often occurring in association with the kidney. A characteristic finding is their reactivity with HMB-45, a monoclonal antibody to the melanocyte-associated antigen gp-100. We tested 18 angiomyolipomas for their reactivity with A103, a monoclonal antibody to Melan-A (MART-1), another melanocyte-associated marker, and compared it with HMB-45. All cases were positive with both antibodies, yet most cases showed a more homogeneous staining pattern with A103. Normal kidney was immunohistochemically negative for both antibodies. We also performed RT-PCR assays for gp-100 and Melan-A in 4 of the 18 angiomyolipoma samples and in three normal kidney samples. All 4 angiomyolipoma specimens revealed mRNA for both melanocyte differentiation markers. gp-100 mRNA was found in the samples of normal kidney, but Melan-A mRNA was not. Our study shows that angiomyolipomas express the melanocyte-associated antigens Melan-A and gp-100 at the protein and at the mRNA level, suggesting a true expression of these antigens rather than cross-reacting epitopes. Based on the mRNA expression pattern, immunohistochemical analysis is the preferred method for the detection of gp-100, while Melan-A can be used at the protein and mRNA levels. Our study demonstrates that A103 is a useful marker for the diagnosis of angiomyolipomas.

Angiomyolipoma↗

PubMed1998

Demonstration of an interferon gamma-dependent tumor surveillance system in immunocompetent mice.

This study demonstrates that endogenously produced interferon gamma (IFN-gamma) forms the basis of a tumor surveillance system that controls development of both chemically induced and spontaneously arising tumors in mice. Compared with wild-type mice, mice lacking sensitivity to either IFN-gamma (i.e., IFN-gamma receptor-deficient mice) or all IFN family members (i.e., Stat1-deficient mice) developed tumors more rapidly and with greater frequency when challenged with different doses of the chemical carcinogen methylcholanthrene. In addition, IFN-gamma-insensitive mice developed tumors more rapidly than wild-type mice when bred onto a background deficient in the p53 tumor-suppressor gene. IFN-gamma-insensitive p53(-/-) mice also developed a broader spectrum of tumors compared with mice lacking p53 alone. Using tumor cells derived from methylcholanthrene-treated IFN-gamma-insensitive mice, we found IFN-gamma's actions to be mediated at least partly through its direct effects on the tumor cell leading to enhanced tumor cell immunogenicity. The importance and generality of this system is evidenced by the finding that certain types of human tumors become selectively unresponsive to IFN-gamma. Thus, IFN-gamma forms the basis of an extrinsic tumor-suppressor mechanism in immunocompetent hosts.

Adenocarcinoma↗

PubMed1998

Identification of multiple cancer/testis antigens by allogeneic antibody screening of a melanoma cell line library.

Cancer/testis (CT) antigens-immunogenic protein antigens that are expressed in testis and a proportion of diverse human cancer types-are promising targets for cancer vaccines. To identify new CT antigens, we constructed an expression cDNA library from a melanoma cell line that expresses a wide range of CT antigens and screened the library with an allogeneic melanoma patient serum known to contain antibodies against two CT antigens, MAGE-1 and NY-ESO-1. cDNA clones isolated from this library identified four CT antigen genes: MAGE-4a, NY-ESO-1, LAGE-1, and CT7. Of these four, only MAGE-4a and NY-ESO-1 proteins had been shown to be immunogenic. LAGE-1 is a member of the NY-ESO-1 gene family, and CT7 is a newly defined gene with partial sequence homology to the MAGE family at its carboxyl terminus. The predicted CT7 protein, however, contains a distinct repetitive sequence at the 5' end and is much larger than MAGE proteins. Our findings document the immunogenicity of LAGE-1 and CT7 and emphasize the power of serological analysis of cDNA expression libraries in identifying new human tumor antigens.

Amino Acid Sequence↗

PubMed1998

Characterization of human colon cancer antigens recognized by autologous antibodies.

The screening of cDNA expression libraries derived from human tumors with autologous antibody (SEREX) has proven to be a powerful method for defining the structure of tumor antigens recognized by the humoral immune system. In the present study, 48 distinct antigens (NY-CO-1-NY-CO-48) reactive with autologous IgG were identified by SEREX analysis in 4 patients with colon cancer. Sequencing analysis showed that 17 of the cDNA clones were previously uncharacterized molecules and 31 represented known gene products. The individual cDNA clones were analyzed in the following manner: a search for mutations or other structural changes; an analysis of mRNA expression in a panel of normal tissues; and a frequency analysis of the antibody response to the expressed product in the sera of colon cancer patients and normal individuals. The initial analysis showed NY-CO-13 to be a mutated version of the p53 tumor suppressor gene. Three of the 48 antigens showed a differential pattern of mRNA expression, with NY-CO-27 (galectin-4) expressed primarily in gastrointestinal tract, and NY-CO-37 and -38 showing a pattern of tissue-specific isoforms. With regard to immunogenicity, 20 of the 48 antigens were detected by allogeneic sera; 14 of these were reactive with sera from both normal donors and cancer patients, and 6 other clones (NY-CO-8, -9, -13, -16, -20 and -38) reacted exclusively with sera from colon cancer patients (ranging from 14% to 27%). Our results on colon cancer illustrate both the complexity and the potential of the SEREX approach for analysis of the humoral immune response against human cancer.

Adult↗

PubMed1998

A survey of the humoral immune response of cancer patients to a panel of human tumor antigens.

Evidence is growing for both humoral and cellular immune recognition of human tumor antigens. Antibodies with specificity for antigens initially recognized by cytotoxic T lymphocytes (CTLs), e.g., MAGE and tyrosinase, have been detected in melanoma patient sera, and CTLs with specificity for NY-ESO-1, a cancer-testis (CT) antigen initially identified by autologous antibody, have recently been identified. To establish a screening system for the humoral response to autoimmunogenic tumor antigens, an enzyme-linked immunosorbent assay (ELISA) was developed using recombinant NY-ESO-1, MAGE-1, MAGE-3, SSX2, Melan-A, and tyrosinase proteins. A survey of sera from 234 cancer patients showed antibodies to NY-ESO-1 in 19 patients, to MAGE-1 in 3, to MAGE-3 in 2, and to SSX2 in 1 patient. No reactivity to these antigens was found in sera from 70 normal individuals. The frequency of NY-ESO-1 antibody was 9.4% in melanoma patients and 12.5% in ovarian cancer patients. Comparison of tumor NY-ESO-1 phenotype and NY-ESO-1 antibody response in 62 stage IV melanoma patients showed that all patients with NY-ESO-1(+) antibody had NY-ESO-1(+) tumors, and no patients with NY-ESO-1(-) tumors had NY-ESO-1 antibody. As the proportion of melanomas expressing NY-ESO-1 is 20-40% and only patients with NY-ESO-1(+) tumors have antibody, this would suggest that a high percentage of patients with NY-ESO-1(+) tumors develop an antibody response to NY-ESO-1.

Antibodies, Neoplasm↗

PubMed1998

Human lung cancer antigens recognized by autologous antibodies: definition of a novel cDNA derived from the tumor suppressor gene locus on chromosome 3p21.3.

Serological analysis of a recombinant lung cancer cDNA expression library with the autologous patient serum led to the isolation of 20 clones representing 12 different genes: 4 of these were known genes, and the other 8 were previously unknown genes. Of the four known genes, aldolase A (NY-LU-1), previously shown to be overexpressed in lung cancer, was most frequently isolated. The other three genes were annexin XI, human HIV Rev-interacting protein Rip-1, and the human homologue of the ATP-binding arsA component of the bacterial arsenite transporter, all of which are known to be widely expressed in human tissues. Among the eight unknown genes, of most interest was NY-LU-12. Cloning of full-length NY-LU-12 showed that this cDNA was derived from the same gene as g16, a partially sequenced gene that mapped to the lung cancer tumor suppressor gene locus on chromosome 3p21. The reported g16 sequence, however, was significantly shorter (2433 versus 3591 bp). As a result of alternate splicing and subsequent frameshift, the reported g16 protein is 603 amino acids shorter than the NY-LU-12 product (1123 residues) at its COOH terminus and would therefore lack the epitopes recognized by the autologous serum. Analysis of the putative NY-LU-12 protein sequence predicted that it is a nuclear zinc finger protein with two RNA-binding domains, and Southern analysis showed that this gene is partially deleted in the lung cancer line NCI-H740 but not in nine other lung cancer lines. Screening of normal and cancer patient sera showed anti-NY-LU-12 seroreactivity in 2 of 21 allogeneic lung cancer patients but not in 24 patients with other tumors or in 16 sera from healthy donors. Comparison of NY-LU-12 cDNA from Lu15 tumor and normal lung tissue by DNA sequencing and/or single-strand conformation polymorphism analysis showed no evidence of mutation. Considering the high frequency of 3p21 alterations in lung cancer and the fact that the tumor suppressor gene or genes in this locus have not been identified, additional studies on the NY-LU-12 gene and its product are warranted.

Amino Acid Sequence↗

PubMed1998

Simultaneous humoral and cellular immune response against cancer-testis antigen NY-ESO-1: definition of human histocompatibility leukocyte antigen (HLA)-A2-binding peptide epitopes.

A growing number of human tumor antigens have been described that can be recognized by cytotoxic T lymphocytes (CTLs) in a major histocompatibility complex (MHC) class I-restricted fashion. Serological screening of cDNA expression libraries, SEREX, has recently been shown to provide another route for defining immunogenic human tumor antigens. The detection of antibody responses against known CTL-defined tumor antigens, e.g., MAGE-1 and tyrosinase, raised the question whether antibody and CTL responses against a defined tumor antigen can occur simultaneously in a single patient. In this paper, we report on a melanoma patient with a high-titer antibody response against the "cancer-testis" antigen NY-ESO-1. Concurrently, a strong MHC class I-restricted CTL reactivity against the autologous NY-ESO-1-positive tumor cell line was found. A stable CTL line (NW38-IVS-1) was established from this patient that reacted with autologous melanoma cells and with allogeneic human histocompatibility leukocyte antigen (HLA)-A2(-), NY-ESO-1-positive, but not NY-ESO-1-negative, melanoma cells. Screening of NY-ESO-1 transfectants with NW38-IVS-1 revealed NY-ESO-1 as the relevant CTL target presented by HLA-A2. Computer calculation identified 26 peptides with HLA-A2-binding motifs encoded by NY-ESO-1. Of these, three peptides were efficiently recognized by NW38-IVS-1. Thus, we show that antigen-specific humoral and cellular immune responses against human tumor antigens may occur simultaneously. In addition, our analysis provides a general strategy for identifying the CTL-recognizing peptides of tumor antigens initially defined by autologous antibody.

Aged↗

PubMed1998

Immunoreactivity for A103, an antibody to melan-A (Mart-1), in adrenocortical and other steroid tumors.

The Melan-A (MART-1) gene encodes an antigen recognized by cytotoxic T cells. It has been said to be restricted in its expression to melanocytes. However, here we report the presence of immunoreactivity for A103, an antibody to Melan-A, in five adrenocortical adenomas, 16 primary and 13 metastatic adrenocortical carcinomas, four Leydig cell tumors of the testis, and three Sertoli-Leydig cell tumors of the ovary. To evaluate the potential diagnostic role of this antibody, we studied immunoreactivity for A103 in 111 carcinomas, 40 germ cell tumors, and 33 miscellaneous nonmelanocytic epithelioid tumors. All of them were negative for A103. Our findings suggest that once melanoma is excluded, A103 can aid in the recognition of steroid hormone-producing tumors and may be particularly useful in the diagnosis of adrenocortical carcinoma. The presence of immunoreactivity for A103 practically excludes any other carcinoma that may enter into the differential diagnosis of adrenocortical tumors.

Adrenal Cortex Neoplasms↗

PubMed1998

A103: An anti-melan-a monoclonal antibody for the detection of malignant melanoma in paraffin-embedded tissues.

Melan-A is a previously defined, melanocyte differentiation antigen, and an anti-Melan-A murine monoclonal antibody, A103, was recently developed by our group. In this study, we evaluated A103 immunoreactivity on formalin-fixed, paraffin-embedded tissues, exploring the potential of A103 in the diagnosis of metastatic melanoma. Seventy-five metastatic melanomas, 10 primary melanomas, and 10 benign melanocytic nevi were tested. The reactivity of A103 was compared with HMB-4, an anti-gp100 antibody. Results showed that all nevi were A103 positive, and most primary melanomas were A103 and HMB45 positive. Of 75 metastatic melanomas, 61 (81%) were A103 positive, and 56 (75%) were HMB45 positive. Of 19 HMB45-negative lesions, 8 were A103 positive; of 14 A103-negative lesions, 3 were HMB45 positive. Eleven metastatic lesions, as well as 2 of 10 primary melanomas, were dual negative. These negative cases consisted mainly of the spindle cell and desmoplastic variants. Of the positive cases, A103 showed homogeneous staining in a significantly higher proportion of cases than HMB45 (72% versus 52%). In addition, focal staining with less than 5% reactive tumor cells was seen more frequently in HMB45 (12 of 56) than in A103 (5 of 61). These results indicated that A103 can be used as a first-line antibody in the diagnosis of metastatic melanoma. Our results also showed that A103 reacted with angiomyolipoma, which is known to be HMB45 positive. Of normal tissues, unexpected A103 reactivity was observed in the adrenal cortex, granulosa and theca cells of the ovary, and Leydig cells of the testis. This A103 immunoreactivity in benign and neoplastic tissues of nonmelanocytic origin, the basis of which is unclear, could also be of potential diagnostic value.

Angiomyolipoma↗

PubMed1998

Expression of melan-A (MART1) in benign melanocytic nevi and primary cutaneous malignant melanoma.

The Melan-A (MART1) gene encodes an antigen recognized by cytotoxic T cells. Although its expression in metastatic melanoma has been documented in the literature by several investigators, little is known about its distribution in primary melanomas and benign melanocytic nevi. In this study, we evaluated Melan-A expression immunohistochemically on sections from paraffin-embedded material of 50 benign nevi and 40 primary cutaneous melanomas using the monoclonal antibody A103. To evaluate a potential role of A103 in the differential diagnosis of melanocytic from nonmelanocytic tumors, we also analyzed a number of benign and malignant peripheral nerve sheath tumors, fibrohistiocytic tumors, and leiomyosarcomas. Immunoreactivity with A103 was present in all "nonneurotized" nevi and in all nondesmoplastic primary melanomas, both in the intraepidermal and the dermal component. Only two nevi that underwent prominent neurotization showed no staining with A103. Although all melanomas with epithelioid cells tended to be strongly positive with A103, only 4 of 13 spindle cell and desmoplastic melanomas (all positive with anti-S-100 and negative with HMB-45) were immunoreactive with A103 (two focally, two diffusely). None of the nonmelanocytic lesions expressed Melan-A. Our results confirm that Melan-A protein is broadly expressed in the majority of benign and malignant melanocytic lesions and suggest that A103 can be helpful diagnostically, not only for metastatic tumors, but also for primary skin lesions. Its use in distinguishing between melanocytic and peripheral nerve sheath tumors, however, is limited because of the low or absent expression of Melan-A in nevi that underwent neurotization and spindle cell and desmoplastic melanomas.

Antibodies, Monoclonal↗

PubMed1997

A testicular antigen aberrantly expressed in human cancers detected by autologous antibody screening.

Serological analysis of recombinant cDNA expression libraries (SEREX) using tumor mRNA and autologous patient serum provides a powerful approach to identify immunogenic tumor antigens. We have applied this methodology to a case of esophageal squamous cell carcinoma and identified several candidate tumor targets. One of these, NY-ESO-1, showed restricted mRNA expression in normal tissues, with high-level mRNA expression found only in testis and ovary tissues. Reverse transcription-PCR analysis showed NY-ESO-1 mRNA expression in a variable proportion of a wide array of human cancers, including melanoma, breast cancer, bladder cancer, prostate cancer, and hepatocellular carcinoma. NY-ESO-1 encodes a putative protein of Mr 17,995 having no homology with any known protein. The pattern of NY-ESO-1 expression indicates that it belongs to an expanding family of immunogenic testicular antigens that are aberrantly expressed in human cancers in a lineage-nonspecific fashion. These antigens, initially detected by either cytotoxic T cells (MAGE, BAGE, GAGE-1) or antibodies [HOM-MEL-40(SSX2), NY-ESO-1], represent a pool of antigenic targets for cancer vaccination.

Amino Acid Sequence↗

PubMed1996

Serological analysis of Melan-A(MART-1), a melanocyte-specific protein homogeneously expressed in human melanomas.

Recent progress in the structural identification of human melanoma antigens recognized by autologous cytotoxic T cells has led to the recognition of a new melanocyte differentiation antigen, Melan-A(MART-1). To determine the properties of the Melan-A gene product, Melan-A recombinant protein was produced in Escherichia coli and used to generate mouse monoclonal antibodies (mAbs). Two prototype mAbs, A103 and A355, were selected for detailed study. Immunoblotting results with A103 showed a 20-22-kDa doublet In Melan-A mRNA positive melanoma cell lines and no reactivity with Melan-A mRNA-negative cell lines. A355, in addition to the 20-22-kDa doublet, recognized several other protein species in Melan-A mRNA-positive cell lines. Immunocytochemical assays on cultured melanoma cells showed specific and uniform cytoplasmic staining in Melan-A mRNA-positive cell lines. Immunohistochemical analysis of normal human tissues with both mAbs showed staining of adult melanocytes and no reactivity with the other normal tissues tested. Analysis of 21 melanoma specimens showed homogenous staining of tumor cell cytoplasm in 16 of 17 Melan-A mRNA-positive cases and no reactivity with the three Melan-A mRNA-negative cases.

Animals↗

PubMed1996

Induction of antibodies reactive with GM2 ganglioside after immunization with lipopolysaccharides from Camplyobacter jejuni.

Ganglioside GM2, expressed on the surface of some human cancers, is a promising target for immune therapy, since GM2 antibodies are cytotoxic, can be induced in humans by vaccination, and the presence of GM2 antibodies is associated with a better prognosis in melanoma patients. In our efforts to induce long-lived, cytotoxic GM2 antibodies, we investigated lipopolysaccharides (LPS) containing "GM2-like" oligosaccharides. LPS were prepared from Campylobacter jejuni serotypes O:1, O:23, or O:36 (all sharing the oligosaccharide structure GalNAcbeta1-4Gal(113NeuAc)-Hex with ganglioside GM2), and tested for their ability to induce GM2-reactive antibodies. Immunization of NZW rabbits (2 animals per vaccine) with LPS from C. jejuni serotype O:1 in Freund's adjuvant resulted in production of high-titer IgG antibodies reactive with purified bovine brain GM2 in ELISA, dot-blot immune strains and immune thin-layer chromatography, and with GM2 derived from various human tumors by immune thin-layer chromatography. These rabbit antibodies bound to cancer cell lines expressing GM2 on their cell surface, as determined by mixed hemadsorption assays, mediating strong antibody-dependent cellular cytotoxicity (ADCC) with tumor cells expressing cell-surface GM2. Antibodies induced by vaccination with C. jejuni serotype O:1 were higher-titer (IgG ELISA titer > 1:60,000) than antibodies induced by immunization with purified GM2 (IgG ELISA titer > 1:200). Immunization with LPS from C. jejuni serotype O:36 resulted in production of moderately high-titer IgM and low-titer IgG GM2 antibodies. Immunization with LPS from C. jejuni serotype O:23 did not elicit GM2-reactive antibodies. No clinical symptoms were observed in animals immunized with these LPS preparations, with purified GM2 ganglioside, or with LPS derived from C. jejuni serotype O:19 (containing a GM1-like oligosaccharide). Our results indicate that lipopolysaccharides sharing carbohydrate epitopes with gangliosides may be useful immunogens for inducing antibodies to ganglioside antigens.

Animals↗