The insulinoma-associated 1: a novel promoter for targeted cancer gene therapy for small-cell lung cancer.

The insulinoma-associated 1 (INSM1) gene is expressed exclusively during early embryonal development, but has been found re-expressed at high levels in neuroendocrine tumors. The regulatory region of the INSM1 gene is therefore a potential candidate for regulating expression of a therapeutic gene in transcriptionally targeted cancer gene therapy against neuroendocrine tumors. We analyzed expression of a reporter gene from a 1.7 kb region of the INSM1 promoter in a large number of small-cell lung cancer (SCLC) cell lines. This INSM1 promoter region showed very high levels of expression in most of the SCLC cell lines and expression was absent in cell lines of non-neuroendocrine origin. Inclusion of the general transcriptional enhancer from SV40 compromised the specificity of the promoter and did not enhance transcription in most of the SCLC cell lines. For comparison, the region of the gastrin releasing peptide (GRP) previously suggested for SCLC gene therapy was analyzed in a similar manner. High expression was observed for a number of cell lines, but unlike for the INSM1 promoter, reporter gene expression from the GRP promoter did not correlate to the relative GRP mRNA levels, demonstrating that this region may not contain all necessary regulatory elements. Expression of the suicide gene herpes simplex virus thymidine kinase (HSV-TK) from the INSM1 promoter in combination with treatment with the prodrug ganciclovir (GCV) caused a significant increase in GCV sensitivity specifically in INSM1-expressing cell lines. The INSM1 promoter is therefore a potential novel tool for transcriptionally targeted gene therapy for neuroendocrine tumors.

IA-2, a transmembrane protein tyrosine phosphatase, is expressed in human lung cancer cell lines with neuroendocrine phenotype.

IA-2 is a transmembrane protein tyrosine phosphatase isolated recently from a human insulinoma subtraction library. Its expression in normal human tissues is restricted primarily to the pancreatic islets and brain. In this report, we describe the expression of IA-2 mRNA in a panel consisting of 20 lung tumor cell lines with neuroendocrine and non-neuroendocrine phenotype and 17 non-lung tumor cell lines. IA-2 mRNA was detected in 8 of 11 neuroendocrine small cell lung carcinomas, 4 of 4 non-small cell lung carcinomas with neuroendocrine phenotype, and 11 of 12 non-lung neuroendocrine tumor cell lines. In contrast, IA-2 mRNA was not detected in five non-neuroendocrine lung carcinomas, nor in a panel of other non-neuroendocrine tumor cell lines. The expression pattern of IA-2 mRNA suggests that IA-2 may represent a new marker for neuroendocrine differentiation In human lung cancer cells and perhaps other neuroendocrine tumors.

Polypeptide core of a human pancreatic tumor mucin antigen.

This work describes the molecular properties of the polypeptide core of a human pancreatic mucin antigen isolated from a human pancreatic adenocarcinoma cell line, HPAF. Pancreatic tumor mucin was isolated by a combination of molecular sieve chromatography and CsCl/4 M guanidine-HCl density gradient ultracentrifugation. Trifluoromethane sulfonic acid was used to remove carbohydrate units from purified mucin molecules. A rabbit monospecific polyclonal antibody was generated against pancreatic apomucin and reacted with a Mr greater than 200,000 species. The antibody binding data indicated that the rabbit antiserum raised against pancreatic apomucin cross-reacted with a breast mucin synthetic peptide. Northern blot and immunodot blot analyses of various cell line extracts revealed that a tandem repeat sequence and a similar mRNA were detected in both pancreatic and breast mucin-producing cell lines. These results suggest that pancreatic apomucin and breast apomucin share some similarity in tandem repeated nucleic acid and protein sequences.

Isolation and properties of a human pancreatic adenocarcinoma-associated antigen, DU-PAN-2.

This work describes the molecular properties of a human pancreatic adenocarcinoma-associated mucin-like antigen defined by a murine monoclonal antibody (DU-PAN-2). DU-PAN-2 antigen is a large molecule, readily detected in the body fluids of patients with pancreatic adenocarcinoma and sensitive to neuraminidase treatment and alkaline reduction. The antigen binding activity of the DU-PAN-2 immunoglobulin M antibody is markedly reduced when coupled to an insoluble matrix. Therefore, the antigen was partially purified from the serum and ascites of patients with pancreatic adenocarcinoma by ammonium sulfate fractionation and Affi-Gel-Blue chromatography to remove most of the serum proteins. Noncovalently associated proteins were further separated on CsCl/CsBr density gradients and noncovalently associated lipids removed by organic solvent extraction. DU-PAN-2 antigen was monitored by a solid-phase competition radioimmunoassay. We have been able to detect antigen reactivity and analyze its electrophoretic pattern following transfer from 1% agarose gel onto nitrocellulose paper and immunoblotting with DU-PAN-2 antibody. Antigen was also labeled metabolically with various radioactive monosaccharides and sulfates. Radioimmunoprecipitation of labeled antigen with DU-PAN-2 antibody showed two distinct broad antigen bands consistent with the immunoblotting signals. The heavily glycosylated and polydisperse nature of this antigen and the results of various enzyme treatments suggest that the DU-PAN-2 epitope is expressed on a mucin-like molecule.

IA-1, a new marker for neuroendocrine differentiation in human lung cancer cell lines.

IA-1 is a recently isolated novel complementary DNA which encodes a protein of 510 amino acids that contains both a zinc finger DNA-binding domain and a putative prohormone domain. mRNA expression of IA-1 has been found thus far only in tumors of neuroendocrine origin. In this report we describe the expression of IA-1 mRNA in a panel of 64 human lung cancer cell lines. IA-1 mRNA was detected by Northern blot analysis in 97% (30 of 31) of small cell lung cancer cell lines. In contrast, IA-1 mRNA was detected in only 13% (4 of 30) of non-small cell lung cancer cell lines. Nine of the 30 (30%) expressed either chromogranin A mRNA or produced L-dopa decarboxylase. Four of these 9 (44%) had detectable levels of IA-1 mRNA. In most of the lung cancer cell lines examined, IA-1 showed high concordance with the other neuroendocrine markers, L-dopa decarboxylase, and chromogranin A. The one exception was a variant small cell lung cancer cell line which expressed low or nondetectable levels of L-dopa decarboxylase. IA-1 is a candidate marker of neuroendocrine differentiation of human lung tumors.

Autoantibodies to IA-2 in IDDM: location of major antigenic determinants.

Thirty-three IDDM sera that immunoprecipitated full-length IA-2 were tested for reactivity with different fragments of the IA-2 molecule. The fragments were prepared by PCR amplification of IA-2 cDNA and by expression in a rabbit reticulocyte transcription/translation system. Whereas all 33 sera reacted with the intracellular domain (amino acid 604 to 979), none of the sera reacted with the extracellular domain of IA-2 (amino acid 31 to 577). Analysis of the reactivity of IDDM sera with the different regions of the intracellular domain showed that 94% (31 of the 33) reacted with the COOH-terminus (amino acid 771 to 979), 40% reacted with the NH2-terminus (amino acid 604 to 776), and 40% reacted with the middle portion (amino acid 692 to 875). Of the 31 sera that reacted with the COOH-terminus, 14 of these reacted only with the COOH-terminus and with no other region. Of the 13 sera that reacted with the NH2-terminus, only one reacted exclusively with the NH2-terminus. Treatments of the different domains of IA-2 with trypsin showed that only the COOH-terminus was resistant to trypsin, arguing that it is from this region of the IA-2 molecule that the 40-kDa tryptic fragment from insulinoma cells is derived. From these experiments, it is concluded that the major antigenic determinant of IA-2 is located at the COOH-terminus and that minor antigenic determinants are located at the NH2-terminus and middle portion of the intracellular domain.

Value of antibodies to islet protein tyrosine phosphatase-like molecule in predicting type 1 diabetes.

Islet antigens associated with type 1 diabetes include a recently identified protein tyrosine phosphatase-like molecule IA-2, which contains the intracellular fragment IA-2ic. To determine whether combinations of antibodies including those to IA-2 characterize and predict type 1 diabetes, we studied antibodies to IA-2, IA-2ic, glutamic acid decarboxylase (GAD65), and islet cell antibodies (ICAs) in 1) 60 newly diagnosed type 1 diabetic patients followed for 1 year, 2) 31 monozygotic twin pairs discordant for type 1 diabetes followed up to 12 years (11 twins developed diabetes), 3) 18 dizygotic twin pairs discordant for type 1 diabetes, and 4) normal healthy control subjects. Newly diagnosed type 1 diabetic patients frequently had antibodies to IA-2 (62%), IA-2ic (67%), GAD65 (77%), and ICAs (85%). The intracellular fragment of IA-2 probably contains the immunodominant epitope as 137 of 143 samples with IA-2 antibodies from type 1 diabetic patients also had IA-2ic antibodies. Monozygotic twins were usually discordant for antibody specificities. Concordance was higher in monozygotic than matched dizygotic twins for both antibody combinations (33 vs. 6%, P < 0.05) and the development of diabetes (33 vs. 0%, P < 0.01). In monozygotic twins, all the antibodies were highly predictive of type 1 diabetes (positive predictive values all >87%), although antibodies were also detected in twins at low risk of disease. In summary, IA-2 emerges as a major antigen associated with type 1 diabetes and distinct from GAD65. Type 1 diabetes-associated autoimmunity, which is probably induced by environmental factors, does not necessarily herald progression to the disease. However, genetic factors may influence the development of combinations of disease-associated antibodies and the progression to type 1 diabetes.

The relationship between humoral and cellular immunity to IA-2 in IDDM.

Autoantibodies to the neuroendocrine protein insulinoma-associated protein 2 (IA-2), a member of the tyrosine phosphatase family, have been observed in individuals with or at increased risk for IDDM. Because this disease is thought to result from a T-cell-mediated autoimmune destruction of the insulin-producing pancreatic beta-cells, we analyzed humoral and cellular immune reactivity to this autoantigen to further define its role in the pathogenesis of IDDM. Peripheral blood mononuclear cells (PBMC) from individuals with newly diagnosed IDDM or at varying levels of risk for the disease were stimulated in vitro with the entire 42-kDa internal domain of IA-2 (amino acids 603-979), a series of control antigens (glutathionine-S-transferase, tetanus toxoid, Candida albicans, mumps, bovine serum albumin), and a mitogen (phytohemagglutinin). The frequency and mean stimulation index of PBMC proliferation against IA-2 was significantly higher in newly diagnosed IDDM subjects (14 of 33 [42%]; 3.8+/-4.5 at 10 microg/ml) and autoantibody-positive relatives at increased risk for IDDM (6 of 9 [66%]; 3.9+/-3.2) compared with autoantibody-negative relatives (1 of 15 [7%]; 1.8+/-1.0) or healthy control subjects (1 of 12 [8%]; 1.5+/-1.0). The frequencies of cellular immune reactivities to all other antigens were remarkably similar between each subject group. Sera from 58% of the newly diagnosed IDDM patients tested were IA-2 autoantibody positive. Despite investigations suggesting an inverse association between humoral and cellular immune reactivities against islet-cell-associated autoantigens, no such relationship was observed (rs=0.18, P=0.39) with respect to IA-2. These studies support the autoantigenic nature of IA-2 in IDDM and suggest the inclusion of cellular immune responses as an adjunct marker for the disease.

L-Arginine regulates the expression of the T-cell receptor zeta chain (CD3zeta) in Jurkat cells.

L-Arginine is a versatile amino acid that plays a central role in the normal function of several organ systems including the immune system. Its availability is tightly controlled and varies significantly in different organs and tissues in the body. L-Arginine plays an important role in supporting T-cell proliferation. Its depletion in certain disease states results in a diminished T-cell response. The main purpose of this study was to determine the effect of the depletion of L-arginine on the expression of the T-cell receptor (TCR) proteins. When the helper T-cell line Jurkat was cultured in arginine-free medium, there was a preferential decrease in the expression of the TCR zeta chain (CD3zeta). The reduced expression of CD3zeta was observed within 24 h of culture in L-arginine-free medium and was completely reversed with the replenishment of L-arginine. Furthermore, the absence of L-arginine blocked the normal re-expression of the TCR that had been internalized after antigen stimulation. There also was a significant decrease in proliferation of Jurkat cells in the absence of L-arginine; however, L-arginine depletion did not prevent the up-regulation of the interleukin 2 receptor chains upon stimulation, nor did it significantly diminish the production of interleukin 2. The changes in the expression of CD3zeta chain were not induced by apoptosis. Thus, the availability of L-arginine in the microenvironment may play a significant role in regulating the expression of the TCR.

The human leukocyte antigen HLA DRB3*020/DQA1*0501 haplotype is associated with Graves' disease in African Americans.

Information on genetic susceptibility to Graves' disease in African Americans is limited. We studied DRB1, DQB1, DRB3 subtypes, DQA1*0501, DQA1*0201, and CTLA-4 polymorphisms in 49 African American patients with adult onset Graves' disease and 47 racially-matched controls using PCR-based sequence-specific priming methods. There were no significant differences in DRB1 or DQB1 allelic frequencies or CTLA-4 polymorphisms between patients and controls. However, we found that the frequency of DRB3 was significantly increased in the patients (75.5% vs. 57.4%, P = 0.006, X2 = 3.52), especially for the DRB3*0202 subtype (53.1% vs. 23.4, P = 0.003, X2 = 8.91). In this one respect, the finding was in concordance with our previous observations in Caucasian patients with adult-onset Graves' disease. In addition, whereas the frequency of DQA1*0501 was increased (P = 0.018, X2 = 5.63) in our patients, the haplotype of DRB3/DQA1*0501, or DRB3*0202/DQA1*0501 was found to be more strongly associated (P = 0.008, X2 = 7.0; P = 0.0008, X2 = 11.34, respectively). These data suggest that DRB3*0202, particularly when found with DQA1*0501 in a haplotype is a susceptible gene(s) for Graves' disease in adult African Americans. Considering these data with those in Caucasian patients, our results would suggest that the primary Graves susceptible locus is likely DRB3 and not DRB1.

Molecular cloning and identification of a receptor-type protein tyrosine phosphatase, IA-2, from human insulinoma.

A novel 3.6-kb cDNA, IA-2, with a 2,937-bp open reading frame was isolated from a human insulinoma subtraction library (ISL-153). The predicted amino acid sequence and in vitro-translated product of IA-2 cDNA revealed a 979-amino-acid protein with a pI value of 7.09 and a molecular mass of 105,847 daltons. The protein sequence is consistent with a signal peptide, an extracellular domain, a transmembrane region, and an intracellular domain. The extracellular domain contains an unusual cysteine-rich region following the signal peptide. The intracellular cytoplasmic domain of IA-2 possesses highly conserved regions similar to the catalytic domains found in members of the protein tyrosine phosphatase (PTP) family. Northern blot analysis showed that IA-2 mRNA was expressed in five of five freshly isolated human insulinomas, rat and mouse insulinoma cell lines, and enriched normal mouse islets. It also was found in normal human brain, pituitary, pancreas, and brain tumor cell lines, but not in a variety of other normal or tumor tissues. Based on the sequence and expression data, it appears that IA-2 is a new member of the receptor-type PTP family that is expressed in islet and brain tissues.

Molecular characterization of a pancreas-specific protein disulfide isomerase, PDIp.

A novel tissue-specific cDNA, PDIp, was previously isolated from human pancreas. It encodes a protein that is structurally and functionally related to protein disulfide isomerase (PDI). To compare the expression pattern of PDI and PDIp in human pancreas and liver tissues, we prepared rabbit polyclonal antiserum against a recombinant glutathione-S-transferase-coupled PDIp fusion protein. Western blot analysis revealed that pancreas expresses both PDI and PDIp, whereas liver only expresses PDI. Rabbit antiserum raised against recombinant PDIp immunostained specifically to the acinar cells of human pancreas. Treatment of PDIp with peptide:N-glycosidase F caused PDIp down shift in the NaDodSO4-PAGE gel, indicating that PDIp is a glycoprotein. A 2.0-kb message was detected from mouse pancreas using a human PDIp cDNA probe. Similarly, PDIp glycoprotein was detected in mouse pancreas extract by anti-human PDIp antiserum, suggesting that PDIp is highly conserved in human and mouse pancreas. From these studies, we conclude that the pancreas expresses two members of PDI and that PDIp is a glycoprotein specifically expressed in pancreatic acinar cells.

Characterization and chromosomal localization of a new protein disulfide isomerase, PDIp, highly expressed in human pancreas.

Protein disulfide isomerase (PDI) catalyzes protein folding and thiol-disulfide interchange reactions. The enzyme is localized in the lumen of endoplasmic reticulum (ER) and is abundant in secretory cells of various tissues. In this study we describe the isolation and characterization from human pancreas of a new protein, PDIp, that is structurally and functionally related to PDIs. PDIp cDNA is 1,659 bp in length and predicts a protein with an open reading frame of 511 amino acids. PDIp amino acid sequence shows 46% identity and 66% similarity to that of human PDI. PDIp possesses two thioredoxin-like active sites (WCGHCQ and WCTHCK) and an endoplasmic reticulum retention signal sequence, KEEL, at the carboxyl terminus. Northern analysis of normal human tissues and various human tumor cell lines revealed PDIp mRNA (2.0 kb) expression only in the normal pancreas. Recombinant PDIp protein catalyzed reductive cleavage of insulin and renaturation of reduced RNaseA. Somatic cell genetics and fluorescence in situ hybridization localized the PDIp gene to the short arm of human chromosome 16. It is concluded that PDIp is a new member of the PDI family and is highly expressed in human pancreas.

Profile and differential expression of protein tyrosine phosphatases in mouse pancreatic islet tumor cell lines.

Protein tyrosine phosphatases (PTPs) play important roles in cell growth and differentiation of normal and tumor cells. In this study, we analyzed the PTP profile in two pancreatic islet tumor cell lines. Transcripts were isolated from alphaTC-1 (glucagon-secreting) and betaTC-1 (insulin-secreting) cell lines for templates. A pair of degenerative primers, based on the conserved regions of known PTPs, was used to amplify the transcripts by polymerase chain reaction. A total of 1,620 clones was examined by restriction enzyme analysis and cDNA sequencing. Twenty-one PTPs were identified, including nine cytosolic PTPs (TcPTP, P19PTP, PTP1B, PTPMEG, PTP1C, SYP, PTPH1, PTPL1, and PTPD1), nine transmembrane PTPs (PTPdelta, PTPgamma, PTPkappa, DEP-1, IA-2, LAR, PTPalpha, PTPNE3, and PTPepsilon), and three new PTPs--PTPmu-like PTPkappa-like, and IA-2beta. An RNase protection assay demonstrated that some of these PTPs were expressed predominantly in glucagonoma (i.e., PTPdelta and IA-2) and others in insulinoma (i.e., PTP1C, PTPkappa, and PTPNE3) cells. In this report, we present the first profile of PTPs in alpha and beta tumor cell lines.

Modifications to the INSM1 promoter to preserve specificity and activity for use in adenoviral gene therapy of neuroendocrine carcinomas.

The INSM1 gene encodes a transcriptional repressor that is exclusively expressed in neuronal and neuroendocrine tissue during embryonic development that is re-activated in neuroendocrine tumors. Using the 1.7 kbp INSM1 promoter, an adenoviral HSV thymidine kinase gene therapy was tested for the treatment of neuroendocrine tumors. An unforeseen interference on the INSM1 promoter specificity from the adenoviral genome was observed. Attempts were made to protect the INSM1 promoter from the influence of essential adenoviral sequences and to further enhance the tissue specificity of the INSM1 promoter region. Using the chicken ß-globin HS4 insulator sequence, we eliminated off-target tissue expression from the Ad-INSM1 promoter-luciferase2 constructs in vivo. In addition, inclusion of two copies of the mouse nicotinic acetylcholine receptor (n(AchR)) neuronal-restrictive silencer element (NRSE) reduced nonspecific activation of the INSM1 promoter both in vitro and in vivo. Further, inclusion of both the HS4 insulator with the n(AchR) 2 × NRSE modification showed a two log increase in luciferase activity measured from the NCI-H1155 xenograft tumors compared with the original adenovirus construct. The alterations increase the therapeutic potential of adenoviral INSM1 promoter-driven suicide gene therapy for the treatment of a variety of neuroendocrine tumors.

Hypermethylation of hepatic Gck promoter in ageing rats contributes to diabetogenic potential.

AIMS/HYPOTHESIS: Hepatic glucokinase (GCK) is a key enzyme in glucose utilisation. Downregulation of its activity is associated with insulin resistance and type 2 diabetes mellitus. However, it is unknown whether hepatic Gck expression is influenced by age and is involved in ageing-mediated diabetes, and whether the degree of methylation of the hepatic Gck promoter is correlated with the transcription of Gck. To address the question, we evaluated hepatic Gck transcription and promoter methylation in young (14 weeks), adult (40 weeks) and aged (80 weeks) rats. METHODS: Hepatic glycogen, Gck expression and the kinase activity of GCK were measured in three age groups. The CpG methylation status was determined by both bisulphite direct sequencing and clone sequencing of the PCR amplificates of Gck promoter. The causal relationship between Gck methylation and mRNA expression was confirmed by treating rat primary hepatocytes with 5-aza-2'-deoxycytidine (5-Aza-CdR). RESULTS: We have shown an age-associated decline in hepatic glycogen, Gck expression levels and the kinase activity of hepatic GCK. The eleven CpG sites studied displayed age-related progressive methylation changes in hepatic Gck promoter, which were confirmed by two methods: direct and clone sequencing. After 5-Aza-CdR treatment of rat primary hepatocytes, there was a fourfold increase in Gck expression. CONCLUSIONS/INTERPRETATION: Our results demonstrate that an age-related increase in methylation is negatively associated with hepatic Gck expression, suggesting that DNA methylation could be involved in increasing age-dependent susceptibility to hepatic insulin resistance and diabetes. Thus, the epigenetic modification of the hepatic Gck promoter may represent an important marker for diabetogenic potential during the ageing process.

Molecular characterization of the promoter region of a neuroendocrine tumor marker, IA-1.

IA-1 is an intronless gene, which encodes a 510 amino acid protein with a zinc-finger DNA-binding motif that is expressed in tumors of neuroendocrine origin. The 5'-upstream region of the IA-1 gene was recently sequenced. In this paper, the regulatory elements and the promoter region of the 5'-upstream region were analyzed by use of a series of deletion mutants (ranging from +26 bp to -2090 bp upstream of the IA-1 gene), which were tested in a pituitary tumor cell line, AtT-20, and Hela cells by transient transfection assays. These experiments showed that a 506 base pair upstream sequence was sufficient for maximal expression of a reporter gene. Multiple known regulatory elements were found within this region including three E boxes and a clustered Sp-1 site. In addition, Southwestern blot analysis, using a radiolabeled promoter sequence (extending from -108 bp to -66 bp) and nuclear extracts from both neuroendocrine and non-neuroendocrine cell lines, revealed four promoter binding proteins designated PBP1, PBP2, PBP3 and PBP4 with molecular weights of 55 kD, 32 kD, 29 kD, and 27/28 kD, respectively. These studies suggest that several different regulatory elements in the 5'-upstream region of the IA-1 gene and at least four different nuclear proteins may be involved in the cell-specific expression of IA-1.

Isolation, sequence and expression of a novel mouse brain cDNA, mIA-2, and its relatedness to members of the protein tyrosine phosphatase family.

This study describes the isolation of a putative transmembrane protein tyrosine phosphatase (PTP), mIA-2, from a mouse brain cDNA library. The cDNA encodes 979 amino acids containing a unique extracellular domain and a single intracellular catalytic domain. Expression of mIA-2 was found primarily in the central nervous system and in neuroendocrine cells. The sequence shares a high degree of homology with its human counterpart (92% identity), especially in the intracellular domain, which shows 99.3% identity between the two species. In both human and mouse IA-2, several substitutions were found in the highly conserved regions including an Ala to Asp substitution in the core sequence. Bacterial expression of a glutathione S-transferase fusion protein showed that mIA-2 had no enzyme activity with conventional substrates such as Raytide, myelin basic protein, angiotensin, RR-src and pNpp. When tested with the total tyrosine-phosphorylated cellular proteins isolated on an anti-phosphotyrosine antibody column, it also showed little, if any, enzyme activity. These findings suggest that mIA-2 is a new member of the transmembrane PTP family that either has very narrow substrate specificity perhaps requiring post-translational modification for enzyme activity or has a still unknown biological function.

IA-2 and IA-2beta: the immune response in IDDM.

Pancreatic islet cell autoantigens associated with insulin-dependent diabetes mellitus (IDDM) include a recently identified family of protein tyrosine phosphatase-like molecules, notably IA-2 and IA-2beta. IA-2 is a 979 amino acid transmembrane protein located on human chromosome 2q35, whereas IA-2beta is 986 amino acids long located on human chromosome 7q36. Comparison of human IA-2 and IA-2beta showed 74% identity within the intercellular domains, but only 27% indentify within the extracellular domains. These IA-2 molecules are expressed predominantly in cells of neuroendocrine origin, particularly pancreatic islets and brain. Radioimmunoprecipitation with recombinant IA-2 and IA-2beta has been used to measure autoantibodies to these molecules and their intracellular fragments. Autoantibodies to IA-2 are detected in the majority (60% to 80%) of newly diagnosed IDDM patients and in less than 2% of controls. The major antigenic determinants of both IA-2 and IA-2beta reside within the C-terminus of their intracellular domains. In first-degree relatives of IDDM patients, the presence of autoantibodies to IA-2 is predictive of IDDM and in combination with autoantibodies to glutamic acid decarboxylase (GAD) the positive predictive value is in the 50% range. The role of IA-2 and IA-2beta in the pathogenesis of IDDM is still unclear. Identification of these antigens has extended our ability to predict the disease and may be valuable in the search for antigen-specific therapies to prevent IDDM.

Specific, major histocompatibility complex-unrestricted recognition of tumor-associated mucins by human cytotoxic T cells.

We have previously reported the establishment of cytotoxic T-cell lines from pancreatic cancer patients, by continuously stimulating tumor-draining lymph node cells with allogeneic pancreatic tumor cell lines. After the preliminary characterization of their phenotype and tumor specificity, detailed studies performed with one of the cell lines, W.D., show that it recognizes a specific antigen, a large and heavily glycosylated mucin molecule, expressed on pancreatic and breast tumors and tumor cell lines. Although this recognition appears major histocompatibility complex (MHC)-unrestricted, the antigen receptor used by the cytotoxic T cell is the alpha/beta heterodimer, typically found on MHC-restricted T cells. The target antigen is atypical, however, in its ability to directly bind and activate the T cells in the absence of self MHC, presumably by abundant and regularly repeated antigenic epitopes. These findings are important because they demonstrate a specific T-cell response against a human tumor-associated antigen. In addition to pancreatic and breast tumors, various mucin molecules are known to be produced by other tumors of epithelial cell origin and could be expected to stimulate similar T-cell-mediated immune responses.