The mechanisms that lead to loss of MHC class I expression in different types of tumors are not yet fully known. Accordingly, we studied colorectal carcinomas to elucidate the specific mechanisms of evasion of the T-cell immune response. We selected tumors with total loss of MHC class I expression and studied 124 colorectal carcinomas with immunohistochemical staining and anti-HLA monoclonal antibodies (mAb). Fourteen of 124 (11%) tumors exhibited a phenotype with HLA class I total loss. Microsatellite instability (MSI) analysis was also carried out in the same tumor samples. The expression of beta2-microglobulin (beta2m), HLA-A, B, and C antigens, transporter associated with antigen processing 1 (TAP1), TAP2, low-molecular-weight protein 2 (LMP2), and LMP7 were analyzed using reverse-transcription polymerase chain reaction (RT-PCR) in microdissected tumor samples. Four of 14 microsatellite instability-positive (MSI+) and W6/32 mAb-negative tumors showed biallelic inactivation of beta2m and accumulation of HLA class I heavy chain in the cytoplasm. MSI-negative (MSI-)/W6/32 mAb-negative tumors presented alterations in the expression of components of the antigen processing machinery (APM). Nine of 10 tumor samples showed LMP7 gene downregulation, and four of 10 presented TAP2 dysregulation. This group apparently expressed normal levels of heavy chain and beta2m mRNA. Two major mechanisms in colorectal cancer appear to be responsible for the total loss of MHC surface expression (beta2m mutations and LMP7/TAP2 downregulation) that may contribute to the failure of T lymphocyte recognition during an immune response. The precise identification of the molecular defects that underlie HLA class I abnormalities will have important implications for patients receiving T-cell-based specific immunotherapy.

Mutations have been identified in the beta2-microglobulin gene of tumor cells of two metastatic melanoma patients who received immunizations with MAGE peptides. One mutation abolishes the start codon whereas the other introduces a premature stop codon. The second beta2-microglobulin allele of both tumors appears to be lost on the basis of sequence data and loss of microsatellite heterozygosity. The lack of beta2-microglobulin gene product results in the absence of HLA class I antigens on the surface of the tumor cells. This may explain why the tumors of both patients progressed despite the immunization treatment and shows the necessity of analyzing in depth the antigen presentation capability of the tumor cells for the interpretation of clinical trials involving anti-tumor vaccination.

Helicobacter pylori establishes life-long infections in the gastric mucosa of over 1 billion people worldwide. In many cases, without specific antimicrobial intervention, H. pylori infected individuals will develop type B gastritis, chronic peptic ulcers and, more rarely, gastric neoplasias. Conventional antimicrobial therapy has been complicated by dramatic increases in resistance to macrolides, metronidazole and fluoroquinolones. Here, we report the development of novel therapeutics that specifically target the unique flavodoxin component of an essential metabolic pathway of H. pylori. With the use of high-throughput screening methodology, we have tested 10,000 chemicals and have identified 29 compounds that bind flavodoxin, four of which interrupted in vitro electron transfer to flavodoxin physiological partners. Three of these compounds are bactericidal and promisingly selective for H. pylori. The minimal inhibitory concentrations of two of them are 10 times lower than their minimal cytotoxic concentrations for HeLa cells. Importantly, neither of the four inhibitors is toxic for mice after administration of 1-10 mg kg(-1) doses twice a day for 5 days. Enzymatic, thermodynamic and structural characterization of the inhibitor-flavodoxin complexes suggests these compounds could act by modifying the redox potentials of flavodoxin. These newly discovered inhibitors represent promising selective leads against the different diseases associated to H. pylori infection.

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Key words: HLA; ␤2m; antigen processing machinery; bladder carcinoma; tumor escape Downregulation of HLA class I molecules is a frequent event observed in human tumors, and it is believed to be related with tumor immune escape. Several types of HLA class I downregulation have been described including total, locus, allele and haplotype loss. 1 Total HLA class I loss is a relatively frequent phenotype (9 -52%) detected in tumors derived from different tissues such as melanoma, head-neck, colorectal, prostate and breast cancer. 2 Molecular mechanisms that have been shown to be responsible for this phenotype include the following. 1) ␤2-microglobulin (␤2-m) gene mutations, which play an important role in assembly of the HLA complex (HLA heavy chain, ␤2-m and peptide). ␤2-microglobulin gene mutations result in failure to assemble the class I molecules for cell surface expression and are therefore responsible for total HLA class I loss. Frequent ␤2-m mutations have been described in colorectal and gastric cancers of the microsatellite mutator phenotype (MMP), 3,4 which exhibit a type of genetic instability characterized by the accumulation of somatic mutations in repeated sequences. 5 The ␤2-m gene contains several repeat sequences and so is a mutational target for mismatch repair deficiency. The frequency of ␤2-m mutations in other tumor types that infrequently display MMP (breast cancer and melanoma) is lower than in colorectal carcinomas. 3 2) Downregulation of proteins involved in the antigen processing pathway is responsible for total HLA class I loss due to a deficiency in peptide formation, loading of MHC molecules and transport. MHC class I presentation of peptides is a multistep process involving several molecules: catalytic ␤ subunits of the proteasome (LMP2, LMP7 and LMP10), transporter associated with antigen processing (TAP-1 and TAP-2) and various endoplasmic reticulum chaperones. Numerous studies have demonstrated deficiencies in the expression of antigen-processing genes in tumor cell lines. 6 -10 Most of the deficits described are combined deficiencies comprising multiple components, 6,9,10 though selective deficiencies have also been described. 7,10 Most of these alterations are corrected by cytokine treatment, particularly by IFNgamma. In contrast, structural defects in antigen-processing genes are very uncommon and only 2 point mutations in TAP-1 gene have been described. 11,12 3) Finally, hypermethylation of HLA class I promotor genes can also repress HLA class I expression, as has been demonstrated in melanoma cell lines. 13 Although HLA class I total loss can be found in histologically different tumor types, carcinogenesis in each tissue present particular characteristics. In despite of the great advances in our knowledge of molecular lesions that produce HLA class I total loss in tumor cell lines, in some particular human tumor tissues the underlying mechanisms remain to be established.Bladder cancer is one of the most common malignancies worldwide, with transitional cell carcinoma (TCC) being ...

Our results show that, at least in some white populations, the contribution of the cytokine gene polymorphisms evaluated in this study (IL-1B, IL-1RN, IL-12p40, LTA, IL-10, IL-4, and TGF-B1) to GC susceptibility may be less relevant than previously reported.

Alterations in the surface expression of HLA class I molecules have been described as a strategy of tumors to evade recognition by cytotoxic T cells. We detected complete loss of HLA class I antigen presentation for 2 tumor cell lines from 1 melanoma patient, the first originated from a regional lymph node lesion diagnosed simultaneously with the primary tumor and the second established 8 months later from a metastatic pleural effusion sample. Antigen presentation was not inducible with IFN-␥ but could be restored after transfection of tumor cells with b2m cDNA, indicating a defect in b2m expression. Analysis of the nature of this defect revealed that it originated from at least 2 mutational events affecting both copies of the b2m gene: a microdeletion of 498 bp in one b2m gene, including its entire exon 1, and a macrodeletion involving the entire copy of the second b2m gene. Microsatellite analysis pointed to the macrodeletion by demonstrating LOH for several specific markers on the long arm (q) of chromosome 15. Structural imbalance of 15q was verified by FISH. FISH studies also indicated the coexistence of a structurally abnormal variant of chromosome 15q with 2 apparently entire chromosomes 15q harboring the homozygous b2m microdeletion. Block of b2m expression in tumor cells builds a barrier to immunotherapy of cancer patients, and its early incidence should be of major consideration in the development and design of immunotherapeutic strategies.

The consistent dysregulation of HLA expression in cervical neoplasia is likely to influence the natural history of the disease and prospects for cell-mediated vaccine therapies. We have studied the underlying mechanisms in eight new cervical cancer cell lines derived from primary tumour biopsies. At least five independent mechanisms leading to changes in HLA expression were seen: HLA class I allelic transcription but no protein; abnormal HLA class I allelic transcription; no HLA-B locus transcription; loss of heterozygosity (LOH); no gammaIFN-mediated upregulation of HLA class I expression, and/or no interferon-gamma (gammaIFN)-mediated HLA class II induction. These were evident in different combinations in 7/8 cell lines showing that multiple, mostly irreversible mechanisms not overridden by gammaIFN, are responsible for HLA dysregulation in cervical neoplasia. Point mutations were responsible for lack of HLA-A2 expression in two cases. In cell line 808, the mutation encodes a stop codon in exon 3; in cell line 778, mutation of the first intron acceptor site leads to use of an alternative AG site in exon 2, resulting in a frameshift and a stop codon after the translation of only 38 amino acids. Tumour cells showing specific HLA class I loss may have selective advantage in the face of tumour-specific cytotoxic T cells (CTL). Such immune escape mechanisms present a major obstacle for the success of CTL-mediated therapies in cervical cancer.