Higher prevalence of LAP+ (Latency TGFß-Associated Peptide) T cells at the tissue level in patients with early gastric cancer.

The presence of cells with regulatory functions in patients with cancer is one of the mechanisms whereby the immune system cannot confront tumor growth. We sought to determine the prevalence of immunoregulatory T-cell subpopulations, expressing the latency TGFß-associated peptide (LAP), in patients with gastric adenocarcinoma. T cells were enriched from blood or gastric tissue (tumoral, TT or tumor-free, TF) samples from 22 patients, 6 with early (EGC) and 16 with advanced gastric cancer (AGC). CD4, CD8, LAP, FoxP3 and IFN-γ were measured by cytometry. CD8 + LAP + cells were increased at tumoral sites, especially in early stages of the disease, as compared to tumor-free explants (EGC 5.28 % [4.67-6.64]*; AGC 2.90 % [1.37-4.44]; TF 3.14 % [2.33-4.16]; *p < 0.05 vs TF). Likewise, the LAP+/CD8 + LAP- ratio is increased in gastric samples from patients with early disease (EGC 0.38 [0.30-0.45]*, AGC 0.12 [0.07-0.14]; TF 0.12 [0.09-0.31]; *p < 0.05 vs AGC).Disease progression is accompanied by decreased LAP membrane expression and, probably, increased LAP secretion, therefore limiting the response to the tumor.

Evolution and molecular interactions of major histocompatibility complex (MHC)-G, -E and -F genes.

Classical HLA (Human Leukocyte Antigen) is the Major Histocompatibility Complex (MHC) in man. HLA genes and disease association has been studied at least since 1967 and no firm pathogenic mechanisms have been established yet. HLA-G immune modulation gene (and also -E and -F) are starting the same arduous way: statistics and allele association are the trending subjects with the same few results obtained by HLA classical genes, i.e., no pathogenesis may be discovered after many years of a great amount of researchers' effort. Thus, we believe that it is necessary to follow different research methodologies: (1) to approach this problem, based on how evolution has worked maintaining together a cluster of immune-related genes (the MHC) in a relatively short chromosome area since amniotes to human at least, i.e., immune regulatory genes (MHC-G, -E and -F), adaptive immune classical class I and II genes, non-adaptive immune genes like (C2, C4 and Bf) (2); in addition to using new in vitro models which explain pathogenetics of HLA and disease associations. In fact, this evolution may be quite reliably studied during about 40 million years by analyzing the evolution of MHC-G, -E, -F, and their receptors (KIR-killer-cell immunoglobulin-like receptor, NKG2-natural killer group 2-, or TCR-T-cell receptor-among others) in the primate evolutionary lineage, where orthology of these molecules is apparently established, although cladistic studies show that MHC-G and MHC-B genes are the ancestral class I genes, and that New World apes MHC-G is paralogous and not orthologous to all other apes and man MHC-G genes. In the present review, we outline past and possible future research topics: co-evolution of adaptive MHC classical (class I and II), non-adaptive (i.e., complement) and modulation (i.e., non-classical class I) immune genes may imply that the study of full or part of MHC haplotypes involving several loci/alleles instead of single alleles is important for uncovering HLA and disease pathogenesis. It would mainly apply to starting research on HLA-G extended haplotypes and disease association and not only using single HLA-G genetic markers.

HLA alleles and haplotypes in Iran Tabriz Azeris population: genes and languages do not correlate.

Azeri people are at present day mainly living in an area which comprises North (Azerbaijan) and South (Azeri Iran provinces) parts, living the biggest population in Azeri Iran provinces with about 17-20 million people. They were studied HLA-A, -B, -DRB1 and -DQB1 allele and extended haplotype frequencies in unrelated Iranian Tabriz Azeris from a rural area close to Tabriz City. The HLA extended haplotypes with highest frequencies are: 1) HLA- A*24:02-B*35:01-DRB1*11:01-DQB1*03:01, shared with Mediterraneans and southern Russians (Chuvash, which also show Mediterranean characters); and 2) HLA-A*01:02-B*08:01-DRB1*03:01-DQB1*02:01, found also in Chuvash and other Azeri samples from Tabriz. Neí's DA HLA-DRB1 genetic distances, HLA-DRB1 Neighbour-Joining dendrogram and Vista analyses show that population with closest distance is Kurdish, followed by Iranian Gorgan and Southern Russia/ North Caucasus Chuvash; probably these latter groups and Azeris were populating North Mesopotamia/ Caucasus Mts. since prehistoric times. Kurds (in Iraq and Iran) do not speak Turk while Azeris do: they are both genetically close, but they are not genetically close to present day Anatolia (Turkey) Turks who also speak Turk language and show a typical Mediterranean HLA profile. In summary, Azeri population studies show examples that genes and languages do not correlate, contradicting the postulate asserted by others.

HLA-G: Too Much or Too Little? Role in Cancer and Autoimmune Disease.

HLA-G is a non-classical HLA class I molecule with immunomodulatory properties. It was initially described at the maternal-fetal interface, and it was later found that this molecule was constitutively expressed on certain immuneprivileged tissues, such as cornea, endothelial and erythroid precursors, and thymus. The immunosuppressive effect of HLA-G is exerted through the interaction with its cognate receptors, expressed on immunocompetent cells, like ILT2, expressed on NK, B, T cells and APCs; ILT4, on APCs; KIR, found on the surface of NK cells; and finally, the co-receptor CD8. Because of these immunomodulatory functions, HLA-G has been involved in several processes, amongst which organ transplantation, viral infections, cancer progression, and autoimmunity. HLA-G neo-expression on tumors has been recently described in several types of malignancies. In fact, tumor progression is tightly linked to the presence of the molecule, as it exerts its tolerogenic function, inhibiting the cells of the immune system and favoring tumor escape. Several polymorphisms in the 3'UTR region condition changes in HLA-G expression (14bp and +3142C/G, among others), which have been associated with both the development and outcome of patients with different tumor types. Also, in recent years, several studies have shown that HLA-G plays an important role in the control of autoimmune diseases. The ability of HLA-G to limit the progression of these diseases has been confirmed and, in fact, levels of the molecule and several of its polymorphisms have been associated with increased susceptibility to the development of autoimmune diseases, as well as increased disease severity. Thus, modulating HLA-G expression in target tissues of oncology patients or patients with autoimmune diseases may be potential therapeutic approaches to treat these pathological conditions.

HLA-G 3'UTR Polymorphisms Are Linked to Susceptibility and Survival in Spanish Gastric Adenocarcinoma Patients.

HLA-G is a non-classical class I HLA molecule that induces tolerance by acting on receptors of both innate and adaptive immune cells. When overexpressed in tumors, limits surveillance by the immune system. The HLA-G gene shows several polymorphisms involved in mRNA and protein levels. We decided to study the implication of two polymorphisms (rs371194629; 14bp INS/DEL and rs1063320; +3142 C/G) in paired tissue samples (tumoral and non-tumoral) from 107 Spanish patients with gastric adenocarcinoma and 58 healthy control individuals, to assess the possible association of the HLA-G gene with gastric adenocarcinoma susceptibility, disease progression and survival. The presence of somatic mutations involving these polymorphisms was also analyzed. The frequency of the 14bp DEL allele was increased in patients (70.0%) compared to controls (57.0%, p=0.025). In addition, the haplotype formed by the combination of the 14bp DEL/+3142 C variants is also increased in patients (54.1% vs 44.4%, p=0.034, OR=1.74 CI95% 1.05-2.89). Kaplan-Meier analysis revealed that 14bp DEL/DEL patients showed lower 5-year life-expectancy than INS/DEL or INS/INS (p=0.041). Adjusting for TNM staging (Cox regression analysis) disclosed a significant difference in death risk (p=0.03) with an expected hazard 2.6 times higher. Finally, no somatic mutations were found when comparing these polymorphisms in tumoral vs non-tumoral tissues, which indicates that this is a preexisting condition in patients and not a de novo, tumor-restricted, event. In conclusion, the variants predominant in patients were those increasing HLA-G mRNA stability and HLA-G expression, clearly involving this molecule in gastric adenocarcinoma susceptibility, disease progression and survival and making it a potential target for immunotherapeutic approaches.

A Reliable and Standardizable Differential PCR and qPCR Methodology Assesses HER2 Gene Amplification in Gastric Cancer.

We have applied two PCR techniques, differential PCR (diffPCR) and qPCR for the identification of HER2 gene amplifications in genomic DNA of tumor and distal gastric samples from patients with gastric cancer. The diffPCR technique consists of the simultaneous amplification of the HER2 gene and a housekeeping gene by conventional PCR and the densitometric analysis of the bands obtained. We established a cut-off point based on the mean and standard deviation analyzing the DNA of 30 gastric tissues from patients undergoing non-cancer gastrectomy. diffPCR and qPCR yielded consistent results. HER2-overexpression was detected in 25% of patients and was further confirmed by immunohistochemistry and immunofluorescence. The approaches herein described may serve as complementary and reliable methods to assess HER2 amplification.