Identifying genetic traces of historical expansions: Phoenician footprints in the Mediterranean

The Phoenicians were the dominant traders in the Mediterranean Sea two thousand to three thousand years ago and expanded from their homeland in the Levant to establish colonies and trading posts throughout the Mediterranean, but then they disappeared from history. We wished to identify their male genetic traces in modern populations. Therefore, we chose Phoenician-influenced sites on the basis of well-documented historical records and collected new Y-chromosomal data from 1330 men from six such sites, as well as comparative data from the literature. We then developed an analytical strategy to distinguish between lineages specifically associated with the Phoenicians and those spread by geographically similar but historically distinct events, such as the Neolithic, Greek, and Jewish expansions. This involved comparing historically documented Phoenician sites with neighboring non-Phoenician sites for the identification of weak but systematic signatures shared by the Phoenician sites that could not readily be explained by chance or by other expansions. From these comparisons, we found that haplogroup J2, in general, and six Y-STR haplotypes, in particular, exhibited a Phoenician signature that contributed > 6% to the modern Phoenician-influenced populations examined. Our methodology can be applied to any historically documented expansion in which contact and noncontact sites can be identified.     

Intracellular retention of the NKG2D ligand MHC class I chain-related gene A in human melanomas confers immune privilege and prevents NK cell-mediated cytotoxicity

Most tumors grow in immunocompetent hosts despite expressing NKG2D ligands (NKG2DLs) such as the MHC class I chain-related genes A and B (MICA/B). However, their participation in tumor cell evasion is still not completely understood. Here we demonstrate that several human melanomas (cell lines and freshly isolated metastases) do not express MICA on the cell surface but have intracellular deposits of this NKG2DL. Susceptibility to NK cell-mediated cytotoxicity correlated with the ratio of NKG2DLs to HLA class I molecules but not with the amounts of MICA on the cell surface of tumor cells. Transfection-mediated overexpression of MICA restored cell surface expression and resulted in an increased in vitro cytotoxicity and IFN-gamma secretion by human NK cells. In xenografted nude mice, these melanomas exhibited a delayed growth and extensive in vivo apoptosis. Retardation of tumor growth was due to NK cell-mediated antitumor activity against MICA-transfected tumors, given that this effect was not observed in NK cell-depleted mice. Also, mouse NK cells killed MICA-overexpressing melanomas in vitro. A mechanistic analysis revealed the retention of MICA in the endoplasmic reticulum, an effect that was associated with accumulation of endoH-sensitive (immature) forms of MICA, retrograde transport to the cytoplasm, and degradation by the proteasome. Our study identifies a novel strategy developed by melanoma cells to evade NK cell-mediated immune surveillance based on the intracellular sequestration of immature forms of MICA in the endoplasmic reticulum. Furthermore, this tumor immune escape strategy can be overcome by gene therapy approaches aimed at overexpressing MICA on tumor cells.     

Autoimmune disease-associated histamine receptor H1 alleles exhibit differential protein trafficking and cell surface expression

Structural polymorphisms (L263P, M313V, and S331P) in the third intracellular loop of the murine histamine receptor H(1) (H(1)R) are candidates for Bphs, a shared autoimmune disease locus in experimental allergic encephalomyelitis and experimental allergic orchitis. The P-V-P haplotype is associated with increased disease susceptibility (H(1)R(S)) whereas the L-M-S haplotype is associated with less severe disease (H(1)R(R)). In this study, we show that selective re-expression of the H(1)R(S) allele in T cells fully complements experimental allergic encephalomyelitis susceptibility and the production of disease-associated cytokines while selective re-expression of the H(1)R(R) allele does not. Mechanistically, we show that the two H(1)R alleles exhibit differential cell surface expression and altered intracellular trafficking, with the H(1)R(R) allele being retained within the endoplasmic reticulum. Moreover, we show that all three residues (L-M-S) comprising the H(1)R(R) haplotype are required for altered expression. These data are the first to demonstrate that structural polymorphisms influencing cell surface expression of a G protein-coupled receptor in T cells regulates immune functions and autoimmune disease susceptibility.     

Plasma fatty acid binding protein 4 is associated with atherogenic dyslipidemia in diabetes

The aim of this study was to evaluate the impact of adipocyte fatty acid binding protein 4 (FABP4) on the lipid profile in type 2 diabetic subjects. Plasma levels of FABP4 and adiponectin and an extensive lipid profile were analyzed in 169 type 2 diabetic subjects and 105 controls. Type 2 diabetic subjects were categorized according the presence of atherogenic dyslipidemia. Univariate statistical analyses, partial correlation tests, and binary logistic regression models were applied. In type 2 diabetic subjects, FABP4 was positively correlated with plasma triglycerides (P = 0.007), apolipoprotein C-III (apoC-III) (P = 0.009), and all the components of triglyceride-rich lipoproteins, including VLDL triglycerides (P = 0.002), VLDL-cholesterol (P = 0.001), and VLDL apoB (P = 0.001). FABP4 was inversely correlated with apoA-I (P = 0.038), HDL-cholesterol (P = 0.002), and HDL apoA-I (P = 0.010) in type 2 diabetic subjects. These correlations are not significantly affected by age, gender, body mass index, adiponectin, insulin, or any pharmacological treatment. The associations are even stronger when the FABP4/adiponectin ratio is considered. None of these associations were observed in controls. High FABP4 and low adiponectin levels are independent predictors of atherogenic dyslipidemia. In conclusion, FABP4 plasma concentrations hold strong potential for development as a clinical biomarker for atherogenic dyslipidemia, independent of obesity and insulin resistance, in type 2 diabetic subjects.     

Genetically Engineered Vesicular Stomatitis Virus in Gene Therapy: Application for Treatment of Malignant Disease

We report here the generation of recombinant vesicular stomatitis virus (VSV) able to produce the suicide gene product thymidine kinase (TK) or cytokine interleukin 4 (IL-4). In vitro cells infected with the engineered viruses expressed remarkably high levels of biologically active TK or IL-4 and showed no defects in replication compared to the wild-type virus. Recombinant viruses retained their ability to induce potent apoptosis in a variety of cancer cells, while normal cells were evidently more resistant to infection and were completely protected by interferon. Significantly, following direct intratumoral inoculation, VSV expressing either TK or IL-4 exhibited considerably more oncolytic activity against syngeneic breast or melanoma tumors in murine models than did the wild-type virus or control recombinant viruses expressing green fluorescent protein (GFP). Complete regression of a number of tumors was achieved, and increased granulocyte-infiltrating activity with concomitant, antitumor cytotoxic T-cell responses was observed. Aside from discovering greater oncolytic activity following direct intratumoral inoculation, however, we also established that VSV expressing IL-4 or TK, but not GFP, was able to exert enhanced antitumor activity against metastatic disease. Following intravenous administration of the recombinant viruses, immunocompetent BALB/c mice inoculated with mammary adenocarcinoma exhibited prolonged survival against lethal lung metastasis. Our data demonstrate the validity of developing novel types of engineered VSV for recombinant protein production and as a gene therapy vector for the treatment of malignant and other disease.     

Enhancing the early performance of the leguminous shrub Retama sphaerocarpa (L.) Boiss.: fertilisation versus Rhizobium inoculation

We have investigated the effect on growth of fertilisation versus biological nitrogen fixation by rhizobial nodules in Retama sphaerocarpa(L.) Boiss, a leafless leguminous shrub native to the Iberian Peninsula and North-West Africa that has generated interest for revegation of dry Mediterranean habitats. Our main objective was to optimise the formation of root nodules under nursery conditions and to evaluate their influence on the first year of seedling growth in comparison with standard fertilisation. Seedlings of R. sphaerocarpa from two Spanish localities were grown under two levels of fertilisation, and half of each were inoculated with rhizobia isolated from adult Retama, Cytisus and Adenocarpusplants in the field. Although some promiscuity was observed, nodulation was significantly successful with specific rhizobia. At the end of the experiment, highly fertilised plants were taller and heavier and exhibited larger photosynthetic rates than either nodulated or non-nodulated plants under low fertilisation. High fertilisation enhanced seedling growth but inhibited both the nodulation and the nitrogenase activity of the nodules. Thus, physiological differences between nodulated and non-nodulated plants were observed in the low but not in the high fertilisation treatment. Nitrogen uptake and use was enhanced by root nodules, which translated into enhanced photosynthesis and growth. Since inoculation is simple, environmentally friendly and cheap, and nodulated plants are more likely to overcome transplant stress than non-nodulated ones, our results suggest that inoculation together with low, background fertilisation (instead of high fertilisation) should be used when producing high quality seedlings of this autochthonous Mediterranean shrub.