Antagonistic effects of oxidized low density lipoprotein and alpha-tocopherol on CD36 scavenger receptor expression in monocytes: involvement of protein kinase B and peroxisome proliferator-activated receptor-gamma

Vitamin E deficiency increases expression of the CD36 scavenger receptor, suggesting specific molecular mechanisms and signaling pathways modulated by alpha-tocopherol. We show here that alpha-tocopherol down-regulated CD36 expression (mRNA and protein) in oxidized low density lipoprotein (oxLDL)-stimulated THP-1 monocytes, but not in unstimulated cells. Furthermore, alpha-tocopherol treatment of monocytes led to reduction of fluorescent oxLDL-3,3'-dioctadecyloxacarbocyanine perchlorate binding and uptake. Protein kinase C (PKC) appears not to be involved because neither activation of PKC by phorbol 12-myristate 13-acetate nor inhibition by PKC412 was affected by alpha-tocopherol. However, alpha-tocopherol could partially prevent CD36 induction after stimulation with a specific agonist of peroxisome proliferator-activated receptor-gamma (PPARgamma; troglitazone), indicating that this pathway is susceptible to alpha-tocopherol action. Phosphorylation of protein kinase B (PKB) at Ser473 was increased by oxLDL, and alpha-tocopherol could prevent this event. Expression of PKB stimulated the CD36 promoter as well as a PPARgamma element-driven reporter gene, whereas an inactive PKB mutant had no effect. Moreover, coexpression of PPARgamma and PKB led to additive induction of CD36 expression. Altogether, our results support the existence of PKB/PPARgamma signaling pathways that mediate CD36 expression in response to oxLDL. The activation of CD36 expression by PKB suggests that both lipid biosynthesis and fatty acid uptake are stimulated by PKB.     

Metazoan parasite fauna as a biological tag for the habitat of the flounder Hippoglossina macrops from northern Chile, in a depth gradient

Quantitative changes in the parasite communities of the flounder Hippoglossina macrops (Steindachner) were studied along a depth gradient. Samples were obtained from the waters off Coquimbo, Chile (29 degrees 18'S to 30 degrees 50'S), at depths ranging from 160 to 342 m. Samples were assigned to 3 depths, i.e., shallow water (less than 200 m), midwater (from 200 to 300 m), and deep water (more than 300 m). Twelve parasite species were recorded: Neoheterobothrium chilensis and Entobdella hippoglossi (Monogenea); Holobomolochus chilensis and Protochondria longicauda (Copepoda); Gliptonobdella sp. (Hirudinea); Nybelinia surmenicola, Scolex pleuronectis, and Neobothriocephalus adspinosus (Cestoda); Floridosentis sp. and Corynosoma australe (Acanthocephala); Anisakidae (Nematoda); and an unidentified hemiurid (Digenea). Univariate analyses showed that C. australe has its highest prevalence and mean intensity of infection in hosts from shallow waters. Floridosentis sp. showed significant differences along the depth gradient, with higher mean intensity and prevalence of infection in fish from midwater. Among the ectoparasites, only N. chilensis exhibited differences in mean intensity with depth, where intensity of infection increased with depth of host habitat. A canonical multivariate analysis demonstrated that parasite burdens can be a good predictor of the environment (shallow, mid-, or deep water) occupied by the flounders.     

Influence of {\text{NH - }}{{\text{S}}^\gamma } bonding interactions on the structure and dynamics of metallothioneins

Mammalian metallothioneins ( \textM₇^\textIIMTs {\text{M}}_7^{\text{IIMTs}} ) show a clustered arrangement of the metal ions and a nonregular protein structure. The solution structures of Cd₃-thiolate cluster containing β-domain of mouse β-MT-1 and rat β-MT-2 show high structural similarities, but widely differing structure dynamics. Molecular dynamics simulations revealed a substantially increased number of \textNH - \textS^g {\text{NH - }}{{\text{S}}^\gamma } hydrogen bonds in β-MT-2, features likely responsible for the increased stability of the Cd₃-thiolate cluster and the enfolding protein domain. Alterations in the \textNH - \textS^g {\text{NH - }}{{\text{S}}^\gamma } hydrogen-bonding network may provide a rationale for the differences in dynamic properties encountered in the β-domains of MT-1, -2, and -3 isoforms, believed to be essential for their different biological function.     

Conformational variation of surface class II MHC proteins during myeloid dendritic cell differentiation accompanies structural changes in lysosomal MIIC

Dendritic cells (DC), uniquely among APC, express an open/empty conformation of MHC class II (MHC-II) proteins (correctly folded molecules lacking bound peptides). Generation and trafficking of empty HLA-DR during DC differentiation are investigated here. HLA-DR did not fold as an empty molecule in the endoplasmic reticulum/trans-Golgi network, did not derived from MHC/Ii complexes trafficking to the cell surface, but was generated after invariant chain degradation within lysosomal-like MHC-II rich compartments (MIIC). In pre-DC, generated from monocytes cultured in the presence of GM-CSF, Lamp-1(+)MHC-II(+) compartments are predominantly electron dense and, in these cells, empty MHC-II molecules accounts for as much as 20% of total surface HLA-DR. In immature DC, generated in presence of GM-CSF and IL-4, empty HLA-DR reside in multilamellar MIIC, but are scarcely observed at the cell surface. Thus, the morphology/composition of lysosomal MIIC at different DC maturational stages appear important for surface egression or intracellular retention of empty HLA-DR. Ag loading can be achieved for the fraction of empty HLA-DR present in the "peptide-receptive" form. Finally, in vivo, APC-expressing surface empty HLA-DR were found in T cell areas of secondary lymphoid organs.     

Age-related changes of liver antioxidant enzymes and 8-hydroxy-2'-deoxyguanosine during fetal-neonate transition and early rat development

We have studied the pro-antioxidant status of the rat liver on the last day of gestation and at 1, 15, and 30 days of extrauterine life. Representative variables, such as activities of superoxide dismutase (SOD), catalase, and glutathione peroxidase and concentrations of reduced glutathione and 8-hydroxy-2'-deoxyguanosine, were determined in liver to assess the degree of birth-associated oxidative stress during the fetal-neonatal transition and early development of the rat. Percentages by which liver Cu/ZnSOD activity increased over the basal value of the fetal liver were 54%, 95%, and 127% at neonatal days 1, 15, and 30, respectively. There was a lack of induction in the development profile of MnSOD. Catalase activity was clearly and progressively induced with time from the fetal state up to the neonatal age of 1 month. Glutathione peroxidase activity and glutathione content showed a tendency to decline during the first day after birth, though they increased to significantly higher values on days 15 and 30. However, the amount of rat liver 8-hydroxy-2'-deoxyguanosine did not increase. These results suggest that the induced antioxidant activities may be responsible for maintaining DNA stability during the perinatal development of the rat liver.     

Feasibility and safety of adoptive immunotherapy with ex vivo-generated autologous, cytotoxic T lymphocytes in patients with solid tumor

Background aimsAdoptive T-cell therapy with tumor-specific T cells has emerged as a potentially useful approach for treating patients with advanced malignancies. We have demonstrated previously the feasibility of obtaining large numbers of autologous anti-tumor-specific cytotoxic T lymphocytes (CTL) generated by stimulation of patients' peripheral blood mononuclear cells with dendritic cells pulsed with apoptotic tumor cellsMethodsSix patients with progressing metastatic solid tumors (one renal cell carcinoma, two ovarian cancers, two extraosseous peripheral neuroectodermal tumors, one soft tissue sarcoma) not eligible for conventional therapies were treated with adoptive immunotherapy. Anti-tumor CTL, proven to be reactive in vitro against patient tumor cells, but not against normal cells, were infused following lymphodepleting chemotherapy administered to favor T-cell proliferation in vivo.ResultsPatients received a median of nine CTL infusions (range 2–19). The median number of CTL administered per infusion was 11 × 10⁸ (range 1–55 × 10⁸). No patient experienced acute or late adverse events related to CTL infusion, even when large numbers of cells were given. Post-infusion laboratory investigations demonstrated an increase in the frequency of circulating anti-tumor T-cells and, in patients with a longer follow-up receiving two CTL infusions/year, a stabilization of these values.ConclusionsOur study demonstrates that autologous ex vivo-generated anti-tumor CTL can be administered safely in patients with advanced solid tumors and can improve the immunologic reactivity of recipients against tumor. These preliminary results provide a rationale for evaluating the clinical efficacy of this immunotherapeutic approach in phase I/II studies.     

Receptor-binding profiles of H7 subtype influenza viruses in different host species

Influenza viruses of gallinaceous poultry and wild aquatic birds usually have distinguishable receptor-binding properties. Here we used a panel of synthetic sialylglycopolymers and solid-phase receptor-binding assays to characterize receptor-binding profiles of about 70 H7 influenza viruses isolated from aquatic birds, land-based poultry, and horses in Eurasia and America. Unlike typical duck influenza viruses with non-H7 hemagglutinin (HA), all avian H7 influenza viruses, irrespective of the host species, displayed a poultry-virus-like binding specificity, i.e., preferential binding to sulfated oligosaccharides Neu5Acα2-3Galβ1-4(6-O-HSO(3))GlcNAc and Neu5Acα2-3Galβ1-4(Fucα1-3)(6-O-HSO(3))GlcNAc. This phenotype correlated with the unique amino acid sequence of the amino acid 185 to 189 loop of H7 HA and seemed to be dependent on ionic interactions between the sulfate group of the receptor and Lys193 and on the lack of sterical clashes between the fucose residue and Gln222. Many North American and Eurasian H7 influenza viruses displayed weak but detectable binding to the human-type receptor moiety Neu5Acα2-6Galβ1-4GlcNAc, highlighting the potential of H7 influenza viruses for avian-to-human transmission. Equine H7 influenza viruses differed from other viruses by preferential binding to the N-glycolyl form of sialic acid. Our data suggest that the receptor-binding site of contemporary H7 influenza viruses in aquatic and terrestrial birds was formed after the introduction of their common precursor from ducks to a new host, presumably, gallinaceous poultry. The uniformity of the receptor-binding profile of H7 influenza viruses in various wild and domestic birds indicates that there is no strong receptor-mediated host range restriction in birds on viruses with this HA subtype. This notion agrees with repeated interspecies transmission of H7 influenza viruses from aquatic birds to poultry.     

Epstein syndrome: another renal disorder with mutations in the nonmuscle myosin heavy chain 9 gene

Epstein syndrome (EPTS) is an autosomal dominant disease characterized by nephritis, mild hearing loss, and thrombocytopenia with giant platelets. Renal and hearing abnormalities are indistinguishable from those observed in Fechtner syndrome (FTNS), an Alport-like variant. EPTS macrothrombocytopenia is similar to that described in FTNS, May-Hegglin anomaly (MHA), and Sebastian syndrome (SBS), three disorders caused by mutations in the nonmuscle heavy chain myosin IIA ( MYH9). Unlike FTNS, MHA, and SBS, EPTS does not show inclusion bodies in the leukocytes. The clinical features of EPTS and the chromosomal localization of the respective gene in the same region as MYH9 suggest that this disorder is allelic with the other giant platelet disorders. We identified a MYH9 missense mutation in two EPTS familial cases. In both families, an R702H substitution was found, probably inducing conformational changes to the myosin head. A different amino acid substitution at the same codon (R702C) has been previously identified in FTNS. On the basis of predictions from molecular modeling of the X-ray crystallographic structure of chick smooth muscle myosin, the mutated thiol reactive group of R702C may lead to intermolecular disulfide bridges, with the consequent formation of the inclusions typical of FTNS. On the contrary, the R702H mutation does not allow the protein to aggregate and thus to generate "D?hle-like" bodies, which are indeed absent in EPTS. In conclusion, our results extend the allelic heterogeneity of MYH9 mutations to another clinical syndrome and contribute to the clarification of the pathogenesis of the various inherited giant platelet disorders.     

Potential role of cholinesterases in the invasive capacity of the freshwater bivalve, Anodonta woodiana (Bivalvia: Unionacea): a comparative study with the indigenous species of the genus, Anodonta sp

To address the potential role of cholinesterase enzymes in the invasive capacity of species, the present study investigated ChE activity in the invasive freshwater bivalve Anodonta woodiana (Lea, 1834) comparing it with that of the indigenous species, Anodonta sp. (Linnaeus, 1758). The invasive capacity of pests has often been linked to their ecological plasticity and high intrinsic genetic variability; however the role played by molecular and cellular mechanisms, generally known as an organism's response to pollution, is unclear. Different substrates and selective ChE enzyme inhibitors were investigated in digestive gland, foot, gonad, adductor muscle and gill tissues while sensitivity to four organophosphate (OP) insecticides was investigated in vitro only in adductor muscle. The invasive species (A. woodiana) showed significantly greater (at least one order of magnitude) ChE activity than the endemic species (Anodonta sp.) (p<0.05) using acetylthiocholine (ASCh) as substrate and the activity was more widely distributed in tissues involved in movement (adductor muscle and foot), respiration, feeding (gills) and reproduction (gonads). Moreover, only the invasive species, A. woodiana, showed detectable ChE (vs. ASCh) activity in gill tissue. No substrate specificity was observed in any tissue of either species as already described for other bivalve species. ChE activity was not inhibited by Iso-OMPA but showed high sensitivity to BW248c51 and eserine. Both species showed moderate to low sensitivities in vitro to OP insecticides in the range 10(-7)-10(-2) M. Calculated IC(50) for fenitrothion and chlorpyrifos was in the range 10(-6)-10(-3) M in muscle of A. woodiana while a higher inhibition was observed for fenitrothion (10(-7) M) and lower for chlorpyrifos (10(-2) M) in the indigenous species Anodonta sp. Similar IC(50) of 10(-5)-10(-6) M were observed for DFP and azamethiphos in both species. The hypotheses of other authors that acetylcholinesterase (AChE) is involved in the control of many essential functions, such as frontal ciliary activity of gill epithelium, temperature resistance, ciliary activity for transport of suspended particulate, valve opening and embryo development, suggest that the high catalytic efficiency of the invasive species may endow it with a competitive advantage over the endemic species. In view of the peculiar reproductive strategy of these mussels, higher ChE vs. ASCh activity in gonads of the invasive species could also favour glochidium production and embryo development under a wider range of environmental conditions.