Luis F. Montaño

Influenza control in the 21st century: Optimizing protection of older adults.

Older adults (> or =65 years of age) are particularly vulnerable to influenza illness. This is due to a waning immune system that reduces their ability to respond to infection, which leads to more severe cases of disease. The majority ( approximately 90%) of influenza-related deaths occur in older adults and, in addition, catastrophic disability resulting from influenza-related hospitalization represents a significant burden in this vulnerable population. Current influenza vaccines provide benefits for older adults against influenza; however, vaccine effectiveness is lower than in younger adults. In addition, antigenic drift is also a concern, as it can impact on vaccine effectiveness due to a mismatch between the vaccine virus strain and the circulating virus strain. As such, vaccines that offer higher and broader protection against both homologous and heterologous virus strains are desirable. Approaches currently available in some countries to meet this medical need in older adults may include the use of adjuvanted vaccines. Future strategies under evaluation include the use of high-dose vaccines; novel or enhanced adjuvantation of current vaccines; use of live attenuated vaccines in combination with current vaccines; DNA vaccines; recombinant vaccines; as well as the use of different modes of delivery and alternative antigens. However, to truly evaluate the benefits that these solutions offer, further efficacy and effectiveness studies, and better correlates of protection, including a precise measurement of the T cell responses that are markers for protection, are needed. While it is clear that vaccines with greater immunogenicity are required for older adults, and that adjuvanted vaccines may offer a short-term solution, further research is required to exploit the many other new technologies.

17β-estradiol inhibits the adhesion of leukocytes in TNF-α stimulated human endothelial cells by blocking IL-8 and MCP-1 secretion, but not its transcription

Inflammation, and especially mononuclear cell adhesion to endothelium, is an important physiopathological component of atherosclerosis. Since coronary heart disease in women of reproductive age and/or with estrogen replacement therapy is reduced, our aim was to determine if 17β-estradiol had a regulatory effect on the adhesion of lymphocytes to the endothelium. We performed U-937 cells adhesion assays in TNF-α-stimulated HUVECs, and we also quantitated IL-8 and MCP-1 in culture supernatants, in the presence or not of 17β-estradiol. The presence of α- and β-estrogen receptors was determined by Western blot and RT-PCR, respectively, whereas the transcription of both chemokines was evaluated by RT-PCR. The results showed a 35% decrease in the adhesion of U-937 monocyte cells to TNF-α-stimulated HUVECs, and a 54% and 65% inhibition of TNF-α-induced IL-8 and MCP-1 secretion by physiological and physiologically high doses of 17β-estradiol. The hormone did not affect the transcription of both chemokine genes. Tamoxifen reverted the inhibitory effect induced by 17βestradiol. In conclusion, 17β-estradiol modifies the adhesion of leukocytes to endothelial cells by inhibiting the secretion, but not the gene transcription, of proinflammatory chemokines.

Statin-induced inhibition of MCF-7 breast cancer cell proliferation is related to cell cycle arrest and apoptotic and necrotic cell death mediated by an enhanced oxidative stress.

Statins have antiproliferative and anti-tumoral effects in MCF-7 cells. We determined the effect of statins upon MCF-7 cell cycle, toxicity, cell death, reactive oxygen species (ROS) production and mitochondrial membrane potential. Fluvastatin, simvastatin and atorvastatin inhibited cell proliferation. Antiproliferation was associated with a decrease in the DNA synthesis and a cell cycle arrest in the G1 and G2/M phases. A loss in the mitochondrial membrane potential was observed with fluvastatin. Statins induced increase in ROS production that was associated with cell death, which was abrogated by the antioxidant NAC. Our results suggest that the cytotoxic effect observed is mediated by an oxidative stress.

Claudin-6, 7, or 9 Overexpression in the Human Gastric Adenocarcinoma Cell Line AGS Increases Its Invasiveness, Migration, and Proliferation Rate

Altered claudin expression is related to metastatic potential, poor prognosis, or tumor recurrence. We analyzed if the overexpression of claudin-6, claudin-7, or claudin-9 in AGS cells altered cell motility, invasiveness, or proliferation rate. Claudin-7, claudin-9, and claudin-6 enhanced their invasive potential by 3.4-fold, 1.6-fold, and 2.0-fold, respectively. Claudin-6 and claudin-9 enhanced cell migration, while the proliferation rate of claudin-6-, claudin-7-, and claudin-9-transfected cells increased by 12.7%, 9.0%, and 13.3%, respectively. Claudin-7 and claudin-9 overexpression increased claudin-1 and zonula occludens-1 levels. In summary, individual increased expression of claudin-6, claudin-7, or claudin-9 is sufficient to enhance tumorigenic properties of a gastric adenocarcinoma cell line.

Dehydroepiandrosterone inhibits the proliferation of human umbilical vein endothelial cells by enhancing the expression of p53 and p21, restricting the phosphorylation of retinoblastoma protein, and is androgen- and estrogen-receptor independent.

Dehydroepiandrosterone (DHEA), a steroid hormone, modified the proliferation of human umbilical vein endothelial cells in a dose‐dependent manner. Its inactive sulfate ester (DHEA‐S) and two of its metabolites – estradiol and testosterone – had no inhibitory effect at physiological concentrations. Antiproliferation was associated with arrest in the G1 phase of the cell cycle, but not with cell death, as evaluated by cleavage of poly(ADP‐ribose) polymerase and exposure of phosphatidylserine. The effect was not blocked by inhibitors of androgen or estrogen receptors. DHEA diminished the levels of phosphorylated retinoblastoma protein and increased the expression of p53 and p21 mRNAs. These results show that DHEA inhibits endothelial cell proliferation by regulating cell cycle relevant proteins through a cytoplasmic steroid hormone‐independent pathway.

Ceramide promotes the death of human cervical tumor cells in the absence of biochemical and morphological markers of apoptosis.

C8-ceramide, a synthetic cell-permeable analog of endogenous ceramides, interfered with cell proliferation, and was cytotoxic to papilloma virus-containing human cervix carcinoma cells, CALO, INBL, and HeLa, that match two clinical stages of tumor progression. C8-ceramide (3 microM) markedly reduced the tumor cell number after 48 h of treatment, an effect that endured even after the removal of C8-ceramide. The carcinoma cells showed morphologic changes, characteristic of necrosis and released lactate dehydrogenase (LDH). A biologically inactive analog C8-dihydro-ceramide had no effect on cell viability in any of the cell lines tested. Seventy-two hours after C8-ceramide treatment none of the biochemical and morphological markers characteristic of apoptosis: (a) nuclear chromatin condensation, (b) DNA fragmentation, (c) proteolysis of the caspase-3 substrate poly-(ADP-ribose)-polymerase (PARP), and (d) appearance of phosphatidylserine on the external cell membrane, were observed. C8-ceramide had no effect on human cervix fibroblasts and induced a mild reduction (30%) in the proliferation of normal human cervix epithelia and HeLa cells (IV-B metastatic stage). The cytotoxicity of C8-ceramide was restricted to CALO (early II-B) and INBL (IV-A non-metastatic) carcinoma cells. The possible application of ceramide in the treatment of early stages of cervical cancer is discussed.

Purification and characterization of a lectin from Macrobrachium rosenbergh (Crustacea, Decapoda) hemolymph

1. 1. We have purified a 19 kDa lectin from the freshwater prawn Macrobrachium rosenbergii by single-step affinity chromatography on a rat stroma column. 2. 2. The lectin is a glycoprotein composed of two monomeric subunits of 9.6 kDa bound by a disulfide bridge; 7% of its contents are carbohydrates. 3. 3. It has an S20, w value of 1.4, a pI of 5.4–6.1, and is rich in glycine, serine, glutamic and aspartic acid residues. 4. 4. It requires divalent cations to function. 5. 5. It recognizes sialic acid. 6. 6. The hemagglutinating and hapten inhibition assays strongly suggest that it is 9-O-acetylsialic acid residue-specific.

Comparative evaluation of the CD4+CD8+ and CD4+CD8- lymphocytes in the immune response to porcine rubulavirus.

The porcine immune system is unique in the expression of CD4+CD8+ (double-positive, DP) lymphocytes. These cells have been associated with immunological memory due to their gradual increase with age, the expression of memory phenotype and their ability to respond to recall viral antigen. This work analyzes the biological function of CD4+CD8- and CD4+CD8+ lymphocytes in the immune response to porcine rubulavirus (PRv). CD4+CD8- cells isolated from pigs 3 weeks after infection with porcine rubulavirus proliferated in response to homologous virus and generated lymphoblasts which were predominantly of the CD4+CD8+ phenotype, whereas stimulation with mitogen induced proliferation but did not switch the phenotype. CD4+CD8- lymphocytes isolated after 10 weeks of infection proliferated in response to phytohemagglutinin (PHA) but did not proliferate in response to homologous virus and did not change their phenotype, whereas CD4+CD8+ lymphocytes proliferated in response to PHA and to viral antigen. The cytokine profile of both lymphocyte populations showed the presence of IL-2 and IL-10 transcripts, quantitation demonstrated that CD4+CD8+ cells expressed mainly IL-10, whereas CD4+CD8- lymphocytes expressed primarily IL-2. Our results show that CD4+CD8- lymphocytes in the early phase of porcine rubulavirus infection can be converted to double-positive cells expressing IL-10 in an antigen-dependent manner, and that CD4+CD8- T-cells late in infection do not acquire CD8.

Distribution and Expression Pattern of Claudins 6, 7, and 9 in Diffuse- and Intestinal-Type Gastric Adenocarcinomas

IntroductionIntestinal- and diffuse-type gastric adenocarcinomas differ in clinical outcome and genetic profile. Abnormal claudin expression has been well documented in several malignancies. Our aim was to find specific claudin markers for each type.MethodsFifty paraffin-embedded tissue blocks of diffuse- and intestinal-type gastric adenocarcinomas and fresh gastric biopsies obtained endoscopically from 20 patients with a presumptive diagnosis of gastric cancer were analyzed. Claudin-specific polyclonal and monoclonal antibodies were used for immunohistochemistry and Western blot analysis in total lysate and subcellular fractions.ResultsClaudin-6 expression was high in both types. Claudin-7 was expressed mainly in the diffuse-type whereas claudin-9 was mainly found in the apical membrane of the gland cells in the intestinal-type. Strong claudin-9 expression was associated with higher mortality rate (66%) in the diffuse type vs the intestinal type (25%) after a 2-year follow-up.ConclusionClaudins 6, 7, and 9 expressions are closely related to gastric carcinogenesis, and their detection is a useful prognostic marker in “intestinal-” and “diffuse-type” gastric adenocarcinomas.

Specificity of Amaranthus leucocarpus lectin

We have demonstrated thatAmaranthus leucocarpus lectin hemagglutinating activity was powerfully inhibited by the T-antigen, containing Gal(β1–3)GalNAc(α1–3)Ser/Thr, and the Tn-antigen, which contains GalNAc(α1–3)Ser/Thr. This suggests that the acetamido group at C-2 and the axial -OH at C-4 of theN-acetyl-D-galactopyranosylamine ring are important for lectin binding. The hemagglutination assays also established that desialylated and Pronase-treated human typeO erythrocytes with an M phenotype were better recognized than erythrocytes from all other blood groups. The recognition was dependent on pH and ionic strength.