Miguel Relloso

Development of sulphated and naphthylsulphonated carbosilane dendrimers as topical microbicides to prevent HIV-1 sexual transmission.

Objectives:For the last 20 years, the idea of alternative prevention strategies based on the use of topical vaginally products to inhibit HIV-1 infection in women has been established. The concept of a ‘microbicide’ product has been born out of the unavailability of a vaccine against HIV-1 and the problems of women in negotiating the use of preventive prophylaxis by their partners, especially in developing countries. Design:We have developed and evaluated polyanionic carbosilane dendrimers G3-S16 and G2-NF16 with sulphated and naphthylsulphonated end groups as nonspecific microbicides. Methods:Cellular in-vitro or in-vivo models were used to evaluate the safety, biocompatibility and anti-HIV ability of two polyanionic carbosilane dendrimers. Results:Both dendrimers showed high biosafety in human epithelial cell lines derived from uterus and vagina and in primary blood human cells (PBMC). These dendrimers not only have a partial capacity to block the entry of different X4 and R5 HIV-1 isolates inside epithelial cells but protect the epithelial monolayer from cell disruption and also reduce HIV-1 infection of activated PBMC. Additionally, treatment of epithelial cells with G3-S16 or G2-NF16 dendrimers did not produce changes in proinflammatory cytokines profile, in proliferation of PBMC, on microbiota or sperm survival. Finally, no irritation or vaginal lesions were detected in female CD1(ICR) mice after dendrimers vaginal administration. Conclusion:These interesting results suggest that G3-S16 or G2-NF16 could be effective to inhibit HIV infection and transmission within genital mucosa as well as the spread of HIV transmission to human PBMC.

Estradiol impairs the Th17 immune response against Candida albicans

Candida albicans is a commensal opportunistic pathogen that is also a member of gastrointestinal and reproductive tract microbiota. Exogenous factors, such as oral contraceptives, hormone replacement therapy, and estradiol, may affect susceptibility to Candida infection, although the mechanisms involved in this process have not been elucidated. We used a systemic candidiasis model to investigate how estradiol confers susceptibility to infection. We report that estradiol increases mouse susceptibility to systemic candidiasis, as in vivo and ex vivo estradiol‐treated DCs were less efficient at up‐regulating antigen‐presenting machinery, pathogen killing, migration, IL‐23 production, and triggering of the Th17 immune response. Based on these results, we propose that estradiol impairs DC function, thus explaining the increased susceptibility to infection during estrus.

Combined Inactivation of the Candida albicans GPR1 and TPS2 Genes Results in Avirulence in a Mouse Model for Systemic Infection

ABSTRACT Inhibition of the biosynthesis of trehalose, a well-known stress protectant in pathogens, is an interesting approach for antifungal or antibacterial therapy. Deletion of TPS2, encoding trehalose-6-phosphate (T6P) phosphatase, results in strongly reduced virulence of Candida albicans due to accumulation of T6P instead of trehalose in response to stress. To further aggravate the deregulation in the pathogen, we have additionally deleted the GPR1 gene, encoding the nutrient receptor that activates the cyclic AMP-protein kinase A signaling pathway, which negatively regulates trehalose accumulation in yeasts. A gpr1 mutant is strongly affected in morphogenesis on solid media as well as in vivo in a mouse model but has only a slightly decreased virulence. The gpr1 tps2 double mutant, on the other hand, is completely avirulent in a mouse model for systemic infection. This strain accumulates very high T6P levels under stress conditions and has a growth defect at higher temperatures. We also show that a tps2 mutant is more sensitive to being killed by macrophages than the wild type or the gpr1 mutant. A double mutant has susceptibility similar to that of the single tps2 mutant. For morphogenesis on solid media, on the other hand, the gpr1 tps2 mutant shows a phenotype similar to that of the single gpr1 mutant. Taken together these results show that there is synergism between Gpr1 and Tps2 and that their combined inactivation results in complete avirulence. Combination therapy targeting both proteins may prove highly effective against pathogenic fungi with increased resistance to the currently used antifungal drugs.

Mesenchymal Contribution to Recruitment, Infiltration, and Positioning of Leukocytes in Human Melanoma Tissues

To understand factors that regulate leukocyte entry and positioning within human melanoma tissues, we performed a multiparametric quantitative analysis of two separated regions: the intratumoral area and the peritumoral stroma. Using two mesenchymal markers, fibroblast activation protein (FAP) and CD90, we identified three subsets of mesenchymal cells (MCs): (i) intratumoral FAP(+)CD90(low/-) MC, (ii) peritumoral FAP(+)CD90(+) MC, and (iii) FAP(-)CD90(+) perivascular MC. We characterized CD90(+) MCs, which showed a stable CCL2-secretory phenotype when long-term expanded ex vivo, and heavily surrounded peritumoral Duffy antigen receptor for chemokine(+) (DARC) postcapillary venules, supporting a role for these vessels in peritumoral inflammatory leukocyte recruitment. Conversely, the intratumoral area was variably invaded by FAP(+)CD90(low/-) MCs that colocalized with a distinct extracellular matrix (ECM) network. A positive correlation was observed between intratumoral stromal cell/ECM networks and leukocyte infiltration among tumor cells (TCs), as well as in a stroma-dependent xenograft tumor model. Adoptively transferred T lymphocytes preferentially infiltrated tumors composed of TC+MC, compared with TCs only. Altogether, our results suggest that a variety of MCs contribute to regulate different steps of leukocyte tumor infiltration, that is, CD90(+) cells surrounding peritumoral vessels secrete CCL2 to recruit CCR2(+) leukocytes at the tumor periphery, whereas intratumoral FAP(+) cells organize a stromal scaffold that contact guide further invasion among densely packed tumor cells.

The inhibition of Th17 immune response in vitro and in vivo by the carbosilane dendrimer 2G-NN16

We evaluated the 2G-NN16-carbosilane dendrimer activities in Th17 response as a potential therapy for Th17 deregulated pathologies. IL17A, IL17F, IL22, IL23 and other interleukins secreted by Th17 cells CD4+ cells were down regulated when cells were cultured in the presence of this dendrimer. Furthermore, IL17F and IL17A protein levels in splenocytes from mice pretreated with 2G-NN16 dendrimer in a Th17 induction mouse model were lower than those corresponding to PBS treated mice. Treatment of mice with 2G-NN16 inhibited the Th17 response causing much more pathogenicity as indicated by the increase in the number of Candida albicans colonies in the kidneys as compared to PBS-treated mice. All these results suggest a potential pharmacological application for this dendrimer in the therapy of Th17-mediated diseases.

A new chimeric protein represses HIV-1 LTR-mediated expression by DNA methylase

Once the human immunodeficiency virus (HIV) genome is inserted into the host genome, the virus cannot be removed, which results in latency periods and makes it difficult to eradicate. The majority of strategies to eradicate HIV have been based on preventing virus latency, thereby enabling antiretroviral drugs to act against HIV replication. Another innovative strategy is permanently silencing the integrated virus to prevent the spread of infection. Epigenetic processes are natural mechanisms that can silence viral replication. We describe a new chimeric protein (IN3b) that consists of a HIV-1 integrase domain, which recognises the HIV long terminal repeat (LTR) and the catalytic domain of DNA methyltransferase DNMT3b. Our objective was to silence HIV replication by the specific delivery of the catalytic methyltransferase domain to the LTR promoter to induce its methylation. We found that our IN3b chimeric protein was expressed in the nucleus and decreased LTR-associated HIV genome expression and HIV replication. Therefore, the IN3b chimeric protein may be an effective tool against HIV replication and maybe used in a new line of research to induce or maintain HIV latency.

A Conserved GPG-Motif in the HIV-1 Nef Core Is Required for Principal Nef-Activities

To find out new determinants required for Nef activity we performed a functional alanine scanning analysis along a discrete but highly conserved region at the core of HIV-1 Nef. We identified the GPG-motif, located at the 121–137 region of HIV-1 NL4.3 Nef, as a novel protein signature strictly required for the p56Lck dependent Nef-induced CD4-downregulation in T-cells. Since the Nef-GPG motif was dispensable for CD4-downregulation in HeLa-CD4 cells, Nef/AP-1 interaction and Nef-dependent effects on Tf-R trafficking, the observed effects on CD4 downregulation cannot be attributed to structure constraints or to alterations on general protein trafficking. Besides, we found that the GPG-motif was also required for Nef-dependent inhibition of ring actin re-organization upon TCR triggering and MHCI downregulation, suggesting that the GPG-motif could actively cooperate with the Nef PxxP motif for these HIV-1 Nef-related effects. Finally, we observed that the Nef-GPG motif was required for optimal infectivity of those viruses produced in T-cells. According to these findings, we propose the conserved GPG-motif in HIV-1 Nef as functional region required for HIV-1 infectivity and therefore with a potential interest for the interference of Nef activity during HIV-1 infection.