María Jesús Serramía

Carbosilane dendrimer nanotechnology outlines of the broad HIV blocker profile.

Researchers have been working hard for more than 20 years to develop safe and effective microbicides to empower women to better control their own sexual life and to protect themselves against HIV and other sexually transmitted infections (STIs). Microbicide classes include moderately specific macromolecular anionic polymers that block HIV and other STIs, and HIV specific drugs that inhibit viral entry and reverse transcription. Based on innovative nanotechnology design, we showed a novel water-soluble anionic carbosilane dendrimer (2G-S16) as a propitious molecule against HIV-infection. A state-of-the-art research was accomplished that focused on biomedical cutting-edge techniques such as in vitro and in vivo cytotoxicity assays performed on female rabbit genital tracts, simulate in vitro model of vaginal epithelium in order to evaluate HIV transmission blockade through the monolayer, complete gene expression profiling experiment to study deregulated genes after 2G-S16 exposition, molecular dynamics simulation of 2G-S16 molecule against principal proteins of HIV particles and pro- and anti-inflammatory cytokine profile study. Therefore, a high-throughput study and detailed analysis of the results were achieved in this article. We provided promising outcomes to encourage 2G-S16 as a hopeful microbicide.

Gene Therapy in HIV‐Infected Cells to Decrease Viral Impact by Using an Alternative Delivery Method

The ability of dendrimer 2G‐[Si{O(CH2)2N(Me)2+(CH2)2NMe3+(I−)2}]8 (NN16) to transfect a wide range of cell types, as well as the possible biomedical application in direct or indirect inhibition of HIV replication, was investigated. Cells implicated in HIV infection such as primary peripheral blood mononuclear cells (PBMC) and immortalized suspension cells (lymphocytes), primary macrophages and dendritic cells, and immortalized adherent cells (astrocytes and trophoblasts) were analyzed. Dendrimer toxicity was evaluated by mitochondrial activity, cell membrane rupture, release of lactate dehydrogenase, erythrocyte hemolysis, and the effect on global gene expression profiles using whole‐genome human microarrays. Cellular uptake of genetic material was determined using flow cytometry and confocal microscopy. Transfection efficiency and gene knockdown was investigated using dendrimer‐delivered antisense oligonucleotides and small interfering RNA (siRNA). Very little cytotoxicity was detected in a variety of cells relevant to HIV infection and erythrocytes after NN16 dendrimer treatment. Imaging of cellular uptake showed high transfection efficiency of genetic material in all cells tested. Interestingly, NN16 further enhanced the reduction of HIV protein 24 antigen release by antisense oligonucleotides due to improved transfection efficiency. Finally, the dendrimer complexed with siRNA exhibited therapeutic potential by specifically inhibiting cyclooxygenase‐2 gene expression in HIV‐infected nervous system cells. NN16 dendrimers demonstrated the ability to transfect genetic material into a vast array of cells relevant to HIV pathology, combining high efficacy with low toxicity. These results suggest that NN16 dendrimers have the potential to be used as a versatile non‐viral vector for gene therapy against HIV infection.

Triple combination of carbosilane dendrimers, tenofovir and maraviroc as potential microbicide to prevent HIV-1 sexual transmission.

AIM To research the synergistic activity by triple combinations of carbosilane dendrimers with tenofovir and maraviroc as topical microbicide. METHODS Cytotoxicity, anti-HIV-1 activity, vaginal irritation and histological analysis of triple combinations were determined. Analysis of combined effects and the median effective concentration were performed using CalcuSyn software. RESULTS Combinations showed a greater broad-spectrum anti-HIV-1 activity than the single-drug, and preserved this activity in acid environment or seminal fluid. The strongest combinations were G2-STE16/G2-S24P/tenofovir, G2-STE16/G2-S16/maraviroc and G2-STE16/tenofovir/maraviroc at 2:2:1, 10:10:1 10:5:1 ratios, respectively. They demonstrated strong synergistic activity profile due to the weighted average combination indices varied between 0.06 and 0.38. No irritation was detected in female BALB/c mice. CONCLUSION The three-drug combination increases their antiviral potency and act synergistically as potential microbicide.

Synergistic Activation of Latent HIV-1 Expression by Novel Histone Deacetylase Inhibitors and Bryostatin-1.

Viral reactivation from latently infected cells has become a promising therapeutic approach to eradicate HIV. Due to the complexity of the viral latency, combinations of efficient and available drugs targeting different pathways of latency are needed. In this work, we evaluated the effect of various combinations of bryostatin-1 (BRY) and novel histone deacetylase inhibitors (HDACIs) on HIV-reactivation and on cellular phenotype. The lymphocyte (J89GFP) or monocyte/macrophage (THP89GFP) latently infected cell lines were treated with BRY, panobinostat (PNB) and romidepsin (RMD) either alone or in combination. Thus, the effect on the viral reactivation was evaluated. We calculated the combination index for each drug combination; the BRY/HDACIs showed a synergistic HIV-reactivation profile in the majority of the combinations tested, whereas non-synergistic effects were observed when PNB was mixed with RMD. Indeed, the 75% effective concentrations of BRY, PNB and RMD were reduced in these combinations. Moreover, primary CD4 T cells treated with such drug combinations presented similar activation and proliferation profiles in comparison with single drug treated cells. Summing up, combinations between BRY, PNB and/or RMD presented a synergistic profile by inducing virus expression in HIV-latently infected cells, rendering these combinations an attractive novel and safe option for future clinical trials.

Prevention vaginally of HIV-1 transmission in humanized BLT mice and mode of antiviral action of polyanionic carbosilane dendrimer G2-S16

UNLABELLED The development of a safe, effective, and low-priced topical microbicide to prevent HIV-1 sexual transmission is urgently needed. The emerging field of nanotechnology plays an important role in addressing this challenge. We demonstrate that topical vaginal administration of 3% G2-S16 prevents HIV-1JR-CSF transmission in humanized (h)-BLT mice in 84% with no presence of HIV-1 RNA and vaginal lesions. Second-generation polyanionic carbosilane dendrimer G2-S16 with silica core and 16 sulfonate end-groups exerts anti-HIV-1 activity at an early stage of viral replication, blocking the gp120/CD4 interaction, acting on the virus, and inhibiting the cell-to-cell HIV-1 transmission, confirming its multifactorial and non-specific ability. This study represents the first demonstration that transmission of HIV-1 can be efficiently blocked by vaginally applied G2-S16 in h-BLT mice. These findings provide a step forward in the development of G2-S16-based vaginal microbicides to prevent vaginal HIV-1 transmission in humans. FROM THE CLINICAL EDITOR HIV infections remain a significant problem worldwide and the major route of transmission is through sexual activity. In this article, the authors developed an antiviral agent containing polyanionic carbosilane dendrimer with silica core and 16 sulfonate end-groups. When applied vaginally, this was shown to exert anti-HIV protection. These positive findings may offer hope in the fight against the spread of HIV epidemic.

Progesterone Inhibits HIV-1 Replication in Human Trophoblast Cells through Inhibition of Autocrine Tumor Necrosis Factor Secretion

BACKGROUND Progesterone levels are higher in placental barriers during pregnancy, but the effect of progesterone on human immunodeficiency virus type 1 (HIV-1) infection in placental cells has not been addressed. We hypothesize that progesterone may affect HIV infection. METHODS Purified trophoblastic cells and trophoblastic cell lines were infected or transfected with HIV-1, and the effect of progesterone was analyzed. Viral replication was measured by viral p24 or viral load quantification. Nuclear factor kappa -B (NF- kappa B) or long terminal repeat (LTR)-dependent transcription was measured by luciferase assays. Expression of chemokine receptors was analyzed by flow cytometry. Tumor necrosis factor (TNF) messenger RNA was assessed by reverse-transcription polymerase chain reaction (RT-PCR) and quantitative RT-PCR. RESULTS Progesterone inhibits HIV-1 replication in placental cells at the concentration found in the placental interface, at a postentry step, and does not affect cell surface expression of chemokine receptors. Progesterone did not inhibit basal or induced LTR transcription or NF- kappa B activation. TNF synthesis in placental cells is induced by HIV-1 infection that, in an autocrine manner, activates viral replication, because neutralizing anti-TNF antibodies block it. Progesterone inhibits the induction of TNF synthesis by viral infection and virus or gp-120-induced TNF transcription. CONCLUSION Our results demonstrate that progesterone inhibits HIV-1 replication in placental cells by reducing TNF levels, which are required for optimal viral replication.

Prostaglandin E2 reduces the release and infectivity of new cell-free virions and cell-to-cell HIV-1 transfer.

Background The course of human immunodeficiency virus type-1 (HIV-1) infection is influenced by a complex interplay between viral and host factors. HIV infection stimulates several proinflammatory genes, such as cyclooxigense-2 (COX-2), which leads to an increase in prostaglandin (PG) levels in the plasma of HIV-1-infected patients. These genes play an indeterminate role in HIV replication and pathogenesis. The effect of prostaglandin E2 (PGE2) on HIV infection is quite controversial and even contradictory, so we sought to determine the role of PGE2 and the signal transduction pathways involved in HIV infection to elucidate possible new targets for antiretrovirals. Results Our results suggest that PGE2 post-infection treatment acts in the late stages of the viral cycle to reduce HIV replication. Interestingly, viral protein synthesis was not affected, but a loss of progeny virus production was observed. No modulation of CD4 CXCR4 and CCR5 receptor expression, cell proliferation, or activation after PGE2 treatment was detected. Moreover, PGE2 induced an increase in intracellular cAMP (cyclic AMP) levels through the EP2/EP4 receptors. PGE2 effects were mimicked by dbcAMP and by a specific Epac (exchange protein directly activated by cyclic AMP) agonist, 8-Cpt-cAMP. Treatment with PGE2 increased Rap1 activity, decreased RhoA activity and subsequently reduced the polymerization of actin by approximately 30% compared with untreated cells. In connection with this finding, polarized viral assembly platforms enriched in Gag were disrupted, altering HIV cell-to-cell transfer and the infectivity of new virions. Conclusions Our results demonstrate that PGE2, through Epac and Rap activation, alters the transport of newly synthesized HIV-1 components to the assembly site, reducing the release and infectivity of new cell-free virions and cell-to-cell HIV-1 transfer.

Cationic Carbosilane Dendrimers as Non‐viral Vectors of Nucleic Acids (Oligonucleotide or siRNA) for Gene Therapy Purposes

A new approach to develop novel non‐viral vector for HIV inhibition based on carbosilane dendrimers is described. Cationic ammonium‐terminated carbosilane dendrimers have been synthesized via alcoholysis of dendritic Si‐Cl bonds or hydrosylilation reactions with dendritic Si‐H bonds. Ammonium‐terminated carbosilane dendrimers are able to form dendriplexes which showed good biocompatibility profiles in vitro, and protect nucleic acids from binding to serum proteins or RNases and degradation. Finally, they were found to be a successful and efficient vehicle for the transport and delivery of genetic material (ODN or siRNA) to a wide range of immune cells, and according to this, a specific reduction of HIV p24 antigen release by anti‐HIV ODN or siRNA was observed.Therefore, the carbosilane dendrimers presented here are in general good candidates as non‐viral vectors in the context of biological applications of small nucleic acids, and in particular, they may present a new interesting approach for the treatment of HIV infection.

Polycationic carbosilane dendrimer decreases angiogenesis and tumor-associated macrophages in tumor-bearing mice

Therapies against cancer have been improved and progressed during recent decades, initially were using chemotherapeutic drugs, which directly affect tumor cells, but nowadays are focused in cellular therapies aimed at treating the tumor stroma, because tumor and stromal cells jointly control development and tumor progression. Immunotherapy is of great relevance because it could modify the tumor stroma, controlling tumor growth. Tumor-associated macrophages have been proposed as target cells owing to the positive correlation between the high content of macrophages and the adverse prognosis of tumor development. 2G-03NN24 dendrimer had previously shown immunomodulatory effects by reducing the functional capabilities of human anti-inflammatory macrophages, leading them to a pro-inflammatory state, and thereby helping to control tumor development. New dendrimer capabilities against tumor mass are described and presented in in vivo studies using tumor-bearing mice. MC38 cells were used to induce tumors in C57BL/6 mice. Tumor growth was evaluated during 21 days and tumors were stained with hematoxylin/eosin to analyze the histopathology features. Tumor histopathology studies show that 2G-03NN24 dendrimer decreases the tumor size and the number of intratumoral blood vessels. Furthermore, cellular populations on tumor mass were analyzed by an immunofluorescence assay. Evaluation of tumor-associated macrophages indicates that 2G-03NN24 dendrimer reduces the amount of tumor-associated macrophages, creating a more favorable microenvironment within tumors. Data defines 2G-03NN24 as a candidate for finding a new antitumor compound based on cellular therapies.