Targeting the Gut Microbiota of Vertically HIV-Infected Children to Decrease Inflammation and Immunoactivation: A Pilot Clinical Trial.

Aims: Children with HIV exhibit chronic inflammation and immune dysfunction despite antiretroviral therapy (ART). Strategies targeting persistent inflammation are needed to improve health in people living with HIV. The gut microbiota likely interacts with the immune system, but the clinical implications of modulating the dysbiosis by nutritional supplementation are unclear. Methods: Pilot, double-blind, randomized placebo-controlled trial in which 24 HIV-infected on ART were randomized to supplementation with a daily mixture of symbiotics, omega-3/6 fatty acids and amino acids, or placebo four weeks, in combination with ART. We analyzed inflammatory markers and T-cell activation changes and their correlations with shifts in fecal microbiota. Results: Twenty-four HIV-infected children were recruited and randomized to receive a symbiotic nutritional supplement or placebo. Mean age was 12 ± 3.9 years, 62.5% were female. All were on ART and had HIV RNA < 50/mL. We did not detect changes in inflammatory (IL-6, IL-7, IP-10), microbial translocation (sCD14), mucosal integrity markers (IFABP, zonulin) or the kynurenine to tryptophan ratio, or changes in markers of the adaptive immune response in relation to the intervention. However, we found correlations between several key bacteria and the assessed inflammatory and immunological parameters, supporting a role of the microbiota in immune modulation in children with HIV. Conclusions: In this exploratory study, a four-week nutritional supplementation had no significant effects in terms of decreasing inflammation, microbial translocation, or T-cell activation in HIV-infected children. However, the correlations found support the interaction between gut microbiota and the immune system.

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.

Antiretroviral drugs do not interfere with bryostatin-mediated HIV-1 latency reversal.

Although an effective combination of antiretroviral therapy (cART) controls HIV-1 viraemia in infected patients, viral latency established soon after infection hinders HIV-1 eradication. It has been shown that bryostatin-1 (BRY) inhibits HIV-infection in vitro and reactivates the latent virus through the protein kinase C-NF-κB pathway. We determined the in vitro potential effect of BRY in combination with currently used antiretroviral drugs. BRY alone or in combination with maraviroc (MVC)/Atripla (ATP) was tested for its capacity to reactivate latent virus and inhibit new infections. JLTRG-R5 cells and two latent HIV-1-infected cell lines, J89GFP and THP89GFP, were used as latency models. To quantify HIV infection, the reporter cell line TZM-bl was used. We found that BRY reactivates HIV-1 even in combination with MVC or ATP. Antiretroviral combinations with BRY do not interfere with BRY activity (i.e., the reactivation of latently infected cells) or with the antiviral activity of antiretroviral drugs. In addition, BRY-mediated down-modulation of surface CD4 and CXCR4 was not affected when it was used in combination with other antiretrovirals, and no hyperactivation or high-proliferation effects were observed in primary T cells. Moreover, the BRY treatment was able to reactivate HIV-1 in CD4+ T cells from HIV-1-infected patients under cART. Thus, we propose the use of BRY to purge the viral reservoir and recommend its combination with current antiretroviral treatments.

In vivo delivery of siRNA to the brain by carbosilane dendrimer.

Nanotechnology offers a new platform for therapeutic delivery of antiretrovirals to the central nervous system (CNS). Nanoformulated antiretroviral drugs offer multifunctionality, that is, the ability to package multiple diagnostic and therapeutic agents in the same nanocompose, along with the added provisions of site-directed delivery, delivery across the blood-brain-barrier (BBB), and controlled release of therapeutics. We studied the viability of dendrimers and dendriplexes in human primary astrocytes, as well as their uptake by these astrocytes. Functional validation was performed by using specific siRNA against HIV-1 Nef to interfere to HIV-1 infectivity. A high efficiency in Nef silencing, reducing HIV-1 infectivity was observed in astrocytes treated with dendriplexes compared with control or siRandom treated astrocytes. More interestingly, we studied the biodistribution of the second generation of carbosilane dendrimer loaded with FITC (2G-(SNMe3I)11-FITC) in vivo, in BALB/c mice. Dendriplexes were inoculated into BALB/c mice by the retro-orbital venous plexus, and their localization was determined after 1 and 24h post-injection. Dendriplexes were detected inside the brain by a sensitive imaging system of fluorescent imaging in vivo (IVIS Lumina), and by confocal microscopy analysis of sections of OCT-embedded tissues. The 2G-(SNMe3I)11-FITC dendrimer transported efficiently siRNA into the brain, crossing the BBB. Moreover, this dendrimer successfully delivered and transfected siRNA to HIV-infected human primary astrocytes and achieved gene silencing without causing cytotoxicity. These results highlight the potential of this nanoformulation in the treatment of neurological disorders.

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.

Carbosilane dendrimers to transfect human astrocytes with small interfering RNA targeting human immunodeficiency virus.

BACKGROUND: HIV infection of the CNS is the principle cause of HIV-associated dementia in adults and encephalopathy in children. Gene therapy techniques such as small interfering RNA (siRNA) possess great potential in drug development, but first they must overcome the key obstacle of reaching the interior of the affected cells. A successful delivery vector for anti-HIV drugs that is capable of crossing the blood-brain barrier (BBB) could provide a way of addressing this issue. Non-viral vectors such as dendrimers offer a means for effectively delivering and transfecting siRNA to the target cells. OBJECTIVE: To evaluate the application of gene therapy for reducing HIV replication in human astrocytes. METHODS: We used the 2G-NN16 amino-terminated carbosilane dendrimer as a method for delivering siRNA to HIV-infected human astrocytes. We tested the cytotoxicity in human astrocytoma cells caused by 2G-NN16 and dendriplexes formed with siRNA (siRNA/2G-NN16) by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium-bromide (MTT) and lactate dehydrogenase assays. The ability to transfect human astrocytes with siRNA/2G-NN16 dendriplexes was tested by flow cytometry and immunofluorescence microscopy. To assess the potential capability of siRNA/2G-NN16 dendriplexes for crossing the BBB, we used an in vitro transcytosis assay with bovine brain microvascular endothelial cells. HIV-1 inhibition assays using 2G-NN16 and siRNA/2G-NN16 dendriplexes were determined by quantification of the viral load from culture supernatants of the astrocytes. RESULTS: A gradual time-controlled degradation of the 2G-NN16 dendrimer and liberation of its siRNA cargo between 12 and 24 hours was observed via gel electrophoresis. There was no cytotoxicity in HIV-infected or non-infected human astrocytoma cells when treated with up to 24 microg/mL of 2G-NN16 dendrimer or siRNA/2G-NN16 dendriplexes, and siRNA/2G-NN16 dendriplexes were seen to successfully transfect human astrocytes even after crossing an in vitro BBB model. More interestingly, transfected siRNA was observed to exert a biologic effect, as dendriplexes were shown to down-regulate the housekeeping gene GAPDH and to reduce replication of HIV-1 strains X4-HIV NL4-3 and R5-HIV BaL in human astrocytes. CONCLUSIONS: The 2G-NN16 dendrimer successfully delivers and transfects siRNA to HIV-infected human astrocytes and achieves gene silencing without causing cytotoxicity.

Gene therapy in HIV-infected cells to decrease viral impact by using an alternative delivery method.

The ability of dendrimer 2G-[Si{O(CH(2))(2)N(Me)(2) (+)(CH(2))(2)NMe(3) (+)(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.

Highly efficient transfection of rat cortical neurons using carbosilane dendrimers unveils a neuroprotective role for HIF-1alpha in early chemical hypoxia-mediated neurotoxicity.

PURPOSE: To study the effect of a non-viral vector (carbosilane dendrimer) to efficiently deliver small interfering RNA to postmitotic neurons to study the function of hypoxia-inducible factor-1alpha (HIF1-alpha) during chemical hypoxia-mediated neurotoxicity. METHODS: Chemical hypoxia was induced in primary rat cortical neurons by exposure to CoCl(2). HIF1-alpha levels were determined by Western Blot and toxicity was evaluated by both MTT and LDH assays. Neurons were incubated with dendriplexes containing anti-HIF1-alpha siRNA and both uptake and HIF1-alpha knockdown efficiency were evaluated. RESULTS: We report that a non-viral vector (carbosilane dendrimer) can deliver specific siRNA to neurons and selectively block HIF1-alpha synthesis with similar efficiency to that achieved by viral vectors. Using this method, we have found that this transcription factor plays a neuroprotective role during the early phase of chemical hypoxia-mediated neurotoxicity. CONCLUSION: This work represents a proof-of-concept for the use of carbosilane dendrimers to deliver specific siRNA to postmitotic neurons to block selected protein synthesis. This indicates that this type of vector is a good alternative to viral vectors to achieve very high transfection levels in neurons. This also suggests that carbosilane dendrimers might be very useful for gene therapy.

Characterization of carbosilane dendrimers as effective carriers of siRNA to HIV-infected lymphocytes.

One of the primary limitations of RNA interference as a technique for gene regulation is effective delivery of siRNA into the target cells. Dendrimers are nanoparticles that are increasingly being used as oligonucleotide and drug delivery vehicles. We have developed amino-terminated carbosilane dendrimers (CBS) as a means to protect and transport siRNA. Initially, stability studies showed that CBS bind siRNA via electrostatic interactions. Dendrimer-bound siRNA was found to be resistant to degradation by RNase. Cytotoxicity assays of CBS/siRNA dendriplexes with peripheral blood mononuclear cells (PBMC) and the lymphocytic cell line SupT1 revealed a maximum safe dendrimer concentration of 25 microg/ml. Next, utilizing flow cytometry and confocal microscopy, lymphocytes were seen to be successfully transfected by fluorochrome-labeled siRNA either naked or complexed with CBS. Dendriplexes with +/- charge ratio of 2 were determined to have the highest transfection efficiency while maintaining a low level of toxicity in these systems including hard-to-transfect HIV-infected PBMC. Finally, CBS/siRNA dendriplexes were shown to silence GAPDH expression and reduce HIV replication in SupT1 and PBMC. These results point to the possibility of utilizing dendrimers such as CBS to deliver and transfect siRNA into lymphocytes thus allowing the use of RNA interference as a potential alternative therapy for HIV infection.