Effect of the use of Galectin-9 and blockade of TIM-3 receptor in the latent cellular reservoir of HIV-1.

Reactivation of latent HIV-1 is a necessary step for the purging of the viral reservoir, although it does not seem to be enough. The stimulation of HIV-1 specific cytotoxic T lymphocytes (CTL) may be just as essential for this purpose. In this study, we aimed to show the effect of galectin-9 (Gal-9), known to revert HIV-1 latency, in combination with the blockade of TIM-3, a natural receptor for Gal-9 and an exhaustion marker. We confirmed the ability of Gal-9 to reactivate latent HIV-1 in Jurkat-LAT-GFP cells, as well as in an IL-7-based cellular model. This reactivation was not mediated via the TIM-3 receptor, but rather by the recognition of the Gal-9 of a specific oligosaccharide pattern of resting memory CD4+ T cells' surfaces. The potency of Gal-9 in inducing transcription of latent HIV-1 was equal to or greater than that of other latency-reversing agents (LRA). Furthermore, the combination of Gal-9 with other LRA did not show synergistic effects in the reactivation of the latent virus. To evaluate the impact of TIM-3 inhibition on the CTL-response, different co-culture experiments with CD4+T, CD8+ T, and NK cells were performed. Our data showed that blocking TIM-3 was associated with control of viral replication in both in vitro and ex vivo models in cells from PLWH on antiretroviral therapy. A joint strategy of the use of Gal-9 to reactivate latent HIV-1 and the inhibition of TIM-3 to enhance the HIV-1 CTL specific-response was associated with control of the replication of the virus that was being reactivated, thus potentially contributing to the elimination of the viral reservoir. Our results place this strategy as a promising approach to be tested in future studies. Reactivation of latent-HIV-1 by Gal-9 and reinvigoration of CD8+ T cells by TIM-3 blockade could be used separately or in combination.ImportanceHIV-1 infection is a health problem of enormous importance that still causes significant mortality. Antiretroviral treatment (ART) has demonstrated efficacy in the control of HIV-1 replication, decreasing the morbidity and mortality of the infection, but it cannot eradicate the virus. In our work, we tested a protein, galectin-9 (Gal-9), an HIV-1 latency-reversing agent, using an in vitro cellular model of latency and in cells from people living with HIV-1 (PLWH) on antiretroviral therapy. Our results confirmed the potential role of Gal-9 as a molecule with a potent HIV-1 reactivation capacity. More importantly, using a monoclonal antibody against T cell immunoglobulin and the mucin domain-containing molecule 3 (TIM-3) receptor we were able to enhance the HIV-1 cytotoxic T lymphocytes (CTL) specific response to eliminate the CD4+ T cells in which the virus had been reactivated. When used together, i.e., Gal-9 and TIM-3 blockade, control of the replication of HIV-1 was observed, suggesting a decrease in the cellular reservoir.

Microbial fuel cell scale-up options: Performance evaluation of membrane (c-MFC) and membrane-less (s-MFC) systems under different feeding regimes.

In recent years, bioelectrochemical systems have advanced towards upscaling applications and tested during field trials, primarily for wastewater treatment. Amongst reported trials, two designs of urine-fed microbial fuel cells (MFCs) were tested successfully on a pilot scale as autonomous sanitation systems for decentralised area. These designs, known as ceramic MFCs ( c -MFCs) and self-stratifying MFCs ( s -MFC), have never been calibrated under similar conditions. Here, the most advanced versions of both designs were assembled and tested under similar feeding conditions. The performance and efficiency were evaluated under different hydraulic retention times (HRT), through chemical oxygen demand measures and polarisation experiments. Results show that c -MFCs displayed constant performance independently from the HRT (32.2 ± 3.9 W m-3) whilst displaying high energy conversion efficiency at longer HRT (NER COD  = 2.092 ± 0.119 KWh.Kg COD -1, at 24h HRT). The s -MFC showed a correlation between performance and HRT. The highest performance was reached under short HRT (69.7 ± 0.4 W m-3 at 3h HRT), but the energy conversion efficiency was constant independently from the HRT (0.338 ± 0.029 KWh.Kg COD -1). The c -MFCs and s -MFCs similarly showed the highest volumetric efficiency under long HRT (65h) with NER V of 0.747 ± 0.010 KWh.m-3 and 0.825 ± 0.086 KWh.m-3, respectively. Overall, c -MFCs seems more appropriate for longer HRT and s -MFCs for shorter HRT.

Maraviroc Is Associated with Latent HIV-1 Reactivation through NF-κB Activation in Resting CD4+ T Cells from HIV-Infected Individuals on Suppressive Antiretroviral Therapy.

Maraviroc is a CCR5 antagonist used in the treatment of HIV-1 infection. We and others have suggested that maraviroc could reactivate latent HIV-1. To test the latency-reversing potential of maraviroc and the mechanisms involved, we performed a phase II, single-center, open-label study in which maraviroc was administered for 10 days to 20 HIV-1-infected individuals on suppressive antiretroviral therapy (EudraCT registration no. 2012-003215-66). All patients completed full maraviroc dosing and follow-up. The primary endpoint was to study whether maraviroc may reactivate HIV-1 latency, eliciting signaling pathways involved in the viral reactivation. An increase in HIV-1 transcription in resting CD4+ T cells, estimated by levels of HIV-1 unspliced RNA, was observed. Moreover, activation of the NF-κB transcription factor was observed in these cells. To elucidate the mechanism of NF-κB activation by maraviroc, we have evaluated in HeLa P4 C5 cells, which stably express CCR5, whether maraviroc could be acting as a partial CCR5 agonist, with no other mechanisms or pathways involved. Our results show that maraviroc can induce NF-κB activity and that NF-κB targets gene expression by CCR5 binding, since the use of TAK779, a CCR5 inhibitor, blocked NF-κB activation and functionality. Taking the results together, we show that maraviroc may have a role in the activation of latent virus transcription through the activation of NF-κB as a result of binding CCR5. Our results strongly support a novel use of maraviroc as a potential latency reversal agent in HIV-1-infected patients.IMPORTANCE HIV-1 persistence in a small pool of long-lived latently infected resting CD4+ T cells is a major barrier to viral eradication in HIV-1-infected patients on antiretroviral therapy. A potential strategy to cure HIV-1-infection is the use of latency-reversing agents to eliminate the reservoirs established in resting CD4+ T cells. As no drug has been shown to be completely effective so far, the search for new drugs and combinations remains a priority for HIV cure. We examined the ability of maraviroc, a CCR5 antagonist used as an antiretroviral drug, to activate latent HIV-1 in infected individuals on antiretroviral therapy. The study showed that maraviroc can activate NF-κB and, subsequently, induce latent HIV-1-transcription in resting CD4+ T cells from HIV-1-infected individuals on suppressive antiretroviral therapy. Additional interventions will be needed to eliminate latent HIV-1 infection. Our results suggest that maraviroc may be a new latency-reversing agent to interfere with HIV-1 persistence during antiretroviral therapy.

Effect of Deuteration on Phase Behavior of Supported Phospholipid Bilayers: A Spectroelectrochemical Study.

Differences in molecular organization of two sides of a chemically symmetric, planar bilayer supported on a Au(111) substrate have been monitored with charge density measurements and in situ polarization modulation infrared reflection-absorption spectroscopy (PM-IRRAS). Isotopic substitution of the hydrogen atoms in the hydrocarbon chains with deuterium atoms in one monolayer was employed to allow the monitoring of C-H vibrations from that monolayer alone. Charge density measurements of bilayers formed from dimyristoylphosphatidylethanolamine (DMPE) showed that the effect of placing the deuterated layer next to the substrate or electrolyte had little impact on the electrical barrier properties. In situ PM-IRRAS studies revealed that the structure of the two monolayers was the same at negative potentials, where the bilayer is separated from the Au substrate, but different at more positive potentials or small charge densities, where the bilayer is expected to be directly adsorbed on the Au surface. Thus, the differences observed for the related molecule dimyristoylphosphatidylcholine (DMPC) persist in planar structures, although to a lesser extent. A small but observable variation in the tilt angle was also apparent in the spectra of both isotopically asymmetric DMPE bilayers during the electrochemical phase transition. The fact that this effect was not previously observed for hydrogenous bilayers means that the dynamic behavior of deuterated DMPE and/or of bilayers composed of different monolayers is different from that of hydrogenous DMPE bilayers. These results have implications for future studies in which isotopic substitution is used to extract selectively information from one layer or component of lipid bilayers in spectroscopic or neutron measurements.

Virological response to short-course maraviroc monotherapy does not predict viral tropism in HIV-1-infected treatment-naive patients.

OBJECTIVES: We aimed to evaluate whether virological response to a short course of maraviroc monotherapy could predict HIV-1 tropism. METHODS: A clinical trial was performed in HIV-1 treatment-naive patients infected with R5- or non-R5-tropic virus determined using the Trofile(®) assay, with >1000 HIV-1 RNA copies/mL. Maraviroc was administered for 10 days. Viral load was measured at baseline and days 4, 7, 10 and 28. The main outcome measurement was the decline in HIV-1 RNA at day 10. The trial was registered in the ClinicalTrials.gov database (NCT01060618; TROPISMVC). RESULTS: Forty patients [30 R5 and 10 dual/mixed (D/M)] were recruited. There was a significant decrease in HIV-1 RNA after 10 days of maraviroc treatment in patients with R5-tropic virus (median 1.52 log10 RNA copies/mL; 95% CI 1.23-1.63; P < 0.0001), but also in patients with D/M-tropic virus (median 1.62 log(10) RNA copies/mL; 95% CI 0.33-1.88; P = 0.00024). The difference in the HIV-1 RNA decrease (-0.16 log(10) RNA copies/mL; 95% CI -0.53 to 0.22) was not significant (P = 0.410). A decrease >0.5 log(10) RNA copies/mL was found in 96.3% of patients with R5-tropic virus and in 70% of patients with D/M-tropic virus (P = 0.052). The differences were not significant when a decline of 1 log(10) RNA copies/mL was considered (92.6% versus 70%; P = 0.11). CONCLUSIONS: Treatment-naive patients infected with R5- or D/M-tropic virus have similar virological responses to a short course of maraviroc monotherapy. This clinical test thus cannot be used as a surrogate marker of viral tropism in this population.

Raltegravir pharmacokinetics in HIV/HCV-coinfected patients with advanced liver cirrhosis (Child-Pugh C).

OBJECTIVES: To describe raltegravir pharmacokinetics at steady-state in HIV/hepatitis C virus (HCV)-coinfected patients under antiretroviral (ARV) treatment with (n = 5) and without (n = 5) advanced liver cirrhosis (Child-Pugh C). METHODS: This was a non-randomized, Phase I, parallel-assignment, open-label pharmacokinetic study in HIV/HCV-coinfected patients with Child-Pugh grade C hepatic cirrhosis. We recruited clinically stable HIV/HCV-coinfected adult patients with controlled HIV viraemia (<50 copies/mL) for at least 6 months. Raltegravir (400 mg twice daily) was added under fasting conditions for 5 days to the successful ritonavir-boosted protease inhibitor-based ARV regimen. The trial was registered in the ClinicalTrials.gov database (NCT01289951) (LIVERAL). RESULTS: Raltegravir AUC0-12 and C12 were increased 1.72-fold (90% CI, 1.02 to 2.92) and 6.58-fold (90% CI, 2.92 to14.85), respectively, in patients with advanced liver cirrhosis. No safety issues were identified and raltegravir was well tolerated by all patients. CONCLUSIONS: Raltegravir plasma levels are increased in HIV/HCV-coinfected patients with advanced liver cirrhosis (Child-Pugh C). Despite the higher exposure, raltegravir was safe and well tolerated.

Metastable ionic diodes derived from an amine-based polymer of intrinsic microporosity.

A highly rigid amine-based polymer of intrinsic microporosity (PIM), prepared by a polymerization reaction involving the formation of Tröger's base, is demonstrated to act as an ionic diode with electrolyte-dependent bistable switchable states.

Effect of headgroup on the physicochemical properties of phospholipid bilayers in electric fields: size matters.

The effect of molecular structure on ensemble structure of phospholipid films has been investigated. Bilayers of dimyristoyl phosphatidylethanolamine (DMPE) were prepared on Au(111) electrodes using Langmuir-Blodgett and Langmuir-Schaeffer deposition. Capacitance and charge density measurements were used to investigate the adsorption behavior and barrier properties of the lipid bilayers. In situ polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) was employed to investigate the organization of the molecules within the bilayer. DMPE bilayers exhibit lower capacitance than bilayers formed from the related lipid, dimyristoyl phosphatidylcholine (DMPC). The infrared data show that these results can be explained by structural differences between the bilayers formed from each molecule. DMPE organizes into bilayers with hydrocarbon chains tilted at a smaller angle to the surface normal, which results in a thicker film. The hydrocarbon chains contain few conformational defects. Spectra in the carbonyl and phosphate stretching mode regions indicate low solvent content of DMPE films. Both of these effects combine to produce films with lower capacitance and enhanced barrier properties. The results are explained in terms of the differences in structure between the constituent molecules.

Risk analysis of human anisakidosis through the consumption of the blue whiting, Micromesistius poutassou, sold at Spanish supermarkets.

European legislation directed at the catering industry concerning the prevention of anisakidosis proposes efficient measures to avoid human infestation, but this legislation does not directly address the consumer at the household level. Assessing the anisakidosis risk for consumers who buy fresh fish at supermarkets in Spain, 284 blue whiting, Micromesistius poutassou, specimens, originating from two fishing zones and seasons of capture, sold at five nationwide Spanish supermarket chains, were examined to identify the presence of anisakid species and analyze their prevalence and abundance in viscera and flesh. The potential influence of intrinsic (length and weight) and extrinsic (origin, season, and days after catch) factors was statistically analyzed. The only two species detected were Anisakis simplex complex and Hysterothylacium sp. Total anisakid prevalence was 55.6%, with A. simplex being more prevalent (53.9%) and abundant (3.9 helminths/fish), and also carrying the biggest risk for consumers. Origin (Atlantic and Mediterranean) and season (spring and autumn) were the most influential factors among those considered. Results suggest that specimens originating from the Atlantic, of greater weight and length, and caught in spring pose the highest transmission risk of anisakidosis. Moreover, the days passed between the catch and consumption should be considered as a risk factor. Thus, in addition to the recommendation of freezing fish at -20°C for at least 24 h (in case the fish is consumed raw or poorly cooked), new and easy-to-follow recommendations are being proposed to avoid anisakidosis at home, which emphasize the importance of the information provided on fish crate labels.

Nematode Parasites of the European Pilchard, Sardina pilchardus (Walbaum, 1792): A Genuine Human Hazard?

The European pilchard is one of the most frequently consumed fish species in Mediterranean countries, especially in Italy and Spain, and has been reported as the cause of at least eight human anisakidosis cases in Spain. With the aim to shed light on the potential human parasitosis risk posed by nematode larvae belonging to families Anisakidae or Raphidascarididae, a total of 350 sardines captured in the Atlantic Ocean (175 specimens) and the Mediterranean Sea (175 specimens), acquired in various Spanish nationwide supermarket chains, were helminthologically analyzed. The statistical analysis of some helminth parameters revealed a higher presence of nematodes belonging to the genus Hysterothylacium (prevalence 24.29%; mean abundance of 2.36), usually considered non-parasitic for humans (only three cases reported worldwide), when compared to nematodes of the genus Anisakis (5.71%; 0.16). The human anisakidosis risk after the consumption of raw or undercooked sardines and the role of Hysterothylacium, the most frequent nematode, is discussed, providing information to consumers. To avoid human infection by anisakid larval nematodes, the established preventive measures are confirmed and new ones are proposed, such as the consumption of sardines preferably caught in the Mediterranean and of small-sized specimens available, and the immediate evisceration after fishing.

Anisakid Nematodes and Potential Risk of Human Anisakiasis through the Consumption of Hake, Merluccius spp., Sold Fresh in Spanish Supermarkets.

Nematode parasite species belonging to the Anisakis simplex complex are the most important cause of human anisakiasis through the consumption of (mainly) undercooked, previously not frozen, or conveniently treated fish. In Spain, the consumption of hake has been recognized as an important source of this parasitosis. With the aim of shedding light on the risk factors that can influence the potential risk of human anisakiasis in Spain through the consumption of fresh hake sold by nationwide supermarket chains, a total of 536 small hake specimens belonging to the species Merluccius bilinearis caught off the Northeast American coasts and Merluccius merluccius caught in the Northeast Atlantic and Mediterranean waters was analysed. Anisakids morphologically identified as Anisakis type I were found as the most prevalent and the most abundant species and were considered the main potential cause of human anisakiasis. Intrinsic and extrinsic factors concerning the hake, such as its origin and season of capture, its size, as well as the days passed between its capture and consumption, should be taken into account to avoid this human parasitosis. It is essential that consumers have access to fish label information which should include, as regulated by the European Commission, traceability data.

Selective miRNA inhibition in CD8+ cytotoxic T lymphocytes enhances HIV-1 specific cytotoxic responses.

miRNAs dictate relevant virus-host interactions, offering new avenues for interventions to achieve an HIV remission. We aimed to enhance HIV-specific cytotoxic responses-a hallmark of natural HIV control- by miRNA modulation in T cells. We recruited 12 participants six elite controllers and six patients with chronic HIV infection on long-term antiretroviral therapy ("progressors"). Elite controllers exhibited stronger HIV-specific cytotoxic responses than the progressors, and their CD8+T cells showed a miRNA (hsa-miR-10a-5p) significantly downregulated. When we transfected ex vivo CD8+ T cells from progressors with a synthetic miR-10a-5p inhibitor, miR-10a-5p levels decreased in 4 out of 6 progressors, correlating with an increase in HIV-specific cytotoxic responses. The effects of miR-10a-5p inhibition on HIV-specific CTL responses were modest, short-lived, and occurred before day seven after modulation. IL-4 and TNF-α levels strongly correlated with HIV-specific cytotoxic capacity. Thus, inhibition of miR-10a-5p enhanced HIV-specific CD8+ T cell capacity in progressors. Our pilot study proves the concept that miRNA modulation is a feasible strategy to combat HIV persistence by enhancing specific cytotoxic immune responses, which will inform new approaches for achieving an antiretroviral therapy-free HIV remission.

Mechanisms of resistance and susceptibility to experimental visceral leishmaniosis: BALB/c mouse versus Syrian hamster model.

Several animal models have been established to study visceral leishmaniosis (VL), a worldwide vector-borne disease affecting humans and domestic animals that constitutes a serious public health problem. BALB/c mice and Syrian hamsters are the most widely used experimental models. In this paper, we summarize the advantages and disadvantages of these two experimental models and discuss the results obtained using these models in different studies of VL. Studies using the BALB/c mouse model have underscored differences between the liver and spleen in the course of VL, indicating that pathological evaluation of the visceral organs is essential for understanding the immune mechanisms induced by Leishmania infantum infection. The main goal of this review is to collate the relevant literature on Leishmania pathogenesis into a sequence of events, providing a schematic view of the main components of adaptive and innate immunity in the liver and spleen after experimental infection with L. infantum or L. donovani. This review also presents several viewpoints and reflections about some controversial aspects of Leishmania research, including the choice of experimental model, route of administration, inoculum size and the relevance of pathology (intimately linked to parasite persistence): a thorough understanding of which is essential for future VL research and the successful development of efficient control strategies for Leishmania spp.

Silymarin secretion and its elicitation by methyl jasmonate in cell cultures of Silybum marianum is mediated by phospholipase D-phosphatidic acid.

The flavonolignan silymarin is released to the extracellular medium of Silybum marianum cultures and its production can be stimulated by the elicitor methyljasmonate (MeJA). The sequence of the signalling processes leading to this response is unknown at present. It is reported in this work that MeJA increased the activity of the enzyme phospholipase D (PLD). Treatment with mastoparan (Mst), a PLD activity stimulator, also enhanced PLD and caused a substantial increase in silymarin production. The application of the product of PLD activity, phosphatidic acid (PA) promoted silymarin accumulation. Altering PLD activity by introducing in cultures n-butanol (nBuOH), which inhibits PA production by PLD, prevented silymarin elicitation by MeJA or Mst and also impeded its release in non-elicited cultures. Treatment with iso-, sec- or tert- butanol had no effect on silymarin production. The exogenous addition of PA reversed the inhibitory action of nBuOH, both in control and MeJA-treated cultures. These results suggest that the enzyme PLD and its product PA mediate silymarin secretion to the medium of S. marianum cultures.

Indirect photo-electrochemical detection of carbohydrates with Pt@g-C3N4 immobilised into a polymer of intrinsic microporosity (PIM-1) and attached to a palladium hydrogen capture membrane.

An "indirect" photo-electrochemical sensor is presented for the measurement of a mixture of analytes including reducing sugars (e.g. glucose, fructose) and non-reducing sugars (e.g. sucrose, trehalose). Its innovation relies on the use of a palladium film creating a two-compartment cell to separate the electrochemical and the photocatalytic processes. In this original way, the electrochemical detection is separated from the potential complex matrix of the analyte (i.e. colloids, salts, additives, etc.). Hydrogen is generated in the photocatalytic compartment by a Pt@g-C3N4 photocatalyst embedded into a hydrogen capture material composed of a polymer of intrinsic microporosity (PIM-1). The immobilised photocatalyst is deposited onto a thin palladium membrane, which allows rapid pure hydrogen diffusion, which is then monitored by chronopotentiometry (zero current) response in the electrochemical compartment. The concept is demonstrated herein for the analysis of sugar content in commercial soft drinks. There is no requirement for the analyte to be conducting with electrolyte or buffered. In this way, samples (biological or not) can be simply monitored by their exposition to blue LED light, opening the door to additional energy conversion and waste-to-energy applications.

Maraviroc reactivates HIV with potency similar to that of other latency reversing drugs without inducing toxicity in CD8 T cells.

Human immunodeficiency virus (HIV) remains incurable due to latent reservoirs established in non-activated CD4 T cells. Current efforts to achieve a functional cure rely on immunomodulatory strategies focused on enhancing the functions of cytotoxic cells. Implementation of these actions requires a coordinated activation of the viral transcription in latently infected cells so that the reservoir became visible and accessible to cytotoxic cells. As no latency reversing agent (LRA) has been shown to be completely effective, new combinations are of increasing importance. Recent data have shown that maraviroc is a new LRA. In this work, we have explored how the combination of maraviroc with other LRAs influences on HIV reactivation using in vitro latency models as well as on the cell viability of CD8 T cells from ART-treated patients. Maraviroc reactivated HIV with a potency similar to other LRAs. Triple combinations resulted toxic and were rejected. No dual combination was synergistic. The combination with panobinostat or disulfiram maintained the effect of both drugs without inducing cell proliferation or toxicity. Maraviroc does not alter the viability of CD8 T cells isolated from patients under antiretroviral treatment. This finding enhances the properties of maraviroc as a LRA.

Prolonged administration of maraviroc reactivates latent HIV in vivo but it does not prevent antiretroviral-free viral rebound.

Human immunodeficiency virus (HIV) remains incurable due to latent viral reservoirs established in non-activated CD4 T cells that cannot be eliminated via antiretroviral therapy. Current efforts to cure HIV are focused on identifying drugs that will induce viral gene expression in latently infected cells, commonly known as latency reversing agents (LRAs). Some drugs have been shown to reactivate latent HIV but do not cause a reduction in reservoir size. Therefore, finding new LRAs or new combinations or increasing the round of stimulations is needed to cure HIV. However, the effects of these drugs on viral rebound after prolonged treatment have not been evaluated. In a previous clinical trial, antiretroviral therapy intensification with maraviroc for 48 weeks caused an increase in residual viremia and episomal two LTR-DNA circles suggesting that maraviroc could reactivate latent HIV. We amended the initial clinical trial to explore additional virologic parameters in stored samples and to evaluate the time to viral rebound during analytical treatment interruption in three patients. Maraviroc induced an increase in cell-associated HIV RNA during the administration of the drug. However, there was a rapid rebound of viremia after antiretroviral therapy discontinuation. HIV-specific T cell response was slightly enhanced. These results show that maraviroc can reactivate latent HIV in vivo but further studies are required to efficiently reduce the reservoir size.

Gz mediates the long-lasting desensitization of brain CB1 receptors and is essential for cross-tolerance with morphine.

BACKGROUND: Although the systemic administration of cannabinoids produces antinociception, their chronic use leads to analgesic tolerance as well as cross-tolerance to morphine. These effects are mediated by cannabinoids binding to peripheral, spinal and supraspinal CB1 and CB2 receptors, making it difficult to determine the relevance of each receptor type to these phenomena. However, in the brain, the CB1 receptors (CB1Rs) are expressed at high levels in neurons, whereas the expression of CB2Rs is marginal. Thus, CB1Rs mediate the effects of smoked cannabis and are also implicated in emotional behaviors. We have analyzed the production of supraspinal analgesia and the development of tolerance at CB1Rs by the direct injection of a series of cannabinoids into the brain. The influence of the activation of CB1Rs on supraspinal analgesia evoked by morphine was also evaluated. RESULTS: Intracerebroventricular (icv) administration of cannabinoid receptor agonists, WIN55,212-2, ACEA or methanandamide, generated a dose-dependent analgesia. Notably, a single administration of these compounds brought about profound analgesic tolerance that lasted for more than 14 days. This decrease in the effect of cannabinoid receptor agonists was not mediated by depletion of CB1Rs or the loss of regulated G proteins, but, nevertheless, it was accompanied by reduced morphine analgesia. On the other hand, acute morphine administration produced tolerance that lasted only 3 days and did not affect the CB1R. We found that both neural mu-opioid receptors (MORs) and CB1Rs interact with the HINT1-RGSZ module, thereby regulating pertussis toxin-insensitive Gz proteins. In mice with reduced levels of these Gz proteins, the CB1R agonists produced no such desensitization or morphine cross-tolerance. On the other hand, experimental enhancement of Gz signaling enabled an acute icv administration of morphine to produce a long-lasting tolerance at MORs that persisted for more than 2 weeks, and it also impaired the analgesic effects of cannabinoids. CONCLUSION: In the brain, cannabinoids can produce analgesic tolerance that is not associated with the loss of surface CB1Rs or their uncoupling from regulated transduction. Neural specific Gz proteins are essential mediators of the analgesic effects of supraspinal CB1R agonists and morphine. These Gz proteins are also responsible for the long-term analgesic tolerance produced by single doses of these agonists, as well as for the cross-tolerance between CB1Rs and MORs.

NMDAR-nNOS generated zinc recruits PKCgamma to the HINT1-RGS17 complex bound to the C terminus of Mu-opioid receptors.

In neurons, the C terminus of the Mu-opioid receptor (MOR) binds to the protein kinase C-interacting protein/histidine triad nucleotide binding protein 1 (PKCI/HINT1) which in turn binds the regulator of G-protein signalling RGSZ1/Z2 (RGSZ) protein. In this study, we found that intracerebroventricular (icv) administration of morphine recruits PKC isoforms, mostly PKCgamma, to the MOR via the HINT1/RGSZ complex. There, diacylglycerol (DAG) activates this PKCgamma to phosphorylate the MOR and thus, its signal strength was reduced. When PKCI/HINT1 expression is depressed, morphine produces stronger analgesic effects and neither the PKCgamma-MOR complex nor serine phosphorylation of this receptor is detected. This MOR-PKC association involves the cysteine rich domains (CRDs) in the regulatory C1 region of PKC, as well as requiring free zinc ions, HINT1 and RGSZ proteins. Increasing the availability of this metal ion recruits inactive PKCgamma to the MOR, while phorbol esters prevent this binding and even disrupt it. The nitric oxide donor (S)-Nitroso-N-acetylpenicillamine (SNAP) foments the association of PKCgamma with the MORs, effect that was prevented by the heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN), suggesting a role for endogenous zinc and neural nitric oxide synthase. The N-methyl-D-aspartate receptor (NMDAR) antagonist, MK801, also prevented PKCgamma recruitment to MORs and serine phosphorylation of the receptors following icv morphine. These results indicate that the NMDAR/nNOS cascade, activated via MORs, provide the free zinc ions required for inactive PKCgamma to bind to HINT1/RGSZ complex at the C terminus of the receptor.

Selective miRNA Modulation Fails to Activate HIV Replication in In Vitro Latency Models.

HIV remains incurable because of viral persistence in latent reservoirs that are inaccessible to antiretroviral therapy. A potential curative strategy is to reactivate viral gene expression in latently infected cells. However, no drug so far has proven to be successful in vivo in reducing the reservoir, and therefore new anti-latency compounds are needed. We explored the role of microRNAs (miRNAs) in latency maintenance and their modulation as a potential anti-latency strategy. Latency models based on treating resting CD4 T cells with chemokine (C-C motif) ligand 19 (CCL19) or interleukin-7 (IL7) before HIV infection and next-generation sequencing were used to identify the miRNAs involved in HIV latency. We detected four upregulated miRNAs (miRNA-98, miRNA-4516, miRNA-4488, and miRNA-7974). Individual or combined inhibition of these miRNAs was performed by transfection into cells latently infected with HIV. Viral replication, assessed 72 h after transfection, did not increase after miRNA modulation, despite miRNA inhibition and lack of toxicity. Furthermore, the combined modulation of five miRNAs previously associated with HIV latency was not effective in these models. Our results do not support the modulation of miRNAs as a useful strategy for the reversal of HIV latency. As shown with other drugs, the potential of miRNA modulation as an HIV reactivation strategy could be dependent on the latency model used.