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To study the activation of HIV by human spumaretrovirus (HSRV) the long terminal repeats (LTRs) of HSRV, HIVl and HIV2 were examined with respect to their ability to function as transcriptional promoters in virus infected and uninfected cells. Transient transfections using plasmids in which the L TRs of the three viruses were coupled to the bacterial chloramphenicol acetyltransferase (CA T) gene revealed (i) the level of cat gene expression directed by the HSRV LTR was markedly increased in HSRV infected cells compared to uninfected cells, (ii) cat gene expression driven by the HIV1 LTR, but not by the HIV2 LTR could be enhanced upon HSRV infection, whereas (iii) neither in HIV1 nor in HIV2 infected cells an effect on HSRV LTR driven cat geneexpression was detected.
Malaria and HIV are among the most important global health problems of our time and together are responsible for approximately 3 million deaths annually. These two diseases overlap in many regions of the world including sub-Saharan Africa, Southeast Asia and South America, leading to a higher risk of co-infection. In this study, we generated and characterized hybrid molecules to target P. falciparum and HIV simultaneously for a potential HIV/malaria combination therapy. Hybrid molecules were synthesized by covalent fusion between azidothymidine (AZT) and dihydroartemisinin (DHA), tetraoxane or chloroquine (CQ); and a small library was generated and tested for antiviral and antimalarial activity. Our data suggest that dihyate is the most potent molecule in vitro, with antiplasmodial activity comparable to that of DHA (IC50 = 26 nM, SI > 3000), a moderate activity against HIV (IC50 = 2.9 µM; SI > 35) and safe to HeLa cells at concentrations used in the assay (CC50 > 100 µM). Pharmacokinetic studies further revealed that dihyate is metabolically unstable and is cleaved following an O-dealkylation once in contact with cytochrome P450 enzymes. The later further explains the uneffectiveness of dihyate against the CQ-sensitive P. berghei N strain in mice when administered by oral route at 20 mg/kg. Here, we report on a first approach to develop antimalarial/anti-HIV hybrid molecules and future optimization efforts will aim at producing second generation hybrid molecules to improve activity against HIV as well as compound bioavailability. With the emergence of resistant parasites against all the counterpart drugs of artemisinin derivatives used in artemisinin based combination therapies (ACTs), the introduction of antibiotics in the treatment of malaria has renewed interest on the identification of antibiotics with potent antimalarial properties. In this study we also investigated the antiplasmodial potential of thiostrepton and derivatives, synthesized using combinations of tail truncation, oxidation, and addition of lipophilic thiols to the terminal dehydroamino acid. We showed that derivatives SS231 and SS234 exhibit a better antiplasmodial activity (IC50 = 1 µM SI > 59 and SI > 77 respectively) than thiostrepton (IC50 = 8.95 µM, SI = 1.7). The antiplasmodial activity of these derivatives was observed at concentrations which are not hemolytic and non-toxic to human cell lines. Thiostrepton and derivatives appeared to exhibit transmission blocking properties when administered at their IC50 or IC90 concentrations and our data also showed that they attenuate proteasome activity of Plasmodium, which resulted in an accumulation of ubiquitinated proteins after incubation with their IC80 concentrations. Our results indicate that the parasite’s proteasome could be an attractive target for therapeutic intervention. In this regard, thiostrepton derivatives are promising candidates by dually acting on two independent targets, the proteasome and the apicoplast, with the capacity to eliminate both intraerythrocytic asexual and transmission stages of the parasite. To further support our findings, we evaluated the activity of a new class of antimalarial and proteasome inhibitors namely peptidyl sulfonyl fluorides on gametocyte maturation and analogues AJ34 and AJ38 were able to completely suppress gametocytogenesis at IC50 concentrations (0.23 µM and 0.17 µM respectively) suggesting a strong transmission blocking potential. The proteasome, a major proteolytic complex, responsible for the degradation and re-cycling of non-functional proteins has been studied only indirectly in P. falciparum. In addition, an apparent proteasome-like protein with similarity to bacterial ClpQ/hslV threonine-peptidases was predicted in the parasite. Antibodies were generated against the proteasome subunits alpha type 5 (α5-SU), beta type 5 (β5-SU) and pfhslV in mice and we showed that the proteasome is expressed in both sexual and asexual blood stages of P. falciparum, where they localize in the nucleus and in the cytoplasm. However, expression of PfhslV was only observed in trophozoites and shizonts. The trafficking of the studied proteasome subunits was further investigated by generating parasites expressing GFP tagged proteins. The expression of α5-SU-GFP in transgenic parasite appeared to localize abundantly in the cytoplasm of all blood stages, and no additional information was obtained from this parasite line. In conclusion, our data highlight two new tools towards combination therapy. Hybrid molecules represent promising tools for the cure of co-infected individuals, while very potent antibiotics with a wide scope of activities could be useful in ACTs by eliminating resistant parasites and limiting transmission of both, resistances and disease.
Background: Opioids may have effects on susceptibility to HIV-infection, viral replication and disease progression. Injecting drug users (IDU), as well as anyone receiving opioids for anesthesia and analgesia may suffer the clinical consequences of such interactions. There is conflicting data between in vitro experiments showing an enhancing effect of opioids on HIV replication and clinical data, mostly showing no such effect. For clarification we studied the effects of the opioids heroin and morphine on HIV replication in cultured CD4-positive T cells at several concentrations and we related the observed effects with the relevant reached plasma concentrations found in IDUs.
Methods: Latently-infected ACH-2 T lymphoblasts were incubated with different concentrations of morphine and heroine. Reactivation of HIV was assessed by intracellular staining of viral Gag p24 protein and subsequent flow cytometric quantification of p24-positive cells. The influence of the opioid antagonist naloxone and the antioxidants N-acetyl-cysteine (NAC) and glutathione (GSH) on HIV reactivation was determined. Cell viability was investigated by 7-AAD staining and flow cytometric quantification.
Results: Morphine and heroine triggered reactivation of HIV replication in ACH-2 cells in a dose-dependent manner at concentrations above 1 mM (EC50 morphine 2.82 mM; EC50 morphine 1.96 mM). Naloxone did not interfere with heroine-mediated HIV reactivation, even at high concentrations (1 mM). Opioids also triggered necrotic cell death at similar concentrations at which HIV reactivation was observed. Both opioid-mediated reactivation of HIV and opioid-triggered cell death could be inhibited by the antioxidants GSH and NAC.
Conclusions: Opioids reactivate HIV in vitro but at concentrations that are far above the plasma levels of analgesic regimes or drug concentrations found in IDUs. HIV reactivation was mediated by effects unrelated to opioid-receptor activation and was tightly linked to the cytotoxic activity of the substances at millimolar concentrations, suggesting that opioid-mediated reactivation of HIV was due to accompanying effects of cellular necrosis such as activation of reactive oxygen species and NF-kB.
As viruses are obligatory intracellular parasites, any step during their life cycle strictly depends on successful interaction with their particular host cells. In particular, their interaction with cellular membranes is of crucial importance for most steps in the viral replication cycle. Such interactions are initiated by uptake of viral particles and subsequent trafficking to intracellular compartments to access their replication compartments which provide a spatially confined environment concentrating viral and cellular components, and subsequently, employ cellular membranes for assembly and exit of viral progeny. The ability of viruses to actively modulate lipid composition such as sphingolipids (SLs) is essential for successful completion of the viral life cycle. In addition to their structural and biophysical properties of cellular membranes, some sphingolipid (SL) species are bioactive and as such, take part in cellular signaling processes involved in regulating viral replication. It is especially due to the progress made in tools to study accumulation and dynamics of SLs, which visualize their compartmentalization and identify interaction partners at a cellular level, as well as the availability of genetic knockout systems, that the role of particular SL species in the viral replication process can be analyzed and, most importantly, be explored as targets for therapeutic intervention.
This study describes a simple technique that improves a recently developed 3D sub-diffraction imaging method based on three-photon absorption of commercially available quantum dots. The method combines imaging of biological samples via tri-exciton generation in quantum dots with deconvolution and spectral multiplexing, resulting in a novel approach for multi-color imaging of even thick biological samples at a 1.4 to 1.9-fold better spatial resolution. This approach is realized on a conventional confocal microscope equipped with standard continuous-wave lasers. We demonstrate the potential of multi-color tri-exciton imaging of quantum dots combined with deconvolution on viral vesicles in lentivirally transduced cells as well as intermediate filaments in three-dimensional clusters of mouse-derived neural stem cells (neurospheres) and dense microtubuli arrays in myotubes formed by stacks of differentiated C2C12 myoblasts.
Background: In a 2008-10 study, we found a pretreatment HIV drug resistance (PDR) prevalence of 18.2% in patients at Bugando Medical Centre (BMC) in Mwanza, Tanzania.
Objectives: To determine the prevalence of PDR and transmitted HIV drug resistance (TDR) in patients visiting the BMC from 2013 to 2015.
Methods: Adult outpatients were sequentially enrolled into two groups, separated by whether they were initiating ART. Previous exposure to antiretroviral drugs, except for prevention of mother-to-child transmission, was an exclusion criterion. HIV pol sequences were analysed according to WHO guidelines for surveillance of PDR and TDR.
Results: Two hundred and thirty-five sequences were analysed (138 ART initiators, 97 non-initiators). The prevalence of PDR was 4.7% (95% CI 2.6%-8.2%) overall, 3.1% (95% CI 1.1%-8.7%) for non-initiators and 5.8% (95% CI 3.0%-11.0%) for ART initiators. PDR to NNRTIs and nucleoside or nucelotide reverse transcriptase inhibitors was found in 3.0% (95% CI 1.5%-6.0%) and 1.7% (95% CI 0.7%-4.3%) of patients, respectively. Resistance to PIs was not observed. The prevalence of TDR was 6.0% (95% CI 3.6%-9.8%).
Conclusions: Prevalence of PDR significantly decreased compared with 2008-10 and was below the WHO-defined threshold for triggering a public health response. National and systematic surveillance is needed to inform Tanzania's public health strategy.
Background:
While HIV, AIDS and atypical Mycobacterium infections are closely linked, the use of Integrase-Inhibitor based cART, notably raltegravir-based regimens is more widespread. RAL should be double-dosed to 800 mg semi-daily in situation of rifampicin co-medication, because RAL is more rapidly metabolized due to rifampicin-induced Uridine-5'-diphosph-gluronosyl-transferase (UGT1A1). Recently, it was speculated that chewed RAL might lead to increased absorption, which might compensate the inductive effect of rifampicin-rapid metabolized RAL, as part of cost-saving effects in countries with high-tuberculosis prevalence and less economic power.
Methods:
We report measurement of raltegravir pharmacokinetics in a 34-year AIDS-patient suffering from disseminated Mycobacterium avium infection with necessity of parenteral rifampicin treatment. RAL levels were measured with HPLC (internal standard: carbamazepine, LLQ 11 ng/ml, validation with Valistat 2.0 program (Arvecon, Germany)). For statistical analysis, a two-sided Wilcoxon signed rank test for paired samples was used.
Results:
High intra-personal variability in raltegravir serum levels was seen. Comparable C\(_{max}\) concentrations were found for 800 mg chewed and swallowed RAL, as well as for 400 mg chewed and swallowed RAL. While C\(_{max}\) seems to be more dependent from overall RAL dosing than from swallowed or chewed tablets, increased AUC(12) is clearly linked to higher RAL dosages per administration. Anyway, chewed raltegravir showed a rapid decrease in serum levels.
Conclusions:
We found no evidence that chewed 400 mg semi-daily raltegravir in rifampicin co-medication leads to optimized pharmacokinetics. There is need for more data from randomized trials for further recommendations.
A 21 year old MSM patient with newly diagnosed HIV infection was hospitalized in our department after ingestion of an overdose of his antiretroviral therapy (ART) comprising dolutegravir (DTG - Tivicay\(^{®}\)) and tenofovir disaproxil fumarate/emtricitabine (Truvada\(^{®}\)) in suicidal intention. On admission, the patient did not show any clinical signs of intoxication and laboratory findings were unremarkable. After 6 hours of intensive care monitoring, the patient was referred to a psychiatric clinic. 5 days after the day of intoxication, serum creatinine levels increased to high normal values (1.2 mg/dl). However, levels never exceeded the upper threshold. 8 and 12 weeks later, serum creatinine normalized to levels measured prior to the intoxication. No other adverse events occurred, and the patient does not suffer from permanent impairments.
Background:
HIV-associated neurocognitive disorder (HAND) remains highly prevalent despite effective anti-retroviral therapy (ART). A number of adjunctive pharmacotherapies for HAND have been studied with disappointing results, but preliminary data suggest that lithium may provide clinical benefit. In addition, the low cost of lithium would facilitate access in low- and middle-income countries which carry the greatest burden of HIV.
Methods:
Our objective was to evaluate the 24-week efficacy and safety of lithium in patients with moderate to severe HAND. Our primary efficacy endpoint was the change in Global Deficit Score (GDS) from baseline to 24 weeks, whereas our secondary endpoint was the change in proton magnetic resonance spectroscopy (1H-MRS) brain metabolite concentrations. We conducted a 24-week randomized placebo-controlled trial of lithium as adjunctive pharmacotherapy. We enrolled participants with moderate to severe HAND, on ART for at least 6 months, with suppressed viral loads and attending public sector primary care clinics in Cape Town, South Africa. We randomized 66 participants to lithium (n = 32) or placebo (n = 34). Lithium or placebo was dosed 12-hourly and titrated to achieve the maintenance target plasma concentration of 0.6 to 1.0 mmol/L. Sham lithium concentrations were generated for participants receiving placebo.
Results:
Totally 61 participants completed the study (lithium arm = 30; placebo arm = 31). Participants at enrolment had a mean age of 40 years and a median CD4+ T-cell count of 500 cells/μL. The median change in GDS between baseline and week 24 for the lithium and placebo arms were –0.57 (95% confidence interval [CI] –0.77, –0.32) and –0.56 (–0.69, –0.34) respectively, with a mean difference of –0.054 (95% CI –0.26, 0.15); P = 0.716. The improvement remained similar when analyzed according to age, severity of impairment, CD4+ count, time on ART, and ART regimen. Standard 1H-MRS metabolite concentrations were similar between the treatment arms. The study drug was well tolerated in both study arms. Six serious adverse events occurred, but none were considered related to the study drug.
Conclusion:
Adjunctive lithium pharmacotherapy in patients on ART with HAND was well tolerated but had no additional benefit on neurocognitive impairment.