Systemic HIV and SIV latency reversal via non-canonical NF-κB signalling in vivo.

Long-lasting, latently infected resting CD4+ T cells are the greatest obstacle to obtaining a cure for HIV infection, as these cells can persist despite decades of treatment with antiretroviral therapy (ART). Estimates indicate that more than 70 years of continuous, fully suppressive ART are needed to eliminate the HIV reservoir1. Alternatively, induction of HIV from its latent state could accelerate the decrease in the reservoir, thus reducing the time to eradication. Previous attempts to reactivate latent HIV in preclinical animal models and in clinical trials have measured HIV induction in the peripheral blood with minimal focus on tissue reservoirs and have had limited effect2-9. Here we show that activation of the non-canonical NF-κB signalling pathway by AZD5582 results in the induction of HIV and SIV RNA expression in the blood and tissues of ART-suppressed bone-marrow-liver-thymus (BLT) humanized mice and rhesus macaques infected with HIV and SIV, respectively. Analysis of resting CD4+ T cells from tissues after AZD5582 treatment revealed increased SIV RNA expression in the lymph nodes of macaques and robust induction of HIV in almost all tissues analysed in humanized mice, including the lymph nodes, thymus, bone marrow, liver and lung. This promising approach to latency reversal-in combination with appropriate tools for systemic clearance of persistent HIV infection-greatly increases opportunities for HIV eradication.

Molecular profiling of postreperfusion milieu determines acute kidney injury after liver transplantation: A prospective study.

Acute kidney injury (AKI) after liver transplantation (LT) is a common event, but its pathogenesis remains unclear. The aim of this prospective study is to investigate the potential relationship between postreperfusion gene expression, serum mediators, and the onset of AKI after LT. Sixty-five consecutive patients undergoing LT were included in the study. Reverse transcription polymerase chain reaction (PCR) was performed on liver biopsies. Gene expression of 23 genes involved in ischemia/reperfusion injury (IRI) was evaluated. The serum concentrations of endothelin (ET)-1 and inflammatory cytokines were analyzed. AKI after LT developed in 21 (32%) recipients (AKI group). Reverse transcription PCR of reperfusion biopsy in the AKI group showed higher expression of several genes involved in IRI compared with the non-AKI group. Fold changes in the gene expression of ET-1, interleukin (IL) 18, and tumor necrosis factor α (TNF-α) were associated with creatinine peak value. AKI patients also had significantly higher ET-1, IL18, and TNF-α postoperative serum levels. Multivariate analysis showed that ET-1 (odds ratio [OR], 16.7; 95% confidence interval [CI], 3.34-83.42; P = 0.001) and IL18 (OR, 5.27; 95% CI, 0.99-27.82, P = 0.048) serum levels on postoperative day 1 were independently predictive of AKI. Receiver operating characteristic analysis demonstrated that the combination of biomarkers ET-1+IL18 was highly predictive of AKI (area under the receiver operating characteristic curve, 0.91; 95% CI, 0.83-0.99). Early allograft dysfunction and chronic kidney disease stage ≥ 2 occurred more frequently in AKI patients. These results suggest that the graft itself, rather than intraoperative hemodynamic instability, plays a main role in AKI after LT. These data may have mechanistic and diagnostic implications for AKI after LT. Liver Transplantation 24 922-931 2018 AASLD.

Postreperfusion microcirculatory derangements after liver transplantation: Relationship to hemodynamics, serum mediators, and outcome.

Despite the growing data supporting the role of microcirculation in regulating liver function, little of this knowledge has been translated into clinical practice. The aim of this study is to quantify hepatic microcirculation in vivo using sidestream dark field (SDF) imaging and correlate these findings with hepatic blood flow, hemodynamic parameters, and soluble mediators. Postreperfusion hepatic microcirculation was assessed using SDF imaging. Hepatic microcirculation measurements included functional sinusoidal density (cm/cm2 ), sinusoidal diameter (µm), red blood cell velocity (µm/second), volumetric blood flow (pl/second), and flow heterogeneity (FH) index. The serum concentrations of endothelin 1 (ET-1) and other inflammatory markers were analyzed with Luminex technology. Portal venous and hepatic artery flows were measured using a flowmeter. Twenty-eight patients undergoing cadaveric liver transplantations have been included in this study. Early allograft dysfunction (EAD) occurred in 7 (25%) patients and was associated with microcirculatory dysfunction. Low arterial and portal flow, high dose of inotropes, cold ischemia time, steatosis, and high ET-1 levels were all associated with impaired microcirculation. The time interval between portal venous and hepatic arterial reperfusion significantly correlated with the changes of the liver grafts' microcirculation. EAD patients tended to have higher serum levels of ET-1 on postoperative days 1, 2, 5, and 7 (all P < 0.01). Serum levels of ET-1 correlated significantly with microcirculation parameters. In conclusion, postreperfusion hepatic microcirculation is a determinant of organ dysfunction after liver reperfusion and could be used to identify very early patients at risk of EAD. Liver Transplantation 23 527-536 2017 AASLD.

The use of biological grafts for reconstruction of the inferior vena cava is a safe and valid alternative: results in 32 patients in a single institution.

BACKGROUND: Resection and reconstruction of the inferior vena cava (IVC) is occasionally required in the surgical treatment of intra-abdominal tumours. IVC reconstruction can be performed with biological or synthetic graft material, with most centres preferring synthetic grafts. In spite of the potential advantages of biological grafts in terms of handling characteristics, and safety, very limited data are available about their use in patients requiring an IVC resection. METHODS: Medical records of 32 patients who underwent an IVC resection and reconstruction from 1990 and 2011 with autogenous peritoneo-fascial (N = 22) and bovine pericardial (N = 10) grafts were reviewed. RESULTS: A tangential resection with patch repair was performed in 10 patients, whereas in the remaining 22 it was necessary to resect and replace a segment or all of the retrohepatic IVC. A concomitant liver resection was performed in 14 patients, nephrectomy in 10 and pancreaticoduodenectomy in 2 patients. There were no acute or late complications related to graft thrombosis or infection. Three patients died as a consequence of multi-organ failure. Overall survival at 1 and 5 years was 78% and 48%, respectively. CONCLUSIONS: The preferential use of synthetic grafts in IVC replacement is not evidence based. Selection of an appropriate prosthetic graft for IVC reconstruction should be based on the safety and its handling features. The use of biological grafts for IVC repair is a valid alternative to current synthetic materials and may in fact be superior in terms of biocompatability, ease of handling, reduced rate of infection and improved long-term patency without permanent anticoagulation.

Synthesis of betulinic acid derivatives as entry inhibitors against HIV-1 and bevirimat-resistant HIV-1 variants.

Betulinic acid derivatives modified at the C28 position are HIV-1entry inhibitors such as compound A43D; however, modified at the C3 position instead of C28 give HIV-1 maturation inhibitor such as bevirimat. Bevirimat exhibited promising pharmacokinetic profiles in clinical trials, but its effectiveness was compromised by the high baseline drug resistance of HIV-1 variants with polymorphism in the putative drug binding site. In an effort to determine whether the viruses with bevirimat resistant polymorphism also altered their sensitivities to the betulinic acid derivatives that inhibit HIV-1 entry, a series of new betulinic acid entry inhibitors were synthesized and tested for their activities against HIV-1 NL4-3 and NL4-3 variants resistant to bevirimat. The results show that the bevirimat resistant viruses were approximately 5- to10-fold more sensitive to three new glutamine ester derivatives (13, 15 and 38) and A43D in an HIV-1 multi-cycle replication assay. In contrast, the wild type NL4-3 and the bevirimat resistant variants were equally sensitive to the HIV-1 RT inhibitor AZT. In addition, these three new compounds markedly improved microsomal stability compared to A43D.

Synthesis and proteasome inhibition of lithocholic acid derivatives.

A new class of proteasome inhibitors was synthesized using lithocholic acid as a scaffold. Modification at the C-3 position of lithocholic acid with a series of acid acyl groups yielded compounds with a range of potency on proteasome inhibition. Among them, the phenylene diacetic acid hemiester derivative (13) displayed the most potent proteasome inhibition with IC(50) = 1.9 µM. Enzyme kinetic analysis indicates that these lithocholic acid derivatives are noncompetitive inhibitors of the proteasome.

Anti-AIDS agents 79. Design, synthesis, molecular modeling and structure-activity relationships of novel dicamphanoyl-2',2'-dimethyldihydropyranochromone (DCP) analogs as potent anti-HIV agents.

In a continued study, 23 3'R,4'R-di-O-(-)-camphanoyl-2',2'-dimethyldihydropyrano[2,3-f]chromone (DCP) derivatives (5-27) were synthesized, and screened for anti-HIV activity against both a non-drug-resistant NL4-3 strain and multiple reverse transcriptase (RT) inhibitor-resistant (RTMDR-1) strain, using 2-EDCP (4) and 2-MDCP (35) as controls. New DCP analogs 5, 9, 14, and 22 exhibited potent anti-HIV activity against HIVNL4-3 with EC50 and therapeutic index (TI) values ranging from 0.036 microM to 0.14 microM and from 110 to 420, respectively. Compounds 5 and 9 also exhibited good activity against RTMDR-1 (EC50 0.049 and 0.054 microM; TI 310 and 200, respectively), and were twofold more potent than the leads 4 and 35 (EC50 0.11 and 0.19 microM; TI 60 and 58, respectively). Evaluation of water solubility showed that 5 and 22 were 5-10 times more water soluble than 4. Quantitative structure-activity relationship (QSAR) modeling results were first performed on this compound type, and the models should aid in design of future anti-HIV DCP analogs and potential clinical drug candidates.

Ultra-long-acting tunable biodegradable and removable controlled release implants for drug delivery.

Here we report an ultra-long-acting tunable, biodegradable, and removable polymer-based delivery system that offers sustained drug delivery for up to one year for HIV treatment or prophylaxis. This robust formulation offers the ability to integrate multiple drugs in a single injection, which is particularly important to address the potential for drug resistance with monotherapy. Six antiretroviral drugs were selected based on their solubility in N-methyl-2-pyrrolidone and relevance as a combination therapy for HIV treatment or prevention. All drugs released with concentrations above their protein-adjusted inhibitory concentration and retained their physical and chemical properties within the formulation and upon release. The versatility of this formulation to integrate multiple drugs and provide sustained plasma concentrations from several weeks to up to one year, combined with its ability to be removed to terminate the treatment if necessary, makes it attractive as a drug delivery platform technology for a wide range of applications.

Synthesis and proteasome inhibition of glycyrrhetinic acid derivatives.

This study discovered that glycyrrhetinic acid inhibited the human 20S proteasome at 22.3microM. Esterification of the C-3 hydroxyl group on glycyrrhetinic acid with various carboxylic acid reagents yielded a series of analogs with marked improved potency. Among the derivatives, glycyrrhetinic acid 3-O-isophthalate (17) was the most potent compound with IC(50) of 0.22microM, which was approximately 100-fold more potent than glycyrrhetinic acid.

Systemic multilineage engraftment in mice after in utero transplantation with human hematopoietic stem cells.

IUHCT of human cord blood-derived CD34+ cells into fetal NSG mice results in systemic multilineage engraftment with human cells.Preconditioning with in utero injection of an anti-c-Kit receptor antibody (ACK2) results in an improved rate of engraftment.

Activation and inhibition of the proteasome by betulinic acid and its derivatives.

This study discovered that betulinic acid (BA) is a potent proteasome activator that preferentially activates the chymotrypsin-like activity of the proteasome. Chemical modifications can transform BA into proteasome inhibitors. Chemical modifications at the C-3 position of BA resulted in compounds, such as dimethylsuccinyl BA (DSB), with various inhibitory activities against the human 20S proteasome. Interestingly, the proteasomal activation by BA and the inhibitory activity of DSB could be abrogated by introducing a side chain at the C-28 position. In summary, this study discovered a class of small molecules that can either activate or inhibit human proteasome activity depending on side chain modifications.

Induction of a nonproductive conformational change in gp120 by a small molecule HIV type 1 entry inhibitor.

Conformational changes in HIV-1 envelope glycoproteins, gp120 and gp41, is a dynamic process essential for HIV-1 entry. Here we show that a small molecule HIV-1 entry inhibitor, IC9564, induces a conformational change in gp120. The conformational change in gp120 is evidenced by a significant increase in the binding of a conformational monoclonal antibody 17b. As a result of the conformational effect, IC9564 significantly enhances the neutralizing activity of 17b. Unlike CD4, IC9564 does not trigger conformational changes in gp41. In fact, IC9564 inhibits CD4-induced conformational changes in gp41. Thus, IC9564 exploits the dynamic nature of gp120 by inducing a nonproductive gp120 conformation that is not able to trigger a conformational change in gp41 for membrane fusion.

Predicting HIV Pre-exposure Prophylaxis Efficacy for Women using a Preclinical Pharmacokinetic-Pharmacodynamic In Vivo Model.

The efficacy of HIV pre-exposure prophylaxis (PrEP) relies on adherence and may also depend on the route of HIV acquisition. Clinical studies of systemic tenofovir disoproxil fumarate (TDF) PrEP revealed reduced efficacy in women compared to men with similar degrees of adherence. To select the most effective PrEP strategies, preclinical studies are critically needed to establish correlations between drug concentrations (pharmacokinetics [PK]) and protective efficacy (pharmacodynamics [PD]). We utilized an in vivo preclinical model to perform a PK-PD analysis of systemic TDF PrEP for vaginal HIV acquisition. TDF PrEP prevented vaginal HIV acquisition in a dose-dependent manner. PK-PD modeling of tenofovir (TFV) in plasma, female reproductive tract tissue, cervicovaginal lavage fluid and its intracellular metabolite (TFV diphosphate) revealed that TDF PrEP efficacy was best described by plasma TFV levels. When administered at 50 mg/kg, TDF achieved plasma TFV concentrations (370 ng/ml) that closely mimicked those observed in humans and demonstrated the same risk reduction (70%) previously attained in women with high adherence. This PK-PD model mimics the human condition and can be applied to other PrEP approaches and routes of HIV acquisition, accelerating clinical implementation of the most efficacious PrEP strategies.

In Vivo Suppression of HIV Rebound by Didehydro-Cortistatin A, a "Block-and-Lock" Strategy for HIV-1 Treatment.

HIV-1 Tat activates viral transcription and limited Tat transactivation correlates with latency establishment. We postulated a "block-and-lock" functional cure approach based on properties of the Tat inhibitor didehydro-Cortistatin A (dCA). HIV-1 transcriptional inhibitors could block ongoing viremia during antiretroviral therapy (ART), locking the HIV promoter in persistent latency. We investigated this hypothesis in human CD4+ T cells isolated from aviremic individuals. Combining dCA with ART accelerates HIV-1 suppression and prevents viral rebound after treatment interruption, even during strong cellular activation. We show that dCA mediates epigenetic silencing by increasing nucleosomal occupancy at Nucleosome-1, restricting RNAPII recruitment to the HIV-1 promoter. The efficacy of dCA was studied in the bone marrow-liver-thymus (BLT) mouse model of HIV latency and persistence. Adding dCA to ART-suppressed mice systemically reduces viral mRNA in tissues. Moreover, dCA significantly delays and reduces viral rebound levels upon treatment interruption. Altogether, this work demonstrates the potential of block-and-lock cure strategies.

Efficient Inhibition of HIV Replication in the Gastrointestinal and Female Reproductive Tracts of Humanized BLT Mice by EFdA.

BACKGROUND: The nucleoside reverse transcriptase inhibitor (NRTI) 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) in preclinical development exhibits improved safety and antiviral activity profiles with minimal drug resistance compared to approved NRTIs. However, the systemic antiviral efficacy of EFdA has not been fully evaluated. In this study, we utilized bone marrow/liver/thymus (BLT) humanized mice to investigate the systemic effect of EFdA treatment on HIV replication and CD4+ T cell depletion in the peripheral blood (PB) and tissues. In particular, we performed a comprehensive analysis of the female reproductive tract (FRT) and gastrointestinal (GI) tract, major sites of transmission, viral replication, and CD4+ T cell depletion and where some current antiretroviral drugs have a sub-optimal effect. RESULTS: EFdA treatment resulted in reduction of HIV-RNA in PB to undetectable levels in the majority of treated mice by 3 weeks post-treatment. HIV-RNA levels in cervicovaginal lavage of EFdA-treated BLT mice also declined to undetectable levels demonstrating strong penetration of EFdA into the FRT. Our results also demonstrate a strong systemic suppression of HIV replication in all tissues analyzed. In particular, we observed more than a 2-log difference in HIV-RNA levels in the GI tract and FRT of EFdA-treated BLT mice compared to untreated HIV-infected control mice. In addition, HIV-RNA was also significantly lower in the lymph nodes, liver, lung, spleen of EFdA-treated BLT mice compared to untreated HIV-infected control mice. Furthermore, EFdA treatment prevented the depletion of CD4+ T cells in the PB, mucosal tissues and lymphoid tissues. CONCLUSION: Our findings indicate that EFdA is highly effective in controlling viral replication and preserving CD4+ T cells in particular with high efficiency in the GI and FRT tract. Thus, EFdA represents a strong potential candidate for further development as a part of antiretroviral therapy regimens.

N-acetylcysteine reduces contrast-associated nephropathy but not clinical events during long-term follow-up.

BACKGROUND: Contrast-associated nephropathy (CAN) is associated with increased morbidity and mortality following percutaneous coronary intervention (PCI). N-acetylcysteine (NAC) has been shown to reduce the risk of nephropathy; however, the impact of NAC on long-term clinical outcomes has not been assessed. METHODS: This randomized, double-blind, placebo-controlled trial enrolled 180 patients with moderate renal dysfunction undergoing PCI or coronary angiography with a high likelihood of ad hoc PCI; 171 patients completed the clinical follow-up. Patients received oral NAC (2000 mg/dose, n = 95) or placebo (n = 85) twice a day for 3 doses if randomized the night prior to the procedure, and 2 doses if randomized the day of the procedure. The primary end point was the incidence of a > or =25% increase in serum creatinine level 48 to 72 hours after PCI. Secondary end points were the inhospital incidence of death, nonfatal myocardial infarction, or urgent dialysis, and the 9-month incidence of death, nonfatal myocardial infarction, need for dialysis, or repeat hospitalization for cardiac reasons. RESULTS: CAN occurred in 9.6% of patients assigned to NAC and 22.2% of patients assigned to placebo (P =.04); 1 patient receiving NAC required urgent dialysis. The inhospital composite end point occurred in 7 (7.4%) NAC-treated and 3 (3.5%) placebo-treated patients, P = NS. At 9 months, the composite end point occurred in 23 (24.2%) NAC-treated patients and 18 (21.2%) placebo-treated, P = NS. CONCLUSION: Although high-dose NAC prevented periprocedural CAN, this benefit did not translate into a decrease in adverse outcomes over 9 months. Further studies to determine the clinical utility of this drug are required.

Acute myocardial infarction and cardiac arrest in a patient receiving paclitaxel.

A 68-year-old man suffered an acute myocardial infarction while receiving an intravenous infusion of paclitaxel. Coronary angiograms revealed only mild plaquing of the infarct-related artery without significant angiographic obstructions. The authors postulate that paclitaxel may have caused coronary vasospasm and myocardial infarction.

New betulinic acid derivatives for bevirimat-resistant human immunodeficiency virus type-1.

Bevirimat (1, BVM) is an anti-HIV agent that blocks HIV-1 replication by interfering with HIV-1 Gag-SP1 processing at a late stage of viral maturation. However, clinical trials of 1 have revealed a high baseline drug resistance that is attributed to naturally occurring polymorphisms in HIV-1 Gag. To overcome the drug resistance, 28 new derivatives of 1 were synthesized and tested against compound 1-resistant (BVM-R) HIV-1 variants. Among them, compound 6 exhibited much improved activity against several HIV-1 strains carrying BVM-R polymorphisms. Compound 6 was at least 20-fold more potent than 1 against the replication of NL4-3/V370A, which carries the most prevalent clinical BVM-R polymorphism in HIV-1 Gag-SP1. Thus, compound 6 merits further development as a potential anti-AIDS clinical trial candidate.

In-depth analysis of the interaction of HIV-1 with cellular microRNA biogenesis and effector mechanisms.

UNLABELLED: The question of how HIV-1 interfaces with cellular microRNA (miRNA) biogenesis and effector mechanisms has been highly controversial. Here, we first used deep sequencing of small RNAs present in two different infected cell lines (TZM-bl and C8166) and two types of primary human cells (CD4(+) peripheral blood mononuclear cells [PBMCs] and macrophages) to unequivocally demonstrate that HIV-1 does not encode any viral miRNAs. Perhaps surprisingly, we also observed that infection of T cells by HIV-1 has only a modest effect on the expression of cellular miRNAs at early times after infection. Comprehensive analysis of miRNA binding to the HIV-1 genome using the photoactivatable ribonucleoside-induced cross-linking and immunoprecipitation (PAR-CLIP) technique revealed several binding sites for cellular miRNAs, a subset of which were shown to be capable of mediating miRNA-mediated repression of gene expression. However, the main finding from this analysis is that HIV-1 transcripts are largely refractory to miRNA binding, most probably due to extensive viral RNA secondary structure. Together, these data demonstrate that HIV-1 neither encodes viral miRNAs nor strongly influences cellular miRNA expression, at least early after infection, and imply that HIV-1 transcripts have evolved to avoid inhibition by preexisting cellular miRNAs by adopting extensive RNA secondary structures that occlude most potential miRNA binding sites. IMPORTANCE: MicroRNAs (miRNAs) are a ubiquitous class of small regulatory RNAs that serve as posttranscriptional regulators of gene expression. Previous work has suggested that HIV-1 might subvert the function of the cellular miRNA machinery by expressing viral miRNAs or by dramatically altering the level of cellular miRNA expression. Using very sensitive approaches, we now demonstrate that neither of these ideas is in fact correct. Moreover, HIV-1 transcripts appear to largely avoid regulation by cellular miRNAs by adopting an extensive RNA secondary structure that occludes the ability of cellular miRNAs to interact with viral mRNAs. Together, these data suggest that HIV-1, rather than seeking to control miRNA function in infected cells, has instead evolved a mechanism to become largely invisible to cellular miRNA effector mechanisms.