K+ Efflux-Independent NLRP3 Inflammasome Activation by Small Molecules Targeting Mitochondria.

Imiquimod is a small-molecule ligand of Toll-like receptor-7 (TLR7) that is licensed for the treatment of viral infections and cancers of the skin. Imiquimod has TLR7-independent activities that are mechanistically unexplained, including NLRP3 inflammasome activation in myeloid cells and apoptosis induction in cancer cells. We investigated the mechanism of inflammasome activation by imiquimod and the related molecule CL097 and determined that K+ efflux was dispensable for NLRP3 activation by these compounds. Imiquimod and CL097 inhibited the quinone oxidoreductases NQO2 and mitochondrial Complex I. This induced a burst of reactive oxygen species (ROS) and thiol oxidation, and led to NLRP3 activation via NEK7, a recently identified component of this inflammasome. Metabolic consequences of Complex I inhibition and endolysosomal effects of imiquimod might also contribute to NLRP3 activation. Our results reveal a K+ efflux-independent mechanism for NLRP3 activation and identify targets of imiquimod that might be clinically relevant.

Host AKT-mediated phosphorylation of HIV-1 accessory protein Vif potentiates infectivity via enhanced degradation of the restriction factor APOBEC3G.

HIV-1 encodes accessory proteins that neutralize antiviral restriction factors to ensure its successful replication. One accessory protein, the HIV-1 viral infectivity factor (Vif), is known to promote ubiquitination and proteasomal degradation of the antiviral restriction factor apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G), a cytosine deaminase that leads to hypermutations in the viral DNA and subsequent aberrant viral replication. We have previously demonstrated that the HIV-1 viral transcription mediator Tat activates the host progrowth PI-3-AKT pathway, which in turn promotes HIV-1 replication. Because the HIV-1 Vif protein contains the putative AKT phosphorylation motif RMRINT, here we investigated whether AKT directly phosphorylates HIV-1 Vif to regulate its function. Coimmunoprecipitation experiments showed that AKT and Vif interact with each other, supporting this hypothesis. Using in vitro kinase assays, we further showed that AKT phosphorylates Vif at threonine 20, which promotes its stability, as Vif becomes destabilized after this residue is mutated to alanine. Moreover, expression of dominant-negative kinase-deficient AKT as well as treatment with a chemical inhibitor of AKT increased K48-ubiquitination and proteasomal degradation of HIV-1 Vif. In contrast, constitutively active AKT (Myr-AKT) reduced K48-ubiquitination of Vif to promote its stability. Finally, inhibition of AKT function restored APOBEC3G levels, which subsequently reduced HIV-1 infectivity. Thus, our results establish a novel mechanism of HIV-1 Vif stabilization through AKT-mediated phosphorylation at threonine 20, which reduces APOBEC3G levels and potentiates HIV-1 infectivity.

MALT1 protease function in regulatory T cells induces MYC activity to promote mitochondrial function and cellular expansion.

Regulatory T cells (Tregs) are essential for the inhibition of immunity and the maintenance of tissue homeostasis. Signals from the T-cell antigen receptor (TCR) are critical for early Treg development, their expansion, and inhibitory activity. Although TCR-engaged activation of the paracaspase MALT1 is important for these Treg activities, the MALT1 effector pathways in Tregs remain ill-defined. Here, we demonstrate that MALT1 protease activity controls the TCR-induced upregulation of the transcription factor MYC and the subsequent expression of MYC target genes in Tregs. These mechanisms are important for Treg-intrinsic mitochondrial function, optimal respiratory capacity, and homeostatic Treg proliferation. Consistently, conditional deletion of Myc in Tregs results similar to MALT1 inactivation in a lethal autoimmune inflammatory syndrome. Together, these results identify a MALT1 protease-mediated link between TCR signaling in Tregs and MYC control that coordinates metabolism and Treg expansion for the maintenance of immune homeostasis.

Osteoprogenitor SFRP1 prevents exhaustion of hematopoietic stem cells via PP2A-PR72/130-mediated regulation of p300.

Remodeling of the bone marrow microenvironment in chronic inflammation and in aging reduces hematopoietic stem cell (HSC) function. To assess the mechanisms of this functional decline of HSC and find strategies to counteract it, we established a model in which the Sfrp1 gene was deleted in Osterix+ osteolineage cells (OS1Δ/Δ mice). HSC from these mice showed severely diminished repopulating activity with associated DNA damage, enriched expression of the reactive oxygen species pathway and reduced single-cell proliferation. Interestingly, not only was the protein level of Catenin beta-1 (bcatenin) elevated, but so was its association with the phosphorylated co-activator p300 in the nucleus. Since these two proteins play a key role in promotion of differentiation and senescence, we inhibited in vivo phosphorylation of p300 through PP2A-PR72/130 by administration of IQ-1 in OS1Δ/Δ mice. This treatment not only reduced the b-catenin/phosphop300 association, but also decreased nuclear p300. More importantly, in vivo IQ-1 treatment fully restored HSC repopulating activity of the OS1Δ/Δ mice. Our findings show that the osteoprogenitor Sfrp1 is essential for maintaining HSC function. Furthermore, pharmacological downregulation of the nuclear b-catenin/phospho-p300 association is a new strategy to restore poor HSC function.

Identification of invariant chain CD74 as a functional receptor of tissue inhibitor of metalloproteinases-1 (TIMP-1).

Multifunctionality of tissue inhibitor of metalloproteinases-1 (TIMP-1) comprising antiproteolytic as well as cytokinic activity has been attributed to its N-terminal and C-terminal domains, respectively. The molecular basis of the emerging proinflammatory cytokinic activity of TIMP-1 is still not completely understood. The cytokine receptor invariant chain (CD74) is involved in many inflammation-associated diseases and is highly expressed by immune cells. CD74 triggers zeta chain-associated protein kinase-70 (ZAP-70) signaling-associated activation upon interaction with its only known ligand, the macrophage migration inhibitory factor. Here, we demonstrate TIMP-1-CD74 interaction by coimmunoprecipitation and confocal microscopy in cells engineered to overexpress CD74. In silico docking in HADDOCK predicted regions of the N-terminal domain of TIMP-1 (N-TIMP-1) to interact with CD74. This was experimentally confirmed by confocal microscopy demonstrating that recombinant N-TIMP-1 lacking the entire C-terminal domain was sufficient to bind CD74. Interaction of TIMP-1 with endogenously expressed CD74 was demonstrated in the Namalwa B lymphoma cell line by dot blot binding assays as well as confocal microscopy. Functionally, we demonstrated that TIMP-1-CD74 interaction triggered intracellular ZAP-70 activation. N-TIMP-1 was sufficient to induce ZAP-70 activation and interference with the cytokine-binding site of CD74 using a synthetic peptide-abrogated TIMP-1-mediated ZAP-70 activation. Altogether, we here identified CD74 as a receptor and mediator of cytokinic TIMP-1 activity and revealed TIMP-1 as moonlighting protein harboring both cytokinic and antiproteolytic activity within its N-terminal domain. Recognition of this functional TIMP-1-CD74 interaction may shed new light on clinical attempts to therapeutically target ligand-induced CD74 activity in cancer and other inflammatory diseases.

Role of NRF2 in immune modulator expression in developing lung.

After birth, the alveolar epithelium is exposed to environmental pathogens and high O2 tensions. The alveolar type II cells may protect this epithelium through surfactant production. Surfactant protein, SP-A, an immune modulator, is developmentally upregulated in fetal lung with surfactant phospholipid synthesis. Herein, we observed that the redox-regulated transcription factor, NRF2, and co-regulated C/EBPß and PPARγ, were markedly induced during cAMP-mediated differentiation of cultured human fetal lung (HFL) epithelial cells. This occurred with enhanced expression of immune modulators, SP-A, TDO2, AhR, and NQO1. Like SP-A, cAMP induction of NRF2 was prevented when cells were exposed to hypoxia. NRF2 knockdown inhibited induction of C/EBPß, PPARγ, and immune modulators. Binding of endogenous NRF2 to promoters of SP-A and other immune modulator genes increased during HFL cell differentiation. In mouse fetal lung (MFL), a developmental increase in Nrf2, SP-A, Tdo2, Ahr, and Nqo1 and decrease in Keap1 occurred from 14.5 to 18.5 dpc. Developmental induction of Nrf2 in MFL was associated with increased nuclear localization of NF-κB p65, a decline in p38 MAPK phosphorylation, increase in the MAPK phosphatase, DUSP1, induction of the histone acetylase, CBP, and decline in the histone deacetylase, HDAC4. Thus, together with surfactant production, type II cells protect the alveolar epithelium through increased expression of NRF2 and immune modulators to prevent inflammation and oxidative stress. Our findings further suggest that lung cancer cells have usurped this developmental pathway to promote immune tolerance and enhance survival.

Dengue Virus Degrades USP33-ATF3 Axis via Extracellular Vesicles to Activate Human Microglial Cells.

Dengue virus (DENV) infection disrupts host innate immune signaling at various checkpoints. Cellular levels and stability of intermediate signaling molecules are a crucial hijacking point for a successful viral pathogenesis. Stability and turnover of all the cellular proteins including intermediate signaling molecules are principally regulated by proteasomal degradation pathway. In this study, we show that how DENV infection and particularly DENV-NS1 can modulate the host extracellular vesicle (EV) cargo to manipulate the deubiquitination machinery of the human microglial cell (CHME3). We have performed EV harvesting, size analysis by nanoparticle tracking analysis, identification of cargo microRNA via quantitative PCR, microRNA target validation by overexpression, and knockdown via mimics and anti-miRs, immunoblotting, dual luciferase reporter assay, in vivo ubiquitination assay, chase assay, and promoter activity assay to reach the conclusion. In this study, we show that DENV-infected monocytes and DENV-NS1-transfected cells release high amounts of EVs loaded with miR-148a. These EVs get internalized by human microglial cells, and miR-148a suppresses the ubiquitin-specific peptidase 33 (USP33) protein expression levels via binding to its 3' untranslated region. Reduced USP33 in turn decreases the stability of cellular ATF3 protein via deubiquitylation. ATF3 acts as a suppressor of major proinflammatory gene expression pathways of TNF-α, NF-κB, and IFN-ß. Our mechanistic model explains how DENV uses the EV pathway to transfer miR-148a for modulating USP33 and downstream ATF3 levels in human microglial cells and contributes in neuroinflammation within the CNS.

Decreased 11ß-hydroxysteroid dehydrogenase 1 in lungs of steroid receptor coactivator (Src)-1/-2 double-deficient fetal mice is caused by impaired glucocorticoid and cytokine signaling.

Our previous research revealed that steroid receptor coactivators (Src)-1 and -2 serve a critical cooperative role in production of parturition signals, surfactant protein A and platelet-activating factor, by the developing mouse fetal lung (MFL). To identify the global landscape of genes in MFL affected by Src-1/-2 double-deficiency, we conducted RNA-seq analysis of lungs from 18.5 days post-coitum (dpc) Src-1-/- /-2-/- (dKO) vs. WT fetuses. One of the genes most highly downregulated (~4.8 fold) in Src-1/-2 dKO fetal lungs encodes 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1), which catalyzes conversion of inactive 11-dehydrocorticosterone to the glucocorticoid receptor (GR) ligand, corticosterone. Glucocorticoids were reported to upregulate 11ß-HSD1 expression in various cell types via induction of C/EBP transcription factors. We observed that C/ebpα and C/ebpß mRNA and protein were markedly reduced in Src-1/-2 double-deficient (Src-1/-2d/d ) fetal lungs, compared to WT. Moreover, glucocorticoid induction of 11ß-hsd1, C/ebpα and C/ebpß in cultured MFL epithelial cells was prevented by the SRC family inhibitor, SI-2. Cytokines also contribute to the induction of 11ß-HSD1. Expression of IL-1ß and TNFα, which dramatically increased toward term in lungs of WT fetuses, was markedly reduced in Src-1/-2d/d fetal lungs. Our collective findings suggest that impaired lung development and surfactant synthesis in Src-1/-2d/d fetuses are likely caused, in part, by decreased GR and cytokine induction of C/EBP and NF-κB transcription factors. This results in reduced 11ß-HSD1 expression and glucocorticoid signaling within the fetal lung, causing a break in the glucocorticoid-induced positive feedforward loop.

Impact of metformin therapy on health-related quality of life outcomes in tuberculosis patients with diabetes mellitus in India: A prospective study.

OBJECTIVE: To assess the impact of metformin use on health-related quality of life (HRQoL) in tuberculosis (TB) patients who are presented with type 2 diabetes mellitus (T2DM). METHODOLOGY: In this community-based prospective study, TB patients attending Hakeem Abdul Hameed Centenary Hospital, New Delhi (India) and had comorbidity of T2DM between April 2018 and July 2019 were enrolled. Patients were divided into metformin users and metformin non-users on the basis of the presence of metformin in their routine as antidiabetic drug(s). HRQoL was determined using a validated TB-specific tool (Dhingra and Rajpal-12 scale ie, DR-12) consists of symptom and socio-psychological and exercise adaptation domains. The HRQoL scores were compared at pretreatment (1st visit), end of intensive phase (2nd visit) and end of treatment (3rd visit) between the two groups. RESULTS: A total of 120 patients were enrolled, of which 24 were excluded as they did not respond at follow-up visits. Among the metformin users (n = 48) the mean age of patients was 47.56 years and 62.50% was males. Among the metformin non-users (n = 48), the mean age of patients was 49.02 years and 54.10% was males. The baseline characteristics were similar in both groups except for the substance used history (P = .025), literacy level (P = .048) and BMI (P = .028). Metformin users demonstrated significant improvement in symptom scores (2nd visit: P < .001; 3rd visit: P = .001) and socio-psychological and exercise adaptation scores (2nd visit: P < .0001; 3rd visit: P < .0001) as compared with metformin non-users at 2nd visit and 3rd visit. Overall, scores were also found to be significantly improved in metformin users (2nd visit: P < .001; 3rd visit: P = .001). CONCLUSION: Metformin therapy exerted favourable effects on HRQoL in patients with TB and T2DM and can be recommended as an adjuvant antitubercular drug in TB patients with co-morbidity of T2DM, unless contraindicated.

HIV-1 Nef promotes ubiquitination and proteasomal degradation of p53 tumor suppressor protein by using E6AP.

Human Immunodeficiency Virus-1 (HIV-1) Nef promotes p53 protein degradation to protect HIV-1 infected cells from p53 induced apoptosis. We found that Nef mediated p53 degradation is accomplished through ubiquitin proteasome pathway in an Mdm2-independent manner. By GST pulldown and immunoprecipitation assays, we have shown that Nef interacts with E3 ubiquitin ligase E6AP in both Nef transfected HEK-293T cells and HIV-1 infected MOLT3 cells. The p53 ubiquitination and degradation was found to be enhanced by Nef with E6AP but not by Nef with E6AP-C843A, a dominant negative E6AP mutant. We show that Nef binds with E6AP and promotes E6AP dependent p53 ubiquitination. Further, Nef inhibits apoptosis of p53 null H1299 cells after exogenous expression of p53 protein. The p53 dependent apoptosis of H1299 cells was further reduced after the expression of Nef with E6AP. However, Nef mediated reduction in p53 induced apoptosis of H1299 cells was restored when Nef was co-expressed with E6AP-C843A. Thus, Nef and E6AP co-operate to promote p53 ubiquitination and degradation in order to suppress p53 dependent apoptosis. CHME3 cells, which are a natural host of HIV-1, also show p53 ubiquitination and degradation by Nef and E6AP. These results establish that Nef induces p53 degradation via cellular E3 ligase E6AP to inhibit apoptosis during HIV-1 infection.

Inhibition of PLK1 by capped-dose volasertib exerts substantial efficacy in MDS and sAML while sparing healthy haematopoiesis.

INTRODUCTION: Targeting the cell cycle machinery represents a rational therapeutic approach in myelodysplastic syndromes (MDS) and secondary acute myeloid leukemia (sAML). Despite substantial response rates, clinical use of the PLK inhibitor volasertib has been hampered by elevated side effects such as neutropenia and infections. OBJECTIVES: The primary objective was to analyse whether a reduced dose of volasertib was able to limit toxic effects on the healthy haematopoiesis while retaining its therapeutic effect. METHODS: Bone marrow mononuclear cells (BMMNCs) of patients with MDS/sAML (n = 73) and healthy controls (n = 28) were treated with volasertib (1 µM to 1 nM) or vehicle control. Short-term viability analysis was performed by flow cytometry after 72 hours. For long-term viability analysis, colony-forming capacity was assessed after 14 days. Protein expression of RIPK3 and MCL-1 was quantified via flow cytometry. RESULTS: Reduced dose levels of volasertib retained high cell death-inducing efficacy in primary human stem and progenitor cells of MDS/sAML patients without affecting healthy haematopoiesis in vitro. Interestingly, volasertib reduced colony-forming capacity and cell survival independent of clinical stage or mutational status. CONCLUSIONS: Volasertib offers a promising therapeutic approach in patients with adverse prognostic profile. RIPK3 and MCL-1 might be potential biomarkers for sensitivity to volasertib treatment.

RAD51 interconnects between DNA replication, DNA repair and immunity.

RAD51, a multifunctional protein, plays a central role in DNA replication and homologous recombination repair, and is known to be involved in cancer development. We identified a novel role for RAD51 in innate immune response signaling. Defects in RAD51 lead to the accumulation of self-DNA in the cytoplasm, triggering a STING-mediated innate immune response after replication stress and DNA damage. In the absence of RAD51, the unprotected newly replicated genome is degraded by the exonuclease activity of MRE11, and the fragmented nascent DNA accumulates in the cytosol, initiating an innate immune response. Our data suggest that in addition to playing roles in homologous recombination-mediated DNA double-strand break repair and replication fork processing, RAD51 is also implicated in the suppression of innate immunity. Thus, our study reveals a previously uncharacterized role of RAD51 in initiating immune signaling, placing it at the hub of new interconnections between DNA replication, DNA repair, and immunity.

Inhibition of protein synthesis leading to unfolded protein response is the major event in abrin-mediated apoptosis.

Abrin obtained from the plant Abrus precatorius inhibits protein synthesis and also triggers apoptosis in cells. Previous studies from our laboratory suggested a link between these two events. Using an active site mutant of abrin A-chain which exhibits 225-fold lower protein synthesis inhibitory activity than the wild-type abrin A-chain, we demonstrate in this study that inhibition of protein synthesis induced by abrin is the major factor triggering unfolded protein response leading to apoptosis. Since abrin A-chain requires the B-chain for internalization into cells, the wild-type and mutant recombinant abrin A-chains were conjugated to native ricin B-chain to generate hybrid toxins, and the toxic effects of the two conjugates were compared. The rate of inhibition of protein synthesis mediated by the mutant ricin B-rABRA (R167L) conjugate was slower than that of the wild-type ricin B-rABRA conjugate as expected. The mutant conjugate activated p38MAPK and caspase-3 similar to its wild-type counterpart although at later time points. Overall, these results confirm that inhibition of protein synthesis is the major event contributing to abrin-mediated apoptosis.

Yeast dynamin Vps1 and amphiphysin Rvs167 function together during endocytosis.

Dynamins are a conserved family of proteins involved in many membrane fusion and fission events. Previously, the dynamin-related protein Vps1 was shown to localize to endocytic sites, and yeast carrying deletions for genes encoding both the BAR domain protein Rvs167 and Vps1 had a more severe endocytic scission defect than either deletion alone. Vps1 and Rvs167 localize to endocytic sites at the onset of invagination and disassemble concomitant with inward vesicle movement. Rvs167-GFP localization is reduced in cells lacking vps1 suggesting that Vps1 influences Rvs167 association with the endocytic complex. Unlike classical dynamins, Vps1 does not have a proline-arginine domain that could interact with SH3 domain-containing proteins. Thus, while Rvs167 has an SH3 domain, it is not clear how an interaction would be mediated. Here, we demonstrate an interaction between Rvs167 SH3 domain and the single type I SH3-binding motif in Vps1. Mutant Vps1 that cannot bind Rvs167 rescues all membrane fusion/fission functions associated with Vps1 except for endocytic function, demonstrating the specificity and mechanistic importance of the interaction. In vitro, an Rvs161/Rvs167 heterodimer can disassemble Vps1 oligomers. Overall, the data support the idea that Vps1 and the amphiphysins function together to mediate scission during endocytosis in yeast.

HIV-1 Tat C modulates expression of miRNA-101 to suppress VE-cadherin in human brain microvascular endothelial cells.

HIV-1 infection leads to the development of HIV-associated neurological disorders. The HIV-1 Tat protein has been reported to exert an adverse effect on blood-brain barrier integrity and permeability. Perturbation in permeability is mainly caused by disruptions in adherens junctions and tight junction proteins. We have identified HIV-1 Tat C-induced disruption of VE-cadherin mediated by miRNA-101 in human brain microvascular endothelial cells (BMVECs). HIV-1 Tat C increased the expression of miR-101, which led to downregulation of VE-cadherin. Overexpression of miR-101 resulted into the suppression of VE-cadherin. Inhibition of miR-101 by the miRNA inhibitor enhanced the expression of VE-cadherin. We have demonstrated that VE-cadherin is a direct target of miR-101 using a luciferase reporter assay, which showed that mutated VE-cadherin 3'UTR and miR-101 cotransfection did not change luciferase activity. By overexpression and knockdown of miR-101, we have demonstrated that the expression level of claudin-5 is governed by the expression of VE-cadherin. These findings demonstrate a novel mechanism for the regulation of barrier permeability by miR-101 via posttranscriptional regulation of VE-cadherin in human BMVECs exposed to the HIV-1 Tat C protein.

Regulatory role of TRIM21 in the type-I interferon pathway in Japanese encephalitis virus-infected human microglial cells.

BACKGROUND: Japanese encephalitis virus (JEV) infection leads to Japanese encephalitis (JE) in humans. JEV is transmitted through mosquitoes and maintained in a zoonotic cycle. This cycle involves pigs as the major reservoir, water birds as carriers and mosquitoes as vectors. JEV invasion into the central nervous system (CNS) may occur via antipodal transport of virions or through the vascular endothelial cells. Microglial cells get activated in response to pathogenic insults. JEV infection induces the innate immune response and triggers the production of type I interferons. The signaling pathway of type I interferon production is regulated by a number of molecules. TRIM proteins are known to regulate the expression of interferons; however, the involvement of TRIM genes and their underlying mechanism during JEV infection are not known. METHODS: Human microglial cells (CHME3) were infected with JEV to understand the role of TRIM21 in JEV infection and its effect on type I interferon (IFN-ß) production. Cells were infected in presence and absence of exogenous TRIM21 as well as after knocking down the TRIM21 mRNA. Levels of activated IRF3 expression were measured through Western blot analyses of anti-p-IRF3 antibody, and IFN-ß production was measured by using IFN-ß real-time PCR and luciferase activity analyses. RESULTS: JEV infection increased expression of TRIM21 in CHME3 cells. JEV induced an innate immune response by increasing production of IFN-ß via IRF3 activation and phosphorylation. Overexpression of TRIM21 resulted in downregulation of p-IRF3 and IFN-ß, while silencing led to increased production of p-IRF3 and IFN-ß in JEV-infected CHME3 cells. CONCLUSION: This report demonstrates TRIM21 as a negative regulator of interferon-ß (IFN-ß) production mediated by IRF-3 during JEV infection in human microglial cells.

HIV-1 Tat C phosphorylates VE-cadherin complex and increases human brain microvascular endothelial cell permeability.

BACKGROUND: Human brain microvascular endothelial cells (hBMVECs) are integral part of the blood brain barrier. Post-translational modifications of adherens junction proteins regulate the permeability of human brain microvascular endothelial cells. Pro-inflammatory signals can induce tyrosine phosphorylation of adherens junction proteins. The primary objective of this work is to provide a molecular model; how the HIV-1 Tat protein can compromise the BBB integrity and eventually lead to neurological consequences. We exposed hBMVECs to recombinant HIV-1 clade C Tat protein to study the effect of HIV-1 Tat C on permeability of hBMVECs. Trans-endothelial electrical resistance and fluorescent dye migration assay have been used to check the permeability of hBMVECs. DCFDA staining has been used for intracellular reactive oxygen species (ROS) detection. Western blotting has been used to study the expression levels and co-immunoprecipitation has been used to study the interactions among adherens junction proteins. RESULTS: HIV-1 Tat C protein induced NOX2 and NOX4 expression level and increased intracellular ROS level. Redox-sensitive kinase; PYK2 activation led to increased tyrosine phosphorylation of VE-cadherin and ß-catenin, leading to disruption of junctional assembly. The dissociation of tyrosine phosphatases VE-PTP and SHP2 from cadherin complex resulted into increased tyrosine phosphorylation of VE-cadherin and ß-catenin in HIV-1 Tat C treated hBMVECs. CONCLUSION: Unrestricted phosphorylation of junctional proteins in hBMVECs, in response to HIV-1 Tat C protein; leads to the disruption of junctional complexes and increased endothelial permeability.

HIV risk behaviors of male injecting drug users and associated non-condom use with regular female sexual partners in north-east India.

BACKGROUND: In the context of increasing HIV prevalence among women in regular sexual partnerships, this paper examines the relationship between male injecting drug users' (IDUs) risky injecting practices and sexual risk behaviors with casual partners and inconsistent condom use with regular partners. METHODS: Data were drawn from the behavioral tracking survey, conducted in 2009 with 1,712 male IDUs in two districts each of Manipur and Nagaland states, in north-east India. IDUs' risky behaviors were determined using two measures: ever shared needles/syringes and engaged in unprotected sex with casual paid/unpaid female partners in the past 12 months. Inconsistent condom use with regular sexual partners (wife/girlfriend) in the past 12 months was assessed in terms of non-condom use in any sexual encounter. RESULTS: More than one-quarter of IDUs had shared needles/syringes, and 40% had a casual sexual partner. Among those who had casual sexual partners, 65% reported inconsistent condom use with such partners. IDUs who shared needles/syringes were more likely to engage in unprotected sex with their regular partners (95% vs 87%; adjusted OR = 2.31, 95% CI = 1.30-4.09). Similarly, IDUs who reported inconsistent condom use with casual partners were more likely to report unprotected sex with their regular partners (97% vs 66%; adjusted OR = 18.14, 95% CI = 6.82-48.21). CONCLUSION: IDUs who engage in risky injecting and/or sexual behaviors with casual partners also report non-condom use with their regular sex partners, suggesting the high likelihood of HIV transmission from IDUs to their regular sexual partners. Risk reduction programs for IDUs need to include communication about condom use in all relationships in an effort to achieve the goal of zero new infections.

Scale-up of a comprehensive harm reduction programme for people injecting opioids: lessons from north-eastern India.

PROBLEM: Harm reduction packages for people who inject illicit drugs, including those infected with human immunodeficiency virus (HIV), are cost-effective but have not been scaled up globally. In the north-eastern Indian states of Manipur and Nagaland, the epidemic of HIV infection is driven by the injection of illicit drugs, especially opioids. These states needed to scale up harm reduction programmes but faced difficulty doing so. APPROACH: In 2004, the Bill & Melinda Gates Foundation funded Project ORCHID to scale up a harm reduction programme in Manipur and Nagaland. LOCAL SETTING: In 2003, an estimated 10 000 and 16 000 people were injecting drugs in Manipur and Nagaland, respectively. The prevalence of HIV infection among people injecting drugs was 24.5% in Manipur and 8.4% in Nagaland. RELEVANT CHANGES: By 2012, the harm reduction programme had been scaled up to an average of 9011 monthly contacts outside clinics (80% of target); an average of 1709 monthly clinic visits (15% of target, well above the 5% monthly goal) and an average monthly distribution of needles and syringes of 16 each per programme participant. Opioid agonist maintenance treatment coverage was 13.7% and retention 6 months after enrolment was 63%. Antiretroviral treatment coverage for HIV-positive participants was 81%. LESSONS LEARNT: A harm reduction model consisting of community-owned, locally relevant innovations and business approaches can result in good harm reduction programme scale-up and influence harm reduction policy. Project ORCHID has influenced national harm reduction policy in India and contributed to the development of harm reduction guidelines.

Central composite design-based optimization, fabrication, and pharmacodynamic assessment of sulfasalazine-loaded lipoidal nanoparticle-based hydrogel for the management of rheumatoid arthritis.

Rheumatoid arthritis is a progressive, chronic, immunological, and inflammatory disorder that is distinguished by joint inflammation, joint tenderness, and synovial joint destruction. The study aimed to fabricate sulfasalazine-loaded solid lipid nanoparticle (SLN)-based gels for rheumatoid arthritis management. The SLNs were fabricated with the melt emulsification technique by employing central composite design (CCD) for SLNs optimization. The optimized formulation of SLNs (FF-1) showed particle size and drug entrapment efficiency of 117.25 nm ± 1.67 and 94.05% ± 1.05, respectively. To scrutinize the outcome of the independent variable on responses; model graphs and the polynomial equation obtained from the Design-Expert were used. The surface morphology studies of SLNs revealed a smooth surface with a slightly asymmetric shape. In vitro drug release of the optimized formulation (FF1) had shown a maximum release of up to ~ 91.89% ± 2.12 over 24 h. The optimized FF1 formulation was subsequently gelled using 1% w/v Carbopol 934 and subjected to ex vivo permeation that displayed 8.01 mg/cm2 ± 0.24 and 7.49 mg/cm2 ± 0.86 amount of drug permeated up to 24 h and 10 h from SLNs gel and plain gel, respectively. In vivo studies manifested a considerable reduction in the paw thickness (*p < 0.0001) and an arthritic score (*p < 0.0001) of the sulfasalazine SLN gel as compared to plain gel. Further, pro-inflammatory cytokines, viz. TNF-α, IL-1, and IL-6 levels, were significantly inhibited (p < 0.0001) by sulfasalazine SLN-based gel that exhibited substantial anti-inflammatory effects. In conclusion, sulfasalazine-loaded SLN-based gel showed sustained release of drug for up to 24 h and can be considered suitable as a topical application for rheumatoid arthritis management.