Vaginal microbicides represent a promising approach for preventing heterosexual HIV transmission. However, preclinical evaluation should be conducted to ensure that microbicides will be safe for human cells and healthy microflora of the female reproductive tract. One microbicide candidate, RC-101, has been effective and well-tolerated in preliminary cell culture and macaque models. However, the effect of RC-101 on primary vaginal tissues and resident vaginal microflora requires further evaluation.
Method of Study
We treated primary vaginal tissues and vaginal bacteria, both pathogenic and commensal, with RC-101 to investigate effects of this microbicide.
RC-101 was well-tolerated by host tissues, and also by commensal vaginal bacteria. Simultaneously, pathogenic vaginal bacteria, which are known to increase susceptibility to HIV acquisition, were inhibited by RC-101.
By establishing vaginal microflora, the specific antibacterial activity of RC-101 may provide a dual mechanism of HIV protection. These findings support advancement of RC-101 to clinical trials.
Bacterial Vaginosis; Lactobacilli; RC-101; Theta-Defensin; Vaginal Microbicide
Host cell microRNAs (miRNAs) have been shown to regulate the expression of both cellular and viral RNAs, in particular impacting both Hepatitis C Virus (HCV) and Human Immunodeficiency Virus (HIV). To investigate the role of miRNAs in regulating replication of the simian immunodeficiency virus (SIV) in macrophage lineage cells, we used primary macrophages to study targeting of SIV RNA by miRNAs. We examined whether specific host miRNAs directly target SIV RNA early in infection and might be induced via type I interferon pathways.
miRNA target prediction programs identified miRNA binding sites within SIV RNA. Predicted binding sites for miRs-29a, -29b, -9 and -146a were identified in the SIV Nef/U3 and R regions, and all four miRNAs decreased virus production and viral RNA expression in primary macrophages. To determine whether levels of these miRNAs were affected by SIV infection, IFNβ or TNFα treatments, miRNA RT-qPCR assays measured miRNA levels after infection or treatment of macrophages. SIV RNA levels as well as virus production was downregulated by direct targeting of the SIV Nef/U3 and R regions by four miRNAs. miRs-29a, -29b, -9 and -146a were induced in primary macrophages after SIV infection. Each of these miRNAs was regulated by innate immune signaling through TNFα and/or the type I IFN, IFNβ.
The effects on miRNAs caused by HIV/SIV infection are illustrated by changes in their cellular expression throughout the course of disease, and in different patient populations. Our data demonstrate that levels of primary transcripts and mature miRs-29a, -29b, -9 and -146a are modulated by SIV infection. We show that the SIV 3′ UTR contains functional miRNA response elements (MREs) for all four miRNAs. Notably, these miRNAs regulate virus production and viral RNA levels in macrophages, the primary cells infected in the CNS that drive inflammation leading to HIV-associated neurocognitive disorders. This report may aid in identification miRNAs that target viral RNAs and HIV/SIV specifically, as well as in identification of miRNAs that may be targets of new therapies to treat HIV.
MicroRNA; MiR-29a; MiR-29b; MiR-9; MiR-146a; IFNb; TNFa; HIV; SIV; Macrophages
The main objective of the present study was to assess culturable airborne fungal concentrations, and types in different seasons. Two-stage viable impactor samplers were used with malt extract agar medium as the collection media. Culturable airborne fungal concentrations were collected indoors and outdoors of 43 homes in urban and rural environments from November 2008 to October 2009 in Egypt. Fungal concentrations were significantly higher in the rural environment than the urban environment. The median indoor and outdoor total fungal concentrations were 608 and 675 CFU/m3 in the urban environment and 1,932 and 1,872 CFU/m3 in the rural environment, respectively. The greatest concentrations were found in the autumn and spring season. Indoor and outdoor concentrations were significantly correlated (P < 0.001). The highest concentrations were observed in the fungal size range of <8 µm (fine fraction). The indoor/outdoor (I/O) ratios were not statistically different between seasons. Alternaria, Aspergillus, Cladosporium, Penicillium and yeasts were the predominant genera indoors and outdoors, and the abundance of genera varied by season and region. This study is of a potential interest as little reported research on the indoor fungal air quality from Egypt.
air; fungi; homes; rural; urban
One major activity of chemokines is recruiting immune cells to sites of infection and inflammation. CD4+ Th1 cells play critical roles in host defense against pathogens and in the pathogenesis of many immune-mediated diseases. It has been reported that epigallocatechin-3-gallate (EGCG) exhibits anti-inflammatory properties, but the mechanisms have not been completely defined. In this study, we found that EGCG markedly decreased recruitment of murine ovalbumin (OVA)-specific Th1 cells and other inflammatory cells into the airways in a Th1 adoptive transfer mouse model. In vitro analysis revealed that EGCG inhibits CXCR3 ligand driven chemotaxis of murine and human cells. Surface plasmon resonance studies revealed that EGCG bound directly to chemokines CXCL9, CXCL10 and CXCL11. These results indicate that one anti-inflammatory mechanism of EGCG is binding of proinflammatory chemokines and limiting their biological activities. These findings support further development of EGCG as a potent therapeutic for inflammatory diseases.
An urgent need exists for HIV-1 microbicides. Here, we describe the in vivo testing of lactic acid bacteria bioengineered to secrete cyanovirin-N. We fed pigtail macaques a yogurt formulation that used bioengineered strains as a starter culture. Cyanovirin-N expression could be detected in the rectal vault during and immediately after feeding. Ex vivo viral challenge of rectal tissue biopsies revealed that peak viral burden was significantly lower in tissue obtained from experimental animals compared to control animals. Formulation of candidate compounds in lactic acid bacteria and their oral administration appears to be a feasible strategy for mucosal delivery of microbicides.
HIV-1; cyanovirin-N; C V-N; lactic acid bacteria; microbicide; pigtail macaque
MicroRNA profiling of diseased/non-diseased tissue has identified expression signatures associated with a wide range of pathogenic conditions including malignancy. For example, colon cancer is associated with the under expression of miRNA-143 yet the molecular etiology of under expression is unknown. The K-Ras oncogene is a target of miRNA-143. Here, we show that the ecotropic viral integration site 1 oncoprotein (Evi1) is a transcriptional suppressor of the miRNA-143 gene. We find an indirect relationship between miRNA-143 and Evi1 expression. A complex molecular axis linking Evi1, miRNA-143 is operational in human colon cancer.
microRNA; Evi1; colon cancer; RNA interference
Decreases in alpha myosin heavy chain (α-MHC) is a common feature of human heart failure (HF), while α-MHC overexpression in transgenic (TG) rabbits is cardioprotective against tachycardia-induced cardiomyopathy (TIC). Hypothesizing that MHC isoform content alterations would impact sarcomere and mitochondrial energetics protein complement, we investigated the impact of α-MHC overexpression on global cardiac protein expression.
Protein expression was assessed by two-dimensional gel electrophoresis and mass spectrometry on extracts from TG and non-transgenic (NTG) rabbits under TIC or sham-operated conditions.
We observed significant changes in the levels of actin, myosin light chain 2 and desmin between the LV tissue of TG and NTG animals. The proteome was broadly impacted, with significant changes in mitochondrial energetics and chaperone protein families. No changes were observed in total cellular MHC or in myofibril-associated MHC. In myofibrils isolated from TGSham animals, only actin levels were altered in TGsham compared to NTGsham animals, suggesting careful myofibril assembly regulation.
Conclusions /Clinical Relevance
These data suggest myofibril protein composition may protect against TIC, emphasizing protein interconnectivity and demonstrating the need for broad-based proteomic studies in understanding targeted genetic manipulations. This study identifies targets for future development of cardioprotective agents and elucidates tachycardia-induced HF pathways.
two dimensional gel; mass spectrometry; myofilament; α myosin heavy chain
Both antiretroviral therapy and the human coreceptor polymorphism CCR2-V64I slow progression of human immunodeficiency virus type 1 (HIV-1) disease. To examine the effect of V64I on disease progression in patients receiving therapy, we determined CCR2 genotypes in the Women’s Interagency HIV Study cohort. We studied 2047 HIV-1–infected women, most of whom initiated treatment during the study. No association was seen between CCR2 genotype and either disease progression or therapeutic response, suggesting that the benefits of treatment most likely overshadow the salutary effects of the V64I polymorphism.
In the era of highly active antiretroviral therapy (HAART) the prevalence of HIV-associated CNS disease has increased despite suppression of plasma viremia.
Using an SIV model system where all animals develop AIDS and 90% develop CNS disease by three months postinoculation (p.i.), pigtailed macaques were treated with a regimen of tenofovir disoproxil fumarate, saquinavir, atazanavir, and an integrase inhibitor starting at 12 days and euthanized at ∼175 days p.i.
Plasma and CSF viral loads declined rapidly after initiating HAART. Brain viral RNA was undetectable at necropsy but viral DNA levels were not different from untreated SIV-infected macaques. CNS inflammation was significantly reduced, with decreased brain expression of MHC Class II and GFAP and reduced CSF CCL2 and IL-6. Brain from treated macaques had significantly lower levels of IFNβ, the Type I IFN-inducible gene myxovirus (influenza) resistance A (MxA), and indolamine 2,3-dioxygenase (IDO) mRNA suggesting suppressed immune hyperactivation, and fewer CD4+ and CD8+ T cells, suggesting reduced trafficking of T cells from peripheral blood. Brain levels of CD68 protein and TNFα and IFNγ RNA, while reduced, were not significantly lower, indicating continued CNS inflammation.
These data, generated in a rigorous, high viral load, SIV/macaque model showed benefits of HAART therapy on CNS virus replication and inflammation but no change in the levels of viral DNA and continued CNS inflammation in some individuals.
SIV; HIV; AIDS; HAART; CNS; IFN; TNFα; immune activation; NeuroAIDS
To characterize the regenerative pattern of cutaneous nerves in SIV-infected and uninfected macaques, excisional axotomies were performed in non-glabrous skin at 14-day intervals. Samples were examined after immunostaining for the pan-axonal marker PGP 9.5 and the Schwann cell marker p75 nerve growth factor receptor. Collateral sprouting of axons from adjacent uninjured superficial dermal nerve bundles was the initial response to axotomy. Both horizontal collateral sprouts and dense vertical regeneration of axons from the deeper dermis led to complete, rapid reinnervation of the epidermis at the axotomy site. In contrast to the slower, incomplete reinnervation previously noted in humans after this technique, in both SIV-infected and uninfected macaques, epidermal reinnervation was rapid and completed by 56 days post-axotomy. p75 was densely expressed on the Schwann cells of uninjured nerve bundles along the excision line and on epidermal Schwann cell processes. In both SIV-infected and uninfected macaques, Schwann cell process density was highest at the earliest time points post-axotomy and then declined at a similar rate. However, SIV-infection delayed epidermal nerve fiber regeneration and remodeling of new sprouts at every time point post-axotomy, and SIV-infected animals consistently had lower mean epidermal Schwann cell densities suggesting that Schwann cell guidance and support of epidermal nerve fiber regeneration may account for altered nerve regeneration. The relatively rapid regeneration time and the completeness of epidermal reinnervation in this macaque model provides a useful platform for assessing the efficacy of neurotrophic or regenerative drugs for sensory neuropathies including those caused by HIV, diabetes mellitus, medications, and toxins.
SIV; epidermal axons; Schwann cells; sprouting axon; p75; PGP9.5
Central nervous system (CNS) invasion during acute-stage HIV-infection has been demonstrated in a small number of individuals, but there is no evidence of neurological impairment at this stage and virus infection in brain appears to be controlled until late-stage disease. Using our reproducible SIV macaque model to examine the earliest stages of infection in the CNS, we identified immune responses that differentially regulate inflammation and virus replication in the brain compared to the peripheral blood and lymphoid tissues. SIV replication in brain macrophages and in brain of SIV-infected macaques was detected at 4 days post-inoculation (p.i.). This was accompanied by upregulation of innate immune responses, including IFNβ, IFNβ-induced gene MxA mRNA, and TNFα. Additionally, IL-10, the chemokine CCL2, and activation markers in macrophages, endothelial cells, and astrocytes were all increased in the brain at four days p.i. We observed synchronous control of virus replication, cytokine mRNA levels and inflammatory markers (MHC Class II, CD68 and GFAP) by 14 days p.i.; however, control failure was followed by development of CNS lesions in the brain. SIV infection was accompanied by induction of the dominant-negative isoform of C/EBPβ, which regulates SIV, CCL2, and IL6 transcription, as well as inflammatory responses in macrophages and astrocytes. This synchronous response in the CNS is in part due to the effect of the C/EBPβ on virus replication and cytokine expression in macrophage-lineage cells in contrast to CD4+ lymphocytes in peripheral blood and lymphoid tissues. Thus, we have identified a crucial period in the brain when virus replication and inflammation are controlled. As in HIV-infected individuals, though, this control is not sustained in the brain. Our results suggest that intervention with antiretroviral drugs or anti-inflammatory therapeutics with CNS penetration would sustain early control. These studies further suggest that interventions should target HIV-infected individuals with increased CCL2 levels or HIV RNA in the CNS.
Regulatory T cells (Treg) play key roles in immune regulation through multiple modes of suppression. The effects of HIV-1 infection on Treg levels in lymphoid tissues remain incompletely understood. To explore this issue, we have measured the levels of forkhead box protein 3 (FOXP3)-positive cells and associated immunomodulatory genes in a pathogenic simian immunodeficiency virus (SIV)/macaque model and found that a loss of Treg in lymph nodes (LNs) occurred following SIV infection. Changes in expression of the ligands for CXCR3, CCR4, and CCR7, and the cytokines TGF-β and IL-2 were all linked to this loss of Treg, which in turn was linked with increased levels of cellular activation. Our findings identify three mechanisms that likely contribute to SIV-driven loss of Treg, including reduced levels of cytokines associated with Treg differentiation and altered expression of agonist and antagonist chemokines. The loss of Treg and the associated cellular activation in lymphoid tissues is consistent with the events in HIV-1 infected individuals and suggest that components of the Treg differentiation and trafficking network could be targets for therapeutic intervention. This is an author-produced version of a manuscript accepted for publication in The Journal of Immunology (The JI). The American Association of Immunologists, Inc. (AAI), publisher of The JI, holds the copyright to this manuscript. This version of the manuscript has not yet been copyedited or subjected to editorial proofreading by The JI; hence, it may differ from the final version published in The JI (online and in print). AAI (The JI) is not liable for errors or omissions in this author-produced version of the manuscript or in any version derived from it by the U.S. National Institutes of Health or any other third party. The final, citable version of record can be found at www.jimmunol.org.
Human immunodeficiency virus (HIV) infection frequently causes neurologic disease even with anti-retroviral treatment. Although associations between MHC class I alleles and acquired immunodeficiency syndrome (AIDS) have been reported, the role MHC class I alleles play in restricting development of HIV-induced organ-specific diseases, including neurologic disease, has not been characterized. This study examined the relationship between expression of the MHC class I allele Mane-A*10 and development of lentiviral-induced central nervous system (CNS) disease using a well-characterized simian immunodeficiency (SIV)/pigtailed macaque model. The risk of developing CNS disease (SIV encephalitis) was 2.5 times higher for animals that did not express the MHC class I allele Mane-A*10 (P = 0.002; RR = 2.5). Animals expressing the Mane-A*10 allele had significantly lower amounts of activated macrophages, SIV RNA, and neuronal dysfunction in the CNS than Mane-A*10 negative animals (P<0.001). Mane-A*10 positive animals with the highest CNS viral burdens contained SIV gag escape mutants at the Mane-A*10-restricted KP9 epitope in the CNS whereas wild type KP9 sequences dominated in the brain of Mane-A*10 negative animals with comparable CNS viral burdens. These concordant findings demonstrate that particular MHC class I alleles play major neuroprotective roles in lentiviral-induced CNS disease.
Algorithms for theoretical reverse translation have direct applications in degenerate PCR. The conventional practice is to create several degenerate primers each of which variably encode the peptide region of interest. In the current work, for each codon we have analyzed the flanking residues in proteins and determined their influence on codon choice. From this, we created a method for theoretical reverse translation that includes information from flanking residues of the protein in question. Our method, named the neighbor correlation method (NCM) and its enhancement, the consensus-NCM (c-NCM) performed significantly better than the conventional codon-usage statistic method (CSM). Using the methods NCM and c-NCM, we were able to increase the average sequence identity from 77% up to 81%. Furthermore, we revealed a significant increase in coverage, at 80% identity, from < 20% (CSM) to > 75% (c-NCM). The algorithms, their applications and implications are discussed herein.
In this study we evaluated the levels of antibiotic- and multidrug-resistant
bacteria in bioaerosols upwind, within, and downwind at locations 25 m, 50 m, 100 m, and 150 m from a swine confined animal feeding operation.
We used Andersen two-stage samplers to collect bacterial samples, the replicate
plate method to isolate organisms, and the Kirby-Bauer disk diffusion
method to determine antibiotic resistance.
The percentage of organisms resistant to at least two antibiotic classes
and all four classes evaluated were, respectively, 2.1 and 3.0 times
higher inside (n = 69) than upwind (n = 59) of the facility. Staphylococcus aureus was the most prevalent organism recovered. Concentrations of antibiotic-resistant S. aureus decreased with increasing distance from the facility. Using Fisher’s
exact methods, the change in distribution of antibiotic resistance
profiles for each antibiotic was statistically significant (oxytetracycline, p = 0.010; tetracycline, p = 0.014; ampicillin, p = 0.007; erythromycin, p = 0.035); however, this relationship was not seen with lincomycin
and penicillin (p > 0.05). In addition, the levels of antibiotic-resistant S. aureus 25 m downwind were significantly greater than the levels from samples
taken upwind from the facility for the same four antibiotics (p < 0.05). The percentage of resistant group A streptococci and fecal
coliform increased within the facility compared with upwind values for
all antibiotics evaluated, except for lincomycin. The percentage of
resistant total coliform organisms increased within the facility compared
with upwind values for oxytetracycline and tetracycline.
Bacterial concentrations with multiple antibiotic resistances or multidrug
resistance were recovered inside and outside to (at least) 150 m downwind
of this facility at higher percentages than upwind. Bacterial
concentrations with multiple antibiotic resistances were found within
and downwind of the facility even after subtherapeutic antibiotics were
discontinued. This could pose a potential human health effect for those
who work within or live in close proximity to these facilities.
antibiotic resistant; bioaerosols; CAFO; confined/concentrated animal feeding operation; microorganisms
The lentiviral Gag polyprotein (Pr55Gag) is cleaved by the viral protease during the late stages of the virus life cycle. Proteolytic cleavage of Pr55Gag is necessary for virion maturation, a structural rearrangement required for infectivity that occurs in budded virions. In this study, we investigate the relationship between phosphorylation of capsid (CA) domains in Pr55Gag and its cleavage intermediates and their cleavage by the viral protease in simian immunodeficiency virus (SIV). First, we demonstrate that phosphorylated forms of Pr55Gag, several CA-containing cleavage intermediates of Pr55Gag, and the free CA protein are detectable in SIV virions but not in virus-producing cells, indicating that phosphorylation of these CA-containing Gag proteins may require an environment that is unique to the virion. Second, we show that the CA domain of Pr55Gag can be phosphorylated in budded virus and that this phosphorylation does not require the presence of an active viral protease. Further, we provide evidence that CA domains (i.e., incompletely cleaved CA) are phosphorylated to a greater extent than free (completely cleaved) CA and that CA-containing Gag proteins can be cleaved by the viral protease in SIV virions. Finally, we demonstrate that Pr55Gag and several of its intermediates, but not free CA, are actively phosphorylated in budded virus. Taken together, these data indicate that, in SIV virions, phosphorylation of CA domains in Pr55Gag and several of its cleavage intermediates likely precedes the cleavage of these domains by the viral protease.
An accelerated, consistent macaque simian immunodeficiency virus (SIV) model in which over 90% of pigtailed macaques (Macaca nemestrina) coinoculated with SIV/17E-Fr and SIV/DeltaB670 developed encephalitis was used to determine whether central nervous system (CNS) lesions are associated with the replication of specific genotypes in the brain and, more specifically, in the microglia. Ten of 11 inoculated macaques had severe (n = 3), moderate (n = 5), or mild (n = 2) encephalitis at 3 months postinoculation. To compare actively replicating viral genotypes in the CNS and in microglia with those in the periphery, the V1 region of the SIV envelope gene was amplified and sequenced from RNA extracted from basal ganglia, from microglial cells isolated from the brain, and from peripheral blood mononuclear cells (PBMC) isolated from blood at the time of death. To distinguish between actively replicating with latent viral genotypes in the CNS, viral genotypes in RNA and DNA from basal ganglia were compared. Two macrophage-tropic, neurovirulent viruses, SIV/17E-Fr and SIV/DeltaB670 Cl-2, predominated in the brain RNA of macaques with encephalitis, comprising 95% of the genotypes detected. The same two viral genotypes were present at the same frequencies in microglial cell RNA, suggesting that microglia are pivotal in the selective replication of neurovirulent viruses. There was a significantly greater number of viral genotypes in DNA than there were in RNA in the brain (P = 0.004), including those of both the macrophage- and lymphocyte-tropic viral strains. Furthermore, significantly fewer viral genotypes were detected in brain RNA than in PBMC RNA at the time of death (P = 0.004) and the viral strain that predominated in the brain frequently was different from that which predominated in the PBMC of the same animal. These data suggest that many viral genotypes enter the brain, but only a limited subset of macrophage-tropic, neurovirulent viruses replicate terminally in the brains of macaques with encephalitis. They further suggest that the selection of macrophage-tropic, neurovirulent viruses occurs not at the level of the blood-brain barrier but at a stage after virus entry and that microglial cells may play an important role in that selection process.
HIV-infected individuals, even with antiretroviral therapy, often display cognitive, behavioral and motor abnormalities and have decreased dopamine (DA) levels. Minocycline prevents encephalitis and neurodegeneration in SIV models, suggesting that it might also protect against nigrostriatal dopaminergic system dysfunction. Using an SIV/macaque model of HIV-associated CNS disease, we demonstrated that striatal levels of DA were significantly lower in macaques late in infection and that levels of the metabolite DOPAC also tended to be lower. DA levels declined more than its metabolites, indicating a dysregulation of DA production or catabolism. Minocycline treatment beginning at 12 but not 21 days postinoculation prevented striatal DA loss. DA decline was not due to direct loss of dopaminergic projections to the basal ganglia as there was no difference in tyrosine hydroxylase, dopamine transporter, vesicular monoamine transporter 2 or synaptophysin between minocycline-treated and untreated macaques. SIV-infected macaques had significantly higher monoamine oxidase (MAO) activity than uninfected macaques, although MAO activity was not affected by minocycline. Oxidative/nitrosative stress was examined by nitrotyrosine staining in the deep white matter and was lower in SIV-infected, minocycline-treated macaques compared with untreated macaques. These data suggest that minocycline, which has antioxidant activity, has a protective effect on DA homeostasis when administered at an appropriate time in SIV neuropathogenesis.
Minocycline; Dopamine; SIV; HIV; Oxidative stress; Monoamine oxidase
Fusion inhibitors are a class of antiretroviral drugs used to prevent entry of HIV into host cells. Many of the fusion inhibitors being developed, including the drug enfuvirtide, are peptides designed to competitively inhibit the viral fusion protein gp41. With the emergence of drug resistance, there is an increased need for effective and unique alternatives within this class of antivirals. One such alternative is a class of cyclic, cationic, antimicrobial peptides known as θ-defensins, which are produced by many non-human primates and exhibit broad-spectrum antiviral and antibacterial activity. Currently, the θ-defensin analog RC-101 is being developed as a microbicide due to its specific antiviral activity, lack of toxicity to cells and tissues, and safety in animals. Understanding potential RC-101 resistance, and how resistance to other fusion inhibitors affects RC-101 susceptibility, is critical for future development. In previous studies, we identified a mutant, R5-tropic virus that had evolved partial resistance to RC-101 during in vitro selection. Here, we report that a secondary mutation in gp41 was found to restore replicative fitness, membrane fusion, and the rate of viral entry, which were compromised by an initial mutation providing partial RC-101 resistance. Interestingly, we show that RC-101 is effective against two enfuvirtide-resistant mutants, demonstrating the clinical importance of RC-101 as a unique fusion inhibitor. These findings both expand our understanding of HIV drug-resistance to diverse peptide fusion inhibitors and emphasize the significance of compensatory gp41 mutations.