Adeno-Associated Virus (AAV)-Delivered Exosomal TAT and BiTE Molecule CD4-αCD3 Facilitate the Elimination of CD4 T Cells Harboring Latent HIV-1.

Combinatorial antiretroviral therapy (cART) has transformed HIV infection from a death sentence to a controllable chronic disease, but cannot eliminate the virus. Latent HIV-1 reservoirs are the major obstacles to cure HIV-1 infection. Previously, we engineered exosomal Tat (Exo-Tat) to reactivate latent HIV-1 from the reservoir of resting CD4+ T cells. Here, we present an HIV-1 eradication platform, which uses our previously described Exo-Tat to activate latent virus from resting CD4+ T cells guided by the specific binding domain of CD4 in interleukin 16 (IL16), attached to the N-terminus of exosome surface protein lysosome-associated membrane protein 2 variant B (Lamp2B). Cells with HIV-1 surface protein gp120 expressed on the cell membranes are then targeted for immune cytolysis by a BiTE molecule CD4-αCD3, which colocalizes the gp120 surface protein of HIV-1 and the CD3 of cytotoxic T lymphocytes. Using primary blood cells obtained from antiretroviral treated individuals, we find that this combined approach led to a significant reduction in replication-competent HIV-1 in infected CD4+ T cells in a clonal in vitro cell system. Furthermore, adeno-associated virus serotype DJ (AAV-DJ) was used to deliver Exo-Tat, IL16lamp2b and CD4-αCD3 genes by constructing them in one AAV-DJ vector (the plasmid was named pEliminator). The coculture of T cells from HIV-1 patients with Huh-7 cells infected with AAV-Eliminator viruses led to the clearance of HIV-1 reservoir cells in the in vitro experiment, which could have implications for reducing the viral reservoir in vivo, indicating that Eliminator AAV viruses have the potential to be developed into therapeutic biologics to cure HIV-1 infection.

Cigarette smoke-induced extracellular vesicles from dendritic cells alter T-cell activation and HIV replication.

Despite decreased rates of tobacco smoking in many areas, cigarette smoking remains a major contributor to many health problems. Cigarette smoking can reduce immune system functioning while concurrently increasing inflammation. Dendritic cells in the lung exposed to cigarette smoke become stimulated and go on to activate T-cells. Extracellular vesicles (EVs) are nano-sized particles released by cells. They participate in intercellular communication by transferring functional proteins and nucleic acids to recipient cells and have been implicated in immune responses. For example, they can display MHC-peptide complexes to activate T-cells. In the current study, we sought to understand the role of cigarette smoke extract (CSE) on dendritic cell-derived EVs and their capacity to activate and differentiate T-cells. Primary human dendritic cells (iDCs) were exposed to CSE and EVs were separated and characterized. We exposed autologous primary CD4 + T-cells to iDC-EVs and observed T helper cell populations skewing towards Th1 and Th17 phenotypes. As HIV + individuals are disproportionately likely to be current smokers, we also examined the effects of iDC-EVs on acutely infected T-cells as well as on a cell model of HIV latency (ACH-2). We found that in most cases, iDC-CSE EVs tended to reduce p24 release from the acutely infected primary T-cells, albeit with great variability. We did not observe large effects of iDC-EVs or direct CSE exposure on p24 release from the ACH-2 cell line. Together, these data suggest that iDC-CSE EVs have the capacity to modulate the immune responses, in part by pushing T-cells towards Th1 and Th17 phenotypes.

Estimating Health Risks to Children Associated with Recreational Play on Oil Spill-Contaminated Beaches.

The human health impact from exposure to contaminated shorelines following an oil spill event has been investigated to some extent. However, the health risks to children have largely been characterized through the use of surveys and extrapolation from adult health outcomes. There is limited information on children's behaviors during beach play requiring assumptions made based on observations from play activities in home settings. The Beach Exposure and Child Health Study (BEACHES) quantified specific beach activities that can be used to inform human health risk assessments of children playing on beaches impacted by oil spills. The results of this study characterize children's risk of cancer from exposure to oil spill chemicals by incorporating exposure-related information collected from the BEACHES study and by assuming oral, dermal, and inhalation exposure routes. Point risk estimates are compared with a previous, similar study that applied default exposure parameter values obtained from the published literature. The point risk estimates informed by BEACHES data are one order of magnitude lower compared with the previous risk assessment, with dermal exposures the overall risk driver in both. Additional Monte Carlo simulations evaluating the BEACHES data provide ranges of health risks with the highest estimates associated with dermal and oral exposure routes.

Microbial risk assessment in recreational freshwaters from southern Brazil.

In this study, total coliforms (TC), Escherichia coli, enterovirus (EV), rotavirus (RV), and human mastadenovirus species C and F (HAdV-C and HAdV-F) were evaluated in water samples from Belo Stream. For HAdV-C and F, the infectivity was assessed by integrated cell culture quantitative real-time polymerase chain reaction (ICC-qPCR). Samples were collected monthly (May/2015 to April/2016) at four sites. Viral analyses were performed for both ultracentrifuge-concentrated and unconcentrated samples. For site P4 (used for recreational purposes), QMRA was applied to estimate health risks associated with exposure to E. coli and HAdV-C and F. TC and E. coli were present throughout the collection period. EV and RV were not detected. HAdV-C were present in 8.51% (1.89E + 06 to 2.28E + 07 GC (Genomic Copies)/L) and 21.27% (2.36E + 05 to 1.29E + 07 GC/L) for unconcentrated and concentrated samples, respectively. For HAdV-F were 12.76% (2.77E + 07 to 3.31E + 08 GC/L) and 48.93% (1.10E + 05 to 4.50E + 08 GC/L) for unconcentrated and concentrated samples, respectively. For unconcentrated samples, infectivity for HAdV-C was detected in 37.20% (1st ICC-qPCR) and 25.58% (2nd ICC-qPCR). For HAdV-F, infectivity was detected in 6.97% (1st ICC-qPCR) and 6.97% (2nd ICC-qPCR). For concentrated samples, HAdV-C infectious was observed in 17.02% (1st ICC-qPCR) and in 8.51% (2nd ICC-qPCR). For HAdV-F, were present in 8.51% for both 1st and 2nd ICC-qPCR. Statistical analyzes showed significant difference between the collection sites when analyzed the molecular data of HAdV-F, data of TC and E. coli. Correlation tests showed direct correlation between HAdV-F with E. coli and TC. E. coli concentrations translated to the lowest estimates of infection risks (8.58E-05 to 2.17E-03). HAdV-F concentrations were associated with the highest infection risks at 9.99E-01 and for group C, 1.29E-01 to 9.99E-01. These results show that commonly used bacterial indicators for water quality may not infer health risks associated with viruses in recreational freshwaters.

Cessation from Smoking Improves Innate Host Defense and Clearance of Experimentally Inoculated Nasal Staphylococcus aureus.

Staphylococcus aureus nasal carriage is transient in most humans and usually benign, but dissemination of S. aureus to extranasal sites causes the majority of clinical infections, and S. aureus is a major cause of serious infections in the United States. A better understanding of innate nasal decolonization mechanisms is urgently needed, as are relevant models for studying S. aureus clearance. Here, we screened a population of healthy smokers for nasal S. aureus carriage and compared the participants' abilities to clear experimentally applied nasal S. aureus before and after completion of a smoking cessation program. We determined that cigarette smoking increases the mean nasal S. aureus load (2.6 × 104 CFU/swab) compared to the load observed in healthy nonsmokers (1.7 × 103 CFU/swab) and might increase the rate of S. aureus nasal carriage in otherwise-healthy adults: 22 of 99 smokers carried S. aureus at the screening visit, while only 4 of 30 nonsmokers screened positive during the same time period. Only 6 of 19 experimental inoculation studies in active smokers resulted in S. aureus clearance within the month of follow-up, while in the cessation group, 6 of 9 subjects cleared nasal S. aureus and carriage duration averaged 21 ± 4 days. Smoking cessation associated with enhanced expression of S. aureus-associated interleukin-1ß (IL-1ß) and granulocyte colony-stimulating factor (G-CSF) in nasal fluids. Participants who failed to clear S. aureus exhibited a higher nasal S. aureus load and elevated nasal interleukin-1 receptor antagonist (IL-1RA) expression at the preexperiment study visits. We conclude that smokers exhibit higher S. aureus loads than nonsmokers and that innate immune pathways, including G-CSF expression and signaling through the IL-1 axis, are important mediators of nasal S. aureus clearance.

Central nervous system-specific consequences of simian immunodeficiency virus Gag escape from major histocompatibility complex class I-mediated control.

In the fourth decade of the HIV epidemic, the relationship between host immunity and HIV central nervous system (CNS) disease remains incompletely understood. Using a simian immunodeficiency virus (SIV)/macaque model, we examined CNS outcomes in pigtailed macaques expressing the MHC class I allele Mane-A1*084:01 which confers resistance to SIV-induced CNS disease and induces the prototypic viral escape mutation Gag K165R. Insertion of gag K165R into the neurovirulent clone SIV/17E-Fr reduced viral replication in vitro compared to SIV/17E-Fr. We also found lower cerebrospinal fluid (CSF), but not plasma, viral loads in macaques inoculated with SIV/17E-Fr K165R versus those inoculated with wildtype. Although escape mutation K165R was genotypically stable in plasma, it rapidly reverted to wildtype Gag KP9 in both CSF and in microglia cultures. We induced robust Gag KP9-specific CTL tetramer responses by vaccinating Mane-A*084:01-positive pigtailed macaques with a Gag KP9 virus-like particle (VLP) vaccine. Upon SIV/17E-Fr challenge, vaccinated animals had lower SIV RNA in CSF compared to unvaccinated controls, but showed no difference in plasma viral loads. These data clearly demonstrate that viral fitness in the CNS is distinct from the periphery and underscores the necessity of understanding the consequences of viral escape in CNS disease with the advent of new therapeutic vaccination strategies.

Inappropriate utilization of SPECT myocardial perfusion imaging on the USA-Mexico border.

BACKGROUND: The American College of Cardiology/American Society of Nuclear Cardiology published revised appropriate use criteria (AUC) for SPECT MPI in 2009. We assessed adherence to these guidelines and factors associated with inappropriate utilization at the University Medical Center. METHODS: The AUC was applied retrospectively to 420 SPECT MPI studies. Two-sample t test, Fisher's exact test, and multivariable logistic regression models were used for analysis. RESULTS: There were 322 appropriate (86%) and 54 (14%) inappropriate studies. The odds of having an inappropriate test increased with younger age (P < .001) and female gender (P < .001). Subjects with diabetes (P = .007) and chest pain (P < .001) were less likely to have an inappropriate test. Academic outpatients were three times more likely to have an inappropriate study (P = .123), while community PCPs were 5.6 times (P = .011) and community cardiologists eight times more likely to order inappropriate tests (P = .031). CONCLUSIONS: Inappropriate SPECT MPI in low risk younger women is an important issue on the USA-Mexico border. Initiatives to reduce inappropriate SPECT MPI should focus on a few indications and evaluation of cardiovascular symptoms in younger age women in outpatient/community practices.

A retrospective look at replacing face-to-face embryology instruction with online lectures in a human anatomy course.

Embryology is integrated into the Clinically Oriented Anatomy course at the Texas Tech University Health Sciences Center School of Medicine. Before 2008, the same instructor presented embryology in 13 face-to-face lectures distributed by organ systems throughout the course. For the 2008 and 2009 offerings of the course, a hybrid embryology instruction model with four face-to-face classes that supplemented online recorded lectures was used. One instructor delivered the lectures face-to-face in 2007 and by online videos in 2008-2009, while a second instructor provided the supplemental face-to-face classes in 2008-2009. The same embryology learning objectives and selected examination questions were used for each of the three years. This allowed direct comparison of learning outcomes, as measured by examination performance, for students receiving only face-to-face embryology instruction versus the hybrid approach. Comparison of the face-to-face lectures to the hybrid approach showed no difference in overall class performance on embryology questions that were used all three years. Moreover, there was no differential effect of the delivery method on the examination scores for bottom quartile students. Students completed an end-of-course survey to assess their opinions. They rated the two forms of delivery similarly on a six-point Likert scale and reported that face-to-face lectures have the advantage of allowing them to interact with the instructor, whereas online lectures could be paused, replayed, and viewed at any time. These experiences suggest the need for well-designed prospective studies to determine whether online lectures can be used to enhance the efficacy of embryology instruction.

Neuroprotective maraviroc monotherapy in simian immunodeficiency virus-infected macaques: reduced replicating and latent SIV in the brain.

OBJECTIVE: HIV-associated neurocognitive deficits remain a challenge despite suppressive combined antiretroviral therapy. Given the association between HIV-induced central nervous system (CNS) disease and replication of HIV in immune-activated macrophages, CCR5 antagonists may attenuate CNS disease by modulating inflammatory signaling and by limiting viral replication. DESIGN: To establish whether initiating CCR5 inhibition during early infection altered CNS disease progression, outcomes were compared between simian immunodeficiency virus (SIV)-infected macaques treated with maraviroc (MVC) versus untreated SIV-infected macaques. METHODS: Six SIV-infected rhesus macaques were treated with MVC monotherapy for 5 months beginning 24 days postinoculation; 22 SIV-infected animals served as untreated controls. SIV RNA levels in plasma, cerobrospinal fluid, and brain, and CNS expression of TNFα and CCL2 were measured by qRT-PCR. Immunostaining for CD68 and amyloid precursor protein in the brain was measured by image analysis. Plasma sCD163 was measured by ELISA. RESULTS: SIV RNA and proviral DNA levels in brain were markedly lower with MVC treatment, demonstrating CCR5 inhibition reduces CNS replication of SIV and may reduce the CNS latent viral reservoir. MVC treatment also lowered monocyte and macrophage activation, represented by CNS CD68 immunostaining and plasma sCD163 levels, and reduced both TNFα and CCL2 RNA expression in brain. Treatment also reduced axonal amyloid precursor protein immunostaining to levels present in uninfected animals, consistent with neuroprotection. CONCLUSION: CCR5 inhibitors may prevent neurologic disorders in HIV-infected individuals by reducing inflammation and by limiting viral replication in the brain. Furthermore, CCR5 inhibitors may reduce the latent viral reservoir in the CNS. Adding CCR5 inhibitors to combined antiretroviral regimens may offer multiple neuroprotective benefits.

SIV replication is directly downregulated by four antiviral miRNAs.

BACKGROUND: 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. RESULTS: 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, IFNß 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, IFNß. CONCLUSIONS: 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.

A compensatory mutation provides resistance to disparate HIV fusion inhibitor peptides and enhances membrane fusion.

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.

The anti-HIV microbicide candidate RC-101 inhibits pathogenic vaginal bacteria without harming endogenous flora or mucosa.

PROBLEM: 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. RESULTS: 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. CONCLUSIONS: 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.

Diastolic dysfunction is associated with myocardial viral load in simian immunodeficiency virus-infected macaques.

OBJECTIVE: To establish the relationship between HIV-induced cardiac diastolic dysfunction, immune responses, and virus replication in the heart using the simian immunodeficiency virus (SIV)/macaque model. DESIGN: Cardiac diastolic dysfunction is common in HIV-infected individuals including asymptomatic patients and those treated with combination antiretroviral therapy. SIV-infected macaques develop cardiac dysfunction, serving as a useful model to establish mechanisms underlying HIV-induced cardiac dysfunction. To understand the relationship between functional cardiac impairment, viral replication in the heart, and associated host inflammatory responses, cardiac function was evaluated in SIV-infected macaques and functional decline was correlated with features of the host immune response and the extent of viral replication in both the myocardium and plasma. METHODS: Cardiac function was evaluated longitudinally in 22 SIV-infected and eight uninfected macaques using mitral inflow and tissue Doppler echocardiography. Myocardial macrophage populations were evaluated by CD68 and CD163 immunostaining. SIV RNA levels in both myocardium and plasma were measured by qRT-PCR. RESULTS: Echocardiographic abnormalities developed in SIV-infected macaques that closely resembled diastolic dysfunction reported in asymptomatic HIV-infected individuals. Although CD68 and CD163 were upregulated in the myocardium of SIV-infected animals, neither macrophage marker correlated with functional decline. SIV-induced diastolic dysfunction was strongly correlated with extent of SIV replication in the myocardium, implicating virus or viral proteins in the initiation and progression of cardiac dysfunction. CONCLUSION: This study demonstrated a strong correlation between cardiac functional impairment and extent of SIV replication in the myocardium, suggesting that persistent viral replication in myocardial macrophages induces cardiomyocyte damage manifest as diastolic dysfunction.

Macrophage-mediated dorsal root ganglion damage precedes altered nerve conduction in SIV-infected macaques.

Peripheral neuropathy is the most common neurological complication of HIV-1 infection, affecting over one-third of infected individuals, including those treated with antiretroviral therapy. To study the pathogenesis of HIV-induced peripheral nervous system disease, we established a model in which SIV-infected macaques developed changes closely resembling alterations reported in components of the sensory pathway in HIV-infected individuals. Significant declines in epidermal nerve fiber density developed in SIV-infected macaques, similar to that of HIV-infected individuals with neuropathy. Changes in dorsal root ganglia (DRG) included macrophage infiltration, SIV replication in macrophages, immune activation of satellite cells, and neuronal loss. To determine whether dorsal root ganglion damage was associated with altered nerve function, we measured unmyelinated C-fiber conduction velocities (CV) in nerves of SIV-infected macaques and compared CV changes with DRG alterations. Twelve weeks postinoculation, SIV-infected macaques had significantly lower C-fiber conduction velocity in sural nerves than uninfected animals and the magnitude of conduction velocity decline correlated strongly with extent of DRG macrophage infiltration. Thus, injury to neurons in the DRG-mediated by activated macrophages-preceded altered conduction of unmyelinated nerve fibers in SIV-infected macaques, suggesting that macrophage-mediated DRG damage may be the initiating event in HIV-induced sensory neuropathy.

The Evi1, microRNA-143, K-Ras axis in colon cancer.

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.

Simian immunodeficiency virus-infected macaques treated with highly active antiretroviral therapy have reduced central nervous system viral replication and inflammation but persistence of viral DNA.

BACKGROUND: During the era of highly active antiretroviral therapy (HAART), the prevalence of HIV-associated central nervous system (CNS) disease has increased despite suppression of plasma viremia. METHODS: In a simian immunodeficiency virus (SIV) model system in which all animals develop AIDS and 90% develop CNS disease by 3 months after inoculation, pigtailed macaques were treated with a regimen of tenofovir disoproxil fumarate, saquinavir, atazanavir, and an integrase inhibitor starting at 12 days after inoculation and were euthanized at approximately 175 days after inoculation. RESULTS: Plasma and cerebrospinal fluid (CSF) viral loads declined rapidly after the initiation of HAART. Brain viral RNA was undetectable at necropsy, but viral DNA levels were not different from those in untreated SIV-infected macaques. CNS inflammation was significantly reduced, with decreased brain expression of major histocompatibility complex class II and glial fibrillary acidic protein and reduced levels of CSF CCL2 and interleukin 6. Brain from treated macaques had significantly lower levels of interferon beta, type 1 interferon-inducible gene myxovirus (influenza) resistance A, and indolamine 2,3-dioxygenase messenger RNA, suggesting that immune hyperactivation was suppressed, and fewer CD4(+) and CD8(+) T cells, suggesting that trafficking of T cells from peripheral blood was reduced. Brain levels of CD68 protein and tumor necrosis factor alpha and interferon gamma RNA were reduced but were not significantly lower, indicating continued CNS inflammation. CONCLUSIONS: These data, generated in a rigorous, high-viral-load SIV-infected macaque model, showed that HAART provided benefits with respect to CNS viral replication and inflammation but that no change in the level of viral DNA and continued CNS inflammation occurred in some macaques.

HIV and SIV induce alterations in CNS CaMKII expression and activation: a potential mechanism for cognitive impairment.

The molecular mechanisms underlying learning and memory impairment in patients with HIV-associated neurological disease have remained unclear. Calcium/calmodulin-dependent kinase II (CaMKII) has key roles in synaptic potentiation and memory storage in neurons and also may have immunomodulatory functions. To determine whether HIV and simian immunodeficiency virus (SIV) induce alterations in CaMKII expression and/or activation (autophosphorylation) in the brain, we measured CaMKII alterations by quantitative immunoblotting in both an in vitro HIV/neuronal culture model and in vivo in an SIV-infected macaque model of HIV-associated neurological damage. Using primary rat hippocampal neuronal cultures treated with culture supernatants harvested from HIV-1-infected human monocyte-derived macrophages (HIV/MDM), we found that CaMKII activation declined after exposure of neurons to HIV/MDM. Consistent with our in vitro measurements, a significant decrease in CaMKII activation was present in both the hippocampus and frontal cortex of SIV-infected macaques compared with uninfected animals. In SIV-infected animals, total CaMKII expression in the hippocampus correlated well with levels of synaptophysin. Furthermore, CaMKII expression in both the hippocampus and frontal cortex was inversely correlated with viral load in the brain. These findings suggest that alterations in CaMKII may compromise synaptic function in the early phases of chronic neurodegenerative processes induced by HIV.

Coordinated regulation of SIV replication and immune responses in the CNS.

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 IFNbeta, IFNbeta-induced gene MxA mRNA, and TNFalpha. 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/EBPbeta, 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/EBPbeta 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.

Subversion of interleukin-1-mediated host defence by a nasal carrier strain of Staphylococcus aureus.

Staphylococcus aureus, a major source of nosocomial and community-acquired infections, has a nasal carriage rate exceeding 25% in the human population. To elucidate host-pathogen interactions pertaining to nasal carriage, we examined the role of interleukin-1 (IL-1) in the colonization of human nasal epithelial cells (NEC) by a nasal carrier strain and a non-carrier strain of S. aureus. Using an organotypic model of the nasal epithelium, we observed that inoculation with a non-carrier strain of S. aureus induced production of IL-1 from NEC, but the expression of this cytokine was significantly reduced when NEC were inoculated with a carrier strain. Moreover, both IL-1alpha and IL-1beta significantly decreased the growth of the nasal carrier strain of S. aureus (P < 0.001, n = 17 to n = 25); however the growth of the non-carrier strain was unaffected. Interestingly, it was found that several nasal carrier strains of S. aureus form quorum-dependent biofilms, which can be partially inhibited when preincubated with IL-1alpha. Taken together these data suggest that, although nasal carrier strains of S. aureus are sensitive to IL-1, they display a significant colonization advantage by both preventing the host from expressing IL-1 and elaborating a protective biofilm.

Pathogenesis of simian immunodeficiency virus-induced alterations in macaque trigeminal ganglia.

Peripheral neuropathy is the most frequent neurologic complication associated with human immunodeficiency virus (HIV) infection, yet its pathogenesis remains poorly understood. To study the mechanisms causing HIV-induced peripheral nervous system disease, we examined trigeminal ganglia obtained from simian immunodeficiency virus (SIV)-inoculated macaques. SIV-infected macaques developed multifocal trigeminal ganglionitis of varying severity characterized by multifocal mononuclear infiltrates, neuronophagia, and neuronal loss resembling reports of HIV-associated changes present in dorsal root ganglia. Neuronal density, measured by calculating the fractional area of trigeminal ganglia occupied by neurons, was significantly lower in SIV-infected macaques versus uninfected macaques (p = 0.001). To characterize the inflammatory cell population and measure productive viral infection in ganglia, trigeminal ganglia from SIV-infected macaques were immunostained for macrophage or cytotoxic lymphocyte markers and for SIV gp41. The extent of macrophage infiltration in trigeminal ganglia was inversely correlated with neuronal loss (p = 0.001), whereas cytotoxic lymphocyte infiltration was not associated with neuronal loss. These studies demonstrate that alterations in the somatosensory ganglia of SIV-infected macaques closely parallel those observed in HIV-infected individuals and show that study of SIV-infected macaques may help elucidate the pathophysiology of HIV-induced peripheral neuropathy.