An exploratory deep learning approach to investigate tuberculosis pathogenesis in nonhuman primate model: Combining automated radiological analysis with clinical and biomarkers data.

BACKGROUND: Tuberculosis (TB) kills approximately 1.6 million people yearly despite the fact anti-TB drugs are generally curative. Therefore, TB-case detection and monitoring of therapy, need a comprehensive approach. Automated radiological analysis, combined with clinical, microbiological, and immunological data, by machine learning (ML), can help achieve it. METHODS: Six rhesus macaques were experimentally inoculated with pathogenic Mycobacterium tuberculosis in the lung. Data, including Computed Tomography (CT), were collected at 0, 2, 4, 8, 12, 16, and 20 weeks. RESULTS: Our ML-based CT analysis (TB-Net) efficiently and accurately analyzed disease progression, performing better than standard deep learning model (LLM OpenAI's CLIP Vi4). TB-Net based results were more consistent than, and confirmed independently by, blinded manual disease scoring by two radiologists and exhibited strong correlations with blood biomarkers, TB-lesion volumes, and disease-signs during disease pathogenesis. CONCLUSION: The proposed approach is valuable in early disease detection, monitoring efficacy of therapy, and clinical decision making.

Lipid Mediators and Cytokines/Chemokines Display Differential Profiles in Severe versus Mild/Moderate COVID-19 Patients.

Host immune responses play a key role in COVID-19 pathogenesis. The underlying phenomena are orchestrated by signaling molecules such as cytokines/chemokines and lipid mediators. These immune molecules, including anti-SARS-CoV-2 antibodies, interact with immune cells and regulate host responses, contributing to inflammation that drives the disease. We investigated 48 plasma cytokines/chemokines, 21 lipid mediators, and anti-S protein (RBD) antibodies in COVID-19 patients (n = 56) and non-COVID-19 respiratory disease controls (n = 49), to identify immune-biomarker profiles. Cytokines/chemokines (IL-6, CXCL-10 (IP-10), HGF, MIG, MCP-1, and G-CSF) and lipid mediators (TxB2, 11-HETE, 9-HODE, 13-HODE, 5-HETE, 12-HETE, 15-HETE, 14S-HDHA, 17S-HDHA, and 5-oxo ETE) were significantly elevated in COVID-19 patients compared to controls. In patients exhibiting severe disease, pro-inflammatory cytokines/chemokines (IL-6, CXCL-10, and HGF) and anti-SARS-CoV-2 antibodies were significantly elevated. In contrast, lipid mediators involved in the reduction/resolution of inflammation, in particular, 5-HETE, 11-HETE, and 5-oxoETE, were significantly elevated in mild/moderate disease. Taken together, these immune-biomarker profiles provide insight into immune responses related to COVID-19 pathogenesis. Importantly, our findings suggest that elevation in plasma concentrations of IL-6, CXCL-10, HGF, and anti-SARS-CoV-2 antibodies can predict severe disease, whereas elevation in lipid mediators peaks early (compared to cytokines) and includes induction of mechanisms leading to reduction of inflammation, associated complications, and maintenance of homeostasis.

Quantitative Imaging Analysis of the Spatial Relationship between Antiretrovirals, Reverse Transcriptase Simian-Human Immunodeficiency Virus RNA, and Fibrosis in the Spleens of Nonhuman Primates.

Although current antiretroviral therapy (ART) has increased life expectancy, a cure for human immunodeficiency virus (HIV) remains elusive due to the persistence of the virus in tissue reservoirs. In the present study, we sought to elucidate the relationship between antiretrovirals (ARVs) and viral expression in the spleen. We performed mass spectrometry imaging (MSI) of 6 different ARVs, RNAscope in situ hybridization of viral RNA, and immunohistochemistry of three different fibrosis markers in the spleens of 8 uninfected and 10 reverse transcriptase simian-human immunodeficiency virus (RT-SHIV)-infected rhesus macaques (infected for 6 weeks) that had been dosed for 10 days with combination ART. Using MATLAB, computational quantitative imaging analysis was performed to evaluate the spatial and pharmacological relationships between the 6 ARVs, viral RNA, and fibrotic deposition. In these spleens, >50% of the spleen tissue area was not covered by any detectable ARV response (any concentration above the limits of detection for individual ARVs). The median spatial ARV coverage across all tissues was driven by maraviroc followed by efavirenz. Yet >50% of RNA-positive cells were not exposed to any detectable ARV. Quantifiable maraviroc and efavirenz colocalization with RNA-positive cells was usually greater than the in vitro concentration inhibiting 50% replication (IC50). Fibrosis markers covered more than 50% of the spleen tissue area and had negative relationships with cumulative ARV coverages. Our findings suggest that a heterogeneous ARV spatial distribution must be considered when evaluating viral persistence in lymphoid tissue reservoirs.

NOD1 and NOD2 signalling links ER stress with inflammation.

Endoplasmic reticulum (ER) stress is a major contributor to inflammatory diseases, such as Crohn disease and type 2 diabetes. ER stress induces the unfolded protein response, which involves activation of three transmembrane receptors, ATF6, PERK and IRE1α. Once activated, IRE1α recruits TRAF2 to the ER membrane to initiate inflammatory responses via the NF-κB pathway. Inflammation is commonly triggered when pattern recognition receptors (PRRs), such as Toll-like receptors or nucleotide-binding oligomerization domain (NOD)-like receptors, detect tissue damage or microbial infection. However, it is not clear which PRRs have a major role in inducing inflammation during ER stress. Here we show that NOD1 and NOD2, two members of the NOD-like receptor family of PRRs, are important mediators of ER-stress-induced inflammation in mouse and human cells. The ER stress inducers thapsigargin and dithiothreitol trigger production of the pro-inflammatory cytokine IL-6 in a NOD1/2-dependent fashion. Inflammation and IL-6 production triggered by infection with Brucella abortus, which induces ER stress by injecting the type IV secretion system effector protein VceC into host cells, is TRAF2, NOD1/2 and RIP2-dependent and can be reduced by treatment with the ER stress inhibitor tauroursodeoxycholate or an IRE1α kinase inhibitor. The association of NOD1 and NOD2 with pro-inflammatory responses induced by the IRE1α/TRAF2 signalling pathway provides a novel link between innate immunity and ER-stress-induced inflammation.

Quantitative Imaging Analysis of the Spatial Relationship between Antiretrovirals, Reverse Transcriptase Simian-Human Immunodeficiency Virus RNA, and Collagen in the Mesenteric Lymph Nodes of Nonhuman Primates.

Human immunodeficiency virus (HIV) persistence in tissue reservoirs is a major barrier to HIV cure. While antiretrovirals (ARVs) suppress viral replication, antiretroviral therapy (ART) interruption results in rapid rebound viremia that may originate from lymphoid tissues. To understand the relationship between anatomic distribution of ARV exposure and viral expression in lymph nodes, we performed mass spectrometry imaging (MSI) of 6 ARVs, RNAscope in situ hybridization for viral RNA (vRNA), and immunohistochemistry of collagen in mesenteric lymph nodes from 8 uninfected and 10 reverse transcriptase simian/human immunodeficiency virus (RT-SHIV)-infected rhesus macaques dosed to steady state with combination ART. MATLAB-based quantitative imaging analysis was used to evaluate spatial and pharmacological relationships between these ARVs, viral RNA (both vRNA+ cells and follicular dendritic cell [FDC]-bound virions), and collagen deposition. Using MSI, 31% of mesenteric lymph node tissue area was found to be not covered by any ARV. Additionally, 28% of FDC-trapped virions and 21% of infected cells were not exposed to any detected ARV. Of the 69% of tissue area that was covered by cumulative ART exposure, nearly 100% of concentrations were greater than in vitro 50% inhibitory concentration (IC50) values; however, 52% of total tissue coverage was from only one ARV, primarily maraviroc. Collagen covered ∼35% of tissue area but did not influence ARV distribution heterogeneity. Our findings are consistent with our hypothesis that ARV distribution, in addition to total-tissue drug concentration, must be considered when evaluating viral persistence in lymph nodes and other reservoir tissues.

Antiretroviral Penetration and Drug Transporter Concentrations in the Spleens of Three Preclinical Animal Models and Humans.

Adequate antiretroviral (ARV) concentrations in lymphoid tissues are critical for optimal antiretroviral therapy (ART). While the spleen contains 25% of the body's lymphocytes, there are minimal data on ARV penetration in this organ. This study quantified total and protein-unbound splenic ARV concentrations and determined whether drug transporters, sex, or infection status were modifiers of these concentrations in animal models and humans. Two humanized mice models (hu-HSC-Rag [n = 36; 18 HIV-positive (HIV+) and 18 HIV-negative (HIV-)] and bone marrow-liver-thymus [n = 13; 7 HIV+ and 6 HIV-]) and one nonhuman primate (NHP) model (rhesus macaque [n = 18; 10 SHIV+ and 8 SHIV-]) were dosed to steady state with ARV combinations. HIV+ human spleens (n = 14) from the National NeuroAIDS Tissue Consortium were analyzed postmortem (up to 24 h postdose). ARV concentrations were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS), drug transporter concentrations were measured with LC-MS proteomics, and protein binding in NHP spleens was determined by rapid equilibrium dialysis. Mice generally had the lowest splenic concentrations of the three species. Protein binding in splenic tissue was 6 to 96%, compared to 76 to 99% in blood plasma. NHPs had quantifiable Mrp4, Bcrp, and Ent1 concentrations, and humans had quantifiable ENT1 concentrations. None significantly correlated with tissue ARV concentrations. There was also no observable influence of infection status or sex. With these dosing strategies, NHP splenic penetration most closely resembled that of humans. These data can inform tissue pharmacokinetic scaling to humans to target HIV reservoirs by identifying important species-related differences.

Antiretroviral Penetration across Three Preclinical Animal Models and Humans in Eight Putative HIV Viral Reservoirs.

For HIV cure strategies like "kick and kill" to succeed, antiretroviral (ARV) drugs must reach effective concentrations in putative viral reservoirs. We characterize penetration of six ARVs in three preclinical animal models and humans. We found that standard dosing strategies in preclinical species closely mimicked tissue concentrations in humans for some, but not all, ARVs. These results have implications for interpreting HIV treatment, prevention, or cure interventions between preclinical and clinical models.

Antiretroviral Drug Concentrations in Lymph Nodes: A Cross-Species Comparison of the Effect of Drug Transporter Expression, Viral Infection, and Sex in Humanized Mice, Nonhuman Primates, and Humans.

In a "kick and kill" strategy for human immunodeficiency virus (HIV) eradication, protective concentrations of antiretrovirals (ARVs) in the lymph node are important to prevent vulnerable cells from further HIV infection. However, the factors responsible for drug distribution and concentration into these tissues are largely unknown. Although humanized mice and nonhuman primates (NHPs) are crucial to HIV research, ARV tissue pharmacology has not been well characterized across species. This study investigated the influence of drug transporter expression, viral infection, and sex on ARV penetration within lymph nodes of animal models and humans. Six ARVs were dosed for 10 days in humanized mice and NHPs. Plasma and lymph nodes were collected at necropsy, 24 hours after the last dose. Human lymph node tissue and plasma from deceased patients were collected from tissue banks. ARV, active metabolite, and endogenous nucleotide concentrations were measured by liquid chromatography-tandem mass spectrometry, and drug transporter expression was measured using quantitative polymerase chain reaction and quantitative targeted absolute proteomics. In NHPs and humans, lymph node ARV concentrations were greater than or equal to plasma, and tenofovir diphosphate/deoxyadenosine triphosphate concentration ratios achieved efficacy targets in lymph nodes from all three species. There was no effect of infection or sex on ARV concentrations. Low drug transporter expression existed in lymph nodes from all species, and no predictive relationships were found between transporter gene/protein expression and ARV penetration. Overall, common preclinical models of HIV infection were well suited to predict human ARV exposure in lymph nodes, and low transporter expression suggests primarily passive drug distribution in these tissues. SIGNIFICANCE STATEMENT: During human immunodeficiency virus (HIV) eradication strategies, protective concentrations of antiretrovirals (ARVs) in the lymph node prevent vulnerable cells from further HIV infection. However, ARV tissue pharmacology has not been well characterized across preclinical species used for HIV eradication research, and the influence of drug transporters, HIV infection, and sex on ARV distribution and concentration into the lymph node is largely unknown. Here we show that two animal models of HIV infection (humanized mice and nonhuman primates) were well suited to predict human ARV exposure in lymph nodes. Additionally, we found that drug transporter expression was minimal and-along with viral infection and sex-did not affect ARV penetration into lymph nodes from any species.

Antiretroviral concentrations and surrogate measures of efficacy in the brain tissue and CSF of preclinical species.

1. Antiretroviral concentrations in cerebrospinal fluid (CSF) are used as surrogate for brain tissue, although sparse data support this. We quantified antiretrovirals in brain tissue across preclinical models, compared them to CSF, and calculated 90% inhibitory quotients (IQ90) for nonhuman primate (NHP) brain tissue. Spatial distribution of efavirenz was performed by mass-spectrometry imaging (MSI). 2. HIV or RT-SHIV-infected and uninfected animals from two humanized mouse models (hemopoietic-stem cell/RAG2-, n = 36; bone marrow-liver-thymus/BLT, n =13) and an NHP model (rhesus macaque, n =18) were dosed with six antiretrovirals. Brain tissue, CSF (NHPs), and plasma were collected at necropsy. Drug concentrations were measured by LC-MS/MS. Rapid equilibrium dialysis determined protein binding in NHP brain. 3. Brain tissue penetration of most antiretrovirals were >10-fold lower (p < 0.02) in humanized mice than NHPs. NHP CSF concentrations were >13-fold lower (p <0.02) than brain tissue with poor agreement except for efavirenz (r = 0.91, p = 0.001). Despite 97% brain tissue protein binding, efavirenz achieved IQ90>1 in all animals and 2-fold greater white versus gray matter concentration. 4. Brain tissue penetration varied across animal models for all antiretrovirals except raltegravir, and extrapolating brain tissue concentrations between models should be avoided. With the exception of efavirenz, CSF is not a surrogate for brain tissue concentrations.

Mass spectrometry imaging reveals heterogeneous efavirenz distribution within putative HIV reservoirs.

Persistent HIV replication within active viral reservoirs may be caused by inadequate antiretroviral penetration. Here, we used mass spectrometry imaging with infrared matrix-assisted laser desorption-electrospray ionization to quantify the distribution of efavirenz within tissues from a macaque dosed orally to a steady state. Intratissue efavirenz distribution was heterogeneous, with the drug concentrating in the lamina propria of the colon, the primary follicles of lymph nodes, and the brain gray matter. These are the first imaging data of an antiretroviral drug in active viral reservoirs.

Enhanced antiretroviral therapy in rhesus macaques improves RT-SHIV viral decay kinetics.

Using an established nonhuman primate model, rhesus macaques were infected intravenously with a chimeric simian immunodeficiency virus (SIV) consisting of SIVmac239 with the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase from clone HXBc2 (RT-SHIV). The impacts of two enhanced (four- and five-drug) highly active antiretroviral therapies (HAART) on early viral decay and rebound were determined. The four-drug combination consisted of an integrase inhibitor, L-870-812 (L-812), together with a three-drug regimen comprising emtricitabine [(-)-FTC], tenofovir (TFV), and efavirenz (EFV). The five-drug combination consisted of one analog for each of the four DNA precursors {using TFV, (-)-FTC, (-)-ß-D-(2R,4R)-1,3-dioxolane-2,6-diaminopurine (amdoxovir [DAPD]), and zidovudine (AZT)}, together with EFV. A cohort treated with a three-drug combination of (-)-FTC, TFV, and EFV served as treated controls. Daily administration of a three-, four-, or five-drug combination of antiretroviral agents was initiated at week 6 or 8 after inoculation and continued up to week 50, followed by a rebound period. Plasma samples were collected routinely, and drug levels were monitored using liquid chromatography-tandem mass spectrometry (LC-MS-MS). Viral loads were monitored with a standard TaqMan quantitative reverse transcriptase PCR (qRT-PCR) assay. Comprehensive analyses of replication dynamics were performed. RT-SHIV infection in rhesus macaques produced typical viral infection kinetics, with untreated controls establishing persistent viral loads of >10(4) copies of RNA/ml. RT-SHIV loads at the start of treatment (V0) were similar in all treated cohorts (P > 0.5). All antiretroviral drug levels were measureable in plasma. The four-drug and five-drug combination regimens (enhanced HAART) improved suppression of the viral load (within 1 week; P < 0.01) and had overall greater potency (P < 0.02) than the three-drug regimen (HAART). Moreover, rebound viremia occurred rapidly following cessation of any treatment. The enhanced HAART (four- or five-drug combination) showed significant improvement in viral suppression compared to the three-drug combination, but no combination was sufficient to eliminate viral reservoirs.

Microbial profiling of combat wound infection through detection microarray and next-generation sequencing.

Combat wound healing and resolution are highly affected by the resident microbial flora. We therefore sought to achieve comprehensive detection of microbial populations in wounds using novel genomic technologies and bioinformatics analyses. We employed a microarray capable of detecting all sequenced pathogens for interrogation of 124 wound samples from extremity injuries in combat-injured U.S. service members. A subset of samples was also processed via next-generation sequencing and metagenomic analysis. Array analysis detected microbial targets in 51% of all wound samples, with Acinetobacter baumannii being the most frequently detected species. Multiple Pseudomonas species were also detected in tissue biopsy specimens. Detection of the Acinetobacter plasmid pRAY correlated significantly with wound failure, while detection of enteric-associated bacteria was associated significantly with successful healing. Whole-genome sequencing revealed broad microbial biodiversity between samples. The total wound bioburden did not associate significantly with wound outcome, although temporal shifts were observed over the course of treatment. Given that standard microbiological methods do not detect the full range of microbes in each wound, these data emphasize the importance of supplementation with molecular techniques for thorough characterization of wound-associated microbes. Future application of genomic protocols for assessing microbial content could allow application of specialized care through early and rapid identification and management of critical patterns in wound bioburden.

Proteomic sample preparation for blast wound characterization.

BACKGROUND: Blast wounds often involve diverse tissue types and require substantial time and treatment for appropriate healing. Some of these subsequent wounds become colonized with bacteria requiring a better understanding of how the host responds to these bacteria and what proteomic factors contribute wound healing outcome. In addition, using reliable and effective proteomic sample preparation procedures can lead to novel biomarkers for improved diagnosis and therapy. RESULTS: To address this need, suitable sample preparation for 2-D DIGE proteomic characterization of wound effluent and serum samples from combat-wounded patients was investigated. Initial evaluation of crude effluent and serum proved the necessity of high abundant protein depletion. Subsequently, both samples were successfully depleted using Agilent Multiple Affinity Removal system and showed greatly improved 2-D spot maps, comprising 1,800 and 1,200 protein spots, respectively. CONCLUSION: High abundant protein removal was necessary for both wound effluent and serum. This is the first study to show a successful method for high abundant protein depletion from wound effluent which is compatible with downstream 2-D DIGE analysis. This development allows for improved biomarker discovery in wound effluent and serum samples.

Plasma antibody profiles in non-human primate tuberculosis.

BACKGROUND: Tuberculosis (TB) in non-human primates (NHPs) is highly contagious, requiring efficient identification of animals infected with Mycobacterium tuberculosis. Tuberculin skin test is usually used but lacks desirable sensitivity/specificity and efficiency. METHODS: We aimed to develop an immunoassay for plasma antibodies against M. tuberculosis. A key challenge is that not all infected animals contain antibodies against the same M. tuberculosis antigen. Therefore, a multiplex panel of 28 antigens (Luminex(®) -Platform) was developed. RESULTS: Data revealed antibodies against eight antigens (Rv3875, Rv3875-Rv3874 fusion, Rv3874, Rv0934, Rv3881, Rv1886c, Rv2031, Rv3841) in experimentally infected (M. tuberculosis strains: Erdman and H37Rv) NHPs (rhesus and cynomolgus macaques). In a naturally acquired M. tuberculosis infection, rhesus macaques (n = 15) with lung TB pathology (n = 10) contained antibodies to five additional antigens (Rv0831, Rv2220, Rv0054, Rv1099, and Rv0129c). CONCLUSIONS: Results suggest that this user-friendly and easily implementable multiplex panel, containing 13 M. tuberculosis antigens, may provide a high-throughput alternative for NHP TB screening.

Protein arginine methyltransferase 1-directed methylation of Kaposi sarcoma-associated herpesvirus latency-associated nuclear antigen.

The Kaposi sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) is a multifunctional protein with roles in gene regulation and maintenance of viral latency. Post-translational modification of LANA is important for functional diversification. Here, we report that LANA is subject to arginine methylation by protein arginine methyltransferase 1 in vitro and in vivo. The major arginine methylation site in LANA was mapped to arginine 20. This site was mutated to either phenylalanine (bulky hydrophobic, constitutive methylated mimetic) or lysine (positively charged, non-arginine methylatable) residues. The significance of the methylation in LANA function was examined in both the isolated form and in the context of the viral genome through the generation of recombinant KSHV. In addition, authentic LANA binding sites on the KSHV episome in naturally infected cells were identified using a whole genome KSHV tiling array. Although mutation of the methylation site resulted in no significant difference in KSHV LANA subcellular localization, we found that the methylation mimetic mutation resulted in augmented histone binding in vitro and increased LANA occupancy at identified LANA target promoters in vivo. Moreover, a cell line carrying the methylation mimetic mutant KSHV showed reduced viral gene expression relative to controls both in latency and in the course of reactivation. These results suggest that residue 20 is important for modulation of a subset of LANA functions and properties of this residue, including the hydrophobic character induced by arginine methylation, may contribute to the observed effects.

Wound outcome in combat injuries is associated with a unique set of protein biomarkers.

BACKGROUND: The ability to forecast whether a wound will heal after closure without further debridement(s), would provide substantial benefits to patients with severe extremity trauma. METHODS: Wound effluent is a readily available material which can be collected without disturbing healthy tissue. For analysis of potential host response biomarkers, forty four serial combat wound effluent samples from 19 patients with either healing or failing traumatic- and other combat-related wounds were examined by 2-D DIGE. Spot map patterns were correlated to eventual wound outcome (healed or wound failure) and analyzed using DeCyder 7.0 and differential proteins identified via LC-MS/MS. RESULTS: This approach identified 52 protein spots that were differentially expressed and thus represent candidate biomarkers for this clinical application. Many of these proteins are intimately involved in inflammatory and immune responses. Furthermore, discriminate analysis further refined the 52 differential protein spots to a smaller subset of which successfully differentiate between wounds that will heal and those that will fail and require further surgical intervention with greater than 83% accuracy. CONCLUSION: These results suggest candidates for a panel of protein biomarkers that may aid traumatic wound care prognosis and treatment. We recommend that this strategy be refined, and then externally validated, in future studies of traumatic wounds.

FIV establishes a latent infection in feline peripheral blood CD4+ T lymphocytes in vivo during the asymptomatic phase of infection.

BACKGROUND: Feline immunodeficiency virus (FIV) is a lentivirus of cats that establishes a lifelong persistent infection with immunologic impairment. RESULTS: In an approximately 2 year-long experimental infection study, cats infected with a biological isolate of FIV clade C demonstrated undetectable plasma viral loads from 10 months post-infection onward. Viral DNA was detected in CD4+CD25+ and CD4+CD25- T cells isolated from infected cats whereas viral RNA was not detected at multiple time points during the early chronic phase of infection. Viral transcription could be reactivated in latently infected CD4+ T cells ex vivo as demonstrated by detectable FIV gag RNA and 2-long terminal repeat (LTR) circle junctions. Viral LTR and gag sequences amplified from peripheral blood mononuclear cells during early and chronic stages of infection demonstrated minimal to no viral sequence variation. CONCLUSIONS: Collectively, these findings are consistent with FIV latency in peripheral blood CD4+ T cells isolated from chronically infected cats. The ability to isolate latently FIV-infected CD4+ T lymphocytes from FIV-infected cats provides a platform for the study of in vivo mechanisms of lentiviral latency.

Histone demethylase JMJD2A regulates Kaposi's sarcoma-associated herpesvirus replication and is targeted by a viral transcriptional factor.

The switch between the latency and lytic cycles of Kaposi's sarcoma-associated herpesvirus (KSHV) is accompanied by specific alterations of histone codes. Recently, comprehensive analysis of histone modifications of KSHV showed the deposition of H3K27me3 across the KSHV genome with two specific regions occupied by the heterochromatin marker H3K9me3. Here, we show that knockdown of JMJD2A, an H3K9me3 demethylase, attenuates viral titers, whereas its overexpression increases KSHV reactivation. JMJD2A is localized in regions of latent viral chromosomes that are deficient in the H3K9me3 mark, indicating that JMJD2A may be responsible for the low level of this mark on viral chromatin. The presence of JMJD2A on the latent genome maintains H3K9 in unmethylated form and signals the readiness of specific sets of viral genes to be reactivated. The demethylase activity of JMJD2A is important for KSHV reactivation, because a demethylase-deficient mutant cannot restore the JMJD2A knockdown phenotype. Interestingly, we found that the KSHV encoded K-bZIP associated with JMJD2A, resulting in the inhibition of demethylase activity of JMJD2A both in vivo and in vitro. Inhibition of JMJD2A by K-bZIP is likely due to a physical interaction which blocks substrate accessibility. A consequence of such an inhibition is increasing global levels of H3K9me3 and gene silencing. Consistently, K-bZIP overexpression resulted in a repression of ∼80% of the ≥2-fold differentially regulated genes compared to results for the uninduced control cells. The consequences of K-bZIP targeting JMJD2A during viral replication will be discussed. To our knowledge, this is the first description of a viral product shown to be a potent inhibitor of a host cellular histone demethylase.

Cluster analysis of host cytokine responses to biodefense pathogens in a whole blood ex vivo exposure model (WEEM).

BACKGROUND: Rapid detection and therapeutic intervention for infectious and emerging diseases is a major scientific goal in biodefense and public health. Toward this end, cytokine profiles in human blood were investigated using a human whole blood ex vivo exposure model, called WEEM. RESULTS: Samples of whole blood from healthy volunteers were incubated with seven pathogens including Yersinia pseudotuberculosis, Yersinia enterocolitica, Bacillus anthracis, and multiple strains of Yersinia pestis, and multiplexed protein expression profiling was conducted on supernatants of these cultures with an antibody array to detect 30 cytokines simultaneously. Levels of 8 cytokines, IL-1α, IL-1ß, IL-6, IL-8, IL-10, IP-10, MCP-1 and TNFα, were significantly up-regulated in plasma after bacterial exposures of 4 hours. Statistical clustering was applied to group the pathogens based on the host response protein expression profiles. The nearest phylogenetic neighbors clustered more closely than the more distant pathogens, and all seven pathogens were clearly differentiated from the unexposed control. In addition, the Y. pestis and Yersinia near neighbors were differentiated from the B. anthracis strains. CONCLUSIONS: Cluster analysis, based on host response cytokine profiles, indicates that distinct patterns of immunomodulatory proteins are induced by the different pathogen exposures and these patterns may enable further development into biomarkers for diagnosing pathogen exposure.

Randomized pilot trial of a synbiotic dietary supplement in chronic HIV-1 infection.

BACKGROUND: Infection with HIV-1 results in marked immunologic insults and structural damage to the intestinal mucosa, including compromised barrier function. While the development of highly active antiretroviral therapy (HAART) has been a major advancement in the treatment of HIV-1 infection, the need for novel complementary interventions to help restore intestinal structural and functional integrity remains unmet. Known properties of pre-, pro-, and synbiotics suggest that they may be useful tools in achieving this goal. METHODS: This was a 4-week parallel, placebo-controlled, randomized pilot trial in HIV-infected women on antiretroviral therapy. A synbiotic formulation (Synbiotic 2000®) containing 4 strains of probiotic bacteria (10(10) each) plus 4 nondigestible, fermentable dietary fibers (2.5 g each) was provided each day, versus a fiber-only placebo formulation. The primary outcome was bacterial translocation. Secondary outcomes included the levels of supplemented bacteria in stool, the activation phenotype of peripheral T-cells and monocytes, and plasma levels of C-reactive protein and soluble CD14. RESULTS: Microbial translocation, as measured by plasma bacterial 16S ribosomal DNA concentration, was not altered by synbiotic treatment. In contrast, the synbiotic formulation resulted in significantly elevated levels of supplemented probiotic bacterial strains in stool, including L. plantarum and P. pentosaceus, with the colonization of these two species being positively correlated with each other. T-cell activation phenotype of peripheral blood lymphocytes showed modest changes in response to synbiotic exposure, with HLA-DR expression slightly elevated on a minor population of CD4+ T-cells which lack expression of HLA-DR or PD-1. In addition, CD38 expression on CD8+ T-cells was slightly lower in the fiber-only group. Plasma levels of soluble CD14 and C-reactive protein were unaffected by synbiotic treatment in this study. CONCLUSIONS: Synbiotic treatment for 4 weeks can successfully augment the levels of probiotic species in the gut during chronic HIV-1 infection. Associated changes in microbial translocation appear to be absent, and markers of systemic immune activation appear largely unchanged. These findings may help inform future studies aimed at testing pre- and probiotic approaches to improve gut function and mucosal immunity in chronic HIV-1 infection. TRIAL REGISTRATION: Clinical Trials.gov: NCT00688311.