Programmatic Implications of National Recent HIV Infection Surveillance in Cambodia.

We compared characteristics of HIV diagnosis and recent HIV infection (ie, likely acquired within the last year) in Cambodia. We included individuals ≥ 15 years old accessing HIV testing. From August 2020 to August 2022, 53 031 people were tested for HIV, 6868 were newly diagnosed, and 192 were recently infected. We found differences in geographical burden and risk behaviors with diagnosis and recency (eg, men who have sex with men, transgender women, and entertainment workers had a nearly 2-fold increased odds of testing positive for recent infection compared to being diagnosed with HIV). Recent infection surveillance may provide unique insights into ongoing HIV acquisition to inform programs.

Chlamydia evasion of neutrophil host defense results in NLRP3 dependent myeloid-mediated sterile inflammation through the purinergic P2X7 receptor.

Chlamydia trachomatis infection causes severe inflammatory disease resulting in blindness and infertility. The pathophysiology of these diseases remains elusive but myeloid cell-associated inflammation has been implicated. Here we show NLRP3 inflammasome activation is essential for driving a macrophage-associated endometritis resulting in infertility by using a female mouse genital tract chlamydial infection model. We find the chlamydial parasitophorous vacuole protein CT135 triggers NLRP3 inflammasome activation via TLR2/MyD88 signaling as a pathogenic strategy to evade neutrophil host defense. Paradoxically, a consequence of CT135 mediated neutrophil killing results in a submucosal macrophage-associated endometritis driven by ATP/P2X7R induced NLRP3 inflammasome activation. Importantly, macrophage-associated immunopathology occurs independent of macrophage infection. We show chlamydial infection of neutrophils and epithelial cells produce elevated levels of extracellular ATP. We propose this source of ATP serves as a DAMP to activate submucosal macrophage NLRP3 inflammasome that drive damaging immunopathology. These findings offer a paradigm of sterile inflammation in infectious disease pathogenesis.

High levels of HIV drug resistance among adults failing second-line antiretroviral therapy in Namibia.

To support optimal third-line antiretroviral therapy (ART) selection in Namibia, we investigated the prevalence of HIV drug resistance (HIVDR) at time of failure of second-line ART. A cross-sectional study was conducted between August 2016 and February 2017. HIV-infected people ≥15 years of age with confirmed virological failure while receiving ritonavir-boosted protease inhibitor (PI/r)-based second-line ART were identified at 15 high-volume ART clinics representing over >70% of the total population receiving second-line ART. HIVDR genotyping of dried blood spots obtained from these individuals was performed using standard population sequencing methods. The Stanford HIVDR algorithm was used to identify sequences with predicted resistance; genotypic susceptibility scores for potential third-line regimens were calculated. Two hundred thirty-eight individuals were enrolled; 57.6% were female. The median age and duration on PI/r-based ART at time of enrolment were 37 years and 3.46 years, respectively. 97.5% received lopinavir/ritonavir-based regimens. The prevalence of nucleoside reverse transcriptase inhibitor (NRTI), non-nucleoside reverse transcriptase inhibitor (NNRTI), and PI/r resistance was 50.6%, 63.1%, and 13.1%, respectively. No significant association was observed between HIVDR prevalence and age or sex. This study demonstrates high levels of NRTI and NNRTI resistance and moderate levels of PI resistance in people receiving PI/r-based second-line ART in Namibia. Findings underscore the need for objective and inexpensive measures of adherence to identify those in need of intensive adherence counselling, routine viral load monitoring to promptly detect virological failure, and HIVDR genotyping to optimize selection of third-line drugs in Namibia.

Chlamydia trachomatis Plasmid Gene Protein 3 Is Essential for the Establishment of Persistent Infection and Associated Immunopathology.

Chlamydia trachomatis is an obligate intracellular bacterial pathogen that causes blinding trachoma and sexually transmitted disease afflicting hundreds of millions of people globally. A fundamental but poorly understood pathophysiological characteristic of chlamydial infection is the propensity to cause persistent infection that drives damaging inflammatory disease. The chlamydial plasmid is a virulence factor, but its role in the pathogenesis of persistent infection capable of driving immunopathology is unknown. Here, we show by using mouse and nonhuman primate infection models that the secreted plasmid gene protein 3 (Pgp3) is essential for establishing persistent infection. Ppg3-dependent persistent genital tract infection resulted in a severe endometritis caused by an intense infiltration of endometrial submucosal macrophages. Pgp3 released from the cytosol of lysed infected oviduct epithelial cells, not organism outer membrane-associated Pgp3, inhibited the chlamydial killing activity of antimicrobial peptides. Genetic Pgp3 rescue experiments in cathelin-related antimicrobial peptide (CRAMP)-deficient mice showed Pgp3-targeted antimicrobial peptides to subvert innate immunity as a pathogenic strategy to establish persistent infection. These findings provide important advances in understanding the role of Pgp3 in the pathogenesis of persistent chlamydial infection and associated immunopathology.IMPORTANCEChlamydia trachomatis can cause persistent infection that drives damaging inflammatory responses resulting in infertility and blindness. Little is known about chlamydial genes that cause persistence or factors that drive damaging pathology. In this work, we show that the C. trachomatis plasmid protein gene 3 (Pgp3) is the essential virulence factor for establishing persistent female genital tract infection and provide supportive evidence that Pgp3 functions similarly in a nonhuman primate trachoma model. We further show that persistent Ppg3-dependent infection drives damaging immunopathology. These results are important advances in understanding the pathophysiology of chlamydial persistence.

Compromise of Second-Line Antiretroviral Therapy Due to High Rates of Human Immunodeficiency Virus Drug Resistance in Mozambican Treatment-Experienced Children With Virologic Failure.

BACKGROUND: Virologic failure (VF) is highly prevalent in sub-Saharan African children on antiretroviral therapy (ART) and is often associated with human immunodeficiency virus drug resistance (DR). Most children still lack access to routine viral load (VL) monitoring for early identification of treatment failure, with implications for the efficacy of second-line ART. METHODS: Children aged 1 to 14 years on ART for ≥12 months at 6 public facilities in Maputo, Mozambique were consecutively enrolled after informed consent. Chart review and caregiver interviews were conducted. VL testing was performed, and specimens with ≥1000 copies/mL were genotyped. RESULTS: Of the 715 children included, the mean age was 103 months, 85.8% had no immunosuppression, 73.1% were taking stavudine/lamivudine/nevirapine, and 20.1% had a history prevention of mother-to-child transmission exposure. The mean time on ART was 60.0 months. VF was present in 259 patients (36.3%); 248 (95.8%) specimens were genotyped, and DR mutations were found in 238 (96.0%). Severe immunosuppression and nutritional decline were associated with DR. M184V and Y181C were the most common mutations. In the 238 patients with DR, standard second-line ART would have 0, 1, 2, and 3 effective antiretrovirals in 1 (0.4%), 74 (31.1%), 150 (63.0%), and 13 (5.5%) patients, respectively. CONCLUSION: This cohort had high rates of VF and DR with frequent compromise of second-line ART. There is urgent need to scale-up VL monitoring and heat-stable protease inhibitor formulations or integrase inhibitorsfor a more a durable first-line regimen that can feasibly be implemented in developing settings.

Chlamydia trachomatis Lipopolysaccharide Evades the Canonical and Noncanonical Inflammatory Pathways To Subvert Innate Immunity.

Chlamydia trachomatis is the most common bacterial cause of sexually transmitted infections. C. trachomatis sexually transmitted infections are commonly asymptomatic, implying a pathogenic strategy for the evasion of innate inflammatory immune responses, a paradox as the C. trachomatis outer membrane contains lipopolysaccharide (LPS), a known potent agonist of inflammatory innate immunity. Here, we studied the ability of chlamydial LPS to activate the proinflammatory canonical and noncanonical inflammasome pathways in mouse bone marrow-derived macrophages (BMDM). We show, in comparison to Escherichiacoli LPS, that C. trachomatis LPS-treated BMDM produce significantly less IL-6, TNF, and type I interferon mRNA, indicating that downstream signaling through the canonical TLR4 myddosome and triffosome pathways was blocked. We confirmed this in C. trachomatis LPS-treated BMDM by showing the lack of NF-κB and IRF3 phosphorylation, respectively. Interestingly, C. trachomatis LPS bound CD14 and promoted its endocytosis; however; it did not promote efficient TLR4/MD-2 dimerization or endocytosis, known requirements for myddosome and triffosome signaling pathways. We further found that transfection of BMDM with C. trachomatis LPS did not cause pyroptotic cell ballooning, cytotoxicity, or IL-1ß secretion, all characteristic features of noncanonical inflammasome activation. Western blotting confirmed that cytosolic C. trachomatis LPS failed to signal through caspase-11, as shown by the lack of gasdermin D, caspase-1, or IL-1ß proteolytic cleavage. We propose that chlamydiae evolved a unique LPS structure as a pathogenic strategy to avoid canonical and noncanonical innate immune signaling and conclude that this strategy might explain the high incidence of asymptomatic infections.IMPORTANCEChlamydia trachomatis is the most common bacterial cause of sexually transmitted infections (STI). C. trachomatis STI are commonly asymptomatic, implying a pathogenic strategy for the evasion of innate inflammatory immune responses, a paradox as the C. trachomatis outer membrane contains lipopolysaccharide (LPS), a known potent agonist of inflammatory innate immunity. Here, we found that C. trachomatis LPS is not capable of engaging the canonical TLR4/MD-2 or noncanonical caspase-11 inflammatory pathways. The inability of C. trachomatis LPS to trigger innate immunity inflammatory pathways is related to its unique fatty acid structure. Evolutionary modification of the LPS structure likely evolved as a pathogenic strategy to silence innate host defense mechanisms. The findings might explain the high incidence of asymptomatic chlamydial genital infection.

Circumcision status at HIV infection is not associated with plasma viral load in men: analysis of specimens from a randomized controlled trial.

BACKGROUND: Male circumcision provides men with approximately 60% protection from acquiring HIV infection via heterosexual sex, and has become a key component of HIV prevention efforts in sub-Saharan Africa. Possible mechanisms for this protection include removal of the inflammatory anaerobic sub-preputial environment and the high concentration of Langerhans cells on the inside of the foreskin, both believed to promote local vulnerability to HIV infection. In people who do acquire HIV, viral load is partially determined by infecting partner viral load, potentially mediated by size of infecting inoculum. By removing a portal for virion entry, prior male circumcision could decrease infecting inoculum and thus viral load in men who become HIV-infected, conferring the known associated benefits of slower progression to disease and decreased infectiousness. METHODS: We performed an as-treated analysis of plasma samples collected under a randomized controlled trial of male circumcision for HIV prevention, comparing men based on their circumcision status at the time of HIV acquisition, to determine whether circumcision is associated with lower viral load. Eligible men were seroconverters who had at least one plasma sample available drawn at least 6 months after infection, reported no potential exposures other than vaginal sex and, for those who were circumcised, were infected more than 6 weeks after circumcision, to eliminate the open wound as a confounder. Initial viral load testing indicated that quality of pre-2007 samples might have been compromised during storage and they were excluded, as were those with undetectable or unquantifiable results. Log viral loads were compared between groups using univariable and multivariable linear regression, adjusting for sample age and sexually transmitted infection diagnosis with 3.5 months of seroconversion, with a random effect for intra-individual clustering for samples from the same man. A per-protocol analysis was also performed. RESULTS: There were no viral load differences between men who were circumcised and uncircumcised at the time of HIV infection (means 4.00 and 4.03 log10 copies/mL respectively, p = .88) in any analysis. CONCLUSION: Circumcision status at the time of HIV infection does not affect viral load in men. TRIAL REGISTRATION: The original RCT which provided the samples was ClinicalTrials.gov trial NCT00059371 .

Plasmid Negative Regulation of CPAF Expression Is Pgp4 Independent and Restricted to Invasive Chlamydia trachomatis Biovars.

Chlamydia trachomatis is an obligate intracellular bacterial pathogen that causes blinding trachoma and sexually transmitted disease. C. trachomatis isolates are classified into 2 biovars-lymphogranuloma venereum (LGV) and trachoma-which are distinguished biologically by their natural host cell infection tropism. LGV biovars infect macrophages and are invasive, whereas trachoma biovars infect oculo-urogenital epithelial cells and are noninvasive. The C. trachomatis plasmid is an important virulence factor in the pathogenesis of these infections. Central to its pathogenic role is the transcriptional regulatory function of the plasmid protein Pgp4, which regulates the expression of plasmid and chromosomal virulence genes. As many gene regulatory functions are post-transcriptional, we employed a comparative proteomic study of cells infected with plasmid-cured C. trachomatis serovars A and D (trachoma biovar), a L2 serovar (LGV biovar), and the L2 serovar transformed with a plasmid containing a nonsense mutation in pgp4 to more completely elucidate the effects of the plasmid on chlamydial infection biology. Our results show that the Pgp4-dependent elevations in the levels of Pgp3 and a conserved core set of chromosomally encoded proteins are remarkably similar for serovars within both C. trachomatis biovars. Conversely, we found a plasmid-dependent, Pgp4-independent, negative regulation in the expression of the chlamydial protease-like activity factor (CPAF) for the L2 serovar but not the A and D serovars. The molecular mechanism of plasmid-dependent negative regulation of CPAF expression in the LGV serovar is not understood but is likely important to understanding its macrophage infection tropism and invasive infection nature.IMPORTANCE The Chlamydia trachomatis plasmid is an important virulence factor in the pathogenesis of chlamydial infection. It is known that plasmid protein 4 (Pgp4) functions in the transcriptional regulation of the plasmid virulence protein 3 (Pgp3) and multiple chromosomal loci of unknown function. Since many gene regulatory functions can be post-transcriptional, we undertook a comparative proteomic analysis to better understand the plasmid's role in chlamydial and host protein expression. We report that Pgp4 is a potent and specific master positive regulator of a common core of plasmid and chromosomal virulence genes shared by multiple C. trachomatis serovars. Notably, we show that the plasmid is a negative regulator of the expression of the chlamydial virulence factor CPAF. The plasmid regulation of CPAF is independent of Pgp4 and restricted to a C. trachomatis macrophage-tropic strain. These findings are important because they define a previously unknown role for the plasmid in the pathophysiology of invasive chlamydial infection.

The Chlamydia trachomatis Plasmid and CT135 Virulence Factors Are Not Essential for Genital Tract Infection or Pathology in Female Pig-Tailed Macaques.

The Chlamydia trachomatis plasmid and inclusion membrane protein CT135 are virulence factors in the pathogenesis of murine female genital tract infection. To determine if these virulence factors play a similar role in female nonhuman primates, we infected pig-tailed macaques with the same C. trachomatis strains shown to be important in the murine model. Wild-type C. trachomatis and its isogenic mutant strain deficient in both plasmid and CT135 were used to infect macaques. Macaques were given primary and repeated cervicovaginal challenges with the wild-type and mutant strains. The infection rate, infection duration, and antibody response were similar among macaques infected with both strains. Unexpectedly, colposcopy, laparoscopy, and histologic analysis revealed no substantial genital tract pathology following either primary or repeated cervicovaginal challenges. Cytokine analysis of cervicovaginal secretions from both challenged groups revealed low concentrations of interleukin 1ß (IL-1ß) and elevated levels of the interleukin 1 receptor agonist (IL-1RA). We propose that an imbalance of IL-1ß and IL-1RA in macaques is the reason for the mild inflammatory responses observed in infected urogenital tissues. Thus, understanding the pathobiology of chlamydial infection requires a better understanding of host epigenetic and chlamydial genetic factors. Our findings also have implications for understanding the high frequency of asymptomatic infections in humans.

Current Status of Point-of-Care Testing for Human Immunodeficiency Virus Drug Resistance.

Healthcare delivery has advanced due to the implementation of point-of-care testing, which is often performed within minutes to hours in minimally equipped laboratories or at home. Technologic advances are leading to point-of-care kits that incorporate nucleic acid-based assays, including polymerase chain reaction, isothermal amplification, ligation, and hybridization reactions. As a limited number of single-nucleotide polymorphisms are associated with clinically significant human immunodeficiency virus (HIV) drug resistance, assays to detect these mutations have been developed. Early versions of these assays have been used in research. This review summarizes the principles underlying each assay and discusses strategic needs for their incorporation into the management of HIV infection.

Decoding noises in HIV computational genotyping.

Lack of a consistent and reliable genotyping system can critically impede HIV genomic research on pathogenesis, fitness, virulence, drug resistance, and genomic-based healthcare and treatment. At present, mis-genotyping, i.e., background noises in molecular genotyping, and its impact on epidemic surveillance is unknown. For the first time, we present a comprehensive assessment of HIV genotyping quality. HIV sequence data were retrieved from worldwide published records, and subjected to a systematic genotyping assessment pipeline. Results showed that mis-genotyped cases occurred at 4.6% globally, with some regional and high-risk population heterogeneities. Results also revealed a consistent mis-genotyping pattern in gp120 in all studied populations except the group of men who have sex with men. Our study also suggests novel virus diversities in the mis-genotyped cases. Finally, this study reemphasizes the importance of implementing a standardized genotyping pipeline to avoid genotyping disparity and to advance our understanding of virus evolution in various epidemiological settings.

Estimating False-Recent Classification for the Limiting-Antigen Avidity EIA and BED-Capture Enzyme Immunoassay in Vietnam: Implications for HIV-1 Incidence Estimates.

Laboratory tests that can distinguish recent from long-term HIV infection are used to estimate HIV incidence in a population, but can potentially misclassify a proportion of long-term HIV infections as recent. Correct application of an assay requires determination of the proportion false recents (PFRs) as part of the assay characterization and for calculating HIV incidence in a local population using a HIV incidence assay. From April 2009 to December 2010, blood specimens were collected from HIV-infected individuals attending nine outpatient clinics (OPCs) in Vietnam (four from northern and five from southern Vietnam). Participants were living with HIV for ≥1 year and reported no antiretroviral (ARV) drug treatment. Basic demographic data and clinical information were collected. Specimens were tested with the BED capture enzyme immunoassay (BED-CEIA) and the Limiting-antigen (LAg)-Avidity EIA. PFR was estimated by dividing the number of specimens classified as recent by the total number of specimens; 95% confidence intervals (CI) were calculated. Specimens that tested recent had viral load testing performed. Among 1,813 specimens (north, n = 942 and south, n = 871), the LAg-Avidity EIA PFR was 1.7% (CI: 1.2-2.4) and differed by region [north 2.7% (CI: 1.8-3.9) versus south 0.7% (CI: 0.3-1.5); p = .002]. The BED-CEIA PFR was 2.3% (CI: 1.7-3.0) and varied by region [north 3.4% (CI: 2.4-4.7) versus south 1.0% (CI: 0.5-1.2), p < .001]. Excluding specimens with an undetectable VL, the LAg-Avidity EIA PFR was 1.2% (CI: 0.8-1.9) and the BED-CEIA PFR was 1.7% (CI: 1.2-2.4). The LAg-Avidity EIA PFR was lower than the BED-CEIA PFR. After excluding specimens with an undetectable VL, the PFR for both assays was similar. A low PFR should facilitate the implementation of the LAg-Avidity EIA for cross-sectional incidence estimates in Vietnam.

Field Evaluation of Dried Blood Spots for HIV-1 Viral Load Monitoring in Adults and Children Receiving Antiretroviral Treatment in Kenya: Implications for Scale-up in Resource-Limited Settings.

BACKGROUND: The World Health Organization recommends viral load (VL) as the preferred method for diagnosing antiretroviral therapy failure; however, operational challenges have hampered the implementation of VL monitoring in most resource-limited settings. This study evaluated the accuracy of dried blood spot (DBS) VL testing under field conditions as a practical alternative to plasma in determining virologic failure (VF). METHODS: From May to December 2013, paired plasma and DBS specimens were collected from 416 adults and 377 children on antiretroviral therapy for ≥6 months at 12 clinics in Kenya. DBSs were prepared from venous blood (V-DBS) using disposable transfer pipettes and from finger-prick capillary blood using microcapillary tubes (M-DBS) and directly spotting (D-DBS). All samples were tested on the Abbott m2000 platform; V-DBS was also tested on the Roche COBAS Ampliprep/COBAS TaqMan (CAP/CTM) version 2.0 platform. VF results were compared at 3 DBS thresholds (≥1000, ≥3000, and ≥5000 copies/mL) and a constant plasma threshold of ≥1000 copies/mL. RESULTS: On the Abbott platform, at ≥1000-copies/mL threshold, sensitivities, specificities, and kappa values for VF determination were ≥88.1%, ≥93.1%, and ≥0.82%, respectively, for all DBS methods, and it had the lowest percentage of downward misclassification compared with higher thresholds. V-DBS performance on CAP/CTM had significantly poorer specificity at all thresholds (1000%-33.0%, 3000%-60.9%, and 5000%-77.0%). No significant differences were found between adults and children. CONCLUSIONS: VL results from V-DBS, M-DBS, and D-DBS were comparable with those from plasma for determining VF using the Abbott platform but not with CAP/CTM. A 1000-copies/mL threshold was optimal and should be considered for VF determination using DBS in adults and children.

Chlamydial Lytic Exit from Host Cells Is Plasmid Regulated.

UNLABELLED: Chlamydia trachomatis is an obligate intracellular bacterium that is a globally important human pathogen. The chlamydial plasmid is an attenuating virulence factor, but the molecular basis for attenuation is not understood. Chlamydiae replicate within a membrane-bound vacuole termed an inclusion, where they undergo a biphasic developmental growth cycle and differentiate from noninfectious into infectious organisms. Late in the developmental cycle, the fragile chlamydia-laden inclusion retains its integrity by surrounding itself with scaffolds of host cytoskeletal proteins. The ability of chlamydiae to developmentally free themselves from this cytoskeleton network is a fundamental virulence trait of the pathogen. Here, we show that plasmidless chlamydiae are incapable of disrupting their cytoskeletal entrapment and remain intracellular as stable mature inclusions that support high numbers of infectious organisms. By using deletion mutants of the eight plasmid-carried genes (Δpgp1 to Δpgp8), we show that Pgp4, a transcriptional regulator of multiple chromosomal genes, is required for exit. Exit of chlamydiae is dependent on protein synthesis and is inhibited by the compound C1, an inhibitor of the type III secretion system (T3S). Exit of plasmid-free and Δpgp4 organisms, which failed to lyse infected cells, was rescued by latrunculin B, an inhibitor of actin polymerization. Our findings describe a genetic mechanism of chlamydial exit from host cells that is dependent on an unknown pgp4-regulated chromosomal T3S effector gene. IMPORTANCE: Chlamydia's obligate intracellular life style requires both entry into and exit from host cells. Virulence factors that function in exiting are unknown. The chlamydial inclusion is stabilized late in the infection cycle by F-actin. A prerequisite of chlamydial exit is its ability to disassemble actin from the inclusion. We show that chlamydial plasmid-free organisms, and also a plasmid gene protein 4 (pgp4) null mutant, do not disassociate actin from the inclusion and fail to exit cells. We further provide evidence that Pgp4-regulated exit is dependent on the chlamydial type III secretion system. This study is the first to define a genetic mechanism that functions in chlamydial lytic exit from host cells. The findings also have practical implications for understanding why plasmid-free chlamydiae are highly attenuated and have the ability to elicit robust protective immune responses.

TLR3 signaling in macrophages is indispensable for the protective immunity of invariant natural killer T cells against enterovirus 71 infection.

Enterovirus 71 (EV71) is the most virulent pathogen among enteroviruses that cause hand, foot and mouth disease in children but rarely in adults. The mechanisms that determine the age-dependent susceptibility remain largely unclear. Here, we found that the paucity of invariant natural killer T (iNKT) cells together with immaturity of the immune system was related to the susceptibility of neonatal mice to EV71 infection. iNKT cells were crucial antiviral effector cells to protect young mice from EV71 infection before their adaptive immune systems were fully mature. EV71 infection led to activation of iNKT cells depending on signaling through TLR3 but not other TLRs. Surprisingly, iNKT cell activation during EV71 infection required TLR3 signaling in macrophages, but not in dendritic cells (DCs). Mechanistically, interleukin (IL)-12 and endogenous CD1d-restricted antigens were both required for full activation of iNKT cells. Furthermore, CD1d-deficiency led to dramatically increased viral loads in central nervous system and more severe disease in EV71-infected mice. Altogether, our results suggest that iNKT cells may be involved in controlling EV71 infection in children when their adaptive immune systems are not fully developed, and also imply that iNKT cells might be an intervention target for treating EV71-infected patients.

Functionalized nanoscale micelles with brain targeting ability and intercellular microenvironment biosensitivity for anti-intracranial infection applications.

Due to complication factors such as blood-brain barrier (BBB), integrating high efficiency of brain target ability with specific cargo releasing into one nanocarrier seems more important. A brain targeting nanoscale system is developed using dehydroascorbic acid (DHA) as targeting moiety. DHA has high affinity with GLUT1 on BBB. More importantly, the GLUT1 transportation of DHA represents a "one-way" accumulative priority from blood into brain. The artificial micelles are fabricated by a disulfide linkage, forming a bio-responsive inner barrier, which can maintain micelles highly stable in circulation and shield the leakage of entrapped drug before reaching the targeting cells. The designed micelles can cross BBB and be further internalized by brain cells. Once within the cells, the drug release can be triggered by high intracellular level of glutathione (GSH). Itraconazole (ITZ) is selected as the model drug because of its poor brain permeability and low stability in blood. It demonstrates that the functionalized nanoscale micelles can achieve highly effective direct drug delivery to targeting site. Based on the markedly increased stability in blood circulation and improved brain delivery efficiency of ITZ, DHA-modified micelles show highly effective in anti-intracranial infection. Therefore, this smart nanodevice shows a promising application for the treatment of brain diseases.

HCV J6/JFH1 tilts the capability of myeloid-derived dendritic cells to favor the induction of immunosuppression and Th17-related inflammatory cytokines.

PURPOSE: How HCV virus affects the function of dendritic cells (DCs) and their ability to induce CD4+ T cell response remains not fully understood. This study was done to elucidate the impact of HCV on the function of DCs and on DC's capability to induce CD4+ T-cell response. METHODS: Monocyte-derived DCs (MoDCs) were treated with cell-culture HCV (HCVcc). The effects of HCVcc on DC maturation, CD40L-induced DC maturation, and cytokine production and the capacity of DCs to induce Th cytokine production of allogeneic CD4+ T cells were evaluated. RESULTS: HCVcc exposure increased expression of both IL-6 and IL-10 by MoDCs. HCV-exposed MoDCs also selectively facilitated allogeneic CD4+ T cells to further produce Th17-related cytokines interleukin 1 (IL-1), IL-6, and IL-17A. Pretreatment of IL-17A inhibited HCV production in Huh7.5 cells, suggesting that induction of Th17 cells may be beneficial to host anti-HCV immunity. Paradoxically, induction of IL-10 expression and the failure of HCV-exposed MoDCs to facilitate other Th cell development may hinder the anti-viral immunity. CONCLUSIONS: This study highlights both the therapeutic potential of IL-17A in treating HCV infection and the cautious consideration of HCV-induced immunosuppression in DC-based therapy.

Limited utility of dried-blood- and plasma spot-based screening for antiretroviral treatment failure with Cobas Ampliprep/TaqMan HIV-1 version 2.0.

The 2013 WHO antiretroviral therapy (ART) guidelines recommend dried blood spots (DBS) as an alternative specimen type for viral load (VL) monitoring. We assessed the programmatic utility of screening for antiretroviral (ARV) treatment failure (TF) at 5,000 and 1,000 copies/ml using DBS and dried plasma spots (DPS) with a commonly used VL assay, the Roche Cobas Ampliprep/Cobas TaqMan V.2.0 (CAP/CTM). Plasma, DBS, and DPS were prepared from 839 whole-blood specimens collected from patients on ART for ≥ 6 months at three public facilities in Namibia. Using the CAP/CTM test, VL were measured in plasma, DBS, and DPS, and the results were compared using the plasma VL as the reference standard. The clinical sensitivities, specificities, and positive (PPV) and negative predictive values (NPV) of DBS at ARV TF diagnostic thresholds of 5,000 copies/ml and 1,000 copies/ml were 0.99, 0.55, 0.33, and 0.99 and 0.99, 0.26, 0.29, and 0.99, respectively, and for DPS at TF diagnostic thresholds of 5,000 copies/ml and 1,000 copies/ml, they were 0.88, 0.98, 0.92, and 0.97 and 0.91, 0.96, 0.89, and 0.97, respectively. The prevalences of TF were overestimated in DBS by 33% and 57% at these two thresholds, respectively. A high rate of false-positive results would occur if the CAP/CTM with DBS were to be used to screen for ARV TF. WHO recommendations for DBS-based VL monitoring should be specific to the VL assay version and type. Despite the better performance of DPS, the programmatic utility for TF screening may be limited by requirements for processing the whole blood at the collection site.

SSC(high)CD11b(high)Ly-6C(high)Ly-6G(low) myeloid cells curtail CD4 T cell response by inducible nitric oxide synthase in murine hepatitis.

Myeloid-derived suppressor cells (MDSCs) play an important role in maintaining immune tolerance in response to tumors and inflammatory diseases. Several liver MDSCs have been described in hepatitis in humans and mouse models. Although all the murine MDSCs are CD11b(+)Gr-1(+), their true phenotype and mechanism of suppression remain elusive. This study revealed that SSC(high)CD11b(high)Ly-6C(high)Ly-6G(low) monocytic cells but not the other liver-infiltrating, CD11b(+)Gr-1(+) subsets could suppress CD4 T cell responses. Their suppressive activity was remarkably effective even at a ratio of 1:50 when co-cultured with CD4 T cells. Mechanistically, the suppression was dependent on nitric oxide production by inducible nitric oxide synthase (iNOS). Furthermore, the suppressive function by these liver MDSCs was found to require direct contact with activated CD4 T cells. Adoptive transfer experiments demonstrate that these liver MDSCs can dramatically ameliorate concanavalin A (Con A)-induced fulminant hepatitis in mice. Finally, MDSC-mediated suppression in vivo was dependent on iNOS expression. Altogether, SSC(high)CD11b(high)Ly-6C(high)Ly-6G(low) cells represent authentic MDSCs in the inflammatory liver and may function to minimize collateral damage caused by an overzealous CD4 T cell response following hepatitis infection.

Th2-type inflammation under conditions of pre-existing chronic disease is associated with liver damage in patients with avian influenza H7N9 virus.

Most patients infected with avian H7N9 influenza virus develop severe disease, including respiratory failure, acute respiratory distress syndrome (ARDS), and multi-organ failure. The pathogenesis of H7N9 infection is not fully understood. This study revealed that H7N9-infected patients who had fatal outcomes or critical illness all had pre-existing chronic diseases. The patients did not have obvious systemic inflammation compared to the healthy controls. However, their fatal outcomes and critically severe illness were significantly associated with high serum levels of tumor necrosis factor (TNF)-α. Interestingly, the degree of liver damage in these patients significantly correlated with their serum levels of Th2 cytokines, interleukin (IL)-4, and IL-9. Taken together, our results suggest that Th2-type inflammation in H7N9-infected patients with pre-existing chronic diseases likely contributes to the pathogenesis of H7N9 infection and is linked to poor clinical outcomes.