Inhibition of HIV-1 infection of primary CD4+ T-cells by gene editing of CCR5 using adenovirus-delivered CRISPR/Cas9.

CCR5 serves as an essential coreceptor for human immunodeficiency virus type 1 (HIV-1) entry, and individuals with a CCR5(Δ32) variant appear to be healthy, making CCR5 an attractive target for control of HIV-1 infection. The CRISPR/Cas9, which functions as a naturally existing adaptive immune system in prokaryotes, has been recently harnessed as a novel nuclease system for genome editing in mammalian cells. Although CRISPR/Cas9 can be readily delivered into cell lines, due to the large size of the Cas9 protein, efficient delivery of CCR5-targeting CRISPR/Cas9 components into primary cells, including CD4(+) T-cells, the primary target for HIV-1 infection in vivo, remains a challenge. In the current study, following design of a panel of top-ranked single-guided RNAs (sgRNAs) targeting the ORF of CCR5, we demonstrate that CRISPR/Cas9 can efficiently mediate the editing of the CCR5 locus in cell lines, resulting in the knockout of CCR5 expression on the cell surface. Next-generation sequencing revealed that various mutations were introduced around the predicted cleavage site of CCR5. For each of the three most effective sgRNAs that we analysed, no significant off-target effects were detected at the 15 top-scoring potential sites. More importantly, by constructing chimeric Ad5F35 adenoviruses carrying CRISPR/Cas9 components, we efficiently transduced primary CD4(+) T-lymphocytes and disrupted CCR5 expression, and the positively transduced cells were conferred with HIV-1 resistance. To our knowledge, this is the first study establishing HIV-1 resistance in primary CD4(+) T-cells utilizing adenovirus-delivered CRISPR/Cas9.

CCL19 and CCL28 augment mucosal and systemic immune responses to HIV-1 gp140 by mobilizing responsive immunocytes into secondary lymph nodes and mucosal tissue.

Induction of broad and potent neutralizing Abs at the mucosal portals of entry remains a primary goal for most vaccines against mucosally acquired viral infections. Selection of appropriate adjuvants capable of promoting both systemic and mucosal responses will be crucial for the development of effective immunization strategies. In this study, we investigated whether plasmid codelivery of cytokines APRIL, CCL19, or CCL28 can enhance Ag-induced immune responses to HIV-1 gp140. Our results demonstrated that pCCL19 and pCCL28, but not pAPRIL, significantly enhanced Ag-specific systemic and mucosal Ab responses. gp140-specific Abs in serum enhanced by pCCL19 or pCCL28 were broadly distributed across all four IgG subclasses, of which IgG1 was predominant. The enhanced systemic and mucosal Abs showed increased neutralizing activity against both homologous and heterologous HIV-1, and potency correlated with gp140-specific serum IgG and vaginal IgA levels. Measurement of gp140-specific cytokines produced by splenocytes demonstrated that pCCL19 and pCCL28 augmented balanced Th1/Th2 responses. pCCL19 and pCCL28 also increased IgA(+) cells in colorectal mucosal tissue. pCCL19 codelivery resulted in an increase of CCR7(+) CD11c(+) cells in mesenteric lymph nodes and both CCR7(+) CD11c(+) cells and CCR7(+) CD3e(+) cells in spleen, whereas pCCL28 codelivery resulted in an augment of CCR10(+) CD19(+) cells in both spleen and mesenteric lymph nodes. Together, our data indicate that pCCL19 and pCCL28 can enhance HIV-1 envelope-specific systemic and mucosal Ab responses, as well as T cell responses. Such enhancements appear to be associated with mobilization of responsive immunocytes into secondary lymphoid organs and mucosal tissues through interactions with corresponding receptors.

Herpes simplex virus type 2 infection of human epithelial cells induces CXCL9 expression and CD4+ T cell migration via activation of p38-CCAAT/enhancer-binding protein-ß pathway.

Recruitment of CD4(+) T cells to infection areas after HSV-2 infection may be one of the mechanisms that account for increased HIV-1 sexual transmission. Lymphocytes recruited by chemokine CXCL9 are known to be important in control of HSV-2 infection in mice, although the underlying mechanism remains to be addressed. Based on our observation that CXCL9 expression is augmented in the cervical mucus of HSV-2-positive women, in this study we demonstrate that HSV-2 infection directly induces CXCL9 expression in primary cervical epithelial cells and cell lines, the principal targets of HSV-2, at both mRNA and protein levels. Further studies reveal that the induction of CXCL9 expression by HSV-2 is dependent upon a binding site for C/EBP-ß within CXCL9 promoter sequence. Furthermore, CXCL9 expression is promoted at the transcriptional level through phosphorylating C/EBP-ß via p38 MAPK pathway, leading to binding of C/EBP-ß to the CXCL9 promoter. Chemotaxis assays indicate that upregulation of CXCL9 expression at the protein level by HSV-2 infection enhances the migration of PBLs and CD4(+) T cells, whereas neutralization of CXCL9 or inhibition of p38-C/EBP-ß pathway can significantly decrease the migration. Our data together demonstrate that HSV-2 induces CXCL9 expression in human cervical epithelial cells by activation of p38-C/EBP-ß pathway through promoting the binding of C/EBP-ß to CXCL9 promoter, which may recruit activated CD4(+) T cells to mucosal HSV-2 infection sites and potentially increase the risk of HIV-1 sexual transmission.

Bifunctional CD4-DC-SIGN fusion proteins demonstrate enhanced avidity to gp120 and inhibit HIV-1 infection and dissemination.

Early stages of mucosal infection are potential targets for HIV-1 prevention. CD4 is the primary receptor in HIV-1 infection whereas DC-SIGN likely plays an important role in HIV-1 dissemination, particularly during sexual transmission. To test the hypothesis that an inhibitor simultaneously targeting both CD4 and DC-SIGN binding sites on gp120 may provide a potent anti-HIV strategy, we designed constructs by fusing the extracellular CD4 and DC-SIGN domains together with varied arrangements of the lengths of CD4, DC-SIGN and the linker. We expressed, purified and characterized a series of soluble CD4-linker-DC-SIGN (CLD) fusion proteins. Several CLDs, composed of a longer linker and an extra neck domain of DC-SIGN, had enhanced affinity for gp120 as evidenced by molecular-interaction analysis. Furthermore, such CLDs exhibited significantly enhanced neutralization activity against both laboratory-adapted and primary HIV-1 isolates. Moreover, CLDs efficiently inhibited HIV-1 infection in trans via a DC-SIGN-expressing cell line and primary human dendritic cells. This was further strengthened by the results from the human cervical explant model, showing that CLDs potently prevented both localized and disseminated infections. This is the first time that soluble DC-SIGN-based bifunctional proteins have demonstrated anti-HIV potency. Our study provides proof of the concept that targeting both CD4 and DC-SIGN binding sites on gp120 represents a novel antiviral strategy. Given that DC-SIGN binding to gp120 increases exposure of the CD4 binding site and that the soluble forms of CD4 and DC-SIGN occur in vivo, further improvement of CLDs may render them potentially useful in prophylaxis or therapeutics.

Removal of two high-mannose N-linked glycans on gp120 renders human immunodeficiency virus 1 largely resistant to the carbohydrate-binding agent griffithsin.

High-mannose N-linked glycans recognized by carbohydrate-binding agents (CBAs) are potential targets for topical microbicides. To better understand the mechanisms by which CBAs inhibit human immunodeficiency virus (HIV)-1 infection at the molecular level, we systematically analysed the contribution of site-specific glycans to the anti-HIV activity of CBAs by site-directed mutagenesis. Our results demonstrate that a single deglycosylation at N295 or N448 in a range of primary and T-cell-line-adapted HIV-1 isolates resulted in marked resistance to griffithsin (GRFT) but maintained the sensitivity to cyanovirin (CV-N), Galanthus nivalis agglutinin (GNA) and a range of neutralizing antibodies. Unlike CV-N and GNA, the interaction between GRFT and gp120 appeared to be dependent on the specific trimeric 'sugar tower' including N295 and N448. This was further strengthened by the results of GRFT-Env binding experiments. Our study identifies GRFT-specific gp120 glycans and may provide information for the design of novel CBA antiviral strategies.

Impact of early life exposures to geohelminth infections on the development of vaccine immunity, allergic sensitization, and allergic inflammatory diseases in children living in tropical Ecuador: the ECUAVIDA birth cohort study.

BACKGROUND: Geohelminth infections are highly prevalent infectious diseases of childhood in many regions of the Tropics, and are associated with significant morbidity especially among pre-school and school-age children. There is growing concern that geohelminth infections, particularly exposures occurring during early life in utero through maternal infections or during infancy, may affect vaccine immunogenicity in populations among whom these infections are endemic. Further, the low prevalence of allergic disease in the rural Tropics has been attributed to the immune modulatory effects of these infections and there is concern that widespread use of anthelmintic treatment in high-risk groups may be associated with an increase in the prevalence of allergic diseases. Because the most widely used vaccines are administered during the first year of life and the antecedents of allergic disease are considered to occur in early childhood, the present study has been designed to investigate the impact of early exposures to geohelminths on the development of protective immunity to vaccines, allergic sensitization, and allergic disease. METHODS/DESIGN: A cohort of 2,403 neonates followed up to 8 years of age. Primary exposures are infections with geohelminth parasites during the last trimester of pregnancy and the first 2 years of life. Primary study outcomes are the development of protective immunity to common childhood vaccines (i.e. rotavirus, Haemophilus influenzae type B, Hepatitis B, tetanus toxoid, and oral poliovirus type 3) during the first 5 years of life, the development of eczema by 3 years of age, the development of allergen skin test reactivity at 5 years of age, and the development of asthma at 5 and 8 years of age. Potential immunological mechanisms by which geohelminth infections may affect the study outcomes will be investigated also. DISCUSSION: The study will provide information on the potential effects of early exposures to geohelminths (during pregnancy and the first 2 years of life) on the development of vaccine immunity and allergy. The data will inform an ongoing debate of potential effects of geohelminths on child health and will contribute to policy decisions on new interventions designed to improve vaccine immunogenicity and protect against the development of allergic diseases. TRIAL REGISTRATION: Current Controlled Trials ISRCTN41239086.

Blockade of attachment and fusion receptors inhibits HIV-1 infection of human cervical tissue.

Identification of cellular factors involved in HIV-1 entry and transmission at mucosal surfaces is critical for understanding viral pathogenesis and development of effective prevention strategies. Here we describe the evaluation of HIV-1 entry inhibitors for their ability to prevent infection of, and dissemination from, human cervical tissue ex vivo. Blockade of CD4 alone or CCR5 and CXCR4 together inhibited localized mucosal infection. However, simultaneous blockade of CD4 and mannose-binding C-type lectin receptors including dendritic cell-specific intercellular adhesion molecule-grabbing integrin was required to inhibit HIV-1 uptake and dissemination by migratory cells. In contrast, direct targeting of HIV-1 by neutralizing mAb b12 and CD4-IgG2 (PRO-542) blocked both localized infection and viral dissemination pathways. Flow cytometric analysis and immunostaining of migratory cells revealed two major populations, CD3(+)HLA-DR(-) and CD3(-)HLA-DR(+) cells, with a significant proportion of the latter also expressing dendritic cell-specific intercellular adhesion molecule-grabbing integrin. Bead depletion studies demonstrated that such HLA-DR(+) cells accounted for as much as 90% of HIV-1 dissemination. Additional studies using immature monocyte-derived dendritic cells demonstrated that although mannose-binding C-type lectin receptors and CD4 are the principal receptors for gp120, other mechanisms may account for virus capture. Our identification of the predominant receptors involved in HIV-1 infection and dissemination within human cervical tissue highlight important targets for microbicide development.

Rapid turnover of effector-memory CD4(+) T cells in healthy humans.

Memory T cells can be divided into central-memory (T(CM)) and effector-memory (T(EM)) cells, which differ in their functional properties. Although both subpopulations can persist long term, it is not known whether they are maintained by similar mechanisms. We used in vivo labeling with deuterated glucose to measure the turnover of CD4(+) T cells in healthy humans. The CD45R0(+)CCR7(-) T(EM) subpopulation was shown to have a rapid proliferation rate of 4.7% per day compared with 1.5% per day for CD45R0(+)CCR7(+) T(CM) cells; these values are equivalent to average intermitotic (doubling) times of 15 and 48 d, respectively. In contrast, the CD45RA(+)CCR7(+) naive CD4(+) T cell population was found to be much longer lived, being labeled at a rate of only 0.2% per day (corresponding to an intermitotic time of approximately 1 yr). These data indicate that human CD4(+) T(EM) cells constitute a short-lived cell population that requires continuous replenishment in vivo.

Characterization of Zika Virus Endocytic Pathways in Human Glioblastoma Cells.

Zika virus (ZIKV) infections can cause microcephaly and neurological disorders. However, the early infection events of ZIKV in neural cells remain to be characterized. Here, by using a combination of pharmacological and molecular approaches and the human glioblastoma cell T98G as a model, we first observed that ZIKV infection was inhibited by chloroquine and NH4Cl, indicating a requirement of low intracellular pH. We further showed that dynamin is required as the ZIKV entry was affected by the specific inhibitor dynasore, small interfering RNA (siRNA) knockdown of dynamin, or by expressing the dominant-negative K44A mutant. Moreover, the ZIKV entry was significantly inhibited by chlorpromazine, pitstop2, or siRNA knockdown of clathrin heavy chain, indicating an involvement of clathrin-mediated endocytosis. In addition, genistein treatment, siRNA knockdown of caveolin-1, or overexpression of a dominant-negative caveolin mutant impacted the ZIKV entry, with ZIKV particles being observed to colocalize with caveolin-1, implying that caveola endocytosis can also be involved. Furthermore, we found that the endocytosis of ZIKV is dependent on membrane cholesterol, microtubules, and actin cytoskeleton. Importantly, ZIKV infection was inhibited by silencing of Rab5 and Rab7, while confocal microscopy showed that ZIKV particles localized in Rab5- and Rab7-postive endosomes. These results indicated that, after internalization, ZIKV likely moves to Rab5-positive early endosome and Rab7-positive late endosomes before delivering its RNA into the cytoplasm. Taken together, our study, for the first time, described the early infection events of ZIKV in human glioblastoma cell T98G.

Environmental determinants of total IgE among school children living in the rural Tropics: importance of geohelminth infections and effect of anthelmintic treatment.

BACKGROUND: The environmental factors that determine the elevated levels of polyclonal IgE observed in populations living in the Tropics are poorly understood but may include geohelminth infections. We investigated the association between geohelminth infections and total IgE levels in school children in rural tropical Ecuador, and assessed the effect on IgE of repeated anthelmintic treatments over a period of 12 months. The study was nested within a cluster-randomized study that randomized 68 schools to receive either 400 mg of albendazole every 2 months over a year or no treatment. We studied random samples of children completing follow-up and representing four groups stratified by the presence of geohelminth infection at baseline and treatment allocation. We measured levels of total IgE and anti-A. lumbricoides IgG (used as a measure of past and current geohelminth infectious exposure) in blood samples collected at the start of the study and after 12 months. RESULTS: We observed elevated levels of total IgE (compared to standard reference values) at the start of the study in this population of school children (geometric mean, 1,004 IU/mL, range 12 to 22,608 IU/mL)) and baseline IgE levels were strongly associated with parameters of geohelminth infection but not with age, nutritional and socioeconomic status. After 12 months, levels of IgE fell significantly in the treatment (by 35.1%) and no treatment (by 10.4%) groups, respectively, but the fall was significantly greater in the treatment group. Falls in IgE were independently associated with albendazole treatment, having a baseline geohelminth infection and with high baseline levels of anti-A. lumbricoides IgG. Increases in IgE at 12 months were associated with the presence of geohelminth infections and increasing levels of anti-A. lumbricoides IgG at 12 months independent of treatment allocation. CONCLUSION: The data provide evidence that geohelminth infections are an important determinant of total IgE in school children in the rural Tropics and that periodic anthelmintic treatments over 12 months are associated with reductions in IgE. The failure of anthelmintic treatment to reduce IgE levels to that considered normal in industrialized countries may be attributed to continued exposure of children to geohelminths or to the effects of infections in early life in programming a long-lasting Th2-biassed immunity.

HIV enteropathy: crypt stem and transit cell hyperproliferation induces villous atrophy in HIV/Microsporidia-infected jejunal mucosa.

OBJECTIVES: The study aim was to analyse the kinetics of stem and transit cells in the crypts of jejunal mucosa infected with HIV and Microsporidia. DESIGN: The size of villi, depth of crypts and proliferative activity of transit and stem cells in jejunal mucosa were measured using morphometric techniques. METHODS: The surface area/volume ratio (S/V) of jejunal biopsies was estimated under light microscopy using a Weibel graticule. Crypt length was measured by counting enterocytes along the crypt side from the base to the villus junction, and the mean crypt length was calculated. The S/V and crypt lengths of the jejunal mucosa of 21 HIV and Microsporidia-infected test cases were compared with 14 control cases. The labelling index in relation to the crypt cell position of 10 of the test cases was analysed compared with 13 control cases. RESULTS: Differences were found in the S/V and crypt length, and there was a negative correlation between S/V and crypt length in test and control cases combined. Cell labelling indices fell into low and high proliferation groups. There were significant differences in labelling indices between low proliferation test cases and controls, between high proliferation test cases and controls, and between high and low proliferation test cases. CONCLUSION: Villous atrophy induced by HIV and Microsporidia is attributed to crypt cell hyperplasia and the encroachment of crypt cells onto villi. These infections induce crypt hypertrophy by stimulating cell mitosis predominantly in transit cells but also in stem cells. Increased stem cell proliferation occurs only in high proliferation cases.

Long-term effects of perinatal nutrition on T lymphocyte kinetics in young Gambian men.

BACKGROUND: Nutritional status is highly dependent on season in countries such as The Gambia. In a rural Gambian setting, individuals born during periods of seasonal nutritional deprivation ("hungry seasons") are susceptible to mortality from infectious diseases in adult life. OBJECTIVE: We investigated the hypothesis that impaired immunocompetence in those born in the hungry season results from an underlying defect in immunologic memory, similar to the immunosenescence of old age, which is likely to be reflected in the phenotype and kinetics of T lymphocytes in young adults. DESIGN: T cell phenotype in terms of CD3, CD4, CD8, CD45RA, and CD45R0 expression and in vivo dynamics measured by stable isotope labeling of T cell subsets combined with gas chromatography-mass spectrometry and frequency of T cell receptor excision circles were measured in 25 young (18-24-y-old) Gambian men. Thirteen of these 25 men were exposed to perinatal malnutrition as defined by birth season and birth weight. RESULTS: In persons born in the hungry season with low birth weight, no differences in the proportions of memory or naive T cells were found. Kinetic analysis showed higher proliferation rates in memory (CD45R0(+)) subsets of T cells than in naïve (CD45R0(-)) cells, which is consistent with previous studies, but no evidence was found for an effect of birth weight or season on T lymphocyte proliferation and disappearance rates. No significant correlations were found between in vivo T cell kinetics and frequency of T cell receptor excision circles. Only absolute numbers of granulocytes were elevated in those born in the nutritionally deprived season. CONCLUSION: In healthy young Gambian men, T lymphocyte homeostasis is extremely robust regardless of perinatal nutritional compromise.

Effect of albendazole treatments on the prevalence of atopy in children living in communities endemic for geohelminth parasites: a cluster-randomised trial.

BACKGROUND: Epidemiological studies have shown inverse associations between geohelminth (intestinal helminth) infection and atopy, leading to the suggestion that geohelminths might protect against allergy. Periodic deworming of school children with anthelmintics is a widely implemented intervention and has raised concerns that such programmes could increase allergy. We investigated the effect of repeated anthelmintic treatments with albendazole over 12 months on the prevalence of atopy and clinical indices of allergy. METHODS: We did a cluster-randomised controlled trial in schoolchildren from 68 rural schools. Children were randomly assigned by school to either albendazole (34 schools, 1164 children) every 2 months for 12 months, or to no intervention (34 schools, 1209 children). The intervention schools received a total of seven albendazole treatments. The primary outcome was atopy at 12 months (allergen skin-test reactivity), and analysis was by intention-to-treat for whole-school analyses and per protocol for children. This study is registered as an International Standard Randomised Controlled Trial, number ISRCTN61195515. FINDINGS: Data for analysis were available for all schools and from 67.4% (784 of 1164) and 70.1% (848 of 1209) of children in albendazole and no-treatment groups, respectively. Albendazole treatment caused large reductions in geohelminth prevalence over the study period (adjusted odds ratio 0.13, 95% CI 0.09-0.19, p<0.001), but there was no evidence that treatment was associated with an increase in atopy prevalence (0.97, 0.68-1.39, p=0.862), or clinical allergy (wheeze, 1.07, 0.54-2.11, p=0.848) in the albendazole compared with the no-treatment group. INTERPRETATION: We saw no increase in the prevalence of atopy or clinical allergy associated with albendazole treatment. Deworming programmes for schoolchildren are unlikely to be accompanied by an increase in allergy.

Chemokine/cytokine production by mononuclear cells from human lymphoid tissues and their modulation by Mycobacterium tuberculosis antigens.

The majority of knowledge about the role of cytokines and chemokines in controlling Mycobacterium tuberculosis infection mainly derives from animal models. In humans, this knowledge is still mainly limited to the blood compartment or accessible lymphoid organs, such as tonsils. Here, we studied cytokine and chemokine production and their modulation by M. tuberculosis antigens in mononuclear cells from human blood, spleen and hilar lung lymph nodes. Results show that the kinetics and magnitude of cytokine and chemokine production varied according to the tissue of cell origin. Mycobacterium tuberculosis antigens enhanced cytokine and chemokine production in blood, but the enhancement was restricted in spleen and hilar lung lymph node cells. We show, for the first time in humans, differences in cytokine and chemokine microenvironments according to lymphoid tissues, and suggest that these differences may affect the way cells respond to M. tuberculosis infection.

Restricted variable residues in the C-terminal segment of HIV-1 V3 loop regulate the molecular anatomy of CCR5 utilization.

The V3 loop of the HIV-1 envelope glycoprotein (Env) is the major determinant for coreceptor utilization, but the structural basis for this specificity remains to be defined. By characterizing a set of naturally occurring R5 Env variants, we demonstrate that Asp324 in the conserved IIGDIR motif of the V3 loop (CTRPN(300)NNTRKSIHIGP(311)GRAFYTTGEIIGD(324)IRQAHC) C-terminal segment regulates the molecular anatomy of CCR5 utilization. Whereas gp120 subunits with Asp or Asn at position 324 were fusogenic with coreceptor chimeras containing either the N-terminal domain or the body of CCR5, substitution of charged (Glu, Lys) or small hydrophobic (Gly, Ala) residues resulted in complete loss of fusogenic activity with the N terminus and markedly reduced utilization of the body of CCR5, although their ability to use wild-type CCR5 was unchanged. This phenotypic conversion was confirmed in both gain and loss of function experiments using Env from multiple subtypes. Alignment of sequences of R5 V3 loops (n=599) from the HIV database revealed that the mutation of Asp324 in the conserved IIGDIR motif is restricted to Asn324, with proportions of 71.5% and 28%, respectively. Infection of primary CD4(+)T cells demonstrated that Env bearing Asp324 was less sensitive to RANTES, suggesting that Asp or Asn in this position may be crucial for viral fitness. The CD4-dependent gp120 binding to CCR5 was decreased when Asp324 was replaced with a charged or hydrophobic residue, but unchanged when replaced with Asn. Molecular modeling analyses predicted that Asp/Asn324 forms a critical H-bond with Asn300. These findings indicate that Asp or Asn at position 324 of the V3 stem stabilizes the conformation of V3 loop and hence influences the intensities of interaction between CD4-activated gp120 and CCR5 which results in viral entry.

Combination antifungal therapies for HIV-associated cryptococcal meningitis: a randomised trial.

BACKGROUND: It frequently takes more than 2 weeks for drug treatments for cryptococcal meningitis to sterilise cerebrospinal fluid (CSF). In-vitro and animal studies lend support to the use of combinations of amphotericin B, flucytosine, and fluconazole for treatment of cryptococcosis. We compared the fungicidal activity of combinations of these drugs for initial treatment of patients with cryptococcal meningitis. METHODS: 64 patients with a first episode of HIV-associated cryptococcal meningitis were randomised to initial treatment with: amphotericin B (0.7 mg/kg daily); amphotericin B plus flucytosine (100 mg/kg daily); amphotericin B plus fluconazole (400 mg daily); or triple therapy with amphotericin B, flucytosine, and fluconazole. Our primary endpoint was fungicidal activity, measured by the rate of reduction in CSF cryptococcal colony-forming units (CFU) from serial quantitative CSF cultures on days 3, 7, and 14 of treatment. FINDINGS: Baseline CSF CFU counts were an important prognostic factor. Clearance of cryptococci from the CSF was exponential and was significantly faster with amphotericin B plus flucytosine than with amphotericin B alone (p=0.0006), amphotericin B plus fluconazole ( p=0.02), or triple therapy (p=0.02). INTERPRETATION: At these doses, amphotericin B plus flucytosine is the most rapidly fungicidal regimen. Quantification of CSF cultures provides a powerful new means to accurately assess the fungicidal activity of new treatment regimens for cryptococcal meningitis.

Measuring lymphocyte kinetics in tropical field settings.

Studies involving in-vivo labelling of lymphocyte DNA with 6,6-2H2-glucose to track T-cell turnover have contributed to understanding lymphocyte homeostasis in health and disease. Applying such studies in tropical settings (where diseases that affect T-cells are prevalent) requires protocol modifications including non-intravenous label administration, applicability in outpatient facilities, and T-cell sorting methods independent of a fluorescence activated cell sorter (FACS). Such protocols were validated in UK pilot studies and applied in The Gambia. Healthy adult subjects (n=12) were recruited from three Gambian villages. 6,6-2H2-glucose was administered orally in an outpatient clinic and T-cell subpopulations isolated from peripheral blood using plastic adherence, and Multisorttrade mark magnetic cell sorting (MACStrade mark) to obtain CD8+CD45R0+, CD8-CD45R0+, CD8+CD45R0- and CD8-CD45R0- subsets. To achieve high cell purity and yield, CD45R0- cells were reincubated with CD45R0 beads. T-cell proliferation and disappearance were quantified using gas chromatography mass spectrometry. Results were consistent with those of other studies showing higher turnover in memory (CD45R0+) than in naïve (CD45R0-) T-cell subsets, and an association between recent cell proliferation and susceptibility to cell death. Cell kinetics research is applicable in tropical settings, and can contribute to further understanding the regulation of adaptive immunity in response to infections and other insults.

Effect of recombinant human growth hormone on exercise capacity in patients with HIV-associated wasting on HAART.

Over 700 patients with HIV-associated wasting while receiving HAART were randomly assigned to double-blind treatment for 12 weeks with recombinant human growth hormone (rhGH) daily or on alternate days, or to placebo. Maximum exercise intensity increased by a median of 2.35kJ in the alternate-days group and 2.60 kJ in the daily group but decreased by 0.25kJ in the placebo group. The median difference between the daily and placebo groups was 2.85 kJ (P < .0001). These improvements suggest that rhGH treatment may enable patients with wasting to perform activities of daily living that would be exhausting without rhGH treatment.

Tetherin restricts HSV-2 release and is counteracted by multiple viral glycoproteins.

Tetherin has been defined as a restriction factor of HIV-1 and several other enveloped viruses. However, the significance of tetherin in viral infection remains to be further addressed. Here, we investigated whether tetherin plays a role in HSV-2 infection. Our study revealed that overexpression of tetherin restricted the release of HSV-2 into the extracellular medium, while knockdown of tetherin by siRNA enhanced its release. We further demonstrated that HSV-2 infection and viral glycoproteins gB, gD, gH and gL but not gM significantly downregulated the endogenous expression of tetherin. Additional study indicated that tetherin likely physically interacted with gB, gD, gH and gL. This is the first time that tetherin has been shown to be counteracted by multiple viral components of a virus. Our findings inform the complexity of HSV-2-host interactions, providing basis for understanding the role of tetherin as a viral restriction factor and the mechanisms underlying viral countermeasures.