The effect of varying analytical methods on estimates of anti-malarial clinical efficacy

<p>Abstract</p> <p>Background</p> <p>Analytical approaches for the interpretation of anti-malarial clinical trials vary considerably. The aim of this study was to quantify the magnitude of the differences between efficacy estimates derived from these approaches and identify the factors underlying these differences.</p> <p>Methods</p> <p>Data from studies conducted in Africa and Thailand were compiled and the risk estimates of treatment failure, adjusted and unadjusted by genotyping, were derived by three methods (intention to treat (ITT), modified intention to treat (mITT) and per protocol (PP)) and then compared.</p> <p>Results</p> <p>29 clinical trials (15 from Africa and 14 from Thailand) with a total of 65 treatment arms (38 from Africa and 27 from Thailand) were included in the analysis. Of the 15,409 patients enrolled, 2,637 (17.1%) had incomplete follow up for the unadjusted analysis and 4,489 (33.4%) for the adjusted analysis. Estimates of treatment failure were consistently higher when derived from the ITT or PP analyses compared to the mITT approach. In the unadjusted analyses the median difference between the ITT and mITT estimates was greater in Thai studies (11.4% [range 2.1–31.8]) compared to African Studies (1.8% [range 0–11.7]). In the adjusted analyses the median difference between PP and mITT estimates was 1.7%, but ranged from 0 to 30.9%. The discrepancy between estimates was correlated significantly with the proportion of patients with incomplete follow-up; p < 0.0001. The proportion of studies with a major difference (> 5%) between adjusted PP and mITT was 28% (16/57), with the risk difference greater in African (37% 14/38) compared to Thai studies (11% 2/19). In the African studies, a major difference in the adjusted estimates was significantly more likely in studies in high transmission sites (62% 8/13) compared to studies in moderate transmission sites (24% 6/25); p = 0.035.</p> <p>Conclusion</p> <p>Estimates of anti-malarial clinical efficacy vary significantly depending on the analytical methodology from which they are derived. In order to monitor temporal and spatial trends in anti-malarial efficacy, standardized analytical tools need to be applied in a transparent and systematic manner.</p

The association between malnutrition and the incidence of malaria among young HIV-infected and -uninfected Ugandan children: a prospective study

BACKGROUND: In sub-Saharan Africa, malnutrition and malaria remain major causes of morbidity and mortality in young children. There are conflicting data as to whether malnutrition is associated with an increased or decreased risk of malaria. In addition, data are limited on the potential interaction between HIV infection and the association between malnutrition and the risk of malaria. METHODS: A cohort of 100 HIV-unexposed, 203 HIV-exposed (HIV negative children born to HIV-infected mothers) and 48 HIV-infected children aged 6 weeks to 1 year were recruited from an area of high malaria transmission intensity in rural Uganda and followed until the age of 2.5 years. All children were provided with insecticide-treated bed nets at enrolment and daily trimethoprim-sulphamethoxazole prophylaxis (TS) was prescribed for HIV-exposed breastfeeding and HIV-infected children. Monthly routine assessments, including measurement of height and weight, were conducted at the study clinic. Nutritional outcomes including stunting (low height-for-age) and underweight (low weight-for-age), classified as mild (mean z-scores between -1 and -2 during follow-up) and moderate-severe (mean z-scores < -2 during follow-up) were considered. Malaria was diagnosed when a child presented with fever and a positive blood smear. The incidence of malaria was compared using negative binomial regression controlling for potential confounders with measures of association expressed as an incidence rate ratio (IRR). RESULTS: The overall incidence of malaria was 3.64 cases per person year. Mild stunting (IRR = 1.24, 95% CI 1.06-1.46, p = 0.008) and moderate-severe stunting (IRR = 1.24, 95% CI 1.03-1.48, p = 0.02) were associated with a similarly increased incidence of malaria compared to non-stunted children. Being mildly underweight (IRR = 1.09, 95% CI 0.95-1.25, p = 0.24) and moderate-severe underweight (IRR = 1.12, 95% CI 0.86-1.46, p = 0.39) were not associated with a significant difference in the incidence of malaria compared to children who were not underweight. There were no significant interactions between HIV-infected, HIV-exposed children taking TS and the associations between malnutrition and the incidence of malaria. CONCLUSIONS: Stunting, indicative of chronic malnutrition, was associated with an increased incidence of malaria among a cohort of HIV-infected and -uninfected young children living in an area of high malaria transmission intensity. However, caution should be made when making causal inferences given the observational study design and inability to disentangle the temporal relationship between malnutrition and the incidence of malaria. TRIAL REGISTRATION: ClinicalTrials.gov: NCT00527800

Proteomic Profiling of the TRAF3 Interactome Network Reveals a New Role for the ER-to-Golgi Transport Compartments in Innate Immunity

<div><p>Tumor Necrosis Factor receptor-associated factor-3 (TRAF3) is a central mediator important for inducing type I interferon (IFN) production in response to intracellular double-stranded RNA (dsRNA). Here, we report the identification of Sec16A and p115, two proteins of the ER-to-Golgi vesicular transport system, as novel components of the TRAF3 interactome network. Notably, in non-infected cells, TRAF3 was found associated with markers of the ER-Exit-Sites (ERES), ER-to-Golgi intermediate compartment (ERGIC) and the cis-Golgi apparatus. Upon dsRNA and dsDNA sensing however, the Golgi apparatus fragmented into cytoplasmic punctated structures containing TRAF3 allowing its colocalization and interaction with Mitochondrial AntiViral Signaling (MAVS), the essential mitochondria-bound RIG-I-like Helicase (RLH) adaptor. In contrast, retention of TRAF3 at the ER-to-Golgi vesicular transport system blunted the ability of TRAF3 to interact with MAVS upon viral infection and consequently decreased type I IFN response. Moreover, depletion of Sec16A and p115 led to a drastic disorganization of the Golgi paralleled by the relocalization of TRAF3, which under these conditions was unable to associate with MAVS. Consequently, upon dsRNA and dsDNA sensing, ablation of Sec16A and p115 was found to inhibit IRF3 activation and anti-viral gene expression. Reciprocally, mild overexpression of Sec16A or p115 in Hec1B cells increased the activation of IFNβ, ISG56 and NF-κB -dependent promoters following viral infection and ectopic expression of MAVS and Tank-binding kinase-1 (TBK1). In line with these results, TRAF3 was found enriched in immunocomplexes composed of p115, Sec16A and TBK1 upon infection. Hence, we propose a model where dsDNA and dsRNA sensing induces the formation of membrane-bound compartments originating from the Golgi, which mediate the dynamic association of TRAF3 with MAVS leading to an optimal induction of innate immune responses.</p> </div

Sec16A and p115 are required for the proper positioning of TRAF3 along the mitochondrial network.

<p>(<b>A</b>) Confocal microscopy of HeLa cells transfected with 40 nM nonsilencing RNA duplexes (panels 1 and 2) or 40 nM siRNA duplexes that specifically target Sec16A (panels 3 and 4) or p115 (panels 5 and 6) and stained for MAVS and endogenous TRAF3 upon no treatment (panels 1, 3 and 5) or SeV infection (200 HAU/ml) for 4 h (panels 2, 4 and 6). Arrows indicate the colocalization of TRAF3 with MAVS. Bars represent 5 µm. One of three independent experiments with similar results is shown. (<b>B</b>) p115 and Sec16A were silenced in HeLa cells as described in (<b>A</b>) and infected with SeV for indicated periods of time. Whole-cell lysates were subjected to immunoprecipitation using an anti-TRAF3 (H-20) antibody followed by immunoblotting for the presence of MAVS and TRAF3. Immunoblot analysis against p115, Sec16A, TRAF3 and SeV proteins are also shown (Input). One of two independent experiments with similar results is shown. (<b>C</b>) Densitometric analysis of the binding activity of MAVS to TRAF3 presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002747#ppat-1002747-g006" target="_blank">Figures 6B</a>. Data represent the ratio of immunoprecipitated MAVS over immunoprecipitated TRAF3 and are means +/− S.D. of two experiments.</p

p115 and Sec16A are required for optimal IRF-3 activation in response to activation of cytosolic RNA and DNA sensors.

<p>HeLa cells were infected with lentiviral vectors encoding shRNA targeting p115 (<b>A</b>) or Sec16A (<b>B</b>) and non-targeting (NT) control shRNA for 24 h followed by puromycin selection (1.5 µg/ml) for 5 days. Cells were left untreated or stimulated with poly I:C (1 µg/ml), poly dA:dT (1 µg/ml) or SeV (200 HAU/ml) for 16 h. Whole-cell lysates were prepared and subjected to immunoblot analysis with indicated antibodies. One of two independent experiments with similar results is shown.</p

TRAF3 localizes to the ER-to-Golgi transport compartments and behaves like a cis-Golgi protein.

<p>(<b>A</b>) Confocal microscopy performed in HeLa cells on FLAG-TRAF3 and Myc-p115 (panel 1), FLAG-p115 and GM130 (panel 2), FLAG-TRAF3 and GM130 (panel 3), FLAG-p115 and ERGIC53 (panel 4), FLAG-TRAF3 and ERGIC53 (panel 5), FLAG-TRAF3 and Calnexin (panel 6), FLAG-TRAF3 and lysotracker (panel 7) or mitotracker (panel 8). The nuclei were stained utilizing DAPI. One of three independent experiments with similar results is shown. Bars represent 10 µm. (<b>B</b>) Confocal microscopy performed in HeLa cells on EGFP-Sec16A and calnexin (panel 1), EGFP-Sec16A and FLAG-p115 (panel 2), EGFP-Sec16A and FLAG-TRAF3 (panel 3) and FLAG-TRAF3 and endogenous Sec16A (panel 4). (<b>C</b>) HeLa cells were stained for endogenous TRAF3 and GM130 (panel 1) or endogenous TRAF3 and ERGIC53 (panel 2) before analysis via confocal microscopy. The nuclei were stained utilizing DAPI. Bars represent 5 µm. One of three independent experiments with similar results is shown.</p

Sec16A and p115 influence the type I IFN antiviral response at the transcriptional level.

<p>(<b>A–C</b>) Hec1B cells were co-transfected with the indicated luciferase reporter genes together with 125 ng of empty vector, Myc-p115 or EGFP-Sec16A. Data are expressed as fold-induction following SeV infection (16 h) over the corresponding non-infected condition. (<b>D–F</b>) Hec1B cells were co-transfected with pGL3-IFNβ-luciferase reporter gene together with 125 ng of empty vector, Myc-p115 or Sec16A and 15 ng of FLAG-MAVS (<b>D</b>), 100 ng of FLAG-TBK1 (<b>E</b>), or 15 ng of His-TRIF (<b>F</b>). Data represent the fold-activation over the corresponding vector control. Each value represents the mean +/− S.D. of triplicate determinations. The data are representative of at least four different experiments with similar results. (<b>G</b>) TRAF3 knockout MEF cells were co-transfected with 250 ng of luciferase reporter plasmid pGL3-IFNβ, 500 ng of pcDNA3 or FLAG-TRAF3 plasmid and 375 ng of indicated plasmids. At 24 h post-transfection, cells were left uninfected or infected with SeV (200 HAU/ml) for 16 h and relative luciferase activity was measured as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002747#s4" target="_blank">Materials and Methods</a>. Mean values +/− S.D. of triplicate determinations are shown (** P<0.01). One of three independent experiments with similar results is shown.</p

Enforced retention of TRAF3 at the ER-to-Golgi compartment negatively regulates type I IFN response.

<p>(<b>A</b>) Confocal microscopy analysis of FLAG-tag and GM130 performed in HeLa cells expressing FLAG-TRAF3 (panels 1 and 2) or FLAG-TRAF3-AKKFF (panels 3 and 4) upon no infection (panels 1 and 3) or SeV infection (200 HAU/ml) (panels 2 and 4) for 4 h. The nuclei were stained with DAPI. Arrows indicate TRAF3 aggregates. One of two independent experiments with similar results is shown. Bars represent 10 µm. (<b>B</b>) Hec1B cells were co-transfected with luciferase reporter plasmid pGL3-IFNβ (250 ng) and indicated plasmids (250 ng) for 24 h and infected with SeV (200 HAU/ml) for 16 h. Hec1B cells were also co-transfected with luciferase reporter plasmid pGL3-IFNβ (250 ng), empty vector or MAVS or TRIF (25 ng) along with indicated plasmids (250 ng) for 24 h. Relative luciferase activity was measured as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002747#s4" target="_blank">Materials and Methods</a>. Mean values +/− S.D. of triplicate determinations are shown (*** P<0.001). One of four independent experiments with similar results is shown. Cellular extracts from transfected Hec1B cells were also prepared and subjected to immunoblot analysis using indicated antibodies (right lower panel).</p