Whole blood interferon-gamma responses to mycobacterium tuberculosis antigens in young household contacts of persons with tuberculosis in Uganda

Background

Due to immunologic immaturity, IFN-γ-producing T cell responses may be decreased in young children compared to adults, thus we hypothesized that IFN-γ responses to mycobacterial antigens in household contacts exposed to Mycobacterium tuberculosis (Mtb) would be impaired in young children relative to adults. The objective of this study was to compare whole blood IFN-γ production in response to mycobacterial antigens between children and adults in Uganda.

Methodology/Principal Findings

We studied household contacts of persons with culture-positive pulmonary tuberculosis (TB) enrolled in a cohort study conducted in Kampala, Uganda. Whole blood IFN-γ production in response to Mtb culture-filtrate antigens was measured by ELISA and compared between infants (<2 years old, n = 80), young children (2 <5 years old, n = 216), older children (5 <15 years old, n = 443) and adults (≥15 years old, n = 528). We evaluated the relationship between IFN-γ responses and the tuberculin skin test (TST), and between IFN-γ responses and epidemiologic factors that reflect exposure to Mtb, and the effect of prior BCG vaccination on IFN-γ responses. Young household contacts demonstrated robust IFN-γ responses comparable to those of adults that were associated with TST and known risk factors for infection. There was no effect of prior BCG immunization on the IFN-γ response.

Conclusions/Significance

Young children in a TB endemic setting can mount robust IFN-γ responses generally comparable to those of adults, and as in adults, these responses correlated with the TST and known epidemiologic risk factors for Mtb infection.     

Structure of the Pseudomonas aeruginosa XcpT pseudopilin,a major component of the type II secretion system

The bacterial type II protein secretion (T2S) and type IV piliation (T4P) systems share several common features. In particular, it is well established that the T2S system requires the function of a pilus-like structure, called pseudopilus, which is built upon assembly of pilin-like subunits, called pseudopilins. Pilins and pseudopilins have a hydrophobic N-terminal region, which precedes an extended hydrophilic C-terminal region. In the case of pilins, it was shown that oligomerisation and formation of helical fibers, takes place through interaction between the hydrophobic domains. XcpT, is the most abundant protein of the Pseudomonas aeruginosa T2S, and was proposed to be the main component in the pseudopilus. In this study we present the high-resolution NMR structure of the hydrophilic domain of XcpT (XcpTp). XcpTp is lacking the C-terminal disulfide bridged “D” domain found in type IV pilins and likely involved in receptor binding. This is in agreement with the idea that the XcpT-containing pseudopilus is required for protein secretion and not for bacterial attachment. Interestingly, by solving the 3D structure of XcpTp we revealed that the previously called αβ-loop pilin region is in fact highly conserved among major type II pseudopilins and constitutes a specific consensus motif for identifying major pseudopilins, which belong to this family.     

The XcpV/GspI Pseudopilin Has a Central Role in the Assembly of a Quaternary Complex within the T2SS Pseudopilus

Gram-negative bacteria use the sophisticated type II secretion system (T2SS) to secrete a large number of exoproteins into the extracellular environment. Five proteins of the T2SS, the pseudopilins GspG-H-I-J-K, are proposed to assemble into a pseudopilus involved in the extrusion of the substrate through the outer membrane channel. Recent structural data have suggested that the three pseudopilins GspI-J-K are organized in a trimeric complex located at the tip of the GspG-containing pseudopilus. In the present work we combined two biochemical techniques to investigate the protein-protein interaction network between the five Pseudomonas aeruginosa Xcp pseudopilins. The soluble domains of XcpT-U-V-W-X (respectively homologous to GspG-H-I-J-K) were purified, and the interactions were tested by surface plasmon resonance and affinity co-purification in all possible combinations. We found an XcpV_I-W_J-X_K complex, which demonstrates that the crystallized trimeric complex also exists in the P. aeruginosa T2SS. Interestingly, our systematic approach revealed an additional and yet uncharacterized interaction between XcpU_H and XcpW_J. This observation suggested the existence of a quaternary, rather than ternary, complex (XcpU_H-V_I-W_J-X_K) at the tip of the pseudopilus. The assembly of this quaternary complex was further demonstrated by co-purification using affinity chromatography. Moreover, by testing various combinations of pseudopilins by surface plasmon resonance and affinity chromatography, we were able to dissect the different possible successive steps occurring during the formation of the quaternary complex. We propose a model in which XcpV_I is the nucleator that first binds XcpX_K and XcpW_J at different sites. Then the ternary complex recruits XcpU_H through a direct interaction with XcpW_J.     

Intermittent preventive treatment of malaria provides substantial protection against malaria in children already protected by an insecticide-treated bednet in Burkina Faso: a randomised, double-blind, placebo-controlled trial

Background

Intermittent preventive treatment of malaria in children (IPTc) is a promising new approach to the control of malaria in areas of seasonal malaria transmission but it is not known if IPTc adds to the protection provided by an insecticide-treated net (ITN).

Methods and Findings

An individually randomised, double-blind, placebo-controlled trial of seasonal IPTc was conducted in Burkina Faso in children aged 3 to 59 months who were provided with a long-lasting insecticide-treated bednet (LLIN). Three rounds of treatment with sulphadoxine pyrimethamine plus amodiaquine or placebos were given at monthly intervals during the malaria transmission season. Passive surveillance for malaria episodes was established, a cross-sectional survey was conducted at the end of the malaria transmission season, and use of ITNs was monitored during the intervention period. Incidence rates of malaria were compared using a Cox regression model and generalized linear models were fitted to examine the effect of IPTc on the prevalence of malaria infection, anaemia, and on anthropometric indicators. 3,052 children were screened and 3,014 were enrolled in the trial; 1,505 in the control arm and 1,509 in the intervention arm. Similar proportions of children in the two treatment arms were reported to sleep under an LLIN during the intervention period (93%). The incidence of malaria, defined as fever or history of fever with parasitaemia ≥5,000/µl, was 2.88 (95% confidence interval [CI] 2.70–3.06) per child during the intervention period in the control arm versus 0.87 (95% CI 0.78–0.97) in the intervention arm, a protective efficacy (PE) of 70% (95% CI 66%–74%) (p<0.001). There was a 69% (95% CI 6%–90%) reduction in incidence of severe malaria (p = 0.04) and a 46% (95% CI 7%–69%) (p = 0.03) reduction in the incidence of all-cause hospital admissions. IPTc reduced the prevalence of malaria infection at the end of the malaria transmission season by 73% (95% CI 68%–77%) (p<0.001) and that of moderately severe anaemia by 56% (95% CI 36%–70%) (p<0.001). IPTc reduced the risks of wasting (risk ratio [RR] = 0.79; 95% CI 0.65–1.00) (p = 0.05) and of being underweight (RR = 0.84; 95% CI 0.72–0.99) (p = 0.03). Children who received IPTc were 2.8 (95% CI 2.3–3.5) (p<0.001) times more likely to vomit than children who received placebo but no drug-related serious adverse event was recorded.

Conclusions

IPT of malaria provides substantial protection against malaria in children who sleep under an ITN. There is now strong evidence to support the integration of IPTc into malaria control strategies in areas of seasonal malaria transmission.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00738946","term_id":"NCT00738946"}}NCT00738946 Please see later in the article for the Editors'' Summary     

Targeting of PP2A enzymes by viral proteins and cancer signalling

Protein phosphatase 2A (PP2A) is a large family of holoenzymes that comprises 1% of total cellular proteins and accounts for the majority of Ser/Thr phosphatase activity in eukaryotic cells. PP2A proteins are made of a core dimer, composed of a catalytic (C) subunit and a structural (A) subunit, in association with a third variable -regulatory (B) subunit. Although initially considered as a constitutive housekeeping enzyme, PP2A is indeed highly regulated by post-translational modifications of its catalytic subunit or by the identity of a regulatory type B subunit, which determines substrate specificity, subcellular localization and enzymatic activity of a defined holoenzyme. During the two last decades, multiple studies of structural and functional regulation of PP2A holoenzymes by viral proteins led to the identification of critical pathways for both viral biology and tumorigenesis. To date a dozen of different viruses (ADN/ARN or retrovirus) have been identified that encode viral proteins associated to PP2A. In this review, we analyze a biological strategy, used by various viruses based on the targeting of PP2A enzymes by viral proteins, in order to specifically deregulate cellular pathways of their hosts. The impact of such PP2A targeting for biomedical search, and in further therapeutic developments against cancer, will also be discussed.     

Neutrophil extracellular traps impair regeneration

Fibrosis is a major health burden across diseases and organs. To remedy this, we study wound-induced hair follicle neogenesis (WIHN) as a model of non-fibrotic healing that recapitulates embryogenesis for de novo hair follicle morphogenesis after wounding. We previously demonstrated that TLR3 promotes WIHN through binding wound-associated dsRNA, the source of which is still unclear. Here, we find that multiple distinct contexts of high WIHN all show a strong neutrophil signature. Given the correlation between neutrophil infiltration and endogenous dsRNA release, we hypothesized that neutrophil extracellular traps (NETs) likely release nuclear spliceosomal U1 dsRNA and modulate WIHN. However, rather than enhance regeneration, we find mature neutrophils inhibit WIHN such that mice with mature neutrophil depletion exhibit higher WIHN. Similarly, Pad4 null mice, which are defective in NET production, show augmented WIHN. Finally, using single-cell RNA sequencing, we identify a dramatic increase in mature and activated neutrophils in the wound beds of low regenerating Tlr3−/− mice. Taken together, these results demonstrate that although mature neutrophils are stimulated by a common pro-regenerative cue, their presence and NETs hinder regeneration.     

Muscarinic binding sites in a catecholaminergic human neuroblastoma cell line

Tyrosine hydroxylase (TH) a characteristic enzyme activity for the catecholaminergic clonal cell line LA-N-1 and choline acetyltransferase (ChAT) a characteristic enzyme activity for the cholinergic clonal cell line LA-N-2 were previously shown to be increased in these cells exposed to 10^–5 M retinoic acid (RA) as differentiating agent. An investigation of the receptor characteristics suggests a complementarity between the two cell lines. The binding of QNB, a muscarinic ligand, was undetectable with the LA-N-2 cells but was present in the LA-N-1 cells and possessed a kD of 1.8 nM and 2.2 nM and a B_max of 0.56 and 0.68 for control and RA grown cells respectively. There was a gradual increase in QNB binding to LA-N-1 cells from 2 days in vitro (DIV) until 6 DIV in both control and RA grown cells. An IC₅₀ of 2.5×10^–8 M and 0.9×10^–8 M for atropine inhibition was obtained for the control and RA grown cells respectively. The corresponding values for carbachol inhibition were 7×10^–2 M and 3×10^–2 M respectively. The inhibition by the agonist oxotremorine is comparable to that of carbachol and 1 mM pilocarpine inhibited the binding by 21%. QNB binding showed a low affinity for pirenzepine and for AF-DX-116 but was inhibited with a rather high affinity by 4-DAMP (IC₅₀:110 M) thus suggesting the presence of an M₃ receptor. Acetylcholine (100 M) plus eserine (50 M) and BW284c55 (1 M), an acetylcholinesterase inhibitor, reduced the binding of QNB by approximately 25%. Nicotine (1 mM) caused a 36% reduction of binding and hemicholinium-3 (HC-3) (1 M), an inhibitor of choline uptake, inhibited the binding by 53%. There was a down regulation of QNB binding observed with cells grown for 24 hours with either the antagonist atropine or the agonists carbachol or oxotremorine. Low amounts of -bungarotoxin (-BgTx) binding sites were barely detectable in both LA-N-1 and LA-N-2 cells. The LA-N-1 muscarinic receptor is coupled to polyphosphoinositide hydrolysis without increased cyclic AMP formation further suggesting its being an M₃ receptor.