Decline of FoxP3+ Regulatory CD4 T Cells in Peripheral Blood of Children Heavily Exposed to Malaria

FoxP3+ regulatory CD4 T cells (T_regs) help to maintain the delicate balance between pathogen-specific immunity and immune-mediated pathology. Prior studies suggest that T_regs are induced by P. falciparum both in vivo and in vitro; however, the factors influencing T_reg homeostasis during acute and chronic infections, and their role in malaria immunopathogenesis, remain unclear. We assessed the frequency and phenotype of T_regs in well-characterized cohorts of children residing in a region of high malaria endemicity in Uganda. We found that both the frequency and absolute numbers of FoxP3+ T_regs in peripheral blood declined markedly with increasing prior malaria incidence. Longitudinal measurements confirmed that this decline occurred only among highly malaria-exposed children. The decline of T_regs from peripheral blood was accompanied by reduced in vitro induction of T_regs by parasite antigen and decreased expression of TNFR2 on T_regs among children who had intense prior exposure to malaria. While T_reg frequencies were not associated with protection from malaria, there was a trend toward reduced risk of symptomatic malaria once infected with P. falciparum among children with lower T_reg frequencies. These data demonstrate that chronic malaria exposure results in altered T_reg homeostasis, which may impact the development of antimalarial immunity in naturally exposed populations.     

Development of high-throughput methods to quantify cysts of Toxoplasma gondii

Toxplasma is a protozoan parasite, which forms persistent cysts in tissues of chronically infected animals and humans. Cysts can reactivate leading to severe pathologies. They also contribute to the transmission of Toxoplasma infection in humans by ingestion of undercooked meat. Classically, the quantification of cyst burden in tissues uses microscopy methods, which are laborious and time consuming. Here, we have developed automated protocols to quantify cysts, based on flow cytometry or high-throughput microscopy. Brains of rodents infected with cysts of Prugniaud strain were incubated with the FITC-Dolichos biflorus lectin and analyzed by flow cytometry and high-throughput epifluorescence microscopy. The comparison of cyst counts by manual epifluorescence microscopy to flow cytometry or to high-throughput epifluorescence microscopy revealed a good correlation (r = 0.934, r = 0.993, P < 0.001 respectively). High-throughput epifluorescence microscopy was found to be more specific and sensitive than flow cytometry and easier to use for large series of samples. This reliable and easy protocol allow the specific detection of Toxoplasma cysts in brain, even at low concentrations; it could be a new way to detect them in water and in contaminate food.     

Growth cones turn and migrate up an immobilized gradient of the laminin IKVAV peptide

Growth cone navigation is guided by extrinsic environmental proteins, called guidance cues. Many in vitro studies have characterized growth cone turning up and down gradients of soluble guidance cues. Although previous studies have shown that axonal elongation rates can be regulated by gradients of surface-bound molecules, there are no convincing demonstrations of growth cones turning to migrate up a surface-bound gradient of an adhesive ligand or guidance cue. In order to test this mode of axonal guidance, we used a photo-immobilization technique to create grids and gradients of an adhesive laminin peptide on polystyrene culture dish surfaces. Chick embryo dorsal root ganglia (DRGs) were placed on peptide grid patterns containing surface-bound gradients of the IKVAV-containing peptide. DRG growth cones followed a path of surface-bound peptide to the middle of a perpendicularly oriented gradient with a 25% concentration difference across 30 microm. The majority of growth cones turned and migrated up the gradient, turning until they were oriented directly up the gradient. Growth cones slowed their migration when they encountered the gradient, but growth cone velocity returned to the previous rate after turning up or down the gradient. This resembles in vivo situations where growth cones slow at a choice point before changing the direction of axonal extension. Thus, these results support the hypothesis that mechanisms of axonal guidance include growth cone orientation by gradients of surface-bound adhesive molecules and guidance cues.     

Effects of age, gender and time on receptor expression and anti-Aspergillus functions of human phagocytes

Phagocytes play a central role in immune defense. Their dysfunction predisposes to infections. This study determined the expression level of nine receptors involved in Aspergillus immune response as well as the values of phagocytosis and production of radical oxygen species after Aspergillus stimulation, in a healthy adult population.The expression values of the CD11b, CD11c, CD14, CD18, CD35, CD181, CD182, CD282 and CD284 receptors on peripheral human monocytes and granulocytes was established. A heterogenous expression of the CD282 on granulocytes was observed as CD181, CD182 and CD284 on monocytes. Similarly, we observed considerable variation in the expression of these receptors over time. Only CD282 on granulocytes varied with sex. No variation with age was observed. Adherence of Aspergillus conidia to phagocytes was dependent of individual, sex, age and time. A better characterization of these innate immunity parameters is necessary to develop in the future an immunologic surveillance strategy for transplant recipients.     

Protective Efficacy and Safety of Three Antimalarial Regimens for the Prevention of Malaria in Young Ugandan Children: A Randomized Controlled Trial

Background

Chemoprevention offers a promising strategy for prevention of malaria in African children. However, the optimal chemoprevention drug and dosing strategy is unclear in areas of year-round transmission and resistance to many antimalarial drugs. To compare three available regimens, we conducted an open-label randomized controlled trial of chemoprevention in Ugandan children.

Methods and Findings

This study was conducted between June 28, 2010, and September 25, 2013. 400 infants were enrolled and 393 randomized at 6 mo of age to no chemoprevention, monthly sulfadoxine-pyrimethamine (SP), daily trimethoprim-sulfamethoxazole (TS), or monthly dihydroartemisinin-piperaquine (DP). Study drugs were administered at home without supervision. Piperaquine (PQ) levels were used as a measure of compliance in the DP arm. Participants were given insecticide-treated bednets, and caregivers were encouraged to bring their child to a study clinic whenever they were ill. Chemoprevention was stopped at 24 mo of age, and participants followed-up an additional year. Primary outcome was the incidence of malaria during the intervention period. During the intervention, the incidence of malaria in the no chemoprevention arm was 6.95 episodes per person-year at risk. Protective efficacy was 58% (95% CI, 45%–67%, p<0.001) for DP, 28% (95% CI, 7%–44%, p = 0.01) for TS, and 7% for SP (95% CI, −19% to 28%, p = 0.57). PQ levels were below the detection limit 52% of the time when malaria was diagnosed in the DP arm, suggesting non-adherence. There were no differences between the study arms in the incidence of serious adverse events during the intervention and the incidence of malaria during the 1-y period after the intervention was stopped.

Conclusions

For preventing malaria in children living in an area of high transmission intensity, monthly DP was the most efficacious and safe, although adherence may pose a problem. Monthly SP and daily TS may not be appropriate in areas with high transmission intensity and frequent resistance to antifolates.

Trial registration

www.ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00948896","term_id":"NCT00948896"}}NCT00948896 Please see later in the article for the Editors'' Summary     

Polymorphisms in K13 and Falcipain-2 Associated with Artemisinin Resistance Are Not Prevalent in Plasmodium falciparum Isolated from Ugandan Children

The emergence of resistance to artemisinin derivatives in Southeast Asia, manifested as delayed clearance of Plasmodium falciparum following treatment with artemisinins, is a major concern. Recently, the artemisinin resistance phenotype was attributed to mutations in portions of a P. falciparum gene (PF3D7_1343700) encoding kelch (K13) propeller domains, providing a molecular marker to monitor the spread of resistance. The P. falciparum cysteine protease falcipain-2 (FP2; PF3D7_1115700) has been shown to contribute to artemisinin action, as hemoglobin degradation is required for potent drug activity, and a stop mutation in the FP2 gene was identified in parasites selected for artemisinin resistance. Although delayed parasite clearance after artemisinin-based combination therapy (ACT) has not yet been noted in Uganda and ACTs remain highly efficacious, characterizing the diversity of these genes is important to assess the potential for resistance selection and to provide a baseline for future surveillance. We therefore sequenced the K13-propeller domain and FP2 gene in P. falciparum isolates from children previously treated with ACT in Uganda, including samples from 2006–7 (n = 49) and from 2010–12 (n = 175). Using 3D7 as the reference genome, we identified 5 non-synonymous polymorphisms in the K13-propeller domain (133 isolates) and 35 in FP2 (160 isolates); these did not include the polymorphisms recently associated with resistance after in vitro selection or identified in isolates from Asia. The prevalence of K13-propeller and FP2 polymorphisms did not increase over time, and was not associated with either time since prior receipt of an ACT or the persistence of parasites ≥2 days following treatment with an ACT. Thus, the K13-propeller and FP2 polymorphisms associated with artemisinin resistance are not prevalent in Uganda, and we did not see evidence for selection of polymorphisms in these genes.     

Successful Treatment of Severe Tungiasis in Pigs Using a Topical Aerosol Containing Chlorfenvinphos,Dichlorphos and Gentian Violet

BackgroundIn endemic communities, zoonotic tungiasis, a severe skin disease caused by penetrating female sand fleas, is a public health hazard causing significant human and animal morbidity. No validated drugs are currently available for treatment of animal tungiasis. Due to the reservoir in domestic animals, integrated management of human and animal tungiasis is required to avert its negative effects.ConclusionsThe study demonstrates that a topical treatment based on chlorfenvinphos, dichlorphos and gentian violet is highly effective against pig tungiasis. Due to its simplicity, the new approach can be used for the treatment of individual animals as well as in mass campaigns.     

Metabolic studies on rabbit bladder smooth muscle and mucosa

Recent studies indicate that the mucosa of the urinary bladder may play a major role in the maintenance of normal bladder function. The mucosal surface of the urinary bladder serves as a protective layer against the irritative solutes found in the urine. The integrity of this barrier can be broken by overdistension, anoxia, detergents, alcohols, bacterial infection and by contact with agents to which the mucosa has been sensitized.In view that both anoxia and ischemia can mediate a breakdown in the role of the mucosal layer as a permeability barrier, it is reasonable to assume that this function is dependent on cellular metabolism. As an initial investigation we have compared a variety of biochemical and metabolic parameters between the mucosal layer (consisting of the lamina propria, urothelium, and any connective tissue and vascular tissue within this layer); and the muscularis layer.The results of these studies demonstrated that the rate of glucose metabolism to lactic acid (LA) of the mucosa was more than three-fold greater than that of the smooth muscle. The rate of CO₂ production of the mucosa was 60% greater than that of the unstimulated smooth muscle. The maximal activity of the mitochondrial enzyme citrate synthase was significantly greater in the mucosa than in the smooth muscle, however, the activity of malate dehydrogenase was similar for both tissues. The maximal activity of the cytosolic enzyme creatine kinase was more than two-fold greater in the bladder smooth muscle than in the mucosa; although the affinities of the creatine kinase isoforms of the mucosa were sigificantly greater than those of the muscle.Although the concentrations of ATP and ADP were similar in both muscle and mucosa, the level of creatine phosphate (CP) was over four-fold greater in the bladder muscle while the level of AMP in the muscle was only 58% of that in the mucosal epithelium.In summary, the rate of glucose metabolism was greater in the mucosa than in the smooth muscle although the concentrations of high energy phosphates (ATP+CP) was significantly greater in the smooth muscle. Future studies will be directed at identifying the specific cellular processes within the mucosal layer that relate to the function of the urothelium as a permeability barrier.     

Assessing the Threat of Amphibian Chytrid Fungus in the Albertine Rift: Past,Present and Future

Batrachochytrium dendrobatidis (Bd), the cause of chytridiomycosis, is a pathogenic fungus that is found worldwide and is a major contributor to amphibian declines and extinctions. We report results of a comprehensive effort to assess the distribution and threat of Bd in one of the Earth’s most important biodiversity hotspots, the Albertine Rift in central Africa. In herpetological surveys conducted between 2010 and 2014, 1018 skin swabs from 17 amphibian genera in 39 sites across the Albertine Rift were tested for Bd by PCR. Overall, 19.5% of amphibians tested positive from all sites combined. Skin tissue samples from 163 amphibians were examined histologically; of these two had superficial epidermal intracorneal fungal colonization and lesions consistent with the disease chytridiomycosis. One amphibian was found dead during the surveys, and all others encountered appeared healthy. We found no evidence for Bd-induced mortality events, a finding consistent with other studies. To gain a historical perspective about Bd in the Albertine Rift, skin swabs from 232 museum-archived amphibians collected as voucher specimens from 1925–1994 were tested for Bd. Of these, one sample was positive; an Itombwe River frog (Phrynobatrachus asper) collected in 1950 in the Itombwe highlands. This finding represents the earliest record of Bd in the Democratic Republic of Congo. We modeled the distribution of Bd in the Albertine Rift using MaxEnt software, and trained our model for improved predictability. Our model predicts that Bd is currently widespread across the Albertine Rift, with moderate habitat suitability extending into the lowlands. Under climatic modeling scenarios our model predicts that optimal habitat suitability of Bd will decrease causing a major range contraction of the fungus by 2080. Our baseline data and modeling predictions are important for comparative studies, especially if significant changes in amphibian health status or climactic conditions are encountered in the future.