Molecular Identification of a Malaria Merozoite Surface Sheddase

Proteolytic shedding of surface proteins during invasion by apicomplexan parasites is a widespread phenomenon, thought to represent a mechanism by which the parasites disengage adhesin-receptor complexes in order to gain entry into their host cell. Erythrocyte invasion by merozoites of the malaria parasite Plasmodium falciparum requires the shedding of ectodomain components of two essential surface proteins, called MSP1 and AMA1. Both are released by the same merozoite surface “sheddase,” but the molecular identity and mode of action of this protease is unknown. Here we identify it as PfSUB2, an integral membrane subtilisin-like protease (subtilase). We show that PfSUB2 is stored in apical secretory organelles called micronemes. Upon merozoite release it is secreted onto the parasite surface and translocates to its posterior pole in an actin-dependent manner, a trafficking pattern predicted of the sheddase. Subtilase propeptides are usually selective inhibitors of their cognate protease, and the PfSUB2 propeptide is no exception; we show that recombinant PfSUB2 propeptide binds specifically to mature parasite-derived PfSUB2 and is a potent, selective inhibitor of MSP1 and AMA1 shedding, directly establishing PfSUB2 as the sheddase. PfSUB2 is a new potential target for drugs designed to prevent erythrocyte invasion by the malaria parasite

Global distribution of two fungal pathogens threatening endangered sea turtles

This work was supported by grants of Ministerio de Ciencia e Innovación, Spain (CGL2009-10032, CGL2012-32934). J.M.S.R was supported by PhD fellowship of the CSIC (JAEPre 0901804). The Natural Environment Research Council and the Biotechnology and Biological Sciences Research Council supported P.V.W. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Thanks Machalilla National Park in Ecuador, Pacuare Nature Reserve in Costa Rica, Foundations Natura 2000 in Cape Verde and Equilibrio Azul in Ecuador, Dr. Jesus Muñoz, Dr. Ian Bell, Dr. Juan Patiño for help and technical support during samplingPeer reviewedPublisher PD

Trauma history and depression predict incomplete adherence to antiretroviral therapies in a low income country.

As antiretroviral therapy (ART) for HIV becomes increasingly available in low and middle income countries (LMICs), understanding reasons for lack of adherence is critical to stemming the tide of infections and improving health. Understanding the effect of psychosocial experiences and mental health symptomatology on ART adherence can help maximize the benefit of expanded ART programs by indicating types of services, which could be offered in combination with HIV care. The Coping with HIV/AIDS in Tanzania (CHAT) study is a longitudinal cohort study in the Kilimanjaro Region that included randomly selected HIV-infected (HIV+) participants from two local hospital-based HIV clinics and four free-standing voluntary HIV counselling and testing sites. Baseline data were collected in 2008 and 2009; this paper used data from 36 month follow-up interviews (N = 468). Regression analyses were used to predict factors associated with incomplete self-reported adherence to ART. INCOMPLETE ART ADHERENCE WAS SIGNIFICANTLY MORE LIKELY TO BE REPORTED AMONGST PARTICIPANTS WHO EXPERIENCED A GREATER NUMBER OF CHILDHOOD TRAUMATIC EVENTS: sexual abuse prior to puberty and the death in childhood of an immediate family member not from suicide or homicide were significantly more likely in the non-adherent group and other negative childhood events trended toward being more likely. Those with incomplete adherence had higher depressive symptom severity and post-traumatic stress disorder (PTSD). In multivariable analyses, childhood trauma, depression, and financial sacrifice remained associated with incomplete adherence.\ud This is the first study to examine the effect of childhood trauma, depression and PTSD on HIV medication adherence in a low income country facing a significant burden of HIV. Allocating spending on HIV/AIDS toward integrating mental health services with HIV care is essential to the creation of systems that enhance medication adherence and maximize the potential of expanded antiretroviral access to improve health and reduce new infections

Construction of Transgenic Plasmodium berghei as a Model for Evaluation of Blood-Stage Vaccine Candidate of Plasmodium falciparum Chimeric Protein 2.9

BACKGROUND:The function of the 19 kDa C-terminal region of the merozoite surface protein 1 (MSP1-19) expressed by Plasmodium has been demonstrated to be conserved across distantly related Plasmodium species. The green fluorescent protein (GFP) is a reporter protein that has been widely used because it can be easily detected in living organisms by fluorescence microscopy and flow cytometry. METHODOLOGY AND RESULTS:In this study, we used gene targeting to generate transgenic P. berghei (Pb) parasites (designated as PfMSP1-19Pb) that express the MSP1-19 of P. falciparum (Pf) and the GFP reporter protein simultaneously. The replacement of the PbMSP1-19 locus by PfMSP1-19 was verified by PCR and Southern analysis. The expression of the chimeric PbfMSP-1 and the GFP was verified by Western blot and fluorescence microscopy, respectively. Moreover, GFP-expressing transgenic parasites in blood stages can be readily differentiated from other blood cells using flow cytometry. A comparison of growth rates between wild-type and the PfMSP1-19Pb transgenic parasite indicated that the replacement of the MSP1-19 region and the expression of the GFP protein were not deleterious to the transgenic parasites. We used this transgenic mouse parasite as a murine model to evaluate the protective efficacy in vivo of specific IgG elicited by a PfCP-2.9 malaria vaccine that contains the PfMSP1-19. The BALB/c mice passively transferred with purified rabbit IgG to the PfCP-2.9 survived a lethal challenge of the PfMSP1-19Pb transgenic murine parasites, but not the wild-type P. berghei whereas the control mice passively transferred with purified IgG obtained from adjuvant only-immunized rabbits were vulnerable to both transgenic and wild-type infections. CONCLUSIONS:We generated a transgenic P. berghei line that expresses PfMSP1-19 and the GFP reporter gene simultaneously. The availability of this parasite line provides a murine model to evaluate the protective efficacy in vivo of anti-MSP1-19 antibodies, including, potentially, those elicited by the PfCP-2.9 malaria vaccine in human volunteers

Juxtamembrane Shedding of Plasmodium falciparum AMA1 Is Sequence Independent and Essential, and Helps Evade Invasion-Inhibitory Antibodies

The malarial life cycle involves repeated rounds of intraerythrocytic replication interspersed by host cell rupture which releases merozoites that rapidly invade fresh erythrocytes. Apical membrane antigen-1 (AMA1) is a merozoite protein that plays a critical role in invasion. Antibodies against AMA1 prevent invasion and can protect against malaria in vivo, so AMA1 is of interest as a malaria vaccine candidate. AMA1 is efficiently shed from the invading parasite surface, predominantly through juxtamembrane cleavage by a membrane-bound protease called SUB2, but also by limited intramembrane cleavage. We have investigated the structural requirements for shedding of Plasmodium falciparum AMA1 (PfAMA1), and the consequences of its inhibition. Mutagenesis of the intramembrane cleavage site by targeted homologous recombination abolished intramembrane cleavage with no effect on parasite viability in vitro. Examination of PfSUB2-mediated shedding of episomally-expressed PfAMA1 revealed that the position of cleavage is determined primarily by its distance from the parasite membrane. Certain mutations at the PfSUB2 cleavage site block shedding, and parasites expressing these non-cleavable forms of PfAMA1 on a background of expression of the wild type gene invade and replicate normally in vitro. The non-cleavable PfAMA1 is also functional in invasion. However – in contrast to the intramembrane cleavage site - mutations that block PfSUB2-mediated shedding could not be stably introduced into the genomic pfama1 locus, indicating that some shedding of PfAMA1 by PfSUB2 is essential. Remarkably, parasites expressing shedding-resistant forms of PfAMA1 exhibit enhanced sensitivity to antibody-mediated inhibition of invasion. Drugs that inhibit PfSUB2 activity should block parasite replication and may also enhance the efficacy of vaccines based on AMA1 and other merozoite surface proteins

Gender and respiratory factors associated with dyspnea in chronic obstructive pulmonary disease

RATIONALE: We had shown that COPD women expressed more dyspnea than men for the same degree of airway obstruction. OBJECTIVES: Evaluate gender differences in respiratory factors associated with dyspnea in COPD patients. METHODS: In a FEV(1 )% matched population of 100 men and women with COPD we measured: age, MMRC, FEV(1), FVC, TLC, IC/TLC, PaO(2), PaCO(2), D(LCO), P(imax), P(0.1), Ti/Ttot, BMI, ffmi, 6MWD and VAS scale before and after the test, the Charlson score and the SGRQ. We estimated the association between these parameters and MMRC scores. Multivariate analysis determined the independent strength of those associations. RESULTS: MMRC correlated with: BMI (men:-0.29, p = 0.04; women:-0.28, p = 0.05), ffmi (men:-0.39, p = 0.01), FEV(1 )% (men:-0.64, p < 0.001; women:-0.29, p = 0.04), FVC % (men:-0.45, p = 0.001; women:-0.33, p = 0.02), IC/TLC (men:-0.52, p < 0.001; women: -0.27, p = 0.05), PaO(2 )(men:-0.59, p < 0.001), PaCO(2 )(men:0.27, p = 0.05), D(LCO )(men:-0.54, p < 0.001), P(0.1)/P(imax )(men:0.46, p = 0.002; women:0.47, p = 0.005), dyspnea measured with the Visual Analog Scale before (men:0.37, p = 0.04; women:0.52, p = 0.004) and after 6MWD (men:0.52, p = 0.002; women:0.48, p = 0.004) and SGRQ total (men:0.50, p < 0.001; women:0.59, p < 0.001). Regression analysis showed that P(0.1)/P(imax )in women (r(2 )= 0.30) and BMI, DL(CO), PaO(2 )and P(0.1)/P(imax )in men (r(2 )= 0.81) were the strongest predictors of MMRC scores. CONCLUSION: In mild to severe COPD patients attending a pulmonary clinic, P(0.1)/P(imax )was the unique predictor of MMRC scores only in women. Respiratory factors explain most of the variations of MMRC scores in men but not in women. Factors other than the respiratory ones should be included in the evaluation of dyspnea in women with COPD

Shortening of atrioventricular delay at increased atrial paced heart rates improves diastolic filling and functional class in patients with biventricular pacing

<p>Abstract</p> <p>Background</p> <p>Use of rate adaptive atrioventricular (AV) delay remains controversial in patients with biventricular (Biv) pacing. We hypothesized that a shortened AV delay would provide optimal diastolic filling by allowing separation of early and late diastolic filling at increased heart rate (HR) in these patients.</p> <p>Methods</p> <p>34 patients (75 ± 11 yrs, 24 M, LVEF 34 ± 12%) with Biv and atrial pacing had optimal AV delay determined at baseline HR by Doppler echocardiography. Atrial pacing rate was then increased in 10 bpm increments to a maximum of 90 bpm. At each atrial pacing HR, optimal AV delay was determined by changing AV delay until best E and A wave separation was seen on mitral inflow pulsed wave (PW) Doppler (defined as increased atrial duration from baseline or prior pacemaker setting with minimal atrial truncation). Left ventricular (LV) systolic ejection time and velocity time integral (VTI) at fixed and optimal AV delay was also tested in 13 patients. Rate adaptive AV delay was then programmed according to the optimal AV delay at the highest HR tested and patients were followed for 1 month to assess change in NYHA class and Quality of Life Score as assessed by Minnesota Living with Heart Failure Questionnaire.</p> <p>Results</p> <p>81 AV delays were evaluated at different atrial pacing rates. Optimal AV delay decreased as atrial paced HR increased (201 ms at 60 bpm, 187 ms at 70 bpm, 146 ms at 80 bpm and 123 ms at 90 bpm (ANOVA F-statistic = 15, p = 0.0010). Diastolic filling time (P < 0.001 vs. fixed AV delay), mitral inflow VTI (p < 0.05 vs fixed AV delay) and systolic ejection time (p < 0.02 vs. fixed AV delay) improved by 14%, 5% and 4% respectively at optimal versus fixed AV delay at the same HR. NYHA improved from 2.6 ± 0.7 at baseline to 1.7 ± 0.8 (p < 0.01) 1 month post optimization. Physical component of Quality of Life Score improved from 32 ± 17 at baseline to 25 ± 12 (p < 0.05) at follow up.</p> <p>Conclusions</p> <p>Increased heart rate by atrial pacing in patients with Biv pacing causes compromise in diastolic filling time which can be improved by AV delay shortening. Aggressive AV delay shortening was required at heart rates in physiologic range to achieve optimal diastolic filling and was associated with an increase in LV ejection time during optimization. Functional class improved at 1 month post optimization using aggressive AV delay shortening algorithm derived from echo-guidance at the time of Biv pacemaker optimization.</p

Attenuation of acute lung inflammation induced by cigarette smoke in CXCR3 knockout mice

Background: CD8+ T cells may participate in cigarette smoke (CS) induced-lung inflammation in mice. CXCL10/IP-10 (IFNγ-inducible protein 10) and CXCL9/Mig (monokine induced by IFN-$\gamma$) are up-regulated in CS-induced lung injury and may attract T-cell recruitment to the lung. These chemokines together with CXCL11/ITAC (IFN-inducible T-cell alpha chemoattractant) are ligands for the chemokine receptor CXCR3 which is preferentially expressed chiefly in activated CD8+ T cells. The purpose of this investigation was to study the contribution of CXCR3 to acute lung inflammation induced by CS using CXCR3 knockout (KO) mice. Methods: Mice (n = 8 per group) were placed in a closed plastic box connected to a smoke generator and were exposed whole body to the tobacco smoke of five cigarettes four times a day for three days. Lung pathological changes, expression of inflammatory mediators in bronchoalveolar lavage (BAL) fluid and lungs at mRNA and protein levels, and lung infiltration of CD8+ T cells were compared between CXCR3-/- mice and wild type (WT) mice. Results: Compared with the WT littermates, CXCR3 KO mice showed less CS-induced lung inflammation as evidenced by less infiltration of inflammatory cells in airways and lung tissue, particularly fewer CD8+ T cells, lower levels of IFNγ and CXCR3 ligands (particularly CXCL10). Conclusion: Our findings show that CXCR3 is important in promoting CD8+ T cell recruitment and in initiating IFNγ and CXCL10 release following CS exposure. CXCR3 may represent a promising therapeutic target for acute lung inflammation induced by CS

Aging syndrome genes and premature coronary artery disease

BACKGROUND: Vascular disease is a feature of aging, and coronary vascular events are a major source of morbidity and mortality in rare premature aging syndromes. One such syndrome is caused by mutations in the lamin A/C (LMNA) gene, which also has been implicated in familial insulin resistance. A second gene related to premature aging in man and in murine models is the KLOTHO gene, a hypomorphic variant of which (KL-VS) is significantly more common in the first-degree relatives of patients with premature coronary artery disease (CAD). We evaluated whether common variants at the LMNA or KLOTHO genes are associated with rigorously defined premature CAD. METHODS: We identified 295 patients presenting with premature acute coronary syndromes confirmed by angiography. A control group of 145 patients with no evidence of CAD was recruited from outpatient referral clinics. Comprehensive haplotyping of the entire LMNA gene, including the promoter and untranslated regions, was performed using a combination of TaqMan(® )probes and direct sequencing of 14 haplotype-tagging single nucleotide polymorphisms (SNPs). The KL-VS variant of the KLOTHO gene was typed using restriction digest of a PCR amplicon. RESULTS: Two SNPs that were not in Hardy Weinberg equilibrium were excluded from analysis. We observed no significant differences in allele, genotype or haplotype frequencies at the LMNA or KLOTHO loci between the two groups. In addition, there was no evidence of excess homozygosity at the LMNA locus. CONCLUSION: Our data do not support the hypothesis that premature CAD is associated with common variants in the progeroid syndrome genes LMNA and KLOTHO