How can malaria rapid diagnostic tests achieve their potential? A qualitative study of a trial at health facilities in Ghana

Malaria is still a major public health problem in Brazil, with approximately 306 000 registered cases in 2009, but it is estimated that in the early 1940s, around six million cases of malaria occurred each year. As a result of the fight against the disease, the number of malaria cases decreased over the years and the smallest numbers of cases to-date were recorded in the 1960s. From the mid-1960s onwards, Brazil underwent a rapid and disorganized settlement process in the Amazon and this migratory movement led to a progressive increase in the number of reported cases. Although the main mosquito vector (Anopheles darlingi) is present in about 80% of the country, currently the incidence of malaria in Brazil is almost exclusively (99,8% of the cases) restricted to the region of the Amazon Basin, where a number of combined factors favors disease transmission and impair the use of standard control procedures. Plasmodium vivax accounts for 83,7% of registered cases, while Plasmodium falciparum is responsible for 16,3% and Plasmodium malariae is seldom observed. Although vivax malaria is thought to cause little mortality, compared to falciparum malaria, it accounts for much of the morbidity and for huge burdens on the prosperity of endemic communities. However, in the last few years a pattern of unusual clinical complications with fatal cases associated with P. vivax have been reported in Brazil and this is a matter of concern for Brazilian malariologists. In addition, the emergence of P. vivax strains resistant to chloroquine in some reports needs to be further investigated. In contrast, asymptomatic infection by P. falciparum and P. vivax has been detected in epidemiological studies in the states of Rondonia and Amazonas, indicating probably a pattern of clinical immunity in both autochthonous and migrant populations. Seropidemiological studies investigating the type of immune responses elicited in naturally-exposed populations to several malaria vaccine candidates in Brazilian populations have also been providing important information on whether immune responses specific to these antigens are generated in natural infections and their immunogenic potential as vaccine candidates. The present difficulties in reducing economic and social risk factors that determine the incidence of malaria in the Amazon Region render impracticable its elimination in the region. As a result, a malaria-integrated control effort - as a joint action on the part of the government and the population - directed towards the elimination or reduction of the risks of death or illness, is the direction adopted by the Brazilian government in the fight against the disease.     

Refinement of Pig Retroperfusion Technique: Global Retroperfusion with Ligation of the Azygos Connection Preserves Hemodynamic Function in an Acute Infarction Model in Pigs (Sus scrofa domestica)

In ischemic hearts, venous retroperfusion is a potential myocardial revascularization strategy. This study aimed to refine the technical and functional aspects of a pig model of acute myocardial infarction and retroperfusion with respect to the azygos connection. Global retroperfusion after ligation of the ramus interventricularis paraconalis (equivalent to the left anterior descending artery in humans) was performed in 16 Landrace pigs (Sus scrofa domestica). Coronary sinus perfusion was performed in 8 pigs (P+) but not in the other 8 (P–), and the azygos vein was ligated (L+) 4 of the 8 pigs in each of these groups but left open (L–) in the remaining animals. Hemodynamic performance (for example, cardiac output, stroke volume) was significantly better in P+L+ pigs that underwent coronary sinus perfusion with ligation of the azygos vein compared with all other animals. In addition, troponin I release was significant lower in P+L+ pigs (1.7 ± 1.3 ng/mL) than in P–L– (5.47 ± 2.1 ng/mL), P–L+ (6.63 ± 2.4 ng/mL), and P+L– (4.81 ± 2.3 ng/mL) pigs. Effective retrograde flow and thus hemodynamic stability was achieved by ligation of the azygos vein. Therefore, experiments focusing on global retroperfusion will benefit from effective inhibition of the blood flow through the azygos vein.Abbreviations: ACS, aorta-to-coronary–sinus shunt, CS, coronary sinus, L, ligation, LAD, left anterior descending artery, P, perfusionAnimal models are used frequently to investigate myocardial revascularization techniques, and researchers have studied global or selective venous retroperfusion in dogs,²² pigs,⁹ and sheep.³³ The goal underlying retrograde coronary sinus (CS) perfusion is perfusion of the ischemic myocardium proximal to the occlusion or stenosis. This method frequently is used for delivering cardioplegic solutions during cardiac surgery. In addition, both clinical^{2,3,6,16,28,30} and experimental ^{11,21,25,34,37,42} studies have validated the efficiency of CS retroperfusion.Interpreting the results from experimental animal models and follow-up examinations of patients who have undergone venous revascularization has led to controversy.^9,37 In particular, technical problems with some studies have been identified. Previous animal studies on interspecies anatomic differences in mammals^4,7,19 have concentrated on the venous connections of the vessels draining the myocardium and have demonstrated a need for further feasibility studies of the pig model (German Landrace pigs, Sus scrofa domestica) that focus on hemodynamic performance.We wanted to characterize in detail the contribution of the azygos vein connection in swine during retroperfusion after myocardial infarction and hypothesized that ligation of the azygos vein would preserve hemodynamic function after ligation of the left anterior descending artery (LAD) in a global retroperfusion model in pigs.     

Synergy between docosahexaenoic acid and butyrate elicits p53-independent apoptosis via mitochondrial Ca(2+) accumulation in colonocytes

Butyrate, a short-chain fatty acid fiber fermentation product, induces colonocyte apoptosis in part via a Fas-mediated (extrinsic) pathway. In previous studies, we demonstrated that docosahexaenoic acid (DHA, 22:6(Delta4,7,10,13,16,19)) enhances the effect of butyrate by increasing mitochondrial lipid oxidation and mitochondrial Ca(2+)-dependent apoptosis in the colon. In this study, we further examined the mechanism of DHA-butyrate synergism in 1) human colon tumor (HCT-116 isogenic p53+/+ vs. p53-/-) cells and 2) primary cultures of rat colonic crypts. Herein, we show that DHA and butyrate promote apoptosis by enhancing mitochondrial Ca(2+) accumulation in both isogenic cell lines. Ca(2+) accumulation and apoptosis were inhibited by blockade of mitochondrial uniporter-mediated Ca(2+) uptake. In addition, Mito-Q, a mitochondria-targeted antioxidant, also blocked apoptosis induced by DHA and butyrate. In complementary experiments, rats were fed diets supplemented with either corn oil (control, contains no DHA) or fish oil (contains DHA). Colonic crypts were isolated and incubated with or without butyrate, after which the mitochondria-to-cytosol Ca(2+) ratio and crypt viability were measured. No significant difference (P > 0.05) in basal mitochondrial Ca(2+) levels was observed between fish oil- or corn oil-fed animals. In contrast, when fish oil was the dietary lipid source, crypts incubated with butyrate exhibited a significant increase (3.6-fold, P < 0.001) in mitochondrial Ca(2+) compared with corn oil plus butyrate treatment. On the basis of these data, we propose that the combination of DHA and butyrate compared with butyrate alone further enhances colonocyte apoptosis by inducing a p53-independent, oxidation-sensitive, mitochondrial Ca(2+) -dependent (intrinsic) pathway.     

Type 1 IFN mediates cross-talk between innate and adaptive immunity that abrogates transplantation tolerance

TLR activation of innate immunity prevents the induction of transplantation tolerance and shortens skin allograft survival in mice treated with costimulation blockade. The mechanism by which TLR signaling mediates this effect has not been clear. We now report that administration of the TLR agonists LPS (TLR4) or polyinosinic:polycytidylic acid (TLR3) to mice treated with costimulation blockade prevents alloreactive CD8(+) T cell deletion, primes alloreactive CTLs, and shortens allograft survival. The TLR4- and MyD88-dependent pathways are required for LPS to shorten allograft survival, whereas polyinosinic:polycytidylic acid mediates its effects through a TLR3-independent pathway. These effects are all mediated by signaling through the type 1 IFN (IFN-alphabeta) receptor. Administration of IFN-beta recapitulates the detrimental effects of TLR agonists on transplantation tolerance. We conclude that the type 1 IFN generated as part of an innate immune response to TLR activation can in turn activate adaptive immune responses that abrogate transplantation tolerance. Blocking of type 1 IFN-dependent pathways in patients may improve allograft survival in the presence of exogenous TLR ligands.     

Effects of cellular cholesterol loading on macrophage foam cell lysosome acidification

Macrophages incubated with mildly oxidized low density lipoprotein (OxLDL), aggregated low density lipoprotein (AggLDL), or cholesteryl ester-rich lipid dispersions (DISPs) accumulate cholesterol in lysosomes followed by an inhibition of lysosomal cholesteryl ester (CE) hydrolysis. The variety of cholesterol-containing particles producing inhibition of hydrolysis suggests that inhibition may relate to general changes in lysosomes. Lysosome pH is a key mediator of activity and thus is a potential mechanism for lipid-induced inhibition. We investigated the effects of cholesterol accumulation on THP-1 macrophage lysosome pH. Treatment with OxLDL, AggLDL, and DISPs resulted in inhibition of the lysosome's ability to maintain an active pH and concomitant decreases in CE hydrolysis. Consistent with an overall disruption of lysosome function, exposure to OxLDL or AggLDL reduced lysosomal apolipoprotein B degradation. The lysosomal cholesterol sequestration and inactivation are not observed in cholesterol-equivalent cells loaded using acetylated low density lipoprotein (AcLDL). However, AcLDL-derived cholesterol in the presence of progesterone (to block cholesterol egression from lysosomes) inhibited lysosome acidification. Lysosome inhibition was not attributable to a decrease in the overall levels of vacuolar ATPase. However, augmentation of membrane cholesterol in isolated lysosomes inhibited vacuolar ATPase-dependent pumping of H+ ions into lysosomes. These data indicate that lysosomal cholesterol accumulation alters lysosomes in ways that could exacerbate foam cell formation and influence atherosclerotic lesion development.     

Innate recognition network driving herpes simplex virus-induced corneal immunopathology: role of the toll pathway in early inflammatory events in stromal keratitis

Ocular infection with herpes simplex virus (HSV) sets off an array of events that succeed in clearing virus from the cornea but leaves the tissue with a CD4(+) T-cell-orchestrated chronic inflammatory lesion that impairs vision. We demonstrate that Toll-like receptor (TLR) signaling forms a part of the recognition system that induces the syndrome that eventually culminates in immunopathology. Accordingly, in a comparison of the outcomes of infection in wild-type (WT) mice and those lacking TLR function, it was apparent that the absence of TLR2 and, to a lesser extent, TLR9 resulted in significantly diminished lesions. Similarly, mice lacking the adapter molecule MyD88 were resistant to lesion development, but such animals were also unable to control infection, with most succumbing to lethal encephalitis. The susceptibility of TLR4(-/-) animals was also evaluated. These animals developed lesions, which were more severe, more rapidly than did WT animals. We discuss the possible mechanisms by which early recognition of HSV constituents impacts the subsequent development of immunopathological lesions.     

Regulation of hepcidin in HepG2 and RINm5F cells

Despite the high impact of the antimicrobial peptide hepcidin in iron homeostasis, the regulation of this hormone is still not completely understood. Studies concerning hepcidin regulation are performed at the mRNA level. For the first time we analyzed the regulation of hepcidin not only at mRNA, but also at protein level in a hepatoma and a pancreatic beta cell line using quantitative RT-PCR and immunoblot analysis. Our data show, that hepcidin is present in HepG2 and RINm5F cells. A significant up-regulation of hepcidin was observed in both cell lines by the inflammatory cytokine interleukin-6, lipopolysaccharide, and a slight upregulation by deferoxamine. A down-regulation was detected after stimulation with erythropoietin. Hepcidin was regulated by iron in a dose dependent manner: low doses up to 3 microM increased hepcidin expression, high doses of iron (65 microM) revealed a switch-over to down-regulation of hepcidin expression. Regulation of hepcidin in HepG2 and RINm5F cells at mRNA and protein level by these substances indicates its involvement in inflammation and iron metabolism.     

Abortive phage resistance mechanism AbiZ speeds the lysis clock to cause premature lysis of phage-infected Lactococcus lactis

The conjugative plasmid pTR2030 has been used extensively to confer phage resistance in commercial Lactococcus starter cultures. The plasmid harbors a 16-kb region, flanked by insertion sequence (IS) elements, that encodes the restriction/modification system LlaI and carries an abortive infection gene, abiA. The AbiA system inhibits both prolate and small isometric phages by interfering with the early stages of phage DNA replication. However, abiA alone does not account for the full abortive activity reported for pTR2030. In this study, a 7.5-kb region positioned within the IS elements and downstream of abiA was sequenced to reveal seven additional open reading frames (ORFs). A single ORF, designated abiZ, was found to be responsible for a significant reduction in plaque size and an efficiency of plaquing (EOP) of 10(-6), without affecting phage adsorption. AbiZ causes phage phi31-infected Lactococcus lactis NCK203 to lyse 15 min early, reducing the burst size of phi31 100-fold. Thirteen of 14 phages of the P335 group were sensitive to AbiZ, through reduction in either plaque size, EOP, or both. The predicted AbiZ protein contains two predicted transmembrane helices but shows no significant DNA homologies. When the phage phi31 lysin and holin genes were cloned into the nisin-inducible shuttle vector pMSP3545, nisin induction of holin and lysin caused partial lysis of NCK203. In the presence of AbiZ, lysis occurred 30 min earlier. In holin-induced cells, membrane permeability as measured using propidium iodide was greater in the presence of AbiZ. These results suggest that AbiZ may interact cooperatively with holin to cause premature lysis.