Neither dectin-2 nor the mannose receptor is required for resistance to Coccidioides immitis in mice

ABSTARCT: We investigated the roles of the mannose receptor (MR) and Dectin-2 in resistance to pulmonary coccidioidomycosis in C57BL/6 (B6) mice and in the interaction of myeloid cells with spherules, using B6 mice with targeted mutations in Mrc1 and Clec4n. Spherules are the tissue form of Coccidioides, and we determined that the MR on bone marrow-derived dendritic cells (BMDC) was important for recognition of spherules (formalin-killed spherules [FKS]) and for secretion of interleukin 10 (IL-10) and proinflammatory cytokines in response to FKS by both elicited macrophages and BMDC. Infected MR knockout (KO) mice produced more IL-10 in their lungs than did B6 mice, and MR KO mice also made more protective Th-17 cytokines. In contrast to the MR, Dectin-2 was not required for recognition of FKS by BMDC or for the production of cytokines by BMDC in response to FKS. However, Dectin-2 KO was required for stimulation of elicited peritoneal macrophages. Despite that, lung cytokine levels were not significantly different in Dectin-2 KO mice and B6 mice 14 days after infection, except for IL-1β, which was higher in Dectin-2 KO lungs. Although both Dectin-2(-/-) and MR(-/-) myeloid cells had reduced proinflammatory cytokine responses to FKS in vitro, neither MR nor Dectin-2 deficiency reduced the resistance of B6 mice to pulmonary coccidioidomycosis

The N-terminus of CD14 acts to bind apoptotic cells and confers rapid-tethering capabilities on non-myeloid cells:CD14 and rapid tethering of apoptotic cells

Cell death and removal of cell corpses in a timely manner is a key event in both physiological and pathological situations including tissue homeostasis and the resolution of inflammation. Phagocytic clearance of cells dying by apoptosis is a complex sequential process comprising attraction, recognition, tethering, signalling and ultimately phagocytosis and degradation of cell corpses. A wide range of molecules acting as apoptotic cell-associated ligands, phagocyte-associated receptors or soluble bridging molecules have been implicated within this process. The role of myeloid cell CD14 in mediating apoptotic cell interactions with macrophages has long been known though key molecules and residues involved have not been defined. Here we sought to further dissect the function of CD14 in apoptotic cell clearance. A novel panel of THP-1 cell-derived phagocytes was employed to demonstrate that CD14 mediates effective apoptotic cell interactions with macrophages in the absence of detectable TLR4 whilst binding and responsiveness to LPS requires TLR4. Using a targeted series of CD14 point mutants expressed in non-myeloid cells we reveal CD14 residue 11 as key in the binding of apoptotic cells whilst other residues are reported as key for LPS binding. Importantly we note that expression of CD14 in non-myeloid cells confers the ability to bind rapidly to apoptotic cells. Analysis of a panel of epithelial cells reveals that a number naturally express CD14 and that this is competent to mediate apoptotic cell clearance. Taken together these data suggest that CD14 relies on residue 11 for apoptotic cell tethering and it may be an important tethering molecule on so called 'non-professional' phagocytes thus contributing to apoptotic cell clearance in a non-myeloid setting. Furthermore these data establish CD14 as a rapid-acting tethering molecule, expressed in monocytes, which may thus confer responsiveness of circulating monocytes to apoptotic cell derived material. © 2013 Thomas et al

An open randomized clinical trial in comparing two artesunate-based combination treatments on Plasmodium falciparum malaria in Nigerian children: artesunate/sulphamethoxypyrazine/pyrimethamine (fixed dose over 24 hours) versus artesunate/amodiaquine (fixed dose over 48 hours)

<p>Abstract</p> <p>Background</p> <p>Several studies have demonstrated the efficacy of artemisinin-combination therapy (ACT) across malaria zones of the world. Fixed dose ACT with shorter courses and fewer tablets may be key determinants to ease of administration and compliance.</p> <p>Methods</p> <p>Children aged one year to 13 years presenting with uncomplicated <it>Plasmodium falciparum </it>malaria were recruited in Ibadan, south-western Nigeria. A total of 250 children each were randomly assigned to receive three doses of artesunate/sulphamethoxypyrazine/pyrimethamine (AS + SMP) (12 hourly doses over 24 hours) or three doses of artesunate/amodiaquine (AS + AQ) (daily doses over 48 hours). Efficacy and safety of the two drugs were assessed using a 28-day follow-up and the primary outcome was PCR- corrected parasitological cure rate and clinical response.</p> <p>Results</p> <p>There were two (0.4%) early treatment failures, one in each treatment arm. The PCR corrected cure rates for day 28 was 97.9% in the AS + AQ arm and 95.6% in the AS + SMP arm (p = 0.15). The re-infection rate was 1.7% in the AS + AQ arm and 5.7% in the AS + SMP arm (p = 0.021). The fever clearance time was similar in the two treatment groups: 1 - 2 days for both AS + SMP and AS + AQ (p = 0.271). The parasite clearance time was also similar in the two treatment groups with 1 - 7 days for AS + SMP and 1 - 4 days for AS + AQ (p = 0.941). The proportion of children with gametocytes over the follow-up period was similar in both treatment groups. Serious Adverse Events were not reported in any of the patients and in all children, laboratory values (packed cell volume, liver enzymes, bilirubin) remained within normal levels during the follow-up period but the packed cell volume was significantly lower in the AS + SMP group.</p> <p>Conclusions</p> <p>This study demonstrates that AS + SMP FDC given as three doses over 24 hours (12-hour intervals) has similar efficacy as AS + AQ FDC given as three doses over 48 hours (24-hour interval) for the treatment of uncomplicated <it>Plasmodium falciparum </it>malaria in children in Nigeria. Both drugs also proved to be safe. Therefore, AS + SMP could be an alternative to currently recommended first-line ACT with continuous resistance surveillance.</p

Respiratory epithelial cells require Toll-like receptor 4 for induction of Human β-defensin 2 by Lipopolysaccharide

BACKGROUND: The respiratory epithelium is a major portal of entry for pathogens and employs innate defense mechanisms to prevent colonization and infection. Induced expression of human β-defensin 2 (HBD2) represents a direct response by the epithelium to potential infection. Here we provide evidence for the critical role of Toll-like receptor 4 (TLR4) in lipopolysaccharide (LPS)-induced HBD2 expression by human A549 epithelial cells. METHODS: Using RTPCR, fluorescence microscopy, ELISA and luciferase reporter gene assays we quantified interleukin-8, TLR4 and HBD2 expression in unstimulated or agonist-treated A549 and/or HEK293 cells. We also assessed the effect of over expressing wild type and/or mutant TLR4, MyD88 and/or Mal transgenes on LPS-induced HBD2 expression in these cells. RESULTS: We demonstrate that A549 cells express TLR4 on their surface and respond directly to Pseudomonas LPS with increased HBD2 gene and protein expression. These effects are blocked by a TLR4 neutralizing antibody or functionally inactive TLR4, MyD88 and/or Mal transgenes. We further implicate TLR4 in LPS-induced HBD2 production by demonstrating HBD2 expression in LPS non-responsive HEK293 cells transfected with a TLR4 expression plasmid. CONCLUSION: This data defines an additional role for TLR4 in the host defense in the lung

Characterization of Within-Host Plasmodium falciparum Diversity Using Next-Generation Sequence Data

Our understanding of the composition of multi-clonal malarial infections and the epidemiological factors which shape their diversity remain poorly understood. Traditionally within-host diversity has been defined in terms of the multiplicity of infection (MOI) derived by PCR-based genotyping. Massively parallel, single molecule sequencing technologies now enable individual read counts to be derived on genome-wide datasets facilitating the development of new statistical approaches to describe within-host diversity. In this class of measures the FWS metric characterizes within-host diversity and its relationship to population level diversity. Utilizing P. falciparum field isolates from patients in West Africa we here explore the relationship between the traditional MOI and FWS approaches. FWS statistics were derived from read count data at 86,158 SNPs in 64 samples sequenced on the Illumina GA platform. MOI estimates were derived by PCR at the msp-1 and -2 loci. Significant correlations were observed between the two measures, particularly with the msp-1 locus (P = 5.92×10−5). The FWS metric should be more robust than the PCR-based approach owing to reduced sensitivity to potential locus-specific artifacts. Furthermore the FWS metric captures information on a range of parameters which influence out-crossing risk including the number of clones (MOI), their relative proportions and genetic divergence. This approach should provide novel insights into the factors which correlate with, and shape within-host diversity

Selective C-Rel Activation via Malt1 Controls Anti-Fungal TH-17 Immunity by Dectin-1 and Dectin-2

C-type lectins dectin-1 and dectin-2 on dendritic cells elicit protective immunity against fungal infections through induction of TH1 and TH-17 cellular responses. Fungal recognition by dectin-1 on human dendritic cells engages the CARD9-Bcl10-Malt1 module to activate NF-κB. Here we demonstrate that Malt1 recruitment is pivotal to TH-17 immunity by selective activation of NF-κB subunit c-Rel, which induces expression of TH-17-polarizing cytokines IL-1β and IL-23p19. Malt1 inhibition abrogates c-Rel activation and TH-17 immunity to Candida species. We found that Malt1-mediated activation of c-Rel is similarly essential to induction of TH-17-polarizing cytokines by dectin-2. Whereas dectin-1 activates all NF-κB subunits, dectin-2 selectively activates c-Rel, signifying a specialized TH-17-enhancing function for dectin-2 in anti-fungal immunity by human dendritic cells. Thus, dectin-1 and dectin-2 control adaptive TH-17 immunity to fungi via Malt1-dependent activation of c-Rel

Multiplicity of Plasmodium falciparum infection in asymptomatic children in Senegal: relation to transmission, age and erythrocyte variants

<p>Abstract</p> <p>Background</p> <p>Individuals living in malaria endemic areas generally harbour multiple parasite strains. Multiplicity of infection (MOI) can be an indicator of immune status. However, whether this is good or bad for the development of immunity to malaria, is still a matter of debate. This study aimed to examine the MOI in asymptomatic children between two and ten years of age and to relate it to erythrocyte variants, clinical attacks, transmission levels and other parasitological indexes.</p> <p>Methods</p> <p>Study took place in Niakhar area in Senegal, where malaria is mesoendemic and seasonal. Three hundred and seventy two asymptomatic children were included. Sickle-cell trait, G6PD deficiency (A- and Santamaria) and α⁺-thalassaemia (-α^3.7type) were determined using PCR. Multiplicity of <it>Plasmodium falciparum </it>infection, i.e. number of concurrent clones, was defined by PCR-based genotyping of the merozoite surface protein-2 (<it>msp2</it>), before and at the end of the malaria transmission season. The χ²-test, ANOVA, multivariate linear regression and logistic regression statistical tests were used for data analysis.</p> <p>Results</p> <p>MOI was significantly higher at the end of transmission season. The majority of PCR positive subjects had multiple infections at both time points (64% before and 87% after the transmission season). MOI did not increase in α-thalassaemic and G6PD mutated children. The ABO system and HbAS did not affect MOI at any time points. No association between MOI and clinical attack was observed. MOI did not vary over age at any time points. There was a significant correlation between MOI and parasite density, as the higher parasite counts increases the probability of having multiple infections.</p> <p>Conclusion</p> <p>Taken together our data revealed that α-thalassaemia may have a role in protection against certain parasite strains. The protection against the increase in MOI after the transmission season conferred by G6PD deficiency is probably due to clearance of the malaria parasite at early stages of infection. The ABO system and HbAS are involved in the severity of the disease but do not affect asymptomatic infections. MOI was not age-dependent, in the range of two to ten years, but was correlated with parasite density. However some of these observations need to be confirmed including larger sample size with broader age range and using other <it>msp2 </it>genotyping method.</p

Haemoglobin C and S Role in Acquired Immunity against Plasmodium falciparum Malaria

A recently proposed mechanism of protection for haemoglobin C (HbC; β6Glu→Lys) links an abnormal display of PfEMP1, an antigen involved in malaria pathogenesis, on the surface of HbC infected erythrocytes together with the observation of reduced cytoadhesion of parasitized erythrocytes and impaired rosetting in vitro. We investigated the impact of this hypothesis on the development of acquired immunity against Plasmodium falciparum variant surface antigens (VSA) encoding PfEMP1 in HbC in comparison with HbA and HbS carriers of Burkina Faso. We measured: i) total IgG against a single VSA, A4U, and against a panel of VSA from severe malaria cases in human sera from urban and rural areas of Burkina Faso of different haemoglobin genotypes (CC, AC, AS, SC, SS); ii) total IgG against recombinant proteins of P. falciparum asexual sporozoite, blood stage antigens, and parasite schizont extract; iii) total IgG against tetanus toxoid. Results showed that the reported abnormal cell-surface display of PfEMP1 on HbC infected erythrocytes observed in vitro is not associated to lower anti- PfEMP1 response in vivo. Higher immune response against the VSA panel and malaria antigens were observed in all adaptive genotypes containing at least one allelic variant HbC or HbS in the low transmission urban area whereas no differences were detected in the high transmission rural area. In both contexts the response against tetanus toxoid was not influenced by the β-globin genotype. These findings suggest that both HbC and HbS affect the early development of naturally acquired immunity against malaria. The enhanced immune reactivity in both HbC and HbS carriers supports the hypothesis that the protection against malaria of these adaptive genotypes might be at least partially mediated by acquired immunity against malaria

Breed and adaptive response modulate bovine peripheral blood cells’ transcriptome

Background: Adaptive response includes a variety of physiological modifications to face changes in external or internal conditions and adapt to a new situation. The acute phase proteins (APPs) are reactants synthesized against environmental stimuli like stress, infection, inflammation. Methods: To delineate the differences in molecular constituents of adaptive response to the environment we performed the whole-blood transcriptome analysis in Italian Holstein (IH) and Italian Simmental (IS) breeds. For this, 663 IH and IS cows from six commercial farms were clustered according to the blood level of APPs. Ten extreme individuals (five APP+ and APP- variants) from each farm were selected for the RNA-seq using the Illumina sequencing technology. Differentially expressed (DE) genes were analyzed using dynamic impact approach (DIA) and DAVID annotation clustering. Milk production data were statistically elaborated to assess the association of APP+ and APP- gene expression patterns with variations in milk parameters. Results: The overall de novo assembly of cDNA sequence data generated 13,665 genes expressed in bovine blood cells. Comparative genomic analysis revealed 1,152 DE genes in the comparison of all APP+ vs. all APP- variants; 531 and 217 DE genes specific for IH and IS comparison respectively. In all comparisons overexpressed genes were more represented than underexpressed ones. DAVID analysis revealed 369 DE genes across breeds, 173 and 73 DE genes in IH and IS comparison respectively. Among the most impacted pathways for both breeds were vitamin B6 metabolism, folate biosynthesis, nitrogen metabolism and linoleic acid metabolism. Conclusions: Both DIA and DAVID approaches produced a high number of significantly impacted genes and pathways with a narrow connection to adaptive response in cows with high level of blood APPs. A similar variation in gene expression and impacted pathways between APP+ and APP- variants was found between two studied breeds. Such similarity was also confirmed by annotation clustering of the DE genes. However, IH breed showed higher and more differentiated impacts compared to IS breed and such particular features in the IH adaptive response could be explained by its higher metabolic activity. Variations of milk production data were significantly associated with APP+ and APP- gene expression patterns

Dectin-1: a role in antifungal defense and consequences of genetic polymorphisms in humans

The clinical relevance of fungal infections has increased dramatically in recent decades as a consequence of the rise of immunocompromised populations, and efforts to understand the underlying mechanisms of protective immunity have attracted renewed interest. Here we review Dectin-1, a pattern recognition receptor involved in antifungal immunity, and discuss recent discoveries of polymorphisms in the gene encoding this receptor which result in human disease