Characterization of Undiscovered miRNA Involved in Tumor Necrosis Factor Alpha-Induced Atrophy in Mouse Skeletal Muscle Cell Line.

Muscular atrophy is a complex catabolic condition that develops due to several inflammatory-related disorders, resulting in muscle loss. Tumor necrosis factor alpha (TNF-α) is believed to be one of the leading factors that drive inflammatory response and its progression. Until now, the link between inflammation and muscle wasting has been thoroughly investigated, and the non-coding RNA machinery is a potential connection between the candidates. This study aimed to identify specific miRNAs for muscular atrophy induced by TNF-α in the C2C12 murine myotube model. The difference in expression of fourteen known miRNAs and two newly identified miRNAs was recorded by next-generation sequencing between normal muscle cells and treated myotubes. After validation, we confirmed the difference in the expression of one novel murine miRNA (nov-mmu-miRNA-1) under different TNF-α-inducing conditions. Functional bioinformatic analyses of nov-mmu-miRNA-1 revealed the potential association with inflammation and muscle atrophy. Our results suggest that nov-mmu-miRNA-1 may trigger inflammation and muscle wasting by the downregulation of LIN28A/B, an anti-inflammatory factor in the let-7 family. Therefore, TNF-α is involved in muscle atrophy through the modulation of the miRNA cellular machinery. Here, we describe for the first time and propose a mechanism for the newly discovered miRNA, nov-mmu-miRNA-1, which may regulate inflammation and promote muscle atrophy.

Iron-Dependent Cell Death: A New Treatment Approach against Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating tumor type where a very high proportion of people diagnosed end up dying from cancer. Surgical resection is an option for only about 20% of patients, where the 5-year survival increase ranges from 10 to 25%. In addition to surgical resection, there are adjuvant chemotherapy schemes, such as FOLFIRINOX (a mix of Irinotecan, oxaliplatin, 5-Fluorouraci and leucovorin) or gemcitabine-based treatment. These last two drugs have been compared in the NAPOLI-3 clinical trial, and the NALIRIFOX arm was found to have a higher overall survival (OS) (11.1 months vs. 9.2 months). Despite these exciting improvements, PDAC still has no effective treatment. An interesting approach would be to drive ferroptosis in PDAC cells. A non-apoptotic reactive oxygen species (ROS)-dependent cell death, ferroptosis was first described by Dixon et al. in 2012. ROS are constantly produced in the tumor cell due to high cell metabolism, which is even higher when exposed to chemotherapy. Tumor cells have detoxifying mechanisms, such as Mn-SOD or the GSH-GPX system. However, when a threshold of ROS is exceeded in the tumor cell, the cell's antioxidant systems are overwhelmed, resulting in lipid peroxidation and, ultimately, ferroptosis. In this review, we point out ferroptosis as an approach to consider in PDAC and propose that altering the cellular ROS balance by combining oxidizing agents or with inhibitors of the main cellular detoxifiers triggers ferroptosis in PDAC.

The Impact of Maternal Human Immunodeficiency Virus Infection on the Burden of Respiratory Syncytial Virus Among Pregnant Women and Their Infants, Western Kenya.

BACKGROUND: Respiratory syncytial virus (RSV) is an important cause of respiratory illness worldwide; however, burden data on mother-infant pairs remain sparse in sub-Saharan Africa, where human immunodeficiency virus (HIV) is prevalent. We evaluated the impact of maternal HIV infection on the burden of RSV among mothers and their infants in western Kenya. METHODS: We enrolled pregnant women (≤20 weeks' gestation) and followed them and their newborns weekly for up to 3-6 months postpartum, to document cases of acute respiratory illness (ARI). Nasal/oropharyngeal swabs were collected and tested for RSV using polymerase chain reaction. Analyses were stratified by maternal HIV status and incidence was computed per 1000 person-months. RESULTS: Compared to RSV-negative ARI cases, RSV-positive cases were associated with cough, apnea, and hospitalization among infants. RSV incidence per 1000 person-months among mothers was 4.0 (95% confidence interval [CI], 3.2-4.4), and was twice that among the HIV-infected mothers (8.4 [95% CI, 5.7-12.0]) compared to the HIV-uninfected mothers (3.1 [95% CI, 2.3-4.0]). Among infants, incidence per 1000 person-months was 15.4 (95% CI, 12.5-18.8); incidence did not differ by HIV exposure or prematurity. CONCLUSIONS: HIV infection may increase the risk of RSV illness among pregnant women. Future maternal RSV vaccines may have added benefit in areas with high HIV prevalence.

TNF-α Induced Myotube Atrophy in C2C12 Cell Line Uncovers Putative Inflammatory-Related lncRNAs Mediating Muscle Wasting.

BACKGROUND: Muscle atrophy is a complex catabolic condition developing under different inflammatory-related systemic diseases resulting in wasting of muscle tissue. While the knowledge of the molecular background of muscle atrophy has developed in recent years, how the atrophic conditions affect the long non-coding RNA (lncRNAs) machinery and the exact participation of the latter in the mediation of muscle loss are still unknown. The purpose of the study was to assess how inflammatory condition developing under the tumor necrosis factor alpha (TNF-α) treatment affects the lncRNAs' expression in a mouse skeletal muscle cell line. MATERIALS AND METHOD: A C2C12 mouse myoblast cell line was treated with TNF-α to develop atrophy, and inflammatory-related lncRNAs mediating muscle loss were identified. Bioinformatics was used to validate and analyze the discovered lncRNAs. The differences in their expression under different TNF-α concentrations and treatment times were investigated. RESULTS: Five lncRNAs were identified in a discovery set as atrophy related and then validated. Three lncRNAs, Gm4117, Ccdc41os1, and 5830418P13Rik, were selected as being significant for inflammatory-related myotube atrophy. Dynamics changes in the expression of lncRNAs depended on both TNF-α concentration and treatment time. Bioinformatics analysis revealed the mRNA and miRNA target for selected lncRNAs and their putative involvement in the molecular processes related to muscle atrophy. CONCLUSIONS: The inflammatory condition developing in the myotube under the TNF-α treatment affects the alteration of lncRNAs' expression pattern. Experimental and bioinformatics testing suggested the prospective role of lncRNAs in the mediation of muscle loss under an inflammatory state.

Rifampicin and clarithromycin (extended release) versus rifampicin and streptomycin for limited Buruli ulcer lesions: a randomised, open-label, non-inferiority phase 3 trial.

BACKGROUND: Buruli ulcer is a neglected tropical disease caused by Mycobacterium ulcerans infection that damages the skin and subcutis. It is most prevalent in western and central Africa and Australia. Standard antimicrobial treatment with oral rifampicin 10 mg/kg plus intramuscular streptomycin 15 mg/kg once daily for 8 weeks (RS8) is highly effective, but streptomycin injections are painful and potentially harmful. We aimed to compare the efficacy and tolerability of fully oral rifampicin 10 mg/kg plus clarithromycin 15 mg/kg extended release once daily for 8 weeks (RC8) with that of RS8 for treatment of early Buruli ulcer lesions. METHODS: We did an open-label, non-inferiority, randomised (1:1 with blocks of six), multicentre, phase 3 clinical trial comparing fully oral RC8 with RS8 in patients with early, limited Buruli ulcer lesions. There were four trial sites in hospitals in Ghana (Agogo, Tepa, Nkawie, Dunkwa) and one in Benin (Pobè). Participants were included if they were aged 5 years or older and had typical Buruli ulcer with no more than one lesion (caterories I and II) no larger than 10 cm in diameter. The trial was open label, and neither the investigators who took measurements of the lesions nor the attending doctors were masked to treatment assignment. The primary clinical endpoint was lesion healing (ie, full epithelialisation or stable scar) without recurrence at 52 weeks after start of antimicrobial therapy. The primary endpoint and safety were assessed in the intention-to-treat population. A sample size of 332 participants was calculated to detect inferiority of RC8 by a margin of 12%. This study was registered with ClinicalTrials.gov, NCT01659437. FINDINGS: Between Jan 1, 2013, and Dec 31, 2017, participants were recruited to the trial. We stopped recruitment after 310 participants. Median age of participants was 14 years (IQR 10-29) and 153 (52%) were female. 297 patients had PCR-confirmed Buruli ulcer; 151 (51%) were assigned to RS8 treatment, and 146 (49%) received oral RC8 treatment. In the RS8 group, lesions healed in 144 (95%, 95% CI 91 to 98) of 151 patients, whereas lesions healed in 140 (96%, 91 to 99) of 146 patients in the RC8 group. The difference in proportion, -0·5% (-5·2 to 4·2), was not significantly greater than zero (p=0·59), showing that RC8 treatment is non-inferior to RS8 treatment for lesion healing at 52 weeks. Treatment-related adverse events were recorded in 20 (13%) patients receiving RS8 and in nine (7%) patients receiving RC8. Most adverse events were grade 1-2, but one (1%) patient receiving RS8 developed serious ototoxicity and ended treatment after 6 weeks. No patients needed surgical resection. Four patients (two in each study group) had skin grafts. INTERPRETATION: Fully oral RC8 regimen was non-inferior to RS8 for treatment of early, limited Buruli ulcer and was associated with fewer adverse events. Therefore, we propose that fully oral RC8 should be the preferred therapy for early, limited lesions of Buruli ulcer. FUNDING: WHO with additional support from MAP International, American Leprosy Missions, Fondation Raoul Follereau France, Buruli ulcer Groningen Foundation, Sanofi-Pasteur, and BuruliVac.

Socio-demographic and sexual practices associated with HIV infection in Kenyan injection and non-injection drug users.

BACKGROUND: Substance use is increasingly becoming prevalent on the African continent, fueling the spread of HIV infection. Although socio-demographic factors influence substance consumption and risk of HIV infection, the association of these factors with HIV infection is poorly understood among substance users on the African continent. The objective of the study was to assess socio-demographic and sexual practices that are associated with HIV infection among injection drug users (IDUs), non-IDUs, and non-drug users (DUs) at an urban setting of coastal Kenya. METHODS: A cross-sectional descriptive study was conducted among 451 adults comprising HIV-infected and -uninfected IDUs (n = 157 and 39); non-IDUs (n = 17 and 48); and non-DUs (n = 55 and 135); respectively at coastal, Kenya. Respondent driven sampling, snowball and makeshift methods were used to enroll IDUs and non-IDUs. Convenience and purposive sampling were used to enroll non-DUs from the hospital's voluntary HIV testing unit. Participant assisted questionnaire was used in collecting socio-demographic data and sexual practices. RESULTS: Binary logistic regression analysis indicated that higher likelihood of HIV infection was associated with sex for police protection (OR, 9.526; 95% CI, 1.156-78.528; P = 0.036) and history of sexually transmitted infection (OR, 5.117; 95% CI, 1.924-13.485; P = 0.001) in IDUs; divorced, separated or widowed marital status (OR, 6.315; 95% CI, 1.334-29.898; P = 0.020) in non-IDUs; and unemployment (OR, 2.724; 95% CI, 1.049-7.070; P = 0.040) in non-drug users. However, never married (single) marital status (OR, 0.140; 95% CI, 0.030-0.649; P = 0.012) was associated with lower odds for HIV infection in non-drug users. CONCLUSION: Altogether, these results suggest that socio-demographic and sexual risk factors for HIV transmission differ with drug use status, suggesting targeted preventive measures for drug users.

DLL3 Is a Prognostic and Potentially Predictive Biomarker for Immunotherapy Linked to PD/PD-L Axis and NOTCH1 in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive neoplasm with very poor patient survival outcomes despite available treatments. There is an urgent need for new potential treatment options and novel biomarkers for these patients. Delta-like canonical Notch ligand 3 (DLL3) interacts with the Notch receptor and causes inhibition of Notch signaling, which confers a survival advantage to PDAC cells. Thus, DLL3 expression could affect cell survival, and its inhibition could increase a patient's survival. To test this hypothesis, a survival analysis was conducted using the progression-free and overall survival from two independent datasets of PDAC patients, with one using mRNA z-score levels and the other using the Hscore protein expression level; both were carried out using a log-rank test and plotted using Kaplan-Meier curves. DLL3 at the mRNA expression level showed an association between high mRNA expression and both a longer progression-free survival (PFS) and overall survival (OS) of patients. Then, we designed a retrospective study with resected PDAC samples. Our primary objective with this dataset was to assess the relationship between PFS and OS and DLL3 protein expression. The secondary assessment was to provide a rationale for the use of anti-DLL3-based treatments in combination with immunotherapy that is supported by the link between DLL3 and other factors that are involved in immune checkpoints. The survival analyses revealed a protective effect of high DLL3 protein expression levels in both PFS and OS. Interestingly, high DLL3 protein expression levels were significantly correlated with PD-L1/2 and negatively correlated with NOTCH1. Therefore, DLL3 could be considered a biomarker for better prognosis in resectable PDAC patients as well as a therapeutic biomarker for immunotherapy response. These facts set a rationale for testing anti-DLL3-based treatments either alone or combined with immunotherapy or other NOTCH1 inhibitors.

Blood Circulating Non-Coding RNAs for the Clinical Management of Triple-Negative Breast Cancer.

Triple negative breast cancer (TNBC) represents the most aggressive subtype of breast cancer, and is related to unfavorable prognosis and limited treatment strategies. Currently, there is a lack of reliable biomarkers allowing for the clinical management of TNBC. This is probably caused by a complex molecular background, leading to the development and establishment of a unique tumor phenotype. Recent studies have reported non-coding RNAs (ncRNAs) not only as the most promising class of molecular agents with a high applicability to manage human cancers, including TNBC, but also as robust and non-invasive biomarkers that are able to be monitored in blood circulation, with the application of liquid biopsy. There is a lack of papers discussing the role of blood-circulating ncRNAs as diagnostic, predictive, and prognostic biomarkers for TNBC. In this paper, we summarized the available literature reports on the utility of blood-circulating ncRNAs for TNBC management. Additionally, we supplemented this review by bioinformatics analysis, for better understanding of the role of ncRNAs' machinery in the development of a unique TNBC phenotype.

The levels of CD16/Fc gamma receptor IIIA on CD14+ CD16+ monocytes are higher in children with severe Plasmodium falciparum anemia than in children with cerebral or uncomplicated malaria.

Fc gamma receptor IIIA (CD16/Fc gamma RIIIA) on monocytes/macrophages may play an important role in the pathogenesis of severe malarial anemia (SMA) by promoting phagocytosis of IgG-coated uninfected red cells and by allowing the production of tumor necrosis factor alpha (TNF-alpha) upon cross-linking by immune complexes (ICs). However, not much is known about the differential expression of this receptor on monocytes of children with severe malaria and uncomplicated malaria. Therefore, we investigated the expression of CD16/Fc gamma RIIIA on monocytes of children with SMA, cerebral malaria (CM), and their age-matched uncomplicated malaria controls by flow cytometry. Since CD14 low (CD14(+)) monocytes are considered more mature and macrophage-like than CD14 high (CD14(++)) monocytes, we also compared the level of expression of CD16/Fc gamma RIIIA according to the CD14 level and studied the relationship between CD16/Fc gamma RIIIA expression and intracellular TNF-alpha production upon stimulation by ICs. CD16/Fc gamma RIIIA expression was the highest overall on CD14(+) CD16(+) monocytes of children with SMA at enrollment. At convalescence, SMA children were the only ones to show a significant decline in the same parameter. In contrast, there were no significant differences among groups in the expression of CD16/Fc gamma RIIIA on CD14(++) CD16(+) monocytes. A greater percentage of CD14(+) CD16(+) monocytes produced TNF-alpha upon stimulation than any other monocyte subset, and the amount of intracellular TNF-alpha correlated positively with CD16/Fc gamma RIIIA expression. Furthermore, there was an inverse correlation between hemoglobin levels and CD16/Fc gamma RIIIA expression in children with SMA and their controls. These data suggest that monocytes of children with SMA respond differently to Plasmodium falciparum infection by overexpressing CD16/Fc gamma RIIIA as they mature, which could enhance erythrophagocytosis and TNF-alpha production.

A novel functional variant in the stem cell growth factor promoter protects against severe malarial anemia.

Plasmodium falciparum malaria is a leading global cause of infectious disease burden. In areas in which P. falciparum transmission is holoendemic, such as western Kenya, severe malarial anemia (SMA) results in high rates of pediatric morbidity and mortality. Although the pathophysiological basis of SMA is multifactorial, we recently discovered that suppression of unexplored hematopoietic growth factors that promote erythroid and myeloid colony development, such as stem cell growth factor (SCGF) (C-type lectin domain family member 11A [CLEC11A]), was associated with enhanced development of SMA and reduced erythropoietic responses. To extend these investigations, the relationships between a novel SCGF promoter variant (-539C/T, rs7246355), SMA (hemoglobin [Hb] < 6.0 g/dl), and reduced erythropoietic responses (reticulocyte production index [RPI], <2.0) were investigated with Kenyan children (n = 486) with falciparum malaria from western Kenya. Circulating SCGF was positively correlated with hemoglobin levels (r = 0.251; P = 0.022) and the reticulocyte production index (RPI) (r = 0.268; P = 0.025). Children with SMA also had lower SCGF levels than those in the non-SMA group (P = 0.005). Multivariate logistic regression analyses controlling for covariates demonstrated that individuals with the homologous T allele were protected against SMA (odds ratio, 0.57; 95% confidence interval [95% CI] 0.34 to 0.94; P = 0.027) relative to CC (wild-type) carriers. Carriers of the TT genotype also had higher SCGF levels in circulation (P = 0.018) and in peripheral blood mononuclear cell culture supernatants (P = 0.041), as well as an elevated RPI (P = 0.005) relative to individuals with the CC genotype. The results presented here demonstrate that homozygous T at -539 in the SCGF promoter is associated with elevated SCGF production, enhanced erythropoiesis, and protection against the development of SMA in children with falciparum malaria.

The impact of maternal HIV and malaria infection on the prevalence of congenital cytomegalovirus infection in Western Kenya.

BACKGROUND: Data on congenital cytomegalovirus (CMV) infection in Africa are limited. OBJECTIVE: To describe the prevalence of congenital CMV infection in a population with high prevalence of maternal HIV and malaria infection in western Kenya. STUDY DESIGN: We screened newborns for CMV by polymerase chain reaction assay of saliva swabs and dried blood spots (DBS), and assessed maternal CMV immunoglobulin G (IgG) status by testing serum eluted from newborn's DBS. We calculated adjusted prevalence ratios (aPRs) using log-binomial regression models. RESULTS: Among 1066 mothers, 210 (19·7%) had HIV infection and 207 (19·4%) had malaria infection; 33 (3·1%) mothers had both. Maternal CMV IgG prevalence was 93·1% (95% confidence interval [CI]: 88·3%-96·0%). Among 1078 newborns (12 sets of twins), 39 (3·6%, 95% CI: 2·7-4·9%) were CMV positive. The prevalence of congenital CMV infection by maternal HIV and malaria infection status was 5·0% (95% CI: 2·7-9·2%) for HIV only, 5·1% (95% CI: 2·7-9·4%) for malaria only, 8·8 (95% CI: 3·1-23·0) for HIV and malaria co-infection, and 2·6% (95% CI: 1·7-4·1%) for none. Congenital CMV infection was independently associated with maternal HIV infection (aPR=2·1; 95% CI: 1·0-4·2), adjusting for maternal age, parity, and malaria infection. CONCLUSIONS: The prevalence of congenital CMV infection was higher than the 0·2-0·7% in developed countries. Maternal HIV infection may increase the risk of congenital CMV infection, but the role of maternal malaria on intrauterine transmission of CMV remains unclear.

Haplotypes of IL-10 promoter variants are associated with susceptibility to severe malarial anemia and functional changes in IL-10 production.

Plasmodium falciparum malaria is one of the leading global causes of morbidity and mortality with African children bearing the highest disease burden. Among the various severe disease sequelae common to falciparum malaria, severe malarial anemia (SMA) in pediatric populations accounts for the greatest degree of mortality. Although the patho-physiological basis of SMA remains unclear, dysregulation in inflammatory mediators, such as interleukin (IL)-10, appear to play an important role in determining disease outcomes. Since polymorphic variability in innate immune response genes conditions susceptibility to malaria, the relationship between common IL-10 promoter variants (-1,082A/G, -819T/C, and -592A/C), SMA (Hb < 6.0 g/dL), and circulating inflammatory mediator levels (i.e., IL-10, TNF-alpha, IL-6 and IL-12) were investigated in parasitemic Kenyan children (n = 375) in a holoendemic P. falciparum transmission area. Multivariate logistic regression analyses demonstrated that the -1,082G/-819C/-592C (GCC) haplotype was associated with protection against SMA (OR; 0.68, 95% CI, 0.43-1.05; P = 0.044) and increased IL-10 production (P = 0.029). Although none of the other haplotypes were significantly associated with susceptibility to SMA, individuals with the -1,082A/-819T/-592A (ATA) haplotype had an increased risk of SMA and reduced circulating IL-10 levels (P = 0.042). Additional results revealed that the IL-10:TNF-alpha ratio was higher in the GCC group (P = 0.024) and lower in individuals with the ATA haplotype (P = 0.034), while the IL-10:IL-12 ratio was higher in ATA haplotype (P = 0.006). Results presented here demonstrate that common IL-10 promoter haplotypes condition susceptibility to SMA and functional changes in circulating IL-10, TNF-alpha, and IL-12 levels in children with falciparum malaria.

Systematic review of clinical trials assessing the therapeutic efficacy of visceral leishmaniasis treatments: A first step to assess the feasibility of establishing an individual patient data sharing platform.

BACKGROUND: There are an estimated 200,000 to 400,000 cases of visceral leishmaniasis (VL) annually. A variety of factors are taken into account when considering the best therapeutic options to cure a patient and reduce the risk of resistance, including geographical area, malnourishment and HIV coinfection. Pooled analyses combine data from many studies to answer specific scientific questions that cannot be answered with individual studies alone. However, the heterogeneity of study design, data collection, and analysis often makes direct comparison difficult. Individual Participant Data (IPD) files can be standardised and analysed, allowing detailed analysis of this merged larger pool, but only a small fraction of systematic reviews and meta-analyses currently employ pooled analysis of IPD. We conducted a systematic literature review to identify published studies and studies reported in clinical trial registries to assess the feasibility of developing a VL data sharing platform to facilitate an IPD-based analysis of clinical trial data. Studies conducted between 1983 to 2015 that reported treatment outcome were eligible. PRINCIPAL FINDINGS: From the 2,271 documents screened, 145 published VL clinical trials were identified, with data from 26,986 patients. Methodologies varied for diagnosis and treatment outcomes, but overall the volume of data potentially available on different drugs and dose regimens identified hundreds or possibly thousands of patients per arm suitable for IPD pooled meta-analyses. CONCLUSIONS: A VL data sharing platform would provide an opportunity to maximise scientific use of available data to enable assessment of treatment efficacy, contribute to evidence-based clinical management and guide optimal prospective data collection.

CD4 T-cell expression of IFN-γ and IL-17 in pediatric malarial anemia.

In Plasmodium falciparum holoendemic transmission regions of western Kenya, life-threatening pediatric malaria manifests primarily as severe malarial anemia (SMA, Hb≤6.0 g/dL with any density parasitemia). To determine the role that CD4+ T-cell-driven inflammatory responses have in the pathogenesis of SMA, peripheral CD4+ T-cell populations and their intracellular production of pro-inflammatory cytokines (IFN-γ and IL-17) were characterized in children aged 12-36 months of age stratified into two groups: non-severe malarial anemia (non-SMA, Hb≥6.0 g/dL, n = 50) and SMA (n = 39). In addition, circulating IFN-γ and IL-17 were measured as part of a Cytokine 25-plex Antibody Bead Kit, Human (BioSource™ International). Children with SMA had higher overall proportions of circulating lymphocytes (P = 0.003) and elevated proportions of lymphocytes expressing IFN-γ (P = 0.014) and comparable IL-17 (P = 0.101). In addition, SMA was characterized by decreased memory-like T-cells (CD4+CD45RA-) expressing IL-17 (P = 0.009) and lower mean fluorescence intensity in memory-like CD4+ T-cells for both IFN-γ (P = 0.063) and IL-17 (P = 0.006). Circulating concentrations of IFN-γ were higher in children with SMA (P = 0.009), while IL-17 levels were comparable between the groups (P = 0.164). Furthermore, circulating levels of IFN-γ were negatively correlated with IL-17 levels in both groups of children (SMA: r = -0.610, P = 0.007; and non-SMA: r = -0.516, P = 0.001), while production of both cytokines by lymphocytes were positively correlated (SMA: r = 0.349, P = 0.037; and non-SMA: r = 0.475, P = 0.001). In addition, this correlation was only maintained by the memory-like CD4+ T cells (r = 0.365, P = 0.002) but not the naïve-like CD4+ T cells. However, circulating levels of IFN-γ were only associated with naïve-like CD4+ T cells producing IFN-γ (r = 0.547, P = 0.028), while circulating levels of IL-17 were not associated with any of the cell populations. Taken together, these results suggest that enhanced severity of malarial anemia is associated with higher overall levels of circulating lymphocytes, enhanced intracellular production of IFN-γ by peripheral lymphocytes and high circulating IFN-γ levels. In addition, the observed inverse relationship between the circulating levels of IFN-γ and IL-17 together with the reduction in the levels of memory-like CD4+ T cells expressing IL-17 in children with SMA may suggest possible relocation of these cells in the deeper tissues for their pathological effect.

Association of FCgamma receptor IIA (CD32) polymorphism with malarial anemia and high-density parasitemia in infants and young children.

Protective immunity against Plasmodium falciparum is partially mediated through binding of malaria-specific IgG antibodies to Fcgamma receptors. Polymorphic variability in Fcgamma RIIa (H/R-131) is associated with differential binding of IgG subtypes and malaria disease outcomes. However, the role of Fcgamma RIIa-131 variability in conditioning susceptibility to severe malarial anemia, the primary manifestation of severe malaria in holoendemic P. falciparum transmission areas, is largely undefined. Thus, Fcgamma RIIa-H131R polymorphism was investigated in 493 children who came to a hospital with acute malaria. Variation in Fcgamma RIIa-131 was not significantly associated with severe malarial anemia (hemoglobin [Hb] < 6.0 g/dL) or malaria anemia (Hb < 8.0 g/dL). However, relative to the heterozygous genotype, homozygotes for the R131 alleles were protected against high-density parasitemia (>or= 10,000 parasites/microL; odds ratio [OR] = 0.58, 95% confidence interval [CI] = 0.37-0.92, P = 0.02), while homozygotes for the H131 alleles were mildly protective (OR = 0.71, 95% CI = 0.45-1.13, P = 0.14). Additional multivariate analyses showed that infection with human immunodeficiency virus type 1 did not influence the associations between FcgammaRIIa-H131R polymorphism and malaria disease outcomes. Genotypic results presented here parallel data illustrating that parasite density is unrelated to the severity of anemia in children with acute malaria. Thus, although homozygosity for the R131 allele protects against high-density parasitemia, FcgammaRIIa-131 polymorphism does not protect against malaria anemia.

Whole genome characterization of human influenza A(H1N1)pdm09 viruses isolated from Kenya during the 2009 pandemic.

An influenza pandemic caused by a novel influenza virus A(H1N1)pdm09 spread worldwide in 2009 and is estimated to have caused between 151,700 and 575,400 deaths globally. While whole genome data on new virus enables a deeper insight in the pathogenesis, epidemiology, and drug sensitivities of the circulating viruses, there are relatively limited complete genetic sequences available for this virus from African countries. We describe herein the full genome analysis of influenza A(H1N1)pdm09 viruses isolated in Kenya between June 2009 and August 2010. A total of 40 influenza A(H1N1)pdm09 viruses isolated during the pandemic were selected. The segments from each isolate were amplified and directly sequenced. The resulting sequences of individual gene segments were concatenated and used for subsequent analysis. These were used to infer phylogenetic relationships and also to reconstruct the time of most recent ancestor, time of introduction into the country, rates of substitution and to estimate a time-resolved phylogeny. The Kenyan complete genome sequences clustered with globally distributed clade 2 and clade 7 sequences but local clade 2 viruses did not circulate beyond the introductory foci while clade 7 viruses disseminated country wide. The time of the most recent common ancestor was estimated between April and June 2009, and distinct clusters circulated during the pandemic. The complete genome had an estimated rate of nucleotide substitution of 4.9×10(-3) substitutions/site/year and greater diversity in surface expressed proteins was observed. We show that two clades of influenza A(H1N1)pdm09 virus were introduced into Kenya from the UK and the pandemic was sustained as a result of importations. Several closely related but distinct clusters co-circulated locally during the peak pandemic phase but only one cluster dominated in the late phase of the pandemic suggesting that it possessed greater adaptability.

Naturally acquired hemozoin by monocytes promotes suppression of RANTES in children with malarial anemia through an IL-10-dependent mechanism.

Regulated upon activation, normal T-cell expressed, and secreted (RANTES, CCL-5) is an important immunoregulatory mediator that is suppressed in children with malarial anemia (MA). Although pro-inflammatory (e.g., TNF-alpha, IL-1beta and IFN-gamma) and anti-inflammatory (e.g., IL-4, IL-10 and IL-13) cytokines regulate RANTES production, their effect on RANTES in children with MA has not been determined. Since intraleukocytic malarial pigment, hemozoin (Hz), causes dysregulation in chemokine and cytokine production, the impact of naturally acquired Hz (pfHz) on RANTES and RANTES-regulatory cytokines (TNF-alpha, IFN-gamma, IL-1beta, IL-4, IL-10, and IL-13) was examined. Circulating RANTES levels progressively declined with increasing levels of pigment-containing monocytes (PCM) (P=0.035). Additional experiments in cultured peripheral blood mononuclear cells (PBMC) showed that monocytic acquisition of pfHz (in vivo) was associated with suppression of RANTES under baseline (P=0.001) and stimulated conditions (P=0.072). Although high PCM levels were associated with decreased circulating IFN-gamma (P=0.003) and IL-10 (P=0.010), multivariate modeling revealed that only PCM (P=0.048, beta=-0.171) and IL-10 (P<0.0001, beta=-0.476) were independently associated with RANTES production. Subsequent in vitro experiments revealed that blockade of endogenous IL-10 significantly increased RANTES production (P=0.028) in PBMC from children with naturally acquired Hz. Results here demonstrate that monocytic acquisition of Hz suppresses RANTES production in children with MA through an IL-10-dependent mechanism.

Polymorphic variability in the interleukin (IL)-1beta promoter conditions susceptibility to severe malarial anemia and functional changes in IL-1beta production.

Interleukin (IL)-1beta is a cytokine released as part of the innate immune response to Plasmodium falciparum. Because the role played by IL-1beta polymorphic variability in conditioning the immunopathogenesis of severe malarial anemia (SMA) remains undefined, relationships between IL-1beta promoter variants (-31C/T and -511A/G), SMA (hemoglobin [Hb] level <6.0 g/dL), and circulating IL-1beta levels were investigated in children with parasitemia (n= 566) from western Kenya. The IL-1beta promoter haplotype -31C/-511A (CA) was associated with increased risk of SMA (Hb level <6.0 g/dL; odds ratio [OR], 1.98 [95% confidence interval {CI}, 1.55-2.27]; P < .05) and reduced circulating IL-1beta levels (p <.05). The TA (-31T/-511A) haplotype was nonsignificantly associated with protection against SMA (OR, 0.52 [95% CI, 0.18-1.16]; p =.11) and elevated IL-1beta production ( p<.05). Compared with the non-SMA group, children with SMA had significantly lower IL-1beta levels and nonsignificant elevations in both IL-1 receptor antagonist (IL-1Ra) and the ratio of IL-1Ra to IL-1beta. The results presented demonstrate that variation in IL-1beta promoter conditions susceptibility to SMA and functional changes in circulating IL-1beta levels.

Combining evidence of natural selection with association analysis increases power to detect malaria-resistance variants.

Statistical power to detect disease variants can be increased by weighting candidates by their evidence of natural selection. To demonstrate that this theoretical idea works in practice, we performed an association study of 10 putative resistance variants in 471 severe malaria cases and 474 controls from the Luo in Kenya. We replicated associations at HBB (P=.0008) and CD36 (P=.03) but also showed that the same variants are unusually differentiated in frequency between the Luo and Yoruba (who historically have been exposed to malaria) and the Masai and Kikuyu (who have not been exposed). This empirically demonstrates that combining association analysis with evidence of natural selection can increase power to detect risk variants by orders of magnitude--up to P=.000018 for HBB and P=.00043 for CD36.