Ready-to-use food supplement, with or without arginine and citrulline, with daily chloroquine in Tanzanian children with sickle-cell disease: a double-blind, random order crossover trial

Background: Sickle cell disease increases malnutrition risk. Low arginine and nitric oxide [NO] bioavailability are implicated in sickle-related morbidity. Simple interventions are required, especially in low-income settings. We aimed to test the hypotheses: (1) supplementary arginine, citrulline and daily chloroquine increases bioavailable arginine and flow-mediated-dilatation (FMDmax%; a measure of NO-dependent endothelial function), and (2); protein energy supplementation in the form of ready-to-use supplementary-food (RUSF) improves nutritional status in children with sickle cell disease. Methods: A random-order, double-blind, cross-over trial with two four-month intervention periods (each followed by four-months wash-out) was conducted in Dar-es-Salaam, Tanzania. 119 children aged 8-12 years, naïve to hydroxyurea, were enrolled from the Muhimbili National Hospital Sickle Cohort. The random order sequence and allocation codes were generated centrally. Two formulations of RUSF (500kcal/day) were tested: ‘basic’ with weekly chloroquine (150/225mg base, depending on weight) (RUSF-b) and ‘vascular’ (RUSF-v) fortified with arginine, citrulline designed to achieve mean intakes of 0.2g/0.1g/kg/day and daily chloroquine (max 3mg base/kg/day). The primary outcomes of the comparison of the 2 RUSF formulations were mean FMDmax%, mean plasma arginine to ornithine ratio and mean plasma arginine to asymmetric-di-methylated-arginine (ADMA) ratio. The primary outcomes of the combined effect of both RUSF interventions were mean height and body mass index for age z-scores with analysis by intention to treat. Trial registration: ISRCTN74331412 Findings: 114/119 children had complete data for all reported endpoints. There was no treatment effect of RUSF-v compared to RUSF-b on the ratio of arginine to ornithine (mean within individual difference -0.09, 95% CI -0.03/0.2, p=0.12), or on FMDmax% (-1.00 95% CI -2.47/0.47, p=0.18) but the arginine:ADMA ratio was significantly increased (-0.56, 95% CI -0.81/-0.31, P<0.001). In planned analyses using random effects models to estimate the effect of each intervention compared to baseline/washout, the arginine:ADMA ratio increased following both RUSF-v or RUSF-b (+86%, p<0.001; +41%, p<0.001). Similarly, FMDmax% was higher after 2 RUSF-v (+0.92, p<0.001) but not after RUSF-b intervention (+0.39, p=0.22). Adjusted for covariates, effect estimates for FMDmax% increased: RUSF-v (+1.19, p<0.001) and RUSF-b (+0.93, p=0.008). Following either intervention (RUSF-b and RUSF-v pooled) compared to baseline/wash-outs, body-mass-index-z-score (+0.091, P=0.001) and height-for-age-z-score (+0.013, P=0.081) increased. There were 71 and 81 adverse events of which 21 and 26 were serious during intervention and washout (P=0.31) in 83 participants, 1 of whom died in the 2nd washout period. Interpretation: RUSF providing 500kcal/day results in small weight gains in children with sickle cell disease. However, RUSF even without arginine and citrulline fortification improves arginine dysregulation and may improve endothelial function. Long-term studies are required to assess if these physiological effects translate to improved clinical outcomes and better growth and development in sickle cell disease

Baseline characteristics of patients at the time of initiation of Tuberculosis (TB) therapy (N = 1245).

<p>Baseline characteristics of patients at the time of initiation of Tuberculosis (TB) therapy (N = 1245).</p

Predictors of delayed sputum smear conversion among Tuberculosis patients (n = 1245)^*.

<p>*Additionally controlled for marital status, education status, monthly income, daily expenditure on food and serum creatinine, none of which were significantly associated with delayed sputum smear conversion.</p

The association between delayed sputum conversion and anemia among Tuberculosis patients (N = 1245)^*.

<p>*Two separate models for (anemia and anemia severity) were each adjusted for: age, sex, HIV status, smoking, alcohol, drug abuse, body mass index (BMI), duration of illness, lymphocytes, platelets count and resistance to rifampicin and/or Isoniazid.</p><p><b>**</b>Trend test p-value.</p

MOESM1 of A robust mass spectrometry method for rapid profiling of erythrocyte ghost membrane proteomes

Additional file 1: Figure 1 Histogram showing the distribution of the Intensity Based Absolute Quantification (iBAQ) values for identified peptides across samples 1 to 10 (represented by iBAQ 1-10 respectively) listed in Table 1

Transcriptional regulatory subnetwork based on microarray meta-analysis.

<p>Regulatory network analysis was performed using RNEA R/package to determine the regulation complexes upstream of DEGs identified in the meta-analysis. Genes carrying in their promoter, a significant regulatory SNPs are marked by a yellow star. Each node represents a DEG or enriched transcription factor, depending on their shapes. The node size indicate greater significance of the enrichment. The edges reflect the relationships between the nodes.</p

Characteristics of included individual microarray dataset.

<p>Characteristics of included individual microarray dataset.</p

Over-representation of pathways and GO categories in biological networks identified by the meta-analysis.

<p>Network representation of an enriched pathway integrating biological processes on the DEG list according to the ClueGO Cytoscape plugin. Hypergeometric (right-handed) enrichment distribution tests were conducted with a p value significance level of ≤0.05, followed by the Bonferroni adjustment for the terms, and thus leading term groups were selected based on the highest significance. Each node represents a biological process. The edges reflect the relationships between the terms based on the similarity of their associated genes. The node size and deeper color indicate greater significance of the enrichment.</p

A PRISMA flow diagram of a systematic database search.

<p>The selection process of eligible microarray datasets for meta-analysis of the shared transcriptomic signatures between Sickle cell disease patients, according to Prisma 2009 flow diagram.</p

Downregulated and upregulated DEGs involved in apoptosis pathways.

<p>The inner ring is a bar plot where the height of the bar indicates the significance of the term (−log₁₀ adjusted <i>p</i> value), and color corresponds to the <i>z</i>-score. The outer ring displays scatterplots of the expression levels (log2FC) for the genes in each term.</p