Nutritional status of HIV-infected patients during the first year HAART in two West African cohorts

Objective: To examine the association between nutritional markers at initiation and during follow up in two different cohorts of HIV-infected adults initiating highly active antiretroviral therapy (HAART) in West Africa. Methods: The ATARAO study was a one year prospective study carried in Mali. It consisted of a sample of consecutive patients initiating HAART in one of four participating centers during that period. Data were collected at time of treatment initiation (baseline) and every 3 months thereafter. The ANRS 1290 study followed Senegalese patients recruited in similar conditions. Bivariate analyses were used to identify nutritional and immunological covariates of malnutrition at baseline. Longitudinal trajectories of body mass index, hemoglobin and albumin, and their associated factors, were evaluated using mixed linear models. Results: In ATARAO, 250 participants were retained for analyses; of which, 36% had a BMI < 18.5 kg/m2, nearly 60% were anemic and 47.4% hypoalbuminemic at time of treatment initiation. At baseline, low hemoglobin, hypoalbuminemia and low CD4 levels were associated with a BMI < 18.5 kg/m2. Similarly, low BMI, low albumin and low CD4 counts were linked to anemia; while, hypoalbuminemia was associated with low hemoglobin levels and CD4 counts. In ANRS, out of the 372 participants retained for analyses, 31% had a low BMI and almost 70% were anemic. At baseline, low BMI was associated with low hemoglobin levels and CD4 counts, while anemia was associated with low CD4 counts and female sex. While treatment contributed to early gains in BMI, hemoglobin and albumin in the first 6 months of treatment, initial improvements plateaued or subsided thereafter. Despite HAART, malnutrition persisted in both cohorts after one year, especially in those who were anemic, hypoalbuminemic or had a low BMI at baseline. Conclusion: In ATARAO and ANRS, malnutrition was common across all indicators (BMI, hemoglobin, albumin) and persisted despite treatment. Low BMI, anemia and hypoalbuminemia were associated with attrition, and with a deficient nutritional and immunological status at baseline, as well as during treatment. In spite of therapy, malnutrition is associated with negative clinical and treatment outcomes which suggests that HAART may not be sufficient to address co-existing nutritional deficiencies

Mitochondrial Vulnerability and Increased Susceptibility to Nutrient-Induced Cytotoxicity in Fibroblasts from Leigh Syndrome French Canadian Patients

<div><p>Mutations in LRPPRC are responsible for the French Canadian variant of Leigh Syndrome (LSFC), a severe disorder characterized biochemically by a tissue-specific deficiency of cytochrome c oxidase (COX) and clinically by the occurrence of severe and deadly acidotic crises. Factors that precipitate these crises remain unclear. To better understand the physiopathology and identify potential treatments, we performed a comprehensive analysis of mitochondrial function in LSFC and control fibroblasts. Furthermore, we have used this cell-based model to screen for conditions that promote premature cell death in LSFC cells and test the protective effect of ten interventions targeting well-defined aspects of mitochondrial function. We show that, despite maintaining normal ATP levels, LSFC fibroblasts present several mitochondrial functional abnormalities under normal baseline conditions, which likely impair their capacity to respond to stress. This includes mitochondrial network fragmentation, impaired oxidative phosphorylation capacity, lower membrane potential, increased sensitivity to Ca²⁺-induced permeability transition, but no changes in reactive oxygen species production. We also show that LSFC fibroblasts display enhanced susceptibility to cell death when exposed to palmitate, an effect that is potentiated by high lactate, while high glucose or acidosis alone or in combination were neutral. Furthermore, we demonstrate that compounds that are known to promote flux through the electron transport chain independent of phosphorylation (methylene blue, dinitrophenol), or modulate fatty acid (L-carnitine) or Krebs cycle metabolism (propionate) are protective, while antioxidants (idebenone, N-acetyl cysteine, resveratrol) exacerbate palmitate plus lactate-induced cell death. Collectively, beyond highlighting multiple alterations in mitochondrial function and increased susceptibility to nutrient-induced cytotoxicity in LSFC fibroblasts, these results raise questions about the nature of the diets, particularly excess fat intake, as well as on the use of antioxidants in patients with LSFC and, possibly, other COX defects.</p></div

Stress-induced cell death in control and LSFC fibroblasts.

<p><b>(A)</b> Lactate dehydrogenase (LDH) release in control and LSFC fibroblasts exposed for 48 h to factors relevant to acidotic crises. <b>(B)</b> Mean LDH release (<i>n</i> = 5), <b>(C)</b> Caspase 3/7 activity (<i>n</i> = 7), <b>(D)</b> Cellular ATP content (<i>n</i> = 7) and <b>(E)</b> COX/CS activity ratio (<i>n</i> = 3) assessed at baseline and after exposure to PL (palmitate 1 mM; lactate 10 mM) for 34 h (LDH release) or 24 h (other parameters). All experiments were performed in one control (EBS-4) and one LSFC (AL-006) cell line. Data are expressed as means ± S.E. Statistical significance of differences between groups or conditions was assessed with a two-way ANOVA for repeated measures followed by a Bonferroni post hoc test. Significantly different from the control cells in the same experimental condition: * <i>p</i> < 0.05, ** <i>p</i> < 0.01. Significantly different from baseline within the same experimental group: ^ρ<i>p</i> < 0.05, ^ρρ<i>p</i> < 0.01, ^ρρρ<i>p</i> < 0.001.</p

Control and Leigh Syndrome French Canadian (LSFC) skin fibroblast cell lines.

<p>Control and Leigh Syndrome French Canadian (LSFC) skin fibroblast cell lines.</p

Effect of the LRPPRC A354V mutation on LRPPRC content, mitochondrial content and cellular ATP levels.

<p><b>(A)</b> Immunoblot and densitometric analysis of LRPPRC content in whole cell lysates from control and LSFC fibroblasts (n = 4). Representative immunofluorescence images of control <b>(B;C)</b> and LSFC fibroblasts <b>(D;E)</b> labeled with anti-LRPPRC (green) and anti-pyruvate dehydrogenase (red) antibodies. Overlay images (yellow), and line scan analysis show the cellular distribution of LRPPRC in the mitochondrial compartment. <b>(F)</b> Cytochrome c oxidase (COX) enzyme activity in whole cell lysates from control and LSFC fibroblasts was normalized to that of the mitochondrial marker citrate synthase (CS) to take into account possible differences in mitochondrial content (n = 4). <b>(G)</b> Immunoblot and densitometric analysis of VDAC, a mitochondrial marker protein, in whole cell lysates from control and LSFC fibroblasts (n = 3). <b>(H)</b> Citrate synthase activity in whole cell lysates from control and LSFC fibroblasts (n = 4). <b>(I)</b> Cellular ATP content in control and LSFC fibroblasts (n = 4). Data are expressed as means ± S.E. Experiments were performed in one control (EBS-4) and one LSFC cell line (AL-006). Difference between control and LSFC cells was assessed with a paired t-test. ** Significantly different from the control group <i>p</i> ≤ 0.01. Statistical power for LRPPRC and COX was 97%.</p

Effect of the LRPPRC A354V mutation on basal mitochondrial network morphology and functions.

<p>Representative live cell images of MTG-loaded control <b>(A)</b> and LSFC <b>(B)</b> fibroblasts used for quantitative analysis of mitochondrial network morphology. <b>(C)</b> Form Factor (FF) values calculated using the equation FF = 4π*Area/perimeter2 (n = 6). Representative live cell images of control <b>(D)</b> and LSFC <b>(E)</b> fibroblasts labeled with TMRE (red) and MTG (green). <b>(F)</b> Mitochondrial membrane (ΔΨ) potential expressed as the ratio of TMRE to MTG (n = 5). Lower values are indicative of reduced ΔΨ. <b>(G)</b> Mean fluorescence intensity of the mitochondria-specific superoxide probe MitoSOX in control and LSFC fibroblasts (n = 5). <b>(H)</b> Maximal ADP-driven respiration in digitonin-permeabilized fibroblasts energized with complex I (5 mM glutamate—2.5 mM malate; Glut-Mal; n = 15) or complex II substrates in presence of the complex I inhibitor rotenone (5 mM succinate + 1 μM rotenone; Succ+Rot; n = 14). Inset shows representative respirometry traces confirming that respiratory rates increased promptly in response to the addition of respiratory substrates, and were potently inhibited by complex I (rotenone), and complex II (malonate) blockers. <b>(I)</b> Mitochondrial calcium retention capacity (CRC) in control and LSFC fibroblasts exposed to progressive Ca2+ loading (n = 8). Inset shows representative Ca2+ kinetic tracings observed in control and LSFC fibroblasts. Tracings show progressive Ca2+ accumulation followed by PTP-induced release of accumulated Ca2+. Each spike indicates the addition of a calcium pulse of 83 nmoles. All experiments were performed in one control (EBS-4) and one LSFC (AL-006) cell line, except for the determination of ΔΨ, which was performed in EBS-3 and AL-002. Data are expressed as means ± S.E. Difference between control and LSFC cells was assessed with a paired t-test. Significantly different from the control group: * <i>p</i> < 0.05, ** <i>p</i> ≤ 0.01. Statistical power: C: 92%; F: 85%; G: 80%; H: Glut-Mal 80%; Succ+Rot: 96%; I: 73%.</p

A Metabolic Signature of Mitochondrial Dysfunction Revealed through a Monogenic Form of Leigh Syndrome

A decline in mitochondrial respiration represents the root cause of a large number of inborn errors of metabolism. It is also associated with common age-associated diseases and the aging process. To gain insight into the systemic, biochemical consequences of respiratory chain dysfunction, we performed a case-control, prospective metabolic profiling study in a genetically homogenous cohort of patients with Leigh syndrome French Canadian variant, a mitochondrial respiratory chain disease due to loss-of-function mutations in LRPPRC. We discovered 45 plasma and urinary analytes discriminating patients from controls, including classic markers of mitochondrial metabolic dysfunction (lactate and acylcarnitines), as well as unexpected markers of cardiometabolic risk (insulin and adiponectin), amino acid catabolism linked to NADH status (α-hydroxybutyrate), and NAD+ biosynthesis (kynurenine and 3-hydroxyanthranilic acid). Our study identifies systemic, metabolic pathway derangements that can lie downstream of primary mitochondrial lesions, with implications for understanding how the organelle contributes to rare and common diseases