Paris Jafari

The unsolved puzzle of neuropathogenesis in glutaric aciduria type I

Glutaric aciduria type I (GA-I) is a cerebral organic aciduria caused by deficiency of glutaryl-Co-A dehydrogenase (GCDH). GCDH deficiency leads to accumulation of glutaric acid (GA) and 3-hydroxyglutaric acid (3-OHGA), two metabolites that are believed to be neurotoxic, in brain and body fluids. The disorder usually becomes clinically manifest during a catabolic state (e.g. intercurrent illness) with an acute encephalopathic crisis that results in striatal necrosis and in a permanent dystonic-dyskinetic movement disorder. The results of numerous in vitro and in vivo studies have pointed to three main mechanisms involved in the metabolite-mediated neuronal damage: excitotoxicity, impairment of energy metabolism and oxidative stress. There is evidence that during a metabolic crisis GA and its metabolites are produced endogenously in the CNS and accumulate because of limiting transport mechanisms across the blood-brain barrier. Despite extensive experimental work, the relative contribution of the proposed pathogenic mechanisms remains unclear and specific therapeutic approaches have yet to be developed. Here, we review the experimental evidence and try to delineate possible pathogenetic models and approaches for future studies.

Ammonium Accumulation and Cell Death in a Rat 3D Brain Cell Model of Glutaric Aciduria Type I

Glutaric aciduria type I (glutaryl-CoA dehydrogenase deficiency) is an inborn error of metabolism that usually manifests in infancy by an acute encephalopathic crisis and often results in permanent motor handicap. Biochemical hallmarks of this disease are elevated levels of glutarate and 3-hydroxyglutarate in blood and urine. The neuropathology of this disease is still poorly understood, as low lysine diet and carnitine supplementation do not always prevent brain damage, even in early-treated patients. We used a 3D in vitro model of rat organotypic brain cell cultures in aggregates to mimic glutaric aciduria type I by repeated administration of 1 mM glutarate or 3-hydroxyglutarate at two time points representing different developmental stages. Both metabolites were deleterious for the developing brain cells, with 3-hydroxyglutarate being the most toxic metabolite in our model. Astrocytes were the cells most strongly affected by metabolite exposure. In culture medium, we observed an up to 11-fold increase of ammonium in the culture medium with a concomitant decrease of glutamine. We further observed an increase in lactate and a concomitant decrease in glucose. Exposure to 3-hydroxyglutarate led to a significantly increased cell death rate. Thus, we propose a three step model for brain damage in glutaric aciduria type I: (i) 3-OHGA causes the death of astrocytes, (ii) deficiency of the astrocytic enzyme glutamine synthetase leads to intracerebral ammonium accumulation, and (iii) high ammonium triggers secondary death of other brain cells. These unexpected findings need to be further investigated and verified in vivo. They suggest that intracerebral ammonium accumulation might be an important target for the development of more effective treatment strategies to prevent brain damage in patients with glutaric aciduria type I.

Brain damage in methylmalonic aciduria: 2-methylcitrate induces cerebral ammonium accumulation and apoptosis in 3D organotypic brain cell cultures

BackgroundMethylmalonic aciduria is an inborn error of metabolism characterized by accumulation of methylmalonate (MMA), propionate and 2-methylcitrate (2-MCA) in body fluids. Early diagnosis and current treatment strategies aimed at limiting the production of these metabolites are only partially effective in preventing neurological damage.MethodsTo explore the metabolic consequences of methylmalonic aciduria on the brain, we used 3D organotypic brain cell cultures from rat embryos. We challenged the cultures at two different developmental stages with 1 mM MMA, propionate or 2-MCA applied 6 times every 12 h. In a dose–response experiment cultures were challenged with 0.01, 0.1, 0.33 and 1 mM 2-MCA. Immunohistochemical staining for different brain cell markers were used to assess cell viability, morphology and differentiation. Significant changes were validated by western blot analysis. Biochemical markers were analyzed in culture media. Apoptosis was studied by immunofluorescence staining and western blots for activated caspase-3.ResultsAmong the three metabolites tested, 2-MCA consistently produced the most pronounced effects. Exposure to 2-MCA caused morphological changes in neuronal and glial cells already at 0.01 mM. At the biochemical level the most striking result was a significant ammonium increase in culture media with a concomitant glutamine decrease. Dose–response studies showed significant and parallel changes of ammonium and glutamine starting from 0.1 mM 2-MCA. An increased apoptosis rate was observed by activation of caspase-3 after exposure to at least 0.1 mM 2-MCA.ConclusionSurprisingly, 2-MCA, and not MMA, seems to be the most toxic metabolite in our in vitro model leading to delayed axonal growth, apoptosis of glial cells and to unexpected ammonium increase. Morphological changes were already observed at 2-MCA concentrations as low as 0.01 mM. Increased apoptosis and ammonium accumulation started at 0.1 mM thus suggesting that ammonium accumulation is secondary to cell suffering and/or cell death. Local accumulation of ammonium in CNS, that may remain undetected in plasma and urine, may therefore play a key role in the neuropathogenesis of methylmalonic aciduria both during acute decompensations and in chronic phases. If confirmed in vivo, this finding might shift the current paradigm and result in novel therapeutic strategies.

Effect of Human Burn Wound Exudate on Pseudomonas aeruginosa Virulence

Microbial infection of severe burn wounds is currently a major medical challenge. Of the infections by bacteria able to colonize such injuries, those by Pseudomonas aeruginosa are among the most severe, causing major delays in burn patient recovery or leading to fatal issues. In this study, we investigated the growth properties of several burn wound pathogens in biological fluids secreted from human burn wounds. We found that P. aeruginosa strains were able to proliferate but not those of the other pathogens tested. In addition, burn wound exudates (BWEs) stimulate the expression of virulence factors in P. aeruginosa. The chemical composition analysis of BWEs enabled us to determine the major components of these fluids. These data are essential for the development of an artificial medium mimicking the burn wound environment and for in vitro analysis of the initial step in the development of burn wound infections. ABSTRACT Burn wound sepsis is currently the main cause of morbidity and mortality after burn trauma. Infections by notorious pathogens such as Pseudomonas aeruginosa, Staphylococcus aureus, and Acinetobacter baumannii impair patient recovery and can even lead to fatality. In this study, we investigated the effect of burn wound exudates (BWEs) on the virulence of those pathogens. BWEs were collected within 7 days after burn trauma from 5 burn patients. We first monitored their effect on pathogen growth. In contrast to A. baumannii and S. aureus, P. aeruginosa was the only pathogen able to grow within these human fluids. Expression of typical virulence factors such as pyocyanin and pyoverdine was even enhanced compared the levels seen with standard laboratory medium. A detailed chemical composition analysis of BWE was performed, which enabled us to determine the major components of BWE and underline the metabolic modifications induced by burn trauma. These data are essential for the development of an artificial medium mimicking the burn wound environment and the establishment of an in vitro system to analyze the initial steps of burn wound infections. IMPORTANCE Microbial infection of severe burn wounds is currently a major medical challenge. Of the infections by bacteria able to colonize such injuries, those by Pseudomonas aeruginosa are among the most severe, causing major delays in burn patient recovery or leading to fatal issues. In this study, we investigated the growth properties of several burn wound pathogens in biological fluids secreted from human burn wounds. We found that P. aeruginosa strains were able to proliferate but not those of the other pathogens tested. In addition, burn wound exudates (BWEs) stimulate the expression of virulence factors in P. aeruginosa. The chemical composition analysis of BWEs enabled us to determine the major components of these fluids. These data are essential for the development of an artificial medium mimicking the burn wound environment and for in vitro analysis of the initial step in the development of burn wound infections.

Anti-Microbial Dendrimers against Multidrug-Resistant P. aeruginosa Enhance the Angiogenic Effect of Biological Burn-wound Bandages.

Multi-drug resistant Pseudomonas aeruginosa has increased progressively and impedes further regression in mortality in burn patients. Such wound infections serve as bacterial reservoir for nosocomial infections and are associated with significant morbidity and costs. Anti-microbial polycationic dendrimers G3KL and G3RL, able to kill multi-drug resistant P. aeruginosa, have been previously developed. The combination of these dendrimers with a class of biological bandages made of progenitor skin cells, which secrete growth factors, could positively impact wound-healing processes. However, polycations are known to be used as anti-angiogenic agents for tumor suppression. Since, neovascularization is pivotal in the healing of deep burn-wounds, the use of anti-microbial dendrimers may thus hinder the healing processes. Surprisingly, we have seen in this study that G3KL and G3RL dendrimers can have angiogenic effects. Moreover, we have shown that a dendrimer concentration ranging between 50 and 100 μg/mL in combination with the biological bandages can suppress bacterial growth without altering cell viability up to 5 days. These results show that antimicrobial dendrimers can be used in combination with biological bandages and could potentially improve the healing process with an enhanced angiogenesis.

Topical Negative Pressure on Burns: An Innovative Method for Wound Exudate Collection

Summary: Burn wound exudate is an important source of information on the wound-healing process and systemic improvement of burn patients. Identification of biomarkers for the prediagnosis of local or systemic complications in patients will have a great impact on adapting personalized procedures in burn treatment. No efficient exudate collection method exists that offers a direct and continuous collection over time. We developed an innovative system based on the negative pressure wound therapy technique to directly collect exudate from burn wounds over several days after burn. This method did not cause any complication or pain for patients, and positive influence on wound healing was seen. Exudate samples were further used in different projects for studying biochemical profile, trace element content, kinetics of bacterial growth, and cell cytotoxicity.

Immunolocalization of glutaryl-CoA dehydrogenase (GCDH) in adult and embryonic rat brain and peripheral tissues

Glutaryl-CoA dehydrogenase (GCDH) is a mitochondrial enzyme that is involved in the degradation of tryptophan, lysine and hydroxylysine. Deficient enzyme activity leads to glutaric aciduria type-I (GA-I). This neurometabolic disease usually manifests with acute encephalopathic crises and striatal neuronal death in early childhood leading to an irreversible dystonic-dyskinetic movement disorder. Fronto-temporal atrophy and white matter changes are already present in the pre-symptomatic period. No detailed information on GCDH expression during embryonic development and in adulthood was available so far. Using immunofluorescence microscopy and cell-type-specific markers to localize GCDH in different tissues, we describe the differential cellular localization of GCDH in adult rat brain and peripheral organs as well as its spatiotemporal expression pattern. During embryonic development GCDH was predominantly expressed in neurons of the central and peripheral nervous system. Significant expression levels were found in epithelial cells (skin, intestinal and nasal mucosa) of rat embryos at different developmental stages. Besides the expected strong expression in liver, GCDH was found to be significantly expressed in neurons of different brain regions, renal proximal tubules, intestinal mucosa and peripheral nerves of adult rats. GCDH was found widely expressed in embryonic and adult rat tissues. In rat embryos GCDH is predominantly expressed in brain implying an important role for brain development. Interestingly, GCDH was found to be significantly expressed in different other organs (e.g. kidney, gut) in adult rats probably explaining the evolving phenotype in GA-I patients.

Transcriptome Analysis of Pseudomonas aeruginosa Cultured in Human Burn Wound Exudates

Pseudomonas aeruginosa is a severe opportunistic pathogen and is one of the major causes of hard to treat burn wound infections. Herein we have used an RNA-seq transcriptomic approach to study the behavior of P. aeruginosa PAO1 growing directly on human burn wound exudate. A chemical analysis of compounds used by this bacterium, coupled with kinetics expression of central genes has allowed us to obtain a global view of P. aeruginosa physiological and metabolic changes occurring while growing on human burn wound exudate. In addition to the numerous virulence factors and their secretion systems, we have found that all iron acquisition mechanisms were overexpressed. Deletion and complementation with pyoverdine demonstrated that iron availability was a major limiting factor in burn wound exudate. The quorum sensing systems, known to be important for the virulence of P. aeruginosa, although moderately induced, were activated even at low cell density. Analysis of bacterial metabolism emphasized importance of lactate, lipid and collagen degradation pathways. Overall, this work allowed to designate, for the first time, a global view of P. aeruginosa characteristics while growing in human burn wound exudate and highlight the possible therapeutic approaches to combat P. aeruginosa burn wound infections.

First Insights into Human Fingertip Regeneration by Echo-Doppler Imaging and Wound Microenvironment Assessment

Fingertip response to trauma represents a fascinating example of tissue regeneration. Regeneration derives from proliferative mesenchymal cells (blastema) that subsequently differentiate into soft and skeletal tissues. Clinically, conservative treatment of the amputated fingertip under occlusive dressing can shift the response to tissue loss from a wound repair process towards regeneration. When analyzing by Immunoassay the wound exudate from occlusive dressings, the concentrations of brain-derived neurotrophic factor (BDNF) and leukemia inhibitory factor (LIF) were higher in fingertip exudates than in burn wounds (used as controls for wound repair versus regeneration). Vascular endothelial growth factor A (VEGF-A) and platelet-derived growth factor (PDGF) were highly expressed in both samples in comparable levels. In our study, pro-inflammatory cytokines were relatively higher expressed in regenerative fingertips than in the burn wound exudates while chemokines were present in lower levels. Functional, vascular and mechanical properties of the regenerated fingertips were analyzed three months after trauma and the data were compared to the corresponding fingertip on the collateral uninjured side. While sensory recovery and morphology (pulp thickness and texture) were similar to uninjured sides, mechanical parameters (elasticity, vascularization) were increased in the regenerated fingertips. Further studies should be done to clarify the importance of inflammatory cells, immunity and growth factors in determining the outcome of the regenerative process and its influence on the clinical outcome.