The microbiological signature of human cutaneous leishmaniasis lesions exhibits restricted bacterial diversity compared to healthy skin

Localised cutaneous leishmaniasis (LCL) is the most common form of cutaneous leishmaniasis characterised by single or multiple painless chronic ulcers, which commonly presents with secondary bacterial infection. Previous culture-based studies have found staphylococci, streptococci, and opportunistic pathogenic bacteria in LCL lesions, but there have been no comparisons to normal skin. In addition, this approach has strong bias for determining bacterial composition. The present study tested the hypothesis that bacterial communities in LCL lesions differ from those found on healthy skin (HS). Using a high throughput amplicon sequencing approach, which allows for better populational evaluation due to greater depth coverage and the Quantitative Insights Into Microbial Ecology pipeline, we compared the microbiological signature of LCL lesions with that of contralateral HS from the same individuals.Streptococcus, Staphylococcus,Fusobacterium and other strict or facultative anaerobic bacteria composed the LCL microbiome. Aerobic and facultative anaerobic bacteria found in HS, including environmental bacteria, were significantly decreased in LCL lesions (p < 0.01). This paper presents the first comprehensive microbiome identification from LCL lesions with next generation sequence methodology and shows a marked reduction of bacterial diversity in the lesions.

The NLRP3 inflammasome: a sensor for metabolic danger?

Interleukin-1beta (IL-1beta), reactive oxygen species (ROS), and thioredoxin-interacting protein (TXNIP) are all implicated in the pathogenesis of type 2 diabetes mellitus (T2DM). Here we review mechanisms directing IL-1beta production and its pathogenic role in islet dysfunction during chronic hyperglycemia. In doing so, we integrate previously disparate disease-driving mechanisms for IL-1beta, ROS, and TXNIP in T2DM into one unifying model in which the NLRP3 inflammasome plays a central role. The NLRP3 inflammasome also drives IL-1beta maturation and secretion in another disease of metabolic dysregulation, gout. Thus, we propose that the NLRP3 inflammasome contributes to the pathogenesis of T2DM and gout by functioning as a sensor for metabolic stress.

New insights into pathophysiology of vestibular migraine.

Vestibular migraine (VM) is a common disorder in which genetic, epigenetic, and environmental factors probably contribute to its development. The pathophysiology of VM is unknown; nevertheless in the last few years, several studies are contributing to understand the neurophysiological pathways involved in VM. The current hypotheses are mostly based on the knowledge of migraine itself. The evidence of trigeminal innervation of the labyrinth vessels and the localization of vasoactive neuropeptides in the perivascular afferent terminals of these trigeminal fibers support the involvement of the trigemino-vascular system. The neurogenic inflammation triggered by activation of the trigeminal-vestibulocochlear reflex, with the subsequent inner ear plasma protein extravasation and the release of inflammatory mediators, can contribute to a sustained activation and sensitization of the trigeminal primary afferent neurons explaining VM symptoms. The reciprocal connections between brainstem vestibular nuclei and the structures that modulate trigeminal nociceptive inputs (rostral ventromedial medulla, ventrolateral periaqueductal gray, locus coeruleus, and nucleus raphe magnus) are critical to understand the pathophysiology of VM. Although cortical spreading depression can affect cortical areas involved in processing vestibular information, functional neuroimaging techniques suggest a dysmodulation in the multimodal sensory integration and processing of vestibular and nociceptive information, resulting from a vestibulo-thalamo-cortical dysfunction, as the pathogenic mechanism underlying VM. The elevated prevalence of VM suggests that multiple functional variants may confer a genetic susceptibility leading to a dysregulation of excitatory-inhibitory balance in brain structures involved in the processing of sensory information, vestibular inputs, and pain. The interactions among several functional and structural neural networks could explain the pathogenic mechanisms of VM.

Alloimmunity and nonimmunologic risk factors in cardiac allograft vasculopathy.

Graft vasculopathy is an accelerated form of coronary artery disease that occurs in transplanted hearts. Despite major advances in immunosuppression, the prevalence of the disease has remained substantially unchanged during the last two decades. According to the 'response to injury' paradigm, graft vasculopathy is the result of a continuous inflammatory response to tissue injury initiated by both alloantigen-dependent and independent stress responses. Experimental evidence suggests that these responses may become self-sustaining, as allograft re-transplantation into the donor strain at a later stage fails to prevent disease progression. Histological evidence of endothelitis and arteritis, in association with intima fibrosis and atherosclerosis, reflects the central role of alloimmunity and inflammation in the development of arterial lesions. Experimental results in gene-targeted mouse models indicate that cellular and humoral immune responses are both involved in the pathogenesis of graft vasculopathy. Circulating antibodies against donor endothelium are found in a significant number of patients, but their pathogenic role is still controversial. Alloantigen-independent factors include donor-transmitted coronary artery disease, surgical trauma, ischaemia-reperfusion injury, viral infections, hyperlipidaemia, hypertension, and glucose intolerance. Recent therapeutic advances include the use of novel immunosuppressive agents such as sirolimus (rapamycin), HMG-CoA reductase inhibitors, calcium channel blockers, and angiotensin converting enzyme inhibitors. Optimal treatment of cardiovascular risk factors remains of paramount importance.

Serum albumin promotes ATP-binding cassette transporter-dependent sterol uptake in yeast.

Sterol uptake in fungi is a multistep process that involves interaction between external sterols and the cell wall, incorporation of sterol molecules into the plasma membrane, and subsequent integration into intracellular membranes for turnover. ATP-binding cassette (ABC) transporters have been implicated in sterol uptake, but key features of their activity remain to be elucidated. Here, we apply fluorescent cholesterol (NBD-cholesterol) to monitor sterol uptake under anaerobic and aerobic conditions in two fungal species, Candida glabrata (Cg) and Saccharomyces cerevisiae (Sc). We found that in both fungal species, ABC transporter-dependent uptake of cholesterol under anaerobic conditions and in mutants lacking HEM1 gene is promoted in the presence of the serum protein albumin that is able to bind the sterol molecule. Furthermore, the C. glabrata ABC transporter CgAus1p expressed in S. cerevisiae requires the presence of serum or albumin for efficient cholesterol uptake. These results suggest that albumin can serve as sterol donor in ABC transporter-dependent sterol uptake, a process potentially important for growth of C. glabrata inside infected humans.

Neuroprotection in cerebral ischemia with XG-102, a new peptide inhibitor of JNK

Abstract Stroke or cerebrovascular accident, whose great majority is of ischemic nature, is the third leading cause of mortality and long lasting disability in industrialised countries. Resulting from the loss of blood supply to the brain depriving cerebral tissues of oxygen and glucose, it induces irreversible neuronal damages. Despite the large amount of research carried out into the causes and pathogenic features of cerebral ischemia the progress toward effective treatments has been poor. Apart the clot-busting drug tissue-type plasminogen activator (tPA) as effective therapy for acute stroke (reperfusion by thrombolysis) but limited to a low percentage of patients, there are currently no other approved medical treatments. The need for new therapy strategies is therefore imperative. Neuronal death in cerebral ischemia is among others due to excitotoxic mechanisms very early after stroke onset. One of the main involved molecular pathways leading to excitotoxic cell death is the c-Jun NH2-terminal kinase (JNK) pathway. Several studies have already shown the efficacy of a neuroprotective agent of a new type, a dextrogyre peptide synthesized in the retro inverso form (XG102, formerly D-JNKI1), which is protease-resistant and cell-penetrating and that selectively and strongly blocks the access of JNK to many of its targets. A powerful protection was observed with this compound in several models of ischemia (Borsello et al. 2003;Hirt et al. 2004). This chimeric compound, made up of a 10 amino acid TAT transporter sequence followed by a 20 amino acids JNK binding domain (JBD) sequence from JNK inhibitor protein (JIP) molecule, induced both a major reduction in lesion size and improved functional outcome. Moreover it presents a wide therapeutic window. XG-102 has proved its powerful efficacy in an occlusion model of middle cerebral artery in mice with intracérebroventricular (i.c.v.) injection but in order to be able to consider the development of this drug for human ischemic stroke it was therefore necessary to determine the feasibility of its systemic administration. The studies being the subject of this thesis made it possible to show a successful neuroprotection with XG-102 administered systemically after transient mouse middle cerebral artery occlusion (MCAo). Moreover our data. provided information about the feasibility to combine XG-102 with tPA without detrimental action on cell survival. By combining the benefits from a reperfusion treatment with the effects of a neuroprotective compound, it would represent the advantage of bringing better chances to protect the cerebral tissue. Résumé L'attaque cérébrale ou accident vasculaire cérébral, dont la grande majorité est de nature ischémique, constitue la troisième cause de mortalité et d'infirmité dans les pays industrialisés. Résultant de la perte d'approvisionnement de sang au cerveau privant les tissus cérébraux d'oxygène et de glucose, elle induit des dommages neuronaux irréversibles. En dépit du nombre élevé de recherches effectuées pour caractériser les mécanismes pathogènes de l'ischémie. cérébrale, les progrès vers des traitements efficaces restent pauvres. Excepté l'activateur tissulaire du plasminogène (tPA) dont le rôle est de désagréger les caillots sanguins et employé comme thérapie efficace contre l'attaque cérébrale aiguë (reperfusion par thrombolyse) mais limité à un faible pourcentage de patients, il n'y a actuellement aucun autre traitement médical approuvé. Le besoin de nouvelles stratégies thérapeutiques est par conséquent impératif. La mort neuronale dans l'ischémie cérébrale est entre autres due à des mécanismes excitotoxiques survenant rapidement après le début de l'attaque cérébrale. Une des principales voies moléculaires impliquée conduisant à la mort excitotoxique des cellules est la voie de la c-Jun NH2terminal kinase (JNK). Plusieurs études ont déjà montré l'efficacité d'un agent neuroprotecteur d'un nouveau type, un peptide dextrogyre synthétisé sous la forme retro inverso (XG-102, précédemment D-JNKI1) résistant aux protéases, capable de pénétrer dans les cellules et de bloquer sélectivement et fortement l'accès de JNK à plusieurs de ses cibles. Une puissante protection a été observée avec ce composé dans plusieurs modèles d'ischémie (Borsello et al. 2003;Hirt et al. 2004). Ce composé chimérique, construit à partir d'une séquence TAT de 10 acides aminés suivie par une séquence de 20 acides aminés d'un domaine liant JNK (JBD) issu de la molécule JNK protéine inhibitrice. (JIP), induit à la fois une réduction importante de la taille de lésion et un comportement fonctionnel amélioré. De plus il présente une fenêtre thérapeutique étendue. XG-102 a prouvé sa puissante efficacité dans un modèle d'occlusion de l'artère cérébrale moyenne chez la souris avec injection intracerebroventriculaire (i.c.v.) mais afin de pouvoir envisager le développement de ce composé pour l'attaque cérébrale chez l'homme, il était donc nécessaire de déterminer la faisabilité de son administration systémique. Les études faisant l'objet de cette thèse ont permis de montrer une neuroprotection importante avec XG-102 administré de façon systémique après l'occlusion transitoire de l'artère cérébrale moyenne chez la souris (MCAo). De plus nos données ont fourni des informations quant à la faisabilité de combiner XG-102 et tPA, démontrant une protection efficace par XG-102 malgré l'action nuisible du tPA sur la survie des cellules. En combinant les bénéfices de la reperfusion avec les effets d'un composé neurooprotecteur, cela représenterait l'avantage d'apporter des meilleures chances de protéger le tissu cérébral.

Anticorps antineuronaux: un domaine en plein développement [Anti-neuronal antibodies: a rapidly developing field].

Anti-neuronal antibodies are implicated in various neurological syndromes that are sometimes associated with tumors. Depending on the antigenic target (nuclear, cytoplasmic or extracellular cell-surface or synaptic) the clinical presentation is different. In neurological syndromes associated with antibodies specific for intracellular antigens, the T-cell mediated immunological response predominates as pathogenic effector and the response to treatment is typically poor. In contrast, in syndromes related to antibodies against extracellular targets, the role of the antibodies is pathogenic and the neurological syndrome often responds better to immunomodulatory treatment, associated or not with an anti-tumoral treatment. We review the spectrum of anti-neuronal antibodies and their corresponding clinical and therapeutic characteristics.

TRPV4-associated skeletal dysplasias.

Dominant mutations in the TRPV4 gene result in a bone dysplasia family and form a continuous phenotypic spectrum that includes, in decreasing severity, lethal, and nonlethal metatropic dysplasia (MD), spondylometaphyseal dysplasia Kozlowski type (SMDK), and autosomal dominant brachyolmia. Several rare variant phenotypes that have some overlap but deviate in some ways from the general pattern have also been described. The known variant phenotypes are spondyloepiphyseal dysplasia Maroteaux type (Pseudo-Morquio type 2), parastremmatic dysplasia, and familial digital arthropathy with brachydactyly. Interestingly, different TRPV4 mutations have been associated with dominantly inherited neurologic disorders such as congenital spinal muscular atrophy and hereditary motor and sensory neuropathy. Finally, a small number of patients have been identified in whom a TRPV4 mutation results in a phenotype combining skeletal dysplasia with peripheral neuropathy. The TRPV4 gene encodes a regulated calcium channel implicated in multiple and diverse cellular processes. Over 50 different TRPV4 mutations have been reported, with two codons appearing to be mutational hot spots: P799 in exon 15, mostly associated with MD, and R594 in exon 11, associated with SMDK. While most pathogenic mutations tested so far result in activation of the calcium channel in vitro, the mechanisms through which TRPV4 activation results in skeletal dysplasia and/or peripheral neuropathy remain unclear and the genotype-phenotype correlations in this group of disorders remains somewhat mysterious. Since the phenotypic expression of most mutations seems to be relatively constant, careful clinical and radiographic assessment is useful in directing molecular analysis. © 2012 Wiley Periodicals, Inc.

Point Mutations in the Monocarboxylate Transporter SLC16A12 Lead to Juvenile and Age-Related Cataract

Purpose: Previously we reported on a premature termination mutation in SLC16A12 that leads to dominant juvenile cataract and renal glucosuria. To assess the mutation rate and genotype-phenotype correlations of SLC16A12 in juvenile or age-related forms of cataract, we performed a mutation screen in cataract patients. Methods: Clinical data of approximately 660 patients were collected, genomic DNA was isolated and analyzed. Exons 3 to 8 including flanking intron sequences of SLC16A12 were PCR amplified and DNA sequence was determined. Selected mutations were tested by cell culture assays, in silico analysis and RT-PCR. Results: We found sequence alterations at a rate of approximately 1/75 patients. None of them was found in 360 control alleles. Alterations affect splice site and regulatory region but most mutations caused an amino acid substitution. The majority of the coding region mutations maps to trans-membrane domains. One mutation located to the 5'UTR. It affects translational efficiency of SLC16A12. In addition, we identified a cataract-predisposing SNP in the non-coding region that causes allele-specific splicing of the 5'UTR region. Conclusions: Altered translational efficiency of the solute carrier SLC16A12 and its allele-specific splicing strongly support a model of challenged homeostasis to cause various forms of cataract. In addition, the pathogenic property of the here reported sequence alterations is supported by the lack of known sequence variations within the coding region of SLC16A12. Due to the relatively high mutation rate, we suggest to include SLC16A12 in diagnostic cataract screening. Generally, our data recommend the assessment of regulatory sequences for diagnostic purposes.

Hypertension portate et infection par le VIH [Portal hypertension and HIV infection]

In the era of antiretroviral therapies, the outcome of patients with chronic HIV infection has considerably changed and their prolonged survival allows the development of chronic liver diseases as a major cause of mortality. Although viral hepatitis, alcoholic and non alcoholic steatohepatitis account forthe majority of chronic liver damage in these patients, there is a growing number of cases with unexplained liver disease, many of which are associated with clinical manifestations of portal hypertension. Inthissituation, nodularregenerative hyperplasia is a frequent finding, characterized at histology by the presence of a nodular architecture in the absence of significant fibrosis, resulting from progressive obliteration of small portal veins. This article describes the clinical presentation, diagnostic aspects, pathogenic mechanisms, as well as the management of this emergent non cirrhotic liver disease in HIV-infected patients.

Exome sequencing of index patients with retinal dystrophies as a tool for molecular diagnosis.

BACKGROUND: Retinal dystrophies (RD) are a group of hereditary diseases that lead to debilitating visual impairment and are usually transmitted as a Mendelian trait. Pathogenic mutations can occur in any of the 100 or more disease genes identified so far, making molecular diagnosis a rather laborious process. In this work we explored the use of whole exome sequencing (WES) as a tool for identification of RD mutations, with the aim of assessing its applicability in a diagnostic context. METHODOLOGY/PRINCIPAL FINDINGS: We ascertained 12 Spanish families with seemingly recessive RD. All of the index patients underwent mutational pre-screening by chip-based sequence hybridization and resulted to be negative for known RD mutations. With the exception of one pedigree, to simulate a standard diagnostic scenario we processed by WES only the DNA from the index patient of each family, followed by in silico data analysis. We successfully identified causative mutations in patients from 10 different families, which were later verified by Sanger sequencing and co-segregation analyses. Specifically, we detected pathogenic DNA variants (∼50% novel mutations) in the genes RP1, USH2A, CNGB3, NMNAT1, CHM, and ABCA4, responsible for retinitis pigmentosa, Usher syndrome, achromatopsia, Leber congenital amaurosis, choroideremia, or recessive Stargardt/cone-rod dystrophy cases. CONCLUSIONS/SIGNIFICANCE: Despite the absence of genetic information from other family members that could help excluding nonpathogenic DNA variants, we could detect causative mutations in a variety of genes known to represent a wide spectrum of clinical phenotypes in 83% of the patients analyzed. Considering the constant drop in costs for human exome sequencing and the relative simplicity of the analyses made, this technique could represent a valuable tool for molecular diagnostics or genetic research, even in cases for which no genotypes from family members are available.

Genetic structure of the genus Leptospira by multilocus enzyme electrophoresis

Thirty strains from the 11 species of the genus Leptospira were studied by multilocus enzyme electrophoresis at 12 enzyme loci, all of which were polymorphic. The mean number of alleles per locus was 6.5. Twenty-five electrophoretic types were distinguished. Grouping of the strains by cluster analysis was in general agreement with species delineation as determined by DNA-DNA hybridization, except for the strains of Leptospira meyeri and Leptospira inadai, which were scattered throughout the genus, reflecting previously recognized taxonomic uncertainties. Analysis of the clonality within Leptospira interrogans sensu stricto indicated that this population was relatively heterogeneous and a lack of gene linkage disequilibrium could not be excluded. There was a genetic discrimination between the pathogenic species and the saprophytic ones. The phenotypically intermediate species (L. inadai and Leptospira fainei) were also genetically separated and were probably closer to the saprophytes than to the pathogens.

African tick-bite fever: a new entity in the differential diagnosis of multiple eschars in travelers. Description of five cases imported from South Africa to Switzerland.

African tick-bite fever (ATBF) is a newly described spotted fever rickettsiosis that frequently presents with multiple eschars in travelers returning from sub-Saharan Africa and, to a lesser extent, from the West Indies. It is caused by the bite of an infected Amblyomma tick, whose hunting habits explain the typical presence of multiple inoculation skin lesions and the occurrence of clustered cases. The etiological agent of ATBF is Rickettsia africae, an emerging tick-borne pathogenic bacterium. We describe herein a cluster of five cases of ATBF occurring in Swiss travelers returning from South Africa. The co-incidental infections in these five patients and the presence of multiple inoculation eschars, two features pathognomonic of this rickettsial disease, suggested the diagnosis of ATBF. Indeed, the presence of at least one inoculation eschar is observed in 53-100% of cases and multiple eschars in 21-54%. Two patients presented regional lymphadenitis and one a mild local lymphangitis. Though a cutaneous rash is described in 15-46% of cases, no rash was observed in our series. ATBF was confirmed by serology. Thus, ATBF has recently emerged as one of the most important causes of flu-like illness in travelers returning from Southern Africa. The presence of one or multiple eschars of inoculation is an important clinical clue to the diagnosis. It can be confirmed by serology or by PCR of a biopsy of the eschar. Culture can also be done in reference laboratories. Dermatologists and primary care physicians should know this clinical entity, since an inexpensive and efficient treatment is available.

Impact of salt on cardiac differential gene expression and coronary lesion in normotensive mineralocorticoid-treated mice.

We previously reported that excess of deoxycorticosterone-acetate (DOCA)/salt-induced cardiac hypertrophy in the absence of hypertension in one-renin gene mice. This model allows us to study molecular mechanisms of high-salt intake in the development of cardiovascular remodeling, independently of blood pressure in a high mineralocorticoid state. In this study, we compared the effect of 5-wk low- and high-salt intake on cardiovascular remodeling and cardiac differential gene expression in mice receiving the same amount of DOCA. Differential gene and protein expression was measured by high-density cDNA microarray assays, real-time PCR and Western blot analysis in DOCA-high salt (HS) vs. DOCA-low salt (LS) mice. DOCA-HS mice developed cardiac hypertrophy, coronary perivascular fibrosis, and left ventricular dysfunction. Differential gene and protein expression demonstrated that high-salt intake upregulated a subset of genes encoding for proteins involved in inflammation and extracellular matrix remodeling (e.g., Col3a1, Col1a2, Hmox1, and Lcn2). A major subset of downregulated genes encoded for transcription factors, including myeloid differentiation primary response (MyD) genes. Our data provide some evidence that vascular remodeling, fibrosis, and inflammation are important consequences of a high-salt intake in DOCA mice. Our study suggests that among the different pathogenic factors of cardiac and vascular remodeling, such as hypertension and mineralocorticoid excess and sodium intake, the latter is critical for the development of the profibrotic and proinflammatory phenotype observed in the heart of normotensive DOCA-treated mice.

Red blood cell microparticles and blood group antigens: an analysis by flow cytometry.

BACKGROUND: The storage of blood induces the formation of erythrocytes-derived microparticles. Their pathogenic role in blood transfusion is not known so far, especially the risk to trigger alloantibody production in the recipient. This work aims to study the expression of clinically significant blood group antigens on the surface of red blood cells microparticles. MATERIAL AND METHODS: Red blood cells contained in erythrocyte concentrates were stained with specific antibodies directed against blood group antigens and routinely used in immunohematology practice. After inducing erythrocytes vesiculation with calcium ionophore, the presence of blood group antigens was analysed by flow cytometry. RESULTS: The expression of several blood group antigens from the RH, KEL, JK, FY, MNS, LE and LU systems was detected on erythrocyte microparticles. The presence of M (MNS1), N (MNS2) and s (MNS4) antigens could not be demonstrated by flow cytometry, despite that glycophorin A and B were identified on microparticles using anti-CD235a and anti-MNS3. DISCUSSION: We conclude that blood group antigens are localized on erythrocytes-derived microparticles and probably keep their immunogenicity because of their capacity to bind specific antibody. Selective segregation process during vesiculation or their ability to elicit an immune response in vivo has to be tested by further studies.