Identification des dermatophytes causant des teignes de la peau glabre (Tinea glabra) et du cuir chevelu (Tinea capitis) par PCR

Contexte: Le nombre de teignes du cuir chevelu et de la peau glabre étant en nette augmentation, l'identification du pathogène qui est indispensable pour un traitement ciblé, a, par conséquence, un grand intérêt pour la santé publique. Dans divers cas, un animal de compagnie peut être identifié en tant que source du pathogène. La fréquence de cultures restant stériles est particulièrement élevée en cas de prétraitement antifongique. Objectif: Le but de ce travail est de mettre au point une méthode rapide d'identification du dermatophyte pathogène in situ par PCR/séquençage dans les cas de teignes du cuir chevelu et/ou de la peau glabre. Matériel et méthodes : De l'ADN a été extrait de squames (N=5) et cheveux (N=21) dont l'examen direct démontrait une infection fongique (N=26) ou se révèlait négatif (N=1). Ensuite, une première PCR du segment 28s de l'ADN ribosomale fongique a été effectuée, suivie par une PCR nichée intérieure à ce segment. L'amplicon a été séquencé et le champignon est identifié par alignement. Résultats : Seule la PCR enchainée a permis d'obtenir une quantité suffisante d'amplicon pour permettre le séquençage. Dans 4 cas sur 5 de tinea pedis, 10 sur 12 de tinea glabra, respectivement 4 sur 4 de tinea capitis, dans lesquels un dermato- phyte a été identifié en culture, le même dermatophyte a été identifié par PCR/séquençage. Une fois sur 27 prélèvements, un autre dermatophyte a été identifié par PCR/séquençage. Ce résultat pourrait être dû à une fausse identification du champignon en culture. Dans un cas de tinea pedis et un cas de tinea corporis, la culture est restée stérile, mais un dermatophyte a pu être identifié par PCR et séquençage. Conclusions : La méthode décrite est à la fois rapide (24 h au lieu de deux semaines pour la culture), sensible et très spécifique. Elle est particulièrement utile dans les cas de teigne du cuir chevelu, dans lesquels le traitement est différent selon l'espèce de dermatophyte et où il s'agit d'un traitement systémique lourd, souvent chez l'enfant.

Characterizing aging in the human brainstem using quantitative multimodal MRI analysis.

Aging is ubiquitous to the human condition. The MRI correlates of healthy aging have been extensively investigated using a range of modalities, including volumetric MRI, quantitative MRI (qMRI), and diffusion tensor imaging. Despite this, the reported brainstem related changes remain sparse. This is, in part, due to the technical and methodological limitations in quantitatively assessing and statistically analyzing this region. By utilizing a new method of brainstem segmentation, a large cohort of 100 healthy adults were assessed in this study for the effects of aging within the human brainstem in vivo. Using qMRI, tensor-based morphometry (TBM), and voxel-based quantification (VBQ), the volumetric and quantitative changes across healthy adults between 19 and 75 years were characterized. In addition to the increased R2* in substantia nigra corresponding to increasing iron deposition with age, several novel findings were reported in the current study. These include selective volumetric loss of the brachium conjunctivum, with a corresponding decrease in magnetization transfer and increase in proton density (PD), accounting for the previously described "midbrain shrinkage." Additionally, we found increases in R1 and PD in several pontine and medullary structures. We consider these changes in the context of well-characterized, functional age-related changes, and propose potential biophysical mechanisms. This study provides detailed quantitative analysis of the internal architecture of the brainstem and provides a baseline for further studies of neurodegenerative diseases that are characterized by early, pre-clinical involvement of the brainstem, such as Parkinson's and Alzheimer's diseases.

Repeated unidirectional introgression towards Populus balsamifera in contact zones of exotic and native poplars.

As the evolutionary significance of hybridization is largely dictated by its extent beyond the first generation, we broadly surveyed patterns of introgression across a sympatric zone of two native poplars (Populus balsamifera, Populus deltoides) in Quebec, Canada within which European exotic Populus nigra and its hybrids have been extensively planted since the 1800s. Single nucleotide polymorphisms (SNPs) that appeared fixed within each species were characterized by DNA-sequencing pools of pure individuals. Thirty-five of these diagnostic SNPs were employed in a high-throughput assay that genotyped 635 trees of different age classes, sampled from 15 sites with various degrees of anthropogenic disturbance. The degree of admixture within sampled trees was then assessed through Bayesian clustering of genotypes. Hybrids were present in seven of the populations, with 2.4% of all sampled trees showing spontaneous admixture. Sites with hybrids were significantly more disturbed than pure stands, while hybrids comprised both immature juveniles and trees of reproductive age. All three possible F1s were detected. Advanced-generation hybrids were consistently biased towards P. balsamifera regardless of whether hybridization had occurred with P. deltoides or P. nigra. Gene exchange between P. deltoides and P. nigra was not detected beyond the F1 generation; however, detection of a trihybrid demonstrates that even this apparent reproductive isolation does not necessarily result in an evolutionary dead end. Collectively, results demonstrate the natural fertility of hybrid poplars and suggest that introduced genes could potentially affect the genetic integrity of native trees, similar to that arising from introgression between natives.

Differentially expressed gene profile in the 6-hydroxy-dopamine-induced cell culture model of Parkinson's disease.

Parkinson's disease (PD) is a chronic neurodegenerative disorder characterized by progressive loss of dopaminergic (DA) neurons of the substantia nigra pars compacta with unknown aetiology. 6-Hydroxydopamine (6-OHDA) treatment of neuronal cells is an established in vivo model for mimicking the effect of oxidative stress found in PD brains. We examined the effects of 6-OHDA treatment on human neuroblastoma cells (SH-SY5Y) and primary mesencephalic cultures. Using a reverse arbitrarily primed polymerase chain reaction (RAP-PCR) approach we generated reproducible genetic fingerprints of differential expression levels in cell cultures treated with 6-OHDA. Of the resulting sequences, 23 showed considerable homology to known human coding sequences. The results of the RAP-PCR were validated by reverse transcription PCR, real-time PCR and, for selected genes, by Western blot analysis and immunofluorescence. In four cases, [tomoregulin-1 (TMEFF-1), collapsin response mediator protein 1 (CRMP-1), neurexin-1, and phosphoribosylaminoimidazole synthetase (GART)], a down-regulation of mRNA and protein levels was detected. Further studies will be necessary on the physiological role of the identified proteins and their impact on pathways leading to neurodegeneration in PD.

Lentiviral gene transfer to the nonhuman primate brain.

Lentiviral vectors infect quiescent cells and allow for the delivery of genes to discrete brain regions. The present study assessed whether stable lentiviral gene transduction can be achieved in the monkey nigrostriatal system. Three young adult Rhesus monkeys received injections of a lentiviral vector encoding for the marker gene beta galatosidase (beta Gal). On one side of the brain, each monkey received multiple lentivirus injections into the caudate and putamen. On the opposite side, each animal received a single injection aimed at the substantia nigra. The first two monkeys were sacrificed 1 month postinjection, while the third monkey was sacrificed 3 months postinjection. Robust incorporation of the beta Gal gene was seen in the striatum of all three monkeys. Stereological counts revealed that 930,218; 1,192,359; and 1,501,217 cells in the striatum were beta Gal positive in monkeys 1 (n = 2) and 3 (n = 1) months later, respectively. Only the third monkey had an injection placed directly into the substantia nigra and 187,308 beta Gal-positive cells were identified in this animal. The injections induced only minor perivascular cuffing and there was no apparent inflammatory response resulting from the lentivirus injections. Double label experiments revealed that between 80 and 87% of the beta Gal-positive cells were neurons. These data indicate that robust transduction of striatal and nigral cells can occur in the nonhuman primate brain for up to 3 months. Studies are now ongoing testing the ability of lentivirus encoding for dopaminergic trophic factors to augment the nigrostriatal system in nonhuman primate models of Parkinson's disease.

Evaluation of a PCR-RFLP assay for the identification of dermatophytes in situ

Dermatophytes are the main cause of superficial mycoses. These fungi have the capacity to invade keratinized tissue of humans or animals to produce infections that are generally restricted to the corneocytes of the skin, hair, and nails. Nevertheless, it is common to obtain negative results from fungal cultures of dermatological specimens where direct mycological examination showed fungal elements (30-40%). However, correct identification of the isolated dermatophytes from Tinea is important to choose the appropriate treatment. Therefore, we aim to develop a rapid polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay based on 28S rDNA that is able to identify dermatophytes species in positive dermatological samples. PCR-RFLP identification of dermatophytes in skin or hair allowed validation of the results obtained in culture. It was also possible to identify the infectious dermatophytes when direct hair/ skin mycological examination showed fungal elements, but negative results were obtained from fungal culture. As a conclusion, PCR methods may provide significant benefits in the rapid diagnosis of Tinea. First, there is an increase in sensitivity of dermatophytes identification when enough material is available. Secondly, identification of the infecting agent can be obtained in 24 hours with PCR-RFLP or sequencing, whereas results from fungal cultures can take 2-3 weeks.

Dermatophyte identification in skin and hair samples using a simple and reliable nested polymerase chain reaction assay.

BACKGROUND: Dermatophyte identification in tinea capitis is essential for choosing the appropriate treatment and in tinea infections to identify the possible source. The failure of fungi to grow in cultures frequently occurs, especially in cases of previous antifungal therapy. OBJECTIVES: To develop a rapid polymerase chain reaction (PCR) sequencing assay for dermatophyte identification in tinea capitis and tinea corporis. MATERIAL AND METHODS: Fungal DNA was extracted from hair and skin samples that were confirmed to be positive by direct mycological examination. Dermatophytes were identified by the sequence of a 28S ribosomal DNA subunit amplicon generated by nested PCR. RESULTS: Nested PCR was found to be necessary to obtain amplicons in substantial amounts for dermatophyte identification by sequencing. The results agreed with those of classical mycological identification in 14 of 23, 6 of 10, and 20 of 23 cases of tinea capitis, tinea corporis and tinea pedis, respectively, from which a dermatophyte was obtained in culture. In seven of the 56 cases, another dermatophyte was identified, revealing previous misidentification. A dermatophyte was identified in 12 of 18, three of five, and four of nine cases of tinea capitis, tinea corporis and tinea pedis, respectively, in cases in which no dermatophyte grew in culture. CONCLUSIONS: Although the gold standard dermatophyte identification from clinical samples remains fungal cultures, the assay developed in the present study is especially suitable for tinea capitis. Improved sensitivity for the identification of dermatophyte species was obtained as it is possible to identify the dermatophyte when the fungus fails to grow in cultures.

Time of injection determines the effect of alpha-MSH antiserum on DA neurons in psychological stress

Male rats were subjected to "psychological stress" which consisted in 10 sec footshock on the first day followed 24 hr later by a 10 sec stay in the experimental chamber without shock. Intravenous antiserum against alpha-MSH markedly changed the functional state of mesencephalic and hypothalamic DA neurons (assessed by histochemical microfluorimetry) when administered before the second session but not when given before the first session. These observations reveal an interesting parallelism in the temporal characteristics of the effects of alpha-MSH on avoidance behavior and central DA systems.

Differential transgene expression profiles from rAAV2/1 vectors using the tetON and CMV promoters in the rat brain.

The biodistribution of transgene expression in the CNS after localized stereotaxic vector delivery is an important issue for safety of gene therapy for neurological diseases. The cellular specificity of transgene expression from rAAV2/1 vectors using the tetON expression cassette in comparison with the CMV promoter was investigated in the rat nigrostriatal pathway. After intrastriatal injection, although GFP was mainly expressed into neurons with both vectors, the relative proportions of DARPP-32+ projection neurons and parvalbumin+ interneurons were respectively 13:1 and 2:1 for the CMV and tetON vectors. DARP32+ neurons projecting to the globus pallidus were strongly GFP+ with both vectors, whereas those projecting to the substantia nigra pars reticulata (SNpr) were efficiently labeled by the CMV but poorly by the tetON vector. Numerous GFP+ cells were evidenced in the subventricular zone with both vectors. However, in the olfactory bulb (OB), GFP+ neurons were observed with the CMV but not the tetON vector. We conclude that the absence of significant amounts of transgene product in distant regions (SN and OB) constitutes a safety advantage of the AAV2/1-tetON vector for striatal gene therapy. Midbrain injections resulted in selective GFP expression in tyrosine hydroxylase+ neurons by the tetON vector whereas with the CMV vector, GFP+ cells covered a widespread area of the midbrain. The biodistribution of GFP protein corresponded to that of the transcripts and not of the viral genomes. We conclude that the rAAV2/1-tetON vector constitutes an interesting tool for specific transgene expression in midbrain dopaminergic neurons.

Molecular chaperones and associated cellular clearance mechanisms against toxic protein conformers in Parkinson's disease.

Parkinson's disease (PD) is a slowly progressive neurodegenerative disorder marked by the loss of dopaminergic neurons (in particular in the substantia nigra) causing severe impairment of movement coordination and locomotion, associated with the accumulation of aggregated α-synuclein (α-Syn) into proteinaceous inclusions named Lewy bodies. Various early forms of misfolded α-Syn oligomers are cytotoxic. Their formation is favored by mutations and external factors, such as heavy metals, pesticides, trauma-related oxidative stress and heat shock. Here, we discuss the role of several complementing cellular defense mechanisms that may counteract PD pathogenesis, especially in youth, and whose effectiveness decreases with age. Particular emphasis is given to the 'holdase' and 'unfoldase' molecular chaperones that provide cells with potent means to neutralize and scavenge toxic protein conformers. Because chaperones can specifically recognize misfolded proteins, they are key specificity factors for other cellular defenses, such as proteolysis by the proteasome and autophagy. The efficiency of the cellular defenses decreases in stressed or aging neurons, leading to neuroinflammation, apoptosis and tissue loss. Thus, drugs that can upregulate the molecular chaperones, the ubiquitin-proteasome system and autophagy in brain tissues are promising avenues for therapies against PD and other mutation-, stress- or age-dependent protein-misfolding diseases.

The dermatophyte species Arthroderma benhamiae: intraspecies variability and mating behaviour.

Arthroderma benhamiae is a zoophilic dermatophyte belonging to the Trichophyton mentagrophytes species complex. Here, a population of A. benhamiae wild strains from the same geographical area (Switzerland) was studied by comparing their morphology, assessing their molecular variability using internal transcribed spacer (ITS) and 28S rRNA gene sequencing, and evaluating their interfertility. Sequencing of the ITS region and of part of the 28S rRNA gene revealed the existence of two infraspecific groups with markedly different colony phenotypes: white (group I) and yellow (group II), respectively. For all strains, the results of mating type identification by PCR, using HMG (high-mobility group) and α-box genes in the mating type locus as targets, were in total accordance with the results of mating type identification by strain confrontation experiments. White-phenotype strains were of mating type + (mt+) or mating type - (mt-), whilst yellow-phenotype strains were all mt-. White and yellow strains were found to produce fertile cleistothecia after mating with A. benhamiae reference tester strains, which belonged to a third group intermediate between groups I and II. However, no interfertility was observed between yellow strains and white strains of mt+. A significant result was that white strains of mt- were able to mate and produce fertile cleistothecia with the white A. benhamiae strain CBS 112371 (mt+), the genome of which has recently been sequenced and annotated. This finding should offer new tools for investigating the biology and genetics of dermatophytes using wild-type strains.

Intracisternal delivery of NFκB-inducible scAAV2/9 reveals locoregional neuroinflammation induced by systemic kainic acid treatment.

We have previously demonstrated disease-dependent gene delivery in the brain using an AAV vector responding to NFκB activation as a probe for inflammatory responses. This vector, injected focally in the parenchyma prior to a systemic kainic acid (KA) injection mediated inducible transgene expression in the hippocampus but not in the cerebellum, regions, respectively, known to be affected or not by the pathology. However, such a focal approach relies on previous knowledge of the model parameters and does not allow to predict the whole brain response to the disease. Global brain gene delivery would allow to predict the regional distribution of the pathology as well as to deliver therapeutic factors in all affected brain regions. We show that self-complementary AAV2/9 (scAAV2/9) delivery in the adult rat cisterna magna allows a widespread but not homogenous transduction of the brain. Indeed, superficial regions, i.e., cortex, hippocampus, and cerebellum were more efficiently transduced than deeper regions, such as striatum, and substantia nigra. These data suggest that viral particles penetration from the cerebrospinal fluid (CSF) into the brain is a limiting factor. Interestingly, AAV2/9-2YF a rationally designed capsid mutant (affecting surface tyrosines) increased gene transfer efficiency approximately fivefold. Neurons, astrocytes, and oligodendrocytes, but not microglia, were transduced in varying proportions depending on the brain region and the type of capsid. Finally, after a single intracisternal injection of scAAV2/9-2YF using the NFκB-inducible promoter, KA treatment induced transgene expression in the hippocampus and cortex but not in the cerebellum, corresponding to the expression of the CD11b marker of microglial activation. These data support the use of disease-inducible vectors administered in the cisterna magna as a tool to characterize the brain pathology in systemic drug-induced or transgenic disease models. However, further improvements are required to enhance viral particles penetration into the brain.

Gene expression profiling in the human pathogenic dermatophyte Trichophyton rubrum during growth on proteins.

Dermatophytes are highly specialized filamentous fungi which cause the majority of superficial mycoses in humans and animals. The high secreted proteolytic activity of these microorganisms during growth on proteins is assumed to be linked to their particular ability to exclusively infect keratinized host structures such as the skin stratum corneum, hair, and nails. Individual secreted dermatophyte proteases were recently described and linked with the in vitro digestion of keratin. However, the overall adaptation and transcriptional response of dermatophytes during protein degradation are largely unknown. To address this question, we constructed a cDNA microarray for the human pathogenic dermatophyte Trichophyton rubrum that was based on transcripts of the fungus grown on proteins. Profiles of gene expression during the growth of T. rubrum on soy and keratin protein displayed the activation of a large set of genes that encode secreted endo- and exoproteases. In addition, other specifically induced factors potentially implicated in protein utilization were identified, including heat shock proteins, transporters, metabolic enzymes, transcription factors, and hypothetical proteins with unknown functions. Of particular interest is the strong upregulation of key enzymes of the glyoxylate cycle in T. rubrum during growth on soy and keratin, namely, isocitrate lyase and malate synthase. This broad-scale transcriptional analysis of dermatophytes during growth on proteins reveals new putative pathogenicity-related host adaptation mechanisms of these human pathogenic fungi.