Altered distribution of juxtaparanodal kv1.2 subunits mediates peripheral nerve hyperexcitability in type 2 diabetes mellitus.

Peripheral nerve hyperexcitability (PNH) is one of the distal peripheral neuropathy phenotypes often present in patients affected by type 2 diabetes mellitus (T2DM). Through in vivo and ex vivo electrophysiological recordings in db/db mice, a model of T2DM, we observed that, in addition to reduced nerve conduction velocity, db/db mice also develop PNH. By using pharmacological inhibitors, we demonstrated that the PNH is mediated by the decreased activity of K(v)1-channels. In agreement with these data, we observed that the diabetic condition led to a reduced presence of the K(v)1.2-subunits in juxtaparanodal regions of peripheral nerves in db/db mice and in nerve biopsies from T2DM patients. Together, these observations indicate that the T2DM condition leads to potassium channel-mediated PNH, thus identifying them as a potential drug target to treat some of the DPN related symptoms.

Arterial properties in relation to genetic variations in the adducin subunits in a white population.

BACKGROUND: Adducin is a membrane skeleton protein, which consists of either alpha- and beta- or alpha- and gamma-subunits. We investigated whether arterial characteristics might be related to the genes encoding ADD1 (Gly460Trp-rs4961), ADD2 (C1797T-rs4984), and ADD3 (IVS11+386A>G-rs3731566). METHODS: We randomly recruited 1,126 Flemish subjects (mean age, 43.8 years; 50.3% women). Using a wall-tracking ultrasound system, we measured the properties of the carotid, femoral, and brachial arteries. We studied multivariate-adjusted phenotype-genotype associations, using a population- and family-based approach. RESULTS: In single-gene analyses, brachial diameter was 0.15 mm (P = 0.0022) larger, and brachial distensibility and cross-sectional compliance were 1.55 x 10(-3)/kPa (P = 0.013) and 0.017 mm(2)/kPa (P = 0.0029) lower in ADD3 AA than ADD3 GG homozygotes with an additive effect of the G allele. In multiple-gene analyses, the association of brachial diameter and distensibility with the ADD3 G allele occurred only in ADD1 GlyGly homozygotes. Otherwise, the associations between the arterial phenotypes in the three vascular beds and the ADD1 or ADD2 polymorphisms were not significant. In family-based analyses, the multivariate-adjusted heritability was 0.52, 0.38, and 0.30 for brachial diameter, distensibility, and cross-sectional compliance, respectively (P < 0.001). There was no evidence for population stratification (0.07 < or = P < or = 0.96). Transmission of the mutated ADD3 G allele was associated with smaller brachial diameter in 342 informative offspring (-0.12 +/- 0.04 mm; P = 0.0085) and in 209 offspring, who were ADD1 GlyGly homozygotes (-0.14 +/- 0.06 mm; P = 0.018). CONCLUSIONS: In ADD1 GlyGly homozygotes, the properties of the brachial artery are related to the ADD3 (A386G) polymorphism, but the underlying mechanism needs further clarification.

The IC3D classification of the corneal dystrophies.

BACKGROUND: The recent availability of genetic analyses has demonstrated the shortcomings of the current phenotypic method of corneal dystrophy classification. Abnormalities in different genes can cause a single phenotype, whereas different defects in a single gene can cause different phenotypes. Some disorders termed corneal dystrophies do not appear to have a genetic basis. PURPOSE: The purpose of this study was to develop a new classification system for corneal dystrophies, integrating up-to-date information on phenotypic description, pathologic examination, and genetic analysis. METHODS: The International Committee for Classification of Corneal Dystrophies (IC3D) was created to devise a current and accurate nomenclature. RESULTS: This anatomic classification continues to organize dystrophies according to the level chiefly affected. Each dystrophy has a template summarizing genetic, clinical, and pathologic information. A category number from 1 through 4 is assigned, reflecting the level of evidence supporting the existence of a given dystrophy. The most defined dystrophies belong to category 1 (a well-defined corneal dystrophy in which a gene has been mapped and identified and specific mutations are known) and the least defined belong to category 4 (a suspected dystrophy where the clinical and genetic evidence is not yet convincing). The nomenclature may be updated over time as new information regarding the dystrophies becomes available. CONCLUSIONS: The IC3D Classification of Corneal Dystrophies is a new classification system that incorporates many aspects of the traditional definitions of corneal dystrophies with new genetic, clinical, and pathologic information. Standardized templates provide key information that includes a level of evidence for there being a corneal dystrophy. The system is user-friendly and upgradeable and can be retrieved on the website www.corneasociety.org/ic3d.

Evolution of a cellular immune response in Drosophila: a phenotypic and genomic comparative analysis.

Understanding the genomic basis of evolutionary adaptation requires insight into the molecular basis underlying phenotypic variation. However, even changes in molecular pathways associated with extreme variation, gains and losses of specific phenotypes, remain largely uncharacterized. Here, we investigate the large interspecific differences in the ability to survive infection by parasitoids across 11 Drosophila species and identify genomic changes associated with gains and losses of parasitoid resistance. We show that a cellular immune defense, encapsulation, and the production of a specialized blood cell, lamellocytes, are restricted to a sublineage of Drosophila, but that encapsulation is absent in one species of this sublineage, Drosophila sechellia. Our comparative analyses of hemopoiesis pathway genes and of genes differentially expressed during the encapsulation response revealed that hemopoiesis-associated genes are highly conserved and present in all species independently of their resistance. In contrast, 11 genes that are differentially expressed during the response to parasitoids are novel genes, specific to the Drosophila sublineage capable of lamellocyte-mediated encapsulation. These novel genes, which are predominantly expressed in hemocytes, arose via duplications, whereby five of them also showed signatures of positive selection, as expected if they were recruited for new functions. Three of these novel genes further showed large-scale and presumably loss-of-function sequence changes in D. sechellia, consistent with the loss of resistance in this species. In combination, these convergent lines of evidence suggest that co-option of duplicated genes in existing pathways and subsequent neofunctionalization are likely to have contributed to the evolution of the lamellocyte-mediated encapsulation in Drosophila.

Demyelination in mild cognitive impairment suggests progression path to Alzheimer's disease.

The preclinical Alzheimer's disease (AD) - amnestic mild cognitive impairment (MCI) - is manifested by phenotypes classified into exclusively memory (single-domain) MCI (sMCI) and multiple-domain MCI (mMCI). We suggest that typical MCI-to-AD progression occurs through the sMCI-to-mMCI sequence as a result of the extension of initial pathological processes. To support this hypothesis, we assess myelin content with a Magnetization Transfer Ratio (MTR) in 21 sMCI and 21 mMCI patients and in 42 age-, sex-, and education-matched controls. A conjunction analysis revealed MTR reduction shared by sMCI and mMCI groups in the medial temporal lobe and posterior structures including white matter (WM: splenium, posterior corona radiata) and gray matter (GM: hippocampus; parahippocampal and lingual gyri). A disjunction analysis showed the spread of demyelination to prefrontal WM and insula GM in executive mMCI. Our findings suggest that demyelination starts in the structures affected by neurofibrillary pathology; its presence correlates with the clinical picture and indicates the method of MCI-to-AD progression. In vivo staging of preclinical AD can be developed in terms of WM/GM demyelination.

Escherichia coli variants in periprosthetic joint infection: diagnostic challenges with sessile bacteria and sonication.

The diagnostic yield of prosthetic joint-associated infection is hampered by the phenotypic change of bacteria into a sessile and resistant form, also called biofilm. With sonication, adherent bacteria can be dislodged from the prosthesis. Species identification may be difficult because of their variations in phenotypic appearance and biochemical reaction. We have studied the phenotypic, genotypic, and biochemical properties of Escherichia coli variants isolated from a periprosthetic joint infection. The strains were collected from synovial fluid, periprosthetic tissue, and fluid from the explanted and sonicated prosthesis. Isolates from synovial fluid revealed a normal phenotype, whereas a few variants from periprosthetic tissue and all isolates from sonication fluid showed different morphological features (including small-colony variants). All isolates from sonication fluid were beta-galactosidase negative and nonmotile; most were indole negative. Because of further variations in biochemical properties, species identification was false or not possible in 50% of the isolates included in this study. In contrast to normal phenotypes, variants were resistant to aminoglycosides. Typing of the isolates using pulsed-field gel electrophoresis yielded nonidentical banding patterns, but all strains were assigned to the same clonal origin when compared with 207 unrelated E. coli isolates. The bacteria were repeatedly passaged on culture media and reanalyzed. Thereafter, most variants reverted to normal phenotype and regained their motility and certain biochemical properties. In addition, some variants displayed aminoglycoside susceptibility after reversion. Sonication of an explanted prosthesis allows insight into the lifestyle of bacteria in biofilms. Since sonication fluid also reveals dislodged sessile forms, species identification of such variants may be misleading.

Susceptibility of Colombian Plasmodium falciparum isolates to 4-aminoquinolines and the definition of amodiaquine resistance in vitro

There are wide variations in the threshold used to define in vitro resistance of Plasmodium falciparum to amodiaquine (AQ), probably due to differences in methodology and interpretation. In vitro susceptibility data of Colombian P. falciparum strains to AQ and N-desethylamodiaquine is used to illustrate the need to standardized methodologies and compare inhibitory concentrations, instead of resistant/susceptible phenotypes, when studying the mechanisms of resistance to AQ and monitoring drug susceptibility trends in the field.

Electronic monitoring of compliance to lipid-lowering therapy in clinical practice

Nonadherence to treatment is a common problem in the clinical management of hypercholesterolemic patients. This study was carried out with the aim of monitoring the daily compliance to a 6-month course of lipid-lowering therapy, using a microelectronic device, the Medication Event Monitoring System (MEMS), versus pill count. Forty men with primary hypercholesterolemia were prescribed fluvastatin 1 x 40 mg daily, provided in a MEMS package to record the date and time of each opening of the pillbox. Thirty-nine of 40 patients (98%) completed the study. Total cholesterol and LDL cholesterol levels decreased significantly (18% and 25%, p < 0.001) during the 6-month therapy period. A high mean rate of compliance was achieved by MEMS using the following three indexes--compliance to total prescribed dose (88.8% +/- 13.5%), compliance to prescribed days (82.4% +/- 19.5%), and compliance to prescribed time of day (81.86% +/- 19.5%)--and by pill count (93.4% +/- 9.5%). In addition, the MEMS provided some patterns of nonadherence to medication, undetectable by pill count alone, such as a drug holiday in 38% of cases, a drug omission for more than 7 consecutive days in 9% of cases, and, conversely, use of more than the one prescribed daily dose in 47% of cases. A significant correlation between the rate of compliance and the decrease in LDL cholesterol was observed only when the compliance was assessed by MEMS. The results indicate that MEMS is a useful tool for monitoring compliance in clinical practice and may possibly increase adherence to long-term lipid-lowering therapy.

Susceptibility of Anopheles aquasalis and An. darlingi to Plasmodium vivax VK210 and VK247

The susceptibility of Anopheles aquasalis (F3 generation) and An. darlingi (F1 generation) to Plasmodium vivax circumsporozoite protein phenotypes from a limited number of blood samples of malaria patients in Belém, state of Pará, Brazil, was examined. A polymerase chain reaction was used to determine the P. vivax phenotypes in blood samples and the blood-fed infected mosquitoes were dissected and tested by ELISA. In all patient infections, more infected An. aquasalis and An. darlingi were positive for VK210 compared with VK247.

The Growing Importance of CNVs: New Insights for Detection and Clinical Interpretation.

Differences between genomes can be due to single nucleotide variants, translocations, inversions, and copy number variants (CNVs, gain or loss of DNA). The latter can range from sub-microscopic events to complete chromosomal aneuploidies. Small CNVs are often benign but those larger than 500 kb are strongly associated with morbid consequences such as developmental disorders and cancer. Detecting CNVs within and between populations is essential to better understand the plasticity of our genome and to elucidate its possible contribution to disease. Hence there is a need for better-tailored and more robust tools for the detection and genome-wide analyses of CNVs. While a link between a given CNV and a disease may have often been established, the relative CNV contribution to disease progression and impact on drug response is not necessarily understood. In this review we discuss the progress, challenges, and limitations that occur at different stages of CNV analysis from the detection (using DNA microarrays and next-generation sequencing) and identification of recurrent CNVs to the association with phenotypes. We emphasize the importance of germline CNVs and propose strategies to aid clinicians to better interpret structural variations and assess their clinical implications.

Increased resistance to first-line agents among bacterial pathogens isolated from urinary tract infections in Latin America: time for local guidelines?

Emerging resistance phenotypes and antimicrobial resistance rates among pathogens recovered from community-acquired urinary tract infections (CA-UTI) is an increasing problem in specific regions, limiting therapeutic options. As part of the SENTRY Antimicrobial Surveillance Program, a total of 611 isolates were collected in 2003 from patients with CA-UTI presenting at Latin American medical centers. Each strain was tested in a central laboratory using Clinical Laboratory Standard Institute (CLSI) broth microdilution methods with appropriate controls. Escherichia coli was the leading pathogen (66%), followed by Klebsiella spp. (7%), Proteus mirabilis (6.4%), Enterococcus spp. (5.6%), and Pseudomonas aeruginosa (4.6%). Surprisingly high resistance rates were recorded for E. coli against first-line orally administered agents for CA-UTI, such as ampicillin (53.6%), TMP/SMX (40.4%), ciprofloxacin (21.6%), and gatifloxacin (17.1%). Decreased susceptibility rates to TMP/SMX and ciprofloxacin were also documented for Klebsiella spp. (79.1 and 81.4%, respectively), and P. mirabilis (71.8 and 84.6%, respectively). For Enterococcus spp., susceptibility rates to ampicillin, chloramphenicol, ciprofloxacin, and vancomycin were 88.2, 85.3, 55.9, and 97.1%, respectively. High-level resistance to gentamicin was detected in 24% of Enterococcus spp. Bacteria isolated from patients with CA-UTI in Latin America showed limited susceptibility to orally administered antimicrobials, especially for TMP/SMX and fluoroquinolones. Our results highlight the need for developing specific CA-UTI guidelines in geographic regions where elevated resistance to new and old compounds may influence prescribing decisions.

Binding of d-methadone, l-methadone, and dl-methadone to proteins in plasma of healthy volunteers: role of the variants of alpha 1-acid glycoprotein.

The plasma concentrations of alpha 1-acid glycoprotein (AAG), albumin, triglycerides, cholesterol, and total proteins, as well as the plasma binding of racemic, d-methadone, and l-methadone were measured in 45 healthy subjects. The AAG phenotypes and the concentrations of AAG variants were also determined. The measured free fractions for racemic, d-methadone, and l-methadone were, respectively, 12.7% +/- 3.3%, 10.0% +/- 2.9%, and 14.2% +/- 3.2% (mean +/- SD). A significant correlation was obtained between the binding ratio (B/F) for dl-methadone and the total AAG concentration (r = 0.724; p less than 0.001). A multiple stepwise regression analysis showed that AAG was the main explanatory variable for the binding of the racemate. When concentrations of AAG variants were considered, a significant correlation was obtained between the binding ratio of dl-methadone and orosomucoid2 A concentration (r = 0.715; p less than 0.001), a weak correlation between dl-methadone and orosomucoid1 S concentration (r = 0.494; p less than 0.001), and no correlation between dl-methadone and orosomucoid1 F1 concentration (r = 0.049; not significant). Similar findings were obtained with the enantiomers. This study shows the importance of considering not only total AAG but also concentrations of AAG variants when measuring the binding of methadone and possibly of other drugs in plasma.

PPAR beta a player in astrocyte metabolism and morphology

AbstractPPARP is a nuclear receptor responding in vivo to several free fatty acids, and implicated in cell metabolism, differentiation and survival. PPARp is ubiquitously expressed but shows high expression in the developing and adult brain. PPARp is expressed in different cell types such as neurons and astrocytes, where it might play a role in metabolism. To study this nuclear receptor the laboratory engineered a PPARP -/- mouse model. The aim of my PhD was to dissect the role of PPARP in astrocytes.Experiments in primary culture revealed that cortical astrocytes from PPARP -/- mouse have an impaired energetic metabolism. Unstimulated PPARP -/- astrocytes exhibit a 30% diminution in glucose uptake, correlating to a 30% decrease in lactate release and intracellular glucose. After acute stimulation by D- aspartate mimicking glutamate exposure, both WT and -/- astrocytes up-regulate their metabolism to respond to the increasing energy needed (ATP) for glutamate uptake. According to the Astrocyte Neuron Lactate Shuttle Hypothesis (ANLSH), the ratio between glucose uptake/ lactate release is 1. However, stimulated PPARp -/- astrocytes display a higher increase in lactate release than glucose uptake which remains lower than in WT. The extra glucose equivalents could come from the degradation of intra cellular glycogen stores, which indeed decrease in PPARP -/- cells upon stimulation. Lower glucose metabolism correlates with a decreased acute glutamate uptake in PPARP -/- astrocytes. Reciprocally, we also observed an increase of glutamate uptake and ATP production after treatment of WT astrocytes with a PPARp agonist. Glutamate transporter protein expression is not affected. However, their trafficking and localization might be altered as PPARp -/- astrocytes have higher cholesterol levels, which may also affect proper transporter structure in the membrane.Metabolism, transporter localization and cholesterol levels are respectively linked to cell mobility, cell cytoskeleton and cellular membrane composition. All three functions are important in astrocytes to in vivo acquire star shaped morphology, in a process known as stellation. PPARP -/- astrocytes showed an impaired acquired stellation in presence of neurons or chemical stimuli, as well as more actin stress fibers and cell adhesion structures. While non stellation of astrocytes is mainly an in vitro phenomenon, it reveals PPARp -/- primary astrocytes inability to respond to different exterior stimuli. These morphological phenotypes correlate with a slower migration in cell culture wound healing assays.This thesis work demonstrates that PPARp is implicated in cortical astrocyte glucose metabolism. PPARp absence leads to an unusual intracellular glycogen use. Added to the effect on acute glutamate uptake and astrocyte migration, PPARp could be an interesting target for neuroprotection therapies.RésuméPPARP est un récepteur nucléaire qui a pour ligands naturels certains acides gras libres. Il est impliqué dans le métabolisme, la différentiation et la survie des cellules. PPARP est ubiquitaire, et a une expression élevée dans le cerveau en développement ainsi qu'adulte. PPARp est exprimé dans différents types cellulaires tels que les neurones et les astrocytes, où il régule potentiellement leurs métabolismes. Pour étudier ce récepteur nucléaire, le laboratoire a créé un modèle de souris PPARp -/-. L'objectif de ma thèse est de comprendre le rôle de PPARp dans les astrocytes.Les expériences montrent un défaut du métabolisme énergétique dans les astrocytes corticaux primaires tirés de souris PPARp -/-. Sans stimulation, l'entrée du glucose dans les astrocytes PPARP -/- est diminuée de 30% ce qui correspond à une diminution de 30% du relargage du lactate. Après stimulation par du D-Aspartate qui mime une exposition au glutamate, les astrocytes WT et -/- augmentent leur métabolisme en réponse à la demande accrue en énergie (ATP) due à l'entrée du glutamate. D'après l'Astrocyte Neuron Lactate Shuttle Hypothesis (ANLSH), le ratio entre le glucose entrant et le lactate sortant est de 1. Cependant le relargage du lactate dans les astrocytes PPARP-/- est plus élevé que l'entrée du glucose. L'apport supplémentaire de glucose transformé en lactate pourrait provenir de la dégradation des stocks de glycogène intracellulaire, qui sont partiellement diminués après stimulation dans les cellules PPARP -/-. Un métabolisme plus faible du glucose corrèle avec une réduction de l'import du glutamate dans les astrocytes PPARp -/-. Réciproquement, nous observons une augmentation de l'import du glutamate et de la production d'ATP après traitement avec l'agoniste pour PPARp. Bien que l'expression des transporteurs de glutamate ne soit pas affectée, nous ne pouvons pas exclure que leur localisation et leur structure soient altérées du fait du niveau élevé de cholestérol dans les astrocytes PPARp -/-.Le métabolisme, la localisation des transporteurs et le niveau de cholestérol sont tous liés au cytosquelette, à la mobilité, et à la composition des membranes cellulaires. Toutes ces fonctions sont importantes pour les astrocytes pour acquérir leur morphologie in vivo. Les astrocytes PPARP -/- présentent un défaut de stellation, aussi bien en présence de neurones que de stimuli chimiques, ainsi qu'un plus grand nombre de fibres de stress (actine) et de structures d'adhésion cellulaire. Bien que les astrocytes non stellaires soient principalement observés in vitro, le défaut de stellation des astrocytes primaires PPARp -/- indique une incapacité à répondre aux différents stimuli extérieurs. Ces phénotypes morphologiques corrèlent avec une migration plus lente en cas de lésion de la culture.Ce travail de thèse a permis de démontrer l'implication de PPARP dans le métabolisme du glucose des astrocytes corticaux. L'absence de ce récepteur nucléaire amène à l'utilisation du glucose intracellulaire, auquel s'ajoutent les effets sur l'import du glutamate et la migration des astrocytes. PPARp aurait des effets neuroprotecteurs, et de ce fait pourrait être utilisé à des fins thérapeutiques.

Hepatic stereology of schistosomiasis mansoni infected-mice fed a high-fat diet

High-fat diets induce weight gain and fatty liver in wild-type mice. Schistosomiasis mansoni infection also promotes hepatic injury. This study was designed to quantify hepatic alterations in schistosomiasis mansoni-infected mice fed a high fat-rich chow compared to mice fed a standard rodent chow, using stereology. Female SW mice fed each either high-fat diet (29% lipids) or standard chow (12% lipids) over 8 months, and then were infected with Schistosoma mansoni cercariae. Four experimental groups were studied: infected mice fed a high-fat diet (IHFC) or standard chow (ISC), uninfected mice fed a high-fat diet (HFC) or standard chow (SC). Mice were sacrificed during early infection (9 weeks from exposure). The following hepatic biometry and the stereology parameters were determined: volume density (hepatocytes [h], sinusoids [s], steatosis [st] and hepatic fibrosis [hf]); numerical density (hepatocyte nuclei - Nv[h]); absolute number of total hepatocyte N[h], normal hepatocyte N[nh], and binucleated hepatocyte N[bh], percentage of normal hepatocyte P[nh] and binucleated hepatocyte P[bh]. IHFC and HFC groups exhibited TC, HDL-C, LDL-C, and body mass significantly greater (p < 0.05) than control group. No significant differences were found regards liver volume (p = 0.07). Significant differences were observed regards P[nh] (p = 0.0045), P[bh] (p = 0.0045), Nv[h] (p = 0.0006), N[h] (p = 0.0125), N[bh] (p = 0.0164) and N[nh] (p = 0.0078). IHFC mice group presented 29% of binucleated hepatocytes compared to HFC group (19%), ISC group (17%) and SC (6%). Volume density was significantly different between groups: Vv[h] (p = 0.0052), Vv[s] (p = 0.0025), Vv[st] (p = 0.0004), and Vv[hf] (p = 0.0007). In conclusion, schistosomiasis mansoni infection with concurrent high-fat diet promotes intensive quantitative changes in hepatic structure, contributing to an increasing on hepatic regeneration.

Evaluation of phenotypic and genotypic alterations induced by long periods of subculturing of Cryptococcus neoformans strains

Cryptococcus neoformans is an encapsulated fungal organism that can cause disease in apparently immunocompetent, as well as immunocompromised, hosts. Since 1930, successive subculture has been used to preserve C. neoformans isolates in our Fungus Collection. In the 1970s, some of these Fungus Collection samples were selected to be subjected to a different methods of maintenance - that of lyophilized. Our objective was to analyze C. neoformans isolates in order to make a comparative evaluation between these two methods of preservation. The overall aim of this study was to qualify the preservation technique used in our mycology laboratory since the technique used might affect the survival, stability and purity of the primary isolates in culture. The samples were analyzed using classical mycology methods and using the randomly amplified polymorphic DNA technique In the analysis of phenotypes and genotypes, the typical characteristics of C. neoformans were found to differ in relation to the different methods of preservation employed. The aim of this study was to demonstrate the importance of selecting the appropriate method of preservation for fungus collections. This selection can affect the survival and purity of the cultures, and preserve the stability of their physiological, biochemical, and genetic characteristics.