Pharmacogenetics of OATP Transporters Reveals That SLCO1B1 c.388A > G Variant Is Determinant of Increased Atorvastatin Response

Aims: The relationship between variants in SLCO1B1 and SLCO2B1 genes and lipid-lowering response to atorvastatin was investigated. Material and Methods: One-hundred-thirty-six unrelated individuals with hypercholesterolemia were selected and treated with atorvastatin (10 mg/day/4 weeks). They were genotyped with a panel of ancestry informative markers for individual African component of ancestry (ACA) estimation by SNaPshot (R) and SLCO1B1 (c.388A>G, c.463C>A and c.521T>C) and SLCO2B1 (-71T>C) gene polymorphisms were identified by TaqMan (R) Real-time PCR. Results: Subjects carrying SLCO1B1 c.388GG genotype exhibited significantly high low-density lipoprotein (LDL) cholesterol reduction relative to c.388AA+c.388AG carriers (41 vs. 37%, p = 0.034). Haplotype analysis revealed that homozygous of SLCO1B1*15 (c.521C and c.388G) variant had similar response to statin relative to heterozygous and non-carriers. A multivariate logistic regression analysis confirmed that c.388GG genotype was associated with higher LDL cholesterol reduction in the study population (OR: 3.2, CI95%: 1.3-8.0, p < 0.05). Conclusion: SLCO1B1 c.388A>G polymorphism causes significant increase in atorvastatin response and may be an important marker for predicting efficacy of lipid-lowering therapy.

Adaptive Immunity against Leishmania Nucleoside Hydrolase Maps Its C-Terminal Domain as the Target of the CD4+ T Cell-Driven Protective Response

Nucleoside hydrolases (NHs) show homology among parasite protozoa, fungi and bacteria. They are vital protagonists in the establishment of early infection and, therefore, are excellent candidates for the pathogen recognition by adaptive immune responses. Immune protection against NHs would prevent disease at the early infection of several pathogens. We have identified the domain of the NH of L. donovani (NH36) responsible for its immunogenicity and protective efficacy against murine visceral leishmaniasis (VL). Using recombinant generated peptides covering the whole NH36 sequence and saponin we demonstrate that protection against L. chagasi is related to its C-terminal domain (amino-acids 199-314) and is mediated mainly by a CD4+ T cell driven response with a lower contribution of CD8+ T cells. Immunization with this peptide exceeds in 36.73 +/- 12.33% the protective response induced by the cognate NH36 protein. Increases in IgM, IgG2a, IgG1 and IgG2b antibodies, CD4+ T cell proportions, IFN-gamma secretion, ratios of IFN-gamma/IL-10 producing CD4+ and CD8+ T cells and percents of antibody binding inhibition by synthetic predicted epitopes were detected in F3 vaccinated mice. The increases in DTH and in ratios of TNF alpha/IL-10 CD4+ producing cells were however the strong correlates of protection which was confirmed by in vivo depletion with monoclonal antibodies, algorithm predicted CD4 and CD8 epitopes and a pronounced decrease in parasite load (90.5-88.23%; p = 0.011) that was long-lasting. No decrease in parasite load was detected after vaccination with the N-domain of NH36, in spite of the induction of IFN-gamma/IL-10 expression by CD4+ T cells after challenge. Both peptides reduced the size of footpad lesions, but only the C-domain reduced the parasite load of mice challenged with L. amazonensis. The identification of the target of the immune response to NH36 represents a basis for the rationale development of a bivalent vaccine against leishmaniasis and for multivalent vaccines against NHs-dependent pathogens.

Transcriptional profiling reveals the expression of novel genes in response to various stimuli in the human dermatophyte Trichophyton rubrum

Background: Cutaneous mycoses are common human infections among healthy and immunocompromised hosts, and the anthropophilic fungus Trichophyton rubrum is the most prevalent microorganism isolated from such clinical cases worldwide. The aim of this study was to determine the transcriptional profile of T. rubrum exposed to various stimuli in order to obtain insights into the responses of this pathogen to different environmental challenges. Therefore, we generated an expressed sequence tag (EST) collection by constructing one cDNA library and nine suppression subtractive hybridization libraries. Results: The 1388 unigenes identified in this study were functionally classified based on the Munich Information Center for Protein Sequences (MIPS) categories. The identified proteins were involved in transcriptional regulation, cellular defense and stress, protein degradation, signaling, transport, and secretion, among other functions. Analysis of these unigenes revealed 575 T. rubrum sequences that had not been previously deposited in public databases. Conclusion: In this study, we identified novel T. rubrum genes that will be useful for ORF prediction in genome sequencing and facilitating functional genome analysis. Annotation of these expressed genes revealed metabolic adaptations of T. rubrum to carbon sources, ambient pH shifts, and various antifungal drugs used in medical practice. Furthermore, challenging T. rubrum with cytotoxic drugs and ambient pH shifts extended our understanding of the molecular events possibly involved in the infectious process and resistance to antifungal drugs.

Proteasome Inhibition Represses Unfolded Protein Response and Nox4, Sensitizing Vascular Cells to Endoplasmic Reticulum Stress-Induced Death

Background: Endoplasmic reticulum (ER) stress has pathophysiological relevance in vascular diseases and merges with proteasome function. Proteasome inhibition induces cell stress and may have therapeutic implications. However, whether proteasome inhibition potentiates ER stress-induced apoptosis and the possible mechanisms involved in this process are unclear. Methodology/Principal Findings: Here we show that proteasome inhibition with MG132, per se at non-lethal levels, sensitized vascular smooth muscle cells to caspase-3 activation and cell death during ER stress induced by tunicamycin (Tn). This effect was accompanied by suppression of both proadaptive (KDEL chaperones) and proapoptotic (CHOP/GADD153) unfolded protein response markers, although, intriguingly, the splicing of XBP1 was markedly enhanced and sustained. In parallel, proteasome inhibition completely prevented ER stress-induced increase in NADPH oxidase activity, as well as increases in Nox4 isoform and protein disulfide isomerase mRNA expression. Increased Akt phosphorylation due to proteasome inhibition partially offset the proapoptotic effect of Tn or MG132. Although proteasome inhibition enhanced oxidative stress, reactive oxygen species scavenging had no net effect on sensitization to Tn or MG132-induced cell death. Conclusion/Relevance: These data indicate unfolded protein response-independent pathways whereby proteasome inhibition sensitizes vascular smooth muscle to ER stress-mediated cell death. This may be relevant to understand the therapeutic potential of such compounds in vascular disease associated with increased neointimal hyperplasia.

Differential regulation of PGC-1 alpha expression in rat liver and skeletal muscle in response to voluntary running

Background: The beneficial actions of exercise training on lipid, glucose and energy metabolism and insulin sensitivity appear to be in part mediated by PGC-1 alpha. Previous studies have shown that spontaneously exercised rats show at rest enhanced responsiveness to exogenous insulin, lower plasma insulin levels and increased skeletal muscle insulin sensitivity. This study was initiated to examine the functional interaction between exercise-induced modulation of skeletal muscle and liver PGC-1 alpha protein expression, whole body insulin sensitivity, and circulating FFA levels as a measure of whole body fatty acid (lipid) metabolism. Methods: Two groups of male Wistar rats (2 Mo of age, 188.82 +/- 2.77 g BW) were used in this study. One group consisted of control rats placed in standard laboratory cages. Exercising rats were housed individually in cages equipped with running wheels and allowed to run at their own pace for 5 weeks. At the end of exercise training, insulin sensitivity was evaluated by comparing steady-state plasma glucose (SSPG) concentrations at constant plasma insulin levels attained during the continuous infusion of glucose and insulin to each experimental group. Subsequently, soleus and plantaris muscle and liver samples were collected and quantified for PGC-1 alpha protein expression by Western blotting. Collected blood samples were analyzed for glucose, insulin and FFA concentrations. Results: Rats housed in the exercise wheel cages demonstrated almost linear increases in running activity with advancing time reaching to maximum value around 4 weeks. On an average, the rats ran a mean (Mean +/- SE) of 4.102 +/- 0.747 km/day and consumed significantly more food as compared to sedentary controls (P < 0.001) in order to meet their increased caloric requirement. Mean plasma insulin (P < 0.001) and FFA (P < 0.006) concentrations were lower in the exercise-trained rats as compared to sedentary controls. Mean steady state plasma insulin (SSPI) and glucose (SSPG) concentrations were not significantly different in sedentary control rats as compared to exercise-trained animals. Plantaris PGC-1 alpha protein expression increased significantly from a 1.11 +/- 0.12 in the sedentary rats to 1.74 +/- 0.09 in exercising rats (P < 0.001). However, exercise had no effect on PGC-1 alpha protein content in either soleus muscle or liver tissue. These results indicate that exercise training selectively up regulates the PGC-1 alpha protein expression in high-oxidative fast skeletal muscle type such as plantaris muscle. Conclusion: These data suggest that PGC-1 alpha most likely plays a restricted role in exercise-mediated improvements in insulin resistance (sensitivity) and lowering of circulating FFA levels.

Changes in cardiac heparan sulfate proteoglycan expression and streptozotocin-induced diastolic dysfunction in rats

Background: Changes in the proteoglycans glypican and syndecan-4 have been reported in several pathological conditions, but little is known about their expression in the heart during diabetes. The aim of this study was to investigate in vivo heart function changes and alterations in mRNA expression and protein levels of glypican-1 and syndecan-4 in cardiac and skeletal muscles during streptozotocin (STZ)-induced diabetes. Methods: Diabetes was induced in male Wistar rats by STZ administration. The rats were assigned to one of the following groups: control (sham injection), after 24 hours, 10 days, or 30 days of STZ administration. Echocardiography was performed in the control and STZ 10-day groups. Western and Northern blots were used to quantify protein and mRNA levels in all groups. Immunohistochemistry was performed in the control and 30-day groups to correlate the observed mRNA changes to the protein expression. Results: In vivo cardiac functional analysis performed using echocardiography in the 10-day group showed diastolic dysfunction with alterations in the peak velocity of early (E) diastolic filling and isovolumic relaxation time (IVRT) indices. These functional alterations observed in the STZ 10-day group correlated with the concomitant increase in syndecan-4 and glypican-1 protein expression. Cardiac glypican-1 mRNA and skeletal syndecan-4 mRNA and protein levels increased in the STZ 30-day group. On the other hand, the amount of glypican in skeletal muscle was lower than that in the control group. The same results were obtained from immunohistochemistry analysis. Conclusion: Our data suggest that membrane proteoglycans participate in the sequence of events triggered by diabetes and inflicted on cardiac and skeletal muscles.

Lack of Galectin-3 Drives Response to Paracoccidioides brasiliensis toward a Th2-Biased Immunity

There is recent evidence that galectin-3 participates in immunity to infections, mostly by tuning cytokine production. We studied the balance of Th1/Th2 responses to P. brasiliensis experimental infection in the absence of galectin-3. The intermediate resistance to the fungal infection presented by C57BL/6 mice, associated with the development of a mixed type of immunity, was replaced with susceptibility to infection and a Th2-polarized immune response, in galectin-3-deficient (gal3(-/-)) mice. Such a response was associated with defective inflammatory and delayed type hypersensitivity (DTH) reactions, high IL-4 and GATA-3 expression and low nitric oxide production in the organs of infected animals. Gal3(-/-) macrophages exhibited higher TLR2 transcript levels and IL-10 production compared to wild-type macrophages after stimulation with P. brasiliensis antigens. We hypothesize that, during an in vivo P. brasiliensis infection, galectin-3 exerts its tuning role on immunity by interfering with the generation of regulatory macrophages, thus hindering the consequent Th2-polarized type of response.

Transmission potential, skin inflammatory response, and parasitism of symptomatic and asymptomatic dogs with visceral leishmaniasis

Background: Visceral leishmaniasis in Brazil is caused by the protozoan Leishmania (Leishmania) chagasi and it is transmitted by sandfly of the genus Lutzomyia. Dogs are an important domestic reservoir, and control of the transmission of visceral leishmaniasis (VL) to humans includes the elimination of infected dogs. However, though dogs are considered to be an important element in the transmission cycle of Leishmania, the identification of infected dogs representing an immediate risk for transmission has not been properly evaluated. Since it is not possible to treat infected dogs, they are sacrificed when a diagnosis of VL is established, a measure that is difficult to accomplish in highly endemic areas. In such areas, parameters that allow for easy identification of reservoirs that represents an immediate risk for transmission is of great importance for the control of VL transmission. In this study we aimed to identify clinical parameters, reinforced by pathological parameters that characterize dogs with potential to transmit the parasite to the vector. Results: The major clinical manifestations of visceral leishmaniasis in dogs from an endemic area were onicogriphosis, skin lesions, conjunctivitis, lymphadenopathy, and weight loss. The transmission potential of these dogs was assessed by xenodiagnosis using Lutzomyia longipalpis. Six of nine symptomatic dogs were infective to Lutzomyia longipalpis while none of the five asymptomatic dogs were infective to the sandfly. Leishmania amastigotes were present in the skin of all clinically symptomatic dogs, but absent in asymptomatic dogs. Higher parasite loads were observed in the ear and ungueal region, and lower in abdomen. The inflammatory infiltrate was more intense in the ears and ungueal regions of both symptomatic and asymptomatic dogs. In clinically affected dogs in which few or none Leishmania amastigotes were observed, the inflammatory infiltrate was constituted mainly of lymphocytes and macrophages. When many parasites were present, the infiltrate was also comprised of lymphocytes and macrophages, as well as a larger quantity of polymorphonuclear neutrophils (PMNs). Conclusion: Dogs that represent an immediate risk for transmission of Leishmania in endemic areas present clinical manifestations that include onicogriphosis, skin lesions, conjunctivitis, lymphadenopathy, and weight loss. Lymphadenopathy in particular was a positive clinical hallmark since it was closely related to the positive xenodiagnosis.

Coactivation of the shoulder and arm muscles during closed kinetic chain exercises on an unstable surface

Introduction: The purpose of this study was to compare the electromyography index of muscle coactivation of the following muscle pairs: posterior deltoid and pectoralis major (PD/PM); triceps brachii and biceps brachii (TB/BB); and serratus anterior and upper trapezius (SA/UT) during three different closed kinetic chain exercises (wall-press, bench-press and push-up) on an unstable surface at the maximal load. Methods: A total of 20 healthy sedentary men participated in the study. Integral linear values were obtained from three sustained contractions of six seconds each for the three proposed exercises. Mean coactivation index values were compared using the mixed-effects linear model, with a five percent significance level. Results: Electromyography indexes of muscle coactivation showed significant differences for the PD/PM and TB/BB muscle pairs. No differences were found between exercises for the SA/UT muscle pair. Conclusion: Our results seem to differ from those of previous studies, which reported that the similarity in exercises performed is responsible for the comparable muscle activation levels.

Genome Survey Sequence Analysis and Identification of Homologs of Major Surface Protease (gp63) Genes in Trypanosoma rangeli

In this study, 222 genome survey sequences were generated for Trypanosoma rangeli strain P07 isolated from an opossum (Didelphis albiventris) in Minas Gerais State, Brazil. T. rangeli sequences were compared by BLASTX (Basic Local Alignment Search Tool X) analysis with the assembled contigs of Leishmania braziliensis, Leishmania infantum, Leishmania major, Trypanosoma brucei, and Trypanosoma cruzi. Results revealed that 82% (182/222) of the sequences were associated with predicted proteins described, whereas 18% (40/222) of the sequences did not show significant identity with sequences deposited in databases, suggesting that they may represent T. rangeli-specific sequences. Among the 182 predicted sequences, 179 (80.6%) had the highest similarity with T. cruzi, 2 (0.9%) with T. brucei, and 1 (0.5%) with L. braziliensis. Computer analysis permitted the identification of members of various gene families described for trypanosomatids in the genome of T. rangeli, such as trans-sialidases, mucin-associated surface proteins, and major surface proteases (MSP or gp63). This is the first report identifying sequences of the MSP family in T. rangeli. Multiple sequence alignments showed that the predicted MSP of T. rangeli presented the typical characteristics of metalloproteases, such as the presence of the HEXXH motif, which corresponds to a region previously associated with the catalytic site of the enzyme, and various cysteine and proline residues, which are conserved among MSPs of different trypanosomatid species. Reverse transcriptase-polymerase chain reaction analysis revealed the presence of MSP transcripts in epimastigote forms of T. rangeli.

Determination of Threshold Dose of Photodynamic Therapy to Measure Superficial Necrosis

Background Data: Photodynamic therapy (PDT) involves the photoinduction of cytotoxicity using a photosensitizer agent, a light source of the proper wavelength, and the presence of molecular oxygen. A model for tissue response to PDT based on the photodynamic threshold dose (Dth) has been widely used. In this model cells exposed to doses below Dth survive while at doses above the Dth necrosis takes place. Objective: This study evaluated the light Dth values by using two different methods of determination. One model concerns the depth of necrosis and the other the width of superficial necrosis. Materials and Methods: Using normal rat liver we investigated the depth and width of necrosis induced by PDT when a laser with a gaussian intensity profile is used. Different light doses, photosensitizers (Photogem, Photofrin, Photosan, Foscan, Photodithazine, and Radachlorin), and concentrations were employed. Each experiment was performed on five animals and the average and standard deviations were calculated. Results: A simple depth and width of necrosis model analysis allows us to determine the threshold dose by measuring both depth and surface data. Comparison shows that both measurements provide the same value within the degree of experimental error. Conclusion: This work demonstrates that by knowing the extent of the superficial necrotic area of a target tissue irradiated by a gaussian light beam, it is possible to estimate the threshold dose. This technique may find application where the determination of Dth must be done without cutting the tissue.

Contralateral cortical response of a subpopulation of interneurons and the glial glutamate transporter GLT1 to an ischemic core

Introduction: Cerebral ischemia is an important cause of brain lesion in humans. The target in research has been the ischemic core or the penumbra zones; little attention has been given to areas outside the core or the penumbra but connected with the primary site of injury. Objective: Evaluate the laminar response of a subpopulation of gabaergic cells, those that are parvalbumin (PV) positive and the astrocytes through the expression of the glial transporter GLT1 on the contralateral cortex to an ischemic core. Methodology: For this purpose we used the medial cerebral artery occlusion model in rats. The artery was occluded for 90 minutes and the animals were sacrificed at 24 and 72 hours post-ischemia. The brains were removed, cut in a vibratome at 50 microns and incubated with the primary antibodies against PV or GLT1. Sections were developed using the vectastain Kit. In control tissue the primary antibody was omitted. Results: When compared with control animals, treated ones show a decrease in the expression of GLT1, especially in layers III and IV of the contralateral cortex to the ischemic core. PV positive cells increases in layers II and V. Conclusion: Increases in the expression of PV cells could correspond to an adaptation associated with glutamate increases in the synaptic compartment. These increases may be due to decreases in the expression of GLT1 transporter, that could not remove the glutamate present in the synaptic cleft, generating hyperactivity in the contralateral cortex. These changes could represent an example of neuronal and glial plasticity in remote areas to an ischemic core but connected to the primary site of injury.

In Vitro Adhesion of Streptococcus sanguinis to Dentine Root Surface After Treatment with Er:Yag Laser, Ultrasonic System, or Manual Curette

Objective: The purpose of this in vitro study was to evaluate the dentine root surface roughness and the adherence of Streptococcus sanguinis (ATCC 10556) after treatment with an ultrasonic system, Er:YAG laser, or manual curette. Background Data: Bacterial adhesion and formation of dental biofilm after scaling and root planing may be a challenge to the long-term stability of periodontal therapy. Materials and Methods: Forty flattened bovine roots were randomly assigned to one of the following groups: ultrasonic system (n = 10); Er:YAG laser (n = 10); manual curette (n = 10); or control untreated roots (n = 10). The mean surface roughness (Ra, mu m) of the specimens before and after exposure to each treatment was determined using a surface profilometer. In addition, S. sanguinis was grown on the treated and untreated specimens and the amounts of retained bacteria on the surfaces were measured by culture method. Results: All treatments increased the Ra; however, the roughest surface was produced by the curettes. In addition, the specimens treated with curettes showed the highest S. sanguinis adhesion. There was a significant positive correlation between roughness values and bacterial cells counts. Conclusion: S. sanguinis adhesion was the highest on the curette-treated dentine root surfaces, which also presented the greatest surface roughness.

Adhesion of Endodontic Sealers to Human Root Dentine Submitted to Different Surface Treatments

Objective: To evaluate the adhesion of the endodontic sealers Epiphany, Apexit Plus, and AH Plus to root canal dentin submitted to different surface treatments, by using the push-out test. Methods: One hundred twenty-eight root cylinders obtained from maxillary canines were embedded in acrylic resin, had the canals prepared, and were randomly assigned to four groups (n = 32), according to root dentin treatment: (I) distilled water (control), (II) 17% EDTAC, (III) 1% NaOCl and (IV) Er:YAG laser with 16-Hz, 400-mJ input (240-mJ output) and 0.32-J/cm(2) energy density. Each group was divided into four subgroups (n = 8) filled with Epiphany (either dispensed from the automix syringe supplied by the manufacturer or prepared by hand mixing), Apexit Plus, or AH Plus. Data (MPa) were analyzed by ANOVA and Tukey's test. Results: A statistically significant difference (p < 0.01) was found among the root-canal sealers, except for the Epiphany subgroups, which had statistically similar results to each other (p > 0.01): AH Plus (4.77 +/- 0.85), Epiphany/hand mixed (3.06 +/- 1.34), Epiphany/automix syringe (2.68 +/- 1.35), and Apexit Plus (1.22 +/- 0.33). A significant difference (p < 0.01) was found among the dentin surface treatments. The highest adhesion values were obtained with AH Plus when root dentin was treated with Er: YAG laser and 17% EDTAC. Epiphany sealer presented the lowest adhesion values to root dentin treated with 17% EDTAC. Conclusions: The resin-based sealers had different adhesive behaviors, depending on the treatment of root canal walls. The mode of preparation of Epiphany (automix syringe or hand mixing) did not influence sealer adhesion to root dentin.

Observational Characterization of the Downward Atmospheric Longwave Radiation at the Surface in the City of Sao Paulo

This work describes the seasonal and diurnal variations of downward longwave atmospheric irradiance (LW) at the surface in Sao Paulo, Brazil, using 5-min-averaged values of LW, air temperature, relative humidity, and solar radiation observed continuously and simultaneously from 1997 to 2006 on a micrometeorological platform, located at the top of a 4-story building. An objective procedure, including 2-step filtering and dome emission effect correction, was used to evaluate the quality of the 9-yr-long LW dataset. The comparison between LW values observed and yielded by the Surface Radiation Budget project shows spatial and temporal agreement, indicating that monthly and annual average values of LW observed in one point of Sao Paulo can be used as representative of the entire metropolitan region of Sao Paulo. The maximum monthly averaged value of the LW is observed during summer (389 +/- 14 W m(-2): January), and the minimum is observed during winter (332 +/- 12 W m(-2); July). The effective emissivity follows the LW and shows a maximum in summer (0.907 +/- 0.032; January) and a minimum in winter (0.818 +/- 0.029; June). The mean cloud effect, identified objectively by comparing the monthly averaged values of the LW during clear-sky days and all-sky conditions, intensified the monthly average LW by about 32.0 +/- 3.5 W m(-2) and the atmospheric effective emissivity by about 0.088 +/- 0.024. In August, the driest month of the year in Sao Paulo, the diurnal evolution of the LW shows a minimum (325 +/- 11 W m(-2)) at 0900 LT and a maximum (345 12 W m-2) at 1800 LT, which lags behind (by 4 h) the maximum diurnal variation of the screen temperature. The diurnal evolution of effective emissivity shows a minimum (0.781 +/- 0.027) during daytime and a maximum (0.842 +/- 0.030) during nighttime. The diurnal evolution of all-sky condition and clear-sky day differences in the effective emissivity remain relatively constant (7% +/- 1%), indicating that clouds do not change the emissivity diurnal pattern. The relationship between effective emissivity and screen air temperature and between effective emissivity and water vapor is complex. During the night, when the planetary boundary layer is shallower, the effective emissivity can be estimated by screen parameters. During the day, the relationship between effective emissivity and screen parameters varies from place to place and depends on the planetary boundary layer process. Because the empirical expressions do not contain enough information about the diurnal variation of the vertical stratification of air temperature and moisture in Sao Paulo, they are likely to fail in reproducing the diurnal variation of the surface emissivity. The most accurate way to estimate the LW for clear-sky conditions in Sao Paulo is to use an expression derived from a purely empirical approach.