INCREASED SARCOLEMMAL PERMEABILITY AS AN EARLY EVENT IN EXPERIMENTAL SEPTIC CARDIOMYOPATHY: A POTENTIAL ROLE FOR OXIDATIVE DAMAGE TO LIPIDS AND PROTEINS

This study describes increased sarcolemmal permeability and myofilamentar damage that occur together with lipid peroxidation and protein nitration in the myocardium in severe sepsis induced by cecal ligation and puncture. Male C57BL/6 mice were submitted to moderate and severe septic injury and sham operation. Using light and laser confocal microscopy, diffuse foci of myocytolysis associated with focal disruption of the actin/myosin contractile apparatus could be seen in hearts with severe septic injury. The myocardial expressions of the sarcomeric proteins myosin and actin were downregulated by both severe and moderate injuries. The detection of albumin staining in the cytoplasm of myocytes to evaluate sarcolemmal permeability provided evidence of severe and mild injury of the plasma membrane in hearts with severe and moderate septic injury, respectively. The administration of a superoxide scavenger caused marked reduction of sarcolemmal permeability, indicating the involvement of free radicals in its genesis. On electron microscopy, these changes were seen to correspond to spread blocks of a few myocytes with fragmentation and dissolution of myofibrils, intracellular edema, and, occasionally, rupture of the sarcolemma. In addition, oxidative damage to lipids, using anti-4-hydroxynonenal, an indicator of oxidative stress and disruption of plasma membrane lipids, and to proteins, using antinitrotyrosine, a stable biomarker of peroxynitrite-mediated protein nitration, was demonstrated. These findings make plausible the hypothesis that increased sarcolemmal permeability might be a primary event in myocardial injury in severe sepsis possibly due to oxidative damage to lipids and proteins that could precede phenotypic changes that characterize a septic cardiomyopathy.

Detection of Auditory Cortex Activity by fMRI Using a Dependent Component Analysis

Functional MRI (fMRI) data often have low signal-to-noise-ratio (SNR) and are contaminated by strong interference from other physiological sources. A promising tool for extracting signals, even under low SNR conditions, is blind source separation (BSS), or independent component analysis (ICA). BSS is based on the assumption that the detected signals are a mixture of a number of independent source signals that are linearly combined via an unknown mixing matrix. BSS seeks to determine the mixing matrix to recover the source signals based on principles of statistical independence. In most cases, extraction of all sources is unnecessary; instead, a priori information can be applied to extract only the signal of interest. Herein we propose an algorithm based on a variation of ICA, called Dependent Component Analysis (DCA), where the signal of interest is extracted using a time delay obtained from an autocorrelation analysis. We applied such method to inspect functional Magnetic Resonance Imaging (fMRI) data, aiming to find the hemodynamic response that follows neuronal activation from an auditory stimulation, in human subjects. The method localized a significant signal modulation in cortical regions corresponding to the primary auditory cortex. The results obtained by DCA were also compared to those of the General Linear Model (GLM), which is the most widely used method to analyze fMRI datasets.

Tactile threshold detection in leprosy patients with an electronic algometer

Objective: To propose an electronic method for sensitivity evaluation in leprosy and to compare it to the Semmes-Weinstein monofilaments. Methods:Thirty patients attending the Dermatology outpatient clinic of HCFMRP-USP were consecutively evaluated by both the electronic aesthesiometer and Semmes-Weinstein monofilaments on hand and foot test points. The intraclass correlation coefficient (ICC) was calculated to determine the variability of the electronic measures and the Kappa coefficient was calculated to determine the agreement between methods according to their categories (altered and non-altered tactile sensitivity). Results: The ICC was approximately 1, demonstrating repeatability. The Kappa coefficient showed more than 75 and 63% agreement on the hand and foot points, respectively. The mean agreement between the 2 methods for the 7 points of the right and left hand was 77.14 and 75.71%, respectively. The mean agreement for all 10 points was 74.33 and 63.66% on the right and left foot, respectively. In cases of disagreement the detection of altered tactile sensitivity by the electronic esthesiometer on the right and left foot was 90.91 and 84.25%, respectively, with no detection by the monofilaments. Conclusion: The results suggest that the electronic esthesiometer is a reliable and easy application, capable of evaluating alterations of tactile sensitivity in leprosy patients. (C) 2009 Elsevier B.V. All rights reserved.

Is pain relief equally efficacious and free of side effects with repeated doses of oral sucrose in preterm neonates?

The aim of the present study was to examine the efficacy and potential side effects of repeated doses of oral sucrose for pain relief during procedures in NICU. Thirty-three preterm neonates were randomly allocated in blind fashion into two groups, the sucrose group (SG = 17) and the control group (CG = 16). The responses of neonates to pain and distress were assessed during blood collection on four consecutive assessment (ass.) days. For the first assessment, the neonates did not receive any solution before the blood collection procedure. During the next three days, the SG received oral sucrose (25%; 0.5 ml/kg) and the CG received sterile water, 2 min before each minor acute painful procedure. The neonates were evaluated during blood collection each morning. The assessment was divided into five phases: Baseline (BL), Antisepsis (A), Puncture (P), Dressing (D), and Recovery (R). The neonates` facial activity (NFCS), behavioral state, and heart rate were evaluated. The data analysis used cut-off scores for painful and distressful responses. No side effects of using sucrose were detected. There were significantly fewer SG neonates with facial actions signaling pain than CG neonates in P (ass.2) and in A (ass.3). We found significantly fewer SG neonates in the awake state than CG neonates in P (ass.2 and ass.4). There were significantly fewer SG neonates crying during A (ass.2), P (ass.2 and ass.4), and D (ass.3). There was no statistical difference between-groups for physiological response. The efficacy of sucrose was maintained for pain relief in preterm neonates with no side effects. (C) 2007 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

Articular Cartilage in the Knee: Current MR Imaging Techniques and Applications in Clinical Practice and Research

Magnetic resonance (MR) imaging is the most important imaging modality for the evaluation of traumatic or degenerative cartilaginous lesions in the knee. It is a powerful noninvasive tool for detecting such lesions and monitoring the effects of pharmacologic and surgical therapy. The specific MR imaging techniques used for these purposes can be divided into two broad categories according to their usefulness for morphologic or compositional evaluation. To assess the structure of knee cartilage, standard spin-echo (SE) and gradient-recalled echo (GRE) sequences, fast SE sequences, and three-dimensional SE and GRE sequences are available. These techniques allow the detection of morphologic defects in the articular cartilage of the knee and are commonly used in research for semiquantitative and quantitative assessments of cartilage. To evaluate the collagen network and proteoglycan content in the knee cartilage matrix, compositional assessment techniques such as T2 mapping, delayed gadolinium-enhanced MR imaging of cartilage (or dGEMRIC), T1 rho imaging, sodium imaging, and diffusion-weighted imaging are available. These techniques may be used in various combinations and at various magnetic field strengths in clinical and research settings to improve the characterization of changes in cartilage. (C)RSNA, 2011 , radiographics.rsna.org

Subchondral Cystlike Lesions Develop Longitudinally in Areas of Bone Marrow Edema-like Lesions in Patients with or at Risk for Knee Osteoarthritis: Detection with MR Imaging-The MOST Study

Purpose: To assess the association of prevalent bone marrow edema-like lesions (BMLs) and full-thickness cartilage loss with incident subchondral cyst-like lesions (SCs) in the knee to evaluate the bone contusion versus synovial fluid intrusion theories of SC formation. Materials and Methods: The Multicenter Osteoarthritis study is a longitudinal study of individuals who have or are at risk for knee osteoarthritis. The HIPAA-compliant protocol was approved by the institutional review boards of all participating centers, and written informed consent was obtained from all participants. Magnetic resonance images were acquired at baseline and 30-month follow-up and read semiquantitatively by using the Whole-Organ Magnetic Resonance Imaging Score system. The tibiofemoral and patellofemoral joints were subdivided into 14 subregions. BMLs and SCs were scored from 0 to 3. Cartilage morphology was scored from 0 to 6. The association of prevalent BMLs and full-thickness cartilage loss with incident SCs in the same subregion was assessed by using logistic regression with mutual adjustment for both predictors. Results: A total of 1283 knees were included. After adjustment for full-thickness cartilage loss, prevalent BMLs showed a strong and significant association with incident SCs in the same subregion, with an odds ratio of 12.9 (95% confidence interval [CI]: 8.9, 18.6). After adjustment for BMLs, prevalent full-thickness cartilage loss showed a significant but much less important association with incident SCs in the same subregion (odds ratio, 1.4; 95% CI: 1.0, 2.0). There was no apparent relationship between severity of full-thickness cartilage loss at baseline and incident SCs. Conclusion: Prevalent BMLs strongly predict incident SCs in the same subregion, even after adjustment for full-thickness cartilage loss, which supports the bone contusion theory of SC formation. (C) RSNA, 2010

Ecological aspects of the free-living ticks (Acari: Ixodidae) on animal trails within Atlantic rainforest in south-eastern Brazil

In a recent ecological study of the ticks on animal trails within an area of Atlantic rainforest in south-eastern Brazil, Amblyomma aureolatum, A. brasiliense, A. incisum, A. ovale and Haemaphysalis juxtakochi were found questing on the vegetation. Most of the ticks recorded by a small, man-made dam on the forest border were A. dubitatum but a few A. brasiliense and A. cajennense, one A. incisum and one H. juxtakochi were also found. The seasonal activity of the ticks indicated that A. incisum and A. brasiliense had one generation/year. On the animal trails, most tick species and stages quested on the vegetation at a height of 30-40 cm above ground level. The questing larvae and adults of A. incisum tended to be found higher, however, with the greatest numbers recorded 40-50 cm (larvae) or 60-70 cm (adults) above ground level. Most of the adult ticks (81.1% -100%), nymphs (78.6%-100%) and larval clusters (100%) found on a forest trail remained questing at the same location over a 24-h period. Carbon-dioxide traps in the rainforest attracted, 50% of the ticks observed questing on the nearby vegetation and, curiously, the CO(2) traps set deep in the forest attracted far fewer ticks than similar traps set by the dam. The ecological relationships between the ticks, their hosts and the rainforest environment are discussed.

The effect of temperature and fasting period on the viability of free-living females of Rhipicephalus sanguineus (Acari : Ixodidae) under laboratory conditions

Little is known about the effect of temperature on viability of free-living phases of the life cycle of Rhipicephalus sanguineus (Latreille, 1806) despite of its importance as vector of several pathogens. Knowledge of the effect of abiotic factors on the capacity of a given tick species to infest new hosts is important for routine experimental activities under laboratory conditions, and may be relevant to understand the transmission of pathogens. The study evaluates the viability of R. sanguineus females held at 18 +/- 1, 27 +/- 1 and 32 +/- 1 degrees C and 80 +/- 5% RH (saturation deficits of 3.0, 5.3 and 7.2 mmHg, respectively) for three fasting periods (3 and 20 days and the day when female mortality reached approximately 50% after ecdysis), under laboratory conditions. In general, the best result on viability was obtained when rabbits were infested with unfed female ticks after three or 20 fasting days at both 27 +/- 1 and 32 +/- 1 degrees C and 80 +/- 5% RH.

Detection of Leptospira spp. in semen and vaginal fluids of goats and sheep by polymerase chain reaction

Thirteen goat herds and seven sheep flocks in the state of Rio de Janeiro, Brazil were screened for leptospirosis. From the three herds and three flocks with greatest seroreactivity, 19 goats (16 females and three bucks) and 40 sheep (26 ewes and 14 rams) that were seropositive (specific anti-Leptospira titres >= 400, based on a microscopic agglutination test), were selected for more detailed studies. From those animals, samples of vaginal fluids or semen were collected for bacteriological and molecular assays. For both species of animals, the most prevalent reactions were to serovars Hardjo, Shermani, and Grippotyphosa. Although leptospires were detected by darkfield microscopy in three vaginal fluid samples (from two goats and one ewe), pure isolates were not obtained by bacteriological culture of vaginal fluids or semen. However, seven vaginal fluid samples (from four goats and three ewes) and six semen samples (all from rams) were positive on polymerase chain reaction (PCR). Based on these findings, in addition to analogous findings in cattle, we inferred that there is potential for venereal transmission of leptospirosis in small ruminants. (c) 2008 Elsevier Inc. All rights reserved.

Evaluation of IFA, MAT, ELISAs and immunoblotting for the detection of anti-Toxoplasma gondii antibodies in paired serum and aqueous humour samples from experimentally infected pigs

The study evaluated the efficiency of diagnostic laboratory methods to detect anti-Toxoplasma gondii antibodies in paired serum and aqueous humour samples from experimentally infected pigs. 18-mixed breed pigs were used during the experiment; these were divided into two groups, G I (infected group, n = 10) and G2 (uninfected group, n = 8). Infection was performed with 4 x 10(4) VEG strain oocysts at day 0 by the oral route in G1 animals. All pigs were euthanized at day 60, when retina, aqueous humour, and blood samples were collected. Anti-T gondii antibody levels were assessed in serum (s) and aqueous humour (ah) by indirect immunofluorescence assay (IFA), modified agglutination test (MAT), m-ELISA (using crude membranes from T gondii tachyzoites as antigen) and r-ELISA (using rhoptries from T gondii tachyzoites as antigen). Polymerase chain reactions (PCR) of samples from the retina were performed by using Tox4 and Tox5 primers. Antibody titers of G1 animals ranged from 128 to 1024 and from 16 to 256 in serum and aqueous humour, respectively. There were differences in the correlation coefficients between IFA(s) x IFA (ah) (r = 0.62, P = 0.05), MAT(s) x MAT (ah) (r = 0.97, P < 0.0001); however, there was no significant difference between r-ELISA(s) x r-ELISA (ah) (r = 0. 14, P = 0.7). Antibodies present in serum and aqueous humour recognized similar antigens. Samples of retina were positive by PCR in 30% (3/10) of infected pigs. G2 animals remained without antibody levels and were PCR negative throughout the experiment. These results suggest that the use of a combination of tests and immunoblotting for paired aqueous humour and serum samples could improve the sensitivity and specificity for the diagnosis of ocular toxoplasmosis. (c) 2007 Elsevier Ltd. All rights reserved.

Genotyping of Toxoplasma gondii isolates from free range chickens in the Pantanal area of Brazil

The aim of this paper was to genetically characterize Toxoplasma gondii isolates from free range chickens in regions of Brazilian territory in the state of Mato Grosso do Sul (MS) where T. gondii strains have never been studied. In total, T. gondii isolates from 22 free range chickens were included in this study. Fifty chickens from Eldorado, thirty from Rio Verde and ten from Aquidauana were sampled between January and April 2007. In relation to the genetic diversity of T. gondii isolates from chickens in MS, the magnitude of the diversity in the isolates sampled in this study was comparable to the overall diversity in a composite data set. These 22 isolates in MS revealed 11 genotypes, whereas the 321 isolates ever genotyped in Brazil have revealed 95 genotypes. The values of Simpson`s Diversity Index for the whole population of T. gondii isolates in Brazil, the whole population of T. gondii isolates from chickens in Brazil and the population surveyed in this study were 0.97, 0.95 and 0.90, respectively. Seven of the 11 genotypes revealed from chicken isolates from MS are newly described genotypes and six of them each have a single isolate. In conclusion, the results obtained from isolates in MS corroborate previous studies on T. gondii isolates in Brazil, thus confirming their diversity and atypicality. Nonetheless, the applicability of PCR-RFLP markers for epidemiological inferences remains controversial. (c) 2011 Elsevier B.V. All rights reserved.

Multiplex semi-nested RT-PCR with exogenous internal control for simultaneous detection of bovine coronavirus and group A rotavirus

Neonatal calf diarrhea is a multi-etiology syndrome of cattle and direct detection of the two major agents of the syndrome, group A rotavirus and Bovine coronavirus (BCoV) is hampered by their fastidious growth in cell culture. This study aimed at developing a multiplex semi-nested RT-PCR for simultaneous detection of BCoV (N gene) and group A rotavirus (VP1 gene) with the addition of an internal control (mRNA ND5). The assay was tested in 75 bovine feces samples tested previously for rotavirus using PAGE and for BCoV using nested RT-PCR targeted to RdRp gene. Agreement with reference tests was optimal for BCoV (kappa = 0.833) and substantial for rotavirus detection (kappa = 0.648). the internal control, ND5 mRNA, was detected successfully in all reactions. Results demonstrated that this multiplex semi-nested RT-PCR was effective in the detection of BCoV and rotavirus, with high sensitivity and specificity for simultaneous detection of both viruses at a lower cost, providing an important tool for studies on the etiology of diarrhea in cattle. (C) 2010 Elsevier B.V. All rights reserved.

A method for simultaneous detection and identification of Brazilian dog- and vampire bat-related rabies virus by reverse transcription loop-mediated isothermal amplification assay

At present, the sporadic occurrence of human rabies in Brazil can be attributed primarily to dog- and vampire bat-related rabies viruses. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) was employed as a simultaneous detection method for both rabies field variants within 60 min. Vampire bat-related rabies viruses could be distinguished from dog variants by digesting amplicons of the RT-LAMP reaction using the restriction enzyme Alwl. Amplification and digestion could both be completed within 120 min after RNA extraction. In addition, the RI-LAMP assay also detected rabies virus in isolates from Brazilian frugivorous bats and Ugandan dog, bovine and goat samples. In contrast, there were false negative results from several Brazilian insectivorous bats and all of Chinese dog, pig, and bovine samples using the RI-LAMP assay. This study showed that the RT-LAMP assay is effective for the rapid detection of rabies virus isolates from the primary reservoir in Brazil. Further improvements are necessary so that the RT-LAMP assay can be employed for the universal detection of genetic variants of rabies virus in the field. (C) 2010 Elsevier B.V. All rights reserved.

Detection and molecular characterization of a canine piroplasm from Brazil

In the beginning of the 20th century, a new canine disease was reported in Brazil under the name ""nambiuvu"", whose etiological agent was called Rangelia vitalii, a distinct piroplasm that was shown to parasitize not only erythrocytes, but also leucocytes and endothelial cells. In this new century, more publications on R. vitalii were reported from Brazil, including an extensive study on its ultrastructural analysis, in addition to clinical, pathological, and epidemiological data on nambiuvu. However, a molecular analysis of R. vitalii has not been performed to date. In the present study, we performed molecular phylogenetic analyses of R. vitalii based on fragments of the genes 18S rRNA and the heat shock protein 70 (hsp70), amplified by PCR performed on blood samples derived from five clinical cases of dogs presumably infected with R. vitalii in southern Brazil. In addition, we examined Giemsa-stained thin blood smears from these same dogs. DNA sequences (604-bp) of the 18S rRNA gene obtained from the five dogs were identical to each other, and by Blast analysis, this sequence shared the highest degree of sequence identity (95%) with Babesia sp. China-BQ1. DNA sequences (1056-bp) of the hsp70 gene obtained from the five dogs were identical to each other, and by Blast analysis, this sequence shared the highest degree of sequence identity (87%) with Babesia bigemina. Phylogenetic analyses inferred from either of the two genes resulted in the newly genotype being placed in the Babesia spp.sensu stricto clade with very high bootstrap support (95-100%) in three analyses (Neighbor-Joining, Maximum parsimony, and Maximum likelihood). Giemsa-stained thin blood smears from the dogs were shown to contain piroplasm organisms within erythrocytes, monocytes and neutrophils (individual forms), and schizont-like forms within neutrophils, in accordance with literature reports of R. vitalii. Based on these results, we conclude that R. vitalii, the etiological agent of ""nambiuvu"" in southern Brazil, is a valid species of piroplasm. Further studies are required to evaluate the validity of the genus Rangelia. (C) 2011 Elsevier B.V. All rights reserved.