Detection of Spinal Muscular Atrophy Carriers in a Sample of the Brazilian Population

Background: Spinal muscular atrophy is a common autosomal recessive neuromuscular disorder caused by mutations in the SMN1 gene. Identification of spinal muscular atrophy carriers has important implications for individuals with a family history of the disorder and for genetic counseling. The aim of this study was to determine the frequency of carriers in a sample of the nonconsanguineous Brazilian population by denaturing high-performance liquid chromatography (DHPLC). Methods: To validate the method, we initially determined the relative quantification of DHPLC in 28 affected patients (DHPLC values: 0.00) and 65 parents (DHPLC values: 0.49-0.69). Following quantification, we studied 150 unrelated nonconsanguineous healthy individuals from the general population. Results: Four of the 150 healthy individuals tested (with no family history of a neuromuscular disorder) presented a DHPLC value in the range of heterozygous carriers (0.6-0.68). Conclusions: Based on these results, we estimated there is a carrier frequency of 2.7% in the nonconsanguineous Brazilian population, which is very similar to other areas of the world where consanguineous marriage is not common. This should be considered in the process of genetic counseling and risk calculations. Copyright (C) 2011 S. Karger AG, Basel

Detection of Clonal Immunoglobulin and T-Cell Receptor Gene Rearrangements in Childhood Acute Lymphoblastic Leukemia Using a Low-Cost PCR Strategy

Background Imunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements function as specific markers for minimal residual disease (MRD) which is one of the best predictors of outcome in childhood acute lymphoblastic leukemia (ALL) We recently reported on the prognostic value of MRD during the induction of remission through a simplified PCR method Here we report on gene rearrangement frequencies and offer guidelines for the application of the technique Procedure Two hundred thirty three children had DNA extracted from bone marrow Ig and TCR gene rearrangements were amplified using consensus primers and conventional PCR PCR products were submitted to homo/heteroduplex analysis A computer program was designed to define combinations of targets for clonal detection using a minimum set of primers and reactions Results At least one clonal marker could be detected in 98% of the patients and two markers in approximately 80% The most commonly rear ringed genes in precursor B cell ALL were IgH (75%) TCRD (59%) IgK (55%), and TCRG (54%) The most commonly rearranged genes for TALL were TCRG (100%) and TCRD (24%) The sensitivity of primers was limited to the detection of 1 leukemic cell among 100 normal cells Conclusions We propose that eight PCR reactions per ALL subtype would allow for the detection of two markers in most cases In addition these reactions ire suitable for MRD monitoring especially when aiming the selection of patients with high MRD levels (>= 10(-2)) at the end of induction therapy Such an approach would be very useful in centers with limited financial resources Pediatr Blood Cancer 2010 55 1278-1286 (C) 2010 Wiley Liss Inc

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.

Development of chronic cardiomyopathy in canine Chagas disease correlates with high IFN-gamma, TNF-alpha, and low IL-10 production during the acute infection phase

When infected with Trypanosoma cruzi, Beagle dogs develop symptoms similar to those of Chagas disease in human beings, and could be an important experimental model for a better understanding of the immunopathogenic mechanisms involved in chronic chagasic infection. This study evaluates IL-10, IFN-gamma and TNF-alpha production in the sera, culture supernatant, heart and cervical lymph nodes and their correlation with cardiomegaly, cardiac inflammation and fibrosis in Beagle dogs infected with T. cruzi. Pathological analysis showed severe splenomegaly, lymphadenopathy and myocarditis in all infected dogs during the acute phase of the disease, with cardiomegaly, inflammation and fibrosis observed in 83% of the animals infected by T. cruzi during the chronic phase. The data indicate that infected animals producing IL-10 in the heart during the chronic phase and showing high IL-10 production in the culture supernatant and serum during the acute phase had lower cardiac alterations (myocarditis, fibrosis and cardiomegaly) than those with high IFN-gamma and TNF-alpha levels. These animals produced low IL-10 levels in the culture supernatant and serum during the acute phase and did not produce IL-10 in the heart during the chronic phase of the disease. Our findings showed that Beagle dogs are a good model for studying the immunopathogenic mechanism of Chagas disease, since they reproduce the clinical and immunological findings described in chagasic patients. The data suggest that the development of the chronic cardiac form of the disease is related to a strong Th1 response during the acute phase of the disease, while the development of the indeterminate form results from a blend of Th1 and Th2 responses soon after infection, suggesting that the acute phase immune response is important for the genesis of chronic cardiac lesions. Crown Copyright (C) 2009 Published by Elsevier B.V. All rights reserved.

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.

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

Short-Term Effects of Hydroxyethylstarch Resuscitation on Systemic and Regional Hemodynamics and Metabolism in a Brain-Dead Canine Model

Background. Hydroxyethylstarch (HES) is a synthetic polymer of glucose that has been suggested for therapeutic use in long-term plasma expansion. The aim of this study was to test the hypothesis that the infusion of a small volume of HES may provide benefits in systemic and regional hemodynamics and metabolism in a brain-dead canine model compared with large volume crystalloid resuscitation. Methods. Fourteen mongrel dogs were subjected to a brain-death protocol by consecutive insufflations of a balloon catheter in the epidural space. One hour after induction of brain-death, the animals were randomly assigned to two groups: NS (0.9% NaCl, 33mL/kg), and HES (6% HES 450/0.7, 17mL/Kg). Systemic and regional hemodynamics were evaluated using Swan-Ganz, ultrasonic flowprobes, and arterial catheters. Serial blood samples were collected for blood gas, electrolyte, and serum chemistry analysis. Systemic, hepatic, and splanchnic O(2)-derived variables were also calculated. Results. Epidural balloon insufflations induced a significant increase in mean arterial pressure, cardiac output (MAP and CO, respectively), regional blood flow, and systemic vascular resistance. Following the hyperdynamic phase, severe hypotension with normalization of systemic and regional blood flow was observed. Fluid resuscitation induced a prompt increase in MAP, CO, and portal vein blood flow, and a significant reduction in systemic and pulmonary vascular resistance. There were no differences between groups in metabolic indices, liver function tests (LFTs), or renal function tests. HES was more effective than NS in restoring cardiac performance in the first 2h after fluid resuscitation (P < 0.05). Both tested solutions partially and temporarily restored systemic and regional oxygen delivery. Conclusion. Small volumes of 6% HES 450/0.7 improved cardiovascular performance and provided the same regional hemodynamic and metabolic benefits of large volumes of isotonic crystalloid solutions. (C) 2011 Elsevier Inc. All rights reserved.

Canine Distemper Virus in a Crab-eating Fox (Cerdocyon thous) in Brazil: Case Report and Phylogenetic Analyses

Although canine distemper is enzootic worldwide and has a wide host range, there are no reports of canine distemper virus there are no reports of canine distemper virus provide information on virus phylogeny and histopathologic lesions. The objective of this study is report and describe canine distemper in a crab-eating fox (C. thous), with a focus oil the phylogeny of the virus strain and the histopathologic lesions in the animal.

Canine population dynamics: the potential effect of sterilization campaigns

Objective. To analyze, through mathematical modeling, the potential ability of sterilization campaigns to reduce the population density of pet dogs. Methods. Mathematical models were constructed to simulate the canine population dynamics and project the results of control strategies based on several sterilization rates. Results. Even at high sterilization rates (for example, 0.80 year(-1)), it would take approximately 5 years to reduce density by 20%. Even so, other sources of population growth, such as the importing of dogs from other geographic areas, could outweigh the effects of a sterilization program. Conclusions. A program`s effectiveness is contingent upon not only on the sterilization rate, but also the rate of population growth. Sterilization campaigns may potentially reduce population density, but this reduction may not be immediately evident.

Canine infection by Rickettsiae and Ehrlichiae in southern Brazil

This study evaluated the infection caused by Rickettsia and Ehrlichia agents among dogs in southern Brazil. A total of 389 dogs were tested by the indirect immunofluorescence assay (IFA) for Rickettsia rickettsii, Rickettsia parkeri, Rickettsia amblyommii, Rickettsia rhipicephali, Rickettsia bellii, and Ehrlichia canis. Overall, 42.4% (165/389) of the dogs were seroreactive to at least one Rickettsia species, but only 11 canine sera reacted with another Rickettsia species without reacting with R. parkeri. A total of 100 (25.7%) canine sera showed titers to R. parkeri at least 4-fold higher than those to any of the other rickettsial antigens, allowing us to consider that these dogs were infected by R. parkeri. Dogs that had direct contact with pasture or forest areas were > 2 times more likely to be seroreactive to Rickettsia than dogs with no such direct contact. Only 19 (4.8%) of the 389 dogs were seroreactive to E. canis.

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.

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.