Evaluation of an Enzyme-Linked Immunosorbent Assay Based on Araraquara Virus Recombinant Nucleocapsid Protein

Laboratory diagnosis of hantavirus cardiopulmonary syndrome (HCPS) in Brazil has been performed mostly by a detection of IgM antibodies to recombinant antigen purified from Sin Nombre virus and Andes Virus (ANDV). Recently, a recombinant nucleocapsid (rN) protein of Argentina virus (ARAV), a Brazilian hantavirus, was Obtained in Escherichia coli. To evaluate ARAV rN as antigen for antibody detection, serum samples from 30 patients front Argentina seropositive for hantavirus were tested. All samples were positive for IgG and IgM by enzyme-linked immunosorbent assay (ELISA) using either ARAV rN or ANDV rN antigens. In Brazil, six of 00 serum samples from patients With suspected HCPS (10%) were positive for IgM by ELISA Using ARAV rN antigen and 7 were positive Using ANDV rN antigen. For results obtained with 90 serum samples analyzed by IgM ELISA with ANDV rN antigen, the sensitivity of the IgM ELISA using ARAV rN antigen was 97.2%,, the specificity was 100%, the positive predictive value was 100% and the negative predictive value was 98.1%. The results show that ARAV rN is a Suitable antigen for diagnosis Of hantavirus infection in Brazil and Argentina.

Enantioselective Determination of Mexiletine and Its Metabolites p-Hydroxymexiletine and Hydroxymethylmexiletine in Rat Plasma by Normal-Phase Liquid Chromatography-Tandem Mass Spectrometry: Application to Pharmacokinetics

Mexiletine (MEX), hydroxymethylmexiletine (HMM) and P-hydroxy-mexiletine (PHM) were analyzed in rat plasma by LC-MS/MS. The plasma samples were prepared by liquid-liquid extraction using methyl-tert-butyl ether as extracting solvent. MEX, HMM, and PHM enantiomers were resolved on a Chiralpak (R) AD column. Validation of the method showed a relative standard deviation (precision) and relative errors (accuracy) of less than 15% for all analytes studied. Quantification limits were 0.5 ng ml(-1) for the MEX and 0.2 ng ml(-1) for the HMM and PHM enantiomers. The validated method was successfully applied to quantify the enantiomers of MEX and its metabolites in plasma samples of rats (n = 6) treated with a single oral dose of racemic MEX. Chirality 21:648-656, 2009. (C) 2008 Wiley-Liss, Inc.

Ictal technetium-99 m ethyl cysteinate dimer single-photon emission tomographic findings in epileptic patients with polymicrogyria syndromes: A Subtraction of ictal-interictal SPECT coregistered to MRI study

Purpose To describe the ictal technetium-99 m-ECD SPECT findings in polymicrogyria syndromes (PMG) during epileptic seizures. Methods We investigated 17 patients with PMG syndromes during presurgical workup, which included long-term video-electroencephalographic (EEG) monitoring, neurological and psychiatry assessments, invasive EEG, and the subtraction of ictal-interictal SPECT coregistered to magnetic resonance imaging (MRI) (SISCOM). Results The analysis of the PMG cortex, using SISCOM, revealed intense hyperperfusion in the polymicrogyric lesion during epileptic seizures in all patients. Interestingly, other localizing investigations showed heterogeneous findings. Twelve patients underwent epilepsy surgery, three achieved seizure-freedom, five have worthwhile improvement, and four patients remained unchanged. Conclusions Our study strongly suggests the involvement of PMG in seizure generation or early propagation. Both conventional ictal single-photon emission computed tomography (SPECT) and SISCOM appeared as the single contributive exam to suggest the localization of the epileptogenic zone. Despite the limited number of resective epilepsy surgery in our study (n=9), we found a strong prognostic role of SISCOM in predicting surgical outcome. This result may be of great value on surgical decision-making of whether or not the whole or part of the PMG lesion should be surgically resected.

Efficiency of the DNA repair and polymorphisms of the XRCC1, XRCC3 and XRCC4 DNA repair genes in systemic lupus erythematosus

Impaired DNA repair efficiency in systematic lupus erythematosus (SLE) patients has been reported ill some studies, mainly regarding the repair of oxidative damage, but little is known about repair kinetics towards primarily single-stranded DNA breaks. In the present study, we aimed to investigate: (a) the efficiency of SLE peripheral blood leucocytes in repairing DNA damage induced by ionizing radiation and (b) the association of DNA repair gene (XRCC1 Arg399Gln, XRCC3 Thr241Met and XRCC4 Ile401Thr) polymorphisms in SLE patients, considering the whole group, or stratified sub-groups according to clinical and laboratory features. A total of 163 SLE patients and 125 healthy control were studied. The kinetics of DNA strand break repair was evaluated by the comet assay, and genotyping for DNA repair genes was performed by PCR-RFLP. Compared with controls. SLE leucocytes exhibited decreased efficiency of DNA repair evaluated at 30 min following irradiation. A significant association with DNA repair gene polymorphisms was not observed for the whole group of SLE patients; however, the XRCC1Arg399Gln polymorphism was associated with the presence of anti-dsDNA antibody. The concomitance of two DNA repair polymorphic sites was associated with the presence of neuropsychiatric manifestations and antiphospholipid antibody syndrome. Taken together, these results indicated that SLE leucocytes repair less efficiently the radiation-induced DNA damage, and DNA repair polymorphic sites may predispose to the development of particular clinical and laboratory features. Lupus (2008) 17, 988-995.

A single nucleotide deletion at the C1 inhibitor gene as the cause of hereditary angioedema: insights from a Brazilian family

Background: Hereditary angioedema is an autosomal dominant disease characterized by episodes of subcutaneous and submucosal edema. It is caused by deficiency of the C1 inhibitor protein, leading to elevated levels of bradykinin. More than 200 mutations in C1 inhibitor gene have been reported. The aim of this study was to analyze clinical features of a large family with an index case of hereditary angioedema and to determine the disease-causing mutation in this family. Methods: Family pedigree was constructed with 275 individuals distributed in five generations. One hundred and sixty-five subjects were interviewed and investigated for mutation at the C1 inhibitor gene. Subjects reporting a history of recurrent episodes of angioedema and/or abdominal pain attacks underwent evaluation for hereditary angioedema. Results: We have identified a novel mutation at the C1 inhibitor gene, c.351delC, which is a single-nucleotide deletion of a cytosine on exon 3, resulting in frameshift with premature stop codon. Sequencing analysis of the hypothetical truncated C1 inhibitor protein allowed us to conclude that, if transcription occurs, this protein has no biological activity. Twenty-eight members of the family fulfilled diagnostic criteria for hereditary angioedema and all of them presented the c.351delC mutation. Variation in clinical presentation and severity of disease was observed among these patients. One hundred and thirty-seven subjects without hereditary angioedema did not have the c.351delC mutation. Conclusion: The present study provides definitive evidence to link a novel genetic mutation to the development of hereditary angioedema in patients from a Brazilian family.

Human leukocyte antigen (HLA) and single nucleotide polymorphisms (SNPs) tumor necrosis factor (TNF)-alpha-238 and-308 as genetic markers of susceptibility to psoriasis and severity of the disease in a long-term follow-up Brazilian study

Background The strongest genetic marker for psoriasis is Cw*06. Polymorphisms in the tumor necrosis factor (TNF)-alpha promoter region, especially replacement of guanine with adenine in positions -238 and -308 are related to higher TNF-alpha production and higher risk for psoriasis in Caucasoid populations, not found in Asians. We performed a case-control study of 69 patients with psoriasis type I and 70 controls, characterized clinical progression along 10-years of follow-up in mild or severe disease and determined HLA class I, II, and TNF single nucleotide polymorphisms (SNPs) -238 and -308 polymorphisms to demonstrate whether these polymorphisms may be genetic risk for susceptibility to psoriasis or severity of the disease in Brazilians. Methods Polymorphisms were identified using PCR/SSP. Alleles, genotypes, and haplotypes frequencies were compared using Fisher`s test. Results More severe disease was found in male patients. It may be suggested that alleles B*37, Cw*06, Cw*12, and DRB1*07 were associated with severe disease course, while B*57 with mild disease. No statistical difference was found between the patients and controls regarding polymorphisms frequencies in TNF SNPs. This study pointed to a higher TNF-238 G/G genotype frequency (OR: 3.21; CI: 1.06-9.71; P = 0.04) in the group with severe disease. Conclusions Polymorphisms in the TNF-alpha SNPs do not seem to be a more important genetic risk factor for psoriasis than the already known Cw*06 in Brazilian patients, but these markers may be related to clinical manifestations.

A reassessment of the in vitro RBC haemolysis assay with defibrinated sheep blood for the determination of the ocular irritation potential of cosmetic products: Comparison with the in vivo Draize rabbit test

We examined the correlation between results obtained from the in vivo Draize test for ocular irritation and in vitro results obtained from the sheep red blood cell (RBC) haemolytic assay, which assesses haemolysis and protein denaturation in erythrocytes, induced by cosmetic products. We sought to validate the haemolytic assay as a preliminary test for identifying highly-irritative products, and also to evaluate the in vitro test as alternative assay for replacement of the in vivo test. In vitro and in vivo analyses were carried out on 19 cosmetic products, in order to correlate the lesions in the ocular structures with three in vitro parameters: (i) the extent of haemolysis (H50); (ii) the protein denaturation index (131); and (iii) the H50/DI ratio, which reflects the irritation potential (IP). There was significant correlation between maximum average scores (MAS) and the parameters determined in vitro (r = 0.752-0.764). These results indicate that the RBC assay is a useful and rapid test for use as a screening method to assess the IP of cosmetic products, and for predicting the IP value with a high level of concordance (94.7%). The assay showed high sensitivity and specificity rates of 91.6% and 100%, respectively.

Comparative analysis of two immunohistochemical methods for antigen retrieval in the optical lobe of the honeybee Apis mellifera: Myosin-v assay

The present study compared two heating methods currently used for antigen retrieval (AR) immunostaining: the microwave oven and the steam cooker. Myosin-V, a molecular motor involved in vesicle transport, was used as a neuronal marker in honeybee Apis mellifera brains fixed in formalin. Overall, the steam cooker showed the most satisfactory AR results. At 100 degrees C, tissue morphology was maintained and revealed epitope recovery, while evaporation of the AR solution was markedly reduced; this is important for stabilizing the sodium citrate molarity of the AR buffer and reducing background effects. Standardization of heat-mediated AR of formalin-fixed and paraffin-embedded tissue sections results in more reliable immunostaining of the honeybee brain.

A single amino acid substitution in one of the lipases of Aspergillus nidulans confers resistance to the antimycotic drug undecanoic acid

A plausible approach to evaluate the inhibitory action of antifungals is through the investigation of the fungal resistance to these drugs. We describe here the molecular cloning and initial characterization of the A. nidulans lipA gene, where mutation (lipA1) conferred resistance to undecanoic acid, the most fungitoxic fatty acid in the C(7:0)-C(18:0) series. The lipA gene codes for a putative lipase with the sequence consensus GVSIS and WIFGGG as the catalytic signature. Comparison of the wild-type and LIP1 mutant strain nucleotide sequences showed a G -> A change in lipA1 allele, which results in a Glu(214) -> Lys substitution in LipA protein. This ionic charge change in a conserved LipA region, next to its catalytic site, may have altered the catalytic properties of this enzyme resulting in resistance to undecanoic acid.

Mitochondrial population genomics supports a single pre-Clovis origin with a coastal route for the peopling of the Americas

It is well accepted that the Americas were the last continents reached by modern humans, most likely through Beringia. However, the precise time and mode of the colonization of the New World remain hotly disputed issues. Native American populations exhibit almost exclusively five mitochondrial DNA (mtDNA) haplogroups (A-D and X). Haplogroups A-D are also frequent in Asia, suggesting a northeastern Asian origin of these lineages. However, the differential pattern of distribution and frequency of haplogroup X led some to suggest that it may represent an independent migration to the Americas. Here we show, by using 86 complete mitochondrial genomes, that all Native American haplogroups, including haplogroup X, were part of a single founding population, thereby refuting multiple-migration models. A detailed demographic history of the mtDNA sequences estimated with a Bayesian coalescent method indicates a complex model for the peopling of the Americas, in which the initial differentiation from Asian populations ended with a moderate bottleneck in Beringia during the last glacial maximum (LGM), around similar to 23,000 to similar to 19,000 years ago. Toward the end of the LGM, a strong population expansion started similar to 18,000 and finished similar to 15,000 years ago. These results support a pre-Clovis occupation of the New World, suggesting a rapid settlement of the continent along a Pacific coastal route.

MHM assay: molecular sexing based on the sex-specific methylation pattern of the MHM region in chickens

Bird sex determination using molecular methods has proved to be a valuable tool in different studies. Although it is possible to sex most birds by coupling the CHD assay with others available methods, no sex-determining gene like SRY in mammalians has been identified in birds. The male hypermethylated (MHM) region on the Z chromosome has been found to be hypermethylated in males and hypomethylated in females in birds of the order Galliformes. We analyzed the DNA from feathers of 50 adult chickens to verify the methylation pattern of the MHM region by PCR and the restriction enzyme HpaII (a method named MHM assay). The results, visualized in agarose gel, were compared with PCR amplification of the CHD-Z and CHD-W genes (polyacrylamide gel) and with the birds` phenotype. All males (25) showed hypermethylation of the MHM region, and all females (25) showed hypomethylation. The sexing by MHM assay was in according with phenotype and CHD sexing. To our knowledge, this is the first study that uses the MHM region for sexing birds. Although the real role of the MHM region in the sex determination is still unclear, this could be a universal marker for sexing birds and may be involved in sex determination by its influence on transcriptional processes. The MHM assay could be a good alternative for CHD assay in developmental studies.

Treatment for low-risk gestational trophoblastic disease: Comparison of single-agent methotrexate, dactinomycin and combination regimens

Objectives. To compare the efficacy of three different standard chemotherapy regimens for low-risk gestational trophoblastic disease according to the FIGO staging system in a single-institute setting. Methods. From 1980 until 2002, we retrospectively reviewed 108 cases with low-risk persistent gestational trophoblastic disease who were treated with first-line chemotherapy. Patients were divided in three groups according to chemotherapy regimen: patients treated with methotrexate (MTX group; n=42), patients treated with dactinomycin (ACT group; n=42) and patients treated with methotrexate and dactinomycin in combination (MACT group; n=24). We compared the number of chemotherapy courses for achieving remission, the duration of treatment, the adverse side effects, the efficacy of the treatment and the need for performing a hysterectomy among the groups Results. The complete remission rates were 69%, 61.4% and 79.1% for methotrexate (MTX), dactinomycin (ACT) and the combination regimen (MACT) treated groups, respectively (p=0.7). The duration of the treatment and the number of chemotherapy courses were similar among the groups (p = 0.2 and p = 0.4, respectively). Adverse side effects rate was reported to be 62.5% in the MACT group, 28.6% in the MTX group and 19.1% in the ACT group (p=0.0003). Second-line chemotherapy was indicated for 30 patients. Hysterectomy was performed in 21 patients overall, and there was no difference among the groups (P=0.6). Conclusion. Our analysis indicates that single-agent chemotherapy regimens are as effective as combination chemotherapy for low-risk gestational trophoblastic disease. Dactinomycin is a less toxic drug and might offer the best cost-effective treatment option. Methotrexate must be considered as the regimen of choice for low resource areas because of the feasibility of its administration. (c) 2007 Elsevier Inc. All rights reserved.

Impact of maternal vitamin A supplementation on the mother-infant pair in Brazil

Background/Objectives: Vitamin A deficiency (VAD) is a major public health problem. The supplementation of lactating women could be an effective strategy to combat it. The objective of this study was to assess the impact of maternal vitamin A supplementation on the mother-infant pair. Subjects/Methods: This was a double blind, placebo-controlled randomized clinical assay in which 33 women received 200 000 IU of vitamin A and 33 women received soy oil between 20th and 30th postpartum days. Maternal blood and milk samples were collected immediately before supplementation and 3 months after delivery, when blood was also collected from the babies. Retinol concentrations <= 0.70 mu mol/l in serum and 1.05 mu mol/l in milk were considered to indicate VAD. Results: Increase in serum retinol level was observed in the supplemented group compared with the pre-supplementation levels (1.05 and 1.17 mu mol/l, respectively; P = 0.026) and to the post-supplementation levels of the control group (1.02 mu mol/l; P = 0.032). Reduction in breast milk retinol was observed in the control group compared with the pre-supplementation levels (1.93 and 1.34 mu mol/l, respectively; P<0.0001) and to the post-supplementation levels of the supplemented group (1.56 mu mol/l; P = 0.0003). There was significant difference in the prevalence of VAD in breast milk after supplementation, 55.6% (15/27) in the control group and 16.1% (5/31) in the supplemented group (P = 0.002). VAD was present in 66.1% (39/59) of infants, with mean serum retinol levels of 0.64 +/- 0.30 mu mol/l in the control group and of 0.69 +/- 0.26 mu mol/l in the supplemented group. Conclusions: Supplementation had a positive impact on maternal vitamin A status. No effect on infant status was detectable 2 months after supplementation with a single dose. European Journal of Clinical Nutrition (2010) 64, 1302-1307; doi: 10.1038/ejcn.2010.165; published online 15 September 2010

Safety and Immunogenicity of Varicella Vaccine in Patients With Juvenile Rheumatic Diseases Receiving Methotrexate and Corticosteroids

Objective. To evaluate the safety and immunogenicity of varicella vaccine (VV) in susceptible patients with juvenile rheumatic diseases receiving methotrexate and corticosteroids. Methods. Twenty-five patients with juvenile rheumatic diseases (ages 2-19 years) and 18 healthy children and adolescents (ages 3-18 years) received a single dose of VV. All 25 patients were receiving methotrexate; 13 were also receiving prednisone and 5 were also receiving other disease-modifying antirheumatic drugs. None of the vaccinated patients or controls had a previous history of varicella. Anti-varicella-zoster virus IgG antibody (anti-VZV-IgG) titers were measured by enzyme-linked immunosorbent assay immediately before, 4-6 weeks after, and 1 year after vaccination. The patients were monitored prospectively for adverse reactions related to the vaccine, exposure, and occurrence of varicella. Disease activity was assessed 3 months before and 3 months after VV. Results. Twenty patients and all of the controls had negative preimmunization titers of VZV-IgG, and 5 patients had equivocal levels. Positive VZV-IgG titers were detected in 10 (50%) of 20 seronegative patients and 13 (72.2%) of 18 controls 4-6 weeks after VV (P = 0.2). One year after vaccination, 8 of 10 patients maintained positive VZV-IgG titers. No overt varicella episodes and no severe adverse reactions were observed during the followup period. No worsening of clinical parameters and no flares of juvenile rheumatic diseases or changes in doses of medications used were detected after vaccination. In fact, the number of active joints in patients with juvenile idiopathic arthritis was significantly lower after VV (P = 0.009). Conclusion. VV appears to be safe in patients with juvenile rheumatic diseases receiving methotrexate, as long as continuous prospective vigilance for side effects is performed.

Treatment of Hepatocellular Carcinoma With Liver Transplantation: A Single-Center Experience From Brazil

Introduction. Orthotopic liver transplantation (OLT) is the treatment of choice of hepatocellular carcinoma (HCC) for patients with cirrhosis, mainly those with early HCC. Herein we have present the clinical characteristics and outcomes of cirrhotic patients with HCC who underwent OLT from cadaveric donors in our institution. Methods. From May 2001 to May 2009, we performed 121 OLT including 24 patients (19.8%) with cirrhosis and HCC within the Milan criteria. In 4 cases, HCC was an incidental finding in the explants. Results. The patients` average age was 55 +/- 10 years, including 82% men. Fifty percent of patients were Child class B or C. The average Model for End Stage Liver Disease for Child A, B, and C categories were 11, 15, and 18, respectively. The HCC diagnosis was made by 2 dynamic images in 16 cases; 1 dynamic image plus alphafetoprotein >400 ng/mL in 4; and 4 by histologic confirmation. Twenty patients received a locoregional treatment before OLT: 6 percutaneous ethanol injection, 9 transarterial chemoembolization, 1 transarterial embolization, and 4 a combination of these modalities. The median follow-up after OLT was 19.7 months (range, 1-51). A vascular invasion was observed in the explant of 1 patient, who developed an HCC recurrence and succumbed at 8 months after OLT. Two further patients, without vascular invasion or satellite tumor displayed tumor recurrences at 7 and 3 months after OLT, and death at 2 and 1 month after the diagnosis. The remaining 25 patients have not shown a tumor recurrence. Conclusion. In the present evaluation, OLT patients with early HCC and no vascular invasion showed satisfactory results and good disease-free survival. Strictly following the Milan criteria for liver transplantation in patients with HCC greatly reduces but does not completely avoid, the chances of tumor recurrence.