Glutathione peroxidase induction protects Saccharomyces cerevisiae sod1deltasod2delta double mutants against oxidative damage

Saccharomyces cerevisiae mutants deficient in superoxide dismutase genes (sod1delta, sod2delta and the double mutant) were subjected to H2O2 stress in the stationary phase. The highest sensitivity was observed in the sod2delta mutant, while the sod1deltasod2delta double mutant was not sensitive. Sod mutants had lower catalase activity (44%) than wild-type cells, independent of H2O2 stress. Untreated cells of sod1deltasod2delta double mutants showed increased glutathione peroxidase activity (126%), while sod1delta had lower activity (77%) than the wild type. Glutathione levels in sod1delta were increased (200-260%) after exposure to various H2O2 concentrations. In addition, the highest malondialdehyde levels could be observed without H2O2 treatment in sod1delta (167%) and sod2delta (225%) mutants. In contrast, the level of malondialdehyde in the sod1deltasod2delta double mutant was indistinguishable from that of the wild type. These results suggest that resistance to H2O2 by sod1deltasod2delta cells depends on the induction of glutathione peroxidase and is independent of catalase, and that glutathione is a primary antioxidant in the defense against H2O2 in stationary phase sod1delta mutants.

The emergence of YMDD mutants precedes biochemical flare by 19 weeks in lamivudine-treated chronic hepatitis B patients: an opportunity for therapy reevaluation

Given the loss of therapeutic efficacy associated with the development of resistance to lamivudine (LMV) and the availability of new alternative treatments for chronic hepatitis B patients, early detection of viral genotypic resistance could allow the clinician to consider therapy modification before viral breakthrough and biochemical relapse occur. To this end, 28 LMV-treated patients (44 ± 12 years; 24 men), on their first therapy schedule, were monitored monthly at four Brazilian centers for the emergence of drug resistance using the reverse hybridization-based INNO-LiPA HBV DR assay and occasionally sequencing (two cases). Positive viral responses (HBV DNA clearance) after 6, 12, and 18 months of therapy were achieved by 57, 68, and 53% of patients, while biochemical responses (serum alanine aminotransferase normalization) were observed in 82, 82, and 53% of cases. All viral breakthrough cases (N = 8) were related to the emergence of YMDD variants observed in 7, 21, and 35% of patients at 6, 12, and 18 months, respectively. The emergence of these variants was not associated with viral genotype, HBeAg expression status, or pretreatment serum alanine aminotransferase levels. The detection of resistance-associated mutations was observed before the corresponding biochemical flare (41 ± 14 and 60 ± 15 weeks) in the same individuals. Then, if highly sensitive LMV drug resistance testing is carried out at frequent and regular intervals, the relatively long period (19 ± 2 weeks) between the emergence of viral resistance and the onset of biochemical relapse can provide clinicians with ample time to re-evaluate drug therapy.

Hospitalization of older adults due to ambulatory care sensitive conditions

OBJECTIVE To analyze the temporal evolution of the hospitalization of older adults due to ambulatory care sensitive conditions according to their structure, magnitude and causes. METHODS Cross-sectional study based on data from the Hospital Information System of the Brazilian Unified Health System and from the Primary Care Information System, referring to people aged 60 to 74 years living in the state of Rio de Janeiro, Souhteastern Brazil. The proportion and rate of hospitalizations due to ambulatory care sensitive conditions were calculated, both the global rate and, according to diagnoses, the most prevalent ones. The coverage of the Family Health Strategy and the number of medical consultations attended by older adults in primary care were estimated. To analyze the indicators’ impact on hospitalizations, a linear correlation test was used. RESULTS We found an intense reduction in hospitalizations due to ambulatory care sensitive conditions for all causes and age groups. Heart failure, cerebrovascular diseases and chronic obstructive pulmonary diseases concentrated 50.0% of the hospitalizations. Adults older than 69 years had a higher risk of hospitalization due to one of these causes. We observed a higher risk of hospitalization among men. A negative correlation was found between the hospitalizations and the indicators of access to primary care. CONCLUSIONS Primary healthcare in the state of Rio de Janeiro has been significantly impacting the hospital morbidity of the older population. Studies of hospitalizations due to ambulatory care sensitive conditions can aid the identification of the main causes that are sensitive to the intervention of the health services, in order to indicate which actions are more effective to reduce hospitalizations and to increase the population’s quality of life.

Hospitalizations for ambulatory care-sensitive conditions, Minas Gerais, Southeastern Brazil, 2000 and 2010

OBJECTIVE To analyze hospitalization rates and the proportion of deaths due to ambulatory care-sensitive hospitalizations and to characterize them according to coverage by the Family Health Strategy, a primary health care guidance program. METHODS An ecological study comprising 853 municipalities in the state of Minas Gerais, under the purview of 28 regional health care units, was conducted. We used data from the Hospital Information System of the Brazilian Unified Health System. Ambulatory care-sensitive hospitalizations in 2000 and 2010 were compared. Population data were obtained from the demographic censuses. RESULTS The number of ambulatory care-sensitive hospitalizations declined from 20.75/1,000 inhabitants [standard deviation (SD) = 10.42) in 2000 to 14.92/thousand inhabitants (SD = 10.04) in 2010 Heart failure was the most frequent cause in both years. Hospitalizations rates for hypertension, asthma, and diabetes mellitus, decreased, whereas those for angina pectoris, prenatal and birth disorders, kidney and urinary tract infections, and other acute infections increased. Hospitalization durations and the proportion of deaths due to ambulatory care-sensitive hospitalizations increased significantly. CONCLUSIONS Mean hospitalization rates for sensitive conditions were significantly lower in 2010 than in 2000, but no correlation was found with regard to the expansion of the population coverage of the Family Health Strategy. Hospitalization rates and proportion of deaths were different between the various health care regions in the years evaluated, indicating a need to prioritize the primary health care with high efficiency and quality.

Study of antiretroviral mutants in HIV patients with treatment failures and the effect of risk factors in the virological failures

INTRODUCTION: Information about HIV phenotypes of resistant to available ART and the influence of different risk factors on virological failures (VF) in Costa Rican HIV positive patients prior or during HAART is unknown. MATERIALS AND METHODS: Eighty nine samples, 72 VF and 17 basal (before treatment) were analyzed by examining resistant mutants in reverse transcriptase (RT) and protease (PT) regions using Trugene or LIPA genotyping kits. Sixty eight control patients were selected and relevant information was collected in a questionnaire. RESULTS: Poor adherence, presence of resistant mutations and number of treatment's changes were the only significant factors found (p = 0.006, 0.04 and 0.01 respectively). From 66 sequenced samples, 78%, 50% and 50% showed resistance to NRTI (nucleoside reverse transcriptase inhibitors), NNRT (non-nucleoside reverse transcriptase inhibitors) and PI (protease inhibitors), respectively. The most frequent mutations were M41L, M184V, and T215FY in RT and L62PI, L10FIRV and M36I in PT. DISCUSSION: The most important factor related to treatment response in this study was adherence to treatment. Mutations in RT were related to the treatment failure while the ones found in PT were secondary mutations which have been previously described to influence the selection of primary resistance mutations in these regions. The study reveals the urgency to detect resistant mutations in VF to be considered by physicians for selection of treatment schedule, to analyze basal HIV patients for monitoring of the spread of resistant mutations and the importance to reinforce the adherence in the patients for overall treatment outcome.

HIGH SENSITIVE PCR METHOD FOR DETECTION OF PATHOGENIC Leptospira spp. IN PARAFFIN-EMBEDDED TISSUES

This study describes the development and application of a new PCR assay for the specific detection of pathogenic leptospires and its comparison with a previously reported PCR protocol. New primers were designed for PCR optimization and evaluation in artificially-infected paraffin-embedded tissues. PCR was then applied to post-mortem, paraffin-embedded samples, followed by amplicon sequencing. The PCR was more efficient than the reported protocol, allowing the amplification of expected DNA fragment from the artificially infected samples and from 44% of the post-mortem samples. The sequences of PCR amplicons from different patients showed >99% homology with pathogenic leptospires DNA sequences. The applicability of a highly sensitive and specific tool to screen histological specimens for the detection of pathogenic Leptospira spp. would facilitate a better assessment of the prevalence and epidemiology of leptospirosis, which constitutes a health problem in many countries.

Glucantime resistant Leishmania promastigotes are sensitive to pentostam

Growth inhibition in vitro tests were used to study the susceptibility to pentostam of different Leishmania strains involved in cutaneous and mucocutaneos leishmaniasis - one glucantime sensitive strain, three naturally glucantime resistant strains and one glucantime resistant line developed by in vitro drug exposure. Contrasting with the high degree , of glucantime resistance, all strains were sensitive to pentostam. These differences suggest that there is some relationship between chemical structure and in vitro activity for these antimonial compounds. These data justify a clinical re-evaluation to compare therapeutic efficacy of glucantime and pentostam in the treatment of leishmaniasis.

Comparative evaluation of a simple and sensitive assay for detection of orthomyxo and paramyxoviruses

Studies were done to evaluate comparatively the traditional HA assay and a more recently introduced lectin-neuraminidase (LN) methodologyin search of a simple and sensitive assay for virus detection during laboratorial diagnosis. The results proved the value of LN assay as a sensitive methodologyfor detection of virus particles, presenting results at least equal to those obtained by HA (hemagglutination) assay, with significant values of accumulated frequencies for LN/HA factors (ratios between LN and HA titers) higher than two. The accumulated values of frequencies for LN/HA factors as high as four were very significant, 72.7 (per cent) for influenzavirus and 60.7 (per cent) for Newcastle disease virus (NDV), moreover accumulated frequencies for LN/HA factors even as high as 32 were due to influenzavirus (45.4 per cent) and NDV (7.2 per cent) samples. After the storage period, most of those concentraded samples that even did not present HA titers could be detected through LN assay, demonstrating a lower threshold for virus detection.

Role of the cyclosporin-sensitive transcription factor NFAT1 in the allergic response

Proteins belonging to the NFAT (nuclear factor of activated T cells) family of transcription factors are expressed in most immune cell types, and play a central role in the transcription of cytokine genes, such as IL-2, IL-4, IL-5, IL-13, IFN-gamma, TNF-alpha, and GM-CSF. The activity of NFAT proteins is regulated by the calcium/calmodulin-dependent phosphatase calcineurin, a target for inhibition by CsA and FK506. Recently, two different groups have described that mice lacking the NFAT1 transcription factor show an enhanced immune response, with tendency towards the development of a late Th2-like response. This review evaluates the possible role of NFAT proteins in the Th2 immune response and in the eosinophil-mediated allergic response.

Genome comparison of progressively drug resistant Plasmodium falciparum lines derived from drug sensitive clone

Chloroquine has been the mainstay of malaria chemotherapy for the past five decades, but resistance is now widespread. Pyrimethamine or proguanil form an important component of some alternate drug combinations being used for treatment of uncomplicated Plasmodium falciparum infections in areas of chloroquine resistance. Both pyrimethamine and proguanil are dihydrofolate reductase (DHFR) inhibitors, the proguanil acting primarily through its major metabolite cycloguanil. Resistance to these drugs arises due to specific point mutations in the dhfr gene. Cross resistance between cycloguanil and pyrimethamine is not absolute. It is, therefore, important to investigate mutation rates in P. falciparum for pyrimethamine and proguanil so that DHFR inhibitor with less mutation rate is favored in drug combinations. Hence, we have compared mutation rates in P. falciparum genome for pyrimethamine and cycloguanil. Using erythrocytic stages of P. falciparum cultures, progressively drug resistant lines were selected in vitro and comparing their RFLP profile with a repeat sequence. Our finding suggests that pyrimethamine has higher mutation rate compared to cycloguanil. It enhances the degree of genomic polymorphism leading to diversity of natural parasite population which in turn is predisposes the parasites for faster selection of resistance to some other antimalarial drugs.

IS6110 fingerprinting of sensitive and resistant strains (1991-1992) of Mycobacterium tuberculosis in Colombia

The standardized method to study the polymorphism of IS 6110 was used to characterize 53 isolates of Mycobacterium tuberculosis obtained during 1991-1992 from 14 regions in Colombia. In Valle region cluster rate was 25% (4/16). The mean number of IS6110 band was 10 ± 3. Similarity between strains was of 60% in 81% of strains and this tended to be correlated with geographic origin. For the first time M. tuberculosis without IS6110 bands in restriction fragment length polymorphism analysis was found in Colombia. Additional studies are necessaries in order to best characterize the situation in relation to human immunodeficiency virus epidemic and recent changes in tuberculosis control program.

Functional complementation of a yeast knockout strain by Schistosoma mansoni Rho1 GTPase in the presence of caffeine, an agent that affects mutants defective in the protein kinase C signal transduction pathway

In a previous study, the Schistosoma mansoni Rho1 protein was able to complement Rho1 null mutant Saccharomyces cerevisiae cells at restrictive temperatures and under osmotic stress (low calcium concentration) better than the human homologue (RhoA). It is known that under osmotic stress, the S. cerevisiae Rho1 triggers two distinct pathways: activation of the membrane 1,3-beta-glucan synthase enzymatic complex and activation of the protein kinase C1 signal transduction pathway, promoting the transcription of response genes. In the present work the SmRho1 protein and its mutants smrho1E97P, smrho1L101T, and smrho1E97P, L101T were used to try to clarify the basis for the differential complementation of Rho1 knockout yeast strain by the human and S. mansoni genes. Experiments of functional complementation in the presence of caffeine and in the presence of the osmotic regulator sorbitol were conducted. SmRho1 and its mutants showed a differential complementation of the yeast cells in the presence of caffeine, since smrho1E97P and smrho1E97P, L101T mutants showed a delay in the growth when compared to the yeast complemented with the wild type SmRho1. However, in the presence of sorbitol and caffeine the wild type SmRho1 and mutants showed a similar complementation phenotype, as they allowed yeast growth in all caffeine concentrations tested.

Using a single tablet daily to treat latent tuberculosis infection in Brazil: bioequivalence of two different isoniazid formulations (300 mg and 100 mg) demonstrated by a sensitive and rapid high-performance liquid chromatography-tandem mass spectrometry method in a randomised, crossover study

The recommended treatment for latent tuberculosis (TB) infection in adults is a daily dose of isoniazid (INH) 300 mg for six months. In Brazil, INH was formulated as 100 mg tablets. The treatment duration and the high pill burden compromised patient adherence to the treatment. The Brazilian National Programme for Tuberculosis requested a new 300 mg INH formulation. The aim of our study was to compare the bioavailability of the new INH 300 mg formulation and three 100 mg tablets of the reference formulation. We conducted a randomised, single dose, open label, two-phase crossover bioequivalence study in 28 healthy human volunteers. The 90% confidence interval for the INH maximum concentration of drug observed in plasma and area under the plasma concentration vs. time curve from time zero to the last measurable concentration “time t” was 89.61-115.92 and 94.82-119.44, respectively. The main limitation of our study was that neither adherence nor the safety profile of multiple doses was evaluated. To determine the level of INH in human plasma, we developed and validated a sensitive, simple and rapid high-performance liquid chromatography-tandem mass spectrometry method. Our results showed that the new formulation was bioequivalent to the 100 mg reference product. This finding supports the use of a single 300 mg tablet daily strategy to treat latent TB. This new formulation may increase patients’ adherence to the treatment and quality of life.