Selective Decrease of Components of the Creatine Kinase System and ATP Synthase Complex in Chronic Chagas Disease Cardiomyopathy

Background: Chronic Chagas disease cardiomyopathy (CCC) is an inflammatory dilated cardiomyopathy with a worse prognosis than other cardiomyopathies. CCC occurs in 30 % of individuals infected with Trypanosoma cruzi, endemic in Latin America. Heart failure is associated with impaired energy metabolism, which may be correlated to contractile dysfunction. We thus analyzed the myocardial gene and protein expression, as well as activity, of key mitochondrial enzymes related to ATP production, in myocardial samples of end-stage CCC, idiopathic dilated (IDC) and ischemic (IC) cardiomyopathies. Methodology/Principal Findings: Myocardium homogenates from CCC (N = 5), IC (N = 5) and IDC (N = 5) patients, as well as from heart donors (N = 5) were analyzed for protein and mRNA expression of mitochondrial creatine kinase (CKMit) and muscular creatine kinase (CKM) and ATP synthase subunits aplha and beta by immunoblotting and by real-time RT-PCR. Total myocardial CK activity was also assessed. Protein levels of CKM and CK activity were reduced in all three cardiomyopathy groups. However, total CK activity, as well as ATP synthase alpha chain protein levels, were significantly lower in CCC samples than IC and IDC samples. CCC myocardium displayed selective reduction of protein levels and activity of enzymes crucial for maintaining cytoplasmic ATP levels. Conclusions/Significance: The selective impairment of the CK system may be associated to the loss of inotropic reserve observed in CCC. Reduction of ATP synthase alpha levels is consistent with a decrease in myocardial ATP generation through oxidative phosphorylation. Together, these results suggest that the energetic deficit is more intense in the myocardium of CCC patients than in the other tested dilated cardiomyopathies.

Urban air pollution and chronic obstructive pulmonary disease-related emergency department visits

Background: Patients with chronic obstructive pulmonary disease (COPD) can have recurrent disease exacerbations triggered by several factors, including air pollution. Visits to the emergency respiratory department can be a direct result of short-term exposure to air pollution. The aim of this study was to investigate the relationship between the daily number of COPD emergency department visits and the daily environmental air concentrations of PM(10), SO(2), NO(2), CO and O(3) in the City of Sao Paulo, Brazil. Methods: The sample data were collected between 2001 and 2003 and are categorised by gender and age. Generalised linear Poisson regression models were adopted to control for both short-and long-term seasonal changes as well as for temperature and relative humidity. The non-linear dependencies were controlled using a natural cubic spline function. Third-degree polynomial distributed lag models were adopted to estimate both lag structures and the cumulative effects of air pollutants. Results: PM(10) and SO(2) readings showed both acute and lagged effects on COPD emergency department visits. Interquartile range increases in their concentration (28.3 mg/m(3) and 7.8 mg/m(3), respectively) were associated with a cumulative 6-day increase of 19% and 16% in COPD admissions, respectively. An effect on women was observed at lag 0, and among the elderly the lag period was noted to be longer. Increases in CO concentration showed impacts in the female and elderly groups. NO(2) and O(3) presented mild effects on the elderly and in women, respectively. Conclusion: These results indicate that air pollution affects health in a gender-and age-specific manner and should be considered a relevant risk factor that exacerbates COPD in urban environments.

Challenges and opportunities for primary, secondary, and tertiary prevention of Chagas' disease

A century after its discovery, Chagas' disease still represents a major public health challenge in Latin America. Moreover, because of growing population movements, an increasing number of cases of imported Chagas' disease have now been detected in non-endemic areas, such as North America and some European countries. This parasitic zoonosis, caused by Trypanosoma cruzi, is transmitted to humans by infected Triatominae insects, or occasionally by non-vectorial mechanisms, such as blood transfusion, mother to fetus, or oral ingestion of materials contaminated with parasites. Following the acute phase of the infection, untreated individuals enter a chronic phase that is initially asymptomatic or clinically unapparent. Usually, a few decades later, 40-50% of patients develop progressive cardiomyopathy and/or motility disturbances of the oesophagus and colon. In the last decades several interventions targeting primary, secondary and tertiary prevention of Chagas' disease have been attempted. While control of both vectorial and blood transfusion transmission of T cruzi (primary prevention) has been successful in many regions of Latin America, early detection and aetiological treatment of asymptomatic subjects with Chagas' disease (secondary prevention) have been largely underutilised. At the same time, in patients with established chronic disease, several pharmacological and non-pharmacological interventions are currently available and have been increasingly used with the intention of preventing or delaying complications of the disease (tertiary prevention). In this review we discuss in detail each of these issues.

Endothelial Nitric Oxide Genotypes and Haplotypes Are Not Associated with End-Stage Renal Disease

The identification of genetic markers associated with chronic kidney disease (CKD) may help to predict its development. Because reduced nitric oxide (NO) bioavailability and endothelial dysfunction are involved in CKD, genetic polymorphisms in the gene encoding the enzyme involved in NO synthesis (endothelial NO synthase [eNos]) may affect the susceptibility to CKD and the development of end-stage renal disease (ESRD). We compared genotype and haplotype distributions of three relevant eNOS polymorphisms (T(-786) C in the promoter region, Glu298Asp in exon 7, and 4b/4a in intron 4) in 110 healthy control subjects and 127 ESRD patients. Genotypes for the T(-786) C and Glu298Asp polymorphisms were determined by TaqMan (R) Allele Discrimination assay and real-time polymerase chain reaction. Genotypes for the intron 4 polymorphism were determined by polymerase chain reaction and fragment separation by electrophoresis. The software program PHASE 2.1 was used to estimate the haplotypes frequencies. We considered significant a probability value of p < 0.05/number of haplotypes (p < 0.05/8 = 0.0063). We found no significant differences between groups with respect to age, ethnicity, and gender. CKD patients had higher blood pressure, total cholesterol, and creatinine levels than healthy control subjects (all p < 0.05). Genotype and allele distributions for the three eNOS polymorphisms were similar in both groups (p > 0.05). We found no significant differences in haplotype distribution between groups (p > 0.05). The lack of significant associations between eNOS polymorphisms and ESRD suggests that eNOS polymorphisms may not be relevant to the genetic component of CKD that leads to ESRD.

Interethnic Differences in the Distribution of Matrix Metalloproteinases Genetic Polymorphisms Are Consistent with Interethnic Differences in Disease Prevalence

Interethnic differences exist in disease prevalence, especially with regard to cancer and cardiovascular diseases, which involve altered expression or activity of matrix metalloproteinases (MMPs). The hypothesis being tested in this study is that interethnic differences exist between blacks and whites with regard to the distribution of genetic variants of MMP polymorphisms and haplotypes. We examined the distribution of polymorphisms of MMP-2 and MMP-9 genes in 177 black and 140 white subjects. We studied the following polymorphisms: the C(-1306)T in the promoter of the MMP-2 gene, the C(-1562)T and a microsatellite -90(CA)(14-24) in the promoter, and the Q279R in exon 6 of the MMP-9 gene. We have also compared our results with those from Hapmap or Seattle SNPs Projects and estimated the haplotype frequency in these two ethnic groups. The ""C'' allele for the C(-1306)T polymorphism was more common in blacks (91.5%) than in whites (80.4%; p<0.0001). The ""T'' allele for the C(-1562)T polymorphism was more common in blacks (15.0%) than in whites (8.9%; p=0.0279), as well as the alleles with >21 repeats for the -90(CA)(14-24) were more common in blacks than in whites (61.9% in blacks and 49.3% in whites; p=0.0017). We found no interethnic differences for the Q279R polymorphism. Moreover, two haplotypes that combine ""detrimental'' alleles were found at higher frequencies in blacks than in whites (31% vs. 16.4%, respectively; p<0.05). The interethnic differences being reported here replicate those previously found with smaller number of subjects in the Hapmap or Seattle SNPs data and may help explain the higher prevalence of cancer and cardiovascular diseases in blacks compared with whites. Our findings suggest a proportional significance of these polymorphisms in each ethnic group.

A simplified minimal residual disease polymerase chain reaction method at early treatment points can stratify children with acute lymphoblastic leukemia into good and poor outcome groups

Background Minimal residual disease is an important independent prognostic factor in childhood acute lymphoblastic leukemia. The classical detection methods such as multiparameter flow cytometry and real-time quantitative polymerase chain reaction analysis are expensive, time-consuming and complex, and require considerable technical expertise. Design and Methods We analyzed 229 consecutive children with acute lymphoblastic leukemia treated according to the GBTLI-99 protocol at three different Brazilian centers. Minimal residual disease was analyzed in bone marrow samples at diagnosis and on days 14 and 28 by conventional homo/heteroduplex polymerase chain reaction using a simplified approach with consensus primers for IG and TCR gene rearrangements. Results At least one marker was detected by polymerase chain reaction in 96.4%, of the patients. By combining the minimal residual disease results obtained on days 14 and 28, three different prognostic groups were identified: minimal residual disease negative on days 14 and 28, positive on day 14/negative on day 28, and positive on both. Five-year event-free survival rates were 85%, 75.6%,, and 27.8%, respectively (p<0.0001). The same pattern of stratification held true for the group of intensively treated children. When analyzed in other subgroups of patients such as those at standard and high risk at diagnosis, those with positive B-derived CD10, patients positive for the TEL/AML1 transcript, and patients in morphological remission on a day 28 marrow, the event-free survival rate was found to be significantly lower in patients with positive minimal residual disease on day 28. Multivariate analysis demonstrated that the detection of minimal residual disease on day 28 is the most significant prognostic factor. Conclusions This simplified strategy for detection of minimal residual disease was feasible, reproducible, cheaper and simpler when compared with other methods, and allowed powerful discrimination between children with acute lymphoblastic leukemia with a good and poor outcome.

Aspirin Treatment of Mice Infected with Trypanosoma cruzi and Implications for the Pathogenesis of Chagas Disease

Chagas disease, caused by infection with Trypanosoma cruzi, is an important cause of cardiovascular disease. It is increasingly clear that parasite-derived prostaglandins potently modulate host response and disease progression. Here, we report that treatment of experimental T. cruzi infection (Brazil strain) beginning 5 days post infection (dpi) with aspirin (ASA) increased mortality (2-fold) and parasitemia (12-fold). However, there were no differences regarding histopathology or cardiac structure or function. Delayed treatment with ASA (20 mg/kg) beginning 60 dpi did not increase parasitemia or mortality but improved ejection fraction. ASA treatment diminished the profile of parasite-and host-derived circulating prostaglandins in infected mice. To distinguish the effects of ASA on the parasite and host bio-synthetic pathways we infected cyclooxygenase-1 (COX-1) null mice with the Brazil-strain of T. cruzi. Infected COX-1 null mice displayed a reduction in circulating levels of thromboxane (TX)A(2) and prostaglandin (PG)F(2 alpha). Parasitemia was increased in COX-1 null mice compared with parasitemia and mortality in ASA-treated infected mice indicating the effects of ASA on mortality potentially had little to do with inhibition of prostaglandin metabolism. Expression of SOCS-2 was enhanced, and TRAF6 and TNF alpha reduced, in the spleens of infected ASA-treated mice. Ablation of the initial innate response to infection may cause the increased mortality in ASA-treated mice as the host likely succumbs more quickly without the initiation of the ""cytokine storm'' during acute infection. We conclude that ASA, through both COX inhibition and other ""off-target'' effects, modulates the progression of acute and chronic Chagas disease. Thus, eicosanoids present during acute infection may act as immunomodulators aiding the transition to and maintenance of the chronic phase of the disease. A deeper understanding of the mechanism of ASA action may provide clues to the differences between host response in the acute and chronic T. cruzi infection.

Synergistic effect of simvastatin and ezetimibe on lipid and pro-inflammatory profiles in pre-diabetic subjects

Background: Ezetimibe specifically blocks the absorption of dietary and biliary cholesterol and plant sterols. Synergism of ezetimibe-statin therapy on LDL-cholesterol has been demonstrated, but data concerning the pleiotropic effects of this combination are controversial. Objective: This open-label trial evaluated whether the combination of simvastatin and ezetimibe also results in a synergistic effect that reduces the pro-inflammatory status of pre-diabetic subjects. Methods: Fifty pre-diabetic subjects were randomly assigned to one of 2 groups, one receiving ezetimibe (10 mg/day), the other, simvastatin (20 mg/d) for 12 weeks, followed by an additional 12-week period of combined therapy. Blood samples were collected at baseline, 12 and 24 weeks. RESULTS: Total cholesterol, LDL-cholesterol and apolipoprotein B levels decreased in all the periods analyzed (p < 0.01), but triglycerides declined significantly only after combined therapy. Both drugs induced reductions in C-reactive protein, reaching statistical significance after combining ezetimibe with the simvastatin therapy (baseline 0.59 +/- 0.14, simvastatin monotherapy 0.48 +/- 0.12 mg/dL and 0.35 +/- 0.12 mg/dL, p < 0.023). Such a reduction was independent of LDL-cholesterol change. However, mean levels of TNF-alpha and interleukin-6 and leukocyte count did not vary during the whole study. Conclusion: Expected synergistic lowering effects of a simvastatin and ezetimibe combination on LDL-cholesterol, apolipoprotein B and triglycerides levels were confirmed in subjects with early disturbances of glucose metabolism. We suggest an additive effect of this combination also on inflammatory status based on the reduction of C-reactive protein. Attenuation of pro-inflammatory conditions may be relevant in reducing cardiometabolic risk.

Fat and fiber consumption are associated with peripheral arterial disease in a cross-sectional study of a Japanese-Brazilian population

Background The Western diet plays a role for the epidemics of obesity and related diseases. This study examined a possible association between peripheral arterial disease (PAD) and the dietary components of Japanese immigrants living in Brazil. Methods and Results In this cross-sectional study, 1,267 subjects (aged 30 years) with complete dietary, clinical and laboratory data were studied according to a standardized protocol. Ankle-to-brachial index was used to identify subjects with PAD. The overall prevalence of PAD was 14.6%. Subjects with PAD were older, had lower education and higher mean values of blood pressure, triglycerides, and fasting and 2-h plasma glucose levels compared with those without the disease. Among the subjects with PAD, the consumption of fiber from whole grains (3.0 vs 3.4g, p=0.001) and linoleic acids (11.0 vs 11.7g, p=0.017) were lower and intake of total (72.8 vs 69.1 a, p=0.016) and saturated fatty acids (17.4 vs 16.3g, p=0.012) were higher than those without PAD. Results of multiple logistic regression analysis showed a significant association between PAD with high total fat intake, low intake of fiber from fruit and oleic acid, independently of other variables. Conclusions Despite limitations in examining the cause-effect relationship, the data support the notion that diet could be important in reducing the occurrence of PAD.

Artificial Lighting as a Vector Attractant and Cause of Disease Diffusion

BACKGROUND: Traditionally, epidemiologists have considered electrification to be a positive factor. In fact, electrification and plumbing are typical initiatives that represent the integration of an isolated population into modern society, ensuring the control of pathogens and promoting public health. Nonetheless, electrification is always accompanied by night lighting that attracts insect vectors and changes people's behavior. Although this may lead to new modes of infection and increased transmission of insect-borne diseases, epidemiologists rarely consider the role of night lighting in their surveys. OBJECTIVE: We reviewed the epidemiological evidence concerning the role of lighting in the spread of vector-borne diseases to encourage other researchers to consider it in future studies. DISCUSSION: We present three infectious vector-borne diseases-Chagas, leishmaniasis, and malaria-and discuss evidence that suggests that the use of artificial lighting results in behavioral changes among human populations and changes in the prevalence of vector species and in the modes of transmission. CONCLUSION: Despite a surprising lack of studies, existing evidence supports our hypothesis that artificial lighting leads to a higher risk of infection from vector-borne diseases. We believe that this is related not only to the simple attraction of traditional vectors to light sources but also to changes in the behavior of both humans and insects that result in new modes of disease transmission. Considering the ongoing expansion of night lighting in developing countries, additional research on this subject is urgently needed.

Antimicrobial activities of Garcinia kola on oral Fusobacterium nucleatum and biofilm

The extracts from the root, bark and seed of Garcinia kola are currently used in traditional medicine in Nigeria. The aim of this study was to evaluate the inhibitory activity of crude extracts of G. kola on Fusobacterium nucleatum isolated from the oral cavity. Methanol and aqueous extracts were prepared from the seed and the minimal inhibitory concentration was evaluated by the agar dilution method, using a Wilkins-Chalgren agar supplemented with horse blood (5%), hemin (5 mu g/ml) and menadione (1 mu g/ml). Antimicrobial activity of plant extracts on microbial biofilms was determined in microtiter plates. The seed of G. kola demonstrated significant inhibitory action on F. nucleatum isolates at a concentration of 1.25 and 12.5 mg/ml for amoxicillin resistant strain. It was able to inhibit the microbial biofilm formed by the association of F. nucleatum with Porphyromonas gingivalis ATCC 33277, Aggregatibacter actinomycetemcomitans ATCC 33384 and Prevotella intermedia ATCC 2564 at a concentration of 25 mg/ml. The in-vitro inhibitory effect of G. kola on F. nucleatum population suggests a potential role for its use in oral hygiene.

Optical fiber laser induced fluorescence spectroscopy as a citrus canker diagnostic

Citrus canker is a serious disease caused by Xanthomonas citri subsp. citri bacteria, which infects citrus plants (Citrus spp.) leading to large economic losses in citrus production worldwide. In this work, laser induced fluorescence spectroscopy (LIF) was investigated as a diagnostic technique for citrus canker disease in citrus trees at an orchard using a portable optical fiber based spectrometer. For comparison we have applied LIF to leaves contaminated with citrus canker, citrus scab, citrus variegates chlorosis, and Huanglongbing (HLB, Greening). In order to reduce the noise in the data, we collected spectra from ten leaves with visual symptoms of diseases and from five healthy leaves per plant. This procedure is carried out in order to minimize the environmental effect on the spectrum (water and nutrient supply) of each plant. Our results show that this method presents a high sensitivity (similar to 90%), however it does present a low specificity (similar to 70%) for citrus canker diagnostic. We believe that such poor performance is due to the fact that the optical fiber collects light from only a small part of the leaf. Such results may be improved using the fluorescence imaging technique on the whole leaf. (C) 2010 Optical Society of America

Molecular determinants of improved cathepsin B inhibition by new cystatins obtained by DNA shuffling

Background: Cystatins are inhibitors of cysteine proteases. The majority are only weak inhibitors of human cathepsin B, which has been associated with cancer, Alzheimer's disease and arthritis. Results: Starting from the sequences of oryzacystatin-1 and canecystatin-1, a shuffling library was designed and a hybrid clone obtained, which presented higher inhibitory activity towards cathepsin B. This clone presented two unanticipated point mutations as well as an N-terminal deletion. Reversing each point mutation independently or both simultaneously abolishes the inhibitory activity towards cathepsin B. Homology modeling together with experimental studies of the reverse mutants revealed the likely molecular determinants of the improved inhibitory activity to be related to decreased protein stability. Conclusion: A combination of experimental approaches including gene shuffling, enzyme assays and reverse mutation allied to molecular modeling has shed light upon the unexpected inhibitory properties of certain cystatin mutants against Cathepsin B. We conclude that mutations disrupting the hydrophobic core of phytocystatins increase the flexibility of the N-terminus, leading to an increase in inhibitory activity. Such mutations need not affect the inhibitory site directly but may be observed distant from it and manifest their effects via an uncoupling of its three components as a result of increased protein flexibility.

Novel insights into the genomic basis of citrus canker based on the genome sequences of two strains of Xanthomonas fuscans subsp aurantifolii

Background: Citrus canker is a disease that has severe economic impact on the citrus industry worldwide. There are three types of canker, called A, B, and C. The three types have different phenotypes and affect different citrus species. The causative agent for type A is Xanthomonas citri subsp. citri, whose genome sequence was made available in 2002. Xanthomonas fuscans subsp. aurantifolii strain B causes canker B and Xanthomonas fuscans subsp. aurantifolii strain C causes canker C. Results: We have sequenced the genomes of strains B and C to draft status. We have compared their genomic content to X. citri subsp. citri and to other Xanthomonas genomes, with special emphasis on type III secreted effector repertoires. In addition to pthA, already known to be present in all three citrus canker strains, two additional effector genes, xopE3 and xopAI, are also present in all three strains and are both located on the same putative genomic island. These two effector genes, along with one other effector-like gene in the same region, are thus good candidates for being pathogenicity factors on citrus. Numerous gene content differences also exist between the three cankers strains, which can be correlated with their different virulence and host range. Particular attention was placed on the analysis of genes involved in biofilm formation and quorum sensing, type IV secretion, flagellum synthesis and motility, lipopolysacharide synthesis, and on the gene xacPNP, which codes for a natriuretic protein. Conclusion: We have uncovered numerous commonalities and differences in gene content between the genomes of the pathogenic agents causing citrus canker A, B, and C and other Xanthomonas genomes. Molecular genetics can now be employed to determine the role of these genes in plant-microbe interactions. The gained knowledge will be instrumental for improving citrus canker control.

Structure-Function Analysis of the HrpB2-HrcU Interaction in the Xanthomonas citri Type III Secretion System

Bacterial type III secretion systems deliver protein virulence factors to host cells. Here we characterize the interaction between HrpB2, a small protein secreted by the Xanthomonas citri subsp. citri type III secretion system, and the cytosolic domain of the inner membrane protein HrcU, a paralog of the flagellar protein FlhB. We show that a recombinant fragment corresponding to the C-terminal cytosolic domain of HrcU produced in E. coli suffers cleavage within a conserved Asn264-Pro265-Thr266-His267 (NPTH) sequence. A recombinant HrcU cytosolic domain with N264A, P265A, T266A mutations at the cleavage site (HrcU(AAAH)) was not cleaved and interacted with HrpB2. Furthermore, a polypeptide corresponding to the sequence following the NPTH cleavage site also interacted with HrpB2 indicating that the site for interaction is located after the NPTH site. Non-polar deletion mutants of the hrcU and hrpB2 genes resulted in a total loss of pathogenicity in susceptible citrus plants and disease symptoms could be recovered by expression of HrpB2 and HrcU from extrachromossomal plasmids. Complementation of the Delta hrcU mutant with HrcU(AAAH) produced canker lesions similar to those observed when complemented with wild-type HrcU. HrpB2 secretion however, was significantly reduced in the Delta hrcU mutant complemented with HrcU(AAAH), suggesting that an intact and cleavable NPTH site in HrcU is necessary for total functionally of T3SS in X. citri subsp. citri. Complementation of the Delta hrpB2 X. citri subsp. citri strain with a series of hrpB2 gene mutants revealed that the highly conserved HrpB2 C-terminus is essential for T3SS-dependent development of citrus canker symptoms in planta.