Hipometilação do LINE-1 está Associada ao Risco de Doença Cardíaca Coronariana na População Chinesa

Fundamentos: O nível de metilação global do ADN de leucócitos no sangue tem sido associado ao risco de doença arterial coronariana (DAC), com resultados inconsistentes em diferentes populações. Faltam dados semelhantes da população chinesa, onde diferentes fatores genéticos, de estilo de vida e ambientais podem afetar a metilação do ADN e sua relação com o risco de DCC. Objetivos: Analisar se a metilação global está associada ao risco de doença coronariana na população chinesa. Métodos: Foram incluídos um total de 334 casos de DCC e 788 controles saudáveis. A metilação global do ADN de leucócitos de sangue foi estimada por meio da análise das repetições do LINE-1 usando pirosequenciamento de bissulfito. Resultados: Em uma análise inicial restrita aos controles o nível do LINE-1 diminui significativamente com a idade avançada, colesterol total elevado, e diagnóstico de diabetes. Na análise de caso-controle, a redução da metilação do LINE-1 foi associada ao aumento do risco de DCC, tendo a análise por quartil revelado uma odds ratio (IC 95%) de 0,9 (0,6-1,4), 1,9 (1,3-2,9) e 2,3 (1,6 3.5) para o terceiro, segundo e primeiro (o mais baixo) quartil (P da tendência < 0,001), respectivamente, em comparação com o quarto (o mais alto) quartil. A metilação inferior (< mediana) do LINE-1 esteve associada a 2,2 vezes (IC 95% = 1,7-3,0) o aumento de risco de doença coronariana. As estimativas de risco de DCC menores relacionadas com o LINE-1 tenderam a ser mais fortes entre os indivíduos com maior tercil de homocisteína (P interação = 0,042) e naqueles com diagnóstico de hipertensão arterial (P interação = 0,012). Conclusão: A hipometilação do LINE-1 está associada ao risco de doença coronariana na população chinesa. Fatores de risco de DCC potenciais tais como a idade avançada, colesterol total elevado, e diagnóstico de diabetes podem ter impacto na metilação global do ADN, exercendo, portanto, o seu efeito sobre o risco de doença coronariana.

Prognostic Value of TIMI Score versus GRACE Score in ST-segment Elevation Myocardial Infarction

Background: The TIMI Score for ST-segment elevation myocardial infarction (STEMI) was created and validated specifically for this clinical scenario, while the GRACE score is generic to any type of acute coronary syndrome. Objective: Between TIMI and GRACE scores, identify the one of better prognostic performance in patients with STEMI. Methods: We included 152 individuals consecutively admitted for STEMI. The TIMI and GRACE scores were tested for their discriminatory ability (C-statistics) and calibration (Hosmer-Lemeshow) in relation to hospital death. Results: The TIMI score showed equal distribution of patients in the ranges of low, intermediate and high risk (39 %, 27 % and 34 %, respectively), as opposed to the GRACE Score that showed predominant distribution at low risk (80 %, 13 % and 7%, respectively). Case-fatality was 11%. The C-statistics of the TIMI score was 0.87 (95%CI = 0.76 to 0.98), similar to GRACE (0.87, 95%CI = 0.75 to 0.99) - p = 0.71. The TIMI score showed satisfactory calibration represented by χ2 = 1.4 (p = 0.92), well above the calibration of the GRACE score, which showed χ2 = 14 (p = 0.08). This calibration is reflected in the expected incidence ranges for low, intermediate and high risk, according to the TIMI score (0 %, 4.9 % and 25 %, respectively), differently to GRACE (2.4%, 25% and 73%), which featured middle range incidence inappropriately. Conclusion: Although the scores show similar discriminatory capacity for hospital death, the TIMI score had better calibration than GRACE. These findings need to be validated populations of different risk profiles.

Very Long-Term Prognostic Role of Admission BNP in Non-ST Segment Elevation Acute Coronary Syndrome

Abstract Background: BNP has been extensively evaluated to determine short- and intermediate-term prognosis in patients with acute coronary syndrome, but its role in long-term mortality is not known. Objective: To determine the very long-term prognostic role of B-type natriuretic peptide (BNP) for all-cause mortality in patients with non-ST segment elevation acute coronary syndrome (NSTEACS). Methods: A cohort of 224 consecutive patients with NSTEACS, prospectively seen in the Emergency Department, had BNP measured on arrival to establish prognosis, and underwent a median 9.34-year follow-up for all-cause mortality. Results: Unstable angina was diagnosed in 52.2%, and non-ST segment elevation myocardial infarction, in 47.8%. Median admission BNP was 81.9 pg/mL (IQ range = 22.2; 225) and mortality rate was correlated with increasing BNP quartiles: 14.3; 16.1; 48.2; and 73.2% (p < 0.0001). ROC curve disclosed 100 pg/mL as the best BNP cut-off value for mortality prediction (area under the curve = 0.789, 95% CI= 0.723-0.854), being a strong predictor of late mortality: BNP < 100 = 17.3% vs. BNP ≥ 100 = 65.0%, RR = 3.76 (95% CI = 2.49-5.63, p < 0.001). On logistic regression analysis, age >72 years (OR = 3.79, 95% CI = 1.62-8.86, p = 0.002), BNP ≥ 100 pg/mL (OR = 6.24, 95% CI = 2.95-13.23, p < 0.001) and estimated glomerular filtration rate (OR = 0.98, 95% CI = 0.97-0.99, p = 0.049) were independent late-mortality predictors. Conclusions: BNP measured at hospital admission in patients with NSTEACS is a strong, independent predictor of very long-term all-cause mortality. This study allows raising the hypothesis that BNP should be measured in all patients with NSTEACS at the index event for long-term risk stratification.

On the current existence of a coccidial line of development in the malaria parasites: a theory

Most opinion favors the origin of the malaria parasites from a coccidial ancestor. It is assumed that whatever the process through which the coccidia differentiated into a Plasmodium this phenomenon very probably occured millions of year ago, and during that differentiation process the original coccidia vanished. Therefore it has never repeated. At the light of some experiments the existence, at the present time, of a coccidial cycle of development in the malaria parasites, is proposed. The conection routes and mechanisms through which the malaria parasite changes to a coccidial life, and the routes in reverse are exposed. Transmission of the malaria-coccidial forms is suggested.

Establishment and Characterization of a Cell Line from the Mosquito Anopheles albimanus (Diptera: Culicidae)

A new cell line designated LSB-AA695BB, was established from embryos of the mosquito Anopheles albimanus. The primary culture was initiated in April, 1995, and the first passage was made 48 days later. Serial subcultures of the cells have been carried through 90 passages from Abril 1995 to February 1996. The cells were grown at 28°C in MK/VP12 medium, supplemented with 20% fetal bovine serum; the pH tolerance ranged between 6.8 to 7.0. The cells have also been adapted to MM/VP12 medium under the same pH, temperature and serum concentration. The majority of the cells were a fibroblast-type. Isozyme characterization showed a pattern similar to that of An. albimanus pupae and adults but distinct from Ae. taeniorhynchus and Ae. albopictus (C6/36) mosquito cell lines. The culture was shown to be free of mycoplasma, bacteria and fungi. Microsporidia contamination of transovarial transmission was controlled with 6.0 mg/ml of albendazole

Establishment and characterization of a new continuous cell line from Lutzomyia longipalpis (Diptera: Psychodidae) and its susceptibility to infections with arboviruses and Leishmania chagasi

Embryonic tissue explants of the sand fly Lutzomyia longipalpis (Lutz & Neiva 1912) the main vector of Leishmania chagasi (Cunha and Chagas), were used to obtain a continuous cell line (Lulo). The tissues were seeded in MM/VP12 medium and these were incubated at 28ºC. The first subculture was obtained 45 days after explanting and 96 passages have been made to date. Lulo is composed of epithelioid cells, showed a 0.04 generations/hour exponential growth rate and population doubling time at 24.7 h. The cell line isoenzymatic profiles were determined by using PGI, PGM, MPI and 6-PGDH systems, coinciding with patterns obtained from the same species and colony's pupae and adults. The species karyotype characteristics were recognized (2n = 8), in which pair 1 is subtelocentric and pairs 2, 3 and 4 are metacentric. Lulo was free from bacterial, fungal, mycoplasmic and viral infection. Susceptibility to five arbovirus was determined, the same as Lulo interaction with Leishmania promastigotes.

A new continuous cell line from the mosquito Psorophora confinnis (Diptera: Culicidae) and its susceptibility to infections with some arboviruses

A new cell line, PC-0199-BR, was established from embryonated eggs of the mosquito Psorophora confinnis. To date (September 2000) it has had 62 continuous passages. This is the first report of a cell line of mosquitoes belonging to the genus Psorophora. Cell growth initially was achieved in the MM/VP12 medium, supplemented with 20% fetal bovine serum; however, the subcultures were later adapted to Grace's medium with 10% fetal bovine serum. Cell morphology in the primary cultures was heterogeneous; but later in the established cell line, the predominant cell type was epithelioid. Cultured cells were predominantly diploid (2n=6); however, chromosome abnormalities were observed in a small proportion of the cells in later passages. C and G band patterns were also determined in the karyotype. The cell line isozyme profiles coincided with pupae and adult samples of the species taken from the same colony. A preliminary arbovirus susceptibility study for the cell line was undertaken. No evidence was observed of contamination of the cell line with bacteria, fungi or mycoplasma.

Laboratory diagnosis of Schistosomiasis in areas of low transmission: a review of a line of research

After 57 years of successful control of schistosomiasis in Venezuela, the prevalence and intensity of infection have declined. Approximately 80% of the individuals eliminate less than 100 eggs/g of stools, therefore morbidity is mild and the majority are asymptomatic. The sensitivity of Kato-Katz decreases to approximately 60%. Available serological methods for the detection of circulating antigens only reach a 70% of sensitivity. Tests based on the detection of antibodies by immunoenzymatic assays have been improved. The circumoval precipitine test has shown a high sensitivity (97%), specificity (100%), and correlation with oviposition, being considered the best confirmatory diagnostic test. Additionally to the classical immunoenzymatic assays, the development of the alkaline phosphatase immunoassay, allowed to reach a 100% specificity with an 89% sensitivity. Recently, we have developed a modified ELISA in which the soluble egg antigen is treated with sodium metaperiodate (SMP-ELISA) in order to eliminate the glycosilated epitopes responsible for the false positive reactions. The specificity and sensitivity reaches 97% and 99%, respectively. Synthetic peptides from the excretory-secretory enzymes, cathepsin B (Sm31) legumain (Sm32) and cathepsin D (Sm45), have been synthesized. The combination of two peptides derived from the Sm31 have been evaluated, reaching a sensitivity of 96% when analyzed independently and with a 100% specificity. Antibodies raised in rabbits against peptides derived from the Sm31 and Sm32 are currently evaluated in two different antigen-capture-based assays. The development of a simple, cheap and reliable test that correlates with parasite activity is a major goal.

Evaluation of indirect susceptibility testing of Mycobacterium tuberculosis to the first- and second-line, and alternative drugs by the newer MB/BacT system

In order to evaluate the Organon Teknika MB/BacT system used for testing indirect susceptibility to the alternative drugs ofloxacin (OFLO), amikacin (AMI), and rifabutin (RIF), and to the usual drugs of standard treatment regimes such as rifampin (RMP), isoniazid (INH), pyrazinamide (PZA), streptomycin (SM), ethambutol (EMB), and ethionamide (ETH), cultures of clinical specimens from 117 patients with pulmonary tuberculosis under multidrug-resistant investigation, admitted sequentially for examination from 2001 to 2002, were studied. Fifty of the Mycobacterium tuberculosis cultures were inoculated into the gold-standard BACTEC 460 TB (Becton Dickinson) for studying resistance to AMI, RIF, and OFLO, and the remaining 67 were inoculated into Lowenstein Jensen (LJ) medium (the gold standard currently used in Brazil) for studying resistance to RMP, INH, PZA, SM, EMB, and ETH. We observed 100% sensitivity for AMI (80.8-100), RIF (80.8-100), and OFLO (78.1-100); and 100% specificity for AMI (85.4-100), RIF (85.4-100), and OFLO (86.7-100) compared to the BACTEC system. Comparing the results obtained in LJ we observed 100% sensitivity for RMP (80-100), followed by INH - 95% (81.8-99.1), EMB - 94.7% (71.9-99.7), and 100% specificity for all drugs tested except for PZA - 98.3 (89.5-99.9) at 95% confidence interval. The results showed a high level of accuracy and demonstrated that the fully automated, non-radiometric MB/BacT system is indicated for routine use in susceptibility testing in public health laboratories.

Rabies neutralizing antibody detection by indirect immunperoxidase serum neutralization assay performed on chicken embryo related cell line

The aim of this study was to evaluate the indirect immunoperoxidase virus neutralization (IPVN) and mouse neutralization test (MNT) to detect antibodies against rabies virus from vaccinated dogs and cattle. The IPVN was set up for the ability to measure 0.5 International Units/ml (IU) of antibody required by the World Health Organization and the Office International des Epizooties as the minimum response for proof of rabies immunization. IPVN was developed and standardized in chicken embryo related (CER) cell line when 141 dog and 110 cattle sera were applied by serial five-fold dilutions (1:5, 1:25, 1:125) as well as the positive and negative reference controls, all added in four adjacent wells, of 96-well microplates. A 50 µl amount of CVS32 strain dilution containing 50-200 TCID50/ml was mixed to each serum dilution, and after 90 min 50 µl of 3 x 10(5) cells/mlcell suspension added to each well. After five days of incubation, the monolayers were fixed and the IPVN test performed. The correlation coefficient between the MNT and IPVN performed in CER cells was r = 0.9949 for dog sera (n = 100) and r = 0.9307 for cattle sera (n = 99), as well as good specificity (94.7%), sensitivity (87.5%), and agreement (96.6%) were also obtained. IPVN technique can adequately identify vaccinated and unvaccinated animals, even from low-responding vaccinated animals, with the advantage of low cost and faster then MNT standard test.

Experimental infection of Leishmania (L.) chagasi in a cell line derived from Lutzomyia longipalpis (Diptera:Psychodidae)

The present work describes the in vitro infection of a cell line Lulo, derived from Lutzomyia longipalpis embryonic tissue, by Leishmania chagasi promastigotes. This infection process is compared with a parallel one developed using the J774 cell line. The L. chagasi MH/CO/84/CI-044B strain was used for experimental infection in two cell lines. The cells were seeded on glass coverslips in 24-well plates to reach a final number of 2 x 10(5) cells/well. Parasites were added to the adhered Lulo and J774 cells in a 10:1 ratio and were incubated at 28 and 37ºC respectively. After 2, 4, 6, 8, and 10 days post-infection, the cells were extensively washed with PBS, fixed with methanol, and stained with Giemsa. The number of internalized parasites was determined by counting at least 400 cultured cells on each coverslip. The results showed continuous interaction between L. chagasi promastigotes with the cell lines. Some ultrastructural characteristics of the amastigote forms were observed using transmission electron microscopy. The highest percentage of infection in Lulo cells was registered on day 6 post-infection (29.6%) and on day 4 in the J774 cells (51%). This work shows similarities and differences in the L. chagasi experimental infection process in the two cell lines. However, Lulo cells emerge as a new model to study the life-cycle of this parasite.

Increased resistance to first-line agents among bacterial pathogens isolated from urinary tract infections in Latin America: time for local guidelines?

Emerging resistance phenotypes and antimicrobial resistance rates among pathogens recovered from community-acquired urinary tract infections (CA-UTI) is an increasing problem in specific regions, limiting therapeutic options. As part of the SENTRY Antimicrobial Surveillance Program, a total of 611 isolates were collected in 2003 from patients with CA-UTI presenting at Latin American medical centers. Each strain was tested in a central laboratory using Clinical Laboratory Standard Institute (CLSI) broth microdilution methods with appropriate controls. Escherichia coli was the leading pathogen (66%), followed by Klebsiella spp. (7%), Proteus mirabilis (6.4%), Enterococcus spp. (5.6%), and Pseudomonas aeruginosa (4.6%). Surprisingly high resistance rates were recorded for E. coli against first-line orally administered agents for CA-UTI, such as ampicillin (53.6%), TMP/SMX (40.4%), ciprofloxacin (21.6%), and gatifloxacin (17.1%). Decreased susceptibility rates to TMP/SMX and ciprofloxacin were also documented for Klebsiella spp. (79.1 and 81.4%, respectively), and P. mirabilis (71.8 and 84.6%, respectively). For Enterococcus spp., susceptibility rates to ampicillin, chloramphenicol, ciprofloxacin, and vancomycin were 88.2, 85.3, 55.9, and 97.1%, respectively. High-level resistance to gentamicin was detected in 24% of Enterococcus spp. Bacteria isolated from patients with CA-UTI in Latin America showed limited susceptibility to orally administered antimicrobials, especially for TMP/SMX and fluoroquinolones. Our results highlight the need for developing specific CA-UTI guidelines in geographic regions where elevated resistance to new and old compounds may influence prescribing decisions.

Hitchhiking Trypanosoma cruzi minicircle DNA affects gene expression in human host cells via LINE-1 retrotransposon

The horizontal transfer of Trypanosoma cruzi mitochondrial minicircle DNA to the genomes of naturally infected humans may play an important role in the pathogenesis of Chagas disease. Minicircle integrations within LINE-1 elements create the potential for foreign DNA mobility within the host genome via the machinery associated with this retrotransposon. Here we document integration of minicircle DNA fragments in clonal human macrophage cell lines and their mobilization over time. The movement of an integration event in a clonal transfected cell line was tracked at three months and three years post-infection. The minicircle sequence integrated into a LINE-1 retrotransposon; one such foreign fragment subsequently relocated to another genomic location in association with associated LINE-1 elements. The p15 locus was altered at three years as a direct effect of minicircle/LINE-1 acquisition, resulting in elimination of p15 mRNA. Here we show for the first time a molecular pathology stemming from mobilization of a kDNA/LINE-1 mutation. These genomic changes and detected transcript variations are consistent with our hypothesis that minicircle integration is a causal component of parasite-independent, autoimmune-driven lesions seen in the heart and other target tissues associated with Chagas disease.