Blood cell adhesion to arterial filters analysis by scanning electron microscopy and real-time PCR assay: observational clinical study in cardiac surgery patients.

INTRODUCTION: Arterial filter is the part of the cardiopulmonary bypass circuit where blood cells are exposed to high mechanical stress and where cellular aggregates may fasten in large quantities. The aim of this study was to analyse blood cell adhesiveness in the arterial filter through scanning electron microscopy and real-time PCR assay. METHODS: Prospective, clinical and observational study performed on 28 patients undergoing cardiac surgery with cardiopulmonary bypass. Arterial filters were analysed by scanning electron microscopy. Real-time PCR assay was performed in extracted material from the arterial filters for analysis of platelet GPIb and CD45 leucocyte gene expression. Blood coagulation was analysed during cardiopulmonary bypass. Patients were followed until hospital discharge or 28 days after surgery. RESULTS: All studied arterial filters used in the subject patients showed a degree of adhesion from blood elements at scanning electron microscopy. All studied filters were positive for platelets GPIb gene expression and 15% had CD45 leucocyte gene expression. The GPIb platelet gene expression in blood lowered at the end of cardiopulmonary bypass (p = 0.019). There was negative correlation between blood GPIb platelet gene expression and Clot SR (HEPSCREEN2 ReoRox®) (rho = 0.635; p = 0.027). The filter fields count was correlated to the D-dimer dosage (rho = 0.828; p < 0.001). CONCLUSION: There was adhesion of blood elements, especially nucleated platelets, on all arterial filters studied. Although the arterial filter worked as a safety device, that possibly prevented arterial embolisation, it may also have caused greater hyperfibrinolysis during cardiopulmonary bypass.

Usefulness of PCR for Trypanosoma cruzi DNA in blood and endomyocardial biopsies for detection of Chagas disease reactivation after heart transplantation: A comparative study.

BACKGROUND: Chagas disease reactivation (CDR) after heart transplantation is characterized by relapse of the infectious disease with proliferation and dissemination of Trypanosoma cruzi parasites. Serial blood PCR testing is consensually recommended for CDR monitoring, but there is uncertainty about the incremental value in performing the molecular tests in endomyocardial biopsies (EMB). METHODS: We compared qualitative and quantitative results of PCR for T cruzi DNA in 62 pairs of blood and EMB collected with a maximum time interval of 7 days, from 34 heart-transplanted, chagasic patients. RESULTS: Blood PCR resulted positive in 39/62 (62.9%) samples, with PL ranging from 0.14 to 1610.73 (median: 3.31). PCR resulted positive in 8/60 (13.3%) EMB, with PL ranging from 2.82 to 1670.55 (median: 65.63). All blood samples which tested negative presented a paired EMB which also tested negative. However, 31/39 (79.5%) blood samples which tested positive presented a paired EMB which tested negative. There was poor agreement between blood and EMB PCR (kappa = 0.153). CDR affecting the myocardium (myo-CDR) was diagnosed in three occasions. PCR resulted positive in both blood and EMB at the time of myo-CDR, with PL ranging from 0.61 to 1610.73 in blood and 13.8 to 1670.55 in EMB. CONCLUSIONS: Negative PCR for T cruzi in blood rules out myo-CDR, with no value of testing EMB. Positive PCR in blood with high PL is diagnostic for myo-CDR. If PCR in blood results positive with low PL, testing EMB is useful: negative PCR turns unlikely, and positive PCR reinforces greatly the possibility of myo-CDR.

Sequential measurement of Trypanosoma cruzi parasitic load in endomyocardial biopsies for early detection and follow-up of Chagas disease reactivation after heart transplantation.

BACKGROUND: Reactivation of Chagas disease after heart transplantation is characterized by proliferation and dissemination of Trypanosoma cruzi parasites to several organs. Reactivation affecting the allograft can simulate acute cellular rejection, from which it should be distinguished through the analysis of endomyocardial biopsies (EMB). METHODS: We evaluated retrospectively 100 EMB collected in the first year of follow-up from 13 heart-transplanted, chagasic patients who presented reactivation and were successfully treated. Additionally, 37 EMB from 8 patients who did not present reactivation constituted the control group. We reviewed histopathology and performed a real-time PCR-based assay in order to evaluate the T cruzi parasitic load of each EMB. RESULTS: The parasitic load of the EMB at the time of reactivation ranged from 22.80 to 190 000/106 cells (median: 1555). In 6 patients, none of the EMB obtained prior to reactivation amplified T cruzi DNA. On the other hand, 10 EMB from 7 patients, obtained 9-105 days before reactivation (median: 26 days), showed parasitic load ranging from 8.25 to 625/106 cells (median: 167.55). In all patients, the parasitic load increased at the time of reactivation, usually sharply. After initiation of treatment, all patients showed negative PCR or a dramatic reduction of the parasitic load in the following EMB. None of the EMB from the control group amplified T cruzi DNA. CONCLUSIONS: Sequential measurement of T cruzi parasitic load in EMB is useful for monitoring Chagas disease reactivation after heart transplantation. Its increase suggests imminent reactivation and its decrease after treatment indicates favorable evolution for cure of the episode of reactivation.

Down-regulation of fibroblast growth factor 2 and its co-receptors heparan sulfate proteoglycans by resveratrol underlies the improvement of cardiac dysfunction in experimental diabetes.

Cardiac remodeling in diabetes involves cardiac hypertrophy and fibrosis, and fibroblast growth factor 2 (FGF2) is an important mediator of this process. Resveratrol, a polyphenolic antioxidant, reportedly promotes the improvement of cardiac dysfunction in diabetic rats. However, little information exists linking the amelioration of the cardiac function promoted by resveratrol and the expression of FGF2 and its co-receptors, heparan sulfate proteoglycans (HSPGs: Glypican-1 and Syndecan-4), in cardiac muscle of Type 2 diabetic rats. Diabetes was induced experimentally by the injection of streptozotocin and nicotinamide, and the rats were treated with resveratrol for 6 weeks. According to our results, there is an up-regulation of the expression of genes and/or proteins of Glypican-1, Syndecan-4, FGF2, peroxisome proliferator-activated receptor gamma and AMP-activated protein kinase in diabetic rats. On the other hand, resveratrol treatment promoted the attenuation of left ventricular diastolic dysfunction and the down-regulation of the expression of all proteins under study. The trigger for the changes in gene expression and protein synthesis promoted by resveratrol was the presence of diabetes. The negative modulation conducted by resveratrol on FGF2 and HSPGs expression, which are involved in cardiac remodeling, underlies the amelioration of cardiac function.

Gene mutations of platelet glycoproteins and response to tirofiban in acute coronary syndrome / Mutações gênicas das glicoproteínas plaquetárias e resposta ao tirofiban na síndrome coronariana aguda

CONTEXT AND OBJECTIVES: Glycoprotein inhibitors (abciximab, eptifibatide and tirofiban) are used in patients with unstable angina and non-ST-segment elevation myocardial infarction before percutaneous coronary intervention. Of these, tirofiban is the least effective. We hypothesized that the response to tirofiban might be associated with glycoprotein gene mutations. DESIGN AND SETTING: Prospective study at Emergency Unit, Heart Institute (InCor), University of São Paulo. METHOD: Intrahospital evolution and platelet aggregation in response to tirofiban were analyzed in relation to four glycoprotein mutations in 50 patients indicated for percutaneous coronary intervention: 17 (34%) with unstable angina and 33 (66%) with non-ST-segment elevation myocardial infarction. Platelet aggregation was analyzed using the Born method. Blood samples were obtained before and one hour after tirofiban infusion. Glycoproteins Ia (807C/T ), Ib (Thr/Met) , IIb (Ile/Ser ) and IIIa (PIA ) were the mutations selected. RESULTS: Hypertension, dyslipidemia, diabetes, smoking, previous coronary artery disease and stroke were similar between the groups. Mutant glycoprotein IIIa genotypes had lower platelet aggregation before tirofiban administration than that of the wild genotype (41.0% ± 22.1% versus 55.9% ± 20.8%; P = 0.035). Mutant glycoprotein IIIa genotypes correlated moderately with lower platelet inhibition (r = -0.31; P = 0.030). After tirofiban administration, platelet glycoprotein Ia, Ib, IIb and IIIa mutations did not influence the degree of inhibition of platelet aggregation or intrahospital mortality. CONCLUSIONS: Mutations of glycoproteins Ia, Ib, IIb and IIIa did not influence platelet aggregation in response to tirofiban in patients with unstable angina and non-ST-segment elevation myocardial infarction.

RESUMO CONTEXTO E OBJETIVOS: Inibidores da glicoproteína (abciximab, eptifibatide, tirofiban) são utilizados em pacientes com angina instável e infarto do miocárdio sem elevação do segmento ST (IAMSSST) antes da intervenção coronária percutânea. Dentre eles, o tirofiban é o menos eficaz. Nossa hipótese é que a resposta ao tirofiban possa estar associada a mutações no gene da glicoproteína. DESENHO E LOCAL: Estudo prospectivo na Unidade de Emergência do Instituto do Coração (InCor), Universidade de São Paulo (USP). MÉTODOS: Foram analisadas a evolução intra-hospitalar e agregabilidade plaquetária em resposta ao tirofiban de 4 mutações da glicoproteína em 50 pacientes com indicação para intervenção coronária percutânea, 17 (34%) com angina instável e 33 (66%) com IAMSSST. A agregação plaquetária foi analisada pelo método de Born. Amostras de sangue foram obtidas antes e uma hora após infusão do tirofiban. As glicoproteínas Ia (807C/T ), Ib (Thr/Met ), IIb (Ile/Ser ) e IIIa (PIA ) foram as mutações selecionadas. RESULTADOS: Hipertensão, dislipidemia, diabetes, tabagismo, doença coronariana e acidente vascular cerebral prévios foram semelhantes entre os grupos. Observou-se menor agregabilidade plaquetária dos genótipos mutantes da glicoproteína IIIa antes da administração de tirofiban do genótipo selvagem (41% ± 22% versus 56% ± 21%; P = 0,035). Genótipos mutantes da glicoproteína IIIa correlacionaram-se moderadamente com menor inibição plaquetária (r = -0,31; P = 0,030). Após a administração tirofiban, as mutações das glicoproteínas Ia, Ib, IIb, e IIIa não influenciaram o grau de inibição da agregação plaquetária e mortalidade intra-hospitalar. CONCLUSÕES: Mutações das glicoproteínas Ia, Ib, IIb e IIIa não influenciaram a agregação plaquetária em resposta ao tirofiban nos pacientes com angina instável e IAMSSST.

Gene mutations of platelet glycoproteins and response to tirofiban in acute coronary syndrome.

CONTEXT AND OBJECTIVES: Glycoprotein inhibitors (abciximab, eptifibatide and tirofiban) are used in patients with unstable angina and non-ST-segment elevation myocardial infarction before percutaneous coronary intervention. Of these, tirofiban is the least effective. We hypothesized that the response to tirofiban might be associated with glycoprotein gene mutations. DESIGN AND SETTING: Prospective study at Emergency Unit, Heart Institute (InCor), University of São Paulo. METHOD: Intrahospital evolution and platelet aggregation in response to tirofiban were analyzed in relation to four glycoprotein mutations in 50 patients indicated for percutaneous coronary intervention: 17 (34%) with unstable angina and 33 (66%) with non-ST-segment elevation myocardial infarction. Platelet aggregation was analyzed using the Born method. Blood samples were obtained before and one hour after tirofiban infusion. Glycoproteins Ia (807C/T ), Ib (Thr/Met) , IIb (Ile/Ser ) and IIIa (PIA ) were the mutations selected. RESULTS: Hypertension, dyslipidemia, diabetes, smoking, previous coronary artery disease and stroke were similar between the groups. Mutant glycoprotein IIIa genotypes had lower platelet aggregation before tirofiban administration than that of the wild genotype (41.0% ± 22.1% versus 55.9% ± 20.8%; P = 0.035). Mutant glycoprotein IIIa genotypes correlated moderately with lower platelet inhibition (r = -0.31; P = 0.030). After tirofiban administration, platelet glycoprotein Ia, Ib, IIb and IIIa mutations did not influence the degree of inhibition of platelet aggregation or intrahospital mortality. CONCLUSIONS: Mutations of glycoproteins Ia, Ib, IIb and IIIa did not influence platelet aggregation in response to tirofiban in patients with unstable angina and non-ST-segment elevation myocardial infarction.

Correlation of bacterial coinfection versus matrix metalloproteinase 9 and tissue inhibitor of metalloproteinase 1 expression in aortic aneurysm and atherosclerosis.

BACKGROUND: We searched for any relationship between Chlamydophila pneumoniae, Mycoplasma pneumoniae, matrix metalloproteinase 9 (MMP-9), and tissue inhibitor of metalloproteinase 1 (TIMP-1) in aneurysmatic atherosclerotic lesions, and whether this relationship differed from that in atherosclerotic nonaneurysmatic lesions. METHODS: Twenty-eight tissue samples paired by age and sex were grouped as follows: group 1 included 14 nonaneurysmal atherosclerotic fragments obtained from abdominal aortas collected from necropsies; group 2 included 14 aneurysmatic atherosclerotic aortic fragments obtained from patients during corrective surgery. Immunohistochemistry reactions were evaluated for C pneumoniae, M pneumoniae, MMP-9, and TIMP-1 antigens. Both groups were compared using the Mann-Whitney test, and the correlations among variables were obtained using the Spearman correlation test. P ≤ 0.05 was considered statistically significant. RESULTS: C pneumoniae and M pneumoniae antigens were detected in 100% of cases. A higher amount of C pneumoniae (P = 0.005), M pneumoniae (P = 0.002), and MMP-9 (P = 0.021) was found in adventitia of group 2 with aneurysm. A positive correlation was found in the aneurysm group, as follows: intima C pneumoniae versus adventitia thickness (r = 0.70; P = 0.01), media C pneumoniae versus adventitia C pneumoniae (r = 0.75; P = 0.002), intima C pneumoniae versus media C pneumoniae (r = 0.8; P = 0.00), and adventitia C pneumoniae versus intima M pneumoniae (r = 0.54; P = 0.05); negative correlations were as follows: adventitia thickness and adventitia M pneumoniae (r = -0.65; P = 0.01), media MMP-9 and media thickness (r = -0.55; P = 0.04), TIMP-1 media versus adventitia C pneumoniae (r = -0.86; P = 0.00), and TIMP-1 media versus M pneumoniae intima (r = -0.67; P = 0.03). Nonaneurysmal atherosclerotic group 1 results are as follows: adventitia C pneumoniae versus TIMP-1 media (r = 0.75; P = 0.01) and media C pneumoniae and adventitia C pneumoniae (r = 0.59; P = 0.03). CONCLUSIONS: The present work favors a role for coinfection of both M pneumoniae and C pneumoniae in the development of aortic atherosclerotic aneurysm, with increased adventitial inflammation, inhibition of TIMP-1 activity, and increased collagen degradation.

Usefulness of qualitative polymerase chain reaction for Trypanosoma cruzi DNA in endomyocardial biopsy specimens of chagasic heart transplant patients.

BACKGROUND: Chagas' disease reactivation (CDR) after heart transplantation is characterized by relapse of the infectious disease, with direct detection of Trypanosoma cruzi parasites in blood, cerebrospinal fluid, or tissues. CDR affecting the myocardium induces lymphocytic myocarditis and should be distinguished from acute cellular rejection in endomyocardial biopsy (EMB) specimens. METHODS: We performed retrospectively qualitative polymerase chain reaction for T cruzi DNA using 2 sets of primers targeting nuclear DNA (nDNA) or kinetoplast DNA (kDNA) in 61 EMB specimens of 11 chagasic heart transplant recipients who presented with CDR. Thirty-five EMB specimens were obtained up to 6 months before (pre-CDR group) and 26 up to 2 years after the diagnosis of CDR. The control group consisted of 6 chagasic heart transplant recipients with 18 EMB specimens who never experienced CDR. RESULTS: Amplification of kDNA occurred in 8 of 35 (22.9%) EMB specimens of the pre-CDR group, in 5 of 18 (27.8%) of the control group, and in 17 of 26 (65.4%) EMB specimens obtained after the successful treatment of CDR. Amplification of nDNA occurred in 3 of 35 (8.6%) EMB specimens of the pre-CDR group, 0 of 18 (0%) of the control group, and 6 of 26 (23.1%) EMB specimens obtained after the successful treatment of CDR. CONCLUSIONS: Amplification of kDNA in EMB specimens is not specific for the diagnosis of CDR, occurring also in patients with no evidence of CDR (control group). However, amplification of nDNA occurred in a few EMB specimens obtained before CDR, but in none of the control group specimens. Qualitative PCR for T cruzi DNA in EMB specimens should not be used as a criterion for cure of CDR because it can persist positive despite favorable clinical evolution of the patients.

A role for archaeal organisms in development of atherosclerotic vulnerable plaques and myxoid matrices

PURPOSE: Vulnerable plaques are characterized by a myxoid matrix, necrotic lipidic core, reactive oxygen species, and high levels of microorganisms. Aerobic microbes such as Chlamydophila pneumoniae and Mycoplasma pneumoniae usually do not survive in oxidative stress media. Archaea are anaerobic microbes with powerful anti-oxidative enzymes that allow detoxification of free radicals whose presence might favor the survival of aerobic microorganisms. We searched for archaeal organisms in vulnerable plaques, and possible associations with myxoid matrix, chlamydia, and mycoplasma bodies. METHODS: Twenty-nine tissue samples from 13 coronary artherectomies from large excentric ostial or bifurcational lesions were studied using optical and electron microscopy. Infectious agents compatible with archaea, chlamydia, and mycoplasma were semiquantified using electron micrographs and correlated with the amounts of fibromuscular tissue, myxoid matrix, and foam cells, as determined from semi-thin sections. Six of the cases were also submitted to polymerase chain reaction with archaeal primers. RESULTS: All 13 specimens showed archaeal-compatible structures and chlamydial and mycoplasmal bodies in at least 1 sample. There was a positive correlation between extent of the of myxoid matrix and archaeal bodies (r = 0.44, P = 0.02); between archaeal and mycoplasmal bodies (r = 0.41, P = 0.03), and between chlamydial bodies and foam cells (r = 0.42; P = 0.03). The PCR test was positive for archaeal DNA in 4 of the 6 fragments. DISCUSSION: DNA and forms suggestive of archaea are present in vulnerable plaques and may have a fundamental role in the proliferation of mycoplasma and chlamydia. This seems to be the first description of apparently pathogenic archaea in human internal organ lesions.

PROPOSTA: Placas vulneráveis são caracterizadas por matriz mixomatosa, centro lipídico necrótico, espécies reativas de oxigênio e alto níveis de microorganismos. Micróbios aeróbicos como Chlamydophila pneumoniae e Mycoplasma pneumoniae usualmente não sobrevivem em meio de estresse oxidativo. Arquéias são microorganismos anaeróbicos com poderosas enzimas anti-oxidantes que permitem detoxificação de radicais livres e a presença delas poderia favorecer a sobrevivência de micróbios aeróbicos. Pesquisamos por elementos de arquéia em placas vulneráveis e sua possível associação com degeneração mixomatosa da matriz e aumento do número de clamídias e micoplasmas. MÉTODOS: Vinte e nove amostras de 13 produtos de aterotomia de lesões grandes e excêntricas de óstio ou bifurcação de coronárias foram estudadas pela microscopia óptica e eletrônica. Agentes compatíveis com arquéia, clamídia e micoplasma foram semiquantificados pela microscopia eletrônica e correlacionados com quantidade de tecido fibromuscular, matriz mixomatosa e células xantomatosas. Seis casos foram também submetidos à reação em cadeia da polimerase com oligonucleotídeos de arquéia. RESULTADOS: Os 13 casos foram positivos para estruturas sugestivas de arquéia, micoplasma ou clamídia, em pelo menos uma amostra. Houve correlação positiva entre intensidade de matriz mixomatosa versus arquéia (r=0.44, p=0.02); arquéia versus micoplasma (r=0.41, p=0.03) e clamídia versus células xantomatosas r=0,42; 0.03). PCR foi positiva para DNA de arqueia em 4 dos 6 fragmentos. DISCUSSÃO: DNA e formas compatíveis com arquéia estão presentes em placas vulneráveis e podem ter papel fundamental na proliferação de micoplasma e clamídia. Este parece ser o primeiro relato de arquéia aparentemente patogênica em lesões de órgãos internos humanos.

Polymerase chain reaction in endomyocardial biopsies for monitoring reactivation of Chagas' disease in heart transplantation: a case report and review of the literature.

Polymerase chain reaction (PCR) has been used to detect microbiological agent recurrence after heart transplantation of viral-induced cardiomyopathies. We report a case of reactivation of Chagas' disease after heart transplantation in which parasites could be detected in the endomyocardial biopsy using hematoxylin-eosin-stained sections, immunohistochemistry, and PCR for Trypanosoma cruzi DNA. Interestingly, PCR results remained positive in the endomyocardial biopsy 53 days after the beginning of successful treatment, pointing to the possibility of chronic persistence of parasites in the myocardium after the reactivation of Chagas' disease.