• Differential gene expression profiles of hepatocellular carcinomas associated or not with viral infection Biochemistry and Molecular Biology

    Bellodi-Privato, M.; Kubrusly, M.S.; Stefano, J.T.; Soares, I.C.; Wakamatsu, A.; Oliveira, A.C.; Alves, V.A.F.; Bacchella, T.; Machado, M.C.C.; D’Albuquerque, L.A.C.

    Abstract in English:

    Chronic hepatitis B (HBV) and C (HCV) virus infections are the most important factors associated with hepatocellular carcinoma (HCC), but tumor prognosis remains poor due to the lack of diagnostic biomarkers. In order to identify novel diagnostic markers and therapeutic targets, the gene expression profile associated with viral and non-viral HCC was assessed in 9 tumor samples by oligo-microarrays. The differentially expressed genes were examined using a z-score and KEGG pathway for the search of ontological biological processes. We selected a non-redundant set of 15 genes with the lowest P value for clustering samples into three groups using the non-supervised algorithm k-means. Fisher’s linear discriminant analysis was then applied in an exhaustive search of trios of genes that could be used to build classifiers for class distinction. Different transcriptional levels of genes were identified in HCC of different etiologies and from different HCC samples. When comparing HBV-HCC vs HCV-HCC, HBV-HCC/HCV-HCC vs non-viral (NV)-HCC, HBC-HCC vs NV-HCC, and HCV-HCC vs NV-HCC of the 58 non-redundant differentially expressed genes, only 6 genes (IKBKβ, CREBBP, WNT10B, PRDX6, ITGAV, and IFNAR1) were found to be associated with hepatic carcinogenesis. By combining trios, classifiers could be generated, which correctly classified 100% of the samples. This expression profiling may provide a useful tool for research into the pathophysiology of HCC. A detailed understanding of how these distinct genes are involved in molecular pathways is of fundamental importance to the development of effective HCC chemoprevention and treatment.
  • Expression of E-cadherin, Snail and Hakai in epithelial cells isolated from the primary tumor and from peritumoral tissue of invasive ductal breast carcinomas Biochemistry and Molecular Biology

    Makdissi, F.B.A.; Machado, L.V.S.T.; Oliveira, A.G.C.; Benvenuti, T.T.; Katayama, M.L.H.; Brentani, M.M.; Osório, C.A.B.T.; Mourão Netto, M.; Lyra, E.C.; Carvalho, F.; Góes, J.C.S.; Folgueira, M.A.A.K.

    Abstract in English:

    Epithelial intercellular cohesion, mainly mediated by E-cadherin (CDH1) expression and function, may be deregulated during cancer cell invasion of adjacent tissues and lymphatic and vascular channels. CDH1 expression is down-modulated in invasive lobular breast carcinomas but its regulation in invasive ductal carcinomas (IDC) is less clear. CDH1 expression is repressed by transcription factors such as Snail (SNAI1) and its product is degraded after Hakai ubiquitination. We compared CDH1, SNAI1 and HAKAI mRNA expression in IDC and paired adjacent normal breast tissue and evaluated its relation with node metastasis and circulating tumor cells. Matched tumor/peritumoral and blood samples were collected from 30 patients with early IDC. Epithelial cells from each compartment (tumor/peritumoral) were recovered by an immunomagnetic method and gene expression was determined by real time RT-PCR. There were no differences in CDH1, SNAI1 and HAKAI mRNA expression between tumor and corresponding peritumoral samples and no differential tumoral gene expression according to nodal involvement. Another 30 patients with a long-term follow-up (at least 5 years) and a differential prognosis (good or poor, as defined by breast cancer death) had E-cadherin and Snail protein detected by immunohistochemistry in tumor samples. In this group, E-cadherin-positive expression, but not Snail, may be associated with a better prognosis. This is the first report simultaneously analyzing CDH1, SNAI1 and HAKAI mRNA expression in matched tumor and peritumoral samples from patients with IDC. However, no clear pattern of their expression could distinguish the invasive tumor compartment from its adjacent normal tissue.
  • Effect of estrogen receptor-alpha (ESR1) gene polymorphism on high density lipoprotein levels in response to hormone replacement therapy Biochemistry and Molecular Biology

    Nogueira-de-Souza, N.C.; Guerreiro da Silva, I.D.C.; de Carvalho, C.V.; Pulchinelli, A.; Haidar, M.A.; Baracat, E.C.; Massad-Costa, A.M.

    Abstract in English:

    Studies have shown that estrogen replacement therapy and estrogen plus progestin replacement therapy alter serum levels of total, LDL and HDL cholesterol levels. However, HDL cholesterol levels in women vary considerably in response to hormone replacement therapy (HRT). A significant portion of the variability of these levels has been attributed to genetic factors. Therefore, we investigated the influence of estrogen receptor-alpha (ESR1) gene polymorphisms on HDL levels in response to postmenopausal HRT. We performed a prospective cohort study on 54 postmenopausal women who had not used HRT before the study and had no significant general medical illness. HRT consisted of conjugated equine estrogen and medroxyprogesterone acetate continuously for 1 year. The lipoprotein levels were measured from blood samples taken before the start of therapy and after 1 year of HRT. ESR1 polymorphism (MspI C>T, HaeIII C>T, PvuII C>T, and XbaI A>G) frequencies were assayed by restriction fragment length polymorphism. A general linear model was used to describe the relationships between HDL levels and genotypes after adjusting for age. A significant increase in HDL levels was observed after HRT (P = 0.029). Women with the ESR1 PvuII TT genotype showed a statistically significant increase in HDL levels after HRT (P = 0.032). No association was found between other ESR1 polymorphisms and HDL levels. According to our results, the ESR1 PvuII TT genotype was associated with increased levels of HDL after 1 year of HRT.
  • Effects of strain and age on ear wound healing and regeneration in mice Cell Biology

    Costa, R.A.; Ruiz-de-Souza, V.; Azevedo Jr., G.M.; Vaz, N.M.; Carvalho, C.R.

    Abstract in English:

    Round holes in the ears of MRL mice tend to close with characteristics of regeneration believed to be absent in other mouse strains (e.g., C57BL/6). We evaluated the kinetics and the histopathology of ear wound closure in young (8 weeks old) C57BL/6 and BALB/c mice. We also used middle-aged (40 weeks old) C57BL/6 mice to evaluate the influence of aging on this process. A circular through-and-through hole was made in the ear, photographs were taken at different times after injury and wound area was measured with digital analysis software. The percentages of closed area measured on day 100 were: 23.57 ± 8.66% for young BALB/c mice, 56.47 ± 7.39% for young C57BL/6 mice, and 75.31 ± 23.65% for middle-aged C57BL/6 mice. Mice were sacrificed on days 1, 3, 5, 25, 44, and 100 for histological evaluation with hematoxylin and eosin, Gomori’s trichrome, periodic acid-Schiff, or picrosirius red staining. In young mice of both strains, healing included re-epithelialization, chondrogenesis, myogenesis, and collagen deposition. Young C57BL/6 and BALB/c mice differed in the organization of collagen fibers visualized using picrosirius-polarization. Sebaceous glands and hair follicles regenerated and chondrogenesis was greater in young C57BL/6 mice. In middle-aged C57BL/6 mice all aspects of regeneration were depressed. The characteristics of regeneration were present during ear wound healing in both young BALB/c and young C57BL/6 mice although they differed in intensity and pattern. Greater ear wound closure in middle-aged C57BL/6 mice was not correlated with regeneration.
  • Collagen XVIII/endostatin expression in experimental endotoxemic acute renal failure Experimental Biology

    Cichy, M.C.; Rocha, F.G.G.; Tristão, V.R.; Pessoa, E.A.; Cenedeze, M.A.; Nürmberg Junior, R.; Schor, N.; Bellini, M.H.

    Abstract in English:

    Acute renal failure (ARF) is a frequent complication of Gram-negative sepsis, with a high risk of mortality. Lipopolysaccharide (LPS)-induced ARF is associated with hemodynamic changes that are strongly influenced by the overproduction of nitric oxide (NO) through the cytokine-mediated up-regulation of inducible NO synthase. LPS-induced reductions in systemic vascular resistance paradoxically culminate in renal vasoconstriction. Collagen XVIII is an important component of the extracellular matrix expressed in basement membranes. Its degradation by matrix metalloproteases, cathepsins and elastases results in the formation of endostatin, claimed to have antiangiogenic activity and to be a prominent vasorelaxing agent. We evaluated the expression of endostatin/collagen XVIII in an endotoxemic ARF model. ARF was induced in C57BL/6 mice by intraperitoneal injection of LPS (10 mg/kg) followed by sacrifice 4 and 12 h later. Kidney tissue was the source of RNA and protein and the subject of histological analysis. As early as 4 h after LPS administration, blood urea, creatinine and NO levels were significantly increased compared to control. Endostatin/collagen XVIII mRNA levels were 0.71 times lower than sham-inoculated mice 4 h after LPS inoculation, returning to normal levels 12 h after LPS inoculation. Immunohistological examination revealed that acute injury caused by LPS leads to an increase of endostatin basement membrane staining in association with the decrease of CD31 endothelial basement membrane staining. These results indicate that in the early phase of endotoxemic ARF the endostatin levels were not regulated by gene expression, but by the metabolism of collagen XVIII.
  • Tracheal cryopreservation: caspase-3 immunoreactivity in tracheal epithelium and in mixed glands Experimental Biology

    Sotres-Vega, A.; Baltazares-Lipp, M.; Villalba-Caloca, J.; Gaxiola-Gaxiola, M.O.; Santibañez-Salgado, J.A.; Olmos-Zúñiga, J.R.; Jasso-Victoria, R.

    Abstract in English:

    Cryopreservation has an immunomodulating effect on tracheal tissue as a result of class II antigen depletion due to epithelium exfoliation. However, not all epithelium is detached. We evaluated the role of apoptosis in the remaining epithelium of 30 cryopreserved tracheal grafts. Caspase-3 immunoreactivity of tracheal epithelium was studied in canine tracheal segments cryopreserved with F12K medium, with or without subsequent storage in liquid nitrogen at -196°C for 15 days. Loss of structural integrity of tracheal mixed glands was observed in all cryopreserved tracheal segments. Caspase-3 immunoreactivity in tracheal mucosa and in mixed glands was significantly decreased, in contrast to the control group and to cryopreserved tracheal segments in which it remained high, due to the effect of storage in liquid nitrogen (P < 0.05, ANOVA and Tukey test). We conclude that apoptosis can be triggered in epithelial cells during tracheal graft harvesting even prior to cryopreservation, and although the epithelial caspase-3 immunoreactivity is reduced in tracheal cryopreservation, this could be explained by increased cell death. Apoptosis cannot be stopped during tracheal cryopreservation.
  • The effect of thyroid hormones on the white adipose tissue gene expression of PAI-1 and its serum concentration Experimental Biology

    Biz, C.; Oliveira, C.; Mattos, A.B.M.; Oliveira, J.; Ribeiro, E.B.; Oller do Nascimento, C.M.; Oyama, L.M.

    Abstract in English:

    Metabolic syndrome is associated with an increased risk of developing cardiovascular diseases and Plasminogen activator inhibitor 1 (PAI-1) overexpression may play a significant role in this process. A positive correlation between adipose tissue gene expression of PAI-1 and its serum concentration has been reported. Furthermore, high serum levels of thyroid hormones (T3 and T4) and PAI-1 have been observed in obese children. The present study evaluates the impact of thyroid hormone treatment on white adipose tissue PAI-1 gene expression and its serum concentration. Male Wistar rats (60 days old) were treated for three weeks with T4 (50 µg/day, Hyper) or with saline (control). Additionally, 3T3-L1 adipocytes were treated for 24 h with T4 (100 nM) or T3 (100 nM). PAI-1 gene expression was determined by real-time PCR, while the serum concentration of PAI-1 was measured by ELISA using a commercial kit (Innovative Research, USA). Both the serum concentration of PAI-1 and mRNA levels were similar between groups in retroperitoneal and epididymal white adipose tissue. Using 3T3-L1 adipocytes, in vitro treatment with T4 and T3 increased the gene expression of PAI-1, suggesting non-genomic and genomic effects, respectively. These results demonstrate that thyroid hormones have different effects in vitro and in vivo on PAI-1 gene expression in adipocytes.
  • Fish oil ingestion reduces the number of aberrant crypt foci and adenoma in 1,2-dimethylhydrazine-induced colon cancer in rats Experimental Biology

    Moreira, A.P.B.; Sabarense, C.M.; Dias, C.M.G.C.; Lunz, W.; Natali, A.J.; Glória, M.B.A.; Peluzio, M.C.G.

    Abstract in English:

    We determined the effect of fish oil (FO) ingestion on colonic carcinogenesis in rats. Male Wistar rats received 4 subcutaneous injections (40 mg/kg body weight each) of 1,2-dimethylhydrazine (DMH) at 3-day intervals and were fed a diet containing 18% by weight FO (N = 10) or soybean oil (SO, N = 10) for 36 weeks. At sacrifice, the colon was removed, aberrant crypt foci were counted and the fatty acid profile was determined. Intestinal tumors were removed and classified as adenoma or carcinoma. Liver and feces were collected and analyzed for fatty acid profile. FO reduced the mean (± SEM) number of aberrant crypt foci compared to SO (113.55 ± 6.97 vs 214.60 ± 18.61; P < 0.05) and the incidence of adenoma (FO: 20% vs SO: 100%), but carcinoma occurred equally in FO and SO rats (2 animals per group). The polyunsaturated fatty acid (PUFA) profile of the colon was affected by diet (P < 0.05): total ω-3 (FO: 8.18 ± 0.97 vs SO: 1.71 ± 0.54%) and total ω-6 (FO: 3.83 ± 0.59 vs SO: 10.43 ± 1.28%). The same occurred in the liver (P < 0.05): total ω-3 (FO: 34.41 ± 2.6 vs SO: 6.46 ± 0.59%) and total ω-6 (FO: 8.73 ± 1.37 vs SO: 42.12 ± 2.33%). The PUFA profile of the feces and liver polyamine levels did not differ between groups (P > 0.05). In conclusion, our findings indicate that chronic FO ingestion protected against the DMH-induced preneoplastic colon lesions and adenoma development, but not against carcinoma in rats.
  • Effect of curcumin on the proliferation and apoptosis of hepatic stellate cells Pharmacology

    Shu, J.C.; He, Y.J.; Lv, X.; Zhao, J.R.; Zhao, J.; Shen, Y.; Ye, G.R.; Wang, L.X.

    Abstract in English:

    This study was designed to investigate the effect of curcumin (diferuloylmethane) on the proliferation and apoptosis of hepatic stellate cells (HSC). The cell line HSC-T6 (1.25 x 10(5) cells/mL) was incubated with curcumin and HSC proliferation was detected by a methyl thiazolyl tetrazolium colorimetric assay. HSC apoptosis was detected by flow cytometry, transmission electron microscope and agarose gel electrophoresis. HSC proliferation was significantly inhibited in a concentration-dependent manner (10.6 to 63.5%) after incubation with 20-100 μM curcumin, compared with a control group. At 20, 40, and 60 μM, after 24 h of incubation, curcumin was associated with a significant increase in the number of HSC in the G2/M phase, and a significant decrease in cell numbers in the S phase (P < 0.05). At these concentrations, curcumin was also associated with an increase in the apoptosis index of 15.3 ± 1.9, 26.7 ± 2.8, and 37.6 ± 4.4%, respectively, compared to control (1.9 ± 0.6%, P < 0.01). At 40 μM, the curcumin-induced apoptosis index at 12, 24, 36, and 48 h of incubation was 12.0 ± 2.4, 26.7 ± 3.5, 33.8 ± 1.8, and 49.3 ± 1.6%, respectively (P < 0.01). In conclusion, curcumin inhibits the in vitro proliferation of HSCs in the G2/M phase of the cell cycle and also induces apoptosis in a concentration- and time-dependent manner. The in vivo effect of curcumin on HSCs requires further investigation.
  • Pharmacogenetic polymorphisms in Brazilian-born, first-generation Japanese descendants Pharmacology

    Perini, J.A.; Vargens, D.D.; Santana, I.S.C.; Moriguchi, E.H.; Ribeiro-dos-Santos, A.K.C.; Tsutsumi, M.; Suarez-Kurtz, G.

    Abstract in English:

    Brazil hosts the largest Japanese community outside Japan, estimated at 1.5 million individuals, one third of whom are first-generation, Brazilian-born with native Japanese parents. This large community provides a unique opportunity for comparative studies of the distribution of pharmacogenetic polymorphisms in native Japanese versus their Brazilian-born descendants. Functional polymorphisms in genes that modulate drug disposition (CYP2C9, CYP2C19 and GSTM3) or response (VKORC1) and that differ significantly in frequency in native Japanese versus Brazilians with no Japanese ancestry were selected for the present study. Healthy subjects (200 native Japanese and 126 first-generation Japanese descendants) living in agricultural colonies were enrolled. Individual DNA was genotyped using RFLP (GSTM3*A/B) or TaqMan Detection System assays (CYP2C9*2 and *3; CYP2C19*2 and *3; VKORC1 3673G>A, 5808T>G, 6853G>C, and 9041G>A). No difference was detected in the frequency of these pharmacogenetic polymorphisms between native Japanese and first-generation Japanese descendants. In contrast, significant differences in the frequency of each polymorphism were observed between native or first-generation Japanese and Brazilians with no Japanese ancestry. The VKORC1 3673G>A, 6853G>C and 9041G>A single nucleotide polymorphisms were in linkage disequilibrium in both native and first-generation Japanese living in Brazil. The striking similarity in the frequency of clinically relevant pharmacogenetic polymorphisms between Brazilian-born Japanese descendants and native Japanese suggests that the former may be recruited for clinical trials designed to generate bridging data for the Japanese population in the context of the International Conference on Harmonization.
  • Effect of immobilization stress on gene expression of catecholamine biosynthetic enzymes in heart auricles of socially isolated rats Physiology and Biophysics

    Gavrilovic, L.; Spasojevic, N.; Zivkovic, M.; Dronjak, S.

    Abstract in English:

    Chronic stress is associated with the development of cardiovascular diseases. The sympathoneural system plays an important role in the regulation of cardiac function both in health and disease. In the present study, the changes in gene expression of the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH), dopamine-β-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT) and protein levels in the right and left heart auricles of naive control and long-term (12 weeks) socially isolated rats were investigated by Taqman RT-PCR and Western blot analysis. The response of these animals to additional immobilization stress (2 h) was also examined. Long-term social isolation produced a decrease in TH mRNA level in left auricles (about 70%) compared to the corresponding control. Expression of the DBH gene was markedly decreased both in the right (about 62%) and left (about 81%) auricles compared to the corresponding control, group-maintained rats, whereas PNMT mRNA levels remained unchanged. Exposure of group-housed rats to acute immobilization for 2 h led to a significant increase of mRNA levels of TH (about 267%), DBH (about 37%) and PNMT (about 60%) only in the right auricles. Additional 2-h immobilization of individually housed rats did not affect gene expression of these enzymes in either the right or left auricle. Protein levels of TH, DBH and PNMT in left and right heart auricles were unchanged either in both individually housed and immobilized rats. The unchanged mRNA levels of the enzymes examined after short-term immobilization suggest that the catecholaminergic system of the heart auricles of animals previously exposed to chronic psychosocial stress was adapted to maintain appropriate cardiovascular homeostasis.
  • Oral rapamycin attenuates atherosclerosis without affecting the arterial responsiveness of resistance vessels in apolipoprotein E-deficient mice Physiology and Biophysics

    Gadioli, A.L.N.; Nogueira, B.V.; Arruda, R.M.P.; Pereira, R.B.; Meyrelles, S.S.; Arruda, J.A.; Vasquez, E.C.

    Abstract in English:

    The objective of the present study was to assess the effects of the immunosuppressant rapamycin (Rapamune®, Sirolimus) on both resistance vessel responsiveness and atherosclerosis in apolipoprotein E-deficient 8-week-old male mice fed a normal rodent diet. Norepinephrine (NE)-induced vasoconstriction, acetylcholine (ACh)- and sodium nitroprusside (SNP)-induced vasorelaxation of isolated mesenteric bed, and atherosclerotic lesions were evaluated. After 12 weeks of orally administered rapamycin (5 mg·kg-1·day-1, N = 9) and compared with untreated (control, N = 9) animals, rapamycin treatment did not modify either NE-induced vasoconstriction (maximal response: 114 ± 4 vs 124 ± 10 mmHg, respectively) or ACh- (maximal response: 51 ± 8 vs 53 ± 5%, respectively) and SNP-induced vasorelaxation (maximal response: 73 ± 6 vs 74 ± 6%, respectively) of the isolated vascular mesenteric bed. Despite increased total cholesterol in treated mice (982 ± 59 vs 722 ± 49 mg/dL, P < 0.01), lipid deposition on the aorta wall vessel was significantly less in rapamycin-treated animals (37 ± 12 vs 68 ± 8 µm² x 10³). These results indicate that orally administered rapamycin is effective in attenuating the progression of atherosclerotic plaque without affecting the responsiveness of resistance vessels, supporting the idea that this immunosuppressant agent might be of potential benefit against atherosclerosis in patients undergoing therapy.
  • Consequences of cerebroventricular insulin injection on renal sodium handling in rats: effect of inhibition of central nitric oxide synthase Physiology and Biophysics

    Oliveira, P.C.; Michelotto, J.B.; Zapparoli, A.; Boer, P.A.; Gontijo, J.A.R.

    Abstract in English:

    In the present study, we investigated the effects of acute intracerebroventricular (icv) insulin administration on central mechanisms regulating urinary sodium excretion in simultaneously centrally NG-nitro-L-arginine methylester (L-NAME)-injected unanesthetized rats. Male Wistar-Hannover rats were randomly assigned to one of five groups: a) icv 0.15 M NaCl-injected rats (control, N = 10), b) icv dose-response (1.26, 12.6 and 126 ng/3 µL) insulin-injected rats (N = 10), c) rats icv injected with 60 µg L-NAME in combination with NaCl (N = 10) or d) with insulin (N = 10), and e) subcutaneously insulin-injected rats (N = 5). Centrally administered insulin produced an increase in urinary output of sodium (NaCl: 855.6 ± 85.1 Δ%/min; 126 ng insulin: 2055 ± 310.6 Δ%/min; P = 0.005) and potassium (NaCl: 460.4 ± 100 Δ%/min; 126 ng insulin: 669.2 ± 60.8 Δ%/min; P = 0.025). The urinary sodium excretion response to icv 126 ng insulin microinjection was significantly attenuated by combined administration of L-NAME (126 ng insulin: 1935 ± 258.3 Δ%/min; L-NAME + 126 ng insulin: 582.3 ± 69.6 Δ%/min; P = 0.01). Insulin-induced natriuresis occurred by increasing post-proximal sodium excretion, despite an unchanged glomerular filtration rate. Although the rationale for decreased urinary sodium excretion induced by combined icv L-NAME and insulin administration is unknown, it is tempting to suggest that perhaps one of the efferent signals triggered by insulin in the CNS may be nitrergic in nature.
  • Detection of patients with functional dyspepsia using wavelet transform applied to their electrogastrogram Analytical, Diagnostic an Therapeutic Techniques and Instruments

    Chacón, M.; Curilem, G.; Acuña, G.; Defilippi, C.; Madrid, A.M.; Jara, S.

    Abstract in English:

    The aim of the present study was to develop a classifier able to discriminate between healthy controls and dyspeptic patients by analysis of their electrogastrograms. Fifty-six electrogastrograms were analyzed, corresponding to 42 dyspeptic patients and 14 healthy controls. The original signals were subsampled, filtered and divided into the pre-, post-, and prandial stages. A time-frequency transformation based on wavelets was used to extract the signal characteristics, and a special selection procedure based on correlation was used to reduce their number. The analysis was carried out by evaluating different neural network structures to classify the wavelet coefficients into two groups (healthy subjects and dyspeptic patients). The optimization process of the classifier led to a linear model. A dimension reduction that resulted in only 25% of uncorrelated electrogastrogram characteristics gave 24 inputs for the classifier. The prandial stage gave the most significant results. Under these conditions, the classifier achieved 78.6% sensitivity, 92.9% specificity, and an error of 17.9 ± 6% (with a 95% confidence level). These data show that it is possible to establish significant differences between patients and normal controls when time-frequency characteristics are extracted from an electrogastrogram, with an adequate component reduction, outperforming the results obtained with classical Fourier analysis. These findings can contribute to increasing our understanding of the pathophysiological mechanisms involved in functional dyspepsia and perhaps to improving the pharmacological treatment of functional dyspeptic patients.
  • Normal range values for thromboelastography in healthy adult volunteers Analytical, Diagnostic an Therapeutic Techniques and Instruments

    Scarpelini, S.; Rhind, S.G.; Nascimento, B.; Tien, H.; Shek, P.N.; Peng, H.T.; Huang, H.; Pinto, R.; Speers, V.; Reis, M.; Rizoli, S.B.

    Abstract in English:

    Thromboelastography (TEG®) provides a functional evaluation of coagulation. It has characteristics of an ideal coagulation test for trauma, but is not frequently used, partially due to lack of both standardized techniques and normal values. We determined normal values for our population, compared them to those of the manufacturer and evaluated the effect of gender, age, blood type, and ethnicity. The technique was standardized using citrated blood, kaolin and was performed on a Haemoscope 5000 device. Volunteers were interviewed and excluded if pregnant, on anticoagulants or having a bleeding disorder. The TEG® parameters analyzed were R, K, α, MA, LY30, and coagulation index. All volunteers outside the manufacturer’s normal range underwent extensive coagulation investigations. Reference ranges for 95% for 118 healthy volunteers were R: 3.8-9.8 min, K: 0.7-3.4 min, α: 47.8-77.7 degrees, MA: 49.7-72.7 mm, LY30: -2.3-5.77%, coagulation index: -5.1-3.6. Most values were significantly different from those of the manufacturer, which would have diagnosed coagulopathy in 10 volunteers, for whom additional investigation revealed no disease (81% specificity). Healthy women were significantly more hypercoagulable than men. Aging was not associated with hypercoagulability and East Asian ethnicity was not with hypocoagulability. In our population, the manufacturer’s normal values for citrated blood-kaolin had a specificity of 81% and would incorrectly identify 8.5% of the healthy volunteers as coagulopathic. This study supports the manufacturer’s recommendation that each institution should determine its own normal values before adopting TEG®, a procedure which may be impractical. Consideration should be given to a multi-institutional study to establish wide standard values for TEG®.
  • Multigenerational Brazilian family with malignant hyperthermia and a novel mutation in the RYR1 gene Analytical, Diagnostic an Therapeutic Techniques and Instruments

    Matos, A.R.; Sambuughin, N.; Rumjanek, F.D.; Amoedo, N.D.; Cunha, L.B.P.; Zapata-Sudo, G.; Sudo, R.T.

    Abstract in English:

    Malignant hyperthermia (MH) is a pharmacogenetic disease triggered in susceptible individuals by the administration of volatile halogenated anesthetics and/or succinylcholine, leading to the development of a hypermetabolic crisis, which is caused by abnormal release of Ca2+ from the sarcoplasmic reticulum, through the Ca2+ release channel ryanodine receptor 1 (RyR1). Mutations in the RYR1 gene are associated with MH in the majority of susceptible families. Genetic screening of a 5-generation Brazilian family with a history of MH-related deaths and a previous MH diagnosis by the caffeine halothane contracture test (CHCT) in some individuals was performed using restriction and sequencing analysis. A novel missense mutation, Gly4935Ser, was found in an important functional and conserved locus of this gene, the transmembrane region of RyR1. In this family, 2 MH-susceptible individuals previously diagnosed with CHCT carry this novel mutation and another 24 not previously diagnosed members also carry it. However, this same mutation was not found in another MH-susceptible individual whose CHCT was positive to the test with caffeine but not to the test with halothane. None of the 5 MH normal individuals of the family, previously diagnosed by CHCT, carry this mutation, nor do 100 controls from control Brazilian and USA populations. The Gly4932Ser variant is a candidate mutation for MH, based on its co-segregation with disease phenotype, absence among controls and its location within the protein.
  • Cystatin C and renal function in pediatric kidney transplant recipients Blood, Immunology and Organ Transplantation

    Franco, M.C.P.; Nagasako, S.S.; Machado, P.G.; Nogueira, P.C.K.; Pestana, J.O.M.; Sesso, R.

    Abstract in English:

    In clinical practice, the glomerular filtration rate (GFR) is often determined with serum creatinine. However, studies have shown cystatin C to be a better parameter for the diagnosis of impaired renal function. We compared GFR estimated by plasma cystatin C with GFR estimated by serum creatinine in a sample of 50 pediatric renal transplant recipients and 24 healthy children. The correlation between GFR estimated by serum creatinine and by cystatin C was significant (r = 0.75; P < 0.001, Person’s correlation); however, in pediatric kidney transplant recipients, the GFR was 6.7 mL/min lower when determined using cystatin C rather than serum creatinine. Moreover, using GFR estimated by cystatin C we found that 42% of the pediatric kidney transplant recipients had an estimated GFR <60 mL·min-1·1.73 (m²)-1, whereas when GFR was estimated by the serum creatinine formula only 16% of the children had values below this cutoff point indicative of chronic kidney disease (P < 0.001). We conclude that, in pediatric kidney transplant recipients, estimation of GFR yields lower values when cystatin C is used rather than serum creatinine.
  • Treadmill exercise testing of asymptomatic men and women without evidence of heart disease Cardiovascular, Respiratory and Sport Medicine

    Chalela, W.A.; Fukushima, R.B.; Araujo, F.; Lima, A.C.P.; Moffa, P.J.; Mansur, A.J.

    Abstract in English:

    The aim of this study was to test the hypothesis of differences in performance including differences in ST-T wave changes between healthy men and women submitted to an exercise stress test. Two hundred (45.4%) men and 241 (54.6%) women (mean age: 38.7 ± 11.0 years) were submitted to an exercise stress test. Physiologic and electrocardiographic variables were compared by the Student t-test and the chi-square test. To test the hypothesis of differences in ST-segment changes, data were ranked with functional models based on weighted least squares. To evaluate the influence of gender and age on the diagnosis of ST-segment abnormality, a logistic model was adjusted; P < 0.05 was considered to be significant. Rate-pressure product, duration of exercise and estimated functional capacity were higher in men (P < 0.05). Sixteen (6.7%) women and 9 (4.5%) men demonstrated ST-segment upslope ≥0.15 mV or downslope ≥0.10 mV; the difference was not statistically significant. Age increase of one year added 4% to the chance of upsloping of segment ST ≥0.15 mV or downsloping of segment ST ≥0.1 mV (P = 0.03; risk ratio = 1.040, 95% confidence interval (CI) = 1.002-1.080). Heart rate recovery was higher in women (P < 0.05). The chance of women showing an increase of systolic blood pressure ≤30 mmHg was 85% higher (P = 0.01; risk ratio = 1.85, 95%CI = 1.1-3.05). No significant difference in the frequency of ST-T wave changes was observed between men and women. Other differences may be related to different physical conditioning.
  • C-reactive protein in acute coronary syndrome: association with 3-year outcomes Cardiovascular, Respiratory and Sport Medicine

    Schaan, B.D.; Pellanda, L.C.; Maciel, P.T.; Duarte, E.R.; Portal, V.L.

    Abstract in English:

    Inflammatory markers have been associated with clinical outcome in patients with acute coronary syndrome (ACS). The present study evaluated the role of high-sensitivity C-reactive protein (CRP) measurements as a predictor of late cardiovascular outcomes after ACS. One hundred and ninety-nine ACS patients in a Coronary Care Unit from March to November 2002 were included and were reassessed clinically after ~3 years. Clinical variables and CRP levels were evaluated as predictors of major cardiovascular events (MACE, defined as the occurrence of cardiac death, ischemic stroke or myocardial infarction) and mortality. Statistical analyses included Cox multivariable analysis and survival curves (Kaplan-Meier). Of the 199 patients, 11 died within 1 month (5.5%). Of the 188 remaining patients, 22 died after a mean follow-up of 2.9 ± 0.5 years. Baseline CRP levels for patients with MACE (N = 57) were significantly higher than those of patients with no events (median = 0.67 mg/L; 25th-75th percentiles = 0.32 and 1.99 mg/L vs median = 0.45 mg/L; 25th-75th percentiles = 0.24 and 0.83 mg/L; P < 0.001). Patients with CRP levels >3 mg/L had a significantly lower survival than the other two groups (1-3 and <1 mg/L; P = 0.001, log-rank test). The odds ratio for MACE was 7.41 (2.03-27.09) for patients with CRP >3 mg/L compared with those with CRP <1 mg/L. For death by any cause, the hazard ratio was 4.58 (1.93-10.86). High CRP levels predicted worse long-term outcomes (MACE and death by any cause) in patients with ACS.
  • Pattern of functional antibody activity against Haemophilus influenzae type b (Hib) in infants immunized with diphtheria-tetanus-pertussis/Hib Brazilian combination vaccine Infectious Agents and Diseases

    Matos, D.C.S.; Silva, A.M.V.; Neves, P.C.C.; Martins, R.M.; Homma, A.; Marcovistz, R.

    Abstract in English:

    We evaluated the functional activity of Haemophilus influenzae B (Hib) antibodies elicited in a group of infants immunized with the diphtheria-tetanus-pertussis vaccine combined with an Hib vaccine produced totally in Brazil after technological transfer of Hib vaccine production from Glaxo SmithKline, Belgium. Blood samples from immunized infants (N = 985) were collected for the determination of Hib antibodies. Total Ig and IgM and IgG subclasses of antibodies against polyribosyl ribitol phosphate (PRP) were analyzed by ELISA. Almost all vaccinees (97.56%, 961/985) developed a strong anti-PRP IgG antibody response (≥1.0 μg/mL), while an anti-PRP IgM response was observed in 64.24% (634/985) of them (≥0.15 μg/mL). Only 18.88% (186/985) of the infants in the group with high PRP antibody IgG concentrations (≥1.0 μg/mL) developed a high IgM antibody response. Anti-PRP IgG antibody levels were significantly higher than anti-PRP IgM. These results demonstrate the predominance of IgG antibodies over IgM antibodies in response to PRP, with a ratio of 17:1. IgG antibodies were predominantly of the IgG1 subclass. An increase in IgG avidity was also observed during the course of immunization.
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