Action potential variability in human pluripotent stem cell-derived cardiomyocytes obtained from healthy donors.

Human pluripotent stem cells (PSC) have been used for disease modelling, after differentiation into the desired cell type. Electrophysiologic properties of cardiomyocytes derived from pluripotent stem cells are extensively used to model cardiac arrhythmias, in cardiomyopathies and channelopathies. This requires strict control of the multiple variables that can influence the electrical properties of these cells. In this article, we report the action potential variability of 780 cardiomyocytes derived from pluripotent stem cells obtained from six healthy donors. We analyze the overall distribution of action potential (AP) data, the distribution of action potential data per cell line, per differentiation protocol and batch. This analysis indicates that even using the same cell line and differentiation protocol, the differentiation batch still affects the results. This variability has important implications in modeling arrhythmias and imputing pathogenicity to variants encountered in patients with arrhythmic diseases. We conclude that even when using isogenic cell lines to ascertain pathogenicity to variants associated to arrythmias one should use cardiomyocytes derived from pluripotent stem cells using the same differentiation protocol and batch and pace the cells or use only cells that have very similar spontaneous beat rates. Otherwise, one may find phenotypic variability that is not attributable to pathogenic variants.

Generation of patient-specific induced pluripotent stem cell lines from one patient with Jervell and Lange-Nielsen syndrome, one with type 1 long QT syndrome and two healthy relatives.

Four human iPSC cell lines (one Jervell and Lange-Nielsen Syndrome, one Long QT Syndrome-type 1 and two healthy controls) were generated from peripheral blood obtained from donors belonging to the same family. CytoTune™-iPS 2.0 Sendai Reprogramming Kit (containing OCT3/4, KLF4, SOX2 and cMYC as reprogramming factors) was used to generate all cell lines. The four iPSCs have normal karyotype, express pluripotency markers as determined by RT-PCR and flow cytometry and differentiated spontaneously in vitro into cells of the three germ layers, confirming their pluripotent capacity.

Validation of methylation markers for diagnosis of oral cavity cancer.

PURPOSE: Activation of proto-oncogenes and inactivation of tumour suppressor genes are the major genetic alterations involved in carcinogenesis. The increase in methylation at the promoter region of a tumour suppressor gene can lead to gene inactivation, selecting cells with proliferative advantage. Thus, promoter hypermethylation is considered a marker in a variety of malignant tumours, including oral cavity. EXPERIMENTAL DESIGN: The methylation pattern of eight genes was evaluated in 40 oral cavity squamous cell carcinomas (OSCCs) and 40 saliva samples from healthy individuals by Q-MSP. Different combinations of genes were also assessed in order to identify gene panels that could better distinguish between OSCC and saliva samples. RESULTS: CCNA1, DAPK, DCC and TIMP3 methylation were highly specific for being found in the OSCC samples. Moreover, the combination of these genes improved detection when compared with single markers, reaching values of 92.5% for sensitivity and specificity (when using the panel CCNA1, DCC, TIMP3). Moreover, DAPK, DCC and TIMP3 were hypermethylated in nearly 90% of clinically T1 and T2 cases. CONCLUSION: The pursuing of this panel of hypermethylated genes is an important tool for the detection of individuals with OSCC. Moreover, the identification of these markers in early stages of OSCC shows the feasibility of using the panel on saliva as possible biomarkers for early diagnosis. The lack of association between the methylation status of these genes and clinical characteristics shows that they are able to distinguish OSCC cases irrespective of social and clinical factors (gender, age, human papillomavirus (HPV) status, clinical stage, vascular embolisation and perineural invasion).

Methylation as a biomarker for head and neck cancer.

Head and neck cancer is a collective term that describes malignant tumors of the oral cavity, pharynx, and larynx characterized by high incidence and mortality rates. Although most HNSCC originate from the mucosal surface of the upper aerodigestive tract, where they can be easily detected during a routine clinical examination. Often the definitive diagnosis is delayed because of the difficulty in differentiating from other similar lesions. Activation of proto-oncogenes and inactivation of tumor suppressor genes are the major molecular alterations involved in carcinogenesis. In addition, epigenetic changes can alter the expression of critical genes important in the development of a variety of cancers. The detection of aberrant gene promoter methylation as a tool for the detection of tumors or its use as prognostic marker have been described for many different cancers including HNSCC. The search for biomarkers has as its main aim the evaluation and measurement of the status of normal and pathological biological processes as well as pharmacological responses to certain treatments. The tracking of these biomarkers is an important part for the identification of individuals in the early stages of head and neck cancer for its diagnostic and prognostic relevance reflecting in high survival rates, better quality of life and less cost to the healthcare system. Therefore, assuming that cancer results from genetic and epigenetic changes, analyzes based on gene methylation profile in combination with the pathological diagnosis would be useful in predicting the behavior of these head and neck tumors.

Soluble factors from multipotent mesenchymal stromal cells have antinecrotic effect on cardiomyocytes in vitro and improve cardiac function in infarcted rat hearts.

The mechanisms underlying the functional improvement after injection of multipotent mesenchymal stromal cells (MSCs) in infarcted hearts remain incompletely understood. The aim of this study was to investigate if soluble factors secreted by MSCs promote cardioprotection. For this purpose, conditioned medium (CM) was obtained after three passages from MSC cultures submitted to 72 h of conditioning in serum-free DMEM under normoxia (NCM) or hypoxia (HCM) conditions. CM was concentrated 25-fold before use (NCM-25X, concentrated normoxia conditioned medium; HCM-25X, concentrated hypoxia conditioned medium). The in vitro cardioprotection was evaluated in neonatal ventricular cardiomyocytes by quantifying apoptosis after 24 h of serum deprivation associated with hypoxia (1% O(2)) in the absence or presence of NCM and HCM (nonconcentrated and 25-fold concentrated). The in vivo cardioprotection of HCM was tested in a model of myocardial infarction (MI) induced in Wistar male rats by permanent left coronary occlusion. Intramyocardial injection of HCM-25X (n = 14) or nonconditioned DMEM (n = 16) was performed 3 h after coronary occlusion and cardiac function was evaluated 19-21 days after medium injection. Cardiac function was evaluated by electro- and echocardiogram, left ventricular catheterization, and treadmill test. The in vitro results showed that HCM was able to decrease cardiomyocyte necrosis. The in vivo results showed that HCM-25X administered 3 h after AMI was able to promote a significant reduction (35%) in left ventricular end-diastolic pressure and improvement of cardiac contractility (15%) and relaxation (12%). These results suggest that soluble factors released in vitro by MSCs are able to promote cardioprotection in vitro and improve cardiac function in vivo.

Cell therapy in dilated cardiomyopathy: from animal models to clinical trials

Dilated cardiomyopathy can be the end-stage form and common denominator of several cardiac disorders of known cause, such as hypertensive, ischemic, diabetic and Chagasic diseases. However, some individuals have clinical findings, such as an increase in ventricular chamber size and impaired contractility (classical manifestations of dilated cardiomyopathy) even in the absence of a diagnosed primary disease. In these patients, dilated cardiomyopathy is classified as idiopathic since its etiology is obscure. Nevertheless, regardless of all of the advances in medical, pharmacological and surgical procedures, the fate of patients with dilated cardiomyopathy (of idiopathic or of any other known cause) is linked to arrhythmic episodes, severe congestive heart failure and an increased risk of sudden cardiac death. In this review, we will summarize present data on the use of cell therapies in animal models of dilated cardiomyopathies and will discuss the few clinical trials that have been published so far involving patients affected by this disease. The animal models discussed here include those in which the cardiomyopathy is produced by genetic manipulation and those in which disease is induced by chemical or infectious agents. The specific model used clearly creates restrictions to translation of the proposed cell therapy to clinical practice, insofar as most of the clinical trials performed to date with cell therapy have used autologous cells. Thus, translation of genetic models of dilated cardiomyopathy may have to wait until the use of allogeneic cells becomes more widespread in clinical trials of cell therapies for cardiac diseases.

Cell therapy in dilated cardiomyopathy: from animal models to clinical trials.

Dilated cardiomyopathy can be the end-stage form and common denominator of several cardiac disorders of known cause, such as hypertensive, ischemic, diabetic and Chagasic diseases. However, some individuals have clinical findings, such as an increase in ventricular chamber size and impaired contractility (classical manifestations of dilated cardiomyopathy) even in the absence of a diagnosed primary disease. In these patients, dilated cardiomyopathy is classified as idiopathic since its etiology is obscure. Nevertheless, regardless of all of the advances in medical, pharmacological and surgical procedures, the fate of patients with dilated cardiomyopathy (of idiopathic or of any other known cause) is linked to arrhythmic episodes, severe congestive heart failure and an increased risk of sudden cardiac death. In this review, we will summarize present data on the use of cell therapies in animal models of dilated cardiomyopathies and will discuss the few clinical trials that have been published so far involving patients affected by this disease. The animal models discussed here include those in which the cardiomyopathy is produced by genetic manipulation and those in which disease is induced by chemical or infectious agents. The specific model used clearly creates restrictions to translation of the proposed cell therapy to clinical practice, insofar as most of the clinical trials performed to date with cell therapy have used autologous cells. Thus, translation of genetic models of dilated cardiomyopathy may have to wait until the use of allogeneic cells becomes more widespread in clinical trials of cell therapies for cardiac diseases.

Effect of long-time administration of tibolone on vaginal cytology of castrated rats.

PURPOSE: To evaluate the estrogenic effect of tibolone administered at high-dose and long-term through cytological examination of vaginal epithelium of castrated rats. METHODS: 15 adult Wistar rats were submitted to bilateral oophorectomy 30 days before starting the experiment. The rats were then randomly divided in two groups. Experimental rats (n = 9) orally received 1 mg tibolone/day; control rats (n = 6) just received carboxymethylcellulose. Vaginal smears were collected from all rats on days 0, 1-6, 30, 60, 90 and 120 of the experiment. RESULTS: On day 0, smears from all rats were atrophic, classified as anestrus, and remained this type in the control group until day 120. In the tibolone group, on day 3 all the rats had vaginal cytology similar to estrus and maintained the same aspect till day 90. CONCLUSION: Tibolone has estrogenic action in the vaginal epithelium which is already evident after the first dose and remains without major changes over time.

A safety and feasibility study of cell therapy in dilated cardiomyopathy

The aim of this study was to determine if bone marrow mononuclear cell (BMMC) transplantation is safe for moderate to severe idiopathic dilated cardiomyopathy (IDC). Clinical trials have shown that this procedure is safe and effective for ischemic patients, but little information is available regarding non-ischemic patients. Twenty-four patients with IDC, optimized therapy, age 46 ± 11.6 years, 17 males, NYHA classes II-IV, and left ventricular ejection fraction <35 percent were enrolled in the study. Clinical evaluation at baseline and 6 months after stem cell therapy to assess heart function included echocardiogram, magnetic resonance imaging, cardiopulmonary test, Minnesota Quality of Life Questionnaire, and NYHA classification. After cell transplantation 1 patient showed a transient increase in enzyme levels and 2 patients presented arrhythmias that were reversed within 72 h. Four patients died during follow-up, between 6 and 12 weeks after therapy. Clinical evaluation showed improvement in most patients as reflected by statistically significant decreases in Minnesota Quality of Life Questionnaire (63 ± 17.9 baseline vs 28.8 ± 16.75 at 6 months) and in class III-IV NYHA patients (18/24 baseline vs 2/20 at 6 months). Cardiopulmonary exercise tests demonstrated increased peak oxygen consumption (12.2 ± 2.4 at baseline vs 15.8 ± 7.1 mL·kg-1·min-1 at 6 months) and walked distance (377.2 ± 85.4 vs 444.1 ± 77.9 m at 6 months) in the 6-min walk test, which was not accompanied by increased left ventricular ejection fraction. Our findings indicate that BMMC therapy in IDC patients with severe ventricular dysfunction is feasible and that larger, randomized and placebo-controlled trials are warranted.

A safety and feasibility study of cell therapy in dilated cardiomyopathy.

The aim of this study was to determine if bone marrow mononuclear cell (BMMC) transplantation is safe for moderate to severe idiopathic dilated cardiomyopathy (IDC). Clinical trials have shown that this procedure is safe and effective for ischemic patients, but little information is available regarding non-ischemic patients. Twenty-four patients with IDC, optimized therapy, age 46 ± 11.6 years, 17 males, NYHA classes II-IV, and left ventricular ejection fraction <35% were enrolled in the study. Clinical evaluation at baseline and 6 months after stem cell therapy to assess heart function included echocardiogram, magnetic resonance imaging, cardiopulmonary test, Minnesota Quality of Life Questionnaire, and NYHA classification. After cell transplantation 1 patient showed a transient increase in enzyme levels and 2 patients presented arrhythmias that were reversed within 72 h. Four patients died during follow-up, between 6 and 12 weeks after therapy. Clinical evaluation showed improvement in most patients as reflected by statistically significant decreases in Minnesota Quality of Life Questionnaire (63 ± 17.9 baseline vs 28.8 ± 16.75 at 6 months) and in class III-IV NYHA patients (18/24 baseline vs 2/20 at 6 months). Cardiopulmonary exercise tests demonstrated increased peak oxygen consumption (12.2 ± 2.4 at baseline vs 15.8 ± 7.1 mL·kg⁻¹·min⁻¹ at 6 months) and walked distance (377.2 ± 85.4 vs 444.1 ± 77.9 m at 6 months) in the 6-min walk test, which was not accompanied by increased left ventricular ejection fraction. Our findings indicate that BMMC therapy in IDC patients with severe ventricular dysfunction is feasible and that larger, randomized and placebo-controlled trials are warranted.

Cardiac gene expression and systemic cytokine profile are complementary in a murine model of post-ischemic heart failure

After myocardial infarction (MI), activation of the immune system and inflammatory mechanisms, among others, can lead to ventricular remodeling and heart failure (HF). The interaction between these systemic alterations and corresponding changes in the heart has not been extensively examined in the setting of chronic ischemia. The main purpose of this study was to investigate alterations in cardiac gene and systemic cytokine profile in mice with post-ischemic HF. Plasma was tested for IgM and IgG anti-heart reactive repertoire and inflammatory cytokines. Heart samples were assayed for gene expression by analyzing hybridization to AECOM 32k mouse microarrays. Ischemic HF significantly increased the levels of total serum IgM (by 5.2-fold) and total IgG (by 3.6-fold) associated with a relatively high content of anti-heart specificity. A comparable increase was observed in the levels of circulating pro-inflammatory cytokines such as IL-1â (3.8X) and TNF-á (6.0X). IFN-ã was also increased by 3.1-fold in the MI group. However, IL-4 and IL-10 were not significantly different between the MI and sham-operated groups. Chemokines such as MCP-1 and IL-8 were 1.4- and 13-fold increased, respectively, in the plasma of infarcted mice. We identified 2079 well annotated unigenes that were significantly regulated by post-ischemic HF. Complement activation and immune response were among the most up-regulated processes. Interestingly, 21 of the 101 quantified unigenes involved in the inflammatory response were significantly up-regulated and none were down-regulated. These data indicate that post-ischemic heart remodeling is accompanied by immune-mediated mechanisms that act both systemically and locally.

Cardiac gene expression and systemic cytokine profile are complementary in a murine model of post-ischemic heart failure.

After myocardial infarction (MI), activation of the immune system and inflammatory mechanisms, among others, can lead to ventricular remodeling and heart failure (HF). The interaction between these systemic alterations and corresponding changes in the heart has not been extensively examined in the setting of chronic ischemia. The main purpose of this study was to investigate alterations in cardiac gene and systemic cytokine profile in mice with post-ischemic HF. Plasma was tested for IgM and IgG anti-heart reactive repertoire and inflammatory cytokines. Heart samples were assayed for gene expression by analyzing hybridization to AECOM 32k mouse microarrays. Ischemic HF significantly increased the levels of total serum IgM (by 5.2-fold) and total IgG (by 3.6-fold) associated with a relatively high content of anti-heart specificity. A comparable increase was observed in the levels of circulating pro-inflammatory cytokines such as IL-1beta (3.8X) and TNF-alpha (6.0X). IFN-gamma was also increased by 3.1-fold in the MI group. However, IL-4 and IL-10 were not significantly different between the MI and sham-operated groups. Chemokines such as MCP-1 and IL-8 were 1.4- and 13-fold increased, respectively, in the plasma of infarcted mice. We identified 2079 well annotated unigenes that were significantly regulated by post-ischemic HF. Complement activation and immune response were among the most up-regulated processes. Interestingly, 21 of the 101 quantified unigenes involved in the inflammatory response were significantly up-regulated and none were down-regulated. These data indicate that post-ischemic heart remodeling is accompanied by immune-mediated mechanisms that act both systemically and locally.

An ultrasound and histomorphological analysis of experimental liver cirrhosis in rats.

We investigated whether liver injury by dual exposure to ethanol and carbon tetrachloride (EtOH + CCl4) for 15 weeks would persist after hepatotoxic agents were removed (EtOH + CCl4/8wR). After 15 weeks of hepatic injury with ethanol (5.5%, m/v) and carbon tetrachloride (0.05, mL/kg, ip), 5 of 11 female Wistar rats were sacrificed. The other 6 rats were maintained for an additional 8 weeks without hepatotoxic agents. Ultrasonography showed increased liver echogenicity and dilation of portal vein caliber in both groups (EtOH + CCl4: 0.22 +/- 0.01 cm, P < 0.001; EtOH + CCl4/8wR: 0.21 +/- 0.02 cm, P < 0.01) vs control (0.16 +/- 0.02 cm). Histopathology showed regenerative nodules in both experimental groups. Histomorphometry revealed increased fibrosis content in both groups (EtOH + CCl4: 12.6 +/- 2.64%, P < 0.001; EtOH + CCl4/8wR: 10.4 +/- 1.36%, P < 0.05) vs control (2.2 +/- 1.21%). Collagen types I and III were increased in groups EtOH + CCl4 (collagen I: 2.5 +/- 1.3%, P < 0.01; collagen III: 1.3 +/- 0.2%, P < 0.05) and EtOH + CCl4/8wR (collagen I: 1.8 +/- 0.06%, P < 0.05; collagen III: 1.5 +/- 0.8%, P < 0.01) vs control (collagen I: 0.38 +/- 0.11%; collagen III: 0.25 +/- 0.06%). Tissue transglutaminase increased in both groups (EtOH + CCl4: 66.4 +/- 8%, P < 0.01; EtOH + CCl4/8wR: 58.8 +/- 21%, P < 0.01) vs control (7.9 +/- 0.8%). Cirrhosis caused by the association of CCl4-EtOH remained for at least 8 weeks after removal of these hepatotoxic agents. Ultrasound images can be a useful tool to evaluate advanced hepatic alterations.

Bone marrow cell transplant does not prevent or reverse murine liver cirrhosis.

We tested the effect of bone marrow cell (BMC) transplantation in either preventing or reversing cirrhosis on an experimental model of chronic liver disease. Female Wistar rats were fed a liquid alcohol diet and received intraperitoneal injections of carbon tetrachloride (CCl4) over 15 weeks. Ten animals (cell-treated group) received five injections of BMCs during the cirrhosis induction protocol (on the 4th, 6th, 8th, 10th, and 12th weeks) and four animals received the cells after liver injury was established through tail vein. Nine animals (nontreated group) were submitted to the previously described protocols; however, they received vehicle injections. Analyses were performed to verify whether the infusion of cells was effective in preventing the development of cirrhosis in our model of induction, and if the cells could reverse cirrhosis once it was established. Hepatic architecture and fibrotic septa were analyzed in liver slices stained with hematoxilin & eosin and Sirius red, respectively. Fibrosis quantification was measured by Sirius red histomorphometry. Indirect immunofluorescence was performed to detect the amount of tissue transglutaminase 2. Blood analyses were performed to assess liver injury and function by the assessment of alanine aminotransferase and albumin. Ultrasound was performed to analyze the portal vein caliber and presence of ascitis. Cirrhosis features (regenerative nodules and fibrous septa) were observed in histopathology after 15 weeks of continuous hepatic injury in nontreated and cell-treated groups. Collagen content, immunofluorescence analysis, and biochemical and ultrasound parameters were similar in nontreated and cell-treated groups; however, both groups showed significant differences compared to a normal control group. Cell infusions with bone marrow-derived cells seem to be ineffective in improving morphofunctional parameters of the liver when applied to chronic cases either during or after establishment of the hepatic lesion.

An ultrasound and histomorphological analysis of experimental liver cirrhosis in rats

We investigated whether liver injury by dual exposure to ethanol and carbon tetrachloride (EtOH + CCl4) for 15 weeks would persist after hepatotoxic agents were removed (EtOH + CCl4/8wR). After 15 weeks of hepatic injury with ethanol (5.5 percent, m/v) and carbon tetrachloride (0.05, mL/kg, ip), 5 of 11 female Wistar rats were sacrificed. The other 6 rats were maintained for an additional 8 weeks without hepatotoxic agents. Ultrasonography showed increased liver echogenicity and dilation of portal vein caliber in both groups (EtOH + CCl4: 0.22 ± 0.01 cm, P < 0.001; EtOH + CCl4/8wR: 0.21 ± 0.02 cm, P < 0.01) vs control (0.16 ± 0.02 cm). Histopathology showed regenerative nodules in both experimental groups. Histomorphometry revealed increased fibrosis content in both groups (EtOH + CCl4: 12.6 ± 2.64 percent, P < 0.001; EtOH + CCl4/8wR: 10.4 ± 1.36 percent, P < 0.05) vs control (2.2 ± 1.21 percent). Collagen types I and III were increased in groups EtOH + CCl4 (collagen I: 2.5 ± 1.3 percent, P < 0.01; collagen III: 1.3 ± 0.2 percent, P < 0.05) and EtOH + CCl4/8wR (collagen I: 1.8 ± 0.06 percent, P < 0.05; collagen III: 1.5 ± 0.8 percent, P < 0.01) vs control (collagen I: 0.38 ± 0.11 percent; collagen III: 0.25 ± 0.06 percent). Tissue transglutaminase increased in both groups (EtOH + CCl4: 66.4 ± 8 percent, P < 0.01; EtOH + CCl4/8wR: 58.8 ± 21 percent, P < 0.01) vs control (7.9 ± 0.8 percent). Cirrhosis caused by the association of CCl4-EtOH remained for at least 8 weeks after removal of these hepatotoxic agents. Ultrasound images can be a useful tool to evaluate advanced hepatic alterations.

Characterization of cardiopulmonary function and cardiac muscarinic and adrenergic receptor density adaptation in C57BL/6 mice with chronic Trypanosoma cruzi infection.

Circulating antibodies in chagasic patients interact with myocardial beta adrenergic and muscarinic cholinergic receptors, triggering intracellular signals that alter cardiac function along the course of the disease. However, until now, experimental data in models of chronically infected chagasic mice linking the effects on myocardial beta adrenergic and muscarinic receptors to cardiopulmonary dysfunction is lacking. Thus, we studied C57BL/6 mice 8 months after intraperitoneal injection of 100 trypomastigote forms of the Colombian strain of T. cruzi. Uninfected mice, matched in age, were used as controls. Histopathological analyses (inflammation and fibrosis) and radio-ligand binding assays for estimation of muscarinic and adrenergic receptor density were performed in myocardium tissue samples. When compared to controls, infected mice had electrical conduction disturbances, diastolic dysfunction, lower O2 consumption and anaerobic threshold. In addition, hearts of chronic chagasic mice had intense inflammation and fibrosis, and decreased beta adrenergic and increased muscarinic receptor densities than normal controls. Our data suggest that chronic T. cruzi infection causes alterations in cardiac receptor density and fibrosis deposition which can be associated with cardiac conduction abnormalities, diastolic dysfunction and lower exercise capacity, associating for the first time all these functional and histopathological alterations in chagasic mice.

Cardiac effects of oxytocin: is there a role for this peptide in cardiovascular homeostasis?

Oxytocin is well known for its role in reproduction. However, evidence has emerged suggesting a role in cardiovascular and hydroelectrolytic homeostasis. Although its renal effects have been characterized, the cardiac ones have not been much studied. Therefore, we aimed to investigate the cardiac effects of oxytocin both in vivo and in vitro. In unanesthetized rats (n=6) intravenous oxytocin (1 mug) decreased dP/dt(max) by 15% (P<0.05) and heart rate by 20% (P<0.001), at the first minute after injection. dP/dt(max) was still lower in OT-treated rats than in controls (n=8) after 15 min (P<0.05), while heart rate returned to control values after 5 min. In isolated hearts, oxytocin was able to promote negative inotropic and chronotropic effects. Perfusion with 10(-5), 10(-6) and 10(-7)M oxytocin resulted in approximately 60% (P<0.01), 25% (P<0.01) and 10% (P<0.05) reduction of left ventricle developed pressure, without effect in lower concentrations (10(-10) to 10(-8) M). Also, dP/dt(max) was reduced by 45 and 20% (10(-5) e 10(-6) M; P<0.01), while diastolic pressure raised and heart rate fell only with 10(-5)M oxytocin (P<0.05). Intravenous oxytocin (1 mug; n=6) increased arterial pressure by 22% at the first minute (+23+/-3 mm Hg; P<0.001), returning to control value thereafter. Thus, oxytocin is able to promote directly negative inotropic and chronotropic effects, but its in vivo effect also involves a reflex mechanism, originated from its pressor effect.

Correlation between conformation and antibody binding: NMR structure of cross-reactive peptides from T. cruzi, human and L. braziliensis.

The structure of peptides corresponding to the C-terminal residues from Trypanosoma cruzi (R13), human (H13) and Leishmania braziliensis (A13) ribosomal proteins were determined using nuclear magnetic resonance. Although there is only one amino acid difference between them, the peptides present distinct structures in solution: R13 adopts a random coil conformation while H13 and A13 form a bend. Interaction of these peptides with polyclonal antibodies from chronic Chagas' disease patients and a monoclonal antibody raised against T. cruzi ribosomal P2beta protein was probed by transferred NOE. The results show that the flexibility of R13 is fundamental for the binding to the antibody.