Talassemia beta maior e gestação na adolescência: relato de dois casos

A talassemia beta maior é uma doença hematológica hereditária rara em que deficiência na síntese de cadeias globínicas beta causa anemia grave. O tratamento consiste de transfusão sanguínea e quelação de ferro. Descrevemos dois casos de adolescentes com talassemia beta maior, com gestação não planejada e início tardio de pré-natal. Uma delas apresentou piora da anemia, necessidade transfusional aumentada, restrição de crescimento fetal e senescência placentária. A outra apresentava também hipotireoidismo e baixo peso materno, e foi internada por duas ocasiões durante a gestação, por choque hemorrágico do dengue e por infecção respiratória associada a vírus influenza H1N1. Uma delas apresentou restrição de crescimento fetal e teve parto vaginal no termo complicado com hipotonia uterina. Ambas necessitaram de transfusão sanguínea no pós-parto e optaram por medroxiprogesterona como método contraceptivo subsequentemente. Esse relato ressalta a importância de orientação contraceptiva para essas mulheres e o papel do cuidado pré-natal especializado em conjunto com hematologista.

Beta-lactamase detection in Staphylococcus aureus and coagulase-negative Staphylococcus isolated from bovine mastitis

The objectives of the study were to evaluate the presence/production of beta-lactamases by both phenotypic and genotypic methods, verify whether results are dependent of bacteria type (Staphylococcus aureus versus coagulase-negative Staphylococcus - CNS) and verify the agreement between tests. A total of 200 bacteria samples from 21 different herds were enrolled, being 100 CNS and 100 S. aureus. Beta-lactamase presence/detection was performed by different tests (PCR, clover leaf test - CLT, Nitrocefin disk, and in vitro resistance to penicillin). Results of all tests were not dependent of bacteria type (CNS or S. aureus). Several S. aureus beta-lactamase producing isolates were from the same herd. Phenotypic tests excluding in vitro resistance to penicillin showed a strong association measured by the kappa coefficient for both bacteria species. Nitrocefin and CLT are more reliable tests for detecting beta-lactamase production in staphylococci.

Resposta de plantas de beterraba (Beta vulgaris) e de cenoura (Daucus carota) à deriva simulada de glyphosate e clomazone

Várias espécies de hortaliças são de muita importância para a alimentação humana e tornam-se alvos da deriva de herbicidas, pois comumente são cultivadas nas proximidades de culturas como arroz, soja e milho, pulverizadas com esses produtos. Neste trabalho, objetivou-se verificar possíveis efeitos de doses reduzidas dos herbicidas glyphosate e clomazone sobre plantas de beterraba (Beta vulgaris) e de cenoura (Daucus carota), em diferentes fases de desenvolvimento. As doses avaliadas dos herbicidas foram de 0, 5, 10, 15 e 20% da dose recomendada, equivalentes a 0, 63, 126, 189 e 252 g ha-1 de glyphosate e 0, 14,4, 28,8, 43,2 e 57,6 g ha-1 de clomazone, respectivamente, aplicadas aos 20, 30 e 40 dias após a emergência das culturas. Observou-se aumento no percentual de fitotoxicidade do glyphosate com o incremento na dose do herbicida, e a maior suscetibilidade ocorreu com a deriva nos estádios mais precoces, em ambas as espécies. As doses de clomazone não causaram qualquer sintoma detectável visualmente para as plantas de beterraba e de cenoura. Os resultados sugerem que o herbicida glyphosate causa injúrias às plantas de beterraba e cenoura, independentemente do estádio em que ocorre a interceptação do produto. No entanto, o herbicida clomazone não interfere no desenvolvimento inicial de plantas de beterraba e cenoura.

Lead (Pb2+) and cadmium (Cd2+) inhibit the dipsogenic action of central beta-adrenergic stimulation by isoproterenol

We have previously demonstrated that acute third ventricle injections of both Pb2+ and Cd2+ impair the dipsogenic response elicited by three different situations: dehydration and central cholinergic or angiotensinergic stimulation. ß-Adrenergic activation is part of the multifactorial integrated systems operating in drinking behavior control in the central nervous system. In the present study acute third ventricle injections of Pb2+ (3, 30 and 300 pmol/rat) or Cd2+ (0.3, 3 and 30 pmol/rat) blocked the dipsogenic response induced by third ventricle injections of isoproterenol (ISO; 160 nmol/rat) in a dose-dependent manner. Normohydrated animals receiving ISO + NaAc (sodium acetate) or saline (controls) displayed a high water intake after 120 min (ISO + saline = 5.78 ± 0.54 ml/100 g; ISO + NaAc = 6.00 ± 0.6 ml/100 g). After the same period, animals receiving ISO but pretreated with PbAc at the highest dose employed (300 pmol/rat) drank 0.78 ± 0.23 ml/100 g while those receiving ISO and pretreated with the highest dose of CdCl2 (30 pmol/rat) presented a water intake of 0.7 ± 0.30 ml/100 g. Third ventricle injections of CdCl2 (3 nmol/rat) or PbAc (3 nmol/rat) did not modify food intake in rats deprived of food for 24 h. Thus, general central nervous system depression explaining the antidipsogenic action of the metals can be safely excluded. It is concluded that both Pb2+ and Cd2+ inhibit water intake induced by central ß-adrenergic stimulation

The left ventricular contractility of the rat heart is modulated by changes in flow and a1-adrenoceptor stimulation

Myocardial contractility depends on several mechanisms such as coronary perfusion pressure (CPP) and flow as well as on a1-adrenoceptor stimulation. Both effects occur during the sympathetic stimulation mediated by norepinephrine. Norepinephrine increases force development in the heart and produces vasoconstriction increasing arterial pressure and, in turn, CPP. The contribution of each of these factors to the increase in myocardial performance needs to be clarified. Thus, in the present study we used two protocols: in the first we measured mean arterial pressure, left ventricular pressure and rate of rise of left ventricular pressure development in anesthetized rats (N = 10) submitted to phenylephrine (PE) stimulation before and after propranolol plus atropine treatment. These observations showed that in vivo a1-adrenergic stimulation increases left ventricular-developed pressure (P<0.05) together with arterial blood pressure (P<0.05). In the second protocol, we measured left ventricular isovolumic systolic pressure (ISP) and CPP in Langendorff constant flow-perfused hearts. The hearts (N = 7) were perfused with increasing flow rates under control conditions and PE or PE + nitroprusside (NP). Both CPP and ISP increased (P<0.01) as a function of flow. CPP changes were not affected by drug treatment but ISP increased (P<0.01). The largest ISP increase was obtained with PE + NP treatment (P<0.01). The results suggest that both mechanisms, i.e., direct stimulation of myocardial a1-adrenoceptors and increased flow, increased cardiac performance acting simultaneously and synergistically.

Transforming growth factor beta activity in urine of patients with type 2 diabetes and diabetic nephropathy

Diabetic nephropathy (DN) is characterized structurally by progressive mesangial deposition of extracellular matrix (ECM). Transforming growth factor-ß (TGF-ß) is considered to be one of the major cytokines involved in the regulation of ECM synthesis and degradation. Several studies suggest that an increase in urinary TGF-ß levels may reflect an enhanced production of this polypeptide by the kidney cells. We evaluated TGF-ß in occasional urine samples from 14 normal individuals and 23 patients with type 2 diabetes (13 with persistent proteinuria >500 mg/24 h, DN, 6 with microalbuminuria, DMMA, and 4 with normal urinary albumin excretion, DMN) by enzyme immunoassay. An increase in the rate of urinary TGF-ß excretion (pg/mg UCreat.) was observed in patients with DN (296.07 ± 330.77) (P<0.001) compared to normal individuals (17.04 ± 18.56) (Kruskal-Wallis nonparametric analysis of variance); however, this increase was not observed in patients with DMMA (25.13 ± 11.30) or in DMN (18.16 ± 11.82). There was a positive correlation between the rate of urinary TGF-ß excretion and proteinuria (r = 0.70, a = 0.05) (Pearson's analysis), one of the parameters of disease progression.

Bradykinin enhances membrane electrical activity of pancreatic beta cells in the presence of low glucose concentrations

In most of cells bradykinin (BK) induces intracellular calcium mobilization. In pancreatic beta cells intracellular calcium is a major signal for insulin secretion. In these cells, glucose metabolism yields intracellular ATP which blocks membrane potassium channels. The membrane depolarizes, voltage-dependent Ca2+ channels are activated and the intracellular calcium load allows insulin secretion. Repolarization occurs due to activation of the Ca2+-dependent K+ channel. The insulin secretion depends on the integrity of this oscillatory process (bursts). Therefore, we decided to determine whether BK (100 nM) induces bursts in the presence of a non-stimulatory glucose concentration (5.6 mM). During continuous membrane voltage recording, our results showed that bursts were obtained with 11 mM glucose, blocked with 5.6 mM glucose and recovered with 5.6 mM glucose plus 100 nM BK. Thus, the stimulatory process obtained in the presence of BK and of a non-stimulatory concentration of glucose in the present study suggests that BK may facilitate the action of glucose on beta cell secretion.

Impaired beta-adrenergic response and decreased L-type calcium current of hypertrophied left ventricular myocytes in postinfarction heart failure

Infarct-induced heart failure is usually associated with cardiac hypertrophy and decreased ß-adrenergic responsiveness. However, conflicting results have been reported concerning the density of L-type calcium current (I Ca(L)), and the mechanisms underlying the decreased ß-adrenergic inotropic response. We determined I Ca(L) density, cytoplasmic calcium ([Ca2+]i) transients, and the effects of ß-adrenergic stimulation (isoproterenol) in a model of postinfarction heart failure in rats. Left ventricular myocytes were obtained by enzymatic digestion 8-10 weeks after infarction. Electrophysiological recordings were obtained using the patch-clamp technique. [Ca2+]i transients were investigated via fura-2 fluorescence. ß-Adrenergic receptor density was determined by [³H]-dihydroalprenolol binding to left ventricle homogenates. Postinfarction myocytes showed a significant 25% reduction in mean I Ca(L) density (5.7 ± 0.28 vs 7.6 ± 0.32 pA/pF) and a 19% reduction in mean peak [Ca2+]i transients (0.13 ± 0.007 vs 0.16 ± 0.009) compared to sham myocytes. The isoproterenol-stimulated increase in I Ca(L) was significantly smaller in postinfarction myocytes (Emax: 63.6 ± 4.3 vs 123.3 ± 0.9% in sham myocytes), but EC50 was not altered. The isoproterenol-stimulated peak amplitude of [Ca2+]i transients was also blunted in postinfarction myocytes. Adenylate cyclase activation through forskolin produced similar I Ca(L) increases in both groups. ß-Adrenergic receptor density was significantly reduced in homogenates from infarcted hearts (Bmax: 93.89 ± 20.22 vs 271.5 ± 31.43 fmol/mg protein in sham myocytes), while Kd values were similar. We conclude that postinfarction myocytes from large infarcts display reduced I Ca(L) density and peak [Ca2+]i transients. The response to ß-adrenergic stimulation was also reduced and was probably related to ß-adrenergic receptor down-regulation and not to changes in adenylate cyclase activity.

Simultaneous changes in the function and expression of beta 1 integrins during the growth arrest of poorly differentiated colorectal cells (LISP-1)

Cells usually lose adhesion and increase proliferation and migration during malignant transformation. Here, we studied how proliferation can affect the other two characteristics, which ultimately lead to invasion and metastasis. We determined the expression of ß1 integrins, as well as adhesion and migration towards laminin-1, fibronectin, collagens type I and type IV presented by LISP-1 colorectal cancer cells exposed to 2.5% dimethyl sulfoxide (DMSO), an agent capable of decreasing proliferation in this poorly differentiated colorectal cell line. Untreated cells (control), as shown by flow cytometry and monoclonal antibodies, expressed alpha2 (63.8 ± 11.3% positive cells), alpha3 (93.3 ± 7.0%), alpha5 (50.4 ± 12.0%) and alpha6 (34.1 ± 4.9%) integrins but not alpha1, alpha4, alphav or ß4. Cells adhered well to laminin-1 (73.4 ± 6.0%) and fibronectin (40.0 ± 2.0%) substrates but very little to collagens. By using blocking monoclonal antibodies, we showed that alpha2, alpha3 and alpha6 mediated laminin-1 adhesion, but neither alpha3 nor alpha5 contributed to fibronectin adherence. DMSO arrested cells at G0/G1 (control: 55.0 ± 2.4% vs DMSO: 70.7 ± 2.5%) while simultaneously reducing alpha5 (24.2 ± 19%) and alpha6 (14.3 ± 10.8%) expression as well as c-myc mRNA (7-fold), the latter shown by Northern blotting. Although the adhesion rate did not change after exposure to DMSO, alpha3 and alpha5 played a major role in laminin-1 and fibronectin adhesion, respectively. Migration towards laminin-1, which was clearly increased upon exposure to DMSO (control: 6 ± 2 cells vs DMSO: 64 ± 6 cells), was blocked by an antibody against alpha6. We conclude that the effects of DMSO on LISP-1 proliferation were accompanied by concurrent changes in the expression and function of integrins, consequently modulating adhesion/migration, and revealing a complex interplay between function/expression and the proliferative state of cells.

Non-obese adult onset diabetes with oral hypoglycemic agent failure: islet cell autoantibodies or reversible beta cell refractoriness?

Pancreatic ß cell function and insulin sensitivity, analyzed by the homeostasis model assessment, before and after 24 weeks of insulin therapy were studied and correlated with the presence of autoantibodies against ß cells (islet cell and anti-glutamic acid decarboxylase antibodies), in a group of 18 Brazilian lean adult non-insulin-dependent diabetes mellitus (NIDDM) patients with oral hypoglycemic agent failure (OHAF). Median fasting plasma glucose before and after insulin treatment was 19.1 and 8.5 mmol/l, respectively (P < 0.001); median HbA1c was 11.7% before vs 7.2% after insulin treatment (P < 0.001). Forty-four percent of the patients were positive (Ab+) to at least one autoantibody. Fasting C-peptide levels were lower in Ab+ than Ab- patients, both before (Ab+: 0.16 ± 0.09 vs Ab-: 0.41 ± 0.35 nmol/l, P < 0.003) and after insulin treatment (Ab+: 0.22 ± 0.13 vs Ab-: 0.44 ± 0.24 nmol/l, P < 0.03). Improvement of Hß was seen in Ab- (median before: 7.3 vs after insulin therapy: 33.4%, P = 0.003) but not in Ab+ patients (median before: 6.6 vs after insulin therapy: 20.9%). These results show that the OHAF observed in the 18 NIDDM patients studied was due mainly to two major causes: autoantibodies and ß cell desensitization. Autoantibodies against ß cells could account for 44% of OHAF, but Ab- patients may still present ß cell function recovery, mainly after a period of ß cell rest with insulin therapy. However, the effects of ß cell function recovery on the restoration of the response to oral hypoglycemic agents need to be determined.

Effect of cyproterone acetate on alpha1-adrenoceptor subtypes in rat vas deferens

Gonadal hormones regulate the expression of alpha1-adrenoceptor subtypes in several tissues. The present study was carried out to determine whether or not cyproterone acetate, an anti-androgenic agent, regulates the alpha1-adrenoceptor subtypes that mediate contractions of the rat vas deferens in response to noradrenaline. The actions of subtype selective alpha1-antagonists were investigated in vas deferens from control and cyproterone acetate-treated rats (10 mg/day, sc, for 7 days). Prazosin (pA2 ~9.5), phentolamine (pA2 ~8.3) and yohimbine (pA2 ~6.7) presented competitive antagonism consistent with activation of alpha1-adrenoceptors in vas deferens from both control and treated rats. The pA2 values estimated for WB 4101 (~9.5), benoxathian (~9.7), 5-methylurapidil (~8.5), indoramin (~8.7) and BMY 7378 (~6.8) indicate that alpha1A-adrenoceptors are involved in the contractions of the vas deferens from control and cyproterone acetate-treated rats. Treatment of the vas deferens from control rats with the alpha1B/alpha1D-adrenoceptor alkylating agent chloroethylclonidine had no effect on noradrenaline contractions, supporting the involvement of the alpha1A-subtype. However, this agent partially inhibited the contractions of vas deferens from cyproterone acetate-treated rats, suggesting involvement of multiple receptor subtypes. To further investigate this, the actions of WB 4101 and chloroethylclonidine were reevaluated in the vas deferens from rats treated with cyproterone acetate for 14 days. In these organs WB 4101 presented complex antagonism characterized by a Schild plot with a slope different from unity (0.65 ± 0.05). After treatment with chloroethylclonidine, the complex antagonism presented by WB 4101 was converted into classical competitive antagonism, consistent with participation of alpha1A-adrenoceptors as well as alpha1B-adrenoceptors. These results suggest that cyproterone acetate induces plasticity in the alpha1-adrenoceptor subtypes involved in the contractions of the vas deferens.

Identification of clonally rearranged T-cell receptor beta chain genes in HTLV-I carriers as a potential instrument for early detection of neoplasia

We analyzed the genetic recombination pattern of the T-cell receptor beta-chain gene (TCR-beta) in order to identify clonal expansion of T-lymphocytes in 17 human T-lymphotropic virus type I (HTLV-I)-positive healthy carriers, 7 of them with abnormal features in the peripheral blood lymphocytes. Monoclonal or oligoclonal expansion of T-cells was detected in 5 of 7 HTLV-I-positive patients with abnormal lymphocytes and unconfirmed diagnosis by using PCR amplification of segments of TCR-beta gene, in a set of reactions that target 102 different variable (V) segments, covering all members of the 24 V families available in the gene bank, including the more recently identified segments of the Vbeta-5 and Vbeta-8 family and the two diversity beta segments. Southern blots, the gold standard method to detect T-lymphocyte clonality, were negative for all of these 7 patients, what highlights the low sensitivity of this method that requires a large amount of very high quality DNA. To evaluate the performance of PCR in the detection of clonality we also analyzed 18 leukemia patients, all of whom tested positive. Clonal expansion was not detected in any of the negative controls or healthy carriers without abnormal lymphocytes. In conclusion, PCR amplification of segments of rearranged TCR-beta is reliable and highly suitable for the detection of small populations of clonal T-cells in asymptomatic HTLV-I carriers who present abnormal peripheral blood lymphocytes providing an additional instrument for following up these patients with potentially higher risk of leukemia.

Expression of beta 2 integrin (CD18) in embryonic mouse and chicken heart

Integrins are heterodimeric receptors composed of α and β transmembrane subunits that mediate attachment of cells to the extracellular matrix and counter-ligands such as ICAM-1 on adjacent cells. β2 integrin (CD18) associates with four different α (CD11) subunits to form an integrin subfamily, which has been reported to be expressed exclusively on leukocytes. However, recent studies indicate that β2 integrin is also expressed by other types of cells. Since the gene for β2 integrin is located in the region of human chromosome 21 associated with congenital heart defects, we postulated that it may be expressed in the developing heart. Here, we show the results from several different techniques used to test this hypothesis. PCR analyses indicated that β2 integrin and the αL, αM, and αX subunits are expressed during heart development. Immunohistochemical studies in both embryonic mouse and chicken hearts, using antibodies directed against the N- or C-terminal of β2 integrin or against its α subunit partners, showed that β2 integrin, as well as the αL, αM, and αX subunits, are expressed by the endothelial and mesenchymal cells of the atrioventricular canal and in the epicardium and myocardium during cardiogenesis. In situ hybridization studies further confirmed the presence of β2 integrin in these various locations in the embryonic heart. These results indicate that the β2 integrin subfamily may have other activities in addition to leukocyte adhesion, such as modulating the migration and differentiation of cells during the morphogenesis of the cardiac valves and myocardial walls of the heart.

Antifungal activity of the naphthoquinone beta-lapachone against disseminated infection with Cryptococcus neoformans var. neoformans in dexamethasone-immunosuppressed Swiss mice

The in vivo antifungal activity of the naphthoquinone beta-lapachone against disseminated infection by Cryptococcus neoformans was investigated. Swiss mice were immunosuppressed daily with dexamethasone (0.5 mg per mouse) intraperitoneally for 3 days, the procedure was repeated 4 days later, and the animals were then challenged intravenously with C. neoformans (10(6) CFU/mL) 1 week later. Seven days after infection, the mice were divided into groups and treated daily with beta-lapachone (10 mg/kg, iv) for 7 (N = 6) and 14 days (N = 10). Amphotericin B (0.5 mg/kg) was used as comparator drug and an additional group received PBS. Treatment with beta-lapachone cleared the yeast from the spleen and liver, and the fungal burden decreased approximately 10(4) times in the lungs and brain 14 days after infection when compared to the PBS group (P < 0.05). This result was similar to that of the amphotericin B-treated group. Protection was suggestively due to in vivo antifungal activity of this drug and apparently not influenced by activation of the immune response, due to similar leukocyte cell counts among all groups. This study highlights the prospective use of beta-lapachone for treatment of disseminated cryptococcosis.

Effect of methylprednisolone on perivascular pulmonary edema, inflammatory infiltrate, VEGF and TGF-beta immunoexpression in the remaining lungs of rats after left pneumonectomy

Pneumonectomy is associated with high rates of morbimortality, with postpneumonectomy pulmonary edema being one of the leading causes. An intrinsic inflammatory process following the operation has been considered in its physiopathology. The use of corticosteroids is related to prevention of this edema, but no experimental data are available to support this hypothesis. We evaluated the effect of methylprednisolone on the remaining lungs of rats submitted to left pneumonectomy concerning edema and inflammatory markers. Forty male Wistar rats weighing 300 g underwent left pneumonectomy and were randomized to receive corticosteroids or not. Methylprednisolone at a dose of 10 mg/kg was given before the surgery. After recovery, the animals were sacrificed at 48 and 72 h, when the pO2/FiO2 ratio was determined. Right lung perivascular edema was measured by the index between perivascular and vascular area and neutrophil density by manual count. Tissue expression of vascular endothelial growth factor (VEGF) and transforming growth factor-beta (TGF-β) were evaluated by immunohistochemistry light microscopy. There was perivascular edema formation after 72 h in both groups (P = 0.0031). No difference was observed between operated animals that received corticosteroids and those that did not concerning the pO2/FiO2 ratio, neutrophil density or TGF-β expression. The tissue expression of VEGF was elevated in the animals that received methylprednisolone both 48 and 72 h after surgery (P = 0.0243). Methylprednisolone was unable to enhance gas exchange and avoid an inflammatory infiltrate and TGF-β expression also showed that the inflammatory process was not correlated with pulmonary edema formation. However, the overexpression of VEGF in this group showed that methylprednisolone is related to this elevation.