Pythiosis in sheep from Paraná, southern Brazil

Abstract: This paper reports pythiosis in a sheep from southwestern Paraná, Brazil, confirmed by indirect ELISA (Enzime-Linked Immunosorbent Assay) and immunohistochemistry, as well as it describes the macro and microscopic injuries, in order to understand the pathogenicity. A 4-year-old ewe from a flock of 30 Santa Inês sheep, raised semi-extensively with access to a weir, showed cachexia, bilateral enlargement in nasal region, a serous and bloody secretion with a fetid odor from its nose and swollen submandibular and retropharyngeal lymph nodes. Blood collection was performed trough jugular vein puncture in order to make complete blood cell count (CBC) and to obtain serum for the subsequent serological examination. As the hematological counts were within the normal range for sheep, the animal was euthanized and submitted to necropsy. Indirect ELISA resulted positive for pythiosis. Necropsy revealed necrosis of the hard palate with a diameter of 3.5cm and extending up to the nasal cavity, forming a fistula. Submandibular and retropharyngeal lymph nodes were enlarged and edematous on section. Microscopic findings for submandibular and retropharyngeal lymph nodes consisted in moderate infiltration of eosinophils mainly in the subcapsular sinus, characterizing reactive eosinophilic lymphadenitis. The nasal cavity revealed rhinitis and oral cavity stomatitis with necro-eosinophilic and pronounced multifocal granulomatous infiltration and presence of hyphae. Hyphae found in palate and nasal cavity were positive for Pythium insidiosum by Grocott's method and immunohistochemistry, the last one considered to be confirmatory for the pathogen diagnostic. This report has an important epidemiological aspect, as it is the first case of pythiosis in sheep confirmed by serology in South Brazil and an alert of possible infection by the pathogen in floodplains.

Protocol for collection and separation of bone marrow mononuclear cells in Chlorocebus aethiops

Abstract: Chlorocebus aethiops is a species of non-human primate frequently used in biomedical research. Some research involves this species as an experimental model for various diseases and possible treatment with stem cells. The bone marrow is one of the main sources of these cells and provides easy access. The aim of this study was to standardize the protocol of collection and separation of bone marrow in C. aethiops. Ten animals were submitted to puncture of bone marrow with access to the iliac crest and cell separation by density gradient. The bone marrow of C. aethiops had an average of 97% viability. From the results achieved, we can conclude that C. aethiops is an excellent model to obtain and isolate mononuclear cells from bone marrow, fostering several studies in the field of cell therapy.

Partial recovery of erythrocyte glycogen in diabetic rats treated with phenobarbital

Erythrocytes may play a role in glucose homeostasis during the postprandial period. Erythrocytes from diabetic patients are defective in glucose transport and metabolism, functions that may affect glycogen storage. Phenobarbital, a hepatic enzyme inducer, has been used in the treatment of patients with non-insulin-dependent diabetes mellitus (NIDDM), increasing the insulin-mediated glucose disposal. We studied the effects of phenobarbital treatment in vivo on glycemia and erythrocyte glycogen content in control and alloxan-diabetic rats during the postprandial period. In control rats (blood glucose, 73 to 111 mg/dl in femoral and suprahepatic veins) the erythrocyte glycogen content was 45.4 ± 1.1 and 39.1 ± 0.8 µg/g Hb (mean ± SEM, N = 4-6) in the femoral artery and vein, respectively, and 37.9 ± 1.1 in the portal vein and 47.5 ± 0.9 in the suprahepatic vein. Diabetic rats (blood glucose, 300-350 mg/dl) presented low (P<0.05) erythrocyte glycogen content, i.e., 9.6 ± 0.1 and 7.1 ± 0.7 µg/g Hb in the femoral artery and vein, respectively, and 10.0 ± 0.7 and 10.7 ± 0.5 in the portal and suprahepatic veins, respectively. After 10 days of treatment, phenobarbital (0.5 mg/ml in the drinking water) did not change blood glucose or erythrocyte glycogen content in control rats. In diabetic rats, however, it lowered (P<0.05) blood glucose in the femoral artery (from 305 ± 18 to 204 ± 45 mg/dl) and femoral vein (from 300 ± 11 to 174 ± 48 mg/dl) and suprahepatic vein (from 350 ± 10 to 174 ± 42 mg/dl), but the reduction was not sufficient for complete recovery. Phenobarbital also stimulated the glycogen synthesis, leading to a partial recovery of glycogen stores in erythrocytes. In treated rats, erythrocyte glycogen content increased to 20.7 ± 3.8 µg/g Hb in the femoral artery and 30.9 ± 0.9 µg/g Hb in the suprahepatic vein (P<0.05). These data indicate that phenobarbital activated some of the insulin-stimulated glucose metabolism steps which were depressed in diabetic erythrocytes, supporting the view that erythrocytes participate in glucose homeostasis

Monosodium glutamate (MSG)-obese rats develop glucose intolerance and insulin resistance to peripheral glucose uptake

Different levels of insulin sensitivity have been described in several animal models of obesity as well as in humans. Monosodium glutamate (MSG)-obese mice were considered not to be insulin resistant from data obtained in oral glucose tolerance tests. To reevaluate insulin resistance by the intravenous glucose tolerance test (IVGTT) and by the clamp technique, newborn male Wistar rats (N = 20) were injected 5 times, every other day, with 4 g/kg MSG (N = 10) or saline (control; N = 10) during the first 10 days of age. At 3 months, the IVGTT was performed by injecting glucose (0.75 g/kg) through the jugular vein into freely moving rats. During euglycemic clamping plasma insulin levels were increased by infusing 3 mU . kg-1 . min-1 of regular insulin until a steady-state plateau was achieved. The basal blood glucose concentration did not differ between the two experimental groups. After the glucose load, increased values of glycemia (P<0.001) in MSG-obese rats occurred at minute 4 and from minute 16 to minute 32. These results indicate impaired glucose tolerance. Basal plasma insulin levels were 39.9 ± 4 µU/ml in control and 66.4 ± 5.3 µU/ml in MSG-obese rats. The mean post-glucose area increase of insulin was 111% higher in MSG-obese than in control rats. When insulinemia was clamped at 102 or 133 µU/ml in control and MSG rats, respectively, the corresponding glucose infusion rate necessary to maintain euglycemia was 17.3 ± 0.8 mg . kg-1 . min-1 for control rats while 2.1 ± 0.3 mg . kg-1 . min-1 was sufficient for MSG-obese rats. The 2-h integrated area for total glucose metabolized, in mg . min . dl-1, was 13.7 ± 2.3 vs 3.3 ± 0.5 for control and MSG rats, respectively. These data demonstrate that MSG-obese rats develop insulin resistance to peripheral glucose uptake

Validation of transit-time flowmetry for chronic measurements of regional blood flow in resting and exercising rats

The objective of the present study was to validate the transit-time technique for long-term measurements of iliac and renal blood flow in rats. Flow measured with ultrasonic probes was confirmed ex vivo using excised arteries perfused at varying flow rates. An implanted 1-mm probe reproduced with accuracy different patterns of flow relative to pressure in freely moving rats and accurately quantitated the resting iliac flow value (on average 10.43 ± 0.99 ml/min or 2.78 ± 0.3 ml min-1 100 g body weight-1). The measurements were stable over an experimental period of one week but were affected by probe size (resting flows were underestimated by 57% with a 2-mm probe when compared with a 1-mm probe) and by anesthesia (in the same rats, iliac flow was reduced by 50-60% when compared to the conscious state). Instantaneous changes of iliac and renal flow during exercise and recovery were accurately measured by the transit-time technique. Iliac flow increased instantaneously at the beginning of mild exercise (from 12.03 ± 1.06 to 25.55 ± 3.89 ml/min at 15 s) and showed a smaller increase when exercise intensity increased further, reaching a plateau of 38.43 ± 1.92 ml/min at the 4th min of moderate exercise intensity. In contrast, exercise-induced reduction of renal flow was smaller and slower, with 18% and 25% decreases at mild and moderate exercise intensities. Our data indicate that transit-time flowmetry is a reliable method for long-term and continuous measurements of regional blood flow at rest and can be used to quantitate the dynamic flow changes that characterize exercise and recovery

Effect of pyloroplasty and fundectomy on the delay of gastric emptying and gastrointestinal transit of liquid elicited by acute blood volume expansion in awake rats

We evaluated the effects of fundectomy and pyloroplasty on the delay of gastric emptying (GE) and gastrointestinal (GI) transit of liquid due to blood volume (BV) expansion in awake rats. Male Wistar rats (N = 76, 180-250 g) were first submitted to fundectomy (N = 26), Heinecke-Mikulicz pyloroplasty (N = 25) or SHAM laparotomy (N = 25). After 6 days, the left external jugular vein was cannulated and the animals were fasted for 24 h with water ad libitum. The test meal was administered intragastrically (1.5 ml of a phenol red solution, 0.5 mg/ml in 5% glucose) to normovolemic control animals and to animals submitted to BV expansion (Ringer-bicarbonate, iv infusion, 1 ml/min, volume up to 5% body weight). BV expansion decreased GE and GI transit rates in SHAM laparotomized animals by 52 and 35.9% (P<0.05). Fundectomy increased GE and GI transit rates by 61.1 and 67.7% (P<0.05) and prevented the effect of expansion on GE but not on GI transit (13.9% reduction, P<0.05). Pyloroplasty also increased GE and GI transit rates by 33.9 and 44.8% (P<0.05) but did not prevent the effect of expansion on GE or GI transit (50.7 and 21.1% reduction, P<0.05). Subdiaphragmatic vagotomy blocked the effect of expansion on GE and GI transit in both SHAM laparotomized animals and animals submitted to pyloroplasty. In conclusion 1) the proximal stomach is involved in the GE delay due to BV expansion but is not essential for the establishment of a delay in GI transit, which suggests the activation of intestinal resistances, 2) pyloric modulation was not apparent, and 3) vagal pathways are involved

Acute blood volume expansion delays the gastrointestinal transit of a charcoal meal in awake rats

The present study evaluates the effect of blood volume expansion on the gastrointestinal transit of a charchoal meal (2.5 ml of an aqueous suspension consisting of 5% charcoal and 5% gum arabic) in awake male Wistar rats (200-270 g). On the day before the experiments, the rats were anesthetized with ether, submitted to left jugular vein cannulation and fasted with water ad libitum until 2 h before the gastrointestinal transit measurement. Blood volume expansion by iv infusion of 1 ml/min Ringer bicarbonate in volumes of 3, 4 or 5% body weight delayed gastrointestinal transit at 10 min after test meal administration by 21.3-26.7% (P<0.05), but no effect was observed after 1 or 2% body weight expansion. The effect of blood volume expansion (up to 5% body weight) on gastrointestinal transit lasted for at least 60 min (P<0.05). Mean arterial pressure increased transiently and central venous pressure increased and hematocrit decreased (P<0.05). Subdiaphragmatic vagotomy and yohimbine (3 mg/kg) prevented the delay caused by expansion on gastrointestinal transit, while atropine (0.5 mg/kg), L-NAME (2 mg/kg), hexamethonium (10 mg/kg), prazosin (1 mg/kg) or propranolol (2 mg/kg) were ineffective. These data show that blood volume expansion delays the gastrointestinal transit of a charcoal meal and that vagal and yohimbine-sensitive pathways appear to be involved in this phenomenon. The delay in gastrointestinal transit observed here, taken together with the modifications of gastrointestinal permeability to salt and water reported by others, may be part of the mechanisms involved in liquid excess management.

Insulin receptor has tyrosine kinase activity toward Shc in rat liver

Insulin induces tyrosine phosphorylation of Shc in cell cultures and in insulin-sensitive tissues of the intact rat. However, the ability of insulin receptor (IR) tyrosine kinase to phosphorylate Shc has not been previously demonstrated. In the present study, we investigated insulin-induced IR tyrosine kinase activity towards Shc. Insulin receptor was immunoprecipitated from liver extracts, before and after a very low dose of insulin into the portal vein, and incubated with immunopurified Shc from liver of untreated rats. The kinase assay was performed in vitro in the presence of exogenous ATP and the phosphorylation level was quantified by immunoblotting with antiphosphotyrosine antibody. The results demonstrate that Shc interacted with insulin receptor after infusion of insulin, and, more important, there was insulin receptor kinase activity towards immunopurified Shc. The description of this pathway in animal tissue may have an important role in insulin receptor tyrosine kinase activity toward mitogenic transduction pathways.

A simple HPLC-fluorescence method for the measurement of R,S-sotalol in the plasma of patients with life-threatening cardiac arrhythmias

R,S-sotalol, a ß-blocker drug with class III antiarrhythmic properties, is prescribed to patients with ventricular, atrial and supraventricular arrhythmias. A simple and sensitive method based on HPLC-fluorescence is described for the quantification of R,S-sotalol racemate in 500 µl of plasma. R,S-sotalol and its internal standard (atenolol) were eluted after 5.9 and 8.5 min, respectively, from a 4-micron C18 reverse-phase column using a mobile phase consisting of 80 mM KH2PO4, pH 4.6, and acetonitrile (95:5, v/v) at a flow rate of 0.5 ml/min with detection at lex = 235 nm and lem = 310 nm, respectively. This method, validated on the basis of R,S-sotalol measurements in spiked blank plasma, presented 20 ng/ml sensitivity, 20-10,000 ng/ml linearity, and 2.9 and 4.8% intra- and interassay precision, respectively. Plasma sotalol concentrations were determined by applying this method to investigate five high-risk patients with atrial fibrillation admitted to the Emergency Service of the Medical School Hospital, who received sotalol, 160 mg po, as loading dose. Blood samples were collected from a peripheral vein at zero, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 6.0, 8.0, 12.0 and 24.0 h after drug administration. A two-compartment open model was applied. Data obtained, expressed as mean, were: CMAX = 1230 ng/ml, TMAX = 1.8 h, AUCT = 10645 ng h-1 ml-1, Kab = 1.23 h-1, a = 0.95 h-1, ß = 0.09 h-1, t(1/2)ß = 7.8 h, ClT/F = 3.94 ml min-1 kg-1, and Vd/F = 2.53 l/kg. A good systemic availability and a fast absorption were obtained. Drug distribution was reduced to the same extent in terms of total body clearance when patients and healthy volunteers were compared, and consequently elimination half-life remained unchanged. Thus, the method described in the present study is useful for therapeutic drug monitoring purposes, pharmacokinetic investigation and pharmacokinetic-pharmacodynamic sotalol studies in patients with tachyarrhythmias.

Inhibition of gastric emptying and intestinal transit in anesthetized rats by a Tityus serrulatus scorpion toxin

The effects of a fraction (T1) of Tityus serrulatus scorpion venom prepared by gel filtration on gastric emptying and small intestinal transit were investigated in male Wistar rats. Fasted animals were anesthetized with urethane, submitted to tracheal intubation and right jugular vein cannulation. Scorpion toxin (250 µg/kg) or saline was injected iv and 1 h later a bolus of saline (1.0 ml/100 g) labeled with 99m technetium-phytate (10 MBq) was administered by gavage. After 15 min, animals were sacrificed and the radioactivity remaining in the stomach was determined. Intestinal transit was evaluated by instillation of a technetium-labeled saline bolus (1.0 ml) through a cannula previously implanted in the duodenum. After 60 min, the progression of the marker throughout 7 consecutive gut segments was estimated by the geometric center method. Gastric retention of the liquid test meal in rats injected with scorpion toxin (median: 88%; range: 52-95%) was significantly higher (P<0.02) than in controls (54%; 21-76%), an effect which was not modified by gastric secretion blockade with ranitidine. The progression of the isotope marker throughout the small intestine was significantly slower (P<0.05) in rats treated with toxin (1.2; 1.0-2.5) than in control animals (2.3; 1.0-3.2). Inhibition of both gastric emptying and intestinal transit in rats injected with scorpion toxin suggests an increased resistance to aboral flow, which might be caused by abnormal neurotransmitter release or by the local effects of venom on smooth muscle cells.

Indirect effects of oral tolerance to ovalbumin interfere with the immune responses triggered by Schistosoma mansoni eggs

The objective of the present study was to investigate whether the injection of a tolerated protein (indirect effects) affects the formation of granulomas around Schistosoma mansoni eggs trapped in the lungs after intravenous (iv) injection into normal (noninfected) C57BL/6 mice (6 animals per group). To induce oral tolerance to chicken egg ovalbumin a 1/5 dilution of egg white in water was offered ad libitum in a drinking bottle for 3 days. Control mice received water. After 7 days, control and experimental animals were injected iv with 2,000 S. mansoni eggs through a tail vein. In some mice of both groups the iv injection of eggs was immediately followed by intraperitoneal (ip) immunization with 10 µg of dinitrophenylated conjugates of ovalbumin (DNP-Ova) emulsified in complete Freund's adjuvant (CFA) or only CFA; 18 days later, mice were bled and killed by ether inhalation. The lungs were fixed in formalin and embedded in paraffin. Serial sections of 5 µm were stained with Giemsa, Gomori's silver reticulin and Sirius red (pH 10.2). Granuloma diameters were measured in histological sections previously stained with Gomori's reticulin. Anti-DNP and anti-soluble egg antigen (SEA) antibodies were analyzed by ELISA. In mice orally tolerant to ovalbumin the concomitant ip injection of DNP-Ova resulted in significantly lower anti-SEA antibodies (ELISA*: 1395 ± 352 in non-tolerant and 462 ± 146 in tolerant mice) and affected granuloma formation around eggs, significantly decreasing granuloma size (area: 22,260 ± 2478 to 12,993 ± 3242 µm²). Active mechanisms triggered by injection of tolerated antigen (ovalbumin) reduce granuloma formation.

Pentoxifylline decreases tumor necrosis factor and interleukin-1 during high tidal volume

Tumor necrosis factor-alpha (TNF-alpha) is one of the most important proinflammatory cytokines which plays a central role in host defense and in the acute inflammatory response related to tissue injury. The major source of TNF-alpha are immune cells such as neutrophils and macrophages. We tested the hypothesis that pentoxifylline, a methylxanthine derivative, down-regulates proinflammatory cytokine expression during acute lung injury in rats. Male Wistar rats weighing 250 to 450 g were anesthetized ip with 50 mg/kg sodium thiopental and randomly divided into three groups: group 1 (N = 7): tidal volume (V T) = 7 ml/kg, respiratory rate (RR) = 50 breaths/min and normal saline infusion; group 2 (N = 7): V T = 42 ml/kg, RR = 9 breaths/min and normal saline infusion; group 3 (N = 7): V T = 42 ml/kg, RR = 9 breaths/min and pentoxifylline infusion. The animals were ventilated with an inspired oxygen fraction of 1.0, a positive end-expiratory pressure of 3 cmH2O, and normal saline or pentoxifylline injected into the left femoral vein. The mRNA of TNF-alpha rapidly increased in the lung tissue within 180 min of ventilation with a higher V T with normal saline infusion. The concentrations of inflammatory mediators were decreased in plasma and bronchoalveolar lavage (BAL) in the presence of higher V T with pentoxifylline infusion (TNF-alpha: plasma, 102.2 ± 90.9 and BAL, 118.2 ± 82.1; IL-1ß: plasma, 45.2 ± 42.7 and BAL, 50.2 ± 34.9, P < 0.05). We conclude that TNF-alpha produced by neutrophil influx may function as an alert signal in host defense to induce production of other inflammatory mediators.

Alternative parameters for echocardiographic assessment of fetal diastolic function

Alternative methods to assess ventricular diastolic function in the fetus are proposed. Fetal myocardial hypertrophy in maternal diabetes was used as a model of decreased left ventricular compliance (LVC), and fetal respiratory movements as a model of increased LVC. Comparison of three groups of fetuses showed that, in 10 fetuses of diabetic mothers (FDM) with septal hypertrophy (SH), the mean excursion index of the septum primum (EISP) (ratio between the linear excursion of the flap valve and the left atrial diameter) was 0.36 ± 0.09, in 8 FDM without SH it was 0.51 ± 0.09 (P = 0.001), and in the 8 normal control fetuses (NCF) it was 0.49 ± 0.12 (P = 0.003). In another study, 28 fetuses in apnea had a mean EISP of 0.39 ± 0.05 which increased to 0.57 ± 0.07 during respiration (P < 0.001). These two studies showed that the mobility of the septum primum was reduced when LVC was decreased and was increased when LVC was enhanced. Mean pulmonary vein pulsatility was higher in 14 FDM (1.83 ± 1.21) than in 26 NCF (1.02 ± 0.31; P = 0.02). In the same fetuses, mean left atrial shortening was decreased (0.40 ± 0.11) in relation to NCF (0.51 ± 0.09; P = 0.011). These results suggest that FDM may have a higher preload than normal controls, probably as a result of increased myocardial mass and LV hypertrophy. Prenatal assessment of LV diastolic function by fetal echocardiography should include analysis of septum primum mobility, pulmonary vein pulsatility, and left atrial shortening.

Induction of oral tolerance and the effect of interleukin-4 on murine skin allograft rejection

We studied the effect of oral and portal vein administration of alloantigens on mouse skin allograft survival. Graft receptor BALB/c mice received spleen cells (30, 90, 150 or 375 x 10(6)) from donor C57BL/6 mice intragastrically on three successive days, starting seven days before the skin graft. Allograft survival was significantly increased with the feeding of 150 x 10(6) allogeneic spleen cells by one gavage (median survival of 12 vs 14 days, P <= 0.005) or when 300 x 10(6) cells were given in six gavage (12 vs 14 days, P < 0.04). A similar effect was observed when 150 x 10(6) spleen cells were injected into the portal vein (12 vs 14 days, P <= 0.03). Furthermore, prolonged allograft survival was observed with subcutaneous (12 vs 16 days, P <= 0.002) or systemic (12 vs 15 days, P <= 0.016) application of murine interleukin-4 (IL-4), alone or in combination with spleen cell injection into the portal vein (12 vs 18 days, P <= 0.0018). Taken together, these results showed that tolerance induction with spleen cells expressing fully incompatible antigens by oral administration or intraportal injection partially down-modulates skin allograft rejection. Furthermore, these findings demonstrated for the first time the effect of subcutaneous or systemic IL-4 application on allograft skin survival suggesting its use as a beneficial support therapy in combination with a tolerance induction protocol.

Cardiac and vascular effects of diltiazem, dobutamine and amrinone, drugs used after myocardial revascularization

Hemodynamic care during postoperative management of myocardial revascularization should include vasorelaxing drugs to insure adequate graft and coronary flow, and stimulation of stroke volume to maintain vascular perfusion pressure. We tested the cardiac (inotropic and lusitropic) and vascular (relaxant) effects of diltiazem (0.1 nM to 0.1 mM), dobutamine (10 µM to 10 mM) and amrinone (10 µM to 1 mM) on isolated rat atria and thoracic aorta, and also on isolated human saphenous vein (HSV) and human mammary artery (HMA). Dobutamine produced a maximal positive inotropic effect (+dF/dt max = 29 ± 7%) at its ED50 for aortic relaxation (88 ± 7 µM). Conversely, at their ED50 for aortic relaxation diltiazem depressed myocardial contractility and amrinone did not exhibit myocardial effects. In HSV and HMA contracted with 80 mM potassium, diltiazem and dobutamine (but not amrinone) had a vasorelaxant activity similar to that in rat aorta. Norepinephrine-contracted human vessels were significantly more sensitive than potassium-contracted vessels to the relaxant effect of amrinone (ED50 HMA = 15 ± 5 µM, ED50 HSV = 72 ± 31 µM, P < 0.05). We conclude that at concentrations still devoid of myocardial effects dobutamine and amrinone are effective dilators in graft segment vessels and rat aorta contracted by membrane depolarization. If the difference between aortic and myocardial tissue still holds in human tissues, at the appropriate concentrations these drugs should be expected to improve cardiac performance while still contributing to the maintenance of graft patency.