Biodritin microencapsulated human islets of langerhans and their potential for type 1 diabetes mellitus therapy

Background. Microencapsulation of pancreatic islets with polymeric compounds constitutes an attractive alternative therapy for type 1 diabetes mellitus. The major limiting factor is the availability of a biocompatible and mechanically stable polymer. We investigated the potential of Biodritin, a novel polymer constituted of alginate and chondroitin sulfate, for islet microencapsulation. Methods. Biodritin microcapsules were obtained using an air jet droplet generator and gelated with barium or calcium chloride. Microencapsulated rat insulinoma RINm5F cells were tested for viability using the [3-(4,5-dimetyl-thiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide] [MTT] colorimetric assay. Microencapsulated rat pancreatic islets were coincubated with macrophages derived from mouse peritoneal liquid to assess the immunomodulatory potential of the microcapsules, using quantitative real time-PCR (qPCR). Biodritin biocompatibility was demonstrated by subcutaneous injection of empty microcapsules into immunocompetent Wistar rats. Insulin secretion by microencapsulated human pancreatic islets was evaluated using an electrochemoluminescent assay. Microencapsulated human islets transplanted into chemically induced diabetic mice were monitored for reversal of hyperglycemia. Results. The metabolic activity of microencapsulated RINm5F cells persisted for at least 15 days. Interleukin-1 beta expression by macrophages was observed during coculture with islets microencapsulated with Biodritin-CaCl2, but not with Biodritin-BaCl2. No statistical difference in glucose-stimulated insulin secretion was observed between nonencapsulated and microencapsulated islets. Upon microencapsulated islet transplantation, the blood glucose level of diabetic mice normalized; they remained euglycemic for at least 60 days, displaying normal oral glucose tolerance tests. Conclusion. This study demonstrated that Biodritin can be used for islet microencapsulation and reversal of diabetes; however, further investigations are required to assess its potential for long-term transplantation.

The tof-guard neuromuscular transmission monitor and its use in horses

Quando da utilização de bloqueadores neuromusculares, já foi enfatizado na literatura médica humana, que é de vital imporância a monitoração do bloqueio neuromuscular e que estes agentes nunca devem ser utilizados sem a mesma. O objetivo deste estudo foi o de avaliar o uso do monitor da transmissão neuromuscular TOF-Guard em eqüinos. Para tanto, doze eqüinos foram separados aleatoriamente para receberem como bloqueadores neuromusculares o pancurônio ou o atracúrio. Todos os eqüinos foram pré-medicados com romifidina, induzidos com diazepam e quetamina e mantidos com halotano. Foi administrado o atracúrio ou o pancurônio, seguindo-se a apnéia e início da ventilação mecânica controlada. O tempo entre a administração do relaxante muscular e a obtenção de um bloqueio máximo (T1=0), o retorno do T1 para 25% e da razão do TOF para 0,7 e o tempo do retorno do T1 de 25 para 75% foram mensurados. Concluiu-se que é de grande importância a monitoração do bloqueio neuromuscular quando da utilização de um bloqueador neuromuscular não-despolarizante, uma vez que ela torna o ato anestésico e cirúrgico mais seguro, com a utilização de doses adequadas que inviabilizam a superficialização do bloqueio durante o procedimento cirúrgico. O monitor TOF-Guard mostrou ser uma boa opção para a monitoração do bloqueio neuromuscular em eqüinos.

Intraocular pressure evaluation of equines anesthetized with romifidine, tiletamine/zolazepam, halothane and vecuronium

O objetivo deste estudo foi o de avaliar uma associação anestésica com e sem a utilização de relaxante muscular não-despolarizante e seu efeito sobre a pressão intra-ocular de eqüinos. Também objetivou-se uma técnica anestésica sem efeitos adversos que possa ser utilizada em procedimentos e cirurgias oftálmicas nesta espécie animal. Para tanto, dezesseis eqüinos foram divididos aleatoriamente em dois grupos de oito animais cada. Os animais do grupo I foram pré-medicados com romifidina, induzidos com tiletamina/zolazepam e a anestesia foi mantida com halotano e vecurônio. Os animais do grupo II receberam a mesma associação anestésica, com exceção do vecurônio. No decorrer do experimento, a pressão intra-ocular, a pressão arterial e a freqüência cardíaca foram avaliadas em diferentes momentos. A associação anestésica composta pela romifidina, tiletamina/ zolazepam e halotano com e sem vecurônio não promoveu alterações estatisticamente significativas na pressão intra-ocular de eqüinos e o seu uso é exeqüível em procedimentos oftálmicos nesta espécie animal.

Hepatoprotective and anti-inflammatory effects of silibinin on experimental preeclampsia induced by 1-NAME in rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Mineral trioxide aggregate stimulates macrophages and mast cells to release neutrophil chemotactic factors: role of IL-1 beta, MIP-2 and LTB4

Objective. In the present study, the role of macrophages and mast cells in mineral trioxide aggregate (MTA)-induced release of neutrophil chemotactic factor was investigated.Study design. MTA suspension (50 mg/mL) was plated over inserts on macrophages or mast cells for 90 minutes. Untreated cells served as controls. Cells were washed and cultured for 90 minutes in RPMI without the stimuli. Macrophages and mast cell supernatants were injected intraperitoneally (0.5 mL/cavity), and neutrophil migration was assessed 6 hours later. In some experiments, cells were incubated for 30 minutes with dexamethasone (DEX, 10 mu M/well), BWA4C (BW, 100 mu M/well) or U75302 (U75, 10 mu M/well). The concentration of Leukotriene B-4 (LTB4) in the cell-free supernatant from mast cells and macrophage culture was measured by ELISA.Results. Supernatants from MTA-stimulated macrophages and mast cells caused neutrophil migration. The release of neutrophil chemotactic factor by macrophages and mast cells was significantly inhibited by DEX, BW, or U75. Macrophages and mast cells expressed mRNA for interleukin-1 (IL-1)beta and macrophage inflammatory protein-2 (MIP-2) and the pretreatment of macrophages and mast cells with DEX, BW, or U75 significantly altered IL-1 beta and MIP-2 mRNA expression. LTB4 was detected in the MTA-stimulated macrophage supernatant but not mast cells.Conclusions. MTA-induces the release of neutrophil chemotactic factor substances from macrophages and mast cells with participation of IL-1 beta, MIP-2, and LTB4. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010; 109: e135-e142)

Simvastatin therapy in cyclosporine A-induced alveolar bone loss in rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of angiotensin and vasopressin V-1 receptors on water and sodium intake induced by injection of vasopressin into lateral septal area

The specific arginine(8)-vasopressin (AVP) V, receptors antagonist (AAVP) was injected (20, 40 and 80 nmol) into the lateral septal area (LSA) to determine the effects of selective septal V, receptor on water and 3% sodium intake in rats. Was also observed the effects of losartan and CGP42112A (select ligands of the AT(1) and AT(2) ANG II receptors, respectively) injected into LSA prior AVP on the same appetites. Twenty-four hours before the experiments, the rats were deprived of water. The volume of drug solution injected was 0.5 mul. Water and sodium intake were measured at 0.25, 0.5, 1.0 and 2,0 h. Injection of AVP reduced the water and sodium ingestion vs. control (0.15 M saline). Pre-treatment with AAVP (40, 80 and 160 nmol) did not alter the decrease in the water ingestion induced by AVP, whereas AAVP abolished the action of AVP-induced sodium intake. Losartan (40, 80 and 160 nmol) did not alter the effect of AVP on water and sodium intake, whereas CGP42112A (20, 40 and 60 nmol) at the first 30 min increased water ingestion. Losartan and CGP42112A together increased the actions of AVP, showing more pronounced effects than when the two antagonists were injected alone. The results showed that AVP inhibited the appetites and these effects were increased by the AAVP. The involvement of angiotensinergic receptors in the effects of AVP is also suggested. (C) 2004 Elsevier B.V. All rights reserved.

Influence of arginine vasopressin receptors and angiotensin receptor subtypes on the water intake and arterial blood pressure induced by vasopressin injected into the lateral septal area of the rat

In this study we investigated the influence of d(CH2)(5)-Tyr(Me)-[Arg(8)]vasopressin (AAVP) and [adamanteanacetyl(1),0-ET-DTyr(2), Val(4), aminobutyryl(6), Arg(8,9)]-[Arg(8)]vasopressin (ATAVP), which are antagonists of vasopressin V-1 and V-2 receptors, and the effects of losartan, a selective angiotensin AT(1) receptor antagonist, and CGP42112A, a selective AT(2) receptor antagonist, injected into the lateral septal area (LSA) on thirst and hypertension induced by [Arg(8)]vasopressin (AVP). AAVP and ATAVP injected into the LSA reduced the drinking responses elicited by injecting AVP into the LSA. Both the AT(1) and AT(2) ligands administered into the LSA elicited a concentration-dependent decrease in the water intake induced by AVP injected into the LSA, but losartan was more effective than CGP42112A. The increase in MAP, due to injection of AVP into the LSA, was reduced by prior injection of AAVP from 18 +/- 1 to 6 +/- 1 mm Hg. Losartan injected into the LSA prior to AVP reduced the increase in MAP to 7 +/- 0.8 mm Hg. ATAVP and CGP42112A produced no changes in the pressor effect of AVP. These results suggest that the dipsogenic effects induced by injecting AVP into the LSA were mediated primarily by AT(1) receptors. However, doses of losartan were more effective when combined with CGP42112A than when given alone, suggesting that the thirst induced by AVP injections into LSA may involve activation of multiple AVP and angiotensin II receptor subtypes. The pressor response of AVP was reduced by losartan and by AAVP. CGP42112A and ATAVP did not change the AVP pressor response. These results suggest that facilitator effects of AVP on water intake are mediated through the activation of V-1 receptors and that the inhibitory effect requires V-2 receptors. The involvement of AT(1) and AT(2) receptors can be postulated. Based on the present findings, we suggest that the AVP in the LSA may play a role in the control of water and arterial blood pressure balance. (C) 2004 Elsevier B.V. All rights reserved.

Annexin 1 peptides protect against experimental myocardial ischemia-reperfusion: analysis of their mechanism of action

Myocardial reperfusion injury is associated with the infiltration of blood-borne polymorphonuclear leukocytes. We have previous described the protection afforded by annexin 1 (ANXA1) in an experimental model of rat myocardial ischemia-reperfusion (IR) injury. We examined the 1) amino acid region of ANXA1 that retained the protective effect in a model of rat heart IR; 2) changes in endogenous ANXA1 in relation to the IR induced damage and after pharmacological modulation; and 3) potential involvement of the formyl peptide receptor (FPR) in the protective action displayed by ANXA1 peptides. Administration of peptide Ac2-26 at 0, 30, and 60 min postreperfusion produced a significant protection against IR injury, and this was associated with reduced myeloperoxidase activity and IL-1 beta levels in the infarcted heart. Western blotting and electron microscopy analyses showed that IR heart had increased ANXA1 expression in the injured tissue, associated mainly with the infiltrated leukocytes. Finally, an antagonist to the FPR receptor selectively inhibited the protective action of peptide ANXA1 and its derived peptides against IR injury. Altogether, these data provide further insight into the protective effect of ANXA1 and its mimetics and a rationale for a clinical use for drugs developed from this line of research.

INJECTION OF RAMIPRIL INTO THE LATERAL VENTRICLE INTERFERES WITH THE DRINKING RESPONSE INDUCED BY PHARMACOLOGICAL AND NATURAL THIRST STIMULI

We investigated the effects of ramipril, an angiotensin I-converting enzyme (ACE) inhibitor, on water intake by male Holtzman rats (250-300 g) with cannulae implanted into the lateral ventricle. Intracerebroventricular (icv) injection of ramipril (1 mu g/mu l) significantly reduced drinking in response to subcutaneous (sc) injection of isoprenaline (100 mu g/kg) from 8.49 +/- 0.69 to 2.96 +/- 0.36 ml/2 h, polyethyleneglycol (PEG) (30% w/v, 10 ml/kg) from 9.51 +/- 2.20 to 1.6 +/- 0.34 ml/2 h or water deprivation for 24 h from 12.61 +/- 0.83 to 5.10 +/- 1.37 ml/2 h. Ramipril had no effect on water intake induced by cellular dehydration produced by sc injection of hypertonic saline (2 M NaCl). These results are consistent with the hypothesis that ramipril acts as an ACE-blocking agent in the brain. The possibility that ramipril is transformed to ramiprilat, the active drug, by the brain is suggested.

THE USE OF AMPPD AS AN ALTERNATIVE SUBSTRATE FOR AP-MEDIATED DETECTION OF NONRADIOLABELED DNA PROBES IN EUCALYPTUS-SALIGNA

We present a non-radioactive alternative to Southern's (J. Mol. Biol. 98: 503-517, 1975) DNA-DNA hybridization technique. The use of AMPPD - Disodium 3-(4-Methoxyspiro {1,2-dioxetane-3,2'tricyclo[3.3.1.1(3,7)]decan}-4-yl)phyenyl phosphate as an alternative substrate for AP-mediated detection of digoxigenin-11 dUTP-labeled probes made possible the simple and nonhazardous reuse of blots. We used 0.8 % agarose gels containing 30 mug per lane of Eucalyptus saligna DNA, digested with Eco RI, electrophoresed and blotted on to nylon membranes (Hybond-N, Amersham, UK), using the Southern blotting procedure, and UV irradiated for one minute for DNA fixation. The hybridizations were carried out overnight with digoxigenin labeled random inserts of E. saligna DNA by using the Genius Kit (Boehringer Mannheim). Detection of the DNA-DNA hybrids was performed in the presence of 0.5% blocking agent and the substrates NBT/BCIP were replaced by 0.26 mM AMPPD in the final alkaline assay buffer (50 mul/cm2). After membrane incubation for five minutes at room temperature in a sealed plastic bag, the AMPPD solution was retrieved and stored at 4-degrees-C for reuse. A Kodak X-BRAF QA-S film was pressed firmly onto the bag containing the wet membrane, exposed for two to six hours and then developed. After use, the probes were stripped off and the blots reutilized, three times so far, with the same results.

Forebrain angiotensin type 1 receptors and parabrachial serotonin in the control of NaCl and water intake

This study investigated the roles of serotonin (5-HT) receptors in the lateral parabrachial nucleus (LPBN), and brain angiotensin type 1 (AT(1)) receptors in the intake of 0.3 M NaCl and water induced by angiotensin II (ANG II). Rats were implanted with stainless steel cannulas for injections into tho subfornical organ (SFO) and into the LPBN. Bilateral LPBN pretreatment with the nonselective serotonergic 5-HT1/5-HT2 receptor antagonist methysergide (4 mu g/200 nl) markedly enhanced 0.3 M NaCl intake induced by injections of ANG II (20 ng/200 nl) into the SFO. Pretreatment of the SFO with the AT(1) receptor antagonist losartan (1 mu g/200 nl) blocked the intake of 0.3 M NaCl induced by ANG II in combination with LPBN methysergide injections. These results suggest that serotonergic mechanisms associated with the LPBN inhibit the expression of salt appetite induced by ANG II injections into Ihs SFO. In addition, the results indicate that the enhanced NaCl intake generated by central administration of ANG II in the presence of LPBN 5-HT blockade is mediated bg brain ATI receptors.

Reinforcing properties of fencamfamine: Involvement of dopamine and opioid receptors

Fencamfamine (FCF) is a psychostimulant classified as an indirect dopamine agonist. The conditioning place preference (CPP) paradigm was used to investigate the reinforcing properties of FCF. After initial preferences had been determined, animals were conditioned with FCF (1.75, 3.5, or 7.0 mg/kg; IP). Only at the dose of 3.5 mg/kg FCF produced a significant place preference. Pretreatment with SCH23390 (0.05 mg/kg, SC) or naloxone (1.0 mg/kg SC) 10 min before FCF (3.5 mg/kg; IP) blocked both FCF-induced hyperactivity and CPP. Pretreatment with metoclopramide (10.0 mg/kg; IP) or pimozide (1.0 mg/kg, IP), respectively, 30 min or 4 h before FCF (3.5 mg/kg; IP), which blocked the FCF-induced locomotor activity, failed to influence place conditioning produced by FCF. In conclusion, the present study suggests that dopamine D 1 and opioid receptors are related to FCF reinforcing effect, while dopamine D 2 subtype receptor was ineffective in modifying FCF-induced CPP.

On a dual role for clonidine stimulation of the ventromedial nucleus of the hypothalamus in sodium and potassium renal excretions of rats

The effects of clonidine on sodium and potassium excretions were examined after previous administration of prazosin (an α 1-adrenergic receptor antagonist) and yohimbine (an α 2-adrenergic receptor antagonist) into the ventromedial nucleus of the hypothalamus of conscious rats. Clonidine injected into the ventromedial nucleus of the hypothalamus induced inhibitory and facilitatory effects on the urinary sodium and potassium excretions. The results suggest that facilitatory effects of clonidine on natriuresis and kaliuresis are mediated through activation of α 1-adrenoceptors and that inhibitory effects require α(2A)-adrenoceptors.

Tempo de latencia do rocuronio e da succinilcolina e condicoes de intubacao traqueal

Background and Objectives - Pulmonary aspiration of gastric content during induction of anesthesia for emergency surgical procedures is a serious complication; fast endotracheal intubation under these circumstances is of vital importance to secure the airways. Despite its numerous side effects, succinylcholine is used for this purpose. Rocuronium is the most recently introduced aminoesteroid neuromuscular blocking drug with short onset. The objective of this study was to compare the onset time and intubating conditions of rocuronium and succinylcholine. Methods - After informed consent, forty-five patients were randomly allocated into three groups of 15: Group I (GI) = succinylcholine 1 mg.kg-1; Group II (GII) = rocuronium 0.6 mg.kg-1; Group III (GIII) = rocuronium 0.9 mg.kg-1. Every patient was premedicated with midazolam 15 mg per os and induction of anesthesia was made with fentanyl 10 μg.kg-1 and etomidate 0.3 mg.kg-1. The neuromuscular block was monitored with the TOP-Guard neuromuscular transmission monitor. The TOP-Guard neuromuscular monitor uses an accelerometer to measure the response to nerve stimulation. The stimulating electrodes were placed close to the course of the ulnar nerve at the wrist. The onset time was considered as the time between the end of neuromuscular drug injection and the twitch height (T1) decrease to 10%. Heart rate and arterial blood pressure were registered at 6 moments before and after induction of anesthesia. Results - The onset time results were: Group I, 71 s; Group II, 120 s and Group III, 70 s or GI = GIII < GII (F = 8.862; p < 0.01). There were 43 patients exhibiting excellent intubating conditions and 2 with good intubating conditions. Heart rate and arterial blood pressure showed alterations due to induction of anesthesia and intubation. Conclusions - Rocuronium 0.9 mg.kg-1 can be used in rapid sequence induction because it has a short onset time which is similar to that of succnylcholine. It is likely that rocuronium would be a good indication in patients with high intracranial pressure, burns and neuromuscular diseases.