A closed system for islet isolation and purification using the COBE2991 cell processor may reduce the need of clean room facilities.

During the isolation of human islets of Langerhans the digest has repeated direct contact with the ambient atmosphere. In order to fulfill GMP requirements in clinical applications, the entire cell preparation must be performed in clean room facilities. We hypothesized that the use of a closed system, which avoids the direct exposure of tissue to the atmosphere, would significantly ease the preparation procedure. To avoid the direct atmosphere exposure we tested a modification of the isolation and purification process by performing all islet preparation steps in a closed system. In this study we compared the isolation outcome of the traditional open preparation technique with the new closed system. Pancreata from 6-month-old hybrid pigs were procured in the local slaughterhouse. After digestion/filtration the digest was cooled, collected, and concentrated in centrifugation containers and purified thereafter in the COBE2991 by top loading (control). In the control group 502 +/- 253 IEQ per gram pancreas were purified. The total preparation time amounted to 12 h. In the closed system the digest was cooled and directly pumped into the COBE2991 for centrifugation followed by supernatant expelling. Bag filling, centrifugation, and expelling were repeated several times. Islets in pellet form were then purified by adding a gradient (bottom loading). Using this closed system 1098 +/- 489 IEQ per gram pancreas were purified with a total cell viability of 67 +/- 10% and a beta-cell viability of 41 +/- 13%. The total preparation time reduced to 6 h. After 24 h of cell culture the viability of beta-cells was still 56 +/- 10% and was only reduced after the addition of proapoptotic IL-1 and TNF-alpha to 40 +/- 4%, indicating that freshly isolated islets are not apoptotic. In conclusion, the closed system preparation is much faster, more effective, and less expensive than the traditional islet preparation. The closed system may be applicable for human islets preparations to restrict the need of clean room facilities for islet preparations to a minimum and may open the way for islet preparations without clean room demand.

Increased translocator protein (TSPO) mRNA levels in colon but not in rectum carcinoma.

BACKGROUND: The peripheral-type benzodiazepine receptor or translocator protein (TSPO) is an 18-kDa protein involved in cell proliferation and apoptosis. TSPO was shown to be overexpressed in malignant tumors and cancer cell lines, correlating with enhanced malignant behavior. The present study analyzed the role of TSPO in patients with colorectal carcinomas. METHODS: Tumor tissues and corresponding normal mucosa from 55 patients who underwent resection for colorectal carcinomas were analyzed for TSPO expression in correlation to GAPDH expression(glyceraldehyde-3-phosphate dehydrogenase) using a multiplex RT-PCR assay. RESULTS: TSPO was overexpressed in 67% of the tumors in comparison to corresponding normal mucosa, and positivity as well as expression levels in colon carcinomas were significantly higher than in the rectum carcinomas. In contrast, TSPO expression was not different in intermediate versus high-grade tumors or in lymph node-positive versus -negative patients. CONCLUSION: The differences in TSPO expression between colon and rectum carcinoma may imply that these tumors are of different biological behavior.

[Haemoptysis after anesthesia]. / Hémoptysie au réveil anesthésique.

Postobstructive pulmonary oedema is a complication after extubation that occurs rarely . It can be associated with haemoptysis. We report two cases of haemoptysis occuring in ASA 1 otherwise healthy patients who underwent uncomplicated anaesthesia. Understanding of the mechanism and prompt treatment lead to rapid recovery of this dramatic complication.

[Anaesthesia and critical care for scheduled infrarenal abdominal aortic aneurysm surgery]. / Anesthésie et réanimation pour chirurgie réglée de l'anévrisme de l'aorte abdominale.

OBJECTIVES: Patient scheduled for infrarenal abdominal aortic aneurysm surgery carries a high risk of cardiac or respiratory comorbidity. To outline the perioperative management for these patients. METHODS: Review of the literature using MesH Terms "abdominal aortic aneurysm", "anesthesia", "analgesia" "critical care" and/or "surgery" in Medline database. RESULTS: Cardiac preoperative evaluation and management have recently been reviewed. Intermediate and high-risk patients should undergo non-invasive cardiac testing to decide between a preoperative medical strategy (using betablocker+/-statin and aspirin) and an interventional strategy (coronary angioplasty or cardiac surgery). Perioperative myocardial ischaemia should also be investigated by clinical, electrocardiographic and biologic monitoring such as plasmatic troponin Ic dosage. Specific score could also assess the respiratory failure risk preoperatively. Epidural analgesia decreases this risk. There is no evidence that a pharmacological treatment decreases the incidence of acute renal failure after aortic surgery. Endovascular repair is actually recommended for older, higher-risk patients or patients with a hostile abdomen or other technical factors that may complicate standard open repair.

Encapsulation of islets in rough surface, hydroxymethylated polysulfone capillaries stimulates VEGF release and promotes vascularization after transplantation.

The transplantation of encapsulated islets of Langerhans is one approach to treat type 1 diabetes without the need of lifelong immunosuppression. Capillaries have been used for macroencapsulation because they have a favorable surface-to-volume ratio and because they can be refilled. It is unclear at present whether the outer surface of such capillaries should be smooth to prevent, or rough to promote, cell adhesions. In this study we tested a new capillary made of modified polysulfone (MWCO: 50 kDa) with a rough, open-porous outer surface for islet transplantation. Compared with free-floating islets, encapsulation of freshly isolated rat islets affected neither the kinetics nor the efficiency of glucose-induced insulin release in perifusion experiments. Free-floating islets maintained insulin secretion during cell culture but encapsulated islets gradually lost their glucose responsiveness and released VEGF. This indicated hypoxia in the capillary lumen. Transplantation of encapsulated rat islets into diabetic rats significantly reduced blood glucose concentrations from the first week of implantation. This hypoglycaemic effect persisted until explantation 4 weeks later. Transplantation of encapsulated porcine islets into diabetic rats reduced blood glucose concentrations depending on the islet purity. With semipurified islets a transient reduction of blood glucose concentrations was observed (2, 8, 18, 18 days) whereas with highly purified islets a sustained normoglycaemia was achieved (more than 28 days). Explanted capillaries containing rat islets were covered with blood vessels. Vascularization was also observed on capillaries containing porcine islets that were explanted from normoglycaemic rats. In contrast, on capillaries containing porcine islets that were explanted from hyperglycemic rats a fibrous capsule and lymphocyte accumulations were observed. No vascularization on the surface of transplanted capillaries was observed in the absence of islets. In conclusion, encapsulated islets can release VEGF, which appears to be an important signal for the vascularization of the capillary material. The rough, open-porous outer surface of the polysulfone capillary provides a site well suited for vascular tissue formation and may allow a prolonged islet function after transplantation.

Encapsulation of Islets in Rough Surface, Hydroxymethylated Polysulfone Capillaries Stimulates VEGF Release and Promotes Vascularization after Transplantation.

The transplantation of encapsulated islets of Langerhans is one approach to treat type 1 diabetes without the need of lifelong immunosuppression. Capillaries have been used for macroencapsulation because they have a favorable surface-to-volume ratio and because they can be refilled. It is unclear at present whether the outer surface of such capillaries should be smooth to prevent, or rough to promote, cell adhesions. In this study we tested a new capillary made of modified polysulfone (MWCO: 50 kDa) with a rough, open-porous outer surface for islet transplantation. Compared with free-floating islets, encapsulation of freshly isolated rat islets affected neither the kinetics nor the efficiency of glucose-induced insulin release in perifusion experiments. Free-floating islets maintained insulin secretion during cell culture but encapsulated islets gradually lost their glucose responsiveness and released VEGF. This indicated hypoxia in the capillary lumen. Transplantation of encapsulated rat islets into diabetic rats significantly reduced blood glucose concentrations from the first week of implantation. This hypoglycaemic effect persisted until explantation 4 weeks later. Transplantation of encapsulated porcine islets into diabetic rats reduced blood glucose concentrations depending on the islet purity. With semipurified islets a transient reduction of blood glucose concentrations was observed (2, 8, 18, 18 days) whereas with highly purified islets a sustained normoglycaemia was achieved (more than 28 days). Explanted capillaries containing rat islets were covered with blood vessels. Vascularization was also observed on capillaries containing porcine islets that were explanted from normoglycaemic rats. In contrast, on capillaries containing porcine islets that were explanted from hyperglycemic rats a fibrous capsule and lymphocyte accumulations were observed. No vascularization on the surface of transplanted capillaries was observed in the absence of islets. In conclusion, encapsulated islets can release VEGF, which appears to be an important signal for the vascularization of the capillary material. The rough, open-porous outer surface of the polysulfone capillary provides a site well suited for vascular tissue formation and may allow a prolonged islet function after transplantation.

[Increase in bispectral index induced by antihyperalgesic dose of ketamine]. / La kétamine à dose antihyperalgésique augmente l'index bispectral.

We report two cases of sudden increase in Bispectral Index (BIS) after the injection of low-dose ketamine for the prevention of postoperative hyperalgesia. The two patients were anaesthetised with a continuous infusion of remifentanil associated with propofol for one and isoflurane for the other. Changes in BIS occurred while the two patients were in a stable phase of surgery (beginning of parietal closure and suture of an anastomosis) and had a stable target concentration of anaesthetic agents. No others signs of awakening were observed. The BIS value returned progressively to 40-50 despite no increase in target concentration. None of the patients complained of intra-operative recall.

Prevalence of postoperative bladder distension and urinary retention detected by ultrasound measurement.

BACKGROUND: Postoperative bladder distension and urinary retention are commonly underestimated. Ultrasound enables accurate measurement of bladder volume and thus makes it possible to determine the prevalence of postoperative bladder distension. METHODS: Using ultrasound, we measured the volume of the bladder contents at the time of discharge from the recovery room in 177 adult patients who had undergone thoracic, vascular, abdominal, orthopaedic or ENT surgery. RESULTS: Forty-four per cent of the patients had a bladder volume >500 ml and 54% of the 44%, who had no symptoms of bladder distension, were unable to void spontaneously within 30 min. The risk factors for urinary retention were age >60 yr (odds ratio (OR) 2.11, 95% confidence interval (CI) 1.01-4.38), spinal anaesthesia (OR 3.97, 95% CI 1.32-11.89) and duration of surgery >120 min (OR 3.03, 95% CI 1.39-6.61). CONCLUSION: Before discharge from the recovery room it seems worthwhile to systematically check the bladder volume with a portable ultrasound device in patients with risk factors.

Areal density measurement is a convenient method for the determination of porcine islet equivalents without counting and sizing individual islets.

The determination of islet mass is important for the normalization of islet experiments in the laboratory and for the precise dosing of islets for transplantation. The common microscopical analysis is based on individual islet sizing, calculation of the frequency distribution, and conversion into islet equivalents (IEQ), which is the volume of a spherical islet with a diameter of 150 microm. However, islets are of irregular form, which makes this determination user dependent, and the analysis is irreproducible once the original sample is discarded. This routine technique of islet quantification was compared with the analysis of areal density measurements. It was assumed that the entire area occupied by islets can be expressed in IEQ without sizing and counting individual islets. Porcine islets were isolated by continuous digestion/filtration and purified by gradient centrifugation. Purified islets were stained with dithizone and were repeatedly pictured under the microscope with random area selection. A total of 51 pictures was taken from 11 different purifications and stained islets were detected by digital image analysis. The correlation coefficient (r) between bothanalyses was 0.977 with an underestimation of islet yield by areal density detection (slope: 0.75 +/- 0.03). Areal density analysis per picture took about 1 min, which is about 10 times faster than the traditional method without increasing the method error (CV 2.1% vs. 2.7%). In summary, areal density measurements allow a rapid and reproducible estimation of IEQ without counting individual islets. It can be performed in a single step analysis without computer programming and is valuable for online determinations of islet yield preceding transplantation.

The hypoglycemic effect of Nigella sativa oil is mediated by extrapancreatic actions.

A plant mixture containing extracts of Nigella sativa possesses blood glucose lowering effects, but the direct antidiabetic effect of Nigella sativa is not yet established. Therefore, the effect of Nigella sativa oil (NSO) on blood glucose concentrations was studied in streptozotocin diabetic rats. In addition, the effect of NSO, nigellone and thymoquinone were studied on insulin secretion of isolated rat pancreatic islets in the presence of 3, 5.6 or 11.1 mM glucose. NSO significantly lowered blood glucose concentrations in diabetic rats after 2, 4 and 6 weeks. The blood lowering effect of NSO was, however, not paralleled by a stimulation of insulin release in the presence of NSO, nigellone or thymoquinone. The data indicate that the hypoglycemic effect of NSO may be mediated by extrapancreatic actions rather than by stimulated insulin release.

Nigella sativa oil, nigellone and derived thymoquinone inhibit synthesis of 5-lipoxygenase products in polymorphonuclear leukocytes from rats.

In the present study, Nigella sativa oil (NSO), nigellone (polythymoquinone) and derived thymoquinone were studied to evaluate their effect on the formation of 5-lipoxygenase (5-LO) products from polymorphonuclear leukocytes (PMNL).NSO produced a concentration dependent inhibition of 5-LO products and 5-hydroxy-eicosa-tetra-enoic acid (5-HETE) production with half maximal effects (IC(50)) at 25+/-1 micro g/ml, respectively 24+/-1 micro g/ml. Nigellone caused a concentration-related inhibition of 5-HETE production (IC(50): 11.9+/-0.3 micro g/ml). Moreover thymoquinone, the active principle of NSO inhibited the production of 5-LO products (IC(50): 0.26+/-0.02 micro g/ml) and 5-HETE production (IC(50): 0.36+/-0.02 micro g/ml) in a similar way. The effects are probably due to an antioxidative action. The data may in part explain the effect of the oil, its derived thymoquinone and nigellone in ameliorating inflammatory diseases.

[Prevention of pain on injection with propofol in children: comparison of nitrous oxide with lidocaine]. / Prévention de la douleur à l'injection du propofol chez l'enfant: comparaison de l'inhalation de protoxyde d'azote à l'injection de lidocaïne.

INTRODUCTION: Injection pain caused by propofol is an important disadvantage, especially in children, incompletely reduced by adding lidocaine intravenously. Nitrous oxide's analgesic effects, well known, have never been evaluated on pain due to propofol. OBJECTIVE: To compare the effects of nitrous oxide with lidocaine on pain on injection caused by propofol in children. STUDY DESIGN: Double blind, randomised, prospective study. PATIENTS AND METHODS: 48 children aged more than 5 were randomly allocated to one of the 2 groups: N2O group, breathed 50% N2O + 50% O2 than received propofol only and Lido group breathed 100% O2 and received a mixture of propofol with lidocaine. The possible pain was scored during injection by a behavioural scale and once again in the recovery room by the child himself with a VAS. RESULTS: There was no significant difference in behavioural pain scores among the 2 groups; pain was assessed as being moderate or severe in 6/24 patients in N2O group and 10/24 in Lido group (behavioural scores > 1). Significantly more children in the N2O group had low VAS scores compared with the Lido group (no child/24 scored a VAS > 4 and 7/23 in the Lido group) demonstrating that N2O amnesic effects would omit the memory of pain caused by propofol. CONCLUSION: The use of nitrous oxide is an easy, cheap and efficient method to reduce the incidence of pain injection of propofol and his amnesic effects can provide real advantages in paediatric anaesthesia.

Diabetogenic effect of cyclosporin A is mediated by interference with mitochondrial function of pancreatic B-cells.

Treatment of patients after organ transplantation with the immunosuppressive drug cyclosporin A (CsA) is often accompanied by impaired glucose tolerance, thus promoting the development of diabetes mellitus. In the present article we show that 2 to 5 microM CsA diminishes glucose-induced insulin secretion of isolated mouse pancreatic islets in vitro by inhibiting glucose-stimulated oscillations of the cytoplasmic free-Ca(2+) concentration [Ca(2+)](c). This effect is not due to an inhibition of calcineurin, which mediates the immunosuppressive effect of CsA, because other calcineurin inhibitors, deltamethrin and tacrolimus, did not affect the oscillations in [Ca(2+)](c) of the B-cells. The CsA-induced decrease in [Ca(2+)](c) to basal values was not caused by a direct inhibition of L-type Ca(2+) channels. CsA is known to be a potent inhibitor of the mitochondrial permeability transition pore (PTP), which we recently suggested to be involved in the regulation of oscillations. Consequently, CsA also inhibited the oscillations of the cell membrane potential, and it is shown that these effects could not be ascribed to cellular ATP depletion. However, the mitochondrial membrane potential Delta Psi was affected by CsA by inhibiting the oscillations in Delta Psi. Interestingly, the observed reduction in [Ca(2+)](c) could be counteracted by the K(+)(ATP) channel blocker tolbutamide, indicating that the stimulus-secretion coupling was interrupted before the closure of K(+)(ATP) channels. It is concluded that CsA alters B-cell function by inhibiting the mitochondrial PTP. This terminates the oscillatory activity that is indispensable for adequate insulin secretion. Thus, CsA acts on different targets to induce the immunosuppressive and the diabetogenic effect.

Macroencapsulation of rat islets without alteration of insulin secretion kinetics.

Transplantation of encapsulated islets may restore endogenous insulin secretion in type 1 diabetics with no need of lifetime immunosuppression of the recipient. A biomaterial should be developed which combined immunoisolation with rapid and efficient diffusion of glucose and insulin. Rat islets were macroencapsulated in capillaries (molecular cut off 50 kD) of differently modified polysulphone. Macroencapsulated islets were perifused to study the kinetics of glucose induced insulin secretion into the perifusion medium. Blending polysulphone (PSU) with poly vinyl pyrrolidone or sodium dodecyl sulphate was not suited for islet macroencapsulation since glucose induced insulin release was absent after encapsulation. Hydroxy methylation (CH2OH) of PSU improved the secretory behaviour of macroencapsulated islets depending on the degree of substitution (DS). At 0.8 DS glucose induced insulin secretion was delayed and inefficient. At maximal degrees of PSU-substitution (1.8) the kinetics of insulin release and the efficiency of insulin release were very similar to that observed of free floating islets. In conclusion, highly substituted hydroxy methylated polysulphone allows a rapid and efficient insulin release after macroencapsulation and is suited for the further development of a bioartificial pancreas.

Methyl pyruvate initiates membrane depolarization and insulin release by metabolic factors other than ATP.

The role of mitochondria in stimulus-secretion coupling of pancreatic beta-cells was examined using methyl pyruvate (MP). MP stimulated insulin secretion in the absence of glucose, with maximal effect at 5 mM. K+ (30 mM) alone, or in combination with diazoxide (100 microM), failed to enhance MP-induced secretion. Diazoxide (100 microM) inhibited MP-induced insulin secretion. MP depolarized the beta-cell in a concentration-dependent manner (5-20 mM). The sustained depolarization induced by 20 mM MP was not influenced by 100 microM diazoxide, but the continuous spiking activity was suppressed by 500 microM diazoxide. Pyruvate failed to initiate insulin release (5-20 mM) or to depolarize the membrane potential. ATP production in isolated beta-cell mitochondria was detected as accumulation of ATP in the medium during incubation in the presence of malate or glutamate in combination with pyruvate or MP. There was no difference in ATP production induced by pyruvate/malate or MP/malate in isolated beta-cell mitochondria. ATP production by MP/glutamate was higher than that induced by pyruvate/glutamate, but it was much lower than that induced by alpha-ketoisocaproate/glutamate. Pyruvate (5 mM) or MP (5 mM) had no effect on the ATP/ADP ratio in whole islets, whereas glucose (20 mM) significantly increased the whole islet ATP/ADP ratio. It is concluded that MP-induced beta-cell membrane depolarization or insulin release does not relate directly to mitochondrial ATP production. Instead MP may exert a direct extramitochondrial effect, or it may stimulate beta-cell mitochondria to produce coupling factors different from ATP to initiate insulin release.

In vitro test of new biomaterials for the development of a bioartificial pancreas.

The implantation of macroencapsulated islets has the potential to restore endogenous insulin secretion in type 1 diabetics, with no need for lifetime immunosuppression. To match the physiological fluctuations of blood glucose concentrations with appropriate insulin release, the macroencapsulation material must combine immunoprotection with optimal diffusion properties for glucose and insulin. The impact of chemical modifications of polysulphone (PSU) capillary polymers with a cutoff of 50 kD on glucose-induced insulin secretion of macroencapsulated rat islets was studied in perifusion experiments. The insulin release of free-floating islets showed the typical rapid response to glucose stimulation. Total insulin release (AUC between minute 30 and 120 of perifusion) reached 117+/-22 ng/ml. Blending PSU with polyvinylpyrrolidone or sodium-dodecyl-sulfate was not suitable for islet macroencapsulation, since glucose-induced insulin release was absent or disturbed. Hydroxy-methylation (CH2OH) of PSU improved the secretory behavior of macroencapsulated islets depending on the degree of PSU substitution (DS 0.8, AUC 62+/-15 ng/ml; DS 1.8, 111+/-24 ng/ml). In highly substituted PSU-capillaries the kinetics of glucose-induced insulin release was very similar to that observed in free-floating islets. Two consecutive glucose stimulations potentiated insulin release of free-floating islets during the second period of stimulation. Furthermore, freshly isolated macroencapsulated islets responded with more efficient insulin secretion after the initial priming. In conclusion, in vitro membrane screening identified highly substituted hydroxy-methylated PSU as the material of choice for islet encapsulation in a bioartificial pancreas.