Search

Unlabelled Boxce:textbox-body/

Unlabelled Boxce:textbox-body/

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.

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 μm. 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 both analyses 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.

Häufig werden Brandschutzmaßnahmen an Gebäuden abweichend von Normen und Verordnungen geplant, deren Richtigkeit nachträglich bewertet werden muß. Ausgehend von den geregelten Brandschutzanforderungen und Risikobetrachtungen gibt der Beitrag anhand von konkreten Beispielen Hinweise zur Nachweisführung des baulichen Brandschutzes bei Abweichungen von den bauaufsichtlich geregelten Anforderungen. Eine Grundbedingung für die Kompensation von Abweichungen ist ein umfassendes Brandschutzkonzept, das in den Planungsprozeß integriert wird.

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 ngml. 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 ngml; DS 1.8, 111 ± 24 ngml). 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 vitromembrane screening identified highly substituted hydroxy-methylated PSU as the material of choice for islet encapsulation in a bioartificial pancreas.

Ricin acts by translocating to the cytosol the enzymatically active toxin A-chain, which inactivates ribosomes. Retrograde intracellular transport and translocation of ricin was studied under conditions that alter the sensitivity of cells to the toxin. For this purpose tyrosine sulfation of mutant A-chain in the Golgi apparatus, glycosylation in the endoplasmic reticulum (ER) and appearance of A-chain in the cytosolic fraction was monitored. Introduction of an ER retrieval signal, a C-terminal KDEL sequence, into the A-chain increased the toxicity and resulted in more efficient glycosylation, indicating enhanced transport from Golgi to ER. Calcium depletion inhibited neither sulfation nor glycosylation but inhibited translocation and toxicity, suggesting that the toxin is translocated to the cytosol by the pathway used by misfolded proteins that are targeted to the proteasomes for degradation. Slightly acidified medium had a similar effect. The proteasome inhibitor, lactacystin, sensitized cells to ricin and increased the amount of ricin A-chain in the cytosol. Anti-Sec61alpha precipitated sulfated and glycosylated ricin A-chain, suggesting that retrograde toxin translocation involves Sec61p. The data indicate that retrograde translocation across the ER membrane is required for intoxication.

Blood velocities in the human femoral artery were measured using pulsed bidirectional Doppler‐ultrasound equipment before, during and after single isometric contractions of the quadriceps muscle group. After contraction periods lasting more than 20 s (long) and of tensions from 10% up to 75% of maximal voluntary contraction (m.v.c.), an increase in blood velocities of seven to eight times the resting level was observed. Estimated maximal volume flow to the whole leg during the post‐contraction hyperaemic phase calculated from these blood velocity measurements and vessel diameter (measured with echo‐ultrasound equipment) was in two of the subjects 2.4 l/min (female) and 4.4 l/min (male), respectively. In the latter, this estimate fitted very well with results obtained using a venous thermo‐dilution method. When using computer tomography to estimate the volume of the quadriceps muscle group, the calculated maximum flow to this muscle group in the post‐contraction hyperaemic phase was approximately 175 (female) and 185 (male) ml/min. 100 ml muscle, respectively. This was about forty times the estimated resting volume flow to this muscle of 4.7 (female) and 4.5 (male) ml/min. 100 ml muscle. The length of the post‐contraction hyperaemia after short (less than 10 s) contraction periods was 12‐13 s, by which time velocities had reached 25% above the precontraction level. After long contractions, the corresponding values were 23‐25 s. By contrast, previous plethysmographic observations by others indicate that postcontraction hyperaemias following long contractions last 10‐15 min. There was a marked difference between the times taken to reach maximal velocity in the hyperaemic phase when comparing short and long contractions. Maximal velocity was reached four to six cardiac cycles following short periods of contraction but during the very first heart beat after long periods of contractions. The present observations are compatible with the hypothesis that locally released metabolites or hormones play a dominant role in the regulation of the post‐contraction hyperaemia. Since during the short contraction periods maximal velocity was reached only after some seconds, whereas with the longer contraction periods it was reached during the first heart beat, it is suggested that these metabolites are released at some distance from the resistance vessels and that some time is needed for diffusion.

1. Pulsed bidirectional Doppler‐ultrasound equipment was used to measure changes in blood velocities in the femoral artery on a beat to beat basis for consecutive contraction and relaxation phases during voluntary rhythmic exercise of the quadriceps muscle group in man. 2. Rapid and large fluctuations of blood velocities were found, being high during relaxation and low during contraction phases. At the onset of contraction phase, negative velocities were present, indicating retrograde flow. During the rest of the contraction phase, forward flow occurred comparable to the resting flow level even at high loads. 3. Estimated maximal flow to the whole leg during relaxation phase, calculated from these blood velocity measurements and vessel diameter (measured with echo‐ultrasound equipment with high resolution) was in two of the subjects 3.32 l min‐1 (female) and 5.97 l min‐1 (male). When using computer tomography to estimate the volume of the quadriceps muscle group, the calculated maximum flow to this muscle group was 243 (female) and 257 (male) ml min‐1 100 ml muscle‐1. The time‐averaged flow during exercise to the whole leg was 1.51 l min‐1 (female) and 2.47 l min‐1 (male). The calculated time‐averaged flow to the quadriceps muscle group was 101 (female) and 98 (male) ml min‐1 100 ml muscle‐1. 4. The duration of post‐contraction hyperaemia following such rhythmic exercise of up to 6 min duration and up to 75% maximum voluntary contraction was never in excess of 150 s.

1. An improved Doppler ultrasound technique was used to measure stroke volume (SV) and cardiac output (CO) on a beat‐to‐beat basis in a group of supine humans before, during and after periods of standardized, rhythmic exercise, involving the quadriceps muscle groups on both sides. The development of CO on such bouts of exercise was compared to Doppler ultrasound records of the simultaneous femoral arterial flow (FF) response. 2. Records of CO at rest revealed spontaneous fluctuations around a mean level, with differences between the minimal and maximal values of the order of 1 l min‐1. The mean CO level at rest again varied considerably from one day to another and from test run to test run. 3. Upon start of exercise an immediate and rapid increase in heart rate (HR) and CO took place. The entire increase, the size of which varied appreciably from test run to test run, was completed within 10‐15 s. No or only minor changes were seen in the mean SV level during the exercise periods. 4. The time course of the increase in FF was indistinguishable from that of the increase in CO, which occurred without any detectable delay relative to the changes in FF. These closely parallel developments indicate a tight regulatory coupling between the two types of flow changes. 5. In the majority of tests the total and two‐sided increase in FF seen in the steady‐state situation in the last part of an exercise period was significantly larger than the recorded increase in CO. This discrepancy implies that some redistribution of flow from tissues other than the working muscles might take place, even at this moderate level of work. 6. Upon the end of exercise a striking but transient increase in CO occurred, resulting from an increase in SV concomitant with a maintained HR. In the course of five to eight post‐exercise cardiac cycles about 100 extra milliliters of blood were expelled from the heart. This cardiac outflow overshoot was found to occur during a post‐exercise fall in mean arterial blood pressure (MAP).