Pancreatic cell immobilization in alginate beads produced by emulsion and internal gelation.

Alginate has been used to protect transplanted pancreatic islets from immune rejection and as a matrix to increase the insulin content of islet progenitor cells. The throughput of alginate bead generation by the standard extrusion and external gelation method is limited by the rate of droplet formation from nozzles. Alginate bead generation by emulsion and internal gelation is a scaleable alternative that has been used with biological molecules and microbial cells, but not mammalian cells. We describe the novel adaptation of this process to mammalian cell immobilization. After optimization, the emulsion process yielded 90 ± 2% mouse insulinoma 6 (MIN6) cell survival, similar to the extrusion process. The MIN6 cells expanded at the same rate in both bead types to form pseudo-islets with increased glucose stimulation index compared to cells in suspension. The emulsion process was suitable for primary pancreatic exocrine cell immobilization, leading to 67 ± 32 fold increased insulin expression after 10 days of immobilized culture. Due to the scaleability and broad availability of stirred mixers, the emulsion process represents an attractive option for laboratories that are not equipped with extrusion-based cell encapsulators, as well as for the production of immobilized or encapsulated cellular therapeutics on a clinical scale.

Mammalian Cell Encapsulation in Alginate Beads Using a Simple Stirred Vessel.

Cell encapsulation in alginate beads has been used for immobilized cell culture in vitro as well as for immunoisolation in vivo. Pancreatic islet encapsulation has been studied extensively as a means to increase islet survival in allogeneic or xenogeneic transplants. Alginate encapsulation is commonly achieved by nozzle extrusion and external gelation. Using this method, cell-containing alginate droplets formed at the tip of nozzles fall into a solution containing divalent cations that cause ionotropic alginate gelation as they diffuse into the droplets. The requirement for droplet formation at the nozzle tip limits the volumetric throughput and alginate concentration that can be achieved. This video describes a scalable emulsification method to encapsulate mammalian cells in 0.5% to 10% alginate with 70% to 90% cell survival. By this alternative method, alginate droplets containing cells and calcium carbonate are emulsified in mineral oil, followed by a decrease in pH leading to internal calcium release and ionotropic alginate gelation. The current method allows the production of alginate beads within 20 min of emulsification. The equipment required for the encapsulation step consists in simple stirred vessels available to most laboratories.

Stimulation of ornithine decarboxylase activity by insulin in developing rat brain.

Insulin administered ip or intracisternally (ic) increased the activity of ornithine decarboxylase (ODC) in whole brains and brain parts of neonatal rats. Maximal stimulation of activity occurred 4-5 h after ip administration. At the highest doses, insulin stimulated ODC activity by up to 5- and 8-fold after ip and ic injection, respectively. The same amount of insulin given ic caused greater increases in activity than when given ip. Insulin stimulated ODC activity in 2-day-old and in 17- to 60-day-old rats but not in 5- or 9-day old neonates or 80-day-old adults. When insulin-induced hypoglycemia was prevented by giving dextrose, the stimulation of ODC activity was approximately the same as that in animals receiving insulin without dextrose. This indicates that insulin-induced stimulation of brain ODC activity was not caused by insulin-induced hypoglycemia or physiological responses to hypoglycemia. Since ODC is considered an indicator of growth stimulation, these results suggest that insulin or insulin-like peptides have a role in the regulation of brain development.

Bronchoconstriction in asthmatics exposed to sulfur dioxide during repeated exercise.

Young male volunteers with mild asthma and hypersensitivity to methacholine were exposed for 75 min with natural breathing to 0.0, 0.25, 0.5, and 1.0 ppm SO2. Each exposure included three 10-min periods of moderate treadmill exercise (minute ventilation 21 l . m-2 . min-1, O2 consumption 25 ml . kg-1, and heart rate 120/min). Specific airway resistance (sRaw) was not significantly increased after exercise in 0.25 ppm SO2, relative to the control exposure (clean air). In 0.5 and 1.0 ppm SO2, sRaw was increased twofold and threefold above preexposure levels, respectively, corresponding to increases of 3.2 and 9.2 cmH2O . s in excess over the increases seen in clean air (P less than 0.001). There was a broad range of responses to exercise and SO2. The increases in sRaw after the second and third exercises were significantly less than after the first exercise. Respiratory impedance measured by forced random noise suggests that the induced bronchoconstriction was primarily associated with peripheral airways. These results confirm that mild asthmatics selected for methacholine sensitivity have as a group significant bronchoconstriction in response to short-term moderate exercise with natural breathing in 1.0 and 0.5 ppm SO2. In addition, the induced bronchoconstriction is decreased after short-term repeated exercise in SO2.

Axonal transport of proteins and glycoproteins in the rat nigro-striatal pathway and the effects of 6-hydroxydopamine.

Following stereotaxic injection of [35S]methionine into the substantia nigra of adult rats, there was rapid local incorporation of radioactivity into acid-insoluble material. Incorporation peaked by 4 h and then decreased. In contrast, acid-precipitable radioactivity in the corpus striatum (the major projection site of the substantia nigra) rose markedly between 1 and 8 h followed by a plateau period and another even more marked increase between 24 h and 6 days. Experiments involving injection of [3H]fucose gave similar results except that most of the acid-precipitable radioactivity in the striatum appeared in an early wave. In each case radioactivity in the contralateral striatum was less than 11% of that on the ipsilateral side. Stereotaxic injection of colchicine (20 microgram) into the nigrostriatal pathway (within the median forebrain bundle) blocked transport of [35S]protein and [3H]glycoprotein by 90% and 50%, respectively. In animals treated with 6-hydroxydopamine (6-OHDA; treated neonatally or as adults) the accumulation of striatal [35S]protein was reduced to 7 to 26% of control levels; striatal [3H]glycoprotein was also reduced, but not as much (29% to 73% of control). In control experiments, [3H]DOPA wa injected into the substantia nigra, and [3H]dopamine was measured in corpus striatum; 6-OHDA treatment reduced the amounts of striatal [3H]dopamine recovered to 3% of control values. The failure of colchicine or 6-OHDA to block transport of incorporated fucose as effectively as the transport of incorporated methionine is possible due to greater diffusion of fucose away from the injection site to non-dopaminergic nuclei projecting to the striatum. The molecular weight distribution of radioactive proteins at the substantia nigra and corpus striatum was analyzed by polyacrylamide gel electrophoresis. For both [35S]methionine and [3H]fucose, the gel electrophoretic pattern of radioactive proteins in the injection site (substantia nigra) was complex and did not change greatly between 2 h and 6 days. At the projection site (striatum) the electrophoretic distribution pattern was initially different from that of the substantia nigra, and changed markedly over the course of several days. In 6-OHDA-treated animals (treated neonatally or as adults), the bulk of proteins transported in nigro-striatal non-dopaminergic neurons appears to be very similar to that transported in the intact pathway in control rats. However, in striata of 6-OHDA-treated animals, a consistent reduction in striatal 35S- and 3H-radioactivitiy was observed in proteins with molecular weight from about 67,000 to 77,000. Assuming that the 6-OHDA treatment did not substantially affect the non-dopaminergic neurons, we interpret this to mean that some of the proteins in this molecular weight range are transported primarily by dopaminergic neurons.

A survey of effects of gaseous and aerosol pollutants on pulmonary function of normal males.

A total of 231 normal male human subjects were exposed for 4 hr to air, ozone, nitrogen dioxide, or sulfur dioxide; to sulfuric acid, ammonium bisulfate, ammonium sulfate, or ammonium nitrate aerosols; or to mixtures of these gaseous and aerosol pollutants. Only one concentration of each pollutant was used. This study, therefore, represents a preliminary survey, intended to allow direct comparison of studies to plan future research. During exposure each subject had two 15-min exercise sessions on a treadmill at 4 mph and 10% grade. Environmental conditions were mildly stressful, i.e., temperature = 30 degrees C and relative humidity = 60%. A battery of 19 measurements of pulmonary function was performed just prior to exposure (air control); 2 hr into the exposure, following the first exercise session; 4 hr into the exposure, following the second exercise session; and 24 hr after exposure. Significant differences were noted in specific airway resistance (SRAW), forced vital capacity (FVC), and forced expiratory flow at 50% of FVC (FEF50) and in related measurements in those experimental groups exposed to ozone or to ozone plus aerosols. None of the aerosols alone, nitrogen dioxide or sulfur dioxide alone, or mixtures of nitrogen dioxide or sulfur dioxide with aerosols produced significant effects. A distribution analysis of subject responsivity to ozone gave a normal distribution among subjects not exposed to ozone, and a distribution shifted to the right and skewed to the right among those exposed to ozone alone or in mixture, with no evidence of bimodal distribution of ozone sensitivity.

Reversal of diabetes by ßTC3 cells encapsulated in alginate beads generated by emulsion and internal gelation.

Encapsulation of insulin-producing cells in alginate beads could improve the treatment of type 1 diabetes by reducing or eliminating the need for immunosuppression. We have recently adapted an emulsion and internal gelation process to ß-cell encapsulation. This process has the advantages of being well suited for m(3)/h production rates and allowing the use of increased alginate concentrations. Compared with 1.5% alginate beads generated by a standard extrusion process, 5% alginate emulsion-generated beads demonstrated greater in vitro stability and greater volumetric exclusion of antibody-sized pullulan. When ßTC3 cells were transplanted into streptozotocin-induced allogeneic diabetic mice, a significant decrease in the blood glucose levels was seen within 2 days with the 5% emulsion-generated beads but not until >16 days with the 1.5% extrusion-generated beads. This was correlated with higher cell survival and lower graft-specific plasma immunoglobulin levels. These results suggest that higher-concentration alginate beads generated by emulsion and internal gelation have improved graft immunoprotection. The emulsion process is a promising and scalable technology for cellular therapies requiring immune isolation.

Airway sensitivity of asthmatics to sulfur dioxide.

The purpose of this study was to describe for asthmatic subjects the distribution of individual bronchial sensitivity to sulfur dioxide (SO2). Subjects were nonsmoking male asthmatics (n = 27) who were sensitive to inhaled methacholine. None of the subjects used corticosteroids or cromolyn sodium. Oral medications were withheld for 48 hr, inhaled medications for 12 hr prior to all testing. Each subject participated in four separate randomly ordered 10 min exposures to 0.00, 0.25, 0.50 and 1.00 ppm SO2 at 26 degrees C, 70% relative humidity. During exposures, subjects breathed naturally and performed moderate exercise (VE, normalized for body surface area = 21 1/m2 X min). Before and 3 min after exposure, specific airway resistance (SRaw) was measured by body plethysmography. Those subjects whose SRaw was not doubled by exposure to 1.00 ppm were also exposed to 2.00 ppm SO2. Dose response curves (relative change in SRaw, corrected for change in clean air vs SO2 concentration) were constructed for each subject. Bronchial sensitivity to SO2 [PC(SO2)], defined as the concentration of SO2 which provoked an increase in SRaw 100% greater than the response to clean air, was determined. Substantial variability in sensitivity was observed: for 23 subjects, PC(SO2) ranged between 0.28 and 1.90 ppm, while for the remaining 4 subjects, it was greater than 2.00 ppm SO2. The median PC(SO2) was 0.75 ppm SO2, and 6 subjects had a PC(SO2) of less than 0.50 ppm. PC(SO2) was not related (r = 0.31) to airway sensitivity to methacholine.

Differing response of asthmatics to sulfur dioxide exposure with continuous and intermittent exercise.

Ten subjects with mild asthma were initially exposed in an environmental chamber (26 degrees C 70% relative humidity) to clean air and 1.0 ppm SO2 while performing 3 sets of 10-min treadmill exercises (ventilation, 41 L/min) broken by 15-min rest periods. To evaluate the effects of the pattern and duration of exercise on the response to SO2 exposure, the subjects were then exposed to the same environmental conditions while exercising continuously for 30 min. Specific airway resistance (SRaw) was measured by body plethysmography before each exposure and after each exercise. All SO2 responses were significantly greater than the clean air responses. With intermittent exercise and SO2 exposure, mean SRaw measurements (preexposure and after 10, 20, and 30 min of exercise) were 5.4, 14.7, 12.8, and 11.1 cm H2O/s. After SO2 exposure with continuous exercise, the mean SRaw showed an increase from 5.2 to 17.3 cm H2O/s. This increase was significantly (p = 0.018) greater than the response after the third exercise in the intermittent protocol. It appears that asthmatics show an attenuated response to repetitive exercise in an atmosphere of 1.00 ppm SO2 and that the response to SO2 exposure develops rapidly and is maintained during 30 min of continuous exercise.

The relationship between exposure duration and sulfur dioxide-induced bronchoconstriction in asthmatic subjects.

The purpose of this study was to determine the shortest duration of exposure to 1.0 ppm sulfur dioxide (SO2) sufficient to induce bronchoconstriction significantly greater than that observed with exposure to clean air (CA) in exercising SO2-sensitive asthmatics. Asymptomatic, nonmedicated, male asthmatics (n = 12) with airway hyperresponsiveness to both methacholine and SO2 were exposed in a chamber (20 degrees C, 40% relative humidity) for 0.0, 0.5, 1.0, 2.0 and 5.0 min to both CA and 1.0 ppm SO2 on separate days (10 exposures). Just prior to each exposure, subjects walked on a treadmill in CA for 5 min at a predetermined speed/elevation to elicit a target ventilation of about 40 L/min, i.e., a brisk pace up a slight incline. After this walk, subjects rapidly entered an adjoining exposure chamber containing either CA or SO2 and immediately walked at the same speed/elevation for the specified exposure duration. Subjects then rapidly exited the chamber. Specific airway resistance (SRaw) and ratings of respiratory symptoms associated with asthma [shortness of breath/chest discomfort (SB/CD) and wheezing (WHZ)] were measured prior to any exercise and following each exposure. Postexposure SRaw and symptom ratings increased with increased exposure duration in SO2; postexposure SRaw also was increased with increased exposure duration in CA but to a lesser extent. After adjusting for the CA response, significantly greater SO2-induced bronchoconstriction was observed for the 2.0 and 5.0 min exposures as indicated by substantially greater increases in SRaw and substantially higher ratings of respiratory symptoms. The authors conclude that with the above exposure conditions, on average, SO2-sensitive asthmatics exhibit significant bronchoconstriction at exposure durations of 2.0 min or more.

Pulmonary function, airway responsiveness, and respiratory symptoms in asthmatics following exercise in NO2.

Two experiments were conducted to determine respiratory responses of persons with asthma performing intermittent moderate exercise while exposed to low concentrations of NO2. In the first, preliminary experiment, 13 male subjects, aged 19-35, with mild asthma were exposed on separate days in a chamber (natural breathing, 20 degrees C, 40% RH) to 0.30 ppm NO2 and to a control or "clean air" exposure (0.0 ppm NO2). Exposure included three 10-min periods of moderate treadmill exercise (VE = 44.5 liter/min), each followed by symptom measurement and pulmonary function testing. The average decrease in FEV1 following the initial 10 min exercise in 0.30 ppm was 11% which was significantly greater (p less than 0.05) than that observed in clean air (7%). Differences in FVC and SRaw were not significantly different at this time. Slight cough and dry mouth and throat were apparent only after the first exercise in NO2. After the second and third exercises, decreases in FEV1 and FVC and increases in SRaw were significantly greater in 0.30 than in 0.0 ppm NO2. Individual subject responses were variable. These results suggested that some asthmatics who perform moderate exercise while exposed to 0.30 ppm NO2 may experience bronchoconstriction and reduction in spirometric performance. Because of these preliminary findings, a more comprehensive, concentration-response experiment was conducted. Twenty-one male volunteers with mild asthma were exposed for 75 min with natural breathing to 0.0, 0.15, 0.30, and 0.60 ppm NO2. Exposure included three 10-min periods of moderate treadmill exercise (VE = 43 liter/min), each exercise followed by symptoms measurement and pulmonary function testing. In addition, airway responsiveness was measured two hr after each exposure by methacholine bronchial challenge testing. In the control exposures (0.0 ppm NO2), the exercise alone caused substantial decrements in pulmonary function. These decrements (as measured by decreases in FEV1 and FVC, and increases in SRaw) were not increased relative to the control exposure after any exercise session in any concentration of NO2. Furthermore, there was no overall group-averaged indication of a concentration-related effect of the NO2 on pulmonary function. Likewise, symptoms reported after NO2 exposure were not significantly different from those reported in clean air. Group-averaged airway responsiveness after exercise in NO2 was also not different from responsiveness after exercise in clean air. For only two subjects was there any indication of a concentration-related increase in airway responsiveness due to exposure to NO2.(ABSTRACT TRUNCATED AT 400 WORDS)