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A prescreen of the in vitro cytotoxicity of both the primary fabrication components and potential leachables from a bead-formed macroporous poly(2-hydroxyethyl methacrylate), (pHEMA) matrix has been carried out using INVITTOX Neutral red and Kenacid blue R dye binding methods. Of the eluants obtained from 24, 48, and 72-h incubated beads, only the 72-h eluant produced a greater than 20% (ID20) inhibition of 3T3-L1 cell proliferation with values of 20.98 +/- 2.33% and 21.41 +/- 1.37% inhibition for the Neutral red and Kenacid blue R binding methods, respectively. ID50 values for the fabrication components obtained using the Kenacid blue R method were generally higher than those obtained by the Neutral red assay, although the ranking of the chemicals in terms of their relative cytotoxicities was identical by both methods, i.e. ethylene glycol dimethacrylate > uranyl nitrate > purified HEMA > n-hexane > ethylene glycol (mmol 1(-1)). Whilst extended washing of finished PHEMA beads in water will reduce their acute in vitro cytotoxicity, this will only be achieved with some loss of previously encapsulated water soluble macromolecules.

Vascular smooth muscle cell (vSMC) proliferation is important in atherosclerosis. We previously demonstrated that methylamine-activated alpha 2-macroglobulin (alpha 2M) and transforming growth factor beta 1 (TGF-beta 1) cause a synergistic proliferative response in quiescent rat aortic vSMCs [Stouffer, G. A., La-Marre, J., Gonias, S. L. & Owens, G. K. (1993) J. Biol. Chem. 268, 18,340-18,344]. The first goal of this study was to determine whether the synergy is due to the ability of alpha 2M-methylamine (alpha 2M-MeNH2) to bind TGF-beta 1 and target the growth factor to vSMCs that express the alpha 2M receptor. Receptor-recognized alpha 2M derivatives without TGF-beta 1-binding activity, including ternary alpha 2M-trypsin, an 18-kDa proteolytic fragment of the alpha 2M subunit, and the corresponding recombinant receptor-binding fragment (rRBF) increased vSMC [3H]thymidine incorporation and cell number in a manner similar to alpha 2M-MeNH2. In combination with TGF-beta 1, each alpha 2M derivative caused a synergistic vSMC proliferative response. vSMCs responded comparably when treated with alpha 2M-MeNH2 and TGF-beta 1 simultaneously or in sequence. Furthermore, alpha 2M-MeNH2-TGF-beta 1 complexes increased [3H]thymidine incorporation no more than alpha 2M-MeNH2 alone. These results indicate that TGF-beta 1 binding to alpha 2M is not responsible for the synergistic mitogenic activity. Additional studies were undertaken to determine whether activated alpha 2M independently induces a signal-transduction response in vSMCs. alpha 2M-MeNH2 and rRBF caused a rapid, transient increase in vSMC inositol 1,4,5-trisphosphate. This response was pertussis-toxin insensitive. Receptor-associated protein (RAP; 170 nmol/L) inhibited 91-95% of the specific binding of 125I-alpha 2M-MeNH2 and 125I-rRBF to vSMC; however, RAP did not affect the inositol 1,4,5-trisphosphate response or the mitogenic response. These studies suggest that vSMCs express a receptor, other than low-density-lipoprotein-receptor-related protein, that transduces a signal in response to activated alpha 2M. This receptor may mediate the mitogenic activity of alpha 2M in vSMC culture.

Freeze-thaw photopolymerization of a mixed solution of monomers and bovine insulin around frozen ice crystals has been used to generate a bead-formed macroporous hydrophilic matrix of p-HEMA. The largest proportion of beads was 500-1000 microns in size (distribution < 106-1700 microns) with a mean EBC of 71.7 +/- 0.92%. Insulin release was monitored using RIA and insulin bioactivity determined using the rate of insulin stimulated D-[U14C] glucose oxidation to 14CO2. The cumulative insulin release profile was characterized by an initial lag phase followed by an almost linear increase in insulin release for up to 30 days. Insulin release at 4 degrees C was significantly greater than release at 37 degrees C both in the presence and absence of 2.5% thiomersalate as preservative. The latter served to extend the time period over which significant insulin release could be detected. Increasing the monomer concentration decreased the mean equilibrium buffer content (EBC), the total mean cumulative release of insulin, and the proportion of the incorporated insulin load subsequently released at both 4 degrees C and 37 degrees C in the presence of preservative. Insulin determination using RIA and bioassay confirmed that insulin released from beads was bioactive and that immunoreactivity was a reasonably reliable indicator of bioactivity.

Freeze-thaw photopolymerization at low temperature of a mixed solution of 2-hydroxyethyl methacrylate (HEMA), ethylene glycol dimethacrylate (EDM), and either glucose oxidase (GOx) or interleukin 2 (IL-2) around frozen ice crystals has been used to generate a bead-formed macroporous hydrophilic matrix with potential for immobilization and sustained release. The mean equilibrium acetate buffer content (EBC) of unloaded p-HEMA beads at room temperature and controlled humidity was approximately 72%. The incorporation of GOx into beads significantly increased the EBC to approximately 76%. The release of GOx was characterized by a short initial burst release which declined rapidly until by day 14 no further biologically active enzyme release could be detected. Bead size had no significant effect on the total mean cumulative release of GOx at room temperature. Since only approximately 4% of the original therapeutic load of GOx was released over 14 days a substantial proportion of biologically active enzyme had become associated with the hydrogel matrix surface generating a bead formed immobilised enzyme system. Total cumulative release profiles for IL-2 were almost linear and maintained for at least 16 days. In absolute terms, the proportion of the original theoretical incorporated load subsequently released over this period was low. However, such a low level sustained release of IL-2 may lend itself therapeutically to a reduction in unwanted non-specific systemic activity.

Cognitive function and school achievement were studied prospectively over 3 to 4 years in 19 children treated for brain tumors with whole-brain radiotherapy; 14 of 19 also received adjuvant chemotherapy. For the group as a whole, mean IQ fell from a baseline of 104 to 92 at follow-up (p < 0.01). Age was inversely correlated with change in IQ over time (r = 0.71; p < 0.001). Children younger than 7 years at diagnosis had a mean IQ loss of 27 points, while children over 7 years at diagnosis showed no significant decrease in IQ. Decline in IQ occurred between baseline and year 2 of follow-up; none could be documented between years 2 and 4. All children younger than 7 years at diagnosis were receiving special education at follow-up; 50% of the children over 7 years at diagnosis were receiving supplemental educational services.

A process of freeze-thaw polymerization involving the low temperature photopolymerization of a mixed solution of monomers and bovine serum albumin around frozen ice crystals has been used to generate a bead formed macroporous hydrophilic matrix with potential for sustained release. Beads over the size range 100-3000 microns were fabricated with surface and internal pores of between 0.7-2.6 microns whose diameter could be controlled by manipulation of the monomers to solvent ratio. Increasing both the proportion of monomers in the monomer solution and the percentage of BSA incorporated reduced the EWC of beads. The BSA release profile was characterized by an initial burst followed by a lower but sustained release lasting up to 1 month. The total cumulative release of BSA and the proportion of the incorporated BSA load subsequently released were both reduced in physiological saline compared with distilled water but enhanced by freeze drying, mild agitation and incubation at 37 degrees C.

The hypothesis that nifedipine may cause an insidious but reversible change in glucose tolerance, similar to that associated with thiazide therapy, was studied in six nondiabetic and six noninsulin-dependent diabetic patients with hypertension. After medium-term nifedipine therapy (mean duration, 11.5 months) was stopped for one month and then resumed for a month, values for fasting blood glucose, fasting serum insulin, serum fructosamine, glucose tolerance, and insulin release in response to oral glucose were unchanged in both groups. These findings suggest that nifedipine in conventional doses does not have important effects on glucose handling in either diabetic or nondiabetic patients.

Hepatocyte and soleus muscle insulin binding and the rate of insulin stimulated glycogen synthesis were examined in normal lean and genetically obese diabetic (ob/ob) mice with age. In ob/ob mice a significant decrease in the concentration of high affinity hepatocyte insulin receptors and insulin binding to soleus muscle at 10-20 and 10-40 weeks of age respectively was associated with an age related development of insulin resistance characterized by increased body weight, plasma insulin and plasma glucose. In lean mice a significant reduction in the concentration of low affinity hepatocyte receptors and soleus muscle insulin binding was observed at 60 weeks and associated with a marginally increased plasma insulin concentration and unchanged level of glycaemia. Insulin stimulated glycogen synthesis was unchanged with age in lean mouse soleus muscle but in ob/ob was significantly reduced by 5 weeks and preceded the reduction in insulin binding in this tissue. In 40 week ob/ob mice insulin sensitivity was improved and characterized by a reduction in body weight, plasma insulin and glucose, increased hepatocyte high affinity insulin receptor concentration and decreased affinity, Ke. Although insulin binding to soleus muscle was not increased and there was no improvement in insulin stimulated glycogen synthesis at this age, the increased hepatocyte insulin binding was probably not the primary cause of the improved insulin sensitivity which was most likely mediated at post-receptor loci.

Expression of the obese hyperglycaemic (bo/ob) syndrome in mice is modified by the background genome. The Aston colony carries the ob gene on a mixed background which produces a unique combination of different features shown by ob/ob mice on other backgrounds. The maximum body weight of Aston ob/ob mice exceeded that of other colonies, possibly reflecting a trait for higher growth rate in the background genome. The hyperphagia, marked hyperinsulinaemia and moderate hyperglycaemia observed during the development of the syndrome receded in old mice. Plasma glucagon concentrations in the fed state were similar to +/+ mice and did nor Vary throughout life. Hyperplasia of the B-cells increased inordinately during the development of the syndrome, but declined in older mice coincident with progressive intercellular vacuolation and the appearance of acinar-like cells within the islets. A-cell hyperplasia was greater in young mice, and A-cells became relocated throughout the islets of older mice. The distinct pattern of age-related changes in ob/ob mice indicates that experiments using this gene type should define clearly their age as well as genetic background.

The role of cellular insulin receptors in the mechanism of thiazide induced glucose intolerance was studied in 10 non-diabetics and six diet controlled type II diabetics with mild essential hypertension. Glucose tolerance tests (75 g) were performed at the start of the study, after one month of placebo and after one month on bendrofluazide 5 mg daily. Erythrocyte insulin receptor status was measured on each occasion in the fasting state. In non-diabetics, low affinity insulin receptor concentration increased after bendrofluazide but high affinity receptor concentration remained unchanged. In the diabetics, there was no change in either high or low affinity insulin receptor concentration. No change in insulin receptor affinity occurred in either group. In the long term, non-diabetics may maintain normal glucose tolerance on thiazide diuretics by increasing insulin receptor numbers. This adjustment did not occur in diabetic patients which may explain the deterioration in glucose tolerance.

Hyperglycemia associated with the manifestation of the obese diabetic (ob/ob) syndrome in mice and the short and long term streptozotocin treatment of lean MF1 mice was accompanied by a significant decrease in erythrocyte glyoxalase 1 activity and a marked increase in the concentration of the alpha-oxoaldehyde methylglyoxal. Erythrocyte glyoxalase II activity was modestly but significantly elevated in both obese mice and short term streptozotocin treated MF1 mice but no significant changes in S-D-lactoylglutathione concentration could be detected. Modification of the cells glyoxalase system during hyperglycaemia, especially the enhanced production of methylglyoxal may be a significant biochemical factor in the development of diabetic complications.

Diurnal variations in food consumption and plasma concentrations of glucose and insulin were determined at 3-hourly intervals in obese hyperglycaemic mice (C57BL/6J ob/ob) and lean mice (C57BL/6J+/+). In lean mice, food consumption and plasma insulin concentrations increased during the light period and were reduced during the dark period, whereas plasma glucose concentrations were maximal at the beginning of the light period and declined to a minimum during the early dark period. In ob/ob mice, the plasma glucose concentration declined temporarily at the beginning of both the light and the dark period and became elevated towards the ends of these periods, but there were no significant diurnal variations of food consumption or plasma insulin concentrations. These observations indicate differences in the diurnal regulation of glucose homeostasis in lean and ob/ob mice.

Insulin release from statically incubated HIT-T15 cells was maximally stimulated by glucose, L-arginine and L-leucine. L-arginine stimulated insulin release in the absence of glucose. Glucose induced insulin release was potentiated by the addition of L-leucine, L-arginine and the two in combination. Both glibenclamide and chlorpropamide stimulated insulin release from HIT-T15 cells. Glibenclamide was the more potent and equivalent in insulinotrophic action to 7.5 mmol/l glucose. Only chlorpropamide significantly potentiated glucose induced insulin release. Perifused HIT-T15 cells produced a reproducible biphasic insulin response to glucose challenge which was characterised by a pronounced and sustained first phase and a reduced second phase. The stimulation of phase I by glibenclamide alone and the inhibition of phase II of glucose induced insulin release by colchicine suggested the presence of a readily available pool of insulin granules which was not rapidly restored by insulin biosynthesis and granule margination.

A method for isolating plasmids from Escherichia coli which requires less than 8 h from cell pellet to purified plasmid essentially free of protein, RNA, and chromosomal DNA is presented. By this procedure, amplified plasmid pBR322 was isolated from E. coli strain RR1. The final product had no detectable protein or RNA, and plasmid comprised approximately 99% of the total DNA. The procedure includes lysozyme treatment in hypertonic solution followed by lysis with a mild detergent in the presence of high salt and an RNase inhibitor--conditions which prevent unfolding of the bacterial nucleoid. After centrifuging out the nucleoid and cell debris, the nucleic acids are selectively precipitated with a neutral solution of sodium trichloroacetate and ethanol. RNA is degraded with RNase and the degradation products and RNase are eliminated through a second trichloroacetate/ethanol precipitation. Finally, the plasmid is resuspended and passed through a nitrocellulose filter to remove aggregates and any residual protein and single-stranded DNA--giving a plasmid preparation suitable for electrophoretic fractionation or cleavage with restriction nucleases.

We reviewed our experience in 43 consecutive patients with primitive neuroectodermal tumors (medulloblastoma), PNET (MB), treated between 1975 and 1984, to characterize their quality of life and identify factors which impacted on long-term function. Twenty-four of forty-three (56%) of children are alive and free of disease, a median of 4.5 years after diagnosis. The quality of life was analyzed for the 24 long-term survivors. 79% (19 of 24) were functioning well in everyday activities. The median full-scale intelligence quotient (FSIQ), obtained a median of 3.5 years after diagnosis for those tested (n = 17) was 97, with all but 3 (12%) of the patients functioning in the normal range. Specific learning, memory and fine-motor disabilities were found in over one half of patients. Factors associated with poorer performance and lower FSIQ included preoperative obtundation, the need for a permanent shunt, younger age at diagnosis, and a complicated postoperative course. It is concluded that (1) the majority of long-term survivors have 'normal' intellectual function, but may have specific intellectual and academic disabilities, and (2) preoperative and postoperative factors strongly impact on the quality of life of survivors.

Experiments were conducted on 18 hr fasted male Wistar rats at 15 minutes (short-term anaesthetised) and 5 hr (long-term anaesthetised) after the induction of anaesthesia with sodium pentobarbitone, 45 mg/kg body weight intraperitoneally. Long-term anaesthetised animals received supplementary injections of anaesthetic, 10 mg/kg body weight at hourly intervals, or as required to maintain anaesthesia. Normal basal arterial blood glucose and plasma insulin levels were well maintained during 5 hours of anaesthesia. Following an intravenous glucose challenge, plasma insulin levels and insulin-glucose ratios were slightly reduced in the long-term anaesthetised animals, but glucose tolerance was not significantly impaired. The insulin secretory capacity of pancreas pieces isolated from short-term and long-term anaesthetised animals and incubated in vitro in the absence and presence of glucose was not significantly altered. The hypoglycaemic effect of an intravenous injection of insulin was markedly diminished and the rate of elimination of insulin from the circulation was significantly increased in the long-term anaesthetised animals. These observations indicate adjustments of the glucose homeostatic mechanism during prolonged pentobarbitone anaesthesia in fasting rats.

As survival rates have risen for children with malignant primary brain tumors, so has the concern that many survivors have significant permanent cognitive deficits. Cranial irradiation (CRT) has been implicated as the major cause for cognitive dysfunction. To clarify the etiology, incidence, and severity of intellectual compromise in children with brain tumors after CRT, a prospective study was undertaken comparing the neuropsychological outcome in 18 consecutive children with malignant brain tumors treated with CRT to outcome in 14 children harboring brain tumors in similar sites in the nervous system who had not received CRT. Children with cortical or subcortical brain tumors were not eligible for study. Neuropsychological testing was performed after surgery prior to radiotherapy, after radiotherapy, and at 1- and 2-year intervals thereafter. Children who had received CRT had a mean full-scale intelligence quotient (FSIQ) of 105 at diagnosis which fell to 91 by Year 2. Similar declines were noted in their performance intelligence quotient (IQ) and verbal IQ. After CRT, patients demonstrated a statistically significant decline from baseline in FSIQ (p less than 0.02) and verbal IQ (p less than 0.04). Children who had not received CRT did not demonstrate a fall in any cognitive parameter over time. The decline between baseline testing and testing performed at Year 2 in patients who had CRT was inversely correlated with age (p less than 0.02), as younger children demonstrated the greatest loss of intelligence. Children less than 7 years of age at diagnosis had a mean decline in FSIQ of 25 points 2 years posttreatment. No other clinical parameter correlated with the overall IQ or decline in IQ. After CRT, children demonstrated a wide range of dysfunction including deficits in fine motor, visual-motor, and visual-spatial skills and memory difficulties. After CRT, children with brain tumors also demonstrated a fall in a wide range of achievement scores and an increased need, over time, for special help in school. The 2-year results of this study suggest that children with brain tumors treated with CRT are cognitively impaired and that these deficits worsen over time. The younger the child is at the time of treatment, the greater is the likelihood and severity of damage. These children, although not retarded, have a multitude of neurocognitive deficits which detrimentally affects school performance. New treatment strategies are needed for children with malignant brain tumors.

Digitoxose specifically and competitively inhibited glucose stimulated insulin release from islets of both lean and obese mice without affecting either the rate of glucose oxidation or the rate of glucose stimulated oxygen consumption. Obese mouse islets were marginally more resistant to the inhibitory effect of digitoxose than lean mouse islets. Digitoxose provides a means for dissociating glucose stimulated insulin release by isolated islets from their metabolism of glucose confirming that glucose metabolism per se is not a necessary prerequisite for the initiation of insulin release but is required to fuel the insulin secretory process.

Immunoreactive insulin (IRI) was determined in the bile and pancreatic juice of rats. Recovery studies in which known amounts of insulin were added to samples of these fluids indicated that the IRI values corresponded closely to total recovery. The concentration of IRI in bile and pancreatic juice was greater than the concentration in arterial plasma and less than the concentration in hepatic portal venous plasma adjacent to the liver. Elevation of the circulating IRI concentration in fasting anaesthetized animals by intravenously administered glucose or insulin produced a concomitant increase in the concentration of IRI in bile and pancreatic juice. In fasting conscious animals there was a circadian variation in the concentration of IRI in bile and pancreatic juice similar to the variation of IRI in the circulation. The total amount of IRI in the bile and pancreatic juice of fasting conscious rats during 24 h was in excess of 4 mU/kg body weight. These studies demonstrate that considerable quantities of IRI are transferred from the circulation into the bile and pancreatic juice of rats.

The treatment of poorly controlled, non-compliant non-insulin-dependent diabetic subjects for one month with guar granules (Guarem) was associated with significant improvements in fasting serum glucose and insulin and urinary glucose excretion. No significant change was observed in either oral glucose tolerance, erythrocyte insulin receptor binding, serum calcium, cholesterol, triglyceride or HbA1. Subjects reported significant side effects including excessive flatus, increased bowel frequency and fullness. The limited advantages of Guarem treatment must be measured against the possibility of these side effects which to a large extent may be avoided by special attention to the means of administration. Prudent supplementation of the diet with Guarem has undoubted potential for diabetic control.

The present study describes the effect of nutrient secretagogues upon insulin release from an insulinoma derived B-cell line RINm5F. Glucose failed to stimulate insulin release, it did however increase the rate of glucose oxidation by some 16 fold. Glyceraldehyde stimulated insulin release in a concentration dependent manner, maximum stimulation occurring at 20 mmol/l. The response to glyceraldehyde was suppressed by calcium channel blockade and calmodulin inhibition. Insulin release was also stimulated by the amino acids leucine and arginine, maximum stimulation for both amino acids occurring at 15 mmol/l. The present study confirms the usefulness of this cell line as a model for the study of insulin secretion.

Acetylcholine stimulated the release of insulin from cultured HIT-T15 and RINm5F cells in the absence of glucose confirming that the insulin response to cholinergic stimulation is preserved in these two cloned B-cell lines. In addition acetylcholine potentiated glucose stimulated insulin release from HIT-T15 but not from RINm5F cells. Perifused HIT-T15 cells responded to square wave acetylcholine challenge with a monophasic release of insulin while the release profile for RINm5F cells was irregular and reduced. Acetylcholine stimulated insulin release from both cell lines was reduced by atropine but unaffected by hexamethonium confirming a mechanism of action involving muscarinic receptor activation.