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B Formby

Publications and source records attributed to B Formby.

At least 37 records · Page 2Linked to original sources
PubMed1990

Autoantibodies in the sera from newly diagnosed diabetic nod mice: evidence against cross-reactivity with a putative beta-cell surface autoantigen.

In insulin-dependent diabetes mellitus in humans, the BB rat and the NOD mouse, serum has been reported to contain autoantibodies that precipitate a 64,000 Mr protein from (32S) methionine labeled histoincompatible non-autoimmune rat or mouse islet cell proteins. Because experimental data reported recently have brought into question the role of the 64,000 Mr protein in targeting autoantibodies and hence initiate beta-cell destruction, we report differently designed experiments to clarify the apparent 64,000 Mr autoantigen enigma. Using an in vitro model of NOD mouse origin mimicking diabetic insulitis we found that target beta-cells induced a 6-fold increase in proliferative response of splenic L3T4+, Thy-1,2+ T cells. The magnitude of the proliferative response was not affected when target beta-cells were pretreated with 50% (vol/vol) partially purified immunoglobulins ((NH4)2SO4 precipitation at 33% saturation) from sera from newly diagnosed (less than 4d after onset) diabetic NOD mice. Cytofluorimetric analysis of beta-cells pretreated with partially purified immunoglobulins plus FITC-conjugated goat antimouse IgG as a second-step antibody were negative and thus gave no indication of an autoantigen-autoantibody complex formed on the surface of the beta-cells. We conclude from the experimental data that it remains still in question whether an autoantigen is targeted by an islet cell surface specific autoantibody and plays a role as a triggering event in the pathogenesis of diabetes in NOD mice.

Animals↗
PubMed1988

Growth hormone stimulates insulin gene expression in cultured human fetal pancreatic islets.

The total body mass of the human fetus increases about 100-fold from 10-20 weeks of gestation, and peak serum GH concentrations occur at 20 weeks. Since insulin has an essential growth-promoting influence in the fetus, these experiments were designed to determine whether GH can function as a growth factor with insulin-releasing activity by stimulating insulin gene expression during embryogenesis. Pancreatic islets were isolated from human fetuses (n = 36) of 18-22 weeks gestational age. Insulin gene expression was quantified by measuring insulin mRNA by blot hybridization analysis using a [32P] cDNA probe encoding human insulin. We found that by 48 h of culture insulin gene expression was stimulated by 1.25 micrograms recombinant human GH/mL medium to 216% of the control value (n = 2). Insulin secretory capacity was expressed as a fractional stimulation ratio (FSR = F2/F1) of insulin release rates during two successive 1-h static incubations. After 48 h of culture 1.25 micrograms/mL GH stimulated the FSR value to 273% of the control value (n = 5). We conclude that recombinant human GH significantly enhances the steady state level of insulin mRNA concurrent with an increase in insulin secretory capacity, hence providing evidence for a regulatory function of GH on insulin gene expression during fetal development.

Gene Expression Regulation↗
PubMed1988

Adoptive immunotherapy of diabetes in autologous nonobese diabetic mice with lymphoid cells ex vivo exposed to cyclosporin plus interleukin 2.

Between 12 and 26 wk of age, approximately 80% of female nonobese diabetic (NOD) mice from the inbred Sansum colony develop lymphocytic insulitis and become overtly diabetic. The disease in this animal model is similar to human insulin-dependent diabetes mellitus (IDDM) in both genetics and autoimmune pathogenesis. Cyclosporin A (CsA) has been used for immunosuppression in IDDM of recent onset in humans but has several limiting side effects. Therefore, a different regimen for CsA immunosuppression was investigated. Autologous splenic lymphoid cells from 12-wk-old not-yet-diabetic female NOD mice were cultured for 72 h with CsA plus interleukin 2 (IL-2) before reinfusion into the animal from which they were isolated. After this treatment, only 2 (18%) of 11 mice became overtly diabetic during an observation period of 19 wk, while 18 (86%) of 21 age-matched control mice developed diabetes during the same observation period (P less than .001). These data suggest an ex vivo preferential IL-2 activation of specific suppressor cells for the autoimmune process with CsA blockade of cytolytic/helper activities. Because the in vivo concentrations of CsA with this procedure would be negligible, these findings have implications for the potential nontoxic use of CsA in human protocols as well.

Animals↗
PubMed1988

3H-cyclosporine internalization and secretion by human fetal pancreatic islets.

Human fetal pancreatic islets were isolated from 16- to 20-week-old fetuses by a collagenase technique and cultured 48 hr in RPMI 1640 containing 10% human adult serum and unlabeled 0 to 5 micrograms cyclosporine A (CsA)/ml. Insulin secretory capacity of human fetal islets was expressed as a fractional stimulatory ratio FSR = F2/F1 of the fractional secretion rates during two successive 1 hr static incubations first with 2 mM glucose (F1) to stabilize secretion followed by maximal stimulus, i.e., 25 mM glucose plus 10 mM L-leucine and 10 mM L-arginine (F2). Unlabeled CsA at the above concentrations had no significant effects on the insulin secretory capacity expressed by FSR-values. Studies of net uptake of 3H-CsA by islets cultured for varying periods up to 40 hr and expressed as picomole 3H-CsA per picomole islet insulin content demonstrated that uptake rate was slow and did not reach isotopic equilibrium over the 40 hr of culture. When isolated fetal islets were cultured for 48 hr in the presence of 3H-CsA and varying concentrations of unlabeled CsA it was found during two successive 1 hr static incubations that fetal islets secrete insulin concomitantly with 3H-CsA following maximal stimulus for secretion. An optimal secretory molar ratio of 3H-CsA to insulin of 4.0 +/- 1.3 (n = 7) was found after islets were cultured 48 hr in the presence of a saturating 2.128 micrograms 3H-CsA per milliliter culture medium. In three successive 30-min static incubations of 3H-CsA loaded islets, first with low glucose, followed by high glucose plus L-arginine and L-leucine, and finally with high glucose plus L-arginine and L-leucine and 10 mM theophylline, the proportional fractional secretion rates of insulin and 3H-CsA were of the same magnitude. It is concluded that human fetal pancreatic islets during 48 hr of culture in the presence of pharmacologically relevant concentrations of CsA can internalize the drug, which is compartmentalized and concomitantly secreted with insulin following maximal stimuli. Transplanted human fetal islets utilized as delivering units for CsA could be beneficial for the induction of immunotolerance to allografted fetal islets.

Cells, Cultured↗
PubMed1987

The offspring of the female diabetic "nonobese diabetic" (NOD) mouse are large for gestational age and have elevated pancreatic insulin content: a new animal model of human diabetic pregnancy.

Pregnancy in diabetic mothers is associated with intrauterine death, perinatal mortality, and birth weight greater than that of infants born of normal mothers. The use of rodents made diabetic by alloxan or streptozotocin as an animal model for human diabetic pregnancy has been controversial because of the severity of the diabetes as well as the direct effect of diabetogenic drugs on the developing organism. Among our female NOD (nonobese diabetic) mice, insulin-dependent diabetes occurs spontaneously in 9% by 12 weeks and in 80% by 29 weeks of age. Offspring born within 21 days of conception to mildly hyperglycemic NOD pregnant mice between 26 and 52 weeks of age, and prior to the onset of maternal ketonuria are macrosomic with an average of 31% increase in body weight and 44% increase in kidney weight, in comparison to controls. Besides organomegaly, the macrosomic offspring have significantly higher pancreatic insulin content which was elevated 80% when compared with that of controls, and litter sizes are significantly 50% smaller. These results suggest that the mildly hyperglycemic pregnant NOD mouse represents a promising model for the study of pregnancy complicated by diabetes.

Animals↗
PubMed1987

Effects of low-dose cyclosporine prophylaxis in nonobese diabetic mice.

Among female nonobese diabetic mice, ketotic insulin-dependent diabetes mellitus (IDDM) develops spontaneously in 80% between 12 and 26 weeks of age. This condition resembles human type I diabetes. IDDM developed in 0 of 11 (0%) mice prophylactically treated with 10 mg of cyclosporine A (CyA) per kg s.c. every 4th day from 8 to 26 weeks of age; 8 of 10 (80%) of sex- and age-matched controls developed IDDM; 2 of 8 (25%) followed up to 5 months beyond the time of drug administration developed IDDM. The distribution of specific radioactivity ([3H]CyA) was used to calculate the concentrations of CyA in serum, blood cells and various organs. Serum values of CyA produced by radioimmunoassay were higher than those estimated by the [3H]CyA method. Pancreata of CyA-treated mice were histologically normal. Pancreata of control mice showed lymphocytic infiltration of the islets of Langerhans. Neither hepatotoxicity nor nephrotoxicity assessed by biochemical and histological data was detectable in CyA-treated mice. The insulin secretory capacity of trypan blue viable functional pancreatic islets isolated post-treatment; was significantly lower in controls than in CyA-treated mice; islet content of insulin was not statistically different between controls and CyA-treated mice. We conclude that low nontoxic doses of CyA abrogate completely the development of diabetes in the female nonobese diabetic mouse and abolish lymphocytic infiltration of the islets of Langerhans against which there is autoimmunity. The effect of CyA persists well past the duration of therapy.

Animals↗
PubMed1987

Effects of duration of cold storage and gestational age on the insulin secretory capacity of human fetal pancreatic islets.

Studies were performed on human fetal pancreatic tissue to determine viability after long-term cold storage at 0 degrees to 2 degrees C and the effect of gestational age on in vitro insulin secretory capacity. Viability expressed by the insulin secretory capacity was assessed by insulin responses to 2 successive 1 hr static batch incubations. The first incubation (F1) was in low glucose (2mM) medium while the second incubation (F2) included 25 mM glucose and 1 mM 3-isobutyl-1-methylxanthine (IBMX) as potentiator. The fractional stimulatory ratio (FSR defined as F2/F1) and trypan exclusion rates of isolated fetal islets were used as indexes of viability. Cold storage at 0 degrees to 2 degrees C of whole pancreata for period up to 144 hr was not found to alter insulin secretory capacity (FSR-values), but the percentage of dead islets indicated by trypan blue uptake increased. Microscopic examination of dithizone stained pancreatic islets showed intact and well demarcated variable sized islets. Histologically, islets showed well preserved endocrine cells after cold storage for 18 hr followed by culture for 48 hr. Fetal islets isolated from pancreata of 16-18 weeks gestational age were found to have a 2-fold increase in FSR-values when compared to islets isolated from pancreata of 19-24 weeks gestational age. These experiments document the feasibility of obtaining human fetal islet tissue for transplantation at centers widely separated from the site of transplantation. The implication of the enhanced insulin secretory response of islets obtained between 16-18 weeks gestational age remains to be defined.

Cold Temperature↗
PubMed1986

Rapid selection of viable transplantable human fetal pancreatic islets by trypan blue exclusion.

A rapid method of separating viable from nonviable human fetal pancreatic islets prior to transplantation is needed to quantify grafted tissue and optimize the possibility of successful treatment of diabetes mellitus in the recipient. After incubation with 0.04% trypan blue in isotonic Krebs-Ringer buffer solution for 15 min, the percentage of islets that excluded trypan blue was found to correlate well with fractional stimulated insulin secretion rates. The incubation procedure did not alter the subsequent insulin secretory capacity of the islets. Trypan blue exclusion rapidly and reliably identifies viable functional islet tissue prior to transplantation.

Cell Separation↗
PubMed1984

Relationship between insulin release and 65zinc efflux from rat pancreatic islets maintained in tissue culture.

In short-term batch-incubation or perfusion experiments, we studied insulin release and associated 65Zn efflux from rat pancreatic islets loaded with 65Zn by 24-h tissue culture in low-glucose medium. The fractional basal insulin release and 65Zn efflux were 0.4% and 3% of total content/h/islet, respectively. Thus, basal 65Zn efflux was much greater than that to be accounted for if zinc was released proportionally with insulin release only; extragranular zinc flux was suggested. Two millimolar glucose, with or without 1 mM 3-isobutyl-1-methylxanthine (IBMX), affected neither insulin release nor associated 65Zn efflux. Twenty-five millimolar glucose produced a significant threefold increase in insulin release above baseline, but somewhat decreased 65Zn efflux at marginal significance. Glucose (25 mM) plus 1 mM IBMX provoked a high increase in insulin release and an associated 30% increase in fractional 65Zn efflux over basal. Calculations based on previous estimations of 65Zn distribution and equilibrium with islet zinc indicated that molar zinc efflux was more than sufficient to account for a 2-zinc-insulin hexamer. L-leucine (2 or 20 mM) plus 1 mM IBMX caused far greater 65Zn efflux for the amount of insulin released, indicating additional 65Zn mobilization not directly related to insulin secretion. To evaluate 65Zn efflux during inhibited insulin secretion, batch incubations were performed in 100% D2O or at 27 degrees C, conditions that inhibited insulin release stimulated by high glucose plus IBMX. These agents decreased the 65Zn efflux far below the basal value (35% and 50%, respectively) and greater than could be accounted for by the attendent inhibition of insulin secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗
PubMed1980

Ca-ATPases in pancreatic islets.

Mg-independent Ca-ATPase activity was measured in secretory granules, mitochondria and microsomes from albino mouse islets and in secretory granules from noninbred ob/ob mouse islets. The enzyme existed in a high-affinity (Km for Ca2+ approx. 10(-7) M) and a low-affinity (Km approx. 10(-5) M) form. In all subfractions the high-affinity Ca-ATPase was inhibited by cyclic AMP, caffeine and Na+. Alloxan stimulated the microsomal Ca-ATPase by 25%, but had no effect on Ca-ATPase activity in granules and mitochondria. Glucose and glucose metabolites had no effect on Ca-ATPase in the secretory granule fraction from ob/ob mouse islets, whereas NADH inhibited the enzymes by 35%. The secretory granule Ca-ATPase was also inhibited by pCMBS (43%), chlorpromazin (87%) and ruthenium red (23%). 45Ca uptake was studied in secretory granules isolated from ob/ob mouse islets. The uptake was accelerated by addition of ATP, the maximum effect being found at 1 to 2 mM ATP. Omission of MgCl2 decreased the uptake by 25%. 45Ca uptake was abolished in the presence of pCMBS and chlorpromazine, whereas caffeine had no effect. The importance of Ca-ATPase in 45Ca transport and regulation of insulin release is discussed.

Animals↗
PubMed1976

Ca-activated ATPase activity in subcellular fractions of mouse pancreatic islets.

Ca-stimulated ATPase activity has been demonstrated in homogenates of mouse pancreatic islets. On subcellular fractionation Ca-ATPase activity was found in secretory granules, mitochondria, and microsomes, but not in the postmicrosomal fractions. Highest specific activity was found in the granules. In all active subcellular fractions two Km(Ca) values for Ca-ATPase around 7.0 X 10(-6) and 1.8 X 10(-7) M were estimated. Assuming an ATP hydrolysis:Ca pumping ratio of 1:2, the highest capacity for active Ca transport was found in secretory granules and mitochondria. Concentrations of 40 mM or higher of Na and 10(-5) M cyclic AMP inhibited Ca-ATPase in all subfractions. Caffeine at a concentration of 10 mM inhibited Ca-ATPase significantly in secretory granules and microsomes. Also MG-ATPase activity was demonstrated in the various subfractions. This activity was compared with that of Ca-ATPase at identical concentrations of free metal ions and in the absence or presence of various inhibitors. It was concluded that high-affinity Ca-ATPase and Mg-ATPase are two different enzymic entities. Ca-ATPase may tentatively be assumed to participate in active transport of Ca between intracellular compartments and to constitute a Ca-accumulating system which returns the cytosolic free Ca concentration to the resting state after stimulation of the beta-cells by secretagogues. This enzyme may therefore play a significant role in regulation of insulin release.

4-Chloromercuribenzenesulfonate↗