Risk factors for multi-joint disease in patients with glucocorticoid-induced osteonecrosis.

This study investigated risk factors for osteonecrosis involving multiple joints (MJON) among glucocorticoid-treated patients. The best predictor of MJON was cumulative oral glucocorticoid dose. Risk of MJON was 12-fold higher in patients who had a second risk factor for osteonecrosis. Further research is needed into strategies for prevention of MJON. INTRODUCTION: Osteonecrosis (ON) is a debilitating musculoskeletal condition in which bone cell death can lead to mechanical failure. When multiple joints are affected, pain and disability are compounded. Glucocorticoid treatment is one of the most common predisposing factors for ON. This study investigated risk factors for ON involving multiple joints (MJON) among glucocorticoid-treated patients. METHODS: Fifty-five adults with glucocorticoid-induced ON were prospectively enrolled. MJON was defined as ON in ≥ three joints. Route, dose, duration, and timing of glucocorticoid treatment were assessed. RESULTS: Mean age of enrolled subjects was 44 years, 58% were women. Half had underlying conditions associated with increased ON risk: systemic lupus erythematosus (29%), acute lymphoblastic leukemia (11%), HIV (9%), and alcohol use (4%). Mean daily oral dose of glucocorticoids was 29 mg. Average cumulative oral dose was 30 g over 5 years. The best predictor of MJON was cumulative oral glucocorticoid dose. For each increase of 1,000 mg, risk of MJON increased by 3.2% (95% CI 1.03, 1.67). Glucocorticoid exposure in the first 6 months of therapy, peak dose (oral or IV), and mean daily dose did not independently increase risk of MJON. The risk of MJON was 12-fold in patients who had a second risk factor (95% CI 3.2, 44.4). CONCLUSIONS: Among patients with glucocorticoid-induced ON, cumulative oral dose was the best predictor of multi-joint disease; initial doses of IV and oral glucocorticoids did not independently increase risk. Further research is needed to better define optimal strategies for prevention and treatment of MJON.

The relationship between physical activity, structural deformity, and spinal mobility in adolescent idiopathic scoliosis patients.

PURPOSE: Adolescent idiopathic scoliosis (AIS) patients experience structural spinal deformity, but the impact of AIS on physical activity is not widely studied. Reports of physical activity levels between children with AIS and their peers are mixed. This study sought to characterize the relationship between spinal deformity, spinal range of motion, and self-reported physical activity in AIS patients. METHODS: Patients aged 11-21 completed self-reported measures of physical activity using the HSS Pedi-FABS and PROMIS Physical Activity questionnaires. Radiographic measures were obtained from standing biplanar radiographic imaging. Surface topographic (ST) imaging data was obtained using a whole-body ST scanning system. Hierarchical linear regression models analyzed the relationship between physical activity, ST, and radiographic deformity while controlling for age and BMI. RESULTS: 149 patients with AIS (mean age 14.5 ± 2.0 years, mean Cobb angle 39.7° ± 18.9°) were included. In the hierarchical regression predicting physical activity from Cobb angle, no factors were significant predictors of physical activity. When predicting physical activity from ST ROM measurements, age and BMI served as covariates. No covariates or ST ROM measurements were significant predictors of physical activity levels for either activity measure. CONCLUSIONS: Physical activity levels of patients with AIS were not predicted by levels of radiographic deformity or surface topographic range of motion. Although patients may experience severe structural deformity and range of motion limitations, these factors do not appear to be associated with decreased physical activity level utilizing validated patient activity questionnaires. LEVEL OF EVIDENCE: Level II.

Intercellular communication in pancreatic islet monolayer cultures: a microfluorometric study.

Single islet cells in monolayer cultures of neonatal rat pancreas were microinjected with fluorescein and scanned topographically by microfluorometry. Fluorescein spread from an injected islet cell directly into neighboring islet cells, and, in the presence of 16.7 millimolar glucose, significantly more islet cells communicated with the injected cell than in glucose-free medium. Islet cells were also microinjected with glycolytic substrates and activators that produced transient changes in cellular levels of reduced pyridine nucleotides-nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate [NAD(P)H]. Changes in NAD(P)H fluorescence were observed in islet cells incubated first for 18 hours in very low glucose concentrations and then in a glucose-free medium and injected with glycolytic substrates and activators; however, little change of fluorescence occurred in adjacent islet cells. In contrast, after adding 16.7 millimolar glucose to the medium, injection of glycolytic substrates and activators produced transient changes in NAD(P)H fluorescence in the injected cell and in neighboring cells.

Cyclic adenosine-3',5'-monophosphate stimulates islet B cell replication in neonatal rat pancreatic monolayer cultures.

A possible role for cyclic adenosine 3',5'-monophosphate (cAMP) in islet B cell replication was examined in neonatal rat pancreatic monolayer cultures. Islet cells deteriorated and insulin release decreased during 12 d of culture in medium with 5.6 mM glucose, whereas the cells survived and insulin release increased during culture in medium with 5.6 mM glucose plus the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX, 0.1 mM), or in medium with 16.7 mM glucose with or without IBMX. IBMX also increased the mitotic index and stimulated dose-dependent increases in [(3)H]thymidine incorporation in nuclei of islet B cells in aldehydethionine stained radioautographs; maximal stimulation of B cell replication occurred with addition of 0.1 mM IBMX to 5.6 mM glucose (+170%, P < 0.001), and this increase was similar to that observed with 16.7 mM glucose (+185%, P < 0.001). Also, 8-bromo-adenosine-3',5-monophosphate, but not 8-bromo-guanosine-3',5'-monophosphate produced dose-dependent increases in islet B cell replication in medium with 5.6 mM glucose. Measurement of cAMP levels in the cultures revealed dissociations between effects on B cell replication and insulin release. Thus, addition of 0.1 mM IBMX, or 0.1 nM cholera toxin, to 5.6 mM glucose produced slightly greater increases in cAMP levels and B cell replication than did 16.7 mM glucose, whereas insulin release was increased significantly more with 16.7 mM glucose. Also, addition of 0.1 mM IBMX, or 0.1 nM cholera toxin, to 16.7 mM glucose stimulated further increases in cAMP levels and insulin release in the cultures, but no further increases in B cell replication. We conclude that (a) cAMP stimulates islet B cell replication, (b) cAMP may mediate the effects of glucose on B cell replication, and (c) mechanisms regulating B cell replication may be more sensitive to cAMP and/or different from those regulating insulin secretion.

Metabolic clearance and production rates of human growth hormone.

The metabolic clearance rate (MCR) of human growth hormone (HGH) was determined by the constant infusion to equilibrium technique utilizing HGH-(125)I. 22 control subjects had a MCR of 229 +/-52 ml/min (mean +/-SD). No difference was evident between sexes, or between various age groups. Patients with acromegaly demonstrated normal MCR's. Moreover, acute elevations of plasma growth hormone concentrations in normal subjects did not alter the MCR of HGH. The MCR was relatively constant from day to day and within the day when subjects were evaluated in the supine position. In contrast, the assumption of the upright position was associated with a mean 24% decrease in the MCR. These results were contrasted with the MCR of HGH observed in a small number of patients with altered thyroid function or diabetes mellitus. In six patients with hypothyroidism the MCR (131 +/-36 ml/min) was significantly decreased (P < 0.001); whereas the MCR in eight patients with hyperthyroidism (240 +/-57 ml/min) did not differ from control subjects. The MCR in eight patients with insulin-independent diabetes mellitus (IID) (185 +/-41 ml/min) and in eight patients with insulin-dependent diabetes mellitus (IDD) (136 +/-31 ml/min) were significantly different from control subjects (P = < 0.05 and P = < 0.001, respectively). These data were interpreted to indicate that the plasma HGH-removing mechanism(s) is not saturated at physiologic plasma HGH levels, that plasma HGH levels alone may not permit distinction between variations in pituitary release of the hormone and its rate of clearance from the plasma, and that the estimation of the MCR of HGH may help clarify the mechanism of abnormal plasma HGH responses to various stimuli. Production rates of HGH (PR) in control subjects (347 +/-173 mmug/min) were contrasted with hyperthyroid patients (529 +/-242 mmug/min, P < 0.05), hypothyroid patients (160 +/-69 mmug/min, P < 0.02), IID (245 +/-100 mmug/min, NS), and IDD (363 +/-153 mmug/min, NS). Considerable variability in the determination of the concentrations of immunoprecipitable HGH-(125)I and endogenous plasma HGH concentrations was encountered at apparent equilibrium conditions. Since both factors are necessary for the PR calculations, the wide 95% confidence limits of the PR's did not permit a clear interpretation of the significance of these observations.

Fetal insulin and growth hormone metabolism in the subhuman primate.

The concentrations of plasma glucose, insulin, growth hormone, and immunoreactive growth hormone-like substance in subhuman primate fetal and maternal plasma were examined after the intravascular administration of glucose, arginine, or tolbutamide to the fetus. Cannulation of interplacental vessels permitted studies on the fetus in utero without disruption of fetal-placental-maternal anatomic integrity. Single glucose injections, glucose infusions, and arginine infusions into the fetus did not alter fetal plasma insulin concentrations. In contrast, tolbutamide injections elicited an immediate 3-4-fold increase in fetal plasma insulin concentrations. A bidirectional placental transfer of insulin was demonstrated with the use of simultaneously injected insulin-(125)I to the mother and insulin-(131)I to the fetus. Simian fetal plasma contained a substance which cross-reacted with immunologic identity to human growth hormone. In contrast, simian maternal plasma and amniotic fluid reacted with immunologic nonidentity to human growth hormone. Although glucose administration to the fetus did not suppress nor did arginine infusion consistently augment fetal plasma growth hormone levels, the latter were observed to vary in individual experiments. The plasma responses to the same stimuli in the neonate were also examined. In contrast to the fetal experiments, glucose injection in the neonate elicited a delayed rise in the concentration of plasma insulin. Similar to the fetus, the plasma concentration of insulin increased after tolbutamide injection and did not change in response to arginine infusion. The initial concentrations of neonatal plasma growth hormone were significantly lower when contrasted with the initial fetal plasma levels. There was no difference in the responsiveness of the fetal and neonatal growth hormone-releasing mechanisms when challenged by glucose or arginine infusion. The data indicate that the fetal plasma concentration of growth hormone is labile, that fetal growth hormone metabolism may differ from that in the neonate, and that pancreatic islet cell responsiveness rapidly changes after delivery.

Subhuman primate pregnancy complicated by streptozotocin-induced diabetes mellitus.

Polydipsia, polyuria, polyphagia, and glucosuria followed the administration of streptozotocin to 6 nonpregnant and 15 pregnant monkeys (Macaca mulatta) in the first trimester of pregnancy. The diabetogenic action of the drug was also reflected in an induced but variable deterioration in maternal intravenous glucose tolerance and a marked attenuation of maternal plasma insulin responsiveness to intravenous glycemic stimuli. The products of conception were examined in 29 pregnancies. The neonates and the placentas of the streptozotocin-treated pregnant animals were significantly heavier than average for the period of gestation, polyhydramnios was consistently present, and there was an increase in the incidence of third trimester stillbirths. The fetal and maternal plasma glucose, insulin, and growth hormone concentrations were examined after the intravascular administration of glucose or a solution of mixed amino acids to the fetus in the third trimester. The neonatal plasma responses to similar insulinogenic stimuli were also examined.Fetal and neonatal base line plasma insulin concentrations were significantly elevated compared to those of the controls. The administration of intravascular glucose to the fetus, mother, or neonate was associated with a prompt 2-to 5-fold increase in fetal or neonatal plasma insulin concentrations. These findings contrast to the unresponsiveness of the pancreatic islet tissue we reported in normal subhuman primate pregnancy. The intravascular infusion of a relatively low concentration of mixed amino acids (2 mg/min) to the conceptii from the streptozotocin-treated pregnancies was associated with an elevation in fetal and neonatal plasma insulin levels, whereas normal monkey fetuses and neonates required a 10-fold greater concentration of amino acids in the infusate for similar responses. The induced hyperaminoacidemia or hyperglycemia did not consistently alter plasma growth hormone concentrations in the conceptii from normal or streptozotocin-treated pregnancies. These data provide evidence that maternal glucose intolerance during pregnancy is associated with enhanced fetal and neonatal pancreatic islet cell responsiveness to glucose and mixed amino acids. Although the specific mechanism(s) that alters both the sensitivity and responsiveness of the normal pancreatic fetal islet to insulinogenic stimuli remains unclear, the data do indicate that insulin-dependent maternal hyperglycemia and hyperaminoacidemia, separately or in combination could contribute to the fetal hyperinsulinemia of pregnancies complicated by diabetes mellitus. Moreover, the overall experiences with these streptozotocin-treated animals suggest that a subhuman primate model may be available to examine directly the antenatal pathophysiology of abnormal carbohydrate metabolism.

Factors affecting the response to insulin in the normal subhuman pregnant primate.

The concentrations of plasma glucose, free fatty acids, insulin, growth hormone, and placental prolactin in subhuman primate fetal and maternal plasma were examined following intravascular administration of insulin and glucagon to the fetus and mother. The neonatal plasma responses to these same stimuli were also examined. Fetal plasma glucose concentrations were minimally altered by direct fetal insulin injections, whereas neonatal glucose levels declined with similar injections. In both instances, however, plasma free fatty acid levels declined following insulin. When the amount of insulin given the fetus was increased, fetal plasma glucose concentrations did decline. Combined intravascular insulin injections and infusions in the mother were associated with a disappearance of the initial maternal to fetal plasma glucose concentration gradient and a nearly parallel fall in both maternal and fetal plasma glucose levels. It was concluded that insulin was biologically active in the fetus. Obtunded fetal plasma glucose responses to direct fetal insulin administration may be a function of placental transfer of glucose from the maternal pool. Maternal plasma placental prolactin and fetal plasma growth hormone levels were unchanged in the presence of sustained maternal and fetal hypoglycemia. However, neonatal plasma growht hormone levels did increase in response to hypoglycemia. The observed bidirectional placental barrier to transfer of radioisotopically labeled growth hormone indicated that fetal plasma growth hormone was solely of fetal origin. These data suggested further that a change in the growth hormone-releasing mechanism may occur from fetal to neonatal life. Direct maternal intravascular glucagon administration led to augmentation in both maternal and fetal plasma insulin and glucose levels. Direct fetal glucagon injections enhanced both maternal and fetal plasma insulin levels. These simultaneous changes in both plasma pools were consistent with the demonstration of a bidirectional placental transfer of radioisotopically labeled glucagon. The role of endogenously produced glucagon in these studies remains to be clarified.

A ganglioside antigen on the rat pancreatic B cell surface identified by monoclonal antibody R2D6.

In an attempt to identify B cell specific antigens, we have generated a mouse monoclonal antibody, R2D6, which is directed against plasma membranes of rat pancreatic B cells but against no other pancreatic cells. R2D6 crossreacted with mouse and guinea pig B cells, but not with human or dog. The B cell specificity of R2D6 was utilized in fluorescence-activated cell sorting to prepare highly enriched separate populations of viable pancreatic islet B cells and A cells. R2D6 also recognized adrenal chromaffin cells, secretory cells in the anterior pituitary, and the myenteric plexus of the gastrointestinal tract. Trypsin, chymotrypsin, papain, ficin, and pronase had no effect on R2D6-binding to dissociated rat islet cells. However, neuraminidase treatment of intact cells reduced R2D6-binding by 75%. The antigen recognized by R2D6, Ag(R2D6), could be quantitatively extracted from rat islets by dichloromethane/methanol (2:1) and, after drying, was soluble in methanol alone as well as in phosphate-buffered saline. When the dichloromethane/methanol extract (DME) was bound to polyvinylchloride microtiter plates, antigenic activity was retained and remained insensitive to pronase. In this solvent-extracted form, antigenic activity was totally destroyed by neuraminidase. Therefore, sialic acid is either an integral part of, or is related sterically to the binding site (epitope) for R2D6. In high performance thin-layer chromatographs of the DME, developed in 60:40:9 chloroform/methanol/2.5 N ammonia, Ag(R2D6) migrated with a relative mobility (Rf) of 0.54 +/- 0.07 (n = 3), which was a position nearly coincident with the purified brain ganglioside, GD1a. The antigen bound to DEAE-Sephacel, was not inactivated by mild treatment with base (which hydrolyzes phospholipids) and eluted in ganglioside fractions upon C18 Sep-Pak and upon silicic acid chromatography. Hence, the solubility characteristics, enzyme sensitivities, and behavior of Ag(R2D6) in four chromatography systems are consistent with its identification as a ganglioside.

Long-term study of vascularized free-draining intraperitoneal pancreatic segmental allografts in beagle dogs.

The purpose of the present study was to evaluate the significance of immunogenetic factors on the survival of pancreatic allografts in beagle dogs. Donors and recipients were leukocyte antigen (DLA)-typed and mixed lymphocyte culture (MLC)-tested. Recipients were made diabetic by total pancreatectomy and immediately implanted intraperitoneally with a vascularized, free-draining (duct unligated) pancreatic segmental (FDPS) allograft. Two groups of dogs were studied. In group I consisting of donor-recipient littermates, recipients were immunosuppressed with prednisone and azathioprine (n = 16 dogs), or not immunosuppressed (n = 4). In group II, recipients were made specifically unresponsive by total body radiation, autologous marrow implantation, and kidney transplantation from DLA-MLC identical donors, 1 yr before FDPS transplantation from the corresponding original kidney donors. Survival of the FDPS grafts in group I was inversely related to pretransplant MLC reactivity, irrespective of DLA genotyped match between donor and recipient. Thus, immunosuppressed high MLC reactors (n = 8) rejected FDPS grafts between 7 and 14 d, whereas immunosuppressed low MLC reactors (n = 8) accepted grafts for 25 to 260+ days, and nonimmunosuppressed low MLC reactors (n = 4) accepted grafts for 9-55 d. Rejection (hyperglycemia) of FDPS grafts was sudden, permanent, and unpredictable despite weekly intravenous glucose tolerance tests with measurements of glucose disappearance rates and serum insulin responses. Nevertheless, serial in vitro cell-mediated lymphocytotoxicity (CML) assays revealed increases in CML before graft rejection in low MLC reactors, and decreases in both CML and MLC responses before graft rejection in high MLC reactors. FDPS graft survival was indefinite (>6 mo) in group II dogs, despite low-grade MLC reactivity (2:4 dogs) and CML responses (4:4 dogs). Biopsies of FDPS grafts at 6 mo in normoglycemic dogs showed disappearance of exocrine tissue and coalescence of islets in both groups I and II, but with less fibrosis in group I (immunosuppressed). These results indicate that (a) pancreatic islets in vascularized grafts (FDPS) may survive indefinitely in the presence of a good tissue match best predicted by MLC testing, (b) tissue specific histocompatibility factors appear to be common enough between kidney and pancreas to allow for long-term survival of both organs transplanted from the same donor, at least in appropriate recipients (group II), and (c) immunosuppression is associated with less fibrosis in FDPS allografts.

Responses of canine lymphocytes to allogeneic and autologous Islets of Langerhans in mixed cell cultures.

Because successful allotransplantation of islets of Langerhans isolated by collagenase digestion has been difficult in many animal species, we asked whether isolated islet preparations might have tissue specific determinants conferring amplified immunogenicity in vitro. Lymphocyte proliferative responses ([(3)H]thymidine uptake) were studied in beagle dogs in mixed culture combinations of lymphocyte vs. lymphocyte (MLC) and lymphocyte vs. islet (MLIC). In five MLC responder and five nonresponder pairs, peripheral blood lymphocytes of dogs A and B were used as responding cells, and dog B x-irradiated lymphocytes (Bx), x-irradiated (or nonirradiated) islets (BI), or hepatic cells (BH) were used as stimulating cells in primary and secondary reactions. For the secondary reactions, A + Bx, A + BI, or B + BI were incubated for 9 d (A'B, A'BI, B'BI, respectively) before addition of new stimulating cells. The results showed that islets were autostimulatory, eliciting a tissue-specific lymphoproliferative response in a primary MLIC. Thus, B + BI reactivity was evident at 3,5, and 7 d in primary culture, whereas collagenase-digested liver cells, or lymphocytes obtained from collagenase-digested lymph nodes did not stimulate autologous lymphocytes. A separate reactivity was observed in the allogeneic A + BI combination in MLC responder pairs, and the peak response of A + BI at 9 d was markedly greater than that of B + BI, suggesting the presence of major histocompatibility complex lymphocyte-defined locus determinants in the islet preparations, in addition to islet-specific determinants. A secondary reaction was observed if lymphocytes were primed with islets and challenged with islets (A'BI + BI or B'BI + BI), but not if they were challenged with lymphocytes (A'BI + Bx, B'BI + Bx) or hepatic cells (A'BI + BH, B'BI + AH). Furthermore, priming of lymphocytes with autologous islets (B'BI) led to exclusion of any reactivity against allogeneic lymphocytes, i.e., B'BI suppressed A + Bx, and B'BI also markedly suppressed phytohemagglutinin-stimulated lymphoproliferative responses. Experiments were performed that excluded the possibility that the insulin levels present in the MLIC, the presence of passenger lymphocytes in the islets, or the maintenance of islets in tissue culture for 1-7 d affected the observations. These results provide evidence for the existence of alloantigens as well as tissue-specific antigens on collagenase-isolated islets of Langerhans.

Natural history of intrahepatic canine islet cell autografts.

We have serially followed the function of intrahepatic canine islet autografts in 15 beagle dogs for up to 24 mo. Of these, only 20% sustained normal levels of fasting blood glucose for greater than 15 mo posttransplant. Failure of autograft function was accompanied by a preferential loss of well-granulated beta cells in the engrafted islets. The chronic stimulation of an initially marginal intrahepatic beta-cell mass ultimately resulted in metabolic deterioration and loss of beta cells below the minimal threshold required to maintain normal fasting blood glucose levels. It is possible that transplantation of a larger mass of islets would result in indefinite graft function in dogs. However, it remains to be demonstrated in larger mammals, including humans, whether an islet cell mass that is initially adequate in a heterotropic site such as the liver can remain functionally competent over a prolonged period.

Primary structure of the canine CD4 antigen.

After PCR amplification two overlapping cDNA clones encoding the dog homologue of the human CD4 glycoprotein were identified. This internal fragment of the mature protein including the complete extracellular domains, consists of 1297 bp with a deduced amino acid sequence of 432 residues. The dog CD4 molecule differs from the corresponding protein of other species including human in the second domain. We found nine extra residues in the beginning of that domain, a cysteine at position 138, usually involved in a disulfide bridge, is substituted by tryptophan, and three new glycosylation sites are present.

Molecular cloning of the canine CD44 antigen cDNA.

Molecular cloning of the dog homologue of the human CD44 was achieved using RT/PCR. A 1055 bp cDNA has a deduced amino acid sequence of 351 residues, 338 of them correspond to the mature protein. Nine conserved cysteine residues were found. The extracellular region contains a single link superfamily domain on the N-terminal part and potential post-translational modification sites as: N- and O-linked glycosylation sites and chondroitin sulfate attachment sites. Three mRNAs of 2.2, 3.8 and 4.4 kb were identified on Northern blot analysis and Western blot hybridization revealed a 85-90 kDa protein expressed in lymph node tissue.

Factors from fibroblasts promote pancreatic islet B cell survival in tissue culture.

A possible role for fibroblasts in promoting the survival and function of islet B cells in tissue culture was examined by the addition of fibroblasts from a mouse embryo cell line (3T3-L2) to islet cell monolayer cultures prepared from newborn rat pancreases. Co-culture of islet cells with fibroblasts significantly increased the recovery of insulin in medium and cells after 7 days of culture in medium supplemented with 10% serum, and prevented the deterioration of islet cells cultured in serum-free medium. Similarly- serum-free medium, conditioned by cultures of either 3T3-L2 fibroblasts or fibroblasts freshly isolated from newborn rat pancreases, maintained the release and content of insulin in islet cell monolayer cultures at levels four- to eightfold higher than in control serum-free medium. Serum-free, fibroblast-conditioned medium also enhanced the survival of intact islets maintained in free-floating culture for 28 days. The active factor(s) in fibroblast-conditioned medium has a high molecular weight and is heat-stable. We conclude that fibroblastic cells produce a macromolecular factor(s) capable of enhancing the survival of functional islet B cells in tissue culture.

Effect of anti-Ia antibodies, culture, and cyclosporin on prolongation of canine islet allograft survival.

Evidence in rodents suggests that islet pretreatment to reduce islet immunogenicity will also require some form of immunosuppression of the recipient for islet allograft acceptance in highly reactive donor-recipient pairs. We attempted to ascertain whether outbred dogs would also require treatment of both donor islets and the recipient to prolong islet allograft survival. Untreated canine islets are uniformly rejected in 6-10 days in beagles. Tissue culture alone, at 37 degrees C for 7 days, or treatment of freshly prepared islets with anti-Ia monoclonal antibodies (MoAbs) (B1F6 + 7.2) did not prolong canine islet allograft survival. Treatment of culture-maintained canine islets with anti-Ia MoAbs plus complement resulted in prolongation of islet allograft survival for 188 and 368 days in two of seven pancreatectomized nonimmunosuppressed beagles. The administration of low doses of cyclosporin A (CsA) intramuscularly, to recipients of untreated canine islet allografts had no effect on graft survival. By contrast, six of nine CsA-treated recipients of islets that were also treated with anti-Ia MoAbs (B1F6 + 7.2) plus complement showed prolongation of graft survival. Euglycemia was sustained for 19, 34, 89, and 300 days after the CsA was discontinued (day 30) in four of these animals. Two animals had unstable grafts from the beginning that failed 23 and 29 days after transplantation. Our results indicate that simple maneuvers like short-term tissue culture at 37 degrees C and treatment of freshly isolated islets with anti-Ia MoAbs and complement are inadequate to prevent rejection in outbred pancreatectomized beagles.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin and multiplication stimulating activity (an insulin-like growth factor) stimulate islet (beta-cell replication in neonatal rat pancreatic monolayer cultures.

A possible role for insulin in stimulating islet beta-cell replication was examined in neonatal rat pancreatic monolayer cultures. Addition of insulin to serum-free medium increased the mitotic index and stimulated dose-dependent increases in [3H]-thymidine incorporation in nuclei of islet beta-cells in aldehyde-thionine-stained autoradiographs. The effects of insulin were not associated with any significant changes in glucagon or somatostatin levels in the culture media. Multiplication stimulating activity (MSA), an insulin-like growth factor, was about 100-fold more potent than insulin: 3 ng/ml MSA stimulated a half-maximal increase in thymidine labeling of beta-cell (+63%, P less than 0.005), whereas 300 ng/ml insulin was required for a similar effect. The maximal effects of insulin and MSA were similar, and the combination of maximal stimulatory concentrations of MSA (30 ng/ml) and insulin (3000 ng/ml) was not more effective than either substance added alone, suggesting that both peptides act on the same mechanism(s) regulating beta-cell replication. Furthermore, an antibody to the insulin receptor did not prevent the stimulatory effects of either insulin or MSA on thymidine labeling of beta-cells. These results demonstrate that insulin can stimulate islet beta-cell replication directly, possibly through a receptor for MSA or another insulin-like growth factor.

Successful long-term survival of pancreatic islet allografts in spontaneous or pancreatectomy-induced diabetes in dogs. Cyclosporine-induced immune unresponsiveness.

Nineteen pancreatectomized beagles and three spontaneously diabetic dogs were recipients of canine islet allografts from one or more unrelated donors. The islets, enriched 30-45-fold for endocrine cells and contained in a packed cell volume of less than 1.5 ml, were engrafted in the livers of recipient animals. Treatment of diabetic recipients with cyclosporine (CsA) was begun 3-5 days before islet transplantation and the initial dosage was adjusted to attain and maintain CsA serum trough levels between 400 and 600 ng/ml. Five dogs with CsA levels less than this (155 +/- 35 SEM ng/ml) at the time of transplantation promptly rejected their grafts, whereas rejection was encountered in only 1 of 17 diabetic animals in which the initial level exceeded 400 ng/ml. CsA was discontinued 30, 60, or 90 days after continuous therapy in 10 animals. Graft failure was observed 2 mo after stopping CsA in 1 animal and 5 mo in the other. Eight other islet allograft recipients have sustained fasting euglycemia for 7 and 8 mo in 2 and for at least 2 mo in the remainder. These results demonstrate that short-term CsA therapy prolongs survival of islet allografts and induces a state of immune unresponsiveness to islet alloantigens in dogs with experimental and spontaneous diabetes. The findings are unique for a nonrodent mammal and thus hold promise that similar results may be achieved for islet allografts of other mammalian species, including humans.

Experimental diabetes mellitus and age-simulated changes in intact rat dermal collagen.

Alterations in the physical properties of dermal collagen by streptozotocin-induced diabetes mellitus were investigated in young adult Lewis rats by mounting standardized rings of tail skin on an Instron Universal Testing Apparatus and measuring the thermally induced isometric contraction and stress at rupture of the tissue. Diabetes significantly increased the maximum tension (Tmax) of the contraction and raised the temperature for the Tmax while the stress at rupture (TR) was unaffected when compared with values for controls fed ad libitum and controls fed restricted diets for weight loss equivalence. The diabetes-mediated changes in thermal contractility appeared to be independent of the collagen concentration or negative caloric balance and resembled the reported age-related change in rat skin collagen.

Long-term follow-up of canine segmental pancreatic autografts.

The functional and morphologic characteristics of free-draining, pancreatic segmental autografts (FDPS) were studied in 8 beagle dogs that had survived longer than 4 yr. After pancreatectomy, animals received FDPS autografts of the left pancreatic limb, representing approximately one-third of the total pancreas with iliac vessel anastamoses. The grafted recipients were given pancreatic enzyme supplements (Viokase). After transplantation (tx), all 8 animals sustained fasting euglycemia with no evidence of microvascular complications. After 4 yr, IVGTT revealed K-values (%/min) that were not significantly different from age-matched controls (2.9 +/- 0.5 versus 3.7 +/- 0.6, P greater than 0.05). Mean fasting serum insulin levels were significantly greater in the tx animals (49 +/- 5 microU/ml versus 12.2 microU/ml, P less than 0.001), although the incremental response to i.v. glucose (0.5 g/kg) was less than in controls (P less than 0.05). Mean fasting plasma C-peptide levels (0.09 +/- 0.01 pmol/ml versus 0.21 +/- 0.5 pmol/ml) and peak C-peptide responses to i.v. glucose were both significantly less than in controls. Sequential pancreatic biopsies up to 2.5 yr post-tx showed atrophy of the exocrine pancreas with coalescence of islets and mild fibrosis that did not progress with time. Immunoperoxidase stains confirmed the presence of insulin, glucagon, and somatostatin within nests of islet cells. Four years after transplantation of FDPS autografts in pancreatectomized dogs, excellent function is retained. The consequences of peripheral hyperinsulinemia remain to be determined.