Larotrectinib in adult patients with solid tumours: a multi-centre, open-label, phase I dose-escalation study.

BACKGROUND: NTRK1, NTRK2 and NTRK3 gene fusions (NTRK gene fusions) occur in a range of adult cancers. Larotrectinib is a potent and highly selective ATP-competitive inhibitor of TRK kinases and has demonstrated activity in patients with tumours harbouring NTRK gene fusions. PATIENTS AND METHODS: This multi-centre, phase I dose escalation study enrolled adults with metastatic solid tumours, regardless of NTRK gene fusion status. Key inclusion criteria included evaluable and/or measurable disease, Eastern Cooperative Oncology Group performance status 0-2, and adequate organ function. Larotrectinib was administered orally once or twice daily, on a continuous 28-day schedule, in increasing dose levels according to a standard 3 + 3 dose escalation scheme. The primary end point was the safety of larotrectinib, including dose-limiting toxicity. RESULTS: Seventy patients (8 with tumours with NTRK gene fusions; 62 with tumours without a documented NTRK gene fusion) were enrolled to 6 dose cohorts. There were four dose-limiting toxicities; none led to study drug discontinuation. The maximum tolerated dose was not reached. Larotrectinib-related adverse events were predominantly grade 1; none were grade 4 or 5. The most common grade 3 larotrectinib-related adverse event was anaemia [4 (6%) of 70 patients]. A dose of 100 mg twice daily was recommended for phase II studies based on tolerability and antitumour activity. In patients with evaluable TRK fusion cancer, the objective response rate by independent review was 100% (eight of the eight patients). Eight (12%) of the 67 assessable patients overall had an objective response by investigator assessment. Median duration of response was not reached. Larotrectinib had limited activity in tumours with NTRK mutations or amplifications. Pharmacokinetic analysis showed exposure was generally proportional to administered dose. CONCLUSIONS: Larotrectinib was well tolerated, demonstrated activity in all patients with tumours harbouring NTRK gene fusions, and represents a new treatment option for such patients. CLINCALTRIALS.GOV NUMBER: NCT02122913.

Selective RET kinase inhibition for patients with RET-altered cancers.

Background: Alterations involving the RET kinase are implicated in the pathogenesis of lung, thyroid and other cancers. However, the clinical activity of multikinase inhibitors (MKIs) with anti-RET activity in RET-altered patients appears limited, calling into question the therapeutic potential of targeting RET. LOXO-292 is a selective RET inhibitor designed to inhibit diverse RET fusions, activating mutations and acquired resistance mutations. Patients and methods: Potent anti-RET activity, high selectivity, and central nervous system coverage were confirmed preclinically using a variety of in vitro and in vivo RET-dependent tumor models. Due to clinical urgency, two patients with RET-altered, MKI-resistant cancers were treated with LOXO-292, utilizing rapid dose-titration guided by real-time pharmacokinetic assessments to achieve meaningful clinical exposures safely and rapidly. Results: LOXO-292 demonstrated potent and selective anti-RET activity preclinically against human cancer cell lines harboring endogenous RET gene alterations; cells engineered to express a KIF5B-RET fusion protein -/+ the RET V804M gatekeeper resistance mutation or the common RET activating mutation M918T; and RET-altered human cancer cell line and patient-derived xenografts, including a patient-derived RET fusion-positive xenograft injected orthotopically into the brain. A patient with RET M918T-mutant medullary thyroid cancer metastatic to the liver and an acquired RET V804M gatekeeper resistance mutation, previously treated with six MKI regimens, experienced rapid reductions in tumor calcitonin, CEA and cell-free DNA, resolution of painful hepatomegaly and tumor-related diarrhea and a confirmed tumor response. A second patient with KIF5B-RET fusion-positive lung cancer, acquired resistance to alectinib and symptomatic brain metastases experienced a dramatic response in the brain, and her symptoms resolved. Conclusions: These results provide proof-of-concept of the clinical actionability of RET alterations, and identify selective RET inhibition by LOXO-292 as a promising treatment in heavily pretreated, multikinase inhibitor-experienced patients with diverse RET-altered tumors.

TNF-related apoptosis-inducing ligand (TRAIL) protects against diabetes and atherosclerosis in Apoe ⁻/⁻ mice.

AIMS/HYPOTHESIS: TNF-related apoptosis-inducing ligand (TRAIL) is implicated in the regulation of diabetes and is reduced in patients with cardiovascular disease. Although TRAIL receptors are widespread, and TRAIL can promote cell proliferation and apoptosis, it is not known how TRAIL might protect against diabetes and atherosclerosis. METHODS: We examined the development of atherosclerosis and diabetes in Apoe (-/-), Trail (also known as Tnfsf10)( -/- ) Apoe ( -/- ) and Trail ( -/- ) mice that were fed a high-fat diet. Plasma cholesterol, triacylglycerol, glucose and insulin, as well as changes in various metabolic enzymes and regulators were assessed. Glucose and insulin tolerance tests were performed. Pancreatic islets were examined for insulin and beta cell dysfunction (apoptosis and macrophage infiltration). RESULTS: Compared with Apoe ( -/- ) mice, Trail ( -/- ) Apoe ( -/- ) and Trail ( -/- ) mice exhibited several features of diabetes, including increased weight, hyperglycaemia, reduced plasma insulin, impaired glucose tolerance, beta cell dysfunction, reduced islet insulin, macrophage infiltration and increased apoptosis. Trail ( -/- ) Apoe ( -/- ) mice had increased plasma cholesterol, triacylglycerol, and VLDL- and LDL-cholesterol, and increased expression of genes involved in cholesterol synthesis and lipogenesis. Trail ( -/- ) Apoe ( -/- ) mice also had increased atherosclerosis, with several features of plaque instability. CONCLUSIONS/INTERPRETATION: We show for the first time that TRAIL deficiency promotes numerous features of diabetes that are typical of human disease, and are associated with reduced insulin and pancreatic inflammation/apoptosis. TRAIL also regulates cholesterol and triacylglycerol homeostasis in Apoe ( -/- ) mice by increasing the expression of genes involved in (1) cholesterol synthesis and absorption, and (2) triacylglycerol production.

Beneficial effects of desferrioxamine on encapsulated human islets--in vitro and in vivo study.

As many as 2000 IEQs (islet equivalent) of encapsulated human islets are required to normalize glucose levels in diabetic mice. To reduce this number, encapsulated islets were exposed to 100 µM desferrioxamine (DFO) prior to transplantation. Cell viability, glucose-induced insulin secretion, VEGF (Vascular endothelial growth factor), HIF-1α (Hypoxia inducible factor-1 alpha), caspase-3 and caspase-8 levels were assessed after exposure to DFO for 12, 24 or 72 h. Subsequently, 1000, 750 or 500 encapsulated IEQs were infused into peritoneal cavity of diabetic mice after 24 h exposure to DFO. Neither viability nor function in vitro was affected by DFO, and levels of caspase-3 and caspase-8 were unchanged. DFO significantly enhanced VEGF secretion by 1.6- and 2.5-fold at 24 and 72 h, respectively, with a concomitant increase in HIF-1α levels. Euglycemia was achieved in 100% mice receiving 1000 preconditioned IEQs, as compared to only 36% receiving unconditioned IEQs (p < 0.001). Similarly, with 750 IEQ, euglycemia was achieved in 50% mice receiving preconditioned islets as compared to 10% receiving unconditioned islets (p = 0.049). Mice receiving preconditioned islets had lower glucose levels than those receiving unconditioned islets. In summary, DFO treatment enhances HIF-1α and VEGF expression in encapsulated human islets and improves their ability to function when transplanted.

Mapping dispersed repetitive loci using semi-specific PCR cloning and somatic cell hybrid mapping.

A simple and effective method based upon semi-specific PCR followed by cloning has been developed. Chromosomal mapping of the generated fragment on a somatic cell hybrid panel identifies the chromosomal position, and yields a unique sequence tag for the site. Using this method, the chromosomal location of one porcine endogenous retrovirus (PERV) was determined. The porcine genomic sequences were first amplified by PCR using a PERV-specific primer and a porcine short interspersed nuclear element (SINE)-specific primer. PCR products were cloned, and those sequences that contained PERV plus flanking regions were selected using a second round of PCR and cloning. Sequences flanking the PERV were determined and a PERV-B was physically mapped on porcine chromosome 17 using a somatic hybrid panel. The general utility of the method was subsequently demonstrated by locating PERVs in the genome of PERV infected human 293 cells. This method obviates the need for individual library construction or linker/adaptor ligation, and can be used to quickly locate individual sites of moderately repeated, dispersed DNA sequences in any genome.

Glucose regulates the maximal velocities of glucokinase and glucose utilization in the immature fetal rat pancreatic islet.

The cause of the poor secretion of insulin in response to glucose by the beta-cell in the fetal rat pancreas is thought to be immaturity of the metabolism of glucose. Glucokinase (GK), a key enzyme in glycolysis, is the glucose sensor that maintains glucose homeostasis in the adult beta-cell; its role in the fetal beta-cell has not been determined. The aim of this study was to examine whether GK was functional in phosphorylation of glucose in the fetal islet, and if so, to determine what factors regulated this activity. Similar Km values were found in both fetal and adult islets: 7.4 vs. 7.7 mmol/l. The maximal GK velocity (Vmax) of the fetal islet and the contribution of GK to total glucose phosphorylation were also not significantly different from their adult counterparts. Western blot analysis of protein extracts from fetal and adult islets confirmed the presence of GK at 52 kDa. To determine if glucose had any effect on the Vmax of GK, islets were cultured for 7 days in medium containing low (1.4 or 2.8 mmol/l), normal (5.6 mmol/l), or high (11.2 or 16.8 mmol/l) concentrations of glucose. The maximal GK velocity increased linearly with increasing concentrations of glucose (r = 0.93; P < 0.01). To determine whether it was possible to up- and down-regulate Vmax of GK, islets were cultured in either a low (1.4 mmol/l) or high (30 mmol/l) concentration of glucose for 7 days and then switched to the opposite concentration for a further 3 days. The Vmax of GK in the fetal islet was upregulated 3.8-fold when the glucose concentration was raised. Conversely, the Vmax was downregulated 3.6-fold when the glucose concentration was lowered. The same phenomenon was also observed in the adult islet. These data indicate that GK is the glucose sensor for the fetal rat islet, just as it is for the adult islet. Since glucose did not cause insulin secretion from the fetal islet, it was important to examine whether this substrate had any effect on its own metabolism. Glucose utilization was estimated, and its Vmax was found to increase linearly with increasing concentrations of glucose (r = 0.96; P < 0.01). We conclude that the inability of the fetal rat beta-cell to secrete insulin in response to glucose cannot be explained by immaturity of GK or the glycolytic pathway.

Growth and hormonal content of human fetal pancreas passaged in athymic mice.

While the usefulness of the human fetal pancreas transplanted into diabetic humans has yet to be realized, its transplantation into nude mice has revealed some of its potential. In this animal the organ grows in size, during which time differentiation of its endocrine component and maturation of the insulinogenic response to glucose occurs. The results reported in this article expand these results by providing data on the weight and both the insulin and glucagon content of these passaged organs. Human fetal pancreata of gestational age 14-19 wk were implanted subcutaneously (s.c.) in nude mice and maintained in vivo for 5-54 wk (absolute age 19-68 wk). The implants were then removed and their insulin and glucagon content determined. Both the weight of these implants and their insulin content were positively correlated with the absolute age of the tissue (weight: r = 0.61, P less than 0.001; insulin content: r = 0.62, P = 0.0003). The glucagon content bore no relationship to the age. The maximum level of insulin extracted from 30 passaged human fetal pancreata was 0.8 U, a level far below the daily insulin requirements in adult humans. It is suggested that an explanation for this is the site of transplantation used. To compare the s.c. site with the renal subcapsular space, the explants of four fetal pancreata were evenly divided between these two sites. After 11-13 wk, the implants were removed. Those beneath the renal capsule were larger and contained a greater amount of insulin and glucagon than those transplanted s.c.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of endocrine-rich monolayers of human fetal pancreas that display reduced immunogenicity.

Fibroblast-free insulin-secreting monolayers of human fetal pancreas (14-20 wk of gestation) were formed by plating isletlike cell clusters (ICCs) obtained from partially digested pancreases on plates coated with bovine corneal matrix. Human fetal pancreatic cells, freshly digested with collagen, displayed a 17-fold response to human peripheral blood lymphocytes (HPBLs) in mixed-lymphocyte culture. After 14 days in culture, monolayers derived from ICCs exhibited a smaller, twofold response to HPBLs. By comparison, in monolayers produced from single-cell suspensions, fibroblast overgrowth remained a problem. The endocrine component of the monolayers was 65 +/- 13 and 43 +/- 8%, respectively, with the number of beta-cells being 51 and 9%. Cells from both monolayers displayed increased insulin release when exposed to 10 mM theophylline, 10 mM Ca2+, and 0.6-1.3 microM 12-O-tetradecanoylphorbol-13-acetate but not to 20 mM glucose. Monolayers derived from ICCs synthesized DNA, proinsulin, and protein. This study showed that it is possible to establish an endocrine-rich monolayer of human fetal pancreas that has greatly reduced immunogenicity. The existence of residual activity to HPBLs suggests some additional form of immunosuppression is required to prevent rejection of this tissue when grafted into diabetic patients. Subculturing and cryopreservation may also be needed to achieve adequate numbers of beta-cells for clinical transplantation.

Insulin secretagogues, but not glucose, stimulate an increase in [Ca2+]i in the fetal rat beta-cell.

Fetal pancreatic islets release insulin poorly in response to glucose; however, the cellular mechanism for this is controversial. By using fura 2 to measure changes in the cytoplasmic free Ca2+ concentration ([Ca2+]i) in beta-cells, we have examined islets from fetal, neonatal, and adult rats to determine the ability of glucose and other secretagogues to cause an increase in [Ca2+]i. The effects of glucose (20 mmol/l), glyceraldehyde (20 mmol/l), leucine (20 mmol/l), arginine (20 mmol/l), and the channel effectors glipizide (50 mumol/l), BAY K8644 (2 mumol/l), diazoxide (300 mumol/l), and verapamil (20 mumol/l) on changes in [Ca2+]i were studied. In both the fetal and the mature islet, glyceraldehyde, leucine, arginine, glipizide, and BAY K8644 caused an increase in [Ca2+]i. In mature islets, glucose also increased [Ca2+]i; however, in the fetal islet, glucose had no effect on [Ca2+]i. The stimulus-induced increases in [Ca2+]i in fetal and adult islets were both significantly inhibited by the addition of either diazoxide or verapamil. Similar results were obtained when insulin secretion was measured. Our data show that various secretagogues are able to stimulate fetal islets and cause an increase in [Ca2+]i. Glucose, however, fails to cause an increase in [Ca2+]i in the fetal islet. Hence, the immature insulin secretory response to glucose by the fetal islet is due to the inability of the fetal beta-cell to translate glucose stimulation into the increase in [Ca2+]i required for exocytosis of the insulin granule.

Histologic differentiation of human fetal pancreatic explants transplanted into nude mice.

The transplantation of human fetal pancreas has been suggested as a means of treatment of insulin-dependent diabetes in man. We have obtained human fetal pancreata during the second trimester of pregnancy and transplanted 1-mm3 explants subcutaneously (s.c.) into both diabetic and nondiabetic nude mice, some of the tissue being cultured in vitro before implantation. These implants coalesced and grew. They were removed at intervals up to 37 wk later and showed selective differentiation of endocrine tissue that normally occurs in the fetus and neonate, with formation of bipolar, mantle, and mature islets. There was growth of this endocrine tissue with significantly more islets than in the freshly stained fetal pancreas assuming an average dimension larger than 150 micron, which is the reported mean diameter of a neonatal islet. Duct and fibrous tissue remained viable, but there was no definitive acinar tissue seen. The pancreata uncultured before implantation reached a larger size than that attained by those implants cultured before being transplanted, the difference probably being the amount of ductular and mesenchymal tissue still present. Of those glands cultured before transplantation, the longer the period of culture, the smaller the size the implants reached. Culture beyond 3 wk in vitro made it difficult to macroscopically locate the implant. These data show that, in human fetal pancreas removed from its usual environment, both selective differentiation of the endocrine component and growth of the islets can occur.

Glucagon-like peptide-1 enhances production of insulin in insulin-producing cells derived from mouse embryonic stem cells.

Embryonic stem cells (ESCs) can be differentiated into insulin-producing cells by a five-stage procedure involving altering culture conditions and addition of nicotinamide. The amounts of insulin in these cells are lower than those found in pancreatic beta cells. Glucagon-like peptide-1 (GLP-1) induces the differentiation of beta cells from ductal progenitor cells. We examined the possibility of GLP-1, and its long-acting agonist exendin-4, enhancing the differentiation of insulin-producing cells from mouse ESCs (mESCs). A five-stage culturing strategy starting with embryoid bodies (EBs) was used in this study. mRNA for pancreatic duodenal homeobox gene 1 (PDX-1) and neurogenic differentiation (NeuroD) was detected from stage 1, hepatocyte nuclear factor 3 beta (HNF3beta) and insulin 2 from stage 2, Ngn3 and glucose transporter 2 (GLUT2) from stage 3, and insulin 1 and other beta-cell markers, at stages 4-5. Cells at stage 5 secreted C-peptide, being 0.68 +/- 0.01 pmol/10(6) cells per 2 days, and had an immunoreactive insulin content of 13.5 +/- 0.7 pmol/10(6) cells. Addition of GLP-1 (100 nM) and nicotinamide (10 mM) at stage 5 resulted in a 50% and 48% increase in insulin content and C-peptide secretion respectively compared with nicotinamide alone. Glucose-induced insulin secretion was enhanced 4-fold by addition of both growth factors. The GLP-1 receptor was present at all five stages of the culture. Addition of exendin-4 to cells at stage 2 resulted in a 4.9-fold increase in expression of the gene for insulin 1 and a 2-fold increase in insulin content compared with the effect of nicotinamide alone at stage 5. It is concluded that both GLP-1 and exendin-4 enhance the level of expression of insulin in glucose-responsive insulin-producing cells derived from the R1 mESC line.

The role of calcium in insulin release from the human fetal pancreas.

Previous experiments have established that the human fetal pancreas is relatively unresponsive to glucose as regards insulin release, but will secrete this hormone when exposed to agents which increase levels of cAMP or which activate protein kinase C. The current experiments were designed to establish which role another major stimulus, calcium, had in the release of insulin from this organ. For this purpose, cultured explants of human fetal pancreas were exposed to stimuli either in static or dynamic stimulation. The data show that insulin release is enhanced in the presence of 10 mM Ca2+, as well as the calcium ionophores A23187 and ionomycin, the latter agent being effective only if extracellular Ca2+ was present. A biphasic response was seen for Ca2+ but only a second phase response for A23187. Voltage-dependent calcium channels were shown to be present by the ability of the calcium channel blocker, verapamil, to inhibit insulin release caused by an agent that depolarizes membranes, potassium. The essential role of extracellular calcium in the insulinogenic effect of agents which increase cAMP levels--theophylline--and which activate protein kinase C--12-O-tetradecanoylphorbol-13-acetate--was demonstrated by showing (a) partial inhibition of insulin secretion by calcium channel blockers, (b) no enhancement of insulin release in the absence of extracellular calcium and (c) greater enhancement of insulin release in the presence of the calcium channel activator BAY-K-8644, which caused no stimulation by itself. These data put into better perspective our understanding of the mechanisms involved in insulin release from the human fetal pancreas.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression and regulation of glucokinase in rat islet beta- and alpha-cells during development.

Glucokinase (GK) is the rate-limiting enzyme in the glycolytic pathway of the beta-cell and, even in the rat fetus at 22-days gestation, immediately before birth, acts as a sensor of glucose influencing the rate of glucose utilization. However, when GK first appears in islets during beta-cell development is unknown. Whether GK is expressed in fetal glucagon-producing cells is also unknown. To determine this information, fetal rat islets were examined at 16-, 18-, and 22-days gestation. GK was identified immunocytochemically in both beta- and alpha-cells at all these ages, with the number of GK immunoreactive cells positively correlated to the fetal age from 16-22 days. Western blot analysis of islet protein extracts demonstrated the presence of GK, at 52 kDa, at 16 days and thereafter. To determine whether glucose had any effect on regulation of GK biosynthesis, fetal islets were cultured in medium containing a wide range of concentrations of glucose for 7 days. The amount of GK protein was significantly decreased in low concentrations of glucose and augmented at high concentrations. In conclusion, GK was expressed in both beta- and alpha-cells in fetal rat islets during development. GK is an integral part of the function of both of these cells at all stages in the development of the fetal islet.

Secretion of pancreatic icosapeptide from porcine pancreas.

The pancreatic polypeptide cell, the only mature endocrine cell in the fetal pig pancreas, produces equimolar amounts of two peptides, pancreatic polypeptide and pancreatic icosapeptide, from the same precursor. The amino acid sequence of pancreatic polypeptide is more homogeneous among species, whereas pancreatic icosapeptide is heterogeneous. We determined the 19-amino acid sequence of porcine pancreatic icosapeptide, which is markedly different from that of known sequences (e.g. 47% homology with human). We developed an ELISA that can measure porcine pancreatic icosapeptide levels in the range of 7.2-480 pmol/liter. Actual levels of pancreatic icosapeptide in pig sera were 9.6-25 pmol/liter. The assay requires relatively small amounts of nonextracted samples, and human and mouse sera do not cross-react. Levels of pancreatic icosapeptide rose in response to hypoglycemia in pigs and to carbachol in fetal porcine pancreatic cells in vitro. When fetal porcine pancreatic tissue was transplanted into nonobese diabetic-severe combined immune deficiency mice, porcine pancreatic icosapeptide (but not C peptide) was detectable in mouse sera for up to 3 wk after transplantation, with levels highest on d 4. Porcine pancreatic icosapeptide and insulin were detectable in grafts removed from the mice. Therefore, porcine pancreatic icosapeptide may be used as a marker of the viability of xenotransplanted fetal pig pancreatic tissue in the immediate posttransplant period.

Expression of glucokinase in glucose-unresponsive human fetal pancreatic islet-like cell clusters.

Glucokinase (GK) is the glucose sensor in the adult beta-cell, resulting in fuel for insulin synthesis and secretion. Defects in this enzyme in the beta-cell are responsible for the genetic disorder maturity-onset diabetes of the young, with the beta-cell being unable to secrete insulin appropriately when challenged with glucose. The human fetal beta-cell is also unable to secrete insulin when exposed to glucose, but whether GK is present and functional in this developing cell is unknown. To determine the expression of GK in human fetal pancreatic tissue, cytosolic protein was extracted from human fetal islet-like cell clusters (ICCs) at 17-19 weeks gestation and examined for protein content and enzyme activity. On Western blots, a single band corresponding to GK was seen at 52 kDa, and this was similar to that obtained from human adult islets. The maximal velocity (Vmax) of GK was less in fetal ICCs than that in adult islets (8.7 vs. 20.7 nmol/mg protein x h); similar K(m) values were found in both ICCs and islets. No attempt was made to determine which cells in an ICC contained GK. Glucose utilization was determined radiometrically; the Vmax of the high K(m) component was less in ICCs than in islets (31.3 pmol/ICC x h vs. 101.4 pmol/islet.h). Culture of ICCs for 3-7 days in medium containing 11.2 mmol/L glucose resulted in a 3.7-fold increase in the Vmax of GK and a 1.8-fold increase in glucose utilization. These enhanced activities of glucose phosphorylation and glycolysis, however, did not lead to the beta-cell being able to secrete insulin when exposed to glucose. In conclusion, glucokinase is present and functional in human fetal ICCs, but the inability of the human fetal beta-cell to secrete insulin in response to an acute glucose challenge is not due to immaturity of this enzyme.

Role of tumor necrosis factor-alpha and interferon-gamma as growth factors to the human fetal beta-cell.

The effects of the cytokines tumor necrosis factor-alpha and interferon-gamma on the adult beta-cell have been well described: a reduction of insulin secretion and content and death of the cell. For this reason and because these cytokines may be released from activated lymphocytes and macrophages that infiltrate islets in insulin-dependent diabetes, they have been implicated in the pathophysiology of this form of diabetes. As to whether the human fetal beta-cell, which differs from the adult beta-cell in not releasing insulin in response to the nutrient glucose and not being adversely affected by the toxin streptozotocin, is similarly affected is unknown. To examine this question we cultured monolayers of a single cell suspension of human fetal pancreas in the presence or absence of 1000 U/mL of these cytokines for 7 days. Chronic insulin release was enhanced for the first 2 days of culture, but unchanged thereafter. Acute insulin release in response to the secretagogue theophylline (10 mM) was enhanced on day 7, but not earlier. There was an increase in the insulin content of the cells by the fourth day, probably due to an increase in the number of beta-cells present (45 +/- 5% vs. 22 +/- 3%). Microscopically, non-beta-cells also seemed to increase in number; there was an increase in both DNA and cell number by the seventh day. In contrast to these beneficial effects on the human fetal beta-cell, treatment of adult rat insulinoma cells, represented by RIN-m5F cells, resulted in inhibition of insulin secretion during the first day of culture and subsequent death of 86% of the cells by the sixth day of culture. It is hypothesized that the functional immaturity and lack of normal (adult) metabolic activity of the human fetal beta-cell somehow confers protection on these cells from the cytotoxic effects of tumor necrosis factor-alpha and interferon-gamma. Indeed, our findings suggest that these cytokines may be trophic for the developing beta-cell.

Interferon-gamma induces the expression of HLA-A,B,C but not HLA-DR on human pancreatic beta-cells.

We examined the effect of interferon-gamma (IFN gamma) on expression of the major histocompatibility proteins on cultured human islet cells isolated from adult and fetal pancreas and from an insulinoma. While the pancreatic beta-cells from different sources varied in their responses to IFN gamma, in all instances the expression of HLA-A,B,C protein was increased. Pancreatic beta-cells did not express HLA-DR protein, before or after culture of the islets in IFN gamma, although HLA-DR protein expression was induced on some non-beta-cells. These findings are at variance with those reported with thyroid follicular cells, in which IFN gamma induced expression of HLA-DR. We, therefore, conclude that the interaction between the immune and the endocrine systems may be endocrine cell specific. The up-regulation of HLA-A,B,C protein on beta-cells by IFN gamma provides a mechanism for enhanced targetting to the beta-cells of autoreactive cytotoxic T-lymphocytes and, hence, for amplifying beta-cell destruction.

Reversal of diabetes by human fetal pancreas. Optimization of requirements in the hyperglycemic nude mouse.

It has been established previously that the grafted human fetal pancreas takes 2-3 months to reverse diabetes in a recipient hyperglycemic immunoincompetent mouse. The factors responsible for this have never been clarified. It was the aim of these experiments to investigate the effect of grafting various amounts of human fetal pancreas of gestational age 14-20 weeks into these animals, to determine if the time required for normalization of blood glucose levels could be altered. The amount of tissue grafted ranged from 2.6-49 mg (median 12.6 mg), which is the equivalent of 8-100% (median 31%) of a complete pancreas. The 25 diabetic nude mice became normoglycemic 66 days (range 28-147) after administration of streptozotocin, with normal or better-than-normal responses in an oral glucose tolerance test, and detectable human C-peptide levels, which fluctuated in parallel with the glucose levels. The minimal time required for correction of diabetes was 28 days, this occurring when 49 mg of tissue was grafted. Blood glucose levels rose from 2.9 +/- 0.3 mmol/L to 21.3 +/- 1.9 mmol/L after removal of the implants. Analysis of the results by stepwise multiple-linear-regression analysis revealed that the time required for normalization of blood glucose levels was dependent on three unrelated factors: the amount of tissue grafted (P = 0.002); the age of the fetal pancreas, as determined by the heel-toe length (P = 0.042); and the rate of growth of the implant (P = 0.031). These data demonstrate that it is advantageous to graft as much human fetal pancreas as possible, ideally from fetuses as old as possible, in order to achieve the most rapid reversal of diabetes in athymic mice. The clinical relevance of this information remains to be established.

Regulation of blood glucose to human levels by human fetal pancreatic xenografts.

Previous experiments xenografting human fetal pancreas into athymic mice made diabetic with streptozotocin have demonstrated that normoglycemia can be achieved 1-3 months after implantation, but that the blood glucose levels obtained were significantly lower than those of control mice. These lower levels could be due either to genetic regulation by the grafted human beta cell--human neonatal blood glucose levels are lower than those of athymic mice--or to excess release of insulin from the increasing number of beta cells in the implant. In order to address the latter possibility, human fetal pancreas was grafted beneath the renal capsule of athymic mice, the pancreas of which was intact, and they and their ungrafted litter mates monitored during their life span--71 +/- 17 weeks after surgery for the 4 successfully grafted mice. The random blood glucose level in all mice was the same initially and remained so until a significant lowering 20 weeks later--2.0 +/- 0.2 vs. 3.4 +/- 0.4; this difference was maintained thereafter. Data obtained from the oral glucose tolerance tests conducted at 8 weekly intervals paralleled these results. Human C-peptide was detectable in blood by 26 weeks, with a rise demonstrable during the oral glucose tolerance test. Peak levels did not change during the ensuing year. At no stage did any of the mice develop clinical hypoglycemia, despite an annual 23 +/- 6-fold increase in size of the graft and the presence of 0.3-9.2 U insulin therein. The insulin content of the mouse pancreas (0.08 +/- 0.03 U) was unaffected by the presence of the human fetal beta cells. These data show that the xenografted human fetal pancreas grows and releases insulin in a controlled but not excessive manner, with the blood glucose levels probably being determined by genetic information stored in the grafted beta cell.

Effect of interleukin-10 on human anti-porcine xenogeneic cellular response in vitro.

BACKGROUND: Pigs are being used as an alternative source of tissues for humans and we are interested in the xenotransplantation of fetal pig islet-like cell clusters (ICC) into type 1 diabetic patients. Interleukin-(IL) 10 is a Th2 cytokine with immunosuppressive properties that down-regulate the cell-mediated response. In this study, we evaluated the effects of recombinant human IL-10 on human anti-pig xenogeneic cellular response in mixed lymphocyte culture (MLC) and in mixed islet lymphocyte culture (MILC). METHODS: Human peripheral blood mononuclear cells as responder cells were cultured in one-way MLC with pig and human peripheral blood mononuclear cells as stimulant cells in xeno and allo-MLC, respectively, and also with fetal pig ICCs in MILC. IL-10 was added at the time of culture. RESULTS: The addition of IL-10 significantly inhibited the xeno-MLC (human anti-pig) in a dose-dependent manner, the percentage inhibition being 36, 60, and 73% at 1, 10, and 50 ng/ml, respectively. Inhibition in xeno-MLC was significantly lower than that of the allo-MLC (human anti-human) at all concentrations used, the percentage inhibition of the latter being 58, 84, and 92% at 1, 10, and 50 ng/ml, respectively. Further, the addition of IL-10 also significantly inhibited the proliferation of human peripheral blood mononuclear cells when they were cocultured with fetal pig ICCs, the inhibition being 59, 72, and 80% at 1, 10, and 50 ng/ml, respectively. IL-10 was not toxic to ICCs as determined by 3H-thymidine incorporation over 5 days culture. Preincubation of IL-10 with the pig stimulant cells or the human responder cells did not confer additional benefit in the inhibition of xeno-MLC. IL-10 needs to be present at the start or at an early stage (within 4 hr) in the xeno-MLC because if the addition of IL-10 was delayed by 4 hr, the effect was lost. Next, the production of cytokines was examined in MLC and MILC. In xeno-MLC, levels (pg/ml) of tumor necrosis factor-alpha (TNF-alpha) (163+/-17), interferon-gamma (IFN-gamma) (278+/-60), IL-5 (24+/-10), IL-6 (2959+/-923), and IL-10 (17+/-2) were produced in greater amounts than autologous controls (P<0.05). The levels of TNF-alpha, IFN-gamma, IL-6, and IL-10 but not IL-5 were significantly (P<0.05) lower in xeno-MLC than those produced in allo-MLC. All of these cytokines were also produced in MILC when human peripheral blood mononuclear cells (PBMC) were cocultured with ICCs, levels (pg/ml) being TNF-alpha (308+/-47), IFN-gamma (93+/-17), IL-5 (6.2+/-3), IL-6 (5649+/-421), and IL-10 (122+/-18). No detectable levels of IL-2 and IL-4 were produced in the MLC and in MILC. Addition of IL-10 significantly inhibited the production of TNF-alpha, IFN-gamma, IL-5, and IL-6 by 76, 96, 100, and 93%, respectively, in xeno-MLC. Addition of IL-10 also significantly (P<0.05) inhibited the production of TNF-alpha, IFN-gamma, IL-5, and IL-6 by 88, 91, 100, and 96%, respectively, in MILC. Exogenous addition of IL-2 was partially able to reverse the effect of IL-10 although addition of TNF-alpha had no effect on xeno and allo-MLC. Synergism was seen between IL-10 and cyclosporine in the inhibition of xeno and allo-MLC. CONCLUSION: Taken together, the results demonstrated that IL-10 has an immunomodulatory role to play in the inhibition of cellular immune responses associated with the xenotransplantation of fetal pig ICCs.