The safe treatment, monitoring and management of severe traumatic brain injury patients in a monoplace chamber.

This report describes how 27 patients with severe traumatic brain injury were safely treated, monitored and managed in a monoplace chamber that was compressed with air to 1.5 atmospheres absolute (152 kPa). A total of 75 hyperbaric oxygen treatments were delivered using the monoplace system described, with all patients receiving 100% oxygen via mechanical ventilation. Specific pieces of equipment, components and features were selected, and modifications were interfaced to safely and effectively treat these critically ill patients in a monoplace chamber. Patient monitoring included cardiovascular and ventilatory parameters as well as intracranial pressure, brain tissue oxygen levels, brain temperature and cerebral microdialysis. The chamber and all the supporting equipment for ventilating, monitoring and managing the patient functioned well.

Prolonged xenograft survival of islets infected with small doses of adenovirus expressing CTLA4Ig.

BACKGROUND: Systemic administration of the inhibitor of costimulation, CTLA4Ig, has been shown to prolong islet graft survival. The purpose of this study was to compare local and systemic expression of murine CTLA4Ig in transplants of rat islets into mice. METHODS: Murine CTLA4Ig was made by joining two polymerase chain reaction products, the extracellular portion of CTLA4 and the Fc portion of IgG2a. Recombinant adenovirus expressing CTLA4Ig (AdCTLA4Ig) was generated using the strategy of Cre-lox recombination. Isolated rat islets infected with AdCTLA4Ig at multiplicities of infection (MOIs) ranging from 0.1 to 10 were transplanted into streptozocin diabetic male B6AF1 mice. Control islets were mock infected or infected with AdLacZ or AdsIg, a recombinant adenovirus expressing only the Fc portion of IgG2a. Also, AdCTLA4Ig and control viruses were injected intramuscularly into mouse transplant recipients at the time of islet transplantation to provide CTLA4Ig systemically. RESULTS: Control islets transplanted into diabetic mice were rejected in 13-17 days. Islets infected with AdCTLA4Ig had dose-dependent prolongation of graft survival. Prolonged survival was even found with very low MOIs of 0.1 and 0.5, with survivals of 24+/-4.2 and 25+/-2.2 days, respectively. Survival with an MOI of 10 was 39+/-8.7 days. With intramuscular injection, no prolongation was found at the lowest relative MOIs of 0.2 and 1, but there was dose-dependent prolongation of graft survival with larger doses. At the highest relative MOI of 400, survival was prolonged to 58+/-10 days. CONCLUSIONS: Rat islets infected with AdCTLA4Ig transplanted into mice had prolonged graft survival. Prolonged survival with MOIs as low as 0.1 and 0.5 indicates that only a minority of islet cells need to express CTLA4Ig to exert an effect. Moreover, the results suggest that the improved islet graft survival is due to a local influence of CTLA4Ig.

Reversal of hyperglycemia in mice after subcutaneous transplantation of macroencapsulated islets.

BACKGROUND: Macroencapsulated islets can reverse hyperglycemia in diabetic animals when transplanted i.p. or into the fat pad. The s.c. space is an attractive site for such transplantation because macrocapsules can be implanted with local anesthesia and be easily removed or reloaded with fresh islets. METHODS: Immunoprotective 20 microl ported devices were transplanted under the skin of Streptozocin-diabetic nude mice. Devices were loaded with 1200 rat islets in culture medium or in alginate. Empty devices were implanted for 2 weeks and then loaded with islets. Normal mice and mice with islets transplanted under the renal capsule or under the skin were used as controls. Seven weeks after transplantation, an intraperitoneal glucose tolerance test (IPGTT) was performed, followed by implant removal. RESULTS: Three weeks after transplantation, normal blood glucose levels were observed in all animals. Compared with those of normal controls, IPGTTs showed accelerated blood glucose clearance in mice transplanted with islets either within devices or beneath the kidney capsule. Fasted transplanted mice were hypoglycemic before glucose injection and 2 hr later. After removal of the implants, all recipient mice returned to hyperglycemia. Histological evaluation revealed viable islet cells and a network of close vascular structures outside the devices. CONCLUSIONS: Macroencapsulated islets transplanted into the s.c. space were able to survive and regulate blood glucose levels in mice. The observed differences in glucose metabolism between normal and transplanted mice may be attributed to the site of transplantation and to the use of rat islets, which have a different set point for glucose induced insulin release.

Differentiation and expansion of beta cell mass in porcine neonatal pancreatic cell clusters transplanted into nude mice.

Neonatal porcine pancreas has considerable capacity for growth and differentiation, making it an attractive potential source of islet tissue for xenotransplantation. Pancreases from 1-3-day-old newborn pigs were digested with collagenase and cultured for 8 days. The resulting cellular aggregates are called porcine neonatal pancreatic cell clusters (NPCCs). The mean yield of NPCCs from a newborn pig was 28,200 +/- 1700 islet equivalents. Cytokeratin 7 (CK7) was used as a marker for the immunostaining of pancreatic duct cells. In neonatal pancreas, 18% of the insulin-positive cells co-stained for CK7, thus being protodifferentiated. NPCCs also contained protodifferentiated cells; insulin/PP and insulin/somatostatin co-stained cells were more common than insulin/glucagon cells. Between 1 and 8 days of culture, the DNA content of the NPCCs fell to 16% and the insulin content to 33% of the starting value, mainly due to the preferential loss of exocrine cells. Transplantation of 2000 or 4000 NPCCs into diabetic nude mice typically normalized glucose values in 10-20 weeks. Mice with successful grafts had lower fasting blood glucose levels than normal mice and accelerated glucose clearance after an i.p. glucose load. The starting NPCCs consisted of 17% insulin-staining cells, but the grafts of mice with reversed diabetes consisted of 94% beta cells, with some co-stained for CK7, indicating that the grafts still contained immature cells. The mass of insulin-producing cells rose from 0.22 +/- 0.08 mg 1 week after transplantation to 4.34 +/- 0.27 mg in mice sacrificed at 27-35 weeks. In summary, NPCCs contain mostly islet precursor cells, which when transplanted into nude mice undergo striking differentiation and beta cell expansion.

Hypoxia induces vascular endothelial growth factor gene and protein expression in cultured rat islet cells.

The formation of new microvasculature by capillary sprouting at the site of islet transplantation is crucial for the long-term survival and function of the graft. Vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen with potent angiogenic and vascular permeability-inducing properties, may be a key factor in modulating the revascularization of islets after transplantation. In this study, we examined the gene expression of VEGF mRNA in three tumor cell lines and in isolated whole and dispersed rat islets in vitro by Northern blot hybridization in normoxic (5% CO2, 95% humidified air) and hypoxic (1% O2, 5% CO2, 94% N2) culture conditions. Increased expression of VEGF mRNA was observed in beta-TC3, RAW 264.7, and IC-21 tumor cell lines when subjected to hypoxia. With isolated whole islets in normoxic culture, a threefold increase in VEGF mRNA (P < 0.001) was seen at 48 h as compared with freshly isolated islets. This response was similar to the 3.8-fold increase observed with islets subjected to hypoxia. Dispersed rat islet cell clusters cultured on Matrigel for 24 h under hypoxic conditions showed a 3.4-fold increase (P < 0.01) in VEGF mRNA compared with those cultured in normoxia. This correlated with increased VEGF secretion as determined by enzyme-linked immunosorbent assay. Immunohistochemical studies revealed the presence of increased expression of VEGF protein near the center of islets after 24 h of normoxic culture. Islet cell clusters on Matrigel showed intense cellular localization of VEGF in both beta-cells and non-beta-cells. These findings suggest that rat islet cells, when subjected to hypoxia during the first few days after transplantation, may act as a major source of VEGF, thereby initiating revascularization and maintaining the vascular permeability of the grafted islets.

Porcine neonatal pancreatic cell clusters (NPCCs): a potential source of tissue for islet transplantation.

This is a short review of porcine neonatal pancreatic cell clusters (NPCCs) which might eventually be useful for beta cell replacement therapy in people with diabetes. The current success with islet allograft transplantation is reviewed and is problematic because only partial success has been obtained and the shortage of human islet tissue means that only a small fraction of people with diabetes would be able to benefit. For these reasons there is considerable interest in xenotransplantation, with pigs being a particularly attractive source. The relative merits of early fetal, late fetal, neonatal and adult porcine tissue are discussed. Neonatal tissue has several attractive features, with their hardiness and potential for growth being especially noteworthy. NPCCs are harvested after digested and dispersed clumps of cells are kept in culture for 7 days. The NPCCs consist mainly of duct cells, protodifferentiated cells and mature endocrine cells. The protodifferentiated cells are either double or triple stained for insulin, cytokeratin 7, glucagon, pancreatic polypeptide, or somatostatin. When transplanted into diabetic nude mice it usually takes weeks before glucose levels are normalized, and during that time differentiation and growth of the graft can be observed. Potential strategies for controlling xenograft rejection are mentioned, with these being immunosuppression, induction of tolerance, immunobarrier devices, and gene transfer approaches.