Human therapeutic cloning.

Somatic cell nuclear 'reprogramming' in livestock species is now routine in many laboratories. Here, Robert Lanza, Jose Cibelli and Michael West discuss how these techniques may soon be used to clone genetically matched cells and tissues for transplantation into patients suffering from a wide range of disorders that result from tissue loss or dysfunction.

Symbolic Communication Between Two Pigeons, (Columba livia domestica).

Through the use of learned symbols, a pigeon accurately communicated information about hidden colors to another pigeon. Each verbal exchange was initiated with a spontaneous request for information. The two pigeons engaged in a sustained and natural conversation without human intervention.

"Self-awareness" in the pigeon.

Each of three pigeons used a mirror to locate a spot on its body which it could not see directly. Although similar behavior in primates has been attributed to a self-concept or other cognitive process, the present example suggests an account in terms of environmental events.

Extension of cell life-span and telomere length in animals cloned from senescent somatic cells.

The potential of cloning depends in part on whether the procedure can reverse cellular aging and restore somatic cells to a phenotypically youthful state. Here, we report the birth of six healthy cloned calves derived from populations of senescent donor somatic cells. Nuclear transfer extended the replicative life-span of senescent cells (zero to four population doublings remaining) to greater than 90 population doublings. Early population doubling level complementary DNA-1 (EPC-1, an age-dependent gene) expression in cells from the cloned animals was 3.5- to 5-fold higher than that in cells from age-matched (5 to 10 months old) controls. Southern blot and flow cytometric analyses indicated that the telomeres were also extended beyond those of newborn (<2 weeks old) and age-matched control animals. The ability to regenerate animals and cells may have important implications for medicine and the study of mammalian aging.

Encapsulated cell technology.

The potential therapeutic applications of encapsulated cells are enormous. In the US alone, it has been estimated that nearly half-a-trillion dollars are spent each year to care for patients who suffer tissue loss or dysfunction. Over 6 million patients suffer from neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease, over 14 million patients suffer from diabetes, and millions more from liver failure, hemophilia, and other diseases caused by the loss of specific vital cellular functions. It appears likely that by the end of the decade clinical trials of encapsulated cells to treat many of these diseases will become a reality. The Food and Drug Administration has already authorized studies to evaluate the safety and biological activity of several types of systems. A number of issues will have to be addressed, including the sourcing of raw materials, the design and building of manufacturing facilities, the scale-up and optimization process, storage and distribution of the product, and quality control.

Prospects for the use of nuclear transfer in human transplantation.

The successful application of nuclear transfer techniques to a range of mammalian species has brought the possibility of human therapeutic cloning significantly closer. The objective of therapeutic cloning is to produce pluripotent stem cells that carry the nuclear genome of the patient and then induce them to differentiate into replacement cells, such as cardiomyocytes to replace damaged heart tissue or insulin-producing beta cells for patients with diabetes. Although cloning would eliminate the critical problem of immune incompatibility, there is also the task of reconstituting the cells into more complex tissues and organs in vitro. In the review, we discuss recent progress that has been made in this field as well as the inherent dangers and scientific challenges that remain before these techniques can be used to harness genetically matched cells and tissues for human transplantation.

Perspectives in diabetes. Islet transplantation with immunoisolation.

Immunoisolation is a potentially important approach to transplanting islets without need for immunosuppressive drugs. Immunoisolation systems have been conceived in which the transplanted tissue is separated from the immune system of the host by an artificial barrier. These systems offer a solution to the problem of human islet procurement by permitting use of islets isolated from animal pancreases. The devices used are referred to as biohybrid artificial organs because they combine synthetic, selectively permeable membranes that block immune rejection with living transplants. Three major types of biohybrid pancreas devices have been studied. These include devices anastomosed to the vascular system as AV shunts, diffusion chambers, and microcapsules. Results in diabetic rodents and dogs indicate that biohybrid pancreas devices significantly improve glucose homeostasis and can function for more than a year. Recent progress made with this approach is discussed, and some of the remaining problems that must be resolved to bring this technology to clinical reality are addressed.

Treatment of severely diabetic pancreatectomized dogs using a diffusion-based hybrid pancreas.

Long-term survival of dog islet allografts implanted in diabetic pancreatectomized dogs was achieved by islet encapsulation inside cylindrical chambers fabricated from permselective acrylic membranes (nominal M(r) exclusion of 50,000-80,000). Dog islets were isolated from the pancreases of outbred mongrel dogs by collagenase digestion. Chambers containing mean +/- SE 316 +/- 63K islet equivalents (mean islet volume, 558 +/- 111 mm3, purity 90-95%) were peritoneally implanted into six totally pancreatectomized dogs. The dogs were monitored for glycemic control by fasting and postprandial blood glucose determinations, and responses to both intravenous glucose (intravenous glucose tolerance test 0.5 g/kg) and oral glucose (oral glucose tolerance test 1 g/kg). All of the dogs required appreciably lower dosages of exogenous insulin therapy for control of fasting blood glucose levels, with the mean daily insulin dose dropping from 38 +/- 7 to 5 +/- 1 U/day during the 1st wk. Three recipients required no insulin for greater than 82, greater than 68, and 51 days. Intravenous glucose tolerance test K values (decline in glucose levels, %/min) at 1 and 2 mo postimplantation were 2.7 +/- 0.4 and 2.0 +/- 0.5, respectively compared with 3.5 +/- 0.5 before pancreatectomy. The glucose values during oral glucose tolerance tests at 2 wk, although returning to less than 125 mg/dl (less than 7.0 mM) by 2 h, exceeded the normal range, with peak values of 174 to 202 mg/dl (9.7 to 11.3 mM). These preliminary results are encouraging, and represent an important step in determining the feasibility of using this type of diffusion-based hybrid artificial pancreas as treatment for diabetes mellitus in humans.

Transplantation of islets using microencapsulation: studies in diabetic rodents and dogs.

Studies involving the transplantation of human islets in Type I diabetics have been of significant value both in documenting the potential importance of islet transplantation as a therapeutic modality, and in defining some of the problems which must be overcome before this approach can be used in large numbers of patients. The currently limited supply of adult human pancreatic glands, and the fact that chronic immunosuppression is required to successfully transplant islets into patients, indicate that techniques must be further developed and refined for allo- and xenografting of isolated islets from human and animal sources to diabetic patients. An increasing body of evidence using microencapsulation techniques strongly suggests that this will be achieved during the next few years. Data from our laboratory in rodents and dogs indicate that these systems can function for extended periods of time. In one study, insulin independence was achieved in spontaneously diabetic dogs by islet microencapsulation inside uncoated alginate gel spheres (Mr exclusion >600 kD). No synthetic materials or membrane coatings were employed in this study. Spheres containing canine islets were implanted into the peritoneum of 4 diabetic dogs. The animals received low-dose CsA (levels below readable limits by HPLC at 3 weeks). Implantation of these spheres completely supplanted exogenous insulin therapy in the dogs for 60 to >175 days. Blood glucose concentration averaged 122+/-4 mg/dl for these animals during the first 2 months. The glycosylated hemoglobin (HbAIC) levels during this period dropped from 6.7+/-0.5% to 4.2+/-0.2% (P<0.001). IVGTT K-values at 1 and 2 months postimplantation were 1.6+/-0.1 (P<0.002) and 1.9+/-0.1 (P<0.001), respectively compared with 0.71+/-0.3 before implantation. In a second group of studies, bovine islets were immobilized inside a new type of selectively permeable "microreactor" (Mr exclusion <150 kD) and implanted into the peritoneum of 33 STZ-induced diabetic rats without any immunosuppression. Diabetes was promptly reversed, and normoglycemia maintained for periods of several weeks to months. Immunohistochemical staining of microreactors recovered from these animals revealed well-granulated beta-cells consistent with functionally active insulin synthesis and secretion. To test further the secretory function of the islets, some of the explanted microreactors were incubated in media containing either basal or stimulatory concentrations of glucose. The islets responded with an approximately 3- to 5-fold average increase above basal insulin secretion. These results are encouraging, and may have important implications in assessing the potential role of these microencapsulation systems as therapy for human insulin-dependent diabetes.

Uso y prescripción de cannabis medicinal por reumatólogos en Argentina / Use and prescription of medical cannabis by rheumatologists in Argentina

El uso y prescripción del cannabis y sus derivados en Reumatología está aún en discusión. La ley de cannabis fue sancionada hace casi 3 años, aunque la reglamentación recién está comenzando. El objetivo de este estudio fue conocer la forma en que el reumatólogo se posiciona frente al uso de cannabis en el marco de su práctica médica. A través de una encuesta realizada durante el Congreso Argentino de Reumatología 2019 se recabó la opinión de 314 médicos que respondieron preguntas acerca del uso, recomendaciones y posturas respecto a la indicación y uso de cannabinoides en su práctica diaria. El 78,3% (246) conocían la existencia de una ley nacional. El 51,6% (162) se mostró en desacuerdo con el uso de cannabinoides en sus pacientes, mientras que el 36,6% (115) respondió estar de acuerdo, el 3,2% (10) refirió estar muy de acuerdo, y 8,6% (27) estaba muy en desacuerdo. Para pacientes con enfermedades reumáticas autoinmunes, el 94,6% (297) refirió que nunca indica cannabinoides, el 4,1% (13) que los indican pocas veces, y el 1,3% (4) algunas veces. Para las enfermedades reumáticas degenerativas, el 90,4% (284) nunca indica cannabinoides, el 6,7% (21) lo hace pocas veces, y el 2,9% (9) lo hace algunas veces. Para la fibromialgia, el 84,4% (265) nunca indica cannabinoides, el 8,3% (26) los indica pocas veces, el 6,4% (20) los indica algunas veces. El principal obstáculo para la prescripción (permitido más de una respuesta) fue no disponer de la suficiente información científica para prescribir (50,3%, 158), el 47,5% (149) contestó que no conoce los componentes de la preparación, el 47,1% (148) no conoce las dosis o la posología, el 41,4% (130) no le resultan confiables los productores, el 38,9% (122) respondió que no le convencen los estudios clínicos en la especialidad. No hubo diferencias significativas entre las variables edad, género, años en la especialidad o lugar de ejercicio y las respuestas descriptas. Conclusión: El uso de cannabis en Reumatología de acuerdo a los especialistas que ejercen en Argentina requiere de un mayor sustento científico y farmacéutico para poder prescribirlo en un marco seguro.

The use and prescription of cannabis and its derivatives in Rheumatology is still under discussion. The cannabis law was enacted near 3 years ago, although the regulation is just beginning. The objective of this study was to know how rheumatologists positions themselves about the use of cannabis in the framework of his medical practice. Through a survey conducted during the 2019 Argentine Congress of Rheumatology, 314 doctors answered questions about the use, recommendations and opinions regarding the indication and use of cannabinoids in their daily practice. 78.3% (246) knew of the existence of a national law. 51.6% (162) disagreed with the use of cannabinoids by their patients, while 36.6% (115) agreed, 3.2% (10) reported to strongly agree, and 8.6% (27) strongly disagreed. For patients with autoimmune rheumatic diseases, 94.6% (297) reported that they never prescribed cannabinoids, 4.1% (13) prescribed them rarely, and 1.3% (4) sometimes. For degenerative rheumatic diseases, 90.4% (284) never prescribed cannabinoids, 6.7% (21) did it rarely, and 2.9% (9) did so sometimes. For fibromyalgia, 84.4% (265) never prescribed cannabinoids, 8.3% (26) prescribed them rarely, 6.4% (20) sometimes. The main obstacle to prescribing (more than one answer allowed) was not having enough scientific information (50.3%, 158), 47.5% (149) were uncertain about the cannabis preparation, 47.1% (148) had no knowledge about doses or posology, 41.4% (130) didn´t trust the producers, 38.9% (122) were no convince by the trials in the field. There were no significant differences between the variables age, gender, years in the specialty or workplace and the responses described. Conclusion: According to specialists in Argentina, the use of cannabis in rheumatology requires more scientific and pharmaceutical data to prescribe cannabinoids in a safer framework.

Transplantation of encapsulated canine islets into spontaneously diabetic BB/Wor rats without immunosuppression.

Extended survival of canine islet xenografts implanted in spontaneously diabetic BB/Wor rats has been achieved by islet encapsulation inside cylindrical chambers fabricated from permselective acrylic membranes. Intraperitoneal implantation of the encapsulated islets reversed the diabetic state of the 10 recipients within 24 h. Plasma glucose levels declined from a preimplantation level of 459 +/- 30 to 102 +/- 14 mg/dl during the first 10 days. All of the animals sustained these levels for at least 1 month, and 2 animals for at least 2 and 8 months, respectively. To confirm that glucose homeostasis resulted from the encapsulated islet grafts, the implants were removed from 2 rats 1 month postimplantation, whereas a third was removed at 2 months. Hyperglycemia was observed immediately in all 3 animals, with glucose levels rising from 100 +/- 3 to 510 +/- 43 mg/dl within 1 day. In contrast, diabetic control rats (n = 4) receiving nonencapsulated islets became hyperglycemic in less than 1 week. The iv glucose tolerance test K value (decline in glucose levels, percent per min) at 10 days was 2.3 +/- 0.4 compared with 0.6 +/- 0.1 (P less than 0.005) and 3.1 +/- 0.1 (P less than 0.02) for untreated diabetic (n = 4) and normal control (n = 4) groups. Histological analyses and electron microscopy of long term functioning grafts revealed well preserved islets, with hormone-producing alpha-, beta-, and delta-cells; the membranes were generally free of fibrosis and host cell adherence. These results demonstrate that permselective artificial membranes can protect discordant islet xenografts from both graft rejection and autoimmune destruction for more than 1 month in an animal model that is similar in several respects to human type I diabetes.

Prolonged pancreas preservation using a simplified UW solution containing polyethylene glycol.

The University of Wisconsin solution is considered the most effective universal flush and cold storage solution to date, and is now being widely applied clinically in organ transplantation. The results of this study show that Cardiosol, a modified cardioplegic solution containing 5% polyethylene glycol (PEG20M, MW 17,000 daltons), is significantly superior to UW (P less than 0.001) in the flush perfusion and hypothermic storage of pancreases for more than 36 hr prior to transplantation into streptozotocin-induced diabetic rats. When the pancreases were stored in Cardiosol, the 1-week survival rate was 7 of 10 (70%) after 24 hr of preservation; 7 of 12 (58%) after 36 hr; and 3 of 10 (30%) after 48 hr. In contrast, when the pancreases were stored in UW solution, the 1-week survival rate was 8 of 12 (67%) after 24 hr of preservation; after 36 hr, no animal survived (0 of 14). Intravenous glucose tolerance test K-values (decline in glucose concentration, percentage per minute) were normal in both groups receiving 24-hr-preserved pancreases, ranging from 2.60 to 4.16. Of interest, the peak insulin response 1 min following intravenous glucose was significantly higher (P less than 0.01) in the Cardiosol-preserved organs (303 +/- 29.8 microU/ml) (+/- SEM) than in the glands preserved in UW solution (112 +/- 47.9 microU/ml). We conclude that Cardiosol allows prolonged whole organ pancreas preservation in the rat transplant model.

Xenogenic humoral responses to islets transplanted in biohybrid diffusion chambers.

It has been hypothesized that chronic antigen leakage from the hybrid artificial pancreas could stimulate a host humoral response. Such antibodies could be induced by antigens shed from the islet cell surface, or by proteins secreted by live cells or liberated after cell death. To determine if this humoral response occurs, porcine (n = 15) or canine (n = 7) islets were seeded (2-5 x 10(4) equivalent islet number, density 30 islets/mm3) into diffusion chambers fabricated from permselective acrylic membranes (nominal M(r) exclusion of 80,000). The chambers were implanted intraperitoneally into streptozotocin-induced diabetic rats. Sera were collected at various intervals (0-12 weeks) and tested against isolated canine and porcine islets, for tissue specificity and interspecies cross-reactivity by fluorescence immunocytochemistry. No immunofluorescence (or only weak background staining) was obtained when islets were exposed to horse sera, or to sera obtained before to xenodevice implantation. Within 2-6 weeks, however, the postimplantation sera showed strong immunoreactivity. The antibodies were found to be reactive to multiple tissues, and to possess little or no interspecies cross-reactivity. The appearance of these xenoantibodies coincided with the appearance of circulating soluble immune complexes. However, none of the respiratory, cutaneous, or gastrointestinal manifestations that are characteristic of an anaphylactic reaction, or of the diseases of immediate-type hypersensitivity, were observed, even after intraperitoneal injection of additional naked islet tissue. Renal glomeruli did not stain for IgG or C3 in islet recipients. These results suggest that islet cell antigens crossed the membrane and stimulated antibody formation in the host, although they did not appear to cause renal or immune complex disease during the course of this study.

Xenotransplantation of cells using biodegradable microcapsules.

BACKGROUND: The use of immunoisolation to protect transplanted cells from the immune system of the host has broad application to the treatment of major diseases such as diabetes and a wide range of other disorders resulting from functional defects of native cell systems. In most cases, limitations in functional cell longevity will necessitate periodic replenishment of the cells. We describe a hydrogel-based microcapsule that breaks down at a rate that can be adjusted to correspond to the functional longevity of the encapsulated cells. These injectable capsules can be engineered to degrade over several weeks to months for short-term drug delivery, or to remain intact and immunoprotective for more extended periods. When the supply of cells needs to be replenished, no surgery will be required to localize and remove the old capsules. METHODS: Porcine and bovine islets were immobilized in "composite" microcapsules fabricated from alginate and low-relative molecular mass (Mr) poly (L-lysine[PLL]) (Mr exclusion <120 Kd) and implanted into the peritoneum of normal and streptozotocin-induced diabetic rats. In addition to demonstrating long-term islet viability and function, a series of in vitro studies were carried out to determine the permeability and biodegradability of the microcapsules used in the present system. RESULTS: Xenogeneic islets implanted in nonimmunosuppressed rats remained in excellent condition indefinitely (>40 weeks)(viability was comparable to that of preimplant control specimens). In contrast, no islets survived in uncoated alginate spheres after 2 weeks postimplantation. By changing the concentration of the alginate, it was possible to vary the rate of capsule breakdown in rats from mechanically unstable (outer matrix <0.5-0.75% alginate) to stable for >1 year (> or =1.5% alginate). In addition to in vivo breakdown studies, the biodegradability of the capsular components was verified in vitro using a mixture of tritosomes (enzymes isolated from animal cells). CONCLUSIONS: We have designed a microcapsule system with controllable biodegradability which allows breakdown and absorption of implants when the cells die or become functionally inactive. These results may have application to other alginate-PLL encapsulation systems. The ability to cross species lines using these biodegradable microcapsules has the potential to expand dramatically the number of patients and the scope of diseases that can be successfully treated with cellular therapy.

Biohybrid artificial pancreas. Long-term function of discordant islet xenografts in streptozotocin diabetic rats.

Long-term function of canine, bovine, and porcine islet xenografts implanted in streptozotocin-induced diabetic rats has been achieved by islet encapsulation within permselective acrylic membrane chambers. Intraperitoneal implants of 1 x 10(4) (n = 11) or 2 x 10(4) (n = 2) encapsulated canine islets reversed the diabetic state of the recipients within 24 hr, with plasma glucose levels dropping from a preimplantation level of 480 +/- 26 (mean +/- SEM) to 97 +/- 4 mg/dl during the first month. Chambers from 2 of the animals were removed, bisected, and reimplanted at 1 week and 2 months; both animals reverted to hyperglycemia (glucose, > 200 mg/dl) in < 2 weeks. The remaining implants maintained function for a mean time of 138 +/- 16 days, whereas the 2 animals that received the higher islet dose maintained function for > 260 days. Membranes containing 2 x 10(4) bovine (n = 6) or porcine (n = 10) islets also normalized glucose concentrations, with plasma glucose levels dropping from 468 +/- 61 to 91 +/- 10 (bovine) and 97 +/- 11 (porcine) mg/dl during the first month (vs. 94 +/- 3 mg/dl for nondiabetic control rats). Three of the latter implants were removed at 1 month. All 3 animals promptly reverted to diabetes. The 3-, 6-, 9-, and 12-month graft survival rates for the remaining animals were 100%, 100%, 60%, and 40%, and 100%, 75%, 50%, and 25%, respectively. The transplant recipients showed an approximately 38-54% gain in body weight during the first 100 days after implantation, compared with < 1% (P < 0.001) and 86% (P < 0.001) for the untreated diabetic (n = 5) and normal control (n = 6) groups. Immunohistochemical staining of long-term grafts (1-20 months) revealed varying degrees of alpha-, beta-, and delta-cell granulation; the external membrane surfaces were generally free of fibrotic overgrowth and exhibited only occasional host cell adherence. Despite a problem of membrane breakage in long-term implants, these results suggest that prolonged survival of discordant transplants of porcine, bovine, and canine islets in diabetic rats can be achieved without immunosuppression.

Xenotransplantation of porcine and bovine islets without immunosuppression using uncoated alginate microspheres.

Uncoated spherical hydrogel microspheres (calcium alginate, nominal M(r) exclusion of > 600 kD) 800-900 microns in diameter were employed to prevent immune rejection of discordant islet xenografts isolated from pigs and cows. The islets were immobilized in the microspheres and injected into the peritoneum of 14 nonimmunosuppressed streptozotocin (STZ)-induced diabetic C57BL/6J mice. Four recipients received islet grafts from bovine calves, and 10 received islet grafts from pigs. In the control group of 15 diabetic mice implanted with nonencapsulated islets, 6 received i.p. porcine islets and 5 received i.p. bovine islets, whereas remaining 4 received porcine islets under the kidney capsule. Plasma glucose concentrations in recipients of the alginate-encapsulated islets promptly dropped from a preimplantation value of 498 +/- 47 (mean +/- SEM) to 142 +/- 6 (bovine) and 178 +/- 7 mg/dl (porcine) during the first wk. All the animals sustained these levels for at least 1 mo. Two mice implanted with bovine islets subsequently reverted to diabetes (plasma glucose > 250 mg/dl) at 43 days postimplantation. The remaining grafts maintained function for > 10 wk. In contrast, nonencapsulated islets failed to function, or sustained euglycemia for < 4 days. Mice receiving encapsulated islets showed a 23-38% gain in body weight during the first mo after implantation, compared with < 1% (P < 0.002) and 32% (P = 0.84) for the untreated diabetic (n = 6) and normal control (n = 6) groups. Immunohistochemical staining of long-term grafts (> 10 wk) revealed viable islets, with well-granulated alpha, beta, and delta cells; the external surfaces of the microreactors were free of fibrotic overgrowth and exhibited only occasional host cell adherence. Uptake studies with IgG and thyroglobulin (M(r) of 669 kD) suggest that the microreactors were permeable to molecules with a molecular weight of up to > 600 kD (including the various proteins of the complement system, M(r) of 24-570 kD). Spheres implanted in the peritoneum after only 1 wk stained positive for both IgG and for the C3 component of complement. These findings suggest that prolonged survival of discordant xenografts of porcine and bovine islets in the STZ diabetic mouse model can be achieved with uncoated alginate microspheres that are permeable to IgG and complement. The question of whether similar results can be achieved with uncoated alginate microspheres in higher animals remains to be fully determined.

Rapid purification of islets using magnetic microspheres coated with anti-acinar cell monoclonal antibodies.

A simple, rapid method of islet purification is important in large-scale human islet isolation. We have previously identified monoclonal antibodies specific for acinar cells, but not islets, and described an immunologic method of purification by selective lysis of the acinar cells. An attractive alternative to lysis of the acinar cell is depletion by a magnetic immunomicrosphere technique. We report in this study a rapid, reproducible method of rat islet purification utilizing magnetic microspheres coated with acinar-cell-specific monoclonal antibodies. Pancreatic digestion with collagenase followed by depletion of acinar cells with the magnetic immunomicrospheres (MIMS) yields large numbers of intact islets. We compared the islets thus obtained with hand-picked (HP) islets (control) for yield, purity, in vitro insulin secretory capacities, and in vivo functional viability. The islet yield with the MIMS method (n = 35) was 72.7% that obtained with the HP method (n = 6) (378 +/- 8 vs. 519 +/- 31 islets per pancreas). The purity of the MIMS-isolated islets was 84 +/- 1.9%, ranging from 75-95%. Static glucose stimulation showed excellent function (2-3-fold increase of insulin release over basal levels) with no statistical difference in insulin secretion between MIMS and HP islets. Under microscopic examination, both groups revealed a well-preserved structure with healthy endocrine cells. When 1321 +/- 59 MIMS islets were transplanted into streptozotocin-induced diabetic rats (n = 10), normoglycemia (less than 200 mg/dl) was restored in all recipients following transplantation, and 100% of them remained normoglycemic on day 120 postgrafting. In summary, a rapid, consistent, and simple method of isolating viable, purified rat islets is described. The broad interspecies crossreactivity of the McAb suggests that this technique may be generally useful for islet purification in large mammalia, including man.

The effects of brain death and 24 hours' storage by hypothermic perfusion on donor heart function in the pig.

The effects on the myocardium of the agonal period and subsequent management have been studied in the pig. Acute ischemia of the brain led to major temporary hemodynamic changes. Brain death, with or without hemodynamic support of the circulation, led to a significant reduction in subsequent myocardial function, associated with some depletion of the myocardial high-energy phosphate and glycogen reserves, although the rate of this depletion was reduced by anaerobic glycolysis. Although 24 hours' storage by continuous hypothermic perfusion of hearts taken from control animals led to only a minimal reduction in myocardial function, storage increased the reduction in function associated with brain death when intravenous fluid and dobutamine support had been given to maintain the brain dead pig in a normotensive state. Storage, however, reduced the anaerobic metabolism seen in hearts functioning in hypotensive brain dead pigs and led to replenishment of the glycogen stores.

Screening for asbestos-related pleural disease with digital storage phosphor radiography.

Digital storage phosphor radiography (SR) has a wide dynamic range and unique postprocessing capabilities that may improve the performance of screening studies for asbestos-related pleural disease compared with conventional film radiography (FR). In a group of 32 asbestos-exposed and nine control subjects with established pleural data, we compared the screening performance of FR and SR obtained with a single isoexposure, dual-energy technique (system resolution 0.2 mm, 10 bits). Performance was evaluated for 7320 observations by eight readers using a paired t test (P less than .02 with Bonferroni correction) of averaged receiver operating characteristic curve (ROC) areas (Az +/- standard error). We found that SR alone and SR supplemented by dual-energy soft-tissue and calcium images (SRde) were superior to FR in the overall detection of pleural abnormalities (Az = 0.90 +/- 0.01, 0.90 +/- 0.01, and 0.88 +/- 0.01, respectively). In the specific detection of pleural calcification, SRde was superior to FR (Az = 0.91 +/- 0.01 and 0.87 +/- 0.01, respectively; P less than 0.01). Analysis of variance indicated that SRde most closely reproduced an established pleural score based on the International Labor Organization (ILO) classification of the pneumoconioses (P less than 0.05, Scheffé's multiple comparison test). We conclude that isodose SR performs at least as well as FR in screening for asbestos-related pleural disease. SR supplemented by dual-energy images might improve the specific detection of pleural calcifications compared with FR.

Encapsulation technologies.

Encapsulation systems have been developed in which cells are separated from the immune system of the host by permselective barriers. These systems do not require a life-long regimen of high dose immunosuppressive drugs to prevent immune rejection. Furthermore, they offer a solution to the problem of human cell procurement by permitting use of cells and tissues from animal sources. Three major types of immunoisolation devices have been studied by our group. These include perfusion devices anastomosed to the vascular system as atrioventricular (AV) shunts, tubular membrane diffusion chambers, and microreactors. This technology is applicable to treating a number of diseases by transplantation of cells that produce specific bioactive substances. In essence, this approach constitutes a living drug delivery and detoxification system. Our work has focused mainly on developing a new treatment for diabetes using encapsulated pancreatic islets. In the first type of system, canine and porcine islets were distributed in a chamber surrounding a permselective acrylic membrane (nominal M(r) exclusion of 80 kDa), and the devices implanted intraperitoneally as AV shunts into diabetic, totally pancreatectomized dogs without use of immunosuppression. In the second type of system, the islets were sealed within the acrylic membranes and the chambers implanted into the peritoneum of diabetic, pancreatectomized dogs (canine islets), streptozotocin (STZ)-induced diabetic rats (canine, bovine and porcine islets), and spontaneously diabetic BB/Wor rats (canine islets) without use of immunosuppression. In the third type of system (microreactors), the islets were implanted into the peritoneum of STZ-induced diabetic mice without use of immunosuppression, into STZ-induced diabetic rats (bovine and porcine islets) both with and without use of low dose immunosuppression, and into spontaneously diabetic dogs (canine islets) with low dose CsA. Results indicate that all three types of encapsulation systems significantly improve glucose homeostasis and can function for periods of several months to more than a year.