Robert C. Johnson

Local inflammatory response around diffusion chambers containing xenografts. Nonspecific destruction of tissues and decreased local vascularization.

Immunoisolation of xenogeneic pancreatic islets within membrane-bound devices has been proposed as an approach to cure diabetes. We examined the local response to implanted xenografts and allografts in comparison with isografts in diffusion chambers with 0.4-microm pore membranes when implanted into epididymal fat pads of rats. These membranes prevented host cell entry into the device but did not prevent passage of large molecules such as IgG and IgM. Well-differentiated allogeneic tissues (Sprague-Dawley rat embryonic lung implanted into Lewis rats) survived for 1 year when implanted in intact devices, but similar tissues were destroyed within 3 weeks when implanted within devices with holes poked in the membrane to allow host cell contact. In contrast, xenografts (CF1 mouse embryonic lung implanted into Lewis rats) were destroyed within 3 weeks even when implanted in devices with intact membranes. The death of the xenogeneic tissues was accompanied by a severe local accumulation of inflammatory cells and a decrease in local vascularization. When isogeneic tissues (Lewis rat embryonic lung implanted in Lewis rats) were mixed with xenogeneic tissues, a local inflammatory response occurred and both iso- and xenogeneic tissues were destroyed within 5 weeks. These results suggest the possibility that xenografts are killed by local accumulation of inflammatory cells, perhaps mediated by the release of antigens from the tissues within the device and presentation by an indirect pathway. The observation that the local response to xenografts is sufficient to kill isografts complicates issues of immunoprotection, suggesting that successful immunoisolation will require membranes that not only provide protection of the encapsulated tissues from the host immune system but also have properties that diminish the release of xenogeneic antigens.

Survival of macroencapsulated allogeneic parathyroid tissue one year after transplantation in nonimmunosuppressed humans.

The use of immunoisolation devices may allow transplantation without need for immunosuppression and could widen the indications for cell transplantation. In this study, we evaluated the survival of encapsulated parathyroid tissue in nonimmunosuppressed humans. Autologous parathyroid implants: Seven patients under-going parathyroidectomy had devices containing small pieces of their own parathyroid tissue implanted SC. These devices were explanted after 2–4 weeks for histological evaluation. Allogeneic parathyroid implants: Four patients with chronic hypoparathyroidism were transplanted with one to three large (40 μl) and one small (4.5 μl) device filled with meshed parathyroid tissue and implanted SC. The small devices were explanted at 4 weeks, while the large ones were explanted 8.5 to 14 months after implantation. In both studies, control implants were placed in nude mice. Autologous study results: At explantation, the grafts consisted of 22 ± 6% endocrine tissue and 63 ± 7% fibrosis, while 15 ± 5% of the grafts were necrotic. Allogeneic study results: In devices explanted from the patients at 4 weeks, fibrosis dominated and only 1%, 5%, and 23% of the grafts consisted of endocrine tissue. A similar histological appearance was found in grafts from nude mice. In devices explanted at 8.5–14 months, histologically intact endocrine tissue was found in all patients. However, nearly all the tissue consisted of fibrosis. There was no detectable increase in the parathormone (PTH) level in all patients. Macroencapsulated human allogeneic parathyroid tissue can survive up to 1 year after transplantation into nonimmunosuppressed patients. However, marked fibroblast overgrowth occurred, especially in the allogeneic implant study, using meshed parathyroid tissue. This was probably not related to the allo-response, because similar findings were observed in the nude mouse implants. In future studies, better tissue preparation and improvements in the physiological milieu inside the device may help to reduce fibroblast overgrowth and increase survival of the parathyroid cells.

Phosphatidylcholine Synthesis by Peritoneal Mesothelium: Its Implications for Peritoneal Dialysis

This study investigated the possibility that the peritoneum is capable of synthesizing phosphatidylcholine (PC), a lubricant surfactant, in an amount similar to that produced by pulmonary alveoli. The synthesis of PC by rat lung (positive control), liver (negative control), and transparent mesentery (test tissue) was determined by in vitro incubation of these tissues in the presence of (methyl-14C) choline chloride for three hours at 37 degrees C in Warburg flasks. All lipid material present in tissue and incubation media was extracted by the Folch technique. Carrier egg PC was added to each sample and total PC was isolated using high performance thin-layer chromatography. The PC fractions were counted for total radioactivity. No statistically significant difference was observed between the mean radioactive value for mesenteric tissue compared with lung tissue. The mean radioactive value for liver when compared with mesenteric and lung tissue was significantly lower (P less than 0.001). Thus, under the conditions of the experiment, we have demonstrated for the first time that peritoneal tissue is capable of synthesizing PC in amounts similar to that produced by the lung. Electron microscopy of transparent mesentery (test tissue) showed that mesothelium constituted the bulk of the cell population. Therefore, it is most likely that the PC that has been detected in peritoneal fluid is produced by the mesothelial cell. These findings are of significant relevance to developing concepts on the role of surfactant phospholipids in the physiology of peritoneal dialysis.

Correction of diabetic nod mice with insulinomas implanted within Baxter immunoisolation devices.

Insulin replacement by injection is clearly not a cure for Insulin Dependent Diabetes Mellitus (IDDM). Replacement of the destroyed islets by pancreas or islet allograft transplantation can achieve the good metabolic control required to prevent diabetic complications, but tissue supply is limited. The problem of islet supply to treat the 1 million IDDM patients in the USA could be overcome by using immortalized islet β-cells as a donor source. However, before either allogeneic or xenogeneic immortalized β-cells are used, some major problems have to be overcome: control of immortalized cell growth, allograft or xenograft rejection and recurrence of autoimmunity. To tackle these problems we have used a cell impermeable immunoisolation device containing mouse insulinoma cells. Transplantion of devices with insulinomas from NOD mice carrying the Rat-insulin promoter regulated SV40 T-Antigen transgene (RIP-TAg), normalized the blood glucose levels of diabetic NOD mice. Insulinomas from allogeneic CBA/NOD-RIP-TAg mice were also capable of normalizing diabetic NOD mice. Not only were non-fasting blood glucoses normalized but when given an intraperitoneal injection of glucose, the corrected mice had a near normal clearance of glucose from the blood. When the devices were removed from normalized mice they became diabetic again, demonstrating that the immunoisolation device was capable of protecting against both alloimmune and autoimmune destruction. The results with allogeneic mouse β-cells suggest the possiblity that immortalized human β-cells could be an effective source of tissue to correct diabetes in IDDM patients without the use of immunosuppression.

In vivo delivery of recombinant human growth hormone from genetically engineered human fibroblasts implanted within Baxter immunoisolation devices

Continuous delivery of therapeutic peptide to the systemic circulation would be the optimal treatment for a variety of diseases. The Baxter TheraCyte® system is a membrane encapsulation system developed for implantation of tissues, cells such as endocrine cells or cell lines genetically engineered for therapeutic peptide delivery in vivo. To demonstrate the utility of this system, cell lines were developed which expressed human growth hormone (hGH) at levels exceeding 1 microgram per million cells per day. These were loaded into devices which were then implanted into juvenile nude rats. Significant levels of hGH of up to 2.5 ng/ml were detected in plasma throughout the six month duration of the study. In contrast, animals implanted with free cells showed peak plasma levels of 0.5 to 1.2 ng four days after implantation with no detectable hGH beyond 10 days. Histological examination of explanted devices showed they were vascularized and contained cells that were viable and morphologically healthy. After removal of the implants, no hGH could be detected which confirmed that the source of hGH was from cells contained within the device. The long term expression of human growth hormone as a model peptide has implications for the peptide therapies for a variety of human diseases using membrane encapsulated cells.

Dietary supplementation of undernourished rats with soy or safflower oil: Effects on myelin polyunsaturated fatty acids

Undernourished suckling rats were administered, by gastric intubation, either soy oil (which is rich in both linoleic and linolenic acids) or safflower oil (which is rich in linoleic acid but deficient in linolenic acid) to determine (1) if dietary supplementation would offset the hypomyelination characteristic of the undernourished, developing brain and (2) to compare myelin fatty acids in normal, undernourished, and oil-supplemented rats. Myelin recovery was not increased by supplementation with either oil. The proportions of C22∶4 and C22∶6 fatty acids were reduced in myelin of the undernourished rats. Undernourished rats supplemented with either soy or safflower oil had higher than normal proportions of polyunsaturated fatty acids (C20∶4 and C22∶6). The triene-tetraene ratio in the oil-supplemented rats was lower than in normal controls, indicating that the oil-supplemented rats were not deficient in essential fatty acids. No significant differences were observed between the oil-supplemented groups.

Comparison of simulated and in-vivo plasma levels of cilastatin following intravenous in-line drug administration.

The primary objective of this work was to establish a method to simulate the plasma levels of cilastatin, a model drug, following an intravenous in-line delivery scheme. In-vivo data in dogs obtained from this work were used to demonstrate the validity of the proposed approach. The in-line drug delivery system consists of a drug containing device which is placed between a large volume parenteral and a patient. Numerous advantages have been identified for this automatic in-line reconstitution delivery system. The numerical convolution integral algorithm was used in this work to perform plasma profile simulation. The results indicated that the simulated cilastatin plasma profile following in-line delivery closely agreed with the in-vivo data.