G. Basta

Immunoprotection of pancreatic islet grafts within artificial microcapsules.

To circumvent pancreatic islet graft-directed immune destruction we enveloped porcine islets within highly biocompatible and selectively permeable algin/polyaminoacid microcapsules. These special microspheres were deposited between the inner (permeable) and the outer (impermeable) layers of an artificial, coaxial vascular prosthesis, directly anastomized to blood vessels. Five dogs with spontaneous, insulin-dependent diabetes received microencapsulated porcine islets in arterio-vein iliac prosthesis by-passes. One showed complete and the remainder partial, sustained reversal of hyperglycemia. Microencapsulation may be a potential solution to immunological problems related to islet transplantation.

A rapid qualitative method to assess in vitro immunobarrier competence of pancreatic islets containing alginate/polyaminoacidic microcapsules

A quick method for the qualitative evaluation of immunoisolatory properties associated with islet-containing alginate/poly-l-ornithine (AG/PLO) microcapsules is described. In particular, we examined a new AG/PLO coherent microcapsule (CM) prototype that was recently developed in our laboratory, although the procedure could be applicable to other capsule types as well. We observed no binding of immunoglobulins (Ig) contained in islet cell antibody (ICA)-positive human sera (>60 Juvenile Diabetes Foundation units, JDF U) to pig islets, enveloped within AG/PLO CM, under indirect immunofluorescence examination. Also, CM were shown to inhibit human lymphocyte proliferative capacity fully, as assessed by the3H-thymidine incorporation rate, in in vitro mixed xenogeneic pig islet/human lymphocyte co-cultures. These results provided us with a preliminary method to attempt standardization of basic physical/chemical properties which should be associated with an immunoisolatory membrane for islet allo/xenograft immunoprotection.

Transplantation of allogeneic/xenogeneic pancreatic islets containing coherent microcapsules in adult pigs.

WE HAVE PREVIOUSLY described a method for fabrication of alginate/polyaminoacidic CM. Thereafter, we completed assessment of either morphological properties of structural as well as ultrastructural level, or in vitro immunoselectivity, or in vivo post-transplant (Tx) functional performance in diabetic rodents of these new biomembranes for islet Tx immunoprotection in full absence of the recipient’s pharmacological immunosuppression. Because CM, unlike conventional-size microcapsules (CSM), measuring an average 600 to 800 mm in equatorial diameter, hold the unique advantage of occupying a volume which is almost coincident with that of naked islets, these new microimmunobarriers could address a number of unsolved problems with regard to encapsulated islet cell Tx in diabetic large mammalians. One of the most formidable hurdles to this approach for the therapy of insulin-dependent diabetes mellitus has consisted of inadequacy of implant sites for encapsulated islets. Because of the final excessive CSM’s Tx size, these capsules had been only implanted in the peritoneal cavity. Unfortunately, even if highly biocompatible, intraperitoneally grafted, islet-containing microcapsules had often provoked severe, possibly mass-related (60 to 80 mL/diabetic dog) inflammatory cell reaction. Dense connective tissue infiltration invariably resulted in impairment of biochemical exchange, ultimately leading to Tx failure. After testing the new CM in either in vitro functional and immunological or in vivo rodent diabetes-correction studies, we had embarked on assessment of CM biocompatibility, after multiple-site transplantation into adult pigs. Preliminarily, we have shown that empty CM, per se, indeed were biocompatible in this large-size mammal animal model, with a very low degree of sensitization being induced by repeated CM-Tx booster. In an attempt to determine whether CM, because of their minimal size, would permit access to Tx sites so far interdicted to CSM, including parenchymatous organs, with full retention of the encapsulated allogeneic/xenogeneic islet cell viability, we have transplanted porcine or canine islet containing CM into multiple Tx sites, and examined them histologically, at 30 days post-Tx, in normal, adult pigs. MATERIALS AND METHODS Animals

Effects of simulated microgravity on the morphology and function of neonatal porcine cell clusters cultured with and without Sertoli cells.

Human islet allografts are well known to induce full and sustained remission of hyperglycemia, with complete normalization of key metabolic parameters. Nevertheless, acquiring human islets, even from cadaveric human donor pancreases, remains a significant impediment to successful transplantation therapy for diabetes. To overcome this difficulty, neonatal porcine cell clusters (NPCCs) have been considered for human islet substitutes because they are easily obtained by collagenase digestion of the neonatal piglet pancreas. Currently, the major hurdle in using NPCCs for xenograft is the delay (time lag) in achieving the posttransplant normalization of blood glucose levels in animal diabetic recipients. The present work is the first attempt to evaluate whether incubation of NPCCs in simulated microgravity, in the presence or absence of Sertoli cells (SC), may reduce the maturation time lag of β-cells by differentiation acceleration in vitro, thereby expediting production, viability, and acquisition of functional competence of pretransplantation β-cell-enriched islets. Following a 3-day incubation period, NPCCs maintained in conventional culture, NPCCs incubated in simulated microgravity in the HARV biochamber, and NPCCs plus co-incubated SC in simulated microgravity were examined for viability, morphology, and insulin secretion. Results show that NPCCs grown alone in the HARV biochamber are superior in quality, both in terms of viability and functional competence, when compared to other culture pretreatment protocols. This finding strongly suggests that NPCC pretreatment in simulated microgravity may enhance the transplantation success of NPCCs in the diabetic recipient.

Insulin resistance in porphyria cutanea tarda

It has been reported that patients with porphyria cutanea tarda (PCT) develop carbohydrate (CHO) intolerance and manifest diabetes mellitus (DM) more frequently than the normal population. In order to verify whether this is due to insulin resistance we studied 5 patients with PCT and 5 normal subjects matched for age, sex and weight. In all the patients an evaluation consisted of the glycemic curve and insulin response to an iv glucose tolerance test (IVGTT: 0.33 g/kg) as well as of an evaluation of the circulating monocyte insulin receptors. Blood samples were drawn in the basal state to measure plasma levels of NEFA, glycerol, and intermediate metabolites. The patients with PCT showed normal glucose tolerance which was obtained, however, at the expense of the elevated insulin levels: therefore a condition of insulin resistance was demonstrated in these subjects. An involvement of the lipid metabolism, observed by the raised levels of plasma NEFA and glycerol, was also evident. The insulin binding to circulating monocytes was reduced but not enough to justify the degree of insulin resistance observed. Therefore, it could be hypothesized, in agreement with similar studies, that a postreceptor defect is responsible for the insulin-resistance observed in patients with PCT and that the reduction of insulin receptors is determined by the down regulation in response to elevated insulinemic levels. An alteration of the porphyrin metabolism might be responsible for this disorder.

Preventive effects of azathioprine (AZA) on the onset of diabetes mellitus in NOD mice

We have studied the effects of longterm treatment with azathioprine (AZA) vs cyclosporin A (CSA) vs placebo (PL), in three groups of 10 week old, prediabetic NOD mice. One of 8 AZA, none of 8 CSA and 7 of 11 PL treated mice developed overt diabetes (IDDM). Quantitative morphometry analysis conducted on mouse pancreatic histologic sections documented that extent and degree of islet beta-cell damage were incomparably less severe in the mice that received AZA or CSA compared to those treated with PL. Since early and prolonged treatment with AZA seems to prevent the onset of DM in NOD mice as nearly effectively as CSA, AZA, which is significantly safer than CSA, could replace the latter as a potential approach for the immunotherapy of IDDM.

A simple method for the DNA content assay of culture maintained pancreatic islets of Langerhans

A simple method for assaying DNA content of in vitro culture maintained pancreatic islets is described. By employing lyophilization of the islets and subsequent fluorometric analysis, we have been able to measure DNA from relatively small cell samples. The method may result advantageous, over others, previously reported, since it is less subject to experiment-related interfering conditions. This procedure, which is easy and reliable, even for very low DNA concentrations, seems to be very useful for culture maintained pancreatic islets, since in vitro work conducted with this endocrine tissue is usually associated with small size cell samples. The method may help for a simple assessment of the viable islet cell mass in in vitro studies.

Effects of Streptozotocin on In Vitro Long-Term Cultured Human Islet Cell Monolayers

Replacement beta cells may be generated from stem/progenitor cells, possibly residing within the pancreatic islet cells. We sought to investigate the possible use of human islet (HI)-derived cell monolayers as a possible source for beta cells. These cells could be propagated in vitro and potentially induced to acquire glucose-stimulated insulin release (GSIR) ability. Loss of three-dimensional architecture, following monolayer establishment, resulted in either rearrangement of both pancreatic hormone expression and key transcription factors or decrease in insulin production or GSIR disappearance. We showed that cell deregulation/dedifferentiation was reversible by treating the cell monolayers, after five passages with streptozotocin (STZ), a well-known beta-cell toxin. When used at subtoxic concentrations, STZ promoted differentiation of HI-derived cell monolayers and GSIR recovery. This effect allowed production of insulin-secreting cells starting from cell monolayers doubled five times in vitro, meaning a 400-fold increase in the cellular starting material. Such islet cell expansion capability in vitro might elucidate a new source of insulin-secreting cells thereby possibly overcoming the problem posed by searching for human organ donation.

Effects of poly-L-lysine and collagen on FH-B-TPN cell differentiation into endocrine cell phenotype.

Pdx-1 genetically engineered FH-B-TPN cells might represent a source for insulin-secreting cells. We then have tested whether poly-L-lysine (PLL) and collagen (C) exposure in vitro promote three-dimensional particle formation and differentiation toward an endocrine cell phenotype. On these matrices, we observed that FH-B-TPN cells showed a tendency to either aggregate when seeded on PLL or to form uniform cell monolayers, but not to aggregate on C. While insulin was released in any condition, GSIR was only associated with PLL mainly at 24 and 72 hours of culture. Various culture matrices influenced the expression of glucose transporter type 2 and gluco kinase, being they expressed more intensively on PLL rather than C or in controls. mRNA expression for NeuroD/Beta2, Isl-1, Ras, Metalloproteinase-2 (MMP-2), -9 and -7 also were affected, with PLL inducing increased expression of NeuroD/Beta2 of Isl-1, and no difference between C and control. PLL, unlike C, strongly increased Ras through observation times. MPP-2 and -9 were decreased by both PLL and C, whereas MMP-7 was increased by PLL. PLL, usually employed to promote culture cell adhesion, has been proven capable to stimulate pancreatic endocrine function and cell aggregation and to stimulate gene expression of key markers for either insulin transcription or MMP-7.

Transplantation of Allogeneic/Xenogeneic Pancreatic Islets Containing Coherent Microcapsules in Adult Pigs 1

WE HAVE PREVIOUSLY described a method for fabrication of alginate/polyaminoacidic CM. Thereafter, we completed assessment of either morphological properties of structural as well as ultrastructural level, or in vitro immunoselectivity, or in vivo post-transplant (Tx) functional performance in diabetic rodents of these new biomembranes for islet Tx immunoprotection in full absence of the recipient’s pharmacological immunosuppression. Because CM, unlike conventional-size microcapsules (CSM), measuring an average 600 to 800 mm in equatorial diameter, hold the unique advantage of occupying a volume which is almost coincident with that of naked islets, these new microimmunobarriers could address a number of unsolved problems with regard to encapsulated islet cell Tx in diabetic large mammalians. One of the most formidable hurdles to this approach for the therapy of insulin-dependent diabetes mellitus has consisted of inadequacy of implant sites for encapsulated islets. Because of the final excessive CSM’s Tx size, these capsules had been only implanted in the peritoneal cavity. Unfortunately, even if highly biocompatible, intraperitoneally grafted, islet-containing microcapsules had often provoked severe, possibly mass-related (60 to 80 mL/diabetic dog) inflammatory cell reaction. Dense connective tissue infiltration invariably resulted in impairment of biochemical exchange, ultimately leading to Tx failure. After testing the new CM in either in vitro functional and immunological or in vivo rodent diabetes-correction studies, we had embarked on assessment of CM biocompatibility, after multiple-site transplantation into adult pigs. Preliminarily, we have shown that empty CM, per se, indeed were biocompatible in this large-size mammal animal model, with a very low degree of sensitization being induced by repeated CM-Tx booster. In an attempt to determine whether CM, because of their minimal size, would permit access to Tx sites so far interdicted to CSM, including parenchymatous organs, with full retention of the encapsulated allogeneic/xenogeneic islet cell viability, we have transplanted porcine or canine islet containing CM into multiple Tx sites, and examined them histologically, at 30 days post-Tx, in normal, adult pigs. MATERIALS AND METHODS Animals