The ability to transfer immunoregulatory, cytoprotective, or antiapoptotic genes into pancreatic islets (PIs) may allow enhanced post-transplantation survival. The available gene transfer vectors differ greatly in their ability to infect and express genes in different cell types. One limitation associated with the use of viral vectors is related to the virus reliance on the presence of its primary binding site. Tropism of the viral vectors can be altered using retargeting strategies. Results on phage biopanning proved that the RGD motif has in vivo targeting capabilities. This motif interacts especially with cellular integrins of the alphavbeta3 and alphavbeta5 types, highly expressed on pancreatic islets. In this report, we have explored the utility of a retargeted adenovirus vector (Ad) containing an RGD motif in the HI loop of the fiber knob in order to improve the infection efficiency to intact isolated nonhuman primate PIs and reduce toxicity after the genetic modification. Nonhuman primate Pis were isolated by a semi-automated technique. Steptozotocin-induced diabetic mice with severe combined immunodeficiency disease (SCID) were used as recipients. A recombinant Ad containing a heterologous RGD peptide and expressing luciferase (AdRGDLuc) or green fluorescent protein (AdRGDGFP) were generated in our laboratory. Similar Ads without the RGD peptide were used as a control (AdLuc and AdGFP). Higher transfection efficiency was demonstrated using AdRGDGFP compared with AdGFP (>80% of the islet cells were infected at 10 particle-forming units (pfu)/cell using AdRGDGFP vs. 7% after infection with AdGFP).More than 90% of the infected cells were insulin-producing cells. Significantly higher transgene expression was demonstrated after infection with AdRGDLuc compared with AdLuc at different titers. Analysis of the glucose-stimulated insulin response demonstrated better performance of PI transfected with AdRGDLuc at low titers (10 pfu/cell in order to achieve > 80% transfection efficiency) compared with AdLuc at high titers. Finally, long-term euglycemia (>250d) was observed in 89% of the animals that received PI infected with AdRGDLuc compared with none of the animals that received PI infected with AdLuc. The present study provides new information about the possibility of tropism modification of Ad vectors to increase the transfection efficiency and transgene expression to isolated PI. Incorporation of the RGD sequence in the HI loop of the fiber knob allows highly efficient transfection efficiency to nonhuman primate insulin-producing cells and adequate long-term function of the p-cell after transplantation.

Calpain is activated in experimental uremia: Is calpain a mediator of uremia-induced myocardial injury?The cysteine proteases calpain and caspase-3 are known mediators of cell death. The aim of this study was to assess their contribution to the tissue damage found in experimental uremia.Calpain and caspase-3 activities were measured in the hearts of rats that were sham-operated (control), sham-operated and spontaneously hypertensive (SHR), and those rendered uremic by 5/6 nephrectomy (uremic). In an in vitro study, heart myoblasts (Girardi) were incubated with human serum from healthy subjects (control serum conditioned media, CSCM) or uremic patients (uremic serum conditioned media, USCM), in the presence and absence of calpain and caspase-3 inhibitors. After 48 hours the activity of calpain and caspase-3 was measured, and cell injury determined by DNA fragmentation (ELISA) and lactate dehydrogenase (LDH) release. An in situ assay was designed to study how USCM affects calpain activity over time.In the in vivo study, mean calpain activities were almost identical in the control and SHR groups, but calpain and caspase-3 activities were much elevated in the uremic group (P < 0.01 and 0.001 respectively vs. control). The SHR group had significantly higher mean arterial blood pressure (P < 0.001 vs. control, 0.01 vs. uremic). In the in vitro study calpain activity and DNA fragmentation were markedly higher in USCM treated cells compared to CSCM (both P<0.05). Both were reduced in USCM cells containing calpain inhibitors (E64d, calpastatin, or PD 150606). LDH release was raised also in USCM treated cultures (P < 0.05), which only the E64d treatment could significantly reduce (P < 0.02). Caspase-3 activities were similar in USCM and CSCM groups. The in situ assay showed significant increases in calpain activity in USCM treated cells compared to CSCM after just 3.5 hours (P<0.01).In vivo results suggest that the increases in calpain and caspase-3 activity in uremic rat hearts were primarily due to uremia and not to hypertension. In vitro data demonstrate that uremia-induced cell injury can be attenuated by calpain inhibition. Therefore, it is likely that calpain is a mediator of uremia-induced myocardial injury.

Since its clinical inception, our group has worked in clinical and experimental transplantation. The exciting early results of composite tissue allografts (CTA) attracted our attention during the 1980s, when some of the first experimental CTA literature appeared. The recent extraordinary results of clinical CTA grafts, primarily the Louisville and Lyon results, have been highlighted at this meeting. Interestingly, this success followed not long after the first International meeting in Louisville in 1997. This is clearly an exciting new application of transplant techniques to one of the newest and most unique types of transplants. Previous studies used immunosuppression for skin allografting. Early results seem quite exciting and we expect to see a rapid growth of interest and activity in this area. We believe that the entire field would benefit from the novel, highly effective and minimally toxic immune tolerance induction using CD3-immunotoxin (CD3-IT) and deoxyspergualin (DSG).

859 Introduction: Pancreatic islet transplantation (PIT) is an attractive alternative to whole organ pancreas transplant for insulin-dependent diabetic (IDDM) patients. PIT is not yet an effective clinical reality because of high incidence of rejection, early loss of islet mass and detrimental effects of the current immunosuppressive drugs on islet survival and function. In the present study, the effects of XIT on islet mass and metabolic function were assessed in spontaneously diabetic primates. Methods: Four severely insulinopenic non-human primates (3 Macacca fascicularis and 1 Ceropithecus aethiops) were studied. All required twice daily treatment with 4-10 units of insulin, achieving poor to moderate glucose control. All were treated with insulin at least 6 months before transplant. One animal presented in ketotic coma with severe electrolyte abnormalities and neurologic symptoms. All animals tested exhibited ketones in urine and elevated glycosylated hemoglobin with almost undetectable C-peptide levels. For immunosuppression, the animals received anti-CD3-immunotoxin (100 μg/kg/ initially infused 2 hr pretransplant and again on day +1), CsA (20 mg/kg/iv/2 hr pretransplant), Neoral, 60 mg/kg/BID on day +1-3 with dose adjusted by blood levels, and Methylprednisolone (15 mg/kg day 0 to +3). No immunosuppression of any kind was used beyond day +3 following XIT. Each recipient was given islets from a single donor (Macaca mulatta). The islets were prepared by the semi-automated technique using Liberase. A mean of 15,000 IE/kg were infused into the portal vein. A 4th animal (M. fascicularis) was used as a diabetic, non-transplanted control. Several metabolic parameters were evaluated. Results: All transplanted monkeys experienced reversal of IDDM with normalization of all diabetic glycemic parameters. Current survival after transplantation without evidence of rejection is 100, >285, and >450 days. In the non-transplanted primate given the same immunosuppression but no XIT, diabetic metabolic parameters were unchanged after 6 months follow up. In contrast, all 3 XIT recipients established fasting and non-fasting euglycemia within 1-2 weeks, and none required exogenous insulin after day 10. C-peptide levels increased posttransplant (0.63±0.2 to 3.4±0.45 ng/mL at day 30; 3.95±0.2 at 200 days). Normalization of the intravenous glucose tolerance test was observed at day 15, and no significant differences in the glucose disappearance rate (Km) were observed at day 15, 45, 190 and 365 days posttransplant. No significant reduction of islet mass (evaluated by acute insulin response to glucose) was observed (Pre=2.67±1.1; day 15= 38.62±2.8; day 190=34.24±1.1; day 365=38.02). Normal glycosylated hemoglobin levels were observed approx. at day 60 (Pre=9.0±2.4% to 5.0±0.7%). Conclusions: XIT in severely insulinopenic IDDM primates resulted in restoration of normal glycemic parameters and durable islet mass. All monkeys were free from rejection. Tolerance induction was accomplished with only 4 days of drug administration, sparing the animals from chronic exposure to potentially diabetogenic immunosuppressive drugs. These results offer an exciting new potential for IDDM treatment.

472 Recently, our group and others have demonstrated LSCX in the difficult hamster-to-rat presensitized CX model using short (14 day) suppression, which strongly blocks the B cell and antibody responses. Over 70% of these CX will survive from 100 days to indefinitely, indicating operational tolerance (OT). These results are exciting since this model is a presensitized semi-discordant one and is refractory to prolongation by high doses of drugs such as CsA, etc. OT permits reduction or discontinuance of chronic suppressive drugs and/or provides insights into mechanisms of tolerance. We studied whether the OT was due to changes in the graft or the host. To answer this question, a technique was developed to remove the healed and tolerated LSCX and re-transplant the organ to the abdomen of a second naive Lewis Rat. All 10 LSCX that were re-tx were surviving over 70 days since their primary transplantation. A control group of 10 hamster hearts to Lewis Rats using CsA (10mgm/kgm) and cyclophosphamide (CYP, 10mgm/kgm × 5 days) received the same immunosuppression as the re-transplant. The mean survival of these CX control group was 10.3 +/3.8 days and all rejected. The mean graft survival of the retransplanted LSCX was 58 +/-11 days (p>0.01). In addition, a second naïve hamster CX was performed in the neck of the Lewis Rat with the LSCX (n=3). The mean survival time was 11.9 +/- 4.6 days (p>0.10 with primary CX to naïve recipient). Interestingly, the CDC and the ADCC antibody titers were both strikingly diminished following the re-tx of the LSCX in the naïve LR (CDC decreased by 70%, ADCC by 95%, p<0.05 compared to primary grafts). The findings provide straightforward in vivo evidence that the mechanism of the LSCX was associated with intrinsic changes in the graft immunogenicity and are not related to recipient immune accommodation. The LSCX was hypoimmunogenic evidenced by a prolonged survival in a second naïve host and by a decreased evoked antibody response, both CDC and ADCC types. The long survivorship could not be explained by recipient tolerance since syngeneic naïve donor hearts were rejected normally in recipients with LSCX. In summary, a change in intrinsic immunogenicity of LSCX associated with long survival in the host and demonstrated by direct testing was shown. This prolonged survival appeared to be due to re-endotheliazation of the LSCX. In contrast, no changes in the host immune system to explain the long survival were evident since a second CXG in the tolerant host animal was rejected normally.

321 Isolated islet transplantation with tolerance induction which obviates chronic immunosuppression (CI) is an exciting new potential therapy for early treatment of juvenile diabetes. This treatment could be essentially non-invasive, would not require hospitalization and would minimize morbidity and mortality for the juvenile diabetic. To date, long tolerance to unencapsulated islets in a primate model has not been reported. An African Vervet primate developed acute ketotic coma with a blood sugar (BS) of 950mgm%. He was resuscitated but required two daily insulin injections still resulting in poor diabetic control (mean blood sugars of 204 +/-480mgm%). The serum insulin levels were undetectable on 12 occasions when the animal had short-term (48-72 hour) discontinuance of exogenous insulin for metabolic testing. The animal had a normal body mass index with no obesity. C-peptide levels (vs. African Vervet controls) were 0 and stimulated C-Peptide<0.4ngm. After 5 months of difficult treatment, the animal was considered for sacrifice or isolated islet transplant (IIT). IIT was performed by infusion of 50,000 rhesus IE into the portal vein. The recipient was treated with only 3 injections of a novel receptor specific anti-CD3-ε immunotoxin, and 4 days of CyA and steroids. The CD3-IT caused a striking depletion of both circulating and sessile T cells (sessile cells in lymph nodes were decreased by a log 2 factor compared to other T cell ablative agents used). No further immunosuppression was given. The BS returned to the 100-300 range with lowered doses of insulin by day 3 and the animal was euglycemic (BS 45-105) by day 8 off insulin. The recipient is now 120 days post-transplant. He has a normal C-Peptide level, normal first-phase insulin secretion, normal glucose tolerance (both IV and oral) and normal levels of serum insulin (all based on controls for normal African Vervets). Glycosylated Hb was abnormal (>10% pre-transplant) and is now normal(<4%). The animal has had no evident rejection episodes and no toxicity from the anti-CD3-ε immunotoxin. He is on a regular diet and eugycemic by daily testing. There was a mild weight increase with reversal of the diabetes. In summary, IIT with a unique tolerance induction protocol has permitted stable tolerance of xenogenic rhesus islets in a pre-clinical African Vervet primate off all immunosuppression. The animal had a "brittle" severely insulinopenic diabetes which was totally reversed. To our knowledge, this is the only primate currently stable with reversed spontaneous severely insulinopenic diabetes off all immunosuppression following IIT with non-encapsulated xenogenic islets. This study suggests that tolerance induction to isolated allogenic or xenogenic islets is feasible in pre-clinical models and is a demonstration of the potential application of islet tolerance to man.

279 XG studies both, clinical and experimental have now spanned a period of 35 years with slow progress. Newer concepts may be needed to speed ultimate clinical application. We performed a series of studies to test a new hypothesis of GAT simply implying that the immunomodulation resulting in long term cardiac xenograft survival (LSCX) is intrinsic to the XG itself and does not correlate with any known measures of host immunity to the XG. In the well studied hamster to Lewis rat cardiac xenograft (CX) model, immunosuppression with CyA and cyclophosphamide (CP) as well as ATG and DSG and ATG-DSG and splenectomy (SPLX) were able to produce LSCX (>60 days, control 7 ±2 days, p<0.01). In a combined series of 50 CX., the length of survivals and antibodies to donor (CDC and ADCC) varied between series but no consistent patterns were seen. ATG-DSG-SPLX animals (14) had significantly lower levels of antibodies (both CDC and ADCC) but survivals were not significantly different (p>0.10). Re-grafting of the primary XG to a naïve recipient with short-term CsA-CP and ATG-DSG suppression (n=11) gave a mean survival of 67 ±16 days whereas a primary (naïve) XG to a naïve animal with these suppressive protocols give a 19 ±4 day survival (p<0.01). Thus, the most direct test of the GAT hypothesis indicated that long survival associated with operational tolerance (OT) was a property of the graft (GAT) and not immunodeviation of the XG recipient. Importantly, anti-XG antibody levels did not correlate with XG survival or rejection with 4 animals having no detectable antibodies above baseline. Survival in the CyA group with a primary XG correlated precisely with CyA administration with graft rejection occurring 3-7 days after stopping CyA despite no change in AB titers. In summary, these in vivo studies with in vitro correlates designed to directly test the GAT hypothesis indicate the LSCX associated with XG changes but were not explained by anti-donor xenograft antibodies. LSCX status was a function of immunosuppressive protocols such as CyA which do not clearly affect humoral antibodies a factor also not weighted in the HA theory.

813 Introduction: According to current concepts, dendritic cells (DC) in peripheral blood (PB) and in organ transplants represent immature precursors (DCp) that process antigen but lack costimulatory molecules for presentation. DCp maturation is driven by proinflammatory cytokines that depend on activation and nuclear translocation of NF-κB transcription factors, particularly Rel-B. We have observed in nonhuman primates (NHP) that, following depletion of T cells in lymph nodes (LN) and blood after day-of-transplant induction with anti-CD3 immunotoxin (IT), stable tolerance to MHC mismatched renal allografts is favored when proinflammatory cytokine responses are inhibited by brief treatment with the NF-κB inhibitor, 15-deoxyspergualin (DSG). This finding led us to examine whether the synergistic effect of DSG in this model might be to block DCp maturation during the T cell recovery phase after IT induction. Methods: Normal 3 kg male rhesus monkeys received a renal allograft from an unrelated, MHC mismatched donor. Immunosuppression consisted of 3 injections of IT (150 μg/kg total dose over days 0 to +2, methylprednisolone (MP) tapered from 7 mg/kg/d to 0 over days 0 to +3, and DSG (2.5 mg/kg/d) on days 0 to 4 (n=5) or 0 to +14 (n=5). Additional recipients had IT with MP alone (n=3) or MP plus Neoral (120 mg/kg/d) on days 0-4(n=3). Inguinal and axillary LN biopsied at 5, 15, and 30 days were examined by immunohistochemistry for presence of mature DC expressing membrane CD83, DR, and CD86 and nuclear Rel-B. To directly examine DCp maturation, we cultured isolated rhesus PB DCp in medium containing monocyte conditioned medium (MCM) plus TNFα. DSG (0.6-10 mg/ml) was added to DCp cultures prior to adding MCM/TNFα. Expression of DC maturation markers was assessed by flow cytometry and by immunocytochemical staining of cytocentrifuge preparations. Results: Renal allografts survived without histologic or clinical evidence of acute or chronic rejection in 80% of IT-treated recipients given MP plus DSG ×15d, in 40% in those given MP plus DSG × 5d, and in 0% of those given only MP or MP plus Neoral. The 3 longest DSG survivors are>830 days (2.3 years) with normal renal function and immunologic indication of specific tolerance. Compared to LN from transplants without DSG, the day +5 LN tissue from DSG-treated recipients showed extreme reduction in mature DC, i.e., a mean 58-fold±9.9 reduction in membrane expression for CD83, 49-fold ± 9.0 for CD86, and 81-fold±13.5 for DR. Rel-B nuclear positive cells were reduced 46-fold±7.1. Partial recovery occurred by day 15, and by day 30, DR+, CD83+, and Rel-B+ cells were within control range. CD86+ cells were still reduced (by 7.5-fold ±1.8). The results are consistent with NF-κB inhibition and DCp maturation arrest by DSG. A dose-dependent DSG effect was confirmed by in vitro analysis of MCM/TNFα-driven DCp maturation. The results showed a shift from nuclear and membrane staining to cytoplasmic staining of Rel-B and CD83, respectively, reflecting a dominant immature DCp phenotype in the DSG treated cultures. Conclusions: These studies show the NF-κB inhibitor, DSG, blocks maturation of DCp in vivo and in vivo in NHP. We hypothesize that the unusual synergy of IT and DSG in promoting tolerance in this model is due in part to depletion of memory as well as naïve T cells by IT. Conceptually, this would create a transitional circumstance in which repopulating naïve T cells are committed to DC costimulatory requirements for activation, while DCp acquisition of costimulatory function is arrested by DSG. The net result is a fleeting lapse into a "non-dangerous" milieu, akin to the neonate's, coincidentally reduced both in T cell mass and DC maturity, thereby favoring tolerance.

267 The traditional in vivo test for DST is re-transplantation of a second graft from the donor. Usually a donor skin graft (DSG) is the common standard. Recently, a variety of studies from our group and others have suggested wide spreads in the differences between skin and organ graft survivals in a host of DST models. In addition, immunobiological differences in rejection mechanisms of skin and organs such as kidney and heart have been documented. We tested primary and secondary donor grafts in a rat model of DST using pre-transplant donor spleen injections and ATG. Cardiac allografts (ACI→Lewis) were markedly prolonged to 73 ±14 days (ATG control = 26 ±4 days). Subsequent ACI cardiac grafts retransplanted in the abdomen, survived 63 ±16 days (p>0.10 with first cardiac grafts). In contrast, primary ACI skin grafts in recipients treated with a similar ATG-spleen extract tolerance induction protocol gave ACI skin graft survival of 42.3 ±2.6 days (p<0.01 with skin graft controls). Naïve ACI skin grafts transplanted to Lewis recipients harboring a tolerant skin graft were rejected in 9 ±2.4 days. Naïve ACI skin grafts transplanted to Lewis rat recipients harboring a ACI heart graft were rejected at 11.3 ±4 days (p>0.10 with grafts to naïve recipients). Finally, removal of the ACI hearts followed by skin grafts 2 weeks later did not change the skin graft survival (9.6 ±3.0 days). In contrast, third party skin and heart grafts were all rejected in 7-10 days (all skin graft rejections were biopsy-proven). Removal of the tolerant heart did not appear to affect the skin graft survival. Thus, there was an extraordinary spread between a DST challenge skin graft and organ graft survival. Similar disparities have been observed in 4 other models of tolerance including 1 xenogenic model. We conclude that the DST is a suboptimal and often invalid measure of donor-specific tolerance in both allogeneic and xenogenic DST models. This is probably due to a certain uniqueness of skin graft rejection mechanisms demonstrated in other studies. True DST should be validated by placement of a similar second donor organ and not a DST.

477 In clinical islet transplantation for autoimmune diabetes, the majority of islet grafts are lost to early ADR. Thus, ADR prevention is of great importance in promoting long term survival. Discordant xenograft islets (DXI) have been shown to resist ADR in early studies in the NOD mouse. ADR was defined as histopathological damage confined selectively to the B cells whereas in rejection all islet cells were equally damaged. In addition, insulin:glucagon ratios in the stained islets are >2 +/0.6 in rejection but <1 in ADR. In these present studies, we compared allografts, concordant Lewis rat xenografts and discordant xenografts (pig islet xenografts) in NOD mice. In all, 60 mice were transplanted in 3 groups. All had a 3-day course of rabbit antithymocyte globulin (RATG) and 14 days of deoxyspergualin (DSG). Group 1 (N=20) had 500 C3H mouse allogenic islets transplanted. Group 2 animals (n=20) had 500 Lewis Rat islets (concordant xenografts). In group 3, (n=20) animals had 500 pig islets. In the allograft group all 20 grafts were lost by 40 days with 16 of the grafts lost to ADR and 4 grafts lost to rejection as diagnosed by pathology. In the concordant rat xenograft, there were 15 graft losses to ADR and 5 to rejection(p<0.10 with the allografts). In the PIX group, all grafts survived over 100 days and there were no grafts lost to ADR or early rejection (p<0.05 with 2 other groups). Function of the PIX was documented by differential RIA assay at 100 days and by loss of euglycemia after nephrectomy. In summary, the study demonstrates that the important goal of prevention of ADR can be engineered by modification of the target organ antigens. In vitro studies demonstrated that the PIX were resistant to lysis by NOD cells and NOD serum whereas allograft and concordant xenograft islets were susceptible to destruction. The MLC, CML and CTLp were lower to the tolerated PIX cells suggesting that the autoimmune response to transplanted islets involve T cell reactivity. The low rate of discordant graft loss to rejection has been noted in other series and associates with low T cell reactivity between discordant species. The results suggest that an important technique for tolerizing a graft to ADR is the use of resistant target epitopes, especially discordant but not concordant xenografts. This system may be applicable to diabetes, hepatitis and other autoimmune disease, which recurs in transplants.

69 Introduction: Pancreatic islet transplantation (PIT) suffers from a high incidence of rejection, early loss of islet mass and detrimental effects of immunosuppressive drugs on islet viability and function. In this study the effects of XIT on serial islet metabolic function were assessed in Primates with IDDM. Methods: Four severely insulinopenic non-human primates (3 Macacca fascicularis and 1 Ceropithecus aethiops) were studied. All required twice daily treatment with 4-10 units of insulin, achieving poor to moderate glucose control (glucose 300-500 mgm%). All were treated with daily insulin at least 6 months before transplant. One animal presented in ketotic coma with severe electrolyte abnormalities and neurologic symptoms. All animals tested had Ketosis and elevated glycosylated hemoglobin with almost undetectable C-peptide levels. Four animals received anti-CD3-immunotoxin infused 2 hr pre-transplant and again on day +1), CsA (20 mg/kg/iv/ 2 hr pretransplant), Neoral, 60 mg/kg/BID and Methylprednisolone 15 mg/kg on day 0 to +3. No immunosuppression of any kind was used beyond day +3 following XIT. One animal served as a control to rule out suppressive effects on autoimmunity. Three recipients were given islets from a single donor (Macaca mulatta). The islets were prepared by the semi-automated technique using Liberase. A mean of 15,000 IE/kg was infused into the portal vein. Several metabolic parameters were evaluated. Results: All transplanted monkeys experienced reversal of IDDM with normalization of all diabetic glycemic parameters 20 days. Current survival after transplantation without evidence of rejection is 120, >320, and >510 days. In the non-transplanted primate given the same immunosuppression but no XIT, diabetic metabolic parameters were unchanged after 6 months follow up. In contrast, all 3 XIT recipients established fasting and non-fasting euglycemia within 1-2 weeks, and none required exogenous insulin after day 10. C-peptide levels increased post-transplant (0.63±0.2 to 3.4±0.45 ng/mL at day 30; 3.95±0.2 at 200 days). Normalization of the intravenous glucose tolerance test was observed at day 15, and no significant differences in the glucose disappearance rate (Km) were observed at day 15, 45, 190 and 365 days post-transplant. No significant reduction of islet mass (evaluated by acute insulin response to glucose) was observed (Pre=2.67±1.1; day 15=38.62±2.8; day 190=34.24±1.1; day 365=38.02). Normal glycosylated hemoglobin levels were observed approx. at day 60 (Pre=9.0±2.4% to 5.0±0.7%). All animals had stable and normal insulin resistance. Conclusions: XIT in severely insulinopenic IDDM primates resulted in restoration of normal glycemic parameters and durable islet mass. All monkeys were free from rejection. Tolerance induction was accomplished with only 3 days of drug administration, resulting in long-term stable islet function. Biopsies showed islet-secreting tissue in the recipient liver and the native pancreas showed destroyed islets. These results show durability of XIT and offer an exciting new potential for IDDM treatment.

176 Pig Islets (PI) are clearly the xenogeneic donor islet of choice for human islet transplant. Pig insulin is known to work well in the human without antibody problems. Furthermore, newer short-term immunosuppression can markedly reduce early graft loss with PI and tolerance induction can even be demonstrated in some discordant xenografts. Isolation of PI remains a major problem with islet fragmentation and islet loss during digestion. In addition, PI undergo a high rate of apoptosis following isolation in culture which can result in 50-95% loss of PI in 24-48 hours. We initially observed that PI embedded in extracellular adherent matrix exhibited an Anchorage-Dependent cell growth and survival with viability of >80% (Ethidium Bromide/Acridine Orange Assay - EB/AO) at 20 days (p<0.01 with controls). We tested the so-called “Tensegrity Concept” that cell viability and growth in culture depends on adhesion and extracellular integrin structuring and“scaffolding”. Tensegrity is optimal when the cell cytoskeleton and the extracellular matrix integrate to achieve a physiological balance of intra and extracellular mechanical tension. By shortening collagenase digestion to yield PI attached to acinar matrix cell remnants, we sought to provide a potential extracellular structure to develop tensegrity. Control PI digestions showed 95% purity with pure islets free of attached acinar and extracellular matrix elements, but a loss of EB/AO viability to 40% and 10% in 24 and 48 hours. In contrast, the matrix-embedded PI had a purity of only 52% but an EB/AO viability of 93% and 86% at 24 and 48 hours and 65% at 30-35 days(p<0.05) At 1 month, the acinar tissue had largely died but fibrous strands remained attached to the cells. Islet function in static incubation was excellent with a insulin rise of 420% in response to a increased glucose incubation solution (normal for fresh islets=320%,p=>0.10). Thus, incomplete digestion of PI with retention of attached elements of the acinar and extracellular matrices apparently supplies a tensegrity (extracellular matrix scaffolding) which promotes long term growth and viability of PI in culture. We conclude that incomplete digestion of islets and/or imbedding islets in extracellular matrix markedly enhances the viability and long term yield of PI and maybe the technique of choice for PI digestion and preparation of PI for culture and/or transplantation.

471 In contrast to allograft tolerance, LSFT of XG has few defined parameters and few long term XG to permit detailed studies. We developed a model of cardiac xenograft (CXG) tolerance in the hamster - to rat model using 3 doses of a rabbit - anti-thymocyte globulin splenectomy and 14 days of deoxyspergualin (DSG) at 2.5 mgm/kgm. In 10 animals, 6 or 60% survived past 100 days (mean of 142 days) off immunosuppression and thus had optimal tolerance, while 4 animals rejected their [email protected] mean of 48 +/-18 days(p<0.05). Tolerant recipients studied at 100 days showed no correlation between T cell parameters (MLC CML or p-CTL) and survival. A' significant difference between the two groups was the anti-donor antibody parameters. Both CDC and ADCC antibodies were significantly lower in 100% of LSFT animals(mean of 1:256 vs. 1:2048 in the rejecting animals,p value<0.05) at 14-30 days post transplant. Past 36 days neither CDC nor ADCC antibodies were different between the 2 groups (1:1024 vs. 1:2048, p>0.10). In summary, LSFT of XG is characterized by similar T cell parameters suggesting the T cell was not central to XG rejection or tolerance. LSFT animals in contrast to non-tolerant animal groups had an early block in both CDC and ADCC antibodies up to 30 days indicating a positive correlation of LSFT and early CDC and ADCC block demonstrated to be particularly effective in animals using this immunosuppression treatment. The best correlate of LSFT was a low CDC and ADCC at 1-30 days post-transplant. The low levels of anti-donor antibody had a excellent correlation with a low incidence of long term severe vasculopathy (r=0.86) in the LSFT group. Vasculopathy was an invariable correlate of rejection in animals with early graft loss and conversely was minimal in the LSFT animals. These results indicate a close correlation of anearly (0-30 days) block in anti-donor antibody and long term xenograft survival. Later rises in CDC and ADCC past 30-40 days showed no correlation with either rejection or long term tolerance. Thus, anti-donor antibody patterns critical to graft survival are established early after treatment.

307 Both viral and non-viral vectors have been quite limited in their ability to accomplish highly efficient gene delivery to relevant target cellsin vivo. As an alternative vector strategy, parenchymal cells have been employed. In this approach, cells are removed from the body and genes are transferred into the cells ex vivo, followed by reimplantation. Thus, the transduced cell becomes the ultimate vector for gene delivery. The role of circulating CD34+ cells as endothelial progenitors, or angioblasts, has recently been described. We sought to develop a novel cellular vector for gene therapy that may allow the delivery of therapeutic molecules into foci of angiogenesis. METHODS: We isolated CD34+ cells from rhesus monkeys. Bone marrow was obtained after mobilization with G-CSF, and mononuclear cells were isolated by Ficoll sedimentation. CD34+ cells were then purified by column immunoabsorption. Cells were maintained in plastic wells in culture medium enriched with IL-3, IL-6, and SCF at 37C in a humidified, 5% CO2 atmosphere. Cells were transduced ex vivo with TOZ.1, a highly efficient (>99%) and nontoxic herpes vector encoding both the reporter geneLacZ and thymidine kinase. Infection was done with a multiplicity of infection (MOI) of 3 infectious particles per cell for 12 hours. To evaluate the localization capacity of CD34+ cells into areas of angiogenesis, we established an in vivo model based in skin autografts. In addition, we implanted s.c., in a separate region, Matrigel and Gelfoam impregnated with the vascular endothelial growth factors VEGF and basic FGF. Transduced cells were infused intravenously two days after graft and pellet implantation. Biopsies were obtained periodically from the graft and pellets. Transduced cells expressing LacZ-encoded β-galactosidase were revealed by X-gal staining, and an antibody against factor VIII was used as endothelial marker. As a further means to show the expression of genes delivered by CD34+ cells at the angiogenesis areas, treatment with ganciclovir (GCV) was given (5 mg/kg i.v. for 14 days) 15 days after CD34+ cell infusion.RESULTS: Both in the skin graft and implants, numerous small, round, intensely blue cells were identified in neovascular structures stained by factor VIII. After 10 days of treatment with GCV, the subcutaneous pellets disappeared, suggesting conversion of GCV to its toxic metabolites in the angiogenesis sites. Animals nontreated with GCV, in contrast, kept palpable implants during the study time. Importantly, no symptom or sign of toxicity has been observed in three different primates infused with TOZ.1-transduced CD34+ cells. CONCLUSIONS: CD34+ cells can indeed localize into areas of angiogenesis and express reporter and toxin genes therein. This allows evaluating their potential as cellular vehicles with capacity for localization and gene delivery into areas of angiogenesis, including graft implants.

308 PI transplantation (tx) is an attractive alternative for the treatment of diabetes mellitus, has the advantage of a low morbidity tx procedure, relatively low cost, and the possibility of islet genetic modificationex vivo and the potential of cell protection, tolerance induction with eventual tx without immunosuppression. In spite of the attractiveness, it has been difficult to implement and is not a clinical reality. Islet damage and programmed cell death (apoptosis) occurs during pancreas procurement, preservation, islet isolation, culture and early after transplantation until complete vascularization is achieved. The family of Bcl-2 related proteins constitutes one of the most biologically relevant classes of apoptosis-regulatory gene products. In the present study, we study the possibility of cytoprotection of PI induced by genetic modification with an adenovirus-mediated gene transfer of the Bcl-2 gene during long-term islet cultures. METHODS: An adenovirus vector (ΔE1A) containing an expression cassette with the human Bcl-2 gene under the control of a cytomegalovirus promoter (AdCMVhBcl-2) was developed. This vector was validated with endothelial cell apoptosis assays induced. The expression of AdCMVBcl-2 in PI was demonstrated by Western blot. An adenovirus vector with an irrelevant gene (E coli β-galactosidase, AdCMVLacZ) was used as a control. Islets were isolated from normal 25-30 g BALB/c mice with standard collagenase digestion technique, hand picked, and assessed by dithizone staining. Islet suspensions were washed and cultured at 24 °C. For gene transfer 1000 pfu of the different adenoviral vectors were used. After the transfection, the islets were washed and cultured at 37 °C in RPMI with 10% FCS for 7, 13 and 30 days. DNA fragmentation (apoptosis), was determined using a sandwich ELISA (Bohringer-Mannheim), which measures cytosolic histone-associated DNA fragments (mono and oligonucleotides). Three experimental groups were studied: Group 1=Control islets without adenovirus, Group 2=Islets transfected with AdCMVLacZ, and Group 3= Islets transfected with AdCMVhBcl-2. DNA fragmentation values are presented as O.D. at 405nm.RESULTS: The rate of adenoviral transfection of PI, as assessed by the expression of the LacZ marker gene was ≈92% at 1000 pfu. The level of DNA fragmentation compared in the different group is shown in the followingfigure:CONCLUSIONS: PI cytoprotection induced by genetic modification with AdCMVBcl-2 decrease the apoptosis process during culture. Genetic modification of PI with gene therapy can make these cells more resistant to cell death and will allow better results in PI transplantation.

309 Ischemia/reperfusion (I/R) injury to the liver is of major importance in numerous clinical situations including hepatic surgery, liver transplantation(LT), shock states, and thermal injury. It has been demonstrated that apoptosis occurs in the period of cold ischemia, anaerobic rewarming, and reperfusion in LT. Strategies to protect the liver from damage by I/R are essential. In the present study, we examined the possibility of gene transfer of the anti-apoptotic Bcl-2 gene into the liver to reduce the I/R injury.METHODS: Normal C57B1/6 mice were used. An adenovirus (ΔE1) vector containing an expression cassette with the human Bcl-2 gene under the control of a cytomegalovirus promoter (AdCMVhBcl-2) was developed. An adenovirus vector encoding an irrelevant gene (E coliβ-galactosidase, AdCMVLacZ) was used as a control. The expression of AdCMVhBcl2 vector was documented by Western Blot and functionally validated in apoptotic studies in endothelial cells. Taken the advantage of the hepatotropic properties of adenovirus vectors gene transfer experiments were performed with 1 × 109 pfu by i.v. injections via tail vein, 48 hrs before the induction of the injury. I/R injury was induced by temporal and segmental occlusion of the hepatic blood flow. For functional analysis, the medial largest lobe of the liver was clamped at its base using a microaneurysm clamp for 15 min., and then the clamp was gently removed. For survival studies, the superior mesenteric artery (SMA) was occluded (decrease of approx. 80% of the total flow to the liver) for 30 min. After the ischemic period, the abdominal wall was sutured. Sham animals were treated in an identical fashion with the omission of the vascular occlusion. AST and ALT activities were measured using a standard assay with an Ames Seralyzer. Liver biopsy specimens were obtained before and 5 hrs post I/R injury.RESULTS: In the functional studies, animals from the Control Group(Naive) shown a significant increase in AST to 2.58±0.14 UI/g body weight (normal=0.56±0.1) and ALT 1.16±0.1 UI/g body weight(normal=0.46±0.12); animals transfected with an irrelevant adenovirus (Group 2) shown a similar increase in AST (2.58±0.08) and ALT (1.28±0.07). In contrast, animals transfected with the anti-apoptotic hBcl-2 gene shown a significant reduction in the transaminases levels (AST 0.89±0.09 and ALT 0.80±0.02). The expression of hBcl-2 after 6 hrs of the I/R injury was confirmed by Western Blot. Preliminary studies shown that the mortality of normal mice with 30 min of SMA occlusion is 97% at 24 hrs. The survival of sham operated animals was 100%, the AdCMVLacZ group 33% at 24 hrs, 0% at 48 hrs, and animals transfected with AdCMVhBcl-2 survive 100% and are currently alive after 2 months. CONCLUSIONS: In this study, we found the possibility of genetic modification of the liver with an adenovirus vector encoding the expression of the anti-apoptotic gene Bcl-2 to decrease the I/R injury. The genetic modification of the liver to induce cytoprotection has potential applications for prevention of I/R injury in hepatic surgery including LT.

509 The exciting possibility of inducing tolerance to isolated pancreas islets(PI) makes the potential of PI transplantation in young diabetics an ethically realistic therapy since it avoids morbidity and mortality from a pancreas transplant and also avoids chronic immunosuppression (CI). Studies from our group and others have shown prolonged survival of MHC class I and class II deficient (C1D and C2D) transgenic donor islets in incompatible allogeneic and xenogeneic hosts. In allogeneic grafts, none of the grafts survive over 60 days without suppression and all are eventually rejected despite their prolonged survival. In 10 C1D and CD2 donor islets transplanted to normal CS7BL6 mice, low doses of CsA (5mgm/kg day) produced IPI survivals>100 days (p<0.05 with non-suppressed islets) but stopping the CsA resulted in islet rejection in 7-21 days. In contrast, a 21 day treatment(days 0-21) with cyclophosphamide (1mgm/kg) and deoxyspergualin (2.5mgm/kg) resulted in indefinite survival of CD1 and CD2 islets (>300 days in 9 of 10 animals, p<0.05). These results indicate that early short-term treatment (0-21 days) can set the stage for an operational tolerance with long-term graft survival without chronic suppression in C1D and C2D islets. Gene therapy may permit engineering of class I and II deficient islets by genome modification or in vitro treatment of islets before transplant which could then be easily incorporated into a short non-chronic (21 day) immunosuppressive regimen that appears to markedly prolong C1D and C2D islet survival by an early tolerogenic effect on these islets which induces a long term operational tolerance (stable graft function without CI).