Renal Subcapsular Space Research Articles

PROLONGED SURVIVAL OF NEONATAL PORCINE ISLET XENOGRAFTS IN MICE TREATED WITH A DONOR-SPECIFIC TRANSFUSION AND ANTI-CD154 ANTIBODY1

Combined treatment with a single donor-specific transfusion (DST) and a brief course of anti-mouse CD154 monoclonal antibody (mAb) to induce co-stimulation blockade leads to long-term murine islet allograft survival. The authors hypothesized that this protocol could also induce long-term survival of neonatal porcine islet cell clusters (NPCC) in chemically diabetic immunocompetent mice and allow their differentiation into functional insulin-producing cells. Pancreata from 1- to 3-day-old pigs were collagenase digested and cultured for 8 days. NPCC were recovered and transplanted into the renal subcapsular space. Recipients included chemically diabetic nonobese diabetic (NOD)-scid and C57BL/6 mice that were otherwise untreated, treated with anti-CD154 mAb alone, or treated with DST plus anti-CD154 mAb. Plasma glucose concentration and body weight were measured, and xenografts were examined histologically. NPCC fully differentiated and restored normoglycemia in four of five diabetic NOD-scid recipients but were uniformly rejected by diabetic C57BL/6 recipients. Anti-CD154 mAb monotherapy restored normoglycemia in 4 of 10 (40%) NPCC-engrafted, chemically diabetic C57BL/6 mice, but combined treatment with DST and anti-CD154 mAb restored normoglycemia in 12 of 13 (92%) recipients. Reversal of diabetes required 5 to 12 weeks. Surviving grafts were essentially free of inflammatory infiltrates 15 weeks after transplantation. Combination therapy with a single DST and a brief course of anti-mouse CD154 mAb without maintenance immunosuppression permits survival and differentiation of NPCC in diabetic C57BL/6 mice. Successful grafts were associated with durable restoration of normoglycemia and the absence of graft inflammation.

Importance of the Gal alpha1-3 Gal antigen in discordant islet xenotransplantation: immunosuppression, which inhibits porcine islet xenograft rejection in ordinary mice, is equally effective in Gal-knockout mice.

Islet xenotransplantation will most likely be performed in diabetic patients treated with immunosuppressive drugs. The importance of the galactosyl alpha(1-3) galactose (Galalpha1-3Gal) antigen in immunosuppressed islet xenograft recipients has not been studied. Fetal porcine islet-like cell clusters (ICCs) were transplanted into the renal subcapsular space of both Gal-knockout mice and ordinary mice. Transplantations were performed in untreated mice and mice immunosuppressed with cyclosporine A (CsA) plus 15-deoxyspergualin (DSG). Studies were also performed in immunosuppressed Gal-knockout mice that had been actively immunized against Galalpha1-3Gal. Evaluation was performed 12 days after transplantation using morphologic techniques. The levels of serum immunoglobulin (Ig)G and IgM to the Galalpha1-3Gal antigen or to the ICCs were determined. No difference in the morphologic appearance could be seen between ordinary mice and Gal-knockout mice. No deposits of IgG, IgM, or C3 could be detected. Almost no difference could be seen between immunosuppressed Gal-knockout mice and immunosuppressed ordinary mice. In immunosuppressed, immunized Gal-knockout mice, the results were similar. In ordinary mice treated with CsA+DSG, the levels of anti-Gal IgM were lower than they were in untreated mice, whereas the levels of anti-Gal IgG were similar. In Gal-knockout mice (including immunized animals) treated with CsA+DSG, the levels of anti-Gal IgG and IgM were lower than they were in untreated Gal-knockout mice. After renal subcapsular transplantation, antibodies against Galalpha1-3Gal have no major influence on islet xenograft rejection in the pig-to-mouse model. Immunosuppression, which inhibits rejection in the pig-to-mouse model, is equally effective when transplantation is performed across the Galalpha1-3Gal barrier.

Intraportal pig islet xenotransplantation into athymic mice as an in vivo model for the study of the instant blood‐mediated inflammatory reaction

One of the main obstacles to successful intraportal islet transplantation is the instant blood-mediated inflammatory reaction (IBMIR) elicited by the isolated islets when exposed to fresh human blood. In the present study, we investigated whether intraportal transplantation of pig islets into diabetic athymic mice could be used as a small animal model to study xenogeneic IBMIR in vivo. Adult porcine islets (APIs) or rat islets were implanted into the portal vein or under the renal subcapsular space of diabetic athymic mice. Graft survival and morphology were evaluated by measuring blood glucose levels and by performing immunohistochemical staining, respectively. Transplantation of rat islets, irrespective of implantation site, cured all diabetic athymic mice. APIs transplanted subcapsularly also cured all diabetic athymic mice, while none of the animals transplanted with an equivalent amount of APIs via the portal vein remained normoglycemic for more than 10 days after transplantation. Immunohistochemical staining on day 7 showed that most of intraportally transplanted APIs were entrapped in clots and infiltrated with CD11b+ leukocytes. Intraportal transplantation of APIs into athymic mice induced IBMIR, thus providing a small animal model for studying xenogeneic IBMIR.

Piscine islet xenotransplantation.

Tilapia, a teleost fish species with large anatomically discrete islet organs (Brockmann bodies; BBs) that can be easily harvested without expensive and fickle islet isolation procedures, make an excellent donor species for experimental islet xenotransplantation research. When transplanted into streptozotocin-diabetic nude or severe combined immunodeficient mice, BBs provide long-term normoglycemia and mammalian-like glucose tolerance profiles. However, when transplanted into euthymic recipients, the mechanism of islet xenograft rejection appears very similar to that of islets from "large animal" donor species such as the very popular fetal/neonatal porcine islet cell clusters (ICCs). Tilapia islets are more versatile than ICCs and can be transplanted (1) into the renal subcapsular space, the cryptorchid or noncryptorchid testis, or intraportally as neovascularized cell transplants; (2) as directly vascularized organ transplants; or (3) intraperitoneally after microencapsulation. Unlike the popular porcine ICCs, BBs function immediately after transplantation; thus, their rejection can be assessed on the basis of loss of function as well as other parameters. We have also shown that transplantation of tilapia BBs into nude mice can be used to study the possible implications of cross-species physiological incompatibilities in xenotransplantation. Unfortunately, tilapia BBs might be unsuitable for clinical islet xenotransplantation because tilapia insulin differs from human insulin by 17 amino acids and, thus, would be immunogenic and less biologically active in humans. Therefore, we have produced transgenic tilapia that express a "humanized" tilapia insulin gene. Future improvements on these transgenic fish may allow tilapia to play an important role in clinical islet xenotransplantation.

Production of capsular material by equine trophoblast transplanted into immunodeficient mice.

A novel xenogeneic transplantation approach was used to determine whether it is embryonic or maternal tissue that produces the material that gives rise to the mucin-like glycoprotein of the equine embryonic capsule. Endometrial biopsy samples and conceptuses from six mares at days 13-15 after ovulation were prepared as 1 mm(3) grafts of endometrium, trophoblast and capsule for transplantation, alone or in combination, into various sites in 88 immunodeficient (severe combined immunodeficient or RAG2/gamma(c) double mutant) mice. The overall recovery rate of grafts was over 50%, reaching 100% with experience and use of the renal subcapsular space exclusively. Periodic acid-Schiff (PAS) staining demonstrated capsule-like extracellular glycoprotein secretions at the graft site in 11 of 22 sites examined. Strong PAS-positive reactions (5-7 microm thick) were found in four of six sites containing trophoblast alone, five of six endometrium plus trophoblast sites, and zero of eight grafts of endometrium alone. Two recovered grafts of capsule were also PAS-positive. The secreted glycoprotein was identified as equine embryonic capsule material by using a monoclonal antibody (mAb) specific to equine capsule (mAb OC-1) in two experiments. In the first, in cryosections, this antibody bound to 19 of 19 recovered trophoblast graft secretions (including those in 12 from mice that had not received endometrium at any site), ten of ten recovered endometrium plus trophoblast grafts, and zero of 12 recovered endometrial grafts from mice in which trophoblast had been grafted to the same site or another site in the same mouse. In the second experiment, in paraformaldehyde-fixed sections of grafts from 11 mice, specific staining, identical to that shown by grafted capsule, was obtained with grafts of trophoblast (both alone and in combination with endometrium) but not with grafts of endometrium. These results support the contention that trophoblast is the principal source of equine embryonic capsule. In addition, they demonstrate that xenogeneic grafting is a useful means of culturing endometrium and conceptus tissues outside the mare when in vitro techniques do not suffice.

Production of capsular material by equine trophoblast transplanted into immunodeficient mice

A novel xenogeneic transplantation approach was used to determine whether it is embryonic or maternal tissue that produces the material that gives rise to the mucin-like glycoprotein of the equine embryonic capsule. Endometrial biopsy samples and conceptuses from six mares at days 13-15 after ovulation were prepared as 1 mm(3) grafts of endometrium, trophoblast and capsule for transplantation, alone or in combination, into various sites in 88 immunodeficient (severe combined immunodeficient or RAG2/gamma(c) double mutant) mice. The overall recovery rate of grafts was over 50%, reaching 100% with experience and use of the renal subcapsular space exclusively. Periodic acid-Schiff (PAS) staining demonstrated capsule-like extracellular glycoprotein secretions at the graft site in 11 of 22 sites examined. Strong PAS-positive reactions (5-7 microm thick) were found in four of six sites containing trophoblast alone, five of six endometrium plus trophoblast sites, and zero of eight grafts of endometrium alone. Two recovered grafts of capsule were also PAS-positive. The secreted glycoprotein was identified as equine embryonic capsule material by using a monoclonal antibody (mAb) specific to equine capsule (mAb OC-1) in two experiments. In the first, in cryosections, this antibody bound to 19 of 19 recovered trophoblast graft secretions (including those in 12 from mice that had not received endometrium at any site), ten of ten recovered endometrium plus trophoblast grafts, and zero of 12 recovered endometrial grafts from mice in which trophoblast had been grafted to the same site or another site in the same mouse. In the second experiment, in paraformaldehyde-fixed sections of grafts from 11 mice, specific staining, identical to that shown by grafted capsule, was obtained with grafts of trophoblast (both alone and in combination with endometrium) but not with grafts of endometrium. These results support the contention that trophoblast is the principal source of equine embryonic capsule. In addition, they demonstrate that xenogeneic grafting is a useful means of culturing endometrium and conceptus tissues outside the mare when in vitro techniques do not suffice.

Identification of a Specific Sertoli Cell Marker, Sox9, for Use in Transplantation

The immunoprivileged environment of the testes was first described in the 1930s, and the Sertoli cell was later identified as the main cell type responsible for this phenomenon. Recent work has examined the possibility of recreating this immunoprivileged environment at heterotopic sites using isolated Sertoli cells. These studies have focused on protection of pancreatic islets and neuronal cells from immune destruction in the hopes of reversing type I diabetes and Parkinson's disease. The absence of a definitive marker for identifying Sertoli cells at the transplant site has been an obstacle to this research. The current study examines the presence of a nuclear transcription factor, Sox9, which is preferentially expressed in Sertoli cells. Syngeneic Lewis rat Sertoli cells were transplanted into the renal subcapsular space and a subcutaneous site in Lewis female rats and examined histologically 21 days later. In addition, porcine Sertoli cells were transplanted into the renal subcapsular space in female SCID mice. Control testes and the transplant sites were examined immunohistochemically using an antibody to Sox9. The results from the study demonstrate that Sox9 expression is restricted to the Sertoli cells of the neonatal rat and porcine testis, indicating high homology between species. In addition, Sox9 expression was also observed in the testicular-like tubules that formed in both syngeneic and xenogeneic heterotopic transplants in rats and SCID mice. The Sox9 expression was restricted to the regions where Sertoli cells would be found in the native testis. These results suggest that the Sox9 protein is a useful marker in identifying Sertoli cells in heterotopic transplants in a manner similar to insulin as a marker for pancreatic islets.

A removable tubing for implanting islet graft and studying immunosuppression

THE RENAL subcapsular space is the most common implantation site for experimental islet transplantation. However, the islet graft disperses underneath the renal capsule and is therefore not discerniable when the site is dissected for histologic examination. In this study, we examined the feasibility of using removable subcutaneous hybrid tubing for implantation of islet transplants in streptozotocin-induced diabetic mice.

Enforced c-REL deficiency prolongs survival of islet allografts1.

The NF-kappaB/Rel family of transcription factors regulates biologic processes ranging from apoptosis to inflammation and innate immunity. Whether c-Rel, a lymphoid-predominant member of the NF-kappaB/Rel family, is essential for transplantation immunity is not known. We explored the role of c-Rel in the anti-allograft repertory using mice with targeted disruption of the c-Rel gene (c-Rel-/-) as recipients of H-2 mismatched islet allografts. Allogeneic DBA/2 (H-2d) islets were transplanted into the renal subcapsular space of diabetic c-Rel-/- C57BL/6 (H-2b) mice or the c-Rel +/+ C57BL/6 wild-type mice. Islet graft survival, cellular traffic into the islet grafts and their phenotype, and intragraft expression of cytokines and cytotoxic attack molecules were determined at the protein (by immunohistochemistry) and mRNA (by real-time quantitative polymerase chain reaction) levels. We found superior islet graft survival in the c-Rel-/- recipients compared to c-Rel+/+ C57BL/6 recipients. Splenocytes from c-Rel-/- mice proliferated poorly compared to splenocytes from the c-Rel+/+ mice on stimulation with anti-CD3 mAbs or Con A. Peri-islet infiltration composed of T lymphocytes and macrophages was found in both c-Rel+/+ recipients and c-Rel-/- recipients, but intra-islet infiltration was observed only in c-Rel+/+ recipients. Immunohistologic and molecular studies showed impaired T helper-type 1 immunity and decreased intragraft expression of cytotoxic attack molecules perforin and granzyme B in c-Rel-/- recipients as compared to wild-type recipients. Our results demonstrate that c-Rel is essential for robust rejection of islet allografts and support the idea that strategies that impair c-Rel function may be of value for constraining alloimmunity and facilitating survival of allogafts.

The interaction between primate blood and mouse islets induces accelerated clotting with islet destruction.

Mouse islets transplanted under the renal subcapsular space of cynomolgus monkeys are subject to a form of hyperacute rejection, the mechanism of which is unclear. As islets are in contact with whole blood at the time of transplantation, the effect of platelets and the coagulation cascade on islet destruction was assessed. Coagulation was assessed using thromboelastography on citrated/recalcified human blood samples with freshly isolated C57/Bl6 mouse islets. A dynamic islet perifusion system was used to assess the effect of islets on blood cells and coagulation factors. Cytotoxicity was evaluated using (51)Cr labelled islets incubated with human blood and islet destruction was also evaluated using a histological grading system. Continuous PO(2) measurements were made in a static incubation system to assess the role of hypoxia in islet destruction. Mouse islets incubated in human blood induced accelerated coagulation and rapid consumption of platelets within 15 min. Within 1 h of incubation, 52% of mouse islets exposed to xenogeneic human blood showed features of severe damage with necrosis when compared with islets incubated in syngeneic blood. Specific lysis of the xenogeneic islets was demonstrable (Mean percentage lysis: 48%, P < 0.05 vs. control) after 4 h incubation in human blood. Oxygen levels remained constant at a level adequate to maintain islet viability in separate experiments. Mouse islets induce rapid activation of the clotting cascade and platelet consumption in vitro when exposed to human blood, which correlated with histological evidence of significant destruction demonstrable within 1 h of exposure to human or non-human primate blood. This in vitro model has features which appear to correlate with the islet destruction seen in vivo and could be a useful model for the study of the mechanisms underlying the rapid destruction of xenogeneic islets in primate recipients.

ICAM-1 antisense oligodeoxynucleotide improves islet allograft survival and function.

Expression of intercellular adhesion molecule-1 (ICAM-1) and its ligand, leukocyte function antigen-1 (LFA-1), after pancreatic islet transplantation may affect both nonspecific and alloantigen-specific phases of graft destruction. We examined the effects of ICAM-1/LFA-1 blockade on the survival of islet allografts. Fresh C57BL/10 (H2h) pancreatic islets were transplanted under the renal subcapsular space (KC) or embolized into the liver after portal vein (PV) injection to C3H (H2k) mice. Recipients remained untreated or were treated for 7 days by i.p. administration of: ICAM-1 antisense phosphorothioate oligodeoxynucleotide (oligo) alone; anti-1CAM-1 (alphaICAM-1) monoclonal antibody (mAb) alone: alphaLFA-1 mAb alone; ICAM-1 oligo/alphaLFA mAb combination; alphaICAM-1 mAb/alphaLFA-1 mAb combination; or control oligo IP-8997 or IP-1082. In some experiments, donors were pretreated with ICAM-1 oligo. Inhibition of single ligand with 5.0 mg/kg ICAM-1 oligo (25.1 +/- 10.3), 100 microg/daily alphaICAM-1 mAb (24.2 +/- 8.0 days), or 50 microg/daily alphaLFA-1 mAb (42.8 +/- 25.9 days) prolonged the survivals of KC islet allografts in comparison with untreated controls (11.9 +/- 1.0 days; all p < 0.01). However, dual ICAM-1/LFA-1 blockade with either ICAM-1 oligo/alphaLFA-1 mAb (78.3 +/- 16.5 days) or (alphaICAM-1 mAb/aLFA-1 mAb (65.2 +/- 31.3 days) was the most effective therapy. Although pretreatment of donors with ICAM-1 oligo alone was ineffective (12.2 +/- 0.8 days; NS), a combination of donor pretreatment and recipient treatment started 1 day prior to grafting with ICAM-1 oligo (39.2 +/- 14.0 days) was more effective than the recipient treatment alone (24.6 +/- 8.8 days). Furthermore, ICAM-1/LFA-1 blockade improved islet function as evaluated by glucose tolerance test, and decreased inflammation in comparison with untreated controls. Similar in vivo results were obtained following PV administration of islet allografts. Thus, ICAM-1/LFA-1 blockade prolongs the survival of pancreatic islet allografts and improves their early function.

Grafting of mitomycin C-treated islet xenograft under the kidney capsule produces a clear bleb.

We have previously shown that mitomycin C (MMC) treatment of donor tissue resulted in significant prolongation of graft survival in allo- and xenotransplantation models. However, the mechanisms involved in this prolongation are not clearly understood. This study aims to shed light on the immune responses to MMC-treated islet xenografting under the kidney capsule. Collagenase-digested WS (RT1k) rat islets incubated for 30 min with MMC and subsequently cultured for 20 h were transplanted into the renal subcapsular space of streptozotocin-induced diabetic C57BL/6 (B6;H-2b) mice. The grafts were harvested on postgrafting day 7 and sections were prepared and stained by hematoxylin and eosin (H&E). Histological study of the grafts in a group not treated with MMC showed marked cellular infiltration and destruction of islet clusters, whereas that of MMC-treated grafts demonstrated a bleb formation under the kidney capsule, in which islet cell clusters were reorganized, creating a layer of cells fixed to the interior of the bleb. Minimal invasion by inflammatory cells was observed only at the edge of the bleb, and most islet cells were protected from these infiltrating cells. In conclusion, MMC treatment induces remodeling of islet structure and forms a bleb under the kidney capsule, where no inflammatory cell infiltration occurs, suggesting that this site is a kind of immunologically privileged environment for xenografted islets.

Pretransplant Induction of HSP-70 in Isolated Adult Pig Islets Decreases Early Islet Xenograft Survival

The heat-induced HSP-70 expression protects rat islet single cells against lysis mediated by nitric oxide (NO), reactive oxygen, and streptozotocin. The present study was performed to investigate the potential antiinflammatory effect of pretransplant heat shock in adult pig islets for subsequent early islet xenograft survival. Maximum HSP-70 expression in freshly isolated pig islets was induced by hyperthermia at 43 degrees C for 90 min prior to islet regeneration at 37 degrees C for 4-6 h. Heat-stressed and sham-treated islets were incubated in 0.6 mM H2O2 or 1.5 mM Na-nitroprusside at 37 degrees C for 20 h. Early graft survival was evaluated in normoglycemic Lewis rats after simultaneous, contralateral transplantation of heat-shocked islets and sham-treated islets into the renal subcapsular space of the same recipient. Prior hyperthermia significantly reduced specific lysis of islets exposed to NO or H2O2, although protection was only marginal. No differences were observed between viability of heat-shocked and sham-treated islets after NO exposure. In contrast, prior heat shock increased islet viability after H2O2 treatment. The finding that hyperthermia reduced recovery of initially grafted pig insulin 48 h after transplantation by 30% compared to controls contrasted significantly with an increased insulin recovery in heat-exposed islets at the end of simultaneous 37 degrees C culture. The observation, that the heat-induced HSP-70 expression decreases early islet xenograft survival as reflected by recovery of grafted insulin, implies an enhancement of islet immunogenicity and the induction of apoptosis. Future experiments aiming at augmentation of intrinsic defense mechanisms should consider detrimental effects associated with induction of heat shock proteins.

ANERGIC T CELLS GENERATED IN VITRO SUPPRESS REJECTION RESPONSE TO ISLET ALLOGRAFTS

Induction of antigen-specific unresponsiveness to grafts is the ultimate goal for organ transplantation. It has been shown that anergic T cells generated in vivo can be transferred as suppressor cells. Anergic cells generated in vitro have never been successfully used to prevent allograft rejection in vivo. We examined whether anergic cells generated in vitro by blocking CD28/B7 costimulatory pathway can suppress allograft rejection in vivo. Anergic T cells were generated in vitro by the addition of anti-B7-1 and anti-B7-2 monoclonal antibodies (mAbs) to primary mixed lymphocyte reaction (MLR) consisting of C57BL/6 (B6) splenocytes as responder and irradiated BALB/c splenocytes as stimulator. We tested the ability of these cells to respond to various stimuli and to suppress alloreactive T-cell responses in vitro. For in vivo studies, 4x10(7) anergic cells were injected intravenously immediately after transplantation of BALB/c islets under the renal subcapsular space of streptozotocin-induced diabetic and 2.5-Gy X-irradiated B6 mice. Anergic cells treated with both mAbs in the primary MLR did not proliferate in secondary MLR against BALB/c and third-party C3H/He stimulators. The cells also failed to respond to immobilized anti-CD3 mAb, although they proliferated in response to concanavalin A or phorbol myristate acetate + ionomycin. The anergic state was reversed by the addition of exogenous IL-2. Furthermore, these cells suppressed the proliferation of naive B6 T cells against either the same (BALB/c) or third-party (C3H/He) stimulator cells. In in vivo studies, irradiated B6 mice rejected BALB/c islet allografts acutely with a mean survival time of 27.0+/-8.3 days, whereas two of six animals injected with the anergic cells accepted the allografts indefinitely (>100 days) with a mean survival time of 52.0+/-38.2 days. Anergic cells generated in vitro by blocking CD28/B7 costimulatory pathway suppress islet allograft rejection after adoptive transfer. This procedure might be clinically useful for promoting allograft survival.

Induction of unresponsiveness to islet xenograft by MMC treatment of graft and blockage of LFA-1/ICAM-1 pathway.

Induction of unresponsiveness to graft is one of major interest in xenotransplantation. Two different modalities [direct graft treatment by mitomycin C (MMC) and blockage of the lymphocyte function-associated antigen-1/intracellular adhesion molecule-1 (LFA-1/ICAM-1) pathway in recipients by species-specific mAbs] were tested for their ability to produce unresponsiveness to secondary islet xenografts. Collagenase-digested WS (RT1k) rat islets, purified by Ficoll density gradient, were incubated for 30 min with MMC 10 microg/ml, cultured for 20 hr, and transplanted into the renal subcapsular space of streptozotocin-induced diabetic C57BL/6 (H-2b) mice. Recipient mice were divided into experimental groups according to anti-rat ICAM-1 and/or anti-mouse LFA-1 mAb treatment and transplantation of MMC-treated or nontreated islets. MMC pretreatment alone prolonged graft survival, with a mean survival time (MST) of 23.0+/-7.4 days, compared with that of cultured islets (12.4+/-2.7 days; P<0.01). MMC treatment of islets significantly augmented graft survival, compared with that of crude islet grafts under treatment with anti-donor ICAM-1 mAb (MST: >41.3+/-30 vs. 16.6+/-5.4 days, P<0.01), anti-recipient LFA-mAb (MST: >70.3+/-28.9 vs. 30.4+/-10.4 days, P<0.001), or both mAbs (MST: >88.1+/-24.1 vs. 23+/-7.4 days, P<0.0001). One of six, four of nine, and six of eight animals accepted MMC-treated islet xenografts over 100 days after treatment with anti-rat ICAM-1, anti-mouse LFA-1, or both mAbs treatments, respectively, whereas none of the animals accepted nontreated islets under the same treatment. When the mice bearing long-term functioning xenografts were challenged with the secondary graft from the original donor strain, the animals previously treated with anti-recipient LFA-1 and anti-donor ICAM-1 mAbs were more prone to accept it than animals given anti-recipient LFA-1 mAb alone (MST: 55.8+/-25.7 vs. 15+/-2.4 respectively; P<0.001), although they rejected the third-party xenograft and allograft acutely. In the xenogeneic islet transplantation model, MMC graft pretreatment and blockage of the ICAM-1/LFA-1 pathway constitute a potent protocol for inducing unresponsiveness to islet xenografts.

Hepatocyte growth factor is essential for amelioration of hyperglycemia in streptozotocin-induced diabetic mice receiving a marginal mass of intrahepatic islet grafts.

It is crucial for clinical islet transplantation to find a procedure to improve the success rate of insulin independence after islet transplantation. In the present study, we determined whether hepatocyte growth factor (HGF) has a favorable effect on amelioration of hyperglycemia in streptozotocin (STZ, 200 mg/kg)-induced diabetic mice (C57BL/6) receiving a marginal mass of intrahepatic islet isografts. Isolated syngeneic islets were transplanted into the liver of recipients. HGF with dextran sulfate (DS) was administered intraperitoneally once a day at day 0, 2, 4, 6, and 8 relative to islet transplantation. DS has been known to enhance the effect of HGF. It was found that the number of 250 islets was a marginal mass as donor islets in this model, in which 2 out of 14 diabetic mice receiving 250 islets became normoglycemic by 90 days after transplantation. The treatment with HGF (100 microg) in conjunction with DS (200 microg) produced normoglycemia in all mice (n = 5). Morphological study as well as intraperitoneal glucose tolerance test revealed the beneficial effects of HGF. To our surprise, six out of nine mice receiving 250 islets and treated with DS alone became normoglycemic. Additional anti-HGF antibody treatment (100 microg, day -1, 0, 2, 4, 6, and 8) abolished the effects of DS, indicating that the effect by DS is mediated via the endogenous HGF. The effects of DS were not observed when the renal subcapsular space was the site of islet transplantation. There was a significant increase in plasma HGF levels in mice after the intrahepatic grafts but not the renal subcapsular one. These findings demonstrate that HGF is essential for amelioration of hyperglycemia in STZ-induced diabetic mice when a marginal mass of islets was grafted into the liver. As the liver is the site of clinical islet transplantation and the inability to achieve insulin independence after transplantation is a major obstacle for successful transplantation, HGF may facilitate to overcome such an important issue for clinical islet transplantation.

Thymic transplantation in miniature swine. I. Development and function of the "thymokidney".

Previous studies in our laboratory have demonstrated the importance of the thymus for rapid and stable tolerance induction in an allotransplant model. The focus of the present study was to explore the feasibility of autologous thymic transplantation to produce a new transplantable organ (thymokidney) and to examine the function of subsequent vascularized thymokidney transplants in T cell development. Eight juvenile swine received autologous thymic grafts under the renal capsule. Thymic tissue was obtained through a partial (n=6) or complete (n=2) thymectomy, and growth of the autologous thymic graft was compared between partially and completely thymectomized animals. Two of the partially thymectomized animals received irradiated (1000 cGy) as well as non-irradiated autologous thymic grafts. Graft survival, growth and evidence of thymocyte development was determined by (a) macroscopic examination of the implanted tissue, (b) histological examination, and (c) flow cytometry. Naive CD4 SP T cells were identified by CD45RA-expression. Growth of transplanted thymic tissue was demonstrated in all thymic graft recipients. No difference was seen between partially and completely thymectomized animals. By POD 60, the thymic grafts exhibited normal macroscopic and microscopic structure, and normal thymocyte composition. Irradiated thymic tissue displayed a similar pattern of development, but growth was markedly delayed. To evaluate thymic function of the graft, a composite thymokidney was transplanted into a recipient which had previously been thymectomized, had few circulating CD4-single positive cells and had lost MLR reactivity. The number of CD4+/CD45RA+ cells in this animal increased steadily from POD 30 to POD 150, indicating that the thymus of the composite thymokidney allograft was functional; in addition, MLR assays demonstrated that the recipient recovered immunocompetence. The establishment of a thymokidney by thymic autografting to the renal subcapsular space results in normal thymic growth and function, and may provide a valuable tool for studying the role of the thymus in tolerance induction. As far as we are aware, we provide the first evidence of functional vascularized thymic graft reconstituting T cells and leading to a return of a immunocompetence in a large animal model.

Anergic cells generated in vitro suppress rejection response to islet allografts

Background. Induction of antigen-specific unresponsiveness to grafts is the ultimate goal for organ transplantation. It has been shown that anergic T cells generated in vivo can be transferred as suppresser cells. Anergic cells generated in vitro have never been successfully used to prevent allograft rejection in vivo. We examined whether anergic cells generated in vitro by blocking CD28/B7 costimulatory pathway can suppress allograft rejection in vivo. Methods. Anergic T cells were generated in vitro by the addition of anti-B7-1 and anti-B7-2 monoclonal antibodies (mAbs) to primary mixed lymphocyte reaction (MLR) consisting of C57BL/6 (B6) splenocytes as responder and irradiated BALB/c splenocytes as stimulator. We tested the ability of these cells to respond to various stimuli and to suppress alloreactive T-cell responses in vitro. For in vivo studies, 4310 7 anergic cells were injected intravenously immediately after transplantation of BALB/c islets under the renal subcapsular space of streptozotocin-induced diabetic and 2.5-Gy X-irradiated B6 mice. Results. Anergic cells treated with both mAbs in the primary MLR did not proliferate in secondary MLR against BALB/c and third-party C3H/He stimulators. The cells also failed to respond to immobilized antiCD3 mAb, although they proliferated in response to concanavalin A or phorbol myristate acetate 1 ionomycin. The anergic state was reversed by the addition of exogenous IL-2. Furthermore, these cells suppressed the proliferation of naive B6 T cells against either the same (BALB/c) or third-party (C3H/He) stimulator cells. In in vivo studies, irradiated B6 mice rejected BALB/c islet allografts acutely with a mean survival time of 27.068.3 days, whereas two of six animals injected with the anergic cells accepted the allografts indefinitely (>100 days) with a mean survival time of 52.0638.2 days. Conclusions. Anergic cells generated in vitro by blocking CD28/B7 costimulatory pathway suppress islet allograft rejection after adoptive transfer. This procedure might be clinically useful for promoting allograft survival.

Preventing effect of anti-ICAM-1 and anti-LFA-1 monoclonal antibodies on murine islet allograft rejection.

Immunosuppressive potentials of the blockade of intercellular adhesion molecule-1 (ICAM)-1/leukocyte function-associated antigen 1 (LFA-1) were examined in a murine islet allotransplantation model by using blocking monoclonal antibodies (MAbs) against these molecules. Isolated islets from ICR mice were transplanted into the renal subcapsular space of streptozotocin-induced diabetic C57BL/6 mice. Antibodies were administered immediately after transplantation at a dose of 100 micrograms/mouse/d for 3 or 7 d. In non-treated mice, islet grafts were rejected within 16 d, but the treatment with an anti-ICAM-1 MAb (KAT-1) alone, with anti-LFA-1 MAb (KBA) alone, or with both MAbs significantly prolonged the graft survival. In particular, the combination of KAT-1 and KBA in a 7-d course produced a marked prolongation and induced indefinite graft survivals over 100 d in 88% of recipients. Expression of cytokine transcripts within the islet allografts was analyzed by reverse transcriptase polymerase chain reaction (RT-PCR). In the mice treated with KAT-1 and KBA, the transcripts for Th1 cytokines (interleukin 2 [IL-2] and interferon gamma [IFN-gamma]) were not detected, but the expression of Th2 cytokines (IL-4 and IL-10) was enhanced and persisted over 140 d. In contrast, Th1 cytokines were dominantly expressed in the grafts from untreated mice. These results indicate that administration of anti-ICAM-1 and/or anti-LFA-1 MAbs prolongs murine islet allograft survival potentially by indicating a Th2 deviation.

PRETREATMENT OF ISLETS WITH MITOMYCIN C (MMC) AND BLOCKAGE OF DIRECT ANTIGEN PRESENTATION INDUCE UNRESPONSIVENESS TO ISLET XENOGRAFT

124 Introduction: We have previously shown that rat ICAM-1 on APCs can interact with mouse LFA-1 on T cells across species barrier and blockage of this pathway using species specific mAbs produces prolongation of islet xenograft survival. We have also demonstrated that infusion of donor splenocytes pretreated with MMC, induced unresponsiveness to rat cardiac allografts. In this study we try to minimize immunosuppression and induce unresponsiveness to xenograft using blockage of costimulatory pathway with anti-donor ICAM-1 mAb and graft pretreatment with MMC. Materials and Methods: Collagenase-digested WS (RT1k) rat islets, purified by Ficoll density gradient, were incubated for 30 mins with MMC at a dose 10 µg/ml and subsequently cultured for 20 hrs. These crude 300-400 islets were transplanted into the renal subcapsular space of streptozotocin-induced diabetic C57BL/6 (H-2b) mice. Recipients were given anti-recipient (mouse) LFA-1 mAb or/and mouse anti-rat ICAM-1 mAb at a dose of 0.05 mg i.p. on days 0 and 1 after transplantation. Mice bearing xenografts over 100 days were challenged with freshly isolated crude WS islets. Results: Significant prolongation was obtained by transplantation of MMC-treated islets (group 2 vs. 1). When these islets were transplanted into the animals given anti-mouse LFA-1 mAb or combination of anti-mouse LFA-1 and anti-rat ICAM-1 mAbs, graft survival over 100 days was 44% and 75% respectively. Animals given anti-recipient LFA-1 and anti-donor ICAM-1 mAbs were more prone to accept the second graft than animals given anti-recipient LFA-1 mAb alone (group 5 vs. 4; p<0.001). The results were summarized in the table.TableConclusion: Unresponsiveness to crude xenogeneic islets can be induced by graft pretreatment with MMC and blockage of costimulatory pathway using species specific anti-donor ICAM-1 mAb under low-dose immunosupression with anti-recipient LFA-1 mAb.