A review of piscine islet xenotransplantation using wild-type tilapia donors and the production of transgenic tilapia expressing a "humanized" tilapia insulin.

Most islet xenotransplantation laboratories have focused on porcine islets, which are both costly and difficult to isolate. Teleost (bony) fish, such as tilapia, possess macroscopically visible distinct islet organs called Brockmann bodies which can be inexpensively harvested. When transplanted into diabetic nude mice, tilapia islets maintain long-term normoglycemia and provide human-like glucose tolerance profiles. Like porcine islets, when transplanted into euthymic mice, they are rejected in a CD4 T-cell-dependent manner. However, unlike pigs, tilapia are so phylogenetically primitive that their cells do not express α(1,3)Gal and, because tilapia are highly evolved to live in warm stagnant waters nearly devoid of dissolved oxygen, their islet cells are exceedingly resistant to hypoxia, making them ideal for transplantation within encapsulation devices. Encapsulation, especially when combined with co-stimulatory blockade, markedly prolongs tilapia islet xenograft survival in small animal recipients, and a collaborator has shown function in diabetic cynomolgus monkeys. In anticipation of preclinical xenotransplantation studies, we have extensively characterized tilapia islets (morphology, embryologic development, cell biology, peptides, etc.) and their regulation of glucose homeostasis. Because tilapia insulin differs structurally from human insulin by 17 amino acids, we have produced transgenic tilapia whose islets stably express physiological levels of humanized insulin and have now bred these to homozygosity. These transgenic fish can serve as a platform for further development into a cell therapy product for diabetes.

Encapsulated piscine (tilapia) islets for diabetes therapy: studies in diabetic NOD and NOD-SCID mice.

BACKGROUND: Our goal was to improve islet transplantation as a therapy for patients with type I diabetes mellitus. Because human donor islets are scarce, we are studying islet xenografts in the diabetic NOD mouse model. We hypothesize that optimal xenoislet survival will be achieved by the combination of donor islet immunoisolation with recipient immunosuppression. We and others have studied adult and neonatal porcine islets as sources of tissue for microencapsulated islet xenografts, but we believe it is also advantageous to consider using islets from fish, which can be raised in large numbers relatively quickly and economically. Therefore, in this study, we have evaluated the function of microencapsulated xenogeneic piscine (tilapia) islets transplanted intraperitoneally (IP) in NOD mice in the presence of CD4(+) T-cell depletion and/or costimulatory blockade. METHODS: Spontaneously diabetic NOD mice or streptozotocin (STZ)-diabetic NOD-SCID mice were transplanted IP with microencapsulated tilapia islets. Recipient immunosuppression included anti-CD4 mAb, CTLA4-Ig, anti-CD80 mAb, anti-CD86 mAb, or anti-CD154 mAb, alone or in combination. Graft function was evaluated by blood glucose (BG) levels, intravenous (IV) and oral glucose tolerance tests (GTTs), histologic and immunohistochemical analyses of grafts, and flow cytometric analysis of peritoneal cells. RESULTS: Encapsulated tilapia islets normalized random BG levels for up to 210 days in NOD-SCID mice. In diabetic NOD mice, encapsulated tilapia islets were rejected on day 11 ± 4 with a peritoneal infiltrate of macrophages, eosinophils, B cells, occasional neutrophils, but few T cells. Immunohistochemical staining demonstrated the presence of murine IgG on tilapia islets within capsules of rejecting, non-immunosuppressed mice, as well as murine IgG-positive lymphocytes in the layer of host cells surrounding those capsules. These findings suggested that our barium (Ba)-gelled alginate capsules are permeable to IgG and that anti-piscine antibodies may be involved in the rejection of encapsulated tilapia islets in untreated mice. No single immunosuppressive agent prolonged encapsulated tilapia islet survival in NOD mice, but the combination of CTLA4-Ig plus anti-CD154 mAb extended tilapia islet graft survival until rejection at 119 ± 20 days and inhibited host cell recruitment to the peritoneal cavity. Triple treatment with CTLA4-Ig, anti-CD154 mAb, and anti-CD4 mAb allowed graft survival for 157 ± 35 days with little evidence of a host cellular reaction. IV and oral glucose tolerance tests (GTTs) of recipients with functioning xenografts demonstrated remarkably normal metabolic function. CONCLUSIONS: We conclude that microencapsulated tilapia islets can survive long term with excellent metabolic control in diabetic mice given targeted immunosuppression, suggesting that cross-species physiological incompatibility may not compromise the applicability of this novel approach for future clinical applications. We predict that an improved microcapsule that prevents the entrance of IgG will enhance tilapia islet survival in this model, possibly allowing the application of this technique with limited or no immunosuppression.

Local autoantigen expression as essential gatekeeper of memory T-cell recruitment to islet grafts in diabetic hosts.

It is generally believed that inflammatory cues can attract noncognate, "bystander" T-cell specificities to sites of inflammation. We have shown that recruitment of naive and in vitro activated autoreactive CD8⁺ T cells into endogenous islets requires local autoantigen expression. Here, we demonstrate that absence of an autoantigen in syngeneic extrapancreatic islet grafts in diabetic hosts renders the grafts "invisible" to cognate memory (and naive) T cells. We monitored the recruitment of islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)206₋214-reactive CD8⁺ T cells into IGRP206₋214-competent and IGRP206₋214-deficient islet grafts in diabetic wild-type or IGRP206₋214(-/-) nonobese diabetic hosts (harboring either naive and memory T cells or only naive IGRP206₋214-specific T-cells, respectively). All four host-donor combinations had development of recurrent diabetes within 2 weeks. Wild-type hosts recruited IGRP206₋214-specific T cells into IGRP206₋214(+/+) but not IGRP206₋214(-/-) grafts. In IGRP206₋214(-/-) hosts, there was no recruitment of IGRP206₋214-specific T cells, regardless of donor type. Graft-derived IGRP206₋214 activated naive IGRP206₋214-specific T cells, but graft destruction invariably predated their recruitment. These results indicate that recurrent diabetes is exclusively driven by autoreactive T cells primed during the primary autoimmune response, and demonstrate that local antigen expression is a sine qua non requirement for accumulation of memory T cells into islet grafts. These findings underscore the importance of tackling autoreactive T-cell memory after ß-cell replacement therapy.

Cloning and molecular characterization of the glucose transporter 1 in tilapia (Oreochromis niloticus).

Facilitative glucose transporters (GLUTs) are responsible for passively transporting monosaccharides across the plasma membrane. We sequenced and characterized the Nile tilapia (Oreochromis niloticus) GLUT-1 (tGLUT-1) cDNA and genomic DNA. Using rapid amplification of the cDNA ends (RACE), two tGLUT-1 transcripts were detected differing in the length of the 3' untranslated region, 2851 and 4577 bp. Translated tGLUT-1 is a 490 amino acid product, which shares 74% homology with that of humans. Computer analysis of the amino acid sequence predicted 12 transmembrane domains, which are conserved in the GLUT-1 of various species. The tGLUT-1 gene spans more than 11 kb, and similar to the mammalian GLUT-1 genes has a 10 exon, 9 intron organization. Potential promoter regulatory elements have some similarity to those recorded for human, mouse, and rat GLUT-1 genes. Tissue expression studies revealed both GLUT-1 transcripts in liver, Brockmann bodies (BB), heart, small intestine, adipose tissue, white and red muscle, gill, spleen, pituitary gland, and brain. The highest level of expression was detected in tilapia heart, followed by BB, brain, and muscle. Protein based food and glucose had minor or no effects on the level of tGLUT-1 expression in most tissues. The tGLUT-1 mRNA level was significantly induced by glucose and food only in white muscle. Current results suggest that tGLUT-1 is similar to the GLUT-1 of other teleost species and mammals at the genomic, mRNA, and amino acid levels, supporting the concept that tGLUT-1 functions as a ubiquitous basal level glucose transporter.

Ligand-dependent induction of noninflammatory dendritic cells by anergic invariant NKT cells minimizes autoimmune inflammation.

Stimulated by an agonistic ligand, alpha-galactosylceramide (alphaGalCer), invariant NKT (iNKT) cells are capable of both eliciting antitumor responses and suppressing autoimmunity, while they become anergic after an initial phase of activation. It is unknown how iNKT cells act as either activators or regulators in different settings of cellular immunity. We examined effects of alphaGalCer administration on autoimmune inflammation and characterized phenotypes and functional status of iNKT cells and dendritic cells in alphaGalCer-treated NOD mice. Although iNKT cells became and remained anergic after the initial exposure to their ligand, anergic iNKT cells induce noninflammatory DCs in response to alphaGalCer restimulation, whereas activated iNKT cells induce immunogenic maturation of DCs in a small time window after the priming. Induction of noninflammatory DCs results in the activation and expansion of islet-specific T cells with diminished proinflammatory cytokine production. The noninflammatory DCs function at inflammation sites in an Ag-specific fashion, and the persistence of noninflammatory DCs critically inhibits autoimmune pathogenesis in NOD mice. Anergic differentiation is a regulatory event that enables iNKT cells to transform from promoters to suppressors, down-regulating the ongoing inflammatory responses, similar to other regulatory T cells, through a ligand-dependent mechanism.

Things we have learned from tilapia islet xenotransplantation.

An islet xenotransplantation model has been developed using tilapia (Oreochromis niloticus) as the donors. Studies using this model for the treatment of experimental type 1 diabetes in mice have produced promising results including the maintenance of long-term normoglycemia and mammalian-like glucose tolerance profiles in islet graft recipients. Islet encapsulation has also provided a promising method for the prevention of graft rejection, and strains of transgenic tilapia expressing a [desThrB30] human insulin molecule have been produced. In addition to studying islet transplantation for the treatment of type 1 diabetes, these studies have also produced insights into piscine glucose homeostasis. Studies demonstrating the glucose responsiveness of tilapia islets are described. In addition, work performed by our group and by others pertaining to presence and nature of piscine glucose transporters is reviewed. Finally, studies addressing some of the broader challenges of islet xenotransplantation are discussed with particular attention paid to the post-transplantation fate of the various islet cell populations and the proteins they produce.

Rapid destruction of encapsulated islet xenografts by NOD mice is CD4-dependent and facilitated by B-cells: innate immunity and autoimmunity do not play significant roles.

BACKGROUND: Spontaneously diabetic NOD mice rapidly reject microencapsulated islet xenografts via an intense pericapsular inflammatory response. METHODS: Tilapia (fish) islets were encapsulated in 1.5% alginate gel microspheres. Recipients in series 1 were spontaneously diabetic NOD mice and streptozotocin-diabetic nude, euthymic Balb/c, prediabetic NOD, and NOR (a recombinant congenic strain not prone to autoimmune diabetes) mice. Recipients in Series 2 were STZ-diabetic NOD, NOD-scid, NOD CD4 T-cell KO, NOD CD8 T-cell KO, and NOD B-cell KO mice. RESULTS: In Series 1, encapsulated fish islet grafts uniformly survived long-term in nude mice but were rejected in Balb/c and, at a markedly accelerated rate, in spontaneously diabetic NOD, streptozotocin-diabetic NOD and NOR recipients. Histologically, intense inflammation (macrophages and eosinophils) surrounding the microcapsules was seen only in NOD and NOR recipients. In Series 2, encapsulated fish islets uniformly survived long-term in NOD-scid and NOD CD4 KO mice; graft survival was markedly prolonged in B-cell KO (P<0.001) but not CD8 KO mice. CONCLUSIONS: The rapid rejection of alginate encapsulated islet xenografts by NOD mice is not solely a consequence of beta-cell directed autoimmunity nor is it merely a vigorous innate immune response. Graft rejection requires CD4 T-cells, is facilitated by B-cells, and does not require CD8 T-cells.

Effect of glucose toxicity on intraportal tilapia islet xenotransplantation in nude mice.

BACKGROUND: Discordant xenogeneic islets transplanted intraportally into athymic nude rats experience primary non-function and are rapidly destroyed. Recently, it has been reported that adult porcine islets transplanted intraportally into nude mice are also rapidly destroyed and that this constitutes a new model for instant blood-mediated inflammatory reaction (IBMIR). METHODS: Tilapia (fish) islets were harvested, mechanically broken into mammalian islet-sized fragments, cultured for 48 h, and transplanted via the portal vein into athymic or euthymic mice. RESULTS: There were several groups of recipient mice. Streptozotocin-diabetic nude mice received 400 islets via the portal vein (n = 12). Recipients were killed when hyperglycemic (>200 mg/dl); livers and native pancreases were examined histologically. Mean graft survival time, based on function, was 5.4 +/- 1.2 days; at autopsy, histology showed occasional viable islets. We also performed a group of transplants in non-diabetic nude mice (n = 6) and then killed the recipients 2 or 4 weeks later; all had abundant viable, well-granulated islet grafts based on histology. Therefore, the intraportal environs in nude mice are not incompatible with discordant fish islets; rather, it appears as if hyperglycemia adversely affects the intraportal islet grafts (i.e. ''glucose toxicity''). To test this hypothesis, transplants were performed into non-diabetic nude mice and allowed to engraft for either 3 days (n = 6) or 10 days (n = 8) prior to injection of streptozotocin (200 to 220 mg/kg i.v.) to destroy the beta-cells in the recipients' native islets (n.b. tilapia islets are exceedingly resistant to streptozotocin); these recipients were followed for 28 days post-transplantation (or until hyperglycemic) and then killed for histology. Mean graft function exceeded 25 days for both groups and viable well-granulated, tilapia islets grafts were readily identified in all recipients; in all but one, the native pancreases were markedly beta-cell depleted -- confirming that normoglycemia was due to functional fish islet xenografts. CONCLUSIONS: Our results suggest that ''glucose toxicity'' plays a role in the immediate demise of intraportal tilapia islet xenografts.

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.

Tilapia islet grafts are highly alloxan-resistant.

We have previously shown that dose-response studies performed in streptozotocin (STZ)-diabetic nude mouse recipients bearing established, functioning islet xenografts can be used to directly compare in vivo STZ-sensitivity between donor species and that tilapia (fish) islet grafts are exceedingly STZ-resistant. Using this method, we tested whether tilapia islets are sensitive to alloxan. Tilapia or rat islets were transplanted under the renal capsules of STZ-diabetic nude mice. Recipients with normal glucose tolerance tests (GTTs) on day 30-35 were injected with increasing i.v. doses of alloxan and blood glucose levels were followed for 5-7 days and then GTTs were repeated. Next, mice were killed and their grafts/native pancreata examined histologically (including insulin stains). Control nude mice were also injected with increasing i.v. doses of alloxan. Based upon non-fasting blood glucose levels, GTT, and graft histology, the following observations were made: (1) Tilapia islet xenografts were uniformly resistant to i.v. doses of 75 mg/kg (n=3), 150 mg/kg (n=4), and 300 mg/kg (n=3). (2) Rat islet recipients became uniformly severely diabetic after alloxan i.v. doses of 50-70 mg/kg (n=6) (i.e., equivalent to the dosage needed to induce diabetes in rats). (3) Control nude mice were severely diabetic at doses of 75 mg/kg (4/5) and 150 mg/kg (n=3/3). Alloxan dose-response studies were also performed in tilapia. Interestingly, tilapia appeared more sensitive than tilapia islet grafts. Although 75 mg/kg i.v. had little effect in tilapia, higher doses caused severe beta cell necrosis, diabetes, and systemic damage; however, this seeming discrepancy can be explained as tilapia have about one-quarter of the blood volume of mice (i.e., as a percentage of body weight) and so the actual concentration in the blood was about 4-fold higher at each dose. We conclude that tilapia beta cells are highly resistant to the beta cell toxin alloxan.