Pancreatic islet isolation variables in non-human primates (rhesus macaques).

BACKGROUND: Non-human primates (NHPs) are important preclinical models for pancreatic islet transplantation (PIT) because of their close phylogenetic and immunological relationship with humans. However, low availability of NHP tissue, long learning curves and prohibitive expenses constrain the consistency of isolated NHP islets for PIT studies. To advance preclinical studies, we attempted to identify key variables that consistently influence the quantity and quality of NHP islets. METHODS: Seventy-two consecutive pancreatic islet isolations from rhesus macaques were reviewed retrospectively. A scaled down, semi-automated islet isolation method was used, and monkeys with streptozotocin-induced diabetes, weighing 3-7 kg, served as recipients for allotransplantation. We analysed the effects of 22 independent variables grouped as donor factors, surgical factors and isolation technique factors. Islet yields, success of isolation and transplantation results were used as quantitative and qualitative outcomes. RESULTS: In the multivariate analysis, variables that significantly affected islet yield were the type of monkey, pancreas preservation, enzyme lot and volume of enzyme delivered. The variables associated with successful isolation were the enzyme lot and volume delivered. The transplant result was correlated with pancreas preservation, enzyme lot, endotoxin levels and COBE collection method. CONCLUSIONS: Islet quantity and quality are highly variable between isolations. The data reviewed suggest that future NHP isolations should use bilayer preservation, infuse more than 80 ml of Liberase into the pancreas, collect non-fractioned tissue from the COBE, and strictly monitor for infection.

Cloning and characterization of recombinant rhesus macaque IL-10/Fc(ala-ala) fusion protein: a potential adjunct for tolerance induction strategies.

The powerful anti-inflammatory and immunosuppressive activities of IL-10 make it attractive for supplemental therapy in translational tolerance induction protocols. This is bolstered by reports of IL-10-mediated inhibition of innate immunity, association of human stem cell and nonhuman primate (NHP) islet allograft tolerance with elevated serum IL-10, and evidence that systemic IL-10 therapy enhanced pig islets survival in mice. IL-10 has not been examined as adjunctive immunosuppression in NHP. To enable such studies, we cloned and expressed rhesus macaque (RM) IL-10 fused to a mutated hinge region of human IgG1 Fc to generate IL-10/Fc(ala-ala). RM IL-10/Fc(ala-ala) was purified to approximately 98% homogeneity by affinity chromatography and shown to be endotoxin-free (<0.008 EU/microg protein). The biological activity of IL-10/Fc(ala-ala) was demonstrated by (1) costimulation of the mouse mast cell line, MC/9 proliferation in a dose-dependent fashion, (2) suppression of LPS-induced septic shock in mice and (3) abrogation of LPS-induced secretion of proinflammatory cytokines/chemokines in vitro and in vivo in NHP. Notably, RM IL-10/Fc(ala-ala) had significantly greater potency than human IL-10/Fc(ala-ala) and exhibited a circulating half-life of approximately 14 days. The availability of this reagent will facilitate definitive studies to determine whether supplemental therapy with RM IL-10/Fc(ala-ala) can influence tolerance outcomes in NHP.

Insulin secretion from pancreatic islets in fibrin glue clots at different fibrinogen and thrombin concentrations.

BACKGROUND: Fibrin glue has proven to be a good delivery system for cell transplantation but the factors that influence the fibrin-cell relationships are not well understood. The purpose of this study was to assess the effect of different concentrations of fibrin glue components (thrombin and fibrinogen) on the function of pancreatic islets. METHOD: Islets were isolated from rat pancreata and combined with 6 different fibrin glue formulations. Each islet sample was incubated sequentially with RPMI containing low and high glucose, and culture supernatants were harvested for insulin determination using enzyme-linked immunosorbent assay (ELISA). RESULTS: The control group (no fibrin glue) and group 3 (with thrombin 50 U/mL and fibrinogen 10 mg/mL) had the highest insulin secretion in response to glucose stimulation. These were followed by groups 5 and 4 with 2.6 and 1.8 stimulation indexes, respectively. Group 2 (with thrombin 50 U/mL and fibrinogen 5 mg/mL) and group 6 (commercial kit with thrombin 250 U/mL and fibrinogen 75-115 mg/mL) had the lowest insulin response after glucose stimulation. CONCLUSION: This study demonstrates that different fibrin glue formulations significantly impact pancreatic islets function. In the future, when using fibrin glue as a carrier for pancreatic islet transplantation, lower concentrations of fibrinogen and thrombin are recommended to obtain more viable and functional grafts.

Islet yield after different methods of pancreatic Liberase delivery.

OBJECTIVE: Enzymatic digestion of the pancreas is a fundamental step in islet isolation and there are many ways to administer the enzyme during procurement. The aim of this study was to evaluate the influence of different methods of Liberase delivery during pancreas harvest on the quality and quantity of islets. METHODS: Depending on the type of Liberase delivery, 4 groups were created. Group 1 was intraductal, Group 2 was interstitial, Group 3 was intragallbladder, and Group 4 was no infusion of enzyme. After injection, the pancreata were harvested, digested in Liberase solution, mechanically disrupted, and purified using discontinuous gradient centrifugation. After 24-hour culture, the number, purity, and viability of the isolated islets were determined. RESULTS: Intraductal injection of the enzyme yielded statistically significantly more islets per mouse when compared with interstitial, intragallbladder, and no injection administration. Although there was a trend toward better islet purity and viability for Group 1, this was not statistically significant. CONCLUSION: Intraductal administration is the best enzyme delivery method for pancreatic islet isolation. The pancreatic ducts are the most anatomic and physiological way to transport the enzyme uniformly inside the pancreas, determining an adequate digestion and better islet quantity and quality when compared with other delivery methods.

Subcutaneous pancreatic islet transplantation using fibrin glue as a carrier.

BACKGROUND: Pancreatic islet grafts are difficult to manipulate and implant in the recipient site mainly because they are formed by a group of cells suspended in a solution. This physical property determines various characteristics that are unique for pancreatic islet transplantation. The purpose of this study was to evaluate the role of fibrin glue as a delivery method for islet transplantation. METHODS: C3H mouse islets were syngeneically transplanted into streptozotocin-diabetic recipients using fibrin glue in a subcutaneous pocket (Group 1) and using liquid islets injected under the kidney capsule (Group 2). Blood glucose levels were measured during 4 weeks of follow-up and compared against normal (Group 3) and diabetic levels (Group 4). RESULTS: No statistical differences were observed between the normal, kidney capsule, and fibrin glue groups. Only the diabetic group had a statistical difference when compared with the normal control group (P < .01). At the beginning, levels in Group 1 (fibrin glue) were higher than in Group 2 (kidney capsule), but turned into similar values after time and no statistical differences were observed between them during follow-up. CONCLUSIONS: Islet/fibrin glue grafts placed in a subcutaneous pocket obtained the same results as liquid grafts placed under the kidney capsule, proving to be an adequate delivery method for islet transplantation and solving some of the engraftment problems we find with liquid grafts.

Coxsackievirus-adenovirus receptor genetically fused to anti-human CD40 scFv enhances adenoviral transduction of dendritic cells.

A promising approach to immunotherapy involves the loading of dendritic cells (DCs) with genetic material to facilitate sustained expression of a relevant antigen in this population of potent antigen presenting cells (APC). Viral vectors such as adenovirus (Ad) have been used for this purpose. Existing methods for DC infection are limited by lack of specificity and a requirement for DC exposure to high viral doses. Targeting of Ad to DCs with bispecific antibodies has significantly augmented levels of transgene expression. Genetic fusion of the extracellular portion of coxsackievirus-adenovirus receptor (CAR) to cell-specific ligands has also proved successful in targeting Ad to cells of interest. We report here the production and primary characterization of a new fusion protein comprising the ecto-domain of CAR connected to a single chain antibody (scFv) G28-5 against human CD40 present on the surface of DCs. We demonstrate that the fusion protein (CAR/G28) specifically interacts with both recombinant Ad fiber knob and the ecto-domain of human CD40 in a binding assay (ELISA). Finally, we show that the CAR/G28 fusion protein promotes highly efficient transduction of DCs of both rhesus monkey and human origin.

Peritransplant tolerance induction in macaques: early events reflecting the unique synergy between immunotoxin and deoxyspergualin.

BACKGROUND: Day of transplant T cell depletion with anti-CD3 immunotoxin or F(Ab)2 immunotoxin induces stable tolerance to renal allografts in rhesus monkeys given 15-deoxyspergualin (DSG), a NF-kappaB inhibitor that suppresses proinflammatory cytokine (PC) production. Because PC and NF-kappaB are involved in dendritic cell (DC) maturation, we asked if impaired DC maturation and Th2-type cytokine deviation might be related to the synergistic effect of DSG in this novel model. METHODS: Immunosuppression was initiated 4 hr before transplanting a major histocompatibility complex mismatched renal allograft. Some groups received a supplemental 5-day course of cyclosporine A or DSG or a 15-day course of DSG. Peripheral lymph nodes were sequentially examined for presence of mature DC. In vitro effects of DSG on PC-induced maturation of DC were also examined. RESULTS: Allografts survived without rejection in 87% of recipients given immunotoxin or F(Ab)2 immunotoxin with DSG x 15 days, in 50% with DSG x 5 days, and 0% with cyclosporine A. The longest DSG survivors are >1000 days with normal graft function and tolerance validated, including acceptance of challenge second donor kidneys without treatment. DSG-treated recipients were unique in developing polarized Th2-type plasma cytokines. In DSG recipients, mature DC were significantly reduced in day +5 lymph node biopsies, with complete repopulation by 30 days. In vitro studies verified an inhibitory effect of DSG on DC maturation. CONCLUSIONS: The study suggests DSG arrests DC maturation. The unusual synergy of immunotoxin and DSG apparently involves coincidental reduction in lymph node T cell mass and mature DC, a transient circumstance favoring development of stable tolerance.

Immunoregulatory role of CD8alpha in the veto effect.

BACKGROUND: Allogeneic bone marrow cell (allo-BMC) infusion induces tolerance to incompatible renal allografts in rhesus macaques after depletion of peripheral T lymphocytes with cytolytic anti-T cell antibodies. The tolerogenic effect of allo-BMC, ascribed to a veto mechanism, associates with specific functional deletion of antidonor cytotoxic T lymphocyte precursor (CTLp), and is dependent on a CD8+ donor BMC subset. In previous studies, the CD8 molecule was implicated by loss of suppression after blocking interaction between CD8 on allo-BMC and major histocompatibility complex class Ialpha3 domain on CTLp. CD8 cross-linking on BMC induced secretion of active transforming growth factor-beta1 (TGF-beta1), suggesting a regulatory mechanism(s) operating via a CD8-mediated signaling pathway. METHODS: CD8 on rhesus cells was cross-linked using IgG-conjugated beads, and TGF-beta1 mRNA and protein were quantified. CD8+ cells were tested for veto activity by mixed lymphocyte reaction (MLR)-induced cell-mediated lymphocytotoxicity (CML) assay. Activated rhesus T cells exposed to TGF-beta1 were examined for apoptosis by TdT-mediated end-labeling and annexin staining. RESULTS: CD8 cross-linking induces accumulation of TGF-beta1 mRNA and protein. Both CD3- CD8+CD16+ and CD3+ CD8+CD16- subsets of allo-BMC up-regulate TGF-beta1 mRNA after CD8 cross-linking, and exhibit veto activity. The CD3-CD8+CD16+ subset expresses more TGF-beta1 mRNA and increased veto activity at low BMC/CTLp ratios. Exposure of activated T cells to TGF-beta1 induces apoptosis. CONCLUSIONS: CD8+ allo-BMC are enriched for veto activity and activation via CD8 induces TGF-beta1 mRNA and protein accumulation. These results agree with the hypothesis that paracrine TGF-beta1 may be involved in peripheral deletion of alloreactive CTLp by CD8+ allo-BMC. We suggest that TGF-beta1 overexpression by donor lymphohematopoietic cells may enhance tolerance induction.

Molecular cloning, expression and characterization of rhesus macaque Fas ligand cDNA.

The Fas/Fas ligand (FasL) interaction is pivotal in apoptosis-mediated regulation of the immune system. As such, it has relevance in areas of transplantation, gene therapy, AIDS, etc., all of which utilize the rhesus macaque as a preclinical animal model. In order to examine rhesus Fas/FasL, we cloned the rhesus FasL cDNA and have analyzed the function of the cloned gene. Our findings indicate that the rhesus FasL is highly homologous to the human but not the mouse (97% for human, 85% for mouse). In addition, soluble rhesus FasL can induce apoptosis, a property shared with the human soluble protein but not the mouse protein. The deduced protein sequence is 280 amino acids with a calculated Mr. of 31,646. Transfection of COS cells with the full-length cDNA yielded a 40 KD protein, which is in agreement with the size of human FasL. COS cells expressing rhesus FasL induced apoptosis in rhesus PHA blasts and human Fas+ CEM-6 cells. Thus, the cloned rhesus macaque FasL is functional and cross-reacts with human Fas. The cloned functional rhesus FasL cDNA will enable studies of its regulatory role in the nonhuman primate immune system.

Peritransplant tolerance induction with anti-CD3-immunotoxin: a matter of proinflammatory cytokine control.

BACKGROUND: Tolerance is gaining momentum as an approach to reduce lifelong immunosuppressive therapy while improving transplant longevity. Anti-CD3 immunotoxin (IT), FN18-CRM9, has potential to induce tolerance owing to its exceptional ability to deplete sessile lymph node T cells. However, if initiated at the time of transplantation, alpha-CD3-IT alone elicits a proinflammatory cytokine response, precluding establishment of tolerance. METHODS: Four groups of rhesus monkeys received kidney allografts and immunosuppression. Three groups received alpha-CD3-IT alone or alpha-CD3-IT supplemented with 15-deoxyspergualin (DSG) and/or methylprednisolone (MP). One group received alpha-CD3-monoclonal antibody with DSG and MP. Cytokines were measured by enzyme-linked immunosorbent assay. RESULTS: Supplementing peritransplant alpha-CD3-IT treatment with a brief course of DSG and MP promoted rejection-free kidney allograft acceptance in 75% of macaques followed for up to 550 days. Among those given alpha-CD3-IT alone or with MP, none were long-term survivors. Tolerance developed after alpha-CD3-IT, DSG, and MP treatment, but not when the unconjugated a-CD3 monoclonal antibody was substituted for IT. Systemic production of proinflammatory cytokines interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha induced after peritransplant alpha-CD3-IT was prevented only in animals given DSG. Despite high levels of interleukin (IL)-12 in the first month after transplant, tolerant recipients exhibited IL-12 resistance, as evidenced by baseline plasma levels of IFN-gamma but elevated IL-4. DSG was shown to inhibit IL-12-driven IFN-gamma production by a mechanism associated with inhibition of nuclear factor kappa-B. CONCLUSIONS: In this model, peritransplant induction of tolerance is promoted by efficient elimination of sessile lymph node T cells and control of the proinflammatory IFN-gamma response by a mechanism that appears to involve resistance to IL-12.

Complex regulation of CDK2 during phorbol ester-induced hematopoietic differentiation.

Phorbol myristate acetate (PMA) treatment of U937 human leukemic cells results in late G1 cell cycle arrest and terminal monocyte/macrophage-like differentiation. The PMA-induced G1 arrest involves a marked decrease in cdk2 activity, which correlates with total cdk2 dephosphorylation. Here, we show that the levels of cyclin A mRNA and protein markedly decrease during PMA-induced differentiation of U937 cells. In contrast, the level of cyclin E protein remains unchanged and in a complex with cdk2 during the entire course of PMA treatment. During the PMA-induced differentiation, cyclin E-associated cdk2 activity drops markedly. Furthermore, the amount of p27(Kip1) protein associated with cyclin E/cdk2 greatly increases 24 to 72 hours after PMA treatment. The absence of changes in p27(Kip1) mRNA levels by Northern blot suggest that the levels of this protein are controlled by posttranscriptional or posttranslational mechanism(s). These results show that the mechanisms mediating PMA-induced G1 arrest are complex. The inhibition of cdk2 activity is associated with (1) a decrease in cyclin A protein levels, (2) inactivation of cdk2 complexes, and (3) upregulation of p27(Kip1) protein.

Inhibition of leukemic cell growth by the protein kinase C activator bryostatin 1 correlates with the dephosphorylation of cyclin-dependent kinase 2.

Bryostatin 1 is a natural antineoplastic agent that activates protein kinase C. Treatment of U937 human leukemic cells with bryostatin 1 caused a 60% reduction in cell growth, whereas another protein kinase C activator, phorbol myristate acetate (PMA), completely inhibited U937 cell growth. Both bryostatin 1 and PMA induced inhibition of cyclin-dependent kinase 2 (cdk2) activity. The first phase of cdk2 inhibition correlated with the transient induction of p21, a known inhibitor of cdk2. In contrast, the second phase of cdk2 inhibition correlated with the dephosphorylation of cdk2 on threonine-160, which must be phosphorylated for cdk2 activity. The level of growth inhibition induced by these two compounds correlated with the degree of cdk2 dephosphorylation as follows: bryostatin 1, 60%; PMA, 100%.

Increasing binding of a transcription factor immediately downstream of the cap site of a cytomegalovirus gene represses expression.

A closely related family of ubiquitous DNA binding proteins, called MDBP, binds with high affinity to two 14 base pair (bp) sites within the human cytomegalovirus immediate early gene 1 (CMV IE1) enhancer and with low affinity to one site beginning 5 bp downstream of the CMV IE1 transcription start point (+5 site). Unlike several cap position downstream MDBP sites in mammalian genes, these MDBP sites do not require cytosine methylation for optimal binding. Mutation of one of the enhancer MDBP sites to prevent MDBP recognition modestly increased the function of a neighboring CREB binding site in a transient transfection assay in the context of one promoter construct. A much larger effect on reporter gene expression (a 10-fold reduction) was seen when the low affinity MDBP recognition sequence at position +5 was converted to a high affinity site in a plasmid containing the CMV IE1 promoter upstream of the reporter gene. Evidence that the increased binding of MDBP at the mutant site is largely responsible for the observed results was provided by transfection experiments with this high affinity MDBP +5 site re-mutated to a non-binding site and by in vitro transcription assay.

Binding of AP-1/CREB proteins and of MDBP to contiguous sites downstream of the human TGF-beta 1 gene.

Transcription of the human gene encoding transforming growth factor beta 1 (TGF-beta 1), which is a key regulator of cell growth and differentiation, is inducible by phorbol esters. DNA sequences resembling phorbol ester response elements (TREs) are present upstream and downstream of this gene. TREs are recognized by proteins from the AP-1 family of transcription factors. We examined a 16 basepair (bp) sequence downstream of the TGF-beta 1 gene that contains three putative TREs. This sequence had been shown to stimulate reporter gene expression from a downstream location in response to phorbol ester treatment. Electrophoretic mobility shift assays suggest that minor proteins from the related AP-1 and CREB families of transcription factors bind to the overlapping TREs within the 16 bp element. A site beginning at the end of this 16 bp element matches the consensus sequence of a DNA-binding protein called MDBP and was shown to bind to this protein. When the intact MDBP site was present in a reporter gene construct in addition to the TREs, the phorbol ester-induced stimulation of reporter gene expression was no longer observed. This suggests that MDBP can counteract the stimulation of transcription by AP-1/CREB-like proteins binding to this downstream enhancer element.

The major histocompatibility complex class II promoter-binding protein RFX (NF-X) is a methylated DNA-binding protein.

A mammalian protein called RFX or NF-X binds to the X box (or X1 box) in the promoters of a number of major histocompatibility (MHC) class II genes. In this study, RFX was shown to have the same DNA-binding specificity as methylated DNA-binding protein (MDBP), and its own cDNA was found to contain a binding site for MDBP in the leader region. MDBP is a ubiquitous mammalian protein that binds to certain DNA sequences preferentially when they are CpG methylated and to other related sequences, like the X box, irrespective of DNA methylation. MDBP from HeLa and Raji cells formed DNA-protein complexes with X-box oligonucleotides that coelectrophoresed with those containing standard MDBP sites. Furthermore, MDBP and X-box oligonucleotides cross-competed for the formation of these DNA-protein complexes. DNA-protein complexes obtained with MDBP sites displayed the same partial supershifting with an antiserum directed to the N terminus of RFX seen for complexes containing an X-box oligonucleotide. Also, the in vitro-transcribed-translated product of a recombinant RFX cDNA bound specifically to MDBP ligands and displayed the DNA methylation-dependent binding of MDBP. RFX therefore contains MDBP activity and thereby also EF-C, EP, and MIF activities that are indistinguishable from MDBP and that bind to methylation-independent sites in the transcriptional enhancers of polyomavirus and hepatitis B virus and to an intron of c-myc.

Increasing the activity of affinity-purified DNA-binding proteins by adding high concentrations of nonspecific proteins.

A large decrease in the activity of two sequence-specific DNA-binding proteins implicated in transcription control was seen when these were affinity purified and assayed under standard conditions in electrophoretic mobility shift assays. Increasing the concentration of bovine serum albumin in the reaction mixtures from 0.1 to 5 mg/ml stimulated the DNA-binding activity of these affinity-purified proteins, human CREB (cyclic AMP response element binding protein) and MDBP (methylated DNA-binding protein), approximately 5-to more than 20-fold. In the case of affinity-purified MDBP, adding back the affinity flow-through fraction to the assay mixture gave similar extents of stimulation at much lower final protein concentrations. The specific DNA-binding activity of the affinity-purified CREB, but not that of MDBP, was also increased by adding a nonionic detergent to the binding reaction buffer although not as much. The large increase in the amount of MDBP.DNA complex seen upon supplementation of the affinity-purified MDBP with the affinity flow-through fraction or 5 mg/ml of BSA was shown to be due to stimulation, by nonspecific proteins, of specific complex formation and not to prevention of activity losses by adsorption or denaturation during the assay.

End-filling of an oligonucleotide duplex containing an MDBP site in the human HSP70 promoter inhibits protein-DNA complex formation.

A site from the promoter region of the human hsp70 gene binds with a high affinity to the ubiquitous mammalian protein called methylated DNA-binding protein (MDBP) when it is present in a CpG-methylated oligonucleotide duplex with only 14 base-pairs. Binding to this site is dependent upon CpG methylation. Surprisingly, when the same methylated sequence is present in a duplex that has 22 or more base-pairs, binding to this protein is greatly inhibited. Such a requirement for a short duplex region is seen only in certain of the cytosine methylation-dependent binding sites for this protein and is proposed to reflect differences in the conformation of the duplex due to small differences in the nucleotide sequence.

Binding sites in mammalian genes and viral gene regulatory regions recognized by methylated DNA-binding protein.

Methylated DNA-binding protein (MDBP), a ubiquitous mammalian protein, recognizes a variety of related DNA sequences. Some of these sequences require methylation of their CpG dinucleotides for binding and others do not. We report that MDBP binds, in a DNA methylation-independent fashion, to two sites in the mouse polyomavirus enhancer, one in the enhancer of the human hepatitis B virus, and to one in the long terminal repeat of equine infectious anemia proviral DNA. We have also found a number of MDBP sites in human and rodent DNAs which bind much better to MDBP when they are methylated at CpG dinucleotides within the recognition site. These include sites at the beginning of the human genes for hypoxanthine phosphoribosyl transferase, HLA-A2, -A3, and -A25 antigens, and alpha-galactosidase A. In the case of methylation-responsive MDBP sites, changes in their methylation status during differentiation or DNA replication could help drive development by modulating transcription.