Cell-free N-glycosylation of peptides using synthetic lipid-linked hybrid and complex N-glycans.

Cell-free, chemoenzymatic platforms are emerging technologies towards generating glycoconjugates with defined and homogeneous glycoforms. Recombinant oligosaccharyltransferases can be applied to glycosylate "empty," i.e., aglycosyalted, peptides and proteins. While bacterial oligosaccharlytransferases have been extensively investigated, only recently a recombinant eukaryotic single-subunit oligosaccharyltransferase has been successfully used to in vitro N-glycosylate peptides. However, its applicability towards synthesizing full-length glycoproteins and utilizing glycans beyond mannose-type glycans for the transfer have not be determined. Here, we show for the first time the synthesis of hybrid- and complex-type glycans using synthetic lipid carriers as substrates for in vitro N-glycosylation reactions. For this purpose, transmembrane-deleted human ß-1,2 N-acetylglucosamintransferase I and II (MGAT1ΔTM and MGAT2ΔTM) and ß-1,4-galactosyltransferase (GalTΔTM) have been expressed in Escherichia coli and used to extend an existing multi-enzyme cascade. Both hybrid and agalactosylated complex structures were transferred to the N-glycosylation consensus sequence of peptides (10 amino acids: G-S-D-A-N-Y-T-Y-T-Q) by the recombinant oligosaccharyltransferase STT3A from Trypanosoma brucei.

Cell-Free Multi-Enzyme Synthesis and Purification of Uridine Diphosphate Galactose.

High costs and low availability of UDP-galactose hampers the enzymatic synthesis of valuable oligosaccharides such as human milk oligosaccharides. Here, we report the development of a platform for the scalable, biocatalytic synthesis and purification of UDP-galactose. UDP-galactose was produced with a titer of 48 mM (27.2 g/L) in a small-scale batch process (200 µL) within 24 h using 0.02 genzyme /gproduct . Through in-situ ATP regeneration, the amount of ATP (0.6 mM) supplemented was around 240-fold lower than the stoichiometric equivalent required to achieve the final product yield. Chromatographic purification using porous graphic carbon adsorbent yielded UDP-galactose with a purity of 92 %. The synthesis was transferred to 1 L preparative scale production in a stirred tank bioreactor. To further reduce the synthesis costs here, the supernatant of cell lysates was used bypassing expensive purification of enzymes. Here, 23.4 g/L UDP-galactose were produced within 23 h with a synthesis yield of 71 % and a biocatalyst load of 0.05 gtotal_protein /gproduct . The costs for substrates per gram of UDP-galactose synthesized were around 0.26 €/g.

Cell-Free Glycoengineering of the Recombinant SARS-CoV-2 Spike Glycoprotein.

The baculovirus-insect cell expression system is readily utilized to produce viral glycoproteins for research as well as for subunit vaccines and vaccine candidates, for instance against SARS-CoV-2 infections. However, the glycoforms of recombinant proteins derived from this expression system are inherently different from mammalian cell-derived glycoforms with mainly complex-type N-glycans attached, and the impact of these differences in protein glycosylation on the immunogenicity is severely under investigated. This applies also to the SARS-CoV-2 spike glycoprotein, which is the antigen target of all licensed vaccines and vaccine candidates including virus like particles and subunit vaccines that are variants of the spike protein. Here, we expressed the transmembrane-deleted human ß-1,2 N-acetlyglucosamintransferases I and II (MGAT1ΔTM and MGAT2ΔTM) and the ß-1,4-galactosyltransferase (GalTΔTM) in E. coli to in-vitro remodel the N-glycans of a recombinant SARS-CoV-2 spike glycoprotein derived from insect cells. In a cell-free sequential one-pot reaction, fucosylated and afucosylated paucimannose-type N-glycans were converted to complex-type galactosylated N-glycans. In the future, this in-vitro glycoengineering approach can be used to efficiently generate a wide range of N-glycans on antigens considered as vaccine candidates for animal trials and preclinical testing to better characterize the impact of N-glycosylation on immunity and to improve the efficacy of protein subunit vaccines.

Cell-free glycoengineering of the recombinant SARS-CoV-2 spike glycoprotein

The baculovirus-insect cell expression system is readily utilized to produce viral glycoproteins for research as well as for subunit vaccines and vaccine candidates, for instance against SARS-CoV-2 infections. However, the glycoforms of recombinant proteins derived from this expression system are inherently different from mammalian cell-derived glycoforms with mainly complex-type N-glycans attached, and the impact of these differences in protein glycosylation on the immunogenicity is severely underinvestigated. This applies also to the SARS-CoV-2 spike glycoprotein, which is the antigen target of all licensed vaccines and vaccine candidates including virus like particles and subunit vaccines that are variants of the spike protein. Here, we expressed the transmembrane-deleted human {beta}-1,2 N-acetlyglucosamintransferases I and II (MGAT1{triangleup}TM and MGAT2{triangleup}TM) and the {beta}-1,4-galactosyltransferase (GalT{triangleup}TM) in E. coli to in-vitro remodel the N-glycans of a recombinant SARS-CoV-2 spike glycoprotein derived from insect cells. In a cell-free sequential one-pot reaction, fucosylated and afucosylated paucimannose-type N-glycans were converted to complex-type galactosylated N-glycans. In the future, this in-vitro glycoengineering approach can be used to efficiently generate a wide range of N-glycans on antigens considered as vaccine candidates for animal trials and preclinical testing to better characterize the impact of N-glycosylation on immunity and to improve the efficacy of protein subunit vaccines.

Synthesis of lipid-linked oligosaccharides by a compartmentalized multi-enzyme cascade for the in vitro N-glycosylation of peptides.

A wide range of glycoproteins can be recombinantly expressed in aglycosylated forms in bacterial and cell-free production systems. To investigate the effect of glycosylation of these proteins on receptor binding, stability, efficacy as drugs, pharmacodynamics and pharmacokinetics, an efficient glycosylation platform is required. Here, we present a cell-free synthetic platform for the in vitro N-glycosylation of peptides mimicking the endoplasmic reticulum (ER) glycosylation machinery of eukaryotes. The one-pot, two compartment multi-enzyme cascade consisting of eight recombinant enzymes including the three Leloir glycosyltransferases, Alg1, Alg2 and Alg11, expressed in E. coli and S. cerevisiae, respectively, has been engineered to produce the core lipid-linked (LL) oligosaccharide mannopentaose-di-(N-acetylglucosamine) (LL-Man5). Pythanol (C20H42O), a readily available alcohol consisting of regular isoprenoid units, was utilized as the lipid anchor. As part of the cascade, GDP-mannose was de novo produced from the inexpensive substrates ADP, polyphosphate and mannose. To prevent enzyme inhibition, the nucleotide sugar cascade and the glycosyltransferase were segregated into two compartments by a cellulose ester membrane with 3.5 kDa cut-off allowing for the effective diffusion of GDP-mannose across compartments. Finally, as a proof-of-principle, pythanyl-linked Man5 and the single-subunit oligosaccharyltransferase Trypanosoma brucei STT3A expressed in Sf9 insect cells were used to in vitro N-glycosylate a synthetic peptide of ten amino acids bearing the eukaryotic consensus motif N-X-S/T.

Establishment of a five-enzyme cell-free cascade for the synthesis of uridine diphosphate N-acetylglucosamine.

In spite of huge endeavors in cell line engineering to produce glycoproteins with desired and uniform glycoforms, it is still not possible in vivo. Alternatively, in vitro glycoengineering can be used for the modification of glycans. However, in vitro glycoengineering relies on expensive nucleotide sugars, such as uridine 5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) which serves as GlcNAc donor for the synthesis of various glycans. In this work, we present a systematic study for the cell-free de novo synthesis and regeneration of UDP-GlcNAc from polyphosphate, UMP and GlcNAc by a cascade of five enzymes (N-acetylhexosamine kinase (NahK), Glc-1P uridyltransferase (GalU), uridine monophosphate kinase (URA6), polyphosphate kinase (PPK3), and inorganic diphosphatase (PmPpA). All enzymes were expressed in E. coli BL21 Gold (DE3) and purified using immobilized metal affinity chromatography (IMAC). Results from one-pot experiments demonstrate the successful production of UDP-GlcNAc with a yield approaching 100%. The highest volumetric productivity of the cascade was about 0.81 g L-1  h-1 of UDP-GlcNAc. A simple model based on mass action kinetics was sufficient to capture the dynamic behavior of the multienzyme pathway. Moreover, a design equation based on metabolic control analysis was established to investigate the effect of enzyme concentration on the UDP-GlcNAc flux and to demonstrate that the flux of UDP-GlcNAc can be controlled by means of the enzyme concentrations. The effect of temperature on the UDP-GlcNAc flux followed an Arrhenius equation and the optimal co-factor concentration (Mg2+) for high UDP-GlcNAc synthesis rates depended on the working temperature. In conclusion, the study covers the entire engineering process of a multienzyme cascade, i.e. pathway design, enzyme expression, enzyme purification, reaction kinetics and investigation of the influence of basic parameters (temperature, co-factor concentration, enzyme concentration) on the synthesis rate. Thus, the study lays the foundation for future cascade optimization, preparative scale UDP-GlcNAc synthesis and for in situ coupling of the network with UDP-GlcNAc transferases to efficiently regenerate UDP-GlcNAc. Hence, this study provides a further step towards cost-effective in vitro glycoengineering of antibodies and other glycosylated proteins.

One pot synthesis of GDP-mannose by a multi-enzyme cascade for enzymatic assembly of lipid-linked oligosaccharides.

Glycosylation of proteins is a key function of the biosynthetic-secretory pathway in the endoplasmic reticulum (ER) and Golgi apparatus. Glycosylated proteins play a crucial role in cell trafficking and signaling, cell-cell adhesion, blood-group antigenicity, and immune response. In addition, the glycosylation of proteins is an important parameter in the optimization of many glycoprotein-based drugs such as monoclonal antibodies. In vitro glycoengineering of proteins requires glycosyltransferases as well as expensive nucleotide sugars. Here, we present a designed pathway consisting of five enzymes, glucokinase (Glk), phosphomannomutase (ManB), mannose-1-phosphate-guanyltransferase (ManC), inorganic pyrophosphatase (PmPpA), and 1-domain polyphosphate kinase 2 (1D-Ppk2) expressed in E. coli for the cell-free production and regeneration of GDP-mannose from mannose and polyphosphate with catalytic amounts of GDP and ADP. It was shown that GDP-mannose is produced at various conditions, that is pH 7-8, temperature 25-35°C and co-factor concentrations of 5-20 mM MgCl2 . The maximum reaction rate of GDP-mannose achieved was 2.7 µM/min at 30°C and 10 mM MgCl2 producing 566 nmol GDP-mannose after a reaction time of 240 min. With respect to the initial GDP concentration (0.8 mM) this is equivalent to a yield of 71%. Additionally, the cascade was coupled to purified, transmembrane-deleted Alg1 (ALG1ΔTM), the first mannosyltransferase in the ER-associated lipid-linked oligosaccharide (LLO) assembly. Thereby, in a one-pot reaction, phytanyl-PP-(GlcNAc)2 -Man1 was produced with efficient nucleotide sugar regeneration for the first time. Phytanyl-PP-(GlcNAc)2 -Man1 can serve as a substrate for the synthesis of LLO for the cell-free in vitro glycosylation of proteins. A high-performance anion exchange chromatography method with UV and conductivity detection (HPAEC-UV/CD) assay was optimized and validated to determine the enzyme kinetics. The established kinetic model enabled the optimization of the GDP-mannose regenerating cascade and can further be used to study coupling of the GDP-mannose cascade with glycosyltransferases. Overall, the study envisages a first step towards the development of a platform for the cell-free production of LLOs as precursors for in vitro glycoengineering of proteins.

A comparison of the cardiopulmonary effects of pressure controlled ventilation and volume controlled ventilation in healthy anesthetized dogs.

OBJECTIVE: To compare the effects of pressure controlled ventilation (PCV) with volume-controlled ventilation (VCV) on lung compliance, gas exchange, and hemodynamics in isoflurane-anesthetized dogs. DESIGN: Prospective randomized study. SETTING: Veterinary teaching hospital. ANIMALS: Forty client-owned bitches undergoing elective ovariohysterectomy. INTERVENTIONS: Dogs were randomly assigned to be ventilated with 100% oxygen using PCV (n = 20) or VCV (n = 20). The respiratory rate was 20/min and positive end-expiratory pressure (PEEP) was 5 cm H2 O, with a tidal volume of 10 mL/kg. Cardiac output (CO) was measured using thermodilution. Cardiopulmonary and blood gas data were obtained during spontaneous ventilation and after 30 (T30) and 60 minutes (T60) of controlled ventilation. MEASUREMENTS AND MAIN RESULTS: In dogs ventilated with PCV, at T30 and T60, PIP was lower (11.4 ± 1.9 and 11.1 ± 1.5 cm H2 O, respectively) and static compliance (CST ) was higher (51 ± 7 and 56 ± 6 mL/cm H2 O, respectively) than in VCV group (PIP of 14.3 ± 1.3 and 15.5 ± 1.4 cm H2 O; CST of 34 ± 8 and 33 ± 9 mL/cm H2 O, P < 0.0001). Compared with spontaneous ventilation, both groups had decreased alveolar-arterial oxygen difference at T30 and T60 (PCV: 128 ± 32 mm Hg vs 108 ± 20 and 104 ± 16 mm Hg, respectively; VCV: 131 ± 38 mm Hg vs 109 ± 19 and 107 ± 14 mm Hg, respectively; P < 0.01), while CO was maintained at all time points. CONCLUSIONS: Compared to spontaneous ventilation, both ventilatory modes effectively improved gas exchange without hemodynamic impairment. PCV resulted in higher lung CST and lower PIP compared to VCV.

Prevalence of antibodies to serotypes of Bluetongue virus and Anaplasma marginale in Montana feeder cattle: 2002-2003.

In a serologic survey of Montana-source weaned calves and yearling cattle, the apparent prevalence of antibodies to Bluetongue virus was 0.68% and 1.26% in 2002 and 2003, respectively, and to Anaplasma marginale at a positive cutoff at 30% inhibition it was 1.82% and 1.35% in 2002 and 2003, and at a positive cutoff at 42% inhibition it was 0.76% and 0.55% in 2002 and 2003, respectively, suggesting that the risk of importing infected animals was very low.

Gene transfer of the Bcl-2 gene confers cytoprotection to isolated adult porcine pancreatic islets exposed to xenoreactive antibodies and complement.

BACKGROUND: Exposing adult porcine pancreatic islets (PI) to xenoreactive natural antibodies (XNA) induces brisk inflammatory injury that involves activation of the complement system. Gene transfer of Bcl-2 has been shown to protect PI from apoptosis and necrosis in several models. In this study, we investigated the effect of Bcl-2 gene transfer on protection of PI from primate XNA and complement-mediated injury. METHODS: The PI were isolated from adult female sows. Only islet preparations that exhibited >90% viability and purity were used. Fresh rhesus monkey serum served as the XNA source. Gene transfer of Bcl-2 was achieved with an adenoviral vector (AdBcl-2) at 500 particle forming units (pfu)/cell. The Bcl-2 expression was confirmed by Western blot technique. Untransfected and transfected PI were incubated in 50% fresh complete serum (CS) or heat-inactivated (HI) rhesus serum for 24 hours. The PI viability was analyzed with acridine orange and ethidium bromide staining. Antibody and complement-mediated cytotoxicity were tested by intracellular lactate dehydrogenase (LDH) release. The PI function was assessed in vitro by static incubation studies and in vivo after intraportal transplantation in diabetic severe combined immunodeficiency (SCID) mice. RESULTS: The AdBcl-2 gene transfer resulted in Bcl-2 gene expression in >90% of PI cells. Following exposure to XNA, <15% of the untransfected cells were viable. Similar results were obtained in PI transfected with a similar recombinant adenovirus encoding the reporter gene E coli beta-galactosidase (AdLacZ), an irrelevant gene. A significant increase in LDH release was observed in control PI after exposure to CS compared with PI that overexpressed Bcl-2 (82.89% +/- 7.78% vs 34.31% +/- 5.4%, P <.005). Higher insulin release was observed in vitro in PI transfected with Bcl-2 compared with untransfected PI or islets transfected with AdLacZ (stimulation index of 0.9 +/- 0.31, 0.9 +/- 0.3 vs 2.67 +/- 0.4, respectively). Only PI treated with AdBcl-2 were able to achieve euglycemia after exposure to XNA and complement after transplantation. CONCLUSIONS: Transfer of the antiapoptotic and antinecrotic Bcl-2 gene into PI can reduce primate XNA and complement-mediated lysis. Cytoprotection of PI with Bcl-2 has potential to improve survival of PI xenotransplants.

Cytoprotection of pancreatic islets before and soon after transplantation by gene transfer of the anti-apoptotic Bcl-2 gene.

Isolated pancreatic islet transplantation is a promising alternative to conventional insulin-dependent diabetes treatment but is not yet a practical clinical therapy. In the first few days after pancreatic islet transplantation, substantial donor pancreatic islet dysfunction and apoptosis commonly occur. Islet apoptosis has been documented after extracellular matrix disruption and exposure to proinflammatory cytokines, and during hypoxia before islet revascularization and rejection. These studies show that targeting the apoptosis pathway by adenoviral-mediated gene transfer of the anti-apoptotic Bcl-2 gene exerts a major cytoprotective effect on isolated macaque pancreatic islets. Bcl-2 transfection ex vivo protects these islets from apoptosis induced by disruption of the islet extracellular matrix during pancreatic digestion. Additionally, overexpression of Bcl-2 confers long-term, stable protection and maintenance of functional islet mass after transplantation of macaque islets into diabetic severe combined immunodeficency mice. Notably, genetic modification of pancreatic islets also reduced the islet mass required to achieve stable euglycemia. Ex vivo gene transfer of anti-apoptotic genes has potential as a therapeutic approach to both minimize loss of functional islet mass after transplant and reduce the high donor islet requirement currently needed for successful stable reversal of insulin-dependent diabetes.

Successful reversal of streptozotocin-induced diabetes with stable allogeneic islet function in a preclinical model of type 1 diabetes.

The recent focus on islet transplantation as primary therapy for type 1 diabetes has heightened interest in the reversal of type 1 diabetes in preclinical models using minimal immunosuppression. Here, we demonstrated in a preclinical rhesus model a consistent reversal of all measured glycemic patterns of streptozotocin-induced type 1 diabetes. The model used single-donor islet transplantation with induction of operational tolerance. The term "operational tolerance" is used to indicate durable survival of single-donor major histocompatibility complex (MHC)-mismatched islet allografts without maintenance immunosuppressive therapy and without rejection or loss of functional islet mass or insulin secretory reserve. In this operational tolerance model, all immunosuppression was discontinued after day 14 posttransplant, and recipients recovered with excellent health. The operational tolerance induction protocol combined peritransplant anti-CD3 immunotoxin to deplete T-cells and 15-deoxyspergualin to arrest proinflammatory cytokine production and maturation of dendritic cells. T-cell deficiency was specific but temporary, in that T-cell-dependent responses in long-term survivors recovered to normal, and there was no evidence of increased susceptibility to infection. Anti-donor mixed lymphocyte reaction responses were positive in the long-term survivors, but all showed clear evidence of systemic T-helper 2 deviation, suggesting that an immunoregulatory rather than a deletional process underlies this operational tolerance model. This study provides the first evidence that operational tolerance can protect MHC nonhuman primate islets from rejection as well as loss of functional islet mass. Such an approach has potential to optimize individual recipient recovery from diabetes as well as permitting more widespread islet transplantation with the limited supply of donor islets.

STEALTH matters: a novel paradigm of durable primate allograft tolerance.

We review a novel strategy for tolerance induction developed in rhesus macaques and termed STEALTH. We summarize the evolution of the STEALTH model, the results of successful trials in inducing long-term, stable transplant tolerance in rhesus kidney and diabetic islet recipients and discuss information related to the mechanism by which durable tolerance is induced. STEALTH tolerance is induced by a 3-day treatment course of CD3epsilon immunotoxin (IT) combined with a 14-day treatment with deoxyspergualin (DSG). IT causes profound depletion of sessile lymph node T cells as well as the more accessible circulating T cells. DSG, an inhibitor of HSC 70-mediated NF-kappaB nuclear translocation, arrests maturation of myeloid dendritic cells, blocks production of proinflammatory cytokines induced by IT administration, and promotes systemic production of Th2 type cytokines that persist indefinitely. Such Th2 cytokine deviation has not been reported in NHP transplant recipients. These studies provide proof of principle in a preclinical model that prevention of both acute and chronic allograft rejection, for at least 2.2-4.9 years of follow-up, can be achieved in NHP in the absence of chronic immunosuppressive drugs or other interventions. This strategy for inducing NHP tolerance is discussed in relation to current tolerance paradigms.

Experience with renal transplantation in the nonhuman primate: a modified ureteroneocystostomy to prevent urologic complications.

Nonhuman primates provide an optimal model for the evaluation of tolerance in the preclinical setting. Transplantation and management of nonhuman primates are technically demanding, and the purpose of this article is to review our extensive experience in renal transplantation in non-human primates, with particular emphasis on modifications of surgical techniques on urologic complications. We retrospectively reviewed our results with 329 renal transplants in rhesus monkeys over an 18-year period. The surgical technique and, in particular, the ureteroneocystostomy have evolved over this period of time. This review extensively details our current technique, the surgical and urologic complications, and their management. There were 329 renal transplants performed. There were 85 early deaths, or animals euthanized, within 30 days of the transplant operation. In the first 15 years, there were 27 (10.68%) surgical complications that required euthanasia, and in the last 3 years the complication rate has been reduced to 5 (7.3%, p < .05). The routine use of microsurgical techniques has reduced the incidence of arterial thrombosis (6.2% vs. 2.9%, p < .05). The incidence of ureteral strictures (15 vs. 0, p < .005) has been reduced by a modification of the ureteroneocystostomy technique detailed in the text. Renal transplantation in small rhesus monkeys is technically demanding. The routine use of microsurgical techniques and a modified ureteroneocystostomy has reduced the incidence of surgical complications.

Durable donor-specific T and B cell tolerance in rhesus macaques induced with peritransplantation anti-CD3 immunotoxin and deoxyspergualin: absence of chronic allograft nephropathy.

Tolerance induction can prevent acute kidney allograft rejection without chronic immunosuppression. It is uncertain whether specific tolerance can prevent chronic allograft nephropathy (CAN), which involves both nonimmune and immune injury. This report provides evidence that immunologically tolerant macaques, induced with immunotoxin and deoxyspergualin, developed neither acute rejection nor CAN. Long survivors, bearing MHC-mismatched grafts without chronic immunosuppression for 0.8 to 3.4 years, exhibited general immune competence with donor-specific T and B cell tolerance and no functional or histological evidence of CAN. Stringent criteria for tolerance were satisfied by specific prolongation of donor skin grafts with rapid rejection of third-party skin, followed by indefinite acceptance of a second donor kidney graft and establishment of microchimerism. Primate tolerance with documented absence of CAN may give impetus to the clinical application of tolerance.

Long-term functional islet mass and metabolic function after xenoislet transplantation in primates.

BACKGROUND: Pancreatic islet transplantation (PIT) is an attractive alternative for patients with type I diabetes mellitus. PIT is not yet an effective clinical reality due in part to the high incidence of rejection and early loss of functional islet mass. In addition, current immunosuppressive drugs have toxic effects on islets and increase the risk of morbidity and mortality. In the present study, the effects of PIT on glycemic parameters were assessed in spontaneously diabetic primates. METHODS: Five insulinopenic nonhuman primates (three Macacca fascicularis, one Ceropithecus aethiops, and one Macacca mulatta) were studied. All required twice-daily treatment with 4-10 U of insulin. For immunosuppression, the animals received anti-CD3-immunotoxin (100 microg/kg(initially infused 2 hr before transplantation and again on day +1), cyclosporine (CsA) (20 mg/kg(i.v./2 hr before transplantation), cyclosporine microemulsion (Neoral) 60 mg/kg/b.i.d. on days +1 to +3 with dose adjusted by blood levels, and methylprednisolone (15 mg/kg day 0 to +3). Three recipients were given islets from a single donor (M mulatta). The islets were prepared by a semiautomated technique using Liberase. A mean of 13,136 islet equivalents/kg was infused into the portal vein. Two animals (M fascicularis and M mulatta) were used as a diabetic, nontransplanted control. Several metabolic parameters were evaluated. RESULTS: All monkeys that underwent transplantation experienced reversal of diabetes mellitus with normalization of all diabetic glycemic parameters. In the nontransplanted primates given the same immunosuppression but no PIT, diabetic metabolic parameters were unchanged after 9 months of follow-up. In contrast, all three PIT recipients established fasting and nonfasting euglycemia within 1-2 weeks, and none required exogenous insulin after day 10. Normal intravenous glucose tolerance tests were observed at day 15, and no significant differences in the glucose disappearance rate (Kg) were observed at days 15, 45, 190, and 365 days after transplantation. The acute insulin response to glucose indicated no significant reduction of functional islet mass. CONCLUSIONS: PIT in severely insulinopenic type I diabetes mellitus primates resulted in restoration of normal glycemic parameters and durable islet mass. Operational tolerance was achieved 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 type I diabetes mellitus treatment.

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.

Reduction of ischemia-reperfusion injury of the liver by in vivo adenovirus-mediated gene transfer of the antiapoptotic Bcl-2 gene.

OBJECTIVE: To examine the possibility of reducing ischemia-reperfusion injury (I/R injury) to the mouse liver by in vivo adenovirus-mediated gene transfer of the antiapoptotic human Bcl-2 gene. SUMMARY BACKGROUND DATA: Ischemia-reperfusion injury has been demonstrated in a number of clinically relevant diseases such as myocardial infarction, cerebrovascular disease, sepsis, peripheral vascular disease, and organ transplantation. In this regard, apoptosis plays a central role. METHODS: Normal C57BL/6 mice were used. An adenovirus (deltaE1) vector containing the human Bcl-2 gene was developed in the authors' laboratory. An adenovirus vector encoding an irrelevant gene (beta-galactosidase, AdCMVLacZ) was used as a control. Taking advantage of the hepatotropic properties of adenovirus vectors, gene transfer was performed with 1 x 10(9) plaque-forming units by intravenous tail injection, 48 hours before the ischemic injury. Ischemic-reperfusion injury was induced by temporal and segmental occlusion of hepatic blood flow. Aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase activity was measured using standard assays. Liver biopsies were obtained before and 6 hours after I/R injury for morphologic assessment, and apoptosis was determined in situ with a histochemical assay. RESULTS: The expression of AdCMVhBcl-2 vector was confirmed by reverse transcription-polymerase chain reaction and functionally validated in apoptotic studies in endothelial cells. Expression of the Bcl-2 gene protects against I/R injury, as shown by a significant decrease in transaminases (p < 0.05) and necrosis and apoptosis (p < 0.001), and permanent survival (p < 0.0001), compared with sham-operated animals and animals treated with AdCMVLacZ. CONCLUSIONS: Genetic modification of the liver to induce cytoprotection has potential applications to prevent I/R injury to the liver in surgical interventions, including liver transplantation.

Tolerability and side effects of anti-CD3-immunotoxin in preclinical testing in kidney and pancreatic islet transplant recipients.

INTRODUCTION: Anti-CD3-immunotoxin (alpha-CD3-IT) promotes allograft tolerance in nonhuman primates owing to efficient depletion of sessile and circulating T cells. Common side effects of vascular leak syndrome, hepatotoxicity, and nephrotoxicity have limited tolerability of other immunotoxins. We report on preclinical studies of alpha-CD3-IT-related side effects. METHODS: Normal rhesus monkeys received a kidney transplant and alpha-CD3-IT alone (on day -to +2) or in combination with brief peritransplant adjunctive immunosuppressive therapy. Some received donor CD34+ cells. Blood chemistries, complete blood count, weight, liver, and kidney biopsies were examined for immunotoxin-related changes. Five spontaneously diabetic primates also received alpha-CD3-IT, three of whom had a pancreas islet transplant. RESULTS: The main side effect of alpha-CD3-IT, vascular leak syndrome, was entirely prevented by adjunctive immunosuppressive therapy. Renal and liver function tests and biopsies revealed a lack of nephrotoxicity and hepatotoxicity. All had transient weight loss (14+/-5%). Without infusion of donor CD34+ cells, 97% had full weight recovery. Of those given donor CD34+ cells, 50% were euthanized for wasting. CONCLUSIONS: Side effects of alpha-CD3-IT are manageable and should not prevent therapeutic application.