Subcutaneous transplantation of human thyroid tissue into a pre-vascularized Cell Pouch™ device in a Mus musculus model: Evidence of viability and function for thyroid transplantation.

This study aimed to investigate the survival and efficacy indicators of human thyroid tissue transplantation into a retrievable, prevascularized implanted Sernova Corp Cell Pouch™ (CP) device. Thyroid tissue from human donors was transplanted subcutaneously into the pre-implanted CP device or into the subcutaneous (SC) space alone as a control in a nude Mus musculus model. Transplanted M. musculus were monitored for human serum thyroglobulin (TG) levels for 3 months until the transplants were removed for histological assessment. Human thyroid tissue survived and continued to produce TG in transplanted nude M. musculus in the CP, with no adverse events. CP transplants exhibited more persistent and robust production of human TG than tissue placed in the SC space alone from 3 to 13 weeks post transplantation. Fresh thyroid transplants had better survival and function compared to cryopreserved transplants. Thyroid transplant viability correlated with TG levels at 3 months post-transplant (p = 0.03). Immunofluorescence staining of transplants for TG and TPO localized in thyroid follicles. Human thyroid tissue transplanted into the subcutaneously implanted pre-vascularized CP in nude M. musculus survived and continued to produce robust and persistent human TG and warrants further investigation as a treatment for postoperative hypothyroidism.

Efficient and safe correction of hemophilia A by lentiviral vector-transduced BOECs in an implantable device.

Hemophilia A (HA) is a rare bleeding disorder caused by deficiency/dysfunction of the FVIII protein. As current therapies based on frequent FVIII infusions are not a definitive cure, long-term expression of FVIII in endothelial cells through lentiviral vector (LV)-mediated gene transfer holds the promise of a one-time treatment. Thus, here we sought to determine whether LV-corrected blood outgrowth endothelial cells (BOECs) implanted through a prevascularized medical device (Cell Pouch) would rescue the bleeding phenotype of HA mice. To this end, BOECs from HA patients and healthy donors were isolated, expanded, and transduced with an LV carrying FVIII driven by an endothelial-specific promoter employing GMP-like procedures. FVIII-corrected HA BOECs were either directly transplanted into the peritoneal cavity or injected into a Cell Pouch implanted subcutaneously in NSG-HA mice. In both cases, FVIII secretion was sufficient to improve the mouse bleeding phenotype. Indeed, FVIII-corrected HA BOECs reached a relatively short-term clinically relevant engraftment being detected up to 16 weeks after transplantation, and their genomic integration profile did not show enrichment for oncogenes, confirming the process safety. Overall, this is the first preclinical study showing the safety and feasibility of transplantation of GMP-like produced LV-corrected BOECs within an implantable device for the long-term treatment of HA.

Therapeutic control of leishmaniasis by inhibitors of the mammalian target of rapamycin.

Leishmaniasis is a serious global health problem affecting many people worldwide. While patients with leishmaniasis can be treated with several agents, drug toxicicty and the emergence of resistant strains render available treatments ineffective in the long run. Inhibitors of the mammalian target of rapamycin (mTOR) have been demonstrated to exert anti-pathogen properties. In this study, we tested the therapeutic efficacy of several mTOR inhibitors in controlling infection with Leishmania major. Rapamycin, GSK-2126458 and KU-0063794 were administered to BALB/c mice, which had received an intrafootpad injection of the parasite. Footpad swelling and parasite burden were assessed, and cytokine production by mouse splenocytes and phenotypic changes in draining lymph node cells were evaluated. Treatment with a clinically relevant dose of rapamycin or with GSK-2126458, but not with KU-0063794, dramatically lowered both the footpad swelling and the parasite load in the draining lymph node. Importantly, the employed dose of rapamycin did not kill the promastigotes in vitro as judged by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays and electron microscopy. Moreover, the IL-4 production capacity of splenocytes harvested from infected mice that were treated with rapamycin was significantly reduced. Consequently, the IFN-γ:IL-4 production ratio was elevated, suggesting a T helper-type 1 (Th1)-skewed cytokine profile. Finally, the expression level of CD69, an early activation marker, on splenic and lymph node CD4+ and CD8+ T cells was enhanced in rapamycin-treated mice. Taken together, our findings suggest that select mTOR inhibitors may be used in therapeutic settings for the management of leishmaniasis. We propose that the beneficial effects of such inhibitors stem from their immunomodulatory properties. Therefore, the adjuvanticity of mTOR inhibitors may also be considered in vaccination strategies against Leishmania species.

Nasopharyngeal infection by Streptococcus pyogenes requires superantigen-responsive Vß-specific T cells.

The globally prominent pathogen Streptococcus pyogenes secretes potent immunomodulatory proteins known as superantigens (SAgs), which engage lateral surfaces of major histocompatibility class II molecules and T-cell receptor (TCR) ß-chain variable domains (Vßs). These interactions result in the activation of numerous Vß-specific T cells, which is the defining activity of a SAg. Although streptococcal SAgs are known virulence factors in scarlet fever and toxic shock syndrome, mechanisms by how SAgs contribute to the life cycle of S. pyogenes remain poorly understood. Herein, we demonstrate that passive immunization against the Vß8-targeting SAg streptococcal pyrogenic exotoxin A (SpeA), or active immunization with either wild-type or a nonfunctional SpeA mutant, protects mice from nasopharyngeal infection; however, only passive immunization, or vaccination with inactive SpeA, resulted in high-titer SpeA-specific antibodies in vivo. Mice vaccinated with wild-type SpeA rendered Vß8+ T cells poorly responsive, which prevented infection. This phenotype was reproduced with staphylococcal enterotoxin B, a heterologous SAg that also targets Vß8+ T cells, and rendered mice resistant to infection. Furthermore, antibody-mediated depletion of T cells prevented nasopharyngeal infection by S. pyogenes, but not by Streptococcus pneumoniae, a bacterium that does not produce SAgs. Remarkably, these observations suggest that S. pyogenes uses SAgs to manipulate Vß-specific T cells to establish nasopharyngeal infection.

Rapid and Rigorous IL-17A Production by a Distinct Subpopulation of Effector Memory T Lymphocytes Constitutes a Novel Mechanism of Toxic Shock Syndrome Immunopathology.

Toxic shock syndrome (TSS) is caused by staphylococcal and streptococcal superantigens (SAgs) that provoke a swift hyperinflammatory response typified by a cytokine storm. The precipitous decline in the host's clinical status and the lack of targeted therapies for TSS emphasize the need to identify key players of the storm's initial wave. Using a humanized mouse model of TSS and human cells, we herein demonstrate that SAgs elicit in vitro and in vivo IL-17A responses within hours. SAg-triggered human IL-17A production was characterized by remarkably high mRNA stability for this cytokine. A distinct subpopulation of CD4+ effector memory T (TEM) cells that secrete IL-17A, but not IFN-γ, was responsible for early IL-17A production. We found mouse "TEM-17" cells to be enriched within the intestinal epithelium and among lamina propria lymphocytes. Furthermore, interfering with IL-17A receptor signaling in human PBMCs attenuated the expression of numerous inflammatory mediators implicated in the TSS-associated cytokine storm. IL-17A receptor blockade also abrogated the secondary effect of SAg-stimulated PBMCs on human dermal fibroblasts as judged by C/EBP δ expression. Finally, the early IL-17A response to SAgs was pathogenic because in vivo neutralization of IL-17A in humanized mice ameliorated hepatic and intestinal damage and reduced mortality. Together, our findings identify CD4+ TEM cells as a key effector of TSS and reveal a novel role for IL-17A in TSS immunopathogenesis. Our work thus elucidates a pathogenic, as opposed to protective, role for IL-17A during Gram-positive bacterial infections. Accordingly, the IL-17-IL-17R axis may provide an attractive target for the management of SAg-mediated illnesses.

Synthesis, self-assembly, and immunological activity of α-galactose-functionalized dendron-lipid amphiphiles.

Nanoassemblies presenting multivalent displays of biologically active carbohydrates are of significant interest for a wide array of biomedical applications ranging from drug delivery to immunotherapy. In this study, glycodendron-lipid hybrids were developed as a new and tunable class of dendritic amphiphiles. A modular synthesis was used to prepare dendron-lipid hybrids comprising distearylglycerol and 0 through 4th generation polyester dendrons with peripheral protected amines. Following deprotection of the amines, an isothiocyanate derivative of C-linked α-galactose (α-Gal) was conjugated to the dendron peripheries, affording amphiphiles with 1 to 16 α-Gal moieties. Self-assembly in water through a solvent exchange process resulted in vesicles for the 0 through 2nd generation systems and micelles for the 3rd and 4th generation systems. The critical aggregation concentrations decreased with increasing dendron generation, suggesting that the effects of increasing molar mass dominated over the effects of increasing the hydrophilic weight fraction. The binding of the assemblies to Griffonia simplicifolia Lectin I (GSL 1), a protein with specificity for α-Gal was studied by quantifying the binding of fluorescently labeled assemblies to GSL 1-coated beads. It was found that binding was enhanced for amphiphiles containing higher generation dendrons. Despite their substantial structural differences with the natural ligands for the CD1d receptor, the glycodendron-lipid hybrids were capable of stimulating invariant natural killer T (iNKT) cells, a class of innate-like T cells that recognize lipid and glycolipid antigens presented by CD1d and that are implicated in a wide range of diseases and conditions including but not limited to infectious diseases, diabetes and cancer.

Quantification of Alloantibody-Mediated Cytotoxicity In Vivo.

BACKGROUND: Preexisting, donor-specific antibodies (DSAs) are culprits of hyperacute rejection. Donor-specific antibodies are also formed de novo, and their role in acute and chronic rejection is increasingly appreciated. However, it is difficult to assess damage inflicted exclusively by DSAs when alloreactive T cell and B cell responses coincide. We reasoned that allosensitization with "costimulation-deficient" cells should induce DSA synthesis but not naive cytotoxic T lymphocyte (CTL) precursors' priming via direct allorecognition. Accordingly, we have developed a novel model to quantify DSA-mediated cytotoxicity in vivo. METHODS: C57BL/6 (H-2b) mice were sensitized with H-2 kidney epithelial cells, and a cytofluorimetric killing assay was tailored to the measurement of allocytotoxicity. We took cell/complement depletion, costimulation blockade, and serum transfer approaches to reveal the mediators of cytotoxicity. "Third-party" controls and a skin allotransplantation model were used to confirm DSAs' specificity for allo-major histocompatibility complex. We validated our experimental approach in other mouse strains primed with different allogeneic cell types, including endothelial cells. To demonstrate the usefulness of our model/method for drug efficacy testing, we examined the effect of CTLA4-Ig and rapamycin on DSA-mediated cytolysis. RESULTS: Allosensitization of MHC-disparate mouse strains with costimulation-deficient cells led to robust cytotoxicity mediated by complement-fixing DSAs and phagocytic cells. This response was independent of CTLs, natural killer or natural killer T cells. It required CD4 T cell help, CD40 signaling and CD28-based costimulation during allosensitization and could be reversed by sustained rapamycin treatment. CONCLUSIONS: The unique model described herein should enable mechanistic studies on sensitization and effector phases of humoral alloreactivity as well as efficacy testing of future immunotherapies to prevent DSA-induced pathology.

Diabetes Is Reversed in a Murine Model by Marginal Mass Syngeneic Islet Transplantation Using a Subcutaneous Cell Pouch Device.

BACKGROUND: Islet transplantation is a successful ß-cell replacement therapy for selected patients with type 1 diabetes mellitus. Although high rates of early insulin independence are achieved routinely, long-term function wanes over time. Intraportal transplantation is associated with procedural risks, requires multiple donors, and does not afford routine biopsy. Stem cell technologies may require potential for retrievability, and graft removal by hepatectomy is impractical. There is a clear clinical need for an alternative, optimized transplantation site. The subcutaneous space is a potential substitute, but transplantation of islets into this site has routinely failed to reverse diabetes. However, an implanted device, which becomes prevascularized before transplantation, may alter this equation. METHODS: Syngeneic mouse islets were transplanted subcutaneously within Sernova Corp's Cell Pouch (CP). All recipients were preimplanted with CPs 4 weeks before diabetes induction and transplantation. After transplantation, recipients were monitored for glycemic control and glucose tolerance. RESULTS: Mouse islets transplanted into the CP routinely restored glycemic control with modest delay and responded well to glucose challenge, comparable to renal subcapsular islet grafts, despite a marginal islet dose, and normoglycemia was maintained until graft explantation. In contrast, islets transplanted subcutaneously alone failed to engraft. Islets within CPs stained positively for insulin, glucagon, and microvessels. CONCLUSIONS: The CP is biocompatible, forms an environment suitable for islet engraftment, and offers a potential alternative to the intraportal site for islet and future stem cell therapies.

Bacterial superantigens promote acute nasopharyngeal infection by Streptococcus pyogenes in a human MHC Class II-dependent manner.

Establishing the genetic determinants of niche adaptation by microbial pathogens to specific hosts is important for the management and control of infectious disease. Streptococcus pyogenes is a globally prominent human-specific bacterial pathogen that secretes superantigens (SAgs) as 'trademark' virulence factors. SAgs function to force the activation of T lymphocytes through direct binding to lateral surfaces of T cell receptors and class II major histocompatibility complex (MHC-II) molecules. S. pyogenes invariably encodes multiple SAgs, often within putative mobile genetic elements, and although SAgs are documented virulence factors for diseases such as scarlet fever and the streptococcal toxic shock syndrome (STSS), how these exotoxins contribute to the fitness and evolution of S. pyogenes is unknown. Here we show that acute infection in the nasopharynx is dependent upon both bacterial SAgs and host MHC-II molecules. S. pyogenes was rapidly cleared from the nasal cavity of wild-type C57BL/6 (B6) mice, whereas infection was enhanced up to ∼10,000-fold in B6 mice that express human MHC-II. This phenotype required the SpeA superantigen, and vaccination with an MHC -II binding mutant toxoid of SpeA dramatically inhibited infection. Our findings indicate that streptococcal SAgs are critical for the establishment of nasopharyngeal infection, thus providing an explanation as to why S. pyogenes produces these potent toxins. This work also highlights that SAg redundancy exists to avoid host anti-SAg humoral immune responses and to potentially overcome host MHC-II polymorphisms.

Establishment of a stringent large animal model of insulin-dependent diabetes for islet autotransplantation: combination of pancreatectomy and streptozotocin.

OBJECTIVE: A stringent porcine islet autograft diabetes model was developed to enable the assessment of autoislet safety and efficacy in either portal vein or an extrahepatic site. METHODS: A 95% pancreatectomy was performed preserving the pancreaticoduodenal arcade; however, glycemic control was still maintained at 3.3 ± 0.3 days (mean ± SEM), shown by euglycemic fasting blood glucose levels of 4.9 ± 0.8 mmol/L (mean ± SEM, n = 3). To reduce surgical complications and eliminate remaining islets, pigs were dosed intravenously after a modified 90% pancreatectomy, with 150-mg/kg streptozotocin, producing a diabetic state (18.9 ± 1.8 mmol/L [mean ± SEM], n = 8; P < 0.001) within 2.0 ± 0.9 days (mean ± SEM). RESULTS: Animals presented with sustained hyperglycemia, failing a glucose challenge test 12 weeks after diabetic induction, and showed no stimulated C-peptide secretion compared to nondiabetic controls (baseline: 0.479 ± 0.080 ng/mL [mean ± SEM] vs after procedure: 0.219 ± 0.055 ng/mL [mean ± SEM], P = 0.02). Diabetic animals were maintained on daily insulin. Despite an initial decline in body weight acutely after pancreatectomy and streptozotocin administration, the mean body weight increased after induction over the approximately 88-day study, indicating that the animals were in good health. CONCLUSION: This stringent porcine model of diabetic induction should be used to assess autograft transplantation safety and efficacy.

The islet size to oxygen consumption ratio reliably predicts reversal of diabetes posttransplant.

ß-Cell replacement therapy by either whole-organ pancreas or islets of Langerhans transplantation can restore carbohydrate control to diabetic patients and reduces complications associated with the disease. One of the variables inherent in islet transplantation is the isolation of functional islets from donor pancreata. Islet isolations fail to consistently produce good-quality functional islets. A rapid pretransplant assay to determine posttransplant function of islets would be an invaluable tool. We have tested the novel hypothesis that modified oxygen consumption rates (OCR), standardized to DNA quantity (nmol/min-mg DNA), would serve as a pretransplant assessment of the metabolic potency of the islets postisolation. This study compares the ability of current in vitro assays to predict in vivo restoration of normoglycemia in a diabetic nude mouse posttransplantation of adult pig islets. There is known to be a diversity of islet sizes within each preparation. This parameter has not heretofore been effectively considered a critical factor in islet engraftment. Our results suggest a surprising finding that islet size influences the probability of restoring carbohydrate control. Based on this observation, we thus developed a novel predictor of islet graft function that combines the effects of both islet OCR and size. When OCR was divided by the islet index (size), a highly significant predictor of graft function was established (p = 0.0002, n = 75). Furthermore, when OCR/islet index values exceeded 70.0 nmol/min-mg DNA/islet index, an effective threshold of diabetes reversal was observed. This assay can be performed with as few as 1,000 islet equivalents (IEQ) and conducted in less than 60 min. Our data suggest that, using this novel method to assess islet cell function prior to transplantation, OCR/islet index thresholds provide a valuable tool in identifying which islet preparations are most likely to restore glycemic control posttransplant.

Preventing and curing citrulline-induced autoimmune arthritis in a humanized mouse model using a Th2-polarizing iNKT cell agonist.

Invariant natural killer T (iNKT) cells are innate lymphocytes with unique reactivity to glycolipid antigens bound to non-polymorphic CD1d molecules. They are capable of rapidly releasing pro- and/or anti-inflammatory cytokines and constitute attractive targets for immunotherapy of a wide range of diseases including autoimmune disorders. In this study, we have explored the beneficial effects of OCH, a Th2-polarizing glycolipid agonist of iNKT cells, in a humanized mouse model of rheumatoid arthritis (RA) in which citrullinated human proteins are targeted by autoaggressive immune responses in mice expressing an RA susceptibility human leukocyte antigen (HLA) DR4 molecule. We found for the first time that treatment with OCH both prevents and cures citrulline-induced autoimmune arthritis as evidenced by resolved ankle swelling and reversed histopathological changes associated with arthritis. Also importantly, OCH treatment blocked the arthritogenic capacity of citrullinated antigen-experienced splenocytes without compromising their global responsiveness or altering the proportion of splenic naturally occurring CD4(+)CD25(+)FoxP3(+) regulatory T cells. Interestingly, administering the Th1-promoting iNKT cell glycolipid ligand α-C-galactosylceramide into HLA-DR4 transgenic mice increased the incidence of arthritis in these animals and exacerbated their clinical symptoms, strongly suggesting a role for Th1 responses in the pathogenesis of citrulline-induced arthritis. Therefore, our findings indicate a role for Th1-mediated immunopathology in citrulline-induced arthritis and provide the first evidence that iNKT cell manipulation by Th2-skewing glycolipids may be of therapeutic value in this clinically relevant model, a finding that is potentially translatable to human RA.

A novel technique for the transplantation of pancreatic islets within a vascularized device into the greater omentum to achieve insulin independence.

BACKGROUND: The greater omentum with its vascularization and blood flow has been considered as a location for islet transplantation; however, there is a need to provide a controlled and protected site for the islets within the omentum that would be applicable to donor islets and future stem cell technologies. Here we describe the use of a novel device implanted within the omentum with a subcutaneous delivery port that offers an environment for donor islets. METHODS: A prototype cell pouch device was wrapped in the greater omentum and an islet implantation port was exposed subcutaneously in diabetic Lewis rats. After tissue growth throughout the device, islet isografts were implanted and long-term glucose control was evaluated. RESULTS: By using this technique, 7 of 10 diabetic rat recipients showed long-term normal blood glucose levels after minimal islet dose transplants. Histologic assessment revealed collagen formation and vascularization within the device. CONCLUSIONS: The implanted device assessed using this technique provides a safe and efficacious environment for the support of pancreatic islets contained within a removable device as a cell therapy in a highly vascularized setting.

CD1d-independent activation of mouse and human iNKT cells by bacterial superantigens.

Invariant NKT (iNKT) cells are infrequent but important immunomodulatory lymphocytes that exhibit CD1d-restricted reactivity with glycolipid Ags. iNKT cells express a unique T-cell receptor (TCR) composed of an invariant α-chain, paired with a limited range of ß-chains. Superantigens (SAgs) are microbial toxins defined by their ability to activate conventional T cells in a TCR ß-chain variable domain (Vß)-specific manner. However, whether iNKT cells are directly activated by bacterial SAgs remains an open question. Herein, we explored the responsiveness of mouse and human iNKT cells to a panel of staphylococcal and streptococcal SAgs and examined the contribution of major histocompatibility complex (MHC) class II and CD1d to these responses. Bacterial SAgs that target mouse Vß8, such as staphylococcal enterotoxin B (SEB), were able to activate mouse hybridoma and primary hepatic iNKT cells in the presence of mouse APCs expressing human leukocyte antigen (HLA)-DR4. iNKT cell-mediated cytokine secretion in SEB-challenged HLA-DR4-transgenic mice was CD1d-independent and accompanied by a high interferon-γ:interleukin-4 ratio consistent with an in vivo Th1 bias. Furthermore, iNKT cells from SEB-injected HLA-DR4-transgenic mice, and iNKT cells from SEB-treated human PBMCs, showed early activation by intracellular cytokine staining and CD69 expression. Unlike iNKT cell stimulation by α-galactosylceramide, stimulation by SEB did not induce TCR downregulation of either mouse or human iNKT cells. We conclude that Vß8-targeting bacterial SAgs can activate iNKT cells by utilizing a novel pathway that requires MHC class II interactions, but not CD1d. Therefore, iNKT cells fulfill important effector functions in response to bacterial SAgs and may provide attractive targets in the management of SAg-induced illnesses.

Negative modulation of invariant natural killer T cell responses to glycolipid antigens by p38 MAP kinase.

Invariant natural killer T (iNKT) cells are CD1d-restricted, glycolipid-reactive lymphocytes with potent immunoregulatory characteristics. Although recent years have witnessed intensified interest in iNKT cells, little is known about intracellular signaling pathways that control iNKT cell responses, including those mediated by mitogen-activated protein kinases (MAPKs). We employed selective inhibitors of ERK1/2, JNK and p38 to examine the importance of these MAPKs in iNKT cell responses to the prototype glycolipid antigen alpha-galactosylceramide (alpha GC). Activation of DN32.D3 iNKT cells in the presence of PD98059 led to decreased interleukin (IL)-2 production, indicating a role for ERK in mouse iNKT cell responses. In contrast, addition of the JNK inhibitor SP600125 to cultures did not significantly affect cytokine production, suggesting that JNK is not critically needed for iNKT cell responses. Interestingly, selective inhibition of p38 by either SB203580 or SK&F 86002 resulted in augmented IL-2 production by DN32.D3 cells after stimulation with alpha GC. This was also evident when iNKT cells were stimulated with an anti-CD3 monoclonal antibody thus bypassing the requirement for CD1d-mediated antigen presentation, indicating that p38 inhibition affects signal transduction downstream of iNKT cells' T cell receptors. Primary splenic iNKT cells similarly exhibited enhanced cytokine response to alpha GC when cultured in the presence of p38 inhibitors. Importantly, in vivo administration of SB203580 resulted in higher IL-4 and interferon-gamma secretion in alpha GC-treated mice. These results demonstrate that MAPKs play distinct signaling roles in iNKT cells and that both in vitro and in vivo iNKT cell responses to glycolipid antigens can be negatively modulated by p38.

Diabetic rats and mice are resistant to porcine and human insulin: flawed experimental models for testing islet xenografts.

BACKGROUND: Islet transplantation is potentially a promising therapy for the restoration of carbohydrate control to diabetic patients. However, the global application of islet transplantation requires a ubiquitous source of beta cells. The xenotransplantation of porcine islets would provide such a source. Success in porcine islet xenografting has been achieved in diabetic primates. However, there are few reports of reversal of diabetes with porcine islet xenografts in rodent models of diabetes, relative to the number of successful rodent experiments performed as allografts. Here we report for the first time the inability of porcine (and human) insulin to control blood glucose levels in diabetic rodents determined by a series of dose escalating studies. METHODS: Insulin was administered intravenously to streptozotocin induced diabetic Lewis rats, Balb/c and athymic Balb/c mice (n = 5 per group) at the following doses: Group I "physiological dose" (pd) of 0.16 U/kg for a total dose of 40 mU to a 250 g rat. Group II received 0.64 U/kg (4xpd), group III 1.6 U/kg (10xpd) and group IV 6.4 U/kg (40xpd). Blood glucose levels were monitored in each animal at seven time points: 0 (pre-injection), 10 min, 20 min, 30 min, 45 min, 1 h, 1.5 h, 2 h and 3 h post-injection. Serum insulin levels were also determined. RESULTS: Diabetic Lewis rats achieved a maximum reduction in blood glucose from 22.1 +/- 1.8mmol/l to 8.0 +/- 3.1 mmol/l (a 63.7% reduction), 90 minutes post-injection of 6.4 U/kg dose of porcine insulin (40xpd). Human insulin was less effective at reducing blood glucose levels in rats than porcine insulin (P < 0.001). Porcine insulin reduced blood glucose levels in Balb/c mice from a mean of 18.2 +/- 2.1 mmol/l to a hypoglycemic minimum of 1.26 +/- 0.18 mmol/l a reduction of 93.0%, 60 min post-injection of the maximum dose of 6.4 U/kg. Balb/c mice were significantly more responsive to porcine insulin than Lewis rats at doses of 0.64 U/kg (P < 0.001), 1.6 U/kg (P < 0.05) and 6.4 U/kg (P < 0.001). Athymic Balb/c nude mice reached a maximum reduction in blood glucose from 21.6 +/- 1.8 mmol/l to 3.6 +/- 0.9 mmol/l (a 83.4% reduction) 120 min post-injection at a dose of 6.4 U/kg. Overall, athymic Balb/c nude mice were more resistant to porcine insulin than immunocompetent Balb/c mice at doses of 0.64 U/kg (P < 0.001), 1.6 U/kg (P < 0.001) and 6.4 U/kg (P < 0.05). Insulin diluent alone marginally increased blood glucose levels in all animals tested. CONCLUSIONS: Our results suggest that restoration of normoglycemia in diabetic rodents is not ideal for testing porcine islets xenografts since the reversals of diabetes in these species requires 20 to 40 times the dose of porcine insulin used in humans.

BDNF and TrkB in the preterm and near-term ovine fetal brain and the effect of intermittent umbilical cord occlusion.

We have determined the developmental change in immunoreactivity for brain-derived neurotrophic factor and its high-affinity tyrosine kinase receptor, TrkB, in the ovine fetal brain with advancing gestation and in response to intermittent umbilical cord occlusion, which might then contribute to adverse neurodevelopment. Fetal sheep (control and experimental groups at 0.75 and 0.90 of gestation) were studied over 4 days with umbilical cord occlusions performed in the experimental group animals by complete inflation of an occluder cuff for 90 seconds every 30 minutes for 3 to 5 hours each day. Animals were then euthanized and the fetal brains perfusion fixed and prepared for subsequent histology with the distribution of brain-derived neurotrophic factor and tyrosine kinase receptor immunoreactivity determined by immunohistochemistry. In the control group animals brain-derived neurotrophic factor immunoreactivity decreased in the gray matter, thalamus, and hippocampus but increased in the white matter, while tyrosine kinase receptor immunoreactivity decreased in all regions (most P < .01), with advancing gestation consistent with the developmental change from neurogenesis/gliagenesis to myelination over this time period. Intermittent umbilical cord occlusion as studied with severe but limited hypoxemia resulted in a variable decrease in brain-derived neurotrophic factor and tyrosine kinase receptor immunoreactivity for all brain regions in the preterm animals (most P < .01) when protein turnover is higher, but a selective increase in brain-derived neurotrophic factor immunoreactivity in the hippocampus of the near-term animals consistent with a heightened vulnerability for necrotic/apoptotic injury at this time. As such, brain-derived neurotrophic factor-tyrosine kinase receptor in the ovine fetal brain may be altered with intermittent hypoxic insults over the latter part of pregnancy with potential for longer term neurologic consequences.