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Apoptotic beta-cell death is central to the pathogenesis of type 1 diabetes and may be important in islet graft rejection. Despite this, genetic control of beta-cell apoptosis is only poorly understood. We report that inhibition of gene transcription sensitized beta-cells to tumor necrosis factor (TNF)-alpha-induced apoptosis, indicating the presence of a regulated antiapoptotic response. Using oligonucleotide microarrays and real-time PCR, we identified TNFAIP3/A20 as the most highly regulated antiapoptotic gene expressed in cytokine-stimulated human and mouse islets. Cytokine induction of A20 mRNA in primary islets and insulinoma cells was rapid and observed within 1 h, consistent with A20 being an immediate early response gene in beta-cells. Regulation of A20 was nuclear factor-kappaB (NF-kappaB)-dependent, two NF-kappaB sites within the A20 promoter were found to be necessary and sufficient for A20 expression in beta-cells. Activation of NF-kappaB by TNF receptor-associated factor (TRAF) 2, TRAF6, NF-kappaB-inducing kinase, or protein kinase D, which transduce signals downstream of Toll-like receptors, TNF receptors, and free radicals, respectively, were all potent activators of the A20 promoter. Moreover, A20 expression was induced in transplanted islets in vivo. Finally, A20 expression was sufficient to protect beta-cells from TNF-induced apoptosis. These data demonstrate that A20 is the cardinal antiapoptotic gene in beta-cells. Further, A20 expression is NF-kappaB dependent, thus linking islet proinflammatory gene responses with protection from apoptosis. [ABSTRACT FROM AUTHOR]

Under ideal conditions, nanotubes with a fixed negative tube-wall charge will reject anions and transport-only cations. Because many proposed nanofluidic devices are optimized in this ideally cation-permselective state, it is important to know the experimental conditions that produce ideal responses. A parameter called C crit , the highest salt concentration in a contacting solution that still produces ideal cation permselectivity, is of particular importance. Pioneering potentiometric studies on gold nanotubes were interpreted using an electrostatic model that states that C crit should occur when the Debye length in the contacting salt solution becomes equivalent to the tube radius. Since this "double-layer overlap model" (DLOM), treats all same-charge ions as identical point charges, it predicts that all same-charged cations should produce the same C crit . However, the effect of cation on C crit in gold nanotubes was never investigated. This knowledge gap has become important because recent studies with a polymeric cation-permselective nanopore membrane showed that DLOM failed for every cation studied. To resolve this issue, we conducted potentiometric studies on the effect of salt cation on C crit for a 10 nm diameter gold nanotube membrane. C crit for all cations studied were, within experimental error, the same and identical, with values predicted by DLOM. The reason DLOM prevailed for the gold nanotubes but failed for the polymeric nanopores stems from the chemical difference between the fixed negative charges of these two membranes.

A key knowledge gap in the emerging field of nanofluidics concerns how the ionic composition and ion-transport properties of a nanoconfined solution differ from those of a contacting bulk solution. We and others have been using potentiometric concentration cells, where a nanopore or nanotube membrane separates salt solutions of differing concentrations to explore this issue. The membranes studied contained a fixed pore/tube wall anionic charge, which ideally would prohibit anions and salt from entering the pore/tube-confined solution. We have been investigating experimental conditions that allow for this ideally permselective cation state to be achieved. Results of potentiometric investigations of a polymeric nanopore membrane (10 ± 2 nm-diameter pores) with anionic charge due to carbonate are presented here. While studies of this type have been reported using alkaline metal and alkaline earth cations, there have been no analogous studies using organic cations. This paper uses a homologous series of tetraalkylammonium ions to address this knowledge gap. The key result is that, in contrast to the inorganic cations, the ideal cation-permselective state could not be obtained under any experimental conditions for the organic cations. We propose that this is because these hydrophobic cations adsorb onto the polymeric pore walls. This makes ideality impossible because each adsorbed alkylammonium must bring a charge-balancing anion, Cl - , with it into the nanopore solution. The alkylammonium adsorption that occurred was confirmed and quantified by using surface contact angle measurements.

Islets prepared for transplantation into type 1 diabetes patients are exposed to compromising intrinsic and extrinsic factors that contribute to early graft failure, necessitating repeated islet infusions for clinical insulin independence. A lack of reliable pre-transplant measures to determine islet viability severely limits the success of islet transplantation and will limit future beta cell replacement strategies. We applied hyperspectral fluorescent microscopy to determine whether we could non-invasively detect islet damage induced by oxidative stress, hypoxia, cytokine injury, and warm ischaemia, and so predict transplant outcomes in a mouse model. In assessing islet spectral signals for NAD(P)H, flavins, collagen-I, and cytochrome-C in intact islets, we distinguished islets compromised by oxidative stress (ROS) (AUC = 1.00), hypoxia (AUC = 0.69), cytokine exposure (AUC = 0.94), and warm ischaemia (AUC = 0.94) compared to islets harvested from pristine anaesthetised heart-beating mouse donors. Significantly, with unsupervised assessment we defined an autofluorescent score for ischaemic islets that accurately predicted the restoration of glucose control in diabetic recipients following transplantation. Similar results were obtained for islet single cell suspensions, suggesting translational utility in the context of emerging beta cell replacement strategies. These data show that the pre-transplant hyperspectral imaging of islet autofluorescence has promise for predicting islet viability and transplant success.

Aims/hypothesis: NF-κB activation unites metabolic and inflammatory responses in many diseases yet less is known about the role that NF-κB plays in normal metabolism. In this study we investigated how RELA impacts the beta cell transcriptional landscape and provides network control over glucoregulation., Methods: We generated novel mouse lines harbouring beta cell-specific deletion of either the Rela gene, encoding the canonical NF-κB transcription factor p65 (βp65KO mice), or the Ikbkg gene, encoding the NF-κB essential modulator NEMO (βNEMOKO mice), as well as βA20Tg mice that carry beta cell-specific and forced transgenic expression of the NF-κB-negative regulator gene Tnfaip3, which encodes the A20 protein. Mouse studies were complemented by bioinformatics analysis of human islet chromatin accessibility (assay for transposase-accessible chromatin with sequencing [ATAC-seq]), promoter capture Hi-C (pcHi-C) and p65 binding (chromatin immunoprecipitation-sequencing [ChIP-seq]) data to investigate genome-wide control of the human beta cell metabolic programme., Results: Rela deficiency resulted in complete loss of stimulus-dependent inflammatory gene upregulation, consistent with its known role in governing inflammation. However, Rela deletion also rendered mice glucose intolerant because of functional loss of insulin secretion. Glucose intolerance was intrinsic to beta cells as βp65KO islets failed to secrete insulin ex vivo in response to a glucose challenge and were unable to restore metabolic control when transplanted into secondary chemical-induced hyperglycaemic recipients. Maintenance of glucose tolerance required Rela but was independent of classical NF-κB inflammatory cascades, as blocking NF-κB signalling in vivo by beta cell knockout of Ikbkg (NEMO), or beta cell overexpression of Tnfaip3 (A20), did not cause severe glucose intolerance. Thus, basal p65 activity has an essential and islet-intrinsic role in maintaining normal glucose homeostasis. Genome-wide bioinformatic mapping revealed the presence of p65 binding sites in the promoter regions of specific metabolic genes and in the majority of islet enhancer hubs (~70% of ~1300 hubs), which are responsible for shaping beta cell type-specific gene expression programmes. Indeed, the islet-specific metabolic genes Slc2a2, Capn9 and Pfkm identified within the large network of islet enhancer hub genes showed dysregulated expression in βp65KO islets., Conclusions/interpretation: These data demonstrate an unappreciated role for RELA as a regulator of islet-specific transcriptional programmes necessary for the maintenance of healthy glucose metabolism. These findings have clinical implications for the use of anti-inflammatories, which influence NF-κB activation and are associated with diabetes., (© 2023. The Author(s).)

Nuclear factor κB (NF-κB) activation is a deleterious molecular mechanism that drives acute kidney injury (AKI) and manifests in transplanted kidneys as delayed graft function. The TNFAIP3 gene encodes A20, a cytoplasmic ubiquitin ligase and a master negative regulator of the NF- κB signaling pathway. Common population-specific TNFAIP3 coding variants that reduce A20's enzyme function and increase NF- κB activation have been linked to heightened protective immunity and autoimmune disease, but have not been investigated in AKI. Here, we functionally identified a series of unique human TNFAIP3 coding variants linked to the autoimmune genome-wide association studies single nucleotide polymorphisms of F127C; namely F127C;R22Q, F127C;G281E, F127C;W448C and F127C;N449K that reduce A20's anti-inflammatory function in an NF- κB reporter assay. To investigate the impact of TNFAIP3 hypomorphic coding variants in AKI we tested a mouse Tnfaip3 hypomorph in a model of ischemia reperfusion injury (IRI). The mouse Tnfaip3 coding variant I325N increases NF- κB activation without overt inflammatory disease, providing an immune boost as I325N mice exhibit enhanced innate immunity to a bacterial challenge. Surprisingly, despite exhibiting increased intra-kidney NF- κB activation with inflammation in IRI, the kidney of I325N mice was protected. The I325N variant influenced the outcome of IRI by changing the dynamic expression of multiple cytoprotective mechanisms, particularly by increasing NF- κB-dependent anti-apoptotic factors BCL-2, BCL-XL, c-FLIP and A20, altering the active redox state of the kidney with a reduction of superoxide levels and the enzyme super oxide dismutase-1, and enhancing cellular protective mechanisms including increased Foxp3 + T cells. Thus, TNFAIP3 gene variants represent a kidney and population-specific molecular factor that can dictate the course of IRI., (Copyright © 2023 International Society of Nephrology. All rights reserved.)

Intrahepatic islet transplantation for type 1 diabetes is limited by the need for multiple infusions and poor islet viability posttransplantation. The development of alternative transplantation sites is necessary to improve islet survival and facilitate monitoring and retrieval. We tested a clinically proven biodegradable temporizing matrix (BTM), a polyurethane-based scaffold, to generate a well-vascularized intracutaneous "neodermis" within the skin for islet transplantation. In murine models, BTM did not impair syngeneic islet renal-subcapsular transplant viability or function, and it facilitated diabetes cure for over 150 days. Furthermore, BTM supported functional neonatal porcine islet transplants into RAG-1-/- mice for 400 days. Hence, BTM is nontoxic for islets. Two-photon intravital imaging used to map vessel growth through time identified dense vascular networks, with significant collagen deposition and increases in vessel mass up to 30 days after BTM implantation. In a preclinical porcine skin model, BTM implants created a highly vascularized intracutaneous site by day 7 postimplantation. When syngeneic neonatal porcine islets were transplanted intracutaneously, the islets remained differentiated as insulin-producing cells, maintained normal islet architecture, secreted c-peptide, and survived for over 100 days. Here, we show that BTM facilitates formation of an islet-supportive intracutaneous neodermis in a porcine preclinical model, as an alternative islet-transplant site., Article Highlights: Human and porcine pancreatic islets were transplanted into a fully vascularized biodegradable temporizing matrix (Novosorb) that creates a unique intracutaneous site outside of the liver in a large-animal preclinical model. The intracutaneous prevascularized site supported pancreatic islet survival for 3 months in a syngeneic porcine-transplant model. Pancreatic (human and porcine) islet survival and function were demonstrated in an intracutaneous site outside of the liver for the first time in a large-animal preclinical model., (© 2023 by the American Diabetes Association.)

Islets transplanted for type-1 diabetes have their viability reduced by warm ischemia, dimethyloxalylglycine (DMOG; hypoxia model), oxidative stress and cytokine injury. This results in frequent transplant failures and the major burden of patients having to undergo multiple rounds of treatment for insulin independence. Presently there is no reliable measure to assess islet preparation viability prior to clinical transplantation. We investigated deep morphological signatures (DMS) for detecting the exposure of islets to viability compromising insults from brightfield images. Accuracies ranged from 98 % to 68 % for; ROS damage, pro-inflammatory cytokines, warm ischemia and DMOG. When islets were disaggregated to single cells to enable higher throughput data collection, good accuracy was still obtained (83-71 %). Encapsulation of islets reduced accuracy for cytokine exposure, but it was still high (78 %). Unsupervised modelling of the DMS for islet preparations transplanted into a syngeneic mouse model was able to predict whether or not they would restore glucose control with 100 % accuracy. Our strategy for constructing DMS' is effective for the assessment of islet pre-transplant viability. If translated into the clinic, standard equipment could be used to prospectively identify non-functional islet preparations unable to contribute to the restoration of glucose control and reduce the burden of unsuccessful treatments., Competing Interests: No conflict of interest., (© 2023 The Authors.)

Background: Neonatal porcine islets (NPIs) can restore glucose control in mice, pigs, and non-human primates, representing a potential abundant alternative islet supply for clinical beta cell replacement therapy. However, NPIs are vulnerable to inflammatory insults that could be overcome with genetic modifications. Here, we demonstrate in a series of proof-of-concept experiments the potential of the cytoplasmic ubiquitin-editing protein A20, encoded by the TNFAIP3 gene, as an NPI cytoprotective gene., Methods: We forced A20 expression in NPI grafts using a recombinant adenovirus 5 (Ad5) vector and looked for impact on TNF-stimulated NF-κB activation and NPI graft function. As adeno-associated vectors (AAV) are clinically preferred vectors but exhibit poor transduction efficacy in NPIs, we next screened a series of AAV serotypes under different transduction protocols for their ability achieve high transduction efficiency and suppress NPI inflammation without impacting NPI maturation., Results: Forcing the expression of A20 in NPI with Ad5 vector blocked NF-κB activation by inhibiting IκBα phosphorylation and degradation, and reduced the induction of pro-inflammatory genes Cxcl10 and Icam1. A20-expressing NPIs also exhibited superior functional capacity when transplanted into diabetic immunodeficient recipient mice, evidenced by a more rapid return to euglycemia and improved GTT compared to unmodified NPI grafts. We found AAV2 combined with a 14-day culture period maximized NPI transduction efficiency (>70% transduction rate), and suppressed NF-κB-dependent gene expression without adverse impact upon NPI maturation., Conclusion: We report a new protocol that allows for high-efficiency genetic modification of NPIs, which can be utilized to introduce candidate genes without the need for germline engineering. This approach would be suitable for preclinical and clinical testing of beneficial molecules. We also report for the first time that A20 is cytoprotective for NPI, such that A20 gene therapy could aid the clinical development of NPIs for beta cell replacement., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Islet transplantation can restore lost glycemic control in type 1 diabetes subjects but is restricted in its clinical application by a limiting supply of islets and the need for heavy immune suppression to prevent rejection. TNFAIP3, encoding the ubiquitin editing enzyme A20, regulates the activation of immune cells by raising NF-κB signaling thresholds. Here, we show that increasing A20 expression in allogeneic islet grafts resulted in permanent survival for ~45% of recipients, and > 80% survival when combined with subtherapeutic rapamycin. Allograft survival was dependent upon Tregs and was antigen specific, and grafts showed reduced expression of inflammatory factors. Transplantation of islets with A20 containing a loss-of-function variant (I325N) resulted in increased RIPK1 ubiquitination and NF-κB signaling, graft hyperinflammation, and acute allograft rejection. Overexpression of A20 in human islets potently reduced expression of inflammatory mediators, with no impact on glucose-stimulated insulin secretion. Therapeutic administration of A20 raises inflammatory signaling thresholds to favor immune tolerance and promotes islet allogeneic survival. Clinically, this would allow for reduced immunosuppression and support the use of alternate islet sources.

Resisting and tolerating microbes are alternative strategies to survive infection, but little is known about the evolutionary mechanisms controlling this balance. Here genomic analyses of anatomically modern humans, extinct Denisovan hominins and mice revealed a TNFAIP3 allelic series with alterations in the encoded immune response inhibitor A20. Each TNFAIP3 allele encoded substitutions at non-catalytic residues of the ubiquitin protease OTU domain that diminished IκB kinase-dependent phosphorylation and activation of A20. Two TNFAIP3 alleles encoding A20 proteins with partial phosphorylation deficits seemed to be beneficial by increasing immunity without causing spontaneous inflammatory disease: A20 T108A;I207L, originating in Denisovans and introgressed in modern humans throughout Oceania, and A20 I325N, from an N-ethyl-N-nitrosourea (ENU)-mutagenized mouse strain. By contrast, a rare human TNFAIP3 allele encoding an A20 protein with 95% loss of phosphorylation, C243Y, caused spontaneous inflammatory disease in humans and mice. Analysis of the partial-phosphorylation A20 I325N allele in mice revealed diminished tolerance of bacterial lipopolysaccharide and poxvirus inoculation as tradeoffs for enhanced immunity.

Objective: Extensive molecular heterogeneity of pancreatic ductal adenocarcinoma (PDA), few effective therapies and high mortality make this disease a prime model for advancing development of tailored therapies. The p16-cyclin D-cyclin-dependent kinase 4/6-retinoblastoma (RB) protein (CDK4) pathway, regulator of cell proliferation, is deregulated in PDA. Our aim was to develop a novel personalised treatment strategy for PDA based on targeting CDK4., Design: Sensitivity to potent CDK4/6 inhibitor PD-0332991 (palbociclib) was correlated to protein and genomic data in 19 primary patient-derived PDA lines to identify biomarkers of response. In vivo efficacy of PD-0332991 and combination therapies was determined in subcutaneous, intrasplenic and orthotopic tumour models derived from genome-sequenced patient specimens and genetically engineered model. Mechanistically, monotherapy and combination therapy were investigated in the context of tumour cell and extracellular matrix (ECM) signalling. Prognostic relevance of companion biomarker, RB protein, was evaluated and validated in independent PDA patient cohorts (>500 specimens)., Results: Subtype-specific in vivo efficacy of PD-0332991-based therapy was for the first time observed at multiple stages of PDA progression: primary tumour growth, recurrence (second-line therapy) and metastatic setting and may potentially be guided by a simple biomarker (RB protein). PD-0332991 significantly disrupted surrounding ECM organisation, leading to increased quiescence, apoptosis, improved chemosensitivity, decreased invasion, metastatic spread and PDA progression in vivo. RB protein is prevalent in primary operable and metastatic PDA and may present a promising predictive biomarker to guide this therapeutic approach., Conclusion: This study demonstrates the promise of CDK4 inhibition in PDA over standard therapy when applied in a molecular subtype-specific context., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

The small GTPase RhoA is involved in a variety of fundamental processes in normal tissue. Spatiotemporal control of RhoA is thought to govern mechanosensing, growth, and motility of cells, while its deregulation is associated with disease development. Here, we describe the generation of a RhoA-fluorescence resonance energy transfer (FRET) biosensor mouse and its utility for monitoring real-time activity of RhoA in a variety of native tissues in vivo. We assess changes in RhoA activity during mechanosensing of osteocytes within the bone and during neutrophil migration. We also demonstrate spatiotemporal order of RhoA activity within crypt cells of the small intestine and during different stages of mammary gestation. Subsequently, we reveal co-option of RhoA activity in both invasive breast and pancreatic cancers, and we assess drug targeting in these disease settings, illustrating the potential for utilizing this mouse to study RhoA activity in vivo in real time., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

The emerging standard of care for patients with inoperable pancreatic cancer is a combination of cytotoxic drugs gemcitabine and Abraxane, but patient response remains moderate. Pancreatic cancer development and metastasis occur in complex settings, with reciprocal feedback from microenvironmental cues influencing both disease progression and drug response. Little is known about how sequential dual targeting of tumor tissue tension and vasculature before chemotherapy can affect tumor response. We used intravital imaging to assess how transient manipulation of the tumor tissue, or "priming," using the pharmaceutical Rho kinase inhibitor Fasudil affects response to chemotherapy. Intravital Förster resonance energy transfer imaging of a cyclin-dependent kinase 1 biosensor to monitor the efficacy of cytotoxic drugs revealed that priming improves pancreatic cancer response to gemcitabine/Abraxane at both primary and secondary sites. Transient priming also sensitized cells to shear stress and impaired colonization efficiency and fibrotic niche remodeling within the liver, three important features of cancer spread. Last, we demonstrate a graded response to priming in stratified patient-derived tumors, indicating that fine-tuned tissue manipulation before chemotherapy may offer opportunities in both primary and metastatic targeting of pancreatic cancer., (Copyright © 2017, American Association for the Advancement of Science.)

Aims/hypothesis: Administration of anti-CD40 ligand (CD40L) antibodies has been reported to allow long-term islet allograft survival in non-human primates without the need for exogenous immunosuppression. However, the use of anti-CD40L antibodies was associated with thromboembolic complications. Targeting downstream intracellular components shared between CD40 and other TNF family co-stimulatory molecules could bypass these complications. TNF receptor associated factor 2 (TRAF2) integrates multiple TNF receptor family signalling pathways that are critical for T cell activation and may be a central node of alloimmune responses., Methods: T cell-specific Traf2-deficient mice (Traf2TKO) were generated to define the role of TRAF2 in CD4 + T cell effector responses that mediate islet allograft rejection in vivo. In vitro allograft responses were tested using mixed lymphocyte reactions and analysis of IFN-γ and granzyme B effector molecule expression. T cell function was assessed using anti-CD3/CD28-mediated proliferation and T cell polarisation studies., Results: Traf2TKO mice exhibited permanent survival of full MHC-mismatched pancreatic islet allografts without exogenous immunosuppression. Traf2TKO CD4 + T cells exhibited reduced proliferation, activation and acquisition of effector function following T cell receptor stimulation; however, both Traf2TKO CD4 + and CD8 + T cells exhibited impaired alloantigen-mediated proliferation and acquisition of effector function. In polarisation studies, Traf2TKO CD4 + T cells preferentially converted to a T helper (Th)2 phenotype, but exhibited impaired Th17 differentiation. Without TRAF2, thymocytes exhibited dysregulated TNF-mediated induction of c-Jun N-terminal kinase (JNK) and canonical NFκB pathways. Critically, targeting TRAF2 in T cells did not impair the acute phase of CD8-dependent viral immunity. These data highlight a specific requirement for a TRAF2-NFκB and TRAF2-JNK signalling cascade in T cell activation and effector function in rejecting islet allografts., Conclusion/interpretation: Targeting TRAF2 may be useful as a therapeutic approach for immunosuppression-free islet allograft survival that avoids the thromboembolic complications associated with the use of anti-CD40L antibodies.

E-cadherin-mediated cell-cell junctions play a prominent role in maintaining the epithelial architecture. The disruption or deregulation of these adhesions in cancer can lead to the collapse of tumor epithelia that precedes invasion and subsequent metastasis. Here we generated an E-cadherin-GFP mouse that enables intravital photobleaching and quantification of E-cadherin mobility in live tissue without affecting normal biology. We demonstrate the broad applications of this mouse by examining E-cadherin regulation in multiple tissues, including mammary, brain, liver, and kidney tissue, while specifically monitoring E-cadherin mobility during disease progression in the pancreas. We assess E-cadherin stability in native pancreatic tissue upon genetic manipulation involving Kras and p53 or in response to anti-invasive drug treatment and gain insights into the dynamic remodeling of E-cadherin during in situ cancer progression. FRAP in the E-cadherin-GFP mouse, therefore, promises to be a valuable tool to fundamentally expand our understanding of E-cadherin-mediated events in native microenvironments., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

The nuclear factor κB (NF-κB) pathway is a master regulator of inflammatory processes and is implicated in insulin resistance and pancreatic β cell dysfunction in the metabolic syndrome. Whereas canonical NF-κB signaling is well studied, there is little information on the divergent noncanonical NF-κB pathway in the context of pancreatic islet dysfunction. Here, we demonstrate that pharmacological activation of the noncanonical NF-κB-inducing kinase (NIK) disrupts glucose homeostasis in zebrafish in vivo. We identify NIK as a critical negative regulator of β cell function, as pharmacological NIK activation results in impaired glucose-stimulated insulin secretion in mouse and human islets. NIK levels are elevated in pancreatic islets isolated from diet-induced obese (DIO) mice, which exhibit increased processing of noncanonical NF-κB components p100 to p52, and accumulation of RelB. TNF and receptor activator of NF-κB ligand (RANKL), two ligands associated with diabetes, induce NIK in islets. Mice with constitutive β cell-intrinsic NIK activation present impaired insulin secretion with DIO. NIK activation triggers the noncanonical NF-κB transcriptional network to induce genes identified in human type 2 diabetes genome-wide association studies linked to β cell failure. These studies reveal that NIK contributes a central mechanism for β cell failure in diet-induced obesity., (© 2015 Malle et al.)

In this study, a critical and novel role for TNF receptor (TNFR) associated factor 2 (TRAF2) is elucidated for peripheral CD8(+) T-cell and NKT-cell homeostasis. Mice deficient in TRAF2 only in their T cells (TRAF2TKO) show ∼40% reduction in effector memory and ∼50% reduction in naïve CD8(+) T-cell subsets. IL-15-dependent populations were reduced further, as TRAF2TKO mice displayed a marked ∼70% reduction in central memory CD8(+) CD44(hi) CD122(+) T cells and ∼80% decrease in NKT cells. TRAF2TKO CD8(+) CD44(hi) T cells exhibited impaired dose-dependent proliferation to exogenous IL-15. In contrast, TRAF2TKO CD8(+) T cells proliferated normally to anti-CD3 and TRAF2TKO CD8(+) CD44(hi) T cells exhibited normal proliferation to exogenous IL-2. TRAF2TKO CD8(+) T cells expressed normal levels of IL-15-associated receptors and possessed functional IL-15-mediated STAT5 phosphorylation, however TRAF2 deletion caused increased AKT activation. Loss of CD8(+) CD44(hi) CD122(+) and NKT cells was mechanistically linked to an inability to respond to IL-15. The reduced CD8(+) CD44(hi) CD122(+) T-cell and NKT-cell populations in TRAF2TKO mice were rescued in the presence of high dose IL-15 by IL-15/IL-15Rα complex administration. These studies demonstrate a critical role for TRAF2 in the maintenance of peripheral CD8(+) CD44(hi) CD122(+) T-cell and NKT-cell homeostasis by modulating sensitivity to T-cell intrinsic growth factors such as IL-15., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Aryl hydrocarbon receptor nuclear translocator (ARNT) is a transcription factor that binds to partners to mediate responses to environmental signals. To investigate its role in the innate immune system, floxed ARNT mice were bred with lysozyme M-Cre recombinase animals to generate lysozyme M-ARNT (LAR) mice with reduced ARNT expression. Myeloid cells of LAR mice had altered mRNA expression and delayed wound healing. Interestingly, when the animals were rendered diabetic, the difference in wound healing between the LAR mice and their littermate controls was no longer present, suggesting that decreased myeloid cell ARNT function may be an important factor in impaired wound healing in diabetes. Deferoxamine (DFO) improves wound healing by increasing hypoxia-inducible factors, which require ARNT for function. DFO was not effective in wounds of LAR mice, again suggesting that myeloid cells are important for normal wound healing and for the full benefit of DFO. These findings suggest that myeloid ARNT is important for immune function and wound healing. Increasing ARNT and, more specifically, myeloid ARNT may be a therapeutic strategy to improve wound healing., (Copyright © 2014 the American Physiological Society.)

B cells inhabit the normal human thymus, suggesting a role in T cell selection. In this study, we report that B cells can modulate thymic production of CD4+ Foxp3+ T cells (regulatory T cells [Tregs]). Mice with transgenic expression of BAFF (BAFF-Tg) harbor increased numbers of Helios+ Foxp3+ thymic Tregs and, similar to some human autoimmune conditions, also exhibit increased numbers of B cells colonizing the thymus. Distinct intrathymic B cell subpopulations were identified, namely B220+, IgM+, CD23(hi), CD21(int) cells; B220+, IgM+, CD23(lo), CD21(lo) cells; and a population of B220+, IgM+, CD23(lo), CD21(hi) cells. Anatomically, CD19+ B cells accumulated in the thymic medulla region juxtaposed to Foxp3+ T cells. These intrathymic B cells engender Tregs. Indeed, thymic Treg development was diminished in both B cell-deficient BAFF-Tg chimeras, but also B cell-deficient wild-type chimeras. B cell Ag capture and presentation are critical in vivo events for Treg development. In the absence of B cell surface MHC class II expression, thymic expansion of BAFF-Tg Tregs was lost. Further to this, expansion of Tregs did not occur in BAFF-Tg/Ig hen egg lysozyme BCR chimeras, demonstrating a requirement for Ag specificity. Thus, we present a mechanism whereby intrathymic B cells, through the provision of cognate help, contribute to the shaping of the Treg repertoire., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Targeting the BAFF/APRIL system has shown to be effective in preventing T-cell dependent autoimmune disease in the NOD mouse, a spontaneous model of type 1 diabetes. In this study we generated BAFF-deficient NOD mice to examine how BAFF availability would influence T-cell responses in vivo and the development of spontaneous diabetes. BAFF-deficient NOD mice which lack mature B cells, were protected from diabetes and showed delayed rejection of an allogeneic islet graft. Diabetes protection correlated with a failure to expand pathogenic IGRP-reactive CD8(+) T cells, which were maintained in the periphery at correspondingly low levels. Adoptive transfer of IGRP-reactive CD8(+) T cells with B cells into BAFF-deficient NOD mice enhanced IGRP-reactive CD8(+) T-cell expansion. Furthermore, when provoked with cyclophosphamide, or transferred to a secondary lymphopenic host, the latent pool of self-reactive T cells resident in BAFF-deficient NOD mice could elicit beta cell destruction. We conclude that lack of BAFF prevents the procurement of B-cell-dependent help necessary for the emergence of destructive diabetes. Indeed, treatment of NOD mice with the BAFF-blocking compound, BR3-Fc, resulted in a delayed onset and reduced incidence of diabetes., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Chronic hyperglycemia contributes to β-cell dysfunction in diabetes and with islet transplantation, but the mechanisms remain unclear. Recent studies demonstrate that the unfolded protein response (UPR) is critical for β-cell function. Here, we assessed the influence of hyperglycemia on UPR gene expression in transplanted islets. Streptozotocin-induced diabetic or control nondiabetic mice were transplanted under the kidney capsule with syngeneic islets either sufficient or not to normalize hyperglycemia. Twenty-one days after transplantation, islet grafts were excised and RT-PCR was used to assess gene expression. In islet grafts from diabetic mice, expression levels of many UPR genes of the IRE1/ATF6 pathways, which are important for adaptation to endoplasmic reticulum stress, were markedly reduced compared with that in islet grafts from control mice. UPR genes of the PERK pathway were also downregulated. The normalization of glycemia restored the changes in mRNA expression, suggesting that chronic hyperglycemia contributes to the downregulation of multiple arms of UPR gene expression. Similar correlations were observed between blood glucose and mRNA levels of transcription factors involved in the maintenance of β-cell phenotype and genes implicated in β-cell function, suggesting convergent regulation of UPR gene expression and β-cell differentiation by hyperglycemia. However, the normalization of glycemia was not accompanied by restoration of antioxidant or pro-inflammatory cytokine mRNA levels, which were increased in islet grafts from diabetic mice. These studies demonstrate that chronic hyperglycemia contributes to the downregulation of multiple arms of UPR gene expression in transplanted mouse islets. Failure of the adaptive UPR may contribute to β-cell dedifferentiation and dysfunction in diabetes.

Aims/hypothesis: For beta cells, contact with TNF-α triggers signalling cascades that converge on pathways important for cell survival and inflammation, specifically nuclear factor κB (NF-κB), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase pathways. Here, we investigated the function of baculoviral inhibitors of apoptosis repeat containing (BIRC) proteins in regulating TNF signalling cascades., Methods: TNF regulation of Birc genes was studied by mRNA expression and promoter analysis. Birc gene control of cell signalling was studied in beta cell lines, and in islets from Birc2(-/-) and Birc3(-/-) mice, and from Birc3(-/-) Birc2Δ beta cell mice that selectively lack Birc2 and Birc3 (double knockout [DKO]). Islet function was tested by intraperitoneal glucose tolerance test and transplantation., Results: TNF-α selectively induced Birc3 in beta cells, which in turn was sufficient to drive and potentiate NF-κB reporter activity. Conversely, Birc3(-/-) islets exhibited delayed TNF-α-induced IκBα degradation with reduced expression of Ccl2 and Cxcl10. DKO islets showed a further delay in IκBα degradation kinetics. Surprisingly, DKO islets exhibited stimulus-independent and TNF-dependent hyperexpression of TNF target genes A20 (also known as Tnfaip3), Icam1, Ccl2 and Cxcl10. DKO islets showed hyperphosphorylation of the JNK-substrate, c-Jun, while a JNK-antagonist prevented increases of Icam1, Ccl2 and Cxcl10 expression. Proteosome blockade of MIN6 cells phenocopied DKO islets. DKO islets showed more rapid loss of glucose homeostasis when challenged with the inflammatory insult of transplantation., Conclusions/interpretation: BIRC3 provides a feed-forward loop, which, with BIRC2, is required to moderate the normal speed of NF-κB activation. Paradoxically, BIRC2 and BIRC3 act as a molecular brake to rein in activation of the JNK signalling pathway. Thus BIRC2 and BIRC3 fine-tune NF-κB and JNK signalling to ensure transcriptional responses are appropriately matched to extracellular inputs. This control is critical for the beta cell's stress response.

We examined whether hypoxic exposure prior to the event of transplantation would have a positive or negative effect upon later islet graft function. Mouse islets exposed to hypoxic culture were transplanted into syngeneic recipients. Islet graft function, β-cell physiology, as well as molecular changes were examined. Expression of hypoxia-response genes in human islets pre- and posttransplant was examined by microarray. Hypoxia-preexposed murine islet grafts provided poor glycemic control in their syngeneic recipients, marked by persistent hyperglycemia and pronounced glucose intolerance with failed first- and second-phase glucose-stimulated insulin secretion in vivo. Mechanistically, hypoxic preexposure stabilized HIF-1α with a concomitant increase in hypoxic-response genes including LDHA, and a molecular gene set, which would favor glycolysis and lactate production and impair glucose sensing. Indeed, static incubation studies showed that hypoxia-exposed islets exhibited dysregulated glucose responsiveness with elevated basal insulin secretion. Isolated human islets, prior to transplantation, express a characteristic hypoxia-response gene expression signature, including high levels of LDHA, which is maintained posttransplant. Hypoxic preexposure of an islet graft drives a HIF-dependent switch to glycolysis with subsequent poor glycemic control and loss of GSIS. Early intervention to reverse or prevent these hypoxia-induced metabolic gene changes may improve clinical islet transplantation.

Islet grafts can contribute to their own destruction via the elaboration of proinflammatory genes, many of which are transcriptionally regulated by nuclear factor κ-light-chain-enhancer of activated B-cells (NF-κB). Thus, NF-κB constitutes an enticing gene therapy candidate to improve the success of islet transplantation. To test this hypothesis in vivo, we blocked NF-κB in BALB/c (H2(d)) to C57/BL6 (H2(b)) mouse islet allografts by genetically engineering islets to express the NF-κB superrepressor, IκBα. Here we show by microarray and RTqPCR that islets exhibit an intrinsic early immediate proinflammatory response, with the most highly upregulated proinflammatory genes comprising the chemokines Cxcl1, Cxcl2, Cxcl10, and Ccl2; the cytokines Tnf-α and Il-6; and the adhesion molecule Icam1. Overexpression of IκBα inhibited the expression of these genes by 50-95% in islets and MIN6 β-cells in vitro, by inhibiting NF-κB-dependent gene transcription. Histological and RTqPCR analysis at postoperative day (POD) 10 revealed that IκBα-transduced islet allografts exhibited improved islet architecture and strong insulin-labeling with decreased Ccl2 and Il-6 mRNA levels compared to the GFP-transduced control grafts. Despite these protective effects, NF-κB-blocked islet allografts were promptly rejected in our MHC-mismatched mouse model. However, IκBα-expressing grafts did harbor localized "pockets" of Foxp3(+) mononuclear cells not evident in the control grafts. This result suggested that the effect of the NF-κB blockade might synergize with regulatory T-cell-sparing rapamycin. Indeed, combining intragraft IκBα expression with low-dose rapamycin increased the mean survival time of islet allografts from 20 to 81 days, with 20% of the grafts surviving for greater than 100 days. In conclusion, rapamycin unmasks the protective potential of intragraft NF-κB blockade, which can, in some cases, permit permanent allograft survival without continuous systemic immunosuppression.

In the context of islet transplantation, experimental models show that induction of islet intrinsic NF-κB-dependent proinflammatory genes can contribute to islet graft rejection. Isolation of human islets triggers activation of the NF-κB and mitogen-activated kinase (MAPK) stress response pathways. However, the downstream NF-κB target genes induced in human islets during the isolation process are poorly described. Therefore, in this study, using microarray, bioinformatic, and RTqPCR approaches, we determined the pattern of genes expressed by a set of 14 human islet preparations. We found that isolated human islets express a panel of genes reminiscent of cells undergoing a marked NF-κB-dependent proinflammatory response. Expressed genes included matrix metallopeptidase 1 (MMP1) and fibronectin 1 (FN1), factors involved in tissue remodeling, adhesion, and cell migration; inflammatory cytokines IL-1β and IL-8; genes regulating cell survival including A20 and ATF3; and notably high expression of a set of chemokines that would favor neutrophil and monocyte recruitment including CXCL2, CCL2, CXCL12, CXCL1, CXCL6, and CCL28. Of note, the inflammatory profile of isolated human islets was maintained after transplantation into RAG(-/-) recipients. Thus, human islets can provide a reservoir of NF-κB-dependent inflammatory factors that have the potential to contribute to the anti-islet-graft immune response. To test this hypothesis, we extracted rodent islets under optimal conditions, forced activation of NF-κB, and transplanted them into allogenic recipients. These NF-κB activated islets not only expressed the same chemokine profile observed in human islets but also struggled to maintain normoglycemia posttransplantation. Further, NF-κB-activated islets were rejected with a faster tempo as compared to non-NF-κB-activated rodent islets. Thus, isolated human islets can make cell autonomous contributions to the ensuing allograft response by elaborating inflammatory factors that contribute to their own demise. These data highlight the potential importance of islet intrinsic proinflammatory responses as targets for therapeutic intervention.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by the production of autoantibodies. However, the underlying cause of disease appears to relate to defects in T cell tolerance or T cell help to B cells. Transgenic (Tg) mice overexpressing the cytokine B cell-activating factor of the tumor necrosis factor family (BAFF) develop an autoimmune disorder similar to SLE and show impaired B cell tolerance and altered T cell differentiation. We generated BAFF Tg mice that were completely deficient in T cells, and, surprisingly, these mice developed an SLE-like disease indistinguishable from that of BAFF Tg mice. Autoimmunity in BAFF Tg mice did, however, require B cell-intrinsic signals through the Toll-like receptor (TLR)-associated signaling adaptor MyD88, which controlled the production of proinflammatory autoantibody isotypes. TLR7/9 activation strongly up-regulated expression of transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), which is a receptor for BAFF involved in B cell responses to T cell-independent antigens. Moreover, BAFF enhanced TLR7/9 expression on B cells and TLR-mediated production of autoantibodies. Therefore, autoimmunity in BAFF Tg mice results from altered B cell tolerance, but requires TLR signaling and is independent of T cell help. It is possible that SLE patients with elevated levels of BAFF show a similar basis for disease.

Objective: Our previous study has shown that porcine antigen-primed and CD4+ T cells activated macrophages are capable of the Recognition and rejection of porcine xenografts but not mouse allografts, and therefore suggested the involvement of signaling between the graft and macrophages in this specific graft recognition and destruction., Methods: NOD-SCID mice were transplanted with fetal pig pancreatic fragment (FPP) before adoptive transfer with exogenous macrophages isolated from rejecting FPP xenografts of BALB/c recipient mice. The exogenous macrophages were tracked by Ly5.1 surface antigen or via CSFE staining. Gene expression of CCR2 and CCR5 and their chemokines in transplanted FPP xenografts was evaluated by real-time PCR., Results: After the adoptive transfer, recently transplanted but not established FPP xenografts were rejected by exogenous activated macrophages. In the meantime, greater level of chemokine gene expression was detected in recently-transplanted compared with the established xenografts. Furthermore, expression of both CCR2 and CCR5 genes was enhanced significantly in activated macrophages when compared with non-activated macrophages., Conclusion: Upregulated chemokines were associated with macrophage recruitment and destruction of islet xenografts.

Background: Adoptive transfer of antigen-primed T-cell-activated macrophages into NOD-SCID mice within 14 days of foetal porcine pancreatic fragment (FPP) or foetal porcine skin (FPS) transplantation had been shown to cause xenograft rejection. In the present study, it was proposed that signaling between the graft and macrophages promoted specific graft recognition and destruction in this setting., Methods: Exogenous macrophages isolated from rejecting FPP xenografts were transferred to NOD-SCID FPP recipients and tracked by Ly5.1 surface antigen or via CSFE staining. Monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1alpha (MIP-1alpha), macrophage inflammatory protein-1beta (MIP-1beta), regulated upon activation, normal T-cell expressed and secreted (RANTES), chemokine (C-C motif) receptor 2 (CCR2), chemokine (C-C motif) receptor 5 (CCR5), toll-like receptors (TLRs) (1-9) and gene expression in transplanted FPP xenografts was evaluated by real-time polymerase chain reaction. Gene expression of CCR2, CCR5 and TLRs was also analyzed in pooled samples of activated and non-activated macrophages., Results: Exogenous macrophages were shown to track to and reject recently transplanted but not established FPP xenografts. Gene expression for MCP-1, RANTES, MIP-1alpha and MIP-1beta was at least 3-fold greater in recently transplanted compared with established xenografts (P < 0.05), and CCR2 and CCR5 gene expression was 10-fold greater in activated compared non-activated macrophages, suggesting that graft-mediated pro-inflammatory signals were important for macrophage recruitment. Specific graft recognition by macrophages may involve TLR signaling as macrophages exposed to porcine islets had higher levels of TLR gene expression compared with those exposed to allografts regardless of the level of activation., Conclusion: Xenografts provide additional activation signals to macrophages that are not seen following allotransplantation. This study identifies chemokines and TLR as important signals in macrophage-mediated recognition and rejection of islet xenografts.

Objective: To determine whether pancreatic islet transplantation can control diabetes and prevent severe life-threatening hypoglycaemia., Design, Setting and Participants: A single-arm observation study of six patients undergoing islet transplantation. All patients had had type 1 diabetes mellitus for over 5 years and documented episodes of repeated severe hypoglycaemia. Islets were isolated from donor pancreases digested by Liberase. Separated islets were infused into the recipient's liver via the portal vein. Patients were immunosuppressed with daclizumab, sirolimus and tacrolimus. The transplants were performed at Westmead Hospital, NSW, between October 2002 and February 2005., Main Outcome Measures: Normal blood glucose control without administration of exogenous insulin; demonstration of islet function and abolition of hypoglycaemia., Results: Five of the patients received two islet infusions, and the sixth was withdrawn after one infusion following a portal vein thrombosis. Three patients became insulin-independent, with excellent glycaemic control. Two had islet function with circulating C-peptide, improved glycaemic control, reduced insulin requirement and abolition of severe hypoglycaemia. However, over a 2-year period, graft function deteriorated. Recipients who were initially insulin free remained C-peptide positive but required supplemental insulin. Complications included one postoperative bleed, two portal vein thromboses (which resolved completely), presumed recurrence of tuberculosis in one patient, and deterioration in renal function in one patient., Conclusions: Islet transplantation is effective at improving glycaemic control and hypoglycaemia unawareness in the short to medium term. However, problems with long-term safety of immunosuppression, islet-induced thrombosis and early detection of loss of islet function remain to be addressed.

Background: Pigs are an important large animal model for transplantation and a potential source of xenografts. Swine leukocyte antigen (SLA) molecules are strong mediators of alloreactive and xenoreactive immune responses. We have characterized the SLA alleles of a new pig line bred for transplantation research, the Westran (Westmead Hospital transplantation) pig, described in a companion paper., Methods: Three sixth generation inbred Westran pigs and a Large White pig control were used to assess SLA alleles. We examined the SLA-1, SLA-3, SLA-6, SLA-2, DQA1, DQB1, DRA1 and DRB1 loci using reverse transcription-polymerase chain reaction and sequencing-based method., Results: All of the Westran pigs had a single allele at each locus, except for the SLA-1 locus. Typing of the SLA-1 locus in additional animals indicated that this is most likely the result of a duplication of the SLA-1 locus rather than heterozygosity. The lack of SLA heterozygosity is consistent with the previous finding of low microsatellite marker heterozygosity and is the result of both the recent deliberate inbreeding of these pigs and their derivation from a feral stock from Kangaroo Island, South Australia, established by the release of a single pair in 1803., Conclusions: After comparing DNA and protein sequences of the Westran SLA alleles with published GenBank SLA sequences, the SLA class I alleles found in the Westran pigs were all novel, while the SLA-DR and DQB1 alleles have been previously described in other pig breeds. Characterization of the SLA alleles in the Westran pigs has identified novel alleles and will be useful for designing protocols for modulation of immune responses to allografts and xenografts.

Primary nonfunction of transplanted islets results in part from their sensitivity to reactive oxygen species (ROS) generated during the isolation and transplantation process. Our aim was to examine whether coexpression of antioxidant enzymes to detoxify multiple ROS increased the resistance of mouse islets to oxidative stress and improved the initial function of islet grafts. Islets from transgenic mice expressing combinations of human copper/zinc superoxide dismutase (SOD), extracellular SOD, and cellular glutathione peroxidase (Gpx-1) were subjected to oxidative stress in vitro. Relative viability after hypoxanthine/xanthine oxidase treatment was as follows: extracellular SOD + Gpx-1 + Cu/Zn SOD > extracellular SOD + Gpx-1 > extracellular SOD > wild type. Expression of all three enzymes was the only combination protective against hypoxia/reoxygenation. Islets from transgenic or control wild-type mice were then transplanted into streptozotocin-induced diabetic recipients in a syngeneic marginal islet mass model, and blood glucose levels were monitored for 7 days. In contrast to single- and double-transgenic grafts, triple-transgenic grafts significantly improved control of blood glucose compared with wild type. Our results indicate that coexpression of antioxidant enzymes has a complementary beneficial effect and may be a useful approach to reduce primary nonfunction of islet grafts.

Background: The Westran pig has been purposely inbred for use in xenotransplantation. The herd originated in the wild from a limited gene pool and has been inbred by repeated full-sib matings for nine generations., Methods: The aim of this study was to evaluate the level of inbreeding by functional assays, such as bi-directional MLR and reciprocal skin grafts between herd members, and by genetic analysis using highly polymorphic genetic markers to calculate the level of inbreeding., Results: The MLR between herd members were non-reactive whereas there was a prompt response to third party pig lymphocytes, indicative of a normal immune responsiveness in Westran pigs but isogenicity of the major histocompatibility complex. Skin grafts between male siblings or female sibling skin grafts on male recipients showed prolonged survival but with few exceptions did not survive beyond 100 days suggesting that by the fifth generation the Westran herd was still mismatched at minor histocompatibility antigens. This level of functional inbreeding was confirmed by microsatellite analysis of highly polymorphic markers, which showed that 52 of 53 chromosomally dispersed markers were fixed by the ninth generation. This level of fixation was consistent with 19 to 20 generations of full-sibling inbreeding. The calculated inbreeding coefficient at generation 10 was 0.98159., Conclusions: This analysis confirms that the Westran pig is highly inbred and we propose that analysis of chromosomally dispersed highly polymorphic markers is an accurate and reproducible method for assessing the level of inbreeding of a pig herd.

Background: Important phylogenetic differences between pig and human tissues prevent xenotransplantation from becoming a clinically feasible option. Humans lack the galactose-alpha1,3-galactose (alphaGal) epitope on endothelial cell surfaces and therefore have preformed anti-alphaGal antibodies. The role of these antibodies in rejection of non-vascular xenografts remains controversial. This study investigated the role of anti-alphaGal antibodies in rejection of non-vascularized alphaGal+/+ grafts in alphaGal -/- mice., Methods: alphaGal +/+ and alphaGal -/- pancreatic islets were transplanted under the renal capsule of streptozotocin-induced diabetic (1) alphaGal -/- mice and (2) alphaGal +/+ mice. alphaGal -/- recepients were immunized with rabbit red blood cell membranes (RRBCs) to produce elevated anti-alphaGal antibody levels., Results: Six of the 18 alphaGal -/- mice rejected the alphaGal +/+ grafts within 68 days whereas indefinite graft survival was achieved in the control groups. Animals with surviving islet grafts were challenged with alphaGal +/+ skin grafts. Although all alphaGal +/+ skin grafts were rejected within 58 days, the islet grafts remained intact. This observation correlated with the level of alphaGal expression (which was very low on islets compared to skin) rather than the actual titre of anti-alphaGal antibody., Discussion: The results suggest that the level of alphaGal expression plays an important role in graft survival. Therefore, its removal is important in the development of a pig islet donor for future clinical therapy.

Purpose: Age-related maculopathy (ARM) is one of the most common causes of blindness in older adults worldwide. Sequence variants in a gene coding for a retina-specific ATP-binding cassette (ABCA4) transporter protein, which is responsible for a phenotypically similar Mendelian form of retinal disease, were proposed to increase the risk of ARM. To examine the potential relationship of ABCA4 sequence variation and ARM risk in an independent data set, a clinically well-characterized population of 165 multiplex patients with ARM from 70 families, 33 unaffected relatives, and 59 unrelated control subjects with confirmed absence of ARM was screened for variants in any of the 50 exons and exon-intron boundaries of this gene., Methods: A combination of denaturing high-performance liquid chromatography (DHPLC) and bidirectional sequencing was used to detect ABCA4 sequence variants. The data set was analyzed with both case-control and family-based association analysis methods., Results: No evidence was found of significantly different allele frequencies of ABCA4 sequence variants in patients compared with control subjects, and no evidence for association or cosegregation with disease in family-based analyses., Conclusions: This study confirmed the very high degree of ABCA4 sequence polymorphism in the general population, which makes the detection of potential disease-associated alleles particularly challenging. While this study does not definitively exclude ABCA4 from contributing to a small or moderate fraction of ARM, it adds to the body of evidence suggesting that ABCA4 is not a major susceptibility gene for this disorder.

Macrophages have been proposed as the major effector cell in T cell-mediated xenograft rejection. To determine their role in this response, NOD-SCID mice were transplanted with fetal pig pancreas (FPP) before reconstitution with CD4(+) T cells from BALB/c mice. Twelve days after CD4(+) T cell reconstitution, purified macrophages (depleted of T cells) were isolated from CD4(+) T cell-reconstituted FPP recipient mice and adoptively transferred to their nonreconstituted counterparts. After adoptive macrophage transfer, FPP recipient mice transferred with macrophages from CD4(+) T cell-reconstituted mice demonstrated xenograft destruction along with massive macrophage infiltration at day 4 and complete graft destruction at day 8 postmacrophage transfer. By contrast, FPP recipients that received macrophages from nonreconstituted mice showed intact FPP xenografts with few infiltrating macrophages at both days 4 and 8 after macrophage transfer. The graft-infiltrating macrophages showed increased expression of their activation markers. Depletion of endogenous macrophages or any remaining CD4(+) T cells did not delay graft rejection in the macrophage-transferred FPP recipients, whereas depletion of transferred macrophages with clodronate liposomes prevented graft rejection. Our results show that macrophages primed by FPP and activated by CD4(+) T cells were attracted from the peripheral circulation and were capable of specific targeting and destruction of FPP xenografts. This suggests that in xenograft rejection, there are macrophage-specific recognition and targeting signals that are independent of those received by T cells.

Background: In this study, the mechanisms by which CD4+ T cells interact with the innate immune system in xenograft rejection were investigated., Methods: Fetal pig pancreas (FPP) grafts were transplanted into female SCID mice. The FPP recipient SCID mice were reconstituted with exogenous leukocytes obtained from male BALB/c mice., Results: Although nonreconstituted SCID recipients or recipients reconstituted with CD4+ T cell-depleted leukocytes showed indefinite FPP graft survival with very few macrophages infiltrating their grafts, reconstitution of SCID recipients with as few as 2x10(5) CD4+ T cells was sufficient to induce rapid xenograft rejection. CD4+ T cells secreted interferon-gamma but not interleukin-4 and initiated the activation and accumulation of macrophages and natural killer cells, that were responsible for the rapid graft destruction. Suppression of interferon-gamma prolonged graft survival and suppressed the macrophages and natural killer cell accumulation and activation., Conclusions: These results demonstrate that CD4+ T cell-dependent cellular xenograft rejection was a result of macrophage and natural killer cell accumulation and activation, but was not mediated by eosinophils. Consistent with this was the finding that interferon-gamma but not interleukin-4 was in part responsible for mediating this effect.

Previously, we demonstrated that combination CTLA4-Fc and anti-CD40L mAb treatment results in tolerance to concordant, cellular islet xenografts. The aim of this study was to determine its effectiveness in a model of fetal pig pancreas (FPP) xenotransplantation. Survival of FPP fragment grafts were compared to the survival of rat islet or cardiac xenografts following short term CTLA4-Fc and anti-CD40L mAb treatment. Rat islet and FPP fragment grafts survived long-term. However, rat cardiac grafts were rejected by 52-91 days. Both rat islet and FPP grafts showed similar histology with intact islet structures and adjacent 'nests' of lymphocytes. Concordant vascularised rat hearts showed extensive polymorphonuclear infiltrate, concentric vasculitis and a perivascular infiltrate predominantly of CD8+ T cells. This suggests that this therapy is effective for prolonging islet xenografts and demonstrates that the cellular mechanism of rejection for vascularised and non-vascularised xenografts are different.

For islet allotransplantation to become a therapy widely applicable to patients with insulin-dependent diabetes, it will be important to avoid conventional immunosuppression and yet maintain long-term rejection-free islet survival. This possibility was tested in a large-animal model using mixed allogeneic chimeras established using total lymphoid irradiation (TLI) and donor-specific bone marrow transplantation (BMTX). Four recipient sex-mismatched and DLA class II-matched English springer spaniels became chimeric after TLI and donor-specific BMTX. Subsequent donor-specific renal allografts survived for more than a year. Acceptance of a donor-specific skin graft and rejection of a third-party graft demonstrated tolerance with maintenance of immunocompetence. Pancreatic microfragments containing islets were refluxed into the splenic vein of the recipient. Purified islets were placed under the capsule of spleen and liver. After 75 days, recipients underwent total native pancreatectomy. All four chimeric pancreatectomized dogs had functioning islet grafts 75 days after transplantation, evidenced by a prompt rise in serum insulin levels following an IVGTT and histological demonstration of islet tissue at the site of transplantation. After removal of the transplanted islet tissue, no insulin was released after IVGTT. In summary, intrasplenic allogeneic canine islets transplanted into chimeric dogs rendered tolerant to donor MHC survive and function for greater than 75 days in the absence of immunosuppression. This study represents proof of the concept that allogeneic islet transplants have the potential to reverse diabetes without the use of conventional immunosuppression.

The long-term goal of this study is to assess the feasibility of using fetal pig pancreas fragment (FPPF) transplantation to treat patients with type I diabetes. Using the highly inbred Westran Pigs, our initial aim was to establish a rejection-free transplant model of FPPF grafted into sibling recipient pigs without immunosuppression. FPPFs were isolated from 80-100-day-old fetuses of either Westran Pigs or outbred pigs and transplanted into the thymus, spleen, liver, or kidney of the recipient Westran pig. Biopsies were taken from each transplant site at set time points and assessed histologically for islet viability, rejection, and endocrine function. Fifty-eight fetal donors were used to transplant 16 recipient pigs. A nonspecific inflammation was seen for both outbred and inbred FPPF donor tissue at day 3 and was considered a response to ischemic necrosis. However, all the transplanted outbred FPPF donor tissue was acutely rejected and lost by day 10-14. In contrast, inbred FPPF tissue showed little evidence of graft necrosis after 3 days, and growth and formation of epithelial islet cell nest-like structures were seen to 28 days after transplantation. With time after transplantation, increasing amounts of insulin immunoperoxidase staining was seen together with chromogranin and somatostatin staining. In summary, this study confirms the potential of the Westran pig to answer the unproven ability of fetal pancreatic tissue to reverse type I diabetes in a large animal model.

Cytokine-induced expression of inducible nitric oxide synthetase (iNOS) and production of nitric oxide (NO) by pancreatic islet cells has been suggested as one potential mechanism for beta cell destruction. In this study, we investigated the role of iNOS and NO in islet primary non-function. Islets were assessed for their function, viability and expression of iNOS. Adult rat and pig islets isolated by collagenase digestion and fetal pig pancreas (FPP) grafts isolated by collagenase digestion or high oxygen culture were transplanted into C57BL6 mice and nude mice. iNOS protein was detected by immunohistochemistry. iNOS protein was found in normal rat and pig pancreas and adult rat and pig islets that were isolated by collagenase digestion and transplanted into either C57BL6 mice or nude mice. iNOS was not detected in fetal pig islet grafts, regardless of whether collagenase was used in the isolation process. In adult pig islet grafts, the presence of iNOS protein correlated with high levels of islet cell apoptosis and primary non-function. Despite the persistent presence of iNOS in rat islets, there was no evidence that it had a deleterious effect on rat islet viability, or function. Therefore, in isolated adult pig islets, there was a correlation between iNOS expression and apoptosis, suggesting that iNOS activation may be deleterious to the adult pig islets. However, other factors such as the fragility of the islet capsule may be equally important. By contrast, fetal pig islets did not express iNOS and this may be an important reason for their enhanced viability when compared with adult islet tissue.