Australian experience with total pancreatectomy with islet autotransplantation to treat chronic pancreatitis.

BACKGROUND: This study aimed to describe the clinical outcomes of total pancreatectomy with islet autotransplantation (TP-IAT) in Australia. METHODS: Individuals selected for TP-IAT surgery according to the Minnesota Criteria (Appendix) without evidence of diabetes were evaluated including time to transplantation from pancreatectomy, islet numbers infused and post-transplantation HbA1c, C-peptide, total daily insulin and analgesic requirement. RESULTS: Sixteen individuals underwent TP-IAT from Australia and New Zealand between 2010 and 2020. Two recipients are deceased. The median islet equivalents/kg infused was 4244 (interquartile range (IQR) 2290-7300). The median C-peptide 1 month post-TP-IAT was 384 (IQR 210-579) pmol/L and at median 29.5 (IQR 14.5-46.5) months from transplant was 395 (IQR 139-862) pmol/L. Insulin independence was achieved in eight of 15 (53.3%) surviving recipients. A higher islet equivalents transplanted was most strongly associated with the likelihood of insulin independence (P < 0.05). Of the 15 surviving recipients, 14 demonstrated substantial reduction in analgesic requirement. CONCLUSION: The TP-IAT programme in Australia has been a successful new therapy for the management of individuals with chronic pancreatitis including hereditary forms refractory to medical treatment to improve pain management with 50% insulin independence rates.

BK Virus Nephropathy: Histological Evolution by Sequential Pathology.

Reactivation of BK virus in renal allografts causes a destructive chronic infection. This single-center retrospective cohort study describes the evolution of BK virus allograft nephropathy (BKVAN) from 63 kidneys (from 61 patients) using sequential histopathology (454 biopsies, averaging 7.8 ± 2.6 per kidney) followed for 60.1 mo. Uninfected protocol biopsies formulated time-matched control Banff scores (n = 975). Interstitial inflammation occurred in 73% at diagnosis, correlating with viral histopathology (r = 0.413, p = 0.008) and amplifying early injury with accelerated interstitial fibrosis and tubular atrophy (IF/TA, p = 0.017) by 3 mo. Prodromal simian virus 40 large T antigen (SV40T)-negative inflammation with viremia preceded the histological diagnosis in 23.8%. Persistent subacute injury from viral cytopathic effect was associated with acute tubular necrosis and ongoing interstitial inflammation, culminating in IF/TA in 86.9%. Overall, cellular interstitial infiltration mitigated the intensity of subsequent tubular injury, SV40T, and tissue viral load, assessed by sequential paired histology (p < 0.001). Graft loss was predicted by high-level viremia (hazard ratio [HR] 4.996, 95% CI 2.19-11.396, p < 0.001), deceased donor (HR 3.201, 95% CI 1.149-8.915, p = 0.026), and late acute rejection (HR 3.124, 95% CI 1.037-9.413, p = 0.043). Transplant failure occurred in 38.1%, with uncontrolled infection (58.3%) and SV40T-negative chronic rejection (41.7%) causing losses. BKVAN is characterized by subacute virus-induced tubular injury, inflammation, and progressive nephron destruction. Effective antiviral therapy remains an unmet clinical need.

Efficacy and Safety Outcomes of Extended Criteria Donor Kidneys by Subtype: Subgroup Analysis of BENEFIT-EXT at 7 Years After Transplant.

The phase III Belatacept Evaluation of Nephroprotection and Efficacy as First-Line Immunosuppression Trial-Extended Criteria Donors Trial (BENEFIT-EXT) study compared more or less intensive belatacept-based immunosuppression with cyclosporine (CsA)-based immunosuppression in recipients of extended criteria donor kidneys. In this post hoc analysis, patient outcomes were assessed according to donor kidney subtype. In total, 68.9% of patients received an expanded criteria donor kidney (United Network for Organ Sharing definition), 10.1% received a donation after cardiac death kidney, and 21.0% received a kidney with an anticipated cold ischemic time ≥24 h. Over 7 years, time to death or graft loss was similar between belatacept- and CsA-based immunosuppression, regardless of donor kidney subtype. In all three donor kidney cohorts, estimated mean GFR increased over months 1-84 for belatacept-based treatment but declined for CsA-based treatment. The estimated differences in GFR significantly favored each belatacept-based regimen versus the CsA-based regimen in the three subgroups (p < 0.0001 for overall treatment effect). No differences in the safety profile of belatacept were observed by donor kidney subtype.

Evaluation of the effect of tofacitinib exposure on outcomes in kidney transplant patients.

Tofacitinib fixed-dose regimens attained better kidney function and comparable efficacy to cyclosporine (CsA) in kidney transplant patients, albeit with increased risks of certain adverse events. This post-hoc analysis evaluated whether a patient subgroup with an acceptable risk-benefit profile could be identified. Tofacitinib exposure was a statistically significant predictor of serious infection rate. One-hundred and eighty six kidney transplant patients were re-categorized to above-median (AME) or below-median (BME) exposure groups. The 6-month biopsy-proven acute rejection rates in AME, BME and CsA groups were 7.8%, 15.7% and 17.7%, respectively. Measured glomerular filtration rate was higher in AME and BME groups versus CsA (61.2 and 67.9 vs. 53.9 mL/min) at Month 12. Fewer patients developed interstitial fibrosis and tubular atrophy (IF/TA) at Month 12 in AME (20.5%) and BME (27.8%) groups versus CsA (48.3%). Serious infections occurred more frequently in the AME group (53.0%) than in BME (28.4%) or CsA (25.5%) groups. Posttransplant lymphoproliferative disorder (PTLD) only occurred in the AME group. In kidney transplant patients, the BME group preserved the clinical advantage of comparable acute rejection rates, improved renal function and a lower incidence of IF/TA versus CsA, and with similar rates of serious infection and no PTLD.

T lymphocytes possess the machinery for 5-HT synthesis, storage, degradation and release.

AIM: Although activated T lymphocytes express tryptophan hydroxylase 1 and produce 5-HT, the metabolic fate and cellular handling of this 5-HT is unclear. Here, we investigated key proteins in T cells linked to 5-HT metabolism and storage and compare differences in 5-HT synthesis and metabolism between T-cell subsets. METHODS: We cultured human Jurkat T cells and mouse splenic CD3(+) , CD4(+) and CD8(+) T cells with or without T-cell activators (phorbol ester/ionomycin, concavalin A or plate-bound anti-CD3 antibody). Subsequently, we measured mRNA and/or protein for monoamine oxidase A and B, vesicular monoamine transporter 1 and 2, N-acetyl transferase and tryptophan hydroxylase 1. In addition, we measured the release of exogenously loaded [(3) H]5-HT and endogenously synthesized 5-HT from CD4(+) and CD8(+) T-cell subsets. RESULTS: Human and mouse T cells selectively express monoamine oxidase A. Following T-cell activation, mRNA levels of MAO-A increase robustly in parallel with tryptophan hydroxylase 1. Concomitant with these changes, T cells increase the expression of the type 1 vesicular monoamine transporter. Raised intracellular [Ca(2+) ] rapidly releases preloaded [(3) H]5-HT from CD4(+) and CD8(+) T cells indicating that these cells have the capacity for the storage and regulated secretion of 5-HT. Notably, both the expression of tryptophan hydroxylase 1 and monoamine oxidase A, and 5-HT production are significantly greater in CD8(+) compared with CD4(+) T cells. CONCLUSION: These data reveal coordinated changes in 5-HT production, metabolism and storage that may optimize 5-HT secretion from the CD8(+) T cell subset in response to activation stimuli.

Islet cell transplantation in Australia: screening, remote transplantation, and incretin hormone secretion in insulin independent patients.

Islet cell transplantation has emerged as a treatment modality for type 1 diabetes in the last 15 years due to the Edmonton protocol leading to consistent and sustained exogenous insulin independence post-transplantation. In recent years, consortia that involve both local and remote islet cell centers have been established, with local centers responsible for processing and shipping of islet cells, and remote centers only transplanting them. There are, however, few data on patient outcomes at remote centers. A tendency for high fasting glucose despite insulin independence was noted by us and others with an unknown mechanism. This review provides a brief history of islet cell transplantation, and focuses on the South Australian remote center experience: the challenges, screening criteria, and the impact on incretin hormone secretion of insulin independent transplant patients.

Islet product characteristics and factors related to successful human islet transplantation from the Collaborative Islet Transplant Registry (CITR) 1999-2010.

The Collaborative Islet Transplant Registry (CITR) collects data on clinical islet isolations and transplants. This retrospective report analyzed 1017 islet isolation procedures performed for 537 recipients of allogeneic clinical islet transplantation in 1999-2010. This study describes changes in donor and islet isolation variables by era and factors associated with quantity and quality of final islet products. Donor body weight and BMI increased significantly over the period (p<0.001). Islet yield measures have improved with time including islet equivalent (IEQ)/particle ratio and IEQs infused. The average dose of islets infused significantly increased in the era of 2007-2010 when compared to 1999-2002 (445.4±156.8 vs. 421.3±155.4×0(3) IEQ; p<0.05). Islet purity and total number of ß cells significantly improved over the study period (p<0.01 and <0.05, respectively). Otherwise, the quality of clinical islets has remained consistently very high through this period, and differs substantially from nonclinical islets. In multivariate analysis of all recipient, donor and islet factors, and medical management factors, the only islet product characteristic that correlated with clinical outcomes was total IEQs infused. This analysis shows improvements in both quantity and some quality criteria of clinical islets produced over 1999-2010, and these parallel improvements in clinical outcomes over the same period.

The role of macrophages in the development of human renal allograft fibrosis in the first year after transplantation.

The aim of this study was to investigate the role of infiltrating macrophages in renal allograft fibrosis. Forty-six protocol renal allograft biopsies obtained 1 year after transplantation were stained with Sirius red to quantify fibrosis and double stained with CD68 and CD206 to identify the proportion of alternatively activated (M2) macrophages. Biopsies were analyzed for gene expression by microarray, which was correlated with macrophage infiltration and the severity of fibrosis. The number of infiltrating CD68+ cells strongly correlated with the percentage of interstitial fibrosis (r = 0.73, p < 0.0001). Macrophage infiltration at 1 year correlated with renal dysfunction at 1, 12 and 36 months posttransplant (estimated GFR low vs. high: 1 month 78 ± 26 vs. 54 ± 19 mL/min, p < 0.01; 12 months 87 ± 29 vs. 64 ± 19 mL/min, p < 0.05; 36 months 88 ± 33 vs. 60 ± 24 mL/min, p < 0.05). Ninety-two percent of infiltrating macrophages exhibited an M2 phenotype with CD68+ CD206+ dual staining. Gene microarrays demonstrated an alloimmune response with up-regulation of interferon-γ-response genes despite the lack of rejection or inflammatory infiltrate. Consistent with this was the presence of CXCL10 in proximal tubular cells at 3 months. This suggests that M2 macrophage proliferation, or infiltration, was associated with subclinical alloimmune inflammation, tubular injury and progression of fibrosis.

Control of IBMIR in neonatal porcine islet xenotransplantation in baboons.

The instant blood-mediated inflammatory reaction (IBMIR) is a major obstacle to the engraftment of intraportal pig islet xenografts in primates. Higher expression of the galactose-α1,3-galactose (αGal) xenoantigen on neonatal islet cell clusters (NICC) than on adult pig islets may provoke a stronger reaction, but this has not been tested in the baboon model. Here, we report that WT pig NICC xenografts triggered profound IBMIR in baboons, with intravascular clotting and graft destruction occurring within hours, which was not prevented by anti-thrombin treatment. In contrast, IBMIR was minimal when recipients were immunosuppressed with a clinically relevant protocol and transplanted with NICC from αGal-deficient pigs transgenic for the human complement regulators CD55 and CD59. These genetically modified (GM) NICC were less susceptible to humoral injury in vitro than WT NICC, inducing significantly less complement activation and thrombin generation when incubated with baboon platelet-poor plasma. Recipients of GM NICC developed a variable anti-pig antibody response, and examination of the grafts 1 month after transplant revealed significant cell-mediated rejection, although scattered insulin-positive cells were still present. Our results indicate that IBMIR can be attenuated in this model, but long-term graft survival may require more effective immunosuppression or further donor genetic modification.

Infiltrating Foxp3(+) regulatory T cells from spontaneously tolerant kidney allografts demonstrate donor-specific tolerance.

Foxp3(+) regulatory T cells (Tregs) have an essential role in immune and allograft tolerance. However, in both kidney and liver transplantation in humans, FOXP3(+) Tregs have been associated with clinical rejection. Therefore, the role and function of graft infiltrating Tregs have been of great interest. In the studies outlined, we demonstrated that Foxp3(+) Tregs were expanded in tolerant kidney allografts and in draining lymph nodes in the DBA/2 (H-2(d) ) to C57BL/6 (H-2(b) ) mouse spontaneous kidney allograft tolerance model. Kidney allograft tolerance was abrogated after deletion of Foxp3(+) Tregs in DEpletion of REGulatory T cells (DEREG) mice. Kidney allograft infiltrating Foxp3(+) Tregs (K-Tregs) expressed elevated levels of TGF-ß, IL-10, interferon gamma (IFN-γ), the transcriptional repressor B lymphocyte-induced maturation protein-1 (Blimp-1) and chemokine receptor 3 (Cxcr3). These K-Tregs had the capacity to transfer dominant tolerance and demonstrate donor alloantigen-specific tolerance to skin allografts. This study demonstrated the crucial role, potency and specificity of graft infiltrating Foxp3(+) Tregs in the maintenance of spontaneously induced kidney allograft tolerance.

Multicenter Australian trial of islet transplantation: improving accessibility and outcomes.

Whilst initial rates of insulin independence following islet transplantation are encouraging, long-term function using the Edmonton Protocol remains a concern. The aim of this single-arm, multicenter study was to evaluate an immunosuppressive protocol of initial antithymocyte globulin (ATG), tacrolimus and mycophenolate mofetil (MMF) followed by switching to sirolimus and MMF. Islets were cultured for 24 h prior to transplantation. The primary end-point was an HbA1c of <7% and cessation of severe hypoglycemia. Seventeen recipients were followed for ≥ 12 months. Nine islet preparations were transported interstate for transplantation. Similar outcomes were achieved at all three centers. Fourteen of the 17 (82%) recipients achieved the primary end-point. Nine (53%) recipients achieved insulin independence for a median of 26 months (range 7-39 months) and 6 (35%) remain insulin independent. All recipients were C-peptide positive for at least 3 months. All subjects with unstimulated C-peptide >0.2 nmol/L had cessation of severe hypoglycemia. Nine of the 17 recipients tolerated switching from tacrolimus to sirolimus with similar graft outcomes. There was a small but significant reduction in renal function in the first 12 months. The combination of islet culture, ATG, tacrolimus and MMF is a viable alternative for islet transplantation.

Selective depletion of alloreactive T cells leads to long-term islet allograft survival across a major histocompatibility complex mismatch in diabetic mice.

Islet cell transplantation as a therapy for type 1 diabetes has been limited by progressive graft loss. Significant immunosuppression including T-cell ablation has been used in an attempt to limit islet rejection. Here, we show that CD3(+) lymphocytes depleted of alloreactive T cells selected from a mixed lymphocyte reaction (MLR), where responder BALB/c splenocytes stained with carboxyfluorescein succinimidyl ester (CFSE) were stimulated with irradiated C57BL/6 splenocytes for 5 days, infused into diabetic immunodeficient mice are capable of restoring a broad T-cell repertoire and specifically do not reject islet transplants from the strain (C57BL/6) used in the original depletion. These mice demonstrate reconstitution with CD4(+) and CD8(+) T cells, the capacity to reject third-party grafts (CBA), and restoration of interferon-γ (IFN-γ) responses to third-party alloantigens. Over time, both forkhead box P3-positive (Foxp3(+)) T regulatory cells (Tregs) and γδ T cells expand, suggesting a role for peripheral tolerance, in addition to the initial depletion of alloreactive T cells, in long-term islet graft survival. Our results suggest that immune restoration with CD3(+) lymphocytes where alloreactive T cells are removed can restore cognate immunity without islet allograft loss and recurrence of diabetes.

Tirofiban and activated protein C synergistically inhibit the Instant Blood Mediated Inflammatory Reaction (IBMIR) from allogeneic islet cells exposure to human blood.

Instant blood mediated inflammatory reaction (IBMIR) occurs when islets are exposed to blood and manifests clinically as portal vein thrombosis and graft failure. The aim of this study was to determine the impact of recombinant human activated protein C (rhAPC) and platelet inhibition on IBMIR in order to develop a better targeted treatment for this condition. Five thousand human islet cell equivalents (IEQ) were mixed in a PVC loop system with 7 mL of ABO compatible human blood and incubated with rhAPC, either alone or in combination with tirofiban. Admixing human islets and blood caused rapid clot formation, consumption of platelets, leukocytes, fibrinogen, coagulation factors and raised d-dimers. Islets were encased in a fibrin and platelet clot heavily infiltrated with neutrophils. Tirofiban monotherapy was ineffective, whereas rhAPC monotherapy prevented IBMIR in a dose-dependent manner, preserving islet integrity while maintaining platelet and leukocyte counts, fibrinogen and coagulation factor levels, and reducing d-dimer formation. The combination of tirofiban and low-dose rhAPC inhibited IBMIR synergistically with an efficacy equal to high dose rhAPC. Tirofiban and rhAPC worked synergistically to preserve islets, suggesting that co-inhibition of the platelet and coagulation pathways' contribution to thrombin generation is required for the optimal anti-IBMIR effect.

Transplant glomerulopathy: ultrastructural abnormalities occur early in longitudinal analysis of protocol biopsies.

Transplant glomerulopathy (TXG) presents a distinctive pattern of glomerular abnormalities. The aim of this study was to describe its sequential ultrastructural pathology. A paired cohort study of 228 protocol biopsies, from our longitudinal database (n = 1345), compared TXG (7 patients, 95 biopsies) and controls (8 patients, 133 biopsies). Ultrastructural morphometry and C4d immunoperoxidase were evaluated from implantation to 5 years after transplantation against sequential histology and functional changes. TXG was predated by early glomerular endothelial cell activation; typified by vacuolation, hypertrophy, serration and expansion of lamina rara interna from 39 +/- 23 days after transplantation. Endothelial cells were transformed into an activated phenotype, containing numerous mitochondria, Golgi and ribosomes. Transition from fenestrated to continuous endothelium, mesangial matrix expansion and podocyte fusion occurred late. Endothelial cell activation also occurred in peritubular capillaries (PTC) followed by basement membrane multi-lamination (p < 0.05-0.001). Light microscopy changes of TXG occurred at 2.3 years. PTC C4d deposition was intermittently expressed over time, correlating with endothelial abnormalities, glomerular C4d and donor-specific antibodies (DSA) (p < 0.05-0.001). In summary, endothelial and subendothelial ultrastructural abnormalities in glomerular and peritubular capillaries are sensitive, early markers of TXG, likely due to stimulation of endothelial cells into an activated phenotype by antibody-mediated sub-lytic complement deposition.

Mycophenolate mofetil is associated with altered expression of chronic renal transplant histology.

Mycophenolate mofetil (MMF) reduces acute rejection in controlled trials of kidney transplantation and is associated with better registry graft survival. Recent experimental studies have demonstrated additional antifibrotic properties of MMF, however, human histological data are lacking. We evaluated sequential prospective protocol kidney biopsies from two historical cohorts treated with cyclosporine (CSA)-based triple therapy including prednisolone and either MMF (n = 25) or azathioprine (AZA, n = 25). Biopsies (n = 360) were taken from euglycemic kidney-pancreas transplant recipients. Histology was independently assessed by the Banff schema and electron microscopic morphometry. MMF reduced acute rejection and OKT3 use (p < 0.05) compared with AZA. MMF therapy was associated with limited chronic interstitial fibrosis, striped fibrosis and periglomerular fibrosis (p < 0.05-0.001), mesangial matrix accumulation (p < 0.01), chronic glomerulopathy scores (p < 0.05) and glomerulosclerosis (p < 0.05). MMF was associated with delayed expression of CSA nephrotoxicity, reduced arteriolar hyalinosis, striped fibrosis and tubular microcalcification (p < 0.05-0.001). The beneficial effects of MMF remained in recipients without acute rejection. Retrospective analysis shows that MMF therapy was associated with substantially reduced fibrosis in the glomerular, microvascular and interstitial compartments, and a delayed expression of CSA nephrotoxicity. These outcomes may be due to a limitation of immune-mediated injury and suggest a direct effect of reduced fibrogenesis.

Prolongation of allograft survival by administration of anti-CD45RB monoclonal antibody is due to alteration of CD45RBhi: CD45RBlo T-cell proportions.

CD45RB monoclonal antibody (mAb) therapy is capable of prolonging allograft survival. We have previously shown that CD45RB mAb enriches the CD45RBlo T-cell population in vitro and in vivo by preferentially depleting CD45RBhi T cells. The present study assessed the importance of CD45RBhi T-cell depletion in murine cardiac allograft survival by infusion of naive CD45RB T-cell subsets. Here we show that naturally occurring CD45RBloCD4+ T cells express regulatory transcription factor Foxp3 and have regulatory function, whereas CD45RBhiCD4+ T cells express low levels of Foxp3 and have effector function. Infusion of syngeneic CD45RBhi T cells significantly reduced graft survival after depletion of CD45RBhi T cells by CD45RB mAb. Reduction of graft survival also occurred when syngeneic CD45RBhi T cells were infused into rapamycin-treated mice, whereas survival was prolonged when CD45RBlo T cells were added. This indicates that an alteration in the balance between regulatory CD45RBlo and effector CD45RBhi T cells is critical to the immunologic function of CD45RB mAb. A strategy to eliminate effector T cells with consequent enrichment of the regulatory T-cell compartment may be an important new strategy in the prevention of rejection.

A monoclonal antibody to CD11d reduces the inflammatory infiltrate into the injured spinal cord: a potential neuroprotective treatment.

The accumulation of inflammatory cells in the lesion of a spinal cord injury (SCI) enhances secondary damage, resulting in further neurological impairment. High-dose methylprednisolone (MP) treatment is the only accepted treatment for inflammation secondary to human SCI but is minimally effective. Using a rat SCI model, we devised an anti-inflammatory treatment to block the infiltration of neutrophils and hematogenous monocyte/macrophages over the first 2 days postinjury by targeting the CD11dCD18 integrin. Anti-CD11d mAb administration following SCI effectively reduced neutrophil and macrophage infiltrate into lesions by 70% and 36%, respectively, over the first 72 h post-SCI. MP also reduced neutrophil and macrophage infiltrate by 60% and 28%, respectively, but by different mechanisms. The immunosuppression caused by anti-CD11d treatment was not sustained, as inflammatory cell numbers were not different from those observed in untreated SCI control animals at 7 days postinjury. In contrast, in MP-treated animals, the number of macrophages was still suppressed in the lesion while neutrophil numbers were significantly increased. These results suggest that anti-CD11d mAb treatment following SCI will minimize the destructive actions associated with early, uncontrolled leukocyte infiltration into the lesion while permitting the positive wound healing effects of macrophages at later time points.