The Epidemiology and Burden of Childhood Chronic Pancreatitis in South Australia.

OBJECTIVE: To assess longitudinal, population-based data on the prevalence and impact of chronic pancreatitis in children. STUDY DESIGN: Administrative data linkage was used to ascertain an index cohort consisting of all individuals who had an initial diagnosis of chronic pancreatitis before age 19 years in the South Australian public hospital system between June 2000 and June 2019. Age- and sex-matched controls were drawn from the general population of South Australia, children with type 1 diabetes, and children with type 2 diabetes. Main outcomes and measures included hospital visits, days in hospital, emergency department (ED) visits, intensive care unit (ICU) admissions, education comparators, and incidence and prevalence estimates. RESULTS: A total of 73 incident cases were identified. The crude prevalence and incidence of pediatric chronic pancreatitis were estimated at 6.8/100 000 and 0.98/100 000 per year, respectively. Of the index cohort, 24 cases (32.8%) of pediatric chronic pancreatitis were identified as occurring in children of Aboriginal and/or Torres Strait Islander descent. Compared with matched general population controls, children with chronic pancreatitis averaged 11-fold more hospital visits, 5-fold more ED visits, and 9-fold more ICU admissions; spent 10-fold more days in the hospital; and had a 2-fold higher rate of absence from school (P < .001 for all). Similarly, children with chronic pancreatitis used substantially more health resources than children with type 1 or 2 diabetes. CONCLUSIONS: Pediatric patients with chronic pancreatitis consume a high volume of public health services and are significantly impacted in their ability to engage in education.

Prolonged Ischemic Time, Delayed Graft Function, and Graft and Patient Outcomes in Live Donor Kidney Transplant Recipients.

The association between prolonged cold ischemic time (CIT) and graft and patient outcomes in live donor kidney transplant recipients remains unclear. The aims of this study were to examine the association of CIT with delayed graft function and graft loss in live donor kidney transplant recipients and those who participated in the Australian Paired Kidney Exchange program using data from the Australia and New Zealand Dialysis and Transplant (ANZDATA) registry. Of 3717 live donor transplant recipients between 1997 and 2012 who were followed for a median of 6.6 years (25 977 person-years), 224 (25%) experienced CIT >4-8 h. Donor age was an effect modifier between CIT and graft outcomes. In recipients who received kidneys from older donors aged >50 years, every hour of increase in CIT was associated with adjusted odds of 1.28 (95% confidence interval [CI] 1.07-1.53, p = 0.007) for delayed graft function, whereas CIT >4-8 h was associated with adjusted hazards of 1.93 (95% CI 1.21-3.09, p = 0.006) and 1.91 (95% CI 1.05-3.49, p = 0.035) for overall and death-censored graft loss, respectively, compared with CIT of 1-2 h. Attempts to reduce CIT in live donor kidney transplants involving older donor kidneys may lead to improvement of graft outcomes.

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.

BK virus encoded microRNAs are present in blood of renal transplant recipients with BK viral nephropathy.

BK viral infection is an important cause of renal transplant dysfunction and failure. Current strategies utilize surveillance for infection with DNA polymerase chain reaction assays and modulation of immunosuppression. Many viruses including polyomaviruses encode microRNAs (miRNAs). We have detected BK virus (BKV) encoded miRNAs in the blood of infected renal transplant recipients, and see a strong correlation between BKV encoded miRNA and BKV DNA in blood and a relationship between levels of bkv-miR-B1-5p and the presence of biopsy-proven BK viral nephropathy. Further research is needed to determine whether the detection of this and other virally encoded miRNAs may be useful in the diagnosis of active viral replication.

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.

Early exposure of interferon-γ inhibits signal transducer and activator of transcription-6 signalling and nuclear factor κB activation in a short-term monocyte-derived dendritic cell culture promoting 'FAST' regulatory dendritic cells.

Interferon (IFN)-γ is a cytokine with immunomodulatory properties, which has been shown previously to enhance the generation of tolerogenic dendritic cells (DC) when administered early ex vivo in 7-day monocyte-derived DC culture. To generate tolerogenic DC rapidly within 48 h, human monocytes were cultured for 24 h with interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) in the presence (IFN-γ-DC) or absence of IFN-γ (500 U/ml) (UT-DC). DC were matured for 24 h with TNF-α and prostaglandin E(2) (PGE(2) ). DC phenotype, signal transducer and activator of transcription-6 (STAT-6) phosphorylation and promotion of CD4(+) CD25(+) CD127(neg/low) forkhead box P3 (FoxP3)(hi) T cells were analysed by flow cytometry. DC nuclear factor (NF)-κB transcription factor reticuloendotheliosis viral oncogene homologue B (RELB) and IL-12p70 protein expression were also determined. Phenotypically, IFN-γ-DC displayed reduced DC maturation marker CD83 by 62% and co-stimulation molecules CD80 (26%) and CD86 (8%). IFN-γ treatment of monocytes inhibited intracellular STAT6, RELB nuclear translocation and IL-12p70 production. IFN-γ-DC increased the proportion of CD4(+) CD25(+) CD127(neg/low) foxp3(hi) T cells compared to UT-DC from 12 to 23%. IFN-γ-DC primed T cells inhibited antigen-specific, autologous naive T cell proliferation by 70% at a 1:1 naive T cells to IFN-γ-DC primed T cell ratio in suppression assays. In addition, we examined the reported paradoxical proinflammatory effects of IFN-γ and confirmed in this system that late IFN-γ exposure does not inhibit DC maturation marker expression. Early IFN-γ exposure is critical in promoting the generation of regulatory DC. Early IFN-γ modulated DC generated in 48 h are maturation arrested and promote the generation of antigen-specific regulatory T cells, which may be clinically applicable as a novel cellular therapy for allograft rejection.

Curcumin induces maturation-arrested dendritic cells that expand regulatory T cells in vitro and in vivo.

Dendritic cells (DC) and regulatory T cells (T(regs) ) are vital to the development of transplant tolerance. Curcumin is a novel biological agent extracted from Curcuma longa (turmeric), with anti-inflammatory and anti-oxidant activity mediated via nuclear factor (NF)-κB inhibition. We investigated the immunomodulatory effects of curcumin on human monocyte-derived and murine DC. Human monocyte-derived DC (hu-Mo-DC) were generated in the presence (CurcDC) or absence (matDC) of 25 µM curcumin, and matured using lipopolysaccharide (1 µg/ml). DC phenotype and allostimulatory capacity was assessed. CD11c(+) DC were isolated from C57BL/6 mice, pretreated with curcumin and injected into BALB/c mice, followed by evaluation of in vivo T cell populations and alloproliferative response. Curcumin induced DC differentiation towards maturation-arrest. CurcDC demonstrated minimal CD83 expression (<2%), down-regulation of CD80 and CD86 (50% and 30%, respectively) and reduction (10%) in both major histocompatibility complex (MHC) class II and CD40 expression compared to matDC. CurcDC also displayed decreased RelB and interleukin (IL)-12 mRNA and protein expression. Functionally, CurcDC allostimulatory capacity was decreased by up to 60% (P < 0·001) and intracellular interferon (IFN-γ) expression in the responding T cell population were reduced by 50% (P < 0·05). T cell hyporesponsiveness was due to generation of CD4(+) CD25(hi) CD127(lo) forkhead box P3 (FoxP3)(+) T(regs) that exerted suppressive functions on naïve syngeneic T cells, although the effect was not antigen-specific. In mice, in vivo infusion of allogeneic CurcDC promoted development of FoxP3(+) T(regs) and reduced subsequent alloproliferative capacity. Curcumin arrests maturation of DC and induces a tolerogenic phenotype that subsequently promotes functional FoxP3(+) T(regs) in vitro and in vivo.

Development of de novo HLA donor specific antibodies (HLA-DSA), HLA antibodies (HLA-Ab) and allograft rejection post blood transfusion in kidney transplant recipients.

BACKGROUND: Blood transfusion during the post-operative period of kidney transplantation is common as part of a life-saving procedure, especially in the event of acute blood loss. However, there have been conflicting opinions since the pre-cyclosporine era. The risk of sensitization post-transfusion remains the main limiting factor following transfusion in kidney transplant recipients. Thus, the objective of this study is to assess the development of de novo HLA-DSA, HLA-Ab and allograft rejection post blood transfusion. METHODOLOGY: This is a retrospective cohort study recruiting all kidney transplant recipients in South Australia from January 2010 till December 2018. Following that, the incidence of blood transfusion within one week post-operatively were traced (transfusion group). The outcomes were compared with all other transplant recipients (non-transfusion group). Recipient's demographic, donor characteristics and immunological risk profiles were obtained from the transplant unit database, while the biopsy report, history of blood transfusion, latest serum creatinine and follow-up status was gathered from the electronic medical system (OASIS). The HLA-DSA and HLA-Ab results were collected from the NOMS database. Finally, the survival data were merged with the Australia and New Zealand Dialysis and Transplant (ANZDATA) Registry for South Australia recipients graft survival. RESULTS: A total of 699 patients were eligible for analysis. The mean age was 50.64 ± 13.23 years old. There were more elderly (>65 years old) and females who needed transfusion. The majority had glomerulonephritis as the primary disease. There was no statistical difference in donor characteristics, cold ischemic time and immunological risk between the transfusion and non-transfusion group. There was no difference in the development of de novo HLA-DSA, HLA-Ab and rejection episodes between the group and the results were consistent in a model adjusted for all potential confounders. Median graft survival in days between the transfusion vs non-transfusion group was 1845 IQR (961,2430) and 1250 IQR (672,2013). CONCLUSION: Blood transfusion under strong immunosuppressive cover within a one-week post-operative period is safe with no significant association with the development of de novo HLA-DSA, HLA-Ab or clinical rejection.

Anti-HLA donor-specific antibodies detected in positive B-cell crossmatches by Luminex predict late graft loss.

The significance of B-cell crossmatching in kidney transplantation is controversial. Recipients (n = 471) transplanted in a single centre from 1987 to 2005 with complete T- and B-cell crossmatch records were studied. Sera from 83 patients transplanted across a positive B-cell crossmatch, with concomitant negative T-cell crossmatch (T-B+) on either current and/or peak sera were studied using Luminex to determine presence of donor-specific antibodies (DSA). Clinical outcomes of T-B+ patients were compared with 386 T-B- patients. T-B+ predicted vascular (p = 0.01), but not cellular (p = 0.82) or glomerular (p = 0.14) rejection. IgG HLA DSA were found in 33% (n = 27) of the T-B+ patients and were associated with higher risk of any (p = 0.047), vascular (p = 0.01) or glomerular (p < 0.001) rejection at 6 months. Of 27 patients with DSA, 18/21 (86%) were the complement-fixing IgG(1) and/or IgG(3) subclass antibodies. DSA imposed a statistically significant higher risk of graft loss 5 years posttransplant (1.8 [1.0-3.3], p = 0.045). This study showed that only one-third of positive B-cell crossmatch (BXM) was caused by DSA and was associated with late graft loss. Thus, using BXM to preclude kidney transplantation may potentially disadvantage >60% of patients in whom BXM is not indicative of the presence of DSA.

IGF-2 coated porous collagen microwells for the culture of pancreatic islets.

Islet transplantation, the only curative therapy for type I diabetes, requires isolation of the graft in highly specialized facilities for its later dispatch to remote transplantation centres. During transport and culture, many valuable cells are lost due to several factors such as mechanical stress, islet aggregation and dissociation. Here, we evaluate a porous microwell array sheet made of natural collagen type I extracellular matrix (ECM) protein as a novel islet culture substrate. This culture platform can be coated with IGF-2, a growth factor favorable for islet survival, and allows segregation of the islets within the porous microwell sheet, preventing aggregation. This design shows promising results for improving human pancreatic islets viability and function during culture and could form a novel paradigm for the transport of islets between isolation and transplantation centres.

Angiotensin II type-1 receptor antibody (AT1Rab) associated humoral rejection and the effect of peri operative plasma exchange and candesartan.

Angiotensin II type 1 antibodies (AT1Rab) can mediate antibody mediated rejection (AMR). Pre transplant AT1Rab levels, and risk of rejection were assessed in Kidney Transplant Recipients (KTR) transplanted in our centre from 2013 to 2014 (n=145). 14/145 (9.7%) KTR experienced antibody mediated rejection (AMR). The Hazard Ratio for AMR=3.7 [95% CI 2-26] (p=0.009) for KTR with AT1Rab levels >17.5U/ml. 6/11 of KTR with levels >25U/ml experienced AMR. In 2015 (n=80) KTR were transplanted and 6/80 KTR experienced rejection (2 AMR and 4 TCMR with vascular lesions). 7/80 of KTR had AT1Rab 17.5-25U/ml and none experienced rejection and were induced with ATG and candesartan. 7/80 had AT1Rab 25-40U/ml and received pre and post-operative plasma exchange, ATG and candesartan and 1/7 experienced TCMR with a vascular lesion. This perioperative regimen may alter the risk of rejection in patients with high levels of AT1Ab and further studies are needed.

Promotion of long-term heart allograft survival by combination of mobilized donor plasmacytoid dendritic cells and anti-CD154 monoclonal antibody.

Infusion of donor immature dendritic cells (DC) can significantly prolong survival of organ allografts, and this is believed to be due to antigen recognition by T cells in the absence of co-stimulation. In this study we report that a single pre-operative infusion of donor-mobilized immature plasmacytoid dendritic cells (pDCs) is superior to that of other DC sub-sets in suppressing allograft rejection. The combination of pDC infusion with injection of anti-CD154 monoclonal antibody further inhibited graft rejection and, in 50% of the mice, led to indefinite graft survival. This finding suggests a role for the plasmacytoid DC sub-set in facilitating organ transplant survival and also in the treatment of autoimmune disorders.

The effect of calcitriol on fasting urine calcium loss and renal tubular reabsorption of calcium in patients with mild renal failure--actions of a permissive hormone.

AIM: Renal production of 1,25-dihydroxycholecalciferol is attenuated in early renal failure. Renal tubular reabsorption of calcium is diminished in moderate renal failure and we wished to see if this were true in the early stages and whether supplementary calcitriol would bring about correction. We were interested in the idea of 1,25-dihydroxycholecalciferol being a permissive agent, operating indirectly. METHODS: We measured calcium-related variables, including calculated ultrafiltrable serum calcium, before and after calcitriol 0.5 microg daily for six days in 34 subjects with stable mild renal failure. RESULTS: The mean serum creatinine was 0.21 (+/- 0.08) mmol/l. The mean serum Ca++ was normal (1.18 mmol/l) but nine patients had values outside the normal range and in six cases, with low-normal serum Ca++ levels, there was a diminished tubular reabsorption. In five cases, basal serum Ca++ was mildly elevated. The coefficient of variation for serum Ca++ was 4.4%. PTH (1-84) levels were mildly elevated and 1,25-dihydroxycholecalciferol levels low-normal. The urine Ca/Cr, representing net bone resorption, was elevated in six cases. After calcitriol, the mean serum Ca++ level rose slightly and the coefficient of variation decreased to 3.6%. Changes in Ca++ whether upward or downward were accounted for by minor alterations in tubular reabsorption and a tendency to less net bone resorption. The initial Ca++ predicted (negatively) the magnitude of the correction. Neither the prevailing PTH nor the 1,25-dihydroxycholecalciferol levels explained any of the observed changes. CONCLUSION: In early renal failure, there may be impaired regulation of serum Ca++. Despite elevated PTH, mild hypocalcemia may exist in the presence of increased net bone resorption relative to GFR. Hypocalcemia was accounted for by reduced renal tubular reabsorption of calcium which corrected after calcitriol. Net bone resorption tended to fall after calcitriol. Mild hypercalcemia, when present, was corrected by a reduction in tubular reabsorption. Calcitriol did not have a simple unidirectional effect but instead contributed to efficiency of the homeostatic mechanisms controlling the serum Ca++ set-point.

IGF2: an endocrine hormone to improve islet transplant survival.

In the week following pancreatic islet transplantation, up to 50% of transplanted islets are lost due to apoptotic cell death triggered by hypoxic and pro-inflammatory cytokine-mediated cell stress. Thus, therapeutic approaches designed to protect islet cells from apoptosis could significantly improve islet transplant success. IGF2 is an anti-apoptotic endocrine protein that inhibits apoptotic cell death through the mitochondrial (intrinsic pathway) or via antagonising activation of pro-inflammatory cytokine signalling (extrinsic pathway), in doing so IGF2 has emerged as a promising therapeutic molecule to improve islet survival in the immediate post-transplant period. The development of novel biomaterials coated with IGF2 is a promising strategy to achieve this. This review examines the mechanisms mediating islet cell apoptosis in the peri- and post-transplant period and aims to identify the utility of IGF2 to promote islet survival and enhance long-term insulin independence rates within the setting of clinical islet transplantation.

Amelioration of renal ischaemia-reperfusion injury by liposomal delivery of curcumin to renal tubular epithelial and antigen-presenting cells.

BACKGROUND AND PURPOSE: Renal ischaemia-reperfusion (IR) injury is an inevitable consequence of renal transplantation, causing significant graft injury, increasing the risk of rejection and contributing to poor long-term graft outcome. Renal injury is mediated by cytokine and chemokine synthesis, inflammation and oxidative stress resulting from activation of the NF-κB pathway. EXPERIMENTAL APPROACH: We utilized liposomal incorporation of a potent inhibitor of the NF-κB pathway, curcumin, to target delivery to renal tubular epithelial and antigen-presenting cells. Liposomes containing curcumin were administered before bilateral renal ischaemia in C57/B6 mice, with subsequent reperfusion. Renal function was assessed from plasma levels of urea and creatinine, 4 and 24 h after reperfusion. Renal tissue was examined for NF-κB activity and oxidative stress (histology, immunostaining) and for apoptosis (TUNEL). Cytokines and chemokines were measured by RT-PCR and Western blotting. KEY RESULTS: Liposomal curcumin significantly improved serum creatinine, reduced histological injury and cellular apoptosis and lowered Toll-like receptor-4, heat shock protein-70 and TNF-α mRNA expression. Liposomal curcumin also reduced neutrophil infiltration and diminished inflammatory chemokine expression. Curcumin liposomes reduced intracellular superoxide generation and increased superoxide dismutase levels, decreased inducible NOS mRNA expression and 3-nitrotyrosine staining consistent with limitations in nitrosative stress and inhibited renal tubular mRNA and protein expression of thioredoxin-interacting protein. These actions of curcumin were mediated by inhibition of NF-κB, MAPK and phospho-S6 ribosomal protein. CONCLUSIONS AND IMPLICATIONS: Liposomal delivery of curcumin promoted effective, targeted delivery of this non-toxic compound that provided cytoprotection via anti-inflammatory and multiple antioxidant mechanisms following renal IR injury.

Successful control of Scedosporium prolificans septic arthritis and probable osteomyelitis without radical surgery in a long-term renal transplant recipient.

Scedosporium species are increasingly isolated from immunocompromised and immunocompetent patients. Scedosporium infections are generally resistant to multiple antifungals, and Scedosporium prolificans is particularly resistant to all single antifungal agents currently in use with in vitro testing. We report here a long-term renal transplant recipient who developed isolated S. prolificans septic monoarthritis and probable osteomyelitis. The infection was successfully treated with a combination of voriconazole and terbinafine in addition to joint washout but did not require radical surgery. This combination has been shown to have synergistic in vitro effect, and anecdotal in vivo success has also been reported recently. We also review the clinical presentation, treatment, and outcome of S. prolificans infection in patients with solid organ transplantation.

Uremia impairs monocyte and monocyte-derived dendritic cell function in hemodialysis patients.

Patients with chronic renal failure maintained on intermittent hemodialysis have frequent infections and a suboptimal response to vaccinations. Dendritic cells are potent antigen-presenting cells essential for the initiation and maintenance of innate and adaptive immunity. In this study we used uremic sera from hemodialysis patients to measure its impact on monocyte and monocyte-derived dendritic cell function in vitro. Monocytes from healthy and uremic subjects were isolated using immunomagnetic beads and differentiated into dendritic cells in the presence of either complete sera or sera from hemodialysis patients. Dendritic cells from normal patients cultured in uremic sera had decreased endocytosis and impaired maturation. These cells, however, had enhanced IL-12p70 production and increased allogeneic T-cell proliferation compared to cells of normal subjects cultured in normal sera. Monocyte derived dendritic cells of hemodialysis patients cultured in either normal or uremic sera were functionally impaired for endocytosis and maturation but had enhanced IL-12p70 production and allogeneic T-cell proliferation only when cultured with uremic sera. High concentrations of urea in normal sera inhibited all aspects of normal dendritic cell function in vitro. Our study suggests that hemodialysis regimes tailored to remove uremic toxins more efficiently may improve immune functions of these patients.

Uremia impairs blood dendritic cell function in hemodialysis patients.

Patients on hemodialysis have a general immunodeficiency involving both innate and adaptive responses. As the mechanisms contributing to this defect are uncertain, we sought to study the effects of uremia on circulating dendritic cells (DC) in hemodialysis patients. Immunomagnetic beads were used to isolate myeloid and plasmacytoid DCs from healthy donors. Immune-related functions were determined in these cells cultured in either a complete media containing ABO-compatible serum or media containing sera from uremic patients. The myeloid cells were analyzed for costimulatory molecule expression and allo-stimulatory capability following lipopolysaccharide stimulation. The production of interferon-alpha following herpes-simplex virus stimulation by the plasmacytoid cells was also measured. Myeloid DCs incubated with uremic sera demonstrated impaired maturation and decreased allo-stimulatory capacity. Similarly, herpes virus-stimulated plasmacytoid DCs incubated with uremic sera produced significantly less interferon-alpha compared with cells incubated in the complete media. Both small and large molecule uremic toxins inhibited DC functions in vitro. Use of more efficient dialysis to improve small molecule clearance reversed the inhibition of uremic sera on myeloid but not plasmacytoid DC function. We have shown that the immunodeficiency of hemodialysis patients is due to dialyzable uremic toxins.

The common marmoset as a novel preclinical transplant model: identification of new MHC class II DRB alleles and prediction of in vitro alloreactivity.

The difficulties with using nonhuman primate species such as rhesus macaques and baboons have led us to investigate the common marmoset (Callithrix jacchus) as an alternative preclinical model for transplantation research. This requires reliable methods of detecting alloreactivity between donor and recipient pairs, particularly if colonies are inbred and share just a few common alleles for leucocyte antigens. We firstly identified marmoset major histocompatibility complex (MHC) Class II DRB genes (Caja-DRB*W1201, Caja-DRB1*03, Caja-DRB*W16) using sequence-based typing techniques. Genomic DNA (n= 49) was extracted from whole blood or spleen tissue. Exon 2 of target genes was amplified by PCR using primers specific for known marmoset alleles, and then sequenced using ABI PRISM((R)) Big Dye Terminator technology and Assign sequence analysis software. DRB*W1201 was universally present. Eight DRB*W16 alleles and five DRB1*03 alleles were identified in this colony. We also identified two previously unreported DRB*W16 alleles, and confirmed inheritance of these alleles within several sibling groups. Subsequently, we investigated whether matching at MHC Class II DRB loci alone could predict alloreactivity, as assessed in vitro by two-way mixed lymphocyte reactions (MLRs). Fully DRB-matched, partially mismatched and fully mismatched animal pairs were prospectively chosen. MLR was performed using mononuclear cells (MNC) isolated from whole blood by density gradient separation. T-cell proliferation after 5-day culture was measured by (3)H-thymidine incorporation. Combined MNC from fully mismatched and partially mismatched animal pairs exhibited significant in vitro T-cell proliferation above single cell controls (P < 0.01). MNC from fully DRB-matched (but unrelated) animal pairs exhibited no proliferation compared with controls (P= 0.3). Using DRB genotyping, suitably alloreactive donor-recipient pairs may therefore be rapidly and accurately identified for use in further studies of cellular and solid organ transplantation.

Human myeloid dendritic cells transduced with an adenoviral interleukin-10 gene construct inhibit human skin graft rejection in humanized NOD-scid chimeric mice.

Human myeloid DC were generated from peripheral blood mononuclear cells by monocyte adhesion and subsequent culture with rhGM-CSF and rhIL-4. We transduced immature (day 5 of culture) myeloid DC with an E1-deleted replication-deficient adenoviral vector encoding the cytokine IL-10 (AdV IL-10) and a control adenovirus MX-17 (AdV MX 17). Human DC transduced with AdV IL-10 showed inhibition of the mixed leukocyte culture, reduced cell surface expression of co-stimulatory molecules (CD80/CD86) and were unable to produce the potent allo-stimulatory cytokine, interleukin-12. In order to test the in vivo properties of these cells a humanized immunodeficient mouse skin transplantation model was developed. Immunodeficient NOD-scid mice were engrafted with human skin, reconstituted via intraperitoneal injection with allogeneic mononuclear cells (MNC) mixed with 1 x 10(6) DC that were autologous to the skin donor and that had been transduced with either AdV IL-10 or AdV MX-17. Skin grafts were removed at day 7 and 14 after reconstitution and studied histologically for evidence of rejection. In animals that received DC modified with AdV IL-10 there was reduced skin graft rejection as characterized by reduced mononuclear cell infiltration and less dermo-epidermal junction destruction compared with those animals that received DC modified with the control virus alone. Injection of equivalent numbers of donor-derived fibroblasts transduced with AdV IL-10 were ineffective at modifying rejection of skin grafts. Immunosuppressive cytokine gene therapy targeting human DC is a novel means of inhibition of the alloimmune response.