C4d-negative antibody-mediated rejection with high anti-angiotensin II type I receptor antibodies in absence of donor-specific antibodies.

AIMS: Acute antibody-mediated rejection can occur in absence of circulating donor-specific antibodies. Agonistic antibodies targeting the anti-angiotensin II type 1 receptor (anti-AT1 R) are emerging as important non-human leucocyte antigen (HLA) antibodies. Elevated levels of anti-angiotensin II receptor antibodies were first observed in kidney transplant recipients with malignant hypertension and allograft rejection. They have now been studied in three separate kidney transplant populations and associate to frequency of rejection, severity of rejection and graft failure. METHODS: We report 11 cases of biopsy-proven, Complement 4 fragment d (C4d)-negative, acute rejection occurring without circulating donor-specific anti-HLA antibodies. In eight cases, anti-angiotensin receptor antibodies were retrospectively examined. The remaining three subjects were identified from our centre's newly instituted routine anti-angiotensin receptor antibody screening. RESULTS: All subjects fulfilled Banff 2013 criteria for antibody-mediated rejection and all responded to anti-rejection therapy, which included plasma exchange and angiotensin receptor blocker therapy. CONCLUSIONS: These cases support the routine assessment of anti-AT1 R antibodies in kidney transplant recipients to identify subjects at risk. Further studies will need to determine optimal assessment protocol and the effectiveness of pre-emptive treatment with angiotensin receptor blockers.

Calcium supplements lower bone resorption after renal transplant.

AIM: Bone loss in renal transplant (RT) patients is a problem that begins during end-stage kidney disease and persists after transplantation. Suppression of parathyroid hormone (PTH) may decrease bone loss and improve fracture rate. METHODS: A single-group prospective intervention study involving 30 patients was performed at a large RT unit. Investigations included dual-emission X-ray absorptiometry scan, vertebral X-ray, calcium absorption test, 24-h urinary calcium and serum measurements of total and ionized calcium, PTH, C-telopeptide cross-links (CTX), osteocalcin, alkaline phosphatase, 25 hydroxyvitamin D (25[OH]D), and 1,25-dihydroxyvitamin D3. Patients were given 500 mg elemental calcium daily for seven d, and serum measurements were repeated. RESULTS: Two-tailed Wilcoxon rank-sum test showed significant decreases in PTH (p<0.01) and CTX (p<0.01) after calcium load. Dietary calcium, mean calcium absorption, and urinary calcium excretion were below desirable levels. Mean 25 hydroxyvitamin D (25(OH)D) was low, but levels of 1,25-dihydroxyvitamin D3 were normal. Calcium absorption significantly correlated with change in PTH (p<0.001), baseline 25(OH)D (p<0.001), and mycophenolate dose (p=0.024). CONCLUSIONS: Calcium malabsorption is prevalent in RT recipients, contributing to bone destruction and compounded by poor dietary intake and low 25(OH)D. Calcium supplementation appears to help overcome this deficiency and acutely suppress PTH. Calcium may be an effective and inexpensive therapy for bone loss in RT recipients.

Endothelial progenitor cells: novel biomarker and promising cell therapy for cardiovascular disease.

Bone-marrow-derived EPCs (endothelial progenitor cells) play an integral role in the regulation and protection of the endothelium, as well as new vessel formation. Peripheral circulating EPC number and function are robust biomarkers of vascular risk for a multitude of diseases, particularly CVD (cardiovascular disease). Importantly, using EPCs as a biomarker is independent of both traditional and non-traditional risk factors (e.g. hypertension, hypercholesterolaemia and C-reactive protein), with infused ex vivo-expanded EPCs showing potential for improved endothelial function and either reducing the risk of events or enhancing recovery from ischaemia. However, as the number of existing cardiovascular risk factors is variable between patients, simple EPC counts do not adequately describe vascular disease risk in all clinical conditions and, as such, the risk of CVD remains. It is likely that this limitation is attributable to variation in the definition of EPCs, as well as a difference in the interaction between EPCs and other cells involved in vascular control such as pericytes, smooth muscle cells and macrophages. For EPCs to be used regularly in clinical practice, agreement on definitions of EPC subtypes is needed, and recognition that function of EPCs (rather than number) may be a better marker of vascular risk in certain CVD risk states. The present review focuses on the identification of measures to improve individual risk stratification and, further, to potentially individualize patient care to address specific EPC functional abnormalities. Herein, we describe that future therapeutic use of EPCs will probably rely on a combination of strategies, including optimization of the function of adjunct cell types to prime tissues for the effect of EPCs.

A comparison of the inflammatory response following autologous compared with allogenic islet cell transplantation.

BACKGROUND: The initial response to islet transplantation and the subsequent acute inflammation is responsible for significant attrition of islets following both autologous and allogenic procedures. This multicentre study compares this inflammatory response using cytokine profiles and complement activation. METHODS: Inflammatory cytokine and complement pathway activity were examined in two cohorts of patients undergoing total pancreatectomy followed either by autologous (n=11) or allogenic (n=6) islet transplantation. Two patients who underwent total pancreatectomy alone (n=2) served as controls. RESULTS: The peak of cytokine production occurred immediately following induction of anaesthesia and during surgery. There was found to be a greater elevation of the following cytokines: TNF-alpha (P<0.01), MCP-1 (P=0.0013), MIP-1α (P=0.001), MIP-1ß (P=0.00020), IP-10 (P=0.001), IL-8 (P=0.004), IL-1α (P=0.001), IL-1ra (0.0018), IL-10 (P=0.001), GM-CSF (P=0.001), G-CSF (P=0.0198), and Eotaxin (P=0.01) in the allogenic group compared to autografts and controls. Complement activation and consumption was observed in all three pathways, and there were no significant differences in between the groups although following allogenic transplantation ∆IL-10 and ∆VEGF levels were significantly elevated those patients who became insulin-independent compared with those who were insulin-dependent. CONCLUSIONS: The cytokine profiles following islet transplantation suggests a significantly greater acute inflammatory response following allogenic islet transplantation compared with auto-transplantation although a significant, non-specific inflammatory response occurs following both forms of islet transplantation.

Calcific uremic arteriolopathy--the argument for hyperbaric oxygen and sodium thiosulfate.

Calcific uremic arteriolopathy (CUA) is a rare event primarily in patients with end-stage kidney disease which is characterized by small vessel media calcification, panniculitis, dermal necrosis producing exquisitely painful difficult to heal wounds. Mortality rates may be as high as 80%, predominantly due to intervening sepsis. This clinical phenomenon is being increasingly reported and treated with a widening number of agents. Recent case reports highlight the benefit of two modalities that have been employed as adjuvant therapy with significant success in the treatment of CUA. Hyperbaric oxygen (HBO) is capable of enhancing oxygen delivery to the ulcerating lesions that characterize CUA. Chronic hypoxia can be reversed using HBO to facilitate growth factor production, neoangiogenesis, fibroblast proliferation, and collagen synthesis that may facilitate all aspects of wound healing. Sodium thiosulfate appears to chelate and solubilize calcium ions, reducing the calcium vascular load that appears to participate in the obliterative small vessel disease. There is a rapid analgesic effect and slower regression of cutaneous calcific nodules. The authors advocate for aggressive treatment of CUA, using all available therapies.

Review article: Kidney dendritic cells: their role in homeostasis, inflammation and transplantation.

There is definitive experimental proof that a lattice of dendritic cells (DC) exist within the renal parenchyma. Kidney-resident DC (KDC) are an important constituent of passenger leucocytes that initiate the direct component of allograft rejection in transplantation and form a central element of the innate immune response following injurious stimuli to the kidney. DC are recruited to the kidney in pathophysiological states such as glomerulonephritis and ischaemia-reperfusion injury. However, the exact mechanism for engaging and attracting DC to infectious and transplant antigens, and whether specific DC subsets are involved remains unresolved. In addition, the extent to which resident and infiltrating DC contribute to the propagation of injury or rejection is also unclear. Despite consistently expanding published work regarding DC location, phenotype and function, there are a number of deficiencies in our knowledge base, particularly in relation to KDC.

Successful treatment of pulmonary mucormycosis in a renal transplant recipient with limited pulmonary reserve by combined medical and surgical therapy.

Mucormycosis is a rare opportunistic fungal infection in renal transplant recipients which is associated with exceedingly high mortality when inadequately treated. Risk factors for this infection include diabetes, neutropaenia and immunosuppression. We report a case of pulmonary mucormycosis in a renal allograft recipient with type 2 diabetes and limited pulmonary reserve. The patient was successfully treated with lobectomy and liposomal amphotericin B with preservation of pulmonary and allograft functions. Early recognition of this infection is warranted before dissemination, which carries a poor prognosis.

Replica moulded poly(dimethylsiloxane) microwell arrays induce localized endothelial cell immobilization for coculture with pancreatic islets.

PolyJet three-dimensional (3D) printing allows for the rapid manufacturing of 3D moulds for the fabrication of cross-linked poly(dimethylsiloxane) microwell arrays (PMAs). As this 3D printing technique has a resolution on the micrometer scale, the moulds exhibit a distinct surface roughness. In this study, the authors demonstrate by optical profilometry that the topography of the 3D printed moulds can be transferred to the PMAs and that this roughness induced cell adhesive properties to the material. In particular, the topography facilitated immobilization of endothelial cells on the internal walls of the microwells. The authors also demonstrate that upon immobilization of endothelial cells to the microwells, a second population of cells, namely, pancreatic islets could be introduced, thus producing a 3D coculture platform.

Pituitary involvement in Wegener's granulomatosis.

Wegener's granulomatosis (WG) is an anti-neutrophil cytoplasmic antibody (ANCA)-associated systemic vasculitis of small and medium-sized vessels. Pituitary involvement in WG is rare with only 22 previous case reports in the English medical literature between 1966 and 2006. Herein we report another patient with WG-related diabetes insipidus (DI) and partial disruption of the anterior pituitary axes. We also review the clinical features, imaging findings, treatment and outcome of WG-related pituitary involvement. Isolated pituitary involvement in the absence of lung or renal complications in WG is rare and described in only one previous patient. Pituitary involvement in WG is usually associated with other organ involvement (96% of cases)-commonly upper respiratory tract (93%), lungs (73%) and kidneys (67%). Abnormalities are often seen in the hypothalamo-pituitary region on magnetic resonance imaging (MRI) or computed tomography (CT) of the head (90% of cases). In 65% of reported cases, cyclophosphamide-based induction therapy was used with a subsequent relapse rate of 27%, occurring at a median of 10.5 months (range: 7-36 months) after initiation of treatment. In comparison, induction treatment without cyclophosphamide was associated with relapse in 50% at a median of 4.5 months (range: 4-18 months after starting treatment) suggesting more frequent and earlier relapse. Therefore, we recommend treatment with cyclophosphamide-based regimen. Despite treatment of WG, only 17% (4 patients) had full recovery in their pituitary function. The long-term prognosis of patients with WG and pituitary involvement is not known.

Effects of body mass index at transplant on outcomes of kidney transplantation.

BACKGROUND: While obesity increases postoperative complications and cardiovascular risks, its effects on long-term kidney transplant outcomes are less clear. METHODS: We used data from the Australian and New Zealand Dialysis and Transplant (ANZDATA) Registry to examine the relationships between body mass index (BMI, classified according to World Health Organization criteria) at transplant and transplant outcome. Patients starting renal replacement therapy from April 1991 and who received a single-organ, primary kidney transplant (when aged > or =16 years) from April 1991 to December 2004 were included, and followed up to death or December 2005. Survival outcomes adjusted for important covariates were analyzed using Cox models, and cause-specific failures by competing risks analysis. Analysis using BMI at various times posttransplant was also performed. Intermediate outcomes were delayed graft function (DGF) and any acute rejection at 6 months. RESULTS: In all, 5684 patients were included. Obese patients had worse graft and patient survival only in univariate analyses, not in multivariate analyses (adjusted hazard ratio [HR] for graft loss: 1.10 [0.94-1.259], P=0.25; for patient death: 1.02 [0.83-1.25], P=0.87). Underweight patients had greater late (> or =5 years) death-censored graft loss (adjusted HR: 1.70 [1.10-2.64], P=0.02), mainly due to chronic allograft nephropathy. Obesity was associated with greater odds for DGF (adjusted OR: 1.56 [1.23-1.97], P<0.001) and 6-month risk of acute rejection (adjusted OR: 1.25 [1.01-1.54], P=0.04). CONCLUSIONS: Obesity per se was not associated with poorer kidney transplant outcomes, although it was associated with factors that led to poorer graft and patient survival. Underweight was associated with late graft failure, mainly due to chronic allograft nephropathy.

Growth factor-induced mobilization of dendritic cells in kidney and liver of rhesus macaques: implications for transplantation.

Hematopoietic growth factors (HGF) mobilize potential tolerogenic cells in transplant donors. Fms-like tyrosine kinase 3 ligand (Flt3L) mobilizes stem cells and dendritic cells (DCs) in human and nonhuman primate blood. Blood and renal and liver biopsies were obtained from untreated and Flt3L-mobilized rhesus macaques. Flt3L increased the number of myeloid CD11c(hi) and plasmacytoid CD123(hi) precursors in blood and both myeloid CD11c(+) HLA-DR(+) fascin(+) (CD45RA(-)) DCs and putative plasmacytoid CD11c(lo) CD45RA(hi) DC precursors in liver and kidneys, without affecting organ function. DC in Flt3L-treated monkeys were concentrated in the glomeruli and interstitium of kidneys, and in the portal triads and parenchyma of liver. These DCs exhibited the phenotype of immature antigen-presenting cells (APCs; CD83(-) CD86(lo) CCR5(+) CCR7(-)). HGF-induced changes reversed significantly within 7 days of Flt3L withdrawal. Therapeutic protocols that mobilize donor hematopoietic cells should consider the influence of HGF on the APC constituency of prospective organ allografts.

Renal transplantation reverses functional deficiencies in circulating dendritic cell subsets in chronic renal failure patients.

BACKGROUND: Dendritic cell (DC) subsets play critical roles in regulation of innate and adaptive immune responses. These important antigen-presenting cells have not been extensively analyzed in chronic renal failure (CRF), during dialysis, or before and after renal transplantation. METHODS: The incidence of circulating precursor (pre)-DC subsets relative to total peripheral blood mononuclear cells was analyzed in healthy controls, haemodialysis patients, peritoneal dialysis patients, CRF patients, and renal transplant (RT) recipients. DC subsets were identified and characterized phenotypically by multicolour flow cytometric analysis and purified by immunomagnetic bead isolation respectively. Cytokine production and circulating DC mobilizing cytokines were determined by ELISA. RESULTS: The incidence of circulating prePDC was reduced in all patients, but the incidence of circulating preMDC was comparable in RT and dialysis patients compared to healthy controls. CRF patients exhibited the lowest incidence of circulating preMDC and prePDC. Immunomagnetic bead-isolated preMDC and prePDC from haemodialysis patients were functionally impaired (reduced expression of surface costimulatory molecules and interleukin-12p70 production following bacterial lipopolysaccharide stimulation, and reduced interferon-alpha production following herpes simplex virus stimulation respectively, compared to healthy controls and RT recipients. Glomerular filtration rate correlated significantly with the incidence of circulating preMDC, but not prePDC. CONCLUSIONS: Deficiencies in the incidence and function of precursor DC can be reversed with successful renal transplantation achieving normal renal function. However, the finding of reduced incidence of circulating prePDC in the peripheral blood in RT recipients may be of significance in the pathogenesis of infections and malignancies.

Neglected nephropathy.

BACKGROUND: Diabetic nephropathy is a significant contributor to the morbidity, mortality and health care cost among patients with diabetes. With increasing understanding of this problem, the natural progression of diabetic nephropathy can potentially be changed. OBJECTIVE: This article examines the natural history of diabetic nephropathy and provides guidelines on detection, management and future treatment possibilities of this complication. DISCUSSION: Prevention and slowing progression are the most important aspects of the management of diabetic nephropathy. This involves monitoring renal function and risk factors for renal damage and early active intervention. The angiotensin converting enzyme inhibitors and receptor antagonists are important components of therapy, both for controlling hypertension and for slowing the progression of micro and macroalbuminuria. When microalbuminuria occurs, intensive intervention on a range of risk factors can halve the number of those suffering a cardiovascular event or progressing to macroalbuminuria. When renal insufficiency occurs, particularly when creatinine clearance is >30 mL/minute, referral to an nephrologist should be considered.

Endothelial progenitor cells enhance islet engraftment, influence ß-cell function, and modulate islet connexin 36 expression.

The success of pancreatic islet transplantation is limited by delayed engraftment and suboptimal function in the longer term. Endothelial progenitor cells (EPCs) represent a potential cellular therapy that may improve the engraftment of transplanted pancreatic islets. In addition, EPCs may directly affect the function of pancreatic ß-cells. The objective of this study was to examine the ability of EPCs to enhance pancreatic islet transplantation in a murine syngeneic marginal mass transplant model and to examine the mechanisms through which this occurs. We found that cotransplanted EPCs improved the cure rate and initial glycemic control of transplanted islets. Gene expression data indicate that EPCs, or their soluble products, modulate the expression of the ß-cell surface molecule connexin 36 and affect glucose-stimulated insulin release in vitro. In conclusion, EPCs are a promising candidate for improving outcomes in islet transplantation, and their mechanisms of action warrant further study.

Proinsulin-specific, HLA-DQ8, and HLA-DQ8-transdimer-restricted CD4+ T cells infiltrate islets in type 1 diabetes.

Type 1 diabetes (T1D) develops when insulin-secreting ß-cells, found in the pancreatic islets of Langerhans, are destroyed by infiltrating T cells. How human T cells recognize ß-cell-derived antigens remains unclear. Genetic studies have shown that HLA and insulin alleles are the most strongly associated with risk of T1D. These long-standing observations implicate CD4(+) T-cell responses against (pro)insulin in the pathogenesis of T1D. To dissect the autoimmune T-cell response against human ß-cells, we isolated and characterized 53 CD4(+) T-cell clones from within the residual pancreatic islets of a deceased organ donor who had T1D. These 53 clones expressed 47 unique clonotypes, 8 of which encoded proinsulin-specific T-cell receptors. On an individual clone basis, 14 of 53 CD4(+) T-cell clones (26%) recognized 6 distinct but overlapping epitopes in the C-peptide of proinsulin. These clones recognized C-peptide epitopes presented by HLA-DQ8 and, notably, HLA-DQ8 transdimers that form in HLA-DQ2/-DQ8 heterozygous individuals. Responses to these epitopes were detected in the peripheral blood mononuclear cells of some people with recent-onset T1D but not in HLA-matched control subjects. Hence, proinsulin-specific, HLA-DQ8, and HLA-DQ8-transdimer-restricted CD4(+) T cells are strongly implicated in the autoimmune pathogenesis of human T1D.

Manipulation of dendritic cells as an approach to improved outcomes in transplantation.

At the time of organ transplantation, a variety of non-parenchymal cells are transplanted simultaneously with the allograft. Recognition of the importance of these cells as potential immunostimulatory cells lead to the concept of 'passenger leukocytes' as the principal instigators of rejection. Passenger leukocytes include interstitial dendritic cells (DCs) and blood-derived monocytes/macrophages. As investigators have discovered the significance of DCs in influencing graft outcome, so have they begun to determine the best ways to influence DCs themselves. This review discusses the role of DCs in transplantation and then focuses on three different approaches for manipulating DCs to improve allograft survival: (1) targeting of chemokines involved in DC migration, (2) pharmacological arrest of DC maturation, and (3) genetic engineering of DCs.

Pharmacologic, biologic, and genetic engineering approaches to potentiation of donor-derived dendritic cell tolerogenicity.

There are various approaches to the enhancement of dendritic cell (DC) tolerogenicity for the promotion of cell or organ allograft survival. Both pharmacologic and biologic agents, including several commonly used immunosuppressive drugs, and specific anti-inflammatory cytokines inhibit DC maturation, whereas co-stimulation-blocking agents can also promote the induction of antigen-specific T-cell unresponsiveness by DC. Delivery of genes encoding molecules that subvert T-cell responses by various mechanisms, and targeting of DC migration by selective manipulation of chemokine and chemokine receptor expression, represent additional promising strategies. In this short review, the authors consider those approaches that have been used to promote the tolerogenicity of donor-derived DC in experimental models. Whereas most work to date has focused on myeloid DC, manipulation of other DC subsets may also offer potential for improving the outcome of transplantation and enhancing tolerance induction.

In vivo-mobilized kidney dendritic cells are functionally immature, subvert alloreactive T-cell responses, and prolong organ allograft survival.

BACKGROUND: Migratory antigen-presenting cells resident in kidneys may have tolerogenic potential. Difficulties inherent in their isolation have limited their characterization. The authors examined the phenotype and function of murine kidney dendritic cells (DC) mobilized in vivo by systemic administration of fms-like tyrosine 3 kinase ligand (Flt3L). METHODS: Monoclonal antibody staining was used to characterize DC subsets in situ, immediately after their isolation, and after lipopolysaccharide stimulation. Cytokine and CC chemokine receptor (CCR) gene expression was analyzed by RNase protection assay. Mixed leukocyte reactions were performed to assess DC allostimulatory ability and also the function of putative T-regulatory cells. In vivo DC trafficking was monitored by fluorescence imaging of dye-labeled cells and the influence of renal DC on vascularized heart allograft survival was determined. RESULTS: Flt3L induced a marked increase both in CD11cCD8alpha and in CD11cCD8alpha DC within the renal cortex and medulla. Rarer, CD11cB220 (precursor plasmacytoid) DC were also detected. Bulk freshly isolated DC exhibited no interleukin (IL)-12p35 mRNA, low surface co-stimulatory molecule expression, and CCR transcripts, consistent with immaturity. They elicited only weak allogeneic T-cell proliferative responses, and repeated stimulation induced CD4CD25 IL-10 T cells. In vivo, the freshly isolated DC failed to prime T cells of naive allogeneic hosts for anti-donor cytotoxic T-cell responses. When infused systemically, 1 week before organ transplantation, they prolonged graft survival without immunosuppressive therapy. CONCLUSIONS: Hematopoietin-mobilized renal DC are functionally immature and exhibit tolerogenic potential. Mobilization of DC within kidneys is likely to affect their antigen-handling capacity, immunogenicity, and tolerogenic ability.

Strategies for preclinical evaluation of dendritic cell subsets for promotion of transplant tolerance in the nonhuman primate.

A role for dendritic cells (DC) as critical regulators of immune reactivity has become increasingly recognized. There is evidence in rodent models that donor-derived DC, particularly in the immature state, can prolong organ allograft survival and even induce donor-specific tolerance. To allow the potential tolerogenic properties of these cells to be evaluated more fully with a view to clinical testing, it is necessary to identify DC subsets in nonhuman primates. We have identified the putative rhesus monkey equivalents of circulating human DC subset precursors as lineage(-), HLA-DR(+), CD123(lo),CD11c(hi)(pDC1) and lineage(-), HLA-DR(+), CD123(hi),CD11c(lo)(pDC2). Testing of these DC populations both in vitro and in vivo, as well as in transplant models in combination with conventional or experimental immunosuppressive reagents, will aid the development of novel strategies for the promotion of allo-antigen specific tolerance in transplantation.

Transplant glomerulopathy: the interaction of HLA antibodies and endothelium.

Transplant glomerulopathy (TG) is a major cause of chronic graft dysfunction without effective therapy. Although the histological definition of TG is well characterized, the pathophysiological pathways leading to TG development are still poorly understood. Electron microscopy suggests an earlier appearance of TG and suggests that endothelial cell injury is the first sign of the disease. The pathogenic role of human leukocyte antigen (HLA) antibodies in endothelial cells has been described in acute vascular and humoral rejection. However the mechanisms and pathways of endothelial cell injury by HLA antibodies remain unclear. Despite the description of different causes of the morphological lesion of TG (hepatitis, thrombotic microangiopathy), the strong link between TG and chronic antibody mediated rejection suggests a major role for HLA antibodies in TG formation. In this review, we describe the effect of classes I or II HLA-antibodies in TG and especially the implication of donor specific antibodies (DSA). We update recent studies about endothelial cells and try to explain the different signals and intracellular pathways involved in the progression of TG.