Chronic Antibody-Mediated Rejection in Nonhuman Primate Renal Allografts: Validation of Human Histological and Molecular Phenotypes.

Molecular testing represents a promising adjunct for the diagnosis of antibody-mediated rejection (AMR). Here, we apply a novel gene expression platform in sequential formalin-fixed paraffin-embedded samples from nonhuman primate (NHP) renal transplants. We analyzed 34 previously described gene transcripts related to AMR in humans in 197 archival NHP samples, including 102 from recipients that developed chronic AMR, 80 from recipients without AMR, and 15 normal native nephrectomies. Three endothelial genes (VWF, DARC, and CAV1), derived from 10-fold cross-validation receiver operating characteristic curve analysis, demonstrated excellent discrimination between AMR and non-AMR samples (area under the curve = 0.92). This three-gene set correlated with classic features of AMR, including glomerulitis, capillaritis, glomerulopathy, C4d deposition, and DSAs (r = 0.39-0.63, p < 0.001). Principal component analysis confirmed the association between three-gene set expression and AMR and highlighted the ambiguity of v lesions and ptc lesions between AMR and T cell-mediated rejection (TCMR). Elevated three-gene set expression corresponded with the development of immunopathological evidence of rejection and often preceded it. Many recipients demonstrated mixed AMR and TCMR, suggesting that this represents the natural pattern of rejection. These data provide NHP animal model validation of recent updates to the Banff classification including the assessment of molecular markers for diagnosing AMR.

Molecular Assessment of Microcirculation Injury in Formalin-Fixed Human Cardiac Allograft Biopsies With Antibody-Mediated Rejection.

Precise diagnosis of antibody-mediated rejection (AMR) in cardiac allograft endomyocardial biopsies (EMBs) remains challenging. This study assessed molecular diagnostics in human EMBs with AMR. A set of 34 endothelial, natural killer cell and inflammatory genes was quantified in 106 formalin-fixed, paraffin-embedded EMBs classified according to 2013 International Society for Heart and Lung Transplantation (ISHLT) criteria. The gene set expression was compared between ISHLT diagnoses and correlated with donor-specific antibody (DSA), endothelial injury by electron microscopy (EM) and prognosis. Findings were validated in an independent set of 57 EMBs. In the training set (n = 106), AMR cases (n = 70) showed higher gene set expression than acute cellular rejection (ACR; n = 21, p < 0.001) and controls (n = 15, p < 0.0001). Anti-HLA DSA positivity was associated with higher gene set expression (p = 0.01). Endothelial injury by electron microscopy strongly correlated with gene set expression, specifically in AMR cases (r = 0.62, p = 0.002). Receiver operating characteristic curve analysis for diagnosing AMR showed greater accuracy with gene set expression (area under the curve [AUC] = 79.88) than with DSA (AUC = 70.47) and C4d (AUC = 70.71). In AMR patients (n = 17) with sequential biopsies, increasing gene set expression was associated with inferior prognosis (p = 0.034). These findings were confirmed in the validation set. In conclusion, biopsy-based molecular assessment of antibody-mediated microcirculation injury has the potential to improve diagnosis of AMR in human cardiac transplants.

Chemical Basis for Qualitative and Quantitative Differences Between ABO Blood Groups and Subgroups: Implications for Organ Transplantation.

Blood group ABH(O) carbohydrate antigens are carried by precursor structures denoted type I-IV chains, creating unique antigen epitopes that may differ in expression between circulating erythrocytes and vascular endothelial cells. Characterization of such differences is invaluable in many clinical settings including transplantation. Monoclonal antibodies were generated and epitope specificities were characterized against chemically synthesized type I-IV ABH and related glycans. Antigen expression was detected on endomyocardial biopsies (n = 50) and spleen (n = 11) by immunohistochemical staining and on erythrocytes by flow cytometry. On vascular endothelial cells of heart and spleen, only type II-based ABH antigens were expressed; type III/IV structures were not detected. Type II-based ABH were expressed on erythrocytes of all blood groups. Group A1 and A2 erythrocytes additionally expressed type III/IV precursors, whereas group B and O erythrocytes did not. Intensity of A/B antigen expression differed among group A1 , A2 , A1 B, A2 B and B erythrocytes. On group A2 erythrocytes, type III H structures were largely un-glycosylated with the terminal "A" sugar α-GalNAc. Together, these studies define qualitative and quantitative differences in ABH antigen expression between erythrocytes and vascular tissues. These expression profiles have important implications that must be considered in clinical settings of ABO-incompatible transplantation when interpreting anti-ABO antibodies measured by hemagglutination assays with reagent erythrocytes.

Vitamin D in renal transplantation - from biological mechanisms to clinical benefits.

Recent developments in our understanding of vitamin D show that it plays a significant role in immunological health, uniquely occupying both an anti-microbial and immunoregulatory niche. Vitamin D deficiency is widespread amongst renal transplant recipients (RTRs), thus providing one patho-mechanism that may influence the achievement of a successful degree of immunosuppression. It may also influence the development of the infectious, cardiovascular and neoplastic complications seen in RTRs. This review examines the biological roles of vitamin D in the immune system of relevance to renal transplantation (RTx) and evaluates whether vitamin D repletion may be relevant in determining immunologically-related clinical outcomes in RTRs, (including graft survival, cardiovascular disease and cancer). While there are plausible biological and epidemiological reasons to undertake vitamin D repletion in RTRs, there are few randomized-controlled trials in this area. Based on the available literature, we cannot at present categorically make the case for routine measurement and repletion of vitamin D in clinical practice but we do suggest that this is an area in urgent need of further randomized controlled level evidence.

Helicobacter pylori induces in-vivo expansion of human regulatory T cells through stimulating interleukin-1ß production by dendritic cells.

Helicobacter pylori is one of the most common infections in the world. Despite inciting inflammation, immunological clearance of the pathogen is often incomplete. CD4(+) CD25(hi) forkhead box protein 3 (FoxP3(+)) regulatory T cells (T(regs)) are potent suppressors of different types of immune responses and have been implicated in limiting inflammatory responses to H. pylori. Investigating the influence of H. pylori on T(reg) function and proliferation, we found that H. pylori-stimulated dendritic cells (DCs) induced proliferation in T(regs) and impaired their suppressive capability. This effect was mediated by interleukin (IL)-1ß produced by H. pylori-stimulated DCs. These data correlated with in-vivo observations in which H. pylori(+) gastric mucosa contained more T(regs) in active cell division than uninfected stomachs. Inciting local proliferation of T(regs) and inhibiting their suppressive function may represent a mechanism for the chronic gastritis and carcinogenesis attributable to H. pylori.

Microtensile bond strength of self-etch adhesives in different surface conditionings.

AIM: Self-etch bonding systems have been introduced recently and have the advantage of reduced operating time. Because of patient management and isolation difficulties in children, the present study aimed to evaluate microtensile bond strength of two self-etch bonding systems (AdheSE and Xeno® III) on cleaned only, ground and sandblasted enamel. MATERIALS AND METHODS: Thirty intact primary molars were selected and randomly divided into two groups according to the type of bonding used (Xeno® III and AdheSE). The teeth were divided into three subgroups according to enamel surface conditioning method: prophylaxis with pumice, grinding with coarse finishing and polishing disc, and sandblasting with aluminum oxide particles. Tetric Ceram composite then was applied. Samples were sectioned coronally and axially. The interface area of dental sticks was measured and specimens were subjected to a tensile force of 0.5 mm/min until failure. RESULTS: Microtensile bond strength of Xeno® III-sandblasting, AdheSE-sandblasting, AdheSE-grinding, and AdheSE- cleaned only were 25.5±7.7, 21.2±2.3, 12.9±2.2, and 9.1±1.1, respectively. During sectioning, all specimens of Xeno® III-cleaned only and Xeno® III-grinding groups were fractured. CONCLUSIONS: Two step AdheSE yields higher mTBS in comparison to one step Xeno ® III. In regard to surface conditioning, sandblasting increases the mTBS relative to grinding with finishing and polishing disc, and the cleaned-only enamel has the lowest mTBS.

Relative resistance of human CD4(+) memory T cells to suppression by CD4(+) CD25(+) regulatory T cells.

Successful expansion of functional CD4(+) CD25(+) regulatory T cells (T(reg)) ex vivo under good manufacturing practice conditions has made T(reg) -cell therapy in clinical transplant tolerance induction a feasible possibility. In animals, T(reg) cells home to both transplanted tissues and local lymph nodes and are optimally suppressive if active at both sites. Therefore, they have the opportunity to suppress both naïve and memory CD4(+) CD25(-) T cells (Tresp). Clinical transplantation commonly involves depleting therapy at induction (e.g. anti-CD25), which favors homeostatic expansion of memory T cells. Animal models suggest that T(reg) cells are less suppressive on memory, compared with naïve Tresp that mediate allograft rejection. As a result, in the context of human T(reg) -cell therapy, it is important to define the effectiveness of T(reg) cells in regulating naïve and memory Tresp. Therefore, we compared suppression of peripheral blood naïve and memory Tresp by fresh and ex vivo expanded T(reg) cells using proliferation, cytokine production and activation marker expression (CD154) as readouts. With all readouts, naïve human Tresp were more suppressible by approximately 30% than their memory counterparts. This suggests that T(reg) cells may be more efficacious if administered before or at the time of transplantation and that depleting therapy should be avoided in clinical trials of T(reg) cells.

Translational mini-review series on Th17 cells: induction of interleukin-17 production by regulatory T cells.

Uncommitted (naive) CD4(+) T helper cells (Thp) can be induced to differentiate to specific lineages according to the local cytokine milieu, towards T helper type 1 (Th1), Th2, Th17 and regulatory T cell (T(reg)) phenotypes in a mutually exclusive manner. Each phenotype is characterized by unique signalling pathways and expression of specific transcription factors, notably T-bet for Th1, GATA-3 for Th2, forkhead box P3 (FoxP3) for T(regs) and receptor-related orphan receptor (ROR)alpha and RORgammat for Th17 cells. T(regs) and Th17 cells have been demonstrated to arise from common precursors in a reciprocal manner based on exposure to transforming growth factor (TGF)-beta or TGF-beta plus interleukin (IL)-6 and carry out diametrically opposing functions, namely suppression or propagation of inflammation, respectively. However, while epigenetic modifications in Th1 and Th2 differentiated cells prevents their conversion to other phenotypes, Th17 cells generated in vitro using TGF-beta and IL-6 are unstable and can convert to other phenotypes, especially Th1, both in vitro and in vivo. T(regs) are generated from naive precursors both in the thymus (natural, nT(regs)) and in the periphery (induced, iT(regs)). The highly suppressive function of T(regs) enables them to control many inflammatory diseases in animals and makes them particularly attractive candidates for immunotherapy in humans. The stability of the T(reg) phenotype is therefore of paramount importance in this context. Recent descriptions of T(reg) biology have suggested that components of pathogens or inflammatory mediators may subvert the suppressive function of T(regs) in order to allow propagation of adequate immune responses. Unexpectedly, however, a number of groups have now described conversion of T(regs) to the Th17 phenotype induced by appropriate inflammatory stimuli. These observations are particularly relevant in the context of cell therapy but may also explain some of the dysregulation seen in autoimmune diseases. In this paper, we review T(reg) to Th17 conversion and propose some potential mechanisms for this phenomenon.

Successful retransplantation using rapamycin in a patient with previous calcineurin inhibitor-induced posterior leukoencephalopathy syndrome.

Posterior Leukoencephalopathy Syndrome (PLES) is a rare but serious neurological condition with many aetiologies. In the era of organ transplantation there have been sporadic reports of calcineurin-inhibitor associated PLES. We describe a case, with subsequent uneventful retransplantation using sirolimus.

Allorecognition and the alloresponse: clinical implications.

The artificial transfer of tissues or cells between genetically diverse individuals elicits an immune response that is adaptive and specific. This response is orchestrated by T lymphocytes that are recognizing, amongst others, major histocompatibility complex (MHC) molecules expressed on the surface of the transferred cells. Three pathways of recognition are described: direct, indirect and semi-direct. The sets of antigens that are recognized in this setting are also discussed, namely, MHC protein products, the MHC class I-related chain (MIC) system, minor histocompatibility antigens and natural killer cell receptor ligands. The end product of the effector responses are hyperacute, acute and chronic rejection. Special circumstances surround the situation of pregnancy and bone marrow transplantation because in the latter, the transferred cells are the ones originating the immune response, not the host. As the understanding of these processes improves, the ability to generate clinically viable immunotherapies will increase.

The role of T helper 17 (Th17) and regulatory T cells (Treg) in human organ transplantation and autoimmune disease.

Uncommitted (naive) murine CD4+ T helper cells (Thp) can be induced to differentiate towards T helper 1 (Th1), Th2, Th17 and regulatory (Treg) phenotypes according to the local cytokine milieu. This can be demonstrated most readily both in vitro and in vivo in murine CD4+ T cells. The presence of interleukin (IL)-12 [signalling through signal transduction and activator of transcription (STAT)-4] skews towards Th1, IL-4 (signalling through STAT-6) towards Th2, transforming growth factor (TGF)-beta towards Treg and IL-6 and TGF-beta towards Th17. The committed cells are characterized by expression of specific transcription factors, T-bet for Th1, GATA-3 for Th2, forkhead box P3 (FoxP3) for Tregs and RORgammat for Th17 cells. Recently, it has been demonstrated that the skewing of murine Thp towards Th17 and Treg is mutually exclusive. Although human Thp can also be skewed towards Th1 and Th2 phenotypes there is as yet no direct evidence for the existence of discrete Th17 cells in humans nor of mutually antagonistic development of Th17 cells and Tregs. There is considerable evidence, however, both in humans and in mice for the importance of interferon (IFN)-gamma and IL-17 in the development and progression of inflammatory and autoimmune diseases (AD). Unexpectedly, some models of autoimmunity thought traditionally to be solely Th1-dependent have been demonstrated subsequently to have a non-redundant requirement for Th17 cells, notably experimental allergic encephalomyelitis and collagen-induced arthritis. In contrast, Tregs have anti-inflammatory properties and can cause quiescence of autoimmune diseases and prolongation of transplant function. As a result, it can be proposed that skewing of responses towards Th17 or Th1 and away from Treg may be responsible for the development and/or progression of AD or acute transplant rejection in humans. Blocking critical cytokines in vivo, notably IL-6, may result in a shift from a Th17 towards a regulatory phenotype and induce quiescence of AD or prevent transplant rejection. In this paper we review Th17/IL-17 and Treg biology and expand on this hypothesis.

Diabetes, kidney disease and anaemia: time to tackle a troublesome triad?

Both chronic kidney disease (CKD) and type II diabetes mellitus (DM) are increasing in frequency among Western populations and both are potent risk factors for the development of anaemia. The presence of CKD and diabetes together represent the most important aetiopathogenic combination for the development of anaemia. New evidence has highlighted some of the underlying mechanisms which make diabetic patients more susceptible to dyserythropoiesis, particularly once they have developed concomitant CKD. In addition, recent publications from large-scale epidemiological studies have highlighted the impact of anaemia on diabetic patients. The purpose of this review was to focus on the pathophysiology and impact of anaemia in DM.

Intercellular transfer of MHC and immunological molecules: molecular mechanisms and biological significance.

The intercellular transfer of many molecules, including the major histocompatibility complexes (MHC), both class I and II, costimulatory and adhesion molecules, extracellular matrix organization molecules as well as chemokine, viral and complement receptors, has been observed between cells of the immune system. In this review, we aim to summarize the findings of a large body of work, highlight the molecules transferred and how this is achieved, as well as the cells capable of acquiring molecules from other cells. Although a physiological role for this phenomenon has yet to be established we suggest that the exchange of molecules between cells may influence the immune system with respect to immune amplification as well as regulation and tolerance. We will discuss why this may be the case and highlight the influence intercellular transfer of MHC molecules may have on allorecognition and graft rejection.

Anaemia in diabetic patients with chronic kidney disease--prevalence and predictors.

AIMS/HYPOTHESIS: We investigated the relationship between haemoglobin (Hb) and diabetes in patients with renal disease. SUBJECTS, MATERIALS AND METHODS: All adult patients with stable chronic kidney disease attending renal or diabetic outpatient clinics in a six-month period were identified. Patients' notes and electronic patient records were used to build a comprehensive biochemical and clinical database. Results were analysed for the predictors of Hb, the severity of anaemia in both groups and the relative impact of diabetes on haemoglobin and anaemia. RESULTS: The study group consisted of 468 patients, of whom 204 had diabetes and 264 did not. At every level of renal function haemoglobin levels were lower by an average of 10 g/l in subjects with diabetes than in those without. Likewise, anaemia was found to occur at an earlier stage of chronic kidney disease (and to be of greater severity) in diabetic patients. Independent predictors of haemoglobin included female sex, diabetes, renal function and serum albumin, with diabetes and renal function being the greatest predictors. Multiple logistic regression showed that patients with diabetes had an odds ratio of 4 for being anaemic. Had we used an estimated GFR of less than 60 ml/min to trigger investigation of anaemia, we would have detected 85% of anaemic patients. CONCLUSIONS/INTERPRETATION: Anaemia is frequently found in diabetic patients with renal disease, occurs earlier and is more severe than in similar but non-diabetic subjects. In contrast to previous publications, our findings suggest that anaemia is prevalent at the earliest stages of chronic kidney disease. We advocate an estimated GFR threshold of <60 ml/min to trigger investigation for anaemia in diabetic subjects.

Transmission of syphilis by solid organ transplantation.

Two organ recipients developed serologic evidence of syphilis infection after renal transplantation from a common deceased donor with a history of treated syphilis. Testing of donor serum for syphilis, which occurred after transplantation, gave results interpreted as consistent with past infection. However, subsequent serologic results in the recipients suggested transmission of infection at transplantation due to active infection of the donor. This may be explained by recent donor re-infection in view of the current syphilis epidemic in the United Kingdom. An initial error in the treatment of recipients further served to highlight unfamiliarity in managing this resurgent infection in the context of organ transplantation.

Differential expression of beta1 and beta2 integrins and L-selectin on CD4+ and CD8+ T lymphocytes in human blood: comparative analysis between isolated cells, whole blood samples and cryopreserved preparations.

Flow cytometric analysis was used to compare the expression of adhesion molecules on human CD4+ and CD8+ T lymphocytes in isolated blood mononuclear cells (MNCs) in whole blood samples and in cryopreserved MNC preparations. Examination of MNCs revealed that the CD11b and CD11c components of the beta2 integrins were preferentially expressed on CD8+ T cells, whereas CD62L was present on more CD4+ T cells. All CD4+ and CD8+ T lymphocytes were positive for CD11a but the CD8+ population had a higher intensity of expression of CD11a and also CD11b. Virtually identical results were obtained with T cells in whole blood samples. In relation to the beta1 integrins, the only difference between isolated CD4+ and CD8+ T cells was that the latter subset had a greater proportion of cells bearing CD49d. The naive cell marker CD45RA was present on the majority of CD8+ T cells whereas CD45RA and the memory marker CD45RO were evenly distributed within the CD4+ T cell subset. Although cryopreservation of lymphocytes did not modify the expression of beta1 and beta2 integrins it produced a marked reduction in the percentage of CD4+ and CD8+ T cells bearing CD62L. With regard to endothelial interactions, it appears that cryopreserved lymphocytes are suitable for inclusion in studies of integrin-mediated adhesion but not for those relating to tethering or recognition of addressins on high endothelial venules. Differences in adhesion molecule expression between CD4+ and CD8+ T lymphocytes could underlie the selective extravasation of these subsets into sites of infection and inflammation.

What added value does ambulatory blood pressure monitoring brings to the management of post renal transplantation hypertension?

OBJECTIVE: To evaluate BP control, white coat hypertension (WCH) and abnormal circadian variability in a significant outpatient sample of renal transplant (RTx) subjects, normotensive at the last regular visit. METHODS: ABPM (Spacelab 90217) was performed every 15 min between 07:00-23:00 h and every 30 min between 23:01-06.59 h. in all patients (N = 68, 39M, S-Cr. = 153 +/- 49 mumol/l) normotensive at their last regular office BP (O-BP) measurement and with available BP records for the 12 months preceding RTx and 6 months preceding ABPM. RESULTS: BP values were frequently abnormal in this RTx cohort considered to have a satisfactory BP control. O-BP (measured with a Hawksley random-0 sphygmomanometer on the day of ABPM) was 135.5/80.6 mmHg, 47.1% of the patients with abnormal BP values. By comparison, ABPM showed a lower prevalence of uncontrolled BP: 44.1% for 24 h.-BP and only 35.3% for the daytime awake period, with values of 134.5/80.4 and 135.3/81 mmHg respectively (P = NS from O-BP). The prevalence of WCH was 12%. 24-h SBP is related to O-SBP (r = 0.71, P < 0.01) and Bland-Altman analysis demonstrates that > 95.6% of all differences between systolic ABPM and O-BP values are within +/- 2SD of the identity line. However, although 24-h DBP is equally related to O-DBP (r = 0.64, P < 0.01), on Bland-Altman analysis, 8.8% of the differences between diastolic ABPM and O-BP values are outside +/- 2SD of the identity line. Thus, systolic but not diastolic O-BP correlates with, and can be substituted to ABPM derived values. Non-dipping was frequent, regardless of the definition of normal nocturnal BP fall (10 mmHg or 10% of the daytime SBP): 82.4%, 89.7%. Even if normality was strictly defined as a night/day ratio < 0.90 for SBP and < 0.92 for DBP, non-dipping prevalence was high 73.5%, with more than one-third of the RTx patients having nocturnal hypertension (ratio > 1). CONCLUSIONS: BP control is not optimal in one-third of a typical RTx population. Furthermore, nocturnal hypertension is a frequent and underestimated phenomenon in this population. There is a good agreement between ABPM derived and casual systolic values. Office measurements, due to WCH, are under-evaluating the quality and efficacy of the antihypertensive regimens.