The diagnosis of posttransplantation diabetes mellitus: meeting the challenges.

Posttransplantation diabetes mellitus (PTDM) is a major complication after renal transplantation due to its negative impact on patient and graft survival, and affects up to 40% of renal transplant recipients. The generation of evidence regarding its optimal treatment is now progressing with some emphasis on early postoperative insulin treatment that targets ß-cell failure. This therapy seems to benefit renal transplant patients but contrasts with previous PTDM guidelines that were following treatment of type 2 diabetes mellitus (DM): oral antidiabetics first, insulin last. Similarly, in the current PTDM consensus recommendations, diagnostic procedures are in accordance with the American Diabetes Association (ADA) recommendations for diagnosis of DM. PTDM and type 2 DM, however, are distinct disease entities with different pathophysiological backgrounds. This review will discuss the significance of the standard diagnostic criteria for DM in patients after renal transplantation without prior DM. In particular, the role of glycated hemoglobin (HbA1c) and oral glucose tolerance testing (OGTT) will be reviewed. In addition, the potential role of other glycated proteins and continuous glucose monitoring will be covered, although these parameters are not yet part of the consensus recommendations.

Post-transplantation diabetes mellitus: evaluation of treatment strategies.

BACKGROUND: Post-transplantation diabetes mellitus (PTDM) is a serious complication after kidney transplantation, but evidence regarding long-term outcomes of treatment regimens remains scarce. AIM AND METHODS: The aim of this retrospective cohort analysis was to assess the long-term efficiency and safety of antidiabetic treatments in kidney transplant recipients (KTRs), who were diagnosed with PTDM by an oral glucose tolerance test (OGTT). RESULTS: Of 561 KTRs that were screened for PTDM at our outpatient clinic, 71 (13%) had a diabetic OGTT and were included in this study. Mean follow-up was 34.2 ± 16.1 months. Thirty-six PTDM patients (51%) received antidiabetic treatment after diagnosis with either a dipeptidyl peptidase-4 (DPP-4) inhibitor, a sulfonylurea, pioglitazone, or insulin. These patients had significantly higher fasting glucose and two-h plasma glucose (2HPG) values at baseline than those who remained without therapy. In contrast to lifestyle modification alone or sulfonylureas, DPP-4 inhibitors improved glycemic control significantly. Adverse events were generally mild and occurred at similar rates in all groups. CONCLUSION: While sulfonylureas failed to improve glycemic control, DPP-4 inhibitors appeared effective and safe for the therapy of PTDM after kidney transplantation.

Efficacy and safety of vildagliptin in new-onset diabetes after kidney transplantation--a randomized, double-blind, placebo-controlled trial.

New-onset diabetes after transplantation (NODAT) is a serious complication after kidney transplantation, but therapeutic strategies remain underexplored. Dipeptidyl peptidase-4 (DPP-4) inhibitors selectively foster insulin secretion without inducing hypoglycemia, which might be advantageous in kidney transplant recipients (KTRs) with NODAT. We conducted a randomized, double-blind, placebo-controlled, phase II trial to assess safety and efficacy of the DPP-4 inhibitor vildagliptin. Intraindividual differences in oral glucose tolerance test (OGTT)-derived 2-h plasma glucose (2HPG) from baseline to 3 months after treatment served as primary endpoint. Among secondary outcomes, we evaluated HbA1c, metabolic and safety parameters, as well as OGTTs at 1 month after drug discontinuation. Of 509 stable KTRs who were screened in our outpatient clinic, 63 (12.4%) had 2HPG ≥ 200 mg/dL, 33 of them were randomized and 32 completed the study. In the vildagliptin group 2HPG and HbA1c were profoundly reduced in comparison to placebo (vildagliptin: 2HPG = 182.7 mg/dL, HbA1c = 6.1%; placebo: 2HPG = 231.2 mg/dL, HbA1c = 6.5%; both p ≤ 0.05), and statistical significance was achieved for the primary endpoint (vildagliptin: 2HPG-difference -73.7 ± 51.3 mg/dL; placebo: -5.7 ± 41.4 mg/dL; p < 0.01). Adverse events were generally mild and occurred at similar rates in both groups. In conclusion, DPP-4 inhibition in KTRs with overt NODAT was safe and efficient, providing a novel treatment alternative for this specific form of diabetes.

Proceedings from an international consensus meeting on posttransplantation diabetes mellitus: recommendations and future directions.

A consensus meeting was held in Vienna on September 8-9, 2013, to discuss diagnostic and therapeutic challenges surrounding development of diabetes mellitus after transplantation. The International Expert Panel comprised 24 transplant nephrologists, surgeons, diabetologists and clinical scientists, which met with the aim to review previous guidelines in light of emerging clinical data and research. Recommendations from the consensus discussions are provided in this article. Although the meeting was kidney-centric, reflecting the expertise present, these recommendations are likely to be relevant to other solid organ transplant recipients. Our recommendations include: terminology revision from new-onset diabetes after transplantation to posttransplantation diabetes mellitus (PTDM), exclusion of transient posttransplant hyperglycemia from PTDM diagnosis, expansion of screening strategies (incorporating postprandial glucose and HbA1c) and opinion-based guidance regarding pharmacological therapy in light of recent clinical evidence. Future research in the field was discussed with the aim of establishing collaborative working groups to address unresolved questions. These recommendations are opinion-based and intended to serve as a template for planned guidelines update, based on systematic and graded literature review, on the diagnosis and management of PTDM.

Fasting and postprandial liver glycogen content in patients with type 1 diabetes mellitus after successful pancreas-kidney transplantation with systemic venous insulin delivery.

BACKGROUND: In patients with type 1 diabetes mellitus (T1DM), insulin is usually replaced systemically (subcutaneously) and not via the physiological portal route. According to previous studies, the liver's capacity to store glycogen is reduced in T1DM patients, but it remains unclear whether this is due to hyperglycaemia, or whether the route of insulin supply could contribute to this phenomenon. T1DM patients after successful pancreas-kidney transplantation with systemic venous drainage (T1DM-PKT) represent a suitable human model to further investigate this question, because they are normoglycaemic, but their liver receives insulin from the pancreas transplant via the systemic route. MATERIALS AND METHODS: In nine T1DM-PKT, nine controls without diabetes (CON) and seven patients with T1DM (T1DM), liver glycogen content was measured at fasting and after two standardized meals employing (13) C-nuclear-magnetic-resonance-spectroscopy. Circulating glucose and glucoregulatory hormones were measured repeatedly throughout the study day. RESULTS: The mean and fasting concentrations of peripheral plasma glucose, insulin, glucagon and C-peptide were comparable between T1DM-PKT and CON, whereas T1DM were hyperglycaemic and hyperinsulinaemic (P < 0·05 vs T1DM-PKT and CON). Total liver glycogen content at fasting and after breakfast did not differ in the three groups. After lunch, T1DM-PKT and T1DM had a 14% and 21% lower total liver glycogen content than CON (P < 0·02). CONCLUSION: In spite of normalized glycaemic control, postprandial liver glycogen content was reduced in T1DM-PKT with systemic venous drainage. Thus, not even optimized systemic insulin substitution is able to resolve the defect in postprandial liver glycogen storage seen in T1DM patients.

CMV late phase-induced mTOR activation is essential for efficient virus replication in polarized human macrophages.

Human cytomegalovirus (CMV) remains one of the most important pathogens following solid-organ transplantation. Mounting evidence indicates that mammalian target of rapamycin (mTOR) inhibitors may decrease the incidence of CMV infection in solid-organ recipients. Here we aimed at elucidating the molecular mechanisms of this effect by employing a human CMV (HCMV) infection model in human macrophages, since myeloid cells are the principal in vivo targets of HCMV. We demonstrate a highly divergent host cell permissiveness for HCMV with optimal infection susceptibility in M2 but not M1 polarized macrophages. Employing an ultrahigh purified HCMV stock we observed rapamycin-independent viral entry and induction of IFN-ß transcripts, but no proinflammatory cytokines or mitogen-activated protein kinases and mTOR activation early after infection. However, in the late infection phase, sustained mTOR activation was observed in HCMV-infected cells and was required for efficient viral protein synthesis including the viral late phase proteins pUL-44 and pp65. Accordingly, rapamycin strongly suppressed CMV replication 3 and 5 days postinfection in macrophages. In conclusion, these data indicate that mTOR is essential for virus replication during late phases of the viral cycle in myeloid cells and might explain the potent anti-CMV effects of mTOR inhibitors after organ transplantation.

Subject specific finite element modelling of periprosthetic femoral fractures in different load cases.

Periprosthetic femoral fractures (PFF) around total hip replacements are one of the biggest challenges for orthopaedic surgeons. To understand the risk factors and formation of these fractures, the development of a reliable finite element (FE) model incorporating bone failure is essential. Due to the anisotropic and complex hierarchical structure of bone, the mechanical behaviour under large strains is difficult to predict. In this study, a state-of-the-art subject specific FE modelling technique for bone is utilised to generate and investigate PFF. A bilinear constitutive law is applied to bone tissue in subject specific FE models of five human femurs which are virtually implanted with a straight hip stem to numerically analyse PFF. The material parameters of the models are expressed as a function of bone ash density and mapped node wise to the FE mesh. In this way the subject specific, heterogeneous structure of bone is mimicked. For material mapping of the parameters, computed tomography (CT) images of the original fresh-frozen femurs are used. Periprosthetic fractures are generated by deleting elements on the basis of a critical plastic strain failure criterion. The models are analysed under physiological and clinically relevant conditions in two different load cases re-enacting stumbling and a sideways fall on the hip. The results of the analyses are quantified with experimental data from previous work. With regard to fracture pattern, stiffness and failure load the simulations of the load case stumbling delivered the most stable and accurate results. In general, mapping of material properties was found to be an appropriate way to reproduce PFF with finite element models.

The multifunctional role of mTOR in innate immunity: implications for transplant immunity.

The mammalian target of rapamycin (mTOR) is an evolutionary conserved serine-threonine kinase that senses various environmental stimuli in most cells primarily to control cell growth. Restriction of cellular proliferation by mTOR inhibition led to the use of mTOR inhibitors as immunosuppressants in allogeneic transplantation as well as novel anticancer agents. However, distinct inflammatory side effects such as fever, pneumonitis, glomerulonephritis or anemia of chronic disease have been observed under this treatment regime. Apart from the mere cell-cycle regulatory effect of mTOR in dividing cells, recent data revealed a master regulatory role of mTOR in the innate immune system. Hence, inhibition of mTOR promotes proinflammatory cytokines such as IL-12 and IL-1beta, inhibits the anti-inflammatory cytokine IL-10 and boosts MHC antigen presentation via autophagy in monocytes/macrophages and dendritic cells. Moreover, mTOR regulates type I interferon production and the expression of chemokine receptors and costimulatory molecules. These results place mTOR in a complex immunoregulatory context by controlling innate and adaptive immune responses. In this review, we discuss the clinical consequences of mTOR-inhibitor therapy and aim to integrate this recent data into our current view of the molecular mechanisms of clinically employed mTOR inhibitors and discuss their relevance with special emphasis to transplantation.

Late onset Pneumocystis pneumonia in renal transplantation after long-term immunosuppression with belatacept.

Interference with T-cell function increases the risk of infections, especially during the early post-transplant period. Belatacept, a costimulation blocker, is currently being tested in phase III clinical trials. Here we report a renal transplant recipient who received belatacept and developed severe Pneumocystis jirovecii pneumonia (PCP) with fatal superinfections 4 years post transplant. Cytomegalovirus infection preceded PCP, which typically occurs in overimmunosuppressed patients, but has not yet been reported under T-cell costimulation blockade in transplant patients. This case illustrates the possibility of excessive immunosuppression even with a lymphocyte-specific regimen.

The PI3K/Akt/mTOR pathway in innate immune cells: emerging therapeutic applications.

The phosphatidylinositol-3 kinases (PI3Ks) and the mammalian target of rapamycin (mTOR) pathway have long been recognised as critically regulating metabolism, growth or survival. Recent data indicate that these molecules are also integral players in coordinating defence mechanisms in the innate immune system. In this respect, PI3K and mTOR positively regulate immune cell activation in neutrophils and mast cells. In plasmacytoid dendritic cells, these pathways have recently emerged as important regulators for type I interferon production via activation of the interferon-regulatory factor 7. Interestingly, in myeloid immune cells, PI3K and mTOR seem to constrain full immune cell activation by upregulation of the key anti-inflammatory cytokine interleukin 10 and inhibition of proinflammatory cytokines. These new insights into innate immune cell regulation may pave the way for manipulating distinct features of the innate immune system for therapeutic treatment of various inflammatory diseases and for implementation of improved vaccination strategies.

Urinary tract infection in renal transplant recipients.

Urinary tract infection (UTI) is common in renal transplant recipients. Frequency of UTIs depend on many factors such as age, female gender, kidney function, co-morbidity, type and amount of immunosuppression, urological instrumentation and/or the follow-up period (short term or long term) after kidney transplantation. UTI may worsen graft and patient survival. A significant proportion of renal transplant recipients with UTIs may develop acute pyelonephritis (APN), which is an independent risk factor for deterioration of graft function. Renal transplant recipients with UTIs are often clinically asymptomatic as a consequence of immunosuppression. UTI, however, may progress to APN (particularly in the early post-transplant period), bacteraemia and the full blown picture of urosepsis. Strategies for long term prophylaxis and antimicrobial treatment of UTI in renal transplant recipients are discussed.

Current concepts of molecular defence mechanisms operative during urinary tract infection.

Mucosal tissues such as the gastrointestinal tract are typically exposed to a tremendous number of microorganisms and many of them are potentially dangerous to the host. In contrast, the urogenital tract is rather infrequently colonized with bacterial organisms and also devoid of physical barriers as a multi-layered mucus or ciliated epithelia, thereby necessitating separate host defence mechanisms. Recurrent urinary tract infection (UTI) represents the successful case of microbial host evasion and poses a major medical and economic health problem. During recent years considerable advances have been made in our understanding of the mechanisms underlying the immune homeostasis of the urogenital tract. Hence, the system of pathogen-recognition receptors including the Toll-like receptors (TLRs) is able to sense danger signalling and thus activate the host immune system of the genitourinary tract. Additionally, various soluble antimicrobial molecules including iron-sequestering proteins, defensins, cathelicidin and Tamm-Horsfall protein (THP), as well as their role for the prevention of UTI by modulating innate and adaptive immunity, have been more clearly defined. Furthermore, signalling mediators like cyclic adenosine monophosphate (cAMP) or the circulatory hormone vasopressin were shown to be involved in the defence of uropathogenic microbes and maintenance of mucosal integrity. Beyond this, specific receptors e.g. CD46 or beta1/beta 3-integrins, have been elucidated that are hijacked by uropathogenic E. coli to enable invasion and survival within the urogenital system paving the way for chronic forms of urinary tract infection. Collectively, the majority of these findings offer novel avenues for basic and translational research implying effective therapies against the diverse forms of acute and chronic UTI.

The novel immunosuppressant FK778 inhibits formation of the immunologic Synapse.

The malononitrilamide FK778 is a derivative of A77 1726, the active metabolite of the antirheumatic drug leflunomide. A77 1726 inhibits de novo pyrimidine synthesis and activity of Src-family kinases; thus, it may interfere with T-cell proliferation as well as with early T-cell signaling. Formation of a stable interaction between T cells and antigen-presenting cells (APC)--the immunologic synapse--has emerged to be of crucial importance for T-cell activation. Here in we show that FK778 inhibits formation of the immunologic synapse by blocking superantigen-stimulated relocalization of adhesion (LFA-1), and signaling molecules (CD3) to the T-cell/APC contact site. These data show that FK778 affects T-cell/APC interactions, particularly events crucial for T-cell adhesion and formation of stable conjugates underlying sustained and effective T-cell activation. Thus, in this model system close to physiologic T-cell stimulation, FK778 affects critical events in the course of T-cell-mediated immune responses earlier than T-cell proliferation, which may contribute to its immunosuppressive potential.

The malononitrilamide FK778 inhibits activation of NF-kappaB in human dendritic cells.

FK778, a derivative of the active leflunomide-metabolite, A77 1726, has been shown to be a powerful immunosuppressant in several transplantation models, particularly efficient in prevention of chronic allograft rejection. However, the cellular and molecular mechanisms underlying these effects of FK778 have not been investigated yet in detail. Because dendritic cells (DCs) are a crucial cell type in initiation of immune responses including chronic allograft rejection, we investigated whether FK778 affects this peculiar cell population. NF-kappaB is the essential transcription factor involved in DC activation and function. We found that lipopolysaccharide (LPS)-induced activation of NF-kappaB, as assessed using electromobility shift assay, is markedly inhibited by FK778 in human monocyte-derived DCs. Hence, FK778 could exert its immunosuppressive effects via inhibition of activation and thus function of the central antigen-presenting cell, ie, DC.

Suppression of primary T-cell responses and induction of alloantigen-specific hyporesponsiveness in vitro by the Janus kinase inhibitor tyrphostin AG490.

BACKGROUND: Tyrphostin AG490 has recently been shown to block interleukin (IL)-2 receptor gamma-chain-associated Janus kinase 3. Here, we analyzed the effect of AG490 on T-cell alloresponses in vitro. METHODS: For the evaluation of T-cell activation, DNA synthesis, surface marker expression, cytokine secretion, intracellular calcium mobilization, early protein tyrosine phosphorylation, and apoptosis were measured. RESULTS: AG490 effectively inhibited T-cell proliferation in human mixed lymphocyte culture (MLC) even when added 4 days after culture initiation. Inhibition of IL-2-dependent proliferation in T-cell blasts and the incapability of IL-2 or IL-15 to restore proliferation in AG490-treated MLC suggests interference with cytokine receptor signaling. T-cell receptor-triggered early protein tyrosine phosphorylation, calcium mobilization, up-regulation of CD69, and initial CD25 expression were not affected. Interestingly, AG490 substantially inhibited production of IL-2 and interferon-gamma in T cells stimulated with alloantigen or via CD3 and CD28. In CD28-independent activation models (e.g., stimulation with phorbol myristate acetate plus ionomycin), however, cytokine secretion was not inhibited. Pretreatment of primary MLC with AG490 resulted in substantial down-regulation of secondary responses to cells from the original donor as opposed to third-party cells or phytohemagglutinin. Unresponsiveness was induced also in T cells stimulated with CD3 monoclonal antibody. Induction of apoptosis in polyclonally activated T cells and the incapability of IL-2 to reverse specific hyporesponsiveness, suggest programmed cell death as an important mechanism underlying antigen-specific down-regulation of alloresponses. CONCLUSIONS: We demonstrate that AG490 blocks different manifestations of T-cell activation. This and its ability to induce alloantigen-specific hyporesponsiveness point to a potential use for interfering with alloreactivities in vivo.

Role of humoral immune reactions as target for antirejection therapy in recipients of a spousal-donor kidney graft.

Excellent graft outcome has been reported for spousal-donor kidney transplantation. In husband-to-wife transplantation, however, a tendency toward inferior graft survival has been described for recipients who were previously pregnant. In our series of spousal-kidney transplantations (nine transplantations; three female recipients), actual graft survival is 100% (median observation time, 339 days). Five patients experienced early allograft rejection. In four transplant recipients, rejection was easily reversible by conventional antirejection therapy. In a multiparous recipient, however, mild interstitial allograft rejection associated with early graft dysfunction was resistant to anticellular treatment (antilymphocyte antibody, tacrolimus rescue therapy). The particular finding of polymorphonuclear neutrophils in peritubular capillaries and the finding of diffuse capillary deposits of the complement split product, C4d, in a posttransplantation biopsy specimen suggested a role of antibody-mediated graft injury. Retrospective flow cytometry cross-matching showed the presence of preformed immunoglobulin G (IgG) antibodies to HLA class I antigens that were not detectable by pretransplantation lymphocytotoxic cross-match testing or screening for panel reactive antibodies. After transplantation, however, complement-fixing antibodies, also presumably triggered by reexposure to spousal-donor HLA antigens, could be detected in the patient's serum. These findings suggested antibody-mediated allograft rejection and led to the initiation of immunoadsorption therapy (14 sessions) with staphylococcal protein A. Selective removal of recipient IgG resulted in complete reversal of graft dysfunction. Our findings suggest that in husband-to-wife transplantation, donor-specific antibodies, presumably triggered by previous pregnancies, might occasionally induce sustained allograft dysfunction. Thus, in this particular setting, a detailed immunologic and histopathologic work-up regarding antibody-mediated allograft dysfunction is warranted because immunoadsorption may be a highly effective treatment modality.

Stable prodrugs of n-butyric acid: suppression of T cell alloresponses in vitro and prolongation of heart allograft survival in a fully allogeneic rat strain combination.

n-Butyric acid has previously been shown in vitro to suppress T cell alloresponses and beyond that to induce a state of alloantigen-specific hyporesponsiveness suggesting a potential relevance for suppressing alloresponses also in vivo. The clinical use of butyrate salt derivatives, however, is limited by an extremely short half-life due to rapid metabolism. This prompted us to investigate the effect of butyric acid derivatives with prolonged residence time in vivo on T cell alloresponses in vitro and further to explore the immunosuppressive capacity of esterified n-butyric acid in vivo. First, the effect of three butyric acid esters, i.e. glucose pentabutyrate, diacetone glucose butyrate and tributyrin on T cell proliferation in a human mixed lymphocyte culture (MLC) was evaluated. All three derivatives were found to inhibit T cell alloresponses in a concentration-dependent manner. Based on the ED50 values, glucose pentabutyrate was found to be most effective in inhibiting T cell alloreactivity in vitro (11 microM), followed by diacetone glucose butyrate (122 microM), tributyrin (146 microM) and sodium butyrate (539 microM). Because of its favourable in vitro properties, glucose pentabutyrate was chosen for in vivo experiments. To test the effect of this compound on allograft survival in vivo, in the second part of this study, heterotopic heart transplants were performed in a high responder fully allogeneic rat strain combination (Brown Norway to Lewis strain rats). We found that intraperitoneal (i.p.) injection of glucose pentabutyrate at 500 mg/kg/day (day 0 and daily up to 12 days posttransplant) induced a significant prolongation of allograft survival as compared to animals treated with vehicle (glycerol formal, i.p.) alone (14.1+/-6.3 versus 9.6+/-3.2 days, p = 0.036), whereby at lower dosage (100 mg/kg/day) no such effect was observed (10.2+/-2.1 days, p = 0.21). Our findings suggest that stable prodrugs of n-butyric acid might have potential clinical relevance for inhibiting alloresponses in vivo.