End-stage renal disease patients have a dysfunctional, prematurely aged peripheral T-cell system. Here we hypothesized that the degree of premature T-cell ageing before kidney transplantation predicts the risk for early acute allograft rejection (EAR).

Copeptin has recently been identified to be a stable surrogate marker for the unstable hormone arginine vasopressin (AVP). Copeptin has been shown to correlate with disease severity in leptospirosis and bacterial sepsis. Hyponatraemia is common in severe imported malaria and dysregulation of AVP release has been hypothesized as an underlying pathophysiological mechanism. The aim of the present study was to evaluate the performance of copeptin as a predictor of disease severity in imported malaria.

Previously, we and others showed that dietary restriction protects against renal ischemia-reperfusion injury in animals. However, clinical translation of preoperative diets is scarce, and in the setting of kidney transplantation these data are lacking. In this pilot study, we investigated the effects of five days of a preoperative protein and caloric dietary restriction (PCR) diet in living kidney donors on the perioperative effects in donors, recipients and transplanted kidneys. Thirty-five kidney donors were randomized into either the PCR, 30% calorie and 80% protein reduction, or control group without restrictions. Adherence to the diet and kidney function in donors and their kidney recipients were analyzed. Perioperative kidney biopsies were taken in a selected group of transplanted kidneys for gene expression analysis. All donors adhered to the diet. From postoperative day 2 up until month 1, kidney function of donors was significantly better in the PCR-group. PCR-donor kidney recipients showed significantly improved kidney function and lower incidence of slow graft function and acute rejection. PCR inhibited cellular immune response pathways and activated stress-resistance signaling. These observations are the first to show that preoperative dietary restriction induces postoperative recovery benefits in humans and may be beneficial in clinical settings involving ischemia-reperfusion injury.

In solid organ transplant (SOT) recipients, transplant rejection during immune checkpoint inhibitor (ICI) treatment for cancer is a clinical problem. Donor-derived cell-free DNA (dd-cfDNA) can be detected in blood and is a sensitive biomarker for diagnosis of acute rejection in SOT recipients. To our best knowledge, this is the first case report of a kidney transplant recipient with advanced cancer treated with ICI who was monitored with dd-cfDNA.

The endothelium plays a key role in acute and chronic rejection of solid organ transplants. During both processes the endothelium is damaged often with major consequences for organ function. Also, endothelial cells (EC) have antigen-presenting properties and can in this manner initiate and enhance alloreactive immune responses. For decades, knowledge about these roles of EC have been obtained by studying both in vitro and in vivo models. These experimental models poorly imitate the immune response in patients and might explain why the discovery and development of agents that control EC responses is hampered. In recent years, various innovative human 3D in vitro models mimicking in vivo organ structure and function have been developed. These models will extend the knowledge about the diverse roles of EC in allograft rejection and will hopefully lead to discoveries of new targets that are involved in the interactions between the donor organ EC and the recipient's immune system. Moreover, these models can be used to gain a better insight in the mode of action of the currently prescribed immunosuppression and will enhance the development of novel therapeutics aiming to reduce allograft rejection and prolong graft survival.

Background: FoxP3+ follicular regulatory T cells (Tfr) have been identified as the cell population controlling T follicular helper (Tfh) cells and B cells which, are both involved in effector immune responses against transplanted tissue. Methods: To understand the biology of Tfr cells in kidney transplant patients treated with tacrolimus and mycophenolate mofetil (MMF) combination immunosuppression, we measured circulating (c)Tfh and cTfr cells in peripheral blood by flow cytometry in n = 211 kidney transplant recipients. At the time of measurement patients were 5-7 years after transplantation. Of this cohort of patients, 23.2% (49/211) had been previously treated for rejection. Median time after anti-rejection therapy was 4.9 years (range 0.4-7 years). Age and gender matched healthy individuals served as controls. Results: While the absolute numbers of cTfh cells were comparable between kidney transplant recipients and healthy controls, the numbers of cTfr cells were 46% lower in immunosuppressed recipients (p < 0.001). More importantly, in transplanted patients, the ratio of cTfr to cTfh was decreased (median; 0.10 vs. 0.06), indicating a disruption of the balance between cTfr and cTfh cells. This shifted balance was observed for both non-rejectors and rejectors. Previous pulse methylprednisolone or combined pulse methylprednisolone + intravenous immunoglobulin anti-rejection therapy led to a non-significant 30.6% (median) and 51.2% (median) drop in cTfr cells, respectively when compared to cTfr cell numbers in transplant patients who did not receive anti-rejection therapy. A history of alemtuzumab therapy did lead to a significant decrease in cTfr cells of 85.8% (median) compared with patients not treated with anti-rejection therapy (p < 0.0001). No association with tacrolimus or MMF pre-dose concentrations was found. Conclusion: This cross-sectional study reveals that anti-rejection therapy with alemtuzumab significantly lowers the number of cTfr cells in kidney transplant recipients. The observed profound effects by these agents might dysregulate cTfr functions.

For decades, flucloxacillin has been used to treat methicillin-susceptible Staphylococcus aureus (MSSA). Little is still known about its pharmacodynamics (PD). The present study aimed to determine the pharmacokinetic (PK)/PD index and the PD-index value minimally required for efficacy. MICs of 305 MSSA isolates were measured to determine the wild-type distribution. The PD of 8 S. aureus, 1 S. pyogenes, and 1 S. agalactiae isolates were evaluated in a neutropenic murine thigh infection model. Two S. aureus isolates were used in a dose-fractionation study and a dose−response analysis was performed additionally in the in vivo model. Data were analyzed with a population PK and sigmoid maximum effect model. The end of the wild-type distribution was 1 mg/L. The percentage of time the unbound concentration was above MIC (%fT > MIC) was best correlated with efficacy. For S. aureus, median %fT > 0.25 × MIC required for 1-log reduction was 15%. The value for S. pyogenes was 10%fT > MIC and for S. agalactiae 22%fT > 0.25xMIC for a 1-log reduction. The effect of flucloxacillin reached a 2-log reduction of S. aureus at 20%fT > 0.25xMIC and also for S. pyogenes and S. agalactiae, a reduction was reached. These data may serve to optimize dosing regimens currently used in humans.

Tacrolimus (Tac) is well established as main immunosuppressant in most immunosuppressive regimens in solid organ transplantation. Due to the narrow therapeutic window, pre dose Tac levels (C0) are monitored in all patients receiving Tac to reach optimal therapeutic levels. Tac is metabolized in the liver and intestine by the cytochrome P450 3A (CYP3A) isoforms CYP3A4 and CYP3A5. We present a case of an African American woman who underwent a liver transplantation in which adequate Tac levels were difficult to accomplish due to differences in cytochrome P450 3A4/5 (CYP3A4/5) polymorphisms of the transplant recipient and the donor liver graft. This case report highlights that genotyping the liver transplant recipient and the donor liver graft might provide data which could be used to predict the tacrolimus metabolism post transplantation.

Tacrolimus (Tac) is a profoundly effective immunosuppressant that reduces the risk of rejection after solid organ transplantation. However, its use is hampered by its narrow therapeutic window along with its highly variable pharmacological (pharmacokinetic [PK] and pharmacodynamic [PD]) profile. Part of this variability is explained by genetic polymorphisms affecting the metabolic pathway. The integration of CYP3A4 and CY3A5 genotype in tacrolimus population-based PK (PopPK) modeling approaches has been proven to accurately predict the dose requirement to reach the therapeutic window. The objective of the present study was to develop an accurate PopPK model in a cohort of 59 kidney transplant patients to deliver this information to clinicians in a clear and actionable manner. We conducted a non-parametric non-linear effects PopPK modeling analysis in Pmetrics®. Patients were genotyped for the CYP3A4∗22 and CYP3A5∗3 alleles and were classified into 3 different categories [poor-metabolizers (PM), Intermediate-metabolizers (IM) or extensive-metabolizers (EM)]. A one-compartment model with double gamma absorption route described very accurately the tacrolimus PK. In covariate analysis, only CYP3A genotype was retained in the final model (Δ-2LL = -73). Our model estimated that tacrolimus concentrations were 33% IC95%[20-26%], 41% IC95%[36-45%] lower in CYP3A IM and EM when compared to PM, respectively. Virtually, we proved that defining different starting doses for PM, IM and EM would be beneficial by ensuring better probability of target concentrations attainment allowing us to define new dosage recommendations according to patient CYP3A genetic profile.

Humoral alloreactivity has been recognized as a common cause of kidney transplant dysfunction. B-cell activation, differentiation, and antibody production are dependent on IL-21+CXCR5+follicular T-helper (Tfh) cells. Here, we studied whether belatacept, an inhibitor of the costimulatory CD28-CD80/86-pathway, interrupts the crosstalk between Tfh- and B-cells more efficiently than the calcineurin inhibitor tacrolimus. The suppressive effects of belatacept and tacrolimus on donor antigen-driven Tfh-B-cell interaction were functionally studied in peripheral blood mononuclear cells from 40 kidney transplant patients randomized to a belatacept- or tacrolimus-based immunosuppressive regimen. No significant differences in uncultured cells or donor antigen-stimulated cells were found between belatacept- and tacrolimus-treated patients in the CXCR5+Tfh cell generation and activation (upregulation of PD-1). Belatacept and tacrolimus in vitro minimally inhibited Tfh-cell generation (by ~6-7%) and partially prevented Tfh-cell activation (by ~30-50%). The proportion of IL-21+-activated Tfh-cells was partially decreased by in vitro addition of belatacept or tacrolimus (by ~60%). Baseline expressions and proportions of activated CD86+ B-cells, plasmablasts, and transitional B-cells after donor antigen stimulation did not differ between belatacept- and tacrolimus-treated patients. Donor antigen-driven CD86 upregulation on memory B-cells was not fully prevented by adding belatacept in vitro (~35%), even in supratherapeutic doses. In contrast to tacrolimus, belatacept failed to inhibit donor antigen-driven plasmablast formation (~50% inhibition vs. no inhibition, respectively, p < 0.0001). In summary, donor antigen-driven Tfh-B-cell crosstalk is similar in cells obtained from belatacept- and tacrolimus-treated patients. Belatacept is, however, less potent in vitro than tacrolimus in inhibiting Tfh-cell-dependent plasmablast formation.

Alemtuzumab, a monoclonal antibody that depletes CD52-bearing immune cells, is an effective drug for the treatment of severe or glucocorticoid-resistant acute kidney transplant rejection (AR). Patient-specific predictions on treatment response are, however, urgently needed, given the severe side effects of alemtuzumab. This study developed a multidimensional prediction model with the aim of generating clinically useful prognostic scores for the response to alemtuzumab. Clinical and histological characteristics were collected retrospectively from patients who were treated with alemtuzumab for AR. In addition, targeted gene expression profiling of AR biopsy tissues was performed. Least absolute shrinkage and selection operator (LASSO) logistic regression modeling was used to construct the ALEMtuzumab for Acute Rejection (ALEMAR) prognostic score. Response to alemtuzumab was defined as patient and allograft survival and at least once an estimated glomerular filtration rate (eGFR) > 30 mL/min/1.73 m2 during the first 6 months after treatment. One hundred fifteen patients were included, of which 84 (73%) had a response to alemtuzumab. The ALEMAR-score accurately predicted the chance of response. Gene expression analysis identified 13 differentially expressed genes between responders and nonresponders. The combination of the ALEMAR-score and selected genes resulted in improved predictions of treatment response. The present preliminary prediction model is potentially helpful for the development of stratified alemtuzumab treatment for acute kidney transplant rejection but requires validation.

Acute T-cell-mediated rejection (aTCMR) still remains a clinical problem after kidney transplantation despite significant improvements in immunosuppressive regimens. Polyfunctional T cells, i.e. T cells producing multiple pro-inflammatory cytokines, are believed to be the most relevant T cells in an immune response. The aim of this study was to determine whether polyfunctional donor-reactive T cells are associated with aTCMR. In a case-control study, 49 kidney transplant recipients with a biopsy-proven aTCMR in the first year after transplantation were included, as well as 51 controls without aTCMR. Circulating donor-reactive T cells were identified by the expression of CD137 after short-term co-culture with donor antigen-presenting cells. Polyfunctional donor-reactive T cells were further characterized by dissection into different T-cell subsets encompassing the spectrum of naïve to terminally differentiated effector T cells. Prior to kidney transplantation, proportions of donor-reactive CD4+ (0.03% versus 0.02%; P < 0.01) and CD8+ (0.18% versus 0.10%; P < 0.01) CD137++ T cells were significantly higher in recipients with a biopsy-proven aTCMR versus non-rejectors. Polyfunctionality was higher (P = 0.03) in this subset of CD137-expressing T cells. These cells were predominantly of the EM/EMRA-phenotype, with polyfunctional donor-reactive CD137++CD4+ T cells predominantly co-expressing CD28 whereas approximately half of the polyfunctional CD137++CD8+ T cells co-expressed CD28. In addition, at the time of aTCMR, polyfunctional donor-reactive CD137++ CD4+, but not CD8+, T cells, were specifically decreased by 75% compared to before transplantation in recipients with as well as those without an aTCMR. Prior to transplantation, the proportion of polyfunctional donor-reactive CD137++ T cells is associated with the occurrence of a biopsy-proven aTCMR within the first year after transplantation.

The relationship between circulating effector memory T and B cells long after transplantation and their susceptibility to immunosuppression are unknown. To investigate the impact of antirejection therapy on T cell-B cell coordinated immune responses, we assessed IFN-γ-producing memory cells and natural antibodies (nAbs) that potentially bind to autoantigens on the graft.

No abstract available

Extracellular vesicles (EVs) are nanoparticles that transmit molecules from releasing cells to target cells. Recent studies link urinary EVs (uEV) to diverse processes such as infection and rejection after kidney transplantation. This, and the unmet need for biomarkers diagnosing kidney transplant dysfunction, has led to the current high level of interest in uEV. uEV provide non-intrusive access to local protein, DNA, and RNA analytics without invasive biopsy. To determine the added value of uEV measurements for detecting allograft dysfunction after kidney transplantation, we systematically included all related literature containing directly relevant information, with the addition of indirect evidence regarding urine or kidney injury without transplantation. According to their varying characteristics, uEV markers after transplantation could be categorized into kidney-specific, donor-specific, and immune response-related (IR-) markers. A few convincing studies have shown that kidney-specific markers (PODXL, ion cotransporters, SYT17, NGAL, and CD133) and IR-markers (CD3, multi-mRNA signatures, and viral miRNA) could diagnose rejection, BK virus-associated nephropathy, and calcineurin inhibitor nephrotoxicity after kidney transplantation. In addition, some indirect proof regarding donor-specific markers (donor-derived cell-free DNA) in urine has been demonstrated. Together, this literature review provides directions for exploring novel uEV markers' profiling complications after kidney transplantation.

The optimal dose regimen for intravenous (IV) treatment in children with severe acute asthma (SAA) is still a matter of debate. We assessed the efficacy of adding a salbutamol loading dose to continuous infusion with salbutamol in children admitted to a pediatric intensive care unit (PICU) with SAA. This multicentre, placebo-controlled randomized trial in the PICUs of four tertiary care children's hospitals included children (2-18 years) with SAA admitted between 2017 and 2019. Children were randomized to receive either a loading dose IV salbutamol (15 mcg/kg, max. 750 mcg) or normal saline while on continuous salbutamol infusion. The primary outcome was the asthma score (Qureshi) 1 h after the intervention. Analysis of covariance models was used to evaluate sensitivity to change in asthma scores. Serum concentrations of salbutamol were obtained. Fifty-eight children were included (29 in the intervention group). Median baseline asthma score was 12 (IQR 10-13) in the intervention group and 11 (9-12) in the control group (p = 0.032). The asthma score 1 h after the intervention did not differ significantly between the groups (p = 0.508, β-coefficient = 0.283). The median increase in salbutamol plasma levels 10 min after the intervention was 13 μg/L (IQR 5-24) in the intervention group and 4 μg/L (IQR 0-7) in the control group (p = 0.001). Side effects were comparable between both groups.

Extracellular vesicles (EVs) are tissue-specific particles containing valuable diagnostic information. However, single EV analysis in blood is challenging due to their physical properties, the molecular complexity of plasma, and a lack of robust data interpretation methods. We assess the applicability of our recently-developed calibrated Imaging Flow Cytometry (IFCM)-based methodology to detect/characterize circulating tissue-specific EV subsets in the clinical setting of kidney transplantation. Platelet-poor plasma was generated from 36 HLA-A3 mismatched donor (HLA-A3 +) and kidney transplant recipients (KTRs; HLA-A3-). Samples taken before transplantation, 3 days, 7 days, and 6 months after transplantation as well as before 'for-cause' kidney transplant biopsies were stained with anti-CD9 (plasma EV-marker) and anti-HLA-A3. Before transplantation, no significant differences in total CD9 + EV concentrations were detected between donor and KTR samples. Tissue-specific EVs were identified as CD9 + HLA-A3 + . Serial dilution experiments of HLA-A3 + in HLA-A3- PPP showed that single CD9 + HLA-A3 + EVs were detectable down to ~ 1% above the recipient 'self-signal'. After transplantation, CD9 + HLA-A3 + EVs were detected above pre-transplantation concentrations in individuals with stable allograft function, but not in individuals with allograft dysfunction. These results demonstrate the applicability of our calibrated IFCM-based methodology in the direct detection of tissue-specific EV subsets in clinical samples. We believe that this EV methodology is applicable in a variety of clinical contexts.

Antipsychotic drugs are an important part of the treatment of irritability and aggression in children with an autism spectrum disorder (ASD). However, significant weight gain and metabolic disturbances are clinically relevant side effects of antipsychotic use in children. In the SPACe study, we showed positive correlations between both risperidone and aripiprazole plasma trough concentrations and weight gain over a 6-month period. The trial SPACe 2: STAR is designed as a follow-up study, in which we aim to research whether therapeutic drug monitoring in clinical practice can prevent severe weight gain, while retaining clinical effectiveness.

There is an unmet clinical need for immunotherapeutic strategies that specifically target the active immune cells participating in the process of rejection after solid organ transplantation. The monocyte-macrophage cell lineage is increasingly recognized as a major player in acute and chronic allograft immunopathology. The dominant presence of cells of this lineage in rejecting allograft tissue is associated with worse graft function and survival. Monocytes and macrophages contribute to alloimmunity via diverse pathways: antigen processing and presentation, costimulation, pro-inflammatory cytokine production, and tissue repair. Cross talk with other recipient immune competent cells and donor endothelial cells leads to amplification of inflammation and a cytolytic response in the graft. Surprisingly, little is known about therapeutic manipulation of the function of cells of the monocyte-macrophage lineage in transplantation by immunosuppressive agents. Although not primarily designed to target monocyte-macrophage lineage cells, multiple categories of currently prescribed immunosuppressive drugs, such as mycophenolate mofetil, mammalian target of rapamycin inhibitors, and calcineurin inhibitors, do have limited inhibitory effects. These effects include diminishing the degree of cytokine production, thereby blocking costimulation and inhibiting the migration of monocytes to the site of rejection. Outside the field of transplantation, some clinical studies have shown that the monoclonal antibodies canakinumab, tocilizumab, and infliximab are effective in inhibiting monocyte functions. Indirect effects have also been shown for simvastatin, a lipid lowering drug, and bromodomain and extra-terminal motif inhibitors that reduce the cytokine production by monocytes-macrophages in patients with diabetes mellitus and rheumatoid arthritis. To date, detailed knowledge concerning the origin, the developmental requirements, and functions of diverse specialized monocyte-macrophage subsets justifies research for therapeutic manipulation. Here, we will discuss the effects of currently prescribed immunosuppressive drugs on monocyte/macrophage features and the future challenges.

Monocytes and macrophages play key roles in many disease states, including cellular and humoral rejection after solid organ transplantation (SOT). To suppress alloimmunity after SOT, immunosuppressive drug therapy is necessary. However, little is known about the effects of the immunosuppressive drugs tacrolimus and mycophenolic acid (MPA) on monocyte activation and function. Here, the effect of these immunosuppressants on monocytes was investigated by measuring phosphorylation of three intracellular signaling proteins which all have a major role in monocyte function: p38MAPK, ERK and Akt. In addition, biological functions downstream of these signaling pathways were studied, including cytokine production, phagocytosis and differentiation into macrophages. To this end, blood samples from healthy volunteers were spiked with diverse concentrations of tacrolimus and MPA. Tacrolimus (200 ng/ml) inhibited phosphorylation of p38MAPK by 30% (mean) in CD14+ monocytes which was significantly less than in activated CD3+ T cells (max 60%; p < 0.05). This immunosuppressive agent also partly inhibited p-AKT (14%). MPA, at a therapeutic concentration showed the strongest effect on p-AKT (27% inhibition). p-ERK was inhibited with a maximum of 15% after spiking with either tacrolimus or MPA. The production of IL-1β and phagocytosis by monocytes were not affected by tacrolimus concentrations, whereas MPA did inhibit IL-1β production by 50%. Monocyte/macrophage polarization was shifted to an M2-like phenotype in the presence of tacrolimus, while MPA increased the expression of M2 surface markers, including CD163 and CD200R, on M1 macrophages. These results show that tacrolimus and MPA do not strongly affect monocyte function, apart from a change in macrophage polarization, to a clinically relevant degree.