Aims of this study were to investigate gadolinium (Gd) in kidney tissue from a female patient with severe renal failure, who had a magnetic resonance imaging (MRI) with Gd-based contrast agent (GBCA) ...three times prior to kidney transplantation. Secondly to assess (semi-)quantitatively the Gd concentration in renal tissue and the spatial distribution of Gd in association to suspected co-elements such as calcium (Ca) and zinc (Zn). Archival paraffin embedded kidney tissue was investigated by micro Synchrotron X-ray fluorescence (µSRXRF) at the DORIS III storage ring at beamline L, HASYLAB/DESY(Hamburg, Germany). Elementary gadolinium (Gd) could be demonstrated in a near histological resolution in areas of about 2 × 1.5 mm
2
of size. Mean Gd resulted in 200 ppm with a huge width of distribution (Gd-max: 2000 ppm). In kidney cortex Gd was in-homogeneously, but not randomly, distributed. Gd was verified throughout the investigated tissue. Low Gd was predominately concentrated either in areas with focally atrophic tubules or in areas with totally preserved uriniferous tubes. Moreover, strong correlations existed between Gd and calcium (Ca) or Gd and zinc (Zn) or Gd and strontium (Sr) distribution. Throughout our analysed areas copper (Cu) was nearly homogeneously distributed and Cu association to Gd could not be established, and also not for Gd to Fe. Gd in glomeruli was relatively reduced compared with mean Gd-values, while iron (Fe) distribution clearly demarks glomeruli mostly due to red blood cell iron in these capillary convolutes. Quantitative µSRXRF analysis provided an insight in element spatial distribution of Gd in the renal cortex. The strong correlation of the spatial distribution and associations between elements like Ca, Zn and Sr let us suspect that these elements are involved in the cell metabolism of GBCA. Low Gd in areas with extreme fibrosis and tubule atrophy or in areas with histologically intact tubes, let us suspect that on the one side Gd cannot be transported and deposited into these tissue areas and on the other side we assume that intact renal tubes do not reabsorb and store excreted Gd.
Late antibody-mediated rejection (ABMR) is a leading cause of kidney allograft failure. Uncontrolled studies have suggested efficacy of the proteasome inhibitor bortezomib, but no systematic trial ...has been undertaken to support its use in ABMR. In this randomized, placebo-controlled trial (the Bortezomib in Late Antibody-Mediated Kidney Transplant Rejection BORTEJECT Trial), we investigated whether two cycles of bortezomib (each cycle: 1.3 mg/m
intravenously on days 1, 4, 8, and 11) prevent GFR decline by halting the progression of late donor-specific antibody (DSA)-positive ABMR. Forty-four DSA-positive kidney transplant recipients with characteristic ABMR morphology (median time after transplant, 5.0 years; pretransplant DSA documented in 19 recipients), who were identified on cross-sectional screening of 741 patients, were randomly assigned to receive bortezomib (
=21) or placebo (
=23). The 0.5-ml/min per 1.73 m
per year (95% confidence interval, -4.8 to 5.8) difference detected between bortezomib and placebo in eGFR slope (primary end point) was not significant (
=0.86). We detected no significant differences between bortezomib- and placebo-treated groups in median measured GFR at 24 months (33 versus 42 ml/min per 1.73 m
;
=0.31), 2-year graft survival (81% versus 96%;
=0.12), urinary protein concentration, DSA levels, or morphologic or molecular rejection phenotypes in 24-month follow-up biopsy specimens. Bortezomib, however, associated with gastrointestinal and hematologic toxicity. In conclusion, our trial failed to show that bortezomib prevents GFR loss, improves histologic or molecular disease features, or reduces DSA, despite significant toxicity. Our results reinforce the need for systematic trials to dissect the efficiency and safety of new treatments for late ABMR.
Late antibody-mediated rejection (ABMR) is a leading cause of transplant failure. Blocking IL-6 has been proposed as a promising therapeutic strategy.
We performed a phase 2 randomized pilot trial to ...evaluate the safety (primary endpoint) and efficacy (secondary endpoint analysis) of the anti-IL-6 antibody clazakizumab in late ABMR. The trial included 20 kidney transplant recipients with donor-specific, antibody-positive ABMR ≥365 days post-transplantation. Patients were randomized 1:1 to receive 25 mg clazakizumab or placebo (4-weekly subcutaneous injections) for 12 weeks (part A), followed by a 40-week open-label extension (part B), during which time all participants received clazakizumab.
Five (25%) patients under active treatment developed serious infectious events, and two (10%) developed diverticular disease complications, leading to trial withdrawal. Those receiving clazakizumab displayed significantly decreased donor-specific antibodies and, on prolonged treatment, modulated rejection-related gene-expression patterns. In 18 patients, allograft biopsies after 51 weeks revealed a negative molecular ABMR score in seven (38.9%), disappearance of capillary C4d deposits in five (27.8%), and resolution of morphologic ABMR activity in four (22.2%). Although proteinuria remained stable, the mean eGFR decline during part A was slower with clazakizumab compared with placebo (-0.96; 95% confidence interval 95% CI, -1.96 to 0.03 versus -2.43; 95% CI, -3.40 to -1.46 ml/min per 1.73 m
per month, respectively,
=0.04). During part B, the slope of eGFR decline for patients who were switched from placebo to clazakizumab improved and no longer differed significantly from patients initially allocated to clazakizumab.
Although safety data indicate the need for careful patient selection and monitoring, our preliminary efficacy results suggest a potentially beneficial effect of clazakizumab on ABMR activity and progression.
Acute kidney injury (AKI) remains a major clinical event with high mortality rates. We previously identified renal miR-182 as the main driver of post-transplantation AKI. Therefore, we tested the ...causal inference of miR-182 by inhibiting its renal expression in vivo . In 45 rats AKI was induced by right nephrectomy and contralateral clamping of the renal pedicle for 40 minutes. Systemically administered antisense oligonucleotide (ASO) inhibited miR-182 in the kidneys up to 96 hours. The maximum creatinine elevation was on day 2 after injury (ASO 2.5 mg/kg: median, 1.9 mg/dL interquartile range (IQR), 1.3 to 3.2 mg/dL; ASO 25 mg/kg: median, 2.8 mg/dL IQR, 0.7 to 5.0 mg/dL; mismatch oligonucleotide (MM) 25 mg/kg: median, 5.7 mg/dL IQR, 5.0 to 5.8 mg/dL; saline: 4.4 mg/dL IQR, 3.5 to 5.8 mg/dL; P = 0.016, analysis of variance). Blinded semiquantitative histologic evaluation of renal biopsies showed better preserved morphology in both ASO groups than saline- and MM- treated kidneys in overall injury scores, ASO both concentrations: median, 1 (IQR, 1 to 1); saline: median, 3 (IQR, 3 to 3); MM: median, 3 (IQR, 3 to 3); P < 0.001, analysis of variance. ASO facilitated cell proliferation, metabolism, and angiogenesis on a genome-wide level. ASO when applied in normothermic kidney machine perfusion reduced renal miR-182 expression by more than two magnitudes. In summary, we showed that in vivo inhibition of miR-182 by ASO improved kidney function and morphology after AKI. This technique may be applicable to reduce the high rate of AKI in the human renal transplantation setting.
Abstract The efficacy of costimulation blockade with CTLA4-Ig (belatacept) in transplantation is limited due to T cell-mediated rejection, which also persists after induction with anti-thymocyte ...globulin (ATG). Here, we investigate why ATG fails to prevent costimulation blockade-resistant rejection and how this barrier can be overcome. ATG did not prevent graft rejection in a murine heart transplant model of CTLA4-Ig therapy and induced a pro-inflammatory cytokine environment. While ATG improved the balance between regulatory T cells (Treg) and effector T cells in the spleen, it had no such effect within cardiac allografts. Neutralizing IL-6 alleviated graft inflammation, increased intragraft Treg frequencies, and enhanced intragraft IL-10 and Th2-cytokine expression. IL-6 blockade together with ATG allowed CTLA4-Ig therapy to achieve long-term, rejection-free heart allograft survival. This beneficial effect was abolished upon Treg depletion. Combining ATG with IL-6 blockade prevents costimulation blockade-resistant rejection, thereby eliminating a major impediment to clinical use of costimulation blockers in transplantation.
Abstract
Background
Rapid histologic diagnosis of frozen sections is essential for a variety of surgical procedures. Frozen sections however, require specialized lab equipment, are prone to freezing ...artifacts and are not applicable to all types of tissue. Adipose tissue is especially difficult to process in frozen sections. Although these limitations are well known, no alternative method for microscopic tissue analysis that might replace frozen sections could be established. Our objective was to evaluate whether tissue imaging based on ex vivo fluorescent confocal microscopy (FCM) is applicable for rapid microscopic assessment of breast tumors specimens with abundant adipose tissue.
Methods
We evaluated 17 tissue samples from mastectomy specimens, rich in adipose tissue, submitted to the department of pathology at the Medical University of Vienna. We conducted our study on the FCM VivaScope® 2500M-G4 (Mavig GmbH, Munich, Germany; Caliber I.D.; Rochester NY, USA).
Results
When comparing FCM to frozen sections, we found a very similar overall processing time for FCM images and frozen sections respectively. Image quality was mostly superior to frozen sections (especially for adipose tissue and nuclear detail) but inferior to H&E stained FFPE sections. Limitations of the technology were uneven coloring, invisibility of ink applied for marking tissue margins and distortion artifacts if too much pressure is applied to the tissue.
Conclusion
FCM has the potential to expand the application and usefulness of rapid tissue analysis as speed is comparable and quality exceeds that of frozen sections especially in tissues rich in adipose cells such as breast specimen.
Increasing evidence accumulates on the central involvement of microRNAs (miRNAs) in disease pathophysiology. We identified distinctly deregulated miRNAs in human renal allograft biopsies from ...patients undergoing acute cellular rejection, antibody-mediated rejection (ABMR), and delayed graft function (DGF).
Sixty-five posttransplantation kidney biopsy samples covering 41 cases with acute rejection (15 vascular rejection, 15 interstitial rejection, and 11 ABMR), 14 DGF cases, and 10 protocol biopsies serving as controls were analyzed using the Affymetrix GeneChip miRNA Array. Differentially regulated miRNAs were identified by Student's t test and Bonferroni correction. Target genes for the set of miRNAs were retrieved from miRTarBase (experimentally verified targets) as well as by applying the target prediction routines DIANAmT, miRanda, and Targetscan.
Patients with acute cellular rejection, ABMR, and DGF discriminate from the control group (protocol biopsies) in unsupervised clustering of miRNA profiles, clearly identifying deregulated miRNAs in rejection and DGF. Angiogenesis, apoptosis, and transforming growth factor-β signaling were identified as relevant pathways in ischemic response following an integrative analysis of miRNA targets and mRNA expression profiles. Inflammation by chemokine and cytokine signaling, T-cell activation, and B-cell activation were identified as relevant in acute rejection accordingly.
These data suggest that distinct miRNA signatures playing a role in specific biological pathways discriminate acute cellular and humoral rejection and DGF. This finding serves as valuable tool for a rational selection of diagnostic, prognostic, and potentially therapeutic molecular targets of posttransplantation events.
In
kidney transplantation, the CTLA4-Ig fusion protein belatacept is associated with improved graft function but also an increased risk of acute rejection compared to calcineurin inhibitor therapy. ...The combination with a second costimulation blocker could potentially improve outcome while avoiding calcineurin inhibitor toxicity. The aim of this study was to define the conditions under which the combination of CTLA4-Ig and CD40L blockade leads to rejection-free permanent graft survival in a stringent murine heart transplantation model.
Naïve wild-type or CD40L (CD154) knock-out mice received a fully mismatched BALB/c cardiac allograft. Selected induction and maintenance protocols for CTLA4-Ig and blocking αCD40L monoclonal antibodies (mAB) were investigated. Graft survival, rejection severity and donor-specific antibody (DSA) formation were assessed during a 100-day follow-up period.
Administering αCD40L mAb as monotherapy at the time of transplantation significantly prolonged heart allograft survival but did not further improve the outcome when given in addition to chronic CTLA4-Ig therapy (which prolongs graft survival to a median of 22 days). Likewise, chronic αCD40L mAb therapy (0.5mg) combined with perioperative CTLA4-Ig led to rejection in a proportion of mice and extensive histological damage, despite abrogating DSA formation. Only the permanent interruption of CD40-CD40L signaling by using CD40L
recipient mice or by chronic αCD40L administration synergized with chronic CTLA4-Ig to achieve long-term allograft survival with preserved histological graft integrity in all recipients without DSA formation. The combination of α-CD40L and CTLA4-Ig works most effectively when both therapeutics are administered chronically.
Aims: This study aimed to investigate gadolinium (Gd) and bio-metals in a renal allograft of a patient who was shortly after transplantation repeatedly exposed to a Gd-based contrast agent (GBCA), ...with the purpose of determining whether Gd can be proven and spatially and quantitatively imaged. Further elemental associations between Gd and bio-metals were also investigated. Materials and Methods: Archival paraffin-embedded kidney tissue (eight weeks after transplantation) was investigated by microscopic synchrotron X-ray fluorescence (µSRXRF) at the DORIS III storage ring, beamline L, at HASYLAB/DESY (Hamburg, Germany). For the quantification of elements, X-ray spectra were peak-fitted, and the net peak intensities were normalized to the intensity of the incoming monochromatic beam intensity. Concentrations were calculated by fundamental parameter-based program quant and external standardization. Results: Analysis of about 15,000 µSRXRF spectra (comprising allograft tissue of four cm2) Gd distribution could be quantitatively demonstrated in a near histological resolution. Mean Gd resulted in 24 ± 55 ppm with a maximum of 2363 ppm. The standard deviation of ±55 ppm characterized the huge differences in Gd and not in detection accuracy. Gd was heterogeneously but not randomly distributed and was mostly found in areas with interstitial fibrosis and tubular atrophy. Concentrations of all other investigated elements in the allograft resembled those found in normal kidney tissue. No correlations between Gd and bio-metals such as calcium, strontium or zinc below ~40 ppm Gd existed. In areas with extremely high Gd, Gd was associated with iron and zinc. Conclusions: We could show that no dose-dependent association between Gd and bio-metals exists—least in renal tissue—at Gd concentrations below ~40 ppm Gd. This was proven compared with a GBCA-exposed end-stage renal failure in which the mean Gd was ten-fold higher. Our results could shed additional light on Gd metabolism.
Transcript analyses highlight an important contribution of natural killer (NK) cells to microvascular inflammation (MVI) in antibody-mediated rejection (ABMR), but only few immunohistologic studies ...have quantified their spatial distribution within graft tissue. This study included 86 kidney transplant recipients who underwent allograft biopsies for a positive donor-specific antibody (DSA) result. NK cells were visualized and quantified within glomeruli and peritubular capillaries (PTC), using immunohistochemistry for CD34 alongside CD16/T-bet double-staining. Staining results were analyzed in relation to histomorphology, microarray analysis utilizing the Molecular Microscope Diagnostic System, functional NK cell genetics, and clinical outcomes. The number of NK cells in glomeruli per mm 2 glomerular area (NK glom ) and PTC per mm 2 cortical area (NK PTC ) was substantially higher in biopsies with ABMR compared to those without rejection, and correlated with MVI scores (NK glom Spearman’s correlation coefficient SCC = 0.55, p < 0.001, NK PTC 0.69, p < 0.001). In parallel, NK cell counts correlated with molecular classifiers reflecting ABMR activity (ABMR prob : NK glom 0.59, NK PTC 0.75) and showed a trend towards higher levels in association with high functional FCGR3A and KLRC2 gene variants. Only NK PTC showed a marginally significant association with allograft function and survival. Our immunohistochemical results support the abundance of NK cells in DSA-positive ABMR.
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