Sofia Christakoudi

Biomarkers of Tolerance in Kidney Transplantation: Are We Predicting Tolerance or Response to Immunosuppressive Treatment?

We and others have previously described signatures of tolerance in kidney transplantation showing the differential expression of B cell–related genes and the relative expansions of B cell subsets. However, in all of these studies, the index group—namely, the tolerant recipients—were not receiving immunosuppression (IS) treatment, unlike the rest of the comparator groups. We aimed to assess the confounding effect of these regimens and develop a novel IS‐independent signature of tolerance. Analyzing gene expression in three independent kidney transplant patient cohorts (232 recipients and 14 tolerant patients), we have established that the expression of the previously reported signature was biased by IS regimens, which also influenced transitional B cells. We have defined and validated a new gene expression signature that is independent of drug effects and also differentiates tolerant patients from healthy controls (cross‐validated area under the receiver operating characteristic curve [AUC] = 0.81). In a prospective cohort, we have demonstrated that the new signature remained stable before and after steroid withdrawal. In addition, we report on a validated and highly accurate gene expression signature that can be reliably used to identify patients suitable for IS reduction (approximately 12% of stable patients), irrespective of the IS drugs they are receiving. Only a similar approach will make the conduct of pilot clinical trials for IS minimization safe and hence allow critical improvements in kidney posttransplant management.

Sodium ascorbate improves yield of urinary steroids during hydrolysis with Helix pomatia juice.

Urinary steroid profile analysis requires enzymatic hydrolysis of glucuronide and sulfate conjugates and this is achieved simultaneously using Helix pomatia juice (HPJ), but steroids with 3beta-hydroxy-5-ene structure undergo transformation and yield of 5alpha-reduced corticosteroid glucuronides is poor. We describe the use of sodium ascorbate to solve these problems and provide a basis for its mode of action. Steroid conjugates were extracted from urine, hydrolyzed in acetate buffer with HPJ and sodium ascorbate and analyzed as methyloxime-trimethylsilylether derivatives by gas chromatography-mass spectrometry. Ranges of temperature, pH and ascorbate, substrate and HPJ concentrations were compared for urine and pure standards. Activity of other antioxidants and that of bacterial cholesterol oxidase were examined. Helix pomatia enzyme preparations from different commercial sources were compared. Loss of 3beta-hydroxy-5-ene steroids was enzyme-dependant, since it required HPJ, was saturable, subject to substrate competition and heat-inactivated. Products were 3-oxo-4-ene steroids and 4,6-diene and 6-oxy derivatives of these but the latter were not formed from 3-oxo-4-ene precursors. Ascorbate, other antioxidants or oxygen exclusion diminished activity. These characteristics were shared by cholesterol oxidase. Yield of 5alpha-reduced steroids was diminished by pre-incubation of HPJ before ascorbate addition and this was reversed if ascorbate was added to the pre-incubation mixture. We conclude that transformation of 3beta-hydroxy-5-ene steroids by HPJ is due to cholesterol oxidase and is diminished by antioxidants or oxygen denial. Yield of 5alpha-reduced steroids is low due to oxidative damage of beta-glucuronidase during hydrolysis, prevented by ascorbate. These features are shared by most commercial Helix pomatia enzyme preparations tested.

Steroids excreted in urine by neonates with 21-hydroxylase deficiency: characterization, using GC-MS and GC-MS/MS, of the D-ring and side chain structure of pregnanes and pregnenes.

Steroid metabolites in urine from neonates with 21-hydroxylase deficiency are predominantly polyhydroxylated 17-hydroxyprogesterone and androgen metabolites, and most have incompletely defined structure. This study forms part of a comprehensive project to characterize and identify these in order to enhance diagnosis and to further elucidate neonatal types of steroid metabolism. Steroids were analyzed, after extraction and enzymatic conjugate hydrolysis, as methyloxime-trimethylsilyl ether derivatives on gas-chromatographs coupled to quadrupole and ion-trap mass-spectrometers. GC-MS and GC-MS/MS spectra, obtained with constant excitation conditions, were used together to determine the structure of the D-ring and the side chain of 20-oxo and 20-hydroxy pregnane(ene)s without oxo groups on the A-, B-, and C-ring. All possible combinations of D-ring and side chain configuration were considered. Most fragmentations could be interpreted as partial or complete D-ring cleavages with loss of the side chain, aided by comparison with spectra of deuterated derivatives and of borohydride reduced metabolites. Possible rearrangement ions are also discussed. More than 140 endogenous metabolites were characterized. GC-MS/MS was especially beneficial for characterization of compounds with 16,17-dihydroxy-20-oxo structure, interpreted as markers of intra-uterine enzyme induction. It also assisted the differentiation of 16-hydroxy-20-oxo metabolites, present in urine of non-affected neonates, from the diagnostic 17-hydroxy-20-oxosteroids and enabled the detection of 15,17-dihydroxy-20-oxo compounds in low concentrations. The presence of 17,21-dihydroxylated pregnane(ene)s despite the deficit in CYP21A2 is discussed. We conclude that GC-MS combined with GC-MS/MS allows reliable identification of the structure of the D-ring and side chain of pregnane(ene)s without prior isolation, even when in low concentrations in urine.

Hepatic Infiltrates in Operational Tolerant Patients After Liver Transplantation Show Enrichment of Regulatory T Cells Before Proinflammatory Genes Are Downregulated.

Immunosuppression can be discontinued from selected and stable patients after liver transplantation resulting in spontaneous operational tolerance (SOT), although the underlying mechanisms remain elusive. Thus, we analyzed serial liver biopsy specimens from adult liver recipients enrolled in a prospective multicenter immunosuppression withdrawal trial that used immunophenotyping and transcriptional profiling. Liver specimens were collected before the initiation of weaning, at the time of rejection, or at 1 and 3 years after complete drug discontinuation. Unexpectedly, the tolerated grafts developed portal tract expansion with increased T cell infiltration after immunosuppression withdrawal. This was associated with transient and preferential accumulation of CD4+FOXP3+ cells and a trend toward upregulation of immune activation and regulatory genes, without signs of rejection. At the same time, no markers of endothelial damage or activation were noted. Portal infiltrates persisted at 3 years but were characterized by decreased expression of genes associated with chronic immunological damage. Further, SOT was not associated with a progressive liver fibrosis up to 5 years. These data suggest that SOT involves several mechanisms: a long‐lasting local immune cell persistence with a transient regulatory T cells accumulation followed by a downregulation of immune‐activated genes over years. These results have important implications for designs and follow‐up of weaning trials.

Steroids excreted in urine by neonates with 21-hydroxylase deficiency. 2. Characterization, using GC–MS and GC–MS/MS, of pregnanes and pregnenes with an oxo- group on the A- or B-ring ☆

Urine from neonates with 21-hydroxylase deficiency contains a large range of metabolites of 17-hydroxyprogesterone, 21-deoxycortisol and androgens but few have been previously described. We present the second part of a comprehensive project to characterize and identify these in order to enhance diagnosis and to further elucidate neonatal steroid metabolism. Steroids were analyzed, after extraction and enzymatic conjugate hydrolysis, as methyloxime-trimethylsilyl ether derivatives on gas-chromatographs coupled to quadrupole and ion-trap mass-spectrometers. GC-MS and GC-MS/MS spectra were used together to determine the structure of the A- and B-rings containing an oxo group. Fragmentations indicating presence of 3-, 6-, and 7-oxo groups and also 1β-, 2α-, 4β-, and 6β-hydroxyls are presented and discussed for the first time. Interpretation was aided by comparison with spectra of available relevant standards, of oxidation products of standards and urinary metabolites and of deuterated derivatives. Endogenous 1-enes and 2(3)-ene artifacts of non-hydrolyzed 3α-sulfates are also reported. D-ring and side chain structure was determined according to our previously published criteria. Likely metabolic relationships were also explored. We conclude that GC-MS combined with GC-MS/MS allows identification of the A- and B-ring structure of pregnane and pregnenes in the presence of an oxo group on one of these rings. Major oxygenations are 1β, 15β, 16α and 21-hydroxy and 6- and 7-oxo groups. Minor positions of hydroxylation are those at 2α, 4β and 6β. Three major metabolic streams exist in affected neonates in addition to the classical 3α-hydroxy-5β-pregnane pathway, i.e. these of the 3-oxo-4-enes as well as 3α- and 3β-hydroxy-5α-anes.

IL-2 therapy restores regulatory T-cell dysfunction induced by calcineurin inhibitors

Significance Preservation and/or enhancement of Treg function is becoming a key component of modern immunotherapeutic strategies, but the direct influence of many immunosuppressive drugs on Tregs remains unknown. Calcineurin inhibitors (CNIs), which are widely used to treat inflammatory disorders, reduce the size of the Treg pool substantially, and this reduction might hinder their overall beneficial effects. Here we show that the decrease in Treg numbers is caused by increased cell death as a result of the limited availability of the IL-2 growth factor. Hence, the addition of IL-2 restores the survival and suppressive properties of Tregs exposed to CNIs and improves allograft survival. Our data provide a strong rationale for combining CNIs with IL-2 therapy to maximize effective immunosuppression and to promote tolerance acquisition. CD4+CD25+FOXP3+ Tregs constitute a heterogeneous lymphocyte subpopulation essential for curtailing effector T cells and establishing peripheral tolerance. Calcineurin inhibitors (CNIs) are among the most effective agents in controlling effector T-cell responses in humans. However, CNIs also reduce the size of the Treg pool. The functional consequences of this negative effect and the mechanisms responsible remain to be elucidated. We report here that CNIs compromise the overall Treg immunoregulatory capacity to a greater extent than would be predicted by the reduction in the size of the Treg compartment, given that they selectively promote the apoptosis of the resting and activated Treg subsets that are known to display the most powerful suppressive function. These effects are caused by reduced access to IL-2, because Tregs remain capable of translocating NFAT even in the presence of high CNI levels. Exogenous IL-2 restores the phenotypic changes and overall gene-expression effects exerted by CNIs and can even promote Treg expansion by enhancing antiapoptotic Bcl-2 expression. In a skin transplant model, the addition of IL-2 synergizes with CNIs treatment, promoting intragraft accumulation of Tregs and prolonged allograft survival. Hence, the combination of IL-2 and CNIs constitutes an optimal immunomodulatory regimen that enhances the pool of suppressive Treg subsets while effectively controlling cytopathic T cells.

Steroids excreted in urine by neonates with 21-hydroxylase deficiency. 4. Characterization, using GC-MS and GC-MS/MS, of 11oxo-pregnanes and 11oxo-pregnenes.

In 21-hydroxylase deficiency, urinary metabolites of 21-deoxycortisol, mainly derived from its 11oxo form 21-deoxycortisone, are indicators of intra-adrenal overproduction of 17-hydroxyprogesterone. In affected neonates these metabolites are numerous and most have not been previously described. This work forms the concluding part of a comprehensive study of urinary steroids, aiming to enhance the diagnosis of this disorder and to further elucidate steroid metabolism in neonates. Cortisol metabolites found in untreated patients, similarly almost exclusively present in their 11oxo form in neonates, have been included for completeness. Steroids were analyzed, after extraction and enzymatic conjugate hydrolysis, as methyloxime-trimethylsilyl ether derivatives on gas-chromatographs coupled to quadrupole and ion-trap mass-spectrometers. GC-MS and GC-MS/MS spectra were used together to determine the structure of hitherto undescribed 11oxo-pregnane(enes). Few GC-MS features were associated with the presence of the non-derivatizeable 11oxo group in pregnane(ene)s. GC-MS/MS contributed only to the characterization of structures outside the C-ring, as described in the preceding parts of this study. Parallels were found between the metabolism of 21-deoxycortisone and cortisone. The major metabolic pathway was that of classical 3α,5β-reduction with a prominent further hydroxylation, predominantly at C6. Oxidation of the 6-hydroxyl was also common. We conclude that further oxygenated metabolites of 21-deoxycortisone have potential as more reliable markers of 21-hydroxylase deficiency in the early neonatal period, because their levels are higher during that period of life than for the classical marker 11oxo-pregnanetriol.

Steroids excreted in urine by neonates with 21-hydroxylase deficiency. 3. Characterization, using GC-MS and GC-MS/MS, of androstanes and androstenes

Urine from neonates with 21-hydroxylase deficiency contains a large range of androstane(ene)s, many of which have not been previously described. We present their characterization as the third part of a comprehensive study of urinary steroids, aiming to enhance the diagnosis of this disorder and to further elucidate steroid metabolism in neonates. Steroids were analyzed, after extraction and enzymatic conjugate hydrolysis, as methyloxime-trimethylsilyl ether derivatives on gas-chromatographs coupled to quadrupole and ion-trap mass-spectrometers. GC-MS and GC-MS/MS spectra were used together to determine the structure of hitherto undescribed androstane(ene)s. GC-MS/MS was pivotal for the structural characterization of 2-hydroxylated androstenediones but GC-MS was generally more informative for androstane(ene)s, in contrast to 17-hydroxylated pregnane(ene)s. Parallels were found between the GC-MS and GC-MS/MS characteristics of structurally similar androstenediones and progesterones without a substituent on the D-ring, but not with those of 17-hydroxylated progesterones. Assignment of 5α(β) orientation, based on GC-MS characteristics, was possible for 11-oxo-androstanes. The major endogenous 3β-hydroxy-5-enes in 21-hydroxylase deficiency did not differ from those in unaffected neonates. The key qualitative and quantitative differences encompassed 5α(β)-androstanes and 3-oxo-androst-4-enes. Major positions of hydroxylation in these were C(2), C(6), C(11), C(16) and C(18). Additional oxo-groups were common at C(6), C(11) and C(16). We conclude that the presence of multiple further oxygenated metabolites of androstenedione in urine from neonates with 21-hydroxylase deficiency and their pattern indicate a predominance of the classical pathway of androgen synthesis and reflect an increased demand for clearance. The positions of oxygenation in androstane(ene)s are dependent on the configuration at C(3)-C(5).

Reduced Tcr Signaling Contributes to Impaired Th17 Responses in Tolerant Kidney Transplant Recipients

Background The development of spontaneous kidney transplant tolerance has been associated with numerous B cell–related immune alterations. We have previously shown that tolerant recipients exhibit reduced B-cell receptor signalling and higher IL-10 production than healthy volunteers. However, it is unclear whether cluster of differentiation (CD)4+ T cells from tolerant recipients also display an anti-inflammatory profile that could contribute to graft maintenance. Methods CD4+ T cells were isolated from kidney transplant recipients who were identified as being tolerant recipients, patients with chronic rejection or healthy volunteers. CD4+ T cells from the 3 groups were compared in terms of their gene expression profile, phenotype, and functionally upon activation. Results Gene expression analysis of transcription factors and signalling proteins, in addition to surface proteins expression and cytokine production, revealed that tolerant recipients possessed fewer Th17 cells and exhibited reduced Th17 responses, relative to patients with chronic rejection or healthy volunteers. Furthermore, impaired T-cell receptor signalling and altered cytokine cooperation by monocytes contributed to the development of Th17 cells in tolerant recipients. Conclusions These data suggest that defective proinflammatory Th17 responses may contribute to the prolonged graft survival and stable graft function, which is observed in tolerant recipients in the absence of immunosuppressive agents.