mTOR inhibitors improve both humoral and cellular response to SARS-CoV-2 messenger RNA BNT16b2 vaccine in kidney transplant recipients.

Kidney transplant recipients (KTRs) have been considered as patients at higher risk of SARS-CoV-2-related disease severity, thus COVID-19 vaccination was highly recommended. However, possible interferences of different immunosuppression with development of both humoral and T cell-mediated immune response to COVID-19 vaccination have not been determined. Here we evaluated the association between mTOR-inhibitors (mTOR-I) and immune response to mRNA BNT162b2 (Pfizer-BioNTech) vaccine in KTR. To this aim 132 consecutive KTR vaccinated against COVID-19 in the early 2021 were enrolled, and humoral and T cell-mediated immune response were assessed after 4-5 weeks. Patients treated with mTOR-I showed significantly higher anti-SARS-CoV-2 IgG titer (p = .003) and higher percentages of anti-SARS-CoV-2 S1/RBD Ig (p = .024), than those without. Moreover, SARS-CoV-2-specific T cell-derived IFNγ release was significantly increased in patients treated with mTOR-I (p < .001), than in those without. Multivariate analysis confirmed that therapy with mTOR-I gained better humoral (p = .005) and T cell-mediated immune response (p = .005) in KTR. The presence of mTOR-I is associated with a better immune response to COVID-19 vaccine in KTR compared to therapy without mTOR-I, not only by increasing vaccine-induced antibodies but also by stimulating anti-SARS-CoV-2 T cell response. These finding are consistent with a potential beneficial role of mTOR-I as modulators of immune response to COVID-19 vaccine in KTR.

CD40 Cross-Linking Induces Migration of Renal Tumor Cell through Nuclear Factor of Activated T Cells (NFAT) Activation.

CD40 crosslinking plays an important role in regulating cell migration, adhesion and proliferation in renal cell carcinoma (RCC). CD40/CD40L interaction on RCC cells activates different intracellular pathways but the molecular mechanisms leading to cell scattering are not yet clearly defined. Aim of our study was to investigate the main intracellular pathways activated by CD40 ligation and their specific involvement in RCC cell migration. CD40 ligation increased the phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun NH (2)-terminal kinase (JNK) and p38 MAPK. Furthermore, CD40 crosslinking activated different transcriptional factors on RCC cell lines: AP-1, NFkB and some members of the Nuclear Factor of Activated T cells (NFAT) family. Interestingly, the specific inhibition of NFAT factors by cyclosporine A, completely blocked RCC cell motility induced by CD40 ligation. In tumor tissue, we observed a higher expression of NFAT factors and in particular an increased activation and nuclear migration of NFATc4 on RCC tumor tissues belonging to patients that developed metastases when compared to those who did not. Moreover, CD40-CD40L interaction induced a cytoskeleton reorganization and increased the expression of integrin ß1 on RCC cell lines, and this effect was reversed by cyclosporine A and NFAT inhibition. These data suggest that CD40 ligation induces the activation of different intracellular signaling pathways, in particular the NFATs factors, that could represent a potential therapeutic target in the setting of patients with metastatic RCC.

Lysine 63 ubiquitination is involved in the progression of tubular damage in diabetic nephropathy.

The purpose of our study was to evaluate how hyperglycemia (HG) influences Lys63 protein ubiquitination and its involvement in tubular damage and fibrosis in diabetic nephropathy (DN). Gene and protein expression of UBE2v1, a ubiquitin-conjugating E2-enzyme variant that mediates Lys63-linked ubiquitination, and Lys63-ubiquitinated proteins increased in HK2 tubular cells under HG. Matrix-assisted laser desorption/ionization-time of flight/tandem mass spectrometry identified 30 Lys63-ubiquitinated proteins, mainly involved in cellular organization, such as ß-actin, whose Lys63 ubiquitination increased under HG, leading to cytoskeleton disorganization. This effect was reversed by the inhibitor of the Ubc13/UBE2v1 complex NSC697923. Western blot analysis confirmed that UBE2v1 silencing in HK2 under HG, restored Lys63-ß-actin ubiquitination levels to the basal condition. Immunohistochemistry on patients with type 2 diabetic (T2D) revealed an increase in UBE2v1- and Lys63-ubiquitinated proteins, particularly in kidneys of patients with DN compared with control kidneys and other nondiabetic renal diseases, such as membranous nephropathy. Increased Lys63 ubiquitination both in vivo in patients with DN and in vitro, correlated with α-SMA expression, whereas UBE2v1 silencing reduced HG-induced α-SMA protein levels, returning them to basal expression. In conclusion, UBE2v1- and Lys63-ubiquitinated proteins increase in vitro under HG, as well as in vivo in T2D, is augmented in patients with DN, and may affect cytoskeleton organization and influence epithelial-to-mesenchymal transition. This process may drive the progression of tubular damage and interstitial fibrosis in patients with DN.-Pontrelli, P., Conserva, F., Papale, M., Oranger, A., Barozzino, M., Vocino, G., Rochetti, M. T., Gigante, M., Castellano, G., Rossini, M., Simone, S., Laviola, L., Giorgino, F., Grandaliano, G., Di Paolo, S., Gesualdo, L. Lysine 63 ubiquitination is involved in the progression of tubular damage in diabetic nephropathy.

Inflammation induces osteoclast differentiation from peripheral mononuclear cells in chronic kidney disease patients: crosstalk between the immune and bone systems.

Background: Inflammation and immune system alterations contribute to bone damage in many pathologies by inducing the differentiation of osteoclasts (OCs), the bone resorbing cells. This link is largely unexplored in chronic kidney disease (CKD) and haemodialysis (HD) patients, in which reduced renal function is accompanied by an increased inflammatory state and skeletal abnormality. Methods: We used ex vivo culture experiments to investigate the osteoclastogenic potential of peripheral blood mononuclear cells (PBMCs) of CKD and HD patients, focusing on immune cell subsets and inflammatory cytokines such as LIGHT and receptor activator of nuclear factor κB ligand (RANKL). Results: We observed spontaneous osteoclastogenesis with a significant increase in OC formation and bone resorbing activity in late-stage CKD and HD patients when compared with early-stage CKD patients and healthy donors, likely due to an increased expression of RANKL and LIGHT (homologous to Lymphotoxins exhibiting Inducible expression and competing with herpes simplex virus Glycoprotein D for herpes virus entry mediator [HVEM], a receptor expressed by T lymphocytes) in PBMCs. Specific inhibition of these cytokines in PBMCs isolated from CKD stages 3b-5 and HD patients induced the reduction of OC formation in vitro. The phenotypic characterization of peripheral blood cells revealed a significant increase of OC precursors (CD14+CD11b+CD51/61+) and CD14+CD16+ monocytes in advanced CKD and HD patients compared with the control group. Conclusions: Our results suggest that circulating inflammatory monocytes from advanced CKD or HD patients trans differentiate into OCs in vitro and play a relevant role in mineral bone disorders and that LIGHT and RANKL represent new potential therapeutic targets in these settings.

Neutrophil-dependent pentraxin-3 and reactive oxygen species production modulate endothelial dysfunction in haemodialysis patients.

BACKGROUND: The aim of this study was to investigate neutrophil activation and its role in long pentraxin-3 (PTX3) release and oxidative stress generation during haemodialysis (HD) and to correlate neutrophil PTX3 and oxidant expression with endothelial dysfunction. METHODS: Forty-seven uraemic patients on stable HD, 12 healthy subjects and 15 patients with congestive heart failure (New York Heart Association classes III and IV) were enrolled. Neutrophil PTX3 protein expression was evaluated by confocal microscopy. l -selectin expression, intracellular PTX3 localization and reactive oxygen species (ROS) generation in human neutrophils were measured by flow cytometry. NADPH-dependent superoxide generation was investigated by chemiluminescence. PTX3 plasma concentrations were measured by ELISA. Endothelial dysfunction was studied by flow-mediated dilation (FMD). RESULTS: The low baseline levels of FMD significantly improved after HD, but worsened by 24 h. A significant up-regulation of PTX3 protein expression, localized within secondary granules, was detected in neutrophils isolated at 30 and 240 min of HD, along with an increase in l -selectin expression. The up-regulation in intracellular PTX3 in neutrophils was associated with a significant increase in PTX3 plasma concentrations at 240 min. HD increased ROS production and NADPH oxidase activity in neutrophils. In a univariate analysis, pre-treatment with FMD was inversely correlated with PTX3 expression and ROS generation in neutrophils. In a multivariate analysis, both circulating pre-HD PTX3 and intracellular ROS generation by neutrophils were independent predictors of abnormal FMD. CONCLUSIONS: Neutrophil overexpression of PTX3 is associated with ROS overproduction and endothelial dysfunction and may represent an emerging marker of vascular damage progression in HD patients.

miR-29b and miR-198 overexpression in CD8+ T cells of renal cell carcinoma patients down-modulates JAK3 and MCL-1 leading to immune dysfunction.

BACKGROUND: Mammalian microRNAs (miR) regulate the expression of genes relevant for the development of adaptive and innate immunity against cancer. Since T cell dysfunction has previously been reported in patients with renal cell carcinoma (RCC; clear cell type), we aimed to analyze these immune cells for genetic and protein differences when compared to normal donor T cells freshly after isolation and 35 days after in vitro stimulation (IVS) with HLA-matched RCC tumor cells. METHODS: We investigated gene expression profiles of tumor-reactive CD8(+) T cells obtained from RCC patient and compared with their HLA-matched healthy sibling donors using a microarray approach. In addition, miRNAs analysis was performed in a validation cohort of peripheral blood CD8(+) T cells from 25 RCC patients compared to 15 healthy volunteers. RESULTS: We observed that CD8(+) T cells from RCC patients expressed reduced levels of anti-apoptotic and proliferation-associated gene products when compared with normal donor T cells both pre- and post-IVS. In particular, JAK3 and MCL-1 were down-regulated in patient CD8(+) T cells versus their normal counterparts, likely due to defective suppressor activity of miR-29b and miR-198 in RCC CD8(+) T cells. Indeed, specific inhibition of miR-29b or miR-198 in peripheral blood mononuclear cells (PBMCs) isolated from RCC patients, resulted in the up-regulation of JAK3 and MCL-1 proteins and significant improvement of cell survival in vitro. CONCLUSIONS: Our results suggest that miR-29b and miR-198 dysregulation in RCC patient CD8(+) T cells is associated with dysfunctional immunity and foreshadow the development of miR-targeted therapeutics to correct such T cell defects in vivo.

A type I interferon signature characterizes chronic antibody-mediated rejection in kidney transplantation.

Chronic antibody-mediated rejection (CAMR) represents the main cause of kidney graft loss. To uncover the molecular mechanisms underlying this condition, we characterized the molecular signature of peripheral blood mononuclear cells (PBMCs) and, separately, of CD4(+) T lymphocytes isolated from CAMR patients, compared to kidney transplant recipients with normal graft function and histology. We enrolled 29 patients with biopsy-proven CAMR, 29 stable transplant recipients (controls), and 8 transplant recipients with clinical and histological evidence of interstitial fibrosis/tubular atrophy. Messenger RNA and microRNA profiling of PBMCs and CD4(+) T lymphocytes was performed using Agilent microarrays in eight randomly selected patients per group from CAMR and control subjects. Results were evaluated statistically and by functional pathway analysis (Ingenuity Pathway Analysis) and validated in the remaining subjects. In PBMCs, 45 genes were differentially expressed between the two groups, most of which were up-regulated in CAMR and were involved in type I interferon signalling. In the same patients, 16 microRNAs were down-regulated in CAMR subjects compared to controls: four were predicted modulators of six mRNAs identified in the transcriptional analysis. In silico functional analysis supported the involvement of type I interferon signalling. To further confirm this result, we investigated the transcriptomic profiles of CD4(+) T lymphocytes in an independent group of patients, observing that the activation of type I interferon signalling was a specific hallmark of CAMR. In addition, in CAMR patients, we detected a reduction of circulating BDCA2(+) dendritic cells, the natural type I interferon-producing cells, and their recruitment into the graft along with increased expression of MXA, a type I interferon-induced protein, at the tubulointerstitial and vascular level. Finally, interferon alpha mRNA expression was significantly increased in CAMR compared to control biopsies. We conclude that type I interferon signalling may represent the molecular signature of CAMR.

Thrombin may modulate dendritic cell activation in kidney transplant recipients with delayed graft function.

BACKGROUND: Coagulation and complement activation represent key events in ischaemia-reperfusion-induced renal injury leading to delayed graft function (DGF). It is still unclear whether the coagulation cascade may also influence the acquired immunity. The aim of the present study was to investigate the expression of protease-activated receptor 1 (PAR-1), the main thrombin receptor, by graft-infiltrating dendritic cells (DCs), and to evaluate whether thrombin may influence DCs complement production and T-cell response. METHODS: PAR-1, BDCA1, CD11c, BDCA4, fibrin, C3c and C3d protein expression were evaluated by confocal microscopy. Cultured DCs were obtained incubating monocytes (Ms) with IL-4 and GM-CSF. DC maturation was obtained with IFN-g+sCD40L or with a cytokine cocktail (IL-1b, TNF-a, PGE2, IL-6). PAR1 protein expression on cultured DC was evaluated by flow-cytometry. Complement receptors, C3, IL12/IL17p40 and IL10 gene expression was evaluated by qPCR. T cell phenotype was evaluated by ELISPOT. IFN-g protein presence was evaluated by ELISA. RESULTS: PAR-1 was expressed by infiltrating myeloid DCs in pre-transplant and in DGF biopsies. In DGF grafts, myeloid DCs localized within fibrin and C3d deposits and expressed C3c. In vitro, PAR-1 protein expression was increased in monocyte-derived immature DCs and in cytokine-induced mature DCs compared to monocytes. PAR-1 activation caused a time-dependent increase in C3 and complement receptors expression. Moreover, thrombin stimulation, while reducing interleukin-10 mRNA abundance, induced interleukin-12/IL-17 p40 gene expression, and promoted C3a ability to increase interleukin-12/IL17 mRNA abundance. These changes in the DCs' cytokine pattern influenced their ability to induce interferon-g production by T cells, suggesting the activation of a T helper-1 bias. CONCLUSION: Our data suggest that PAR-1 is expressed by DCs in DGF grafts and its activation may induce complement production and a Th1 bias. This observation suggests a potential pathogenic link between DGF and acquired allo-response leading to graft damage.

Rapamycin induces ILT3(high)ILT4(high) dendritic cells promoting a new immunoregulatory pathway.

ILT3(high)ILT4(high) dendritic cells (DCs) may cause anergy in CD4(+)CD45RO(+)CD25(+) T cells transforming them into regulatory T cells (Tregs). Here, we tested whether chronic exposure to rapamycin may modulate this immunoregulatory pathway in renal transplant recipients. Forty renal transplant patients with biopsy-proven chronic allograft nephropathy and receiving calcineurin inhibitors were randomly assigned to either calcineurin inhibitor dose reduction or withdrawal with rapamycin introduction. At conversion and 2 years thereafter, we measured the rapamycin effects on circulating DCs (BDCA1/BDCA2 and ILT3/ILT4 expression), CD4(+)/CD25(high)/Foxp3(+) Tregs, CD8(+)/CD28(-) T cells, and the Th1/Th2 balance in graft biopsies. In rapamycin-treated patients, peripheral BDCA2(+) cells were significantly increased along with ILT3/ILT4(+) DCs. The number of circulating CD4(+)/CD25(high)/Foxp3(+)/CTLA4(+) Tregs, CD8(+)CD28(-) T cells, and HLA-G serum levels were higher in the rapamycin-treated group. The number of ILT3/ILT4(+)BDCA2(+) DC was directly and significantly correlated with circulating Tregs and CD8(+)CD28(-) T cells. ILT3/ILT4 expression was increased in kidney biopsies at the end of the study period along with a significant bias toward a Th2 response within the graft only in the rapamycin-treated patients. Thus, rapamycin induces the upregulation of ILT3 and ILT4 on the DC surface, and this effect is associated with an increase in the number of Tregs and expansion of the CD8(+)CD28(-) T cell population. This suggests that mTOR inhibition may promote a novel immunoregulatory pathway.

Endothelial-to-mesenchymal transition and renal fibrosis in ischaemia/reperfusion injury are mediated by complement anaphylatoxins and Akt pathway.

BACKGROUND: Increasing evidence demonstrates a phenotypic plasticity of endothelial cells (ECs). Endothelial-to-mesenchymal transition (EndMT) contributes to the development of tissue fibrosis. However, the pathogenic factors and signalling pathways regulating this process in ischaemia/reperfusion (I/R) injury are still poorly understood. METHODS: We investigated the possible role of complement in the induction of this endothelial dysfunction in a swine model of renal I/R injury by using recombinant C1 inhibitor in vivo. RESULTS: Here, we showed that I/R injury reduced the density of renal peritubular capillaries and induced tissue fibrosis with generation of CD31(+)/α-SMA(+) and CD31(+)/FPS-1(+) cells indicating EndMT. When we inhibited complement, the process of EndMT became rare, with preserved density of peritubular capillaries and significant reduction in renal fibrosis. When we activated ECs by anaphylatoxins in vitro, C3a and C5a led to altered endothelial phenotype with increased expression of fibroblast markers and decrease expression of specific endothelial markers. The activation of Akt pathway was pivotal for the C3a and C5a-induced EndMT in vitro. In accordance, inhibition of complement in vivo led to the abrogation of Akt signalling, with hampered EndMT and tissue fibrosis. CONCLUSIONS: Our data demonstrate a critical role for complement in the acute induction of EndMT via the Akt pathway. Therapeutic inhibition of these systems may be essential to prevent vascular damage and tissue fibrosis in transplanted kidney.

Endothelial dysfunction and renal fibrosis in endotoxemia-induced oliguric kidney injury: possible role of LPS-binding protein.

INTRODUCTION: The pathophysiology of endotoxemia-induced acute kidney injury (AKI) is characterized by an intense activation of the host immune system and renal resident cells by lipopolysaccharide (LPS) and derived proinflammatory products. However, the occurrence of renal fibrosis in this setting has been poorly investigated. The aim of the present study was to investigate the possible association between endothelial dysfunction and acute development of tissue fibrosis in a swine model of LPS-induced AKI. Moreover, we studied the possible effects of coupled plasma filtration adsorption (CPFA) in this setting. METHODS: After 9 hours from LPS infusion and 6 hours of CPFA treatment, histologic and biochemical changes were analyzed in pigs. Apoptosis and endothelial dysfunction were assessed on renal biopsies. The levels of LPS-binding protein (LBP) were quantified with enzyme-linked immunosorbent assay (ELISA). Endothelial cells (ECs) were stimulated in vitro with LPS and cultured in the presence of swine sera and were analyzed with FACS and real-time RT-PCR. RESULTS: In a swine model of LPS-induced AKI, we observed that acute tubulointerstitial fibrosis occurred within 9 hours from LPS injection. Acute fibrosis was associated with dysfunctional alpha-smooth muscle actin (α-SMA)+ ECs characterized by active proliferation (Ki-67+) without apoptosis (caspase-3-). LPS led to EC dysfunction in vitro with significant vimentin and N-cadherin expression and increased collagen I mRNA synthesis. Therapeutic intervention by citrate-based CPFA significantly prevented acute fibrosis in endotoxemic animals, by preserving the EC phenotype in both peritubular capillaries and renal arteries. We found that the removal of LBP from plasma was crucial to eliminate the effects of LPS on EC dysfunction, by blocking LPS-induced collagen I production. CONCLUSIONS: Our data indicate that EC dysfunction might be pivotal in the acute development of tubulointerstitial fibrosis in LPS-induced AKI. Selective removal of the LPS adaptor protein LBP might represent a future therapeutic option to prevent EC dysfunction and tissue fibrosis in endotoxemia-induced AKI.

Osteoclastogenic potential of peripheral blood mononuclear cells in cleidocranial dysplasia.

Cleidocranial dysplasia (CCD) is an autosomal dominant skeletal dysplasia characterized by hypoplastic or aplastic clavicles, dental abnormalities, and delayed closure of the cranial sutures. In addition, mid-face hypoplasia, short stature, skeletal anomalies and osteoporosis are common. We aimed to evaluate osteoclastogenesis in a child (4 years old), who presented with clinical signs of CCD and who have been diagnosed as affected by deletion of RUNX2, master gene in osteoblast differentiation, but also affecting T cell development and indirectly osteoclastogenesis. The results of this study may help to understand whether in this disease is present an alteration in the bone-resorptive cells, the osteoclasts (OCs). Unfractionated and T cell-depleted Peripheral Blood Mononuclear Cells (PBMCs) from patient were cultured in presence/absence of recombinant human M-CSF and RANKL. At the end of the culture period, OCs only developed following the addition of M-CSF and RANKL. Moreover, real-time PCR experiment showed that freshly isolated T cells expressed the osteoclastogenic cytokines (RANKL and TNFα) at very low level, as in controls. This is in accordance with results arising from flow cytometry experiments demonstrating an high percentage of circulating CD4(+)CD28(+) and CD4(+)CD27(+) T cells, not able to produce osteoclastogenic cytokines. Also RANKL, OPG and CTX serum levels in CCD patient are similar to controls, whereas QUS measurements showed an osteoporotic status (BTT-Z score -3.09) in the patient. In conclusions, our findings suggest that the heterozygous deletion of RUNX2 in this CCD patient did not alter the osteoclastogenic potential of PBMCs in vitro.

High dickkopf-1 levels in sera and leukocytes from children with 21-hydroxylase deficiency on chronic glucocorticoid treatment.

Children with 21-hydroxylase deficiency (21-OHD) need chronic glucocorticoid (cGC) therapy to replace congenital deficit of cortisol synthesis, and this therapy is the most frequent and severe form of drug-induced osteoporosis. In this study, we enrolled 18 patients (9 females) and 18 sex- and age-matched controls. We found in 21-OHD patients high serum and leukocyte levels of dickkopf-1 (DKK1), a secreted antagonist of the Wnt/ß-catenin signaling pathway known to be a key regulator of bone mass. In particular, we demonstrated by flow cytometry, confocal microscopy, and real-time PCR that monocytes, T lymphocytes, and neutrophils from patients expressed high levels of DKK1, which may be related to the cGC therapy. In fact, we showed that dexamethasone treatment markedly induced the expression of DKK1 in a dose- and time-dependent manner in leukocytes. The serum from patients containing elevated levels of DKK1 can directly inhibit in vitro osteoblast differentiation and receptor activator of NF-κB ligand (RANKL) expression. We also found a correlation between both DKK1 and RANKL or COOH-terminal telopeptides of type I collagen (CTX) serum levels in 21-OHD patients on cGC treatment. Our data indicated that DKK1, produced by leukocytes, may contribute to the alteration of bone remodeling in 21-OHD patients on cGC treatment.

Activated innate immunity and the involvement of CX3CR1-fractalkine in promoting hematuria in patients with IgA nephropathy.

A hallmark of immunoglobulin A nephropathy (IgAN) is episodes of gross hematuria coinciding with mucosal infections that can represent the disease-triggering event. Here we performed a whole genomic screen of IgAN patients during gross hematuria to clarify the link between mucosal antigens and glomerular hematuria. Modulated genes showed a clear involvement of the intracellular interferon signaling, antigen-presenting pathway, and the immunoproteasome. The mRNA and protein level of the chemokine receptor characterizing cytotoxic effector lymphocytes, CX3CR1, was upregulated. In vitro antigenic stimulation of peripheral blood mononuclear cells from IgAN patients, healthy blood donors, and other nephropathies with microscopic hematuria showed that only in IgAN patients was CX3CR1 enhanced in a dose-dependent manner. A significantly higher amount of glomerular and urinary fractalkine, the only ligand of CX3CR1, was also found in IgAN patients with recurrent episodes of gross hematuria compared with other patients with microscopic or no hematuria. This suggests a predisposition for cytotoxic cell extravasation only in patients with recurrent gross hematuria. Thus, we found a defect in antigen handling in peripheral blood mononuclear cells of IgAN patients with a specific increase of CX3CR1. This constitutive upregulation of glomerular and urinary fractalkine suggests an involvement of the CX3CR1-fractalkine axis in the exacerbation of gross hematuria.

T helper 1, 2 and 17 cell subsets in renal transplant patients with delayed graft function.

Ischemia-reperfusion injury (IRI) in kidney transplantation is the major cause of delayed graft function (DGF), an event associated with an increased risk of acute rejection. The aim of this study was to evaluate T helper (Th) cell phenotype in renal transplants with DGF. T-bet (Th1), GATA-3 (Th2) and IL-17 (Th17) protein expression was investigated in pretransplant biopsies, DGF and acute tubular damage (ATD) caused by calcineurin-inhibitor toxicity. Intracytofluorimetric analysis of IFN-γ, IL-4 and IL-17 was performed to analyze Th1, Th2 and Th17 responses in peripheral blood mononuclear cells of recipients with early graft function (EGF) and DGF, before (T0) and 24 h after transplantation (T24). In pretransplant biopsies, T-bet(+) , GATA-3(+) and IL-17(+) cells were barely detectable. In DGF, T-bet(+) and IL-17(+) cells were significantly increased compared with pretransplant and ATD. More than 90% of T-bet(+) and less then 5% of IL-17(+) cells were CD4(+) . GATA-3(+) cells were increased to a lower extent. T-bet(+) /GATA-3(+) cell ratio was significantly higher in DGF. Peripheral CD4(+) IFN-γ/IL-4 ratio was significantly decreased in DGF, while CD4(+) /IL-17(+) cells did not differ between T0 and T24 in DGF. Our data suggest that DGF is characterized by a prevalent Th1 phenotype within the graft. This event might represent a link between DGF and acute rejection.

Phenotypical and Functional Characterization of Cytotoxic Unconventional T-Cells.

During the last two decades, the immunology field has been focused on the study of conventional T-cells, leading to important advances in identification of specific subsets involved in promoting and suppressing immune response in patients with cancer, autoimmune disease or transplanted patients. In these recent years, the research on unconventional subset of CD4-CD8- double-negative T-cells is growing. DNTs are a unique subset of T-cells characterized by being CD4-CD8-CD3+ and express either ß or α T-cell receptors (TCR). In this chapter, we describe the methods used to phenotypically characterize and isolate these cells in order to study their functional profile.

CTL ELISPOT Assay and T Cell Detection.

Enzyme-linked immune absorbent spot (Elispot) is a quantitative method for measuring relevant parameters of T-cell activation. The sensitivity of Elispot allows the detection of low-frequency antigen-specific T-cells that secrete cytokines and effector molecules, such as granzyme B and perforin. Cytotoxic T-cell (CTL) studies have taken advantage with this high-throughput technology by providing insights of quantity and immune kinetics. Accuracy, sensitivity, reproducibility, and robustness of Elispot resulted in a wide range of applications in research as well as in diagnostic field. Actually, CTL monitoring by Elispot is a gold standard for the evaluation of antigen-specific T-cell immunity in clinical trials and vaccine candidates where the ability to detect rare antigen-specific T-cells is of relevance for immune diagnostic. The most utilized Elispot assay is the Interferon-gamma (IFN-γ) test, a marker for CD8+ CTL activation, but Elispot can be also used to distinguish different subsets of activated T-cells by using other cytokines such as T-helper (Th) 1 type cells (characterized by the production of IFN-γ, IL-2, IL-6, IL-12, IL-21 and TNF-α), Th2 (producing cytokines like IL-4, IL-5, IL-10 and IL-13), and Th17 (IL-17) cells.The reliability of Elispot generated data, by the evaluation of T-cell frequency recognizing individual antigen/peptide, is the core of this method currently applied widely to investigate specific immune responses in cancer, infections, allergies, and autoimmune diseases. The Elispot Assay is competing with other methods measuring single-cell cytokine production, e.g., intracellular cytokine by FACS or Milteny cytokine secretion assay. Other types of lymphocyte frequency and function assays include limiting dilution assay (LDA), cytotoxic T-cell assay (CTL), and tetramer staining. Compared with respect to sensitivity the Elispot Assay is outranking other methods to define frequency of antigen-specific lymphocytes. The method described herein would like to offer helpful and clear protocols for researchers that apply Elispot. IFN-γ and Perforin Elispot assays will be described.

The Ambivalent Role of miRNAs in Carcinogenesis: Involvement in Renal Cell Carcinoma and Their Clinical Applications.

The analysis of microRNA (miRNAs), small, non-coding endogenous RNA, plays a crucial role in oncology. These short regulatory sequences, acting on thousands of messenger RNAs (mRNAs), modulate gene expression at the transcriptional and post-transcriptional level leading to translational repression or degradation of target molecules. Although their function is required for several physiological processes, such as proliferation, apoptosis and cell differentiation, miRNAs are also responsible for development and/or progression of several cancers, since they may interact with classical tumor pathways. In this review, we highlight recent advances in deregulated miRNAs in cancer focusing on renal cell carcinoma (RCC) and provide an overview of the potential use of miRNA in their clinical settings, such as diagnostic and prognostic markers.

JAK3/STAT5/6 pathway alterations are associated with immune deviation in CD8 T cells in renal cell carcinoma patients.

To investigate the molecular mechanisms underlying altered T cell response in renal cell carcinoma (RCC) patients, we compared autologous and allogeneic CD8(+) T cell responses against RCC line from RCC patients and their HLA-matched donors, using mixed lymphocyte/tumor cell cultures (MLTCs). In addition, we analyzed the expression of molecules associated with cell cycle regulation. Autologous MLTC responder CD8(+) T cells showed cytotoxic activity against RCC cell lines; however the analysis of the distribution of CD8(+) T-cell subsets revealed that allogenic counterparts mediate superior antitumor efficacy. In RCC patients, a decreased proliferative response to tumor, associated with defects in JAK3/STAT5/6 expression that led to increased p27KIP1 expression and alterations in the cell cycle, was observed. These data define a molecular pathway involved in cell cycle regulation that is associated with the dysfunction of tumor-specific CD8(+) effector cells. If validated, this may define a therapeutic target in the setting of patients with RCC.

[Renal and upper urinary tract tumors: what the nephrologist should know]. / Tumori del rene e della via urinaria: cosa deve sapere il nefrologo.

Urinary tract carcinomas are among the most common malignancies and are derived from neoplastic transformation of the urothelium. They can be located in the lower (bladder, urethra) or upper (pyelocaliceal cavities, ureter) urinary tract. Urothelial carcinomas are the fourth most common cancer type after prostate or breast cancer, lung cancer, and colorectal cancer. Bladder cancer accounts for 90-95% of urothelial carcinomas and it is the most common malignancy of the urinary tract. Renal cancer also belongs to the urothelial carcinomas and is a relatively uncommon solid tumor, accounting for about 3% of all adult malignancies, although its incidence is on the rise. The most common histological subtype, renal cell carcinoma (RCC), is a clear cell carcinoma that makes up approximately 70-80% of all renal neoplasms and appears to be the only histological subtype that is partially responsive to immunotherapeutic approaches. The current review gives an overview of upper urinary tract tumors and renal cancer, in particular RCC, highlighting issues related to its molecular pathogenesis and the new immunotherapeutic approaches.