Lucrezia Furian

Evaluation of Cytomegalovirus (CMV)-Specific T Cell Immune Reconstitution Revealed That Baseline Antiviral Immunity, Prophylaxis, or Preemptive Therapy but not Antithymocyte Globulin Treatment Contribute to CMV-Specific T Cell Reconstitution in Kidney Transplant Recipients

BACKGROUND The ultimate goal of organ transplantation is the reestablishment of organ function and the restoration of a solid immunity to prevent the assault of potentially deadly pathogens. T cell immunity is crucial in controlling cytomegalovirus (CMV) infection. It is still unknown how preexisting antiviral T cell levels, prophylaxis, or preemptive antiviral strategies and pharmacological conditioning affect immune reconstitution. METHODS Seventy preemptively treated CMV-seropositive recipients, 13 prophylaxis-treated CMV-seronegative recipients of seropositive donor transplants, 2 seropositive recipients of seronegative donor kidneys, and 27 pretransplant subjects were enrolled in a cross-sectional study and analyzed for CMV viremia (DNAemia) and CMV-specific T cell response (interferon-gamma enzyme-linked immunospot assay) before transplantation and at 30, 60, 90, 180, and 360 days after transplantation. RESULTS CMV-seropositive transplant recipients displayed a progressive but heterogeneous pattern of immune reconstitution starting from day 60 after transplantation. CMV-seronegative recipients did not mount a detectable T cell response throughout the prophylaxis regimen. A single episode of CMV viremia (CMV copy number, 7000-170,000 copies/mL) was sufficient to prime a protective T cell immune response in CMV-seronegative recipients. Antithymocyte globulin treatment did not significantly affect CMV-specific T cell response. CONCLUSIONS Baseline immunity, antiviral therapy but not antithymocyte globulin treatments profoundly influence T cell reconstitution in kidney transplant recipients.

Technical Aspects of Unilateral Dual Kidney Transplantation from Expanded Criteria Donors: Experience of 100 Patients

One option for using organs from donors with a suboptimal nephron mass, e.g. expanded criteria donors (ECD) kidneys, is dual kidney transplantation (DKT). In adult recipients, DKT can be carried out by several techniques, but the unilateral placement of both kidneys (UDKT) offers the advantages of single surgical access and shorter operating time. One hundred UDKT were performed using kidneys from ECD donors with a mean age of 72 years (Group 1). The technique consists of transplanting both kidneys extraperitoneally in the same iliac fossa. The results were compared with a cohort of single kidney transplants (SKT) performed with the same selection criteria in the same study period (Group 2, n = 73). Ninety‐five percent of UDKTs were positioned in the right iliac fossa, lengthening the right renal vein with an inferior vena cava patch. In 69% of cases, all anastomoses were to the external iliac vessels end‐to‐side. Surgical complications were comparable in both groups. At 3‐year follow‐up, patient and graft survival rates were 95.6 and 90.9% in Group 1, respectively. UDKT can be carried out with comparable surgical complication rates as SKT, leaving the contralateral iliac fossa untouched and giving elderly recipients a better chance of receiving a transplant, with optimal results up to 3‐years follow‐up.

Comparison of Cytomegalovirus (CMV) Enzyme-Linked Immunosorbent Spot and CMV Quantiferon Gamma Interferon-Releasing Assays in Assessing Risk of CMV Infection in Kidney Transplant Recipients

ABSTRACT Assessing cytomegalovirus (CMV)-specific cell-mediated immunity (CMI) represents an appealing strategy for identifying transplant recipients at risk of infection. In this study, we compared two gamma interferon-releasing assays (IGRAs), Quantiferon-CMV and CMV enzyme-linked immunosorbent spot (ELISPOT), to determine the ability of each test to predict protective CMV-specific T-cell responses. Two hundred twenty-one Quantiferon-CMV and ELISPOT tests were conducted on 120 adult kidney transplant recipients (KTRs), including 100 CMV-seropositive transplant recipients (R+) and 20 CMV-seronegative transplant recipients of a CMV-positive donor (D+/R−). As a control cohort, 39 healthy adult subjects (including 33 CMV-seropositive and 6 CMV-seronegative subjects) were enrolled. CMV IgG serology was used as a reference for both tests. In the CMV-seropositive individuals, the ELISPOT and Quantiferon-CMV assays provided 46% concordance with the serology, 12% discordance, 18% disagreement between ELISPOT or Quantiferon-CMV and the serology, and 24% gray areas when one or both tests resulted in weak positives. None of the CMV-seronegative subjects showed detectable responses in the ELISPOT or the Quantiferon-CMV test. In transplant recipients, both the ELISPOT and Quantiferon-CMV assays positively correlated with each other and negatively correlated with CMV DNAemia in a significant way (P < 0.05). During the antiviral prophylaxis, all 20 D+/R− KTRs we examined displayed undetectable Quantiferon-CMV and ELISPOT results, and there was no evidence of CMV seroconversion. The receiving operator curve (ROC) statistical analysis revealed similar specificities and sensitivities in predicting detectable viremia (areas under the curve [AUC], 0.66 and 0.62 for Quantiferon-CMV and ELISPOT, respectively). ELISPOT and Quantiferon-CMV values of >150 spots/200,000 peripheral blood mononuclear cells (PBMCs) and >1 to 6 IU gamma interferon (IFN-γ) were associated with protection from CMV infection (odds ratios [OR], 5 and 8.75, respectively). In transplant recipients, the two tests displayed similar abilities for predicting CMV infection. Both the ELISPOT and Quantiferon-CMV assays require several ameliorations to avoid false-negative results.

Perforin, Granzyme B, and fas ligand for molecular diagnosis of acute renal-allograft rejection: analyses on serial biopsies suggest methodological issues.

Background. The Perforin-Granzyme B and Fas/Fas Ligand apoptotic mechanisms are involved in the development of acute renal rejection (AR). We describe our experience of analyzing the expression of cytotoxic T-lymphotoxins (CTL) in biopsies and peripheral blood leukocytes (PBL) for the diagnosis of AR. Methods. We analyzed Perforin (P), Granzyme B (GB) and Fas Ligand (FL) expression in 68 renal biopsies and 64 PBL using comparative kinetic RT-PCR and, for GAPDH and FL, we also replicated with real-time RT-PCR. The levels of expression were measured in different groups, such as T0 (biopsies before reperfusion and PBL in recipient before the transplant [Tx]), Td (biopsies and PBL collected for clinical purposes) and Tp (biopsies and PBL two months after Tx). Results. A higher CTL expression was seen in nonrejecting (NR) biopsies in the first 2 months after Tx. P and FL were significantly more expressed during AR in all biopsies and in Td, while P remained upregulated in Tp. In PBL, there was no significant increase in CTL transcription during AR. A variable expression of CTL emerged in all T0 biopsies. Conclusions. Two lytic pathways are activated in biopsies when AR occurs shortly after Tx, whereas the P/GB mechanism prevails if it occurs later on. Only P and FL in biopsies might be able to predict AR diagnosis, but with a considerable variability in each sample, possibly due to the small portion of tissue core, which may be inadequate for molecular diagnosis. CTL expression in PBL does not correlate with histological AR.

Plasma fibrinolytic capacity in renal transplant recipients: Effect of steroid-free immunosuppression therapy

Background. Cardiovascular disease is the most common cause of death among renal transplant recipients (RTRs). Impaired fibrinolytic capacity caused by an increase in plasminogen activator inhibitor type 1 (PAI-1) levels is involved in the onset of atherosclerosis and thrombotic complications. Long-term steroid treatment may induce arterial hypertension and metabolic and prothrombotic changes (including up-regulation of PAI-1 synthesis), which increase the cardiovascular risk. We evaluated plasma fibrinolytic behavior in two groups of RTRs treated with different immunosuppressive regimens. Methods. Twenty-seven RTRs were randomized to receive long-term (17 patients) or perioperative short-term (10 patients) steroids in addition to immunosuppression with cyclosporine A plus everolimus (Certican; Novartis, Basel, Switzerland) (7 patients) or FK506 plus mycophenolate mofetil (20 patients). In each patient, fibrinolytic capacity was studied with the 20-min venous occlusion test 1 and 6 months after transplantation. The following were assayed: euglobulin lysis time, tissue-type plasminogen activator antigen, and PAI-1 antigen and activity. Results. One month after transplantation, a severe impairment of fibrinolytic capacity, mainly caused by an increase in PAI-1 antigen and activity levels, was seen in patients with and without steroid treatment. Six months after transplantation, an improvement in fibrinolytic potential as the result of a decrease in PAI-1 levels was observed only in patients without steroid therapy. None of the steroid-treated patients demonstrated PAI-1 values correlating with body mass index, blood pressure, and metabolic parameters, thus confirming the effect of exogenous factors on PAI-1 expression. Moreover, all patients revealed a slight impairment of stimulated endothelial tissue-type plasminogen activator release, regardless of any steroid treatment, which was probably attributable to calcineurin inhibitor-induced endothelial dysfunction. Conclusions. Our study suggests that steroid-free immunosuppression is associated with a better fibrinolytic capacity in RTRs. This finding may contribute toward reducing the risk of cardiovascular events.

Calcineurin inhibitor‐free immunosuppression in dual kidney transplantation from elderly donors

Abstract:  Background:  Kidneys from expanded‐criteria donors may be particularly susceptible to calcineurin inhibitor (CI)‐mediated vasoconstriction and nephrotoxicity. In the early post‐transplant phase, using CI may prolong ischemic injury and, in the long term, chronic CI nephrotoxicity is an even greater concern. To avoid the acute and chronic consequences of CI in kidneys from marginal donors, CI‐free protocols have been introduced for maintenance immunosuppressive therapy. A CI‐free protocol of anti‐thymocyte globulin (ATG) induction, sirolimus, mycophenolate mofetil (MMF) and steroids has been adopted at our center in recipients of dual kidney transplantation (DKT) from elderly donors (EDs).

Acute rejection and graft survival in renal transplanted patients with viral diseases

Transplanted patients are susceptible to viral infections; thus, the aim of this study was to evaluate the features of acute rejections and the outcome of the renal graft in transplanted patients with herpes virus diseases. Renal biopsies from 30 renal transplanted patients undergoing early acute rejection (type IA and IB according to the Banff 97 classification) were evaluated. In total, 15 of these patients experienced cytomegalovirus (CMV) or Epstein–Barr virus disease within the first year following transplantation (group I) and 15 patients showed no evidence of viral infection (group II). No significant differences between the groups in terms of age, male/female ratio, living/cadaveric donor ratio, cold ischemia time, HLA A-B matching, pretransplant panel reactive antibody test, occurrence of post-transplant tubular necrosis, plasma levels of cyclosporin A and mean percent increase of serum creatinine at the time of the biopsy were observed. In group I biopsies, the mean number of interstitial plasma cells, as well as the mean number of CD79a-positive cells (B lymphocytes and plasma cells) was significantly higher than in group II (P<0.01 and <0.01, respectively). There was a positive correlation between the number of infections and the number of plasma cells (P<0.05). In transplanted patients, CMV can trigger the formation of anti-endothelial cell antibodies, which have been proposed to play a role in antibody-mediated rejections. We investigated whether a deposition of C4d, a marker of antibody-mediated reactions, was present in renal peritubular capillaries. In group I C4d deposition was found in five cases, while in group II it was not observed (P<0.05). In group I, 7/15 patients developed chronic allograft nephropathy vs 1/15 patients in group II (P<0.05). The estimated 1-, 5- and 8-year cumulative graft survival rates were 80, 66 and 57%, respectively, in group I, while in group II the estimated 8-year cumulative survival rate was 100% (P<0.05). In conclusion, acute rejection biopsies of patients with viral infections displayed plasma cell infiltrates and, in several cases, C4d deposition. Our study suggests a role of B lymphocytes in the pathology of these rejections and confirms the association between viral infections and poor graft survival.

Monolateral placement of both kidneys in dual kidney transplantation: low surgical complication rate and short operating time.

Dual kidney transplantation (DKT) from marginal donors is increasingly used at many centers to help cope with the organ shortage problem. The disadvantages of DKT consist in longer operating times and the risk of surgical complications. DKT can be performed in two ways, i.e. using monolateral or bilateral procedures. From October 1999 to June 2005, 58 DKTs were performed at our unit. In 29 cases (group I), the kidneys were extraperitoneally placed bilaterally in the iliac fossae via two separate incisions; as of June 2003, monolateral kidney placement was preferred in 29 cases, whenever compatible with the recipients morphological status (group II). After a mean follow‐up of 51 ± 19 months for group I and 15 ± 7 months for group II, all patients are alive with 1‐year graft survival rates of 93% and 96%, respectively. Mean operating times were 351 ± 76 min in group I and 261 ± 31 min in group II (P = 0.0001). The mean S‐creatinine levels in groups I and II were 132 ± 47 and 119 ± 36 μmol/l, respectively, at 1 year. We observed eight surgical complications in group I and seven in group II. Both techniques proved safe, with no differences in surgical complication rates. The monolateral procedure has the advantage of a shorter operating time and the contralateral iliac fossa remains available for further retransplantation procedures.

A single-center experience with 200 dual kidney transplantations

This study reports on a large series of 200 dual kidney transplantations (DKTs) from expanded criteria donors (ECDs) and proposes specific ways to optimize outcomes. Data concerning 200 DKTs performed in the last 14 yr were retrospectively analyzed. Kidneys from high‐risk ECD were allocated for use in DKTs on an old‐for‐old basis after histological assessment. Different surgical techniques and immunosuppressant regimens were used over time, and the outcomes are discussed. Donors and recipients were a median 73 (70–77) and a 62 (58–67) yr old, respectively. Delayed graft function occurred in 31.5% of cases, and acute rejection in 13.5%. Patient and graft survival at five yr were 90.4% and 85.8%, respectively. Unilateral kidney placement was preferred for 75% of patients, and was associated with a low rate of surgical complications. Our current standard therapy comprising low‐dose calcineurin inhibitors (CNIs) associated with mammalian target of rapamycin inhibitors (mTOR) and steroids appears to offer the best risk/benefit profile for elderly patients undergoing DKT. In our experience, outcomes after DKT can be improved by: (i) kidney clinical–histological assessment; (ii) unilateral kidney placement; (iii) minimal use of CNI associated with mTOR.

Early activation of fibrogenesis in transplanted kidneys: A study on serial renal biopsies

BACKGROUND/AIMS In kidney transplants, the renin-angiotensin system (RAS) is involved in systemic and local changes that may induce fibrosis. Our aim was to use gene expression and immunohistochemical analysis to investigate the RAS and several factors involved in the fibrogenic cascade in allograft biopsies. METHODS We considered 43 donor biopsies (T0), 18 biopsies obtained for diagnostic purposes (Td) and 24 protocol biopsies (Tp) taken 2 months after transplantation in patients with stable renal function. Morphometric alpha SMA and TGF beta 1 analysis, and Massons Trichrome staining were performed. mRNA levels of angiotensinogen, renin, ACE, AT1-R, AT2-R, TGF beta 1, BMP-7, Coll III, fibronectin and alpha SMA were analyzed by real-time RT/PCR. MDRD a year after the transplant was also considered. RESULTS Significantly higher levels of AT1-R and alpha SMA transcripts were found in Tp than in T0. Regression analysis showed significant TGF beta 1-independent positive correlations between RAS and matrix components in T0 and Tp, but more evident in Tp, where a positive correlation between TGF beta 1 and Massons Trichrome stained areas was also seen. CONCLUSION Our results suggest that RAS and TGF beta 1-related fibrogenic loops are activated as early as 2 months after kidney transplantation.