Josep Maria Cruzado

Subclinical rejection associated with chronic allograft nephropathy in protocol biopsies as a risk factor for late graft loss

Chronic allograft nephropathy (CAN) in protocol biopsies is associated with graft loss while the association between subclinical rejection (SCR) and outcome has yielded contradictory results. We analyze the predictive value of SCR and/or CAN in protocol biopsies on death‐censored graft survival. Since 1988, a protocol biopsy was done during the first 6 months in stable grafts with serum creatinine <300 μmol/L and proteinuria <1 g/day. Biopsies were evaluated according to Banff criteria. Borderline changes and acute rejection were grouped as SCR. CAN was defined as presence of interstitial fibrosis and tubular atrophy. Mean follow‐up was 91 ± 46 months. Sufficient tissue was obtained in 435 transplants. Biopsies were classified as normal (n = 186), SCR (n = 74), CAN (n = 110) and SCR with CAN (n = 65). Presence of SCR with CAN was associated with old donors, percentage of panel reactive antibodies and presence of acute rejection before protocol biopsy. Cox regression analysis showed that SCR with CAN (relative risk [RR]: 1.86, 95% confidence interval [CI]: 1.11–3.12; p = 0.02) and hepatitis C virus (RR: 2.27, 95% CI: 1.38–3.75; p = 0.01) were independent predictors of graft survival. In protocol biopsies, the detrimental effect of interstitial fibrosis/tubular atrophy on long‐term graft survival is modulated by SCR.

Steroid withdrawal in mycophenolate mofetil-treated renal allograft recipients

BACKGROUND Acute rejection is an inherent risk of the withdrawal of steroids in renal allograft recipients. Mycophenolate mofetil is a potent immunosuppressant that, when given with cyclosporine (CsA), reduces the incidence of acute rejection and may facilitate discontinuation of steroids without increasing the risk of rejection. METHODS In an open pilot study, steroids were withdrawn from 26 adult cadaveric kidney transplant recipients. Corticosteroids were discontinued between 4 and 30 (mean 17) months after transplantation, and steroid-free follow-up ranged from 7 to 18 (mean 10) months. RESULTS Mean CsA doses, CsA blood levels, and serum creatinine at the time of steroid withdrawal and at last patient visit after cessation of steroids were 4.2+/-1.2 mg/kg/day and 3+/-0.8 mg/kg/day (P<0.001), 170+/-53 ng/ml and 113+/-34 ng/ml (P<0.001), and 133+/-36 microM/L and 130+/-37 microM/L (NS), respectively. No rejection episodes occurred after steroid withdrawal. CONCLUSIONS This open study shows that corticosteroids can be safely and successfully withdrawn from renal allograft recipients receiving CsA and mycophenolate mofetil.

Cross-validation of IFN-γ Elispot assay for measuring alloreactive memory/effector T cell responses in renal transplant recipients.

Assessment of donor‐specific alloreactive memory/effector T cell responses using an IFN‐γ Elispot assay has been suggested to be a novel immune‐monitoring tool for evaluating the cellular immune risk in renal transplantation. Here, we report the cross‐validation data of the IFN‐γ Elispot assay performed within different European laboratories taking part of the EU RISET consortium. For this purpose, development of a standard operating procedure (SOP), comparisons of lectures of IFN‐γ plates assessing intra‐ and interlaboratory assay variability of allogeneic or peptide stimuli in both healthy and kidney transplant individuals have been the main objectives. We show that the use of a same SOP and count‐settings of the Elispot bioreader allow low coefficient variation between laboratories. Frozen and shipped samples display slightly lower detectable IFN‐γ frequencies than fresh samples. Importantly, a close correlation between different laboratories is obtained when measuring high frequencies of antigen‐specific primed/memory T cell alloresponses. Interestingly, significant high donor‐specific alloreactive T cell responses can be similarly detected among different laboratories in kidney transplant patients displaying histological patterns of acute T cell mediated rejection. In conclusion, assessment of circulating alloreactive memory/effector T cells using an INF‐γ Elispot assay can be accurately achieved using the same SOP, Elispot bioreader and experienced technicians in kidney transplantation.

Pretransplant Immediately Early-1-Specific T Cell Responses Provide Protection for CMV Infection After Kidney Transplantation

Cytomegalovirus (CMV) infection is still a major complication after kidney transplantation. Although cytotoxic CMV‐specific T cells play a crucial role controlling CMV survival and replication, current pretransplant risk assessment for CMV infection is only based on donor/recipient (IgG)‐serostatus. Here, we evaluated the usefulness of monitoring pre‐ and 6‐month CMV‐specific T cell responses against two dominant CMV antigens (IE‐1 and pp65) and a CMV lysate, using an IFN‐γ Elispot, for predicting the advent of CMV infection in two cohorts of 137 kidney transplant recipients either receiving routine prophylaxis (n = 39) or preemptive treatment (n = 98). Incidence of CMV antigenemia/disease within the prophylaxis and preemptive group was 28%/20% and 22%/12%, respectively. Patients developing CMV infection showed significantly lower anti‐IE‐1‐specific T cell responses than those that did not in both groups (p < 0.05). In a ROC curve analysis, low pretransplant anti‐IE‐1‐specific T cell responses predicted the risk of both primary and late‐onset CMV infection with high sensitivity and specificity (AUC > 0.70). Furthermore, when using most sensitive and specific Elispot cut‐off values, a higher than 80% and 90% sensitivity and negative predictive value was obtained, respectively. Monitoring IE‐1‐specific T cell responses before transplantation may be useful for predicting posttransplant risk of CMV infection, thus potentially guiding decision‐making regarding CMV preventive treatment.

Risk Factors and Outcomes of Bacteremia Caused by Drug-Resistant ESKAPE Pathogens in Solid-Organ Transplant Recipients

Background Although infections due to the six ESKAPE pathogens have recently been identified as a serious emerging problem, information regarding bacteremia caused by these organisms in solid-organ transplant (SOT) recipients is lacking. We sought to determine the frequency, risk factors, and outcomes of bacteremia due to drug-resistant ESKAPE (rESKAPE) organisms in liver, kidney, and heart adult transplant recipients. Methods All episodes of bacteremia prospectively documented in hospitalized SOT recipients from 2007 to 2012 were analyzed. Results Of 276 episodes of bacteremia, 54 (19.6%) were due to rESKAPE strains (vancomycin-resistant Enterococcus faecium [0], methicillin-resistant Staphylococcus aureus [5], extended-spectrum &bgr;-lactamase–producing Klebsiella pneumoniae [10], carbapenem-resistant Acinetobacter baumannii [8], carbapenem- and quinolone-resistant Pseudomonas aeruginosa [26], and derepressed chromosomal &bgr;-lactam and extended-spectrum &bgr;-lactamase–producing Enterobacter species [5]). Factors independently associated with rESKAPE bacteremia were prior transplantation, septic shock, and prior antibiotic therapy. Patients with rESKAPE bacteremia more often received inappropriate empirical antibiotic therapy than the others (41% vs. 21.6%; P=0.01). Overall case-fatality rate (30 days) was higher in patients with rESKAPE bacteremia (35.2% vs. 14.4%; P=0.001). Conclusions Bacteremia due to rESKAPE pathogens is frequent in SOT recipients and causes significant morbidity and mortality. rESKAPE organisms should be considered when selecting empirical antibiotic therapy for hospitalized SOT recipients presenting with septic shock, particularly those with prior transplantation and antibiotic use.

Intragraft regulatory T cells in protocol biopsies retain foxp3 demethylation and are protective biomarkers for kidney graft outcome.

Presence of subclinical rejection (SCR) with IF/TA in protocol biopsies of renal allografts has been shown to be an independent predictor factor of graft loss. Also, intragraft Foxp3+ Treg cells in patients with SCR has been suggested to differentiate harmful from potentially protective infiltrates. Nonetheless, whether presence of Foxp3 Treg cells in patients with SCR and IF/TA may potentially protect from a deleterious graft outcome has not yet been evaluated. This is a case‐control study in which 37 patients with the diagnosis of SCR and 68 control patients with no cellular infiltrates at 6‐month protocol biopsies matched for age and time of transplantation were evaluated. We first confirmed that numbers of intragraft Foxp3‐expressing T cells in patients with SCR positively correlates with Foxp3 demethylation at the Treg‐specific demethylation region. Patients with SCR without Foxp3+ Treg cells within graft infiltrates showed significantly worse 5‐year graft function evolution than patients with SCR and Foxp3+ Treg cells and those without SCR. When presence of SCR and IF/TA were assessed together, presence of Foxp3+ Treg could discriminate a subgroup of patients showing the same graft outcome as patients with a normal biopsy. Thus, presence of Foxp3+ Treg cells in patients with SCR even with IF/TA is associated with a favorable long‐term allograft outcome.

New immunosuppresor strategies in the treatment of murine lupus nephritis

Renal involvement in systemic lupus erythematosus is a common complication that significantly worsens morbidity and mortality. Although treatment with corticosteroids and cytotoxic drugs may be useful in many cases, morbidity associated with these drugs and the relapsing nature of the disease make it necessary to develop new treatment strategies. Five-month old female NZB/W F1 mice were divided into the following groups: CYP group (n = 10), cyclophosphamide (CYP) 50 mg/kg intraperitoneally every 10 days; RAPA 1 group (n = 10) oral daily sirolimus (SRL), 1 mg/kg; RAPA 12 group (n = 13), oral daily SRL, 12 mg/kg; FTY group (n = 10), oral fingolimod (FTY720), 2 mg/kg three times per week. An additional group of 13 non-treated mice were used as a control (control group). Follow-up was performed over four months. Animal survival, body weight, anti-DNA antibodies and proteinuria were determined. Kidneys were processed for conventional histology and immunofluorescence for IgG and complement. Total histological score (HS) was the sum of mesangial expansion, endocapillary proliferation glomerular deposits, extracapillary proliferation, interstitial infiltrates, tubular atrophy and interstitial fibrosis. All treated groups had lower proteinuria at the end of the follow-up with respect to the control group (P < 0.0001). Serum anti-DNA antibodies were appropriately controlled in RAPA 1 and CYP groups, but not in FTY or RAPA 12 groups. SRL and CYP arrested, and perhaps reversed almost all histological lesions. FTY720 ameliorated histological lesions but did not control mesangial expansion or interstitial infiltrates. SRL produces great improvement in murine lupus nephritis, while FTY720 seems a promising alternative if used in appropriate doses.

Long-Term Results of Biopsy-Guided Selection and Allocation of Kidneys From Older Donors in Older Recipients

In our old‐for‐old program, we discard or allocate older extended criteria donor kidneys to single (SKT) or dual kidney transplantation (DKT) depending on histological Remuzzis score in recipients older than 60 years. Here, we analyze the long‐term results of this program and try to identify independent predictors of patient and graft survival. Between December 1996 and January 2008, we performed 115 SKT and 88 DKT. Discard rate was 15%. Acute rejection incidence was higher in SKT than in DKT (22.6% vs. 11.4%, p = 0.04). Renal function was better in DKT than in SKT up to 5 years after transplantation. Surgical complications were frequent in DKT. Ten‐year cumulative graft survival was significantly lower in the SKT group (31% vs. 53%, p = 0.03). In SKT, histological score 4 provided similar graft survival than 3 or less, whereas in DKT score 4, 5 or 6 displayed similar outcome. Finally, independent predictors of graft survival were history of major adverse cardiac event and 1‐year serum creatinine, rather than SKT or DKT. In conclusion, this biopsy‐guided old‐for‐old strategy resulted in acceptable long‐term graft survival. Our results suggest that DKT should be considered for scores of 5 or 6 only.

Direct electrotransfer of hHGF gene into kidney ameliorates ischemic acute renal failure.

In the early phase of kidney transplantation, the transplanted kidney is exposed to insults like ischemia/reperfusion, which is a leading cause of acute renal failure (ARF). ARF in the context of renal transplantation predisposes the graft to developing chronic damage and to long-term graft loss. Hepatocyte growth factor (HGF) has been suggested to support the intrinsic ability of the kidney to regenerate in response to injury by its morphogenic, mitogenic, motogenic and antiapoptotic activities. In the present paper, we examine whether human HGF (hHGF) gene electrotransfer helps in the recovery from ARF in a model of rat renal warm ischemia. We also assess the advantages of this form of gene therapy by direct electroporation of the kidney, given that transplantation offers the possibility of manipulating the organ in vivo. We have compared the therapeutic efficiency of two electroporation methodologies in a rat ARF model. Although they both targeted the same organ, the two methods were applied to different parts of the animal: muscle and kidney. Kidney direct electrotransfer was shown to be more efficient not only in pharmacokinetic but also in therapeutic terms, so it may become a clinically practical alternative in renal transplantation.

Preformed circulating HLA-specific memory B cells predict high risk of humoral rejection in kidney transplantation

The accurate evaluation of donor-specific antibodies (DSAs) has allowed a precise identification of sensitized patients at risk of antibody-mediated rejection (ABMR). However, the scale of the humoral response is not always fully addressed, as it excludes the complete memory B-cell (mBC) pool such as that caused by antigen-specific mBC. Using a novel B-cell ELISpot assay approach, we assessed circulating mBC frequencies against class I and II HLA antigens in highly sensitized and nonsensitized patients in the waiting list for kidney transplantation. Also, kidney transplant patients undergoing ABMR were evaluated for the presence of donor-specific mBCs both at the time of rejection and before transplantation. For this purpose, 278 target HLA-sp antigens from 70 patients were studied and compared to circulating HLA-sp antibodies. Both class I and II HLA-sp mBC frequencies were identified in highly sensitized individuals but not in nonsensitized and healthy individuals, many years after first sensitization. Also, high donor-specific mBC responses were clearly found both during ABMR and before transplantation, regardless of circulating DSA. The higher the donor-specific mBC response, the more aggressive the allograft rejection. Thus, assessing donor-specific mBC frequencies may be relevant to better refine patient alloimmune-risk stratification, and provides new insight into the mechanisms of the adaptive humoral alloimmune response taking place in kidney transplantation.