Hypothermic pulsatile perfusion of human pancreas: Preliminary technical feasibility study based on histology.

BACKGROUND: There are currently two approaches to hypothermic preservation for most solid organs: static or dynamic. Cold storage is the main method used for static storage (SS), while hypothermic pulsatile perfusion (HPP) and other machine perfusion-based methods, such as normothermic machine perfusion and oxygen persufflation, are the methods used for dynamic preservation. HPP is currently approved for kidney transplantation. METHODS: We evaluated, for the first time, the feasibility of HPP on 11 human pancreases contraindicated for clinical transplantation because of advanced age and/or history of severe alcoholism and/or abnormal laboratory tests. Two pancreases were used as SS controls, pancreas splitting was performed on 2 other pancreases for SS and HPP and 7 pancreases were tested for HPP. HPP preservation lasted 24 h at 25 mmHg. Resistance index was continuously monitored and pancreas and duodenum histology was evaluated every 6 h. RESULTS: The main finding was the complete absence of edema of the pancreas and duodenum at all time-points during HPP. Insulin, glucagon and somatostatin staining was normal. Resistance index decreased during the first 12 h and remained stable thereafter. CONCLUSION: 24 h hypothermic pulsatile perfusion of marginal human pancreas-duodenum organs was feasible with no deleterious parenchymal effect. These observations encourage us to further develop this technique and evaluate the safety of HPP after clinical transplantation.

Failure of Calcineurin Inhibitor (Tacrolimus) Weaning Randomized Trial in Long-Term Stable Kidney Transplant Recipients.

Long-term renal transplant outcome is limited by side effects of immunosuppressive drugs, particularly calcineurin inhibitor (CNI). We assumed that some patients selected for a "low immunological risk of rejection" could be eligible and benefit from a CNI weaning strategy. We designed a prospective, randomized, multicenter, double-blind placebo-controlled clinical study (Eudract: 2010-019574-33) to analyze the benefit-risk ratio of tacrolimus weaning on highly selected patients (≥4 years of transplantation, normal histology, stable graft function, no anti-HLA immunization). The primary endpoint was improvement of renal function. Fifty-two patients were scheduled in each treatment arm, placebo compared to the CNI maintenance arm. Only 10 patients were eligible and randomized. Five patients were assigned to the placebo arm and five were assigned to the tacrolimus maintenance arm. In the tacrolimus maintenance arm, all patients maintained stable graft function and no immunological events occurred. Contrastingly, in the placebo arm, all five patients had to reintroduce a full dose of tacrolimus since three of them presented an acute rejection episode (one humoral, one mixed, and one borderline) and two displayed anti-HLA antibodies without histological lesion (one donor-specific antibodies [DSA] and one non-DSA). Clearly, tacrolimus withdrawal must be avoided even in long-term highly selective stable kidney recipients.

Bortezomib, C1-inhibitor and plasma exchange do not prolong the survival of multi-transgenic GalT-KO pig kidney xenografts in baboons.

Galactosyl-transferase KO (GalT-KO) pigs represent a potential solution to xenograft rejection, particularly in the context of additional genetic modifications. We have performed life supporting kidney xenotransplantation into baboons utilizing GalT-KO pigs transgenic for human CD55/CD59/CD39/HT. Baboons received tacrolimus, mycophenolate mofetil, corticosteroids and recombinant human C1 inhibitor combined with cyclophosphamide or bortezomib with or without 2-3 plasma exchanges. One baboon received a control GalT-KO xenograft with the latter immunosuppression. All immunosuppressed baboons rejected the xenografts between days 9 and 15 with signs of acute humoral rejection, in contrast to untreated controls (n = 2) that lost their grafts on days 3 and 4. Immunofluorescence analyses showed deposition of IgM, C3, C5b-9 in rejected grafts, without C4d staining, indicating classical complement pathway blockade but alternate pathway activation. Moreover, rejected organs exhibited predominantly monocyte/macrophage infiltration with minimal lymphocyte representation. None of the recipients showed any signs of porcine endogenous retrovirus transmission but some showed evidence of porcine cytomegalovirus (PCMV) replication within the xenografts. Our work indicates that the addition of bortezomib and plasma exchange to the immunosuppressive regimen did not significantly prolong the survival of multi-transgenic GalT-KO renal xenografts. Non-Gal antibodies, the alternative complement pathway, innate mechanisms with monocyte activation and PCMV replication may have contributed to rejection.

FR104, an antagonist anti-CD28 monovalent fab' antibody, prevents alloimmunization and allows calcineurin inhibitor minimization in nonhuman primate renal allograft.

Selective targeting of CD28 might represent an effective immunomodulation strategy by preventing T cell costimulation, while favoring coinhibition since inhibitory signals transmitted through CTLA-4; PD-L1 and B7 would not be affected. We previously showed in vitro and in vivo that anti-CD28 antagonists suppress effector T cells while enhancing regulatory T cell (Treg) suppression and immune tolerance. Here, we evaluate FR104, a novel antagonist pegylated anti-CD28 Fab' antibody fragment, in nonhuman primate renal allotransplantation. FR104, in association with low doses of tacrolimus or with rapamycin in a steroid-free therapy, prevents acute rejection and alloantibody development and prolongs allograft survival. However, when FR104 was associated with mycophenolate mofetil and steroids, half of the recipients rejected their grafts prematurely. Finally, we observed an accumulation of Helios-negative Tregs in the blood and within the graft after FR104 therapy, confirmed by Treg-specific demethylated region DNA analysis. In conclusion, FR104 reinforces immunosuppression in calcineurin inhibitor (CNI)-low or CNI-free protocols, without the need of steroids. Accumulation of intragraft Tregs suggested the promotion of immunoregulatory mechanisms. Selective CD28 antagonists might become an alternative CNI-sparing strategy to B7 antagonists for kidney transplant recipients.

Pretransplant sensitization against angiotensin II type 1 receptor is a risk factor for acute rejection and graft loss.

The angiotensin II type 1 receptor (AT1R) is an emerging target of functional non-HLA antibodies (Ab). We examined the potential of determining the degree of presensitization against AT1R as a risk factor for graft survival and acute rejection (AR). The study included 599 kidney recipients between 1998 and 2007. Serum samples were analyzed in a blinded fashion for anti-AT1R antibodies (AT1R-Abs) using a quantitative solid-phase assay. A threshold of AT1R-Ab levels was statistically determined at 10 U based on the time to graft failure. An extended Cox model determined risk factors for occurrence of graft failure and a first AR episode. AT1R-Abs >10 U were detected in 283 patients (47.2%) before transplantation. Patients who had a level of AT1R-Abs >10 U had a 2.6-fold higher risk of graft failure from 3 years posttransplantation onwards (p = 0.0005) and a 1.9-fold higher risk of experiencing an AR episode within the first 4 months of transplantation (p = 0.0393). Antibody-mediated rejection (AMR) accounted for 1/3 of AR, whereby 71.4% of them were associated with >10 U of pretransplant AT1R-Abs. Pretransplant anti-AT1R-Abs are an independent risk factor for long-term graft loss in association with a higher risk of early AR episodes.

The natural history of clinical operational tolerance after kidney transplantation through twenty-seven cases.

We report here on a European cohort of 27 kidney transplant recipients displaying operational tolerance, compared to two cohorts of matched kidney transplant recipients under immunosuppression and patients who stopped immunosuppressive drugs and presented with rejection. We report that a lower proportion of operationally tolerant patients received induction therapy (52% without induction therapy vs. 78.3%[p = 0.0455] and 96.7%[p = 0.0001], respectively), a difference likely due to the higher proportion (18.5%) of HLA matched recipients in the tolerant cohort. These patients were also significantly older at the time of transplantation (p = 0.0211) and immunosuppression withdrawal (p = 0.0002) than recipients who rejected their graft after weaning. Finally, these patients were at lower risk of infectious disease. Among the 27 patients defined as operationally tolerant at the time of inclusion, 19 still display stable graft function (mean 9 ± 4 years after transplantation) whereas 30% presented slow deterioration of graft function. Six of these patients tested positive for pre-graft anti-HLA antibodies. Biopsy histology studies revealed an active immunologically driven mechanism for half of them, associated with DSA in the absence of C4d. This study suggests that operational tolerance can persist as a robust phenomenon, although eventual graft loss does occur in some patients, particularly in the setting of donor-specific alloantibody.

[Prostatic Stromal Tumors of Uncertain Malignant Potential (STUMP): definition, pathology, prognosis and management]. / Tumeur stromale prostatique à potentiel malin incertain (STUMP) : définition, anatomopathologie, pronostic et prise en charge.

Prostatic Stromal Tumors of Uncertain Malignant Potential (STUMP) are rare tumor of the prostate of mesenchymal origin, accounting, with sarcoma for 0.1-0.2% of all malignant prostatic tumours. They however require to be individualized, to differentiate it from a benign prostatic hyperplasia or a sarcoma of the prostate. The therapeutic management should be made keeping in mind the risk of degeneration towards a malignant shape. Although the appropriate treatment is unknown, radical prostatectomy seem to be the treatment of reference, especially for young patient or for extensive lesion.

Xenotransplantation of galactosyl-transferase knockout, CD55, CD59, CD39, and fucosyl-transferase transgenic pig kidneys into baboons.

Galactosyl-transferase knockout (GT-KO) pigs represent the latest major progress to reduce immune reactions in xenotransplantation. However, their organs are still subject to rapid humoral rejection involving complement activation requiring the ongoing development of further genetic modifications in the pig. In a pig-to-baboon renal transplantation setting, we have used donor pigs that are not only GT-KO, but also transgenic for human CD55 (hCD55), hCD59, hCD39, and fucosyl-transferase (hHT). We studied kidney xenograft survival, physiological and immunologic parameters, xenogeneic rejection characteristics, as well as viral transmission aspects among two groups of baboons: control animals (n = 2), versus those (n = 4) treated with a cocktail of cyclophosphamide, tacrolimus, mycophenolate mofetil, steroids, and a recombinant human C1 inhibitor. Whereas control animals showed clear acute humoral rejection at around day 4, the treated animals showed moderately improved graft survival with rejection at around 2 weeks posttransplantation. Biopsies showed signs of acute vascular rejection (interstitial hemorrhage, glomerular thrombi, and acute tubular necrosis) as well as immunoglobulin (Ig)M and complement deposition in the glomerular and peritubular capillaries. The low level of preformed non-Gal-α1.3Gal IgM detected prior to transplantation increased at 6 days posttransplantation, whereas induced IgG appeared after day 6. No porcine endogenous retrovirus (PERV) transmission was detected in any transplanted baboon. Thus, surprisingly, organs from the GT-KO, hCD55, hCD59, hCD39, and hHT transgenic donors did not appear to convey significant protection against baboon anti-pig antibodies and complement activation, which obviously continue to be significant factors under a suboptimal immunosuppression regimen. The association, timing, and doses of immunosuppressive drugs remain critical. They will have to be optimized to achieve longer graft survivals.

Guidelines for the diagnosis of antibody-mediated rejection in pancreas allografts-updated Banff grading schema.

The first Banff proposal for the diagnosis of pancreas rejection (Am J Transplant 2008; 8: 237) dealt primarily with the diagnosis of acute T-cell-mediated rejection (ACMR), while only tentatively addressing issues pertaining to antibody-mediated rejection (AMR). This document presents comprehensive guidelines for the diagnosis of AMR, first proposed at the 10th Banff Conference on Allograft Pathology and refined by a broad-based multidisciplinary panel. Pancreatic AMR is best identified by a combination of serological and immunohistopathological findings consisting of (i) identification of circulating donor-specific antibodies, and histopathological data including (ii) morphological evidence of microvascular tissue injury and (iii) C4d staining in interacinar capillaries. Acute AMR is diagnosed conclusively if these three elements are present, whereas a diagnosis of suspicious for AMR is rendered if only two elements are identified. The identification of only one diagnostic element is not sufficient for the diagnosis of AMR but should prompt heightened clinical vigilance. AMR and ACMR may coexist, and should be recognized and graded independently. This proposal is based on our current knowledge of the pathogenesis of pancreas rejection and currently available tools for diagnosis. A systematized clinicopathological approach to AMR is essential for the development and assessment of much needed therapeutic interventions.

Banff '09 meeting report: antibody mediated graft deterioration and implementation of Banff working groups.

The 10th Banff Conference on Allograft Pathology was held in Banff, Canada from August 9 to 14, 2009. A total of 263 transplant clinicians, pathologists, surgeons, immunologists and researchers discussed several aspects of solid organ transplants with a special focus on antibody mediated graft injury. The willingness of the Banff process to adapt continuously in response to new research and improve potential weaknesses, led to the implementation of six working groups on the following areas: isolated v-lesion, fibrosis scoring, glomerular lesions, molecular pathology, polyomavirus nephropathy and quality assurance. Banff working groups will conduct multicenter trials to evaluate the clinical relevance, practical feasibility and reproducibility of potential changes to the Banff classification. There were also sessions on quality improvement in biopsy reading and utilization of virtual microscopy for maintaining competence in transplant biopsy interpretation. In addition, compelling molecular research data led to the discussion of incorporation of omics-technologies and discovery of new tissue markers with the goal of combining histopathology and molecular parameters within the Banff working classification in the near future.

Extreme oral manifestations in a Marfan-type syndrome.

A 12-year-old girl with an otherwise typical Marfan syndrome (Ghent criteria fulfilled) presented with highly unusual oral manifestations consisting of supernumerary teeth and severe dental crowding. Pathological examination of the supernumerary teeth revealed an elevated number of pulpoliths. No mutation in the FBN1, TGFBR1 and TGFBR2 genes was identified despite exhaustive screening, suggesting that another gene defect could explain this association of marfanoid features with dental abnormalities.

Indirect CD4+ TH1 response, antidonor antibodies and diffuse C4d graft deposits in long-term recipients conditioned by donor antigens priming.

Priming of recipients by DST induces long-term survival of mismatched allografts in adult rats. Despite these recipients developing inducible T regulatory cells able to transfer long-term graft survival to a secondary host, a state of chronic rejection is also observed. We revisited the molecular donor MHC targets of the cellular response in acute rejection and analyzed the cellular and humoral responses in recipients with long-term graft survival following transplantation. We found three immunodominant peptides, all derived from LEW.1W RT1.D(u) molecules to be involved in acute rejection of grafts from unmodified LEW.1A recipients. Although the direct pathway of allorecognition was reduced in DST-treated recipients, the early CD4+ indirect pathway response to dominant peptides was almost unimpaired. We also detected early and sustained antidonor class I and II antibody subtypes with diffuse C4d deposits on graft vessels. Finally, long-term accepted grafts displayed leukocyte infiltration, endarteritis and fibrosis, which evolved toward vascular narrowing at day 100. Altogether, these data suggest that the chronic graft lesions developed in long-term graft recipients are the result of progressive humoral injury associated with a persisting indirect T helper response. These features may represent a useful model for understanding and manipulating chronic active antibody-mediated rejection in human.

Banff schema for grading pancreas allograft rejection: working proposal by a multi-disciplinary international consensus panel.

Accurate diagnosis and grading of rejection and other pathological processes are of paramount importance to guide therapeutic interventions in patients with pancreas allograft dysfunction. A multi-disciplinary panel of pathologists, surgeons and nephrologists was convened for the purpose of developing a consensus document delineating the histopathological features for diagnosis and grading of rejection in pancreas transplant biopsies. Based on the available published data and the collective experience, criteria for the diagnosis of acute cell-mediated allograft rejection (ACMR) were established. Three severity grades (I/mild, II/moderate and III/severe) were defined based on lesions known to be more or less responsive to treatment and associated with better- or worse-graft outcomes, respectively. The features of chronic rejection/graft sclerosis were reassessed, and three histological stages were established. Tentative criteria for the diagnosis of antibody-mediated rejection were also characterized, in anticipation of future studies that ought to provide more information on this process. Criteria for needle core biopsy adequacy and guidelines for pathology reporting were also defined. The availability of a simple, reproducible, clinically relevant and internationally accepted schema for grading rejection should improve the level of diagnostic accuracy and facilitate communication between all parties involved in the care of pancreas transplant recipients.

Banff 07 classification of renal allograft pathology: updates and future directions.

The 9th Banff Conference on Allograft Pathology was held in La Coruna, Spain on June 23-29, 2007. A total of 235 pathologists, clinicians and scientists met to address unsolved issues in transplantation and adapt the Banff schema for renal allograft rejection in response to emerging data and technologies. The outcome of the consensus discussions on renal pathology is provided in this article. Major updates from the 2007 Banff Conference were: inclusion of peritubular capillaritis grading, C4d scoring, interpretation of C4d deposition without morphological evidence of active rejection, application of the Banff criteria to zero-time and protocol biopsies and introduction of a new scoring for total interstitial inflammation (ti-score). In addition, emerging research data led to the establishment of collaborative working groups addressing issues like isolated 'v' lesion and incorporation of omics-technologies, paving the way for future combination of graft biopsy and molecular parameters within the Banff process.

Induction of tolerance by exosomes and short-term immunosuppression in a fully MHC-mismatched rat cardiac allograft model.

Exosomes are MHC-bearing vesicles secreted by a wide array of cells. We have previously shown that donor-haplotype exosomes from bone marrow dendritic cells (DCs) injected before transplantation significantly prolong heart allograft survival in congenic and fully MHC-mismatched Lewis rats. Here we show that donor exosomes administered after transplantation are similarly able to prolong allograft survival, however, without inducing tolerance. We therefore tested the effect of exosomes combined with short-term LF 15-0195 (LF) treatment, which blocks the maturation of DCs, so that donor-MHC antigens from exosomes could be presented in a more tolerogenic environment. LF treatment does not preclude the development of a strong antidonor cellular response, and while LF, but not exosome, treatment inhibits the antidonor humoral response and decreases leukocyte graft infiltration, allografts from LF-treated recipients were either acutely or strongly chronically rejected. Interestingly, when combined with LF treatment, exosomes induced a donor-specific allograft tolerance characterized by a strong inhibition of the antidonor proliferative response. This donor-specific tolerance was transferable to naïve allograft recipients. Moreover, exosomes/LF treatment prevented or considerably delayed the appearance of chronic rejection. These results suggest that under LF treatment, presentation of donor-MHC antigens (from exosomes) can induce regulatory responses that are able to modulate allograft rejection and to induce donor-specific allograft tolerance.

[Endobronchial stenosis in Wegener's granulomatosis]. / Sténoses endobronchiques au cours de la granulomatose de Wegener.

INTRODUCTION: Endobronchial stenoses are rare in the course of Wegener's granulomatosis, and they occur even more rarely than subglottic stenoses. EXEGESIS: We report seven cases of endobronchial stenoses in the setting of Wegener granulomatosis. Neither the pulmonary symptoms nor the systemic manifestations of vasculitis were specific. However 6/7 patients presented a wheezing or an hemoptysis. Bronchial endoscopy has permitted the diagnosis in all cases. Local evolution was cicatricial and symptomatic stenosis in 3 cases (42,8%). CONCLUSION: Thus these lesions must be research in any case of pulmonary abnormality in the course of Wegener's granulomatosis, because they may lead to a pejorative prognosis. Moreover general and local treatment must be given early (at the inflammatory stage). After this stage, the local treatments are difficult and not efficient.

Loss of NOS1 expression in high-grade renal cell carcinoma associated with a shift of NO signalling.

In normal human kidney, NOS1 and soluble guanylate cyclase (sGC) are expressed in tubular epithelial cells, suggesting a physiological autocrine NO signalling pathway. Therefore, we investigated both NOS1 and sGC expressions in benign and malignant renal tumours. In addition, we examined the pattern of protein tyrosine nitration in normal and tumour tissue. NOS1 expression and activity were found to be downregulated, correlating with the tumour grade, as shown by immunohistochemistry, quantitative RT-PCR analysis, and histochemical detection of the NADPH-diaphorase activity of nitric oxide synthases (NOS). These results show that the autocrine NO signalling pathway is maintained in benign tumours and lost in malignant tumours. In contrast, sGC expression was maintained in renal tumours whatever the tumour type, a finding showing that tumour cells remain sensitive to the bioregulatory role of exogeneous NO(*). Finally, the staining pattern of protein tyrosine nitration, assessed by immunohistochemistry, parallelled that of NOS1 expression in normal renal parenchyma and benign tumours, supporting the concept that protein nitration was accounted for by NOS1 activity. In contrast, in malignant tumours, protein tyrosine nitration was accounted for by the production of reactive nitrogen oxide species by the inflammatory infiltrate. Altogether, these findings argue for a pattern of NO signalling similar in normal kidney and benign renal tumours, whereas it is completely different in malignant renal tumours.

Fatal acute granulomatous pulmonary aspergillosis in a healthy subject after inhalation of vegetal dust.

A previously healthy 28-year-old man presented a few hours after inhalation of vegetal dust with acute bilateral community-acquired pneumonia, which caused death in 10 days despite treatment with broad-spectrum antibiotics, intravenous amphotericin B, inotropic support, and mechanical ventilation. A postmortem lung biopsy indicated miliary granulomatous pulmonary aspergillosis. Six similar previously published cases of acute granulomatous pulmonary aspergillosis are reviewed. This entity in adulthood may reveal a defect in neutrophil or macrophage function, such as late-onset chronic granulomatous disease.

[Bronchopulmonary aspergillosis: new developments]. / Aspergilloses broncho-pulmonaires: les nouveaux enjeux.

Bronchopulmonary aspergillosis are in the news. Invasive pulmonary aspergillosis raise early diagnostic problems and prevention problems in immunocompromised patients. These infections are no unusual in chronic obstructive pulmonary disease. The diagnosis between aspergilloma and chronic necrotizing pulmonary aspergillosis can be difficult. In allergic bronchopulmonary aspergillosis, epidemiology and therapy are questionable. Real progress has been made due to thoracic computed tomographic scan and mycological methods. Better use of amphotericin B, of amphotericin B lipid formulations and of azole antifungal agents, combined with surgical resection if necessary should improve aspergillosis prognosis.