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Outcomes after transplantation of lungs (LuTX) treated with ex-vivo lung perfusion (EVLP) are debated. In a single-center 8 years of retrospective analysis, we compared: donors' and recipients' characteristics, gas exchange and lung mechanics at ICU admission, 3, 6, and 12 months, and patients' survival of LuTX from standard donors compared with EVLP-treated grafts. A total of 193 LuTX were performed. Thirty-one LuTX, out of 50 EVLP procedures, were carried out: 7 from nonheart beating and 24 from extended criteria brain-dead donors. Recipients' characteristics were similar. At ICU admission, compared with standard donors, EVLP patients had worse PaO2 /FiO2 [276 (206; 374) vs. 204 (133; 245) mmHg, P < 0.05], more frequent extracorporeal support (18% vs. 32%, P = 0.053) and longer mechanical ventilation duration [28 days of ventilator-free days: 27 (24; 28) vs. 26 (19; 27), P < 0.05]. ICU length of stay [4 (2; 9) vs. 6 (3; 12) days, P = 0.208], 28-day survival (99% vs. 97%, P = 0.735), and 1-year respiratory function were similar between groups. Log-rank analysis (median follow-up 2.5 years) demonstrated similar patients' survival (P = 0.439) and time free of chronic lung allograft disease (P = 0.484). The EVLP program increased by 16% the number of LuTX. Compared to standard donors, EVLP patients had worse respiratory function immediately after LuTX but similar early and mid-term outcomes.

Background Lung transplantation is an established therapeutic option for patients with end-stage pulmonary disease. In May 2005, the lung allocation score (LAS) was introduced in the United States to maximize the benefit to the recipient population and reduce waiting list mortality. The LAS has been applied in a region of Italy since March 2016 on a provisional basis. The aims of the study were describing waiting list characteristics and short-term outcomes after lung transplantation before and after LAS introduction. Methods All the patients who received transplants between January 1, 2011, and March 15, 2017, were included in our retrospective study. The study population was divided into 2 cohorts (historical cohort and post-LAS cohort) and a comparison among the main perioperative data was performed. Results The historical cohort consisted of 415 patients on the waiting list with 91 deaths and 199 lung transplants; the post-LAS cohort consisted of 134 patients with 10 deaths on the waiting list and 51 transplants. Median waiting time and mortality on the list decreased from 223 to 106 days (P = .03) and from 11.2% to 7.5% (P > .05), respectively. The transplantation rate increased from 25% to 38% (P = .001) and the probability to receive a transplant in the first year in the post-LAS era increased significantly (P = .004). Conclusions The results of the introduction of the LAS system in our region are encouraging and have not shown any adverse short-term effects. The regional coordination decided to prolong the experimental application of LAS in order to accumulate more data and to evaluate medium-term outcomes.

BACKGROUND Identification of heart transplant (HTx) rejection currently relies on immunohistology and immunohistochemistry. We aimed to identify specific sets of microRNAs (miRNAs) to characterize acute cellular rejection (ACR), antibody-mediated rejection (pAMR), and mixed rejection (MR) in monitoring formalin-fixed paraffin-embedded (FFPE) endomyocardial biopsies (EMBs) in HTx patients. METHODS In this study we selected 33 adult HTx patients. For each, we chose the first positive EMB for study of each type of rejection. The next-generation sequencing (NGS) IonProton technique and reverse transcript quantitative polymerase chain reaction (RT-qPCR) analysis were performed on FFPE EMBs. Using logistic regression analysis we created unique miRNA signatures as predictive models of each rejection. In situ PCR was carried out on the same EMBs. RESULTS We obtained >2,257 mature miRNAs from all the EMBs. The 3 types of rejection showed a different miRNA profile for each group. The logistic regression model formed by miRNAs 208a, 126-5p, and 135a-5p identified MR; that formed by miRNAs 27b-3p, 29b-3p, and 199a-3p identified ACR; and that formed by miRNAs 208a, 29b-3p, 135a-5p, and 144-3p identified pAMR. The expression of miRNAs on tissue, through in situ PCR, showed different expressions of the same miRNA in different rejections. miRNA 126-5p was expressed in endothelial cells in ACR but in cardiomyocytes in pAMR. In ACR, miRNA 29b-3p was significantly overexpressed and detected in fibroblasts, whereas in pAMR it was underexpressed and detected only in cardiomyocytes. CONCLUSIONS miRNA profiling on FFPE EMBs differentiates the 3 types of rejection. Localization of expression of miRNAs on tissue showed different expression of the same miRNA for different cells, suggesting different roles of the same miRNA in different rejections.

Purpose Vasculitis had been considered a histopathological marker in severe form of both cellular and humoral rejection. However, in mild forms of rejections, it has not been considered a diagnostic and prognostic histopathologic criterion. Aim of this study is to assess clinical relevance of vasculitis, in terms of mortality and cardiac allograft vasculopathy development, in the mild forms of rejection in heart transplanted patients. Methods We reviewed 2794 monitoring endomyocardial biopsies from 170 adult heart transplanted pts at our center. On endomyocardial biopsies, we evaluated the presence of vasculitis in the different types of rejection . Clinical data were collected during follow up. A Multi-State Markov model was applied to describe the rejection dynamic profile and hazard for mortality, persistence of rejection and CAV development.. We used as reference population the registry data of patients who underwent HTX from 2004 to 2018 provided by the International Society for Heart and Lung Transplantation (ISHLT) Thoracic Organ Transplant Registry Results Vasculitis was detected in 442 endomyocardial biopsies ( 442/2794, 15,8%) with an incremental percent distribution according to the severity of rejection. In ACR, vasculitis significantly increased the risk of mortality by 9% (HR [95% CI]: 1.09; [1.00-1.18]) the hazard of mortality if the patient was negative for ACR and by 14% (HR [95% CI]: 1.14; [1.02-1.37], if the patients was negative for ACR, and by 14% in a mild form of rejection (1R). Similar results were obtained for pAMR and mixed rejection. In ACR, pAMR and mixed rejection patients with vasculitis have a shorter life expectancy than patients without with a loss of four months at ten year follow up for ACR, and of 9 months for pAMR and mixed rejection. Conclusion Vasculitis when present, independently from the grade and type of rejection, carries a negative prognostic value. The worse histopathological feature was represented by the association of pAMR+ACR and vasculitis.

Purpose We present the results of a clinical trial (NCT02061462) comparing outcomes after lung transplantation (LT) of subjects receiving lungs from donation after brain death (DBD) donors with that of subjects receiving lungs from donation after circulatory death (DCD) donors, procured using our original open lung strategy. Briefly, our protocol consists of a non-rapid normothermic open-lung procurement using recruitment manoeuvres, protective ventilation and CPAP without topical cooling. Methods We evaluated patients undergoing bilateral LT from 11/2014 to 07/2019; exclusion criteria: recipients ≤14 years, re-LT, donors ≥65 years, DCD category I and IV. Variables were prospectively recorded as well as respiratory functional parameters in the first year. Results We performed 143 LT; 22 cases were excluded, 121 were enrolled: 11 recipients received lungs from DCD donors (5 and 6 from DCD category II and III, respectively). The two groups had similar features. Most of the subjects had cystic fibrosis (72,7% and 61,9% of DCD and DBD, respectively). DCD donors’ BMI was significantly higher (p=0.022); 15,5% of DBD grafts underwent machine perfusion. Duration of postoperative mechanical ventilation was higher in the DCD group (DCD vs DBD=2 vs 1 day, p= 0.011). Incidence of grade 3 primary graft dysfunction was 27,3% in the DCD group and 18,2% in the DBD group (p=0.742). Cold ischemia and preservation times were significantly higher in the DCD group (p Conclusion Regardless of the small study population, our results are encouraging. Our data show the feasibility of LT from DCD donors using our original protocol, with adequate grafts function and survival.

Purpose Aim of the study to assess the clinical relevance of vasculitis, in terms of survival and cardiac allograft vasculopathy development in the mild forms of rejection in HTx adult patients Methods We review 2639 monitoring EMBs from 172 adult heart transplanted pts at our center from January 2008 to September 2016, median follow up of 4,59 (range 0,13-8,78 years), with a mean of 15 EMB per pts. For each EMB we evaluated vasculitis and types of rejection classified according to 1990 and 2005 ISHLT classification for ACR and 2013 ISHLT classification for AMR. Clinical data were collected during follow up. For statistical analysis we applied the Multi-State Markov (MSM) model to describe the rejection profile of the subjects during the follow-up. Results Four hundred and twelve EMBs were positive for vasculitis, (412 / 2639; 15,6%), in 134 pts (134/172; 78,5%). In acute cellular rejection vasculitis was present in 329/964 (34,1%) in 1R, in 69/132 (52,3%) in 2R and 14/20 (70%) in 3R. In antibody mediated rejection vasculitis was present in 52/144 (36%) of EMBs. Transition probabilities showed that presence of vasculitis associated to ACR carries a risk of persistence of rejections of 78% (95% CI, 74%-82%), whereas vasculitis associated to pAMR carries a risk of persistence of rejection of 50% (95% CI, 30%-70%). The transitions rate risk of develop CAV was 11.01 (95% CI, 8.61-14.84) fold higher for vasculitis with pAMR+ACR compared to any other rejections without vasculitis. The transition rate risk for death was 21.18 (95% CI, 8.61-109.06) fold higher risk for vasculitis + pAMR and 2.83 (95% CI, 1.08-7.94) fold higher risk vasculitis with pAMR+ACR compared to any other rejections without vasculitis. Conclusion Vasculitis was present in any grades and types of rejection and is associated with persistence of rejection, CAV development and death. The worse histopathological features is represented by the association of pAMR+ACR and vasculitis.

Purpose Antibody mediated rejection (AMR) has recently emerged as an important risk factor for lung allograft dysfunction and mortality. Several clinical protocols have been proposed to try to remove donor specific antibodies (DSA) and prevent their future development, including intravenous immunoglobulin (IVIG), therapeutic plasma exchange (TPE), rituximab and other. Unlike traditional TPE, immunoadsorption (IAS) is a blood-purification technique that enables the selective removal of immunoglobulins from separated plasma through high-affinity adsorbers; advantageously, IAS does not remove other plasma components such as fibrinogen and compounds of the coagulation cascade, reducing potential adverse effects. Our centre implemented a multimodality strategy including steroids, immunoadsorbption, IVIG and Rituximab. Methods All adult lung transplant (LuTx) recipients receiving a diagnosis of AMR (based on 2016 ISHLT consensus definition), were considered eligible to receive this treatment: pulsed methylprednisolone (10 mg/Kg), 5 cycles of IAS, IVIG (2 g/Kg) and rituximab (375 mg/m^2). Of note, C4d staining is not currently available at our institution. Results Between 2016 and 2018, four patients received multimodality antibody directed therapy for AMR after LuTx. Details can be found in table 1. 2 out of the 4 patients qualified for chronic lung allograft dysfunction (CLAD) prior to protocol start (patient 1 and 3). No adverse events were registered during IAS procedures, but patient 3 suffered transfusion related acute lung injury while receiving IVIG. However, patient 3′s death was attributed to progressive allograft failure and not to the therapy itself. Conclusion Following this treatment, while the total number of the original DSA and the value of MFI of the originally observed DSA decreased, clinical outcomes were variable: 2 patients are experiencing long term clinical stability, but the others rapidly progressed to severe CLAD.

OBJECTIVES Donation after circulatory death (DCD) potentially provides transplantable lungs suitable for a transplant, but in Italy, the need for 20 min of a no-touch period after cardiac arrest for legal declaration of death poses real challenges to organ preservation. METHODS This is a single-institution, retrospective study using data collected prospectively between October and December 2017. After the approval of the multidisciplinary DCD study group of Regione Lombardia, Maastricht category III DCD donors became eligible for combined procurement of lungs and abdominal organs. Our group subsequently established a dedicated technical protocol. Our protocol consists of a non-rapid normothermic open-lung procurement process that takes place during abdominal normothermic regional perfusion, namely without pleural topical cooling before the start of pneumoplegia. After the lung is procured according to the technique described in the article, lung function is evaluated by ex vivo lung perfusion, which is run with the low-flow, open atrium, low haematocrit technique. RESULTS During the study, we managed 5 controlled DCDs. In 3 cases, the lungs were successfully transplanted. All 3 patients are alive after 1 year, with good respiratory function. CONCLUSIONS Our approach resulted in adequate lung preservation and successful transplants without detrimental effects on abdominal organ procurement, confirming the possibility of overcoming the obstacle of a long no-touch period in a DCD setting.

Purpose Human herpes virus 8 (HHV8) is associated with Kaposi's sarcoma, multicentric Castleman's disease and Primary Effusion Lymphoma. Behaving as an opportunistic virus, immune-compromised patients, including solid organ transplant recipients, are disproportionately affected. The aim of our study was to review all the cases of HHV8 related disease in our lung transplant population. Methods We retrospectively evaluated the incidence of KS and HHV8 associated lymphoproliferative disorders among all lung transplant recipients at our centre, from January 2008 to December 2017. Clinical data of these patients were collected, taking into account time of onset, donor/recipient HHV8 serology, treatments and outcomes. Results Four out of 203 (2%) patients developed KS (Table 1). Disseminated forms of KS occurred through HHV8 infection from a seropositive donor (D+/R-); both patients initially complained of aspecific symptoms (fever and malaise) and presented with pancytopenia and very high HHV8 DNAemia. We observed a very aggressive progression of KS, and patient 3 died 4 weeks after the fourth dose of liposomal doxorubicin due to an untreatable mycotic infection. By contrast, KS due to viral reactivation (i.e., D-/R+) was milder, limited to the skin and responded to modifications in the immunosuppressive regimens. Interestingly, the recipient of the liver harvested from patient 4’s donor developed another serious HHV8 related disease (i.e., Castelman's disease, investigation currently ongoing at another transplant centre). Conclusion We suggest implementation of D/R serological screening for HHV-8 before transplantation, in order to identify patients at risk of developing HHV-8 related diseases, improve clinical and virological surveillance and possibly individualize immune-suppressive therapy. For the time being, in the absence of a systematic screening, we strongly recommend to report index cases as soon as possible to regional allocation systems in order to identy other recipients potentially at risk.

Purpose The development of donor specific human leukocyte antigen (HLA) antibodies (DSA) has already been identified as a risk factor for graft dysfunction and poor survival in the context of lung transplantation (LuTx), but available evidence is not conclusive. The aim of our study was to evaluate the possibly existing correlation between pre-existing and/or post transplant developing DSA and recipients’ outcome. Methods This was a prospective, cohort study on consecutive LuTx recipients from January 2013 to December 2017. Every individual underwent DSA surveillance with blood specimens collection before and after LuTx. Three groups were defined based on pre-formed antibody setting at time of LuTx: silent, if no antibody was found on their blood; reactive, patients with non-DSA antibodies; and activated, patient with DSA antibodies. Correlation between antibodies panel (before and after LuTx) and outcomes (lung allograft dysfunction and survival) was then investigated. Results 132 patients were considered; 70 (53%) males, median age at LuTx 36 (26; 51) years. Among the “silent” patients: 63% remained negative after LuTx, 20% developed non-DSA and 17% DSA. Among “reactive” recipients: 15% became negative after LuTx, 39% maintained non-DSA and 46.2% developed de-novo DSA. Finally, considering “activated” individuals: the vast majority (80%) maintained pre-transplant DSA and developed new ones, while the remaining 20% lost pre-transplant DSA but showed de-novo non DSA. DSA tended to appear much earlier in pre-transplant allosensitized patients (1 vs. 3 months). Image 1 depicts these results. Finally, both pre and post LuTx DSA showed correlation with the development of chronic lung allograft dysfunction (CLAD) (HR 3.43; 1.19-9.89 and HR 3.35; 1.46-7.68). Conclusion These preliminary findings support the role of both pre-existing and post LuTx developed DSA as a potentially dangerous risk factor for CLAD development, while they did not correlate with acute cellular rejection and/or survival.

OBJECTIVES In Europe, the age of heart donors is constantly increasing. Ageing of heart donors limits the probability of success of heart transplantation (HTx). The aim of this study is to compare the outcome of patients with advanced heart failure (HF) treated with a continuous-flow left ventricular assist device (CF-LVAD) with indication as bridge to transplantation (BTT) or bridge to candidacy (BTC) versus recipients of HTx with the donor's age above 55 years (HTx with donors >55 years). METHODS we prospectively evaluated 301 consecutive patients with advanced HF treated with a CF-LVAD (n = 83) or HTx without prior bridging (n = 218) in our hospital from January 2006 to January 2015. We compared the outcome of CF-LVAD-BTT (n = 37) versus HTx with donors >55 years (n = 45) and the outcome of CF-LVAD-BTT plus BTC (n = 62) versus HTx with donors >55 years at the 1- and 2-year follow-up. Survival was evaluated according to the first operation. RESULTS The perioperative (30-day) mortality rate was 0% in the LVAD-BTT group vs 20% (n = 9) in the HTx group with donors >55 years (P = 0.003). Perioperative mortality occurred in 5% of the LVAD-BTT/BTC patients (n = 3) and in 20% of the HTx with donors >55 year group (P = 0.026). Kaplan-Meier curves estimated a 2-year survival rate of 94.6% in CF-LVAD-BTT vs 68.9% in HTx with donors >55 years [age- and sex-adjusted hazard ratio (HR) 0.25; 95% confidence interval (CI) 0.08-0.81; P = 0.02 in favour of CF-LVAD]. Considering the post-HTx outcome, a trend in favour of CF-LVAD-BTT was also observed (age- and sex-adjusted HR 0.45; 95% CI 0.17-1.16; P = 0.09 in favour of CF-LVAD), whereas CF-LVAD-BTT/BTC showed a similar survival at 2 years compared with HTx with donors >55 years, both censoring the follow-up at the time of HTx and considering the post-HTx outcome. CONCLUSIONS Early and mid-term outcomes of patients treated with a CF-LVAD with BTT indication seem better than HTx with old donors. It must be emphasized that up to 19% of patients in the CF-LVAD/BTT group underwent transplantation in an urgent condition due to complications related to the LVAD. At the 2-year follow-up, CF-LVAD with BTT and BTC indications have similar outcome than HTx using old heart donors. These results must be confirmed in a larger and multicentre population and extending the follow-up.

The development of solid-phase assays for antibody detection has aided in the frequent detection of human leukocyte antigen (HLA) antibodies in nonalloimmunized males. Some scientists have reported that these HLA antibodies are produced to pathogens or allergens and the reactivity with HLA coated beads is the result of cross-reactive epitopes. These antibodies may also be directed toward cryptic epitopes exposed on the denatured beads. In this report, we describe the case of a heart transplanted patient who exhibited anti-HLA-A*02:01 donor-specific antibodies detected with a bead-based assay (Luminex) and undetected with the complement-dependent cytotoxicity (CDC) test. Posttransplant monitoring, carried out with CDC and with Luminex on sera from this patient collected at the 2nd, 4th, 8th, and 12th posttransplant weeks and at 1 year confirmed the presence of anti-HLA-A*02:01 in all serum samples. Additional tests carried out with denatured and intact HLA molecules using single antigen beads demonstrated that the antibody was directed toward a cryptic epitope. One year after transplantation the patient is doing well. No sign of antibody-mediated rejection was observed throughout the follow-up. A comprehensive evaluation of the anamnesis and of antibodies is critical to avoid needless exclusion of organ donors.

BACKGROUND The aim of this study was to assess the significance of positive C4d capillary immunostaining of endomyocardial biopsies and its correlation to clinical outcome in adult heart transplant recipients. METHODS Nine hundred eighty-five endomyocardial biopsies from 107 heart transplant recipients were evaluated. Immunostaining for detection of intragraft C4d capillary deposition was performed on paraffin-embedded tissue using anti-human C4d polyclonal antibody. RESULTS Positive staining of C4d was present in 36 patients (34%) and antibody-mediated rejection in eight patients (7%). The patients were subdivided into four groups on the basis of their C4d, circulating antidonor antibodies (donor-specific antibodies [DSAs]), and graft function: group 1=C4d positive, DSA negative, and no graft dysfunction; group 2=C4d positive, DSA positive, and no graft dysfunction; group 3=C4d positive, DSA positive, and signs of graft dysfunction, and group 0 (control)=all negative. An higher mortality risk was found in C4d-positive patients, when compared with negative ones (unadjusted hazard ratios: group 1: 18, group 2: 61, and group 3: 32-fold risk; P

For this study we consulted the Bone Marrow Donors' Registry of Lombardy (Italy) and analyzed 43937 HLA-A,B phenotypes and 13922 HLA-A,B,DR phenotypes. We estimated the HLA-A,B and HLA-A,B,DR haplotype frequencies via the maximum-likelihood method. We analyzed the genetic structure of the 11 provinces of Lombardy by means of Principal Component Analysis and Correspondence Analysis, and estimated the variety of the different haplotypes at provincial level and the percentage of unique phenotypes at village level. We found 11189 different HLA-A,B phenotypes, 661 different HLA-A,B haplotypes and more than 4000 different HLA-A,B,DR haplotypes. We identified 20 villages, in Western Lombardy, very rich in unique/rare phenotypes. Here we report a formula which allows the identification of a putative donor matched for two haplotypes with a recipient. This result may be of great importance for the genetic study of the population of Lombardy and, even more, for bone marrow transplantation programs.

Polymerase chain reaction using sequence-specific oligonucleotide probes (PCR-SSOP) typing was used to analyze HLA class I A, B and C alleles in three different Colombian populations. Fifty-nine samples were from Hispano-American Mestizos living in the urban areas of Cali (referred to here as Aso population). Forty-four and thirty samples were from the African Black populations of Zacarias (Zac) and Punta Soldado (PS), respectively. Samples were selected for expression of HLA-A2 by monoclonal antibody staining and allele-specific hybridization, and their HLA-A2 subtype distribution has been reported previously. Although only a limited number of samples was analyzed, the data suggest the existence of a remarkable degree of HLA class I polymorphism in the populations studied, with representatives of most serological classes. Despite their common African origin, the populations Zac and PS, both resident in malaria endemic regions, showed some striking differences in allelic distribution for all three class I loci. Furthermore, the samples from Aso and PS, but not Zac, showed a low percentage of blank alleles at the HLA-B locus (0 and 0.4%, respectively), suggesting the possibility of a heterozygote advantage for HLA-B alleles in Colombian populations.

This study describes a comprehensive easy to perform PCR-SSOP typing approach suitable for complete genomic subtyping of HLA-A*02. A single 1.6 kb PCR-amplificate spanning exons 2, 3 and 4 of the HLA-A*02 gene was used for hybridization with a panel of twenty-four SSOPs. This allowed unequivocal assignment of all so far known HLA-A2 subtypes, including A*0209 and A*0215N which differ for nucleotide substitutions in exon 4, without the need for two separate amplifications. Using this approach, HLA-A*02 subtype distribution was analyzed in 218 samples from unrelated, healthy individuals from northern Italy enrolled in the Italian Bone Marrow Registry and typed as HLA-A2 by serology or generic molecular analysis. As expected, A*0201 was found in the majority (92.6%) of samples. However, a significant number (6.8%) of individuals carried A*0205. Furthermore, A*0202, A*0208, A*0209 and A*0217, so far not described in Caucasians, were detected in a low number of samples (frequency ranging from 0.45% to 1.8%). Finally, a novel HLA-A*02 subtype, A*0220, was detected in 0.9% of the samples. As confirmed by DNA sequencing of exons 2 and 3, this allele is identical to A*0201 except for a single nucleotide substitution in codon 66 which changes the predicted amino acid sequence form Lys to Asn. The findings of this study have implications for the selection of HLA-A*02+ donors in unrelated bone marrow transplantation and of patients for specific immuno-therapy with HLA-A*02 restricted peptide vaccines.

The feasibility and utility of a lung donor score that has been recently proposed was tested among a pool of lung donors referred to the Nord Italia Transplant program (NITp) organ procurement organization.Each lung donor was assigned an Oto score including, age, smoking history, chest X-ray, secretions and ratio of arterial oxygen tension to inspired oxygen fraction (PaO(2)/FiO(2)). Based on clinical compromise, each variable received a score between 0 and 3, except for PaO(2)/FiO(2), which was scored between 0 and 6 given its overall relevance.Throughout 2010, 201 multiorgan donors were initially considered to be potential lung donors. Among these, 59 (29.4%) eventually yielded 67 lung transplantations (named "Used group"). Among the 142 (70.6%) refused lungs, 28 were not used due to logistic or medical problems ("general exclusion" group, GE) and 114, because of poor lung function ("lung exclusion" group, LE). Median lung donor scores were 1 (range, 0 to 3), 4 (range, 2.5 to 6.5), and 7 (range, 5 to 9) in the Used, GE, and LE groups, respectively (one-way analysis of variance, P.001). Some donors with Oto scores ≤7 worsened over time so that the score had significantly increased by the time of organ retrieval. Overall, subjects who died after lung transplantation were characterized by higher lung donor scores, (2 [1-4] versus 0.5 [0-3], P = .003).Our analysis suggested that the use of a donor score as a dynamic tool over the donation process was of great utility to describe and analyze a pool of lung donors.

Background The aim of our study was to evaluate the role of intravascular macrophages in the diagnosis of early and late antibody-mediated rejection (AMR) on endomyocardial biopsies (EMBs). Methods We reviewed 1,420 consecutive EMBs from 131 patients and selected 75 C4d + EMBs. The C4d + group was compared with a control group (66 patients) matched for age, gender, date of transplantation, follow-up, immunosuppressive regimen and primary heart disease. A total of 141 EMBs were evaluated. Immunoperoxidase staining for C4d and CD68 were performed. Post-transplant IgG anti-HLA reactivity was investigated by Luminex technology. Clinical data were also collected. Fourteen EMBs were available from 11 symptomatic AMR patients. Results Of the 141 EMBs evaluated, 53 were positive for intravascular macrophages (CD68); among them, 32 were also positive for C4d (32 of 53, 60.4%). Of the 88 CD68 − EMBs, 43 were also C4d + (43 of 88, 48.9%). Of the 53 CD68 + EMBs, 30 EMBs were within the first year since transplantation (30 of 53, 57.8%), and among these 21 were also positive for C4d (21 of 30, 70.0%). In the late period, among the 23 CD68 + EMBs (23 of 53, 42.2%) 11 were also positive for C4d (11 of 23, 47.8%). In the early period, intravascular macrophages were more common in symptomatic (3 of 3, 100%) than asymptomatic (3 of 11, 27.3%) patients. Sensitivity and specificity of intravascular macrophages in predicting donor-specific antibodies (DSA) within the first year were 50.0% and 100.0%, respectively. Conclusions Intravascular macrophages predict C4d, DSA and symptoms early after transplantation; however, in the late period, they are unable to identify patients with circulating DSA, C4d and/or symptoms.

Background The present study evaluated if morphologic parameters detect signs of early sub-clinical or latent stages of antibody-mediated rejection (AMR) and their correlation with C4d staining in cardiac transplants recipients. Methods The study reviewed 1,270 endomyocardial biopsies (EMB) from 131 patients. Of these, 61 stained positive for C4d in the absence of acute cellular rejection >2R. Sixty-six EMB specimens negative for C4d were matched for pre-transplant diagnosis, time after transplantation, age, and acute cellular rejection (ACR) grading. Histopathologic evaluation and C4d staining were performed on formalin-fixed, paraffin-embedded sections using the C4d polyclonal antibody. Results Of the 8 histologic characteristics evaluated, only endothelial swelling (78.7% sensitivity, 28.8% specificity; positive likelihood ratio, 1.10) and interstitial edema (77% sensitivity, 31.8% specificity; positive likelihood ratio, 1.13) could be considered fair predictors of C4d capillary positivity. The presence of mononuclear cells in capillaries in relation to C4d positivity showed 39.3% sensitivity and 68.2% specificity. Combining the parameters endothelial swelling and mononuclear cells in capillaries, sensitivity was 31.1% (95% confidence interval [CI] 19.9–44.3) and specificity was 71.2% (95 CI, 58.8–81.7), with a positive likelihood ratio of 1.08 (95% CI, 0.68–1.84). Conclusions Our results showed that histologic parameters did not always detect signs of early sub-clinical or latent stages of AMR. Combining the parameters of endothelial swelling and intracapillary mononuclear cells did not significantly improve the sensitivity or specificity. Screening recommendations should, therefore, be modified to include more sensitive tests such as C4d staining in the routine protocol to improve patient risk stratification.

In Italy, like everywhere in the world, the organ shortage for transplantation is a real problem. It is well known that lung donors (LD) are particularly difficult to procure and that management of the organ do not care during the diagnosis of cerebral death represents a difficult challenge. In this context, the salvage of the so-called "marginal donors" may increase the pool of donors, favoring organ retrieval. To increase lung procurement, the intensivist must recognize "marginal donors," optimizing organ selection and function. The aim of our study was to review LD procured in 2008, as identified by the unrestricted criteria, of the Nord Italian Transplant program Center (NITp). Particularly, the age and habits of donors and the presence of a parenchyma contusion were not sufficient per se to exclude donation. We revisited lung ventilation and monitoring modalities during cerebral death before retrieval. In 2008, the application of enlarged criteria for LD enabled us to collect 21 LD, namely 33% of all cerebral deaths, versus 13% in 2007. Seeking to maintain good gas exchange and lung function, we implemented a safe ventilation program avoided high peak pressures, and fluid therapy properly guided by the cardiac index and extravascular lung water index monitoring. Specific actions to improve LD procurement may help cope with the organ-donor shortage. Although our series was small, our results were encouraging; they underline the necessity to continuously review donor criteria and care, allowing good donor/recipient matching.

Humoral rejection in heart transplantation is associated with graft dysfunction, circulating anti-donor antibodies and C4d deposits in endomyocardial biopsies. Detecting C4d capillary positivity is of diagnostic and prognostic value. C4d positivity can be found in solid-organ transplants in cases of "accommodation," a form of humoral rejection without graft dysfunction. Accommodation might reflect a change in antibodies or in the antigen, or the graft acquiring a resistance to injury by antibodies and complement. We present a case of accommodation in the setting of adult ABO-compatible orthotopic heart transplantation, which was diagnosed according to the recently introduced ISHLT criteria for humoral rejection: despite this immunologic profile, the patient never showed signs of graft dysfunction. Physicians should be aware of the accommodation phenomenon so they can identify this subset of patients and assess its long-term effects on chronic rejection and outcome in transplanted patients.

Sir: In a recent paper Houzvicka and Ponec (1997) examined the various mechanisms of skeletal isomerization of butene proposed in the literature. Among them, the mechanisms which we proposed in the case of ferrierite catalysts, (i) bimolecular with fresh HFER samples (Guisnet et al., 1996a) and (ii) pseudomonomolecular (involving anchored carbocations as sites) with aged HFER samples (Guisnet et al., 1995, 1996b), are rejected by these authors owing to a misinterpretation of our proposals and of those of Meriaudeau et al. (1996). The previously reported observations in favor of these mechanisms are summarized here, and complementary results are presented.

This study describes sequence specific oligonucleotide probe (SSOP) typing of hypervariable regions in exons 2 and 3 of HLA-B locus genes. A single HLA-B specific PCR-product spanning from bp 84 in exon 2 to bp 241 in exon 3 was used for dot blot hybridization to forty-seven chemiluminescent labeled oligonucleotide probes. Thirty-one of these probes were derived from four hypervariability zones in exon 3 of HLA-B genes and covered most known sequence polymorphisms within these regions. In addition, sixteen probes derived from polymorphic regions in exon 2 were used to discriminate alleles not unequivocally characterized by the exon 3 based probes. This SSOP panel gave rise to eighty-six distinct hybridization patterns that could be used to unequivocally define all WHO-designated serological HLA-B specificities except for HLA-B54 in all homo- and heterozygous combinations. Furthermore, sixty-six out of ninety-seven molecularly defined HLA-B subtypes were characterized by unique hybridization patterns in all homozygous and most (possibly all) heterozygous combinations. The reproducibility of these results was confirmed by analysis of forty-four Workshop reference cell lines and of seventy-eight randomly chosen samples (one-hundred forty-seven alleles) from unrelated individuals serologically typed in the laboratory. For sixty-five samples (one-hundred-thirty-three alleles), molecular typing confirmed the results obtained by serology and allowed molecular subtype assignment for ninety-one alleles tested. A serologically blank allele could be defined by molecular analysis in three cases. The method presented here for molecular typing of the HLA-B locus can be used as an alternative to biochemical methods such as one-dimensional isoelectric focusing for assignment of serologically cross-reacting HLA-B molecules as well as for subtype characterization of a large variety of HLA-B alleles.

Insulin-dependent diabetes meUitus (IDDM) is an autoimmune disease that results in the destruction of the pancreatic islet β cells. Glutamic acid decarboxylase (GAD) has been recently indicated as a key autoantigen in the induction of IDDM in nonobese diabetic mice. In human diabetes, the mechanism by which the β cells are destroyed is still unknown. Here we report the first evidence for the presence of GAD-specific cytotoxic T cells in asymptomatic and recent diabetic patients. GAD65 peptides displaying the human histocompatibility leukocyte antigen (HLA)-A*0201 binding motif have been synthesized. One of these peptides, GAD114-123, binds to HLAA* 0201 molecules in an HLA assembly assay. Peripheral blood mononudear cells from individuals with preclinical IDDM, recent-onset IDDM, and from healthy controls were stimulated in vitro with the selected peptide in the presence of autologous antigen-presenting cells. In three cases (one preclinical IDDM and two recent-onset IDDM), we detected specific killing of autologous antigen-presenting cells when incubated with GAD114-123 peptide or when infected with a recombinant vaccinia virus expressing GAD65. These patients were the only three carrying the HLA-A*0201 allele among the subjects studied. Our finding suggests that GAD-specific cytotoxic T lymphocytes may play a critical role in the initial events of IDDM. © 1995, Rockefeller University Press., All rights reserved.

Purpose: According to a recent consensus conference on antibody-mediated rejection (AMR), AMR should be defined solely by pathologic criteria. The presence of donor specific antibody (DSA) was not mandated in the AMR definition as DSA were not always detected during AMR episodes. This may be due to DSA being adhered to the donor heart. It is believed that the detection of DSA during AMR episodes might lead to worse outcome. We reviewed our heart transplant patients with AMR to assess this hypothesis. Methods and Materials: We evaluated 53 patients status post heart transplant from 2003 to 2010 with treated AMR within the first year post transplant. Of these, 14 patients had concomitant circulating DSA and 39 patients did not. Outcomes were compared for subsequent 3-year survival, freedom from cardiac allograft vasculopathy (CAV, stenosis 30%), and freedom from non-fatal major adverse cardiac events (NF-MACE: myocardial infarction, heart failure, coronary stenting, need for defibrillator, stroke, and new peripheral vascular disease). Results: Patients who developed treated AMR with and without circulating DSA were found to have similar subsequent 3-year survival (71% vs. 82%, p 0.37), subsequent 3-year freedom from CAV (80% vs. 86%, p 0.62), and freedom from NF-MACE (100% vs. 92%, p 0.29). There was no difference in the incidence of hemodynamic compromise rejection between groups (7% vs. 8%, p 0.96). Conclusions: The finding of DSA in conjunction with pathology confirmed AMR does not appear to impact 3-year outcome after heart transplantation. Further studies to assess strength and other characteristics of DSA during AMR episodes may shed light to define its role in clinical AMR.

One hundred haemodialysed patients have been treated for 4 h thrice weekly by acetate haemodialysis with high-flux dialysers (HAHD) 1.4-1.8 m2 and automated ultrafiltration control for 18.9 +/- 8.3 months. The aim of the study was to evaluate the efficacy of treatment as regards urea and beta 2-microglobulin removal, cardiovascular stability, acid-base balance and plasma Il-1 variations. Moreover medium-term observation of both lipid profile and basal beta 2-microglobulin concentrations were performed. With our current adequacy criteria (urea clearance greater than or equal to 120 l/week; KT/V greater than or equal to 1.2; beta 2-microglobulin removal greater than or equal to 150 mg/treatment), the occurrence of hypotension was 6% during 24,500 treatments. Cardiovascular stability was preserved by the increase of total peripheral resistances in response to cardiac output decrease. No disturbances of acid-base parameters were observed in spite of plasma acetate concentrations greater than 6 mmol/l. No variations of plasma Il-1 occurred during the session or 2 h later.

Sixty out-patients with acute or sub-acute irritable colon were randomly allocated to receive 3 daily doses of 100 mg tiropramide, 150 mg trimebutine maleate or 20 mg octilonium bromide, orally during 5 consecutive days. Before and after treatment, multiple colonic manometry was performed, monitoring tonus, intensity and frequency of sinusoid contraction waves, transitories and vibrations, as well as the voluntary contraction capacity. Before treatment and after 2 and 5 days, the specific symptoms were also monitored, scored and recorded. Significant variations in tonus were not observed with any drug, but while tiropramide left unmodified the voluntary contractile ability, a significant inhibition was observed with trimebutine and, mainly, with octilonium. The overall power of spontaneous colonic contractions did not vary significantly with any drug. However, while with tiropramide a significant redistribution of muscular power was observed so as to increase propulsion waves and to decrease the ineffective transitory and vibrational contractions, with octilonium and trimebutine no clinically relevant redistribution of the power wasted in transient spasms was observed. Based on these observations, tiropramide was considered to be at least as effective an antispasmodic as octilonium and at least as effective a synchronizer as trimebutine, but was different from both reference drugs because it was the only one to act simultanously as both an antispasmodic and a synchronizer. The three drugs produced an improvement in each and all monitored symptoms as well as in the overall symptom intensity. Tiropramide, however, produced an improvement significantly faster, more progressively and to a greater extent than either reference drug.(ABSTRACT TRUNCATED AT 250 WORDS)