Effects of opioids on proximal renal tubular cells undergoing ATP depletion.

Abstract This study investigated the effect of morphine, fentanyl, butorphanol and buprenorphine on viability and caspase-3 activity in renal proximal tubular cells exposed to opioids for 2 h before or 12 h after chemical anoxia. Cell viability decreased regardless the treatment although intracellular ATP content was elevated in morphine and fentanyl pre-treated cells at 12 h. Anoxia increased caspase activity but this effect was significantly reduced in cells treated before or after with morphine, fentanyl and in cell treated with butorphanol for 12 h. No influence of buprenorphine was detected. Morphine, fentanyl and butorphanol might have protective effects during kidney ischemia

The role of antibody responses against glycans in bioprosthetic heart valve calcification and deterioration

Outcomes research; Risk factorsInvestigación de resultados; Factores de riesgoRecerca dels resultats; Factors de riscBioprosthetic heart valves (BHVs) are commonly used to replace severely diseased heart valves but their susceptibility to structural valve degeneration (SVD) limits their use in young patients. We hypothesized that antibodies against immunogenic glycans present on BHVs, particularly antibodies against the xenoantigens galactose-α1,3-galactose (αGal) and N-glycolylneuraminic acid (Neu5Gc), could mediate their deterioration through calcification. We established a large longitudinal prospective international cohort of patients (n = 1668, 34 ± 43 months of follow-up (0.1–182); 4,998 blood samples) to investigate the hemodynamics and immune responses associated with BHVs up to 15 years after aortic valve replacement. Early signs of SVD appeared in <5% of BHV recipients within 2 years. The levels of both anti-αGal and anti-Neu5Gc IgGs significantly increased one month after BHV implantation. The levels of these IgGs declined thereafter but anti-αGal IgG levels declined significantly faster in control patients compared to BHV recipients. Neu5Gc, anti-Neu5Gc IgG and complement deposition were found in calcified BHVs at much higher levels than in calcified native aortic valves. Moreover, in mice, anti-Neu5Gc antibodies were unable to promote calcium deposition on subcutaneously implanted BHV tissue engineered to lack αGal and Neu5Gc antigens. These results indicate that BHVs manufactured using donor tissues deficient in αGal and Neu5Gc could be less prone to immune-mediated deterioration and have improved durability.This work was supported by the European Union Seventh Framework Program (FP7/2007/2013) under grant agreement no. 603049 for the Translink Consortium. This research was also funded by a European Union H2020 Program grant no. ERC-2016-STG-716220 to V.P-K. and by the Elizabeth and Nicholas Slezak Super Center for Cardiac Research and Medical Engineering (to V.P-K.). This work was supported by an Institut National de la Santé et de la Recherche Médicale translational grant no. 2012-2016 to T.L.T. This work was supported by the Ministerio de Economía y Competitividad-ISCiii (PI15/00181) and the PERIS SLT002/16/00445 funded by the Department of Health of Generalitat de Catalunya (both granted to C.C.), and cofunded by FEDER (European Regional Development Fund), a way to build Europe. This work was supported by an Israel Ministry of Science & Technology PhD fellowship to S.B. We thank L. Adler for her assistance in the affinity purification of anti-Neu5Gc antibodies and IgG subclass analysis. Finally, we thank N. Bovin from the Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, who provided the Bdi-C3 PAA substrate needed to develop the anti-αGal assays

Differential Immune Response to Bioprosthetic Heart Valve Tissues in the α1,3Galactosyltransferase-Knockout Mouse Model

Structural valve deterioration (SVD) of bioprosthetic heart valves (BHVs) has great clinical and economic consequences. Notably, immunity against BHVs plays a major role in SVD, especially when implanted in young and middle-aged patients. However, the complex pathogenesis of SVD remains to be fully characterized, and analyses of commercial BHVs in standardized-preclinical settings are needed for further advancement. Here, we studied the immune response to commercial BHV tissue of bovine, porcine, and equine origin after subcutaneous implantation into adult a1,3-galactosyltransferase-knockout (Gal KO) mice. The levels of serum anti-galactose a1,3-galactose (Gal) and -non-Gal IgM and IgG antibodies were determined up to 2 months post-implantation. Based on histological analyses, all BHV tissues studied triggered distinct infiltrating cellular immune responses that related to tissue degeneration. Increased anti-Gal antibody levels were found in serum after ATS 3f and Freedom/Solo implantation but not for Crown or Hancock II grafts. Overall, there were no correlations between cellular-immunity scores and post-implantation antibodies, suggesting these are independent factors differentially affecting the outcome of distinct commercial BHVs. These findings provide further insights into the understanding of SVD immunopathogenesis and highlight the need to evaluate immune responses as a confounding factor

“Clinical Stability” and Propensity Score Matching in Cardiac Surgery: is the clinical evaluation of treatment efficacy algorithmdependent in small sample size settings?

Background: Propensity score matching represents one of the most popular techniques to deal with treatment allocation bias in observational studies. However, when the number of enrolled patients is very low, the creation of matched set of subjects may highly depend on the model used to estimate individual propensity scores, undermining the stability of consequential clinical findings. In this study, we investigate the potential issues related to the stability of the matched sets created by different propensity score models and we propose some diagnostic tools to evaluate them. Methods: Matched groups of patients were created using five different methods: Logistic Regression, Classification and Regression Trees, Bagging, Random Forest and Generalized Boosted Model. Differences between subjects in the matched sets were evaluated by comparing both pre-treatment covariates and propensity score distributions. We applied our proposal to a cardio-surgical observational study that aims to compare two different procedures of cardiac valve replacement. Results: Both baseline characteristics and propensity score distributions were systematically different across matched samples of patients created with different models used to estimate propensity score. The most relevant differences were observed for the matched set created by estimating individual propensity scores with Classification and Regression Trees algorithm. Conclusion: Clinical stability of matched samples created with different statistical methods should always be evaluated to ensure reliability of final estimates. This work opens the door for future investigations that fully assess the implications of this finding

Spontaneous coronary artery dissection in women with acute myocardial infarction: is there a new role for autoimmunity?

Aims Spontaneous coronary artery dissection (SCAD) is an uncommon cause of acute myocardial infarction in women and has an unclear pathophysiology. Autoantibodies (AAs) targeting angiotensin-II receptor type 1 (AT1R) and endothelin-1 receptor type A (ETAR) have known detrimental effects on endothelial function. We investigated the prevalence of these AAs in SCAD-affected female patients. Methods and results Female patients diagnosed at coronary angiography with myocardial infarction and SCAD were consecutively enrolled. Autoantibodies targeting angiotensin-II receptor type 1 and ETAR-AA titres and seropositivity prevalence were compared between SCAD patients, ST-elevation myocardial infarction (STEMI) patients, and healthy women. Ten women with SCAD and 20 age-matched controls (10 women with STEMI and 10 healthy women) were included. Six out of 10 (60%) women with myocardial infarction and SCAD were seropositive for AT1R-AAs and ETAR-AAs. In contrast, only one (10%) healthy woman and one (10%) STEMI patient were seropositive for AT1R-AAs (P = 0.03 and P = 0.03, respectively). One STEMI patient was seropositive for ETAR-AAs, while none of the healthy women was found to be seropositive (P = 0.03 and P = 0.01, respectively). The median AA titre was significantly higher in SCAD patients than in healthy women (P = 0.01 for AT1R-AAs; P = 0.02 for ETAR-AAs) and STEMI patients (P < 0.001 for AT1R-AAs; P = 0.002 for ETAR-AAs). Conclusion Autoantibodies targeting angiotensin-II receptor type 1 and ETAR-AA seropositivity is significantly higher in SCAD women with myocardial infarction than in healthy women or female patients with STEMI. Our findings, corroborated by previous data in the literature and biological plausibility, suggest a possible role for AT1R-AAs and ETAR-AAs in the pathophysiology of SCAD in women with acute myocardial infarction and should warrant further studies with larger sample sizes

LYMPHOCRYPTOVIRUS-ASSOCIATED PTLD VIRAL TRANSCRIPT EXPRESSION IN 8 XENOTRANSPLANTED CYNOMOLGUS MACAQUES

The aim of this work is to characterize posttransplant lymphoproliferative disorders (PTLD), frequently related to Epstein Barr Virus, observed in 8 adult immunosuppressed cynomolgus macaques, which received neural precursors from CTLA4Ig transgenic pigs to treat a pharmacologically-induced form of Parkinson's disease. The mean time of occurrence of the first clinical signs of PTLD was 177.62 days and the mean survival time was 199.87 days. 87.5% of PTLD occurred in the nasal cavity (50%), intestinal tract (50%), or both (12.5%); 50% with lymph nodal involvement. The histological diagnosis and immunohistochemical profile (anti-CD3, anti-CD5, anti-CD20, anti-CD79cyt, Ki-67) is compatible with a monomorphic PTLD, most frequently diffuse large B-cell lymphoma, high grade. Double-labeled immunohistochemistry for Epstein-Barr virus nuclear antigen (EBNA)-2 and CD20 revealed a high percentage of CD20+ neoplastic cells also positive for EBNA-2, while CD3+ cells in the tumors were negative for EBNA2. RT-PCR of transcripts EBNA-1 and Latent Membrane Protein-1 (LMP-1) have been performed in neoplastic cells from 5/8 PTLD primates and 2 hyperplastic lymph nodes from 2 PTLD+ primates (control). A consensus-PCR with specificity for EBNA-2 gene was performed on PTLD+ samples with a 92% of sequence identity between Rhesus and cynomolgus viruses. EBNA-1 and EBNA-2 expression was demonstrated in all PTLD+ specimens (no expression in controls). Four different RT-PCR assays failed to detect LMP-1 in all specimens. This report identifies in detail the pathological features of PTLD in cynomolgus monkeys in the setting of neuronal transplantation and a specific pattern of viral transcript expression (EBNA-1+ EBNA-2+ LMP-1-) in Lymphocryptovirus-associated PTLD

CARATTERIZZAZIONE IMMUNOISTOCHIMICA E MOLECOLARE DI DISORDINI LINFOPROLIFERATIVI IN PRIMATI XENOTRAPIANTATI CON PRECURSORI NEURONALI

Introduzione: I disordini linfoproliferativi post-trapianto (PTLD) rappresentano un gruppo di malattie che insorgono in corso d\u2019immunosoppressione dopo il trapianto (Tx). La maggior parte dei PTLD nell\u2019uomo e nel primate sono dovuti alla presenza di Epstein-Barr virus e Lymphocryptovirus (MacLCV). Materiali e Metodi: 28 adulti di Macaca fascicularis con malattia di Parkinson farmacologicamente indotta hanno ricevuto precursori neuronali di suini transgenici per CTLA4Ig (n=7) e wild-type (n=2).. La caratterizzazione di PTLD ha riguardato aspetti morfologici, immunoistochimici (IHC) e molecolari. Risultati: i PTLD sono stati diagnosticati in 9 animali, ad una distanza media di 172,7 giorni dal tx.. Le neoplasie erano localizzate nelle cavit\ue0 nasali (n=3), nell\u2019intestino (n=4) e in entrambe le sedi (n=2). Istologia e IHC hanno rivelato un PTLD monomorfo (linfoma a grandi cellule B) di grado elevato. IHC a doppia marcatura per EBNA2 e CD20 ha dimostrato un\u2019elevata percentuale di cellule neoplastiche CD20+ e EBNA2+. RT-PCR e sequenziamento dei trascritti di EBNA-1, EBNA-2 e LMP-1 eseguiti su 5 primati PTLD+ hanno evidenziato che EBNA-1 e EBNA-2 sono sempre presenti, mentre l\u2019espressione di LMP1 \ue8 assente. Il DNA di MacLCV \ue8 stato isolato in tutti i primati riceventi ela viremia \ue8 stata evidenziata circa 60 gg prima dei sintomi clinici. Conclusioni: lo studio identifica un pattern di espressione dei trascritti virali nel PTLD associato a MacLCV: EBNA-1+ EBNA-2+ LMP-1-. Nel ricevente si evidenzia un picco viremico di MacLCV prima dell\u2019insorgenza del PLTD. Tale test potrebbe essere utilizzato come strumento precoce per la diagnosi di PTLD

Bone marrow and haemathological evaluation of 62 xenografted cynomolgus monkeys

Background: Non-human primates (NHP) are used in pre-clinical studies, such as pig-to-NHP xenotransplantation. In this context, the evaluation of clinical-pathological data is essential for monitoring the post-transplantation period. Cytological examination of bone marrow (BM) is a helpful adjunct for a complete interpretation of haematological data. The aim of this work is to evaluate the cytology of BM and haematological data in immunosuppressed (IS) xenotransplanted NHP. Methods: Sixty-two xenografted NHP were included: 36 recipients of porcine renal xenograft (RX group) and 17 recipients of neuronal precursors (NPX group). Both groups received a maintenance immunosuppression with cyclosporine A, sodium mycophenolate and steroids. RX group received an induction therapy consistent of cyclophosphamide and/or GAS914, or rituximab, or IVIG or anti-CD154; NPX received cyclosporine A and GAS914. A complete CBC and femoral BM smears collected at euthanasia were obtained. BM cytological evaluation and differential counts of erythroid, granulocytic, megakaryocytic, lymphocytic and plasmacytic series were performed. The myeloid-to-erythroid ratio (M:E), the erythroid (E-MI) and myeloid (M-MI) maturation indices were calculated. Results: Compare to the reference range, 44 NHP showed an increased M:E ratio (>1.85) due to myeloid hyperplasia and erythroid hypoplasia. In 16 cases the M:E ratio was severely increased (>5.45). Both E-MI and M-MI were raised in 61 and 57 cases, respectively. The highest values of M:E and M-MI were recorded in the RX group. The main haemathological alterations observed were anemia (54/62 NHP), leucopenia (15/62) and lymphopenia (25/62). Cytoplasmatic and nuclear vacuolization, dysplastic changes in granulocytic and erythroid lineages were the main morphological alterations in BM probably due to toxic effects of IS therapy. In spite of IS therapy, xenografted NHP showed a myeloid hyperplasia, mainly in RX group probably due to the marked inflammatory status during acute rejection. Conclusions: The BM evaluation in xenografted IS NHP may provide the basis for a complete interpretation of hematological profiles and represent an important tool for a better design of IS strategies