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Donor brain death-induced lung injury may compromise graft function after transplantation. Establishing strategies to attenuate lung damage remains a challenge because the underlying mechanisms remain uncertain. The effects of tacrolimus pretreatment were evaluated in an experimental model of brain death-induced lung injury. Brain death was induced by slow intracranial infusion of blood in anesthetized pigs after randomization to tacrolimus (orally administered at 0.25 mg. kg-1 BID the day before the experiment and intravenously at 0.05 mg. kg-1 one hour before the experiment; n=8) or placebo (n=9) pretreatment. Hemodynamic measurements were performed 1, 3, 5 and 7 hours after brain death. After euthanasia of the animals, lung tissue was sampled for pathobiological and histological analysis, including lung injury scoring (LIS). Tacrolimus pretreatment prevented increases in pulmonary artery pressure, pulmonary vascular resistance and pulmonary capillary pressure and decreases in systemic artery pressure and thermodilution cardiac output associated with brain death. After brain death, the ratio of the partial arterial O2 pressure to the inspired O2 fraction (PaO2/FiO2) decreased, which was prevented by tacrolimus. Tacrolimus pretreatment prevented increases in the interleukin (IL)-6-to-IL-10 ratio, vascular cell adhesion molecule-1, circulating levels of IL-1β, IL-6-to-IL-10 ratio and glycocalyx-derived molecules. Tacrolimus partially decreased apoptosis [Bax-to-Bcl2 ratio (p=0.07) and the number of apoptotic cells in the lungs (p

Background: Right ventricular (RV) dysfunction remains a major problem after heart transplantation and may be associated with brain death (BD) in a donor. A calcineurin inhibitor tacrolimus was recently found to have beneficial effects on heart function. Here, we examined whether tacrolimus might prevent BD-induced RV dysfunction and the associated pathobiological changes. Methods: After randomized tacrolimus (n = 8; 0.05 mg·kg−1·day−1) or placebo (n = 9) pretreatment, pigs were assigned to a BD procedure and hemodynamically investigated 1, 3, 5, and 7 h after the Cushing reflex. After euthanasia, myocardial tissue was sampled for pathobiological evaluation. Seven pigs were used as controls. Results: Calcineurin inhibition prevented increases in pulmonary vascular resistance and RV-arterial decoupling induced by BD. BD was associated with an increased RV pro-apoptotic Bax-to-Bcl2 ratio and RV and LV apoptotic rates, which were prevented by tacrolimus. BD induced increased expression of the pro-inflammatory IL-6-to-IL-10 ratio, their related receptors, and vascular cell adhesion molecule-1 in both the RV and LV. These changes were prevented by tacrolimus. RV and LV neutrophil infiltration induced by BD was partly prevented by tacrolimus. BD was associated with decreased RV expression of the β-1 adrenergic receptor and sarcomere (myosin heavy chain [MYH]7-to-MYH6 ratio) components, while β-3 adrenergic receptor, nitric oxide-synthase 3, and glucose transporter 1 expression increased. These changes were prevented by tacrolimus. Conclusions: Brain death was associated with isolated RV dysfunction. Tacrolimus prevented RV dysfunction induced by BD through the inhibition of apoptosis and inflammation activation.

Background Acute kidney injury (AKI) requiring renal replacement therapy (RRT) in the ICU portends a poor prognosis. We aimed to better characterize predictors of survival and the mechanism of kidney failure in these patients. Methods Retrospective observational study using clinical and radiological electronic health records, analysed by univariable and multivariable binary logistic regression. Histopathological examination of post-mortem renal tissue was performed. Results Among 157 patients with AKI requiring RRT, higher serum creatinine at RRT initiation associated with increased ICU-survival (OR 0.33, 95%CI 0.17–0.62, p = 0.001) however, muscle mass (a marker of frailty) interacted with creatinine (p = 0.02) and superseded creatinine as a predictor of survival (OR 0.26 95%CI 0.08–0.82; p = 0.02). Achieving lower cumulative fluid balance (mls/kg) predicted ICU-survival (OR 1.01 95%CI 1.00 -1.01, p 500mls/24 hours strongly predicted successful liberation from RRT (OR 0.125, 95%CI 0.05–0.35, p Conclusions Confounding of creatinine by muscle-mass undermines its use as a marker of AKI severity in clinical studies. Volume management and urine-output are key determinants of outcome. Loss of synaptopodin implicates glomerular injury in the pathogenesis of S-AKI.

Ovarian-like epithelial tumors of the testis, including serous borderline tumors, are rare entities. We report the case of a 60-year-old man with a left intratesticular mass who had a radical orchidectomy. Histologically, the tumor was identical to the ovarian counterpart showing a well-delineated cystic lesion characterized by intraluminal papillae. The papillae are lined by atypical cuboidal or ciliated cells and are associated with psammoma bodies. The tumor cells express cytokeratin 7 (CK7), cytokeratin 5-6 (CK5-6), cancer antigen 125 (CA125), estrogen (ER), progesterone (PR), Wilm’s tumor gene (WT1), paired box gene 8 (PAX8), Ber-EP4, and epithelial membrane antigen (EMA). The diagnosis of a serous borderline tumor of the testis was proposed. Mutation testing using next-generation sequencing showed a Q61K KRAS gene mutation. To the best of our knowledge, this is the second case report of a serous borderline tumor of the testis with a Q61K KRAS gene mutation.

Metastatic melanoma is a fatal disease with poor prognosis. Ever since targeted therapy against oncogenic BRAF was approved, molecular profiling has become an integral part of the management of such patients. While molecular testing is not available in all pathology laboratories, immunohistochemistry (IHC) is a reliable screening option. The major objective of the present study was to evaluate whether IHC detection of BRAF and the tumor (suppressor) protein 53 gene (TP53) are reliable surrogates for mutation detection. Formalin-fixed paraffin-embedded samples of melanomas for which molecular data were previously obtained by targeted next-generation sequencing (NGS) between January 2014 and February 2019 were immunostained with BRAF V600E and p53 antibodies. A blinded evaluation of the IHC slides was performed by two pathologists in order to evaluate inter-observer concordance (discordant cases were reviewed by a third observer). The associations between the results of IHC and molecular profiling were evaluated. The study included a series of 37 cases of which 15 harbored a BRAF mutation and five a TP53 mutation. IHC had an overall diagnostic accuracy of 93.9% for BRAF V600E and 68.8% for TP53 compared to NGS. A statistically significant association between the two diagnostic methods was obtained for BRAF V600E (P=0.0004) but not for p53 (P=0.3098) IHC. The κ coefficient for IHC assessment of p53 was 0.55 and that for BRAF V600E was 0.72. In conclusion, the present results evidenced that IHC staining is a reliable surrogate for NGS in identifying the BRAF V600E mutation, which may become an efficient screening tool. Aberrant expression of p53 on IHC is at times associated with TP53 mutations but it was not possible to establish a direct link.

Intramedullary astrocytomas (IMAs) consist of a heterogeneous group of rare central nervous system (CNS) tumors associated with variable outcomes. A DNA methylation-based classification approach has recently emerged as a powerful tool to further classify CNS tumors. However, no DNA methylation-related studies specifically addressing to IMAs have been performed yet. In the present study, we analyzed 16 IMA samples subjected to morphological and molecular analyses, including DNA methylation profiling. Among the 16 samples, only 3 cases were classified in a reference methylation class (MC) with the recommended calibrated score (≥0.9). The remaining cases were either considered "no-match" cases (calibrated score, info:eu-repo/semantics/published

Immunotherapies based on immune checkpoint inhibitors are emerging as an innovative treatment for different types of advanced cancers. While the utility of immune checkpoint inhibitors has been clearly demonstrated, the response rate is highly variable across individuals. Due to the cost and toxicity of these immunotherapies, a critical challenge in this field is the identification of predictive biomarkers to discriminate which patients may respond to immunotherapy. Recently, a high tumor mutational burden (TMB) has been identified as a genetic signature that is associated with a favorable outcome for immune checkpoint inhibitor therapy. The TMB is defined as the total number of nonsynonymous mutations per coding area of a tumor genome. Initially, it was determined using whole exome sequencing, but due to the high costs and long turnaround time of this method, targeted panel sequencing is currently being explored to measure TMB. In the near future, TMB evaluation may play an important role in immuno-oncology, but its implementation in a routine setting involves robust analytical and clinical validation. Standardization is also needed in order to make informed decisions about patients. This review presents the methodologies employed for determining TMB and discusses the factors that may have an impact on its measurement., SCOPUS: re.j, info:eu-repo/semantics/published

Metastatic melanoma is a fatal disease with poor prognosis. Ever since targeted therapy against oncogenic BRAF was approved, molecular profiling has become an integral part of the management of such patients. While molecular testing is not available in all pathology laboratories, immunohistochemistry (IHC) is a reliable screening option. The major objective of the present study was to evaluate whether IHC detection of BRAF and the tumor (suppressor) protein 53 gene (TP53) are reliable surrogates for mutation detection. Formalin-fixed paraffin-embedded samples of melanomas for which molecular data were previously obtained by targeted next-generation sequencing (NGS) between January 2014 and February 2019 were immunostained with BRAF V600E and p53 antibodies. A blinded evaluation of the IHC slides was performed by two pathologists in order to evaluate inter-observer concordance (discordant cases were reviewed by a third observer). The associations between the results of IHC and molecular profiling were evaluated. The study included a series of 37 cases of which 15 harbored a BRAF mutation and five a TP53 mutation. IHC had an overall diagnostic accuracy of 93.9% for BRAF V600E and 68.8% for TP53 compared to NGS. A statistically significant association between the two diagnostic methods was obtained for BRAF V600E (P=0.0004) but not for p53 (P=0.3098) IHC. The κ coefficient for IHC assessment of p53 was 0.55 and that for BRAF V600E was 0.72. In conclusion, the present results evidenced that IHC staining is a reliable surrogate for NGS in identifying the BRAF V600E mutation, which may become an efficient screening tool. Aberrant expression of p53 on IHC is at times associated with TP53 mutations but it was not possible to establish a direct link.

BACKGROUND Acetaminophen overdose is the most frequent cause of drug-induced liver failure in developed countries. Substantial progress has been made in understanding the mechanism of hepatocellular injury, but N-acetylcysteine remains the only effective treatment despite its short therapeutic window. Thus, other hepatoprotective drugs are needed for the delayed treatment of acetaminopheninduced hepatotoxicity. Our interest focused on glycyrrhizin for its role as an inhibitor of high mobility group box 1 (HMGB1) protein, a member of the family of damage-associated molecular pattern, known to play an important pathological role in various diseases. AIM To investigate the efficacy of the N-acetylcysteine/glycyrrhizin combination compared to N-acetylcysteine alone in the prevention of liver toxicity. METHODS Eight-week-old C57BL/6J wild-type female mice were used for all our experiments. Mice fasted for 15 h were treated with acetaminophen (500 mg/kg) or vehicle (phosphate-buffered saline) by intraperitoneal injection and separated into the following groups: Glycyrrhizin (200 mg/kg); N-acetylcysteine (150 mg/kg); and N-acetylcysteine/glycyrrhizin. In all groups, mice were sacrificed 12 h following acetaminophen administration. The assessment of hepatotoxicity was performed by measuring plasma levels of alanine aminotransferase, aspartate aminotransferase and lactate dehydrogenase. Hepatotoxicity was also evaluated by histological examination of hematoxylin and eosin-stained tissues sections. Survival rates were compared between various groups using Kaplan-Meier curves. RESULTS Consistent with data published in the literature, we confirmed that intraperitoneal administration of acetaminophen (500 mg/kg) in mice induced severe liver injury as evidenced by increases in alanine aminotransferase, aspartate aminotransferase and lactate dehydrogenase but also by liver necrosis score. Glycyrrhizin administration was shown to reduce the release of HMGB1 and significantly decreased the severity of liver injury. Thus, the co-administration of glycyrrhizin and N-acetylcysteine was investigated. Administered concomitantly with acetaminophen, the combination significantly reduced the severity of liver injury. Delayed administration of the combination of drugs, 2 h or 6 h after acetaminophen, also induced a significant decrease in hepatocyte necrosis compared to mice treated with N-acetylcysteine alone. In addition, administration of N-acetylcysteine/glycyrrhizin combination was associated with an improved survival rate compared to mice treated with only N-acetylcysteine. CONCLUSION We demonstrate that, compared to N-acetylcysteine alone, co-administration of glycyrrhizin decreases the liver necrosis score and improves survival in a murine model of acetaminophen-induced liver injury. Our study opens a potential new therapeutic pathway in the prevention of acetaminophen hepatotoxicity., SCOPUS: ar.j, info:eu-repo/semantics/published

Post-mortem studies can provide important information for understanding new diseases and small autopsy case series have already reported different findings in COVID-19 patients., info:eu-repo/semantics/published

BackgroundPost-mortem studies can provide important information for understanding new diseases and small autopsy case series have already reported different findings in COVID-19 patients.MethodsWe evaluated whether some specific post-mortem features are observed in these patients and if these changes are related to the presence of the virus in different organs. Complete macroscopic and microscopic autopsies were performed on different organs in 17 COVID-19 non-survivors. Presence of SARS-CoV-2 was evaluated with immunohistochemistry (IHC) in lung samples and with real-time reverse-transcription polymerase chain reaction (RT-PCR) test in lung and other organs.ResultsPulmonary findings revealed early-stage diffuse alveolar damage (DAD) in 15 out of 17 patients and microthrombi in small lung arteries in 11 patients. Late-stage DAD, atypical pneumocytes and/or acute pneumonia were also observed. Four lung infarcts, two acute myocardial infarctions and one ischemic enteritis were observed. There was no evidence of myocarditis, hepatitis or encephalitis. Kidney evaluation revealed the presence of hemosiderin in tubules or pigmented casts in most patients. Spongiosis and vascular congestion were the most frequently encountered brain lesions. No specific SARS-CoV-2 lesions were observed in any organ. IHC revealed positive cells with a heterogeneous distribution in the lungs of 11 of the 17 (65%) patients; RT-PCR yielded a wide distribution of SARS-CoV-2 in different tissues, with 8 patients showing viral presence in all tested organs (i.e. lung, heart, spleen, liver, colon, kidney and brain).ConclusionsIn conclusion, autopsies revealed a great heterogeneity of COVID-19-related organ injury and the remarkable absence of any specific viral lesions, even when RT-PCR identified the presence of the virus in many organs.

In cancer biology, epithelial-to-mesenchymal transition (EMT) is associated with tumorigenesis, stemness, invasion, metastasis, and resistance to therapy. Evidence of co-expression of epithelial and mesenchymal markers suggests that EMT should be a stepwise process with distinct intermediate states rather than a binary switch. In the present study, we propose a morphological approach that enables the detection and quantification of cancer cells with hybrid E/M states, i.e. which combine partially epithelial (E) and partially mesenchymal (M) states. This approach is based on a sequential immunohistochemistry technique performed on the same tissue section, the digitization of whole slides, and image processing. The aim is to extract quantitative indicators able to quantify the presence of hybrid E/M states in large series of human cancer samples and to analyze their relationship with cancer aggressiveness. As a proof of concept, we applied our methodology to a series of about a hundred urothelial carcinomas and demonstrated that the presence of cancer cells with hybrid E/M phenotypes at the time of diagnosis is strongly associated with a poor prognostic value, independently of standard clinicopathological features. Although validation on a larger case series and other cancer types is required, our data support the hybrid E/M score as a promising prognostic biomarker for carcinoma patients., info:eu-repo/semantics/published

Introduction: As only ten cases have been described in current literature, urachal mucinous cystadenoma seems rare. All studied cases concerned adults or adolescents; this paper reports the first infant presenting urachal mucinous cystadenoma. Case report: A seven-month-old boy affected by urachal mucinous cystadenoma, presented as only sign persistent purulent umbilical discharge. Ultrasonography showed nodular structure with liquid content evoking urachal cyst. Radical surgical excision was performed. Histopathology proved diagnosis of urachal mucinous cystadenoma. No complications were observed. Follow-up consultation eight days postoperative was reassuring, evaluating infection and recurrence absence, and scar healing. Discussion: Preservation of urachus lumen may lead to urachal diseases: patent, diverticulum, sinus and cyst. Mucinous cystadenoma is rarely found in urachus. Solely umbilical purulent discharge was observed, in contrast to all reported cases. Same type of investigation, ultrasonography, was conducted to evoke diagnosis. Since urachal mucinous cystadenoma may transform into adenocarcinoma, prophylactic excision is recommended, and prognosis depends on its radicalness. No long-term outcome for infants is available in literature. We propose annual follow-up by ultrasonography. Conclusion: Urachal mucinous cystadenoma in infants is uncommon. Umbilical discharge must indicate ultrasonography. Surgical excision is recommended in case of suspicion. Long term outcomes are yet to be evaluated., SCOPUS: ar.j, info:eu-repo/semantics/published

Research on tumor angiogenesis has mainly focused on the vascular endothelial growth factor (VEGF) family and on methods to block its actions. However, reports on VEGF receptor (VEGFR) expression in tumor-associated endothelial cells (ECs) are limited. Thus, we evaluated VEGF, VEGFR-1 and VEGFR-2 expression in ECs of colorectal cancer (CRC) using immunohistochemistry. VEGF, VEGFR-1 and -2 expression in ECs was quantitatively evaluated by digital image analysis in a retrospective series of 204 tumor tissue samples and related to clinical variables. The data show that the VEGF, VEGFR-1 and VEGFR-2 expression in ECs is heterogeneous. Multivariate analysis including a set of clinicopathological variables reveals that high EC VEGFR-1 expression is an independent prognostic factor for overall survival (OS). The combination of low VEGFR-1 and high VEGFR-2 expression in ECs outperforms models integrating VEGFR-1 and VEGFR-2 as separate markers. Indeed, this VEGFR-1_VEGFR-2 combination is an independent negative prognostic factor for OS (p = 0.012) and metastasis-free survival (p = 0.007). In conclusion, this work illustrates the importance of studying the distribution of VEGF members in ECs of CRC. Interestingly, our preliminary data suggest that high VEGFR-1 and low VEGFR-2 expression in ECs appear to be involved in the progression of CRC, suggesting that targeting EC VEGFR-1 could offer novel opportunities for CRC treatment. However, a prospective validation study is needed., SCOPUS: ar.j, info:eu-repo/semantics/published

SCOPUS: ar.j, info:eu-repo/semantics/published

547 Background: Cisplatin-based NAC followed by surgery is the gold standard treatment of non-metastatic MIBC. However, predictive biomarkers have not been established yet. Here, we addressed the relevance of TILs for NAC response and prognosis in MIBC patients (pts). Methods: We conducted a single center, retrospective cohort analysis of consecutive MIBC pts treated with cisplatin-based NAC followed by surgery. Pts with pathological complete response (pCR; ypT0/isN0) were grouped as responders, pts with pathological residual disease as non responders. TIL stromal count were assessed pre- and post-NAC. Next Generation Sequencing of selected genes was performed on pre-NAC samples. We catalogued the 3-years(y) relapse free rate and 3-y overall survival rate (OS, Kaplan Meier) for the overall cohort and by subgroups. Results: Of the 25 pts who received cisplatin-gemcitabine NAC, a total of 14 (56%) pts achieved a pCR and 11 (44%) had residual disease. NAC was discontinued in 17 (65%) after completing treatment; and in 8 (32%) pts after 2 cycles due to toxicity. Median follow-up was 54.2 months(mos). At baseline, median TIL count was 60% in the overall cohort. Numerically higher TIL count was observed among responders (80%) compared to non-responders (50%) ( p=0.22). Median TIL count post-NAC in residual samples was 40%, not significantly different to matched pre-NAC samples ( p=0.93). Overall, 3-y relapse free rate was 74.7% (95%CI 51.7-87.9); 3-y OS rate was 79.6% (95%CI 57.6-91) with 6 deaths of which 4 were related to metastatic disease. Table reports subset analyses. Most frequently altered genes were PIK3CA and TP53 in the overall cohort (each 20%) and in the responders subgroup (each 29%). Conclusions: In our study, pts who achieved pCR had numerically greater TIL infiltrate in baseline samples and pCR was associated with reduced risk of recurrence. TILs did not increase in residual tumors after NAC. The independent predictive value of TILs warrants assessment in large cohorts.[Table: see text]

YAP and TAZ are key downstream regulators of the Hippo pathway, regulating cell proliferation and differentiation. YAP and TAZ activation has been reported in different cancer types. However, it remains unclear whether they are required for the initiation of major skin malignancies like basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Here, we analyze the expression of YAP and TAZ in these skin cancers and evaluate cancer initiation in knockout mouse models. We show that YAP and TAZ are nuclear and highly expressed in different BCC types in both human and mice. Further, we find that cells with nuclear YAP and TAZ localize to the invasive front in well‐differentiated SCC, whereas nuclear YAP is homogeneously expressed in spindle cell carcinoma undergoing EMT. We also show that mouse BCC and SCC are enriched for YAP gene signatures. Finally, we find that the conditional deletion of YAP and TAZ in mouse models of BCC and SCC prevents tumor formation. Thus, YAP and TAZ are key determinants of skin cancer initiation, suggesting that targeting the YAP and TAZ signaling pathway might be beneficial for the treatment of skin cancers.

The recent developments in anti-angiogenic and immunomodulatory drugs show that the tumour micro-environment (TME) becomes increasingly important in cancer research. Here we investigated the correlation between the Gleason score (GS) and the TME by comparing tissue expression profiles of steroid hormone receptors, cancer activated fibroblast (CAF) markers and vessel densities between different GS groups. Therefore, matched patient cohorts were composed for different GS (6-7-8). Tissue micro-arrays with 6 samples/patient were processed for immunohistochemistry. Stained slides were digitised, stroma and epithelium were selectively annotated, and all selected areas were quantitatively analysed for marker expression. The most striking findings were decreased stromal expression levels of several steroid hormone receptors, increased CAF-phenotypes and increased vessel densities in high GS prostate cancer compared to low GS prostate cancer and paired prostate non-tumour tissue. The present data reveal a complex correlation between prostate cancer differentiation and TME components and suggest that different GS can be associated with different possible actionable targets in the TME. The use of standardised digital image analysis tools generated robust and reproducible quantitative data, which is novel and more informative compared to the classic semi-quantitative and observer-dependent visual scoring of immunohistochemistry., SCOPUS: ar.j, info:eu-repo/semantics/published

Non‑coding RNAs (ncRNAs) have been shown to serve important roles in carcinogenesis via complex mechanisms, including transcriptional and post‑transcriptional regulation, and chromatin interactions. Urothelial carcinoma‑associated 1 (UCA1), a long ncRNA, was recently shown to have tumorigenic properties in urothelial bladder cancer (UBC), as demonstrated by enhanced proliferation, migration, invasion and therapy resistance of UBC cell lines in vitro. These in vitro findings suggested that UCA1 is associated with aggressive tumor behavior and could have prognostic implications in UBC. The aims of the present study were to therefore to investigate the statistical associations between UCA1 RNA expression and UBC pathological features, patient prognosis and p53 and Ki‑67 expression. Chromogenic in situ hybridization and immunohistochemistry were performed on UBC tissue microarrays to characterize UCA1 RNA, and p53 and Ki‑67 expression in 208 UBC cases, including 145 non‑muscle‑invasive and 63 muscle‑invasive cases. UCA1 was observed in the tumor cells of 166/208 (80%) UBC cases tested. No expression was noted in normal stromal and endothelium cells. Patients with UBC that overexpressed UCA1 (35%) had a significantly higher survival rate (P=0.006) compared with that in patients with UBC that did not overexpress UCA1. This prognostic factor was independent of tumor morphology, concomitant carcinoma in situ, tumor grade and tumor stage. In addition, the absence of UCA1 overexpression was significantly associated with a high Ki‑67 proliferative index (P=0.008) and a p53 'mutated' immunoprofile (strong nuclear expression or complete absence of staining; P=0.003). In conclusion, the present results identified UCA1 as potentially being a novel independent prognostic marker in UBC that was associated with a better patient prognosis and that could serve a pivotal role in bladder cancer carcinogenesis., SCOPUS: ar.j, info:eu-repo/semantics/published

We evaluated the relationship between IL-8 and prostate cancer (PCa) with emphasis on diagnosis, aggressiveness and prognosis., info:eu-repo/semantics/published

Glioblastomas (GBMs) are the most common malignant primary brain tumours in adults and are refractory to conventional therapy, including surgical resection, radiotherapy and chemotherapy. The insulin-like growth factor (IGF) system is a complex network that includes ligands (IGFI and IGFII), receptors (IGF-IR and IGF-IIR) and high-affinity binding proteins (IGFBP-1 to IGFBP-6). Many studies have reported a role for the IGF system in the regulation of tumour cell biology. However, the role of this system remains unclear in GBMs., SCOPUS: ar.j, info:eu-repo/semantics/published

Diabetes is characterized by a progressive decline of the pancreatic beta cell mass (BCM), which is responsible for insufficient insulin secretion and hyperglycaemia. There are currently no reliable methods to measure non-invasively the BCM in diabetic patients. Our work describes a phage display-derived peptide (P88) that is highly specific to (FXYD2)γa expressed by human beta cells and is proposed as a molecular vector for the development of functionalized imaging probes. P88 does not bind to the exocrine pancreas and is able to detect down to ~156 human pancreatic islets/mm(3) in vitro after conjugation to ultra-small particles of iron oxide (USPIO), as proven by the R2 measured on MR images. For in vivo evaluation, MRI studies were carried out on nude mice bearing Capan-2 tumours that also express (FXYD2)γa. A strong negative contrast was obtained subsequent to the injection of USPIO-P88, but not in negative controls. On human histological sections, USPIO-P88 seems to be specific to pancreatic beta cells, but not to duodenum, stomach or kidney tissues. USPIO-P88 thus represents a novel and promising tool for monitoring pancreatic BCM in diabetic patients. The quantitative correlation between BCM and R2 remains to be demonstrated in vivo, but the T2 mapping and the black pixel estimation after USPIO-P88 injection could provide important information for the future pancreatic BCM evaluation by MRI.

The incidence of papillary thyroid cancer has increased these last decades due to a better detection. High prevalence of nodules combined with the low incidence of thyroid cancers constitutes an important diagnostic challenge. We propose to develop an alternative diagnostic method to reduce the number of useless and painful thyroidectomies using a vectorized contrast agent for magnetic resonance imaging. Galectin-1 (gal-1), a protein overexpressed in well-differentiated thyroid cancer, has been targeted with a randomized linear 12-mer peptide library using the phage display technique. Selected peptides have been conjugated to ultrasmall superparamagnetic particles of iron oxide (USPIO). Peptides and their corresponding contrast agents have been tested in vitro for their specific binding and toxicity. Two peptides (P1 and P7) were selected according to their affinity toward gal-1. Their binding has been revealed by immunohistochemistry on human thyroid cancer biopsies, and they were co-localized with gal-1 by immunofluorescence on TPC-1 cell line. Both peptides induce a decrease in TPC-1 cells' adhesion to gal-1 immobilized on culture plates. After coupling to USPIO, the peptides preserved their affinity toward gal-1. Their specific binding has been corroborated by co-localization with gal-1 expressed by TPC-1 cells and by their ability to compete with anti-gal-1 antibody. The peptides and their USPIO derivatives produce no toxicity in HepaRG cells as determined by MTT assay. The vectorized contrast agents are potential imaging probes for thyroid cancer diagnosis. Moreover, the two gal-1-targeted peptides prevent cancer cell adhesion by interacting with the carbohydrate-recognition domain of gal-1.

Background: The mechanisms of brain death (BD)-induced lung injury remain incompletely understood, as uncertainties persist about time-course and relative importance of mechanical and humoral perturbations. Methods: Brain death was induced by slow intracranial blood infusion in anesthetized pigs after randomization to placebo (n = 11) or to methylprednisolone (n = 8) to inhibit the expression of pro-inflammatory mediators. Pulmonary artery pressure (PAP), wedged PAP (PAWP), pulmonary vascular resistance (PVR) and effective pulmonary capillary pressure (PCP) were measured 1 and 5 hours after Cushing reflex. Lung tissue was sampled to determine gene expressions of cytokines and oxidative stress molecules, and pathologically score lung injury. Results: Intracranial hypertension caused a transient increase in blood pressure followed, after brain death was diagnosed, by persistent increases in PAP, PCP and the venous component of PVR, while PAWP did not change. Arterial PO2/fraction of inspired O2 (PaO2/FiO2) decreased. Brain death was associated with an accumulation of neutrophils and an increased apoptotic rate in lung tissue together with increased pro-inflammatory interleukin (IL)-6/IL-10 ratio and increased heme oxygenase(HO)-1 and hypoxia inducible factor(HIF)-1 alpha expression. Blood expressions of IL-6 and IL-1β were also increased. Methylprednisolone pre-treatment was associated with a blunting of increased PCP and PVR venous component, which returned to baseline 5 hours after BD, and partially corrected lung tissue biological perturbations. PaO2/ FiO2 was inversely correlated to PCP and lung injury score. Conclusions: Brain death-induced lung injury may be best explained by an initial excessive increase in pulmonary capillary pressure with increased pulmonary venous resistance, and was associated with lung activation of inflammatory apoptotic processes which were partially prevented by methylprednisolone., SCOPUS: ar.j, info:eu-repo/semantics/published

The presence of tumor-infiltrating lymphocytes (TIL), reflecting host immune activity, is frequently correlated with better clinical outcomes, particularly in HER2-positive and triple-negative breast cancer. Recent findings suggest that organization of immune infiltrates in tertiary lymphoid structures also has a beneficial effect on survival. This study investigated inter- and intra-observer variation in TIL assessment using conventional hematoxylin-eosin versus immunohistochemical staining to identify immune cells. Global, intratumoral, and stromal TIL, as well as tertiary lymphoid structures were scored independently by experienced pathologists on full-face tumor sections (n=124). The fidelity of scoring infiltrates in core biopsies compared to surgical specimens, and pathological assessment compared to quantitative digital analysis was also evaluated. The inter-observer concordance correlation coefficient was 0.80 for global, 0.72 for intratumoral, and 0.71 for stromal TIL, while the intra-observer concordance correlation coefficient was 0.90 for global, 0.77 for intratumoral, and 0.89 for stromal TIL using immunohistochemical stains. Correlations were lower with hematoxylin-eosin stains, particularly for intratumoral TIL, while global scores had the highest concordance correlation coefficients. Our study concluded that tertiary lymphoid structures are accurately and consistently scored using immunohistochemical but not hematoxylin-eosin stains. A strong association was observed between TIL in core biopsies and surgical samples (R2=0.74) but this did not extend to tertiary lymphoid structures (R2=0.26). TIL scored by pathologists and digital analysis were correlated but our analysis reveals a constant bias between these methods. These data challenge current criteria for TIL and tertiary lymphoid structure assessment in breast cancer and recommend that how pathologists evaluate immune infiltrates be reexamined for future studies.

Aim. Tracking human cells xenotransplanted into animal models is crucial for studying their in vivo biologic potential and essential to follow their fate in the field of cell therapy or tumor xenografting. Preclinical assessment for cellular therapy requires data on biodistribution, viability, integration into host tissue, differentiation into functional cells, tumorigenic potential and safety of delivery. Often injected in limited amount, these cells, from human origin, must be recognized with high sensitivity. Post-mortem immunohistochemistry is currently the most sensitive method for cell tracking affording the detection of specific cell populations, while preserving both their morphology and that of the surrounding environment. Here, we focused our work on the specific expression throughout the human body of three human-specific biomarkers: Ku80, human mitochondria (hMito) and Alu after transplantation of human stem cell in animals. Results. We showed that Ku80, hMito and Alu biomarkers are broadly expressed in human tissues with no or low cross-reactivity towards rat, mouse or pig tissues. Through in vitro studies, we demonstrated that the expression of Ku80 and Alu biomarkers is stable over time and does not change upon the differentiation process of human-derived induced pluripotent stem cells or glial-restricted precursors. We also showed that these human biomarkers could be reliably used to track human neural or human glial stem cells transplanted in the rodent spinal cord. Using computer-assisted image analysis, we were able to quantify the engraftment of these human stem cells in animal tissue. Human-specific antibodies helping at detecting apoptotic, proliferative or neural-committed cells were also characterized. Conclusions. Based on immunohistochemistry and in situ hybridization, this methodological study assesses the human-species specificity of Ku80, hMito and Alu biomarkers and proposes useful tools for the critical analysis of human stem cells tracking after transplantation, which is needed for both preclinical cell therapy evaluation and tumor xenografting analysis.

Cancer stem cells (CSCs) have been reported in various cancers, including in skin squamous-cell carcinoma (SCC). The molecular mechanisms regulating tumour initiation and stemness are still poorly characterized. Here we find that Sox2, a transcription factor expressed in various types of embryonic and adult stem cells, was the most upregulated transcription factor in the CSCs of squamous skin tumours in mice. SOX2 is absent in normal epidermis but begins to be expressed in the vast majority of mouse and human pre-neoplastic skin tumours, and continues to be expressed in a heterogeneous manner in invasive mouse and human SCCs. In contrast to other SCCs, in which SOX2 is frequently genetically amplified, the expression of SOX2 in mouse and human skin SCCs is transcriptionally regulated. Conditional deletion of Sox2 in the mouse epidermis markedly decreases skin tumour formation after chemical-induced carcinogenesis. Using green fluorescent protein (GFP) as a reporter of Sox2 transcriptional expression (SOX2-GFP knock-in mice), we showed that SOX2-expressing cells in invasive SCC are greatly enriched in tumour-propagating cells, which further increase upon serial transplantations. Lineage ablation of SOX2-expressing cells within primary benign and malignant SCCs leads to tumour regression, consistent with the critical role of SOX2-expressing cells in tumour maintenance. Conditional Sox2 deletion in pre-existing skin papilloma and SCC leads to tumour regression and decreases the ability of cancer cells to be propagated upon transplantation into immunodeficient mice, supporting the essential role of SOX2 in regulating CSC functions. Transcriptional profiling of SOX2-GFP-expressing CSCs and of tumour epithelial cells upon Sox2 deletion uncovered a gene network regulated by SOX2 in primary tumour cells in vivo. Chromatin immunoprecipitation identified several direct SOX2 target genes controlling tumour stemness, survival, proliferation, adhesion, invasion and paraneoplastic syndrome. We demonstrate that SOX2, by marking and regulating the functions of skin tumour-initiating cells and CSCs, establishes a continuum between tumour initiation and progression in primary skin tumours.

Aristolochic acid (AA) causes aristolochic acid nephropathy (AAN), first described in women in Belgium accidently prescribed Aristolochia fangchi in a slimming treatment, and also Balkan endemic nephropathy (BEN), through probable dietary contamination with Aristolochia clematitis seeds. Both nephropathies have a high risk of urothelial cancer, with AA being the causative agent. In tissues of AAN and BEN patients, a distinct DNA adduct, 7-(deoxyadenosin-N6-yl)-aristolactam I (dA-AAI), has been detected. DNA adducts can be removed through DNA repair, they can result in mutations through erroneous DNA replication or they can cause cell death. The dA-AAI adduct induces AT to TA transversions in the tumor-suppressor TP53 gene in experimental systems, matching TP53 mutations observed in urothelial tumors from AAN cancer cases. Using thin-layer chromatography 32P-postlabeling and mass spectrometric analysis we report the detection of dA-AAI in renal DNA from 11 Belgian AAN patients over 20 years after exposure to AA had ceased. Our results showed that dA-AAI is an established biomarker of AA exposure, and that this biomarker can be demonstrated to be persistent decades after a distinct AA exposure. Further, the persistence of dA-AAI adducts appears to be a critical determinant for the AA mutational fingerprint frequently found in oncogenes and tumor suppressor genes recently identified by whole genome sequencing of AA-associated urothelial tumors. The potential for exposure to AA worldwide is high; the unprecedented long-term persistence of dA-AAI provides a useful long-term biomarker of exposure and attests to the role of AA in human urothelial malignancy.

Basal cell carcinoma, the most frequent human skin cancer, arises from activating hedgehog (HH) pathway mutations; however, little is known about the temporal changes that occur in tumour-initiating cells from the first oncogenic hit to the development of invasive cancer. Using an inducible mouse model enabling the expression of a constitutively active Smoothened mutant (SmoM2) in the adult epidermis, we carried out transcriptional profiling of SmoM2-expressing cells at different times during cancer initiation. We found that tumour-initiating cells are massively reprogrammed into a fate resembling that of embryonic hair follicle progenitors (EHFPs). Wnt/ β-catenin signalling was very rapidly activated following SmoM2 expression in adult epidermis and coincided with the expression of EHFP markers. Deletion of β-catenin in adult SmoM2-expressing cells prevents EHFP reprogramming and tumour initiation. Finally, human basal cell carcinomas also express genes of the Wnt signalling and EHFP signatures.

Background: Interleukin-7 receptor alpha (IL-7Rα) represents a biomarker with potential applications in rheumatoid arthritis (RA) diagnosis and therapy. We have therefore searched by phage display potential IL-7Rα specific peptides with the primary goal being to develop in vivo molecular imaging tools. Methods: IL-7Rα-targeted peptides were searched within a disulfide-constrained combinatorial phage displayed library of random linear heptapeptides. The apparent dissociation constant (Kd) and half maximal inhibition constant (IC50) were estimated for phage clones and synthesized peptides by ELISA. We used 5-Aza-2'-deoxycytidine (ADC)-stimulated Jurkat cells and human synovial tissue from patients with RA for in vitro characterization of peptides. For molecular imaging studies performed by magnetic resonance imaging (MRI), experimental arthritis was induced in DBA/1 male mice by immunization with an emulsion of complete Freund's adjuvant and type II collagen from chicken sternal cartilage. Results: After several steps of phage display and peptide screening, two IL-7Rα-specific heptapeptides (P258 and P725) were selected from the initial library, based on their affinity for the target (extracellular domain of IL-7Rα, which contains a fibronectin type III repeat-like sequence). P258 (a linear peptide obtained by removing the Cys-constraint) had the lowest affinity for fibronectin itself and was therefore proposed for molecular imaging. After grafting to ultra-small superparamagnetic particles of iron oxide (USPIO), P258 produced a strong negative contrast on MRI in mice with collagen-induced arthritis (CIA), even at 2 hours post injection. The co-localization of USPIO-P258 with IL-7Rα-expressing cells in the synovial tissue from CIA mice and its ability to discriminate the level of IL-7R expression and the disease severity confirmed its efficacy as an in vivo IL-7Rα imaging agent. Interestingly, the cyclic peptide (P725), which was less adequate for molecular imaging because of higher affinity for fibronectin, had a strong ability to compete with IL-7 for the IL-7Rα binding sites, making it a potential candidate for blocking applications. Accordingly, P725 prevented the signal transducer and activator of transcription 5 (STAT5) activation induced by IL-7 in ADC-stimulated Jurkat cells. Conclusions: The two peptides identified in this work demonstrate that IL-7Rα targeting in RA presents potential applications for in vivo molecular imaging and putative blocking purposes., SCOPUS: ar.j, info:eu-repo/semantics/published

Background Right ventricular (RV) dysfunction remains the leading cause of early death after cardiac transplantation. Methylprednisolone is used to improve graft quality; however, evidence for that remains empirical. We sought to determine whether methylprednisolone, acting on inflammation and apoptosis, might prevent brain death–induced RV dysfunction. Methods After randomization to placebo (n = 11) or to methylprednisolone (n = 8; 15 mg/kg), 19 pigs were assigned to a brain-death procedure. The animals underwent hemodynamic evaluation at 1 and 5 hours after Cushing reflex (i.e., hypertension and bradycardia). The animals euthanized, and myocardial tissue was sampled. This was repeated in a control group (n = 8). Results At 5 hours after the Cushing reflex, brain death resulted in increased pulmonary artery pressure (27 ± 2 vs 18 ± 1 mm Hg) and in a 30% decreased ratio of end-systolic to pulmonary arterial elastances (Ees/Ea). Cardiac output and right atrial pressure did not change. This was prevented by methylprednisolone. Brain death–induced RV dysfunction was associated with increased RV expression of heme oxygenase-1, interleukin (IL)-6, IL-10, IL-1β, tumor necrosis factor (TNF)-α, IL-1 receptor-like (ST)-2, signal transducer and activator of transcription-3, intercellular adhesion molecules-1 and -2, vascular cell adhesion molecule-1, and neutrophil infiltration, whereas IL-33 expression decreased. RV apoptosis was confirmed by terminal deoxynucleotide transferase-mediated deoxy uridine triphosphate nick-end labeling staining. Methylprednisolone pre-treatment prevented RV–arterial uncoupling and decreased RV expression of TNF-α, IL-1 receptor-like-2, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and neutrophil infiltration. RV Ees/Ea was inversely correlated to RV TNF-α and IL-6 expression. Conclusions Brain death–induced RV dysfunction is associated with RV activation of inflammation and apoptosis and is partly limited by methylprednisolone.