Donation after circulatory death and liver transplantation: a cohort study.

Donations after circulatory death (DCD) are still challenging in Italy because of prolonged ischemia time (tWIT) due to the law and logistical issues. This cohort study was primarily aimed at assessing the association between successful transplantation and DCD types in the North Italy Transplant program. Adjusted risk ratios (RR) and 95% confidence intervals (CIs) for type III versus type II DCD were estimated using a Poisson regression model with a robust error variance. All consecutive DCD between 2008 and 2020 were included. Among 142 DCD, 102 were eligible for liver donation, and 96 were proposed: 68/69 (99%) and 28/33 (85%) type III and II DCD, respectively. Sixty-nine livers were recovered, 51/68 (75%) from type III and 18/28 (64%) from type II DCD, respectively (RR: 1.18; 95% CI: 0.87-1.60). After ex-vivo perfusion, 50/68 (74%) and 14/28 (50%) livers from type III and type II DCD were transplanted (RR: 1.49; 95% CI: 1.01-2.19). The estimate decreased after further controlling for tWIT (RR: 1.11; 95% CI: 0.55-2.24). Five patients (7.8%) experienced a PNF, 3/50 and 2/14 from type III and type II DCD, respectively. Type III DCD livers were more likely to be transplanted than type II. Warm ischemia time might explain this difference.

Insulin resistance promotes Lysyl Oxidase Like 2 induction and fibrosis accumulation in non-alcoholic fatty liver disease.

In patients with non-alcoholic fatty liver disease (NAFLD), insulin resistance (IR) associates with fibrosis progression independently of the hepatic inflammation, but the mechanisms are still unclear. We modeled the independent contribution of inflammation (non-alcoholic steatohepatitis: NASH) by exploiting the methionine-choline deficient (MCD) diet, and that of IR by insulin receptor (InsR) haploinsufficiency (InsR+/-) in the pathogenesis of liver fibrosis in C57BL/6 mice. We confirmed the study findings in 96 patients with NAFLD. InsR+/- enhanced hepatic fat content and impaired hepatic insulin signaling leading to Forkhead box protein O1 (FoxO1) accumulation in MCD-fed mice. Remarkably, despite reduced inflammation and hampered transdifferentiation of hepatic stellate cells (HSCs), InsR+/- promoted hepatic fibrosis accumulation, which correlated with the induction of the Lysyl Oxidase Like 2 (Loxl2), involved in matrix stabilization. Loxl2 up-regulation was not a cell autonomous property of insulin resistant HSCs, but was dependent on microparticles (MPs) released specifically by insulin resistant hepatocytes (HEPs) exposed to fatty acids. The mechanism entailed FoxO1 up-regulation, as FoxO1 silencing normalized Loxl2 expression reversing fibrosis in InsR+/- MCD-fed mice. Loxl2 up-regulation was similarly detected during IR induced by obesity, but not by lipogenic stimuli (fructose feeding). Most importantly, LOXL2 up-regulation was observed in NAFLD patients with type 2 diabetes (T2D) and LOXL2 hepatic and circulating levels correlated with histological fibrosis progression. IR favors fibrosis deposition independently of the classic 'inflammation - HSC transdifferentiation' pathway. The mechanism entails a cross-talk between enhanced lipotoxicity in insulin resistant HEPs and Loxl2 production by HSCs, which was confirmed in patients with diabetes, thereby facilitating extracellular matrix (ECM) stabilization.

Ex-vivo characterization of circulating colon cancer cells distinguished in stem and differentiated subset provides useful biomarker for personalized metastatic risk assessment.

BACKGROUND: Circulating tumor cells (CTCs) represent one of the most interesting target in improving diagnosis, prognosis and treatment. Herein we evaluate the possibility of using an emo-cytometric approach on the evaluation of the heterogeneous population of CTCs to improve personalized metastatic risk assessment. We benchmarked ex vivo behavior of distinct subsets of circulating colon tumor cells with correspondent clinical behavior of patients from which we isolated CTCs. METHODS: Isolation and CTC expansion were performed by a gradient protocol. In vitro characterization was determined by flow cytometry, immunofluorescence, western blotting and proteomic profiling. Cell sorter was performed with immunomagnetic beads. Confocal microscopy was used to evaluate tissue sections. Kaplan Mayer curves was cared for through Medcalc program. RESULTS: We collected heterogeneous CTCs, derived from the whole blood of seven patients affected by colon cancer, expressing CD133(pos)CD45(neg) (5 ± 1) and (2 ± 1) and CK20(pos)CD45(neg) of (29 ± 3) (11 ± 1) cells/ml in Dukes D and A stage respectively. Proliferation rate of 57 ± 16 %, expression for CXCR4(pos) of 18 ± 7 % and detectable levels of IL-6, IL-8 and SDF-1 cytokines in conditioned culture medium characterized short-time expanded-CTCs (eCTCs). ECTCs organized in tumor sphere were CD45(neg)CD133(pos) while in adhesion were CXCR4(pos)CK20(pos). These two subsets were separately injected in mice. The first group of xenografts developed superficial lesions within 2 weeks. In the second group, in absence of growing tumour, the survival of injected eCTCs was monitored through SDF-1 serum levels detection. The detection of human cancer cells expressing CK20, in mice tissues sections, suggested a different biological behaviour of injected eCTC-subsets: tumorigenic for the first and disseminating for the second. The benchmarking of the experimental data with the clinical course highlights that patients with prevalence of circulating cancer stem cells (CD45(neg)CD133(pos)) have a lower overall survival. Conversely, patients with prevalence of circulating differentiated cells (CXCR4(pos)CK20(pos)) have a low disease-free survival. CONCLUSION: On the basis of the heterogeneous composition and despite the low number of CTCs, it was possible to distinguish two subgroups of CTCs, suggesting a different clinical outcome. CTC-subsets detailing is useful to better define the metastatic-risk personalized score thus improving disease management and reducing patient care cost.

Endothelial progenitor cells, defined by the simultaneous surface expression of VEGFR2 and CD133, are not detectable in healthy peripheral and cord blood.

Circulating endothelial cells (CEC) and their progenitors (EPC) are restricted subpopulations of peripheral blood (PB), cord blood (CB), and bone marrow (BM) cells, involved in the endothelial homeostasis maintenance. Both CEC and EPC are thought to represent potential biomarkers in several clinical conditions involving endothelial turnover/remodeling. Although different flow cytometry methods for CEC and EPC characterization have been published so far, none of them have reached consistent conclusions, therefore consensus guidelines with respect to CEC and EPC identification and quantification need to be established. Here, we have carried out an in depth investigation of CEC and EPC phenotypes in healthy PB, CB and BM samples, by optimizing a reliable polychromatic flow cytometry (PFC) panel. Results showed that the brightness of CD34 expression on healthy PB and CB circulating cells represents a key benchmark for the identification of CEC (CD45neg/CD34bright/CD146pos) respect to the hematopoietic stem cell (HSC) compartment (CD45dim/CD34pos/CD146neg). This approach, combined with a dual-platform counting technique, allowed a sharp CEC enumeration in healthy PB (n = 38), and resulting in consistent CEC counts with previously reported data (median = 11.7 cells/ml). In parallel, by using rigorous PFC conditions, CD34pos/CD45dim/CD133pos/VEGFR2pos EPC were not found in any healthy PB or CB sample, since VEGFR2 expression was never detectable on the surface of CD34pos/CD45dim/CD133pos cells. Notably, the putative EPC phenotype was observed in all analyzed BM samples (n = 12), and the expression of CD146 and VEGFR2, on BM cells, was not restricted to the CD34bright compartment, but also appeared on the HSC surface. Altogether, our findings suggest that the previously reported EPC antigen profile, defined by the simultaneous expression of VEGFR2 and CD133 on the surface of CD45dim/CD34pos cells, should be carefully re-evaluated and further studies should be conducted to redefine EPC features in order to translate CEC and EPC characterization into clinical practice.

Folic acid functionalized surface highlights 5-methylcytosine-genomic content within circulating tumor cells.

Although the detection of methylated cell free DNA represents one of the most promising approaches for relapse risk assessment in cancer patients, the low concentration of cell-free circulating DNA constitutes the biggest obstacle in the development of DNA methylation-based biomarkers from blood. This paper describes a method for the measurement of genomic methylation content directly on circulating tumor cells (CTC), which could be used to deceive the aforementioned problem. Since CTC are disease related blood-based biomarkers, they result essential to monitor tumor's stadiation, therapy, and early relapsing lesions. Within surface's bio-functionalization and cell's isolation procedure standardization, the presented approach reveals a singular ability to detect high 5-methylcytosine CTC-subset content in the whole CTC compound, by choosing folic acid (FA) as transducer molecule. Sensitivity and specificity, calculated for FA functionalized surface (FA-surface), result respectively on about 83% and 60%. FA-surface, allowing the detection and characterization of early metastatic dissemination, provides a unique advance in the comprehension of tumors progression and dissemination confirming the presence of CTC and its association with high risk of relapse. This functionalized surface identifying and quantifying high 5-methylcytosine CTC-subset content into the patient's blood lead significant progress in cancer risk assessment, also providing a novel therapeutic strategy.

Solid Organ Transplantation in the Coronavirus Disease 2019 Era: "The Great Bet" in the North Italy Transplant Program Area.

INTRODUCTION: Solid organ transplantation is challenging for waitlist patients during the coronavirus disease 2019 (COVID-19) pandemic. AIM: This study investigates COVID-19 incidence and mortality in patients transplanted in the North Italy Transplant program (NITp) during the outbreak. MATERIALS AND METHODS: All consecutive patients transplanted from February 20 to April 3, 2020 (6 weeks), were included in our cohort and were observed for at least 4 weeks. Survival analyses were performed. RESULTS: In this study, 124 patients were transplanted with 12 (9.7%) hearts, 4 (3.2%) lungs, 39 (31.4%) livers, 67 (54%) kidneys, and 2 (1.6%) combined kidney-pancreas. Recipients' mean age was 51 years (standard deviation [SD] ± 16.6), and 76 of 124 (61%) were men. Five (4%) patients developed COVID-19 after a mean of 13 days (SD ± 6.7), with a cumulative incidence of 4.0% (95% confidence interval [CI], 0.5-7.5). During the follow-up period, 5 of 124 (4%) recipients died; overall mortality was 4.3% (95% CI, 0.6-8.0), with only 1 patient dying of COVID-19, for a COVID-19-related mortality of 0.8% (95% CI, 0-6.0). CONCLUSIONS: This study showed a low COVID-19 incidence and COVID-19-related mortality in patients transplanted during the COVID-19 pandemic. Further studies with a longer follow-up period are mandatory to confirm the safety of transplant procedures.

A standardized flow cytometry network study for the assessment of circulating endothelial cell physiological ranges.

Circulating endothelial cells (CEC) represent a restricted peripheral blood (PB) cell subpopulation with high potential diagnostic value in many endothelium-involving diseases. However, whereas the interest in CEC studies has grown, the standardization level of their detection has not. Here, we undertook the task to align CEC phenotypes and counts, by standardizing a novel flow cytometry approach, within a network of six laboratories. CEC were identified as alive/nucleated/CD45negative/CD34bright/CD146positive events and enumerated in 269 healthy PB samples. Standardization was demonstrated by the achievement of low inter-laboratory Coefficients of Variation (CVL), calculated on the basis of Median Fluorescence Intensity values of the most stable antigens that allowed CEC identification and count (CVL of CD34bright on CEC ~ 30%; CVL of CD45 on Lymphocytes ~ 20%). By aggregating data acquired from all sites, CEC numbers in the healthy population were captured (medianfemale = 9.31 CEC/mL; medianmale = 11.55 CEC/mL). CEC count biological variability and method specificity were finally assessed. Results, obtained on a large population of donors, demonstrate that the established procedure might be adopted as standardized method for CEC analysis in clinical and in research settings, providing a CEC physiological baseline range, useful as starting point for their clinical monitoring in endothelial dysfunctions.

Galectin-3 coats the membrane of breast cells and makes a signature of tumours.

Galectin-3, ß-galactoside-binding lectin, coats the membrane of most cancer cells and is involved in metastasis and endothelium recognition as well as in evading immune surveillance through killing of activated T cells. To flag galectin as a biomarker of tumours and metastasis, it is pivotal to understand the role of this protein in different tumours and at different stages. Breast tumours have an anomalous behaviour of the galectin-3 compared to other tumour cells. Herein, FACS sorting and galactoside based assays were used to investigate the role of galectin-3 in metastasis and metastatisation of breast cancer cells. Breast galectin fingerprint at the FACS displayed a higher amount in healthy cells, compared to metastatic cells. The microfluidic assay was able to isolate tumour and metastatic cells more than healthy breast cells. Investigation was performed on samples from patients with breast tumours at stage I and stage III whilst MCF7 and EPH-4 cells were used to perform preliminary investigations. The readout of the conditioned medium (from culturing of stage I cells) fingerprint by FACS evidenced high expression of free galectin. Analysis of the results established that the galectin coating the membrane, by galactoside recognition of the breast cells, and engaged by the cells to form protein-carbohydrate complexes inside the microfluidic assay, resembled the tumour signature of tumours in breast cells whilst the galectin free is independent of those mechanisms.