Sphingosine 1-phosphate receptor 1 is required for MMP-2 function in bone marrow mesenchymal stromal cells: implications for cytoskeleton assembly and proliferation

Bone marrow-derived mesenchymal stromal cell- (BM-MSC-) based therapy is a promising option for regenerative medicine. An important role in the control of the processes influencing the BM-MSC therapeutic efficacy, namely, extracellular matrix remodelling and proliferation and secretion ability, is played by matrix metalloproteinase- (MMP-) 2. Therefore, the identification of paracrine/autocrine regulators of MMP-2 function may be of great relevance for improving BM-MSC therapeutic potential. We recently reported that BM-MSCs release the bioactive lipid sphingosine 1-phosphate (S1P) and, here, we demonstrated an impairment of MMP-2 expression/release when the S1P receptor subtype S1PR1 is blocked. Notably, active S1PR1/MMP-2 signalling is required for F-actin structure assembly (lamellipodia, microspikes, and stress fibers) and, in turn, cell proliferation. Moreover, in experimental conditions resembling the damaged/regenerating tissue microenvironment (hypoxia), S1P/S1PR1 system is also required for HIF-1α expression and vinculin reduction. Our findings demonstrate for the first time the trophic role of S1P/S1PR1 signalling in maintaining BM-MSCs' ability to modulate MMP-2 function, necessary for cytoskeleton reorganization and cell proliferation in both normoxia and hypoxia. Altogether, these data provide new perspectives for considering S1P/S1PR1 signalling a pharmacological target to preserve BM-MSC properties and to potentiate their beneficial potential in tissue repair

Prediction of morbidity and mortality after early cholecystectomy for acute calculous cholecystitis : results of the SPRi.MACC study

Peer reviewe

Textbook outcome in urgent early cholecystectomy for acute calculous cholecystitis: results post hoc of the S.P.Ri.M.A.C.C study

Introduction: A textbook outcome patient is one in which the operative course passes uneventful, without complications, readmission or mortality. There is a lack of publications in terms of TO on acute cholecystitis. Objetive: The objective of this study is to analyze the achievement of TO in patients with urgent early cholecystectomy (UEC) for Acute Cholecystitis. and to identify which factors are related to achieving TO. Materials and methods: This is a post hoc study of the SPRiMACC study. It ́s a prospective multicenter observational study run by WSES. The criteria to define TO in urgent early cholecystectomy (TOUEC) were no 30-day mortality, no 30-day postoperative complications, no readmission within 30 days, and hospital stay ≤ 7 days (75th percentile), and full laparoscopic surgery. Patients who met all these conditions were taken as presenting a TOUEC. Outcomes: 1246 urgent early cholecystectomies for ACC were included. In all, 789 patients (63.3%) achieved all TOUEC parameters, while 457 (36.6%) failed to achieve one or more parameters and were considered non-TOUEC. The patients who achieved TOUEC were younger had significantly lower scores on all the risk scales analyzed. In the serological tests, TOUEC patients had lower values for in a lot of variables than non-TOUEC patients. The TOUEC group had lower rates of complicated cholecystitis. Considering operative time, a shorter duration was also associated with a higher probability of reaching TOUEC. Conclusion: Knowledge of the factors that influence the TOUEC can allow us to improve our results in terms of textbook outcome

DEVELOPMENT & QUALIFICATION OF BIOASSAYS FOR THE DETERMINATION OF THE BIOACTIVITY, PREDICTIVE PHARMACOKINETICS AND POTENTIAL IMMUNOGENICITY OF THERAPEUTIC ANTIBODIES

The critical quality attributes of a given biotherapeutic monoclonal antibody (mAb), its molecular characterization, functional assessment and effector function analysis should be defined and profiled in detail during the life cycle of a biotherapeutic drug. In the past, this product characterization was simple and standardized. In today’s complex world of biologics, success demands a more thoughtful approach and drug developers are investing in advanced analytics much earlier in the development process. Indeed, investing in selected sophisticated and state of the art analytics in early developmental phases of a therapeutic monoclonal antibody may mitigate risks by confirming that the drug candidate has the required basic characteristics and functionality. Therefore, recognizing the added value of an early functional characterization of new biological entities (NBE) in the pharmaceutical industry, this work was focused on the development and qualification of novel methodologies defining the proper analytical characterization panel for a new therapeutic monoclonal antibody. In details, this PhD project was specifically designed to identify, develop and qualify with a stepwise approach in silico tools and in vitro assays aimed at studying the biological activity, predictive pharmacokinetics (PK) and potential immunogenicity of therapeutic drugs in early phase. To this aim Anti-TIGIT mAb was selected as proof of concept molecule. First, an investigation on the mechanism of action of Anti-TIGIT has been carried out and then different approaches were applied to identify the best in vitro assay reflecting the mechanism underlying Anti-TIGIT biological effect. Once selected as the best method a cell-based Anti-TIGIT ligand binding bioassay, it was checked for linearity, precision, accuracy and specificity demonstrating its fitting for the purpose. Different alternative assays for the assessment of the mAb’s effector functions were also presented and qualified showing the ability of Anti-TIGIT to induce antibody-dependent cell-mediated cytotoxicity (ADCC). Then, the attention was focused on the predictive pharmacokinetics of Anti-TIGIT and specifically on in vitro techniques to assess affinity and kinetic of the interaction with neonatal Fc receptor (FcRn) demonstrating that the developed and qualified methods were reliable, precise and accurate. Moreover, forced degradation studies, where the mAb underwent different stresses, were performed demonstrating the assays’ suitability for the purpose highlighting the impact of the applied stresses on both Anti-TIGIT biological activity and PK. In addition, to ensure a good comparison and integration of all the findings, several orthogonal assays were also developed, and the results compared demonstrating the equivalence of the methods. In the end, also an in silico tool was used to predict the potential immunogenicity of the drug. Altogether the findings of this work elucidate and integrate the strategies applied in different pharmaceutical companies for method development and validation and extend the knowledge on the methodologies used for therapeutic monoclonal antibodies characterization and stability studies opening new perspectives for the industrial setting

Endosonography-Guided Versus Percutaneous Gallbladder Drainage Versus Cholecystectomy in Fragile Patients with Acute Cholecystitis—A High-Volume Center Study

Background and Objectives: Acute cholecystitis is a frequent cause of admission to the emergency department, especially in old and frail patients. Percutaneous drainage (PT-GBD) and endosonographic guided drainage (EUS-GBD) could be an alternative option for relieving symptoms or act as a definitive treatment instead of a laparoscopic or open cholecystectomy (LC, OC). The aim of the present study was to compare different treatment groups. Materials and Methods: This is a five-year monocentric retrospective study including patients ≥65 years old who underwent an urgent operative procedure. A descriptive analysis was conducted comparing all treatment groups. A propensity score was estimated based on the ACS score, incorporated into a predictive model, and tested by recursive partitioning analysis. Results: 163 patients were included: 106 underwent a cholecystectomy (81 laparoscopic (LC) and 25 Open (OC)), 33 a PT-GBD and 21 EUS-GBD. The sample was categorized into three prognostic groups according to the adverse event occurrence rate. All patients treated with EUS-GBD or LC resulted in the low risk group, and the adverse event rate (AE) was 10/96 (10.4%). The AE was 4/28 (14.2%) and 21/36 (58.3%) in the middle- and high-risk groups respectively (p Conclusions: Surgery still represents the gold standard for AC treatment. Nevertheless, EUS-GBD is a good alternative to PT-GBD in terms of clinical success, RR and AEs in all kinds of patients

Ultrasound Patterns of Hepatocellular Carcinoma and Their Prognostic Impact: A Retrospective Study

Background: Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death. Abdominal ultrasound (US) is by far the most widely used first-level exam for the diagnosis of HCC. We aimed to assess whether different ultrasound patterns were related to tumor prognosis. Methods: We retrospectively reviewed all patients with a new diagnosis of HCC (single nodule) and undergoing radiofrequency thermal ablation (RFTA) at our clinic between January 2009 and December 2021. Patients were classified according to four HCC ultrasound patterns: 1A, single capsulated nodule; 1B, well capsulated intra-node nodule; 1C, cluster consisting of capsulated nodules; and 2, non-capsulated nodule. Results: 149 patients were analysed; median follow-up time was 43 months. US patterns 1A (32.9%) and 1B (61.1%) were the most commonly seen. Median overall survival (OS) and recurrence-free survival (RFS) from RFTA were 54 months (95% CI, 42–66) and 22 months (95% CI, 12–32), respectively. Pattern 1A showed the best OS. Compared to pattern 1A, 1B was independently associated with worse OS (51 months (95% CI, 34–68) vs. 46 months (95% CI, 18–62)) and RFS (34 months (95% CI, 27–41) vs. 18 months (95% CI, 12–24)). Patterns 1C and 2 were associated with worse RFS compared to 1A, while no difference was seen for OS. Among baseline clinical variables, pattern 1B exhibited higher histological grade (p = 0.048) and tumor dimension (p = 0.034) compared to pattern 1A. Conclusions: Our findings demonstrate that different US patterns correlate with different survival outcomes and tumor behavior in patients with HCC. Prospective studies are needed to confirm these results

Effect of VEGF signaling inhibition, MSC-secreted S1P and exogenous S1P on C2C12 cell proliferation.

<p>C2C12 cells were exposed to MSC-conditioned medium obtained by culturing MSCs for 48 h in differentiation medium (DM) in the absence (MSC-DM) or in the presence of 5 µM iSK (MSC-DMiSK), in the absence and in the presence of KRN633 (170 nM), an ATP competitive inhibitor of VEGFR tyrosine kinase activity, and stimulated or not with 1 µM S1P and then processed for cell proliferation analyses. A) <i>Confocal immunofluorescence analysis of Ki67 expression in C2C12 cells</i>. Quantitative analysis of the percentage of Ki67 positive nuclei performed on digitized confocal fluorescent images of the cells immunostained with antibodies against the nuclear protein Ki67. The percentage of Ki67 positive nuclei is expressed as percentage of the total nuclei number (the nuclei were counterstained with PI). The data shown are mean±SEM and represent the results of at least three independent experiments with similar results. B) <i>[³H]thymidine incorporation assay</i>. The cells were labeled with methyl [³H]thymidine and processed as reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108662#pone-0108662-g002" target="_blank">Figure 2B</a>. Significance of difference (one-way ANOVA and Newman-Keuls multiple comparison test) *p<0.05 <i>vs</i> MSC-DM, ^#p<0.05 <i>vs</i> MSC-DM+KRN633, °p<0.05 <i>vs</i> MSC-DM+KRN633+S1P; ^§p<0.05 <i>vs</i> MSC-DMiSK+KRN633.</p

Effect of S1P secreted by MSCs on C2C12 and satellite cell proliferation: cell counting and [³H]thymidine incorporation.

<p>C2C12 or satellite cells isolated from single muscle fibers were cultured in differentiation medium (DM) or satellite cell proliferation medium (PM) or exposed to MSC-conditioned medium obtained by culturing MSCs for 48 h in DM in the absence (MSC-DM) or in the presence of 5 µM iSK (MSC-DMiSK), or in PM in the absence (MSC-PM) or in the presence of iSK (MSC-PMiSK), respectively. A) <i>Cell counting</i>. After 24 h of culture the cells were trypsinized and counted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108662#s2" target="_blank">Materials and Methods</a> Section. B) <i>[³H]thymidine incorporation assay</i>. Cells were cultured for the last 2 h in the presence of methyl [³H]thymidine. C2C12 and satellite cells were processed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108662#s2" target="_blank">Materials and Methods</a> Section and the recovered radioactivity measured in a beta counter. The results shown are mean±SEM of at least three independent experiments performed in duplicates. Significance of difference (Student's t test): *p<0.05 vs DM or PM; ^§p<0.05 <i>vs</i> MSC-DM or MSC-PM.</p

S1P content in lysate and conditioned medium of [³H]serine pulsed MSCs.

<p>[³H]lipids were extracted by MSCs cultured in the presence or in the absence (control) of 5 µM sphingosine kinase inhibitor (iSK) and successively pulsed with [³H]serine for 2 h.</p><p>[³H]lipids were separated by TLC as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108662#s2" target="_blank">Materials and Methods</a> Section.</p><p>S1P content in lysate and conditioned medium of [³H]serine pulsed MSCs.</p

Schematic drawing summarizing the molecular events involved in the promotion of skeletal myoblast proliferation induced by the MSC paracrine action.

<p>MSCs, mesenchymal stromal cells; SphK, sphingosine kinase; S1P, sphingosine 1-phosphate; iSK, SphK inhibitor; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor; S1PR, S1P receptor subtypes; KRN633, VEGFR inhibitor; MK571, S1P transporter inhibitor; NICD, Notch intracellular domain.</p