Evaluation of laboratory parameters in 1587 COVID-19 patients admitted to metropolitan hospital area of Bologna, Italy.

Since the outbreak of the 2019 pandemic coronavirus disease (COVID-19), great attention has been given to identifying the main clinical features of the disease. Identification of laboratory parameters able to classify patients based on their risk is mandatory to improve their clinical management. We retrospectively evaluated twenty-six laboratory tests measured in COVID-19 positive patients admitted to the hospital in March and April 2020 to find any correlation between their changes and the risk of death. We divided them into surviving and non-surviving patients. A total of 1587 patients were recruited, 854 males with median age of 71 (IQR 56-81) and 733 females with median age of 77 (IQR 61-87). On admission, death was found to be positively correlated with age (p=0.001), but not with sex (p=0.640) or with hospitalization in days (p=0.827). Brain natriuretic peptide (BNP), creatinine, C-reactive protein (CRP), INR, leukocyte count, lymphocyte count, neutrophil count, and procalcitonin (PCT) demonstrated a statistically significant difference between the two groups (p<0.001), suggesting their role as markers of disease severity; only lymphocyte count resulted as an independent risk factor for death.

Fibrinogen Depletion Coagulopathy Predicts Major Bleeding After Thrombolysis for Ischemic Stroke: A Multicenter Study.

BACKGROUND: Symptomatic intracerebral hemorrhage (sICH) and major bleeding can be fatal complications of intravenous thrombolysis (IVT) for acute ischemic stroke. We investigated the impact of early fibrinogen depletion after IVT on major bleeding events. METHODS: This multicenter observational prospective cohort study enrolled 1678 consecutive patients receiving IVT for acute ischemic stroke at 6 Italian centers, undergoing fibrinogen concentration assessment at baseline, 2 hours and 6 hours after IVT. Fibrinogen depletion was defined as a reduction below 200 mg/dL after 2 hours from IVT, or as a reduction below 50% of baseline fibrinogen levels after 2 hours from IVT. Main outcomes were (1) sICH (National Institute of Neurological Disorders and Stroke criteria) and (2) major bleeding defined as fatal bleeding, decrease in the hemoglobin level>2 g/dL/>1 unit transfusion, or bleeding at critical site. Additional outcomes were (1) any ICH, (2) any bleeding, (3) fatal ICH, and (4) sICH according to ECASSII definition. Good functional recovery was defined as modified Rankin Scale score 0 to 2 at 3 months. RESULTS: Overall, 1678 patients were included (mean age 72 years, 46% female). sICH (n=116) and major bleeding (n=297) were associated with lower rate of good functional recovery (P<0.001). Despite similar fibrinogen levels at admission, fibrinogen depletion after 2 hours from IVT was more common in people with sICH, major bleeding and all additional bleeding outcomes. In the backward stepwise multivariable logistic regression model, fibrinogen depletion remained a significant predictor of sICH (OR, 1.55 [95% CI, 1.04-2.32]) and major bleeding (OR, 1.36 [95% CI, 1.03-1.8]). Thirty-one percent of sICH could be attributable to fibrinogen depletion. The association between fibrinogen depletion and worse clinical outcome at 3 months after stroke (P=0.012) was attributable to the higher risk of major bleeding/sICH. CONCLUSIONS: Fibrinogen depletion significantly increases the risk of sICH and major bleeding after IVT for acute ischemic stroke. Fibrinogen depletion represents an independent risk factor for bleeding, and routine assessment could be considered to stratify the risk of ICH. Trials on early fibrinogen repletion are needed to investigate mitigation of bleeding risk.

Expert clinical pharmacological advice may make an antimicrobial TDM program for emerging candidates more clinically useful in tailoring therapy of critically ill patients.

BACKGROUND: Therapeutic drug monitoring (TDM) may represent an invaluable tool for optimizing antimicrobial therapy in septic patients, but extensive use is burdened by barriers. The aim of this study was to assess the impact of a newly established expert clinical pharmacological advice (ECPA) program in improving the clinical usefulness of an already existing TDM program for emerging candidates in tailoring antimicrobial therapy among critically ill patients. METHODS: This retrospective observational study included an organizational phase (OP) and an assessment phase (AP). During the OP (January-June 2021), specific actions were organized by MD clinical pharmacologists together with bioanalytical experts, clinical engineers, and ICU clinicians. During the AP (July-December 2021), the impact of these actions in optimizing antimicrobial treatment of the critically ill patients was assessed. Four indicators of performance of the TDM-guided real-time ECPA program were identified [total TDM-guided ECPAs July-December 2021/total TDM results July-December 2020; total ECPA dosing adjustments/total delivered ECPAs both at first assessment and overall; and turnaround time (TAT) of ECPAs, defined as optimal (< 12 h), quasi-optimal (12-24 h), acceptable (24-48 h), suboptimal (> 48 h)]. RESULTS: The OP allowed to implement new organizational procedures, to create a dedicated pathway in the intranet system, to offer educational webinars on clinical pharmacology of antimicrobials, and to establish a multidisciplinary team at the morning bedside ICU meeting. In the AP, a total of 640 ECPAs were provided for optimizing 261 courses of antimicrobial therapy in 166 critically ill patients. ECPAs concerned mainly piperacillin-tazobactam (41.8%) and meropenem (24.9%), and also other antimicrobials had ≥ 10 ECPAs (ceftazidime, ciprofloxacin, fluconazole, ganciclovir, levofloxacin, and linezolid). Overall, the pre-post-increase in TDM activity was of 13.3-fold. TDM-guided dosing adjustments were recommended at first assessment in 61.7% of ECPAs (10.7% increases and 51.0% decreases), and overall in 45.0% of ECPAs (10.0% increases and 35.0% decreases). The overall median TAT was optimal (7.7 h) and that of each single agent was always optimal or quasi-optimal. CONCLUSIONS: Multidisciplinary approach and timely expert interpretation of TDM results by MD Clinical Pharmacologists could represent cornerstones in improving the cost-effectiveness of an antimicrobial TDM program for emerging TDM candidates.

B4GALT1 as a New Biomarker of Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a disease characterized by progressive scarring of the lung that involves the pulmonary interstitium. The disease may rapidly progress, leading to respiratory failure, and the long-term survival is poor. There are no accurate biomarkers available so far. Our aim was to evaluate the expression of the B4GALT1 in patients with IPF. Analysis of B4GALT1 gene expression was performed in silico on two gene sets, retrieved from the Gene Expression Omnibus database. Expression of B4GALT1 was then evaluated, both at the mRNA and protein levels, on lung specimens obtained from lung biopsies of 4 IPF patients, on one IPF-derived human primary cell and on 11 cases of IPF associated with cancer. In silico re-analysis demonstrated that the B4GALT1 gene was overexpressed in patients and human cell cultures with IPF (p = 0.03). Network analysis demonstrated that B4GALT1 upregulation was correlated with genes belonging to the EMT pathway (p = 0.01). The overexpression of B4GALT1 was observed, both at mRNA and protein levels, in lung biopsies of our four IPF patients and in the IPF-derived human primary cell, in other fibrotic non-lung tissues, and in IPF associated with cancer. In conclusion, our results indicate that B4GALT1 is overexpressed in IPF and could represent a novel marker of this disease.

Interleukin-6 and Outcome of Chronic Hemodialysis Patients with SARS-CoV-2 Pneumonia.

Background and Objectives: Chronic hemodialysis (CHD) patients are at increased risk of SARS-CoV-2 infection and the related complications and mortality of COVID-19 due to the high rate of comorbidities combined with advanced age. This observational study investigated the clinical manifestations of SARS-CoV-2 infection in CHD and the risk factors for patients' death. Materials and Methods: The study included 26 CHD patients with SARS-CoV-2 pneumonia detected by positive RT-PCR on nasopharyngeal swabs and high-resolution computed tomography at hospital admission, aged 71 + 5.9 years, 14 of which (53.8%) were male, 20 (77%) under hemodiafiltration, and 6 (23%) on standard hemodialysis, with a median follow-up of 30 days. Results: Simple logistic regression analysis revealed that the factors associated with a higher risk of death were older age (OR: 1.133; 95%CI: 1.028−1.326, p = 0.0057), IL-6 levels at admission (OR: 1.014; 95%CI: 1.004−1.028, p = 0.0053), and C-reactive protein (OR: 1.424; 95%CI: 1.158−2.044, p < 0.0001). In the multiple logistic regression model, circulating IL-6 values at admission remained the only significant prognosticator of death. The ROC curve indicated the discriminatory cut-off value of 38.20 pg/mL of blood IL-6 for predicting death in chronic hemodialysis patients with SARS-CoV-2 pneumonia (sensitivity: 100%; specificity: 78%; AUC: 0.8750; p = 0.0027). Conclusions: This study identified a threshold of IL-6 levels at hospital admission for death risk in CHD patients with SARS-CoV-2 pneumonia. This might represent a valuable outcome predictor, feasibly better than other clinical, radiological, or laboratory parameters and preceding the IL-6 peak, which is unpredictable.

IL-10, IL-13, Eotaxin and IL-10/IL-6 ratio distinguish breast implant-associated anaplastic large-cell lymphoma from all types of benign late seromas.

Breast implant-associated anaplastic large-cell lymphoma (BI-ALCL) is an uncommon peripheral T cell lymphoma usually presenting as a delayed peri-implant effusion. Chronic inflammation elicited by the implant has been implicated in its pathogenesis. Infection or implant rupture may also be responsible for late seromas. Cytomorphological examination coupled with CD30 immunostaining and eventual T-cell clonality assessment are essential for BI-ALCL diagnosis. However, some benign effusions may also contain an oligo/monoclonal expansion of CD30 + cells that can make the diagnosis challenging. Since cytokines are key mediators of inflammation, we applied a multiplexed immuno-based assay to BI-ALCL seromas and to different types of reactive seromas to look for a potential diagnostic BI-ALCL-associated cytokine profile. We found that BI-ALCL is characterized by a Th2-type cytokine milieu associated with significant high levels of IL-10, IL-13 and Eotaxin which discriminate BI-ALCL from all types of reactive seroma. Moreover, we found a cutoff of IL10/IL-6 ratio of 0.104 is associated with specificity of 100% and sensitivity of 83% in recognizing BI-ALCL effusions. This study identifies promising biomarkers for initial screening of late seromas that can facilitate early diagnosis of BI-ALCL.

Sarcopenia in cardiac surgery: Dual X-ray absorptiometry study from the McGill frailty registry.

BACKGROUND: To determine the prevalence and prognostic value of sarcopenia measured by dual x-ray absorptiometry (DXA) and physical performance tests in patients undergoing coronary artery bypass surgery or heart valve procedures. METHODS: Adults undergoing cardiac surgery were prospectively enrolled and completed a questionnaire, physical performance battery, and a DXA scan (GE Lunar) to measure appendicular muscle mass indexed to height2 (AMMI). Patients were categorized as sarcopenic based on European Working Group 2 guidelines if they had low AMMI defined as <7 kg/m2 for men or <5.5 kg/m2 for women, and low muscle strength defined as 5 chair rise time ≥15 seconds. Cox proportional hazards regression was used to test the association between sarcopenia and all-cause mortality over a median follow-up of 4.3 years. RESULTS: The cohort consisted of 141 patients with a mean age of 69.7 ± 10.0 years and 21% females. The prevalence rates of low AMMI, slow chair rise time, and sarcopenia (low AMMI and slow chair rise time) were 24%, 57%, 13%, respectively. The 4-year survival rate was 79% in the non-sarcopenic group as compared to 56% in the sarcopenic group (Log-rank P = 0.01). In the multivariable model, each standard deviation of decreasing AMMI and increasing chair rise time was associated with a hazard ratio for all-cause mortality of 1.84 (95% CI 1.18, 2.86) and 1.79 (95% CI 1.26, 2.54), respectively. CONCLUSION: Lower-extremity muscle strength and DXA-based muscle mass are objective indicators of sarcopenia that are independently predictive of all-cause mortality in older cardiac surgery patients.

Nonthyroidal illness syndrome (NTIS) in severe COVID-19 patients: role of T3 on the Na/K pump gene expression and on hydroelectrolytic equilibrium.

BACKGROUND: Nonthyroidal Illness Syndrome (NTIS) can be detected in many critical illnesses. Recently, we demonstrated that this condition is frequently observed in COVID-19 patients too and it is correlated with the severity the disease. However, the exact mechanism through which thyroid hormones influence the course of COVID-19, as well as that of many other critical illnesses, is not clear yet and treatment with T4, T3 or a combination of both is still controversial. Aim of this study was to analyze body composition in COVID-19 patients in search of possible correlation with the thyroid function. METHODS AND FINDINGS: We report here our experience performed in 74 critically ill COVID-19 patients hospitalized in the intensive care unit (ICU) of our University Hospital in Rome. In these patients, we evaluated the thyroid hormone function and body composition by Bioelectrical Impedance Analysis (BIA) during the acute phase of the disease at admission in the ICU. To examine the effects of thyroid function on BIA parameters we analyzed also 96 outpatients, affected by thyroid diseases in different functional conditions. We demonstrated that COVID-19 patients with low FT3 serum values exhibited increased values of the Total Body Water/Free Fat Mass (TBW/FFM) ratio. Patients with the lowest FT3 serum values had also the highest level of TBW/FFM ratio. This ratio is an indicator of the fraction of FFM as water and represents one of the best-known body-composition constants in mammals. We found an inverse correlation between FT3 serum values and this constant. Reduced FT3 serum values in COVID-19 patients were correlated with the increase in the total body water (TBW), the extracellular water (ECW) and the sodium/potassium exchangeable ratio (Nae:Ke), and with the reduction of the intracellular water (ICW). No specific correlation was observed in thyroid patients at different functional conditions between any BIA parameters and FT3 serum values, except for the patient with myxedema, that showed a picture similar to that seen in COVID-19 patients with NTIS. Since the Na+/K+ pump is a well-known T3 target, we measured the mRNA expression levels of the two genes coding for the two major isoforms of this pump. We demonstrated that COVID-19 patients with NTIS had lower levels of mRNA of both genes in the peripheral blood mononuclear cells (PBMC)s obtained from our patients during the acute phase of the disease. In addition, we retrieved data from transcriptome analysis, performed on human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM)s treated with T3 and we demonstrated that in these cells T3 is able to stimulate the expression of these two genes in a dose-dependent manner. CONCLUSIONS: In conclusion, we demonstrated that measurement of BIA parameters is a useful method to analyze water and salt retention in COVID-19 patients hospitalized in ICU and, in particular, in those that develop NTIS. Our results indicate that NTIS has peculiar similarities with myxedema seen in severe hypothyroid patients, albeit it occurs more rapidly. The Na+/K+ pump is a possible target of T3 action, involved in the pathogenesis of the anasarcatic condition observed in our COVID-19 patients with NTIS. Finally, measurement of BIA parameters may represent good endpoints to evaluate the benefit of future clinical interventional trials, based on the administration of T3 in patients with NTIS.

Gene signature and immune cell profiling by high-dimensional, single-cell analysis in COVID-19 patients, presenting Low T3 syndrome and coexistent hematological malignancies.

BACKGROUND: Low T3 syndrome is frequent in patients admitted to intensive care units for critical illness and pneumonia. It has been reported also in patients with COVID-19, Hodgkin disease and chronic lymphocytic leukemia. We analyzed the clinical relevance of Low T3 syndrome in COVID-19 patients and, in particular, in those with associated hematological malignancies. METHODS: Sixty-two consecutive patients, hospitalized during the first wave of SARS-CoV-2 outbreak in Sant'Andrea University Hospital in Rome, were subdivided in 38 patients (Group A), showing low levels of FT3, and in 24 patients (Group B), with normal FT3 serum values. During the acute phase of the disease, we measured serum, radiologic and clinical disease severity markers and scores, in search of possible correlations with FT3 serum values. In addition, in 6 COVID-19 patients, 4 with Low T3 syndrome, including 2 with a hematological malignancy, and 2 with normal FT3 values, we performed, high-dimensional single-cell analysis by mass cytometry, multiplex cytokine assay and gene expression profiling in peripheral blood mononuclear cells (PBMC). RESULTS: Low FT3 serum values were correlated with increased Absolute Neutrophil Count, NLR and dNLR ratios and with reduced total count of CD3+, CD4+ and CD8+ T cells. Low FT3 values correlated also with increased levels of inflammation, tissue damage and coagulation serum markers as well as with SOFA, LIPI and TSS scores. The CyTOF analysis demonstrated reduction of the effector memory and terminal effector subtypes of the CD4+ T lymphocytes. Multiplex cytokine assay indicates that mainly IL-6, IP-10 and MCAF changes are associated with FT3 serum levels, particularly in patients with coexistent hematological malignancies. Gene expression analysis using Nanostring identified four genes differently expressed involved in host immune response, namely CD38, CD79B, IFIT3 and NLRP3. CONCLUSIONS: Our study demonstrates that low FT3 serum levels are associated with severe COVID-19. Our multi-omics approach suggests that T3 is involved in the immune response in COVID-19 and coexistent hematological malignancy and new possible T3 target genes in these patients have been identified.

Circulating Vitamin D levels status and clinical prognostic indices in COVID-19 patients.

BACKGROUND: Several immune mechanisms activate in COVID-19 pathogenesis. Usually, coronavirus infection is characterized by dysregulated host immune responses, interleukine-6 increase, hyper-activation of cytotoxic CD8 T lymphocytes. Interestingly, Vitamin D deficiency has been often associated with altered immune responses and infections. In the present study, we evaluated Vitamin D plasma levels in patients affected with different lung involvement during COVID-19 infection. METHODS: Lymphocyte phenotypes were assessed by flow cytometry. Thoracic CT scan involvement was obtained by an image analysis program. RESULTS: Vitamin D levels were deficient in (80%) of patients, insufficient in (6.5%) and normal in (13.5%). Patients with very low Vitamin D plasma levels had more elevated D-Dimer values, a more elevated B lymphocyte cell count, a reduction of CD8 + T lymphocytes with a low CD4/CD8 ratio, more compromised clinical findings (measured by LIPI and SOFA scores) and thoracic CT scan involvement. CONCLUSIONS: Vitamin D deficiency is associated with compromised inflammatory responses and higher pulmonary involvement in COVID-19 affected patients. Vitamin D assessment, during COVID-19 infection, could be a useful analysis for possible therapeutic interventions. TRIAL REGISTRATION: 'retrospectively registered'.

Application of the Sampson equation to estimate LDL-C in children: Comparison with LDL direct measurement and Friedewald equation in the BLIP study.

BACKGROUND AND AIMS: In epidemiological trials and in clinical practices, it is relevant to have affordable and reliable methods to measure the main lipid cardiovascular risk factors, and in particular low-density lipoprotein cholesterol (LDL-C) plasma level. In this context, we aimed to compare the reliability of the Friedewald's (LDL-Cf) and Sampson's (LDL-Cs) equations with the LDL-value dosed by a validated dosage method (LDL-Cd) in a large cohort of children. METHODS AND RESULTS: We considered the lipid values of 145 infants, 278 preschoolers, 810 scholar children, and 1372 adolescents (Total N. 2605, 1291 males, 1314 females), with mean total cholesterol (TC) = 169.8 ± 39.7 mg/dL, HDL-Cholesterol = 50.8 ± 12.7 mg/dL, non HDL-Cholesterol = 118.9 ± 35.9 mg/dL, Triglycerides (TG) = 90.3 ± 77.9 mg/dL, LDL-Cd = 106.2 ± 29.9 mg/dL, LDL-Cf = 100.9 ± 33.8 mg/dL, and LDL-Cs = 102.2 ± 33.4 mg/dL. Comparing the distance to the LDL-Cd, Friedewald's equation mildly but significantly underestimated in infants (3.4 ± 5.3 mg/dL), preschoolers (1.5 ± 7.1 mg/dL). Children (1.2 ± 2.2 mg/dL) and adolescents (1.1 ± 5.9 mg/dL) compared to Sampson's equation (all comparisons, p < 0.001). CONCLUSIONS: Our analysis, being carried out on a large population sample, shows that Sampson's equation is more reliable than Friedewald's one at each considered age class and even for extreme TG values.

CytoMatrix for a reliable and simple characterization of lung cancer stem cells from malignant pleural effusions.

Cancer stem cells (CSCs) are a subpopulation with the properties of extensive self-renewal, capability to generate differentiated cancer cells and resistance to therapies. We have previously shown that malignant pleural effusions (MPEs) from patients with non-small-cell lung cancer (NSCLC) represent a valuable source of cancer cells that can be grown as three-dimensional (3D) spheroids enriched for stem-like features, which depend on the activation of the Yes-associated protein-transcriptional coactivator with PDZ-binding motif (YAP-TAZ)/Wnt-ßcatenin/stearoyl-CoA desaturase 1 (SCD1) axis. Here, we describe a novel support, called CytoMatrix, for the characterization of limited amounts of cancer cells isolated from MPEs of patients with NSCLC. Our results show that this synthetic matrix allows an easy and fast characterization of several epithelial cellular markers. The use of CytoMatrix to study CSCs subpopulation confirms that SCD1 protein expression is enhanced in 3D spheroids when compared with 2D adherent cell cultures. YAP/TAZ nuclear-cytoplasmic distribution analysed by CytoMatrix in 3D spheroids is highly heterogeneous and faithfully reproduces what is observed in tumour biopsies. Our results confirm and extend the robustness of our workflow for the isolation and phenotypic characterization of primary cancer cells derived from the lung MPEs and underscore the role of SCD1.

Reverse transcriptase inhibition potentiates target therapy in BRAF-mutant melanomas: effects on cell proliferation, apoptosis, DNA-damage, ROS induction and mitochondrial membrane depolarization.

Target therapies based on BRAF and MEK inhibitors (MAPKi) have changed the therapeutic landscape for metastatic melanoma patients bearing mutations in the BRAF kinase. However, the emergence of drug resistance imposes the necessity to conceive novel therapeutic strategies capable to achieve a more durable disease control. In the last years, retrotransposons laying in human genome have been shown to undergo activation during tumorigenesis, where they contribute to genomic instability. Their activation can be efficiently controlled with reverse transcriptase inhibitors (RTIs) frequently used in the treatment of AIDS. These drugs have demonstrated anti-proliferative effects in several cancer models, including also metastatic melanoma. However, to our knowledge no previous study investigated the capability of RTIs to mitigate drug resistance to target therapy in BRAF-mutant melanomas. In this short report we show that the non-nucleoside RTI, SPV122 in combination with MAPKi strongly inhibits BRAF-mutant melanoma cell growth, induces apoptosis, and delays the emergence of resistance to target therapy in vitro. Mechanistically, this combination strongly induces DNA double-strand breaks, mitochondrial membrane depolarization and increased ROS levels. Our results shed further light on the molecular activity of RTI in melanoma and pave the way to their use as a novel therapeutic option to improve the efficacy of target therapy. Video Abstract.

Serum or plasma? An old question looking for new answers.

Serum or plasma? An old question looking for new answers. There is a continual debate on what type of sample a clinical laboratory should use. While serum is still considered the gold standard and remains the required sample for some assays, laboratories must consider turn-around time, which is an important metric for laboratory performance and, more importantly, plays a critical role in patient care. In addition, a body of evidence emphasise the choice of plasma in order to prevent modifications of some analytes due to the coagulation process and related interferences. Advantages and disadvantages of serum and plasma are discussed on the basis of current literature and evidence. In addition, data are provided on the current utilisation of the samples (serum or plasma) in Italy and in other countries. Finally, a rationale for a possible switch from serum to plasma is provided.

Multiplexed therapeutic drug monitoring of antipsychotics in dried plasma spots by LC-MS/MS.

In this work, a convenient method for the therapeutic monitoring of seven common antipsychotic drugs in "dried plasma spot" samples has been developed. It is based on the liquid chromatography tandem mass spectrometry technique, operating in multiple reaction monitoring mode, and a straightforward procedure for the simultaneous extraction of all antipsychotics in a single step, with high extraction yield. The method was fully validated with proper accuracy, precision, selectivity and sensitivity, for all the drugs. Limits of quantification were 0.12, 1.09, 1.46, 1.47, 5.70, 1.32, 1.33 µg/L for haloperidol, aripiprazole, olanzapine, quetiapine, clozapine, risperidone, and paliperidone, respectively. Accuracy, intra- and interday precision values were <10% for all drugs at all concentration levels examined. The method was tested in the analysis of 30 plasma samples from real patients for each drug. The proposed analytical approach, by combining practical and logistical advantages of microsampling with liquid chromatography tandem mass spectrometry analytical performance, could offer an ideal strategy for accurate and timely therapeutic drug monitoring of antipsychotic drugs in most clinical settings, even in remote centers and/or in out-patient settings, bringing so many potential improvements in psychiatric patient care.

In Vitro Biophysical and Biological Characterization of Lipid Nanoparticles Co-Encapsulating Oncosuppressors miR-199b-5p and miR-204-5p as Potentiators of Target Therapy in Metastatic Melanoma.

Uncontrolled MAPK signaling is the main oncogenic driver in metastatic melanomas bearing mutations in BRAF kinase. These tumors are currently treated with the combination of BRAF/MEK inhibitors (MAPKi), but this therapy is plagued by drug resistance. In this context we recently discovered that several microRNAs are involved in the development of drug resistance. In particular miR-204-5p and miR-199b-5p were found to function as antagonists of resistance because their enforced overexpression is able to inhibit melanoma cell growth in vitro either alone or in combination with MAPKi. However, the use of miRNAs in therapy is hampered by their rapid degradation in serum and biological fluids, as well as by the poor intracellular uptake. Here, we developed lipid nanoparticles (LNPs) encapsulating miR-204-5p, miR-199b-5p individually or in combination. We obtained LNPs with mean diameters < 200 nm and high miRNA encapsulation efficiency. These formulations were tested in vitro on several melanoma cell lines sensitive to MAPKi or rendered drug resistant. Our results show that LNPs encapsulating combinations of the two oncosuppressor miRNAs are highly efficient in impairing melanoma cell proliferation and viability, affect key signaling pathways involved in melanoma cell survival, and potentiate the efficacy of drugs inhibiting BRAF and MEK. These results warrant further assessment of the anti-tumor efficacy of oncosuppressor miRNAs encapsulating LNPs in in vivo tumor models.

Frailty assessment in older adults undergoing interventions for peripheral arterial disease.

OBJECTIVE: Frailty is a multidimensional syndrome that influences postoperative morbidity and mortality after vascular procedures; however, its integration in clinical practice has been limited, given the lack of consensus on how to measure it. This study sought to compare the incremental predictive value of six different nonphysical performance frailty scales to predict poor outcomes after interventions for peripheral arterial disease (PAD). METHODS: This preplanned analysis of the FRailty Assessment In Lower Extremity arterial Disease (FRAILED) prospective cohort included two centers recruiting patients between July 1, 2015, and October 1, 2016. Individuals who underwent vascular interventions for Rutherford class 3 or higher PAD were enrolled. The following scales were compared: Edmonton Frail Scale, Groningen Frailty Indicator (GFI), modified Essential Frailty Toolset (mEFT), modified Frailty Index, Multidimensional Prognostic Index, and the Risk Analysis Index-C. The primary end point was a composite of all-cause mortality and major disability at 12 months after the procedure. The secondary end point was length of stay. Logistic regression was used to determine the association of frailty with the primary end point after adjusting for confounders. To compare the incremental predictive value of each frailty scale, model performance statistics were calculated. RESULTS: The cohort was composed of 148 patients with a mean age of 70 years. Depending on the scale used, the prevalence of frailty ranged from 16% to 70%. Frailty as measured by the GFI (adjusted odds ratio, 1.76; 95% confidence interval, 1.14-2.72) and mEFT (adjusted odds ratio, 2.71; 95% confidence interval, 1.29-5.73) predicted mortality and worsening disability at 12 months after interventions for PAD. Furthermore, there was statistically significant C-statistic, Bayesian information criterion, and integrated discrimination improvement when the GFI and mEFT were added to the baseline model. Frailty was not associated with length of stay. CONCLUSIONS: Frailty is associated with mortality and worsening disability after interventions for PAD. The GFI and mEFT performed well and identified vulnerable older adults who are at risk of poor outcomes after interventions for PAD and recommended for use in this setting.

Comparative serum bactericidal activity of meropenem-based combination regimens against extended-spectrum beta-lactamase and KPC-producing Klebsiella pneumoniae.

Combination therapies are frequently used in the treatment of multidrug-resistant Klebsiella pneumoniae infection without consensus regarding which combination is the most effective. We compared bactericidal titres from sera collected from critically ill patients receiving meropenem plus tigecycline (n = 5), meropenem plus colistin (n = 5), or meropenem, colistin and tigecycline (n = 5) against K. pneumoniae isolates that included ESBL-producing (n = 7) and KPC-producing strains (n = 14) with varying sensitivity patterns to colistin and tigecycline. Meropenem concentrations (Cmin) were measured in all samples by LC-MS/MS, and indexed to respective pathogen MICs to explore differences in patterns of bactericidal activity for two versus three drug combination regimens. All combination regimens achieved higher SBTs against ESBL (median reciprocal titre 128, IQR 32-256) versus KPC (4, IQR 2-32) strains. Sera from patients treated with meropenem-colistin yielded higher median SBTs (256, IQR 64-512) than either meropenem-tigecycline (32, IQR 8-256; P < 0.001). The addition of tigecycline was associated with a lower probability of achieving a reciprocal SBT above 8 when meropenem concentrations were below the MIC (P = 0.04). Although the clinical significance is unknown, sera from patients receiving tigecycline-based combination regimens produce lower serum bactericidal titres against ESBL or KPC-producing K. pneumoniae. SBTs may represent a useful complimentary endpoint for comparing pharmacodynamics of combinations regimens for MDR Enterobacteriaceae.

miR-579-3p controls melanoma progression and resistance to target therapy.

Therapy of melanoma patients harboring activating mutations in the BRAF (V-raf murine sarcoma viral oncogene homolog B1) oncogene with a combination of BRAF and MEK inhibitors is plagued by the development of drug resistance. Mutational events, as well as adaptive mechanisms, contribute to the development of drug resistance. In this context we uncover here the role of a miRNA, miR-579-3p. We first show that low expression of miR-579-3p is a negative prognostic factor correlating with poor survival. Expression levels of miR-579-3p decrease from nevi to stage III/IV melanoma samples and even further in cell lines resistant to BRAF/MEK inhibitors. Mechanistically, we demonstrate that miR-579-3p acts as an oncosuppressor by targeting the 3'UTR of two oncoproteins: BRAF and an E3 ubiquitin protein ligase, MDM2. Moreover miR-579-3p ectopic expression impairs the establishment of drug resistance in human melanoma cells. Finally, miR-579-3p is strongly down-regulated in matched tumor samples from patients before and after the development of resistance to targeted therapies.

c-Src Recruitment is Involved in c-MET-Mediated Malignant Behaviour of NT2D1 Non-Seminoma Cells.

: c-MET pathway over-activation is the signature of malignancy acquisition or chemotherapy resistance of many cancers. We recently demonstrated that type II Testicular Germ Cell Tumours (TGCTs) express c-MET receptor. In particular, we elucidated that the non-seminoma lesions express c-MET protein at higher level, compared with the seminoma ones. In line with this observation, NTERA-2 clone D1 (NT2D1) non-seminoma cells increase their proliferation, migration and invasion in response to Hepatocyte Growth Factor (HGF). One of the well-known adaptor-proteins belonging to c-MET signaling cascade is c-Src. Activation of c-Src is related to the increase of aggressiveness of many cancers. For this reason, we focused on the role of c-Src in c-MET-triggered and HGF-dependent NT2D1 cell activities. In the present paper, we have elucidated that this adaptor-protein is involved in HGF-dependent NT2D1 cell proliferation, migration and invasion, since Src inhibitor-1 administration abrogates these responses. Despite these biological evidences western blot analyses have not revealed the increase of c-Src activation because of HGF administration. However, notably, immunofluorescence analyses revealed that cytoplasmic and membrane-associated localization of c-Src shifted to the nuclear compartment after HGF stimulation. These results shed new light in the modality of HGF-dependent c-Src recruitment, and put the basis for novel investigations on the relationship between c-Src, and TGCT aggressiveness.