External Quality Assessment (EQA) scheme for serological diagnostic test for SARS-CoV-2 detection in Sicily Region (Italy), in the period 2020-2022.

OBJECTIVES: Since December 2019, worldwide public health has been exposed to a severe acute respiratory syndrome caused by Coronavirus-2. Serological testing is necessary for retrospective assessment of seroprevalence rates, and the determination of vaccine response and duration of immunity. For this reason, it was necessary to introduce a panel of tests able to identify and quantify Covid-19 antibodies. METHODS: As a Regional Reference Centre, the CRQ Laboratory (Regional Laboratory for the Quality Control) developed and conducted an External Quality Assessment (EQA) panel of assays, to evaluate the quality of various methods, that were used by 288 Sicilian laboratories, previously authorized on behalf of the Public Health Service. RESULTS: The performance test was based on pooled samples with different levels of concentration of antibodies. 97 , 98, and 95 % of the participating laboratories tested all samples correctly in 2020, 2021, and 2022 respectively. The best performance was observed in the test of total Ig. The general performance of laboratories improved over the years. CONCLUSIONS: The incorrect diagnosis had and could still have important implications on vaccination cycles. Only through the effort of laboratory professionals, and the extension of the EQA scheme, a better harmonization of methods, protocols, and thus results, to guarantee a better healthcare system, will be possible.

Technical and health governance aspects of the external quality assessment system for classical and new psychoactive substances analysis testing in blood.

New psychoactive substances (NPS) are uncontrolled analogues of existing drugs or newly synthesized chemicals that exhibit psychopharmacological effects. Due to their diverse nature, composition, and increasing prevalence, they present significant challenges to the healthcare system and drug control policies. In response, healthcare system laboratories have developed analytical methods to detect NPS in biological samples. As a Regional Reference Centre, the Sicilian CRQ Laboratory (Regional Laboratory for Quality Control) developed and conducted an External Quality Assessment (EQA) study to assess, in collaboration with the Istituto Superiore di Sanità (ISS), the ability of different Italian laboratories to identify NPS and traditional drugs of abuse (DOA) in biological matrices. Two blood samples were spiked with substances from various drug classes, including synthetic cannabinoids, cathinones, synthetic opiates, and benzodiazepines, at concentrations ranging from 2 to 10 ng/mL. The blood samples were freeze-dried to ensure the stability of DOA and NPS. Twenty-two laboratories from the Italian healthcare system participated in this assessment. The information provided by the laboratories during the registration in an in-house platform included a general description of the laboratory, analytical technique, and the chosen panels of analytes. The same platform was employed to collect and statistically analyze the data and record laboratory feedback and comments. The evaluation of the results revealed that the participating laboratories employed three different techniques for analyzing the samples: GC-MS, LC-MS, and immunoenzymatic methods. Approximately 90 % of the laboratories utilized LC-MS techniques. Around 40 % of false negative results were obtained, with the worst results in the identification of 5-chloro AB PINACA. The results showed that laboratories that used LC-MS methods obtained better specificity and sensitivity compared to the laboratories using other techniques. The results obtained from this first assessment underscore the importance of external quality control schemes in identifying the most effective analytical techniques for detecting trace molecules in biological matrices. Since the judicial authorities have not yet established cut-off values for NPS, this EQA will enable participating laboratories to share their analytical methods and expertise, aiming to establish common criteria for NPS identification.

NAIF: A novel artificial intelligence-based tool for accurate diagnosis of stage F3/F4 liver fibrosis in the general adult population, validated with three external datasets.

OBJECTIVE: The purpose of this study was to determine the effectiveness of a new AI-based tool called NAIF (NAFLD-AI-Fibrosis) in identifying individuals from the general population with advanced liver fibrosis (stage F3/F4). We compared NAIF's performance to two existing risk score calculators, aspartate aminotransferase-to-platelet ratio index (APRI) and fibrosis-4 (Fib4). METHODS: To set up the algorithm for diagnosing severe liver fibrosis (defined as Fibroscan® values E ≥ 9.7 KPa), we used 19 blood biochemistry parameters and two demographic parameters in a group of 5,962 individuals from the NHANES population (2017-2020 pre-pandemic, public database). We then assessed the algorithm's performance by comparing its accuracy, precision, sensitivity, specificity, and F1 score values to those of APRI and Fib4 scoring systems. RESULTS: In a kept-out sub dataset of the NHANES population, NAIF achieved a predictive precision of 72 %, a sensitivity of 61 %, and a specificity of 77 % in correctly identifying adults (aged 18-79 years) with severe liver fibrosis. Additionally, NAIF performed well when tested with two external datasets of Italian patients with a Fibroscan® score E ≥ 9.7 kPa, and with an external dataset of patients with diagnosis of severe liver fibrosis through biopsy. CONCLUSIONS: The results of our study suggest that NAIF, using routinely available parameters, outperforms in sensitivity existing scoring methods (Fib4 and APRI) in diagnosing severe liver fibrosis, even when tested with external validation datasets. NAIF uses routinely available parameters, making it a promising tool for identifying individuals with advanced liver fibrosis from the general population. Word count abstract: 236.

Genetic Biomarkers of Sorafenib Response in Patients with Hepatocellular Carcinoma.

The identification of biomarkers for predicting inter-individual sorafenib response variability could allow hepatocellular carcinoma (HCC) patient stratification. SNPs in angiogenesis- and drug absorption, distribution, metabolism, and excretion (ADME)-related genes were evaluated to identify new potential predictive biomarkers of sorafenib response in HCC patients. Five known SNPs in angiogenesis-related genes, including VEGF-A, VEGF-C, HIF-1a, ANGPT2, and NOS3, were investigated in 34 HCC patients (9 sorafenib responders and 25 non-responders). A subgroup of 23 patients was genotyped for SNPs in ADME genes. A machine learning classifier method was used to discover classification rules for our dataset. We found that only the VEGF-A (rs2010963) C allele and CC genotype were significantly associated with sorafenib response. ADME-related gene analysis identified 10 polymorphic variants in ADH1A (rs6811453), ADH6 (rs10008281), SULT1A2/CCDC101 (rs11401), CYP26A1 (rs7905939), DPYD (rs2297595 and rs1801265), FMO2 (rs2020863), and SLC22A14 (rs149738, rs171248, and rs183574) significantly associated with sorafenib response. We have identified a genetic signature of predictive response that could permit non-responder/responder patient stratification. Angiogenesis- and ADME-related genes correlation was confirmed by cumulative genetic risk score and network and pathway enrichment analysis. Our findings provide a proof of concept that needs further validation in follow-up studies for HCC patient stratification for sorafenib prescription.

Homologous Recombination Deficiency (HRD) Scoring, by Means of Two Different Shallow Whole-Genome Sequencing Pipelines (sWGS), in Ovarian Cancer Patients: A Comparison with Myriad MyChoice Assay.

High-grade serous ovarian cancer (HGSOC) patients carrying the BRCA1/2 mutation or deficient in the homologous recombination repair system (HRD) generally benefit from treatment with PARP inhibitors. Some international recommendations suggest that BRCA1/2 genetic testing should be offered for all newly diagnosed epithelial ovarian cancer, along with HRD assessment. Academic tests (ATs) are continuously under development, in order to break down the barriers patients encounter in accessing HRD testing. Two different methods for shallow whole-genome sequencing (sWGS) were compared to the reference assay, Myriad. All these three assays were performed on 20 retrospective HGSOC samples. Moreover, HRD results were correlated with the progression-free survival rate (PFS). Both sWGS chemistries showed good correlation with each other and a complete agreement, even when compared to the Myriad score. Our academic HRD assay categorized patients as HRD-Deficient, HRM-Mild and HRN-Negative. These three groups were matched with PFS, providing interesting findings in terms of HRD scoring and months of survival. Both our sWGS assays and the Myriad test correlated with the patient's response to treatments. Finally, our AT confirms its capability of determining HRD status, with the advantage of being faster, cheaper, and easier to carry out. Our results showed a prognostic value for the HRD score.

The Suitability of RNA from Positive SARS-CoV-2 Rapid Antigen Tests for Whole Virus Genome Sequencing and Variant Identification to Maintain Genomic Surveillance.

The COVID-19 pandemic has transformed laboratory management, with a surge in demand for diagnostic tests prompting the adoption of new diagnostic assays and the spread of variant surveillance tools. Rapid antigen tests (RATs) were initially used only for screening and later as suitable infection assessment tools. This study explores the feasibility of sequencing the SARS-CoV-2 genome from the residue of the nasopharyngeal swab extraction buffers of rapid antigen tests (RATs) to identify different COVID-19 lineages and sub-lineages. METHODS: Viral RNA was extracted from the residue of the nasopharyngeal swab extraction buffers of RATs and, after a confirmation of positivity through a reaction of RT-PCR, viral genome sequencing was performed. RESULTS: Overall, the quality of the sequences obtained from the RNA extracted from the residue of the nasopharyngeal swab extraction buffers of RATs was adequate and allowed us to identify the SARS-CoV-2 variants' circulation and distribution in a period when the use of molecular swabs had been drastically reduced. CONCLUSIONS: This study demonstrates the potential for genomic surveillance by sequencing SARS-CoV-2 from the residue of the nasopharyngeal swab extraction buffers of RATs, highlighting alternative possibilities for tracking variants.

Nutritional Intervention in Cushing's Disease: The Ketogenic Diet's Effects on Metabolic Comorbidities and Adrenal Steroids.

BACKGROUND: a very low-calorie ketogenic diet (VLCKD) is associated with improvement of metabolic and cardiovascular disorders. We aimed to evaluate the effects of a VLCKD in patients with Cushing's disease (CD) as adjunctive therapy to treatment for the primary disease. METHODS: we evaluated clinical, hormonal and metabolic parameters in 15 patients with CD and 15 controls at baseline after 1 week and 3 weeks of VLCKD and, further, after 2 weeks of a low-carbohydrate ketogenic diet (LCKD). RESULTS: after 5 weeks of diet, a significant decrease in BMI (p = 0.002), waist circumference (WC) (p = 0.024), systolic blood pressure (p = 0.015), diastolic blood pressure (p = 0.005), ACTH (p = 0.026), cortisone (p = 0.025), total cholesterol (p = 0.006), LDL cholesterol (p = 0.017), triglycerides (p = 0.016) and alkaline phosphatase (p = 0.008) and a significant increase in HDL cholesterol (p = 0.017), vitamin D (p = 0.015) and oral disposition index (oDI) (p = 0.004) was observed in the CD patients. A significant decrease in BMI (p = 0.003), WC (p = 0.002), systolic blood pressure (p = 0.025), diastolic (p = 0.007) blood pressure and total cholesterol (p = 0.026) and an increase in HDL cholesterol (p = 0.001) and oDI (p < 0.001) was observed in controls. CONCLUSIONS: the current study confirms that a ketogenic diet is effective in improving metabolic disorders in CD and shows that a nutritional approach may be combined with conventional CD therapy in order to improve metabolic and cardiovascular comorbidities.

The Truth Is Out There: Biological Features and Clinical Indications of Extracellular Vesicles from Human Perinatal Stem Cells.

The potential of perinatal tissues to provide cellular populations to be used in different applications of regenerative medicine is well established. Recently, the efforts of researchers are being addressed regarding the evaluation of cell products (secreted molecules or extracellular vesicles, EVs) to be used as an alternative to cellular infusion. The data regarding the effective recapitulation of most perinatal cells' properties by their secreted complement point in this direction. EVs secreted from perinatal cells exhibit key therapeutic effects such as tissue repair and regeneration, the suppression of inflammatory responses, immune system modulation, and a variety of other functions. Although the properties of EVs from perinatal derivatives and their significant potential for therapeutic success are amply recognized, several challenges still remain that need to be addressed. In the present review, we provide an up-to-date analysis of the most recent results in the field, which can be addressed in future research in order to overcome the challenges that are still present in the characterization and utilization of the secreted complement of perinatal cells and, in particular, mesenchymal stromal cells.

Left ventricular hypertrophy and metabolic resetting in the Notch3-deficient adult mouse heart.

The heart depends on a functional vasculature for oxygenation and transport of nutrients, and it is of interest to learn how primary impairment of the vasculature can indirectly affect cardiac function and heart morphology. Notch3-deficiency causes vascular smooth muscle cell (VSMC) loss in the vasculature but the consequences for the heart remain largely elusive. Here, we demonstrate that Notch3-/- mice have enlarged hearts with left ventricular hypertrophy and mild fibrosis. Cardiomyocytes were hypertrophic but not hyperproliferative, and the expression of several cardiomyocyte markers, including Tnt2, Myh6, Myh7 and Actn2, was altered. Furthermore, expression of genes regulating the metabolic status of the heart was affected: both Pdk4 and Cd36 were downregulated, indicating a metabolic switch from fatty acid oxidation to glucose consumption. Notch3-/- mice furthermore showed lower liver lipid content. Notch3 was expressed in heart VSMC and pericytes but not in cardiomyocytes, suggesting that a perturbation of Notch signalling in VSMC and pericytes indirectly impairs the cardiomyocytes. In keeping with this, Pdgfbret/ret mice, characterized by reduced numbers of VSMC and pericytes, showed left ventricular and cardiomyocyte hypertrophy. In conclusion, we demonstrate that reduced Notch3 or PDGFB signalling in vascular mural cells leads to cardiomyocyte dysfunction.

Effectiveness and Safety of Remdesivir in Treating Hospitalised Patients with COVID-19: A Propensity Score Analysis of Real-Life Data from a Monocentric Observational Study in Times of Health Emergency.

BACKGROUND AND OBJECTIVES: Remdesivir is an antiviral agent, which was shown to be safe and effective in treating early COVID-19, but its favourable impact in hospitalised patients with non-critical disease is still under investigation. The present study aimed to assess the effectiveness and safety of remdesivir as a treatment for hospitalised patients with COVID-19 by a propensity score analysis of observational data. METHODS: In this monocentric retrospective cohort study, the effectiveness and safety of a 5-day course of remdesivir (200 mg intravenously at Day 1, then 100 mg from Days 2-5) in association with the standard of care were assessed in comparison with the standard of care only. The primary endpoint was the proportion of recovery on Day 14. RESULTS: Of 3662 eligible inpatients who tested positive for the severe acute respiratory syndrome coronavirus 2 genome by nasopharyngeal swab at admission, 861 (24%) non-critical patients were included in a propensity score analysis and 281 (33%) were exposed to remdesivir. In total, 242/281 (86.1%) and 435/580 (75.0%) patients recovered in exposed and non-exposed, respectively, with a relative improvement of 11.1% (95% CI + 5.8 to 16.5%; unadjusted odds ratio: 2.07, 95% CI 1.40-3.05, p = 0.0001; after adjustment by propensity score weighting, odds ratio: 1.92, 95% CI 1.30-2.83, p = 0.001). In treated patients, 1 (0.03%) anaphylactic reaction and 1 (0.03%) acute reaction during drug injection were reported, and 24 (8.5%) patients stopped the treatment due to adverse reactions. No significant differences were found with respect to the secondary efficacy endpoints (in-hospital all-cause death, need for intensive care treatments, clinical improvement score at Day 28) and safety endpoints (any and serious adverse reactions). CONCLUSION: A 5-day course of remdesivir in association with the standard of care effectively promoted recovery from COVID-19 among non-critical in-hospital patients and had an acceptable safety profile.

Molecular anatomy of adult mouse leptomeninges.

Leptomeninges, consisting of the pia mater and arachnoid, form a connective tissue investment and barrier enclosure of the brain. The exact nature of leptomeningeal cells has long been debated. In this study, we identify five molecularly distinct fibroblast-like transcriptomes in cerebral leptomeninges; link them to anatomically distinct cell types of the pia, inner arachnoid, outer arachnoid barrier, and dural border layer; and contrast them to a sixth fibroblast-like transcriptome present in the choroid plexus and median eminence. Newly identified transcriptional markers enabled molecular characterization of cell types responsible for adherence of arachnoid layers to one another and for the arachnoid barrier. These markers also proved useful in identifying the molecular features of leptomeningeal development, injury, and repair that were preserved or changed after traumatic brain injury. Together, the findings highlight the value of identifying fibroblast transcriptional subsets and their cellular locations toward advancing the understanding of leptomeningeal physiology and pathology.

A new method for oral cancer biomarkers detection with a non-invasive cyto-salivary sampling and rapid-highly sensitive ELISA immunoassay: a pilot study in humans.

Introduction: Oral squamous cell carcinoma (OSCC) accounts for approximately 90% of oral malignancies and has a 5-year mortality rate close to 50%. A consistent part (70%) of all oral cancers is diagnosed at an advanced stage since available screening techniques are ineffective. Therefore, it would be urgent to improve them. The diagnostic gold standard is tissue biopsy with histological and immunohistochemical assessment. This method presents some limitations. Biopsy is invasive and the histopathological evaluation is semi-quantitative, and the absolute abundance of the target cannot be reliably determined. In addition, tissue is highly processed and may lead to loss of information of the natural state. The search for classical and new clinical biomarkers on fragments of tissue/cells collected with a cytobrush is a highly hopeful technique for early detection and diagnosis of OSCC, because of its non-invasive sampling and easy collection method. Methods: Here we analyzed cytobrush biopsies samples collected from the oral cavity of 15 patients with already diagnosed OSCC by applying an innovative high-sensitivity ELISA technique, in order to verify if this approach may provide useful information for detection, diagnosis, and prognosis of OSCC. To this end, we selected six biomarkers, already used in clinical practice for the diagnosis of OSCC (EGFR, Ki67, p53) or selected based on recent scientific and clinical data which indicate their presence or over-expression in cells undergoing transformation and their role as possible molecular targets in immunecheckpoints blockade therapies (PD-L1, HLA-E, B7-H6). Results: The selected tumor biomarkers were highly expressed in the tumor core, while were virtually negative in healthy tissue collected from the same patients. These differences were highly statistically significant and consistent with those obtained using the gold standard test clearly indicating that the proposed approach, i.e. analysis of biomarkers by a custom ELISA technique, is strongly reliable. Discussion: These preliminary data suggest that this non-invasive rapid phenotyping technique could be useful as a screening tool for phenotyping oral lesions and support clinical practice by precise indications on the characteristics of the lesion, also with a view to the application of new anti-tumor treatments, such as immunotherapy, aimed at OSCC patients.

Facing the Challenges in the COVID-19 Pandemic Era: From Standard Treatments to the Umbilical Cord-Derived Mesenchymal Stromal Cells as a New Therapeutic Strategy.

Coronavirus disease 2019 (COVID-19), the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which counts more than 650 million cases and more than 6.6 million of deaths worldwide, affects the respiratory system with typical symptoms such as fever, cough, sore throat, acute respiratory distress syndrome (ARDS), and fatigue. Other nonpulmonary manifestations are related with abnormal inflammatory response, the "cytokine storm", that could lead to a multiorgan disease and to death. Evolution of effective vaccines against SARS-CoV-2 provided multiple options to prevent the infection, but the treatment of the severe forms remains difficult to manage. The cytokine storm is usually counteracted with standard medical care and anti-inflammatory drugs, but researchers moved forward their studies on new strategies based on cell therapy approaches. The perinatal tissues, such as placental membranes, amniotic fluid, and umbilical cord derivatives, are enriched in mesenchymal stromal cells (MSCs) that exert a well-known anti-inflammatory role, immune response modulation, and tissue repair. In this review, we focused on umbilical-cord-derived MSCs (UC-MSCs) used in in vitro and in vivo studies in order to evaluate the weakening of the severe symptoms, and on recent clinical trials from different databases, supporting the favorable potential of UC-MSCs as therapeutic strategy.

Transplacental permeability of heavy metals in relation to newborn sex - evidence from the neurodevelopment project.

Introduction: Gender medicine is an innovative medical approach that studies how some biological variables are influenced by the male or female sex and gender. This issue is under debate because it characterizes the impact of tailored or individual medicine. In this scenario, the aim of this study is to study the correlation between heavy metal exposure and pathologies of neurodevelopment, according to the sex of newborns. In particular, this is an observational study under the name of the Neurosviluppo Project, involving 217 mother-child couples. Material and methods: The correlation with phenotype small for gestational age and congenital malformations were studied, but above all we focused on the pattern of placental permeability to heavy metals. Results: Our results are specifically related to foetal medicine and investigate the impact of foetal sex in transplacental metal exposure. Our results did not show any significant differences related to foetal sex in terms of congenital malformations or the other variables taken into consideration. However, because these conclusions are the first related to the gender medicine in transplacental foetal medicine, they could be a marked background for further studies. Conclusions: Considering the lack of data in literature regarding foetal sexual medicine and transplacental exposure, these study results are pioneering in terms of sexual foetal medicine. Possibly in the future, studies regarding the correlation between foetal sex and obstetrics outcomes will be performed.

A robust and efficient microvascular isolation method for multimodal characterization of the mouse brain vasculature.

Studying disease-related changes in the brain vasculature is warranted due to its crucial role in supplying oxygen and nutrients and removing waste and due to the anticipated vascular dysfunction in brain diseases. To this end, we have developed a protocol for fast and simple isolation of brain vascular fragments without the use of transgenic reporters. We used it to isolate and analyze 22,515 cells by single-cell RNA sequencing. The cells distributed into 23 distinct clusters corresponding to all known vascular and perivascular cell types in the brain. Western blot analysis also suggested that the protocol is suitable for proteomic analysis. We further adapted it for the establishment of primary cell cultures. The protocol generated highly reproducible results. In conclusion, we have developed a simple and robust brain vascular isolation protocol suitable for different experimental modalities, such as single-cell analyses, western blotting, and primary cell culture.

Technical and health governance aspects of the External Quality Assessment Scheme for the SARS-CoV-2 molecular tests: institutional experience performed in all clinical laboratories of a Regional Health Service.

OBJECTIVES: Since December 2019, the worldwide public health has been threatened by a severe acute respiratory syndrome caused by Coronavirus-2. From the beginning, a turning point has been the identification of new cases of infection, in order to minimize the virus spreading among the population. For this reason, it was necessary introducing a panel of tests able to identify positive cases, which became crucial for all countries. METHODS: As a Regional Reference Centre, the CRQ Laboratory (Regional Laboratory for the Quality Control) developed and conducted an External Quality Assessment (EQA) panel of assay, so as to evaluate the quality of real-time reverse transcription polymerase chain reaction (PCR), which were used by 62 Sicilian laboratories, previously authorized to issue certificates for the COVID-19 diagnosis, on behalf of the Public Health Service. RESULTS: The qualitative performance test was based on pooled samples with different viral loads of SARS-CoV-2 or human Coronavirus OC43. 75% of the participating laboratories tested all core samples correctly, while the remaining 25% interpreted incorrectly the EQA exercise samples matching negatively the standards required. CONCLUSIONS: Subsequent inspection visits confirmed the issue of incorrect positive and negative certifications for COVID-19 by private and public laboratories, despite the possession of the authorization requirements currently provided for by current regulations, with a significant impact on the SSR.

Setting up of a machine learning algorithm for the identification of severe liver fibrosis profile in the general US population cohort.

BACKGROUND: The progress of digital transformation in clinical practice opens the door to transforming the current clinical line for liver disease diagnosis from a late-stage diagnosis approach to an early-stage based one. Early diagnosis of liver fibrosis can prevent the progression of the disease and decrease liver-related morbidity and mortality. We developed here a machine learning (ML) algorithm containing standard parameters that can identify liver fibrosis in the general US population. MATERIALS AND METHODS: Starting from a public database (National Health and Nutrition Examination Survey, NHANES), representative of the American population with 7265 eligible subjects (control population n = 6828, with Fibroscan values E < 9.7 KPa; target population n = 437 with Fibroscan values E ≥ 9.7 KPa), we set up an SVM algorithm able to discriminate for individuals with liver fibrosis among the general US population. The algorithm set up involved the removal of missing data and a sampling optimization step to managing the data imbalance (only âˆ¼ 5 % of the dataset is the target population). RESULTS: For the feature selection, we performed an unbiased analysis, starting from 33 clinical, anthropometric, and biochemical parameters regardless of their previous application as biomarkers of liver diseases. Through PCA analysis, we identified the 26 more significant features and then used them to set up a sampling method on an SVM algorithm. The best sampling technique to manage the data imbalance was found to be oversampling through the SMOTE-NC. For final model validation, we utilized a subset of 300 individuals (150 with liver fibrosis and 150 controls), subtracted from the main dataset prior to sampling. Performances were evaluated on multiple independent runs. CONCLUSIONS: We provide proof of concept of an ML clinical decision support tool for liver fibrosis diagnosis in the general US population. Though the presented ML model represents at this stage only a prototype, in the future, it might be implemented and potentially applied to program broad screenings for liver fibrosis.

Active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common monogenic form of familial small vessel disease; no preventive or curative therapy is available. CADASIL is caused by mutations in the NOTCH3 gene, resulting in a mutated NOTCH3 receptor, with aggregation of the NOTCH3 extracellular domain (ECD) around vascular smooth muscle cells. In this study, we have developed a novel active immunization therapy specifically targeting CADASIL-like aggregated NOTCH3 ECD. Immunizing CADASIL TgN3R182C150 mice with aggregates composed of CADASIL-R133C mutated and wild-type EGF1-5 repeats for a total of 4 months resulted in a marked reduction (38-48%) in NOTCH3 deposition around brain capillaries, increased microglia activation and lowered serum levels of NOTCH3 ECD. Active immunization did not impact body weight, general behavior, the number and integrity of vascular smooth muscle cells in the retina, neuronal survival, or inflammation or the renal system, suggesting that the therapy is tolerable. This is the first therapeutic study reporting a successful reduction of NOTCH3 accumulation in a CADASIL mouse model supporting further development towards clinical application for the benefit of CADASIL patients.

Characterization of Gut Microbiota Composition in Type 2 Diabetes Patients: A Population-Based Study.

(1) Background: A clinical laboratory index to assess gut dysbiosis is the F/B ratio < 0.8. In fact, an elevated proportion of Firmicutes and a reduced population of Bacteroides in diabetes type 2 (T2D) subjects has been observed. This study aimed to detail the dysbiosis status in the Italian population, focusing on some pathogenic spectra (T2D) or metabolic disorders. (2) Material and methods: A quantity of 334 fecal samples was analyzed in order to perform genetic testing and sequencing. (3) Results: A trend in over imbalance was observed in the percentage of Proteobacteria (median value: 6.75%; interquartile range (IQR): 3.57−17.29%). A statistically significant association (χ2p = 0.033) was observed between type of dysbiosis and T2D, corresponding to an Odds Ratio (OR) of 1.86. It was noted that females with cystitis/candidiasis are significantly prevalent in T2D patients (p < 0.01; OR: 3.59; 95% CI: 1.43−8.99). Although, in non-diabetic males, a sugar craving is significantly associated with the rate of dysbiosis in non-diabetic males (p < 0.05; OR 1.07; 95% CI 1.00−1.16). (4) Conclusion: In T2D patients, the Bacteroidetes/Firmicutes ratio was biased in favor of Proteobacteria, to be expected due to the nutritional habits of the patients. Thus, T2D females had altered gut permeability favoring the development of infections in the vaginal tract.

Cigarette Smoke Extract Induces p38 MAPK-Initiated, Fas-Mediated Eryptosis.

Eryptosis is a physiological mechanism for the clearance of senescent or damaged erythrocytes by phagocytes. Excessive eryptosis is stimulated under several pathologies and associated with endothelial injury and thrombosis. Cigarette smoke (CS) is an established risk factor for vascular diseases and cigarette smokers have high-levels of eryptotic erythrocytes. This study, for the first time, investigates the mechanism by which CS damages red blood cells (RBCs). CS extract (CSE) from commercial cigarettes was prepared and standardized for nicotine content. Cytofluorimetric analysis demonstrated that treatment of human RBCs with CSE caused dose-dependent, phosphatidylserine externalization and cell shrinkage, hallmarks of apoptotic death. CSE did not affect cellular levels of Ca2+, reactive oxygen species (ROS) or glutathione (GSH). Immununoprecipitation and immunoblotting revealed the assembly of the death-inducing signaling complex (DISC) and oligomerization of Fas receptor as well as cleaved caspase-8 and caspase-3 within 6 h from the treatment. At the same time-interval, CSE elicited neutral sphyngomielinase (nSMase) activity-dependent ceramide formation and phosphorylation of p38 MAPK. Through specific inhibitors' nSMase, caspase-8 or p38 MAPK activities, we demonstrated that p38 MAPK activation is required for caspase-8-mediated eryptosis and that ceramide generation is initiator caspase-dependent. Finally, ex vivo analysis detected phosphorylated p38 MAPK (p-p38) and Fas-associated signaling complex in erythrocytes from cigarette smokers. In conclusion, our study demonstrates that CSE exposure induces in erythrocytes an extrinsic apoptotic pathway involving p38 MAPK-initiated DISC formation followed by activation of caspase-8/caspase-3 via ceramide formation.