Exploring the Pathophysiologic Cascade Leading to Osteoclastogenic Activation in Gaucher Disease Monocytes Generated via CRISPR/Cas9 Technology.

Gaucher disease (GD) is caused by biallelic pathogenic variants in the acid ß-glucosidase gene (GBA1), leading to a deficiency in the ß-glucocerebrosidase (GCase) enzyme activity resulting in the intracellular accumulation of sphingolipids. Skeletal alterations are one of the most disabling features in GD patients. Although both defective bone formation and increased bone resorption due to osteoblast and osteoclast dysfunction contribute to GD bone pathology, the molecular bases are not fully understood, and bone disease is not completely resolved with currently available specific therapies. For this reason, using editing technology, our group has developed a reliable, isogenic, and easy-to-handle cellular model of GD monocytes (GBAKO-THP1) to facilitate GD pathophysiology studies and high-throughput drug screenings. In this work, we further characterized the model showing an increase in proinflammatory cytokines (Interleukin-1ß and Tumor Necrosis Factor-α) release and activation of osteoclastogenesis. Furthermore, our data suggest that GD monocytes would display an increased osteoclastogenic potential, independent of their interaction with the GD microenvironment or other GD cells. Both proinflammatory cytokine production and osteoclastogenesis were restored at least, in part, by treating cells with the recombinant human GCase, a substrate synthase inhibitor, a pharmacological chaperone, and an anti-inflammatory compound. Besides confirming that this model would be suitable to perform high-throughput screening of therapeutic molecules that act via different mechanisms and on different phenotypic features, our data provided insights into the pathogenic cascade, leading to osteoclastogenesis exacerbation and its contribution to bone pathology in GD.

Cytokines from Bench to Bedside: A Retrospective Study Identifies a Definite Panel of Biomarkers to Early Assess the Risk of Negative Outcome in COVID-19 Patients.

The main aim of this study was to identify the most relevant cytokines which, when assessed in the earliest stages from hospital admission, may help to select COVID-19 patients with worse prognosis. A retrospective observational study was conducted in 415 COVID-19 patients (272 males; mean age 68 ± 14 years) hospitalized between May 2020 and March 2021. Within the first 72 h from hospital admission, patients were tested for a large panel of biomarkers, including C-reactive protein (CRP), Mid-regional proadrenomedullin (MR-proADM), Interferon-γ, interleukin 6 (IL-6), IL-1ß, IL-8, IL-10, soluble IL2-receptor-α (sIL2Rα), IP10 and TNFα. Extensive statistical analyses were performed (correlations, t-tests, ranking tests and tree modeling). The mortality rate was 65/415 (15.7%) and a negative outcome (death and/or orotracheal intubation) affected 98/415 (23.6%) of cases. Univariate tests showed the majority of biomarkers increased in severe patients, but ranking tests helped to select the best variables to put on decisional tree modeling which identified IL-6 as the first dichotomic marker with a cut-off of 114 pg/mL. Then, a good synergy was found between IL-10, MR-proADM, sIL2Rα, IP10 and CRP in increasing the predictive value in classifying patients at risk or not for a negative outcome. In conclusion, beside IL-6, a panel of other cytokines representing the degree of immunoparalysis and the anti-inflammatory response (IP10, sIL2Rα and IL-10) showed synergic role when combined to biomarkers of systemic inflammation and endothelial dysfunction (CRP, MR-proADM) and may also better explain disease pathogenesis and suggests targeted intervention.

Interleukin 6, soluble interleukin 2 receptor alpha (CD25), monocyte colony-stimulating factor, and hepatocyte growth factor linked with systemic hyperinflammation, innate immunity hyperactivation, and organ damage in COVID-19 pneumonia.

BACKGROUND: Patients infected by SARS-CoV-2 can develop interstitial pneumonia, requiring hospitalisation or mechanical ventilation. Increased levels of inflammatory biomarkers are associated with development of acute respiratory distress syndrome (ARDS). The aim of the present study was to determine which cytokines are associated with respiratory insufficiency in patients hospitalised for COVID-19. PATIENTS AND METHODS: Data on 67 consecutive patients were collected between March 8 and March 30, 2020. PaO2/FiO2 ratio (P/F) was calculated at hospital admission. The following cytokines were analysed: interleukin (IL)-6, IL-1α, IL-18, tumour necrosis factor (TNF)-ß, macrophage colony-stimulating factor (M-CSF), macrophage migration inhibitory factor (MIF), soluble IL-2 receptor alpha (sIL-2Rα; CD25), IL-12ß, IL-3, interferon (IFN) α2a, monokine induced by gamma interferon (MIG), monocyte-chemotactic protein 3 (MCP3) and hepatocyte growth factor (HGF). RESULTS: P/F lower than 300 was recorded in 22 out of 67 patients (32.8%). P/F strongly correlated with IL-6 (r = -0.62, P < 0.0001), M-CSF (r = -0.63, P < 0.0001), sIL-2Rα (r = -0.54, P < 0.0001), and HGF (r = -0.53, P < 0.0001). ROC curve analyses for IL-6 (AUC 0.83, 95% CI 0.73-0.93, P < 0.0001), M-CSF (AUC 0.87, 95% CI 0.79-0.96, P < 0.0001), HGF (AUC 0.81, 95% CI 0.70-0.93, P < 0.0001), and sIL-2Rα (AUC 0.80, 95% CI, 0.69-0.90, P < 0.0001) showed that these four soluble factors were highly significant. All four soluble factors correlated with LDH, white blood cell count, neutrophil count, lymphocyte count, and CRP. CONCLUSION: IL-6, M-CSF, sIL-2Rα, and HGF are possibly involved in the main biological processes of severe COVID-19, mirroring the level of systemic hyperinflammatory state, the level of lung inflammation, and the severity of organ damage.

Pro-Inflammatory Microenvironment Modulates the Transfer of Mutated TP53 Mediated by Tumor Exosomes.

Exosomes released from tumor cells are instrumental in shaping the local tumor microenvironment to allow cancer progression. Recently, it has been shown that tumor exosomes carry large fragments of dsDNA, which may reflect the mutational status of parental cells. Although it has been described that a stressful microenvironment can influence exosomal cargo, the effects on DNA packing and its transfer into recipient cells have yet to be investigated. Here, we report that exosomes derived from SW480 (human colorectal adenocarcinoma cell line) cells can carry dsDNA fragments containing the entire coding sequence of both TP53 and KRAS genes, harboring the SW480-related TP53 c.818G > A and KRAS c.35G > T typical mutations. We also report the following: that cell stimulation with lipopolysaccharides (LPS) promotes the selective packaging of the TP53 gene, but not the KRAS gene; that exosomes secreted by SW480 cells efficiently transfer the mutated sequences into normal CCD841-CoN colon epithelial and THLE-2 hepatic cells; that this mechanism is more efficient when the cells had been previously incubated with pro-inflammatory cytokines; that the TP53 gene appears actively transcribed in both recipient cells; and that mutated mRNA levels are not influenced by cytokine treatment. Our data strongly suggest that pro-inflammatory stimulation promotes the horizontal transfer of an oncogene by exosomes, although this remains a rare event. Further studies are needed to assess the impact of the oncogenic transfer by exosomes in malignant transformation and its role in tumor progression.

The Influence of a Stressful Microenvironment on Tumor Exosomes: A Focus on the DNA Cargo.

Exosomes secreted by tumor cells, through the transport of bioactive molecules, reprogram the surroundings, building a microenvironment to support the development of the tumor. The discovery that exosomes carry genomic DNA reflecting that of the tumor cell of origin has encouraged studies to use them as non-invasive biomarkers. The exosome-mediated transfer of oncogenes suggested a new mechanism of malignant transformation that could play a role in the formation of metastases. Several studies have examined the role of tumor exosomes on the modulation of the tumor microenvironment, but relatively few have been directed to assess how stressful stimuli can influence their production and cargo. Understanding the changes in exosome loads and the production pattern of the stressed tumor cell may uncover actionable mechanisms responsible for tumor progression.

Determination of Serum Infliximab Concentration by Point-of-care Devices in Children With Inflammatory Bowel Disease.

OBJECTIVES: Therapeutic drug monitoring is becoming increasingly important in clinical decision-making in children with inflammatory bowel disease (IBD). However, enzyme-linked immunosorbent assay (ELISA) assays do not allow results to be provided in real-time. We sought to compare 2 point-of-care (POC) devices for quantification of serum infliximab concentration with 2 validated ELISA assays in children with IBD. METHODS: We studied 32 serum samples from 19 children with IBD treated with infliximab. Serum samples were collected immediately before drug infusion (trough level). Infliximab was measured using 2 POC infliximab assays, Quantum Blue (POC IFX/QB) and Rida Quick (POC IFX/RQ), and 2 ELISA assays: Lisa-Tracker (used as primary reference), and Promonitor (used as second control). Intraclass correlation coefficient (ICC) was assessed for quantitative comparison. Qualitative analysis was also performed to evaluate whether POC assays would correctly classify infliximab serum according to a target window (between 3 and 7 µg/mL). RESULTS: ICC was 0.82 and 0.87 for POC IFX/QB and POC IFX/RQ with the primary reference ELISA assay, respectively; ICC between the 2 ELISA assays was 0.87. Classification of results according to therapeutic intervals showed good agreement between pairs of assays, with kappa of 0.67 and 0.80 for POC IFX/QB and POC IFX/RQ, respectively, with reference ELISA, and 0.81 between the 2 ELISAs. Accuracy of POC assays was better for drug levels <3 µg/mL. CONCLUSIONS: POC infliximab assays showed good agreement with traditional ELISA assays. POC devices may represent a viable option for real-time therapeutic drug monitoring in children treated with infliximab.

Causes of Treatment Failure in Children With Inflammatory Bowel Disease Treated With Infliximab: A Pharmacokinetic Study.

OBJECTIVES: Anti-tumor necrosis factor antibodies have led to a revolution in the treatment of inflammatory bowel diseases (IBD); however, a sizable proportion of patients does not respond to therapy. There is increasing evidence suggesting that treatment failure may be classified as mechanistic (pharmacodynamic), pharmacokinetic, or immune-mediated. Data regarding the contribution of these factors in children with IBD treated with infliximab (IFX) are still incomplete. The aim was to assess the causes of treatment failure in a prospective cohort of pediatric patients treated with IFX. METHODS: This observational study considered 49 pediatric (median age 14.4) IBD patients (34 Crohn disease, 15 ulcerative colitis) treated with IFX. Serum samples were collected at 6, 14, 22 and 54 weeks, before IFX infusions. IFX and anti-infliximab antibodies (AIA) were measured using enzyme linked immunosorbent assays. Disease activity was determined by Pediatric Crohn's Disease Activity Index or Pediatric Ulcerative Colitis Activity Index. RESULTS: Clinical remission, defined as a clinical score <10, was obtained by 76.3% of patients at week 14 and by 73.9% at week 54. Median trough IFX concentration was higher at all time points in patients achieving sustained clinical remission. IFX levels during maintenance correlated also with C-reactive protein, albumin, and fecal calprotectin. After multivariate analysis, IFX concentration at week 14 >3.11 µg/mL emerged as the strongest predictor of sustained clinical remission. AIA concentrations were correlated inversely with IFX concentrations and directly with adverse reactions. CONCLUSIONS: Most cases of therapeutic failure were associated with low serum drug levels. IFX trough levels at the end of induction are associated with sustained long-term response.

Characterization of the Proinflammatory Profile of Synovial Fluid-Derived Exosomes of Patients with Osteoarthritis.

The purpose of this study is to characterize synovial fluid- (SF-) derived exosomes of patients with gonarthrosis comparing two methods of isolation and to investigate their immune regulatory properties. Extracellular vesicles (EVs) have been isolated from inflamed SF by polymer precipitation method and quantified by Exocet kit and by nanoparticle tracking analysis. Vesicles expressed all the specific exosomal markers by immunoblot and FACS. After isolation with Exoquick, a relevant contamination by immune complexes was detected, which required further magnetic bead-based purification to remove. SF-derived exosomes significantly stimulated the release of several inflammatory cytokines and chemokines and metalloproteinases by M1 macrophages but did not influence the expression of CD80 and CD86 costimulatory molecules. In conclusion, we characterized purified exosomes isolated from inflamed SF and demonstrate that purified exosomes are functionally active in their ability to stimulate the release of proinflammatory factors from M1 macrophages. Our data indicate that SF-derived exosomes from gonarthrosis patients play a role in disease progression.

Brain Endothelial Cells Activate Neuroinflammatory Pathways in Response to Early Cerebral Small Vessel Disease (CSVD) Patients' Plasma.

The pathogenesis of cerebral small vessel disease (CSVD) is largely unknown. Endothelial disfunction has been suggested as the turning point in CSVD development. In this study, we tested the effect of plasma from CSVD patients on human cerebral microvascular endothelial cells with the aim of describing the pattern of endothelial activation. Plasma samples from three groups of young subjects have been tested: PTs (subjects affected by early stage CSVD); CTRLs (control subjects without abnormalities at MRI scanning); BDs (blood donors). Human Brain Endothelial Cells 5i (HBEC5i) were treated with plasma and total RNA was extracted. RNAs were pooled to reduce gene expression-based variability and NGS analysis was performed. Differentially expressed genes were highlighted comparing PTs, CTRLs and BDs with HBEC5i untreated cells. No significantly altered pathway was evaluated in BD-related treatment. Regulation of p38 MAPK cascade (GO:1900744) was the only pathway altered in CTRL-related treatment. Indeed, 36 different biological processes turned out to be deregulated after PT treatment of HBEC5i, i.e., the cytokine-mediated signaling pathway (GO:0019221). Endothelial cells activate inflammatory pathways in response to stimuli from CSVD patients' plasma, suggesting the pathogenetic role of neuroinflammation from the early asymptomatic phases of cerebrovascular disease.

Ceramide releases exosomes with a specific miRNA signature for cell differentiation.

Exosomes are well established effectors of cell-cell communication. Their role on maturation of embryonic cells located in hippocampus, seat of memory, is unknown. Here we show that ceramide facilitates release of exosomes from HN9.10e cells extending information for cell differentiation to neighboring cells. We found only 38 miRNAs differentially expressed in exosomes derived from ceramide-treated cells in comparison with control cells (including 10 up-regulated and 28 down-regulated). Some overexpressed miRNAs (mmu-let-7f-1-3p, mmu-let-7a-1-3p, mmu-let-7b-3p, mmu-let-7b-5p, mmu-miR-330-3p) regulate genes encoding for protein involved in biological, homeostatic, biosynthetic and small molecule metabolic processes, embryo development and cell differentiation, all phenomena relevant for HN9.10e cell differentiation. Notably, the overexpressed mmu-let-7b-5p miRNA appears to be important for our study based on its ability to regulate thirty-five gene targets involved in many processes including sphingolipid metabolism, sphingolipid-related stimulation of cellular functions and neuronal development. Furthermore, we showed that by incubating embryonic cells with exosomes released under ceramide treatment, some cells acquired an astrocytic phenotype and others a neuronal phenotype. We anticipate our study to be a start point for innovative therapeutic strategies to regulate the release of exosomes useful to stimulate delayed brain development in the newborn and to improve the cognitive decline in neurodegenerative disorders.

The role of asymmetric dimethylarginine (ADMA) in COVID-19: association with respiratory failure and predictive role for outcome.

We aimed to assess the potential role of Asymmetric dimethylarginine (ADMA) in conditioning respiratory function and pulmonary vasoregulation during Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2) infection. Within 72 h from admission, samples from 90 COVID-19 patients were assessed for ADMA, SDMA, L-arginine concentrations. In addition to classical statistics, patients were also clustered by a machine learning approach according to similar features. Multivariable analysis showed that C-reactive protein (OR 1.012), serum ADMA (OR 4.652), white blood cells (OR = 1.118) and SOFA (OR = 1.495) were significantly associated with negative outcomes. Machine learning-based clustering showed three distinct clusters: (1) patients with low severity not requiring invasive mechanical ventilation (IMV), (2) patients with moderate severity and respiratory failure whilst not requiring IMV, and (3) patients with highest severity requiring IMV. Serum ADMA concentration was significantly associated with disease severity and need for IMV although less pulmonary vasodilation was observed by CT scan. High serum levels of ADMA are indicative of high disease severity and requirement of mechanical ventilation. Serum ADMA at the time of hospital admission may therefore help to identify COVID-19 patients at high risk of deterioration and negative outcome.

The impact of body composition on mortality of COVID-19 hospitalized patients: A prospective study on abdominal fat, obesity paradox and sarcopenia.

BACKGROUND & AIMS: Obesity has been described as a predisposing risk factor to severe forms of COVID-19, but conflicting results are emerging on its real impact on the mortality of COVID-19. We aimed to compare clinical outcomes and mortality among COVID-19 patients according to obesity, metabolic syndrome and adiposity distribution. METHODS: We conducted a prospective observational study of all consecutive adult patients with a confirmed diagnosis of SARS-CoV-2 infection admitted to the Infectious Diseases Clinic at Udine Hospital, Italy, from January 2021 to February 2021. At admission, the study population was submitted to specific anthropometric, laboratory and bioimpedance analysis (BIA) measurements and divided into five groups according to: 1) BMI < or >30 kg/m2; 2) waist circumference (WC) < or >98 cm for women, < or >102 cm for men; 3) presence or absence of metabolic syndrome (MS); 4) visceral adipose tissue (VAT) distribution; and 5) presence or absence of sarcopenia (SP) both based on BIA. We then compared clinical outcomes (ventilatory support, intensive care unit (ICU) admission, ICU length of stay, total hospital length of stay and mortality), immune and inflammatory makers and infectious and non-infectious acute complications within the five groups. RESULTS: A total of 195 patients were enrolled in the study. The mean age of patients was 71 years (IQR 61-80) and 64.6% (126) were male. The most common comorbidities were hypertension (55.9%) and MS (55.4%). Overall mortality was 19.5%. Abdominal adiposity, measured both with WC and with BIA, and SP were significantly associated with need for increased ventilator support (p = 0.013 for WC; p = 0.037, 0.027 and 0.009 for VAT; p = 0.004 and 0.036 for FMI; and p = 0.051 for SP), but not with ICU admission (WC p = 0.627, VAT p = 0.153, FMI p = 0.519 and SP p = 0.938), length of stay (WC p = 0.345, VAT p = 0.650, FMI p = 0.159 and SP p = 0.992) and mortality (WC p = 0.277, VAT p = 0.533, FMI p = 0.957 and SP p = 0.211). Obesity and MS did not discriminate for the intensity of ventilatory outcome (p = 0.142 and p = 0.198, respectively), ICU admission (p = 0.802 and p = 0.947, respectively), length of stay (p = 0.471 and p = 0.768, respectively) and mortality (p = 0.495 and p = 0.268, respectively). We did not find significant differences in inflammatory markers and secondary complications within the five groups. CONCLUSIONS: In patients admitted with COVID-19, increased WC, visceral abdominal fat and SP are associated with higher need for ventilatory support. However, obesity, MS, SP and abdominal adiposity are not sensitive predictive factors for mortality.

Validation of a One-Step Reverse Transcription-Droplet Digital PCR (RT-ddPCR) Approach to Detect and Quantify SARS-CoV-2 RNA in Nasopharyngeal Swabs.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has rapidly spread worldwide from the beginning of 2020. Quantitative reverse transcription-PCR (RT-qPCR) is, to this day, the preferred methodology for viral RNA detection, even if not without problems. To overcome some of the limitations still existing for the detection and quantification of nucleic acids in various applications, the use of one-step reverse transcription-droplet digital PCR (RT-ddPCR) has been established. The purpose of this study was, then, to evaluate the efficacy of ddPCR for the detection of SARS-CoV-2 RNA in nasopharyngeal swabs, optimizing the detection of low-viral load-burdened samples. METHODS: The RT-ddPCR workflow was validated for sensitivity, specificity, linearity, reproducibility, and precision using samples from 90 COVID-19-infected patients referred to the Department of Laboratory Medicine of the University Hospital of Udine (Italy). RESULTS: The present study shows that RT-ddPCR allows the detection of as low as 10.3 copies of a SARS-COV-2 E-gene per sample with a higher level of accuracy and precision, especially at low concentration. CONCLUSION: During the postpeak phase of the SARS-CoV-2 pandemic, it is essential to rely on a highly robust molecular biology method to identify infected subjects, whether they have symptoms or not, in order to prepare appropriate containment measures.

Pharmacogenetic variants of infliximab response in young patients with inflammatory bowel disease.

Infliximab is commonly used in inflammatory bowel disease (IBD), however, differences in clinical response among patients are common. Several studies have considered the possibility that these differences are caused by genetic variability even if no unique marker has been yet identified in pediatric patients. We evaluated the impact of two candidate single-nucleotide polymorphisms (SNPs) rs396991 in FCGR3A and rs1800629 in TNFα genes on infliximab response in an Italian cohort of 76 pediatric patients with IBD. Results showed that patients with the variant FCGR3A allele had a reduced clinical response at the end of induction (p value = 0.004), at 22 weeks (p value = 0.001), and at 52 weeks of treatment (p value = 0.01). A significant association between the FCGR3A variant and median infliximab levels measured during maintenance therapy was also observed: patients with wild type genotype had higher infliximab levels compared to patient with variant allele. Furthermore, patients with the variant allele had a higher probability to produce antidrug antibodies (ADAs). No association was found among the TNFα SNP, clinical response, and infliximab levels. This study addressed for the first time in pediatric patients with IBD, the association of FCGR3A SNP, infliximab response, and ADA production.

Circulating exosomes express α4ß7 integrin and compete with CD4+ T cells for the binding to Vedolizumab.

Vedolizumab (VDZ) is a therapeutic monoclonal antibody approved for the treatment of inflammatory bowel diseases (IBD). VDZ selectively binds to the α4ß7 integrin and blocks trafficking of a specific subset of gastrointestinal-homing T-lymphocytes to inflamed tissue. Although VDZ has shown promising results in numerous clinical studies a subgroup of patients do not respond adequately. Mechanistic insights and prognostic biomarkers able to predict which patients might benefit from VDZ therapy are currently lacking. Circulating exosomes were isolated from serum of blood donors and VDZ-treated patients by polymer-based precipitation. The surface expression of α4ß7 integrin was evaluated by flow cytometry and the levels of exosome-bound VDZ were investigated by Promonitor-VDZ ELISA kit. The capacity of exosomes to interfere with the adhesion of VDZ-treated CD4+ T cells was assessed by adhesion assay. In this study, we showed that serum exosomes isolated from both blood donor and ulcerative colitis patients express on their surface the VDZ target α4ß7 integrin. We observed an increased exosomal sequestration of VDZ in anti-TNF exposed patients compared to anti- TNFα naïve patients, according to a greater expression of α4ß7 integrin on vesicles surface. Circulating exosomes could compete for VDZ binding with CD4+ T cells since we found that the amount of VDZ bound to T cells was impaired in the presence of exosomes. In addition, we demonstrated that exosomes bind VDZ, which consequently becomes unable to block MadCAM-1-mediated adhesion of lymphocytes. Circulating exosomes might contribute to drug sequestration, possibly affecting the therapeutic efficacy of VDZ in IBD patients. Our data suggest that previous biologic therapy may have altered the sequestration capacity of circulating exosomes, thus reducing the efficacy of VDZ in patients who failed anti-TNF agents.

The Exposure to Osteoarthritic Synovial Fluid Enhances the Immunomodulatory Profile of Adipose Mesenchymal Stem Cell Secretome.

OBJECTIVE: Several clinical studies have proposed the infusion of adipose mesenchymal stem cells (AMSCs) as an alternative therapy for joint diseases with inflammatory components, such as osteoarthritis. Indeed, AMSCs are able to stimulate tissue repair through a paracrine activity and the interaction with the inflammatory microenvironment seems to have a critical role. DESIGN: To reproduce the inflammatory microenvironment, AMSCs were exposed to osteoarthritic synovial fluid (SF) for 48 h and the effect of their secretome on differentiation of monocytes (M0) into macrophages M1-like and mature dendritic cells (mDCs) was evaluated. Furthermore, the effect of the secretome of AMSCs exposed to SF was evaluated on the T cell population in terms of T cell proliferation and expansion of T regulatory cells (T reg). RESULTS: Our data show that the exposure of AMSCs to SF activates cells and promotes the release of immunosuppressive factors, which induce macrophage polarization of M0 into the M2-like phenotype and inhibit differentiation of monocytes into mature dendritic cells (mDCs). Only the secretome of exposed AMSCs was able to inhibit T cell proliferation and promote T reg expansion. CONCLUSIONS: Our results suggest that the microenvironment plays a fundamental role for the development of anti-inflammatory and immunomodulatory properties of AMSCs.

A Streamlined Approach to Rapidly Detect SARS-CoV-2 Infection Avoiding RNA Extraction: Workflow Validation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has rapidly spread worldwide from the beginning of 2020. The presence of viral RNA in samples by nucleic acid (NA) molecular analysis is the only method available to diagnose COVID-19 disease and to assess patients' viral load. Since the demand for laboratory reagents has increased, there has been a worldwide shortage of RNA extraction kits. We, therefore, developed a fast and cost-effective viral genome isolation method that, combined with quantitative RT-PCR assay, detects SARS-CoV-2 RNA in patient samples. The method relies on the addition of Proteinase K followed by a controlled heat-shock incubation and, then, E gene evaluation by RT-qPCR. It was validated for sensitivity, specificity, linearity, reproducibility, and precision. It detects as low as 10 viral copies/sample, is rapid, and has been characterized in 60 COVID-19-infected patients. Compared to automated extraction methods, our pretreatment guarantees the same positivity rate with the advantage of shortening the time of the analysis and reducing its cost. This is a rapid workflow meant to aid the healthcare system in the rapid identification of infected patients, such as during a pathogen-related outbreak. For its intrinsic characteristics, this workflow is suitable for large-scale screenings.

Toll-like Receptor-4 Activation Boosts the Immunosuppressive Properties of Tumor Cells-derived Exosomes.

The biology of tumor-derived exosomes (TEX) is only partially understood and much remains to be studied in order to define the effect that the tumor microenvironment or the activation of tumor cells exerts on their composition and functions. Increased expression and activity of toll-like receptor 4 (TLR4) in chronic infectious and inflammatory conditions is related with cancer progression: its activation induces an inflammatory signaling that increases the tumorigenic potential of cancer cells promoting their immune evasion. We investigated the immune modulatory properties of TEX released upon cell TLR4 activation, and we found that, although differences were observed depending on the type of the tumor, the treatment influences TEX composition and boosts their immunosuppressive ability. Our results suggest that the activation of TLR4 supports tumor progression by stimulating the release of more effective immunosuppressive exosomes, which allow tumor cells to escape immune surveillance and probably even play a role in the metastatic process.

ADMA as a possible marker of endothelial damage. A study in young asymptomatic patients with cerebral small vessel disease.

Sporadic small vessel disease (SVD) has high prevalence in aging population and stroke patients, but also in younger asymptomatic subjects. In this last group it can represents a prelude to stroke and cognitive impairment. Still nowadays, its pathogenesis is unclear. 35 consecutive patients with SVD at brain MRI and 35 age- and sex-matched controls, between January 2016 and February 2018, underwent an extended screening for thrombophilia, autoimmunity and evaluated levels of blood markers of inflammation and endothelial activation. Asymmetric DiMethyl Arginine (ADMA) levels proved higher in patients (70.44 ± 36.25 ng/ml vs. 46.58 ± 30.67 ng/ml; p = 0.004), also after controlling for confounding factors. ADMA levels showed positive correlation with Fazekas score (r = 0.304; p = 0.01). ROC curve analysis showed a moderate accuracy in discriminating patients and controls (AUC = 0.70; CI 0.57-0.82; p = 0.004): a cut-off of 46 ng/ml is associated with 80% sensitivity, but limited (54%) specificity. Higher ADMA levels characterize selected subjects with sporadic SVD, asymptomatic for vascular diseases and without latent inflammatory conditions or coagulopathy. This reinforces the hypothesis of the key role of endothelial dysfunction in SVD. Further studies should explore the cause-effect relationship between ADMA pathway and SVD.