Diagnostic capabilities, clinical features, and longitudinal UBA1 clonal dynamics of a nationwide VEXAS cohort.

VEXAS is a prototypic hemato-inflammatory disease combining rheumatologic and hematologic disorders in a molecularly defined nosological entity. In this nationwide study, we aimed at screenshotting the current diagnostic capabilities and clinical-genomic features of VEXAS, and tracked UBA1 longitudinal clonal dynamics upon different therapeutics, including allogeneic hematopoietic cell transplant. We leveraged a collaboration between the Italian Society of Experimental Hematology and of Rheumatology and disseminated a national survey to collect clinical and molecular patient information. Overall, 13/29 centers performed UBA1 genomic testing locally, including Sanger sequencing (46%), next-generation sequencing (23%), droplet digital polymerase chain reaction (8%), or combination (23%). A total of 41 male patients were identified, majority (51%) with threonine substitutions at Met41 hotspot, followed by valine and leucine (27% and 8%). Median age at VEXAS diagnosis was 67 years. All patients displayed anemia (median hemoglobin 9.1 g/dL), with macrocytosis. Bone marrow vacuoles were observed in most cases (89%). The most common rheumatologic association was polychondritis (49%). A concomitant myelodysplastic neoplasm/syndrome (MDS) was diagnosed in 71% of patients (n = 28), chiefly exhibiting lower Revised International Prognostic Scoring System risk profiles. Karyotype was normal in all patients, except three MDS cases showing -Y, t(12;16)(q13;q24), and +8. The most frequently mutated gene was DNMT3A (n = 10), followed by TET2 (n = 3). At last follow-up, five patients died and two patients progressed to acute leukemia. Longitudinal UBA1 clonal dynamics demonstrated mutational clearance following transplant. We collected a nationwide interdisciplinary VEXAS patient cohort, characterized by heterogeneous rheumatologic manifestations and treatments used. MDS was diagnosed in 71% of cases. Patients exhibited various longitudinal UBA1 clonal dynamics.

Vx-809, a CFTR Corrector, Acts through a General Mechanism of Protein Folding and on the Inflammatory Process.

Correct protein folding is the basis of cellular well-being; thus, accumulation of misfolded proteins within the endoplasmic reticulum (ER) leads to an imbalance of homeostasis that causes stress to the ER. Various studies have shown that protein misfolding is a significant factor in the etiology of many human diseases, including cancer, diabetes, and cystic fibrosis. Misfolded protein accumulation in the ER triggers a sophisticated signal transduction pathway, the unfolded protein response (UPR), which is controlled by three proteins, resident in ER: IRE1α, PERK, and ATF6. Briefly, when ER stress is irreversible, IRE1α induces the activation of pro-inflammatory proteins; PERK phosphorylates eIF2α which induces ATF4 transcription, while ATF6 activates genes encoding ER chaperones. Reticular stress causes an alteration of the calcium homeostasis, which is released from the ER and taken up by the mitochondria, leading to an increase in the oxygen radical species production, and consequently, to oxidative stress. Accumulation of intracellular calcium, in combination with lethal ROS levels, has been associated with an increase of pro-inflammatory protein expression and the initiation of the inflammatory process. Lumacaftor (Vx-809) is a common corrector used in cystic fibrosis treatment which enhances the folding of mutated F508del-CFTR, one of the most prevalent impaired proteins underlying the disease, promoting a higher localization of the mutant protein on the cell membrane. Here, we demonstrate that this drug reduces the ER stress and, consequently, the inflammation that is caused by such events. Thus, this molecule is a promising drug to treat several pathologies that present an etiopathogenesis due to the accumulation of protein aggregates that lead to chronic reticular stress.

Legionella bononiensis sp. nov., isolated from a hotel water distribution system in northern Italy.

Legionella-like isolates, strains 27fs60, 30fs61 and 30cs62T, were isolated from a hotel water distribution system in the Emilia-Romagna region, Italy. Isolates were Gram- and Ziehl Neelsen-stain-negative, rod-shaped, with transitory flagella presence and able to grow at 32-37 °C (with an optimum at 32 °C) on buffered charcoal-yeast extract agar with l-cysteine, glycine-vancomycin-polymyxin B-cycloheximide agar and Wadowsky-Yee medium agar. The strains showed positive reactions for oxidase, hippurate and gelatinase and a weakly positive reaction for catalase. Based on the EUCAST cut-off, strain 30cs62T was resistant to ciprofloxacin (5 mg l-1). The mip and rpoB gene sequences of the three strains showed close matches to those of Legionella quateirensis ATCC 49507T with similarity values of 98.2 and 94.5 %, respectively. Whole genome sequencing of the three strains was performed, resulting in G+C contents of 39.0, 39.1 and 39.0 mol%, respectively. The identity percentage measured by average nucleotide identity between the three strains and their respective closest strains were: 91.32 % L. quateirensis NCTC 12376T, 91.45 % L. quateirensis ATCC 49507T and 91.45 % L. quateirensis ATCC 49507T, respectively. The digital DNA-DNA hybridization analysis demonstrated how the isolates were separated from the most related phylogenetic Legionella species (L. quateirensis ATCC 49507T, ≤40.10 % DNA-DNA relatedness). The concatenated phylogenetic tree based on 16S rRNA, mip, rpoB and rnpB genes, shows a close relationship with L. quateirensis ATCC 49507T. The results obtained confirm the status of an independent species. The name proposed for this species is Legionella bononiensis sp. nov. with 30cs62T (=ATCC TSD-262T=DSM 112526T) as the type strain.

Dynamics of Legionella Community Interactions in Response to Temperature and Disinfection Treatment: 7 Years of Investigation.

In man-made water distribution systems, Legionella community interactions remain unknown, due to their ability to change from sessile to planktonic states or live in viable but non-culturable forms, in response to anthropic and environmental stress. During 7 years of hospital Legionella surveillance, in 191 hot water positive samples, the interactions among the Legionella species, temperature, and disinfection treatment were evaluated. Legionella was isolated following ISO 11731:2017, and identification was performed by mip gene sequencing and sequence-based typing (SBT) for L. anisa or L. rubrilucens and L. pneumophila, respectively. The species with the higher frequency of isolation was L. pneumophila serogroup 1 (78.53%; 4865.36 ± 25,479.11 cfu/L), followed by L. anisa (54.45%; 558.79 ± 2637.41 cfu/L) and L. rubrilucens (21.99%; 307.73 ± 1574.95 cfu/L), which were sometimes present together. Spearman's rho correlation test was conducted among the species with respect to temperature and disinfectant (H2O2/Ag+). The results showed a generally positive interaction among these species sharing the same environment, except for competition between L. anisa and L. rubrilucens. High temperature (48.83 ± 2.59 °C) and disinfection treatment (11.58 ± 4.99 mg/L) affected the presence of these species. An exception was observed with L. anisa, which showed disinfection treatment resistance. For the purposes of environmental surveillance, it is fundamental to better understand the interactions and dynamic of the Legionella community in man-made water systems in order to choose the proper physical or chemical treatments. The simultaneous presence of different Legionella species could result in an increased resistance to high temperature and disinfectant treatment, leading to changes in contamination level and species diversity.

Cytotoxic Sesquiterpenoids from Ammoides atlantica Aerial Parts.

Seven new terpenoids, namely, guaiane (1-4), eudesmane (5), and bisabolane (6) sesquiterpenoids and a furanone (7), were isolated from the aerial parts of Ammoides atlantica, a herbaceous plant growing in Algeria, together with eight known compounds. All metabolites were characterized by their 1D and 2D NMR and HRESIMS data. A combined DFT/NMR method was applied to study the relative configurations of 1-4, 6, and 7. All compounds, except 2, were assayed against MCF-7, A375, A549, HaCaT, and Jurkat cell lines. Compounds 8, 10, and 11 induced a dose-dependent reduction in cell viability with different potency on almost all cell lines used. The most active compounds, 8 and 10, were studied to assess their potential apoptotic effects and cell cycle inhibition.

Combined aCGH and Exome Sequencing Analysis Improves Autism Spectrum Disorders Diagnosis: A Case Report.

Background and Objectives: The development and standardization of genome-wide technologies able to carry out high-resolution, genomic analyses in a cost- and time-affordable way is increasing our knowledge regarding the molecular bases of complex diseases like autism spectrum disorder (ASD). ASD is a group of heterogeneous diseases with multifactorial origins. Genetic factors seem to be involved, albeit they remain still largely unknown. Here, we report the case of a child with a clinical suspicion of ASD investigated by using such a genomic high-resolution approach. Materials and Methods: Both array comparative genomic hybridization (aCGH) and exome sequencing were carried out on the family trio. aCGH was performed using the 4 × 180 K SurePrint G3 Human CGH Microarray, while the Human All Exon V7 targeted SureSelect XT HS panel was used for exome sequencing. Results: aCGH identified a paternally inherited duplication of chromosome 7 involving the CNTNAP2 gene, while 5 potentially clinically-relevant variants were identified by exome sequencing. Conclusions: Within the identified genomic alterations, the CNTNAP2 gene duplication may be related to the patient's phenotype. Indeed, this gene has already been associated with brain development and cognitive functions, including language. The paternal origin of the alteration cannot exclude an incomplete penetrance. Moreover, other genomic factors may act as phenotype modifiers combined with CNTNAP2 gene duplication. Thus, the case reported herein strongly reinforces the need to use extensive genomic analyses to shed light on the bases of complex diseases.

Role of BAG3 in cancer progression: A therapeutic opportunity.

BAG3 is a multifunctional protein that can bind to heat shock proteins (Hsp) 70 through its BAG domain and to other partners through its WW domain, proline-rich (PXXP) repeat and IPV (Ile-Pro-Val) motifs. Its intracellular expression can be induced by stressful stimuli, while is constitutive in skeletal muscle, cardiac myocytes and several tumour types. BAG3 can modulate the levels, localisation or activity of its partner proteins, thereby regulating major cell pathways and functions, including apoptosis, autophagy, mechanotransduction, cytoskeleton organisation, motility. A secreted form of BAG3 has been identified in studies on pancreatic ductal adenocarcinoma (PDAC). Secreted BAG3 can bind to a specific receptor, IFITM2, expressed on macrophages, and induce the release of factors that sustain tumour growth and the metastatic process. BAG3 neutralisation therefore appears to constitute a novel potential strategy in the therapy of PDAC and, possibly, other tumours.

Useful applications of growth factors for cardiovascular regenerative medicine.

Novel advances for cardiovascular diseases (CVDs) include regenerative approaches for fibrosis, hypertrophy, and neoangiogenesis. Studies indicate that growth factor (GF) signaling could promote heart repair since most of the evidence is derived from preclinical models. Observational studies have evaluated GF serum/plasma levels as feasible biomarkers for risk stratification of CVDs. Noteworthy, two clinical interventional published studies showed that the administration of growth factors (GFs) induced beneficial effect on left ventricular ejection fraction (LVEF), myocardial perfusion, end-systolic volume index (ESVI). To date, large scale ongoing studies are in Phase I-II and mostly focussed on intramyocardial (IM), intracoronary (IC) or intravenous (IV) administration of vascular endothelial growth factor (VEGF) and fibroblast growth factor-23 (FGF-23) which result in the most investigated GFs in the last 10 years. Future data of ongoing randomized controlled studies will be crucial in understanding whether GF-based protocols could be in a concrete way effective in the clinical setting.

Tetra-substituted pyrrole derivatives act as potent activators of p53 in melanoma cells.

Cutaneous melanoma, the most aggressive form of skin cancer, is characterized by activating BRAF mutations. Despite the initial success of selective BRAF inhibitors, only few patients exhibited complete responses, whereas many showed disease progression. Melanoma is one of the few types of cancer in which p53 is not frequently mutated, but p53 inactivation can be indirectly achieved by a stable activation of MDM2 induced by a deletion in CDKN2A (Cyclin Dependent Kinase Inhibitor 2A) locus, encoding for p16INK4A and p14ARF, two tumor suppressor genes. In this study, we tested the efficacy of the previously synthesized tetra-substituted pyrrole derivatives, 8 g, 8 h and 8i, in melanoma cell lines, and we compared the effects of the most active of these, the 8i compound, with that exerted by Nutlin 3, a well-known inhibitor of p53-MDM2 interaction. The obtained results showed that 8i potentiates the inhibitory effect of Nutlin 3 and the combined use of 8i and Nutlin 3 triggers apoptosis and significantly impairs melanoma viability. Finally, the 8i compound reduces p53-MDM2 interaction and induces p53-HSP90 complex formation, suggesting that the observed raise in p53 transcriptional activity could be mediated by HSP90. Because the main feature of melanoma is the resistance to most chemotherapeutics, our studies suggest that the 8i tetra-substituted pyrrole derivative, restoring p53 functions and its transcriptional activities, may have potential application, at least as adjuvant, in the treatment of human melanoma.

Platelet Gel in a Non-Regenerating Cryosurgery-Induced Skin Wound in an Old Patient: A Case Report.

OBJECTIVE: To report on the clinical benefits of platelet gel application in a non-regenerating skin wound. CLINICAL PRESENTATION AND INTERVENTION: An 84-year-old man presented with a severe wound with a regular circumference in the frontal region which resulted in a complete loss of epidermis and dermis. The skin lesion, induced by cryosurgery used to remove a basal-cell carcinoma, had previously been treated with a dermal substitute application (Integra®). After the failure of the skin graft, the patient was treated using a platelet gel therapeutic protocol which achieved the complete healing of the injured area. CONCLUSION: This case showed the clinical efficacy of using platelet gel in this elderly patient in whom the dermal substitute graft had been ineffective.

Epigenetic control of autoimmune diseases: from bench to bedside.

Genome-wide association studies have revealed several genes predisposing to autoimmunity, however, concordance rates in monozygotic twins are significantly below 50% for several autoimmune diseases. The limited presence of a strong genetic association only in some patients supports that other non-genetic mechanisms are active in these pathologies. Epigenetic modifications such as DNA methylation, histone modification, and microRNA signaling regulate gene expression and are sensitive to external stimuli and they might be as bridging between genetic and environmental factors. Some evidence has highlighted the involvement of epigenetic alterations in the pathogenesis of various autoimmune diseases giving rise to great expectations among clinicians and researchers. The direct role of these alterations in the initiation/progression of autoimmune diseases is still unclear. The knowledge in depth of these pathogenic and epigenetic mechanisms will increase the possibility of the control and/or prevention of autoimmune diseases through the use of drugs that target epigenetic pathways. Moreover, we could use epigenetic-related biomarkers to follow this complicated framework (for example H3K4me3 and miRNA-155 are among those proposed biomarkers). This article reviews current understanding of the epigenetic involvement in the field of autoimmune diseases especially in systemic lupus erythematosus, rheumatoid arthritis, sclerosis multiple and type 1 diabetes.

Epigenetic reprogramming in atherosclerosis.

Recent data support the involvement of epigenetic alterations in the pathogenesis of atherosclerosis. The most widely investigated epigenetic mechanism is DNA methylation although also histone code changes occur during the diverse steps of atherosclerosis, such as endothelial cell proliferation, vascular smooth muscle cell (SMC) differentiation, and inflammatory pathway activation. In this review, we focus on the main genes that are epigenetically modified during the atherogenic process, particularly nitric oxide synthase (NOS), estrogen receptors (ERs), collagen type XV alpha 1 (COL15A1), vascular endothelial growth factor receptor (VEGFR), and ten-eleven translocation (TET), which are involved in endothelial dysfunction; gamma interferon (IFN-γ), forkhead box p3 (FOXP3), and tumor necrosis factor-α (TNF-α), associated with atherosclerotic inflammatory process; and p66shc, lectin-like oxLDL receptor (LOX1), and apolipoprotein E (APOE) genes, which are regulated by high cholesterol and homocysteine (Hcy) levels. Furthermore, we also discuss the role of non-coding RNAs (ncRNA) in atherosclerosis. NcRNAs are involved in epigenetic regulation of endothelial function, SMC proliferation, cholesterol synthesis, lipid metabolism, and inflammatory response.

Screening tests for hepatitis B virus, hepatitis C virus, and human immunodeficiency virus in blood donors: evaluation of two chemiluminescent immunoassay systems.

Automated chemiluminescent immunoassays (CLIAs) are useful for the detection of hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus 1/2 antigen/antibodies (HIV 1/2 Ag/Ab) in blood donor screening. Eight hundred and forty serum samples were tested for hepatitis B surface antigen (HBsAg), HCV antibodies (anti-HCV), and HIV1/2 Ag/Ab in parallel using 2 different CLIAs (Abbott Architect i2000SR and Roche Cobas e411). The concordance between the 2 systems was high (Cohen's kappa 0.97 for HBsAg, 0.77 for anti-HCV, 0.92 for HIV1/2 Ag/Ab) and the specificity and the positive predictive value were comparable. Among the 12 discrepant results, 11 were false-positive and 1 (reactive by Architect) was true-positive for anti-HCV. Positivity for HBV DNA, HCV RNA, and HIV RNA was recorded in 90.9%, 38.9%, and 100% of true-positive samples, respectively. This study represents the first stringent comparison between Architect i2000SR and Cobas e411 in blood donors. We observed a good correlation and high agreement among HBV, HCV, and HIV with the 2 automated systems.

Characterization of a Novel Species of Legionella Isolated from a Healthcare Facility: Legionella resiliens sp. nov.

Two Legionella-like isolates, 8cVS16T and 9fVS26, were isolated from a water distribution system (WDS) in a healthcare facility. Cells were Gram- and Ziehl Neelsen-stain-negative, rod-shaped, motile, and exhibited a blue-white fluorescence under Wood's lamp at 365 nm. The strains grew in a range of 32-37 °C on BCYE with L-cysteine (Cys+), GVPC, and MWY agar medium, with a positive reaction for oxidase, catalase, and gelatinase. The dominant fatty acids were summed features 3 (C16:1ω7c/C16:1ω6c) (27.7%), C16:0 iso (17.5%), and C16:0 (16.3%), and Q13 as the major ubiquinone. The mip and rpoB gene sequences showed a similarity of 96.7% and 92.4%, with L. anisa (ATCC 35292T). The whole genomes sequencing (WGS) performed displayed a GC content of 38.21 mol% for both. The digital DNA-DNA hybridization (dDDH) analysis demonstrated the separation of the two strains from the phylogenetically most related L. anisa (ATCC 35292T), with ≤43% DNA-DNA relatedness. The Average Nucleotide Identity (ANI) between the two strains and L. anisa (ATCC 35292T) was 90.74%, confirming that the two isolates represent a novel species of the genus Legionella. The name proposed for this species is Legionella resiliens sp. nov., with 8cVS16T (=DSM 114356T = CCUG 76627T) as the type strain.

Role of BAG3 protein in leukemia cell survival and response to therapy.

The ability of BAG3, a member of the BAG family of heat shock protein (Hsp) 70 - cochaperones, to sustain the survival of human primary B-CLL and ALL cells was recognized about nine years ago. Since then, the anti-apoptotic activity of BAG3 has been confirmed in other tumor types, where it has been shown to regulate the intracellular concentration and localization of apoptosis-regulating factors, including NF-κB-activating (IKKγ) and Bcl2-family (Bax) proteins. Furthermore, growing evidences support its role in lymphoid and myeloid leukemia response to therapy. Moreover in the last years, the contribution of BAG3 to autophagy, a process known to be involved in the pathogenesis and response to therapy of leukemia cells, has been disclosed, opening a new avenue for the interpretation of the role of this protein in leukemias' biology.

IKK{gamma} protein is a target of BAG3 regulatory activity in human tumor growth.

BAG3, a member of the BAG family of heat shock protein (HSP) 70 cochaperones, is expressed in response to stressful stimuli in a number of normal cell types and constitutively in a variety of tumors, including pancreas carcinomas, lymphocytic and myeloblastic leukemias, and thyroid carcinomas. Down-regulation of BAG3 results in cell death, but the underlying molecular mechanisms are still elusive. Here, we investigated the molecular mechanism of BAG3-dependent survival in human osteosarcoma (SAOS-2) and melanoma (M14) cells. We show that bag3 overexpression in tumors promotes survival through the NF-kappaB pathway. Indeed, we demonstrate that BAG3 alters the interaction between HSP70 and IKKgamma, increasing availability of IKKgamma and protecting it from proteasome-dependent degradation; this, in turn, results in increased NF-kappaB activity and survival. These results identify bag3 as a potential target for anticancer therapies in those tumors in which this gene is constitutively expressed. As a proof of principle, we show that treatment of a mouse xenograft tumor model with bag3siRNA-adenovirus that down-regulates bag3 results in reduced tumor growth and increased animal survival.

[Clinical significance of Staphylococcus aureus in urine]. / Significado clínico del aislamiento de Staphylococcus aureus en muestras de orina.

INTRODUCTION: To evaluate the clinical significance of the isolation of Staphylococcus aureus in urine samples. METHODS: A retrospective study was performed on adult patients identified from a microbiology database in a 200-bed general hospital between the years 2000 and 2009. The demographic data, comorbidities, and risk factors, were reviewed, particularly those associated with the concomitant isolation of S.aureus in blood cultures. RESULTS: The frequency of S.aureus found in urine samples was 0.63%. A total of 43 patients (mean age 68.7 years [SD±16], and 58.1% males) were identified in the database. A Charlson comorbidity index >3 was observed in 20.9%. Concurrent bacteremia was seen in 48.8%. Two groups of patients were distinguished: with concomitant bacteremia (n=21) or without (n=22). Intervention in the urinary tract significantly predicted (P=.00004) bacteriuria without bacteremia (81.8%), compared to bacteremia cases (19%). The attributable mortality was 47.6% in patients with bacteremia compared to non-bacteremia (no deaths), even though the appropriate antibiotic treatment was more frequent among patients with bacteremia (92% and 60%, respectively). CONCLUSION: The presence of S.aureus in urine was accompanied by bacteremia in half of the cases, but in patients without previous urinary tract intervention such a possibility increased to 81%. Concomitant bacteremia predicts a worse prognosis even with appropriate treatment.