Histone deacetylase inhibition mitigates fibrosis-driven disease progression in recessive dystrophic epidermolysis bullosa.

BACKGROUND: Recessive dystrophic epidermolysis bullosa (RDEB) is a blistering disease caused by mutations in the gene encoding type VII collagen (C7). RDEB is associated with fibrosis, which is responsible for severe complications. The phenotypic variability observed in RDEB siblings suggests that epigenetic modifications contribute to disease severity. Identifying epigenetic changes may help to uncover molecular mechanisms underlying RDEB pathogenesis and new therapeutic targets. OBJECTIVES: To investigate histone acetylation in RDEB skin and to explore histone deacetylase inhibitors (HDACis) as therapeutic molecules capable of counteracting fibrosis and disease progression in RDEB mice. METHODS: Acetylated histone levels were detected in human skin by immunofluorescence and in RDEB fibroblasts by ELISA. The effects of Givinostat and valproic acid (VPA) on RDEB fibroblast fibrotic behaviour were assessed by collagen-gel contraction assay, Western blot and immunocytofluorescence for α-smooth muscle actin, ELISA for released transforming growth factor-ß1 (TGF-ß1). RNA-seq was performed in HDACi- and vehicle-treated RDEB fibroblasts. VPA was systemically administered to RDEB mice, and effects on overt phenotype were monitored. Fibrosis was investigated in the skin using histological and immunofluorescence analyses. Eye and tongue defects were examined microscopically. Mass spectrometry proteomics was performed on skin protein extracts from VPA-treated RDEB and control mice. RESULTS: Histone acetylation decreases in RDEB skin and primary fibroblasts. RDEB fibroblasts treated with HDACis lowered fibrotic traits including contractility, TGF-ß1 release, and proliferation. VPA administration to RDEB mice mitigated severe manifestations affecting eyes and paws. These effects were associated with fibrosis inhibition. Proteomic analysis of mouse skin revealed that VPA almost normalised protein sets involved in protein synthesis and immune response, processes linked to the increased susceptibility to cancer and bacterial infections observed in RDEB patients. CONCLUSIONS: Dysregulated histone acetylation contributes to RDEB pathogenesis by facilitating the progression of fibrosis. Repurposing of HDACi could be considered for disease-modifying treatments of RDEB.

Early administration of nirmatrelvir/ritonavir leads to faster negative SARS-CoV-2 nasal swabs than monoclonal antibodies in COVID 19 patients at high-risk for severe disease.

PURPOSE: Besides the well-established efficacy in preventing severe COVID-19, the impact of early treatments, namely antivirals and monoclonal antibodies (mAbs), on the time length to negativization of SARS-CoV-2 nasal swabs is still unclear. The aim of this study was to compare the efficacy of different early treatments in reducing the SARS-CoV-2 viral shedding, identifying a single drug that might potentially lead to a more rapid negativization of SARS-CoV-2 nasal swab. METHODS: This was a single-centre, retrospective, observational study conducted at Ospedale Luigi Sacco in Milan. Data of high-risk COVID-19 patients who received early treatments between 23 December 2021 and March 2023 were extracted. The comparison across treatments was conducted using the Kruskall-Wallis test for continuous variables. Dunn's test with Bonferroni adjustment was performed for post-hoc comparisons of days to negativization. Secondly, a negative binomial regression adjusted for age, sex, number of comorbidities, immunosuppression, and SARS-CoV-2 vaccination status was implemented. RESULTS: Data from 428 patients receiving early treatments were collected. The majority were treated with Nirmatrelvir/Ritonavir and were affected by SARS-CoV-2 Omicron infection with BA.2 sublineage. The median length time to SARS-CoV-2 nasal swab negativization was 9 days [IQR 7-13 days]. We found that Nirmatrelvir/Ritonavir determined a significant decrease of the length time to SARS-CoV-2 nasal swab negativization compared to mAbs (p = 0.003), but not compared to Remdesivir (p = 0.147) and Molnupiravir (p = 0.156). CONCLUSION: Our findings highlight the importance of promptly treating high-risk COVID-19 patients with Nirmatrelvir/Ritonavir, as it also contributes to achieving a faster time to negative SARS-CoV-2 nasal swabs.

TMBleR: a bioinformatic tool to optimize TMB estimation and predictive power.

MOTIVATION: Tumor mutational burden (TMB) has been proposed as a predictive biomarker for immunotherapy response in cancer patients, as it is thought to enrich for tumors with high neoantigen load. TMB assessed by whole-exome sequencing is considered the gold standard but remains confined to research settings. In the clinical setting, targeted gene panels sampling various genomic sizes along with diverse strategies to estimate TMB were proposed and no real standard has emerged yet. RESULTS: We provide the community with TMBleR, a tool to measure the clinical impact of various strategies of panel-based TMB measurement. AVAILABILITY AND IMPLEMENTATION: R package and docker container (GPL-3 Open Source license): https://acc-bioinfo.github.io/TMBleR/. Graphical-user interface website: https://bioserver.ieo.it/shiny/app/tmbler. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

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.

Nasal Microbiome in COVID-19: A Potential Role of Corynebacterium in Anosmia.

The evolution and the development of the symptoms of Coronavirus disease 19 (COVID-19) are due to different factors, where the microbiome plays a relevant role. The possible relationships between the gut, lung, nasopharyngeal, and oral microbiome with COVID-19 have been investigated. We analyzed the nasal microbiome of both positive and negative SARS-CoV-2 individuals, showing differences in terms of bacterial composition in this niche of respiratory tract. The microbiota solution A (Arrow Diagnostics) was used to cover the hypervariable V1-V3 regions of the bacterial 16S rRNA gene. MicrobAT Suite and MicrobiomeAnalyst program were used to identify the operational taxonomic units (OTUs) and to perform the statistical analysis, respectively. The main taxa identified in nasal microbiome of COVID-19 patients and in Healthy Control subjects belonged to three distinct phyla: Proteobacteria (HC = 14%, Cov19 = 35.8%), Firmicutes (HC = 28.8%, Cov19 = 30.6%), and Actinobacteria (HC = 56.7%, Cov19 = 14.4%) with a relative abundance > 1% in all groups. A significant reduction of Actinobacteria in Cov19 group compared to controls (P < 0.001, FDR = 0.01) was found. The significant reduction of Actinobacteria was identified in all taxonomic levels down to the genus (P < 0.01) using the ANOVA test. Indeed, a significantly reduced relative abundance of Corynebacterium was found in the patients compared to healthy controls (P = 0.001). Reduced abundance of Corynebacterium has been widely associated with anosmia, a common symptom of COVID-19 as suffered from our patients. Contrastingly, the Corynebacterium genus was highly represented in the nasal mucosa of healthy subjects. Further investigations on larger cohorts are necessary to establish functional relationships between nasal microbiota content and clinical features of COVID-19.

Enhanced NAMPT-Mediated NAD Salvage Pathway Contributes to Psoriasis Pathogenesis by Amplifying Epithelial Auto-Inflammatory Circuits.

Dysregulated cross-talk between immune cells and epithelial compartments is responsible for the onset and amplification of pathogenic auto-inflammatory circuits occurring in psoriasis. NAMPT-mediated NAD salvage pathway has been recently described as an immunometabolic route having inflammatory function in several disorders, including arthritis and inflammatory bowel diseases. To date, the role of NAD salvage pathway has not been explored in the skin of patients affected by psoriasis. Here, we show that NAD content is enhanced in lesional skin of psoriatic patients and is associated to high NAMPT transcriptional levels. The latter are drastically reduced in psoriatic skin following treatment with the anti-IL-17A biologics secukinumab. We provide evidence that NAMPT-mediated NAD+ metabolism fuels the immune responses executed by resident skin cells in psoriatic skin. In particular, intracellular NAMPT, strongly induced by Th1/Th17-cytokines, acts on keratinocytes by inducing hyper-proliferation and impairing their terminal differentiation. Furthermore, NAMPT-mediated NAD+ boosting synergizes with psoriasis-related cytokines in the upregulation of inflammatory chemokines important for neutrophil and Th1/Th17 cell recruitment. In addition, extracellular NAMPT, abundantly released by keratinocytes and dermal fibroblasts, acts in a paracrine manner on endothelial cells by inducing their proliferation and migration, as well as the expression of ICAM-1 membrane molecule and chemokines important for leukocyte recruitment into inflamed skin. In conclusion, our results showed that NAMPT-mediated NAD salvage pathway contributes to psoriasis pathogenic processes by amplifying epithelial auto-inflammatory responses in psoriasis.

Evaluation of cutaneous, oral and intestinal microbiota in patients affected by pemphigus and bullous pemphigoid: A pilot study.

BACKGROUND: Significant alterations of the cutaneous microbiota (CM) have been recently demonstrated in bullous pemphigoid (BP). Microbiome data of both oral cavity (OM) and gut (GM) from patients affected by bullous disease are not available yet and, further consistent studies focused on the role of such microbial populations are still missing. OBJECTIVE: Objective: In this pilot study we characterized and compared GM, OM and CM of patients affected by pemphigus vulgaris (PV) and BP to investigate a distinctive microbiome composition in this two rare dermatological disorders. METHODS: High-throughput sequencing of the V1-V3 hyper-variable regions of 16S rRNA was used to compare the bacterial community composition of stool, skin and oral mucosae swabs in a cohort of PV and BP patients. A dedicated bioinformatics software coupled with in-house pipeline was implemented to analyse and compare diseases dataset. RESULTS: GM samples of both PV and BP patients were principally characterized by Firmicutes and Bacteroidetes phyla. Interestingly, the Firmicutes phylum and Staphylococcus genus were mainly represented in cutaneous samples. The diversity of phyla in oral mucosae was higher than those of gut and skin samples and, Bacteroidetes phylum was significantly underrepresented in all PV samples. CONCLUSION: Firmicutes phylum and Staphilococcus genus were the most represented in OM and CM swabs of PV and BP microbial populations. Moreover, we argue the quantitative imbalance linked to the decrease of Bacteriodetes in the oral cavity of PV patients might be associated to disease typical fetor. To shed light on this peculiar feature further studies are still required.

High-resolution melting analysis to screen the ST18 gene functional risk variant for pemphigus vulgaris: The occasion to open a debate on its usefulness in clinical setting.

The ST18 -497-65050 T > C polymorphisms (rs17315309) exhibit a very strong association in the pathogenesis of Pemphigus Vulgaris (PV) and could represent a new potential molecular target for the treatment of disease. The present study aimed to establish a low-cost, sensitive and reliable assay using high-resolution melting curve analysis (HRMA) on magnetic induction rotor-based platform, the Magnetic Induction Cycler (MIC) (Bio molecular Systems). HRMA assay was able to identify easily and unambiguously the c.-497-65050 T > C genotypes evaluating melting curve shape and melting temperature (Tm). The results of HRMA were validated by direct DNA sequencing. The HRMA is rapid, sensitive, low-cost and high-throughput assay to screen the rs17315309 variant and could be used in clinical diagnostic laboratories.

CYP24A1 and SLC34A1 genetic defects associated with idiopathic infantile hypercalcemia: from genotype to phenotype.

Loss of function mutations in the CYP24A1 gene, involved in vitamin D catabolism and in calcium homeostasis, are known to be the genetic drivers of both idiopathic infantile hypercalcemia (IIH) and adult renal stone disease. Recently, also defects in the SLC34A1 gene, encoding for the renal sodium-phosphate transporter NaPi-IIa, were associated with the disease. IIH typically affects infants and pediatric patients with a syndrome characterized by severe hypercalcemia, hypercalciuria, suppressed parathyroid hormone level and nephrolithiasis. In SLC34A1 mutated carriers, hypophosphatemia is also a typical biochemical tract. IIH may also persist undiagnosed into adulthood, causing an increased risk of nephrocalcinosis and renal complication. To note, a clinical heterogeneity characterizes IIH manifestation, principally due to the controversial gene-dose effect and, to the strong influence of environmental factors. The present review is aimed to provide an overview of the current molecular findings on the IIH disorder, giving a comprehensive description of the association between genotype and biochemical and clinical phenotype of the affected patients. We also underline that patients may benefit from genetic testing into a targeted diagnostic and therapeutic workflow.

A Whole Germline BRCA2 Gene Deletion: How to Learn from CNV In Silico Analysis.

BRCA1/2 screening in Hereditary Breast and Ovarian Syndrome (HBOC) is an essential step for effective patients' management. Next-Generation Sequencing (NGS) can rapidly provide high throughput and reliable information about the qualitative and quantitative status of tumor-associated genes. Straightforwardly, bioinformatics methods play a key role in molecular diagnostics pipelines. BRCA1/2 genes were evaluated with our NGS workflow, coupled with Multiplex Amplicon Quantification (MAQ) and Multiplex Ligation-dependent Probe Amplification (MLPA) assays. Variant calling was performed on Amplicon Suite, while Copy Number Variant (CNV) prediction by in house and commercial CNV tools, before confirmatory MAQ/MLPA testing. The germline profile of BRCA genes revealed a unique HBOC pattern. Although variant calling analysis pinpointed heterozygote and homozygote polymorphisms on BRCA1 and BRCA2, respectively, the CNV predicted by our script suggested two conflicting interpretations: BRCA1 duplication and/or BRCA2 deletion. Our commercial software reported a BRCA1 duplication, in contrast with variant calling results. Finally, the MAQ/MLPA assays assessed a whole BRCA2 copy loss. In silico CNV analysis is a time and cost-saving procedure to powerfully identify possible Large Rearrangements using robust and efficient NGS pipelines. Our layout shows as bioinformatics algorithms alone cannot completely and correctly identify whole BRCA1/2 deletions/duplications. In particular, the complete deletion of an entire gene, like in our case, cannot be solved without alternative strategies as MLPA/MAQ. These findings support the crucial role of bioinformatics in deciphering pitfalls within NGS data analysis.

Synergic role of nucleophosmin three-helix bundle and a flanking unstructured tail in the interaction with G-quadruplex DNA.

Nucleophosmin (NPM1) is a nucleocytoplasmic shuttling protein, mainly localized at nucleoli, that plays a number of functions in ribosome biogenesis and export, cell cycle control, and response to stress stimuli. NPM1 is the most frequently mutated gene in acute myeloid leukemia; mutations map to the C-terminal domain of the protein and cause its denaturation and aberrant cytoplasmic translocation. NPM1 C-terminal domain binds G-quadruplex regions at ribosomal DNA and at gene promoters, including the well characterized sequence from the nuclease-hypersensitive element III region of the c-MYC promoter. These activities are lost by the leukemic variant. Here we analyze the NPM1/G-quadruplex interaction, focusing on residues belonging to both the NPM1 terminal three-helix bundle and a lysine-rich unstructured tail, which has been shown to be necessary for high affinity recognition. We performed extended site-directed mutagenesis and measured binding rate constants through surface plasmon resonance analysis. These data, supported by molecular dynamics simulations, suggest that the unstructured tail plays a double role in the reaction mechanism. On the one hand, it facilitates the formation of an encounter complex through long range electrostatic interactions; on the other hand, it directly contacts the G-quadruplex scaffold through multiple and transient electrostatic interactions, significantly enlarging the contact surface.

Nucleophosmin mutations alter its nucleolar localization by impairing G-quadruplex binding at ribosomal DNA.

Nucleophosmin (NPM1) is an abundant nucleolar protein implicated in ribosome maturation and export, centrosome duplication and response to stress stimuli. NPM1 is the most frequently mutated gene in acute myeloid leukemia. Mutations at the C-terminal domain led to variant proteins that aberrantly and stably translocate to the cytoplasm. We have previously shown that NPM1 C-terminal domain binds with high affinity G-quadruplex DNA. Here, we investigate the structural determinants of NPM1 nucleolar localization. We show that NPM1 interacts with several G-quadruplex regions found in ribosomal DNA, both in vitro and in vivo. Furthermore, the most common leukemic NPM1 variant completely loses this activity. This is the consequence of G-quadruplex-binding domain destabilization, as mutations aimed at refolding the leukemic variant also result in rescuing the G-quadruplex-binding activity and nucleolar localization. Finally, we show that treatment of cells with a G-quadruplex selective ligand results in wild-type NPM1 dislocation from nucleoli into nucleoplasm. In conclusion, this work establishes a direct correlation between NPM1 G-quadruplex binding at rDNA and its nucleolar localization, which is impaired in the acute myeloid leukemia-associated protein variants.

Understanding the burden of antibiotic resistance: a decade of carbapenem-resistant Gram-negative bacterial infections in Italian intensive care units.

Introduction: In patients admitted to intensive care units (ICUs), Gram-negative bacteria (GNB) infections pose significant challenges due to their contribution to morbidity, mortality, and healthcare costs. During the SARS-CoV-2 pandemic, Italy witnessed a rise in healthcare-associated infections (HAIs), with GNBs involved in a substantial proportion of cases. Concerningly, carbapenem-resistant GNBs (CR-GNBs) have increased worldwide, posing therapeutic challenges. Methods: Retrospective multicentre study analysing data from over 299,000 patients admitted to Italian ICUs from 2013 to 2022. Results: The study revealed an average of 1.5 infections per patient, with HAIs peaking during the pandemic years. Ventilator associated pneumonia (VAP) emerged as the most common HAI, with Klebsiella spp. and Pseudomonas aeruginosa predominating. Alarmingly, CR-GNBs accounted for a significant proportion of infections, particularly in VAP, bloodstream infections, and intra-abdominal infections. Discussion: Our findings underscore the pressing need for enhanced infection control measures, particularly in the ICU setting, to mitigate the rising prevalence of CR-GNBs and their impact on patient outcomes. The study provides valuable insights into the epidemiology of HAIs in Italian ICUs and highlights the challenges posed by CR-GNBs, especially in the context of the SARS-CoV-2 pandemic, which exacerbated the issue and may serve as a crucial example for the management of future viral pandemics.

Exploring early COVID-19 therapies, variants, and viral clearance dynamics: Insights from a high-risk outpatients study.

This retrospective observational study investigates the impact of early COVID-19 therapies, including antivirals and monoclonal antibodies (mAbs), on time to achieve negative swab results in high-risk outpatients infected with specific Omicron sublineages. The study enrolled 104 patients from Luigi Sacco Hospital in Milan between December 2021 and March 2023, categorizing them based on the Omicron sublineage they were infected with (BA.1, BA.2, BA.4/BA.5) and the early treatment they received (antivirals or mAbs). Key data collected included demographic and clinical characteristics, initial and follow-up cycle threshold (Ct) values from qPCR tests, and the interval between swabs. The median age of the participants was 63 years (Interquartile Range [IQR] 54.0-76.5), and 55.8% were male. Among the patients, 15 received mAbs (14.4%), and 99 received antiviral treatments (95.2%) - specifically, Paxlovid (51.9%), Molnupiravir (21.1%), and Remdesivir (12.5%). No patients required hospitalization or experienced mortality during the one-month follow-up period. Regarding Omicron sublineages, 23 patients (22.1%) were infected with BA.1, 53 (51%) with BA.2, and 28 (26.9%) with BA.4/BA.5. The median interval between the initial and follow-up swabs was 6 days (IQR 6.0-7.0). Initial Ct values had a median of 18.5 (IQR 16.5-22.1), which increased to a median of 30.5 (IQR 27.1-33.0) at follow-up, indicating a reduction in viral load. A non-significant trend suggested that patients infected with BA.2 and BA.4/BA.5 sublineages might experience a faster increase in Ct values-indicating quicker viral load reduction - compared to those infected with BA.1, regardless of treatment type. However, this trend did not achieve statistical significance (p=0.609), likely due to the limited sample size and the absence of a clear trend curve. In summary, the study did not find a significant association between specific early therapies and the time to achieve swab negativization. These findings underscore the complex dynamics of viral clearance and highlight the need for further research with larger patient cohorts to refine treatment protocols for high-risk COVID-19 patients.

One-step NGS molecular analysis of the CFTR gene on newborn dried blood spots gives a higher diagnostic sensitivity in affected and carrier subjects: A pilot study.

BACKGROUND: Cystic fibrosis is the most common hereditary recessive disease with an incidence of about 1:2500/3000. It has long been known that the disease is caused by deleterious mutations in the CFTR gene. Conventionally, the disease is diagnosed in several phases. The analysis of all the possible disease-causing molecular alterations is time consuming and may not lead to a definitive diagnosis in several cases. Consequently, we propose, in this paper, a rapid sequencing method that, in a single procedural asset, reveals the presence of small mutations and also the copy number variants (CNVs) from the DNA extracted from the Guthrie Spot. MATERIALS AND METHODS: We first sequenced 30 blood spots, then we validated the method on 100 spots that underwent both traditional analyses and this complete NGS sequencing, and lastly, we tested the strategy on patients who normally do not reach the molecular sequencing step because of low level of Immune-Reactive Trypsinogen. RESULTS: Using this procedure, we identified 97 variants in the CFTR gene of our samples and 6 CNVs. Notably, the significant data were obtained in the group of patients with borderline or negative IRT who routinely would not undergo molecular testing. We also identified 6 carriers of "disease-causing" variants. CONCLUSION: This method is very robust. Indeed, there was a 100% concordance with Sanger sequencing validation, and 6 mutation carriers were identified who normally escaped molecular testing with actual conventional procedure. There were also 3 duplications of almost the entire gene in heterozygosity, which were not seen with traditional methods. Being quick and easy to perform, we suggest that complete sequencing of the CFTR gene, as in this study be considered for all newborns.

Identification of immunological patterns characterizing immune-related psoriasis reactions in oncological patients in therapy with anti-PD-1 checkpoint inhibitors.

Introduction: Immunotherapy with biologics targeting programmed cell death protein-1 (PD-1) is highly effective in the treatment of various malignancies. Nevertheless, it is frequently responsible for unexpected cutaneous manifestations, including psoriasis-like dermatitis. The pathogenesis of anti-PD-1-induced psoriasis has yet to be clarified, even though it is plausible that some innate and adaptive immunity processes are in common with canonical psoriasis. The genetic predisposition to psoriasis of patients could also be a contributing factor. Here, we investigated the immunological and genetic profiles of two patients with metastatic melanoma and one patient affected by lung cancer, who developed severe psoriasis after receiving anti-PD-1 nivolumab therapy. Methods: The immune patterns of the three patients were compared with those detectable in classical, chronic plaque-type psoriasis or paradoxical psoriasis induced by anti-TNF-α therapy, mostly sustained by adaptive and innate immunity processes, respectively. Therefore, immunohistochemistry and mRNA analyses of innate and adaptive immunity molecules were conducted on skin biopsy of patients. Genetic analysis of polymorphisms predisposing to psoriasis was carried out by NGS technology. Results: We found that anti-PD-1-induced psoriasis showed immunological features similar to chronic psoriasis, characterized by the presence of cellular players of adaptive immunity, with abundant CD3+, CD8+ T cells and CD11c+ dendritic cells infiltrating skin lesions, and producing IL-23, IL-6, TNF-α, IFN-γ and IL-17. On the contrary, a lower number of innate immunity cells (BDCA2+ plasmacytoid dendritic cells, CD15+ neutrophils, CD117+ mast cells) and reduced IFN-α/ß, lymphotoxin (LT)-α/ß, were observed in anti-PD-1-induced psoriasis lesions, as compared with anti-TNF-α-induced paradoxical psoriasis. Importantly, the disintegrin and metalloprotease domain containing thrombospondin type 1 motif-like 5 (ADAMTSL5) psoriasis autoantigen was significantly upregulated in psoriasis lesions of anti-PD-1-treated patients, at levels comparable with chronic plaque-type psoriasis. Finally, NGS analysis revealed that all patients carried several allelic variants in psoriasis susceptibility genes, such as HLA-C, ERAP1 and other genes of the major psoriasis susceptibility PSORS1 locus. Discussion: Our study showed that adaptive immunity predominates over innate immunity in anti-PD-1-induced psoriasis lesions, consistently with the local ADAMTSL5 overexpression. The presence of numerous SNPs in psoriasis susceptibility genes of the three patients also suggested their strong predisposition to the disease.

Evaluating the Clinical Meaning of Dermatology Life Quality Index Scores Between Different Phenotypes of Atopic Dermatitis in Patients Before and After Biologic Therapy With Dupilumab.

Background and Objective: Atopic Dermatitis (AD) is the most prevalent inflammatory skin disorder resulting in an intense impact on patients quality of life. The aim of this study is to evaluate the clinical meaning of the DLQI scores documented between different phenotypes of AD patients under biologic therapy with Dupilumab. Method: We conducted a retrospective analysis of 209 patients with AD treated with Dupilumab for 2 years. These patients were categorized into different clinical phenotypes. Severity of the disease was assessed by using the Eczema Area and Severity Index (EASI), Numerical Scale Rating (NRS) for sleep (NRS sleep), pruritus (NRS pruritus) and Dermatology Life Quality Index (DLQI) at baseline and subsequently at 4,12 and 24 months. Results: Our results show that the higher DLQI scores (mean: 18.6, range:9-30) achieved at T0 are associated with a prurigo nodularis AD pattern, while after 24 months (T3) of therapy with Dupilumab, the worst quality of life index results were reported in Flexural and Head-Neck combined clinical phenotypes. Conclusions: Quality of life is probably what matters most as an overall endpoint in AD. Assessing the clinical meaning of DLQI scores across different AD phenotypes could be a further aid when considering decision making factors in patient management.

Clinical characteristics and outcomes of vaccinated patients hospitalised with SARS-CoV-2 breakthrough infection: Multi-IPV, a multicentre study in Northern Italy.

BACKGROUND: Despite the well-known efficacy of anti-COVID-19 vaccines in preventing morbidity and mortality, several vaccinated individuals are diagnosed with SARS-CoV-2 breakthrough infection, which might require hospitalisation. This multicentre, observational, and retrospective study aimed to investigate the clinical characteristics and outcomes of vaccinated vs. non-vaccinated patients, both hospitalised with SARS-CoV-2 infection in 3 major hospitals in Northern Italy. METHODS: Data collection was retrospective, and paper and electronic medical records of adult patients with a diagnosed SARS-CoV-2 infection were pseudo-anonymised and analysed. Vaccinated and non-vaccinated individuals were manually paired, using a predetermined matching criterion (similar age, gender, and date of hospitalisation). Demographic, clinical, treatment, and outcome data were compared between groups differing by vaccination status using Pearson's Chi-square and Mann-Whitney tests. Moreover, multiple logistic regression analyses were performed to assess the impact of vaccination status on ICU admission or intra-hospital mortality. RESULTS: Data from 360 patients were collected. Vaccinated patients presented with a higher prevalence of relevant comorbidities, like kidney replacement therapy or haematological malignancy, despite a milder clinical presentation at the first evaluation. Non-vaccinated patients required intensive care more often than their vaccinated counterparts (8.8% vs. 1.7%, p = 0.002). Contrariwise, no difference in intra-hospital mortality was observed between the two groups (19% vs. 20%, p = 0.853). These results were confirmed by multivariable logistic regressions, which showed that vaccination was significantly associated with decreased risk of ICU admission (aOR=0.172, 95%CI: 0.039-0.542, p = 0.007), but not of intra-hospital mortality (aOR=0.996, 95%CI: 0.582-1.703, p = 0.987). CONCLUSIONS: This study provides real-world data on vaccinated patients hospitalised with COVID-19 in Northern Italy. Our results suggest that COVID-19 vaccination has a protective role in individuals with higher risk profiles, especially regarding the need for ICU admission. These findings contribute to our understanding of SARS-CoV-2 infection outcomes among vaccinated individuals and emphasise the importance of vaccination in preventing severe disease, particularly in those countries with lower first-booster uptake rates.

Probing the stability of the "naked" mucin-like domain of human α-dystroglycan.

BACKGROUND: α-Dystroglycan (α-DG) is heavily glycosylated within its central mucin-like domain. The glycosylation shell of α-dystroglycan is known to largely influence its functional properties toward extracellular ligands. The structural features of this α-dystroglycan domain have been poorly studied so far. For the first time, we have attempted a recombinant expression approach in E. coli cells, in order to analyze by biochemical and biophysical techniques this important domain of the α-dystroglycan core protein. RESULTS: We expressed the recombinant mucin-like domain of human α-dystroglycan in E. coli cells, and purified it as a soluble peptide of 174 aa. A cleavage event, that progressively emerges under repeated cycles of freeze/thaw, occurs at the carboxy side of Arg461, liberating a 151 aa fragment as revealed by mass spectrometry analysis. The mucin-like peptide lacks any particular fold, as confirmed by its hydrodynamic properties and its fluorescence behavior under guanidine hydrochloride denaturation. Dynamic light scattering has been used to demonstrate that this mucin-like peptide is arranged in a conformation that is prone to aggregation at room temperature, with a melting temperature of ~40°C, which indicates a pronounced instability. Such a conclusion has been corroborated by trypsin limited proteolysis, upon which the protein has been fully degraded in less than 60 min. CONCLUSIONS: Our analysis indirectly confirms the idea that the mucin-like domain of α-dystroglycan needs to be extensively glycosylated in order to reach a stable conformation. The absence/reduction of glycosylation by itself may greatly reduce the stability of the dystroglycan complex. Although an altered pattern of α-dystroglycan O-mannosylation, that is not significantly changing its overall glycosylation fraction, represents the primary molecular clue behind currently known dystroglycanopathies, it cannot be ruled out that still unidentified forms of αDG-related dystrophy might originate by a more substantial reduction of α-dystroglycan glycosylation and by its consequent destabilization.

The performance of multi-gene panels for breast/ovarian cancer predisposition.

BRCA1 and BRCA2 are the most mutated genes in breast cancer. We analyzed 48 breast cancer subjects using two methods that differ in terms of number of genes investigated and strategy used (primers: Panel A - 12 genes - vs probes: Panel B - 48 genes). Both the panels and procedures identified "pathogenic" or "likely pathogenic" variants in TP53, ATM, CHEK2 and BARD1 besides BRCA1 and BRCA2. Panel B identified two other putatively pathogenic variants in RNASEL and in RAD50. Identification of variants other than the BRCA genes can be useful in patient management. A total of 121 variants were distributed within the 12 genes and were correctly detected by both panels. However, the number of calls without divergence, namely ± 0.10 difference of allelic frequency, was 78.3%, while calls with a divergence below 0.10 was 16.7%, thus indicating that only 5% (n = 275) of 5,412 calls had a divergence above 0.10. Although these panels differ from each other, both are useful in different situations, particularly when patients should be tested for genes other than BRCA1/2 (as occurs in patients affected by a so called hereditary syndrome) or for therapeutic purposes.