Modulation of innate immunity related genes resulting in prophylactic antimicrobial and antiviral properties.

BACKGROUND: The innate immunity acts during the early phases of infection and its failure in response to a multilayer network of co-infections is cause of immune system dysregulation. Epidemiological SARS-CoV-2 infections data, show that Influenza Virus (FLU-A-B-C) and Respiratory Syncytial Virus (RSV) are co-habiting those respiratory traits. These viruses, especially in children (mostly affected by 'multi-system inflammatory syndrome in children' [MIS-C] and the winter pandemic FLU), in the aged population, and in 'fragile' patients are causing alteration in immune response. Then, bacterial and fungal pathogens are also co-habiting the upper respiratory traits (e.g., Staphylococcus aureus and Candida albicans), thus contributing to morbidity in those COVID-19 affected patients. METHODS: Liquid chromatography coupled with high-resolution mass spectrometry using the quadrupole orbital ion trap analyser (i.e., UHPLC-Q-Orbitrap HRMS) was adopted to measure the polyphenols content of a new nutraceutical formula (Solution-3). Viral infections with SARS-CoV-2 (EG.5), FLU-A and RSV-A viruses (as performed in BLS3 authorised laboratory) and real time RT-PCR (qPCR) assay were used to test the antiviral action of the nutraceutical formula. Dilution susceptibility tests have been used to estimate the minimum inhibitory and bactericidal concentration (MIC and MBC, respectively) of Solution-3 on a variety of microorganisms belonging to Gram positive/ negative bacteria and fungi. Transcriptomic data analyses and functional genomics (i.e., RNAseq and data mining), coupled to qPCR and ELISA assays have been used to investigate the mechanisms of action of the nutraceutical formula on those processes involved in innate immune response. RESULTS: Here, we have tested the combination of natural products containing higher amounts of polyphenols (i.e., propolis, Verbascum thapsus L., and Thymus vulgaris L.), together with the inorganic long chain polyphosphates 'polyPs' with antiviral, antibacterial, and antifungal behaviours, against SARS-CoV-2, FLU-A, RSV-A, Gram positive/ negative bacteria and fungi (i.e., Candida albicans). These components synergistically exert an immunomodulatory action by enhancing those processes involved in innate immune response (e.g., cytokines: IFNγ, TNFα, IL-10, IL-6/12; chemokines: CXCL1; antimicrobial peptides: HBD-2, LL-37; complement system: C3). CONCLUSION: The prophylactic antimicrobial success of this nutraceutical formula against SARS-CoV-2, FLU-A and RSV-A viruses, together with the common bacteria and fungi co-infections as present in human oral cavity, is expected to be valuable.

Oreoch-1: A broad-spectrum virus and host-targeting peptide against animal infections.

In recent decades, the global rise of viral emerging infectious diseases has posed a substantial threat to both human and animal health worldwide. The rapid spread and accumulation of mutations into viruses, and the limited availability of antiviral drugs and vaccines, stress the urgent need for alternative therapeutic strategies. Antimicrobial peptides (AMPs) derived from natural sources present a promising avenue due to their specificity and effectiveness against a broad spectrum of pathogens. The present study focuses on investigating the antiviral potential of oreochromicin-1 (oreoch-1), a fish-derived AMP obtained from Nile tilapia, against a wide panel of animal viruses including canine distemper virus (CDV), Schmallenberg virus (SBV), caprine herpesvirus 1 (CpHV-1), and bovine herpesvirus 1 (BoHV-1). Oreoch-1 exhibited a strong antiviral effect, demonstrating an inhibition of infection at concentrations in the micromolar range. The mechanism of action involves the interference with viral entry into host cells and a direct interaction between oreoch-1 and the viral envelope. In addition, we observed that the peptide could also interact with the cell during the CDV infection. These findings not only highlight the efficacy of oreoch-1 in inhibiting viral infection but also emphasize the potential of fish-derived peptides, specifically oreoch-1, as effective antiviral agents against viral infections affecting animals, whose potential to spill into humans is high. This research contributes valuable insights to the ongoing quest for novel antiviral drugs with the potential to mitigate the impact of infectious diseases on a global scale.

The antiherpetic and anti-inflammatory activity of the frog-derived peptide Hylin-a1.

AIM: The high incidence of virus-related infections and the large diffusion of drug-resistant pathogens stimulate the search and identification of new antiviral agents with a broad spectrum of action. Antivirals can be designed to act on a single target by interfering with a specific step in the viral lifecycle. On the contrary, antiviral peptides (AVPs) are known for acting on a wide range of viruses, with a diversified mechanism of action targeting virus and/or host cell. In the present study, we evaluated the antiviral potential of the peptide Hylin-a1 secreted by the frog Hypsiobas albopunctatus against members of the Herpesviridae family. METHODS AND RESULTS: The inhibitory capacity of the peptide was evaluated in vitro by plaque assays in order to understand the possible mechanism of action. The results were also confirmed by real-time PCR and Western blot evaluating the expression of viral genes. Hylin-a1 acts to block the herpetic infection interfering at the early stages of both herpes simplex virus type 1 (HSV-1) and type 2 infection. Its mechanism is mainly directed on the membrane, probably by damaging the viral envelope. The same effect was also observed against HSV-1 strains resistant to acyclovir. CONCLUSIONS: The data presented in this study, such as the increased activity of the peptide when combined to acyclovir, a weak hemolytic profile, an anti-inflammatory effect, and a tolerable half-life in serum, indicates Hylin-a1 as a novel antiherpetic molecule with promising potential in the clinical setting.

Aerobic vaginitis: antibiotic resistance trend and future actions of antimicrobial diagnostic stewardship.

The study objective is to examine epidemiological and microbiological aspects of aerobic vaginitis in female patients admitted to University Hospital of Campania "L. Vanvitelli" over five years. The most represented strains were E. coli (n = 153), Citrobacter spp. increasing from 2020, E. faecalis (n = 149), S. haemolitycus (n = 61), and Candida albicans (n = 87). The susceptibility patterns of a selection of gram-negative and gram-positive representative bacterial isolates were examined. Carbapenems, aminoglycosides, and fosfomycin were most effective against gram-negative bacteria, whereas vancomycin, daptomycin, and linezolid exhibited greater efficacy against gram-positive bacteria. None of the E. coli and Citrobacter spp. isolates produced extended-spectrum beta-lactamases, and the S. haemolyticus strains were methicillin-resistant. In gram-positive isolates, gentamicin susceptibility increased in 2020 and 2021 compared to clindamycin; erythromycin showed high resistance rates in 2020. Our findings indicate that integrating proper microbiological cultures into clinical practice could improve the management of aerobic vaginitis. Moreover, they highlight the necessity of establishing a nationwide surveillance guideline to mitigate antimicrobial resistance. Improvement actions in antimicrobial diagnostic stewardship must be considered when seeking the appropriate diagnosis and treatment for aerobic vaginitis.

Antimicrobial Susceptibility Profiles of Klebsiella pneumoniae Strains Collected from Clinical Samples in a Hospital in Southern Italy.

Infections caused by antibiotic-resistant bacteria represent a serious threat to global public health. Recently, due to its increased resistance to carbapenems and ß-lactams, Klebsiella pneumoniae has become one of the main causes of septicemia, pneumonia, and urinary tract infections. It is crucial to take immediate action and implement effective measures to prevent further spread of this issue. This study aims to report the prevalence and antibiotic resistance rates of K. pneumoniae strains isolated from clinical specimens from 2015 to 2020 at the University Hospital of Salerno, Italy. More than 3,800 isolates were collected from urine cultures, blood cultures, respiratory samples, and others. K. pneumoniae isolates showed broad resistance to penicillin and cephalosporins, and increased susceptibility to fosfomycin and gentamicin. Extended spectrum beta-lactamase (ESBL) isolates accounted for 20-22%. A high percentage of strains tested were resistant to carbapenems, with an average of 40% to meropenem and 44% to ertapenem. The production of ESBLs and resistance to carbapenems is one of the major public health problems. Constant monitoring of drug-resistant isolates is crucial for developing practical approaches in implementing antimicrobial therapy and reducing the spread of K. pneumoniae in nosocomial environments.

Cupferron impairs the growth and virulence of Escherichia coli clinical isolates.

AIMS: Multidrug resistance is a worrying problem worldwide. The lack of readily available drugs to counter nosocomial infections requires the need for new interventional strategies. Drug repurposing represents a valid alternative to using commercial molecules as antimicrobial agents in a short time and with low costs. Contextually, the present study focused on the antibacterial potential of the ammonium salt N-nitroso-N-phenylhydroxylamine (Cupferron), evaluating the ability to inhibit microbial growth and influence the main virulence factors. METHODS AND RESULTS: Cupferron cytotoxicity was checked via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and hemolysis assays. The antimicrobial activity was assessed through the Kirby-Bauer disk diffusion test, broth microdilution method, and time-killing kinetics. Furthermore, the impact on different stages of the biofilm life cycle, catalase, swimming, and swarming motility was estimated via MTT and crystal violet (CV) assay, H2O2 sensitivity, and motility tests, respectively. Cupferron exhibited <15% cytotoxicity at 200 µg/mL concentration. The 90% bacterial growth inhibitory concentrations (MIC90) values recorded after 24 hours of exposure were 200 and 100 µg/mL for multidrug-resistant (MDR) and sensitive strains, respectively, exerting a bacteriostatic action. Cupferron-treated bacteria showed increased susceptibility to biofilm production, oxidative stress, and impaired bacterial motility in a dose-dependent manner. CONCLUSIONS: In the new antimicrobial compounds active research scenario, the results indicated that Cupferron could be an interesting candidate for tackling Escherichia coli infections.

Galdieria sulphuraria: An Extremophilic Alga as a Source of Antiviral Bioactive Compounds.

In the last decades, the interest in bioactive compounds derived from natural sources including bacteria, fungi, plants, and algae has significantly increased. It is well-known that aquatic or terrestrial organisms can produce, in special conditions, secondary metabolites with a wide range of biological properties, such as anticancer, antioxidant, anti-inflammatory, and antimicrobial activities. In this study, we focused on the extremophilic microalga Galdieria sulphuraria as a possible producer of bioactive compounds with antiviral activity. The algal culture was subjected to organic extraction with acetone. The cytotoxicity effect of the extract was evaluated by the 2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The antiviral activity was assessed through a plaque assay against herpesviruses and coronaviruses as enveloped viruses and poliovirus as a naked one. The monolayer was treated with different concentrations of extract, ranging from 1 µg/mL to 200 µg/mL, and infected with viruses. The algal extract displayed strong antiviral activity at non-toxic concentrations against all tested enveloped viruses, in particular in the virus pre-treatment against HSV-2 and HCoV-229E, with IC50 values of 1.7 µg/mL and IC90 of 1.8 µg/mL, respectively. However, no activity against the non-enveloped poliovirus has been detected. The inhibitory effect of the algal extract was confirmed by the quantitative RT-PCR of viral genes. Preliminary chemical profiling of the extract was performed using ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS), revealing the enrichment in primary fatty acid amides (PFAA), such as oleamide, palmitamide, and pheophorbide A. These promising results pave the way for the further purification of the mixture to explore its potential role as an antiviral agent.

Hylin-a1: A Pan-Inhibitor against Emerging and Re-Emerging Respiratory Viruses.

Pandemic and epidemic outbreaks of respiratory viruses are a challenge for public health and social care system worldwide, leading to high mortality and morbidity among the human populations. In light of the limited efficacy of current vaccines and antiviral drugs against respiratory viral infections and the emergence and re-emergence of new viruses, novel broad-spectrum antiviral drugs are needed for the prevention and treatment of these infections. Antimicrobial peptides with an antiviral effect, also known as AVPs, have already been reported as potent inhibitors of viral infections by affecting different stages of the virus lifecycle. In the present study, we analyzed the activity of the AVP Hylin-a1, secreted by the frog Hypsiboas albopunctatus, against a wide range of respiratory viruses, including the coronaviruses HCoV-229E and SARS-CoV-2, measles virus, human parainfluenza virus type 3, and influenza virus H1N1. We report a significant inhibitory effect on infectivity in all the enveloped viruses, whereas there was a lack of activity against the naked coxsackievirus B3. Considering the enormous therapeutic potential of Hylin-a1, further experiments are required to elucidate its mechanism of action and to increase its stability by modifying the native sequence.

Microbiota and HPV: The role of viral infection on vaginal microbiota.

The World Health Organization (WHO) estimates that the prevalence of human papillomaviruses (HPV) infection is between 9% and 13% of the world population and only in the United States, more than 6.2 million are positive every year. There are more than 100 types of HPV, among them, two serotypes (16 and 18) are related to 70% of cervical cancers and precancerous cervical lesions. The vaginal microbiota could play a considerable role in HPV infection and the genesis of cervical tumors caused by HPV. Moreover, bacteria are strongly associated with vaginal inflammation and oncogenic mutations in human cells. We aim to investigate whether HPV infection could influence the bacterial microbiota composition in the uterine cervix. A total of 31 women were enrolled in this study. The vaginal swabs were collected; the HPV-DNA was extracted with QIAamp DNA Microbiome. The V3-V4-V6 region of the 16S rDNA gene was amplified by polymerase chain reaction (PCR) followed by sequencing with MiSeq Illumina. The main phylum identified in the vaginal microbiota were Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria. The phylum of Actinobacteria, Proteobacteria, and Bacteroides was more represented in HPV-positive patients. Lactobacilli represented the dominant genus, with a high percentage of Lactobacilli iners, Lactobacilli jensenii, and Lactobacilli crispatus as species. Gardnerella vaginalis, Enterococcus spp., Staphylococcus spp., Proteus spp., and Atopobium were the most represented in HPV-positive patients. An altered vaginal microbiota might play a functional role in HPV cervical infection, progression, and clearance. The relationship between infection and microbiota could spur the development of new probiotics. However, further studies are needed to clarify the role of the vaginal microbiota in HPV infection.

Nasopharyngeal virome analysis of COVID-19 patients during three different waves in Campania region of Italy.

From December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has spread rapidly, leading to a global pandemic. Little is known about possible relationships between SARS-CoV-2 and other viruses in the respiratory system affecting patient prognosis and outcomes. This study aims to characterize respiratory virome profiles in association with SARS-CoV-2 infection and disease severity, through the analysis in 89 nasopharyngeal swabs collected in a patient's cohort from the Campania region (Southern Italy). Results show coinfections with viral species belonging to Coronaviridae, Retroviridae, Herpesviridae, Poxviridae, Pneumoviridae, Pandoraviridae, and Anelloviridae families and only 2% of the cases (2/89) identified respiratory viruses.

Dynamics of nasopharyngeal tract phageome and association with disease severity and age of patients during three waves of COVID-19.

In December 2019, several patients were hospitalized and diagnosed with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, which subsequently led to a global pandemic. To date, there are no studies evaluating the relationship between the respiratory phageome and the SARS-CoV-2 infection. The current study investigated the phageome profiles in the nasopharyngeal swabs collected from 55 patients during the three different waves of coronavirus disease 2019 (COVID-19) in the Campania Region (Southern Italy). Data obtained from the taxonomic profiling show that phage families belonging to the order Caudovirales have a high abundance in the patient samples. Moreover, the severity of the COVID-19 infection seems to be correlated with the phage abundance.

NGS analysis of nasopharyngeal microbiota in SARS-CoV-2 positive patients during the first year of the pandemic in the Campania Region of Italy.

Since its first appearance, the SARS-CoV-2 has spread rapidly in the human population, reaching the pandemic scale with >280 million confirmed infections and more than 5 million deaths to date (https://covid19.who.int/). These data justify the urgent need to enhance our understanding of SARS-CoV-2 effects in the respiratory system, including those linked to co-infections. The principal aim of our study is to investigate existing correlations in the nasopharynx between the bacterial community, potential pathogens, and SARS-CoV-2 infection. The main aim of this study was to provide evidence pointing to possible relationships between components of the bacterial community and SARS-CoV-2 in the nasopharynx. Meta-transcriptomic profiling of the nasopharyngeal microbial community was carried out in 89 SARS-Cov-2 positive subjects from the Campania Region in Italy. To this end, RNA extracted from nasopharyngeal swabs collected at different times during the initial phases of the pandemic was analyzed by Next-Generation Sequencing (NGS). Results show a consistently high presence of members of the Proteobacteria (41.85%), Firmicutes (28.54%), and Actinobacteria (16.10%) phyla, and an inverted correlation between the host microbiome, co-infectious bacteria, and super-potential pathogens such as Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, and Neisseria gonorrhoeae. In depth characterization of microbiota composition in the nasopharynx can provide clues to understand its potential contribution to the clinical phenotype of Covid-19, clarifying the interaction between SARS-Cov-2 and the bacterial flora of the host, and highlighting its dysbiosis and the presence of pathogens that could affect the patient's disease progression and outcome.

Six-month humoral response to BNT162b2 mRNA COVID-19 vaccine in people with multiple sclerosis treated with natalizumab.

BACKGROUND: Few studies investigated the immune response to SARS-CoV-2 vaccine in patients with multiple sclerosis (pwMS) treated with natalizumab (NTZ) and found a short-term efficient humoral response; however, there are no studies assessing the levels of SARS-CoV-2 IgG antibodies in pwMS treated with NTZ over time. METHODS: Humoral immune response to BNT162b2 mRNA COVID-19 vaccine was assessed in a group of 26 pwMS on NTZ up to 6 months after a full COVID-19 vaccination cycle and compared it with 43 age- and sex-matched group of HC. Serum samples were collected before the first dose (T0), and 4 weeks (T1) and 6 months (T2) after the first dose of BNT162b2 mRNA COVID-19 vaccine. The LIAISON® SARS-CoV-2 TrimericS-IgG assay (DiaSorin-S.p.A.) was employed for the detection of IgG antibodies to SARS-CoV-2 spike protein (cutoff for positive IgG antibodies: 33.8 BAU/mL). RESULTS: At T1 and T2, both groups showed an efficient humoral response to BNT162b2 mRNA COVID-19 vaccine. A significant reduction of IgG antibodies to SARS-CoV-2 spike protein was detected at T2 both in pwMS and in HC, but SARS-CoV-2 IgG antibodies were still above the cutoff limit in all participants. CONCLUSIONS: pwMS on NTZ develop and maintain a long-term humoral response after a full COVID-19 vaccination cycle comparable to their healthy peers, and these findings are relevant for clinicians called to counsel about COVID-19 mRNA vaccine timing and booster doses in pwMS treated with NTZ.

Hepatitis B Virus prevalence and serological profiles in a hospital in Southern Italy.

Viral hepatitis still represents a significant worldwide public health issue, being an important cause of morbidity and mortality. The aim of our study is to evaluate the prevalence of Hepatitis B virus (HBV) markers from serologic analysis of hospitalized patients at University Hospital of Campania "Luigi Vanvitelli" and also to investigate the prevalence of HBV/HCV coinfection. We screened serum Anti-Hepatitis B surface antigen (HBsAg), antibody to HBsAg (anti-HBs), antibody to hepatitis B core antigen (anti-HBc), and antibody to Hepatitis C Virus (Anti-HCV) Anti-HCV from January to December 2020. Analyses of HBV serological profile based on age showed that the 51-60 age group was the most numerous and with the highest cases of HBsAg. The 61-70 age group recorded the highest prevalence of anti-HBc while age groups 0-10 years and 31-40 years the highest cases of anti-HBs. Antibody levels decline with time. In subjects older than 20 years, compared to vaccinated cohort individuals, anti-HBc seropositive prevalence increased linearly. This study underlined, in our geographic region, the decreased incidence of hepatitis B and high immunogenicity in the young population. Therefore, administration of HBV vaccine booster dose should be considered for the population rather than vaccination in the first year of life. In conclusion, our findings reaffirm the importance of health surveillance in hospitalized subjects, stressing the need to improve immunized subjects to increase the general population's health.

Antimicrobial resistance changing trends of Klebsiella pneumoniae isolated over the last 5 years.

The aim of this study was to describe the prevalence and epidemiology distribution of K. pneumoniae isolated at University Hospital of Campania "Luigi Vanvitelli," including the susceptibility evolution profile. Data on resistant phenotype strains, such as extended-spectrum-ß-lactamase (ESBL) producers and carbapenem-resistant K. pneumoniae (CRE) isolates, were also reported. K. pneumoniae strains were collected at the Complex Operative Unit (UOC) of Virology and Microbiology from different colonization and infection sites from January 2016 to December 2020. The highest rates of isolation were in urinary samples and in respiratory and wound swabs. Antibiotics susceptibility patterns showed more than 50% of the isolates resistant to cephalosporins, fluoroquinolones and penicillin. On the other hand, from 20% to 40% of K. pneumoniae strains were resistant to carbapenems and aminoglycosides. Based on our analysis, fosfomycin, ceftazidime/avibactam and ceftolozane/tazobactam are still therapeutic alternatives. Data analysis on carbapenem class evolution in 2016-2020 showed a significant increase in resistance rates (p<0.05). Increased rates in CRE and ESBL producing K. pneumoniae since 2017 were reported. Providing information on clinical characteristics and epidemiology data on contemporary K. pneumoniae evolution could help mitigate the spread of these isolates in our hospital and avert the endemic levels that have been observed in Southern Italy and in other European countries.

The Broad-Spectrum Antiviral Potential of the Amphibian Peptide AR-23.

Viral infections represent a serious threat to the world population and are becoming more frequent. The search and identification of broad-spectrum antiviral molecules is necessary to ensure new therapeutic options, since there is a limited availability of effective antiviral drugs able to eradicate viral infections, and consequently due to the increase of strains that are resistant to the most used drugs. Recently, several studies on antimicrobial peptides identified them as promising antiviral agents. In detail, amphibian skin secretions serve as a rich source of natural antimicrobial peptides. Their antibacterial and antifungal activities have been widely reported, but their exploitation as potential antiviral agents have yet to be fully investigated. In the present study, the antiviral activity of the peptide derived from the secretion of Rana tagoi, named AR-23, was evaluated against both DNA and RNA viruses, with or without envelope. Different assays were performed to identify in which step of the infectious cycle the peptide could act. AR-23 exhibited a greater inhibitory activity in the early stages of infection against both DNA (HSV-1) and RNA (MeV, HPIV-2, HCoV-229E, and SARS-CoV-2) enveloped viruses and, on the contrary, it was inactive against naked viruses (PV-1). Altogether, the results indicated AR-23 as a peptide with potential therapeutic effects against a wide variety of human viruses.

Broad-Spectrum Antiviral Activity of the Amphibian Antimicrobial Peptide Temporin L and Its Analogs.

The COVID-19 pandemic has evidenced the urgent need for the discovery of broad-spectrum antiviral therapies that could be deployed in the case of future emergence of novel viral threats, as well as to back up current therapeutic options in the case of drug resistance development. Most current antivirals are directed to inhibit specific viruses since these therapeutic molecules are designed to act on a specific viral target with the objective of interfering with a precise step in the replication cycle. Therefore, antimicrobial peptides (AMPs) have been identified as promising antiviral agents that could help to overcome this limitation and provide compounds able to act on more than a single viral family. We evaluated the antiviral activity of an amphibian peptide known for its strong antimicrobial activity against both Gram-positive and Gram-negative bacteria, namely Temporin L (TL). Previous studies have revealed that TL is endowed with widespread antimicrobial activity and possesses marked haemolytic activity. Therefore, we analyzed TL and a previously identified TL derivative (Pro3, DLeu9 TL, where glutamine at position 3 is replaced with proline, and the D-Leucine enantiomer is present at position 9) as well as its analogs, for their activity against a wide panel of viruses comprising enveloped, naked, DNA and RNA viruses. We report significant inhibition activity against herpesviruses, paramyxoviruses, influenza virus and coronaviruses, including SARS-CoV-2. Moreover, we further modified our best candidate by lipidation and demonstrated a highly reduced cytotoxicity with improved antiviral effect. Our results show a potent and selective antiviral activity of TL peptides, indicating that the novel lipidated temporin-based antiviral agents could prove to be useful additions to current drugs in combatting rising drug resistance and epidemic/pandemic emergencies.

Natural Formulations Based on Olea europaea L. Fruit Extract for the Topical Treatment of HSV-1 Infections.

In the present study, a hydroxytyrosol-rich Olea europaea L. fruit extract (OFE) was added to three thoroughly green formulations-hydrogel, oleogel, and cream-in order to evaluate their antiviral activity against HSV-1. The extract was characterized by different analytical techniques, i.e., FT-IR, XPS, and TGA. HPLC analyses were carried out to monitor the content and release of hydroxytyrosol in the prepared formulations. The total polyphenol content and antioxidant activity were investigated through Folin-Ciocâlteu's reagent, DPPH, and ABTS assays. The ability of the three formulations to convey active principles to the skin was evaluated using a Franz cell, showing that the number of permeated polyphenols in the hydrogel (272.1 ± 1.8 GAE/g) was significantly higher than those in the oleogel and cream (174 ± 10 and 179.6 ± 2 GAE/g, respectively), even if a negligible amount of hydroxytyrosol crossed the membrane for all the formulations. The cell viability assay indicated that the OFE and the three formulations were not toxic to cultured Vero cells. The antiviral activity tests highlighted that the OFE had a strong inhibitory effect against HSV-1 with a 50% inhibitory concentration (IC50) at 25 µg/mL, interfering directly with the viral particles. Among the three formulations, the hydrogel exhibited the highest antiviral activity also against the acyclovir-resistant strain.

SARS-COV-2 colonizes coronary thrombus and impairs heart microcirculation bed in asymptomatic SARS-CoV-2 positive subjects with acute myocardial infarction.

BACKGROUND: The viral load of asymptomatic SAR-COV-2 positive (ASAP) persons has been equal to that of symptomatic patients. On the other hand, there are no reports of ST-elevation myocardial infarction (STEMI) outcomes in ASAP patients. Therefore, we evaluated thrombus burden and thrombus viral load and their impact on microvascular bed perfusion in the infarct area (myocardial blush grade, MBG) in ASAP compared to SARS-COV-2 negative (SANE) STEMI patients. METHODS: This was an observational study of 46 ASAP, and 130 SANE patients admitted with confirmed STEMI treated with primary percutaneous coronary intervention and thrombus aspiration. The primary endpoints were thrombus dimension + thrombus viral load effects on MBG after PPCI. The secondary endpoints during hospitalization were major adverse cardiovascular events (MACEs). MACEs are defined as a composite of cardiovascular death, nonfatal acute AMI, and heart failure during hospitalization. RESULTS: In the study population, ASAP vs. SANE showed a significant greater use of GP IIb/IIIa inhibitors and of heparin (p < 0.05), and a higher thrombus grade 5 and thrombus dimensions (p < 0.05). Interestingly, ASAP vs. SANE patients had lower MBG and left ventricular function (p < 0.001), and 39 (84.9%) of ASAP patients had thrombus specimens positive for SARS-COV-2. After PPCI, a MBG 2-3 was present in only 26.1% of ASAP vs. 97.7% of SANE STEMI patients (p < 0.001). Notably, death and nonfatal AMI were higher in ASAP vs. SANE patients (p < 0.05). Finally, in ASAP STEMI patients the thrombus viral load was a significant determinant of thrombus dimension independently of risk factors (p < 0.005). Thus, multiple logistic regression analyses evidenced that thrombus SARS-CoV-2 infection and dimension were significant predictors of poorer MBG in STEMI patients. Intriguingly, in ASAP patients the female vs. male had higher thrombus viral load (15.53 ± 4.5 vs. 30.25 ± 5.51 CT; p < 0.001), and thrombus dimension (4.62 ± 0.44 vs 4.00 ± 1.28 mm2; p < 0.001). ASAP vs. SANE patients had a significantly lower in-hospital survival for MACE following PPCI (p < 0.001). CONCLUSIONS: In ASAP patients presenting with STEMI, there is strong evidence towards higher thrombus viral load, dimension, and poorer MBG. These data support the need to reconsider ASAP status as a risk factor that may worsen STEMI outcomes.

Preliminary evidence of blunted humoral response to SARS-CoV-2 mRNA vaccine in multiple sclerosis patients treated with ocrelizumab.

OBJECTIVES: Several concerns regard the immunogenicity of SARS-CoV-2 vaccines in people with multiple sclerosis (pwMS), since the majority of them is treated with immunomodulating/immunosuppressive disease modifying therapies. Here we report the first data on the humoral response to mRNA SARS-CoV-2 vaccine in a case series of 4 pwMS treated with ocrelizumab (OCR) as compared to a group of healthy subjects (HS). METHODS: We collected serum samples at 0, 14, 21 days after the first dose and 7 days after the second dose of BNT162b2-mRNA-Covid-19 vaccine from 55 health-care workers and 4 relapsing pwMS on OCR, with no history of Covid-19 infection. Sera were tested using the LIAISON®SARS-CoV-2 TrimericS-IgG assay (DiaSorin-S.p.A.) for the detection of IgG antibodies to SARS-CoV-2 spike protein. The anti-spike IgGtiters were expressed in Binding Antibody Units (BAU), an international standard unit. RESULTS: At baseline all subjects were negative for anti-spike IgG. Seven days after the second dose of vaccine all HS mounted a significant humoral response (geometric mean 2010.4 BAU/mL C.I. 95% 1512.7-2672) while the 4 pwMS showed a lower response (range <4.81-175 BAU/mL). DISCUSSION: Humoral response to BNT162b2-mRNA-vaccine in pwMS treated with OCR was clearly blunted. Further data are urgently needed to confirm and expand these preliminary results and to develop strategies to optimize the response to SARSCoV-2 vaccines in pwMS on OCR.