New Pyrazolyl Thioureas Active against the Staphylococcus Genus.

To meet the urgent need for new antibacterial molecules, a small library of pyrazolyl thioureas (PTUs) was designed, synthesized and tested against difficult-to-treat human pathogens. The prepared derivatives are characterized by a carboxyethyl functionality on C4 and different hydroxyalkyl chains on N1. Compounds 1a-o were first evaluated against a large panel of Gram-positive and Gram-negative pathogens. In particular, the majority of PTUs proved to be active against different species of the Staphylococcus genus, with MIC values ranging from 32 to 128 µg/mL on methicillin-resistant Staphylococcus strains, often responsible for severe pulmonary disease in cystic fibrosis patients. Time-killing experiments were also performed for the most active compounds, evidencing a bacteriostatic mechanism of action. For most active derivatives, cytotoxicity was evaluated in Vero cells, and at the tested concentrations and at the experimental exposure time of 24 h, none of the compounds analysed showed significant toxicity. In addition, favourable drug-like, pharmacokinetic and toxicity properties were predicted for all new synthesized derivatives. Overall, the collected data confirmed the PTU scaffold as a promising chemotype for the development of novel antibacterial agents active against Gram-positive multi-resistant strains frequently isolated from cystic fibrosis patients.

Correction: Alfei et al. 4-Hydroxybenzoic Acid as an Antiviral Product from Alkaline Autoxidation of Catechinic Acid: A Fact to Be Reviewed. Plants 2022, 11, 1822.

In the original publication [...].

4-Hydroxybenzoic Acid as an Antiviral Product from Alkaline Autoxidation of Catechinic Acid: A Fact to Be Reviewed.

The dark brown mixture resulting from the autooxidation of catechinic acid (CA) (AOCA) has been reported to possess antiviral activity against Herpes Simplex Virus 1 and 2 (HSV-1 and HSV-2). Unfortunately, the constituents of AOCA were not separated or identified and the compound(s) responsible for AOCA's antiviral activity remained unknown until recently. Colorless 4-hydroxy benzoic acid (4-HBA) has been reported as the main constituent (75%) of AOCA, and as being responsible for its antiviral activity. The findings seemed not to be reliable because of the existence in the literature of very different findings, because of the high concentration that was attributed to the supposed 4-HBA in the dark mixture, and because of the absence of essential analytical experiments to confirm 4-HBA in AOCA. Particularly, the AOCA chromatograms highlighting a peak attributable to 4-HBA, using commercial 4-HBA as a standard, is missing, as well as investigations concerning the antiviral activity of marketed 4-HBA. Therefore, in this study, to verify the exactness of the recent reports, we prepared CA from catechin and AOCA from CA, and the absence of 4-HBA in the mixture was first established by thin-layer chromatography (TLC), and then was confirmed by UHPLC­MS/MS, UV­Vis, and ATR­FTIR analyses. For further confirmation, the ATR­FTIR spectral data were processed by principal components analysis (PCA), which unequivocally established strong structural differences between 4-HBA and AOCA. Finally, while the antiviral effects of AOCA against HSV-2 were confirmed, a commercial sample of 4-HBA was completely inactive.

In Vitro Reduction of Interleukin-8 Response to Enterococcus faecalis by Escherichia coli Strains Isolated from the Same Polymicrobial Urines.

Urinary tract infections are often polymicrobial and are mainly due to uropathogenic Escherichia coli (UPEC). We previously demonstrated a link among clinical fluoroquinolone susceptible E. coli reducing in vitro urothelial interleukin-8 (CXCL8) induced by E. coli K-12, polymicrobial cystitis, and pyuria absence. Here, we evaluated whether fifteen clinical fluoroquinolone susceptible UPEC were able to reduce CXCL8 induced by Enterococcus faecalis that had been isolated from the same mixed urines, other than CXCL8 induced by E. coli K-12. We also evaluated the connection between fluoroquinolone susceptibility and pathogenicity by evaluating the immune modulation of isogenic gyrA, a mutant UPEC resistant to ciprofloxacin. Using the 5637 bladder epithelial cell line, we observed that lower CXCL8 induced the most UPEC isolates than K-12 and the corresponding E. faecalis. During coinfections of UPEC/K-12 and UPEC/E. faecalis, we observed lower CXCL8 than during infections caused by K-12 and E. faecalis alone. UPEC strains showed host-pathogen and pathogen-pathogen interaction, which in part explained their persistence in the human urinary tract and coinfections, respectively. Mutant UPEC showed lower modulating activity with respect to the wildtypes, confirming the connection between acquired fluoroquinolone resistance and the decrease of innate microbial properties.

Conscientious Objection to Animal Experimentation in Italian Universities.

In Italy, Law 413/1993 states that public and private Italian Institutions, including academic faculties, are obliged to fully inform workers and students about their right to conscientious objection to scientific or educational activities involving animals, hereafter written as "animal CO". However, little monitoring on the faculties' compliance with this law has been performed either by the government or other institutional bodies. Based on this premise, the authors have critically reviewed the existing data and compared them with those emerging from their own investigation to discuss limitations and inconsistencies. The results of this investigation revealed that less than half of Italian academic faculties comply with their duty to inform on animal CO. Non-compliance may substantially affect the right of students to make ethical choices in the field of animal ethics and undermines the fundamental right to express their own freedom of thought. The Italian Ministry of Education, Universities and Research, ethics committees and animal welfare bodies should cooperate to make faculties respect this law. Further research is needed to better understand the reasons for the current trend, as well as to promote the enforcement of Law 413/1993 with particular regard to information on animal CO.

Break with tradition: donating cadavers for scientific purposes and reducing the use of sentient beings.

In recent years, the development of research and the increased awareness of our moral duties beyond the human species have pushed the scientific community to revise widely-accepted ontological reductionist views that regard non-human animals as mere things. The new horizons offered by the development of advanced research methods therefore require an on-going commitment to new perspectives able to find the right balance between the need for scientific knowledge on one hand and the respect for animal life on the other. This is in line with increasing attention to animal welfare and expansion of the "3Rs model": replacement, reduction, refinement.With the view of promoting the adoption of alternative methods, human body donation for research can contribute not only to the acquisition of important information for human health and for doctors' training, but also can reduce significantly the number of animals sacrificed.By investigating the scientific and ethical reasons that may encourage cadaver donation, the authors aim to promote the adoption of the practice in Italy following other European experiences.

An ethics for the living world: operation methods of Animal Ethics Committees in Italy.

INTRODUCTION AND OBJECTIVES: Coinciding with the recent implementation in Italy of the "Directive 2010/63/EU, regarding the protection of animals used for scientific purposes", the Authors would like to analyse the topic of the introduction of ethical committees for animal experimentation in Italy. This paper furthermore aims to underline some critical aspects concerning the actions taken by Italian institutions to comply with the provisions of EU. RESULTS AND DISCUSSION: The implementation of the recent Italian law (Decreto Legislativo n. 26 on 4 March 2014 Implementation of the Directive 2010/63/EU on the protection of animals used for scientific purposes) leans towards a restrictive interpretation of the European provisions about composition and responsibilities of "Ethical Committee for Animal Experimentation". In the composition of the bodies mentioned, we note a tendency to restrict the composition to few professional figures contemplated by Italian law, without guaranteeing the independence of each committee; also, an absence of hierarchical relationship between a research institution and his committee is apparent. Moreover, a critical aspect is the lack of decision-making powers of these new organisms in terms of ethical evaluation of protocols and research projects. CONCLUSIONS: What EU legislation imposes on the member states is to set up an animal-welfare body (art. 26). This represents a strong incentive for Italy to follow the steps of many other European Countries, where ad hoc ethical committees have been working for a long time. The proper functioning of these bodies may contribute to guarantee the safety and welfare of the animals inside the laboratories, and to balance the protection of animal life and the interests of research.

Mechanisms for reduced pulmonary diffusing capacity in haematopoietic stem-cell transplantation recipients.

Lung diffusing capacity for CO (DLCO) is compromised in haematopoietic stem-cell transplantation (HSCT) recipients. We derived alveolar-capillary membrane conductance (DM,CO) and pulmonary capillary volume (VC) from DLCO and diffusing capacity for NO (DLNO). Forty patients were studied before and 6 weeks after HSCT. Before HSCT, DLNO and DLCO were significantly lower than in 30 healthy controls. DM,CO was ∼40% lower in patients than in controls (p<0.001), whereas VC did not differ significantly. After HSCT, DLNO and DM,CO further decreased, the latter by ∼22% from before HSCT (p<0.01) while VC did not change significantly. Lung density, serum CRP and reactive oxygen metabolites were significantly increased, with the latter being correlated (R2=0.71, p<0.001) with the decrement in DLNO. We conclude that DLNO and, to a lesser extent, DLCO are compromised before HSCT mainly due to a DM,CO reduction. A further reduction of DM,CO without VC loss occurs after HSCT, possibly related to development of oedema, or interstitial fibrosis, or both.

Quinolone/fluoroquinolone susceptibility in Escherichia coli correlates with human polymicrobial bacteriuria and with in vitro interleukine-8 suppression.

Urinary tract infections (UTIs) are frequently polymicrobial diseases mainly sustained by Escherichia coli in association with other opportunistic pathogens. Cystitis and pyelonephritis are usually accompanied by an inflammatory response, which includes neutrophil recruitment. Uropathogenic E. coli possess the ability to evade host defenses, modulating the innate immune response. The aim of this study was to determine whether particular E. coli strains correlate with polymicrobial bacteriuria and whether escape from the early host defenses and microbial synergy could lead to mixed UTIs. We evaluated 188 E. coli-positive urine samples and assessed the relationships among polymicrobism, neutrophil presence and several traits of E. coli isolates (virulence factors such as hlyA, fimA, papC and their relative products, i.e. hemolysin, type 1 and P fimbriae, and cnf1, their phylogenetic group) and their ability to suppress cytokine response in 5637 bladder epithelial cells. Escherichia coli susceptibility toward quinolones and fluoroquinolones, known to be linked to the pathogenicity of this species, was also considered. We found significant correlations among polymicrobial bacteriuria, absence of pyuria and quinolone/fluoroquinolone susceptibility of E. coli isolates and their enhanced capability to suppress interleukin-8 urothelial production when compared with the patterns induced by the resistant strains.

A comparative study of leukaemia inhibitory factor and interleukin-1alpha intracellular content in a human keratinocyte cell line after exposure to cosmetic fragrances and sodium dodecyl sulphate.

According to European laws animal testing in cosmetic industry will be prohibited in a few years and it will be replaced by alternative methods based on cell and tissue culture. Many ingredients of cosmetic formulations are potentially causes of skin inflammation and sensibilization. Since cytotoxicity is known, among other factors, to trigger irritation, in an alternative model for evaluation of skin irritation, it can be considered also the precocious release of inflammatory mediators, i.e. cytokines, originating mainly from keratinocytes. In this in vitro study we have analysed some parameters directly or indirectly related to irritation/inflammation, in NCTC 2544 human keratinocytes during short-time exposure to some potential irritants cosmetic fragrances, included in the European Laws 2003/15/EEC. IIC50 was extrapolated by MTT and NRU viability indexes after exposure of cell ultures to Geraniol Limonene and Benzylic Alcohol for 1, 3 and 6h. NCTC cells were then exposed to sub-toxic doses of selected compounds and interleukin-1alpha (IL-1alpha) and leukaemia inhibitory factor (LIF) expressions were analysed as early proinflammatory cytokines. To our knowledge our findings demonstrated for the first time that NCTC cells synthesize and modulate LIF after exposure to selected irritating stimuli. Moreover, our results give evidence on LIF role as in vitro precocious endpoint for the assessment of the risk in cosmetic field, because its response under irritation stimuli is very quick and comparable to IL-1alpha.

Antioxidant status in J774A.1 macrophage cell line during chronic exposure to glycated serum.

Advanced glycation end-products (AGEs) are linked to aging and correlated diseases. The aim of present study was to evaluate oxidative stress related parameters in J774A.1 murine macrophage cells during chronic exposure to a subtoxic concentration of AGE (5% ribose-glycated serum (GS)) and subsequently for 48 h to a higher dose (10% GS). No effects on cell viability were evident in either experimental condition. During chronic treatment, glycative markers (free and bound pentosidine) increased significantly in intra- and extracellular environments, but the production and release of thiobarbituric acid reactive substances (TBARs), as an index of lipid peroxidation, underwent a time-dependent decrease. Exposure to 10% GS evidenced that glycative markers rose further, while TBARs elicited a cellular defence against oxidative stress. Nonadapted cultures showed an accumulation of AGEs, a marked oxidative stress, and a loss of viability. During 10% GS exposure, reduced glutathione levels in adapted cultures remained constant, as did the oxidized glutathione to reduced glutathione ratio, while nonadapted cells showed a markedly increased redox ratio. A constant increase of heat shock protein 70 (HSP70) mRNA was observed in all experimental conditions. On the contrary, HSP70 expression became undetectable for a longer exposure time; this could be due to the direct involvement of HSP70 in the refolding of damaged proteins. Our findings suggest an adaptive response of macrophages to subtoxic doses of AGE, which could constitute an important factor in the spread of damage to other cellular types during aging.