Unraveling the parahormetic mechanism underlying the health-protecting effects of grapeseed procyanidins.

Proanthocyanidins (PACs), the predominant constituents within Grape Seed Extract (GSE), are intricate compounds composed of interconnected flavan-3-ol units. Renowned for their health-affirming properties, PACs offer a shield against a spectrum of inflammation associated diseases, such as diabetes, obesity, degenerations and possibly cancer. While monomeric and dimeric PACs undergo some absorption within the gastrointestinal tract, their larger oligomeric and polymeric counterparts are not bioavailable. However, higher molecular weight PACs engage with the colonic microbiota, fostering the production of bioavailable metabolites that undergo metabolic processes, culminating in the emergence of bioactive agents capable of modulating physiological processes. Within this investigation, a GSE enriched with polymeric PACs was employed to explore in detail their impact. Through comprehensive analysis, the present study unequivocally verified the gastrointestinal-mediated transformation of medium to high molecular weight polymeric PACs, thereby establishing the bioaccessibility of a principal catabolite termed 5-(3',4'-dihydroxyphenyl)-γ-valerolactone (VL). Notably, our findings, encompassing cell biology, chemistry and proteomics, converge to the proposal of the notion of the capacity of VL to activate, upon oxidation to the corresponding quinone, the nuclear factor E2-related factor 2 (Nrf2) pathway-an intricate process that incites cellular defenses and mitigates stress-induced responses, such as a challenge brought by TNFα. This mechanistic paradigm seamlessly aligns with the concept of para-hormesis, ultimately orchestrating the resilience to stress and the preservation of cellular redox equilibrium and homeostasis as benchmarks of health.

Salivary proteomic profile of young healthy subjects.

Background: The incidence of noncommunicable diseases (NCDs) has been rapidly ramped up worldwide. Hence, there is an urgent need to non-invasively detect NCDs possibly by exploiting saliva as a 'liquid biopsy' to identify biomarkers of the health status. Since, the absence of standardized procedures of collection/analysis and the lack of normal ranges makes the use of saliva still tricky, our purpose was to outline a salivary proteomic profile which features healthy individuals. Methods: We collected saliva samples from 19 young blood donors as reference population and the proteomic profile was investigated through mass-spectrometry. Results: We identified 1,004 proteins of whose 243 proteins were shared by all subjects. By applying a data clustering approach, we found a set of six most representative proteins across all subjects including Coronin-1A, F-actin-capping protein subunit alpha, Immunoglobulin J chain, Prosaposin, 78 kDa glucose-regulated protein and Heat shock 70 kDa protein 1A and 1B. Conclusion: All of these proteins are involved in immune system activation, cellular stress responses, proliferation, and invasion thus suggesting their use as biomarkers in patients with NCDs.

Building confidence in skin sensitisation potency assessment using new approach methodologies: report of the 3rd EPAA Partners Forum, Brussels, 28th October 2019.

Skin sensitising substances that induce contact allergy and consequently risk elicitation of allergic contact dermatitis (ACD) remain an important focus regarding the replacement of animal experimentation. Current in vivo methods, notably the local lymph node assay (LLNA) refined and reduced animal usage and led to a marked improvement in hazard identification, characterisation and risk assessment. Since validation, regulatory confidence in the LLNA approach has evolved until it became the first choice assay in most regulated sectors. Currently, hazard identification using the LLNA is being actively replaced by a toolbox of non-animal approaches. However, there remains a need to increase confidence in the use of new approach methodologies (NAMs) as replacements for LLNA sensitiser potency estimation. The EPAA Partners Forum exchanged the current state of knowledge on use of NAMs in various industry sectors and regulatory environments. They then debated current challenges in this area and noted several ongoing needs. These included a requirement for reference standards for potency, better characterisation of applicability domains/technical limitations of NAMs, development of a framework for weight of evidence assessments, and an increased confidence in the characterisation of non-sensitisers. Finally, exploration of an industry/regulator cross-sector user-forum on skin sensitisation was recommended.

Co-administration of H-ferritin-doxorubicin and Trastuzumab in neoadjuvant setting improves efficacy and prevents cardiotoxicity in HER2 + murine breast cancer model.

Neoadjuvant chemotherapy has been established as the standard of care for HER2-positive breast cancer since it allows cancer down-staging, up to pathological complete response. The standard of care in the neoadjuvant setting for HER2-positive breast cancer is a combination of highly cytotoxic drugs such as anthracyclines and the anti-HER2 monoclonal antibody. Despite this cocktail allows a pathological complete response in up to 50%, their co-administration is strongly limited by intrinsic cardiotoxicity. Therefore, only a sequential administration of anthracyclines and the anti-HER2 treatment is allowed. Here, we propose the anthracycline formulation in H-Ferritin nanocages as promising candidate to solve this unmet clinical need, thanks to its capability to increase anthracyclines efficacy while reducing their cardiotoxicity. Treating a murine model of HER2-positive breast cancer with co-administration of Trastuzumab and H-Ferritin anthracycline nanoformulation, we demonstrate an improved tumor penetration of drugs, leading to increased anticancer efficacy and reduced of cardiotoxicity.

In vitro RHE skin sensitisation assays: Applicability to challenging substances.

In the last 20 years, alternative approaches to the identification of skin sensitisation hazards have been at the forefront of the 3Rs and have helped refine the validation and acceptance processes. However, experience with the local lymph node assay showed that, post-validation, challenges still occurred, particularly when a wider diversity of chemical substances was addressed, a situation which will arise with validated in vitro alternatives. In the present work, a range of substances potentially challenging to assess in current nonanimal OECD test guidelines were evaluated in several of the emerging in vitro alternatives. Twelve such substances (of which just over half were known skin sensitisers) were assessed in 4 assays, all based on reconstructed human epidermis (RHE) models. For hazard identification, the overall predictive accuracy ranged around 70% for three assays, although for one (SensCeeTox), it fell below 50% when human data was used as the benchmark. In most cases, sensitivity was high, such that sensitisation was overpredicted. As the substances were challenging to assess in other nonanimal methods, the results indicate that the 3D RHE models may be a useful tool for assessing skin sensitisation potentials without needing to revert to animal use.

Standardisation of defined approaches for skin sensitisation testing to support regulatory use and international adoption: position of the International Cooperation on Alternative Test Methods.

Skin sensitisation is the regulatory endpoint that has been at the centre of concerted efforts to replace animal testing in recent years, as demonstrated by the Organisation for Economic Co-operation and Development (OECD) adoption of five non-animal methods addressing mechanisms under the first three key events of the skin sensitisation adverse outcome pathway. Nevertheless, the currently adopted methods, when used in isolation, are not sufficient to fulfil regulatory requirements on the skin sensitisation potential and potency of chemicals comparable to that provided by the regulatory animal tests. For this reason, a number of defined approaches integrating data from these methods with other relevant information have been proposed and documented by the OECD. With the aim to further enhance regulatory consideration and adoption of defined approaches, the European Union Reference Laboratory for Alternatives to Animal testing in collaboration with the International Cooperation on Alternative Test Methods hosted, on 4-5 October 2016, a workshop on the international regulatory applicability and acceptance of alternative non-animal approaches, i.e., defined approaches, to skin sensitisation assessment of chemicals used in a variety of sectors. The workshop convened representatives from more than 20 regulatory authorities from the European Union, United States, Canada, Japan, South Korea, Brazil and China. There was a general consensus among the workshop participants that to maximise global regulatory acceptance of data generated with defined approaches, international harmonisation and standardisation are needed. Potential assessment criteria were defined for a systematic evaluation of existing defined approaches that would facilitate their translation into international standards, e.g., into a performance-based Test Guideline. Informed by the discussions at the workshop, the ICATM members propose practical ways to further promote the regulatory use and facilitate adoption of defined approaches for skin sensitisation assessments.

Consensus of classification trees for skin sensitisation hazard prediction.

Since March 2013, it is no longer possible to market in the European Union (EU) cosmetics containing new ingredients tested on animals. Although several in silico alternatives are available and achievements have been made in the development and regulatory adoption of skin sensitisation non-animal tests, there is not yet a generally accepted approach for skin sensitisation assessment that would fully substitute the need for animal testing. The aim of this work was to build a defined approach (i.e. a predictive model based on readouts from various information sources that uses a fixed procedure for generating a prediction) for skin sensitisation hazard prediction (sensitiser/non-sensitiser) using Local Lymph Node Assay (LLNA) results as reference classifications. To derive the model, we built a dataset with high quality data from in chemico (DPRA) and in vitro (KeratinoSens™ and h-CLAT) methods, and it was complemented with predictions from several software packages. The modelling exercise showed that skin sensitisation hazard was better predicted by classification trees based on in silico predictions. The defined approach consists of a consensus of two classification trees that are based on descriptors that account for protein reactivity and structural features. The model showed an accuracy of 0.93, sensitivity of 0.98, and specificity of 0.85 for 269 chemicals. In addition, the defined approach provides a measure of confidence associated to the prediction.

An international trial of quantitative PCR for monitoring Legionella in artificial water systems.

AIMS: To perform an international trial to derive alert and action levels for the use of quantitative PCR (qPCR) in the monitoring of Legionella to determine the effectiveness of control measures against legionellae. METHODS AND RESULTS: Laboratories (7) participated from six countries. Legionellae were determined by culture and qPCR methods with comparable detection limits. Systems were monitored over ≥10 weeks. For cooling towers (232 samples), there was a significant difference between the log mean difference between qPCR (GU l(-1) ) and culture (CFU l(-1) ) for Legionella pneumophila (0·71) and for Legionella spp. (2·03). In hot and cold water (506 samples), the differences were less, 0·62 for Leg. pneumophila and 1·05 for Legionella spp. Results for individual systems depended on the nature of the system and its treatment. In cooling towers, Legionella spp. GU l(-1) always exceeded CFU l(-1) , and usually Legionella spp. were detected by qPCR when absent by culture. The pattern of results by qPCR for Leg. pneumophila followed the culture trend. In hot and cold water, culture and qPCR gave similar results, particularly for Leg. pneumophila. There were some marked exceptions with temperatures ≥50°C, or in the presence of supplementary biocides. Action and alert levels for qPCR were derived that gave results comparable to the application of the European Guidelines based on culture. Algorithms are proposed for the use of qPCR for routine monitoring. CONCLUSIONS: Action and alert levels for qPCR can be adjusted to ensure public health is protected with the benefit that remedial actions can be validated earlier with only a small increase in the frequency of action being required. SIGNIFICANCE AND IMPACT OF THE STUDY: This study confirms it is possible to derive guidelines on the use of qPCR for monitoring the control of legionellae with consequent improvement to response and public health protection.

Compost facilities as a reservoir of Legionella pneumophila and other Legionella species.

Data on the presence of Legionellae outside the aquatic environment are scarce. Alternative ecosystems that could act as a reservoir for Legionella spp. have been investigated to identify unconventional contaminated substrates that are able to produce bioaerosols. We considered eight green waste collection sites including three composting facilities. Legionella pneumophila sg 1-15, Legionella bozemanii, Legionella cincinnatiensis, Legionella jamestowniensis, Legionella micdadei and L. oakridgensis were isolated from samples taken at six of the eight sites. The degree of contamination ranged from 10(3) to 10(8) CFU/g. Compost facilities appear to comprise an important reservoir for Legionellae.

An investigation on the Culicoides species composition at seven sites in southern Switzerland.

In the past decade, there have been regular outbreaks of bluetongue (BT) in many parts of Europe. Owing to the presence of BT disease and its vectors in countries adjacent to Switzerland, an initial entomological survey was conducted in 2003, which established the presence of several midges of the genus Culicoides (Diptera: Ceratopogonidae). Subsequently, a sentinel herd monitoring system was established with the primary entomological aim being the determination and further study of Culicoides population compositions. Insects were collected in 2005 and 2006 at seven sentinel herd sites in the south of Switzerland (canton of Ticino) near the border of Italy, using Onderstepoort-type light traps. This region is botanically and zoologically similar to the Mediterranean and is one of the warmest and most humid areas of the country, hence it is considered a potential access path for BT disease into Switzerland. Collections were made at four cattle farms, two equestrian centres and one goat farm. Sites were sampled four times per month from June to October. Traps were operated from dusk until dawn and samples were collected monthly for analysis through microscopy as well as a Culicoides imicola-specific PCR. Results confirmed the absence of C. imicola (Kieffer) and demonstrated that the potential BT virus vectors are highly abundant, notably: Culicoides obsoletus (Meigen), Culicoides scoticus (Downes & Kettle) and Culicoides dewulfi (Goetghebuer) subgenus Avaritia and Culicoides pulicaris (Linnaeus) subgenus Culicoides. These findings expand the current knowledge of Culicoides population composition in the southern part of the Switzerland. Culicoides cataneii (Clastrier), Culicoides flavipulicaris (Dzhafarov), Culicoides indistinctus (Khalaf), Culicoides nubeculosus (Meigen) and species of the Grisescens complex were reported for the first time in Switzerland.

Commercial potting soils as an alternative infection source of Legionella pneumophila and other Legionella species in Switzerland.

Legionella spp. are pathogens that can cause Legionnaires' disease in humans through inhalation of contaminated aerosols. The principal reservoir for these microorganisms is water, but Legionella spp. have been isolated from composted vegetable and plant material, and from many potting mixes as well. In Australia, there have been several cases of Legionnaires' disease in which Legionella longbeachae has been isolated from potting soils. In Switzerland, the source of infection cannot always be identified as water or cooling towers: therefore, we have investigated 46 commercially available potting soils in Switzerland to determine the presence of Legionella spp. We were able to detect Legionella spp. in 45.7% (21/46) of the potting soil samples analysed by culture. Legionella pneumophila was present in 19.6% (9/46) of the samples and L. pneumophila serogroup 1 in 6.5% (3/46). Quantification by both culture and quantitative real-time PCR revealed high concentrations of legionellae in potting soils, ranging between 10(3) CFU/g and 10(5) CFU/g and 10(4) genomic units (GU)/g and 10(6) GU/g, respectively. Thus, potting soils may represent an alternative reservoir for Legionella spp. in Switzerland.

Is fasting glucose variability a risk factor for retinopathy in people with type 2 diabetes?

AIMS: Fasting plasma glucose variability strongly predicts the incidence of cardiovascular events in type 2 diabetic patients. We prospectively assessed whether fasting plasma glucose variability predicts the development/progression of retinopathy in a large cohort of type 2 diabetic outpatients. METHODS: In the period 1996-1999, 1019 type 2 diabetic participants (aged 69+/-11 years) in the Verona Diabetes Study underwent at least 3 fasting plasma glucose (FPG) determinations and an eye examination by retinography. Of these, 746 underwent a 2nd eye examination in the period 2000-2004, while 273 did not (102 patients had died before undergoing the 2nd eye examination). For each patient, the mean (M-FPG) and the coefficient of variation of FPG (CV-FPG) were computed. RESULTS: By the 2nd eye examination, 124 patients had either developed new retinopathy (79 patients) or progressed to a more severe degree of retinopathy (45 patients). In a multivariable logistic regression analysis, the development/progression of retinopathy was independently predicted by average glycaemia over time, expressed as glycated haemoglobin (odds ratio [OR] 1.82, 95%CI 1.40-2.38 for 1 SD increase) or M-FPG (OR 1.88, 1.47-2.41), but not by CV-FPG. Among other independent variables, HDL-cholesterol was inversely associated with the development/progression of retinopathy. CONCLUSIONS: These results suggest that in elderly type 2 diabetic patients the magnitude of hyperglycaemia, but not fasting plasma glucose variability, strongly predicts the development/progression of diabetic retinopathy independently of other known risk factors.

Application of human CFU-Mk assay to predict potential thrombocytotoxicity of drugs.

Megakaryocytopoiesis gives rise to platelets by proliferation and differentiation of lineage-specific progenitors, identified in vitro as Colony Forming Unit-Megakaryocytes (CFU-Mk). The aim of this study was to refine and optimize the in vitro Standard Operating Procedure (SOP) of the CFU-Mk assay for detecting drug-induced thrombocytopenia and to prevalidate a model for predicting the acute exposure levels that cause maximum tolerated decreases in the platelets count, based on the correlation with the maximal plasma concentrations (C max) in vivo. The assay was linear under the SOP conditions, and the in vitro endpoints (percentage of colonies growing) were reproducible within and across laboratories. The protocol performance phase was carried out testing 10 drugs (selected on the base of their recognised or potential in vivo haematotoxicity, according to the literature). Results showed that a relationship can be established between the maximal concentration in plasma (C max) and the in vitro concentrations that inhibited the 10-50-90 percent of colonies growth (ICs). When C max is lower than IC10, it is possible to predict that the chemicals have no direct toxicity effect on CFU-Mk and could not induce thrombocytopenia due to bone marrow damage. When the C max is higher than IC90 and/or IC50, thrombocytopenia can occur due to direct toxicity of chemicals on CFU-Mk progenitors.

Assessment of intraspecific mtDNA variability of European Ixodes ricinus sensu stricto (Acari: Ixodidae).

The Ixodes ricinus complex is composed of 14 species distributed worldwide. Some members of this complex are involved in the transmission of a number of diseases to animals and humans, in particular Lyme borreliosis, tick-borne encephalitis, ehrlichiosis and babesiosis. While the phylogenetic relationships between species of the I. ricinus complex have been investigated in the past, still little is known about the genetic structure within the species I. ricinus sensu stricto. We have investigated the intraspecific variability among 26 I. ricinus s.s. ticks collected in various European countries, including Switzerland, Italy, Austria, Denmark, Sweden, and Finland by using five mitochondrial gene fragments corresponding to the control region, 12S rDNA, cytb, COI, and COII. The five genes considered here showed a low genetic variability (1.6-5%). Our results based on both statistical parsimony (applied to the COI + COII + cytb + 12S + CR data set, for a total of 3423 bp) and maximum parsimony (applied to the COI + COII + cytb + 12S data set, for a total of 2980 bp) did not provide any evidence for a correlation between the identified haplotypes and their geographic origin. Thus, the European I. ricinus s.s. ticks do not seem to show any phylogeography structure.

Mitochondrial function, apoptosis and cell cycle delay in the WEHI-3B leukaemia cell line and its variant Ciprofloxacin-resistant WEHI-3B/CPX.

OBJECTIVE: The susceptibility of two cell lines, WEHI-3B myelomonocytic leukaemia and its variant Ciprofloxacin-resistant WEHI-3B/CPX to undergo apoptosis induced by Ciprofloxacin was studied and compared. MATERIALS AND METHODS: Apoptosis was checked by measuring the DNA fragmentation and determining the ratio of apoptotic/necrotic cells. The relationship between the induction of apoptosis and G(1), S or G(2) block in the cell cycle has also been investigated and cytogenetical evaluation of chromosomal aberrations in both cell lines has been carried out. The regulation of expression of Bax and Bcl-2 was also checked by western blotting after Ciprofloxacin treatment. RESULTS: We observed that the resistance of the subline was caused by a small percentage of cells that underwent apoptosis during continuous exposure to Ciprofloxacin in comparison with the parental cell line, whereas the percentage of necrotic cells remained unchanged. The WEHI-3B cells showed a G(2) block and a higher degree of cytogenetic damage after drug exposure. The two cell lines expressed the same level of Bax and Bcl-2 following stimulation by Ciprofloxacin. Only in the resistant subclone, the ratio Bcl-2/Bax reversed in the anti-apoptotic gene expression. CONCLUSION: The resistance to ciprofloxacin observed is not related to mitochondrial function and although Bcl-2/Bax ratio behaviour does not fully explain the resistance of the WEHI3B/CPX subclone it is consistent with phenotypic character of resistance to CPX. The toxic effect on sensitive cells could be mediated by the cell cycle arrest whereas in the resistant clone, the prolonged G(2) phase could play a key role to favour cell cycle progression and proliferation.

Complete (1)H and (13)C NMR spectral assignment of alpha- and beta-adenosine, 2'-deoxyadenosine and their acetate derivatives.

(1)H and (13)C NMR chemical shifts of alpha- and beta-anomers of adenosine, 2'-deoxyadenosine and their acetate derivatives were completely and definitely assigned using the concerted application of one- and two-dimensional experiments (gCOSY, gNOESY, gHSQC and gHMBC). The influence of the stereochemistry of the purine base on the NMR data of the hydrogen and carbon atoms of the furanose moiety was estimated.

Stakeholder appraisal of policy options for responding to obesity in Italy.

This article deals with the phenomenon of obesity in contemporary Italian society. It is based on the fieldwork carried out during the year 2005 within the European Union-funded project 'PorGrow: policy options for responding to obesity' (see: http://www.sussex.ac.uk/spru/PorGrow). The most recent statistical data on the spread of overweight and obesity in Italian society reveal that the phenomenon has reached a dimension that, albeit not as serious as in other Western countries, constitutes a serious threat to public health and to the national budget. The panel of stakeholders interviewed for this research showed awareness of this issue and generally agreed on the necessity to adopt a very multifaceted portfolio of policy measures to address the problem. Participants frequently regarded educational policies as the highest priority, followed by informational measures and infrastructural actions. Fiscal policy options were widely considered ineffective and unacceptable, and little enthusiasm was shown for technological innovations. In sum, while interviewees saw a real need for improved food habits on the part of Italian citizens, nonetheless in a country affected by many criticalities, the weakness of political will and the pressure of fast food culture remain severe obstacles in the fight against overweight and obesity.

[Autochthonous cases of canine babesiosis in the canton Solothurn]. / Autochthone Fälle von caniner Babesiose im Kanton Solothurn.

Starting in November 2003 a series of five clinical cases of canine babesiosis was registered in the region of Obergösgen (canton Solothurn). All presented dogs showed increased body temperature, thrombocytopenia and hemoglobinuria, and none of the dogs had been abroad or visited endemic regions in the southern or western part of Switzerland so far. Babesia canis was detected in the blood smears of all five patients, but only three had detectable specific antibodies against this parasite. However, seroconversion was found in a second sample collected from the negative dogs at a later timepoint, confirming the diagnosis of canine babesiosis. The blood samples of two parasitized dogs were used for DNA-isolation and were tested with a Babesia-specific PCR, detecting the 18S rRNA-gene. Sequencing of the amplified products revealed a 100% identity with the sub-species B. canis canis. The ticks Rhipicephalus sanguineus and Dermacentor marginatus are potential vectors for B. canis. In the area where the infection with B. canis was suspected a total of 152 ticks was collected and characterized; one was a female R. sanguineus.Although babesia could not be detected in the latter tick and the final prooffor the complete life cycle is still lacking, it is very probable that B. canis has become autochthonous in the canton Solothurn.

Inhibition of CFU-E/BFU-E and CFU-GM colony growth by cyclophosphamide, 5-fluorouracil and taxol: development of a high-throughput in vitro method.

The major side effect of anticancer drugs is damage to the hematopoietic system. These compounds may interfere with cell proliferation and differentiation in different blood lineages causing many diseases such as neutropenia, aplastic anaemia or trombocytopenia. The clonogenic assays are useful in vitro tools for evaluating and predicting acute xenobiotics myelotoxicity. A miniaturisation of these assays, in order to reduce costs and increase the number of compounds that could be tested, is under investigation. The in vitro sensitivity of human burst-forming unit erythroid (BFU-E) and colony-forming unit granulocyte-macrophage (CFU-GM) to three anticancer drugs: cyclophosphamide (CTX), 5-fluorouracil (5-FU) and taxol (TAX) was evaluated both in 35 mm plate and 96-well plate systems and the dose-response curves, IC50 values and IC90 values were compared. The correlation between in vitro data and clinical plasma levels confirms that severe hematotoxicity is the primary adverse effect of these drugs with an evident selectivity on erythroid progenitors for cyclophosphamide. IC50 and IC90 values, calculated on the basis of results obtained with the traditional assay, correlate with those obtained in microplate, as well as the dose-response curves, indicating that the 96 well plate assay could be a useful and reliable tool for high-throughput screening in early stages of drug development.

Complete 1H and 13C NMR spectral assignment of 17-hydroxy epimeric sterols with planar A or A and B rings.

Complete 1H and 13C spectral assignments of 17beta- and 17alpha-hydroxy epimers of three biologically active sterols (boldenone, 3-methoxyestradiol and 3-methoxydihydroequilenin) were achieved making use of one- and two-dimensional NMR techniques (1D-HOHAHA, DEPT, COSY, NOESY, TOCSY, HSQC and COLOC).