In vitro evaluation of toxic effects, bioavailability and bioaccesibility of beauvericin, enniatins and fusaproliferin = Evaluación in vitro de los efectos tóxicos, biodisponibilidad y bioacesibilidad de beuavericina, eniantinas y fusaproliferina

Los hongos de Fusarium pueden producir micotoxinas hexadepsipeptidicas, como beauvericina (BEA) y eniatinas (ENs) e isoprenoides como la fusaproliferina (FUS), las cuales se encuentran de forma natural en los alimentos y piensos. En las últimas décadas se han publicado datos, aunque escasos, sobre su toxicidad potencial en humanos y animales. Los objectivos de esta tesis fueron evaluar: los efectos citotóxicos de las FUS, BEA y ENs A, A1, B y B1 en células Caco-2, CHO-K1 y HT-29; los efectos citotóxicos de las ENs combinadas, la generación de especies reactivas de oxígeno (ROS) y la producción de peroxidación lipídica (LPO) tras exposición a FUS, BEA y ENs en las células Caco-2; la citoprotección del glutatión (GSH) y de polifenoles frente a la citotoxicidad de las ENs y BEA en las células Caco-2 y CHO-K1. Además, se determinó el efecto de la BEA y ENs sobre el ciclo celular, apoptosis/necrosis, la alteración de la membrana mitocondrial y efectos genotoxicos tras su exposición en células Caco-2; la biodisponibilidad de BEA y FUS mediante las células Caco-2 y la bioaccesibilidad de ENs A, A1, B y B1 a partir de cereales contaminados artificialmente. Los resultados demostraron que BEA y las ENs A, A1, B y B1 son citotóxicas dependiendo de la dosis y tiempo en las células Caco-2, HT-29 y CHO-K1. Las células CHO-K1 y Caco-2 fueron más sensibles a las ENs del grupo B y del grupo A, respectivamente. FUS no mostró efectos citotóxicos en el rango de concentraciones ensayadas. Se observó efecto sinérgico con las combinaciones de las ENs A1+B, A1+B1 y A+A1+B1 en las células Caco-2; mientras que la combinación de ENs B+B1 a bajas concentraciones mostraron efecto sinérgico. Se produjo estrés oxidativo en las células Caco-2 tras exponerlas a BEA y ENs en el siguiente orden: EN A1=EN A>EN B1>BEA>EN B. Como consecuencia del daño oxidativo se produjo LPO en el orden: BEA>EN A>EN A1=EN B1>EN B. FUS no produjo daño oxidativo. BEA redujo los niveles de GSH y aumentó los de glutatión oxidado (GSSG) en células Caco-2; mientras que los polifenoles mostraron efecto citoprotector en las células CHO-K1 (t-pterostilbeno>miricetina>rutina>quercetina 3-β-glucosido>quercetina) frente a la citotoxicidad inducida por las ENs. La BEA y ENs detuvieron el ciclo celular en la fase G2/M, lo cual se relaciona con el desequilibrio oxidativo inducido por la BEA y el daño al ADN inducido por las ENs A y A1 en las células Caco-2. Además, se observó muerte celular por apoptosis y necrosis, caracterizadas por alteración del potencial de membrana mitocondrial. Por otra parte, se observaron elevados porcentajes en la biodisponibilidad de BEA y FUS en las células Caco-2, siendo la FUS la más biodisponibles. La bioaccesibilidad de las ENs, mediante el modelo simulado de digestión in vitro, disminuyó siguiendo el orden: EN A=EN B1>EN B>EN A1. Los datos in vitro obtenidos en esta tesis pueden incorporarse a los datos de toxicidad de BEA, EN y FUS disponibles en la literatura y contribuir, junto a los datos in vivo, a su evaluación del riesgo.Fusarium spp. can produce hexadepsipeptidic mycotoxins, such as beauvericin (BEA) and enniatins (ENs) and the isoprenoidic fusaproliferin (FUS), which occurr naturally in food and feed. In the last decades they showed a potential toxicity to human and animal health, so far there are few data on their toxicity. The objectives of the study were to evaluate the cytotoxic effects of FUS, BEA and ENs A, A1 , B and B1 in Caco-2, CHO-K1 and HT-29 cells, the cytotoxic effects of ENs in combination, the generation of reactive oxygen species (ROS); the production of lipid peroxidation (LPO) after exposure to FUS, BEA and ENs in Caco-2 cells; the cytoprotection of glutathione (GSH) and some polyphenols against the cytotoxicity of ENs and BEA in the Caco-2 and CHO-K1 cells; the effect on the cell cycle, apoptosis/necrosis and mitochondrial membrane potential; the genotoxic effects after exposure to BEA and ENs in Caco-2 cells; BEA and FUS bioavailability by using Caco-2 cells and the bioavailability of ENs A, A1, B and B1 from artificially contaminated grain. BEA and ENs A, A1, B and B1 are cytotoxic in a dose and time-dependent manner in Caco-2, HT-29 and CHO-K1 cells. CHO-K1 and Caco-2 cells were more sensitive to the ENs in group B and group A, respectively. FUS had no cytotoxic effect in the concentrations tested. Synergistic effect was obtained after exposure to the combinations of ENs A1+B, A1+B1, A+A1+B1. Antagonism was produced by the combination of ENs B+B1 at low concentrations, in Caco-2 cells. Oxidative stress was observed after exposure to BEA and ENs in Caco-2 cells in the order: EN A1=EN A>EN B1>BEA>EN B. As a result of oxidative damage LPO occurred in the order: BEA>EN A>EN A1=EN B1>EN B. FUS did not produce any oxidative damage. BEA reduced GSH levels and increased oxidized glutathione (GSSG) levels in Caco-2 cells and polyphenols showed cytoprotective effect in CHO -K1 (t-pterostilbene > myricetin > rutin > quercetin 3-β-glucoside > quercetin) against ENs induced cytotoxicity. BEA and ENs arrested the cell cycle at G2/M phase, which is related to the oxidative imbalance induced by BEA and DNA damage induced by ENs A and A1 in Caco-2 cells. Apoptosis and necrosis were induced and characterized by the mitochondrial membrane potential alteration. High percentages of BEA and FUS bioavailability were determined through Caco-2 cells. FUS was more bioavailable than BEA. ENs bioaccessibility was reduced after applying the in vitro digestion method, in the order: EN A=EN B1>EN B>EN A1. In vitro data obtained in this research work can be incorporated into the whole data related to the cytotoxicity of BEA, EN and FUS in literature and, together with in vivo studies, could contribute to a future risk assessment of them

Baseline characteristics associated with NEDA-3 status in fingolimod-treated patients with relapsing-remitting multiple sclerosis

Abstract Background Fingolimod is an efficacious treatment for relapsing-remitting multiple sclerosis (RRMS) and there is class I evidence that it is superior to standard care in reducing relapse rate. However, real-world data investigating its effectiveness and potential predictors of response are still scarce. Objective To estimate (i) the proportion of fingolimod-treated patients who achieved the no evidence of disease activity (NEDA-3) status; and (ii) to determine which baseline (i.e. at treatment start) clinical and magnetic resonance imaging (MRI) variables were associated with better outcomes. Methods We collected clinical and MRI data of RRMS patients treated with fingolimod and followed-up for 24 months. The proportion of patients who had NEDA-3 - i.e. absence of relapses, sustained Expanded Disability Status Scale (EDSS) worsening and radiological activity on MRI - was estimated. A Cox proportional hazard model was carried out to investigate which baseline characteristics were associated with the NEDA status at follow-up. Results We collected data of 201 patients who started fingolimod. Of them, 24 (12%) were treatment-naïve, 115 (58%) were switched after failing a self-injectable drug, and 60 (30%) switching from natalizumab. Five patients who discontinued fingolimod early (within 3 months) (bradycardia, n = 2; leukopaenia, n = 2; macular oedema, n = 1) were removed from the analysis. At follow-up, 118 (60%) patients achieved the NEDA-3 status, while 78 experienced clinical and/or MRI activity. The risk of not achieving the NEDA-3 status was associated with higher baseline EDSS score (hazard ratio [HR] = 1.18, p = 0.024) and more relapses in the 12 months prior to fingolimod start (HR = 1.61, p = 0.014). Conclusion Our findings suggest that fingolimod may lead to a better control of the disease if started in patients with a less aggressive disease (i.e. fewer pre-treatment relapses and milder disability level), thus supporting its possible role as an early treatment for MS

The added value of spinal cord lesions to disability accrual in multiple sclerosis

Spinal cord MRI is not routinely performed for multiple sclerosis (MS) monitoring. Here, we explored whether spinal cord MRI activity offers any added value over brain MRI activity for clinical outcomes prediction in MS. This is a retrospective, monocentric study including 830 MS patients who underwent longitudinal brain and spinal cord MRI [median follow-up 7 years (range: < 1–26)]. According to the presence (or absence) of MRI activity defined as at least one new T2 lesion and/or gadolinium (Gd) enhancing lesion, each scan was classified as: (i) brain MRI negative/spinal cord MRI negative; (ii) brain MRI positive/spinal cord MRI negative; (iii) brain MRI negative/spinal cord MRI positive; (iv) brain MRI positive/spinal cord MRI positive. The relationship between such patterns and clinical outcomes was explored by multivariable regression models. When compared with the presence of brain MRI activity alone: (i) Gd + lesions in the spine alone and both in the brain and in the spinal cord were associated with an increased risk of concomitant relapses (OR = 4.1, 95% CI 2.4–7.1, p < 0.001 and OR = 4.9, 95% CI 4.6–9.1, p < 0.001, respectively); (ii) new T2 lesions at both locations were associated with an increased risk of disability worsening (HR = 1.4, 95% CI = 1.0–2.1, p = 0.05). Beyond the presence of brain MRI activity, new spinal cord lesions are associated with increased risk of both relapses and disability worsening. In addition, 16.1% of patients presented asymptomatic, isolated spinal cord activity (Gd + lesions). Monitoring MS with spinal cord MRI may allow a more accurate risk stratification and treatment optimization

Cost-Effectiveness of Dimethyl Fumarate Compared to Teriflunomide for Relapsing Remitting Multiple Sclerosis Patients in Italy

BACKGROUND: The objective of this economic analysis was to compare the cost-effectiveness of dimethyl fumarate vs teriflunomide for the treatment of adult patients with relapsing-remitting multiple sclerosis (RRMS) in the Italian setting. Additionally, the cost-effectiveness analysis was used to predict some patient-relevant outcomes such as burden of relapses and survival with disability over time.METHODS: A Markov model was used to conduct the cost-effectiveness analysis. The model measured health outcomes and costs of RRMS patients treated with either dimethyl fumarate or teriflunomide. Data from a published mixed treatment comparison were used for efficacy and safety input. Local economic data were used to calculate costs. A supplementary analysis was carried out to assess ICER variability over time from the Italian National Healthcare Service (NHS) and societal perspectives. Further analyses were conducted to compare clinical effectiveness of the alternatives over time, in terms of incidence of relapses, proportion of patients with EDSS (Expanded Disability Status Scale) score ≤3 and EDSS score ≥6.RESULTS: In the base-case analysis (lifetime horizon; societal perspective) dimethyl fumarate was dominant over teriflunomide (6.526 vs 5.953 QALYs – quality-adjusted life-years; € 1.01 M vs € 1.03 M). The most relevant cost savings (per-patient) with dimethyl fumarate were related to relapses (-€ 5,096), inpatient care (-€ 5,767), informal care (-€ 9,603), long-term absence/early retirement (-€ 14,187). The additional analysis of ICER by time horizon shows that dimethyl fumarate is cost-effective vs teriflunomide (i.e., ICER <€ 50,000 per QALY gained) at already 6 years and at 15 years in societal or NHS perspectives, respectively. Results favoured dimethyl fumarate vs teriflunomide also for: cumulative burden of relapses (-0.23 and -1.37 relapses saved per patient already at 1 year and 10 years, respectively), proportion of patients with mild disability (+4.0% at 10 years), proportion of patients with severe disability (-4.0% at 10 years).CONCLUSIONS: Dimethyl fumarate is dominant (societal perspective), or cost-effective (NHS perspective), referring to a threshold of € 50,000 per QALY gained, vs teriflunomide for the first-line treatment of RRMS, in the Italian setting

A Review of the Mycotoxin Enniatin B

Mycotoxin enniatin B (ENN B) is a secondary metabolism product by Fusarium fungi. It is a well-known antibacterial, antihelmintic, antifungal, herbicidal, and insecticidal compound. It has been found as a contaminant in several food commodities, particularly in cereal grains, co-occurring also with other mycotoxins. The primary mechanism of action of ENN B is mainly due to its ionophoric characteristics, but the exact mechanism is still unclear. In the last two decades, it has been a topic of great interest since its potent mammalian cytotoxic activity was demonstrated in several mammalian cell lines. Moreover, the co-exposure in vitro with other mycotoxins enhances its toxic potential through synergic effects, depending on the concentrations tested. Despite its clear cytotoxic effect, European Food Safety Authority stated that acute exposure to ENNs, such as ENN B, does not indicate concern for human health, but a concern might be the chronic exposure. However, given the lack of relevant toxicity data, no firm conclusion could be drawn and a risk assessment was not possible. In fact, very few studies have been carried out in vivo and, in these studies, no adverse effects were observed. So, research on toxicological effects induced by ENN B is still on-going. Recently, some studies are dealing with new advances regarding ENN B. This review summarizes the information on biochemical and biological activity of ENN B, focusing on toxicological aspects and on the latest advances in research on ENN B

The Prognostic Value of Pyrosequencing-Detected MGMT Promoter Hypermethylation in Newly Diagnosed Patients with Glioblastoma

O6-methylguanine-DNA-methyltransferase (MGMT) has emerged as a relevant predictor of therapeutic response and good prognosis in patients with glioblastoma (GBM). Transcriptionally active MGMT rapidly removes the alkyl adducts, preventing the formation of cross-links and thereby causing resistance to alkylating drugs. Studies with pyrosequencing (PSQ) showed that this technique has a higher reproducibility and sensitivity than other techniques. However, the definition of a prognostically relevant threshold for the percentage of MGMT methylation remains one of the most critical issues in the use of PSQ analysis. The aim of this study was to define the cut-off value correlated with good favourable prognostic outcomes. We retrospectively analyzed 51 patients (33 males, 18 females) with GBM who underwent surgery or biopsy. The Receiver Operating Characteristics analysis showed that the best possible criteria for PSQ-detected percentage of MGMT methylation that predicted progression-free survival (PFS) and overall survival (OS) were 19% and 13%, respectively. Patients with ≤19% of PSQ-detected MGMT had a shorter PFS (HR: 0.24, &lt; 0.01); those ones with ≤13% had a shorter OS (HR: 0.33, &lt; 0.05). Our study reinforces the importance of MGMT in the management of GBM patients, but future studies with larger sample sizes are warranted to confirm our findings

The still under-investigated role of cognitive deficits in PML diagnosis

Background: Despite cognitive deficits frequently represent the first clinical manifestations of Progressive Multifocal Leukoencephalopathy (PML) in Natalizumab-treated MS patients, the importance of cognitive deficits in PML diagnosis is still under-investigated. The aim of the current study is to investigate the cognitive deficits at PML diagnosis in a group of Italian patients with PML. Methods: Thirty-four PML patients were included in the study. The demographic and clinical data, the lesion load and localization, and the longitudinal clinical course was compared between patients with (n = 13) and without (n = 15) cognitive deficit upon PML suspicion (the remaining six patients were asymptomatic). Clinical presentation of cognitive symptoms was described in detail. Result: After symptoms detection, the time to diagnosis resulted to be shorter for patients presenting with cognitive than for patients with non cognitive onset (p = 0.03). Within patients with cognitive onset, six patients were presenting with language and/or reading difficulties (46.15%); five patients with memory difficulties (38.4%); three patients with apraxia (23.1%); two patients with disorientation (15.3%); two patients with neglect (15.3%); one patients with object agnosia (7.7%), one patient with perseveration (7.7%) and one patient with dementia (7.7%). Frontal lesions were less frequent (p = 0.03), whereas temporal lesions were slightly more frequent (p = 0.06) in patients with cognitive deficits. The longitudinal PML course seemed to be more severe in cognitive than in non cognitive patients (F = 2.73, p = 0.03), but differences disappeared (F = 1.24, p = 0.29) when balancing for the incidence of immune reconstitution syndrome and for other treatments for PML (steroids, plasma exchange (PLEX) and other therapies (Mefloquine, Mirtazapine, Maraviroc). Conclusion: Cognitive deficits at PML onset manifest with symptoms which are absolutely rare in MS. Their appearance in MS patients should strongly suggest PML. Clinicians should be sensitive to the importance of formal neuropsychological evaluation, with particular focus on executive function, which are not easily detected without a formal assessment

Predictors of lymphocyte count recovery after dimethyl fumarate-induced lymphopenia in people with multiple sclerosis

Background Dimethyl fumarate (DMF) is an oral drug approved for Relapsing Multiple Sclerosis (RMS) patients. Grade III lymphopenia is reported in 5\u201310% DMF-treated patients. Data on lymphocyte count (ALC) recovery after DMF withdrawal following prolonged lymphopenia are still scarce. Objectives To characterize ALC recovery and to identify predictors of slower recovery after DMF interruption. Methods Multicenter data from RMS patients who started DMF and developed lymphopenia during treatment were collected. In patients with grade II\u2013III lymphopenia, ALCs were evaluated from DMF withdrawal until reaching lymphocyte counts &gt; 800/mm3. Results Among 1034 patients who started DMF, we found 198 (19.1%) patients with lymphopenia and 65 patients (6.3%) who discontinued DMF due to persistent grade II\u2013III lymphopenia. Complete data were available for 51 patients. All patients recovered to ALC &gt; 800 cells/mm3 with a median time of 3.4 months. Lower ALCs at DMF suspension (HR 0.98; p = 0.005), longer disease duration (HR 1.29; p = 0.014) and prior exposure to MS treatments (HR 0.03; p = 0.025) were found predictive of delayed ALC recovery. Conclusion ALC recovery after DMF withdrawal is usually rapid, nevertheless it may require longer time in patients with lower ALC count at DMF interruption, longer disease duration and previous exposure to MS treatments, potentially leading to delayed initiation of a new therapy

COVID-19 Severity in Multiple Sclerosis: Putting Data Into Context

Background and objectives: It is unclear how multiple sclerosis (MS) affects the severity of COVID-19. The aim of this study is to compare COVID-19-related outcomes collected in an Italian cohort of patients with MS with the outcomes expected in the age- and sex-matched Italian population. Methods: Hospitalization, intensive care unit (ICU) admission, and death after COVID-19 diagnosis of 1,362 patients with MS were compared with the age- and sex-matched Italian population in a retrospective observational case-cohort study with population-based control. The observed vs the expected events were compared in the whole MS cohort and in different subgroups (higher risk: Expanded Disability Status Scale [EDSS] score &gt; 3 or at least 1 comorbidity, lower risk: EDSS score ≤ 3 and no comorbidities) by the χ2 test, and the risk excess was quantified by risk ratios (RRs). Results: The risk of severe events was about twice the risk in the age- and sex-matched Italian population: RR = 2.12 for hospitalization (p &lt; 0.001), RR = 2.19 for ICU admission (p &lt; 0.001), and RR = 2.43 for death (p &lt; 0.001). The excess of risk was confined to the higher-risk group (n = 553). In lower-risk patients (n = 809), the rate of events was close to that of the Italian age- and sex-matched population (RR = 1.12 for hospitalization, RR = 1.52 for ICU admission, and RR = 1.19 for death). In the lower-risk group, an increased hospitalization risk was detected in patients on anti-CD20 (RR = 3.03, p = 0.005), whereas a decrease was detected in patients on interferon (0 observed vs 4 expected events, p = 0.04). Discussion: Overall, the MS cohort had a risk of severe events that is twice the risk than the age- and sex-matched Italian population. This excess of risk is mainly explained by the EDSS score and comorbidities, whereas a residual increase of hospitalization risk was observed in patients on anti-CD20 therapies and a decrease in people on interferon