Everolimus Nanoformulation in Biological Nanoparticles Increases Drug Responsiveness in Resistant and Low-Responsive Breast Cancer Cell Lines

Everolimus (Eve) is an FDA approved drug that inhibits mammalian target of rapamycin (mTOR). It is employed in breast cancer treatment even if its responsiveness is controversial. In an attempt to increase Eve effectiveness, we have developed a novel Eve nanoformulation exploiting H-ferritin nanocages (HEve) to improve its subcellular delivery. We took advantage of the natural tumor targeting of H-Ferritin, which is mediated by the transferrin receptor-1 (TfR1). Breast cancer cells overexpressing TfR-1 were successfully recognized by H-Ferritin, displaying quick nanocage internalization. HEve has been tested and compared to Eve for in vitro efficacy in sensitive and resistant breast cancer cells. Nanoformulated Eve induced remarkable antiproliferative activity in vitro, making even resistant cell lines sensitive to Eve. Moreover, the antiproliferative activity of HEve is fully in accordance with cytotoxicity observed by cell death assay. Furthermore, the significant increase in anticancer efficacy displayed in HEve-treated samples is due to the improved drug accumulation, as demonstrated by UHPLC-MS/MS quantifications. Our findings suggest that optimizing Eve subcellular delivery, thanks to nanoformulation, determines its improved antitumor activity in a panel of Eve-sensitive or resistant breast cancer cell lines

Validation of the italian version of the Cluster Headache Impact Questionnaire (CHIQ)

Background: The Cluster Headache Impact Questionnaire (CHIQ) is a specific and easy-to-use questionnaire to assess the current impact of cluster headache (CH). The aim of this study was to validate the Italian version of the CHIQ. Methods: We included patients diagnosed with episodic CH (eCH) or chronic CH (cCH) according to the ICHD-3 criteria and included in the “Italian Headache Registry” (RICe). The questionnaire was administered to patients through an electronic form in two sessions: at first visit for validation, and after 7 days for test-retest reliability. For internal consistency, Cronbach’s alpha was calculated. Convergent validity of the CHIQ with CH features and the results of questionnaires assessing anxiety, depression, stress, and quality of life was evaluated using Spearman’s correlation coefficient. Results: We included 181 patients subdivided in 96 patients with active eCH, 14 with cCH, and 71 with eCH in remission. The 110 patients with either active eCH or cCH were included in the validation cohort; only 24 patients with CH were characterized by a stable attack frequency after 7 days, and were included in the test-retest cohort. Internal consistency of the CHIQ was good with a Cronbach alpha value of 0.891. The CHIQ score showed a significant positive correlation with anxiety, depression, and stress scores, while showing a significant negative correlation with quality-of-life scale scores. Conclusion: Our data show the validity of the Italian version of the CHIQ, which represents a suitable tool for evaluating the social and psychological impact of CH in clinical practice and research

Liquid-liquid extraction procedure for trace determination of cyclophosphamide in human urine by high-performance liquid chromatography tandem mass spectrometry

A sensitive, specific and accurate high performance liquid chromatography/ionspray-tandem mass spectrometry procedure (HPLC/MS/MS) has been developed to quantify cyclophosphamide in human urine from hospital personnel involved in drug preparation and administration of antineoplastic alkylating agents. This methodology, which includes liquid-liquid extraction with ethylacetate, requires no derivatization procedures, preventing cyclophosphamide (CP) from possible thermal and chemical decomposition reactions. We detected the excretion of this unmetabolized alkylating drug in 50% of all the study participants. The amount of CP ranged from 0.1 ng microL-1 to 1.9 ng microL-1 urine. This methodology was validated by the use of ifosfamide as internal standard. The assay was linear over the range 0 to 3.2 ng microL-1 urine, with a lower limit of quantification of 0.2 microL-1. The limit of detection was assessed at 0.05 ng microL-1 urine. This method is characterized by a coefficient of variation < 10%. Standard calibration curves, obtained on three different days, had correlation coefficients always greater than 0.998. The intra and interday precision were within 11%, and accuracy was in the range 99-103%. The mean extracted recovery assessed at three different concentrations (0.5, 0.8, 3.2 ng microL-1) was always more than 85%. The extraction efficiency of cyclophosphamide from urine samples was also studied at six different pH values (pH 4, 5, 6, 7, 8, 10). The maximum extraction efficiency was obtained when the pH of urine solutions was adjusted to 7.

Occupational exposure to antineoplastic drugs in four Italian health care settings.

Exposure assessment of health care workers to antineoplastic drugs (ADs) is still an open issue since new, critical, and emerging factors may put pharmacists who prepare hazardous drugs or nurses who administer anti cancer agents to an increased risk of developing adverse health effects. Overall, eight pharmacies and nine patient areas have been surveyed in this study. Wipe and pad samples were experienced during the surveillance program in four Italian health care settings. Urine samples were collected from workers handling ADs. Cyclophosphamide (CP), ifosfamide (IF), and gemcitabine (GEM) were detected in all the work environments by using a LC-MS/MS method-based capable of analysing all the three drugs simultaneously. In total, 54% of wipe samples were positive for at least one drug and 19% of pad samples were shown to be contaminated by cyclophosphamide. Pharmacies were generally more contaminated than patient areas with the exception of one site where a nurse had an acute exposure during the cleaning-up of an hazardous drug solution spill. In total, 22 urine samples collected from pharmacists and 78 urine samples from nurses had no detectable concentrations of any antineoplastic drugs. Despite the adherence to the recommended safety practices residue contamination on surfaces and floors has continued to be assessed in all the investigated sites

Exposure to Antineoplastic Drugs in Occupational Settings: A Systematic Review of Biological Monitoring Data

The high toxicity of antineoplastic drugs (ADs) makes them dangerous not only for patients, but also for exposed workers. Therefore, the aim of this review was to provide an updated overview of the biological monitoring of occupational AD exposure in order to extrapolate information useful to improve risk assessment and management strategies in workplaces. Several studies demonstrated that remarkable portions of healthcare workers may have traces of these substances or their metabolites in biological fluids, although with some conflicting results. Nurses, directly engaged in AD handling, were the occupational category at higher risk of contamination, although, in some cases, personnel not involved in AD-related tasks also showed quantifiable internal doses. Overall, further research carried out on greater sample sizes appears necessary to gain deeper insight into the variability retrieved in the reported results. This may be important to understand the impact of the extent of ADs use, different handling, procedures, and cleaning practices, spill occurrence, training of the workforce, as well as the adoption of adequate collective and personal protective equipment in affecting the occupational exposure levels. This may support the achievement of the greatest clinical efficiency of such therapies while assuring the health and safety of involved workers

The Occurrence Birth-Death Process for combined-evidence analysis in macroevolution and epidemiology

Phylodynamic models generally aim at jointly inferring phylogenetic relationships, model parameters, and more recently, the number of lineages through time, based on molecular sequence data. In the fields of epidemiology and macroevolution these models can be used to estimate, respectively, the past number of infected individuals (prevalence) or the past number of species (paleodiversity) through time. Recent years have seen the development of “total-evidence” analyses, which combine molecular and morphological data from extant and past sampled individuals in a unified Bayesian inference framework. Even sampled individuals characterized only by their sampling time, i.e. lacking morphological and molecular data, which we call occurrences, provide invaluable information to estimate the past number of lineages. Here, we present new methodological developments around the Fossilized Birth-Death Process enabling us to (i) incorporate occurrence data in the likelihood function; (ii) consider piecewise-constant birth, death and sampling rates; and (iii) estimate the past number of lineages, with or without knowledge of the underlying tree. We implement our method in the RevBayes software environment, enabling its use along with a large set of models of molecular and morphological evolution, and validate the inference workflow using simulations under a wide range of conditions. We finally illustrate our new implementation using two empirical datasets stemming from the fields of epidemiology and macroevolution. In epidemiology, we infer the prevalence of the COVID-19 outbreak on the Diamond Princess ship, by taking into account jointly the case count record (occurrences) along with viral sequences for a fraction of infected individuals. In macroevolution, we infer the diversity trajectory of cetaceans using molecular and morphological data from extant taxa, morphological data from fossils, as well as numerous fossil occurrences. The joint modeling of occurrences and trees holds the promise to further bridge the gap between between traditional epidemiology and pathogen genomics, as well as paleontology and molecular phylogenetics

Resposta de anticorpos IgE, IgG1 e IgG4 aos componentes ligantes de Concanavalina A isolados de Blomia tropicalis (Acari: Echimyopodidae) em indivíduos alérgicos e não alérgicos

Blomia tropicalis (Bt) and Dermatophagoides pteronyssinus (Dp) are the most prevalent house dust mites in tropical countries and associated with allergic diseases. Glycosilated antigens are highly immunogenic and involved in different pathologies, including allergies. The aims of this study were to evaluate IgE, IgG1, and IgG4 responses to Concanavalin A-binding components (Bt-ConA) isolated from Bt extract in sera of allergic and non-allergic subjects and to analyze the crossreactivity with Dp extract. Also, IgE-, IgG1- and IgG4-reactive antigenic components from both extracts were analyzed by Immunoblotting. Bt-ConA was obtained from Btwhole extract fractionated on Con-A-Sepharose affinity chromatography and both extracts were evaluated in SDS-PAGE and ELISA for IgE, IgG1, and IgG4 in sera of 121 patients with allergic rhinitis and 36 non-allergic subjects. Subjects were skin prick tested (SPT) to Bt-whole extract. Inhibition and immunoblotting test were performed to analyze IgE, IgG1, and IgG4 responses to both extracts. SPT showed that 58% of patients were sensitized to Bt (Bt+), with 52% reactive to both mites (Bt and Dp) and 6% to Bt only. A broad spectrum of proteins (14-152kDa) were visualized in Bt-whole extract and components >27kDa in Bt-ConA extract. ELISA showed a similar profile of IgE, IgG1 and IgG4 levels to Bt-whole and Bt-ConA extracts in different groups, although Bt+ patients showed a lower IgG4 reactivity to Bt-ConA extract. Specific IgG1 levels were higher in Bt+ patients than control subjects, and IgG4 levels showed no significant difference among the groups. ELISA inhibition showed a partial IgE and total IgG1 and IgG4 cross-reactivity with Dp extract for Bt-whole and Bt-ConA extracts. Immunoblotting revealed ten antigenic components in Bt-whole extract (14-152kDa) recognized by IgE and IgG4 antibodies and five of these components (>50kDa) were recognized by IgG1 in Bt+ patients. Bt- ConA extract showed eight antigenic components (27-152kDa), from which the 152 and 123kDa bands were predominantly recognized by IgE, and only three of these components (93, 123 and 152kDa) were recognized by IgG1 while IgG4 antibodies weakly recognized the 66 and 152kDa components in Bt+ patients. It can be concluded that Con A-binding components isolated from Bt constitute major allergens and are involved in both allergen sensitization (IgE response) and homeostase maintenance (IgG1 and IgG4 responses) and that glycosilated components, particularly those of high molecular weight, are preferentially recognized by IgE and IgG1, but not by IgG4 antibodies.Mestre em Ciências da SaúdeBlomia tropicalis (Bt) e Dermatophagoides pteronyssinus (Dp) são os principais ácaros da poeira domiciliar em países tropicais e subtropicais que estão associados com doenças alérgicas. Antígenos glicosilados são altamente imunogênicos e estão envolvidos em diferentes patologias, incluindo alergias. Os objetivos deste estudo foram avaliar a resposta de anticorpos IgE, IgG1 e IgG4 a componentes ligantes de Concanavalina A (Bt-ConA) isolados do extrato Bt total em soros de indivíduos alérgicos e não alérgicos, bem como a reatividade cruzada com Dp e identificar por immunoblotting componentes protéicos de ambos extratos reconhecidos por anticorpos IgE, IgG1 e IgG4. O extrato Bt-ConA foi obtido por fracionamento do extrato Bt total em cromatografia de afinidade Con A-Sepharose e ambos extratos foram avaliados em SDS-PAGE e ELISA para IgE, IgG1 e IgG4 em 121 soros de pacientes com rinite alérgica e 36 indivíduos não alérgicos. Testes cutâneos de puntura (TCP) foram realizados em todos os indivíduos do estudo. Ensaios de inibição foram realizados para detecção de IgE, IgG1 e IgG4 específica a Bt total e Bt-ConA. Ensaios de immunoblotting foram realizados para detecção de componentes antigênicos de ambos extratos reconhecidos por IgE, IgG1 e IgG4. Do total de 121 pacientes, 58% mostraram-se sensibilizados a Bt (Bt+), com 52% apresentando reatividade cutânea a ambos ácaros (Bt e Dp) e apenas 6% somente a Bt. Um largo espectro de proteínas (14-152kDa) foram visualizadas no extrato Bt total e componentes acima de 27kDa na fração Bt-ConA. ELISA apresentou um perfil similar nos níveis de IgE, IgG1 e IgG4 para os extratos Bt total e Bt-ConA em diferentes grupos, embora pacientes do grupo Bt+ demonstraram uma reatividade menor de IgG4 para o extrato Bt-ConA. Níveis de IgG1 específica foram maiores nos pacientes Bt+ do que nos indivíduos controles e os níveis de IgG4 não apresentaram diferença significativa entre os grupos. ELISA de inibição demonstrou uma reatividade cruzada parcial com o extrato de Dp para IgE específica e total para IgG1 e IgG4 específicas aos extratos de Bt total e Bt-ConA. Immunoblotting revelou dez componentes antigênicos do extrato Bt total (14-152kDa) reconhecidos por anticorpos IgE e IgG4 e cinco destes componentes (>50kDa) foram reconhecidos por IgG1 em pacientes Bt+. O extrato Bt-ConA revelou oito componentes antigênicos (27-152kDa), dos quais as bandas de 152 e 123kDa foram predominantemente reconhecidos por IgE e apenas três destes componentes (93, 123 e 152kDa) foram reconhecidos por IgG1 enquanto anticorpos IgG4 reconheceram fracamente os componentes de 66 e 152kDa em pacientes Bt+. Pode-se concluir que componentes ligantes de Con-A isolados de Bt constituem alérgenos principais e estão envolvidos tanto na sensibilização alergênica (resposta IgE) quanto na manutenção da homeostase (respostas IgG1 e IgG4) e que componentes glicosilados, particularmente os de alto peso molecular, são preferencialmente reconhecidos por anticorpos IgE e IgG1, mas não por anticorpos IgG4