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Three-dimensional culture systems and suitable substrates topographies demonstrated to drive stem cell fate in vitro by mechanical conditioning. For example, the Nichoid 3D scaffold remodels stem cells and shapes nuclei, thus promoting stem cell expansion and stemness maintenance. However, the mechanisms involved in force transmission and in biochemical signaling at the basis of fate determination are not yet clear. Among the available investigation systems, confocal fluorescence microscopy using fluorescent dyes enables the observation of cell function and shape at the subcellular scale in vital and fixed conditions. Contrarily, nonlinear optical microscopy techniques, which exploit multi-photon processes, allow to study cell behavior in vital and unlabeled conditions. We apply confocal fluorescence microscopy, coherent anti-Stokes Raman scattering (CARS), and second harmonic generation (SHG) microscopy to characterize the phenotypic expression of mesenchymal stem cells (MSCs) towards adipogenic and chondrogenic differentiation inside Nichoid scaffolds, in terms of nuclear morphology and specific phenotypic products, by comparing these techniques. We demonstrate that the Nichoid maintains a rounded nuclei during expansion and differentiation, promoting MSCs adipogenic differentiation while inhibiting chondrogenesis. We show that CARS and SHG techniques are suitable for specific estimation of the lipid and collagenous content, thus overcoming the limitations of using unspecific fluorescent probes.
Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Mesenquimais/fisiologia , Alicerces Teciduais/química , Adipogenia/fisiologia , Animais , Células Cultivadas , Condrogênese/fisiologia , Corantes Fluorescentes/metabolismo , Células-Tronco Mesenquimais/metabolismo , Microscopia Confocal/métodos , Ratos , Análise Espectral Raman/métodosRESUMO
Smart biomaterials are increasingly being used to control stem cell fate in vitro by the recapitulation of the native niche microenvironment. By integrating experimental measurements with numerical models, we show that in mesenchymal stem cells grown inside a 3D synthetic niche both nuclear transport of a myogenic factor and the passive nuclear diffusion of a smaller inert protein are reduced. Our results also suggest that cell morphology modulates nuclear proteins import through a partition of the nuclear envelope surface, which is a thin but extremely permeable annular portion in cells cultured on 2D substrates. Therefore, our results support the hypothesis that in stem cell differentiation, the nuclear import of gene-regulating transcription factors is controlled by a strain-dependent nuclear envelope permeability, probably related to the reorganization of stretch-activated nuclear pore complexes.
Assuntos
Transporte Ativo do Núcleo Celular/genética , Núcleo Celular/genética , Células-Tronco Mesenquimais/metabolismo , Proteína MyoD/genética , Diferenciação Celular/genética , Núcleo Celular/metabolismo , Células Cultivadas , Regulação da Expressão Gênica , Humanos , Membrana Nuclear/genética , Membrana Nuclear/metabolismo , Poro Nuclear/genética , Nicho de Células-Tronco/genéticaRESUMO
A recent challenge in the field of bioimaging is to image vital, thick, and complex tissues in real time and in non-invasive mode. Among the different tools available for diagnostics, nonlinear optical (NLO) multi-photon microscopy allows label-free non-destructive investigation of physio-pathological processes in live samples at sub-cellular spatial resolution, enabling to study the mechanisms underlying several cellular functions. In this review, we discuss the fundamentals of NLO microscopy and the techniques suitable for biological applications, such as two-photon excited fluorescence (TPEF), second and third harmonic generation (SHG-THG), and coherent Raman scattering (CRS). In addition, we present a few of the most recent examples of NLO imaging employed as a label-free diagnostic instrument to functionally monitor in vitro and in vivo vital biological specimens in their unperturbed state, highlighting the technological advantages of multi-modal, multi-photon NLO microscopy and the outstanding challenges in biomedical engineering applications.
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This review provides an update for the international research community on the cell modeling tools that could accelerate the understanding of SARS-CoV-2 infection mechanisms and could thus speed up the development of vaccines and therapeutic agents against COVID-19. Many bioengineering groups are actively developing frontier tools that are capable of providing realistic three-dimensional (3D) models for biological research, including cell culture scaffolds, microfluidic chambers for the culture of tissue equivalents and organoids, and implantable windows for intravital imaging. Here, we review the most innovative study models based on these bioengineering tools in the context of virology and vaccinology. To make it easier for scientists working on SARS-CoV-2 to identify and apply specific tools, we discuss how they could accelerate the discovery and preclinical development of antiviral drugs and vaccines, compared to conventional models.
Assuntos
Antivirais/isolamento & purificação , Antivirais/farmacologia , Betacoronavirus , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/prevenção & controle , Pandemias/prevenção & controle , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/prevenção & controle , Vacinas Virais/isolamento & purificação , Vacinas Virais/farmacologia , Betacoronavirus/química , Betacoronavirus/genética , Betacoronavirus/imunologia , Bioengenharia/métodos , Bioengenharia/tendências , Reatores Biológicos , COVID-19 , Vacinas contra COVID-19 , Técnicas de Cultura de Células , Simulação por Computador , Infecções por Coronavirus/imunologia , Descoberta de Drogas/métodos , Descoberta de Drogas/tendências , Avaliação de Medicamentos/métodos , Avaliação de Medicamentos/tendências , Farmacorresistência Viral , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Modelos Biológicos , Organoides/citologia , Organoides/virologia , Pneumonia Viral/imunologia , SARS-CoV-2 , Nanomedicina TeranósticaRESUMO
The performances of surveillance systems for measles in Europe are poorly investigated, despite the fundamental role they should play in the early detection of outbreaks and in the assessment of the progress towards elimination. A new chief complaint syndrome surveillance system has been developed in Genoa, Italy, using data from the Emergency Department records of the regional reference university hospital and its ability to early detect an outbreak of measles that began during the winter months of 2007/2008 was evaluated. For the 23-month period from January 2007 to November 2008, the Emergency Department registration and triage software was used to obtain the time series of daily counts, that were related with cases notified by the statutory notification system and detection and characterization data from the measles regional reference laboratory. One hundred fifty five cases of measles-like illness were identified by the syndrome surveillance system. Two epidemic threshold breakthroughs were able to anticipate the first notified case by 54 and 11 days. Globally, the new syndrome surveillance system allows the activation of the alert state with a specificity of 94.3% and a sensitivity of 91%. Molecular investigation showed the spread of the virus from United Kingdom to Piemonte and then to Liguria and allowed us to exclude the re- circulation of strains circulating in Northern Italy during the previous seasons. Syndrome surveillance integrated with a rapid detection and characterization of the agent responsible for the disease could be an effective, specific and sensitive tool for measles surveillance.
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Controle de Doenças Transmissíveis/métodos , Surtos de Doenças , Sarampo/epidemiologia , Sarampo/virologia , Morbillivirus/classificação , Morbillivirus/genética , Vigilância de Evento Sentinela , Análise por Conglomerados , Coleta de Dados/métodos , Humanos , Itália/epidemiologia , Sarampo/patologia , Sarampo/fisiopatologia , Epidemiologia Molecular , Morbillivirus/isolamento & purificação , Filogenia , RNA Viral , Sensibilidade e Especificidade , Análise de Sequência de DNA , SoftwareRESUMO
High quality large scale fabrication of cellular scaffolds, with three-dimensional resolution comparable to cell size, is an important task to enable regenerative medicine applications with stem cells. We are using two-photon polymerization to produce our stem cell culture substrate called Nichoid, which we already demonstrated capable of stimulating cell proliferation while maintaining their stemness, without the need of dangerous additives. Parallelization of this technique can be achieved with the use of a spatial light modulator: here we show the results obtained combining this device with fast linear stages to produce Nichoid-covered substrates by two-photon polymerization. The well-polymerized structures confirm that this approach is particularly convenient for porous structures, and allows a significant time saving by a factor of almost five, with minor design adjustments. A Live & Dead assay was performed on mesenchymal stem cells cultured into the Nichoid microstructures in order to verify that no difference in cell viability is present, compared to microstructures fabricated by a single focus. This parallel setup opens the possibility to obtain a much larger number of microstructured substrates, that are essential to test new stem cell-based therapies. This approach can be also used for the fast fabrication of other kinds of cell culture devices.
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Polímeros/química , Medicina Regenerativa , Células-Tronco/citologia , Alicerces Teciduais , Materiais Biocompatíveis , HumanosRESUMO
INTRODUCTION: Healthcare workers (HCWs) are exposed to various risk factors and risky behaviours that may seriously affect their health and ability to work. The aim of this protocol is to detail the steps to follow in order to carry out a scoping review to assess the prevalence/incidence of injuries among HCWs. METHODS AND ANALYSIS: The study will be carried out in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses-Protocols guidelines. Studies will be selected according to the following criteria: P (HCWs), E (exposure to injuries), C (different types of exposure and different categories of HCWs) and O (prevalence/incidence and determinants of injuries). A time filter has been set (literature between 2000 and 2018) to enable updated, direct comparison between the findings and the epidemiological data available at national and local 'Istituto Nazionale per l'Assicurazione contro gli Infortuni sul Lavoro' (National Institute for Insurance Against Accidents at Work) centres in Italy. No language restriction will be applied. ETHICS AND DISSEMINATION: Formal ethical approval is not required; primary data will not be collected, as they have already been published. The results will be disseminated through peer-reviewed publication(s), conference presentation(s) and the press.
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Pessoal de Saúde/estatística & dados numéricos , Traumatismos Ocupacionais/epidemiologia , Humanos , Incidência , Exposição Ocupacional/estatística & dados numéricos , Traumatismos Ocupacionais/classificação , Traumatismos Ocupacionais/etiologia , Prevalência , Literatura de Revisão como Assunto , Violência no Trabalho/estatística & dados numéricosRESUMO
BACKGROUND: The aim of the study was to assess radon concentration in schoolrooms in a city located in the midwest of Italy. METHODS: A two-phase environmental study was carried out in 19 school buildings of 16 primary, secondary, and tertiary schools. RESULTS: Median (interquartile range-IQR) indoor radon concentration in schoolrooms was 91.6 (45.0-140.3) Bq/m³. The highest (median 952.8 Bq/m³) radon concentration was found in one (3.6%) classroom, located in a building of a primary school whose median concentration was 185 Bq/m³. Radon concentration was significantly correlated with the number of students and teachers, foundation wall construction material, and with the absence of underground floors. A geopedological survey was performed close to the building with highest radon level, showing the presence of granite and tonalithic granodiorite in the soil. CONCLUSIONS: Radon levels should be routinely assessed where individuals live or work. Schools are susceptible targets, because of childhood stay and the long daily stay of occupants. Low-cost interventions, such as implementation of natural air ventilation and school maintenance, can reduce radon levels, limiting individual exposure.
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Poluentes Radioativos do Ar/análise , Poluição do Ar em Ambientes Fechados/análise , Exposição à Radiação/análise , Radônio/análise , Instituições Acadêmicas , Adolescente , Adulto , Criança , Pré-Escolar , Materiais de Construção , Feminino , Pisos e Cobertura de Pisos , Humanos , Itália , Masculino , Monitoramento de Radiação , Dióxido de Silício , Solo , Estudantes , Adulto JovemRESUMO
Antigenic drift, the evolutionary mechanism of influenza viruses, results in an increased susceptibility of vaccinated subjects against circulating viruses. New vaccines able to grant a broader and cross-reactive immune response against drifted influenza variants are needed. Several strategies were explored to enhance the immunogenicity of plain vaccines: adjuvants, carriers and intradermal administration of influenza vaccine emerge as a promising options. To evaluate the ability of a MF59-adjuvanted and intradermal influenza vaccine to elicit an effective antibody response against circulating viruses presenting antigenic patterns different from those of the vaccine strains, we compared antibody responses elicited by "implemented" vaccines and conventional intramuscular trivalent inactivated vaccine against heterologous circulating influenza A viruses. Different studies, simulating different epidemiological pictures produced by the natural antigenic drift of seasonal influenza viruses, highlighted the superior cross-reactivity of the antibodies elicited by MF59 and intradermal vaccines, compared with subunit or split vaccine against heterologous viruses.
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Adjuvantes Imunológicos/administração & dosagem , Proteção Cruzada , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Orthomyxoviridae/imunologia , Polissorbatos/administração & dosagem , Esqualeno/administração & dosagem , Anticorpos Antivirais/sangue , Humanos , Vacinas contra Influenza/administração & dosagem , Injeções Intradérmicas , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/imunologia , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
In the present study we first compare immunogenicity against vaccine and heterologous circulating A(H1N1)pdm09 strains, tolerability and safety of intradermal Intanza 15 µg and of virosomal adjuvanted, intramuscularly delivered influenza vaccine, Inflexal V, in healthy elderly volunteers. Five-hundred participants were enrolled in the study and randomly assigned to the two vaccine groups to receive either one dose of Intanza 15 µg or Inflexal V vaccine. All subjects reported solicited local and systemic reactions occurred within 7 d after vaccination and unsolicited adverse events up to 21 d post-immunization and any serious adverse event appeared during the study. A subset of 55 participants was randomly selected for immunogenicity and cross-protection evaluations. Serum samples were collected before and 1 and 3 mo after immunization. Antibody responses were measured using hemagglutination inhibition (HI) against all viruses used in the study and neutralization (NT) assays against A(H1N1)pdm09 strains. At least one of the CHMP criteria for influenza vaccine approval in the elderly was met by virosomal vaccine against all the tested viruses; intradermal vaccine met all criteria against all strains. Several parameters of immune response against strains with a different antigenic pattern from that of vaccine A/California/04/09(H1N1)pdm09 were significantly higher in the intradermal vaccine group compared with the virosomal group. Safety and systemic tolerability of both vaccines were excellent, but injection site reactions occurred significantly more frequently in the intradermal vaccination group. Immunogenicity of Intanza 15 µg intradermal vaccine tended to be higher than that of Inflexal V against heterologous strains in healthy elderly.
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Proteção Cruzada , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antivirais/sangue , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/epidemiologia , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/patologia , Feminino , Testes de Inibição da Hemaglutinação , Humanos , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/efeitos adversos , Injeções Intradérmicas , Injeções Intramusculares , Masculino , Pessoa de Meia-Idade , Testes de Neutralização , Vacinas Virossomais/administração & dosagem , Vacinas Virossomais/efeitos adversos , Vacinas Virossomais/imunologiaRESUMO
This study evaluated safety, tolerability and immunogenicity of intradermal (ID) trivalent inactivated split influenza vaccine, with a lower antigen content (9 mcg HA per strain) than the conventional intramuscular one (15 mcg), in HIV-1-infected adults younger than 60 years. A total of 54 HIV-1-positive participants were enrolled and randomly assigned to receive a single dose of either ID-administered low-antigen-content split inactivated vaccine or intramuscularly-administered (IM) standard-dose inactivated split vaccine. Subjects were provided with a diary to monitor any local and/or systemic reactions to the vaccine for 7 days following vaccination. Serum samples were collected before, 28 days and 90 days after immunization. The plasma HIV-RNA and CD4+ T-lymphocyte count were checked at day 0 and day 90. Serum hemagglutination-inhibition (HI) activity for the three influenza strains included in the vaccine composition was measured to assess the antibody response at one month and 3 months after vaccination. Both vaccines showed optimal safety and tolerability profiles. All the three Committee for Medicinal Products for Human Use immunogenicity criteria for vaccine approval in adults younger than 60 were met by both vaccines against A(H1N1) and A(H3N2) viruses. Both vaccines met mean-fold-increase and seroprotection criteria but failed seroconversion criteria against B virus. No difference in terms of post-vaccination geometric mean titers, mean fold increase, seroprotection and seroconversion rates were found comparing ID and IM vaccines. In conclusion, the recently available low-antigen-content ID vaccine is safe, well-tolerated and as immunogenic as IM standard-dose influenza vaccine.
Assuntos
Infecções por HIV/imunologia , Vacinas contra Influenza/efeitos adversos , Vacinas contra Influenza/imunologia , Adulto , Anticorpos Antivirais/sangue , Antígenos Virais/administração & dosagem , Antígenos Virais/imunologia , Contagem de Linfócito CD4 , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/epidemiologia , Feminino , Testes de Inibição da Hemaglutinação , Humanos , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H2N2/imunologia , Vírus da Influenza B/imunologia , Vacinas contra Influenza/administração & dosagem , Injeções Intradérmicas , Injeções Intramusculares , Masculino , Pessoa de Meia-Idade , RNA Viral/sangue , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/efeitos adversos , Vacinas de Produtos Inativados/imunologiaRESUMO
The current public health strategy for the containment of influenza is annual vaccination, which is recommended for the elderly and for those in risk factor categories that present the highest morbidity and mortality. However, because the immune response in the elderly is known to be less vigorous than in younger adults, research in the last decade has focused on improving the immune response to vaccination and increasing the protection of aged populations. The decreased efficacy of vaccines in the elderly is due to several factors, such as a decrease in the number of Langerhans cells, the limited capacity of dendritic cells to present antigen, defects in the expression of Toll-like receptors and the reduced expression of MHC class I and II molecules. Also, production of mature naive T cells by the thymus decreases with age. Among several approaches proposed to address the need for more immunogenic vaccines compared with conventional agents, the most well proven is the use of adjuvants. The first licensed adjuvant, aluminium-based mineral salts (alum), introduced in the 1920s, remains the standard worldwide adjuvant for human use and it has been widely used for almost a century. However, the addition of alum adjuvant to a split or subunit influenza vaccine has induced only marginal improvements. Other adjuvants have been developed and approved for human use since 1997; in particular, MF59, an oil-in-water adjuvant emulsion of squalene, which is able to increase immunogenicity of seasonal, pre-pandemic and pandemic subunit vaccines while maintaining acceptable safety and tolerability profiles. More recently, another oil-in-water emulsion, AS03, has been approved as a component of pre-pandemic H5N1 and pandemic H1N1 2009 vaccines. Besides adjuvants, several other strategies have been assessed to enhance antibody response in the elderly and other less responsive subjects, such as high-dose antigen vaccines, carrier systems (liposomes/virosomes) and the intradermal route of immunization. In particular, the potential of intradermal vaccination is well documented and the recent availability of an appropriate injection system, which combines simplicity, safety and ease of use, has allowed evaluation of the tolerability, safety and immunogenicity of the intradermal influenza vaccine in large numbers of subjects. Data that emerged from large clinical trials showed an improved immunogenicity compared with that of standard vaccine. Observational studies or comparisons between adjuvanted, intradermal or high-dose versus conventional vaccines are needed to evaluate whether the greater immunogenicity observed in a number of recent studies is correlated with greater protection against influenza and influenza-related complications and death.
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Adjuvantes Imunológicos/uso terapêutico , Envelhecimento/imunologia , Vacinas contra Influenza/uso terapêutico , Influenza Humana/prevenção & controle , Idoso , Ensaios Clínicos como Assunto , Eficiência , Humanos , Imunidade/imunologia , Influenza Humana/imunologia , Vacinas de Produtos Inativados/uso terapêuticoRESUMO
Vaccination is considered the most effective means of reducing influenza burden, providing substantial benefits in terms of reduction of morbidity, complications, hospitalizations and deaths, even if vaccines have been associated with a reduced immune response and lower effectiveness in older adults, in particular when a mismatch between the vaccine and the circulating virus strains occurred. Several strategies have been proposed to enhance vaccine protection against drifted strains, including the use of adjuvants. Among oil-emulsion adjuvants, MF-59 was approved for human use more than a decade ago and it is largely used for adjuvantation of influenza vaccine. Recent studies have demonstrated that addition of the MF-59 to subunit influenza vaccine can lead to higher haemagglutination-inhibiting seroprotection rates and to higher neutralization antibody titers against drifted strains not included in the vaccine respect to non-adjuvanted vaccine. Promising results were obtained using a new generation of oil-in-water emulsion adjuvants, named AS, offering cross-protection against heterologous challenge in ferrets.