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1.
Sci Total Environ ; 713: 136707, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32019041

RESUMO

Gut microbes play an essential role in the development and functioning of the human immune system. A disturbed gut microbiota composition is often associated with a number of health disorders including immune-mediated diseases. Differences in host characteristics such as ethnicity, living habit and diet have been used to explain differences in the gut microbiota composition in inter-continental comparison studies. As our previous studies imply that daily skin contact with organic gardening materials modify gut microflora, here we investigated the association between living environment and gut microbiota in a homogenous western population along an urban-rural gradient. We obtained stool samples from 48 native elderly Finns in province Häme in August and November 2015 and identified the bacterial phylotypes using 16S rRNA Illumina MiSeq sequencing. We assumed that yard vegetation and land cover classes surrounding homes explain the stool bacterial community in generalized linear mixed models. Diverse yard vegetation was associated with a reduced abundance of Clostridium sensu stricto and an increased abundance of Faecalibacterium and Prevotellaceae. The abundance of Bacteroides was positively and strongly associated with the built environment. Exclusion of animal owners did not alter the main associations. These results suggest that diverse vegetation around homes is associated with health-related changes in gut microbiota composition. Manipulation of the garden diversity, possibly jointly with urban planning, is a promising candidate for future intervention studies that aim to maintain gut homeostasis.


Assuntos
Microbioma Gastrointestinal , Animais , Bactérias , Bacteroides , Fezes , Humanos , RNA Ribossômico 16S
2.
Viruses ; 12(1)2020 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-31936473

RESUMO

BACKGROUND: Enteroviruses are a group of common non-enveloped RNA viruses that cause symptoms ranging from mild respiratory infections to paralysis. Due to the abundance of enterovirus infections it is hard to distinguish between on-going and previous infections using immunological assays unless the IgM fraction is studied. METHODS: In this study we show using Indirect ELISA and capture IgM ELISA that an IgG antibody response against the nonstructural enteroviral proteins 2A and 3C can be used to distinguish between IgM positive (n = 22) and IgM negative (n = 20) human patients with 83% accuracy and a diagnostic odds ratio of 30. Using a mouse model, we establish that the antibody response to the proteases is short-lived compared to the antibody response to the structural proteins in. As such, the protease antibody response serves as a potential marker for an acute infection. CONCLUSIONS: Antibody responses against enterovirus proteases are shorter-lived than against structural proteins and can differentiate between IgM positive and negative patients, and therefore they are a potential marker for acute infections.

3.
Viruses ; 11(12)2019 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-31795245

RESUMO

Enteroviruses are small RNA viruses that cause diseases with various symptoms ranging from mild to severe. Enterovirus proteins are translated as a single polyprotein, which is cleaved by viral proteases to release capsid and nonstructural proteins. Here, we show that also cellular calpains have a potential role in the processing of the enteroviral polyprotein. Using purified calpains 1 and 2 in an in vitro assay, we show that addition of calpains leads to an increase in the release of VP1 and VP3 capsid proteins from P1 of enterovirus B species, detected by western blotting. This was prevented with a calpain inhibitor and was dependent on optimal calcium concentration, especially for calpain 2. In addition, calpain cleavage at the VP3-VP1 interface was supported by a competition assay using a peptide containing the VP3-VP1 cleavage site. Moreover, a mass spectrometry analysis showed that calpains can cleave this same peptide at the VP3-VP1 interface, the cutting site being two amino acids aside from 3C's cutting site. Furthermore, we show that calpains cannot cleave between P1 and 2A. In conclusion, we show that cellular proteases, calpains, can cleave structural proteins from enterovirus polyprotein in vitro. Whether they assist polyprotein processing in infected cells remains to be shown.

4.
Vaccine ; 37(51): 7509-7518, 2019 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-31585726

RESUMO

Enteric viruses cause diverse infections with substantial morbidity and mortality in children, rotavirus (RV) and norovirus (NoV) being the leading agents of severe pediatric gastroenteritis. Coxsackie B viruses (CVB) are common enteroviruses (EV), associated with increased incidence of severe neonatal CVB disease with potentially fatal consequences. To prevent majority of childhood gastroenteritis, we have developed a non-live NoV-RV combination vaccine consisting of NoV virus-like particles (VLPs) and RV oligomeric rVP6 protein that induced protective immune responses to NoV and RV in mice. Moreover, rVP6 acted as an adjuvant for NoV VLPs. Here, we investigated a possibility to include a third enteric virus-derived antigen in the candidate NoV-RV vaccine, by adding recombinant nanoparticles derived from EV CVB1. To examine immunogenicity of EV-NoV-RV vaccine, BALB/c mice were immunized intramuscularly twice with 10 µg CVB1 VLPs, GII.4 VLPs and rVP6 nanotubes, either separately or combined. To evaluate the adjuvant effect of rVP6 on EV responses, mice received 0.3 µg CVB1 VLPs with or without 10 µg rVP6. Comparable serum IgG antibodies were detected whether the antigens were administered separately or in combination. Each formulation generated IgG1 and IgG2a antibodies, indicating a mixed Th2/Th1-type response. CVB1 VLPs skewed the isotype distribution slightly towards IgG1 subtype, while EV-NoV-RV combination vaccine induced unbiased Th1/Th2 responses to CVB1. Each antigen also induced T cell mediated immunity measured by IFN-γ secretion to specific stimulants ex vivo. Antisera raised by single antigens and combined formulation also exhibited strong neutralizing ability against CVB1 and NoV GII.4. Further, rVP6 showed an adjuvant effect on CVB1 responses, sparing the VLP dose and homogenizing the responses. Finally, the results support inclusion of additional antigens in the candidate NoV-RV combination vaccine to combat severe childhood infections and confirm adjuvant effect of rVP6 nanostructures.

5.
Antiviral Res ; 171: 104595, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31491431

RESUMO

Type B Coxsackieviruses (CVBs) are a common cause of acute and chronic myocarditis, dilated cardiomyopathy and aseptic meningitis. However, no CVB-vaccines are available for human use. We have previously produced virus-like particles (VLPs) for CVB3 with a baculovirus-insect cell production system. Here we have explored the potential of a VLP-based vaccine targeting CVB1 and describe the production of CVB1-VLPs with a scalable VLP purification method. The developed purification method consisting of tangential flow filtration and ion exchange chromatography is compatible with industrial scale production. CVB1-VLP vaccine was treated with UV-C or formalin to study whether stability and immunogenicity was affected. Untreated, UV treated and formalin treated VLPs remained morphologically intact for 12  months  at 4 °C. Formalin treatment increased, whereas UV treatment decreased the thermostability of the VLP-vaccine. High neutralising and total IgG antibody levels, the latter predominantly of a Th2 type (IgG1) phenotype, were detected in female BALB/c mice immunised with non-adjuvanted, untreated CVB1-VLP vaccine. The immunogenicity of the differently treated CVB1-VLPs (non-adjuvanted) were compared in C57BL/6 J mice and animals vaccinated with formalin treated CVB1-VLPs mounted the strongest neutralising and, CVB1-specific IgG and IgG1 antibody responses. This study demonstrates that formalin treatment increases the stability and immunogenicity of CVB1-VLP vaccine and may offer a universal tool for the stabilisation of VLPs in the production of more efficient vaccines.

6.
Vaccine ; 37(40): 5962-5971, 2019 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-31471148

RESUMO

Type B Coxsackieviruses (CVBs) belong to the enterovirus genus, and they cause both acute and chronic diseases in humans. CVB infections usually lead to flu-like symptoms but can also result in more serious diseases such as myocarditis, aseptic meningitis and life-threatening multi-organ infections in young infants. Thus, CVBs have long been considered as important targets of future vaccines. We have previously observed CVB1 capsid disintegration and virus concentration decrease with 12-day long formalin inactivation protocol. Here a scalable ion exchange chromatography purification method was developed, and purified CVB1 was inactivated with UV-C or formalin. Virus morphology and concentration remained unchanged, when the UV (2 min) or formalin (5 days) inactivation were performed in the presence of tween80 detergent. The concentration of the native and UV inactivated CVB1 remained constant at 4 °C during a six months stability study, whereas the concentration of the formalin inactivated vaccine decreased 29% during this time. UV treatment decreased, whereas formalin treatment increased the thermal stability of the capsid. The formalin inactivated CVB1 vaccine was more immunogenic than the UV inactivated vaccine; the protective neutralizing antibody levels were higher in mice immunized with formalin inactivated vaccine. High levels of CVB1 neutralizing antibodies as well as IgG1 antibodies were detected in mice that were protected against viremia induced by experimental CVB1 infection. In conclusion, this study describes a scalable ion exchange chromatography purification method and optimized 5-day long formalin inactivation method that preserves CVB1 capsid structure and immunogenicity. Formalin treatment stabilizes the virus particle at elevated temperatures, and the formalin inactivated vaccine induces high levels of serum IgG1 antibodies (Th2 type response) and protective levels of neutralizing antibodies. Formalin inactivated CVB vaccines are promising candidates for human clinical trials.

7.
Environ Int ; 132: 105069, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31400602

RESUMO

An agricultural environment and exposure to diverse environmental microbiota has been suggested to confer protection against immune-mediated disorders. As an agricultural environment may have a protective role, it is crucial to determine whether the limiting factors in the transfer of environmental microbiota indoors are the same in the agricultural and urban environments. We explored how sampling month, garden diversity and animal ownership affected the indoor-transfer of environmental microbial community. We collected litter from standardized doormats used for 2 weeks in June and August 2015 and February 2016 and identified bacterial phylotypes using 16S rRNA Illumina MiSeq sequencing. In February, the diversity and richness of the whole bacterial community and the relative abundance of environment-associated taxa were reduced, whereas human-associated taxa and genera containing opportunistic pathogens were enriched in the doormats. In summer, the relative abundances of several taxa associated previously with beneficial health effects were higher, particularly in agricultural areas. Surprisingly, the importance of vegetation on doormat microbiota was more observable in February, which may have resulted from snow cover that prevented contact with microbes in soil. Animal ownership increased the prevalence of genera Bacteroides and Acinetobacter in rural doormats. These findings underline the roles of season, living environment and lifestyle in the temporal variations in the environmental microbial community carried indoors. As reduced contact with diverse microbiota is a potential reason for immune system dysfunction, the results may have important implications in the etiology of immune-mediated, non-communicable diseases.


Assuntos
Bactérias/isolamento & purificação , Habitação/estatística & dados numéricos , Microbiota , Microbiologia do Solo , Idoso , Agricultura , Animais , Bactérias/genética , Gatos , Bovinos , Cidades , Cães , Jardins , Humanos , Plantas , RNA Ribossômico 16S/genética , Estações do Ano , Solo
8.
Artigo em Inglês | MEDLINE | ID: mdl-31426345

RESUMO

Nature contacts are recognized as positively contributing to humans' health and well-being. Although there have been projects to green daycare or schoolyards, yard greening and microbial biodiversity have never been studied simultaneously. We asked whether simultaneously increasing biodiversity exposure and greening urban daycare yards affects 3-5 years-old children's physical activity and play, their environmental relationships, and their perceived well-being. For transforming six daycare yards in Finland, we used a forest floor with high biodiversity, sod, peat blocks, and planters for vegetable and flower growing. We used qualitative interview and survey-based data collected from the daycare personnel and parents to analyze how green yards encourage children's engagement with their everyday life-worlds. We identified the functional possibilities provided by the yards and the dynamic aspects related to the greening. Green, biodiverse yards were considered safe, and inspired children's play, diversified their activities, and increased physical activity. The greenery offered embodied experiences of nature and provided the children with multi-sensory exploration and diverse learning situations. The dynamic and emotional ways of engaging with the natural environment increased their well-being. The activities related to caring for the yards and exploring them promoted the development of environmental relationships. The results can be used for designing health-enhancing yards.


Assuntos
Comportamento Infantil , Creches , Meio Ambiente , Natureza , Jogos e Brinquedos , Adulto , Biodiversidade , Pré-Escolar , Exercício Físico , Feminino , Finlândia , Humanos , Masculino , Plantas , Inquéritos e Questionários
9.
Environ Int ; 130: 104894, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31220749

RESUMO

Polycyclic aromatic hydrocarbons (PAHs) are priority environmental pollutants that cause adverse health effects. PAHs belong to endocrine signaling disruptors to which children are sensitive to. Recent evidence suggests that PAH pollution alters the abundance of environmental bacteria that is associated with health outcomes. The alteration of environmental and commensal microbiota by PAH pollution has never been connected to endocrine signaling pathways. To estimate the risk of endocrine disruption in daycare children, we measured PAHs from soil and air of eleven urban daycare centres in Finland. We analyzed daycare yards' soil and children's gut and skin bacterial communities with 16S rRNA gene metabarcoding and used Kyoto Encyclopaedia of Genes and Genomes database to categorize endocrine signaling pathways. We also assessed the PAH hazard to children's health based on the current risk assesments. We observed associations between signaling pathways in endocrine system and gaseous PAH levels in ambient air. Peroxisome proliferator-activated receptor and adipocytokine signaling pathway decreased with higher chrysene concentration in the air. Soil PAH contamination was associated with altered Actinobacteria, Bacteoridetes and Proteobacteria communities on children's skin and in daycare yard soil. However, adjusted genera were not the same in soil and on skin, with the exception of Mycobacterium that was associated with higher PAH concentrations both in soil and on the skin. Even though fluoranhtene levels were above the current threshold values, total PAHs were below safety threshold values and based on current risk assessments there is a minor risk for child health. Our findings indicate that PAH concentrations that are considered safe may interfere with endocrine signaling by commensal microbiota and alter both environmental and commensal bacterial communities. The imbalance in human microbiota and the decrease in signaling pathways may contribute to emerging public health problems, including inflammatory disorders, obesity and diabetes. Therefore, the optimal risk assessments of PAHs and theoretically also other contaminants shaping commensal microbiota may need to take into account the possibility of the disruption of endocrine signaling pathways.

10.
Front Microbiol ; 10: 536, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30967847

RESUMO

Human activities typically lead to simplified urban diversity, which in turn reduces microbial exposure and increases the risk to urban dwellers from non-communicable diseases. To overcome this, we developed a microbial inoculant from forest and agricultural materials that resembles microbiota in organic soils. Three different sand materials (sieved, safety, and sandbox) commonly used in playgrounds and other public spaces were enriched with 5% of the inoculant. Skin microbiota on fingers (identified from bacterial 16S rDNA determined using Illumina MiSeq sequencing) was compared after touching non-enriched and microbial inoculant-enriched sands. Exposure to the non-enriched materials changed the skin bacterial community composition in distinct ways. When the inoculant was added to the materials, the overall shift in community composition was larger and the differences between different sand materials almost disappeared. Inoculant-enriched sand materials increased bacterial diversity and richness but did not affect evenness at the OTU level on skin. The Firmicutes/Bacteroidetes ratio was higher after touching inoculant-enriched compared to non-enriched sand materials. The relative abundance of opportunistic pathogens on skin was 40-50% before touching sand materials, but dropped to 14 and 4% after touching standard and inoculant-enriched sand materials, respectively. When individual genera were analyzed, Pseudomonas sp. and Sphingomonas sp. were more abundant after touching standard, non-enriched sand materials, while only the relative abundance of Chryseobacterium sp. increased after touching the inoculant-enriched materials. As Chryseobacterium is harmless for healthy persons, and as standard landscaping materials and normal skin contain genera that include severe pathogens, the inoculant-enriched materials can be considered safe. Microbial inoculants could be specifically created to increase the proportion of non-pathogenic bacterial taxa and minimize the transfer of pathogenic taxa. We recommend further study into the usability of inoculant-enriched materials and their effects on the bacterial community composition of human skin and on the immune response.

11.
Microbiologyopen ; 8(3): e00645, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-29808965

RESUMO

Immune-mediated diseases have increased during the last decades in urban environments. The hygiene hypothesis suggests that increased hygiene level and reduced contacts with natural biodiversity are related to the increase in immune-mediated diseases. We tested whether short-time contact with microbiologically diverse nature-based materials immediately change bacterial diversity on human skin. We tested direct skin contact, as two volunteers rubbed their hands with sixteen soil and plant based materials, and an exposure via fabric packets filled with moss material. Skin swabs were taken before and after both exposures. Next-generation sequencing showed that exposures increased, at least temporarily, the total diversity of skin microbiota and the diversity of Acidobacteria, Actinobacteria, Bacteroidetes, Proteobacteria and Alpha-, Beta- and Gammaproteobacteria suggesting that contact with nature-based materials modify skin microbiome and increase skin microbial diversity. Until now, approaches to cure or prevent immune system disorders using microbe-based treatments have been limited to use of a few microbial species. We propose that nature-based materials with high natural diversity, such as the materials tested here, might be more effective in modifying human skin microbiome, and eventually, in reducing immune system disorders. Future studies should investigate how long-term changes in skin microbiota are achieved and if the exposure induces beneficial changes in the immune system markers.


Assuntos
Bactérias/classificação , Bactérias/genética , Exposição Ambiental , Microbiota , Pele/microbiologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Metagenoma , Plantas , Solo
12.
Diabetologia ; 61(5): 1193-1202, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29404673

RESUMO

AIMS/HYPOTHESIS: Islet autoimmunity usually starts with the appearance of autoantibodies against either insulin (IAA) or GAD65 (GADA). This categorises children with preclinical type 1 diabetes into two immune phenotypes, which differ in their genetic background and may have different aetiology. The aim was to study whether Coxsackievirus group B (CVB) infections, which have been linked to the initiation of islet autoimmunity, are associated with either of these two phenotypes in children with HLA-conferred susceptibility to type 1 diabetes. METHODS: All samples were from children in the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) study. Individuals are recruited to the DIPP study from the general population of new-born infants who carry defined HLA genotypes associated with susceptibility to type 1 diabetes. Our study cohort included 91 children who developed IAA and 78 children who developed GADA as their first appearing single autoantibody and remained persistently seropositive for islet autoantibodies, along with 181 and 151 individually matched autoantibody negative control children, respectively. Seroconversion to positivity for neutralising antibodies was detected as the surrogate marker of CVB infections in serial follow-up serum samples collected before and at the appearance of islet autoantibodies in each individual. RESULTS: CVB1 infections were associated with the appearance of IAA as the first autoantibody (OR 2.4 [95% CI 1.4, 4.2], corrected p = 0.018). CVB5 infection also tended to be associated with the appearance of IAA, however, this did not reach statistical significance (OR 2.3, [0.7, 7.5], p = 0.163); no other CVB types were associated with increased risk of IAA. Children who had signs of a CVB1 infection either alone or prior to infections by other CVBs were at the highest risk for developing IAA (OR 5.3 [95% CI 2.4, 11.7], p < 0.001). None of the CVBs were associated with the appearance of GADA. CONCLUSIONS/INTERPRETATION: CVB1 infections may contribute to the initiation of islet autoimmunity being particularly important in the insulin-driven autoimmune process.


Assuntos
Infecções por Coxsackievirus/complicações , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/virologia , Insulina/metabolismo , Anticorpos Neutralizantes/química , Autoanticorpos/química , Doenças Autoimunes , Autoimunidade , Criança , Pré-Escolar , Estudos de Coortes , Diabetes Mellitus Tipo 1/complicações , Progressão da Doença , Enterovirus , Feminino , Finlândia , Genótipo , Humanos , Lactente , Ilhotas Pancreáticas/imunologia , Masculino , Risco
13.
J Virol Methods ; 255: 29-37, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29425680

RESUMO

Enteroviruses (EVs), such as the Coxsackie B-viruses (CVBs), are common human pathogens, which can cause severe diseases including meningitis, myocarditis and neonatal sepsis. EVs encode two proteases (2Apro and 3Cpro), which perform the proteolytic cleavage of the CVB polyprotein and also cleave host cell proteins to facilitate viral replication. The 2Apro cause direct damage to the infected heart and tools to investigate 2Apro and 3Cpro expression may contribute new knowledge on virus-induced pathologies. Here, we developed new antibodies to CVB-encoded 2Apro and 3Cpro; Two monoclonal 2Apro antibodies and one 3Cpro antibody were produced. Using cells infected with selected viruses belonging to the EV A, B and C species and immunocytochemistry, we demonstrate that the 3Cpro antibody detects all of the EV species B (EV-B) viruses tested and that the 2Apro antibody detects all EV-B viruses apart from Echovirus 9. We furthermore show that the new antibodies work in Western blotting, immunocyto- and immunohistochemistry, and flow cytometry to detect CVBs. Confocal microscopy demonstrated the expression kinetics of 2Apro and 3Cpro, and revealed a preferential cytosolic localization of the proteases in CVB3 infected cells. In summary, the new antibodies detect proteases that belong to EV species B in cells and tissue using multiple applications.


Assuntos
Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Cisteína Endopeptidases/imunologia , Enterovirus Humano B/imunologia , Infecções por Enterovirus/diagnóstico , Infecções por Enterovirus/imunologia , Proteínas Virais/imunologia , Animais , Antígenos Virais/genética , Células Cultivadas , Clonagem Molecular , Cisteína Endopeptidases/genética , Enterovirus Humano B/enzimologia , Enterovirus Humano B/genética , Infecções por Enterovirus/virologia , Expressão Gênica , Células HeLa , Humanos , Imuno-Histoquímica , Camundongos , Sorogrupo , Proteínas Virais/genética
14.
Front Microbiol ; 9: 84, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29467728

RESUMO

Expanding urbanization is a major factor behind rapidly declining biodiversity. It has been proposed that in urbanized societies, the rarity of contact with diverse environmental microbiota negatively impacts immune function and ultimately increases the risk for allergies and other immune-mediated disorders. Surprisingly, the basic assumption that urbanization reduces exposure to environmental microbiota and its transfer indoors has rarely been examined. We investigated if the land use type around Finnish homes affects the diversity, richness, and abundance of bacterial communities indoors. Debris deposited on standardized doormats was collected in 30 rural and 26 urban households in and near the city of Lahti, Finland, in August 2015. Debris was weighed, bacterial community composition determined by high throughput sequencing of bacterial 16S ribosomal RNA (rRNA) gene on the Illumina MiSeq platform, and the percentage of four different land use types (i.e., built area, forest, transitional, and open area) within 200 m and 2000 m radiuses from each household was characterized. The quantity of doormat debris was inversely correlated with coverage of built area. The diversity of total bacterial, Proteobacterial, Actinobacterial, Bacteroidetes, and Firmicutes communities decreased as the percentage of built area increased. Their richness followed the same pattern except for Firmicutes for which no association was observed. The relative abundance of Proteobacteria and particularly Gammaproteobacteria increased, whereas that of Actinobacteria decreased with increasing built area. Neither Phylum Firmicutes nor Bacteroidetes varied with coverage of built area. Additionally, the relative abundance of potentially pathogenic bacterial families and genera increased as the percentage of built area increased. Interestingly, having domestic animals (including pets) only altered the association between the richness of Gammaproteobacteria and diversity of Firmicutes with the built area coverage suggesting that animal ownership minimally affects transfer of environmental microbiota indoors from the living environment. These results support the hypothesis that people living in densely built areas are less exposed to diverse environmental microbiota than people living in more sparsely built areas.

15.
Sci Rep ; 8(1): 33, 2018 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-29311608

RESUMO

Enteroviruses (EVs) are common RNA viruses that cause diseases ranging from rash to paralytic poliomyelitis. For example, EV-A and EV-C viruses cause hand-foot and mouth disease and EV-B viruses cause encephalitis and myocarditis, which can result in severe morbidity and mortality. While new vaccines and treatments for EVs are under development, methods for studying and diagnosing EV infections are still limited and therefore new diagnostic tools are required. Our aim was to produce and characterize new antibodies that work in multiple applications and detect EVs in tissues and in vitro. Rats were immunized with Coxsackievirus B1 capsid protein VP1 and hybridomas were produced. Hybridoma clones were selected based on their reactivity in different immunoassays. The most promising clone, 3A6, was characterized and it performed well in multiple techniques including ELISA, immunoelectron microscopy, immunocyto- and histochemistry and in Western blotting, detecting EVs in infected cells and tissues. It recognized several EV-Bs and also the EV-C representative Poliovirus 3, making it a broad-spectrum EV specific antibody. The 3A6 rat monoclonal antibody can help to overcome some of the challenges faced with commonly used EV antibodies: it enables simultaneous use of mouse-derived antibodies in double staining and it is useful in murine models.


Assuntos
Anticorpos Monoclonais/imunologia , Proteínas do Capsídeo/imunologia , Enterovirus Humano B/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Proteínas do Capsídeo/química , Enterovirus Humano B/classificação , Enterovirus Humano B/ultraestrutura , Infecções por Enterovirus/imunologia , Infecções por Enterovirus/virologia , Ensaio de Imunoadsorção Enzimática , Epitopos , Humanos , Imuno-Histoquímica , Camundongos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios Proteicos/imunologia , Ratos
16.
Diabetologia ; 61(2): 476-481, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29151123

RESUMO

AIMS/HYPOTHESIS: Epidemiological studies suggest a role for Coxsackievirus B (CVB) serotypes in the pathogenesis of type 1 diabetes, but their actual contribution remains elusive. In the present study, we have produced a CVB1 vaccine to test whether vaccination against CVBs can prevent virus-induced diabetes in an experimental model. METHODS: NOD and SOCS1-tg mice were vaccinated three times with either a formalin-fixed non-adjuvanted CVB1 vaccine or a buffer control. Serum was collected for measurement of neutralising antibodies using a virus neutralisation assay. Vaccinated and buffer-treated mice were infected with CVB1. Viraemia and viral replication in the pancreas were measured using standard plaque assay and PCR. The development of diabetes was monitored by blood glucose measurements. Histological analysis and immunostaining for viral capsid protein 1 (VP1), insulin and glucagon in formalin-fixed paraffin embedded pancreas was performed. RESULTS: The CVB1 vaccine induced strong neutralising antibody responses and protected against viraemia and the dissemination of virus to the pancreas in both NOD mice (n = 8) and SOCS1-tg mice (n = 7). Conversely, 100% of the buffer-treated NOD and SOCS1-tg mice were viraemic on day 3 post infection. Furthermore, half (3/6) of the buffer-treated SOCS1-tg mice developed diabetes upon infection with CVB1, with a loss of the insulin-positive beta cells and damage to the exocrine pancreas. In contrast, all (7/7) vaccinated SOCS1-tg mice were protected from virus-induced diabetes and showed no signs of beta cell loss or pancreas destruction (p < 0.05). CONCLUSIONS/INTERPRETATION: CVB1 vaccine can efficiently protect against both CVB1 infection and CVB1-induced diabetes. This preclinical proof of concept study provides a base for further studies aimed at developing a vaccine for use in elucidating the role of enteroviruses in human type 1 diabetes.


Assuntos
Infecções por Coxsackievirus/complicações , Diabetes Mellitus Tipo 1/etiologia , Diabetes Mellitus Tipo 1/prevenção & controle , Enterovirus Humano B/patogenicidade , Vacinas Virais/uso terapêutico , Animais , Diabetes Mellitus Tipo 1/metabolismo , Modelos Animais de Doenças , Feminino , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Reação em Cadeia da Polimerase
17.
PLoS One ; 12(11): e0187852, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29145477

RESUMO

Long-term exposure to polyaromatic hydrocarbons (PAHs) has been connected to chronic human health disorders. It is also well-known that i) PAH contamination alters soil bacterial communities, ii) human microbiome is associated with environmental microbiome, and iii) alteration in the abundance of members in several bacterial phyla is associated with adverse or beneficial human health effects. We hypothesized that soil pollution by PAHs altered soil bacterial communities that had known associations with human health. The rationale behind our study was to increase understanding and potentially facilitate reconsidering factors that lead to health disorders in areas characterized by PAH contamination. Large containers filled with either spruce forest soil, pine forest soil, peat, or glacial sand were left to incubate or contaminated with creosote. Biological degradation of PAHs was monitored using GC-MS, and the bacterial community composition was analyzed using 454 pyrosequencing. Proteobacteria had higher and Actinobacteria and Bacteroidetes had lower relative abundance in creosote contaminated soils than in non-contaminated soils. Earlier studies have demonstrated that an increase in the abundance of Proteobacteria and decreased abundance of the phyla Actinobacteria and Bacteroidetes are particularly associated with adverse health outcomes and immunological disorders. Therefore, we propose that pollution-induced shifts in natural soil bacterial community, like in PAH-polluted areas, can contribute to the prevalence of chronic diseases. We encourage studies that simultaneously address the classic "adverse toxin effect" paradigm and our novel "altered environmental microbiome" hypothesis.


Assuntos
Bactérias/isolamento & purificação , Hidrocarbonetos Policíclicos Aromáticos/análise , Microbiologia do Solo , Poluentes do Solo/análise , Finlândia , Cromatografia Gasosa-Espectrometria de Massas
18.
J Infect Dis ; 216(10): 1308-1317, 2017 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-28968805

RESUMO

Acute respiratory virus infections predispose the cystic fibrosis (CF) lung to chronic bacterial colonization, which contributes to high mortality. For reasons unknown, respiratory virus infections have a prolonged duration in CF. Here, we demonstrate that mice carrying the most frequent cystic fibrosis transmembrane conductance regulator (CFTR) mutation in humans, ΔF508, show increased morbidity and mortality following infection with a common human enterovirus. ΔF508 mice demonstrated impaired viral clearance, a slower type I interferon response and delayed production of virus-neutralizing antibodies. While the ΔF508 mice had a normal immune cell repertoire, unchanged serum immunoglobulin concentrations and an intact immune response to a T-cell-independent antigen, their response to a T-cell-dependent antigen was significantly delayed. Our studies reveal a novel function for CFTR in antiviral immunity and demonstrate that the ΔF508 mutation in cftr is coupled to an impaired adaptive immune response. This important insight could open up new approaches for patient care and treatment.


Assuntos
Imunidade Adaptativa/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Fibrose Cística/imunologia , Imunidade Inata/genética , Mutação , Viroses/etiologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Códon , Fibrose Cística/complicações , Modelos Animais de Doenças , Resistência à Doença/genética , Resistência à Doença/imunologia , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunização , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Interferon-alfa/biossíntese , Camundongos , Poli I-C/imunologia , Taxa de Sobrevida , Carga Viral
19.
Vaccine ; 35(30): 3718-3725, 2017 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-28579231

RESUMO

Coxsackie B viruses are among the most common enteroviruses, causing a wide range of diseases. Recent studies have also suggested that they may contribute to the development of type 1 diabetes. Vaccination would provide an effective way to prevent CVB infections, and the objective of this study was to develop an efficient vaccine production protocol for the generation of novel CVB vaccines. Various steps in the production of a formalin-inactivated Coxsackievirus B1 (CVB1) vaccine were optimized including the Multiplicity Of Infection (MOI) used for virus amplification, virus cultivation time, type of cell growth medium, virus purification method and formulation of the purified virus. Safety and immunogenicity of the formalin inactivated CVB1 vaccine was characterized in a mouse model. Two of the developed methods were found to be optimal for virus purification: the first employed PEG-precipitation followed by gelatin-chromatography and sucrose cushion pelleting (three-step protocol), yielding 19-fold increase in virus concentration (0.06µg/cm2) as compared to gold standard method. The second method utilized tandem sucrose pelleting without a PEG precipitation step, yielding 83-fold increase in virus concentration (0.24µg/cm2), but it was more labor-intensive and cannot be efficiently scaled up. Both protocols provide radically higher virus yields compared with traditional virus purification protocols involving PEG-precipitation and sucrose gradient ultracentrifugation. Formalin inactivation of CVB1 produced a vaccine that induced a strong, virus-neutralizing antibody response in vaccinated mice, which protected against challenge with CVB1 virus. Altogether, these results provide valuable information for the development of new enterovirus vaccines.


Assuntos
Infecções por Coxsackievirus/prevenção & controle , Enterovirus Humano A/imunologia , Imunogenicidade da Vacina , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Infecções por Coxsackievirus/imunologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Enterovirus Humano A/crescimento & desenvolvimento , Enterovirus Humano A/isolamento & purificação , Feminino , Formaldeído/farmacologia , Camundongos , Polissorbatos/farmacologia , Vacinação , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/imunologia , Vacinas de Produtos Inativados/isolamento & purificação , Células Vero , Vacinas Virais/administração & dosagem , Vacinas Virais/isolamento & purificação , Cultura de Vírus
20.
Diabetologia ; 60(3): 424-431, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28070615

RESUMO

AIMS/HYPOTHESIS: This case-control study was nested in a prospective birth cohort to evaluate whether the presence of enteroviruses in stools was associated with the appearance of islet autoimmunity in the Type 1 Diabetes Prediction and Prevention study in Finland. METHODS: Altogether, 1673 longitudinal stool samples from 129 case children who turned positive for multiple islet autoantibodies and 3108 stool samples from 282 matched control children were screened for the presence of enterovirus RNA using RT-PCR. Viral genotype was detected by sequencing. RESULTS: Case children had more enterovirus infections than control children (0.8 vs 0.6 infections per child). Time-dependent analysis indicated that this excess of infections occurred more than 1 year before the first detection of islet autoantibodies (6.3 vs 2.1 infections per 10 follow-up years). No such difference was seen in infections occurring less than 1 year before islet autoantibody seroconversion or after seroconversion. The most frequent enterovirus types included coxsackievirus A4 (28% of genotyped viruses), coxsackievirus A2 (14%) and coxsackievirus A16 (11%). CONCLUSIONS/INTERPRETATION: The results suggest that enterovirus infections diagnosed by detecting viral RNA in stools are associated with the development of islet autoimmunity with a time lag of several months.


Assuntos
Autoimunidade/imunologia , Enterovirus/imunologia , Enterovirus/patogenicidade , Fezes/virologia , Ilhotas Pancreáticas/imunologia , Autoimunidade/genética , Estudos de Casos e Controles , Pré-Escolar , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Feminino , Genótipo , Humanos , Lactente , Masculino , Estudos Prospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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