RESUMO
Soon after activation, CD4+ T cells are segregated into BCL6+ follicular helper (Tfh) and BCL6- effector (Teff) T cells. Here, we explored how these subsets are maintained during chronic antigen stimulation using the mouse chronic LCMV infection model. Using single cell-transcriptomic and epigenomic analyses, we identified a population of PD-1+ TCF-1+ CD4+ T cells with memory-like features. TCR clonal tracing and adoptive transfer experiments demonstrated that these cells have self-renewal capacity and continue to give rise to both Teff and Tfh cells, thus functioning as progenitor cells. Conditional deletion experiments showed Bcl6-dependent development of these progenitors, which were essential for sustaining antigen-specific CD4+ T cell responses to chronic infection. An analogous CD4+ T cell population developed in draining lymph nodes in response to tumors. Our study reveals the heterogeneity and plasticity of CD4+ T cells during persistent antigen exposure and highlights their population dynamics through a stable, bipotent intermediate state.
Assuntos
Antígenos , Linfócitos T Auxiliares-Indutores , Transferência Adotiva , Animais , Diferenciação Celular , Camundongos , Proteínas Proto-Oncogênicas c-bcl-6/genética , Células-TroncoRESUMO
Epidermal tissues play vital roles in maintaining homeostasis and preventing the dysregulation of the cutaneous barrier. Sphingomyelin (SM), a sphingolipid synthesized by sphingomyelin synthase (SMS) 1 and 2, is involved in signal transduction via modulation of lipid-raft functions. Though the implications of SMS on inflammatory diseases have been reported, its role in dermatitis has not been clarified. In this study, we investigated the role of SM in the cutaneous barrier using a dermatitis model established by employing Sgms1 and 2 deficient mice. SM deficiency impaired the cutaneous inflammation and upregulated signal transducer and activator of transcription 3 (STAT3) phosphorylation in epithelial tissues. Furthermore, using mouse embryonic fibroblast cells, the sensitivity of STAT3 to Interleukin-6 stimulation was increased in Sgms-deficient cells. Using tofacitinib, a clinical JAK inhibitor, the study showed that SM deficiency might participate in STAT3 phosphorylation via JAK activation. Overall, these results demonstrate that SM is essential for maintaining the cutaneous barrier via the STAT3 pathway, suggesting SM could be a potential therapeutic target for dermatitis treatment.
Assuntos
Fator de Transcrição STAT3/fisiologia , Pele/metabolismo , Esfingomielinas/fisiologia , Animais , Células Cultivadas , Dermatite/tratamento farmacológico , Dermatite/etiologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Transdução de Sinais/fisiologia , Esfingomielinas/uso terapêutico , Transferases (Outros Grupos de Fosfato Substituídos)/fisiologiaRESUMO
BACKGROUND: Adolescent pregnancy is a serious reproductive health problem in Tanzania. However, the risk factors for multidimensional attitudes and behaviors of reproductive health toward pregnancy in Tanzanian adolescents remain unexplored. METHODS: We collected baseline characteristics and information on attitudes and behaviors of reproductive health from 4161 Tanzanian adolescents in all 54 primary and secondary schools in the Korogwe district. We applied mixed effect multiple regression analyses stratified by sex to find the factors related to reproductive health attitudes and behaviors toward pregnancy. RESULTS: In female students, regarding the attitudes of reproductive health, higher age, hope for marriage in the future, a talk with a parent about sex or pregnancy, and a higher hope score were significantly associated with a lower score. For the behaviors of reproductive health, higher age, a talk with a parent about sex or pregnancy, time to talk with a parent about daily life, and a higher hope score were significantly associated with a lower score. In male students, regarding the attitudes of reproductive health, a higher hope score was significantly associated with a lower score. For the behaviors of reproductive health, higher age, time to talk with a parent about daily life, and a higher hope score was significantly associated with a lower score. CONCLUSIONS: The heterogeneous factor-outcomes association between female and male students suggested that sex-specialized interventions may be required to change their risky attitudes or behaviors of reproductive health. Although we cannot conclude as points of intervention, our study suggested that it may be practical to improve parent-adolescents communication about sex or reproductive health and change adolescents' views of pregnancy or marriage for gaining financial or social status.
Assuntos
Saúde Reprodutiva , Comportamento Sexual , Gravidez , Humanos , Masculino , Feminino , Adolescente , Estudos Transversais , Tanzânia , Atitude Frente a Saúde , Conhecimentos, Atitudes e Prática em SaúdeRESUMO
T helper type 2 (T(H)2) bias, which is the propensity of naive CD4(+) T cells to differentiate into interleukin 4 (IL-4)-secreting T(H)2 cells, is a genetic trait that affects susceptibility to infectious, autoimmune and allergic diseases. T(H)2 bias correlates with the amount of IL-4 initially secreted by newly activated helper T cells that feeds back positively through the pathway of the IL-4 receptor and the transcription factors STAT6 and GATA-3 to drive T(H)2 development. Here we identify Mina, a member of the jumonji C (JmjC) protein family, as a genetic determinant of T(H)2 bias. Mina specifically bound to and repressed the Il4 promoter. Mina overexpression in transgenic mice impaired Il4 expression, whereas its knockdown in primary CD4(+) T cells led to Il4 derepression. Our findings collectively provide mechanistic insight into an Il4-regulatory pathway that controls helper T cell differentiation and genetic variation in T(H)2 bias.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Interleucina-4/biossíntese , Proteínas de Neoplasias/fisiologia , Proteínas Nucleares/fisiologia , Células Th2/imunologia , Animais , Sequência de Bases , Linfócitos T CD4-Positivos/citologia , Diferenciação Celular , Fator de Transcrição GATA3/metabolismo , Haplótipos , Interleucina-4/genética , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Fator de Transcrição STAT6/metabolismo , Especificidade da Espécie , Células Th2/citologiaRESUMO
Mycoplasma bovis is a destructive pathogen that causes large economic losses in rearing cattle for beef and dairy worldwide. M. bovis causes suppression of and evades the host immune response; however, the mechanisms of host immune function involved in M. bovis mastitis have not been elucidated. The purpose of this study was to elucidate the characteristics of the bovine immune response to mycoplasmal mastitis. We evaluated the responsiveness of the bovine mammary gland following infusion of M. bovis Somatic cell counts and bacterial counts in milk from the infected quarter were increased. However, the proliferation of peripheral blood mononuclear cells (blood MNCs) and mononuclear cells isolated from M. bovis-stimulated mammary lymph nodes (lymph node MNCs) did not differ from that in the unstimulated cells. Transcriptome analysis revealed that the mRNA levels of innate immune system-related genes in blood MNCs, complement factor D (CFD), ficolin 1 (FCN1), and tumor necrosis factor superfamily member 13 (TNFSF13) decreased following intramammary infusion of M. bovis The mRNA levels of immune exhaustion-related genes, programmed cell death 1 (PD-1), programmed cell death-ligand 1 (PD-L1), lymphocyte activation gene 3 (LAG3), and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) of milk mononuclear cells (milk MNCs) in the infected quarter were increased compared with those before infusion. Increase in immune exhaustion-related gene expression and decrease in innate immune response-related genes of MNCs in quarters from cows were newly characterized by M. bovis-induced mastitis. These results suggested that M. bovis-induced mastitis affected the immune function of bovine MNCs, which is associated with prolonged duration of infection with M. bovis.
Assuntos
Imunidade Inata/imunologia , Glândulas Mamárias Animais/imunologia , Mastite Bovina/imunologia , Mycoplasma bovis , Animais , Bovinos , Feminino , Tolerância Imunológica , Peptídeos e Proteínas de Sinalização Intercelular/metabolismoRESUMO
Virulence factors responsible for bacterial pathogenicity are often encoded by plasmids. In Melissococcus plutonius, the causative agent of European foulbrood of honey bees, a putative virulence plasmid (pMP19) possessing mtxA, which encodes a putative insecticidal toxin, was found by comparative genome analyses. However, as the role of pMP19 in the pathogenesis of European foulbrood remains to be elucidated, we generated pMP19 cured-M. plutonius from representative strains of the three genetically distinct groups (CC3, CC12 and CC13) and compared their virulence against Apis mellifera larvae using our in vitro infection model. Under the conditions tested, the loss of pMP19 abrogated the pathogenicity in CC3 strains, and > 94% of pMP19-cured CC3 strain-infected larvae became adult bees, suggesting that pMP19 is a virulence determinant of CC3 strains. However, introduction of mtxA on its own did not increase the virulence of pMP19-cured strains. In contrast to CC3 strains, the representative CC12 strain remained virulent even in the absence of pMP19, whereas the representative CC13 strain was avirulent even in the presence of the plasmid. Thus, pMP19 plays a role in the virulence of M. plutonius; however, its impact on the virulence varies among strains with different genetic backgrounds.
Assuntos
Abelhas/microbiologia , Enterococcaceae/genética , Enterococcaceae/patogenicidade , Virulência/genética , Animais , Toxinas Bacterianas/genética , Genoma Bacteriano/genética , Larva/microbiologia , PlasmídeosRESUMO
Royal jelly (RJ), a brood food of honey bees, has strong antimicrobial activity. Melissococcus plutonius, the causative agent of European foulbrood of honey bees, exhibits resistance to this antimicrobial activity and infects larvae orally. Among three genetically distinct groups (CC3, CC12 and CC13) of M. plutonius, CC3 strains exhibit the strongest RJ resistance. In this study, to identify genes involved in RJ resistance, we generated an RJ-susceptible derivative from a highly RJ-resistant CC3 strain by UV mutagenesis. Genome sequence analysis of the derivative revealed the presence of a frameshift mutation in the putative regulator gene spxA1a. The deletion of spxA1a from a CC3 strain resulted in increased susceptibility to RJ and its antimicrobial component 10-hydroxy-2-decenoic acid. Moreover, the mutant became susceptible to low-pH and oxidative stress, which may be encountered in brood foods. Differentially expressed gene analysis using wild-type and spxA1a mutants revealed that 45 protein-coding genes were commonly upregulated in spxA1a-positive strains. Many upregulated genes were located in a prophage region, and some highly upregulated genes were annotated as universal/general stress proteins, oxidoreductase/reductase, chaperons and superoxide dismutase. These results suggest that SpxA1a is a key regulator to control the tolerance status of M. plutonius against stress in honey bee colonies.
Assuntos
Abelhas/microbiologia , Farmacorresistência Bacteriana/genética , Enterococcaceae/efeitos dos fármacos , Enterococcaceae/genética , Ácidos Graxos Monoinsaturados/farmacologia , Ácidos Graxos/farmacologia , Animais , Antibacterianos/farmacologia , Ácidos Graxos/química , Mutação da Fase de Leitura , Deleção de Genes , Larva/microbiologia , Mutagênese , Raios UltravioletaRESUMO
BACKGROUND: Aspiculuris tetraptera, as a parasitic pinworm, is most frequently detected in laboratory mice, and transmission is mediated by the eggs contained in the faeces of infected mice. A highly sensitive and quantitative faeces-based diagnostic tool would be useful for the early detection of A. tetraptera to inhibit the expansion of infection. In this study, we developed a quantitative assay that exhibits high sensitivity in detecting A. tetraptera in faeces using PCR techniques. RESULTS: Endpoint PCR demonstrated the detection of A. tetraptera DNA in 0.5 ng genomic DNA extracted from the faeces of infected mice. To quantitatively detect the small amount of A. tetraptera DNA, locked nucleic acid (LNA)-based primers and LNA-based TaqMan probes were used for the quantitative PCR assay (qPCR). The combination of LNA-based DNA increased detection sensitivity by more than 100-fold compared to using normal oligo DNAs. The copy number of the A. tetraptera DNA detected was positively related to the infected faeces-derived genomic DNA with a simple linearity regression in the range of 20 pg to 15 ng of the genomic DNA. To more conveniently detect infection using faeces, the LNA-based TaqMan assay was applied to the crude fraction of the faeces without DNA purification. An assay using ethanol precipitation of the faeces yielded results consistent with those of direct microscopic observation. CONCLUSION: The LNA-TaqMan assay developed in this study quantitatively detects A. tetraptera infection in mouse faeces.
Assuntos
DNA de Helmintos/análise , Fezes/parasitologia , Oxyuroidea/isolamento & purificação , Carga Parasitária/normas , Animais , Primers do DNA , DNA de Helmintos/genética , Camundongos , Oligonucleotídeos , Oxyuroidea/genética , Carga Parasitária/métodos , Reação em Cadeia da Polimerase em Tempo Real , Padrões de ReferênciaRESUMO
BACKGROUND: Caudata species such as salamanders are easily affected by environmental changes, which can drastically reduce their population. The effects of acute X-rays and chronic γ-irradiation on Hynobius lichenatus, the Japanese Tohoku hynobiid salamander, are known. However, the expression of radiation-inducible genes, such as the DNA-damage checkpoint response gene p53, has not been analyzed in H. lichenatus. This has not occurred because there is no established method for mRNA quantification in H. lichenatus due to a lack of information on available nucleotide sequences corresponding to both radiation-inducible genes and endogenous control genes such as ACTB (ß-actin). RESULTS: In this study, we aimed to evaluate the effects of radiation on gene expression in H. lichenatus. Using RNA extracted from irradiated salamanders, we performed rapid amplification of cDNA ends (RACE) and cloned H. lichenatus ß-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and p53. We confirmed that the cloned cDNAs were able to synthesize salamander proteins by western blotting after transfection into cultured HEK293 cells. Proliferation assays using HEK293 cells stably expressing H. lichenatus p53 protein showed that this protein has antiproliferative effects, similar to that of mammalian p53. Furthermore, RT-qPCR analysis using gene-specific primers revealed that p53 mRNA expression in H. lichenatus was upregulated upon exposure to radiation. CONCLUSION: Our results suggest that H. lichenatus p53 protein take an important role in regulating the cellular responses to various stimuli as mammalian p53 does. Furthermore, our study provides novel data to select appropriate primers to analyze internal control mRNA expression in H. lichenatus and to evaluate p53 expression as a marker of radiation and environmental stimuli.
Assuntos
Proteínas de Anfíbios/genética , Expressão Gênica/efeitos da radiação , Radiação , Pele/efeitos da radiação , Proteína Supressora de Tumor p53/genética , Urodelos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Células HEK293 , Humanos , Homologia de SequênciaRESUMO
BACKGROUND: The purpose of this study was to clarify the species of Candida that are important for the development of atrophic glossitis in xerostomia patients. METHODS: A total of 231 patients with subjective dry mouth were enrolled in the present study. Logistic regression analysis was performed to clarify the contribution of each Candida species and other variables to the development of atrophic glossitis. The dependent variable was the absence/presence of atrophic glossitis. The Candida colony-forming units (CFU) of C. albicans, C. glabrata, C. tropicalis, and C. krusei, as well as age, gender, resting (RSFR) and stimulated (SSFR) whole salivary flow rate, and denture-wearing status, were treated as explanatory variables. RESULTS: Logistic regression analysis showed that two factors were closely associated with the presence of atrophic glossitis: an increase in C. albicans CFU and a decrease in the SSFR. CONCLUSIONS: C. albicans, but not non-albicans Candida, was associated with atrophic glossitis in xerostomia patients who had no systemic predisposing factors, indicating that C. albicans remains a treatment target for Candida-related atrophic glossitis.
Assuntos
Candida albicans/isolamento & purificação , Candida/isolamento & purificação , Candidíase/microbiologia , Glossite/microbiologia , Língua/microbiologia , Xerostomia/complicações , Idoso , Idoso de 80 Anos ou mais , Atrofia , Candida/patogenicidade , Candidíase/etiologia , Contagem de Colônia Microbiana , Feminino , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-IdadeRESUMO
Estrogen is known to have anti-inflammatory effects, that are thought to be mediated by the classical estrogen receptors (ERs), ERα and ERß. G protein coupled estrogen receptor1 (GPER) is a novel membrane-type estrogen receptor that can mediate non-genomic estrogenic responses. Although there have been several reports asserting that the participation of GPER in anti-inflammatory effects is induced by estrogen, the role of GPER remains poorly understood. In this study, we investigated the involvement of GPER in the regulation of a representative inflammatory cytokine, IL-6. We first examined the expression of IL-6 mRNA by TNFα stimulation in the transfection of GPER-expression plasmid into HeLa cells. Exogenous GPER significantly inhibited TNFα-induced IL-6 expression, and blocked NF-κB promoter activity inducing the expression of IL-6 in a dose-dependent manner. The promoter activity was restored almost to control level by transfection with the C-terminal deletion mutant of GPER. Similar results have been observed in endogenous GPER using SKBR3 cells which do not express the classical ERs. The data have been validated by treatment of GPER with siRNA. These findings indicate that GPER negatively regulates TNFα-induced IL-6 expression, probably through inhibition of NF-κB promoter activity by a signal(s) derived from the C-terminal region of GPER.
Assuntos
Neoplasias da Mama/genética , Interleucina-6/genética , Receptores de Estrogênio/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Fator de Necrose Tumoral alfa/farmacologia , Neoplasias da Mama/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Interleucina-6/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Receptores de Estrogênio/genética , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Células Tumorais CultivadasRESUMO
OBJECTIVE: The purpose of this study was to investigate the procedures for efficiently diagnosing Sjögren's syndrome to reduce patient burden. METHODS: This study analyzed data from 254 Japanese patients diagnosed with Sjögren's syndrome out of 4967 who visited our clinic complaining of xerostomia. RESULTS: Of the 254 Sjögren's syndrome patients, 140 fulfilled the criteria proposed by the Committee on Sjögren's Syndrome of the Ministry of Health and Welfare of Japan, 228 fulfilled the criteria proposed by the American-European Consensus Group, and 69 fulfilled the criteria proposed by the American College of Rheumatology. Numbers of definitive cases varied with each set of criteria. Logistic regression analysis was used to analyze useful examination items for definitive diagnosis of Sjögren's syndrome, demonstrating that anti-Ro/SSA (odds ratio (OR), 7.165), lip biopsy (OR, 4.273), sialography (OR, 2.402), and ANA (OR, 0.678) correlated significantly with definitive diagnosis of Sjögren's syndrome. CONCLUSIONS: These results suggest that the following diagnostic procedure for Sjögren's syndrome would reduce burden on patients. When clinicians choose examination items for diagnosing Sjögren's syndrome, they should first select which criteria to use. Then, to minimize the number of examination items, examinations should be performed in order of anti-SSA antibody, lip biopsy, and parotid gland sialography.
Assuntos
Efeitos Psicossociais da Doença , Síndrome de Sjogren/diagnóstico , Xerostomia/etiologia , Autoanticorpos/sangue , Estudos Transversais , Humanos , Japão , Ribonucleoproteínas/imunologia , Síndrome de Sjogren/complicações , Síndrome de Sjogren/imunologia , Xerostomia/imunologiaRESUMO
Tyramine, a trace monoamine produced from tyrosine by decarboxylation and found naturally in foods, plants, and animals, is a suspected virulence factor of Melissococcus plutonius that causes European foulbrood in honey bee brood. In the present study, we developed a method for quantitative analysis of tyramine in culture medium and honey bee larvae with a limit of quantitation of 3 ng/mL and a recovery rate of >97% using Liquid Chromatography-Mass Spectrometry/Mass Spectrometry and deuterium-labeled tyramine, demonstrating for the first time that a highly virulent M. plutonius strain actually produces tyramine in infected larvae. This method will be an indispensable tool to elucidate the role of tyramine in European foulbrood pathogenesis in combination with exposure bioassays using artificially reared bee larvae.
Assuntos
Enterococcaceae , Larva , Tiramina , Animais , Larva/microbiologia , Abelhas/microbiologia , Tiramina/análise , Enterococcaceae/isolamento & purificação , Cromatografia Líquida/veterinária , Espectrometria de Massas em Tandem/veterináriaRESUMO
OBJECTIVE: The purpose of this study was to investigate the efficacy of Fungiflora Y staining (fluorescent stain) for the diagnosis of erythematous candidiasis. SUBJECTS AND METHODS: This study enrolled 48 patients who were diagnosed with erythematous candidiasis and who underwent fungal culture and microscopic examination of a smear specimen stained with CytoQuick (modification of the Giemsa stain) and Fungiflora Y. Fungiflora Y staining was observed using a portable fluorescent microscope (CyScope(®)). The sensitivity, specificity and positive and negative predictive values were calculated using fungal culture as the gold standard test. Accuracy was calculated, and the difference between the CytoQuick and Fungiflora Y groups was examined using contingency tables and the chi-square test. RESULTS: The sensitivity and specificity of the CytoQuick stain was 0.51 and 0.91, respectively; the positive predictive value was 0.95, and the negative predictive value was 0.36. The sensitivity and specificity of the Fungiflora Y stain was 0.84 and 1.0, respectively; the positive predictive value was 1.00, and the negative predictive value was 0.65. The accuracy of Fungiflora Y (0.88) was superior to that of CytoQuick (0.60) (p = 0.0052). CONCLUSIONS: Microscopic examinations of smear specimens using a combination of Fungiflora Y staining and the CyScope(®) portable fluorescent microscope was found to be useful for the diagnosis of oral erythematous candidiasis.
Assuntos
Candidíase Bucal/diagnóstico , Corantes Fluorescentes , Adulto , Idoso , Idoso de 80 Anos ou mais , Corantes Azur , Candida albicans/isolamento & purificação , Candida glabrata/isolamento & purificação , Candida tropicalis/isolamento & purificação , Corantes , Citodiagnóstico/métodos , Feminino , Humanos , Masculino , Microscopia de Fluorescência/métodos , Pessoa de Meia-Idade , Micologia/métodos , Compostos Orgânicos , Valor Preditivo dos Testes , Sensibilidade e Especificidade , Doenças da Língua/microbiologiaRESUMO
Helicobacter pylori is a well-known pathogen that causes chronic gastritis, leading to the development of gastric cancer. This bacterium has also been detected in dogs, and symptoms similar to those in humans have been reported. The cytotoxin-associated gene A (CagA) is involved in pathogenesis through aberrant activation of host signal transduction, including the nuclear factor-kappa B (NF-κB) pathway. We have previously shown the anti-inflammatory effect of the G-protein-coupled estrogen receptor (GPER) via inhibiting of NF-κB activation in several cells. Therefore, here, we investigated the effect of GPER on CagA-mediated NF-κB promoter activity and showed that CagA overexpression in gastric cancer cells activated the NF-κB reporter and induced interleukin 8 (il-8) expression, both of which were inhibited by the GPER agonist.
Assuntos
Doenças do Cão , Infecções por Helicobacter , Helicobacter pylori , Neoplasias Gástricas , Animais , Cães , Humanos , Citotoxinas/metabolismo , Doenças do Cão/metabolismo , Mucosa Gástrica/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Infecções por Helicobacter/metabolismo , Infecções por Helicobacter/veterinária , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Interleucina-8/genética , NF-kappa B/metabolismo , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/veterináriaRESUMO
Streptococcus pluranimalium, an emerging zoonotic pathogen associated with infections in various animal species and humans, cannot be reliably identified by phenotypic characterization using the commercial kits routinely used in laboratories. We herein developed the first S. pluranimalium-specific PCR assay useful for the easy and reliable identification of this species.
Assuntos
Infecções Estreptocócicas , Animais , Humanos , Infecções Estreptocócicas/diagnóstico , Infecções Estreptocócicas/veterinária , Streptococcus/genética , Técnicas de Amplificação de Ácido Nucleico , Reação em Cadeia da PolimeraseRESUMO
Mast cells are a significant source of cytokines and chemokines that play a role in pathological processes. Gangliosides, which are complex lipids with a sugar chain, are present in all eukaryotic cell membranes and comprise lipid rafts. Ganglioside GM3, the first ganglioside in the synthetic pathway, is a common precursor of the specifying derivatives and is well known for its various functions in biosystems. Mast cells contain high levels of gangliosides; however, the involvement of GM3 in mast cell sensitivity is unclear. Therefore, in this study, we elucidated the role of ganglioside GM3 in mast cells and skin inflammation. GM3 synthase (GM3S)-deficient mast cells showed cytosolic granule topological changes and hyperactivation upon IgE-DNP stimulation without affecting proliferation and differentiation. Additionally, inflammatory cytokine levels increased in GM3S-deficient bone marrow-derived mast cells (BMMC). Furthermore, GM3S-KO mice and GM3S-KO BMMC transplantation showed increased skin allergic reactions. Besides mast cell hypersensitivity caused by GM3S deficiency, membrane integrity decreased and GM3 supplementation rescued this loss of membrane integrity. Additionally, GM3S deficiency increased the phosphorylation of p38 mitogen-activated protein kinase. These results suggest that GM3 increases membrane integrity, leading to the suppression of the p38 signalling pathway in BMMC and contributing to skin allergic reaction.
Assuntos
Gangliosídeo G(M3) , Mastócitos , Camundongos , Animais , Gangliosídeo G(M3)/metabolismo , Mastócitos/metabolismo , Diferenciação Celular , CitocinasRESUMO
Knowledge about bacterial species in bee environments is essential for maintaining healthy honeybee colonies. Therefore, we performed whole-genome sequence analysis on bacteria isolated from honey harvested in Japan. This study reports the genomic sequences of the five bacterial strains identified.
RESUMO
American foulbrood (AFB) is a honeybee disease caused by Paenibacillus larvae, and tylosin is used as the prophylactic in Japan. Honey contains macrolide-resistant bacteria that are a potential source of genes that may confer tylosin resistance to P. larvae. To investigate the potential risk of such genes in Japanese honey, we developed real-time PCR assays for the detection of important macrolide resistance genes, ermC and ermB, and analyzed 116 Japanese honey samples with known contamination status of P. larvae. Consequently, 91.38% of samples contained ermC and/or ermB, and 71.55% of samples contained both ermC and P. larvae, suggesting the possible emergence of tylosin-resistant P. larvae in Japan. Therefore, judicious use of the prophylactic is essential in maintaining its effectiveness.
Assuntos
Mel , Macrolídeos , Abelhas/genética , Estados Unidos , Animais , Macrolídeos/farmacologia , Antibacterianos/farmacologia , Tilosina , Mel/análise , Mel/microbiologia , Japão , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Farmacorresistência Bacteriana/genética , Larva/microbiologiaRESUMO
Paenibacillus larvae and Melissococcus plutonius are the causative agents of American and European foulbroods of honey bees, respectively. Since their virulence and resistance to disinfectants differ depending on the genotypes/phenotypes of the strains, the discrimination of strain types is important for the effective control of these diseases. Methods to detect and differentiate pathogens in honey are useful for surveying the contamination status of beehives/apiaries. In the present study, we selected a sequence (GenBank accession no. FI763267) as the specific target for enterobacterial repetitive intergenic consensus (ERIC) II-type P. larvae strains for the first time and developed a novel multiplex PCR assay that precisely distinguishes between the major types of foulbrood pathogens (ERIC I and II P. larvae and typical and atypical M. plutonius) in one reaction. In addition, we found that commercially available kits designed for DNA extraction from Mycobacterium in feces efficiently extracted DNA from foulbrood pathogens in honey. Using the multiplex PCR assay and DNA extraction kits, all the targeted types of P. larvae and M. plutonius were detected in honey spiked with the pathogens at a concentration of 100 bacterial cells/strain/ml. Moreover, 94% of the Japanese honey samples examined in the present study were contaminated with one or more types of the foulbrood pathogens. These results indicate that the newly developed methods are useful for detecting foulbrood pathogens in honey. The epidemiological information obtained by these methods will contribute to the effective control of foulbroods in apiaries.