RESUMO
Chronic spontaneous urticaria (CSU) is characterized by daily recurring wheal and flare with itch for more than 6 weeks. The extrinsic coagulation system has been shown to be activated in correlation with CSU severity. We have reported that tissue factor (TF), a trigger of the extrinsic coagulation cascade, is synergistically expressed on vascular endothelial cells by simultaneous stimulation with TF inducers (TFI), followed by activation of the extrinsic coagulation cascade and hyper permeability in vitro. However, vascular endothelial cells are not likely to be simultaneously stimulated by multiple TFIs under physiological conditions. Therefore, in order to know whether sequential, rather than simultaneous, stimuli with interval may induce synergistic activation of TF, we investigated the time course of the priming effects of each TFI for synergistic TF expression in vascular endothelial cells (HUVECs). We stimulated HUVECs with a TFI (first stimulation) and then stimulated cells with another TFI at indicated time points (second stimulation) and detected TF expression and activity. The TF expression induced by simultaneous stimulation diminished in a few hours. However, both synergistic enhancement of TF expression and activation level of the coagulation cascade were detected even when the second stimulation was added 18 or 22 h after the first stimulation. Thus, the priming effect of TFI for synergistic TF expression may persist for a half day or longer.
Assuntos
Células Endoteliais , Tromboplastina , Humanos , Tromboplastina/genética , Tromboplastina/metabolismo , Células Endoteliais/metabolismo , Coagulação Sanguínea , Células CultivadasAssuntos
Basófilos/imunologia , Fatores de Coagulação Sanguínea/metabolismo , Urticária Crônica/imunologia , Complemento C5/metabolismo , Mastócitos/imunologia , Receptor da Anafilatoxina C5a/metabolismo , Pele/imunologia , Degranulação Celular , Células Cultivadas , Liberação de Histamina , HumanosAssuntos
Coagulação Sanguínea/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Histamina/farmacologia , Lipopolissacarídeos/toxicidade , Tromboplastina/biossíntese , Receptores Toll-Like/metabolismo , Urticária/metabolismo , Coagulação Sanguínea/genética , Doença Crônica , Células Endoteliais da Veia Umbilical Humana , Humanos , Ligantes , Mastócitos/metabolismo , Mastócitos/patologia , Tromboplastina/genética , Receptores Toll-Like/genética , Urticária/genética , Urticária/patologiaRESUMO
Objective: During the coronavirus disease pandemic in Japan, all patients with respiratory symptoms were initially tested for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This study describes the respiratory pathogens detected from patients who tested negative for SARS-CoV-2 at the Saitama Institute of Public Health from January to December 2020. Methods: We performed pathogen retrieval using multiplex real-time polymerase chain reaction on samples from patients with acute respiratory diseases who tested negative for SARS-CoV-2 in Saitama in 2020 and analysed the results by age and symptoms. Results: There were 1530 patients aged 0-104 years (1727 samples), with 14 pathogens detected from 213 patients (245 samples). Most pathogens were human metapneumovirus (25.4%, 54 cases), rhinovirus (16.4%, 35 cases) and Mycoplasma pneumoniae (13.1%, 23 cases). Human metapneumovirus, human coronavirus (but not NL63) and M. pneumoniae were detected in almost all age groups without any significant bias. Seasonal human coronaviruses, human metapneumovirus, M. pneumoniae and several other pathogens were detected until April 2020. Discussion: Multiple respiratory pathogens were circulating during 2020 in Saitama, including SARS-CoV-2 and influenza viruses. We suggest introducing a system that can comprehensively monitor the regional prevalence of all viruses that cause acute respiratory infections.
Assuntos
COVID-19 , Metapneumovirus , Infecções Respiratórias , Humanos , SARS-CoV-2 , COVID-19/epidemiologia , Japão/epidemiologia , Infecções Respiratórias/epidemiologiaRESUMO
Background: Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by intermittent itchy rash. Type 2 inflammatory cytokines such as interleukin (IL)-4, IL-13, and IL-31 are strongly implicated in AD pathogenesis. Stimulation of IL-31 cognate receptors on C-fiber nerve endings is believed to activate neurons in the dorsal root ganglion (DRG), causing itch. The IL-31 receptor is a heterodimer of OSMRß and IL31RA subunits, and OSMRß can also bind oncostatin M (OSM), a pro-inflammatory cytokine released by monocytes/macrophages, dendritic cells, and T lymphocytes. Further, OSM expression is enhanced in the skin lesions of AD and psoriasis vulgaris patients. Objective: The current study aimed to examine the contributions of OSM to AD pathogenesis and symptom expression. Methods: The expression levels of the OSM gene (OSM) and various cytokine receptor genes were measured in human patient skin samples, isolated human monocytes, mouse skin samples, and mouse DRG by RT-qPCR. Itching responses to various pruritogens were measured in mice by counting scratching episodes. Results: We confirmed overexpression of OSM in skin lesions of patients with AD and psoriasis vulgaris. Monocytes isolated from the blood of healthy subjects overexpressed OSM upon stimulation with IL-4 or GM-CSF. Systemic administration of OSM suppressed IL31RA expression in the mouse DRG and IL-31-stimulated scratching behavior. In contrast, systemic administration of OSM increased the expression of IL-4- and IL-13-related receptors in the DRG. Conclusion: These results suggest that OSM is an important cytokine in the regulation of skin monocytes, promoting the actions of IL-4 and IL-13 in the DRG and suppressing the action of IL-31. It is speculated that OSM released from monocytes in skin modulates the sensitivity of DRG neurons to type 2 inflammatory cytokines and thereby the severity of AD-associated skin itch.
Assuntos
Dermatite Atópica , Psoríase , Humanos , Camundongos , Animais , Oncostatina M/farmacologia , Oncostatina M/metabolismo , Interleucina-4/metabolismo , Gânglios Espinais/metabolismo , Interleucina-13/metabolismo , Prurido/metabolismo , Interleucinas/genética , Interleucinas/metabolismo , Dermatite Atópica/metabolismo , Receptores de Interleucina/metabolismo , Psoríase/metabolismoRESUMO
Propofol is widely used for general anesthesia and sedation; however, the mechanisms of its anesthetic and adverse effects are not fully understood. We have previously shown that propofol activates protein kinase C (PKC) and induces its translocation in a subtype-specific manner. The purpose of this study was to identify the PKC domains involved in propofol-induced PKC translocation. The regulatory domains of PKC consist of C1 and C2 domains, and the C1 domain is subdivided into the C1A and C1B subdomains. Mutant PKCα and PKCδ with each domain deleted were fused with green fluorescent protein (GFP) and expressed in HeLa cells. Propofol-induced PKC translocation was observed by time-lapse imaging using a fluorescence microscope. The results showed that persistent propofol-induced PKC translocation to the plasma membrane was abolished by the deletion of both C1 and C2 domains in PKCα and by the deletion of the C1B domain in PKCδ. Therefore, propofol-induced PKC translocation involves the C1 and C2 domains of PKCα and the C1B domain of PKCδ. We also found that treatment with calphostin C, a C1 domain inhibitor, abolished propofol-induced PKCδ translocation. In addition, calphostin C inhibited the propofol-induced phosphorylation of endothelial nitric oxide synthase (eNOS). These results suggest that it may be possible to modulate the exertion of propofol effects by regulating the PKC domains involved in propofol-induced PKC translocation.
Assuntos
Propofol , Proteína Quinase C , Humanos , Proteína Quinase C/metabolismo , Proteína Quinase C-alfa/metabolismo , Propofol/farmacologia , Células HeLa , Isoenzimas/metabolismo , Transporte ProteicoRESUMO
Many anesthetics, including Propofol, have been reported to induce elevation of intracellular calcium, and we were interested to investigate the possible contribution of calcium elevation to the mechanism of the newly approved remimazolam actions. Remimazolam is an intravenous anesthetic first approved in Japan in July 2020, and is thought to exert its anesthetic actions via γ-aminobutyric acid A (GABAA) receptors; however, the precise mechanisms of how remimazolam elevates intracellular calcium levels remains unclear. We examined the remimazolam-induced elevation of intracellular calcium using SHSY-5Y neuroblastoma cells, COS-7 cells, HEK293 cells, HeLa cells, and human umbilical vein endothelial cells (HUVECs) loaded with fluorescent dyes for live imaging. We confirmed that high concentrations of remimazolam (greater than 300 µM) elevated intracellular calcium in a dose-dependent manner in these cells tested. This phenomenon was not influenced by elimination of extracellular calcium. The calcium elevation was abolished when intracellular or intraendoplasmic reticulum (ER) calcium was depleted by BAPTA-AM or thapsigargin, respectively, suggesting that calcium was mobilized from the ER. Inhibitors of G-protein coupled receptors (GPCRs)-mediated signals, including U-73122, a phospholipase C (PLC) inhibitor and xestospongin C, an inositol 1,4,5-triphosphate receptors (IP3R) antagonist, significantly suppressed remimazolam-induced calcium elevation, whereas dantrolene, a ryanodine receptor antagonist, did not influence remimazolam-induced calcium elevation. Meanwhile, live imaging of ER during remimazolam stimulation using ER-tracker showed no morphological changes. These results suggest that high doses of remimazolam increased intracellular calcium concentration in a dose-dependent manner in each cell tested, which was predicted to be caused by calcium mobilization from the ER. In addition, our studies using various inhibitors revealed that this calcium elevation might be mediated by the GPCRs-IP3 pathway. However, further studies are required to identify which type of GPCRs is involved.
Assuntos
Benzodiazepinas/farmacologia , Cálcio/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Cafeína/farmacologia , Linhagem Celular , Dantroleno/farmacologia , Retículo Endoplasmático/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tapsigargina/farmacologiaRESUMO
BACKGROUND: Oral administration of pulverized Konjac glucomannan (KGM) reduces increased plasma IgE and the amount of epsilon-germline transcript (epsilonGT) in the spleen, as well as preventing the development of dermatitis in mice. To elucidate the mechanism of action of pulverized KGM, we solubilized KGM and studied its effect on IgE in vitro and in vivo. METHODS: Solubilized KGM was prepared by acid hydrolysis, and we analyzed the effective molecular size for the suppression of IgE production and epsilonGT in vitro and the level of plasma IgE induced by immunization with ovalbumin in BALB/c mice. RESULTS: The production of IgE and epsilonGT in splenic cells, but not purified B cells, was inhibited by hydrolyzed KGM (KGM hydrolyzed with 0.25 N HCl; H-KGM) at the optimal size of between 10 and 500 kDa. However, no effect was observed when H-KGM was substituted with unhydrolyzed KGM in vitro. IgE production from purified B cells cocultured with purified monocytes, but not with purified T cells, was inhibited by H-KGM. The release of IFNgamma in cultures of monocytes but in purified B cells with or without T cells was enhanced in the presence of H-KGM. Injection of mice with H-KGM also suppressed the production of plasma IgE and IgG1 but not IgG2a in vivo. CONCLUSION: KGM at an optimal size prevents germline class-switching and IgE production both in vitro and in vivo. H-KGM may be useful as a tool to study the mechanism of action of KGM and as a dietary supplement to prevent atopic diseases.
Assuntos
Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Imunoglobulina E/biossíntese , Mananas/farmacologia , Animais , Linfócitos B/citologia , Técnicas de Cocultura , Citocinas/metabolismo , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Hidrólise , Switching de Imunoglobulina/efeitos dos fármacos , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Cadeias épsilon de Imunoglobulina/genética , Interferon gama/metabolismo , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Masculino , Mananas/química , Camundongos , Camundongos Endogâmicos BALB C , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Ovalbumina/imunologia , Baço/citologia , Linfócitos T/citologiaRESUMO
The pathogenesis of chronic spontaneous urticaria (CSU), also called chronic idiopathic urticaria, has been considered to be associated with the activation of the extrinsic blood coagulation cascade. However, the trigger for the extrinsic coagulation cascade in patients with CSU remains unclear. We previously reported that histamine and lipopolysaccharide (LPS) synergistically induced the expression of tissue factor (TF), a trigger for the extrinsic coagulation cascade, in human umbilical vein endothelial cells (HUVEC). Because the elevation of tumor necrosis factor (TNF)-α, interleukin (IL)-33, IL-1ß and vascular endothelial growth factor (VEGF) in serum has also been observed in patients with CSU, we examined the effects of LPS, TNF-α, IL-33, IL-1ß, VEGF and histamine on TF expression in HUVEC by reverse transcription polymerase chain reaction and flow cytometry, as well as examining the activity that triggers the extrinsic coagulation cascade and induces intercellular gap formation of HUVEC in the presence of plasma by Actochrome® TF activity assay and impedance sensor, respectively. The expression of TF mRNA and surface protein of TF on HUVEC in response to histamine or VEGF were synergistically enhanced by the treatment with LPS, TNF-α, IL-33 or IL-1ß. Moreover, the activation of the extrinsic coagulation pathway and intercellular gap formation of HUVEC in response to histamine or VEGF were also synergistically increased in the presence of TNF-α and LPS. Thus, TF expression on vascular endothelial cells was strongly enhanced by co-stimulation with CSU-related molecules in blood. Blocking a common pathway of LPS, TNF-α, IL-33 and IL-1ß, and/or that of VEGF and histamine may be an effective therapeutic measure for patients with severe and refractory CSU.
Assuntos
Lipopolissacarídeos , Fator de Necrose Tumoral alfa , Células Cultivadas , Endotélio Vascular , Histamina/farmacologia , Humanos , Interleucina-33 , Tromboplastina/genética , Fator A de Crescimento do Endotélio VascularRESUMO
We previously reported that fucoidan, a dietary fiber purified from seaweed, inhibited IgE production by B cells in vitro. In this study, we examined the effect of fucoidan on IgE production in vivo. The OVA-induced increase of plasma IgE was significantly suppressed when fucoidan was intraperitoneally, but not orally, administered prior to the first immunization with OVA. The production of IL-4 and IFN-gamma in response to OVA in spleen cells isolated from OVA-sensitized mice treated with fucoidan in vivo was lower than that from mice treated without fucoidan. Moreover, the flow cytometric analysis and ELISpot assay revealed that the administration of fucoidan suppressed a number of IgE-expressing and IgE-secreting B cells, respectively. These results indicate that fucoidan inhibits the increase of plasma IgE through the suppression of IgE-producing B cell population, and the effect of fucoidan in vivo is crucially dependent on the route and timing of its administration.
Assuntos
Linfócitos B/efeitos dos fármacos , Imunoglobulina E/sangue , Polissacarídeos/administração & dosagem , Animais , Linfócitos B/imunologia , Feminino , Terapia de Imunossupressão , Injeções Intraperitoneais , Interferon gama/imunologia , Interleucina-4/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/imunologiaRESUMO
OBJECTIVES: Progress in antiretroviral treatment has led to fewer virological failure cases, but 10%-20% of treatment-naive HIV/AIDS cases are reported to harbor drug-resistant strains, suggesting transmission of drug-resistant HIV. We aimed to determine the trend in prevalence of transmitted drug-resistant (TDR) HIV in Japan, particularly in recently infected patients. METHODS: Drug-resistance test was performed on 3904 HIV-1-infected cases newly diagnosed between 2007 and 2012. The number of cases infected within 6 months [recent seroconverters (RS)] was estimated by BED assay of 2700 plasma samples. Characteristics of RS cases were further analyzed. RESULTS: The overall prevalence of TDR was 9.1%, ranging from 7.3% in 2008% to 12.5% in 2010. Among 1403 subtype B/E/D cases with >50 CD4 T cell counts and >1000 HIV copies per milliliter, 468 (33.4%) were estimated to be RS. The prevalence of RS was significantly higher among cases who were male, Japanese, and men who have sex with men. The prevalence of TDR did not differ significantly between recent and long-term seroconverters (8.5% vs. 9.2%, respectively, P = 0.68). Common mutations in both groups were M46I/L and T215 revertants. Furthermore, sequences with these mutations, K103N and D30N/N88D formed clusters on phylogenetic trees. CONCLUSION: Our study clarified an increase in prevalence of TDR in Japan from 2007 to 2012. The phylogenetic clustering of cases with M46I/L or T215 revertants suggests that HIV with these mutations have become circulating strains. Furthermore, detailed analyses showed that Japanese men who have sex with men are more aware of their risk of HIV infection.
Assuntos
Fármacos Anti-HIV/farmacologia , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , Adulto , Farmacorresistência Viral , Feminino , Infecções por HIV/epidemiologia , Infecções por HIV/transmissão , HIV-1/classificação , HIV-1/genética , Homossexualidade Masculina , Humanos , Japão/epidemiologia , Masculino , Mutação , Filogenia , PrevalênciaRESUMO
Atopic dermatitis (AD) is a chronic relapsing eczematous skin disease. Certain populations of patients are resistant to standard therapies with topical steroids and/or calcineurin inhibitors, and require systemic medication, such as immunosuppressants. Recently, several reports have shed light on the anti-allergic effects of carotenoids. Therefore, we investigated the effect of p.o. administration of ß-carotene or lycopene on AD-like symptoms of HR-1 hairless mice fed with a low zinc/magnesium diet. Mice were divided into four groups: (i) fed with a standard diet (Co group); (ii) low zinc/magnesium diet (HR group); (iii) low zinc/magnesium and ß-carotene diet (HR-C group); and (iv) low zinc/magnesium and lycopene diet (HR-L group). They were then fed these diets for 8 weeks. Severities of dermatitis were assessed by their appearance, and histopathological and hematological observations. Mice in the HR group developed AD-like dermatitis both clinically and histologically. HR-C and HR-L group mice also developed xerosis and wrinkle-like skin changes, but they were milder than those of HR group mice. Histological analysis revealed that epidermis thickening and inflammatory cell infiltration in the skin of the HR-C and HR-L groups were both statistically less than those of the HR group. The concentration of thymus and activation regulated chemokine in the skin of the HR-L group and the concentration of CCL27 in the skin of the HR-C group were significantly lower than those of the HR group, respectively. In conclusion, p.o. administration of ß-carotene or lycopene prevents AD-like symptoms in association with a suppression of T-helper 2 chemokines in a murine model. Ingestion of carotenoids may be beneficial for patients with AD.
Assuntos
Antialérgicos/uso terapêutico , Carotenoides/uso terapêutico , Dermatite Atópica/prevenção & controle , beta Caroteno/uso terapêutico , Administração Oral , Animais , Antialérgicos/administração & dosagem , Carotenoides/administração & dosagem , Quimiocina CCL27/metabolismo , Dermatite Atópica/etiologia , Dermatite Atópica/patologia , Dieta/efeitos adversos , Suplementos Nutricionais , Modelos Animais de Doenças , Epiderme/metabolismo , Epiderme/patologia , Humanos , Licopeno , Magnésio/metabolismo , Masculino , Camundongos , Camundongos Pelados , Células Th2/metabolismo , Zinco/deficiência , beta Caroteno/administração & dosagemRESUMO
Norwalk-like viruses (NLV) are a major causative agent of nonbacterial gastroenteritis. There are still many NLV strains that are refractory to gene amplification by ordinary reverse transcription-polymerase chain reaction. This is due mainly to the genetic diversity among NLV, especially mismatches in the primer sequences, which limits this technique in clinical utility. In this study, improved primer sets based on the capsid region, to detect both genogroup I and II NLV by genogroup-specific manner, were developed. When stool specimens from gastroenteritis patients, that were positive for NLV by electron microscopy, were tested by this new primer set, all specimens were positive by RT-PCR. Primers described previously for RdRp and capsid protein were capable of amplifying the specimens by 31 and 77%, respectively. Therefore, new primer sets are extremely useful for the amplification and rapid diagnosis of nonbacterial gastroenteritis due to NLV as well as for epidemiological studies.
Assuntos
Infecções por Caliciviridae/virologia , Proteínas do Capsídeo , Capsídeo/genética , Primers do DNA , Surtos de Doenças , Gastroenterite/virologia , Norovirus/isolamento & purificação , RNA Viral/análise , RNA Polimerase Dependente de RNA/genética , Sequência de Bases , Infecções por Caliciviridae/epidemiologia , DNA Viral , Gastroenterite/epidemiologia , Humanos , Japão/epidemiologia , Dados de Sequência Molecular , Norovirus/classificação , Norovirus/genética , Reação em Cadeia da Polimerase/métodos , Sensibilidade e EspecificidadeRESUMO
Although various cell lines have been used for virus isolation, few study of virus isolation using MRC-5 cell, a human embryonic lung fibroblasts, have been reported in Japan. MRC-5 and other cell lines (Caco-2, Vero, RD-18s, LLC-MK2, HeLa, MDCK, FL, B95a and HMV-II), and suckling mouse were compared for isolation of viruses from clinical specimens. A total of 3,284 specimens, collected from clinics and hospitals in Saitama Prefecture between July 1997 and August 2001, were inoculated in these cells. A total of 1,252 viral strains were isolated and 1,190 viral strains of these were identified. MRC-5 detected 209 of specimens positive for various viruses. As for adenovirus, a total of 132 viral strains were isolated using cell lines described above, and 100 of 132 viral strains were isolated in MRC-5. MRC-5 showed the highest sensitivity for isolation of adenovirus 3 and 7 (79.1% and 100%) of all other cells. The sensitivity in isolation of these viruses in HeLa was 58.1% and 50.0%, respectively. It showed that MRC-5 is able to isolate enterovirus, especially coxsackie virus A16 and enterovirus 71 with a high sensitivity (85.7% and 73.7%). RD-18s detected 35.7% and 26.3% of coxsackie virus A16 and enterovirus 71 isolates, LLC-MK2 detected 60.7% and 47.4%, and Vero detected 48.6% and 52.6%, respectively. Coxsackie virus B group was not isolated, except for a few coxsackie virus B 5 strains. Enteroviruses except coxsackie virus A16 and enterovirus 71 were isolated more frequently in Caco-2 and RD-18s. Seven hundred thirteen strains of influenza viruses were isolated in MDCK and Caco-2, but none was isolated in MRC-5. It was probably due to the maintenance medium without trypsin. The isolation rate of herpes simplex virus in Vero was 88.9% and MRC-5 showed 77.8%, it was high secondary to Vero by MRC-5. However, the CPE was detected in a few days in MRC-5, it was earlier than in Vero. The MRC-5 is possible to be maintained without changing the maintenance medium and passaged for 2 weeks, and clear CPE was observed. On the other hand, the disadvantages in using the MRC-5 were that the passage was limited and that the split ratio was only 1:2. However, the MRC-5 was used successfully for virus isolation, especially coxsackie virus A16, enterovirus 71 and adenoviruses, from clinical specimens.
Assuntos
Adenoviridae/isolamento & purificação , Enterovirus/isolamento & purificação , Fibroblastos/virologia , Pulmão/citologia , Humanos , Pulmão/embriologiaRESUMO
Hereditary angioedema (HAE) presents as severe angioedema, which is mostly due to the C1 inhibitor (C1-INH) gene mutations. Environmental factors, minor trauma and oral contraceptives have been reported to induce angioedema attack, but the trigger may often be uncertain. Activated factor XII controlled by C1-INH facilitates bradykinin generation and also regulates coagulation cascade, but the relationship between edema formation and coagulation is still unclear. We have described a 35-year-old female patient with HAE, presenting with frequent angioedema attacks in the absence of an apparent triggering factor. She showed higher levels of FDP and D-dimer during angioedema than those in remission. In addition, tissue factor (TF), an initiator of the extrinsic coagulation cascade, was expressed on the surface of monocytes. It was significantly higher than that of monocytes from healthy controls and tends to further increase during attacks. The expression of TF on monocytes may play a role in the induction of angioedema attacks in HAE by activating the coagulation pathway in association with reduced functions of C1-INH.
Assuntos
Produtos de Degradação da Fibrina e do Fibrinogênio/metabolismo , Angioedema Hereditário Tipos I e II/metabolismo , Monócitos/metabolismo , Proteínas/metabolismo , Tromboplastina/metabolismo , Adulto , Proteína Inibidora do Complemento C1/metabolismo , Complemento C4/metabolismo , Feminino , HumanosRESUMO
In this study, the amorphous calcium phosphate (ACP) method developed previously for calicivirus concentration from water was applied for norovirus detection from food. The viral recovery from cabbage, lettuce, or ham (10g of each) was firstly examined in seeding experiments with feline caliciviruses (FCVs). The viruses were concentrated by viral adsorption to ACP particles (0.3g) in the eluent solution (40ml) from foods, collection of the particles by centrifugation, followed by dissolution of the particles with 3.3M citric acid (3ml). In ham, FCV recovery was improved by addition of ascorbic acids into the eluent solution before ACP-particle adsorption. Quantitative real-time reverse transcription-PCR (qRT-PCR) revealed that FCV recoveries were 32-33%, 50-55%, and 37-46% from cabbage, lettuce, and ham, respectively, when seeded with 10(3)-10(4) viruses, and detection limits were estimated â¼10(3) genomic copies in all 3 foods. Subsequently, the ACP-concentration method was evaluated for norovirus (NoV) detection from these 3 foods. The recoveries and detection limit of NoVs determined by qRT-PCR were 12-41% and 10(3) (genomic copies) from cabbage, 30-57% and 10(3) from lettuce, and 20-26% and 10(4) from ham, when seeded with 10(3)-10(5) viruses. This simple method may be suitable for NoV detection from these foods.
Assuntos
Brassica/virologia , Fosfatos de Cálcio/química , Lactuca/virologia , Carne/virologia , Norovirus/isolamento & purificação , Infecções por Caliciviridae/diagnóstico , Infecções por Caliciviridae/virologia , Calicivirus Felino/isolamento & purificação , Microbiologia de Alimentos , Gastroenterite/diagnóstico , Gastroenterite/virologia , Humanos , RNA Viral/análise , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Surface plasmon resonance (SPR) biosensor detects intracellular signaling events as a change of the angle of resonance (AR). We previously reported that the activation of epidermal growth factor receptor (EGFR) on keratinocytes causes a unique triphasic change of AR, whereas the activation of other receptors, such as IgE receptor and adenosine A3 receptor on mast cells, causes a transient monophasic increase of AR. To study the mechanism of AR changes induced by EGFR activation, we introduced wild and mutated EGFR cDNAs into Chinese hamster ovary (CHO) cells and analyzed changes of AR in response to EGF. CHO cells expressing wild-type EGFR showed a triphasic change of AR, whereas cells expressing kinase-dead EGFR (K721M) showed minimum change of AR. A phosphatidylinositol 3-kinase inhibitor, wortmannin, attenuated the third phase of AR change in CHO cells expressing wild-type EGFR. The pattern of AR change was independent on the concentration of EGF. We also analyzed changes of AR with a nontumorigenic keratinocyte cell line, HaCaT, and several cell lines of carcinoma to explore the feasibility of SPR biosensor as a tool for clinical diagnosis. The activation of HaCaT cells and one out of six carcinoma cell lines showed a full triphasic change of AR. In contrast, five out of the six cell lines showed mono- or bi-phasic change of AR. These results suggest that EGF induces the SPR signals via the phosphorylation of EGFR, and provide a possibility that the SPR biosensor could be applied to the real-time detection and diagnosis of malignant tumors.
Assuntos
Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Neoplasias/metabolismo , Ressonância de Plasmônio de Superfície/métodos , Animais , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Humanos , FosforilaçãoAssuntos
Antígenos Virais/imunologia , Vírus da Influenza A Subtipo H3N2 , Vírus da Influenza B , Influenza Humana/virologia , Linhagem Celular , Pré-Escolar , Testes de Inibição da Hemaglutinação , Humanos , Vírus da Influenza A Subtipo H3N2/imunologia , Vírus da Influenza A Subtipo H3N2/isolamento & purificação , Vírus da Influenza B/imunologia , Vírus da Influenza B/isolamento & purificação , Influenza Humana/diagnóstico , Influenza Humana/epidemiologia , Masculino , Cavidade Nasal/virologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
A novel concentration method using minute particles of amorphous calcium phosphate (ACP) was developed for the detection of caliciviruses including norovirus and sapovirus, agents of human gastroenteritis, from water. In seeding experiments with feline calicivirus (FCV), ACP particles were able to adsorb efficiently the viruses in water, and the FCV-concentrated solution was obtained by dissolution of the virus-adsorbing ACP particles with citric acid after centrifugation. By quantitative real-time RT-PCR, the recovery efficiencies from 300 ml ultrapure water seeded with 10³, 104 and >105 copies of FCV were 48, 68 and >100â%, respectively. A comparative study showed that in the addition of viruses at <105 copies, the recovery efficiency of our method was significantly higher (P<0.05) than that of the similar calcium flocculation-citrate dissolution method. Using our newly developed method, we successfully detected 2.1 x 104 copies l⻹ of norovirus (each of genogroups I and II) and 5.4 x 10³ copies l⻹ of sapovirus (genogroups I, II, IV and V) from river water. The data suggest that our new viral concentration is a rapid, simple, cost efficient and high virus recovery method, and it can be used for routine monitoring of norovirus and sapovirus in water, especially environmental water.