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BACKGROUND AND PILOT STUDY: Recent reports reveal a close relationship between migraine and gastrointestinal disorders (GI), such as celiac disease (CD) and non-celiac gluten sensitivity (NCGS). CD is a genetic autoimmune disorder, which affects the mucosa of the small intestine. Gluten, found in various grains, not only plays a major role in the pathophysiology of CD and NCGS, but also aggravates migraine attacks. Another common food component, which can induce migraine headaches, is histamine. Diamine oxidase (DAO) is an enzyme, which degrades histamine. Reduced activity of DAO means reduced histamine degradation, which can cause histamine build-up and lead to various symptoms, including headaches and migraine. In this paper we propose a hypothesis, that in pathogenesis of migraine, low serum DAO activity is related to CD and NCGS. We also conducted our own pilot study of 44 patients with severe migraine in efforts to evaluate the co-presence of decreased serum DAO activity and celiac disease/NCGS in patients. 44 consecutive migraine patients were divided into 2 groups: decreased DAO activity (group 1; n = 26) and normal DAO activity (group 2; n = 18). All patients were screened for celiac disease. The diagnosis of NCGS was made after exclusion of CD, food allergies and other GI disorders in the presence of gluten sensitivity symptoms. Furthermore, dietary recommendations were given to all participants and their effects were assessed 3 months after the initial evaluation via the MIDAS (Migraine Disability Assessment) questionnaire. RESULTS AND CONCLUSIONS: Only 1 patient fit the criteria for celiac disease, rendering this result inconclusive. Pathological findings of the remainder of patients were attributed to NCGS (n = 10). 9 of 10 patients with NCGS belonged to the decreased serum DAO activity group (group 1; n = 26), suggesting a strong relationship between reduced serum DAO activity and NCGS. MIDAS questionnaire revealed, that patients with decreased serum DAO activity were more severely impacted by migraine than those with normal DAO activity, and this remained so after our interventions. Dietary adjustments significantly reduced the impact of migraine on patients' daily activities after 3 months in both groups. We argue, that migraine, celiac disease and NCGS may benefit from treatment with a multidisciplinary approach, involving neurologists, gastroenterologists and dietitians.
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Amina Oxidase (contendo Cobre) , Doença Celíaca , Hipersensibilidade Alimentar , Transtornos de Enxaqueca , Doença Celíaca/complicações , Glutens/efeitos adversos , Humanos , Transtornos de Enxaqueca/complicações , Projetos PilotoRESUMO
Clinically relevant diagnosis of human bocavirus 1 (HBoV1) is challenging, as the virus is frequently detected in asymptomatic patients, and cofindings with other respiratory viruses are common. The clinical value of current diagnostic methods, such as PCR, is therefore low, and alternative diagnostic strategies are needed. We describe for the first time the use of an antigen detection assay for the rapid identification of HBoV1 in a paediatric patient with respiratory tract infection symptoms. We estimate the duration of active HBoV1 infection to be 6 days.
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BACKGROUND: Human parvovirus 4 (PARV4) is a recently discovered member of the Parvoviridae family, which is not closely related to any previously discovered human parvoviruses. PARV4 has been isolated from the plasma of individuals with symptoms of acute viral infection; however, until recently PARV4 had not been associated with any disease, and its prevalence in the human population is yet to be established. METHODS: The major capsid protein VP2 of PARV4 was generated in the yeast Saccharomyces cerevisiae and used for serological detection of virus-specific IgG and IgM in the sera of low-risk individuals. RESULTS: One hundred and seventy serum specimens obtained from patients with acute respiratory diseases were tested for PARV4-specific IgG and IgM antibodies. Sixteen individuals (9.4%) were diagnosed as seropositive, including 6 IgM and IgG positive, 6 IgM positive/IgG negative and 4 IgG positive/IgM negative. Seven of the 16 seropositive individuals were between the ages of 3 and 11 with no evidence of parenteral exposure to PARV4 infection. CONCLUSION: Our data demonstrate that recombinant yeast-derived VP2 protein, self-assembled to virus-like particles, can represent a useful tool when studying the seroprevalence of PARV4 infection. The presence of PARV4-specific antibodies in a low-risk group may indicate the possibility of alternative routes of virus transmission.
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Anticorpos Antivirais/sangue , Antígenos Virais , Proteínas do Capsídeo , Infecções por Parvoviridae/diagnóstico , Parvovirus/imunologia , Virossomos , Adolescente , Adulto , Antígenos Virais/genética , Proteínas do Capsídeo/genética , Criança , Pré-Escolar , Feminino , Expressão Gênica , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Lactente , Masculino , Pessoa de Meia-Idade , Parvovirus/genética , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Virossomos/genética , Adulto JovemRESUMO
The B-cell response against measles nucleoprotein (MeN) plays an important role in the control of measles infection. However, the data on B cell epitopes of MeN are still limited. The objective of this study was to identify B cell epitopes in MeN using monoclonal and polyclonal antibodies raised against recombinant yeast-expressed MeN (rMeN) as well as human sera from measles-positive individuals. After immunization of mice, 15 monoclonal antibodies (mAbs) against rMeN were generated. The B cell epitopes were localized using recombinant overlapping MeN fragments, PepScan analysis, and competitive ELISA. The epitopes of 14 mAbs were mapped within the C-terminus of MeN between amino acids (aa) 419 and 525. Four mAbs recognized a linear epitope located within a sequence of aa 440-448. Competitive ELISA revealed a cluster of conformational mAb epitopes. Cross-inhibition studies with human sera demonstrated similar localization of B cell epitopes recognized by serum antibodies from naturally infected individuals. Thus, the majority of B cell epitopes are located at the C-terminal domain of MeN. These findings provide new data on the antigenic structure of MeN and are in agreement with recent experimental evidence indicating that the C-terminal domain of MeN is well accessible on the surface of nucleocapsid-like structures.
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Linfócitos B/imunologia , Mapeamento de Epitopos/métodos , Vírus do Sarampo/imunologia , Nucleoproteínas/imunologia , Proteínas Virais/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Anticorpos Antivirais , Antígenos Virais/genética , Ensaio de Imunoadsorção Enzimática , Epitopos/genética , Humanos , Sarampo/imunologia , Sarampo/virologia , Vírus do Sarampo/genética , Camundongos , Microscopia Eletrônica , Proteínas do Nucleocapsídeo , Nucleoproteínas/química , Nucleoproteínas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
Foreign protein sequences presented on hamster polyomavirus (HaPyV) major capsid protein VP1-derived virus-like particles (VLPs) have been demonstrated to be highly immunogenic. The current study was aimed to evaluate VP1-derived chimeric VLPs as tools for hybridoma technology to generate monoclonal antibodies (mAbs) of desired specificity. Chimeric VLPs containing inserts of different size and origin were used as immunogens. Chimeric VLPs carrying a 9 amino acid (aa)-long cytotoxic T-cell epitope (STAPPVHNV) of human mucin 1 (MUC1) elicited a strong epitope-specific humoral immune response in mice and promoted the production of MUC1-specific mAbs. From a total of seven mAbs of IgG isotype generated against the chimeric VLPs, two mAbs were directed against the MUC1 epitope and five mAbs against the VP1-carrier. Two out of five anti-VP1 mAbs recognized epitopes located at the previously defined insertion site #2 (aa 223/224), which confirms its surface-exposed localization. Chimeric VLPs carrying a 120-aa long sequence of Puumala hantavirus (PUUV) nucleocapsid protein (NP) promoted the generation of five mAbs of IgG isotype specific to PUUV NP. All mAbs recognized the full-length NP of different PUUV strains. In contrast, no VP1-specific mAbs were obtained. The ability of chimeric VLPs to activate antigen-presenting cells was evaluated by studying the uptake of chimeric VLPs by murine spleen cell-derived dendritic cells (DCs). Efficient uptake of VLPs and activation of murine DCs were demonstrated, which may represent the basis of the strong immunogenicity of chimeric VLPs. In conclusion, chimeric VLPs effectively stimulated the production of IgG antibodies specific for foreign epitopes presented at surface-exposed regions. Thus, chimeric HaPyV VP1-derived VLPs represent efficient immunogens for hybridoma technology and provide a promising alternative to chemical coupling of synthetic peptides to carrier proteins.
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Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Proteínas do Capsídeo/genética , Polyomavirus/genética , Animais , Especificidade de Anticorpos , Antígenos de Neoplasias , Western Blotting , Proteínas do Capsídeo/imunologia , Células Dendríticas/imunologia , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos , Epitopos/imunologia , Hibridomas/imunologia , Imunoglobulina G/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mucina-1 , Mucinas/imunologia , Proteínas do Nucleocapsídeo/genética , Virus Puumala/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/imunologiaRESUMO
OBJECTIVE: Analysis of cytokine production in patients with acute and chronic reactive arthritis (AcReA/ChrReA) in order to search for new treatment possibilities. METHODS: Cytokine production by peripheral blood and synovial fluid mononuclear cells (PBMCs/SFMCs) of 28 patients with AcReA, 27 patients with ChrReA, 26 patients with rheumatoid arthritis (RA) and 31 healthy controls was analysed by enzyme-linked immunosorbent assay (ELISA) and flow-cytometry. Production of tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma) and interleukin (IL)-10 was measured by ELISA, while the percentages of TNF-alpha-, IFN-gamma- and IL-4-positive CD3+ cells were determined in the same groups of patients and healthy subjects using flow cytometry. RESULTS: Spontaneous TNF-alpha production observed in PBMCs of ChrReA, but not of AcReA, patients was significantly higher (P<0.001) than in healthy controls. The percentages of TNF-alpha-positive CD3+ blood cells in ChrReA exceeded that of RA patients and healthy controls (P<0.05 and P<0.001, respectively). Also, the percentages of IFN-gamma-positive CD3+ cells were significantly higher in peripheral blood and synovial fluid of ChrReA patients (P<0.05 and P<0.05, respectively) as compared with AcReA. In ChrReA spontaneous IL-10 production in PBMCs was similar to that observed in healthy controls, while in RA and AcReA the production of IL-10 was significantly increased (P<0.05 and P<0.05, respectively). IL-4 production was low in all study groups with no significant differences detected. CONCLUSIONS: High production of TNF-alpha and IFN-gamma detected in ChrReA supports the possible use of anti-TNF-alpha treatment in ChrReA.
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Artrite Reativa/metabolismo , Citocinas/biossíntese , Doença Aguda , Adulto , Idoso , Artrite Reativa/sangue , Artrite Reumatoide/sangue , Artrite Reumatoide/metabolismo , Doença Crônica , Citocinas/sangue , Feminino , Citometria de Fluxo/métodos , Antígeno HLA-B27/análise , Humanos , Interferon gama/biossíntese , Interferon gama/sangue , Interleucina-10/biossíntese , Interleucina-10/sangue , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Monócitos/metabolismo , Líquido Sinovial/citologia , Subpopulações de Linfócitos T/metabolismo , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/biossínteseRESUMO
OBJECTIVES: Non-viral methods of gene transfer have been preferred in gene therapy approaches for several reasons, particularly for their safety, simplicity and convenience in introducing heterologous DNA into cells. Polyomavirus virus-like particles (VLPs) represent a promising carrier for encapsidation of foreign nucleic acids for gene therapy. For the development of such gene delivery systems as well as for providing reagents for improving virus diagnostics, an efficient yeast expression system for the generation of different polyomavirus VLPs was established. METHODS: A galactose-inducible Saccharomyces cerevisiae yeast expression system was used. Formation of empty VLPs was confirmed by cesium chloride ultracentrifugation, agarose gel electrophoresis and electron microscopy. Cross-reactivity of the major capsid proteins (VP1) of different polyomaviruses was analyzed by Western blot using rabbit and mice sera raised against the VP1 proteins. RESULTS: VP1 of polyomaviruses from humans (JC polyomavirus and serotypes AS and SB of BK polyomavirus), rhesus monkeys (simian virus 40), hamsters (hamster polyomavirus), mice (murine polyomavirus) and birds (budgerigar fledgling disease virus) were expressed at high levels in yeast. Empty VLPs formed by all yeast-expressed VP1 proteins were dissociated into pentamers and reassociated into VLPs by defined ion and pH conditions. Different patterns of cross-reactivity of the VP1 proteins with heterologous mice and rabbit sera were observed. CONCLUSION: The developed heterologous yeast expression system is suitable for high-level production of polyomavirus VLPs. Yeast-derived VLPs are generally free of toxins, host cell DNA and proteins. These VLPs might be useful for the generation of new diagnostical tools, gene delivery systems and antiviral vaccines.
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Proteínas do Capsídeo/biossíntese , Polyomavirus/fisiologia , Proteínas Recombinantes/biossíntese , Saccharomyces cerevisiae/genética , Vírion/fisiologia , Sequência de Aminoácidos , Animais , Proteínas do Capsídeo/química , Proteínas do Capsídeo/imunologia , Humanos , Dados de Sequência MolecularRESUMO
Pituitary adenylate cyclase activating polypeptide type I receptor (PACAPr) belongs to the novel subfamily of the G-protein coupled receptors with a long extracellular N-terminus, which functions as a major binding site for the PACAP. Three different N-terminal fragments of rat PACAPr were overexpressed in Escherichia coli and purified using His-tags or maltose-binding protein as anchors for affinity chromatography. The purified and refolded proteins were used for the production and screening of monoclonal antibodies (MAbs) to PACAPr. Fifteen hybridoma cell lines producing MAbs specific to PACAPr were generated and characterized. Epitope analysis by competitive enzyme-linked immunoadsorbent assay (ELISA) indicated the presence of two groups of overlapping epitopes in the N-terminal fragment of PACAPr. Reactivity of MAbs with SDS-denaturated and native rat PACAPr was demonstrated by immunoblotting and flow cytometric analysis using transiently transfected COS cells and stably transfected CHO cells expressing rat PACAPr. Each antibody was examined by immunoblotting for the ability to cross react with the human PACAPr in human neuroblastoma NB-OK cells and most of them were shown to recognize human PACAPr as effectively as rat PACAPr. MAbs against the N-terminal extracellular domain of PACAPr can be used for the immunochemical study of the receptor-ligand interaction and for the investigation of PACAPr distribution in normal and tumor tissues.
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Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Receptores do Hormônio Hipofisário/imunologia , Animais , Afinidade de Anticorpos , Ligação Competitiva/imunologia , Células CHO/imunologia , Células COS/imunologia , Cricetinae , Reações Cruzadas/imunologia , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos , Escherichia coli/química , Escherichia coli/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Mitógenos/imunologia , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/genética , Ratos , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Receptores do Hormônio Hipofisário/metabolismo , Proteínas Recombinantes , Transfecção/imunologia , Células Tumorais CultivadasRESUMO
In this study we investigated the effects of two guanine derivatives, 9-benzyl- (I) and 7-benzyl-8-bromoguanines (II) on the proliferation of human T-cell leukemia and T-cell lymphoma, normal human peripheral blood mononuclear cells (PBMC), and mouse Th1 (pGL10) and Th2 (D10.G4.1) clones. We also assessed their effects on cytokine production (IL-3, IL-10 and IFN-gamma) in PBMC, T-cell lymphoma, HUT78 (IL-2), and murine Th1 (IL-2) and Th2 (IL-4 and IL-5) clones. These compounds were synthesize as analog of known inhibitors of purine nucleoside phosphorylase (PNP) 8-amino-9-benzylguanine. These compounds suppressed proliferation of human leukemia MOLT-4 cells, human cutaneous lymphoma HUT78 cells and normal PMBC. Compound II was a significantly more potent inhibitor than compound I. Exogenous recombinant human IL-2 reversed the anti-proliferative effects of both compounds on HUT78 cells. These compounds had low toxicity to human EBV-transformed B-lymphocytes. Both compounds suppressed the production of IL-2 by activated human HUT78 cells, IFN-gamma by PBMC and did not affect IL-3 and IL-10 production in PBMC. Compound I inhibited anti-CD3-activated IL-2 secretion from the murine Th1 clone. The murine Th2 clone was less sensitive to both compounds as compared with Thl. The production of IL-4 and IL-5 by this clone was not suppressed. Thus, it has been shown that not only 9-substituted guanines but also their 7-isomers selectively inhibit T-cell functions and both selectively inhibit Th1-related cytokines secretion.
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Compostos de Benzil/farmacologia , Guanina/farmacologia , Interleucina-2/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Linfoma de Células T/metabolismo , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular Transformada/efeitos dos fármacos , Humanos , Interleucina-2/farmacologia , Leucemia de Células T/metabolismo , Camundongos , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Linfócitos T Auxiliares-Indutores/metabolismo , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismoRESUMO
Interferon gamma (IFN gamma) and interleukin 4, 10 and 12 (IL-4, -10, -12) production was measured in whole peripheral blood (WPB) and peripheral blood mononuclear cells (PBMC) of 10 chronic hepatitis C (CHC) patients. The level of IFN gamma in supernatants in mitogen-activated WPB was lower than in healthy donors. IL-10 served as a possible downregulative factor for IFN gamma, since its spontaneous IL-10 production was enhanced in CHC. Neutralization of IL-10 partly restored IFN gamma response in CHC patients. Recombinant IL-12 (rIL-12) also enhanced IFN gamma of CHC patients, but IL-12 production was decreased in CHC. Thus, IFN gamma production deficiency in CHC patients is secondary to blockage by high levels of IL-10-impaired IL-12 production.