RESUMO
Behçet's disease (BD) is an inflammatory disease mainly affecting men along the ancient Silk Route. In the present study we describe a Dutch family suffering from BD-like disease with extreme pathergic responses, but without systemic inflammation. Genetic assessment revealed a combination of the human leukocyte antigen (HLA)-B*51 risk-allele together with a rare heterozygous variant in the CSF2 gene (c.130A>C, p.N44H) encoding for granulocyte-macrophage colony-stimulating factor (GM-CSF) found by whole exome sequencing. We utilized an over-expression vector system in a human hepatocyte cell line to produce the aberrant variant of GM-CSF. Biological activity of the protein was measured by signal transducer and activator of transcription 5 (STAT-5) phosphorylation, a downstream molecule of the GM-CSF receptor, in wild-type peripheral mononuclear cells (PBMCs) using flow cytometry. Increased STAT-5 phosphorylation was observed in response to mutated GM-CSF when compared to the wild-type or recombinant protein. CSF2 p.N44H results in disruption of one of the protein's two N-glycosylation sites. Enzymatically deglycosylated wild-type GM-CSF also enhanced STAT-5 phosphorylation. The patient responded well to anti-tumor necrosis factor (TNF)-α treatment, which may be linked to the capacity of TNF-α to induce GM-CSF in phorbol 12-myristate 13-acetate (PMA)-treated PBMCs, while GM-CSF itself only induced dose-dependent interleukin (IL)-1Ra production. The identified CSF2 pathway could provide novel insights into the pathergic response of BD-like disease and offer new opportunities for personalized treatment.
Assuntos
Síndrome de Behçet/genética , Mutação com Ganho de Função/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Linhagem Celular , Linhagem Celular Tumoral , Exoma/genética , Feminino , Células Hep G2 , Humanos , Fosforilação/genéticaRESUMO
Signal transducer and activator of transcription 1 (STAT-1) gain-of-function (GOF) mutations cause chronic mucocutaneous candidiasis (CMC), a disease associated with Candida albicans and Staphylococcus aureus infection. Patients suffer from dysegulated immune responses due to aberrant cell programming and function. We investigated the effect of inhibitory molecules targeting histone deacetylases (HDACi) on the immune responses of peripheral blood mononuclear cells (PBMCs) of healthy controls and patients with CMC towards microbes relevant for CMC. PBMCs cells were pretreated with HDACi and challenged with C. albicans or S. aureus. Innate and adaptive cytokines were measured in cell culture supernatants by enzyme-linked immunosorbent assay (ELISA). We assessed the effect of HDAC inhibitors on T helper type 1 (Th1) and Th17 cells and measured STAT-1 and STAT-3 phosphorylation using flow cytometry. Panobinostat, a pan-HDAC inhibitor, strongly inhibits innate and adaptive cytokines upon challenge with C. albicans or S. aureus. Specific inhibitors (entinostat or RGFP966) also had a tendency to lower production of most innate cytokines in CMC patient cells. Entinostat and RGFP966 increased the production of interleukin (IL)-22 specifically after S. aureus challenge in patient cells. In healthy and control cells, entinostat and RGFP966 treatment down-regulated STAT-1 phosphorylation while pSTAT-3 levels remained stable. HDACi modulate cytokine production in response to C. albicans and S. aureus. Pan-inhibitors lower overall cytokine production, whereas specific inhibitors confer a selective effect. Entinostat and RGFP966 are promising therapeutic candidates to treat STAT-1 GOF due to their capacity to restore IL-22 production and decrease STAT-1 phosphorylation; however, their inhibition of innate cytokines poses a possible risk to secondary infections.
Assuntos
Candida albicans/fisiologia , Candidíase Mucocutânea Crônica/imunologia , Inibidores de Histona Desacetilases/farmacologia , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/fisiologia , Células Th1/imunologia , Células Th17/imunologia , Imunidade Adaptativa , Candidíase Mucocutânea Crônica/tratamento farmacológico , Candidíase Mucocutânea Crônica/genética , Células Cultivadas , Regulação para Baixo , Histona Desacetilases/metabolismo , Humanos , Imunidade Inata , Interleucinas/metabolismo , Mutação/genética , Fosforilação , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3/metabolismo , Interleucina 22RESUMO
BACKGROUND: STAT1 mutations cause chronic mucocutaneous candidiasis (CMC), while STAT3 mutations cause hyper-IgE syndrome (HIES). CMC and HIES patients have T helper (Th) 17 defects suffering from mucosal Candida infections, but only patients with HIES show an allergic phenotype with eczema, eosinophilia and high IgE levels. OBJECTIVE: We investigated whether differential Th2 and Th9 responses may explain the clinical differences. METHODS: Peripheral blood mononuclear cells of patients with CMC (n = 4), patients with HIES (n = 4), patients with atopic dermatitis (n = 4) and healthy volunteers (n = 13) were stimulated with Candida and Staphylococcus aureus, with and without IL-4. The cytokines IL-5, IL-13, IL-9, IL-17 and TGFß and regulatory T cells were measured in cell culture supernatants by ELISA or flow cytometry, respectively. RESULTS: Peripheral blood mononuclear cells of patients with CMC showed a significantly impaired production of the Th2 cytokines IL-5 and IL-13, especially in the presence of IL-4. Moreover, IL-9 production was significantly lower in patients with CMC compared to healthy controls. In contrast, patients with HIES and patients with AD showed normal IL-5 and IL-13 production, while IL-9 production was significantly lower in patients with HIES compared to healthy controls. Although TGFß was involved in the IL-4-induced IL-9 production, TGFß levels and the frequency of regulatory T cells did not differ between patients with HIES and controls. Flow cytometry analysis demonstrated an IL-9+ IL-17+ CD4+ subset in healthy controls after stimulation with Candida which was less present in patients with HIES. CONCLUSION: Patients with CMC have a general Th defect including Th2 and Th9, while patients with HIES have normal Th2 cytokines. These differences are in line with their clinical presentation. Surprisingly, the allergic cytokine IL-9 was deficient in both HIES and CMC, suggesting a Th-17-derived origin.
Assuntos
Candidíase Mucocutânea Crônica/diagnóstico , Candidíase Mucocutânea Crônica/imunologia , Síndrome de Job/diagnóstico , Síndrome de Job/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Adulto , Idoso , Biomarcadores , Candidíase Mucocutânea Crônica/metabolismo , Candidíase Mucocutânea Crônica/terapia , Estudos de Casos e Controles , Citocinas/sangue , Citocinas/metabolismo , Eosinófilos/imunologia , Eosinófilos/metabolismo , Feminino , Humanos , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Síndrome de Job/metabolismo , Síndrome de Job/terapia , Contagem de Leucócitos , Masculino , Pessoa de Meia-Idade , Fenótipo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Auxiliares-Indutores/metabolismo , Células Th2/imunologia , Células Th2/metabolismoAssuntos
Hipopigmentação/genética , Ceratodermia Palmar e Plantar/genética , Mutação de Sentido Incorreto/genética , Diester Fosfórico Hidrolases/genética , Pirofosfatases/genética , Somatomedinas/genética , Adolescente , Adulto , Calcinose/genética , Feminino , Antebraço , Heterozigoto , Humanos , Masculino , LinhagemAssuntos
Dermatite/genética , Desmogleína 1/genética , Hipersensibilidade/genética , Mutação/genética , Síndrome de Emaciação/genética , Adulto , Idade de Início , Criança , Desmogleína 1/deficiência , Exoma , Genes Recessivos , Estudo de Associação Genômica Ampla , Humanos , Ceratodermia Palmar e Plantar/genética , Noruega , Linhagem , SíndromeRESUMO
Streptococcus agalactiae (Group B Streptococcus, GBS) is a Gram-positive bacterium, which colonizes the vaginal tract in 10-30% of women. Colonization is transient in nature, and little is known about the host and bacterial factors controlling GBS persistence. Gaining insight into these factors is essential for developing therapeutics to limit maternal GBS carriage and prevent transmission to the susceptible newborn. In this work, we have used human cervical and vaginal epithelial cells, and our established mouse model of GBS vaginal colonization, to characterize key host factors that respond during GBS colonization. We identify a GBS strain that persists beyond a month in the murine vagina, whereas other strains are more readily cleared. Correspondingly, we have detected differential cytokine production in human cell lines after challenge with the persistent strain vs. other GBS strains. We also demonstrate that the persistent strain more readily invades cervical cells compared with vaginal cells, suggesting that GBS may potentially use the cervix as a reservoir to establish long-term colonization. Furthermore, we have identified interleukin-17 production in response to long-term colonization, which is associated with eventual clearance of GBS. We conclude that both GBS strain differences and concurrent host immune responses are crucial in modulating vaginal colonization.
Assuntos
Infecções Estreptocócicas/imunologia , Vagina/imunologia , Vagina/microbiologia , Animais , Western Blotting , Linhagem Celular , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Infecções Estreptocócicas/genética , Streptococcus agalactiae/genética , Streptococcus agalactiae/imunologiaRESUMO
Hepatitis C virus (HCV) results frequently in chronic hepatitis and its sequelae liver cirrhosis and hepatocellular carcinoma. Interferon-alpha is at present the most effective treatment, resulting in a sustained response in about 20-25% of patients. HCV genotype, titer and quasispecies determine the success of treatment. In this study, fluorescent single strand conformation polymorphism (f-SSCP) was evaluated for the analysis of HCV quasispecies. Two sera from a chronically HCV-infected patient, obtained 6 years apart, were examined. The hypervariable region I (HVRI) of the HCV genome was amplified by reverse transcription and PCR. The PCR products were cloned and sequenced or fluorescein-labeled and subjected to f-SSCP. Both methods demonstrated a single HCV species in the early serum and multiple quasispecies in the late serum. Single clones of the heterogeneous virus population were used to optimize conditions for f-SSCP. The most important factors were the gel temperature and virus titer. At the optimal running temperature one base exchange in 218 bases was detectable. Repeat extractions and amplifications gave identical results. Dilution of the serum containing multiple quasispecies resulted in a 'loss' of species. Provided the running temperature is optimal and virus titer is sufficient, f-SSCP is shown to be fast and reliable for HCV quasispecies analysis.
Assuntos
Hepacivirus/genética , Hepatite C/virologia , Polimorfismo Conformacional de Fita Simples , Sequência de Aminoácidos , Sequência de Bases , DNA Viral , Hepacivirus/isolamento & purificação , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Viral/análise , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , Proteínas do Envelope Viral/genéticaRESUMO
Albino rats aged 13 weeks experienced a single training for bright-dark discrimination in a Y-chamber. 24 hours later after a successful training the paramembranaceous structures of the synapses contrasted by phosphotungstic acid were morphometrically analysed. As a result of this investigation a signifikant reduction of the height and the width of the dense projections was to be stated compared to untrained rats. Likewise the length and the width of the postsynaptic ribbon were reduced in trained rats. But this alteration proved not to be significant.
Assuntos
Condicionamento Operante/fisiologia , Aprendizagem por Discriminação/fisiologia , Hipocampo/anatomia & histologia , Sinapses/ultraestrutura , Animais , Masculino , Ratos , Membranas Sinápticas/ultraestruturaRESUMO
The areal portion of the lipofuscin granules and mitochondria contained in ultrathin sections, the length of rough endoplasmic reticulum (RER) and the numerical density of ribosomes were determined in the pyramidal cells of layers III and V of area 10 (Brodmann) of the frontal brains of six newly died persons of different ages. This revealed considerable differences in lipofuscin contents and numerical density of organelles between the pyramidal cells and greater values, on an average, for the pyramidal cells of layer III. The comparison of averages for the single brains did not show the continuity nor the age dependence of changes but rather the individual variability of these processes. The expected decrease of the density of organelles with increasing lipofuscin contents was found more frequently only at extremely high values of lipofuscin contents. It becomes evident that even under the conditions of lipofuscin accumulation the permanently necessary physiological renewal of cell structures involved in metabolism is guaranteed over a longer period of time and that their full performance is almost completely unimpaired during this phase.
Assuntos
Envelhecimento , Lobo Frontal/anatomia & histologia , Lipofuscina/metabolismo , Organoides/ultraestrutura , Pigmentos Biológicos/metabolismo , Adulto , Idoso , Núcleo Celular/ultraestrutura , Grânulos Citoplasmáticos/ultraestrutura , Dendritos/ultraestrutura , Retículo Endoplasmático/ultraestrutura , Humanos , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade , Mitocôndrias/ultraestrutura , Neurônios/ultraestrutura , Ribossomos/ultraestruturaRESUMO
The lipofuscin contents in pyramidal cells of laminae III and V in area 10 (Brodmann) of the frontal brain from 6 men who recently died at different ages without any neurological or psychiatric symptoms were investigated by electronmicroscopic-morphometric methods. The results of this investigation actually do reveal an increase of the lipofuscin with age, but also demonstrate to what extent this process is subject to individual variability. Great differences in the accumulation of the lipofuscin exist among the cells within the same cytoarchitectonic layer. On the other hand wide correspondence between the mean values of both laminae can be stated.
Assuntos
Envelhecimento , Lobo Frontal/metabolismo , Lipofuscina/metabolismo , Pigmentos Biológicos/metabolismo , Adulto , Idoso , Grânulos Citoplasmáticos/ultraestrutura , Lobo Frontal/anatomia & histologia , Humanos , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade , Neurônios/metabolismo , Neurônios/ultraestruturaRESUMO
In a period of 10 years (1981-90) papillary thyroid carcinomas were found in 124 cases. In 95 cases we were able to perform immunohistochemical studies for Leu-M1 antigen and S-100 protein. The examination of these cases has revealed that the presence of S-100 positive Langerhans cells yields no additional information for the prognosis of the papillary thyroid carcinoma. But we observed that the existence of Leu-M1 positive tumour cells is connected with a poor postoperative course of disease in comparison to Leu-M1 negative tumours.
Assuntos
Antígenos CD/análise , Antígenos de Diferenciação Mielomonocítica/análise , Biomarcadores Tumorais/análise , Carcinoma Papilar/patologia , Células de Langerhans/patologia , Proteínas S100/análise , Neoplasias da Glândula Tireoide/patologia , Carcinoma Papilar/imunologia , Carcinoma Papilar/mortalidade , Carcinoma Papilar/cirurgia , Feminino , Humanos , Técnicas Imunoenzimáticas , Células de Langerhans/imunologia , Antígenos CD15 , Masculino , Prognóstico , Taxa de Sobrevida , Glândula Tireoide/imunologia , Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/imunologia , Neoplasias da Glândula Tireoide/mortalidade , Neoplasias da Glândula Tireoide/cirurgiaRESUMO
For the first time in 1975 ultrastructural defects of the sperm tail and the cilia of the respiratory tract were recognized as causing male sterility and chronic bronchitis. A spectrum of ultrastructural defects leading to a restricted mucociliary clearance and chronic bronchitis was found by further investigations. Whereas genetically determined primary structural defects appear generally, secondary ciliary changes caused by chronic infections are found sporadically and not bound to a fixed pattern. Congenital and acquired specific ciliary alterations in the tubulin-dynein-system should be distinguished from nonspecific acquired alterations. A summary of the presently known ultrastructural changes is illustrated by findings of the authors.
Assuntos
Bronquite/patologia , Transtornos da Motilidade Ciliar/patologia , Brônquios/patologia , Criança , Doença Crônica , Cílios/ultraestrutura , Humanos , Microscopia Eletrônica , Otite Média/patologia , Sinusite/patologiaRESUMO
The results of an ultrastructural analysis of cilia in 30 children at the age of 11 month to 16 years are presented. Children with marked symptoms of chronic bronchitis were consciously chosen for examination. One group of 15 children was suffering from chronic bronchitis with bronchiectases. No bronchiectasis was found in the second group of 15 children with chronic bronchitis. Specific changes were more evident in the first group, whereas nonspecific changes were of equal frequency in both groups. Only in 3 patients of group I generalized specific defects could be demonstrated. In these cases mucociliary clearance was nearly not present. Ultrastructural changes of cilia in children with chronic nonspecific lung diseases enclose a wide spectrum. Compared to it generalized structural defects are rare and only in 3 cases of our study demonstrable (negative selection of a bronchological centre).
Assuntos
Bronquiectasia/patologia , Bronquite/patologia , Transtornos da Motilidade Ciliar/patologia , Adolescente , Brônquios/patologia , Criança , Pré-Escolar , Doença Crônica , Cílios/ultraestrutura , Dineínas/metabolismo , Humanos , Lactente , Microscopia Eletrônica , Tubulina (Proteína)/metabolismoRESUMO
Using the E-PTA procedure of Aghajanian and Bloom we determined the numerical density of synapses in the stratum lacunosun-modeculare of the CA1-region in the hippocampus of male albino rats. On this occasion the sources of error both the natural and the methodical were investigated. As natural causes of remarkable scattering and failing correspondence in the findings the individual variability of the synaptic density and the inhomogeneous distribution of synapses must be taken into consideration. Concerning the methodical sources of error the differences in tissue shrinkage become apparent. But faults resulting from applying morphometric methods are the most serious. The counting of synaptic profiles is based on defined morphological criteria. The portion of the synaptic profiles recorded in this way in relation to all those present in the section depends beside other things on the section thickness. Accordingly the correction factors are in correlation with section thickness. The application of the Floderus correction presupposes the determination of the minimum length of the counted profiles. As a result of these investigated the possibilities for reducing the error have been demonstrated.
Assuntos
Hipocampo/citologia , Sinapses , Animais , Técnicas Citológicas , Hipocampo/ultraestrutura , Masculino , Matemática , Ácido Fosfotúngstico , Ratos , Sinapses/ultraestruturaRESUMO
Male albino rats aged 13 weeks experienced a single training for bright -- dark discrimination in a Y-chamber. The numerical density of synapses determined in the stratum lacunosum-moleculare of the hippocampal area CA1 24 hours later turned out to be lower by 3.4 per cent with trained animals compared to passive controls. This difference failed, however, to prove significant.
Assuntos
Condicionamento Clássico/fisiologia , Hipocampo/citologia , Animais , Contagem de Células , Escuridão , Aprendizagem por Discriminação/fisiologia , Hipocampo/fisiologia , Masculino , Ratos , Sinapses/fisiologiaRESUMO
Based on a simple model of the synaptic cleft, some of those procedures that render possible an appraisal of the cleft width starting from cuttings as seen in micrographs are described in this paper. The results of the investigation prove that contrary to figures published previously by other authors, the width of the synaptic cleft is not constant, but exhibits a frequency distribution with a maximum - to - minimum ratio of about ten. Relative frequency in per cent can be approximately expressed as follows: p = 7.7 + 4.1 n with n = 1, 2, 5 ordinal class numbers of cleft width and the according class mid-points 7.62n nm Typflying of synapses according to physiological points of view has turned out to be out of reach.
Assuntos
Encéfalo/anatomia & histologia , Sinapses/ultraestrutura , Animais , Masculino , Microscopia Eletrônica , Ratos , Sinapses/fisiologiaRESUMO
OBJECTIVE: To investigate the effect of pulse cyclophosphamide treatment on hepatitis C virus (HCV) kinetics and quasispecies in interferon alpha (IFNalpha) resistant HCV related cryoglobulinaemic vasculitis. METHODS: Reports on two patients with severe manifestations of HCV related cryoglobulinaemia who failed to respond to interferon alpha are given. Both patients were treated with pulse cyclophosphamide (750-1000 mg/month for six and 11 months, respectively). HCV RNA was quantified and HCV quasispecies determined in cryoprecipitates and supernatants before and during treatment. RESULTS: Cryocrit and complement activation decreased in both patients with rebound of cryocrit in one case during continuing pulse cyclophosphamide treatment. Vasculitic symptoms improved. Alanine aminotransferase (ALT) levels and HCV viral load (0.2-0.4 log) increased slightly and reached pretreatment levels after cyclophosphamide was stopped. A highly heterogeneous quasispecies was found in the cryoprecipitate and supernatant of one patient, whereas the viral population was homogeneous in the other patient. After six cycles of cyclophosphamide, viral distances decreased non-significantly. However, phylogenetic analysis showed the evolution of distinct viral strains in one patient and replacement of the main viral population by another population in the second patient. CONCLUSIONS: Immunosuppressive treatment with pulse cyclophosphamide has a temporary limited effect on HCV associated cryoglobulinaemia and leads to a reversible increase of ALT levels and HCV viral load. Short term immunosuppression does not affect the viral heterogeneity as measured by amino acid and nucleotide distances in the hypervariable region 1 of HCV. A change of quasispecies was observed, but further studies are needed to evaluate if this does affect the outcome of IFNalpha treatment in such patients.