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1.
Clin Gastroenterol Hepatol ; 21(1): 229-231.e1, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-34793965

RESUMO

Helicobacter pylori is the most prevalent bacterial infection, affecting half of the world's population, with a high morbidity and mortality rate.1,2 Several invasive and noninvasive testing procedures are available, and their selective use serves the specific needs of diverse clinical scenarios. For gastric cancer prevention, mass screening is necessary and requires a noninvasive, rapid, accurate and cost-effective test. For this purpose H pylori serology currently seems to be the preferred noninvasive diagnostic method. Population-based testing and treatment for H pylori is cost effective in high-risk countries, but less effective in low- and medium-risk countries.3,4 Many serologic tests are available on the market, with inconsistent performance often being observed. Therefore, international guidelines recommend considering only serologic tests with high accuracy that have been validated in the respective local populations. To date, no rapid point-of-care test (POCT) has reached a sufficient degree of accuracy.


Assuntos
Anticorpos Antibacterianos , Antígenos de Bactérias , Proteínas de Bactérias , Infecções por Helicobacter , Helicobacter pylori , Testes de Diagnóstico Rápido , Humanos , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Infecções por Helicobacter/sangue , Infecções por Helicobacter/diagnóstico , Helicobacter pylori/isolamento & purificação , Sensibilidade e Especificidade , Testes Sorológicos/métodos
2.
Helicobacter ; 27(3): e12888, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35363924

RESUMO

BACKGROUND: Murine Helicobacter species have gained increasing awareness in mouse facilities over the last years. Infections with Helicobacter species may have an impact effect on the health of mice and might pose a zoonotic risk to researchers. To minimize the interference with experiments and hence contribute to the 3Rs, a reliable method of monitoring Helicobacter infections in animal facilities needs to be available. The aim of this study was to improve and validate the detection of the most common murine Helicobacter species. MATERIAL AND METHODS: A multiplex PCR assay was developed for identification of Helicobacter hepaticus, H. bilis, H. muridarum, H. rodentium, and H. typhlonius that could simultaneously detect these five strains in fecal samples. To ensure the quality of the results, the method was validated based on recommendations for in-house developed tests. RESULTS: The method established was highly sensitive and specific. All five strains were detectable with a detection limit of 102 bacteria. Eight different mouse facilities were tested with the validated assay, and the following prevalence were found: H. rodentium 57%, H. hepaticus 46%, H. typhlonius 17%, H. bilis 12%, and H. muridarum 0%. CONCLUSION: The multiplex PCR is a reliable, economic, and time-saving diagnostic tool for routine health monitoring. Further prevalence studies are needed to confirm the high prevalence and hence importance of H. rodentium, as until now this agent is not yet listed in FELASA recommendations.


Assuntos
Infecções por Helicobacter , Helicobacter pylori , Helicobacter , Animais , Fezes/microbiologia , Helicobacter/genética , Infecções por Helicobacter/diagnóstico , Infecções por Helicobacter/microbiologia , Infecções por Helicobacter/veterinária , Humanos , Camundongos , Reação em Cadeia da Polimerase Multiplex
3.
Analyst ; 146(11): 3549-3556, 2021 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-33899848

RESUMO

The detection of pathogens in aquatic environments issues a time-consuming challenge, but it is an essential task to prevent the spread of diseases. We have developed a new point-of-care (POC) method for the fast and efficient detection of Legionella pneumophila in water. The method consists first of the generation of immunocomplexes of bacteria species with its corresponding targeted fluorescence-labelled serogroup-specific antibodies, and second a concentration step of pathogens with a membrane filter. Third, on the filtration membrane, our method can detect the fluorescence intensity corresponding to the pathogen concentration. Thus selective and efficient evidence for the presence of bacteria can be evaluated. We tested our system on fluorescent Escherichia coli bacteria and were able to reach an accurate determination of 1000 cells. The technique was furthermore tested on Legionella pneumophila cells, which were labelled with fluorescence-labelled antibodies as a proof of principle. Furthermore, we were able to verify this method in the presence of other bacteria species. We were able to detect bacteria cells within half an hour, a substantial advancement compared to the prevailling state of the art detection method based on the cultivation of Legionella pneumophila. Hence, this system represents the basis for future developments in analysis of pathogens.


Assuntos
Legionella pneumophila , Microbiologia da Água , Anticorpos , Filtração , Sorogrupo
4.
Carcinogenesis ; 40(4): 551-559, 2019 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-30380024

RESUMO

Ring finger protein 43 (RNF43) is an E3 ubiquitin ligase that has been described to be frequently mutated in gastrointestinal cancers. RNF43 downregulation was associated with distant metastasis, TNM stage and poorer survival in patients with gastric and colorectal cancers. Functional analysis has shown that overexpressed RNF43 negatively regulates Wnt signalling by ubiquitinating Frizzled receptors and targeting them for degradation and by sequestering T-cell factor 4 (TCF4) to the nuclear membrane, thereby inhibiting Wnt-mediated transcription. In the stomach, RNF43 overexpression was shown to impair stem-like properties and to be negatively correlated with expression of Wnt-target genes. In this study, we show that RNF43 knockdown enhances the tumourigenic potential of gastric and colorectal cancer cell lines in vitro and in vivo. Thus, loss of RNF43 leads to increased proliferation and anchorage-independent growth as well as increased invasive capacity. In a xenograft model, RNF43 depletion enhanced tumour growth. Furthermore, we established two mouse models in which mutations in the RING domain of RNF43 were introduced. In the intestine and colon, loss of Rnf43 did not induce changes in epithelial architecture or proliferation. In contrast, in the stomach, thickening of the mucosa, hyperplasia and cellular atypia were observed in these mice. Notably, this was independent of elevated Wnt signalling. Together, our results show that RNF43 plays a tumour suppressive role in gastric and colorectal cancer cells and that the loss of its function alters gastric tissue homeostasis in vivo.


Assuntos
Proliferação de Células/genética , Neoplasias Colorretais/genética , Intestinos/patologia , Neoplasias Gástricas/genética , Estômago/patologia , Ubiquitina-Proteína Ligases/genética , Animais , Células CACO-2 , Carcinogênese/genética , Carcinogênese/patologia , Linhagem Celular , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Células HT29 , Humanos , Camundongos , Mucosa/patologia , Mutação/genética , Neoplasias Gástricas/patologia , Ubiquitinação/genética , Via de Sinalização Wnt/genética
5.
Mediators Inflamm ; 2014: 426309, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24587595

RESUMO

Helicobacter pylori is the most widespread chronic bacterial agent in humans and is well recognized for its association with ulcer disease and gastric cancer, with both representing major global health and socioeconomic issues. Given the high level of adaptation and the coevolution of this bacterium with its human host, a thorough and multidirectional view of the specific microbiological characteristics of this infection as well as the host physiology is needed in order to develop novel means of prevention of therapy. This review aims to pinpoint some of these potentially important angles, which have to be considered mutually when studying H. pylori's pathogenicity. The host's biological changes due to the virulence factors are a valuable pillar of H. pylori research as are the mechanisms by which bacteria provoke these changes. In this context, necessary adhesion molecules and significant virulence factors of H. pylori are discussed. Moreover, metabolism of the bacteria, one of the most important aspects for a better understanding of bacterial physiology and consequently possible therapeutic and prophylactic strategies, is addressed. On the other hand, we discuss the recent experimental proofs of the "hygiene hypothesis" in correlation with Helicobacter's infection, which adds another aspect of complexity to this infection.


Assuntos
Infecções por Helicobacter/imunologia , Helicobacter pylori/patogenicidade , Fatores de Virulência/metabolismo , Adesinas Bacterianas/metabolismo , Aderência Bacteriana , Proteínas da Membrana Bacteriana Externa/metabolismo , Infecções por Helicobacter/patologia , Humanos , Sistema Imunitário , Inflamação , Estômago/microbiologia , Simbiose , Resultado do Tratamento
6.
Proc Natl Acad Sci U S A ; 108(36): 14944-9, 2011 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-21896770

RESUMO

The bacterial pathogen Helicobacter pylori chronically infects the human gastric mucosa and is the leading risk factor for the development of gastric cancer. The molecular mechanisms of H. pylori-associated gastric carcinogenesis remain ill defined. In this study, we examined the possibility that H. pylori directly compromises the genomic integrity of its host cells. We provide evidence that the infection introduces DNA double-strand breaks (DSBs) in primary and transformed murine and human epithelial and mesenchymal cells. The induction of DSBs depends on the direct contact of live bacteria with mammalian cells. The infection-associated DNA damage is evident upon separation of nuclear DNA by pulse field gel electrophoresis and by high-magnification microscopy of metaphase chromosomes. Bacterial adhesion (e.g., via blood group antigen-binding adhesin) is required to induce DSBs; in contrast, the H. pylori virulence factors vacuolating cytotoxin A, γ-glutamyl transpeptidase, and the cytotoxin-associated gene (Cag) pathogenicity island are dispensable for DSB induction. The DNA discontinuities trigger a damage-signaling and repair response involving the sequential ataxia telangiectasia mutated (ATM)-dependent recruitment of repair factors--p53-binding protein (53BP1) and mediator of DNA damage checkpoint protein 1 (MDC1)--and histone H2A variant X (H2AX) phosphorylation. Although most breaks are repaired efficiently upon termination of the infection, we observe that prolonged active infection leads to saturation of cellular repair capabilities. In summary, we conclude that DNA damage followed by potentially imprecise repair is consistent with the carcinogenic properties of H. pylori and with its mutagenic properties in vitro and in vivo and may contribute to the genetic instability and frequent chromosomal aberrations that are a hallmark of gastric cancer.


Assuntos
Aderência Bacteriana , Quebras de DNA de Cadeia Dupla , Infecções por Helicobacter/metabolismo , Helicobacter pylori/metabolismo , Neoplasias Gástricas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Aberrações Cromossômicas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Ilhas Genômicas , Infecções por Helicobacter/complicações , Infecções por Helicobacter/patologia , Histonas/genética , Histonas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/microbiologia , Neoplasias Gástricas/patologia , Transativadores/genética , Transativadores/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53
7.
Genesis ; 51(11): 793-802, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24038996

RESUMO

The HMG-box transcription factor Sox17 is essential for endoderm formation, vascular development, and definitive hematopoiesis. To investigate the fate of distinct Sox17-expressing progenitor cells in a spatiotemporal manner, we generated a hormone-inducible CreERT2 knock-in mouse line. By homologous recombination we fused a codon improved, ligand-dependent estrogen receptor Cre recombinase by an intervening viral T2A sequence for co-translational cleavage to the 3' coding region of Sox17. Induction of Cre activity by administration of tamoxifen at defined time points of early mouse development and subsequent genetic lineage tracing confirmed the inducibility and tissue specificity of Cre recombination. Furthermore, Cre activity could be selectively induced in extra-embryonic and embryonic endoderm lineages, the primitive gut tube, and in endothelial cells of the vascular system as well as in the hemogenic endothelium of the dorsal aorta. The Sox17CreERT2 mouse line therefore represents a new tool for genetic lineage tracing in a tissue-specific manner and in addition enables lineage-restricted functional analysis.


Assuntos
Linhagem da Célula , Células-Tronco Embrionárias/metabolismo , Técnicas de Introdução de Genes , Integrases/metabolismo , Fatores de Transcrição SOXF/genética , Animais , Aorta/metabolismo , Diferenciação Celular , Embrião de Mamíferos , Endoderma/metabolismo , Células Endoteliais/metabolismo , Gástrula/metabolismo , Genótipo , Células-Tronco Hematopoéticas/metabolismo , Integrases/genética , Camundongos , Camundongos Transgênicos , Modelos Animais , Especificidade de Órgãos , Tamoxifeno/farmacologia
8.
Int J Med Microbiol ; 303(8): 618-23, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24103649

RESUMO

Screening for H. pylori in large populations continues to be a challenging task, since available tests have limited sensitivity and specificity, which, in population-based approaches, leads to significant numbers of false positive and false negative results. Various H. pylori proteins associated with virulence are highly immunogenic and therefore candidates to detect the infection. There are currently no defined markers that are recognized in all H. pylori infected patients and that do not show cross-reactivity with other bacterial proteins. We identified the H. pylori "hook-associated protein 2 homologue", FliD (UniProtKB/Swiss-Prot: P96786.4) as a novel marker of infection for serological analysis. The H. pylori FliD protein is an essential element in the assembly of the functional flagella. However, this virulence factor has not yet been tested as a diagnostic marker in serology. For this purpose FliD was recombinantly expressed in E. coli, purified by affinity chromatography and gel filtration and used to coat ELISA plates or immobilized on nitrocellulose stripes. To evaluate its antigenicity we screened a defined panel of patient sera. The recombinant H. pylori FliD protein reacted with a high percentage of human sera. Among 318 samples reported positive by histology, 310 (97.4%) were tested positive by FliD Line assay, and 165 out of 170 samples were tested positive by ELISA (97%). We could also reconfirm 297 out of 300 (99%) negative sera by Line assay and 73 from 76 (96%) by ELISA. Taken together, application of FliD in serological diagnosis of H. pylori infection presents a high specificity of up to 99% and a sensitivity of up to 97%. This makes especially the FliD ELISA a simple, cost effective and highly efficient tool to detect H. pylori infection in developing countries where prevalence is high and other screening methods are either not available or are unaffordable.


Assuntos
Anticorpos Antibacterianos/sangue , Proteínas de Bactérias , Infecções por Helicobacter/diagnóstico , Helicobacter pylori/isolamento & purificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Escherichia coli/genética , Feminino , Helicobacter pylori/imunologia , Humanos , Masculino , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Sensibilidade e Especificidade , Testes Sorológicos/métodos
9.
Heliyon ; 9(9): e19238, 2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37674821

RESUMO

Emerging evidence indicates that fibroblasts play pivotal roles in immunoregulation by producing various proteins under health and disease states. In the present study, for the first time, we compared the proteomes of serum-starved human skin fibroblasts and peripheral blood mononuclear cells (PBMCs) using Nano-LC-ESI-tandem mass spectrometry. This analysis contributes to a better understanding of the underlying molecular mechanisms of chronic inflammation and cancer, which are intrinsically accompanied by growth factor deficiency.The proteomes of starved fibroblasts and PBMCs consisted of 307 and 294 proteins, respectively, which are involved in lymphocyte migration, complement activation, inflammation, acute phase response, and immune regulation. Starved fibroblasts predominantly produced extracellular matrix-related proteins such as collagen/collagenase, while PBMCs produced focal adhesion-related proteins like beta-parvin and vinculin which are involved in lymphocyte migration. PBMCs produced a more diverse set of inflammatory molecules like heat shock proteins, while fibroblasts produced human leukocytes antigen-G and -E that are known as main immunomodulatory molecules. Fifty-four proteins were commonly found in both proteomes, including serum albumin, amyloid-beta, heat shock cognate 71 kDa, and complement C3. GeneMANIA bioinformatic tool predicted 418 functions for PBMCs, including reactive oxygen species metabolic processes and 241 functions for starved fibroblasts such as antigen processing and presentation including non-classical MHC -Ib pathway, and negative regulation of the immune response. Protein-protein interactions network analysis indicated the immunosuppressive function for starved fibroblasts-derived human leucocytes antigen-G and -E. Moreover, in an in vitro model of allogeneic transplantation, the immunosuppressive activity of starved fibroblasts was experimentally documented. Conclusion: Under serum starvation-induced metabolic stress, both PBMCs and fibroblasts produced molecules like heat shock proteins and amyloid-beta, which can have pathogenic roles in auto-inflammatory diseases such as rheumatoid arthritis, type 1 diabetes mellitus, systemic lupus erythematosus, aging, and cancer. However, starved fibroblasts showed immunosuppressive activity in an in vitro model of allogeneic transplantation, suggesting their potential to modify such adverse reactions by down-regulating the immune system.

10.
J Allergy Clin Immunol ; 127(5): 1187-94.e7, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21353297

RESUMO

BACKGROUND: Based on a recent positional cloning approach, it was claimed that the collagen 29A1 gene (COL29A1), which encodes an epidermal collagen, represents a major risk gene for eczema underlying a previously reported linkage to chromosome 3q21. However, thus far, not a single replication attempt has been published, and no definitive functional data have been provided. OBJECTIVES: We aimed to determine whether COL29A1 polymorphisms contribute to eczema susceptibility and whether COL29A1 expression is altered in eczema. METHODS: We investigated the reported association of COL29A1 variants with eczema, subtypes of eczema, and eczema-related traits in 5 independent and large study populations comprehensively phenotyped for allergic diseases: a set of 1687 German patients with eczema and 2387 population control subjects, a collection of 274 German families with eczema-diseases children, a cross-sectional population of German children (n = 3099), the Swedish population-based birth cohort Children Allergy and Milieu in Stockholm, an Epidemiologic Study (BAMSE) (n = 2033), and the European cross-sectional Prevention of Allergy-Risk Factors for Sensitization Related to Farming and Anthroposophic Lifestyle (PARSIFAL) study (n = 3113). An additional set of 19 COL29A1 coding single nucleotide polymorphisms was analyzed in BAMSE and PARSIFAL. COL29A1 expression was investigated by using in situ hybridization. RESULTS: We found no evidence for a relationship between COL29A1 polymorphisms and eczema. The equivalence test rejected the hypothesis of association even excluding small effects. In situ hybridization carried out on biopsy specimens from lesional and nonlesional skin of patients with eczema and from healthy control subjects did not show any differences in the cellular distribution pattern of COL29A1 expression at the mRNA level. CONCLUSIONS: Our results suggest that COL29A1 is unlikely to contain genetic variants that have a major effect on eczema or atopy susceptibility.


Assuntos
Colágeno Tipo VI/genética , Eczema/genética , Predisposição Genética para Doença , Hipersensibilidade Imediata/genética , Adolescente , Estudos de Casos e Controles , Criança , Pré-Escolar , Colágeno Tipo VI/metabolismo , Estudos Transversais , Família , Feminino , Alemanha , Humanos , Hibridização In Situ , Recém-Nascido , Masculino , Polimorfismo de Nucleotídeo Único , Pele/metabolismo , Suécia
11.
Front Immunol ; 13: 1025933, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36908807

RESUMO

Introduction: The microenvironment of solid tumors such as breast cancer is heterogeneous and complex, containing different types of cell, namely, cancer stem cells and immune cells. We previously reported the immunoregulatory behavior of the human immune cell in a solid tumor microenvironment-like culture under serum starvation stress for 96 h. Here, we examined the effect of this culture-derived solution on breast cancer development in rats. Method: Ninety-six-hour starved PBMCs supernatant (96 h-SPS) was collected after culturing human PBMCs for 96 h under serum starvation condition. Breast cancer stem cells, LA7 cell line, was used for in vitro study by analyzing gene expression status and performing cytotoxicity, proliferation, scratch wound healing assays, followed by in vivo tumor induction in three groups of mature female Sprague Dawley rats. Animals were treated with 96 h-SPS or RPMI and normal saline as control, n = 6 for each group. After biochemical analysis of iron, lactate, and pH levels in the dissected tumors, Ki67 antigen expression, angiogenesis, and necrosis evaluation were carried out. Metabolic-related gene expression was assessed using RT-qPCR. Moreover, 96 h-SPS composition was discovered by Nano-LC-ESI-MS/MS. Results: 96 h-SPS solution reduced the LA7 cell viability, proliferation, and migration and Gch1 and Spr genes expression in vitro (p< 0.05), whereas stemness gene Oct4 was upregulated (p< 0.01). The intracellular lactate was significantly decreased in the 96 h-SPS treated group (p = 0.007). In this group, Gch1 and Spr were significantly downregulated (p< 0.05), whereas the Sox2 and Oct4 expression was not changed significantly. The number of vessels and mitosis (Ki67+ cells) in the 96 h-SPS-treated group was significantly reduced (p = 0.024). The increased rate of necrosis in this group was statistically significant (p = 0.04). Last, proteomics analysis revealed candidate effectors' components of 96 h-SPS solution. Conclusion: 96 h-SPS solution may help to prevent cancer stem cell mediated tumor development. This phenomenon could be mediated through direct cytotoxic effects, inhibition of cell proliferation and migration in association with reduction in Gch1 and Spr genes expression, angiogenesis and mitosis rate, and necrosis augmentation. The preliminary data obtained from the present study need to be investigated on a larger scale and can be used as a pilot for further studies on the biology of cancer development.


Assuntos
Neoplasias da Mama , Animais , Feminino , Ratos , Neoplasias da Mama/patologia , Antígeno Ki-67/metabolismo , Leucócitos Mononucleares/metabolismo , Necrose/patologia , Células-Tronco Neoplásicas/metabolismo , Ratos Sprague-Dawley , Espectrometria de Massas em Tandem , Microambiente Tumoral
12.
Cell Rep ; 38(2): 110214, 2022 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-34968416

RESUMO

T cell immunity is crucial for control of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and has been studied widely on a quantitative level. However, the quality of responses, in particular of CD8+ T cells, has only been investigated marginally so far. Here, we isolate T cell receptor (TCR) repertoires specific for immunodominant SARS-CoV-2 epitopes restricted to common human Leukocyte antigen (HLA) class I molecules in convalescent individuals. SARS-CoV-2-specific CD8+ T cells are detected up to 12 months after infection. TCR repertoires are diverse, with heterogeneous functional avidity and cytotoxicity toward virus-infected cells, as demonstrated for TCR-engineered T cells. High TCR functionality correlates with gene signatures that, remarkably, could be retrieved for each epitope:HLA combination analyzed. Overall, our data demonstrate that polyclonal and highly functional CD8+ TCRs-classic features of protective immunity-are recruited upon mild SARS-CoV-2 infection, providing tools to assess the quality of and potentially restore functional CD8+ T cell immunity.


Assuntos
Linfócitos T CD8-Positivos/imunologia , COVID-19/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , SARS-CoV-2/imunologia , Adulto , Células Cultivadas , Reações Cruzadas/imunologia , Epitopos de Linfócito T/imunologia , Feminino , Humanos , Epitopos Imunodominantes/imunologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/virologia , Masculino , Glicoproteína da Espícula de Coronavírus/imunologia , Linfócitos T Citotóxicos/imunologia
13.
Exp Dermatol ; 20(1): 48-52, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20955203

RESUMO

Primary human keratinocytes and immortalized HaCaT cells were analysed for their capacity to bind purified staphylococcal protein A (SpA). Co-incubation with FITC-labelled SpA led to a dose-depending attachment. Pull-down experiments with cellular extracts revealed the TNFα receptor I (TNF RI) as binding partner on keratinocytes. Thus, we next looked for expression of this receptor in human epidermis and cultured keratinocytes. TNF RI is strongly expressed on all keratinocytes analysed, both at the mRNA and protein level and activation by SpA at optimal doses of 50-100 µg/ml resulted in the phosphorylation of the TNF RI downstream kinases MEK1/2, JNK1/2, and p38 subsequently leading to translocation of the p65 NF kappa B subunit and AP-1 into the nucleus. This translocation was then followed by increased expression of IL-8 and COX-2, two known NF kappa B-induced pro-inflammatory genes. To further test the relevance of our findings, we analysed in vitro production of over 100 strains isolated from atopic eczema showing that more than 85% of the tested strains produced extracellular SpA in substantial amounts. Thus, besides superantigens, haemolysins, and other cell wall components, Staphylococcus aureus exerts pro-inflammatory stimuli on human keratinocytes through the production of SpA signalling through TNF RI.


Assuntos
Queratinócitos/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Proteína Estafilocócica A/metabolismo , Fator de Transcrição AP-1/metabolismo , Fator de Transcrição RelA/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Bases , Linhagem Celular , Células Cultivadas , Ciclo-Oxigenase 2/genética , Dermatite Atópica/genética , Dermatite Atópica/metabolismo , Dermatite Atópica/microbiologia , Humanos , Técnicas In Vitro , Mediadores da Inflamação/metabolismo , Interleucina-8/genética , Queratinócitos/imunologia , Queratinócitos/microbiologia , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Proteína Estafilocócica A/biossíntese , Staphylococcus aureus/imunologia , Staphylococcus aureus/isolamento & purificação , Staphylococcus aureus/patogenicidade , Transcrição Gênica
14.
Rep Biochem Mol Biol ; 10(1): 105-118, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-34277874

RESUMO

BACKGROUND: Stem cell differentiation therapy is a promising strategy in cancer treatment. we show that protein cocktail prepared from serum starved fibroblasts has therapeutic potential based on this strategy. METHODS: The condition medium was prepared from foreskin isolated fibroblasts and analyzed by Liquid chromatography electrospray ionization mass spectrometry-mass spectrometry (LC-ESI-MS/MS). LA7 mammary gland cancer stem cells originated tumors were induced in Sprague Dawley rats. The rats treated subcutaneously with DMEM (group A), condition medium (group B), or normal saline (group C) once daily for 7 days. Then the tumors were removed and divided into the two parts, one part was used to quantify gene expression by stem-loop RT-qPCR assay and the other part was used for Hematoxylin & Eosin (H & E), Giemsa, and immunohistochemistry (IHC) staining. RESULTS: All induced tumors appeared as sarcomatoid carcinoma (SC). Immunohistochemistry staining confirmed this conclusion by recognizing the tumor as Ki67+, cytokeratin+, vimentine+, and estrogen receptor negative SC. RT-qPCR analysis revealed that Oct4-, Sox-2, Nanog- gene expression was much reduced in the condition medium treated tumors versus proper controls (p< 0.05). Tissue necrosis was more prevalent in this group while tumors volume was diminished almost by 40%. The LC-ESI-MS/MS analysis unrevealed the stemness reducing and the cell death inducing proteins such as, pigment epithelium-derived factor (PEDF), insulin like growth factor binding protein-5 (IGFBP-5) and -7 (IGFBP-7) in the condition medium. CONCLUSION: This study showed that the substances released from starved human fibroblasts were able to down-regulate the stemness-related genes and induce necrosis in LA7 derived tumors.

15.
Am J Clin Dermatol ; 11(1): 1-10, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20000870

RESUMO

The family of toll-like receptors (TLRs) plays a central role in the cutaneous immune defense system. To date, different TLRs have been found on several major cell populations of the skin, such as keratinocytes, fibroblasts, antigen-presenting cells, and melanocytes. Activation of TLRs leads, via different intracellular signaling pathways, to the production of pro-inflammatory stimuli, and is considered a danger signal that should transform the skin in to the functional state of defense. However, TLRs have also been implicated in tissue homeostasis and renewal. Within the group of TLRs, two types have been identified: surface-expressed TLRs, which are predominantly active against bacterial cell wall compounds; and intracellular receptors, which preferentially recognize virus-associated pattern molecules. In addition, surface-expressed receptors trigger phagocytotic and maturation signals, while the intracellular TLRs lead to the induction of antiviral genes. Our review aims to outline the importance of TLRs in the pathogenesis of numerous skin diseases and the potential of TLR agonists as a treatment option for various skin diseases.


Assuntos
Dermatopatias/imunologia , Pele/imunologia , Receptores Toll-Like/imunologia , Acne Vulgar/imunologia , Células Apresentadoras de Antígenos/imunologia , Doenças Autoimunes/imunologia , Biomarcadores/metabolismo , Infecções por Borrelia/imunologia , Dermatite Atópica/imunologia , Fármacos Dermatológicos/uso terapêutico , Fibroblastos/imunologia , Humanos , Queratinócitos/imunologia , Hanseníase/imunologia , Melanócitos/imunologia , Psoríase/imunologia , Transdução de Sinais/imunologia , Pele/metabolismo , Dermatopatias/tratamento farmacológico , Dermatopatias/metabolismo , Sífilis/imunologia , Receptores Toll-Like/agonistas
16.
J Immunol ; 181(4): 2694-704, 2008 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-18684960

RESUMO

Emerging evidence suggests an important role for human epidermal keratinocytes in innate immune mechanisms against bacterial and viral skin infections. The proinflammatory effect of viral infections can be mimicked by double-stranded RNA (dsRNA). Herein, we demonstrate that keratinocytes express all known dsRNA sensing receptors at a constitutive and inducible level, and that they use several downstream signaling pathways leading to a broad pattern of gene expression, not only proinflammatory and immune response genes under the control of NF-kappaB, but also genes under transcriptional control of IRF3. As a consequence, dsRNA, a stimulus for TLR3, protein kinase R (PKR), and the RNA helicases retinoic acid-inducible gene I (RIG-I) and MDA5, induces a status of antiviral defense in keratinocytes. Using inhibitors for the various dsRNA signaling pathways and specific small interfering RNA for TLR3, RIG-I, and MDA5, we demonstrated that in human keratinocytes, TLR3 seems to be necessary for NF-kappaB but not for IRF3 activation, whereas RIG-I and MDA5 are crucial for IRF3 activation. PKR is essential for the dsRNA response in both signaling pathways and thus represents the central antiviral receptor for dsRNA stimulation. Moreover, human keratinocytes up-regulate TLR7, the receptor for single-stranded RNA, in response to stimulation with dsRNA, which renders keratinocytes functionally responsive to the TLR7 agonist gardiquimod, a member of the imidazoquinoline antiviral immune response modifier family. Thus, in addition to building a physical barrier against infectious pathogens, keratinocytes are specially equipped with a full antiviral defense program that enables them to efficiently target viral infections of the skin.


Assuntos
Antivirais/metabolismo , RNA Helicases DEAD-box/fisiologia , Queratinócitos/virologia , RNA de Cadeia Dupla/fisiologia , Receptores do Ácido Retinoico/fisiologia , Transdução de Sinais/imunologia , Receptor 3 Toll-Like/fisiologia , eIF-2 Quinase/fisiologia , Células Cultivadas , Regulação da Expressão Gênica/imunologia , Humanos , Fator Regulador 3 de Interferon/metabolismo , Fator Regulador 3 de Interferon/fisiologia , Helicase IFIH1 Induzida por Interferon , Queratinócitos/enzimologia , Queratinócitos/imunologia , Queratinócitos/metabolismo , Papillomaviridae/imunologia , Poli I-C/biossíntese , Poli I-C/farmacologia , Receptores de Reconhecimento de Padrão/biossíntese , Transdução de Sinais/genética , Receptor 3 Toll-Like/deficiência , Receptor 3 Toll-Like/genética
17.
Braz J Microbiol ; 51(1): 183-187, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31656022

RESUMO

Noroviruses (NoV) cause the majority of non-bacterial gastroenteritis cases worldwide, with genotype II.4 being the most common. The aim of our study was to quantitate norovirus-specific IgG in immunocompromised patients before and after laboratory-confirmed norovirus infection. A quantitative ELISA was developed by coating ELISA plates with recombinantly expressed P domain of GII.1 capsid protein. After testing mouse sera drawn before and after immunization with GII.1- and GII.4 P domain, sera from GII.1- and GII.4 infected patients were tested. The assay reliably detected preexisting NoV-specific IgG antibodies. Sera drawn after infection showed increased antibody concentrations. Antibodies elicited by GII.1- and GII.4 infections could be detected with coated GII.1 capsid protein. IgG levels remained constant during the first week and then increased in the second week after laboratory diagnosis. The results show that immunocompromised patients elicited IgG responses to NoV infections that could be reliably detected with our quantitative ELISA.


Assuntos
Anticorpos Antivirais/sangue , Infecções por Caliciviridae/imunologia , Hospedeiro Imunocomprometido , Imunoglobulina G/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Infecções por Caliciviridae/virologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Norovirus
18.
Dermatol Clin ; 25(4): 531-40, viii, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17903612

RESUMO

The human skin represents the first line of defense against potentially hazardous environmental threats (ie, infection by microbes, such as viruses, bacteria, and fungi). To fulfill this crucial function and to maintain the integrity of the skin compartment, evolution has equipped the human immune system with a variety of sophisticated tools leading to an efficient defense system of responses to various infectious challenges. The role of the skin within the different defense lines is multifaceted. The central role of the immune defense system is performed by the group of "pathogen-associated pattern recognition receptors," among which the group of Toll-like receptors (TLRs) has evolved as the central family during the last years. Ten TLRs are identified in humans, all of which share similarities in their structure and function, but respond to different microbial components.


Assuntos
Fármacos Dermatológicos/farmacologia , Epiderme/fisiologia , Dermatopatias/fisiopatologia , Receptores Toll-Like/fisiologia , Epiderme/imunologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Ligantes , Polimorfismo Genético , Transdução de Sinais/fisiologia , Dermatopatias/genética , Receptores Toll-Like/efeitos dos fármacos , Receptores Toll-Like/genética
19.
Nat Microbiol ; 2: 16189, 2016 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-27748768

RESUMO

Helicobacter pylori specifically colonizes the human gastric epithelium and is the major causative agent for ulcer disease and gastric cancer development. Here, we identify members of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family as receptors of H. pylori and show that HopQ is the surface-exposed adhesin that specifically binds human CEACAM1, CEACAM3, CEACAM5 and CEACAM6. HopQ-CEACAM binding is glycan-independent and targeted to the N-domain. H. pylori binding induces CEACAM1-mediated signalling, and the HopQ-CEACAM1 interaction enables translocation of the virulence factor CagA into host cells and enhances the release of pro-inflammatory mediators such as interleukin-8. Based on the crystal structure of HopQ, we found that a ß-hairpin insertion (HopQ-ID) in HopQ's extracellular 3+4 helix bundle domain is important for CEACAM binding. A peptide derived from this domain competitively inhibits HopQ-mediated activation of the Cag virulence pathway, as genetic or antibody-mediated abrogation of the HopQ function shows. Together, our data suggest the HopQ-CEACAM1 interaction to be a potentially promising novel therapeutic target to combat H. pylori-associated diseases.


Assuntos
Adesinas Bacterianas/metabolismo , Aderência Bacteriana , Moléculas de Adesão Celular/metabolismo , Helicobacter pylori/fisiologia , Helicobacter pylori/patogenicidade , Interações Hospedeiro-Patógeno , Adesinas Bacterianas/química , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Linhagem Celular , Cristalografia por Raios X , Humanos , Interleucina-8/metabolismo , Ligação Proteica , Conformação Proteica , Transporte Proteico , Virulência
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