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Influenza A virus (IAV) can cause severe respiratory infection leading to significant global morbidity and mortality through seasonal epidemics. Likewise, the constantly increasing number of cancer diseases is a growing problem. Nevertheless, the understanding of the mutual interactions of the immune responses between cancer and infection is still very vague. Therefore, it is important to understand the immunological cross talk between cancer and IAV infection. In several preclinical mouse models of cancer, including melanoma and colorectal cancer, we observed that IAV infection in the lung significantly decreased the tumour burden. Concomitantly, tumour-specific CD8+ T-cells are strongly activated upon infection, both in the tumour tissue and in the lung. CD8+ T-cell depletion during infection reverses the reduced tumour growth. Interestingly, IAV infection orchestrated the migration of tumour-specific CD8+ T-cells from the tumour into the infected lung. Blocking the migration of CD8+ T-cells prevented the anti-tumoural effect. Thus, our findings show that viral respiratory infection has significant impact on the anti-tumour CD8+ T-cell response, which will significantly improve our understanding of the immunological cross talk between cancer and infection.
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Doenças Transmissíveis , Vírus da Influenza A , Influenza Humana , Neoplasias , Infecções por Orthomyxoviridae , Camundongos , Animais , Humanos , Linfócitos T CD8-Positivos , ImunidadeRESUMO
Neuropilin-1 (Nrp-1) expression on CD8+ T cells has been identified in tumor-infiltrating lymphocytes and in persistent murine gamma-herpes virus infections, where it interferes with the development of long-lived memory T cell responses. In parasitic and acute viral infections, the role of Nrp-1 expression on CD8+ T cells remains unclear. Here, we demonstrate a strong induction of Nrp-1 expression on CD8+ T cells in Plasmodium berghei ANKA (PbA)-infected mice that correlated with neurological deficits of experimental cerebral malaria (ECM). Likewise, the frequency of Nrp-1+CD8+ T cells was significantly elevated and correlated with liver damage in the acute phase of lymphocytic choriomeningitis virus (LCMV) infection. Transcriptomic and flow cytometric analyses revealed a highly activated phenotype of Nrp-1+CD8+ T cells from infected mice. Correspondingly, in vitro experiments showed rapid induction of Nrp-1 expression on CD8+ T cells after stimulation in conjunction with increased expression of activation-associated molecules. Strikingly, T cell-specific Nrp-1 ablation resulted in reduced numbers of activated T cells in the brain of PbA-infected mice as well as in spleen and liver of LCMV-infected mice and alleviated the severity of ECM and LCMV-induced liver pathology. Mechanistically, we identified reduced blood-brain barrier leakage associated with reduced parasite sequestration in the brain of PbA-infected mice with T cell-specific Nrp-1 deficiency. In conclusion, Nrp-1 expression on CD8+ T cells represents a very early activation marker that exacerbates deleterious CD8+ T cell responses during both, parasitic PbA and acute LCMV infections.
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Coriomeningite Linfocítica , Malária Cerebral , Parasitos , Camundongos , Animais , Neuropilina-1 , Coriomeningite Linfocítica/patologia , Vírus da Coriomeningite Linfocítica , Linfócitos T CD8-Positivos/patologia , Camundongos Endogâmicos C57BLRESUMO
Decline in immune function during aging increases susceptibility to different aging-related diseases. However, the underlying molecular mechanisms, especially the genetic factors contributing to imbalance of naïve/memory T-cell subpopulations, still remain largely elusive. Here, we show that loss of DJ-1 encoded by PARK7/DJ-1, causing early-onset familial Parkinson's disease (PD), unexpectedly diminished signs of immunoaging in T-cell compartments of both human and mice. Compared with two gender-matched unaffected siblings of similar ages, the index PD patient with DJ-1 deficiency showed a decline in many critical immunoaging features, including almost doubled non-senescent T cells. The observation was further consolidated by the results in 45-week-old DJ-1 knockout mice. Our data demonstrated that DJ-1 regulates several immunoaging features via hematopoietic-intrinsic and naïve-CD8-intrinsic mechanisms. Mechanistically, DJ-1 depletion reduced oxidative phosphorylation (OXPHOS) and impaired TCR sensitivity in naïve CD8 T cells at a young age, accumulatively leading to a reduced aging process in T-cell compartments in older mice. Our finding suggests an unrecognized critical role of DJ-1 in regulating immunoaging, discovering a potent target to interfere with immunoaging- and aging-associated diseases.
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Estresse Oxidativo , Doença de Parkinson , Envelhecimento/genética , Animais , Humanos , Camundongos , Camundongos Knockout , Estresse Oxidativo/genética , Doença de Parkinson/genética , Proteína Desglicase DJ-1/genética , Proteína Desglicase DJ-1/metabolismo , Linfócitos TRESUMO
BACKGROUND: Infective endocarditis (IE) poses a significant health risk, especially in patients with prosthetic heart valves. Despite advances in treatment, mortality rates remain high. This study aims to investigate the antibacterial properties of a copper titanium dioxide (4× Cu-TiO2) coating on cardiovascular implants against Staphylococcus aureus, a common causative agent of IE. METHODS: Titanium oxide carriers functionalized with copper ions were employed as an antibacterial coating for heart and vascular prostheses. The coating's antibacterial efficacy was assessed using S. aureus ATCC 29213. Microscopic evaluations were conducted on both biological and artificial materials. Antibacterial activity was qualitatively assessed via a modified disc diffusion method and quantitatively measured through colony counts in NaCl suspensions. RESULTS: The coating process was successfully applied to all tested cardiovascular prosthetic materials. Qualitative assessments of antibacterial effectiveness revealed an absence of bacterial growth in the area directly beneath the coated valve. Quantitative evaluations showed a significant reduction in bacterial colonization on coated mechanical valves, with 2.95 × 104 CFU per valve, compared to 1.91 × 105 CFU in control valves. CONCLUSIONS: The 4× Cu-TiO2 coating demonstrated promising antibacterial properties against S. aureus, suggesting its potential as an effective strategy for reducing the risk of bacterial colonization of cardiovascular implants. Further studies are needed to assess the longevity of the coating and its efficacy against other pathogens.
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Endocardite Bacteriana , Endocardite , Próteses Valvulares Cardíacas , Humanos , Cobre , Staphylococcus aureus , Projetos Piloto , Materiais Revestidos Biocompatíveis , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Próteses e Implantes , Endocardite Bacteriana/prevenção & controle , TitânioRESUMO
INTRODUCTION: Bacterial biofilm formation on medical devices, such as Cochlear implants (CI), can lead to chronic infections. Not only the inner parts of the implant but also the externally located headpiece might be associated with prolonged superficial skin eczema resulting in the inability of wearing the headpiece. In this study, the surface of three CI headpieces from different manufacturers were examined for bacterial biofilm formation. MATERIALS AND METHODS: Two bacterial species associated with implant-related infections were tested: Pseudomonas aeruginosa (ATCC9027) and Staphylococcus aureus (ATCC6538). Biofilms were formed over 24 h in tryptic soy broth at 36 °C. Biofilm formation was detected in form of biomass measurement by crystal violet staining. CI headpiece dummies of three manufacturers were used. RESULTS: Both tested bacterial species formed biofilms on the examined CI headpiece-surfaces in a species-dependent manner with higher biofilm formation of P. aeruginosa. For both, S. aureus and P. aeruginosa, biofilm formation on the CI components was comparable to a polystyrene control surface. Between the three manufacturers, no significant difference in biofilm formation was found. DISCUSSION: The tested bacteria displayed biofilm formation on the CI headpieces in a species-specific manner with higher amount of biofilm formed by P. aeruginosa. The biofilm formation was comparable between the manufacturers. In this study, an enhanced biofilm formation on CI headpieces could not be demonstrated. These in vitro tests suggest a minor role of bacterial biofilm on the CI headpiece in skin infections under the CI headpiece.
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BACKGROUND AIMS: Extracellular vesicles (EVs) harvested from conditioned media of human mesenchymal stromal cells (MSCs) suppress acute inflammation in various disease models and promote regeneration of damaged tissues. After successful treatment of a patient with acute steroid-refractory graft-versus-host disease (GVHD) using EVs prepared from conditioned media of human bone marrow-derived MSCs, this study focused on improving the MSC-EV production for clinical application. METHODS: Independent MSC-EV preparations all produced according to a standardized procedure revealed broad immunomodulatory differences. Only a proportion of the MSC-EV products applied effectively modulated immune responses in a multi-donor mixed lymphocyte reaction (mdMLR) assay. To explore the relevance of such differences in vivo, at first a mouse GVHD model was optimized. RESULTS: The functional testing of selected MSC-EV preparations demonstrated that MSC-EV preparations revealing immunomodulatory capabilities in the mdMLR assay also effectively suppress GVHD symptoms in this model. In contrast, MSC-EV preparations, lacking such in vitro activities, also failed to modulate GVHD symptoms in vivo. Searching for differences of the active and inactive MSC-EV preparations, no concrete proteins or miRNAs were identified that could serve as surrogate markers. CONCLUSIONS: Standardized MSC-EV production strategies may not be sufficient to warrant manufacturing of MSC-EV products with reproducible qualities. Consequently, given this functional heterogeneity, every individual MSC-EV preparation considered for the clinical application should be evaluated for its therapeutic potency before administration to patients. Here, upon comparing immunomodulating capabilities of independent MSC-EV preparations in vivo and in vitro, we found that the mdMLR assay was qualified for such analyses.
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Vesículas Extracelulares , Doença Enxerto-Hospedeiro , Células-Tronco Mesenquimais , MicroRNAs , Humanos , Animais , Camundongos , Meios de Cultivo Condicionados/metabolismo , Vesículas Extracelulares/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Doença Enxerto-Hospedeiro/terapia , Células-Tronco Mesenquimais/metabolismoRESUMO
Neuropilin-1 (Nrp-1) is a well described marker molecule for CD4+Foxp3+ thymus-derived regulatory T cells (Tregs). In addition, a small population of CD4+Foxp3- conventional (conv) T cells expresses Nrp-1 in naive mice, and Nrp-1 expression has been described to be upregulated on activated CD4+ T cells. However, the function of Nrp-1 expression on CD4+ non-Tregs still remains elusive. In this study, we demonstrate that Nrp-1 expression was induced upon stimulation of CD4+Foxp3- T cells in vitro and during an ongoing immune response in vivo. This activation-induced Nrp-1+CD4+ T cell subset (iNrp-1+) showed a highly activated phenotype in terms of elevated CD25 and CD44 expression, enhanced production of proinflammatory cytokines, and increased proliferation compared with the Nrp-1-CD4+ counterpart. In contrast, Nrp-1+CD4+Foxp3- conv T cells from naive mice (nNrp-1+) were dysfunctional. nNrp-1+CD4+ conv T cells upregulated activation-associated molecules to a lesser extent, exhibited impaired proliferation and produced fewer proinflammatory cytokines than Nrp-1-CD4+ conv T cells upon stimulation in vitro. Moreover, the expression of PD-1 and CTLA-4 was significantly higher on nNrp-1+CD4+Foxp3- T cells compared with iNrp-1+CD4+Foxp3- T cells and Nrp-1-CD4+Foxp3- T cells after stimulation and under homeostatic conditions. Strikingly, transfer of Ag-specific iNrp-1+CD4+ conv T cells aggravated diabetes development, whereas Ag-specific nNrp-1+CD4+ conv T cells failed to induce disease in a T cell transfer model of diabetes. Overall, our results indicate that Nrp-1 expression has opposite functions in recently activated CD4+ non-Tregs compared with CD4+ non-Tregs from naive mice.
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Fatores de Transcrição Forkhead , Neuropilina-1 , Animais , Imunidade , Camundongos , Subpopulações de Linfócitos T , Linfócitos T ReguladoresRESUMO
OBJECTIVES: Interest in aspergillosis has increased over the past decades. An increase in the incidence of azole-resistant Aspergillus fumigatus strains has been reported; therefore, the need for novel therapeutic approaches is urgent. The formation of biofilms contributes to pathogen resistance. We investigated the biofilm formation capabilities of azole-resistant A. fumigatus and analysed the susceptibility of biofilms at various developmental stages to three antifungal agents. METHODS: Biofilm formation of 19 clinical A. fumigatus strains (3 azole-susceptible and 16 azole-resistant strains) was determined by crystal violet staining and by an XTT assay over a period of 48â h. We measured antibiofilm activity of voriconazole, amphotericin B and olorofim. These agents were added before adhesion, after adhesion, after germination and to mature fungal biofilm. Antibiofilm activity was assessed in an XTT assay and in confocal laser scan microscopy. Additionally, a growth-kinetic assay with planktonic A. fumigatus was performed. RESULTS: Each of the antifungal agents inhibited the metabolic activity of A. fumigatus biofilms when applied at early stages of biofilm formation. The mature biofilms were more resistant. Olorofim and voriconazole showed promising effects against A. fumigatus adhesion and germination, whereas the mature biofilm was not affected by treatment. In contrast, the biofilm of A. fumigatus showed amphotericin B susceptibility throughout the entire developmental process. The planktonic cells were susceptible to all three antifungal drug classes with an inhibition peak at 12â h after incubation. CONCLUSIONS: This is the first known study to demonstrate the antibiofilm activity of olorofim, voriconazole and amphotericin B against azole-resistant A. fumigatus.
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Antifúngicos , Aspergillus fumigatus , Acetamidas , Anfotericina B/farmacologia , Antifúngicos/uso terapêutico , Azóis/farmacologia , Biofilmes , Farmacorresistência Fúngica , Proteínas Fúngicas/metabolismo , Testes de Sensibilidade Microbiana , Piperazinas , Pirimidinas , Pirróis , Voriconazol/metabolismo , Voriconazol/farmacologiaRESUMO
Demethylation of FOXP3-TSDR (Treg specific demethylated region) is a hallmark of stable differentiation and suppressive function of regulatory T (Treg) cells. Previous protocols aiming at human naïve T cell differentiation failed to implement a Treg cell specific epigenetic signature. Ten-eleven translocation (TET) enzymes catalyze DNA demethylation. Plasmids towardexpression of a fusion protein encompassing nonfunctional Cas9, the catalytic domain of TET1, blue fluorescent protein, and encoding single guide RNAs (sgRNAs) targeting specific segments of the FOXP3-TSDR were engineered and transfected into Jurkat T cells. FOXP3-TSDR methylation was analyzed by deep-amplicon bisulfite sequencing while cellular Foxp3, Tbet, Gata3, and Rorgt mRNA levels were determined by real-time PCR. Overexpression of dCas9TET1 significantly decreased Jurkat cell FOXP3-TSDR methylation and increased Foxp3 mRNA expression while expressions of master transcription factor mRNAs of other major T cell lineages remained largely unaffected. dCas9-TET1 construct transfection mediated Treg programming of patients' primary T cells might be feasible.
Assuntos
Sistemas CRISPR-Cas/genética , Metilação de DNA/genética , Fatores de Transcrição Forkhead/metabolismo , Edição de Genes/métodos , Oxigenases de Função Mista/genética , Proteínas Proto-Oncogênicas/genética , Linfócitos T Reguladores/citologia , Proteína 9 Associada à CRISPR/genética , Diferenciação Celular/imunologia , Linhagem Celular Tumoral , Desmetilação , Fatores de Transcrição Forkhead/genética , Fator de Transcrição GATA3/genética , Humanos , Células Jurkat , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , RNA Guia de Cinetoplastídeos/genética , RNA Mensageiro/genética , Linfócitos T Reguladores/imunologiaRESUMO
The glycoprotein CD83 is known to be expressed by different immune cells including activated CD4+Foxp3+ regulatory T cells (Tregs) and CD4+Foxp3- conventional T cells. However, the physiological function of endogenous CD83 in CD4+ T cell subsets is still unclear. In this study, we have generated a new CD83flox mouse line on BALB/c background, allowing for specific ablation of CD83 in T cells upon breeding with CD4-cre mice. Tregs from CD83flox/flox/CD4-cretg/wt mice had similar suppressive activity as Tregs from CD83flox/flox/CD4-crewt/wt wild-type littermates, suggesting that endogenous CD83 expression is dispensable for the inhibitory capacity of Tregs. However, CD83-deficient CD4+ conventional T cells showed elevated proliferation and IFN-γ secretion as well as an enhanced capacity to differentiate into Th1 cells and Th17 cells upon stimulation in vitro. T cell-specific ablation of CD83 expression resulted in aggravated contact hypersensitivity reaction accompanied by enhanced CD4+ T cell activation. Moreover, adoptive transfer of CD4+CD45RBhigh T cells from CD83flox/flox/CD4-cretg /wt mice into Rag2-deficient mice elicited more severe colitis associated with increased serum concentrations of IL-12 and elevated CD40 expression on CD11c+ dendritic cells (DCs). Strikingly, DCs from BALB/c mice cocultured with CD83-deficient CD4+ conventional T cells showed enhanced CD40 expression and IL-12 secretion compared with DCs cocultured with CD4+ conventional T cells from CD83flox/flox/CD4-crewt/wt wild-type mice. In summary, these results indicate that endogenous CD83 expression in CD4+ conventional T cells plays a crucial role in controlling CD4+ T cell responses, at least in part, by regulating the activity of CD11c+ DCs.
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Antígenos CD/imunologia , Linfócitos T CD4-Positivos/imunologia , Imunidade/imunologia , Imunoglobulinas/imunologia , Inflamação/imunologia , Glicoproteínas de Membrana/imunologia , Transferência Adotiva/métodos , Animais , Células Dendríticas/imunologia , Feminino , Interferon gama/imunologia , Interleucina-12/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Linfócitos T Reguladores/imunologia , Células Th1/imunologia , Células Th17/imunologia , Antígeno CD83RESUMO
BACKGROUND: COVID-19-associated invasive pulmonary aspergillosis (CAPA) is associated with increased mortality. Cases of CAPA caused by azole-resistant Aspergillus fumigatus strains have been reported. OBJECTIVES: To analyse the twelve-month CAPA prevalence in a German tertiary care hospital and to characterise clinical A. fumigatus isolates from two German hospitals by antifungal susceptibility testing and microsatellite genotyping. PATIENTS/METHODS: Retrospective observational study in critically ill adults from intensive care units with COVID-19 from 17 February 2020 until 16 February 2021 and collection of A. fumigatus isolates from two German centres. EUCAST broth microdilution for four azole compounds and microsatellite PCR with nine markers were performed for each collected isolate (N = 27) and additional for three non-COVID A. fumigatus isolates. RESULTS: welve-month CAPA prevalence was 7.2% (30/414), and the rate of azole-resistant A. fumigatus isolates from patients with CAPA was 3.7% with detection of one TR34/L98H mutation. The microsatellite analysis revealed no major clustering of the isolates. Sequential isolates mainly showed the same genotype over time. CONCLUSIONS: Our findings demonstrate similar CAPA prevalence to other reports and a low azole-resistance rate. Genotyping of A. fumigatus showed polyclonal distribution except for sequential isolates.
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COVID-19 , Aspergilose Pulmonar , Adulto , Antifúngicos/farmacologia , Aspergillus fumigatus , Azóis/farmacologia , Farmacorresistência Fúngica/genética , Proteínas Fúngicas/genética , Humanos , Unidades de Terapia Intensiva , Testes de Sensibilidade Microbiana , Aspergilose Pulmonar/complicações , Aspergilose Pulmonar/epidemiologiaRESUMO
Previous studies have shown that sphingosine kills a variety of pathogenic bacteria, including Pseudomonas aeruginosa and Staphylococcus aureus Sphingosine concentrations are decreased in airway epithelial cells of cystic fibrosis (CF) mice, and this defect has been linked to the infection susceptibility of these mice. Here, we tested whether the genetic overexpression of acid ceramidase rescues cystic fibrosis mice from pulmonary infections with P. aeruginosa We demonstrate that the transgenic overexpression of acid ceramidase in CF mice corresponds to the overexpression of acid ceramidase in bronchial and tracheal epithelial cells and normalizes ceramide and sphingosine levels in bronchial and tracheal epithelial cells. In addition, the expression of ß1-integrin, which is ectopically expressed on the luminal surface of airway epithelial cells in cystic fibrosis mice, an alteration that is very important for mediating pulmonary P. aeruginosa infections in cystic fibrosis, is normalized in cystic fibrosis airways upon the overexpression of acid ceramidase. Most importantly, the overexpression of acid ceramidase protects cystic fibrosis mice from pulmonary P. aeruginosa infections. Infection of CF mice or CF mice that inhaled sphingosine with P. aeruginosa or a P. aeruginosa mutant that is resistant to sphingosine indicates that sphingosine and not a metabolite kills P. aeruginosa upon pulmonary infection. These studies further support the use of acid ceramidase and its metabolite sphingosine as potential treatments of cystic fibrosis.
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Ceramidase Ácida/genética , Ceramidase Ácida/farmacologia , Ceramidase Ácida/uso terapêutico , Fibrose Cística/complicações , Fibrose Cística/tratamento farmacológico , Infecções por Pseudomonas/etiologia , Infecções por Pseudomonas/prevenção & controle , Animais , Fibrose Cística/fisiopatologia , Regulação Bacteriana da Expressão Gênica , Humanos , Camundongos , Modelos Animais , Pseudomonas aeruginosa/efeitos dos fármacos , Virulência/genéticaRESUMO
We describe screening results for detection of co-infections with Legionella pneumophila in patients infected with severe acute respiratory syndrome coronavirus 2. In total, 93 patients were tested; 1 was positive (1.1%) for L. pneumophila serogroup 1. Co-infections with L. pneumophila occur in coronavirus disease patients and should not be missed.
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COVID-19 , Coinfecção , Legionella pneumophila , Alemanha/epidemiologia , Humanos , SARS-CoV-2 , Centros de Atenção TerciáriaRESUMO
Heroin dependence may result in suppression of adaptive immune responses. Previously, we demonstrated an increase in relative numbers of inhibitory CD4+ regulatory T cells (Tregs) and impaired proliferative activity of CD4+ T cells from heroin-addicted patients in contrast to patients in opioid maintenance therapy and healthy controls. However, it remains elusive whether heroin has a direct impact on the CD4+ T cell compartment or whether observed effects result from stressful living conditions. Here, we analyzed the frequencies of Tregs and the proliferation as well as the cytokine production of stimulated CD4+ T cells from heroin-addicted patients with use of illicit heroin, patients in heroin-assisted treatment (HAT), and patients in methadone maintenance therapy (MMT). Relative numbers of CD4+ Tregs were significantly enhanced in patients with illicit heroin abuse compared with patients in HAT or MMT. Notably, CD4+ T cells from patients in HAT and from persons using illicit heroin showed significant reduced proliferation and secretion of the pro-inflammatory cytokines IFN-γ and IL-6 upon stimulation in vitro. From these results, we conclude that structured programs such as HAT and MMT dampen elevated frequencies of Tregs in heroin-addicted patients, whereas chronic heroin administration irrespective of using illicit heroin or receiving HAT has a direct impact on the proliferative activity and cytokine production of CD4+ T cells.
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Proliferação de Células/efeitos dos fármacos , Dependência de Heroína/tratamento farmacológico , Heroína/uso terapêutico , Linfócitos T Reguladores/efeitos dos fármacos , Adulto , Analgésicos Opioides/uso terapêutico , Citocinas/efeitos dos fármacos , Feminino , Humanos , Masculino , Metadona/uso terapêutico , Pessoa de Meia-Idade , Tratamento de Substituição de Opiáceos/métodosRESUMO
A reciprocal interaction exists between the gut microbiota and the immune system. Regulatory T (Treg) cells are important for controlling immune responses and for maintaining the intestinal homeostasis but their precise influence on the gut microbiota is unclear. We studied the effects of Treg cell depletion on inflammation of the intestinal mucosa and analysed the gut microbiota before and after depletion of Treg cells using the DEpletion of REGulatory T cells (DEREG) mouse model. DNA was extracted from stool samples of DEREG mice and wild-type littermates at different time-points before and after diphtheria toxin application to deplete Treg cells in DEREG mice. The V3/V4 region of the 16S rRNA gene was used for studying the gut microbiota with Illumina MiSeq paired ends sequencing. Multidimensional scaling separated the majority of gut microbiota samples from late time-points after Treg cell depletion in DEREG mice from samples of early time-points before Treg cell depletion in these mice and from gut microbiota samples of wild-type mice. Treg cell depletion in DEREG mice was accompanied by an increase in the relative abundance of the phylum Firmicutes and by intestinal inflammation in DEREG mice 20 days after Treg cell depletion, indicating that Treg cells influence the gut microbiota composition. In addition, the variables cage, breeding and experiment number were associated with differences in the gut microbiota composition and these variables should be respected in murine studies.
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Colo/microbiologia , Firmicutes/crescimento & desenvolvimento , Fatores de Transcrição Forkhead/imunologia , Microbioma Gastrointestinal , Depleção Linfocítica , Linfócitos T Reguladores/imunologia , Animais , Cruzamento , Colite/imunologia , Colite/metabolismo , Colite/microbiologia , Colo/imunologia , Colo/metabolismo , Disbiose , Fezes/microbiologia , Feminino , Firmicutes/imunologia , Fatores de Transcrição Forkhead/metabolismo , Interações Hospedeiro-Patógeno , Abrigo para Animais , Masculino , Camundongos , Fatores Sexuais , Linfócitos T Reguladores/metabolismo , Fatores de TempoRESUMO
OBJECTIVES: Patients with immunodeficiency or cystic fibrosis frequently suffer from respiratory fungal infections. In particular, biofilm-associated fungi cause refractory infection manifestations, linked to increased resistance to anti-infective agents. One emerging filamentous fungus is Lomentospora prolificans. Here, the biofilm-formation capabilities of L. prolificans isolates were investigated and the susceptibility of biofilms to various antifungal agents was analysed. METHODS: Biofilm formation of L. prolificans (n = 11) was estimated by crystal violet stain and antibiofilm activity was additionally determined via detection of metabolically active biofilm using an XTT assay. Amphotericin B, micafungin, voriconazole and olorofim were compared with regard to their antibiofilm effects when added prior to adhesion, after adhesion and on mature and preformed fungal biofilms. Imaging via confocal laser scanning microscopy was carried out to demonstrate the effect of drug treatment on the fungal biofilm. RESULTS: Antibiofilm activities of the tested antifungal agents were shown to be most effective on adherent cells whilst mature biofilm was the most resistant. The most promising antibiofilm effects were detected with voriconazole and olorofim. Olorofim showed an average minimum biofilm eradication concentration (MBEC) of 0.06 mg/L, when added prior to and after adhesion. The MBECs of voriconazole were ≤4 mg/L. On mature biofilm the MBECs of olorofim and voriconazole were higher than the previously determined MICs against planktonic cultures. In contrast, amphotericin B and especially micafungin did not exhibit sufficient antibiofilm activity against L. prolificans. CONCLUSIONS: To our knowledge, this is the first study demonstrating the antibiofilm potential of olorofim against the human pathogenic fungus L. prolificans.
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Preparações Farmacêuticas , Scedosporium , Acetamidas , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Biofilmes , Humanos , Testes de Sensibilidade Microbiana , Piperazinas , Pirimidinas , PirróisRESUMO
Since the discovery of T helper type 1 and type 2 effector T cell subsets 20 years ago, inducible regulatory T cells and interleukin 17 (IL-17)-producing T helper cells have been added to the 'portfolio' of helper T cells. It is unclear how many more effector T cell subsets there may be and to what degree their characteristics are fixed or flexible. Here we show that transforming growth factor-beta, a cytokine at the center of the differentiation of IL-17-producing T helper cells and inducible regulatory T cells, 'reprograms' T helper type 2 cells to lose their characteristic profile and switch to IL-9 secretion or, in combination with IL-4, drives the differentiation of 'T(H)-9' cells directly. Thus, transforming growth factor-beta constitutes a regulatory 'switch' that in combination with other cytokines can 'reprogram' effector T cell differentiation along different pathways.
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Diferenciação Celular/imunologia , Interleucina-9/biossíntese , Subpopulações de Linfócitos T/citologia , Células Th2/citologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Linhagem da Célula/imunologia , Citocinas/biossíntese , Camundongos , Camundongos Transgênicos , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Subpopulações de Linfócitos T/imunologia , Células Th2/imunologiaRESUMO
BACKGROUND: Commonly, the application of radiological and clinical criteria and the determination of galactomannan (GM) in respiratory samples are used as a diagnostic tool for the detection of invasive pulmonary aspergillosis (IPA). MATERIALS/METHODS: In this study, two lateral flow assays, OLM Aspergillus lateral flow device (LFD) and IMMY sona Aspergillus Galactomannan lateral flow assay (LFA), were evaluated at two tertiary hospitals in Germany. A total of 200 bronchoalveolar lavage (BAL) samples from patients with suspicion of IPA were analysed retrospectively. LFD and LFA were evaluated against four different criteria: Blot, EORTC/MSG, Schauwvlieghe and extended Blot criteria and additionally against GM. RESULTS: The evaluation of four algorithms for the diagnosis of IPA showed that there exist good diagnostic tools to rule out an IPA even before results of Aspergillus culture are available. Sensitivities and negative predictive values are generally higher for the LFA than for the LFD in all four criteria. Specificity and positive predictive values varied depending on the classification criteria. The total agreement between the GM and the LFA cube reader (cut-off = 1) was 84%. The correlation between the GM and LFA was calculated with r = 0.8. CONCLUSION: The here presented data indicate that a negative LFA result in BAL fluid can reliable rule out an IPA in a heterogeneous group of ICU patients based on the original Blot criteria. LFA seems to be a promising immunochromatographic test exhibiting a good agreement with positive GM values.
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Antígenos de Fungos/análise , Aspergillus/química , Líquido da Lavagem Broncoalveolar/química , Cromatografia de Afinidade/métodos , Aspergilose Pulmonar Invasiva/diagnóstico , Algoritmos , Aspergillus/imunologia , Feminino , Galactose/análogos & derivados , Humanos , Imunoensaio , Masculino , Mananas/análise , Pessoa de Meia-Idade , Estudos Retrospectivos , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Various tools are obtainable for the detection of Pneumocystis jirovecii, among them qPCR promising highest sensitivity. A novel molecular method is commercially available, the loop-mediated isothermal amplification (LAMP) assay. OBJECTIVES: We compared the performance of the LAMP eazyplex® Pneumocystis jirovecii with the RealStar Pneumocystis jirovecii PCR 1.0 qPCR. MATERIAL/METHODS: Overall, 162 lower respiratory tract specimens from 146 critically ill patients were investigated. LAMP assay and qPCR were carried out according to the manufacturer's recommendations. Positive results of the LAMP were described as time to positivity (TTP). The limit of detection (LOD) of the LAMP was analysed using 10-fold serial dilutions of a high positive P jirovecii respiratory sample. For each serial dilution, TTP of the LAMP was plotted against cycle threshold (Ct) values of the qPCR. RESULTS: The LOD of the LAMP was determined to be approximately 4 × 103 copies/mL. While the LAMP revealed 28 (17%) positive signals from 20 patients, by using qPCR 41 (25%) positive samples from 28 patients were identified. Overall agreement with qPCR was 92%. Five false-negative, one false-positive and nine invalid results were detected by the LAMP. Positive and negative predictive values were 96% each, and sensitivity and specificity were 84% and 99%, respectively. There was a low correlation between the TTP and the fungal load. CONCLUSION: The LAMP is a time-saving and easy-to-perform method. It can be used as an alternative diagnostic method. However, for quantification purposes the qPCR is still the gold standard.