RESUMO
According to class M2.1 of the World Anti-Doping Agency (WADA) Prohibited List, the manipulation of doping control urine samples to alter their integrity and validity is prohibited both in- and out-of-competition. However, some paraplegic athletes with an overactive bladder need to be regularly treated with anti-cholinergic and anti-spasmodic drugs such as oxybutynin, which are often administered intravesically to reduce the substantial side effects observed after oral application. So far, it remains unclear whether such bladder instillations have a negative impact on analytical procedures and thus represent an anti-doping rule violation. Within this pilot study, urine samples were collected from five paraplegic athletes before and after an intravesical oxybutynin hydrochloride instillation. The samples were routinely tested for the presence of performance-enhancing drugs and afterwards fortified with 25 model compounds representing different classes of doping agents (anabolic agents, cannabinoids, diuretics, glucocorticoids, hormone and metabolic modulators, and stimulants) at low and medium concentrations. Additionally, the pH value and specific gravity were measured and the presence of oxybutynin was qualitatively determined by gas chromatography-mass spectrometry (GC-MS). In initial testing procedures, all samples were tested negative. Oxybutynin was present in most of the samples but found to have no significant effect on the detectability of the 25 model compounds subsequently added to each urine specimen. Therefore, it can be concluded that intravesical instillations with oxybutynin hydrochloride do not alter the integrity and validity of doping control urine samples.
Assuntos
Ácidos Mandélicos/urina , Substâncias para Melhoria do Desempenho/urina , Detecção do Abuso de Substâncias/métodos , Urinálise/métodos , Agentes Urológicos/urina , Administração Intravesical , Dopagem Esportivo , Feminino , Cromatografia Gasosa-Espectrometria de Massas/métodos , Humanos , Masculino , Ácidos Mandélicos/administração & dosagem , Projetos Piloto , Agentes Urológicos/administração & dosagemRESUMO
The liver bears unique immune properties that support both immune tolerance and immunity, but the mechanisms responsible for clearance versus persistence of virus-infected hepatocytes remain unclear. Here, we dissect the factors determining the outcome of antiviral immunity using recombinant adenoviruses that reflect the hepatropism and hepatrophism of hepatitis viruses. We generated replication-deficient adenoviruses with equimolar expression of ovalbumin, luciferase, and green fluorescent protein driven by a strong ubiquitous cytomegalovirus (CMV) promoter (Ad-CMV-GOL) or by 100-fold weaker, yet hepatocyte-specific, transthyretin (TTR) promoter (Ad-TTR-GOL). Using in vivo bioluminescence to quantitatively and dynamically image luciferase activity, we demonstrated that Ad-TTR-GOL infection always persists, whereas Ad-CMV-GOL infection is always cleared, independent of the number of infected hepatocytes. Failure to clear Ad-TTR-GOL infection involved mechanisms acting during initiation as well as execution of antigen-specific immunity. First, hepatocyte-restricted antigen expression led to delayed and curtailed T-cell expansion-10,000-fold after Ad-CMV-GOL versus 150-fold after Ad-TTR-GOL-infection. Second, CD8 T-cells primed toward antigens selectively expressed by hepatocytes showed high PD-1/Tim-3/LAG-3/CTLA-4/CD160 expression levels similar to that seen in chronic hepatitis B. Third, Ad-TTR-GOL but not Ad-CMV-GOL-infected hepatocytes escaped being killed by effector T-cells while still inducing high PD-1/Tim-3/LAG-3/CTLA-4/CD160 expression, indicating different thresholds of T-cell receptor signaling relevant for triggering effector functions compared with exhaustion. Conclusion: Our study identifies deficits in the generation of CD8 T-cell immunity toward hepatocyte-expressed antigens and escape of infected hepatocytes expressing low viral antigen levels from effector T-cell killing as independent factors promoting viral persistence. This highlights the importance of addressing both the restauration of CD8 T-cell dysfunction and overcoming local hurdles of effector T-cell function to eliminate virus-infected hepatocytes.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Hepatite Viral Animal/imunologia , Hepatócitos/imunologia , Adenoviridae , Animais , Antígenos/metabolismo , Citomegalovirus/genética , Expressão Gênica , Ativação Linfocitária , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pré-Albumina/genética , Regiões Promotoras GenéticasAssuntos
Infecções por Adenoviridae/imunologia , Infecções por Adenoviridae/metabolismo , Pneumonia Viral/imunologia , Pneumonia Viral/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Receptor Toll-Like 9/metabolismo , Adenoviridae , Infecções por Adenoviridae/virologia , Animais , Biomarcadores , Modelos Animais de Doenças , Camundongos , Pneumonia Viral/virologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
OBJECTIVE: Patients with liver cirrhosis suffer from increased susceptibility to life-threatening bacterial infections that cause substantial morbidity. METHODS: Experimental liver fibrosis in mice induced by bile duct ligation or CCl4 application was used to characterise the mechanisms determining failure of innate immunity to control bacterial infections. RESULTS: In murine liver fibrosis, translocation of gut microbiota induced tonic type I interferon (IFN) expression in the liver. Such tonic IFN expression conditioned liver myeloid cells to produce high concentrations of IFN upon intracellular infection with Listeria that activate cytosolic pattern recognition receptors. Such IFN-receptor signalling caused myeloid cell interleukin (IL)-10 production that corrupted antibacterial immunity, leading to loss of infection-control and to infection-associated mortality. In patients with liver cirrhosis, we also found a prominent liver IFN signature and myeloid cells showed increased IL-10 production after bacterial infection. Thus, myeloid cells are both source and target of IFN-induced and IL-10-mediated immune dysfunction. Antibody-mediated blockade of IFN-receptor or IL-10-receptor signalling reconstituted antibacterial immunity and prevented infection-associated mortality in mice with liver fibrosis. CONCLUSIONS: In severe liver fibrosis and cirrhosis, failure to control bacterial infection is caused by augmented IFN and IL-10 expression that incapacitates antibacterial immunity of myeloid cells. Targeted interference with the immune regulatory host factors IL-10 and IFN reconstitutes antibacterial immunity and may be used as therapeutic strategy to control bacterial infections in patients with liver cirrhosis.
Assuntos
Translocação Bacteriana , Imunidade Inata , Interferon Tipo I/metabolismo , Interleucina-10/biossíntese , Listeriose/imunologia , Cirrose Hepática Experimental/imunologia , Células Mieloides/imunologia , Animais , Tetracloreto de Carbono , Imunidade Inata/genética , Listeriose/complicações , Listeriose/metabolismo , Cirrose Hepática Experimental/complicações , Cirrose Hepática Experimental/metabolismo , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Células Mieloides/metabolismo , Células Mieloides/microbiologia , Proteínas de Resistência a Myxovirus/genética , Receptor de Interferon alfa e beta/antagonistas & inibidores , Receptor de Interferon alfa e beta/genética , Receptores de Interleucina-10/antagonistas & inibidores , Receptores de Reconhecimento de Padrão/genética , Transdução de Sinais , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/genética , Receptor 7 Toll-Like/genética , Receptor Toll-Like 9/genéticaRESUMO
The mannose receptor (MR) is an endocytic receptor involved in serum homeostasis and antigen presentation. Here, we identify the MR as a direct regulator of CD8(+) T-cell activity. We demonstrate that MR expression on dendritic cells (DCs) impaired T-cell cytotoxicity in vitro and in vivo. This regulatory effect of the MR was mediated by a direct interaction with CD45 on the T cell, inhibiting its phosphatase activity, which resulted in up-regulation of cytotoxic T-lymphocyte-associated Protein 4 (CTLA-4) and the induction of T-cell tolerance. Inhibition of CD45 prevented expression of B-cell lymphoma 6 (Bcl-6), a transcriptional inhibitor that directly bound the CTLA-4 promoter and regulated its activity. These data demonstrate that endocytic receptors expressed on DCs contribute to the regulation of T-cell functionality.
Assuntos
Antígeno CTLA-4/genética , Lectinas Tipo C/genética , Antígenos Comuns de Leucócito/genética , Ativação Linfocitária/genética , Lectinas de Ligação a Manose/genética , Receptores de Superfície Celular/genética , Animais , Apresentação de Antígeno/genética , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Antígeno CTLA-4/imunologia , Regulação da Expressão Gênica/genética , Humanos , Tolerância Imunológica/genética , Lectinas Tipo C/imunologia , Antígenos Comuns de Leucócito/imunologia , Ativação Linfocitária/imunologia , Receptor de Manose , Lectinas de Ligação a Manose/imunologia , Camundongos , Proteínas Proto-Oncogênicas c-bcl-6/genética , Receptores de Superfície Celular/imunologia , Linfócitos T Citotóxicos/imunologia , Ativação Transcricional/genéticaRESUMO
A main function of splenic red pulp macrophages is the degradation of damaged or aged erythrocytes. Here we show that these macrophages accumulate ferrimagnetic iron oxides that render them intrinsically superparamagnetic. Consequently, these cells routinely contaminate splenic cell isolates obtained with the use of MCS, a technique that has been widely used in immunological research for decades. These contaminations can profoundly alter experimental results. In mice deficient for the transcription factor SpiC, which lack red pulp macrophages, liver Kupffer cells take over the task of erythrocyte degradation and become superparamagnetic. We describe a simple additional magnetic separation step that avoids this problem and substantially improves purity of magnetic cell isolates from the spleen.
Assuntos
Separação Celular/métodos , Eritrócitos/metabolismo , Macrófagos/citologia , Baço/citologia , Animais , Contagem de Células Sanguíneas , Linhagem da Célula , Óxido Ferroso-Férrico/efeitos adversos , Óxido Ferroso-Férrico/química , Citometria de Fluxo , Humanos , Fígado/citologia , Macrófagos/metabolismo , Camundongos , Fagocitose , Baço/metabolismoRESUMO
CD11b+Gr1+ myeloid derived suppressor cells (MDSC) are known to be very potent suppressors of T cell immunity and can be further stratified into granulocytic MDSC and monocytic MDSC in mice based on expression of Ly6G or Ly6C, respectively. Here, using these markers and functional assays, we aimed to identify whether MDSC are induced during chronic inflammation leading to fibrosis in both kidney and liver and whether additional markers could more specifically identify these MDSC subsets. In an adenine-induced model of kidney inflammation/fibrosis suppressive Ly6Gpos MDSC were induced. The suppressive function within the Ly6G+ MDSC population was exclusively present in IFNγRß expressing cells. In contrast, in chronic inflammation in the liver induced by bile duct ligation, suppressive capacity was exclusively present in the Ly6Cpos MDSC subset. Gene expression analyses confirmed the differential origins and regulation of those MDSC subsets. Additionally, depletion of MDSC in either kidney or liver fibrosis enhanced fibrosis markers, indicating a protective role for MDSC in organ fibrosis. Thus, our data demonstrate that during liver inflammation and kidney fibrosis MDSC with similar function arise bearing a distinct marker profile and arising from different cell populations.
Assuntos
Antígenos Ly/metabolismo , Cirrose Hepática/imunologia , Células Mieloides/imunologia , Células Mieloides/metabolismo , Insuficiência Renal Crônica/imunologia , Animais , Biomarcadores/metabolismo , Progressão da Doença , Granulócitos/imunologia , Granulócitos/metabolismo , Inflamação/imunologia , Camundongos , FenótipoRESUMO
BACKGROUND & AIMS: In the liver, antigen-presenting cell populations such as Kupffer cells, liver dendritic cells, and liver sinusoidal endothelial cells (LSECs) participate through cross-presentation to CD8 T cells (CTLs) in hepatic immune-regulation and immune-surveillance. The participation of hepatic stellate cells (HSCs) in immune regulation is controversial. Here we studied HSC's contribution to antiviral CTL immunity. METHODS: Flow cytometric analysis of MHC-I molecules at the cell surface of liver cells from mice with cell-type restricted MHC-I expression. Mice with HSC-restricted MHC-I expression were infected with a hepatotropic virus and analyzed for development of viral hepatitis after CTL transfer. RESULTS: HSCs transferred MHC-I molecules to LSECs and these molecules were employed for LSEC cross-presentation to CTLs. Such transfer of MHC-I molecules was sufficient to support in vivo LSEC cross-presentation of soluble antigens to CTLs. Importantly, this transfer of MHC-I molecules contributed to anti-viral CTL immunity leading to development of immune-mediated hepatitis. CONCLUSIONS: Our findings demonstrate transfer of MHC-I molecules among sinusoidal liver cell populations as a potent mechanism to increase anti-viral CTL effector function. The transfer of MHC-I molecules from HSCs supplies LSECs with additional MHC-I molecules for their own cell-intrinsic cross-presentation. Such cross-allocation of MHC-I molecules in liver cell populations is distinct from cross-dressing that occurs among immune cell populations in lymphoid tissues where peptide-loaded MHC-I molecules are transferred. Our findings thus reveal a novel mechanism that increases local cross-presentation and CTL effector function in the liver, which may be instrumental for immune-surveillance during viral infection of antigen-presenting liver cells.