RESUMO
The pathophysiology of degenerative cervical myelopathy (DCM) is characterized by chronic compression-induced damage to the spinal cord leading to secondary harm such as disruption of the blood spinal cord barrier (BSCB). It is therefore the purpose of this study to analyze BSCB disruption in pre- and postoperative DCM patients and to correlate those with the clinical status and postoperative outcome. This prospectively controlled cohort included 50 DCM patients (21 female; 29 male; mean age: 62.9 ± 11.2 years). As neurological healthy controls, 52 (17 female; 35 male; mean age 61.8 ± 17.3 years) patients with thoracic abdominal aortic aneurysm (TAAA) and indication for open surgery were included. All patients underwent a neurological examination and DCM-associated scores (Neck Disability Index, modified Japanese Orthopaedic Association Score) were assessed. To evaluate the BSCB status, blood and cerebrospinal fluid (CSF) samples (lumbar puncture or CSF drainage) were taken preoperatively and in 15 DCM patients postoperatively (4 female; 11 male; mean age: 64.7 ± 11.1 years). Regarding BSCB disruption, CSF and blood serum were examined for albumin, immunoglobulin (Ig) G, IgA and IgM. Quotients for CSF/serum were standardized and calculated according to Reiber diagnostic criteria. Significantly increased preoperative CSF/serum quotients were found in DCM patients as compared to control patients: AlbuminQ (p < .001), IgAQ (p < .001) and IgGQ (p < .001). IgMQ showed no significant difference (T = - 1.15, p = .255). After surgical decompression, neurological symptoms improved in DCM patients, as shown by a significantly higher postoperative mJOA compared to the preoperative score (p = .001). This neurological improvement was accompanied by a significant change in postoperative CSF/serum quotients for Albumin (p = .005) and IgG (p = .004) with a trend of a weak correlation between CSF markers and neurological recovery. This study further substantiates the previous findings, that a BSCB disruption in DCM patients is evident. Interestingly, surgical decompression appears to be accompanied by neurological improvement and a reduction of CSF/serum quotients, implying a BSCB recovery. We found a weak association between BSCB recovery and neurological improvement. A BSCB disruption might be a key pathomechanism in DCM patients, which could be relevant to treatment and clinical recovery.
Assuntos
Vértebras Cervicais , Doenças da Medula Espinal , Humanos , Masculino , Feminino , Pessoa de Meia-Idade , Idoso , Adulto , Estudos Prospectivos , Vértebras Cervicais/cirurgia , Doenças da Medula Espinal/diagnóstico , Descompressão Cirúrgica/efeitos adversos , Imunoglobulina A , Imunoglobulina M , Resultado do TratamentoRESUMO
BACKGROUND: Bacterial meningitis is still a cause of severe neurological disability. The brain is protected from penetrating pathogens by the blood-brain barrier and the innate immune system. The invading pathogens are recognized by pattern recognition receptors including the G-protein-coupled formyl peptide receptors (FPRs), which are expressed by immune cells of the central nervous system. FPRs show a broad spectrum of ligands, including pro- and anti-inflammatory ones. Here, we investigated the effects of the annexin A1 mimetic peptide Ac2-26 in a mouse model of pneumococcal meningitis. METHODS: Wildtype (WT) and Fpr1- and Fpr2-deficient mice were intrathecally infected with Streptococcus pneumoniae D39 (type 2). Subsequently, the different mice groups were treated by intraperitoneal injections of Ac2-26 (1 mg/kg body weight) 2, 8, and 24 h post-infection. The extent of inflammation was analyzed in various brain regions by means of immunohistochemistry and real-time reverse transcription polymerase chain reaction (RT-PCR) 30 h post-infection. RESULTS: Ac2-26-treated WT mice showed less severe neutrophil infiltration, paralleled by a reduced induction of pro-inflammatory glial cell responses in the hippocampal formation and cortex. While meningitis was ameliorated in Ac2-26-treated Fpr1-deficient mice, this protective effect was not observed in Fpr2-deficient mice. Irrespective of Ac2-26 treatment, inflammation was more severe in Fpr2-deficient compared to Fpr1-deficient mice. CONCLUSIONS: In summary, this study demonstrates anti-inflammatory properties of Ac2-26 in a model of bacterial meningitis, which are mediated via FPR2, but not FPR1. Ac2-26 and other FPR2 modulators might be promising targets for the development of novel therapies for Streptococcus pneumoniae-induced meningitis.
Assuntos
Anexina A1/uso terapêutico , Anti-Inflamatórios/uso terapêutico , Encefalite/tratamento farmacológico , Hipocampo/efeitos dos fármacos , Meningite Pneumocócica/tratamento farmacológico , Infiltração de Neutrófilos/efeitos dos fármacos , Peptídeos/uso terapêutico , Animais , Anexina A1/farmacologia , Anti-Inflamatórios/farmacologia , Camundongos , Camundongos Knockout , Neutrófilos/efeitos dos fármacos , Peptídeos/farmacologia , Receptores de Formil Peptídeo/genética , Resultado do TratamentoRESUMO
PURPOSE: The aim of this study is to detect the presence of blood spinal cord barrier (BSCB) disruption in patients with degenerative cervical myelopathy (DCM). METHODS: In this prospective non-randomized controlled cohort study, 28 patients with DCM were prospectively included. All patients had indication for neurosurgical decompression. Furthermore, 38 controls with thoracic abdominal aortic aneurysm (TAAA) and indication for surgery were included. All patients underwent neurological examination. Regarding BSCB disruption and intrathecal immunoglobulin (Ig) concentrations, cerebrospinal fluid (CSF) and blood serum were examined for albumin, IgG, IgA and IgM. Quotients (Q) (CSF/serum) were standardized and calculated according to Reibers' diagnostic criteria. RESULTS: Patients and controls distinguished significantly in their clinical status. AlbuminQ, as expression of BSCB disruption, was significantly increased in the DCM patients compared to the controls. Quotients of IgG and IgA differed significantly between the groups as an expression of intrathecal diffusion. In the subgroup analysis of patients with mild/moderate clinical status of myelopathy and patients with severe clinical status, the disruption of the BSCB was significantly increased with clinical severity. Likewise, IgAQ and IgGQ presented increased quotients related to the clinical severity of myelopathy. CONCLUSION: In this study, we detected an increased permeability and disruption of the BSCB in DCM patients. The severity of BSCB disruption and the diffusion of Ig are related to the clinical status in our patient cohort. Having documented this particular pathomechanism in patients with DCM, we suggest that this diagnostic tool cloud be an important addition to surgical decision making in the future. These slides can be retrieved under Electronic Supplementary Material.
Assuntos
Vértebras Cervicais , Doenças da Medula Espinal , Estudos de Coortes , Humanos , Estudos Prospectivos , Medula Espinal , Doenças da Medula Espinal/cirurgiaRESUMO
Pattern recognition receptors (PRRs) are key elements in the innate immune response. Formyl peptide receptor (FPR) 2 is a PRR that, in addition to proinflammatory, pathogen-derived compounds, also recognizes the anti-inflammatory endogenous ligand annexin A1 (AnxA1). Because the contribution of this signaling axis in viral infections is undefined, we investigated AnxA1-mediated FPR2 activation on influenza A virus (IAV) infection in the murine model. AnxA1-treated mice displayed significantly attenuated pathology upon a subsequent IAV infection with significantly improved survival, impaired viral replication in the respiratory tract, and less severe lung damage. The AnxA1-mediated protection against IAV infection was not caused by priming of the type I IFN response but was associated with an increase in the number of alveolar macrophages (AMs) and enhanced pulmonary expression of the AM-regulating cytokine granulocyte-M-CSF (GM-CSF). Both AnxA1-mediated increase in AM levels and GM-CSF production were abrogated when mouse (m)FPR2 signaling was antagonized but remained up-regulated in mice genetically deleted for mFPR1, an mFPR2 isoform also serving as AnxA1 receptor. Our results indicate a novel protective function of the AnxA1-FPR2 signaling axis in IAV pathology via GM-CSF-associated maintenance of AMs, expanding knowledge on the potential use of proresolving mediators in host defense against pathogens.-Schloer, S., Hübel, N., Masemann, D., Pajonczyk, D., Brunotte, L., Ehrhardt, C., Brandenburg, L.-O., Ludwig, S., Gerke, V., Rescher, U. The annexin A1/FPR2 signaling axis expands alveolar macrophages, limits viral replication, and attenuates pathogenesis in the murine influenza A virus infection model.
Assuntos
Anexina A1/fisiologia , Vírus da Influenza A/fisiologia , Macrófagos Alveolares/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Receptores de Formil Peptídeo/fisiologia , Replicação Viral , Animais , Modelos Animais de Doenças , Fator Estimulador de Colônias de Granulócitos e Macrófagos/fisiologia , Vírus da Influenza A/patogenicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/fisiologiaRESUMO
Enzalutamide's accepted mode of action is by targeting the androgen receptor's (AR) activity. In clinical practice, enzalutamide demonstrates a good benefit-risk profile for the treatment of advanced prostate cancer (PC), even after poor response to standard antihormonal treatment. However, since both, well-established antiandrogens and enzalutamide, target AR functionality, we hypothesized that additional unknown mechanisms might be responsible for enzalutamide's superior anticancer activity. In the current study, PC cells were incubated with enzalutamide and enzalutamide-dependent modulation of apoptotic mechanisms were assessed via Western blot analysis, TDT-mediated dUTP-biotin nick end-labeling assay, and nuclear morphology assay. Alterations of heat shock protein (HSP), AR, and estrogen receptor (ER) expression were examined by Western blot analysis. Enzalutamide attenuated the proliferation of PC cells in a time- and dose-dependent manner. In the presence of enzalutamide, apoptosis occurred which was shown by increased BAX expression, decreased Bcl-2 expression, nuclear pyknosis, and genomic DNA fragmentation. Moreover, enzalutamide inhibited the expression of HSPs primarily involved in steroid receptor stabilization and suppressed AR and ERß1 expression. This study demonstrates for the first time that enzalutamide treatment of PC cells triggers varying molecular mechanisms resulting in antiproliferative effects of the drug. In addition to the well-characterized antagonistic inhibition of AR functionality, we have shown that enzalutamide also affects the intracellular synthesis of steroid receptor-associated HSPs, thereby diminishing the expression of AR and ERß1 proteins and inducing apoptotic pathways. According to an indirect attenuation of HSP-associated factors such as steroid receptors, endometrial carcinoma, uterine leiomyosarcoma, and mamma carcinoma cells also demonstrated inhibited cell growth in the presence of enzalutamide. Our data, therefore, suggest that enzalutamide's high efficacy is at least partially independent of AR and p53 protein expression, which are frequently lost in advanced PC.
Assuntos
Antagonistas de Receptores de Andrógenos/farmacologia , Apoptose/efeitos dos fármacos , Receptor beta de Estrogênio/biossíntese , Proteínas de Neoplasias , Feniltioidantoína/análogos & derivados , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/biossíntese , Benzamidas , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Masculino , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/biossíntese , Nitrilas , Células PC-3 , Feniltioidantoína/farmacologia , Neoplasias da Próstata/patologiaRESUMO
BACKGROUND/AIM: In prostate cancer (PC), the formation of new blood vessels is stimulated by hypoxic conditions, androgens, and a number of molecular factors including microRNAs. MicroRNA-1 (miR-1) has been characterized in some tumor entities as anti-angiogenic, but this has not yet been investigated in PC. MATERIALS AND METHODS: PC cells stably overexpressing miR-1 (LNCaP-miR-1) were incubated on an in vivo hen's egg test-chorioallantoic membrane (HET-CAM) model and compared to maternal LNCaP cells. Cell growth, blood vessel organisation, and total blood vessel area were analysed. RESULTS: Both matrigel-embedded LNCaP and LNCaP-miR-1 cells formed compact tumor-like cell aggregates on the CAM of the HET-CAM model. Although not quantifiable, bleeding of the CAM and remodelling of the blood vessel network in the CAM indicated an influence of miR-1 on the vascular system. The statistically significant decrease in the total surface area of blood vessels in the visible CAM section to 79.4% of control cells demonstrated the antiangiogenic properties of miR-1 for the first time. CONCLUSION: MiR-1 had a tumor-suppressive and anti-angiogenic effect in an in vivo PC model. In the clinic, miR-1-mediated anti-angiogenesis would result in reduced tumor supply and increased hypoxic stress inside the tumor. Thus, miR-1 restoration by nucleic acid-based miR-1 mimetics would represent a promising option for future PC therapy.
Assuntos
Vasos Sanguíneos/metabolismo , Membrana Corioalantoide/metabolismo , MicroRNAs/genética , Neoplasias da Próstata/genética , Inibidores da Angiogênese/farmacologia , Animais , Vasos Sanguíneos/patologia , Linhagem Celular Tumoral , Galinhas , Colágeno/química , Combinação de Medicamentos , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Laminina/química , Masculino , Próstata/efeitos dos fármacos , Próstata/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteoglicanas/química , Hipóxia TumoralRESUMO
OBJECTIVE: Cytokines and chemokines are widely involved in cancer cell progression and thus represent promising candidate factors for new biomarkers. METHODS: Four renal cell cancer (RCC) cell lines (Caki-1, 786-O, RCC4, and A498) and a nonmalignant renal cell line (RC-124) were examined with respect to their proliferation. The cytokine and chemokine expression pattern was examined by a DNA array (Human Cytokines & Chemokines RT2 Profiler PCR Array; Qiagen, Hilden, Germany), and expression profiles were compared. RESULTS: Caki-1 and 786-O cells exhibited significantly increased proliferation rates, whereas RCC4 and A498 cells demonstrated attenuated proliferation, compared to nonmalignant RC-124 cells. Expression analysis revealed 52 cytokines and chemokines primarily involved in proliferation and inflammation and differentially expressed not only in malignant and nonmalignant renal cells but also in the four RCC cell lines. CONCLUSION: This is the first study examining the expression of 84 cytokines and chemokines in four RCC cell lines compared to that in a nonmalignant renal cell line. VEGFA, NODAL, and BMP6 correlated with RCC cell line proliferation and, thus, may represent putative clinical biomarkers for RCC progression as well as for RCC diagnosis and prognosis.
Assuntos
Quimiocinas/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais/genética , Neoplasias Renais/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Quimiocinas/metabolismo , Humanos , Análise de Componente Principal , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
BACKGROUND/AIM: Due to its poor prognosis, it is increasingly necessary to understand the biology of renal cell cancer (RCC). Therefore, we investigated the role of microRNAs miR-1 and miR-21 in the growth of RCC cells compared to that of non-malignant renal cells. MATERIALS AND METHODS: Four malignant cell lines (Caki-1, 786-O, RCC4, A498) were examined regarding their cell growth, microRNA and telomerase expression, and were compared to non-malignant RC-124 renal cells. RESULTS: Inconsistencies appeared in the panel of RCC cells regarding antiproliferative and proliferative properties of miR-1 and miR-21, respectively. Notably, and most likely due to immortaliziation, non-malignant RC-124 cells exhibited telomerase expression and activity. CONCLUSION: miR-1 and miR-21 functionality in cancer progression, particularly in tumor growth, may be more dependent on the individual cellular context and may reflect RCC heterogeneity. Thus, both microRNAs, in combination with other stratifying biomarkers, may be useful in terms of RCC diagnosis, prognosis, or treatment response.
Assuntos
Carcinoma de Células Renais/genética , Rim/crescimento & desenvolvimento , MicroRNAs/genética , Carcinogênese/genética , Carcinoma de Células Renais/fisiopatologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Rim/citologia , Rim/metabolismo , Invasividade Neoplásica/genética , Telomerase/genéticaRESUMO
Multiple sclerosis (MS) is a chronic degenerative disease of the central nervous system that is characterized by myelin abnormalities, oligodendrocyte pathology, and concomitant glia activation. Unclear are the factors triggering gliosis and demyelination. New findings suggest an important role of the innate immune response in the initiation and progression of active demyelinating lesions. The innate immune response is induced by pathogen-associated or danger-associated molecular patterns, which are identified by pattern recognition receptors (PRRs), including the G-protein coupled with formyl peptide receptors (FPRs). Glial cells, the immune cells of the central nervous system, also express the PRRs. In this study, we used the cuprizone mice model to investigate the expression of the FPR1 in the course of cuprizone-induced demyelination In addition, we used FPR1-deficient mice to analyze glial cell activation through immunohistochemistry and real-time RT-PCR in cuprizone model. Our results revealed a significantly increased expression of FPR1 in the cortex of cuprizone-treated mice. FPR1-deficient mice showed a slight but significant decrease of demyelination in the corpus callosum compared to the wild-type mice. Furthermore, FPR1 deficiency resulted in reduced glial cell activation and mRNA expression of microglia/macrophages markers, as well as pro- and anti-inflammatory cytokines in the cortex, compared to wild-type mice after cuprizone-induced demyelination. Combined together, these results suggest that the FPR1 is an important part of the innate immune response in the course of cuprizone-induced demyelination.
Assuntos
Doenças Desmielinizantes/imunologia , Neuroglia/imunologia , Receptores de Formil Peptídeo/metabolismo , Animais , Corpo Caloso/imunologia , Corpo Caloso/patologia , Cuprizona/toxicidade , Citocinas/genética , Citocinas/metabolismo , Doenças Desmielinizantes/etiologia , Imunidade Inata , Ativação de Macrófagos , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Formil Peptídeo/genéticaRESUMO
Antimicrobial peptides are an important part of the innate immune defense in the central nervous system (CNS). The expression of the antimicrobial peptides psoriasin (S100A7) is up-regulated during bacterial meningitis. However, the exact mechanisms induced by psoriasin to modulate glial cell activity are not yet fully understood. Our hypothesis is that psoriasin induced pro- and anti-inflammatory signaling pathways as well as regenerative factors to contribute in total to a balanced immune response. Therefore, we used psoriasin-stimulated glial cells and analyzed the translocation of the pro-inflammatory transcription factor nuclear factor 'kappa-light-chain-enhancer' of activated B-cells (NFκB) in murine glial cells and the expression of pro- and anti-inflammatory mediators by real time RT-PCR, ELISA technique, and western blotting. Furthermore, the relationship between psoriasin and the antioxidative stress transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) was investigated. Stimulation with psoriasin not only enhanced NFκB translocation and increased the expression of the pro-inflammatory cytokines, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF- α) but also neurotrophin expression. Evidence for functional interactions between psoriasin and Nrf2 were detected in the form of increased antioxidant response element (ARE) activity and induction of Nrf2/ARE-dependent heme oxygenase 1 (HO-1) expression in psoriasin-treated microglia and astrocytes. The results illustrate the ability of psoriasin to induce immunological functions in glia cells where psoriasin exerts divergent effects on the innate immune response.
Assuntos
Imunidade Inata/fisiologia , Neuroglia/imunologia , Neuroglia/metabolismo , Proteínas S100/imunologia , Proteínas S100/farmacologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Feminino , Células HEK293 , Humanos , Imunidade Inata/efeitos dos fármacos , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neuroglia/efeitos dos fármacos , Proteína A7 Ligante de Cálcio S100 , Proteínas S100/biossínteseRESUMO
Bacterial meningitis is - despite therapeutical progress during the last decades - still characterized by high mortality and severe permanent neurogical sequelae. The brain is protected from penetrating pathogens by both the blood-brain barrier and the innate immune system. Invading pathogens are recognized by so-called pattern recognition receptors including the Toll-like receptors (TLR) which are expressed by glial immune cells in the central nervous system. Among these, TLR2 is responsible for the detection of Gram-positive bacteria such as the meningitis-causing pathogen Streptococcus pneumoniae. Here, we used TLR2-deficient mice to investigate the effects on mortality, bacterial growth and inflammation in a mouse model of pneumococcal meningitis. Our results revealed a significantly increased mortality rate and higher bacterial burden in TLR2-deficient mice with pneumococcal meningitis. Furthermore, infected TLR2-deficient mice suffered from a significantly increased pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) and Chemokine (C-C motif) ligand 2 (CCL2) or CCL3 chemokine expression and decreased expression of anti-inflammatory cytokines and antimicrobial peptides. In contrast, glial cell activation assessed by glial cell marker expression was comparable to wildtype mice. Taken together, the results suggest that TLR2 is essential for an efficient immune response against Streptococcus pneumoniae meningitis since lack of the receptor led to a worse outcome by higher mortality due to increased bacterial burden, weakened innate immune response and reduced expression of antimicrobial peptides.
Assuntos
Imunidade Inata/fisiologia , Meningite Pneumocócica/metabolismo , Meningite Pneumocócica/mortalidade , Neuroglia/metabolismo , Receptor 2 Toll-Like/deficiência , Animais , Meningite Pneumocócica/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mortalidade/tendências , Neuroglia/imunologia , Receptor 2 Toll-Like/imunologiaRESUMO
BACKGROUND/AIM: The high variability of primary cells propagated in vitro led us to study the expression patterns of 11 most commonly accepted and widely used biomarkers specific for prostate cancer (PC) cells in primary cell models. MATERIALS AND METHODS: Primary PC cells from five PC patients were partially subjected to RNA preparation immediately and remaining cells were propagated up to 84 days followed by RNA preparation. Subsequently, biomarker mRNA quantification was performed by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and biomarker transcript concentrations before and after cultivation of primary PC cells were compared. RESULTS: Evaluation of androgen receptor, prostate-specific antigen, acid phosphatase, prostate-specific membrane antigen, fatty acid synthase, cytokeratin types 5/8/19, E-cadherin, epithelial cell adhesion molecule and fibroblast-specific protein 1 demonstrated temporal changes, as well as individual differences in expression, during primary PC cell propagation. CONCLUSION: Experimental design, as well as data evaluation, may need to take under consideration the high variability of biomarker expression in primary PC cells.
Assuntos
Biomarcadores Tumorais/biossíntese , Neoplasias da Próstata/genética , RNA Mensageiro/biossíntese , Antígenos CD , Antígenos de Superfície/biossíntese , Antígenos de Superfície/genética , Biomarcadores Tumorais/genética , Caderinas/biossíntese , Caderinas/genética , Molécula de Adesão da Célula Epitelial/biossíntese , Molécula de Adesão da Célula Epitelial/genética , Regulação Neoplásica da Expressão Gênica , Glutamato Carboxipeptidase II/biossíntese , Glutamato Carboxipeptidase II/genética , Humanos , Masculino , Cultura Primária de Células , Antígeno Prostático Específico/biossíntese , Antígeno Prostático Específico/genética , Neoplasias da Próstata/patologia , RNA Mensageiro/genética , Receptores Androgênicos/biossíntese , Receptores Androgênicos/genéticaRESUMO
Short non-coding microRNAs have been identified to orchestrate crucial mechanisms in cancer progression and treatment resistance. MicroRNAs are involved in posttranscriptional modulation of gene expression and therefore represent promising targets for anticancer therapy. As mircoRNA-1 (miR-1) exerted to be predominantly downregulated in the majority of examined tumors, miR-1 is classified to be a tumor suppressor with high potential to diminish tumor development and therapy resistance. Here we review the complex functionality of miR-1 in tumor biology.
Assuntos
MicroRNAs/genética , Neoplasias/genética , Animais , Biomarcadores Tumorais/genética , Morte Celular , Movimento Celular , Humanos , Neoplasias/diagnóstico , Neoplasias/patologia , PrognósticoRESUMO
The most frequent pathogen that causes bacterial meningitis is the Gram-positive bacterium Streptococcus pneumoniae. By entering the brain, host cells will be activated and proinflammatory cytokines like interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) are released. The goal of the current study was to examine the interaction between IL-6 and TNFR1 as receptor for TNF-α and the innate immune response in vivo in a model of Streptococcus pneumoniae-induced meningitis. For the experiments IL-6(-/-), TNFR1(-/-), and TNFR1-IL-6(-/-) KO mice were used. Our results revealed higher mortality rates and bacterial burden after infection in TNFR1(-/-), IL-6(-/-), and TNFR1-IL-6(-/-) mice and a decreased immune response including lower neutrophil infiltration in the meninges of TNFR1(-/-) and TNFR1-IL-6(-/-) mice in contrast to IL-6(-/-) and wild type mice. Furthermore, the increased mortality of TNFR1(-/-) and TNFR1-IL-6(-/-) mice correlated with decreased glial cell activation compared to IL-6(-/-) or wild type mice after pneumococcal meningitis. Altogether, the results show the importance of TNFR1 and IL-6 in the regulation of the innate immune response. The lack of TNFR1 and IL-6 results in higher mortality by weakened immune defence, whereas the lack of TNFR1 results in more severe impairment of the innate immune response than the lack of IL-6 alone.
Assuntos
Imunidade Inata/imunologia , Interleucina-6/deficiência , Meningites Bacterianas/imunologia , Infecções Pneumocócicas/imunologia , Receptores Tipo I de Fatores de Necrose Tumoral/deficiência , Animais , Imunidade Inata/genética , Interleucina-6/genética , Masculino , Meningites Bacterianas/metabolismo , Camundongos , Camundongos Knockout , Infecções Pneumocócicas/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Streptococcus pneumoniae/imunologia , Streptococcus pneumoniae/patogenicidadeRESUMO
INTRODUCTION: Inhibition of androgen synthesis by abiraterone acetate (AA) entails enhanced overall survival rates and clinical benefit for patients with locally advanced and metastasized prostate cancer (PC). The expression of heat shock protein 27 (HSP27) is generally associated with cytoprotection and was demonstrated to mediate chemoresistance under cytostatic therapy, for instance, docetaxel treatment. In this study, we investigated the impact of AA treatment on HSP27 expression and PC cell growth. MATERIALS AND METHODS: HSP27 expression levels in docetaxel and AA-treated PC cell lines LNCaP and PC-3 were determined by SDS PAGE and Western blot analysis. Proliferation assays were performed using a CASY Cell Counter and Analyzer Model TT (Roche Applied Science). RESULTS: Despite significantly increased HSP27 expression in PC cells incubated with docetaxel, Western blot analysis implicated a significant reduction of the cytoprotective HSP27 in AA-treated PC cells. Notably, HSP27 stably overexpressed in PC-3-HSP27 cells did not appear as an HSP27-mediated proliferation benefit in the presence of AA as shown in docetaxel incubation studies. CONCLUSION: In contrast to repeatedly demonstrated HSP27-driven chemoresistance related to chemotherapeutics, our results may constitute a broader molecular mode of action of AA chemotherapy. AA efficacy may exert an HSP27 suppressive role that goes beyond the primarily assumed inhibition of androgen biosynthesis.
Assuntos
Acetato de Abiraterona/farmacologia , Inibidores das Enzimas do Citocromo P-450/farmacologia , Proteínas de Choque Térmico HSP27/antagonistas & inibidores , Neoplasias da Próstata/patologia , Células Tumorais Cultivadas/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Proteínas de Choque Térmico HSP27/biossíntese , Humanos , Masculino , Neoplasias da Próstata/metabolismoRESUMO
BACKGROUND: Remodeling of the tumor environment and the modulation of tumor associated non-malignant cells are essential events in tumor progression. Exosomes are small membranous vesicles of 50-150 nm in diameter, which are secreted into the extracellular space and supposedly serve as vehicles for signal and effector molecules to modulate adjacent target cells. We characterized the mRNA and protein composition as well as cellular functions of prostate cancer cell-derived exosomes. METHODS: Exosomes were prepared from prostate cancer cell culture supernatant by ultracentrifugation and subsequently characterized by dynamic light scattering and electron microscopy. Exosomal mRNA and protein composition were analyzed by DNA microarrays and gel electrophoresis coupled with mass spectrometry. Physiological effects of exosomes were studied by means of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase release cell assays. Using a SILAC approach, putative uptake of exosomal human proteins in canine cells and canine de novo synthesis of proteins specified by exosome-transferred human mRNA was analyzed in MDCK cells via mass spectrometry. RESULTS: Preparations of exosomes revealed typical cup shaped particles of 150 nm in diameter. Analysis of mRNA and protein composition of exosomes exhibited a wide range of mRNA and protein species. Interestingly, the packaging of at least small proteins into exosomes was apparently unspecific, as shown with the example of two model proteins. In cell culture incubation experiments exosomal preparations of prostate cancer cells caused anti-proliferative effects. MS analysis revealed the uptake of exosomal human proteins into canine cells after 6 hr of incubation. CONCLUSIONS: The results reveal a distinct exosomal functionality in the modulation of the prostatic tumor adjacent environment. The multitude of translocated factors implies the induction of numerous effects in tumor-associated target cells, including impact on cellular growth.
Assuntos
Exossomos/fisiologia , Neoplasias da Próstata/ultraestrutura , Proteínas/metabolismo , RNA Mensageiro/metabolismo , Animais , Comunicação Celular/fisiologia , Linhagem Celular Tumoral , Cães , Difusão Dinâmica da Luz , Exossomos/ultraestrutura , Células HEK293 , Humanos , Células Madin Darby de Rim Canino , Masculino , Microscopia Eletrônica de Transmissão , Tamanho da Partícula , Transporte Proteico/fisiologia , Proteínas/análise , Transporte de RNA/fisiologia , RNA Mensageiro/análise , Microambiente TumoralRESUMO
UNLABELLED: Backround/Aim: Physical plasmas are ionized gases containing several biologically-reactive factors that yet exert their anti-microbial and anti-proliferative effects in fields of surface sterilisation, de-contamination and wound healing. MATERIALS AND METHODS: Cold atmospheric plasma (CAP) was generated via the atmospheric pressure plasma jet kINPen09. Apoptotic effects of CAP treatment on the human epithelial prostate cancer cell line LNCaP as a cell culture model for malignant tumor tissue was analyzed by cell counting, western blot and quantitative reverse transcription-polymerase chain reaction analysis. RESULTS: LNCaP cells exhibited significantly reduced cell growth following CAP treatment. We show that most probably the induction of apoptosis is the terminus of CAP treatment illustrated by the pro-apoptotic modulation of p53, p21, caspase-3, Bax, and survivin, as well as morphological changes of cell architecture. CONCLUSION: Our in vitro study offers first indicatory results for molecular response mechanisms after CAP treatment in a suitable LNCaP cell model.
Assuntos
Proliferação de Células/efeitos dos fármacos , Gases em Plasma/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Masculino , Neoplasias da Próstata/metabolismoRESUMO
BACKGROUND: Upon inflammation, myeloid cell generation in the bone marrow (BM) is broadly enhanced by the action of induced cytokines which are produced locally and at multiple sites throughout the body. METHODS: Using microarray studies, we found that Angptl4 is upregulated in the BM during systemic inflammation. RESULTS: Recombinant murine Angptl4 (rmAngptl4) stimulated the proliferation of myeloid colony-forming units (CFUs) in vitro. Upon repeated in vivo injections, rmAngptl4 increased BM progenitor cell frequency and this was paralleled by a relative increase in phenotypically defined granulocyte-macrophage progenitors (GMPs). Furthermore, in vivo treatment with rmAngptl4 resulted in elevated platelet counts in steady-state mice while allowing a significant acceleration of reconstitution of platelets after myelosuppressive therapy. The administration of rmAngptl4 increased the number of CD61(+)CD41(low)-expressing megakaryocytes (MK) in the BM of steady-state and in the spleen of transplanted mice. Furthermore, rmAngptl4 improved the in vitro differentiation of immature MKs from hematopoietic stem and progenitor cells. Mechanistically, using a signal transducer and activator of transcription 3 (STAT3) reporter knockin model, we show that rmAngptl4 induces de novo STAT3 expression in immature MK which could be important for the effective expansion of MKs after myelosuppressive therapy. CONCLUSION: Whereas the definitive role of Angptl4 in mediating the effects of lipopolysaccharide (LPS) on the BM has to be demonstrated by further studies involving multiple cytokine knockouts, our data suggest that Angptl4 plays a critical role during hematopoietic, especially megakaryopoietic, reconstitution following stem cell transplantation.
Assuntos
Angiopoietinas/metabolismo , Plaquetas/metabolismo , Medula Óssea/metabolismo , Células Mieloides/metabolismo , Proteína 4 Semelhante a Angiopoietina , Animais , Feminino , Humanos , Camundongos , Células Mieloides/citologia , Fator de Transcrição STAT3/metabolismo , Regulação para CimaRESUMO
Despite the development of new antibiotic agents, mortality of pneumococcal meningitis remains high. In addition, meningitis results in severe long-term morbidity, most prominently cognitive deficits. Granulocyte colony-stimulating factor (G-CSF) stimulates proliferation and differentiation of hematopoietic progenitor cells and increases the number of circulating neutrophil granulocytes. This study investigated the effect of adjuvant G-CSF treatment on cognitive function after pneumococcal meningitis. C57BL/6 mice were infected by subarachnoid injection of Streptococcus pneumoniae serotype 3 and treated with ceftriaxone and G-CSF subcutaneously or ceftriaxone alone for 5 days. Clinical scores, motor performance, and mortality during bacterial meningitis were unaffected by adjuvant G-CSF treatment. No effect of G-CSF treatment on production of proinflammatory cytokines or activation of microglia or astrocytes was observed. The G-CSF treatment did, however, result in hippocampal neurogenesis and improved spatial learning performance 6 weeks after meningitis. These results suggest that G-CSF might offer a new adjuvant therapeutic approach in bacterial meningitis to reduce long-term cognitive deficits.
Assuntos
Transtornos Cognitivos/tratamento farmacológico , Transtornos Cognitivos/etiologia , Fator Estimulador de Colônias de Granulócitos/uso terapêutico , Hipocampo/efeitos dos fármacos , Meningite Pneumocócica/complicações , Neurogênese/efeitos dos fármacos , Células-Tronco Adultas/efeitos dos fármacos , Animais , Antibacterianos/uso terapêutico , Ceftriaxona/uso terapêutico , Diferenciação Celular/efeitos dos fármacos , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Meningite Pneumocócica/tratamento farmacológico , Camundongos , Camundongos Endogâmicos C57BL , Atividade Motora/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Streptococcus pneumoniae/patogenicidade , Fatores de TempoRESUMO
Prolonged agonist exposure of many G-protein coupled receptors induces a rapid receptor phosphorylation and uncoupling from G-proteins. Resensitization of these desensitized receptors requires endocytosis and subsequent dephosphorylation. Numerous studies show the involvement of phospholipid-specific phosphodiesterase phospholipase D (PLD) in the receptor endocytosis and recycling of many G-protein coupled receptors e.g., opioid, formyl or dopamine receptors. The PLD hydrolyzes the headgroup of a phospholipid, generally phosphatidylcholine (PC), to phosphatidic acid (PA) and choline and is assumed to play an important function in cell regulation and receptor trafficking. Protein kinases and GTP binding proteins of the ADP-ribosylation and Rho families regulate the two mammalian PLD isoforms 1 and 2. Mammalian and yeast PLD are also potently stimulated by phosphatidylinositol 4,5-bisphosphate. The PA product is an intracellular lipid messenger. PLD and PA activities are implicated in a wide range of physiological processes and diseases including inflammation, diabetes, oncogenesis or neurodegeneration. This review discusses the characterization, structure, and regulation of PLD in the context of membrane located G-protein coupled receptor function.