RESUMEN

OBJECTIVE: To observe the effect of moxibustion on proteins related with apoptosis of hippocampal neurons in rats with vascular dementia (VD), and to explore the possible mechanism of moxibustion on improving VD. METHODS: Thirty SD rats were selected from 100 rats (3 rats were excluded) and randomly divided into a normal group and a sham operation group, 15 rats in each group. The remaining 67 rats were treated with ischemia-reperfusion method at bilateral common carotid artery to establish VD model. The 45 rats with successful VD model were randomly divided into a model group, a moxibustion group and a medication group, 15 rats in each group. On the 7th day after successful modeling, the rats in the moxibustion group were treated with suspended moxibustion at "Guanyuan" (CV 4), "Mingmen" (GV 4) and "Dazhui" (GV 14), 15 min per acupoint, once a day; there was 1 d of rest after 6 d of moxibustion, and the treatment was given for 4 weeks. The rats in the medication group was treated with nimodipine tablets by gavage, 2 mg/kg per day, 3 times a day for 4 weeks. Before and after intervention, the Morris water maze test was used to detect the escape latency of rats in each group; after the intervention, the TUNEL method was used to detect the apoptosis rate of neurons in hippocampal CA1 area; the immunofluorescence double labeling method was used to detect the number of co-expression positive cells of B-cell lymphoma-2 (Bcl-2)/neuronal nuclear antigen (NeuN) and Bcl-2-associated X protein (Bax)/NeuN in hippocampal CA1 area; the immunofluorescence single labeling method was used to detect cytochrome C (cytC) and outer mitochondrial membrane receptor Tom20 (Tom20) in hippocampal CA1 area; the Western blot method was used to detect the p53 upregulated modulator of apoptosis (PUMA) in hippocampus. RESULTS: Before intervention, compared with the normal group and the sham operation group, the escape latency in the model group, the moxibustion group and the medication group was prolonged (P<0.01). After intervention, the escape latency in the moxibustion group and the medication group was shorter than that before intervention (P<0.01). Compared with the model group, the escape latency in the moxibustion group and the medication group was shortened (P<0.05); compared with the medication group, the escape latency in the moxibustion group was shortened (P<0.05). Compared with the normal group and the sham operation group, the apoptosis rate of neurons in hippocampal CA1 area was increased, the number of Bcl-2/NeuN co-expression positive cells was decreased, and the number of Bax/NeuN co-expression positive cells was increased in the model group (P<0.01); compared with the model group, the apoptosis rates of hippocampal CA1 neurons were decreased, the number of Bcl-2/NeuN co-expression positive cells was increased, and the number of Bax/NeuN co-expression positive cells was decreased in the moxibustion group and the medication group (P<0.01); compared with the medication group, the apoptosis rate of neurons in hippocampal CA1 area was decreased, the number of Bcl-2/NeuN co-expression positive cells was increased, and the number of Bax/NeuN co-expression positive cells was decreased in the moxibustion group (P<0.01, P<0.05). Compared with the normal group and the sham operation group, the expressions of cytC, Tom20 protein in hippocampal CA1 area and PUMA protein in hippocampal tissue in the model group were increased (P<0.01); compared with the model group, the expressions of cytC, Tom20 protein in hippocampal CA1 area and PUMA protein in hippocampal tissue in the moxibustion group and the medication group were decreased (P<0.01); compared with the medication group, the expressions of cytC, Tom20 protein in hippocampal CA1 area and PUMA protein in hippocampal tissue in the moxibustion group were decreased (P<0.05, P<0.01). CONCLUSION: Moxibustion could improve the cognitive function of VD rats, which may be related to reducing the expression of Bax, cytC, Tom20 and PUMA protein in hippocampal CA1 area, promoting the release of Bcl-2 and inhibiting the apoptosis of hippocampal neurons.

Asunto(s)

Demencia Vascular , Moxibustión , Animales , Apoptosis , Cognición , Demencia Vascular/terapia , Hipocampo , Neuronas , Ratas , Ratas Sprague-Dawley

RESUMEN

BACKGROUND: Efficient therapy and potential prophylaxis are confounded by current ignorance of the pathogenesis of airway remodelling and blockade in COPD. OBJECTIVE: To explore the role of the IL-33/ST2 axis in cigarette smoke (CS) exposure-induced airways remodelling. METHODS: C57BL/6, BALB/c and IL-1RL1 -/- mice exposed to CS were used to establish an animal surrogate of COPD (air-exposed=5~8, CS-exposed=6~12). Hallmarks of remodelling were measured in mice. Cigarette smoke extract (CSE)-induced proliferation and protein production in vitro by fibroblasts in the presence of anti-interleukin-33 (anti-IL-33) or hST2 antibodies were measured. Expression of IL-33 and ST2 and other remodelling hallmarks were measured, respectively, in bronchoalveolar lavage fluid (BALF) (controls=20, COPD=20), serum (controls=59, COPD=90) and lung tissue sections (controls=11, COPD=7) from patients with COPD and controls. RESULTS: Wild-type mice exposed to CS elevated expression of hallmarks of tissue remodelling in the lungs and also in the heart, spleen and kidneys, which were significantly abrogated in the IL-1RL1 -/- mice. Fibroblasts exposed to CSE, compared with control, exhibited early cellular translocation of IL-33, accompanied by proliferation and elevated protein synthesis, all inhabitable by blockade of IL-33/ST2 signalling. Expression of IL-33 and ST2 and hallmarks of tissue remodelling were significantly and proportionally elevated in BALF, serum and tissue samples from patients with COPD. CONCLUSIONS: Exposure to CS induces remodelling changes in multiple organs. The data support the hypothesis that CS-induced lung collagen deposition is at least partly a result of CS-induced IL-33 translocation and release from local fibroblasts.

RESUMEN

OBJECTIVE: To observe the eliminating effects of moxibustion at "Baihui" (GV 20), "Fengfu" (GV 16) and "Dazhui" (GV 14) on amyloid ß-peptide (Aß) in brain of the amyloid precursor protein/presenili1 (APP/PS1) double-transgenic mice with Alzheimer's disease (AD) by regulating the phosphoinositide 3-kinases/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway. METHODS: A total of 60 APP/PS1 double-transgenic mice with AD were randomly divided into a model group, a moxibustion group, a rapamycin group and a combination group (treated with moxibustion and inhibitor), 15 mice in each group, another 15 male C57BL/6J mice with same age and background were selected as the control group. In the moxibustion group, pressing moxibustion was applied at "Baihui" (GV 20) while the mild moxibustion was applied at "Fengfu" (GV 16) and "Dazhui" (GV 14). The treatment was manipulated for 20 min each time, once a day for 2 weeks. In the rapamycin group, rapamycin (2 mg/kg) was given by intraperitoneal injection once a day for 2 weeks. On the basis of the treatment in the moxibustion group, 3-methyladenine (1.5 mg/kg) was given by intraperitoneal injection once a day for 2 weeks. The mice in the control and the model group received normal diet and no intervention was given for 2 weeks. Immunohistochemica method was used to measure the levels of Aß1-42 in the cerebral cortex and hippocampal, transmission electron microscopy was used to observe the formation of autophagosome in hippocampus, and Western blot method was used to observe the levels of PI3K, Akt, p-Akt, mTOR and p-mTOR in hippocampus. RESULTS: Compared with the control group, the levels of Aß1-42 in the cerebral cortex and hippocampal were increased in the model group (P<0.01). Compared with the model group, the levels of Aß1-42 in the cerebral cortex and hippocampal were decreased in the moxibustion group, the rapamycin group and the combination group (all P<0.01), compared with the moxibustion group, the levels of Aß1-42 in the cerebral cortex and hippocampal were increased in the combination group (P<0.01), while there was no significant difference between the moxibustion group and the rapamycin group in the levels of Aß1-42（P>0.05）. Compared with the rapamycin group, the levels of Aß1-42 in the cerebral cortex and hippocampal were increased in the combination group (P<0.01). In the model group, the cytoplasmic utophagic vacuoles and organelles of neuron were reduced. In the moxibustion group, the utophagic vacuoles were increased, and the organelles showed deformation and atrophy. In the rapamycin group, the utophagic vacuoles were widely disturbed and few deformed organelles were found. In the combination group, few utophagic vacuoles were found and additional organelles showed deformation and atrophy. Compared with the control group, the levels of PI3K、Akt、p-Akt、mTOR and p-mTOR were increased in the model group (all P<0.01). Compared with the model group, the levels of PI3K、Akt、p-Akt、mTOR and p-mTOR were reduced in the moxibustion group, the rapamycin group and the combination group (all P<0.01). Compared with the moxibustion group, the levels of PI3K、Akt、and p-mTOR were increased in the rapamycin group and the levels of PI3K、Akt、p-Akt、mTOR and p-mTOR were increased in the combination group (all P<0.01). Compared with the rapamycin group, the levels of PI3K、Akt、p-Akt、mTOR and p-mTOR were increased in the combination group (P<0.01). CONCLUSION: Moxibustion at acupoints of governor vessel can enhance the autophagy process on Aß1-42 in brain of the APP/PS1 double-transgenic AD mice, which may be associated with its effects on inhibiting the abnormal activation of PI3K/Akt/mTOR signaling pathway.

Asunto(s)

Enfermedad de Alzheimer , Autofagia , Moxibustión , Péptidos beta-Amiloides , Animales , Modelos Animales de Enfermedad , Hipocampo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal , Serina-Treonina Quinasas TOR

RESUMEN

OBJECTIVE: To observe the effect of moxibustion of acupoints of the Governor Vessel on the levels of cellular autophagy, ß amyloid protein (Aß) immunoactivity, and expression of LC3-Ⅰ， LC3-Ⅱ， p62 and p-P70S6K proteins in the hippocampal tissue of APPswe/PS1de9 (APP/PS1) double-transgenic Alzheimer's disease (AD) mice, so as to reveal its underlying mechanisms in improving AD. METHODS: APP/PS1 double-transgenic AD mice were randomly divided into AD model, moxibustion, autophagy-inducer (Rapamycin) and autophagy-inhibitor (3-MA)＋moxibustion groups (n＝10 in each group), and other 10 C57BL/6J male mice (the same age) were used as the normal control group. Herbal-cake (made of Chuanwu [Radix Aconiti Praeparata]) partitioned moxibustion was applied to "Baihui"(GV20), moxibustion was applied to "Fengfu"(GV16) and "Dazhui"(GV14), all for 20 min, once daily for 2 weeks, with one day's off between two weeks. For mice of the autophagy-inducer and 3-MA＋moxibustion groups, Rapamycin (2 mg•kg－1•d－1) and 3-MA (1.5 mg•kg－1•d－1) were separately administered by intraperitoneal injection for 2 weeks. The cognitive ability was examined by Morris water maze tests, and the ultrastructural changes (including autophagic lysosomes, etc.) of hippocampal neurons were observed by using transmission electron microscopy. The immunoactivity of cerebral cortex and hippocampal Amyloid ß peptide 1-42 (Aß1-42) was detected by immunohistochemistry, and the expression levels of hippocampal LC3-Ⅰ， LC3-Ⅱ， p62 and p-P70S6K proteins were detected by Western blot. RESULTS: After modeling, the escape latency of Morris water maze tasks was prolonged in the model group than in the normal control group (P<0.05) and obviously shortened in the moxibustion and autophagy-inducer groups (not the autophagy-inhibitor group) than in the model group (P<0.05). Results of transmission electron microscope showed deformed, irregular or atrophic neurons with rough and incomplete and fuzzy nuclear membrane, and decreased intracellular autophagosomes in the hippocampus in the model group, and partial irregular, atrophic neurons with more autophagic vesicles and lysosomes in the moxibustion group. The expression levels of Aß1-42 in both cerebral cortex and hippocampus tissues, and LC3-Ⅰ， p62 and p-P70S6K proteins in the hippocampus were consi-derably up-regulated in the model group relevant to the normal control group (P<0.01), and evidently down-regulated in both moxibustion and autophagy-inducer groups (not the autophagy-inhibitor group) than in the model group (P<0.01), while that of hippocampal LC3-Ⅱ protein and LC3-Ⅱ/Ⅰ ratio levels were obviously down-regulated in the model group relevant to the normal control group (P<0.01), and significantly up-regulated in both moxibustion and autophagy-inducer groups (not the autophagy-inhibitor group) than in the model group (P<0.01)．. CONCLUSION: Moxibustion can improve the cognitive ability of APP/PS1 double-transgenic AD mice, which is associated with its effects in promoting hip-pocampal and cerebral cortex autophagy level, and down-regulating the expression levels of Aß1-42, LC3-Ⅰ， p62 and p-P70S6K proteins in the hippocampus.

Asunto(s)

Enfermedad de Alzheimer , Autofagia , Moxibustión , Péptidos beta-Amiloides , Precursor de Proteína beta-Amiloide , Animales , Proteínas Relacionadas con la Autofagia , Corteza Cerebral , Cognición , Modelos Animales de Enfermedad , Hipocampo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos

RESUMEN

Oxidative stress is known to be associated with various age-related diseases. D-galactose (D-gal) has been considered a senescent model which induces oxidative stress response resulting in memory dysfunction. Pyrroloquinoline quinone (PQQ) is a redox cofactor which is found in various foods. In our previous study, we found that PQQ may be converted into a derivative by binding with amino acid, which is beneficial to several pathological processes. In this study, we found a beneficial glutamate mixture which may diminish neurotoxicity by oxidative stress in D-gal induced mouse. Our results showed that PQQ may influence the generation of proinflammatory mediators, including cytokines and prostaglandins during aging process. D-gal-induced mouse showed increased MDA and ROS levels, and decreased T-AOC activities in the hippocampus, these changes were reversed by PQQ supplementation. Furthermore, PQQ statistically enhanced Superoxide Dismutase SOD2 mRNA expression. PQQ could ameliorate the memory deficits and neurotoxicity induced by D-gal via binding with excess glutamate, which provide a link between glutamate-mediated neurotoxicity, inflammation and oxidative stress. In addition, PQQ reduced the up-regulated expression of p-Akt by D-gal and maintained the activity of GSK-3ß, resulting in a down-regulation of p-Tau level in hippocampus. PQQ modulated memory ability partly via Akt/GSK-3ß pathway.

Asunto(s)

Disfunción Cognitiva/tratamiento farmacológico , Galactosa/toxicidad , Ácido Glutámico/toxicidad , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Proteína Oncogénica v-akt/metabolismo , Cofactor PQQ/administración & dosificación , Transducción de Señal , Animales , Disfunción Cognitiva/inducido químicamente , Citosol/química , Hipocampo/patología , Factores Inmunológicos/administración & dosificación , Ratones , Quinonas/análisis , Especies Reactivas de Oxígeno/análisis , Superóxido Dismutasa/análisis

RESUMEN

OBJECTIVE: To investigate the clinicopathological characteristics and risk factors in patients with lung cancer and COPD. MATERIALS AND METHODS: We retrospectively reviewed the clinical data of 282 patients with lung cancer, including 174 and 108 patients with and without COPD, respectively. Information on age, sex, smoking status, and histologic type was obtained from medical records. RESULTS: Lung cancer patients with COPD and those with the chronic bronchitis (CB) phenotype had higher smoking indices compared to those without COPD (723.95 ± 631.48 and 920.95 ± 712.93 versus 418.40 ± 506.84; P = 0.010; P = 0.001, resp.), and current smokers accounted for significantly higher proportions of lung cancer patients with COPD and the CB phenotype versus without COPD (51.15% and 63.74% versus 35.19%; P = 0.009; P = 0.001, resp.). Adenocarcinoma was significantly more common in lung cancer patients without versus with COPD (48.15% versus 35.63%; P = 0.037), whereas small cell lung cancer was more common in patients with COPD (23.56% versus 13.89%). Among patients with COPD, male sex (odds ratio [OR], 19.946; P < 0.001), current smokers (OR: 6.588; P = 0.001), and age ≥ 75 years (OR: 2.670; P = 0.008) were identified as high-risk factors. CONCLUSION: The risk factors for COPD among lung cancer patients were age ≥ 75 years, current smokers with the CB phenotype, and male sex.

Asunto(s)

Neoplasias Pulmonares/complicaciones , Neoplasias Pulmonares/patología , Enfermedad Pulmonar Obstructiva Crónica/complicaciones , Enfermedad Pulmonar Obstructiva Crónica/patología , Factores de Edad , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Análisis Multivariante , Análisis de Regresión , Factores de Riesgo , Factores Sexuales , Fumar/efectos adversos

RESUMEN

OBJECTIVE: We investigated the role of endoplasmic reticulum stress (ERS) in silica-induced apoptosis in alveolar macrophages in vitro. METHODS: RAW264.7 cells were incubated with 200 µg/mL silica for different time periods. Cell viability was assayed by the MTT assay. Cell apoptosis was evaluated by DAPI staining, flow cytometry analysis, and Western blot analysis of caspase-3. Morphological changes in the endoplasmic reticulum were observed by transmission electron microscopy. The expression of ERS markers binding protein (BiP) and CCAAT-enhancer-binding protein homologous protein (CHOP) was examined by Western blotting and real-time PCR. As an inhibitor of ERS, 4-phenylbutyric acid (4-PBA) was used in the experiments. RESULTS: Silica exposure induced nuclear condensation and caspase-3 expression in RAW264.7 cells. The number of apoptotic cells increased after silica exposure in a time-dependent manner. Silica treatment induced expansion of the endoplasmic reticulum. In addition, the expression of BiP and CHOP increased in silica-stimulated cells. Furthermore, 4-PBA treatment inhibited silica-induced endoplasmic reticulum expansion and the expression of BiP and CHOP. Moreover, 4-PBA treatment attenuated nuclear condensation, reduced apoptotic cells, and downregulated caspase-3 expression in silica-stimulated cells. CONCLUSION: Silica-induced ERS is involved in the apoptosis of alveolar macrophages.

Asunto(s)

Apoptosis/efectos de los fármacos , Estrés del Retículo Endoplásmico/fisiología , Dióxido de Silicio/toxicidad , Animales , Butilaminas , Supervivencia Celular/efectos de los fármacos , Ratones , Células RAW 264.7

Asunto(s)

Evolución Biológica , Virus Zika/patogenicidad , Genes Virales , Mutación , Virus Zika/genética

RESUMEN

OBJECTIVE: To observe the efficacy on primary osteoporosis treated with spreading moxibustion for warming yang and activating blood circulation so as to provide the effective clinical therapeutic methods for osteoporosis. METHODS: Sixty cases of primary osteoporosis were randomized into a spreading moxibustion group (30 cases) and a calcium tablet group (30 cases). In the calcium tablet group, caltrate was prescribed for oral administration, 600 mg per day. In the spreading moxibustion group, on the basis of the treatment as the calcium tablet group, the spreading moxibustion was applied at Dazhui (GV 14) to Yaoshu (GV 2) for warming yang and activating blood circulation. The duration of treatment was 12 weeks. Visual analogue scale (VAS) score, TCM clinical symptom score and bone mineral density (BMD) were observed and compared before and after treatment in the patients between the two groups. RESULTS: VAS scores were reduced apparently after treatment in the two groups (both P < 0.01) and the results in the spreading moxibustion group were obviously superior to that in the calcium tablet group (2.36 +/- 0.43 vs 4.52 +/- 0.35, P < 0.01). BMD were all increased in the two groups (P < 0.05, P < 0.01) and the results in the spreading moxibustion group were superior to those in the calcium tablet group (both P < 0.05). The total clinical effective rate was 86.67% (26/30) in the spreading moxibustion group, apparently better than 63.33% (19/30) in the calcium tablet group (P < 0.05). TCM clinical symptom scores after treatment were all reduced apparently in the two groups (both P < 0.01), and the result in the spreading moxibustion group was obviously superior to that in the calcium tablet group (4.72 +/- 1.90 vs 6.82 +/- 2.30, P < 0.01). The total effective rate of TCM symptoms was 93.33% (28/30) in the spreading moxibustion group, apparently better than 70.00% (21/30) in the calcium tablet group (P < 0.05). CONCLUSION: The combined therapy of spreading moxibustion for warming yang and activating blood circulation and the oral administration of caltrate apparently relieves pain and TCM clinical symptoms, improves BMD in the patients of osteoporosis and achieves definite clinical efficacy in the patients of osteoporosis.

Asunto(s)

Moxibustión , Osteoporosis/terapia , Deficiencia Yang/terapia , Anciano , Circulación Sanguínea , Densidad Ósea , Femenino , Humanos , Masculino , Persona de Mediana Edad , Osteoporosis/fisiopatología , Deficiencia Yang/fisiopatología

RESUMEN

OBJECTIVE: To observe clinical curative effect of cake-separated moxibustion on impaired glucose regulation (IGR) and explore its action mechanism. METHODS: Sixty cases were randomly divided into a simple lifestyle intervention group (control group) and a cake-separated moxibustion combined with lifestyle intervention group (observation group), 30 cases in each one. The control group was treated with lifestyle intervention. Based on lifestyle intervention, cake-separated moxibustion at Pishu (BL 20), Weishu (BL 21) and Yishu (EX-B 3) was applied to the observation group. Fast plasma glucose (FPG), two hours plasma glucose after oral glucose tolerance test (OGTT2hPG), fasting insulin (FINS), homa insulin resistance index (HOMA-IR), blood lipid, body mass index (BMI) and waist circumference (WC) were observed in the two groups before and after treatment. RESULTS: After treatment, the OGTT2hPG and FPG were both decreased significantly (both P<0.05) in the two groups, compared between the two groups, the differences of FPG [(0.41 +/- 0.42) mmol/L vs (0.05 +/- 0.08)mmol/L] and OGTT2hPG [(0.85 +/- 0.53)mmol/L vs (0.17 +/- 0.19)mmol/L] were both statistically significant. There were no significant changes in FINS, HOMA-IR, blood lipid, BMI and WC in the control group before and after treatment (all P>0.05), but FINS, HOMA-IR levels, triglycerides (TG), total cholest-erol (TC), low density lipoprotein (LDL-C), BMI and WC in the observation group were decreased obviously after treatment (all P<0.05), which had statistical differences between the two groups (all P<0.05). CONCLUSION: The cake-separated moxibustion combined with lifestyle intervention can obviously control blood glucose levels, improve insulin resistance and blood lipid levels, decrease BMI and WC.

Asunto(s)

Intolerancia a la Glucosa/terapia , Glucosa/metabolismo , Moxibustión , Adulto , Anciano , Femenino , Intolerancia a la Glucosa/metabolismo , Intolerancia a la Glucosa/fisiopatología , Humanos , Insulina , Masculino , Persona de Mediana Edad , Circunferencia de la Cintura

RESUMEN

We recently reported that administration of low doses of myotoxins at vaccination sites potentiated antigen-specific T-cell immunity induced by genetic cancer vaccines in mice, an effect which was superior to TLR agonists. In the current study, we found unexpectedly that the mechanism of this potent adjuvant effect was immune-mediated. Myotoxins induced sterile inflammation at vaccination sites, associated with a predominant infiltration of dendritic cells (DC). Inhibition of DC recruitment abrogated the immune stimulation effect of myotoxins, suggesting the requirement for DC. Genetic profiling of myotoxin-treated tissues revealed characteristics of an immune microenvironment with up-regulation of chemokines, proinflammatory cytokines, Toll-like receptors (TLR) and their endogenous ligands, and activation of innate immunity. Mechanistic experiments in vivo also elucidated the requirement for genes triggering DC maturation including TLR signaling and CD40. These studies suggest that myotoxins-induced sterile inflammation generates a favorable microenvironment that promotes multiple stages in the development of adaptive immunity. This novel mechanism of immune potentiation may be exploited for development of adjuvants for genetic vaccines against infectious pathogens and cancer.

Asunto(s)

Inmunidad Adaptativa , Adyuvantes Inmunológicos/farmacología , Cardiotoxinas/farmacología , Células Dendríticas/inmunología , Inflamación/inmunología , Animales , Cardiotoxinas/inmunología , Citocinas/inmunología , Inmunidad Innata , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Linfocitos T/inmunología , Receptores Toll-Like/inmunología , Regulación hacia Arriba , Vacunación/métodos , Vacunas de ADN/inmunología

RESUMEN

The development of distinct dendritic cell (DC) subsets is regulated by cytokines. The ligand for the FMS-like tyrosine kinase 3 receptor (Flt3L) is necessary for plasmacytoid DC (pDC) and conventional DC (cDC) maturation. The cytokine GM-CSF inhibits Flt3L-driven pDC production while promoting cDC growth. We show that GM-CSF selectively utilized its signal transducer STAT5 to block Flt3L-dependent pDC development from the lineage-negative, Flt3+ (lin- Flt3+) bone-marrow subset. The signaling molecule STAT3, by contrast, was necessary for expansion of DC progenitors but not pDC maturation. In vivo, STAT5 suppressed pDC formation during repopulation of the DC compartment after bone-marrow ablation. GM-CSF-dependent STAT5 signaling rapidly extinguished pDC-related gene expression in lin- Flt3+ progenitors. Inspection of the Irf8 promoter revealed that STAT5 was recruited during GM-CSF-mediated suppression, indicating that STAT5 directly inhibited transcription of this critical pDC gene. Our results therefore show that GM-CSF controls the production of pDCs by employing STAT5 to suppress IRF8 and the pDC transcriptional network in lin- Flt3+ progenitors.

Asunto(s)

Diferenciación Celular/inmunología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Inhibidores de Crecimiento/fisiología , Factores Reguladores del Interferón/antagonistas & inhibidores , Factor de Transcripción STAT5/fisiología , Transducción de Señal/inmunología , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Diferenciación Celular/genética , Linaje de la Célula/genética , Linaje de la Célula/inmunología , Células Cultivadas , Células Dendríticas/citología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/fisiología , Factores Reguladores del Interferón/biosíntesis , Factores Reguladores del Interferón/fisiología , Ratones , Ratones Noqueados , Células Madre Multipotentes/citología , Células Madre Multipotentes/inmunología , Células Madre Multipotentes/metabolismo , Factor de Transcripción STAT5/deficiencia , Factor de Transcripción STAT5/genética , Transducción de Señal/genética , Tirosina Quinasa 3 Similar a fms/biosíntesis

RESUMEN

Although there is evidence for distinct roles of myeloid dendritic cells (DCs [mDCs]) and plasmacytoid pre-DCs (pDCs) in regulating T cell-mediated adaptive immunity, the concept of functional DC subsets has been questioned because of the lack of a molecular mechanism to explain these differences. In this study, we provide direct evidence that maturing mDCs and pDCs express different sets of molecules for T cell priming. Although both maturing mDCs and pDCs upregulate the expression of CD80 and CD86, only pDCs upregulate the expression of inducible costimulator ligand (ICOS-L) and maintain high expression levels upon differentiation into mature DCs. High ICOS-L expression endows maturing pDCs with the ability to induce the differentiation of naive CD4 T cells to produce interleukin-10 (IL-10) but not the T helper (Th)2 cytokines IL-4, -5, and -13. These IL-10-producing T cells are T regulatory cells, and their generation by ICOS-L is independent of pDC-driven Th1 and Th2 differentiation, although, in the later condition, some contribution from endogenous IL-4 cannot be completely ruled out. Thus, in contrast to mDCs, pDCs are poised to express ICOS-L upon maturation, which leads to the generation of IL-10-producing T regulatory cells. Our findings demonstrate that mDC and pDCs are intrinsically different in the expression of costimulatory molecules that drive distinct types of T cell responses.

Asunto(s)

Células Dendríticas/inmunología , Interleucina-10/biosíntesis , Proteínas/metabolismo , Linfocitos T Reguladores/inmunología , Adulto , Antígenos CD , Diferenciación Celular , Células Dendríticas/clasificación , Células Dendríticas/metabolismo , Humanos , Técnicas In Vitro , Ligando Coestimulador de Linfocitos T Inducibles , Células Mieloides/clasificación , Células Mieloides/inmunología , Células Mieloides/metabolismo , Células Plasmáticas/clasificación , Células Plasmáticas/inmunología , Células Plasmáticas/metabolismo , Células Th2/inmunología , Regulación hacia Arriba

RESUMEN

The caspase-recruitment domain-containing adaptor protein CARD9 regulates the innate signaling responses to fungal infection. Here we show that CARD9 is required for innate immune responses against intracellular pathogens. We generated Card9(-/-) mice and found that CARD9-deficient macrophages had defects in activation of the kinases p38 and Jnk but not of transcription factor NF-kappaB after bacterial and viral infection. CARD9-deficient mice failed to clear infection and showed altered cytokine production after challenge with Listeria monocytogenes. In wild-type cells, we found CARD9 inducibly associated with both the intracellular 'biosensor' Nod2 and the serine-threonine kinase RICK. Our data demonstrate that CARD9 has a critical function in Nod2-mediated activation of p38 and Jnk in innate immune responses to intracellular pathogens.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales/inmunología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Inmunidad Innata/inmunología , Espacio Intracelular/microbiología , Listeria monocytogenes/inmunología , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteínas Adaptadoras de Señalización CARD , Células Cultivadas , Citocinas/metabolismo , Activación Enzimática , Expresión Génica , Ligandos , Listeria monocytogenes/fisiología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/metabolismo , Proteína Adaptadora de Señalización NOD2/metabolismo , Unión Proteica , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/metabolismo , Transducción de Señal , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo

RESUMEN

Mitogen-activated protein/ERK kinase kinase 3 (MEKK3) is a Ser/Thr protein kinase belonging to the MEKK/STE11 subgroup of the MAP3K family. Recently, we found that MEKK3 plays a critical role in interleukin-1 (IL-1) receptor and Toll-like receptor 4 signalling using established primary mouse embryonic fibroblast (MEF) cell lines. However, the function of MEKK3 in immune cells has not been studied because germ-line MEKK3 knockout mice are embryonically lethal between embryonic days 10 and 11. In this study, we used small interference RNA to the mouse Mekk3 gene to specifically knock down MEKK3 expression in the macrophage line Raw264.7. We found that the lipopolysaccharide-induced IL-6 and granulocyte-macrophage colony-stimulating factor (GM-CSF) production was dramatically decreased in MEKK3 knockdown cells whereas the tumour necrosis factor-alpha and IL-1beta production were not affected. We also observed that the ERK1/2, p38 and JNK MAPK induction in MEKK3 knockdown cells were moderately inhibited within the first 60 min of stimulation, while the ERK and p38 were more severely inhibited after 2-4 hr of stimulation. Degradation of IkappaBalpha was also partially blocked in MEKK3 knockdown cells. Notably, the impairment in IL-6 and GM-CSF production in the MEKK3 knockdown cells was restored by reintroducing a human Mekk3 cDNA that could not be targeted by mouse Mekk3-siRNAs. In conclusion, this study showed that MEKK3 is a crucial and specific regulator of the proinflammatory cytokines IL-6 and GM-CSF in macrophages and provided a novel method for investigating MEKK3 function in other immune cells.

Asunto(s)

Factor Estimulante de Colonias de Granulocitos y Macrófagos/biosíntesis , Interleucina-6/biosíntesis , Lipopolisacáridos/inmunología , MAP Quinasa Quinasa Quinasa 3/inmunología , Macrófagos/inmunología , Animales , Línea Celular , Activación Enzimática/inmunología , Vectores Genéticos , Interleucina-1beta/biosíntesis , Lentivirus/genética , MAP Quinasa Quinasa Quinasa 3/genética , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , ARN Interferente Pequeño/genética , Transducción de Señal/inmunología , Receptores Toll-Like/inmunología , Transducción Genética , Factor de Necrosis Tumoral alfa/biosíntesis

RESUMEN

BACKGROUND: P-TEFb, a general RNA polymerase II elongation factor, is composed of CDK9 (cyclin-dependent kinase 9) as a catalytic unit and either cyclin T1, T2 or K as a regulatory subunit. The cyclin T1/P-TEFb complex is targeted by HIV to mediate Tat transactivation. Cyclin T1 protein expression is induced during early macrophage differentiation, suggesting a role in regulation of mRNA expression during the differentiation process. To study the functional significance of cyclin T1 induction during differentiation, we utilized the human Mono Mac 6 (MM6) monocytic cell line. RESULTS: We found that cyclin T1 protein expression is induced by a post-transcriptional mechanism following PMA treatment of MM6 cells, similar to its induction in primary monocytes and macrophages. Also in agreement with findings in primary cells, cyclin T2a is present at relatively high levels in MM6 cells and is not induced by PMA. Although the knock-down of cyclin T1 in MM6 cells by shRNA inhibited HIV-1 Tat transactivation, MM6 cell growth was not affected by the depletion of cyclin T1. Using DNA microarray technology, we found that more than 20% of genes induced by PMA require cyclin T1 for their normal level of induction, and approximately 15% of genes repressed by PMA require cyclin T1 for their normal level of repression. Gene ontology analysis indicates that many of these cyclin T1-dependent genes are related to immune response and signal transduction. CONCLUSION: These results suggest that cyclin T1 serves a critical role in the program of macrophage differentiation, and this raises questions about the feasibility of cyclin T1 serving as an antiviral therapeutic target.

Asunto(s)

Diferenciación Celular/fisiología , Ciclinas/biosíntesis , Regulación de la Expresión Génica/fisiología , Genes tat , VIH-1/genética , Macrófagos/fisiología , Monocitos/fisiología , ARN Mensajero/biosíntesis , Diferenciación Celular/genética , Ciclina T , Ciclinas/genética , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Macrófagos/citología , Macrófagos/efectos de los fármacos , Monocitos/citología , Monocitos/efectos de los fármacos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , ARN Polimerasa II/genética , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Acetato de Tetradecanoilforbol/farmacología , Activación Transcripcional

RESUMEN

Myogenic differentiation is a fundamental biological process that involves a hierarchical series of events that ultimately leads to muscle-specific gene expression and myofiber formation. Posttranslational modifications of the myogenic regulatory factors have been implicated as important regulatory mechanisms in this process. Here we investigate whether covalent protein modification by a small ubiquitin-like modifier (SUMO) that is known to affect transcription factor activity can impact muscle differentiation. We show that the overall load of sumoylated proteins present in myoblasts diminishes progressively throughout myogenesis. Interestingly, knockdown of the SUMO-conjugating enzyme, Ubc9, severely compromises C2C12 muscle cell terminal differentiation. However, it does not affect the expression, the localization and the activation of MyoD and myogenin. These novel results suggest that protein sumoylation plays a pivotal role in myoblast differentiation and is required to regulate the activity of key targets downstream of MyoD and myogenin.

Asunto(s)

Regulación hacia Abajo , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/metabolismo , Enzimas Ubiquitina-Conjugadoras/fisiología , Animales , Técnicas de Cultivo de Célula , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Fusión de Membrana/genética , Ratones , Proteína MioD/metabolismo , Mioblastos/citología , Factores Reguladores Miogénicos/metabolismo , Miogenina/metabolismo , ARN Interferente Pequeño/farmacología , Enzimas Ubiquitina-Conjugadoras/genética

RESUMEN

In vitro interaction of fluconazole and berberine chloride was investigated against 40 fluconazole-resistant clinical isolates of Candida albicans. Synergism in fungistatic activity was found with the checkerboard microdilution assay. The findings of agar diffusion tests and time-kill curves confirmed the synergistic interaction, but no antagonistic action was observed.

Asunto(s)

Antifúngicos/farmacología , Berberina/farmacología , Candida albicans/genética , Farmacorresistencia Fúngica/genética , Fluconazol/farmacología , Candida albicans/efectos de los fármacos , Candida albicans/aislamiento & purificación , Candidiasis/tratamiento farmacológico , Candidiasis/microbiología , Farmacorresistencia Fúngica/efectos de los fármacos , Sinergismo Farmacológico , Humanos , Técnicas In Vitro , Pruebas de Sensibilidad Microbiana

RESUMEN

Immune cells respond to chemotactic signals by means of G protein-coupled receptors. Attempts to elucidate the function of specific G protein family members in these responses is complicated by redundancy among the different G protein isoforms. We have used lentiviral-based RNA interference to eliminate expression of specific G protein subunits selectively in J774A.1 mouse macrophages. The chemotactic response to the complement factors C5a and C3a is ablated in cells lacking G beta(2) but is unaffected in cells lacking G beta(1), G alpha i(2), or G alpha i(3). Similarly, the C5a-mediated calcium response of single cells is either absent or significantly delayed and weakened by G beta(2) knockdown. Assessment of Akt1 phosphorylation levels in response to C5a shows rapid and sustained phosphorylation in both wild-type cells and cells lacking G beta(1). Cells lacking G beta(2) retain the rapid response but cannot sustain phospho-Akt1 levels. The phenotype of cells lacking G beta(2) can be reversed by overexpression of either human G beta(2) or mouse G beta(1). These data demonstrate the usefulness of lentiviral-based RNA interference in the systematic analysis of a signaling pathway, and they suggest that in J774A.1 cells, G beta(2)-derived G beta gamma is the most effective mediator of chemotaxis to C5a.

Asunto(s)

Quimiotaxis de Leucocito/fisiología , Complemento C5a/fisiología , Vectores Genéticos , Lentivirus/genética , ARN Interferente Pequeño/administración & dosificación , Animales , Secuencia de Bases , Línea Celular , Cartilla de ADN , Cinética , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

Double-stranded RNAs approximately 21 nucleotides long [small interfering RNA (siRNA)] are recognized as powerful reagents to reduce the expression of specific genes. To use them as reagents to protect cells against viral infection, effective methods for introducing siRNAs into primary cells are required. Here, we describe success in constructing a lentivirus-based vector to introduce siRNAs against the HIV-1 coreceptor, CCR5, into human peripheral blood T lymphocytes. With high-titer vector stocks, >40% of the peripheral blood T lymphocytes could be transduced, and the expression of a potent CCR5-siRNA resulted in up to 10-fold inhibition of CCR5 expression on the cell surface over a period of 2 weeks in the absence of selection. In contrast, the expression of another major HIV-1 coreceptor, CXCR4, was not affected. Importantly, blocking CCR5 expression by siRNAs provided a substantial protection for the lymphocyte populations from CCR5-tropic HIV-1 virus infection, dropping infected cells by 3- to 7-fold; only a minimal effect on infection by a CXCR4-tropic virus was observed. Thus, our studies demonstrate the feasibility and potential of lentiviral vector-mediated delivery of siRNAs as a general means of intracellular immunization for the treatment of HIV-1 and other viral diseases.

Asunto(s)

Síndrome de Inmunodeficiencia Adquirida/prevención & control , VIH-1/fisiología , ARN Interferente Pequeño/genética , Receptores CCR5/genética , Linfocitos T/inmunología , Antagonistas de los Receptores CCR5 , Células Cultivadas , Vectores Genéticos , Humanos , Lentivirus , ARN Interferente Pequeño/administración & dosificación , Linfocitos T/virología , Virus de la Estomatitis Vesicular Indiana