RESUMO
Objective: To study the surgical safety and efficacy of preoperative neoadjuvant therapy with immune checkpoint inhibitors combined with anti-angiogenic drugs in patients with China liver cancer staging(CNLC)-â ¡b and â ¢a resectable hepatocellular carcinoma. Methods: The data of 129 patients with â ¡b and â ¢a hepatocellular carcinoma who underwent surgery at the First Affiliated Hospital of Nanjing Medical University from January 2018 to December 2020 were analyzed. All patients were divided into two groups: the neoadjuvant therapy group(n=14,13 males and 1 female,aged (55.4±12.6)years(range:34 to 75 years)) received immune combined targeted therapy before surgery,immune checkpoint inhibitor camrelizumab was administered intravenously at a dose of 200 mg each time,every 2 weeks for 3 cycles,anti-angiogenesis drug apatinib was taken orally and continuously with a dose of 250 mg for 3 weeks and the conventional surgery group(n=115,103 males and 12 females,aged (55.8±12.0)years(range:21 to 83 years)) did not receive antitumor systemic therapy before surgery. There were 3 patients with CNLC-â ¡b,11 with CNLC-â ¢a in the neoadjuvant group;28 patients with CNLC-â ¡b,87 with CNLC-â ¢a in the conventional group. Student's t test or rank-sum test was used to compare the differences between two groups for quantitative data, Fisher's exact probability method was used to compare the differences of proportions between two groups, and Log-rank test was used to compare survival differences between two groups. Results: The 1-year recurrence rate in the neoadjuvant group was 42.9%,and the 1-year recurrence rate in the conventional group was 64.0%,with a statistically significant difference between the two groups(χ²=3.850,P=0.050);The 1-year survival rate in the neoadjuvant group was 100% and that in the conventional group was 74.2%,with a statistically significant difference between the two groups(χ²=5.170,P=0.023). According to the stratified analysis of the number of tumors,for single tumor,the 1-year recurrence rate in the neoadjuvant group was 25.0%,and that in the conventional surgery group was 71.0%,and the difference between the two groups was statistically significant(χ²=5.280, P=0.022). For multiple tumors, the 1-year recurrence rate in the neoadjuvant group was 66.7%,and the 1-year recurrence rate in the conventional surgery group was 58.9%,with no significant difference between the two groups(χ²=0.110,P=0.736). The operative time,intraoperative blood loss,and postoperative hospital stay in the neoadjuvant group were similar to those in the conventional group,and their differences were not statistically significant. Conclusions: Immune checkpoint inhibitors combined with anti-angiogenic targeted drugs as a neoadjuvant therapy for resectable hepatocellular carcinoma can reduce the 1-year recurrence rate and improve the 1-year survival rate,especially for those with solitary tumor. Limited by the sample size of the neoadjuvant group,the safety of immune combined targeted therapy before surgery cannot be observed more comprehensively,and further studies will be explored.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Carcinoma Hepatocelular/terapia , Feminino , Humanos , Inibidores de Checkpoint Imunológico , Imunoterapia , Neoplasias Hepáticas/terapia , Masculino , Terapia Neoadjuvante , Estudos Retrospectivos , Resultado do TratamentoRESUMO
Endovascular aortic arch repair provides treatment opportunity for patients with aortic arch dissection and aneurysm who are intolerant to open surgery. The aortic arch branches provide the blood flow for brain, the revascularization of these branches is part and parcel of the endovascular aortic arch repair. The anatomical configuration, high-speed blood flow and long access from femoral artery increase the difficulty of endovascular aortic arch repair. Debranch technique, combined with chimney, scallop, and fenestration, have partially simplified the endovascular aortic arch repair. The dedicated endografts for aortic arch is becoming a research focus. In the design of aortic arch endografts, fenestrated and branched stent-grafts are the two main strategies. A variety of innovative concepts have been applied in the design of aortic arch endografts, including modular and integrated design, inner branch and outer branch design, single branch and multi-branch design, etc. Today, these procedures of complex endovascular aortic arch repair still need to be limited to experienced centers. Endovascular aortic arch repair showed favorable short-term outcomes through the development of strict surgical plans, as well as effective teamwork. Long-term efficacy and safety in larger participants need further investigation.
Assuntos
Aneurisma da Aorta Torácica , Implante de Prótese Vascular , Procedimentos Endovasculares , Aneurisma da Aorta Torácica/cirurgia , Prótese Vascular , Humanos , Desenho de Prótese , Estudos Retrospectivos , Fatores de Risco , Stents , Fatores de Tempo , Resultado do TratamentoRESUMO
AIM: To identify and summarise the common findings from 2019 novel coronavirus (2019-nCoV) infections in children. MATERIALS AND METHODS: The clinical characteristics and radiological findings (chest radiography and chest computed tomography [CT]) of nine children infected with the 2019-nCoV were reviewed in this retrospective case series. RESULTS: Among the children, six had fever (including two children with cough), one had only cough, one had a stuffy nose when initially diagnosed, and one was an asymptomatic carrier. Chest radiographs seemed mostly normal in six cases whereas increased and/or disordered bilateral bronchovascular shadows and dense hilar shadows were seen in three cases. Chest CT exhibited no obvious abnormal signs in four cases. Typical CT findings included patchy, peripheral ground-grass opacities, subpleural lamellar dense shadows, and parenchymal bands. Pleural effusions, mediastinal lymphadenopathy, cavitation, and pleural thickening were absent. CONCLUSION: The clinical manifestations and radiological findings of the 2019-nCoV-infected children were mild and lacked a typical pattern.
Assuntos
Infecções por Coronavirus/diagnóstico por imagem , Infecções por Coronavirus/fisiopatologia , Febre/fisiopatologia , Pulmão/diagnóstico por imagem , Pulmão/fisiopatologia , Pneumonia Viral/diagnóstico por imagem , Pneumonia Viral/fisiopatologia , Radiografia/métodos , Adolescente , Betacoronavirus , COVID-19 , Criança , Pré-Escolar , Infecções por Coronavirus/complicações , Feminino , Febre/complicações , Humanos , Lactente , Masculino , Pandemias , Pneumonia Viral/complicações , Estudos Retrospectivos , SARS-CoV-2 , Tomografia Computadorizada por Raios X/métodosRESUMO
OBJECTIVE: To study the protective mechanism of glutamine (Gln) on myocardial ischemia-reperfusion (IR) injury in rats through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway. MATERIALS AND METHODS: A total of 30 healthy SD rats weighing 200-300 g were used in this experiment. They were randomly divided into 3 groups: sham group (n=10), myocardial IR injury group (IR group, n=10), IR+Gln group (n=10). The protein expression levels of phosphorylated Akt (p-Akt), total Akt (t-Akt), phosphorylated mammalian target of rapamycin (p-mTOR), mTOR, proliferating cell nuclear antigen (PCNA), P21, and Tubulin were determined by Western blotting (WB). Quantitative Polymerase Chain Reaction (qPCR) was applied to detect the messenger ribonucleic acid (mRNA) levels of Akt and mTOR. 3-(4,5)-dimethylthiazol(-z-y1)-3,5-diphenyl tetrazolium bromide (MTT) test was utilized to examine the proliferation ability of cardiomyocytes in vitro. Besides, the contents of the inflammatory cytokines, tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6) were measured via enzyme-linked immunosorbent assay (ELISA). Cell apoptosis in each group was examined through Hoechst staining. RESULTS: Compared with those in the sham group, ratios of p-AKT/AKT, p-mTOR/mTOR, and the level of PCNA extremely significantly decreased, but the level of P21 notably increased in IR group (p<0.01). In comparison with those in the IR group, ratios of p-AKT/AKT, p-mTOR/mTOR, and the level of PCNA were remarkably raised, while the level of P21 was remarkably reduced in IR+Gln group (p<0.05). QRT-PCR results manifested that there were no significant differences in the mRNA levels of Akt and mTOR among the three groups [no significant difference (NS)]. Moreover, the cell proliferation ability in IR group was remarkably lower than that in the sham group (p<0.01), while it was enhanced in the IR+Gln group compared with that in the IR group (p<0.05). Additionally, IR group had significantly elevated expression levels of TNF-α and IL-6 compared with the sham group (p<0.01), whereas the IR+Gln group had notably decreased expression levels of TNF-α and IL-6 compared with IR group (p<0.05). In comparison with that in the sham group, the apoptosis in IR group was significantly raised (p<0.01), and compared with that in the IR group, the apoptosis in the IR+Gln group prominently decreased (p<0.05). The contents of the inflammatory cytokines, TNF-α, and IL-6 presented the same trends among the three groups. CONCLUSIONS: Gln activates the PI3K/Akt signaling pathway by increasing the levels of p-AKT and p-mTOR. Gln can increase the PCNA level and reduce the P21 level, so as to enhance the proliferation ability of cardiomyocytes. Besides, Gln reduces the levels of inflammatory cytokines, TNF-α, and IL-6, and inhibits cell apoptosis. Finally, Gln can protect cells from myocardial IR injury by activating the PI3K/Akt signaling pathway.
Assuntos
Glutamina/farmacologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Fosfatidilinositol 3-Quinases/metabolismo , Substâncias Protetoras/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Fosforilação/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacosRESUMO
Objective: To investigate the perfusion characteristics of arterial spin labeling (ASL) in intracranial tumor and its application value in classification. Methods: The clinical, pathological and imaging data of 44 patients with gliomas confirmed by pathology were analyzed retrospectively, including 9 low grade gliomas, 15 high grade gliomas, 11 cases of meningiomas, 6 cases of neurilemmoma, 3 cases of metastatic tumors.Conventional plain scan, 3D- ASL and MRI dynamic enhanced imaging (DSC-MRI) were performed.The mean maximal cerebral blood flow (CBF) of the solid component of tumor was obtained based on the region of interest.Immunohistochemical staining was performed in 24 patients with glioma.The differences of cerebral blood flow map (CBF) and relative cerebral blood flow (rCBF) in 44 patients with intracranial tumors were compared. The results of paired t test between the tumor area and the contralateral mirror area were measured by the two methods. Results: Taken the normal control-lateral grey matter(GM) as reference to normalize the CBF of tumor, three normalized tumor blood flow (nTBF) acquired by ASL showed statistical difference between low grade and high grade gliomas respectively (P<0.05). While taken the mirror region (M) and normal control-lateral white matter (WM) as reference to normalize the CBF of tumor, it showed no statistical difference (P>0.05). There was no 1p deletion in the cases of ASL perfusion in low-grade glioma group.In the case of 1p deletion in high grade glioma group, ASL was low perfusion, and there was no 1p deletion in the cases of ASL perfusion. Conclusion: 3D ASL can be used to identify high-grade and low-grade gliomas which has important reference value in the qualitative diagnosis of brain tumors and preoperative grading of gliomas.A separate use of 3D-ASL might cause over-or underestimation of tumor diagnosis, therefore a comprehensive analysis is needed.
Assuntos
Neoplasias Encefálicas/diagnóstico por imagem , Glioma/diagnóstico por imagem , Imageamento por Ressonância Magnética , Marcadores de Spin , Artérias , Encéfalo , Neoplasias Encefálicas/irrigação sanguínea , Circulação Cerebrovascular , Glioma/irrigação sanguínea , HumanosRESUMO
We have previously observed that mice exposed to cigarette smoke and treated with exogenous alpha(1)-antitrypsin (A1AT) were protected against the development of emphysema and against smoke-induced increases in serum TNF-alpha. To investigate possible mechanisms behind this latter observation, we cultured alveolar macrophages lavaged from C57 mice. Smoke-conditioned medium caused alveolar macrophages to increase secretion of macrophage metalloelastase (MMP-12) and TNF-alpha, and this effect was suppressed in a dose-response fashion by addition of A1AT. Macrophages from animals exposed to smoke in vivo and then lavaged also failed to increase MMP-12 and TNF-alpha secretion when the animals were pretreated with A1AT. Because proteinase activated receptor-1 (PAR-1) is known to control MMP-12 release, macrophages were treated with the G protein-coupled receptor inhibitor, pertussis toxin; this suppressed both TNF-alpha and MMP-12 release, while a PAR-1 agonist (TRAP) increased TNF-alpha and MMP-12 release. Smoke-conditioned medium caused increased release of the prothrombin activator, tissue factor, from macrophages. Hirudin, a thrombin inhibitor, and aprotinin, an inhibitor of plasmin, reduced smoke-mediated TNF-alpha and MMP-12 release, and A1AT inhibited both plasmin and thrombin activity in a cell-free functional assay. These findings extend our previous suggestion that TNF-alpha production by alveolar macrophages is related to MMP-12 secretion. They also suggest that A1AT can inhibit thrombin and plasmin in blood constituents that leak into the lung after smoke exposure, thereby preventing PAR-1 activation and MMP-12/TNF-alpha release, and decreasing smoke-mediated inflammatory cell influx.
Assuntos
Macrófagos Alveolares/metabolismo , Metaloproteinase 12 da Matriz/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , alfa 1-Antitripsina/metabolismo , Animais , Células Cultivadas , Meios de Cultivo Condicionados , Relação Dose-Resposta a Droga , Fibrinolisina/metabolismo , Fibrinolíticos/metabolismo , Hirudinas/metabolismo , Humanos , Macrófagos Alveolares/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Toxina Pertussis/metabolismo , Fumaça , Fumar/efeitos adversos , Trombina/metabolismo , Nicotiana/efeitos adversos , alfa 1-Antitripsina/farmacologiaAssuntos
Mucosa Respiratória/patologia , Fumar , Fator de Crescimento Transformador beta1/metabolismo , Regulação para Cima/fisiologia , Animais , Broncoconstrição , Células Cultivadas , Fator de Crescimento do Tecido Conjuntivo , Humanos , Proteínas Imediatamente Precoces/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Ratos , Mucosa Respiratória/metabolismo , Fumar/efeitos adversos , Fumar/metabolismo , Fumar/patologia , Fator de Crescimento Transformador beta1/antagonistas & inibidores , alfa-Fetoproteínas/farmacologiaRESUMO
Male sterility of wheat-breeding line 337S (Triticum aestivum L.) is sensitive to both short day-length/low temperature and long day-length/high temperature. 337S was crossed with the common wheat variety, Huamai No. 8 and the F1 was highly fertile. The F2 population segregated in a 15:1 ratio for fertility/sterility in 243 individuals under long day-length/high-temperature. The two thermophotoperiod-responsive male sterile genes were mapped to chromosomes 5B and 2B using Simple Sequence Repeat (SSR) markers and bulked segregant analysis. Partial linkage maps around the sterility loci of chromosomes 2B and 5B were constructed using the 243 individuals in the F2 population. One gene (wptms1) for male sterility was flanked by the SSR markers Xgwm335 and Xgwm371 at a genetic distance in chromosome 5B of 4.1 and 24.4 cM, respectively. The second gene (wptms2) was mapped between markers Xgwm374 and Xgwm120 at a genetic distance of 6.6 and 20.9 cM, respectively. The closest linked markers Xgwm335 (wptms1) and Xgwm374 (wptms2) explained 53 and 38% of phenotypic variation for the fertility. The SSR markers provide a useful tool to transfer the male sterile genes into elite wheat germplasm.
Assuntos
Genes de Plantas , Genes Recessivos , Temperatura , Triticum/genética , Mapeamento Cromossômico , Cromossomos de Plantas , Cruzamentos Genéticos , Fertilidade/genética , Ligação Genética , Marcadores Genéticos , Repetições de Microssatélites , Fotoperíodo , Polimorfismo GenéticoRESUMO
Air pollution particles (PM) are known to elicit an acute inflammatory response in vivo that is mediated in part through PM-induced activation of the NF-kappaB signaling pathway. Many of the details of this process and particularly where in the cell it occurs are unclear. To determine whether contact of PM particles with an epithelial cell surface activates NF-kappaB, rat tracheal explants were exposed to Ottawa Urban Air Particles or iron-loaded fine TiO2, a model PM particle, for up to 2 h. During this period, there was no evidence of particle entry into the tracheal epithelial cells by light or electron microscopy, but both types of particle activated NF-kappaB as assayed by gel shifts. NF-kappaB activation could be inhibited by the active oxygen species scavenger, tetramethylthiourea; the redox-inactive metal chelator, deferoxamine; the Src inhibitor, PP2; and the epidermal growth factor (EGF) receptor inhibitor AG1478. An iron-containing citrate extract of both dusts also produced NF-kappaB activation. Both dusts and a citrate extract caused phosphorylation of the EGF receptor on tyrosine 845, an indicator of Src activity. We conclude that iron-containing PM particles can activate NF-kappaB via a pathway involving Src and the EGF receptor. This process does not require entry of particles into the airway epithelial cells but is dependent on the presence of iron and generation of active oxygen species by the dusts. These findings imply that even brief contact of PM with a pulmonary epithelial cell surface may produce deleterious effects in vivo.
Assuntos
Poluentes Atmosféricos/farmacocinética , Células Epiteliais/metabolismo , NF-kappa B/metabolismo , Traqueia/metabolismo , Poluentes Atmosféricos/análise , Poluentes Atmosféricos/toxicidade , Animais , Ligação Competitiva/efeitos dos fármacos , Transporte Biológico/efeitos dos fármacos , Desferroxamina/farmacologia , Poeira , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/ultraestrutura , Compostos Férricos/análise , Compostos Férricos/farmacocinética , Compostos Férricos/toxicidade , Sequestradores de Radicais Livres/farmacologia , Técnicas In Vitro , Tamanho da Partícula , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Quinazolinas , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Tioureia/análogos & derivados , Tioureia/farmacologia , Fatores de Tempo , Titânio/análise , Titânio/farmacocinética , Titânio/toxicidade , Traqueia/efeitos dos fármacos , Traqueia/ultraestrutura , Tirfostinas/farmacologiaRESUMO
Small airway remodeling (SAR) is an important cause of airflow obstruction in cigarette smokers, but whether SAR represents a response to smoke-evoked inflammation or is directly mediated by smoke-induced growth factor production is disputed. To examine this process, we exposed rat tracheal explants, a model free of exogenous inflammatory cells, to cigarette smoke in vitro. Cigarette smoke caused release of active transforming growth factor (TGF)-beta1, and this was prevented by the oxidant scavenger tetramethythiourea. Nuclear immunostaining for phospho-Smad2, a TGF-beta downstream signaling molecule, was present in epithelial and interstitial cells within 1 h after exposure. Smoke caused upregulation of gene expression of connective tissue growth factor (CTGF), a mediator of TGF-beta fibrogenic effects, within 2 h, and upregulation of procollagen gene expression at 24 h; both changes could be prevented by the TGF-beta antagonist fetuin (alpha2-HS-glycoprotein). In a cell-free system, recombinant human TGF-beta latency-associated peptide was oxidized by cigarette smoke, and smoke released active TGF-beta1 from recombinant latent TGF-beta1 via an oxidant mechanism. These experiments suggest that SAR in cigarette smokers may be caused by direct, smoke-mediated, oxidant-driven induction of growth factor signaling in the airway wall, and that SAR does not necessarily require exogenous inflammatory cells.
Assuntos
Fumar , Traqueia/anatomia & histologia , Traqueia/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Fator de Crescimento do Tecido Conjuntivo , Meios de Cultivo Condicionados , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Macrófagos/metabolismo , Pró-Colágeno/genética , Pró-Colágeno/metabolismo , Ratos , Ratos Sprague-Dawley , Proteína Smad2 , Tioureia/análogos & derivados , Tioureia/metabolismo , Traqueia/patologia , Transativadores/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta1RESUMO
Mice lacking tumor necrosis factor-alpha (TNF-alpha) receptors (TNFRKO mice) do not develop an inflammatory infiltrate or matrix breakdown after a single acute cigarette smoke exposure. To determine the role of TNF-alpha in the long-term development of emphysema, mice were exposed to smoke for 6 months. TNFRKO mice demonstrated an 11% increase in mean linear intercept; wild-type mice had a 38% increase. TNFRKO mice had 65% fewer neutrophils and no increase in macrophages in lavage fluid. Whole lung matrix metalloprotease (MMP)-2, MMP-9, MMP-12, MMP-13, and matrix type-1 (MT1)-MMP proteins were increased in wild-type mice, but smaller increases in MMP-12, MMP-13, and MT1-MMP were also seen in TNFRKO mice. Lavage matrix breakdown products were elevated in wild-type mice and only partially reduced by anti-neutrophil antibody, implying both neutrophil- and non-neutrophil-mediated matrix breakdown. We conclude that TNF-alpha-mediated processes, probably driving neutrophil influx, are responsible for approximately 70% of airspace enlargement and the majority of inflammatory cell influx/matrix breakdown in the mouse model. TNF-alpha causes increased MMP production, but some increased MMP activity is present even in TNFRKO mice. These findings imply a second TNF-alpha-independent process, possibly related to direct MMP attack on matrix, that produces the remaining 30% of airspace enlargement.
Assuntos
Enfisema Pulmonar/etiologia , Receptores do Fator de Necrose Tumoral/fisiologia , Fumar/efeitos adversos , Animais , Desmosina/metabolismo , Hidroxiprolina/metabolismo , Pulmão/metabolismo , Pulmão/patologia , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Enfisema Pulmonar/metabolismo , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
Small airway remodeling ("small airways disease") is a common finding in cigarette smokers and is an important cause of airflow obstruction. Airway remodeling is usually attributed to the effects of cigarette smoke-induced inflammation in the airway wall, but little is actually known about its pathogenesis. We exposed rat tracheal explants to cigarette smoke and then maintained them in air organ culture. At 24 hours after smoke exposure, there was a dose-dependent increase in gene expression of procollagen and a significant increase in tissue hydroxyproline, a measure of collagen content. Greater increases in procollagen gene expression were found with repeated smoke exposures. Increased procollagen gene expression could be prevented with SN50, a selective inhibitor of nuclear factor-kappaB activation, and superoxide dismutase, catalase, and tetramethylthiourea, scavengers of active oxygen species. AG1478, an inhibitor of epidermal growth factor receptor signaling, also prevented increased procollagen gene expression, but PD98059 and SB203580, inhibitors of mitogen-activated protein kinases, did not. These findings indicate that cigarette smoke can directly induce airway remodeling, specifically airway wall fibrosis, probably through active oxygen species-dependent transactivation of the epidermal growth factor receptor and subsequent nuclear factor-kappaB activation. Smoke-evoked inflammatory cells are not required for this process.
Assuntos
Nicotiana/efeitos adversos , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/patologia , Fumaça/efeitos adversos , Traqueia/efeitos dos fármacos , Doenças da Traqueia/patologia , Animais , Técnicas de Cultura , Relação Dose-Resposta a Droga , Expressão Gênica/efeitos dos fármacos , Hidroxiprolina/análise , Hidroxiprolina/genética , Pró-Colágeno/análise , Pró-Colágeno/genética , Ratos , Ratos Sprague-Dawley , Fumar/efeitos adversos , Traqueia/patologia , Doenças da Traqueia/etiologiaRESUMO
Serine elastase inhibitors have been proposed as a treatment for cigarette smoke-induced emphysema, but little is known about whether such agents actually are effective. We recently reported that a synthetic serine elastase inhibitor, ZD0892, provided some protection against emphysema in a guinea pig model. For these experiments, we used transgenic mice that expressed extremely low levels of human alpha-1-antitrypsin (A1AT) but were tolerant of exogenous human A1AT. Mice were exposed to daily cigarette smoke for up to 6 months; some animals received 20 mg of human A1AT (Prolastin) every 48 hours. Treatment with A1AT produced an approximate twofold increase in serum A1AT levels and elastase inhibitory capacity and abolished smoke-induced elevations in lavage neutrophils and matrix breakdown products (desmosine and hydroxyproline) measured from 2 to 30 days of smoke exposure. A1AT oxidized to remove antiproteolytic activity did not increase serum elastase inhibitory capacity but did prevent neutrophil influx. Treatment with A1AT for 6 months provided 63% protection against increased airspace size (emphysema) and abolished smoke-mediated increases in plasma tumor necrosis factor-alpha. We conclude that A1AT therapy ameliorates smoke-induced inflammation and matrix breakdown, possibly via an antiinflammatory mechanism related to tumor necrosis factor-alpha suppression, and provides partial protection against emphysema.
Assuntos
Enfisema Pulmonar/tratamento farmacológico , Enfisema Pulmonar/etiologia , alfa 1-Antitripsina/uso terapêutico , Animais , Humanos , Camundongos , Camundongos Transgênicos , Inibidores de Serina Proteinase/uso terapêutico , Fumaça , Fatores de Tempo , NicotianaRESUMO
The cells and proteases that mediate cigarette smoke-induced emphysema are controversial, with evidence favoring either neutrophils and neutrophil-derived serine proteases or macrophages and macrophage-derived metalloproteases as the important effectors. We recently reported that both macrophage metalloelastase (MMP-12) and neutrophils are required for acute cigarette smoke-induced connective tissue breakdown, the precursor of emphysema. Here we show how these disparate observations can be linked. Both wild-type (MMP-12 +/+) mice and mice lacking MMP-12 (MMP-12 -/-) demonstrated rapid increases in whole-lung nuclear factor-kappaB activation and gene expression of proinflammatory cytokines after cigarette smoke exposure, indicating that a lack of MMP-12 does not produce a global failure to upregulate inflammatory mediators. However, only MMP-12 +/+ mice demonstrated increased whole-lung tumor necrosis factor-alpha (TNF-alpha) protein or release of TNF-alpha from cultured alveolar macrophages exposed to smoke in vitro. Levels of whole-lung E-selectin, an endothelial activation marker, were increased in only MMP-12 +/+ mice. These findings suggest that, acutely, MMP-12 mediates smoke-induced inflammation by releasing TNF-alpha from macrophages, with subsequent endothelial activation, neutrophil influx, and proteolytic matrix breakdown caused by neutrophil-derived proteases. TNF-alpha release may be a general mechanism whereby metalloproteases drive cigarette smoke-induced inflammation.
Assuntos
Metaloendopeptidases/imunologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Fumar/efeitos adversos , Fumar/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Doença Aguda , Animais , Células Cultivadas , Metaloproteinase 12 da Matriz , CamundongosRESUMO
Chemiluminescent probes offer highly sensitive quantitative analyses of proteins blotted from electrophoretic gels onto a supporting matrix (e.g. nitrocellulose or polyvinylidene difluoride). Visualization of signals from probes involves the emission of light that is dependent on a number of variables (e.g. conjugated enzyme activity, antibody titer, hybridization efficiency, substrate concentration, chemical reactivity, temperature, etc.). Thus, it is important to be able to correct for these variations. For example, the exposure time of the blot to the detection medium (e.g., film or digital camera) is a critical variable in the final results. Several protein samples separated on a single blot (e.g. one-dimensional resolution) can be compared from the ratio of the individual proteins, but comparison of separate blots completed on different days requires a chemiluminescent standard. The situation is more complex when only one sample per gel/blot is used (i.e. two-dimensional electrophoresis (2-DE)). This paper describes a method for preparing agarose embedded standardized strips that contain dilutions of antigens that can be visualized with the corresponding probe antibody. This standardization strip can be produced from common laboratory supplies and provides a method to correct for alterations in chemiluminescent intensities from different 2-DE analysis. Several standardization strips can be produced simultaneously and then used during the electroblotting step of different blots on different days.
Assuntos
Western Blotting/normas , Eletroforese em Gel Bidimensional/normas , Medições Luminescentes , Animais , Anticorpos , Antígenos/isolamento & purificação , Humanos , Proteoma , Padrões de ReferênciaRESUMO
The numerical density (Nv) of the alpha-granule (alpha G) and dense granule (dG) measured by using electron microscopic morphometry and the cytosolic free calcium concentration ([Ca2+]i) by using Ca-fluorescent indicator were observed for studying the relationship of [Ca2+]i and Nv of alpha G and dG in platelets activated by A23187, thrombin or ADP. The results showed that the [Ca2+]i rose markedly by A23187. Thrombin or ADP could also induce an increase of [Ca2+]i concentration-dependently. Under 3 different degrees of activation induced by different agonists, the close relationship between Nv of both granules, alpha G and dG, and [Ca2+]i in platelets was found (Pr(alpha G) < 0.05, Pr(dG) < 0.01). These findings indicate that the increase in [Ca2+]i may enhance the secretion of alpha G and dG.
Assuntos
Plaquetas/metabolismo , Cálcio/metabolismo , Selectina-P/metabolismo , Animais , Transporte Biológico Ativo , Citoplasma/metabolismo , Proteínas de Protozoários/metabolismo , CoelhosRESUMO
The cellular retinol binding proteins, CRBP and CRBP II, are implicated in the cellular uptake of retinol and intracellular trafficking of retinol between sites of metabolic processing. 19F-NMR studies of retinol transfer between CRBP and CRBP II and phospholipid vesicles, using either fluorine-labeled ligand or protein, demonstrated that there was significantly more transfer of retinol from CRBP II to lipid vesicles than from CRBP. Differences in how readily protein-bound retinol is released to lipid bilayers may lead to differences in how these two proteins modulate intracellular retinol metabolism.
Assuntos
Lipossomos , Proteínas de Ligação ao Retinol/metabolismo , Vitamina A/metabolismo , Animais , Apoproteínas/metabolismo , Clonagem Molecular , Escherichia coli , Flúor , Espectroscopia de Ressonância Magnética , Ratos , Proteínas Recombinantes/metabolismo , Proteínas de Ligação ao Retinol/química , Proteínas Celulares de Ligação ao RetinolRESUMO
Cellular retinoic acid binding protein-I (CRABP-I) and cellular retinoic acid binding protein-II (CRABP-II) are highly homologous, 15 kDa proteins which bind all-trans-retinoic acid. In the adult, CRABP-II is expressed predominately in the epidermis, while CRAPB-I is expressed in a variety of tissues. To obtain structural information which could aid the design of more selective ligands, isotope-directed NMR methods were employed to observe the CRABP-bound conformation of 13C-labeled retinoic acid and to identify its contact points with neighboring amino acids. Analysis of HMQC, HMQC-TOCSY, and 13C-TOCSY-REVINEPT on CRABP-bound (2,3,6,7,8,9,10,11,19-13C)- and (1,4,5,8,9,16, 17,18,19-13C)-all trans-retinoic acid allowed the unambiguous assignment of all labeled protons and their attached 13C resonances. The volumes of 16 olefinic proton-methyl NOE cross-peaks measured from 30-ms 13C-(omega 2)-filtered 1H NOESY experiments were used to determine the conformations about the 6-, 8-, and 10-single bonds of the retinoic acid polyene chain. These spectra show qualitatively distinct NOE patterns for the two CRABPs. Measured cross-peak volumes for CRABP-II bound retinoic acid were well predicted by a single, static conformational having a 6-s torsion angle of -60 degrees skewed from a cis conformation. In contrast, for CRABP-I no single, static conformation was able to match the pattern of cross-peaks, suggesting motion about the 6-s bond. The measured cross-peaks were best described by 8-s and 10-s torsion angles of 180 degrees +/- 30 degrees, a trans configuration, for both proteins. The pattern of intermolecular NOESY cross-peaks between 13C-labeled protons in the ring portion of retinoic acid and protein protons were different between CRABP-I and CRABP-II. These differences coincide well with nearby amino acid substitutions in the recently reported X-ray structures of crystalline CRABP-I and CRABP-II and may assist rational design of selective ligands.
Assuntos
Receptores do Ácido Retinoico/metabolismo , Tretinoína/metabolismo , Sequência de Aminoácidos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Receptores do Ácido Retinoico/química , Alinhamento de Sequência , Tretinoína/químicaRESUMO
Comparative 19F-NMR studies of fluororetinol analogs with rat cellular retinol binding protein II (CRBP II) and rat cellular retinol-binding protein (CRBP) were performed to probe differences in the binding interactions of these two homologous proteins. Line shape analyses of 19F-NMR spectra of (E,E,Z,E)-6-fluoro-9-(4-methoxy-2,3,6-trimethylphenyl)-3,7-dimethyl- 2,4,6,8-nonatetren-1-ol (ligand 1), (E,E,Z,E)-6-fluoro-9-(2,2' dimethyl-6-methylcyclohexenyl)-3,7- dimethyl-2,4,6,8-nonatetren-1-ol (ligand 2), (E,Z,E,E)-5-fluoro-9-(2,2'- dimethyl-6-methylcyclohexenyl)-3,7-dimethyl-2,4,6,8-nonatetren+ ++-1-ol (ligand 3), when complexed with CRBP II at temperatures ranging from 0-45 degrees C, revealed that the 19F resonances corresponding to the bound ligand were in slow chemical exchange between two resonance frequencies. This was further supported by a 2D-NOESY exchange experiment. The kex at 25 degrees C was estimated from spectral simulation and fitting analyses to be 887 s-1, 1010 s-1 and 771 s-1 for CRBP II complexed 1, 2, and 3, respectively. In contrast, only a single absorption was observed for bound ligands complexed with rat CRBP over this temperature range, suggesting that the conformational dynamics of retinol binding are different for these two closely homologous proteins.
Assuntos
Corantes Fluorescentes , Espectroscopia de Ressonância Magnética , Proteínas de Ligação ao Retinol/metabolismo , Vitamina A/análogos & derivados , Animais , Ligantes , Conformação Proteica , Ratos , Proteínas de Ligação ao Retinol/química , Proteínas Celulares de Ligação ao Retinol , Relação Estrutura-Atividade , Termodinâmica , Vitamina A/química , Vitamina A/metabolismoRESUMO
Effects of kappa-selenocarrageenan (kappa-SeC) on membrane fluidity and ghost reseal ability of rat erythrocyte were studied by measuring the fluorescence polarization and the NADH-cytochrome C oxido-reductase, respectively. After rats were given ig kappa-SeC 140 mg.kg-1.d-1 x 30 d, the fluorescence polarization was decreased in comparison with saline control group (P < 0.05). It suggested that kappa-SeC increased the membrane fluidity. The reseal ability of erythrocyte membrane (ghost) was also elevated after ig kappa-SeC 140 and 70 mg.kg-1.d-1 x 30 d (P < 0.05).