RESUMO
BACKGROUND: Anatomical knowledge of the duodenojejunal flexure is necessary for abdominal surgeries, and also important for physiologic studies about the duodenum. But little is known about the anatomy of this region in mammals. Here, we examined comparative anatomy to understand the anatomical formation of the duodenojejunal flexure in mammals. MATERIALS AND METHODS: The areas around the duonenojejunal flexure were ob-served in mouse, rat, dog, pig, and human, and the anatomical structures around the duodenojejunal junction in the animals were compared with those in human. RESULTS: The superior and inferior duodenal folds, and the superior and inferior duodenal fossae were identified in all examined humans. In pig, the structures were not clearly identified because the duodenum strongly adhered to the retroperitoneum and to the mesocolon. In mouse, rat, and dog, only the plica duodenocolica, which is regarded as the animal counterpart of the superior duo-denal fold in human, was identified, and other folds or fossae were not observed, probably because the duodenum was not fixed to the parietal peritoneum in those animals. Transection of the plica duodenocolica could return the normally rotated intestine back to the state of non-rotation in rat. CONCLUSIONS: This study showed the anatomical similarities and dissimilarities of the duodenojejunal flexure among the mammals. Anatomical knowledge of the area is useful for duodenal and pancreatic surgeries, and for animal studies about the duodenum. (Folia Morphol 2018; 77, 2: 286-292).
Assuntos
Duodeno/anatomia & histologia , Jejuno/anatomia & histologia , Anatomia Comparada , Animais , Cães , Humanos , Ratos , Especificidade da Espécie , SuínosRESUMO
Calcineurin inhibitors (CNIs) have been used off-label for the treatment of refractory Kawasaki disease (KD). However, it remains unknown whether CNIs show protective effects against the development of coronary artery lesions in KD patients. To investigate the effects of CNIs on coronary arteries and the mechanisms of their actions on coronary arteritis in a mouse model of KD, we performed experiments with FK565, a ligand of nucleotide-binding oligomerization domain-containing protein 1 (NOD1) in wild-type, severe combined immunodeficiency (SCID), caspase-associated recruitment domain 9 (CARD9)-/- and myeloid differentiation primary response gene 88 (MyD88)-/- mice. We also performed in-vitro studies with vascular and monocytic cells and vascular tissues. A histopathological analysis showed that both cyclosporin A and tacrolimus exacerbated the NOD1-mediated coronary arteritis in a dose-dependent manner. Cyclosporin A induced the exacerbation of coronary arteritis in mice only in high doses, while tacrolimus exacerbated it within the therapeutic range in humans. Similar effects were obtained in SCID and CARD9-/- mice but not in MyD88-/- mice. CNIs enhanced the expression of adhesion molecules by endothelial cells and the cytokine secretion by monocytic cells in our KD model. These data indicated that both vascular and monocytic cells were involved in the exacerbation of coronary arteritis. Activation of MyD88-dependent inflammatory signals in both vascular cells and macrophages appears to contribute to their adverse effects. Particular attention should be paid to the development of coronary artery lesions when using CNIs to treat refractory KD.
Assuntos
Arterite/tratamento farmacológico , Inibidores de Calcineurina/uso terapêutico , Endotélio Vascular/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Síndrome de Linfonodos Mucocutâneos/tratamento farmacológico , Fator 88 de Diferenciação Mieloide/metabolismo , Oligopeptídeos/uso terapêutico , Animais , Proteínas Adaptadoras de Sinalização CARD/genética , Vasos Coronários/patologia , Citocinas/metabolismo , Modelos Animais de Doenças , Endotélio Vascular/imunologia , Humanos , Mediadores da Inflamação/metabolismo , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos SCID , Fator 88 de Diferenciação Mieloide/genética , Células RAW 264.7 , Transdução de SinaisRESUMO
OBJECTIVE: The aims of this study were to elucidate the association between plasma intestinal fatty acid-binding protein (I-FABP) level and actual pathological damage of intestinal mucosa and its reversibility. METHODS: An intestinal ischemia-reperfusion model was created by temporary occlusion of the descending aorta in 9 pigs which were divided into 3 groups according to the duration of visceral ischemic insult: 15-minute ischemia (n=3), 30-minute ischemia (n=3) and 60-minute ischemia (n=3). Blood samples and short segments of the jejunum for pathological examinations, including immunohistochemical staining of I-FABP, Ki-67 and E-cadherin, were taken at the beginning of the operation (T1) and 15 minutes (T2), 30 minutes (T3), 45 minutes (T4) and 60 minutes (T5) after reperfusion. RESULTS: Plasma I-FABP after 15 minutes of ischemia reached a peak of 1859 ± 1089 pg/ml at T3, while the level after 30 minutes of ischemia achieved a peak level of 5053 ± 1 717 pg/ml at T5. The level after 60 minutes of ischemia demonstrated a rapid increment up to 10734 ± 93 pg/ml at T3. There was a significant difference in the trend of plasma I-FABP levels between 30 minutes and 60 minutes of ischemia (p=0.01). The strongest immunohistochemical staining of the intestinal epithelium for I-FABP was observed at T4 after 30 minutes of ischemia, with the shedding of injured epithelium followed by re-epithelialisation, with sequential up-regulation of Ki67 and E-cadherin. However, the intestinal epithelium after 60 minutes of ischemia demonstrated the lack of I-FABP expression with irreversible damage. CONCLUSION: Plasma I-FABP levels may be a crucial marker to recognize the reversibility of damage of the intestinal epithelium after an ischemic insult and the level of 5000 pg/ml is considered to be the critical borderline for irreversibility, which might prevent diagnostic delay in the clinical setting.
Assuntos
Biomarcadores/sangue , Proteínas de Ligação a Ácido Graxo/sangue , Enteropatias/diagnóstico , Mucosa Intestinal/patologia , Traumatismo por Reperfusão/complicações , Animais , Feminino , Técnicas Imunoenzimáticas , Enteropatias/sangue , Enteropatias/etiologia , Mucosa Intestinal/irrigação sanguínea , Mucosa Intestinal/lesões , Masculino , Valor Preditivo dos Testes , SuínosRESUMO
BACKGROUND: Colorectal cancer (CRC) is the second most common malignancy in Japan. Treatment with inhibitors of the vascular endothelial growth factor (VEGF) signalling pathway has proven benefit in metastatic CRC. Cediranib is an oral highly potent VEGF signalling inhibitor that inhibits all three VEGF receptors. PATIENTS AND METHODS: In this phase II, double-blind, placebo-controlled study, 172 patients with metastatic CRC were randomised to receive once-daily cediranib (20 or 30 mg) or placebo, each combined with modified FOLFOX6 (mFOLFOX6). The primary objective was comparison of progression-free survival (PFS). RESULTS: The comparison of cediranib 20 mg versus placebo met the primary objective of PFS prolongation [hazard ratio = 0.70 (95% confidence interval 0.44-1.11), P = 0.167], which met the protocol-defined criterion of P < 0.2. Median PFS was 10.2 versus 8.3 months, respectively. The PFS comparison for cediranib 30 mg versus placebo did not meet the criterion. The most common adverse events (AEs) in the cediranib-containing groups were diarrhoea and hypertension. CONCLUSIONS: Cediranib 20 mg plus mFOLFOX6 met the predefined criteria in terms of improved PFS compared with placebo plus mFOLFOX6. Cediranib 20 mg was generally well tolerated and the AE profile was consistent with previous studies.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma/tratamento farmacológico , Carcinoma/secundário , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Carcinoma/sangue , Carcinoma/mortalidade , Neoplasias Colorretais/sangue , Neoplasias Colorretais/mortalidade , Intervalo Livre de Doença , Método Duplo-Cego , Feminino , Fluoruracila/administração & dosagem , Humanos , Japão , Estimativa de Kaplan-Meier , Leucovorina/administração & dosagem , Masculino , Pessoa de Meia-Idade , Compostos Organoplatínicos/administração & dosagem , Oxaliplatina , Quinazolinas/administração & dosagem , Resultado do Tratamento , Fator A de Crescimento do Endotélio Vascular/sangueRESUMO
Siglecs (sialic acid binding immunoglobulin (Ig)-like lectins) constitute a group of 15 human and 9 murine cell-surface transmembrane receptors belonging to the I-type lectin family, mostly expressed on innate immune cells and characterized by broadly similar structural features. Here, the prominent inhibitory CD22 (Siglec-2), well known in maintaining tolerance and preventing autoimmune responses on B cells, is studied in its human and murine forms in complex with sialoglycans. In detail, the role of the N-glycolyl neuraminic acid (Neu5Gc) moiety in the interaction with both orthologues was explored. The analysis of the binding mode was carried out by the combination of NMR spectroscopy, computational approaches, and CORCEMA-ST calculations. Our findings provide a first model of Neu5Gc recognition by h-CD22 and show a comparable molecular recognition profile by h- and m-CD22. These data open the way to innovative diagnostic and/or therapeutic methodologies to be used in the modulation of the immune responses.
RESUMO
A 77-year-old female with unstable angina pectoris was referred to our hospital for further evaluation of multiple aortic aneurysms. Computed tomography showed descending thoracic (65 mm), thoracoabdominal (40 mm) and infra-renal abdominal aneurysm (50 mm). Initially, this patient underwent off pump coronary revascularization. On 11 days after initial surgery, descending thoracic aneurysm ruptured, followed by emergent descending thoracic and thoraco-abdominal aneurysm repair. Two months later from this aortic repair, this patient successfully underwent abdominal aortic aneurysm repair. At 3 years and 7th month after the last operation, she is well without limitation of daily activities and any evidence of myocardial ischemia.
Assuntos
Angina Instável/complicações , Aneurisma Aórtico/cirurgia , Idoso , Aorta Abdominal , Aorta Torácica , Feminino , HumanosRESUMO
A 65-year-old man was referred to our hospital to treat recent anterior myocardial infarction. Coronary artery angiography showed acute occlusion of left anterior descending coronary artery (LAD) and chronic occlusion of right coronary artery. After emergent percutaneous coronary intervention for LAD, drug-refractory electrical storm necessitating frequent electrical defibrillating cardioversion occurred. This patient successfully underwent surgical cryoablation, left ventriculoplasty and coronary revascularization. At 2 years and 10th month after the operation, he is well without limitation of daily activities and any evidence of myocardial ischemia and ventricular tachycardia.
Assuntos
Criocirurgia , Infarto do Miocárdio/complicações , Infarto do Miocárdio/cirurgia , Taquicardia Ventricular/etiologia , Fibrilação Ventricular/etiologia , Idoso , Ventrículos do Coração/cirurgia , Humanos , Masculino , Revascularização Miocárdica , Resultado do TratamentoRESUMO
A 78-year-old male with aortitis syndrome was referred to our hospital for the treatment of unstable angina pectoris with ischemic mitral regurgitation, which was diagnosed by transthoracic echocardiography and coronary artery angiography. Computed tomography showed segmental wall thickness of thoracic and abdominal aorta He underwent an emergent coronary artery bypass grafting. The postoperative course was uneventful without any neurological complications. Postoperative echocardiogram and coronary artery angiography showed good mitral valve function and all patent bypass grafts. He was discharged 33 days after surgery. At 26 months after surgery, he is well without limitation of daily activities and any evidence of myocardial ischemia.
Assuntos
Angina Instável/cirurgia , Ponte de Artéria Coronária , Arterite de Takayasu/complicações , Idoso , Humanos , MasculinoRESUMO
Lipopolysaccharide (LPS) is the main inducer of shock and death in Gram-negative sepsis. Recent evidence suggests that LPS-induced signal transduction begins with CD14-mediated activation of 1 or more Toll-like receptors (TLRs). The lipid A analogues lipid IVa and Rhodobacter sphaeroides lipid A (RSLA) exhibit an uncommon species-specific pharmacology. Both compounds inhibit the effects of LPS in human cells but display LPS-mimetic activity in hamster cells. We transfected human TLR4 or human TLR2 into hamster fibroblasts to determine if either of these LPS signal transducers is responsible for the species-specific pharmacology. RSLA and lipid IVa strongly induced NF-kappaB activity and IL-6 release in Chinese hamster ovary fibroblasts expressing CD14 (CHO/CD14), but these compounds antagonized LPS antagonists in CHO/CD14 fibroblasts that overexpressed human TLR4. No such antagonism occurred in cells overexpressing human TLR2. We cloned TLR4 from hamster macrophages and found that human THP-1 cells expressing the hamster TLR4 responded to lipid IVa as an LPS mimetic, as if they were hamster in origin. Hence, cells heterologously overexpressing TLR4 from different species acquired a pharmacological phenotype with respect to recognition of lipid A substructures that corresponded to the species from which the TLR4 transgene originated. These data suggest that TLR4 is the central lipid A-recognition protein in the LPS receptor complex.
Assuntos
Proteínas de Drosophila , Receptores de Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Células CHO , Cricetinae , Glicolipídeos/metabolismo , Humanos , Ligantes , Lipídeo A/análogos & derivados , Lipídeo A/metabolismo , Receptores de Lipopolissacarídeos/genética , Lipopolissacarídeos/antagonistas & inibidores , Macrófagos/citologia , Macrófagos/metabolismo , Glicoproteínas de Membrana/genética , Mimetismo Molecular , Receptores de Superfície Celular/genética , Proteínas Recombinantes/metabolismo , Rhodobacter sphaeroides , Transdução de Sinais , Especificidade da Espécie , Receptor 2 Toll-Like , Receptor 4 Toll-Like , Receptores Toll-LikeRESUMO
A 59-year-old male with congestive heart failure caused by impaired left ventricular function after coronary artery bypass grafting (CABG) was referred to our hospital, and massive ischemic mitral regurgitation was detected by echocardiography. This patient underwent on-pump beating-heart mitral valve repair without aortic cross-clamp successfully through right thoracotomy. Postoperative echocardiography revealed no mitral regurgitation. The patient recovered uneventfully and was discharged on the 17th postoperative day. At 6th month after the operation, he is well without mitral regurgitation.
Assuntos
Ponte de Artéria Coronária , Insuficiência da Valva Mitral/fisiopatologia , Insuficiência da Valva Mitral/cirurgia , Valva Mitral/cirurgia , Complicações Pós-Operatórias/fisiopatologia , Complicações Pós-Operatórias/cirurgia , Volume Sistólico , Procedimentos Cirúrgicos Cardíacos/métodos , Insuficiência Cardíaca/fisiopatologia , Insuficiência Cardíaca/cirurgia , Humanos , Masculino , Pessoa de Meia-Idade , Resultado do Tratamento , Função Ventricular EsquerdaRESUMO
Oral epithelium might be the first barrier against oral bacteria in periodontal tissue. We hypothesized that oral epithelium is endowed with innate immune receptors for bacterial components, which play roles in host defense against bacterial infection without being accompanied by excessive inflammatory responses. We found clear expression of Toll-like receptor (TLR)4 as well as TLR2, and strong expression of NOD1 and NOD2 in normal oral epithelial tissues by immunohistochemical analysis. We also showed that primary oral epithelial cells in culture expressed these molecules using PCR, flow cytometry, and immunostaining. In inflamed oral epithelium, cell-surface localizations of TLR2 and TLR4 were more clearly observed than in healthy tissue. Upon stimulation with synthetic ligands for these receptors, the expression of beta-defensin 2 was markedly up-regulated. These findings indicate that these molecules in oral epithelial cells are functional receptors that induce antibacterial responses.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Gengiva/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Receptores Toll-Like/imunologia , Anti-Infecciosos , Células Cultivadas , Células Epiteliais/imunologia , Citometria de Fluxo , Imunofluorescência , Gengiva/citologia , Humanos , Imuno-Histoquímica , Células KB , Ligantes , Proteína Adaptadora de Sinalização NOD1 , Proteína Adaptadora de Sinalização NOD2 , Periodontite/imunologia , Periodontite/patologia , Reação em Cadeia da Polimerase , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/imunologia , Regulação para Cima , beta-Defensinas/imunologia , beta-Defensinas/metabolismoRESUMO
We studied the effect of hypoxia on X-ray-induced delayed effects in normal human embryo cells to elucidate the role of oxidative stress in the susceptibility of cells to induction of genetic instability by radiation. We examined X-ray-induced delayed cell death, giant cell formation, and chromosome aberrations under normally oxygenated (20%) and hypoxic (2%) conditions at 28-38 population doublings postirradiation. The results revealed that hypoxia reduced the X-ray-induced delayed effects, suggesting that radiation enhances cellular oxidative stress, which plays a significant role in determining the susceptibility of irradiated cells to genetic instability. The present study emphasizes the biological significance of epigenetic effects, such as oxygen tension, as well as direct DNA damage in the induction of genetic instability by radiation.
Assuntos
Hipóxia Celular/fisiologia , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/efeitos da radiação , Raios X , Contagem de Células , Morte Celular/efeitos dos fármacos , Morte Celular/efeitos da radiação , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Aberrações Cromossômicas , Células Clonais/efeitos dos fármacos , Células Clonais/metabolismo , Células Clonais/efeitos da radiação , Relação Dose-Resposta à Radiação , Embrião de Mamíferos/citologia , Células Gigantes/efeitos dos fármacos , Células Gigantes/patologia , Células Gigantes/efeitos da radiação , Humanos , Estresse Oxidativo/fisiologia , Oxigênio/metabolismo , Oxigênio/farmacologia , Tolerância a Radiação/efeitos dos fármacosRESUMO
We previously reported the detection of novel O-linked sugar chains classified as being of the glucosyl-O-serine type [Hase et al. (1988) J. Biochem. 104, 867-868]. The sugar chains are a disaccharide (Xyl alpha 1-3Glc) and a trisaccharide (Xyl alpha 1-3Xyl alpha 1-3 Glc) linked to serine residues in epidermal growth factor-like domains of human and bovine blood coagulation factors. The structures of these sugar chains suggested the presence of an alpha 1-->3xylosyltransferase for their biosynthesis. We report here on the detection of alpha 1-->3xylosyltransferase activity which catalyzes the transfer of xylose to Xyl alpha 1-3Glc in the human hepatoma cell line HepG2. We employed pyridylaminated Xyl alpha 1-3Glc as a fluorescent acceptor and UDP-D-Xyl as a donor. The reaction product was purified by reversed-phase HPLC, and the structure of the transfer product isolated was confirmed to be pyridylaminated Xyl alpha 1-3Xyl alpha 1-3Glc by Smith degradation, mass spectrometry, and alpha- and beta-xylosidase digestions. The apparent K(m) value for pyridylaminated Xyl alpha 1-3Glc was 52 mM and for UDP-D-Xyl 0.28 mM. Optimum pH was 7.2. The enzyme was inactivated by addition of EDTA, and its activity was restored by addition of Mn2+ and Mg2+. These results indicate the presence of a novel enzyme which is able to transfer xylose to Xyl alpha 1-3Glc, forming Xyl alpha 1-3Xyl alpha 1-3Glc in human cells.
Assuntos
Carcinoma Hepatocelular/enzimologia , Pentosiltransferases/análise , Animais , Cátions Bivalentes/farmacologia , Bovinos , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Ácido Edético/farmacologia , Humanos , Solubilidade , Células Tumorais Cultivadas , UDP Xilose-Proteína XilosiltransferaseRESUMO
Nalpha-[3-(Hexadecanoyloxy)hexadecanoyl]-ornithine is a typical bacterial ornithine-containing lipid (OL). The configuration of the 3-hydroxy fatty acids in the OL was proved to be D by using HPLC with chiral column. For this analysis, Nalpha-(D or L)-[3-(hexadecanoyloxy)hexadecanoyl]-L-ornithine were synthesized and used as standards. The typical bacterial OL, as well as the synthesized one, exhibited strong interleukin-1- and prostaglandin E2-inducing activities, and further, it induced the production of high IgG anti-tetanus toxoid antibodies in mice. The typical OL is expected to be utilized as a nontoxic, potent adjuvant.
Assuntos
Adjuvantes Imunológicos , Bactérias Gram-Negativas/química , Lipídeos/imunologia , Ornitina/análise , Animais , Bactérias Gram-Negativas/imunologia , Imunoglobulina G/sangue , Interleucina-1/biossíntese , Lipídeos/química , Lipídeos/isolamento & purificação , Ativação de Macrófagos , Macrófagos Peritoneais/imunologia , Camundongos , Prostaglandinas E/biossíntese , Espectrometria de Massas de Bombardeamento Rápido de Átomos , Toxoide Tetânico/imunologiaRESUMO
A strain of Streptoverticillium cinnamoneum produced a peptide antibiotic named lanthiopeptin, which contained four unusual amino acids, erythro-beta-hydroxyaspartic acid, mesolanthionine, threo-beta-methyllanthionine and lysinoalanine. Lanthiopeptin showed antiviral activity against herpes simplex virus type 1 KOS strain infection in Vero cells by cytopathic effect reduction assay. The structure of lanthiopeptin is similar to that of ancovenin.
Assuntos
Antibacterianos , Antibacterianos/isolamento & purificação , Antivirais/isolamento & purificação , Peptídeos/isolamento & purificação , Simplexvirus/efeitos dos fármacos , Streptomycetaceae/metabolismo , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Antibacterianos/análise , Antibacterianos/biossíntese , Antibacterianos/farmacologia , Antifúngicos/análise , Antifúngicos/biossíntese , Antifúngicos/isolamento & purificação , Antifúngicos/farmacologia , Antivirais/análise , Antivirais/biossíntese , Antivirais/farmacologia , Bactérias/efeitos dos fármacos , Cromatografia em Gel , Efeito Citopatogênico Viral/efeitos dos fármacos , Fermentação , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Biossíntese Peptídica , Peptídeos/análise , Peptídeos/farmacologia , Peptídeos Cíclicos , Microbiologia do Solo , Solubilidade , Esporos Bacterianos , Streptomycetaceae/crescimento & desenvolvimento , Streptomycetaceae/fisiologia , Células VeroRESUMO
The structural prerequisites for lipopolysaccharide (LPS) and its partial structures for the activation of the Limulus clotting cascade (Limulus amebocyte lysate [LAL] test) are described and compared with the corresponding requirements for the activation of human immune cells such as mononuclear cells. A necessary, but not sufficient, structural motif for this is the presence of the 4(')-phosphate-diglucosamine backbone recognition structure ('epitope') in lipid A. High activity is only expressed by assemblies of endotoxins, but this is largely independent of the type of supramolecular aggregate structure. A particular conformation of the epitope within the lipid A assembly must be present, which is influenced by addition of further saccharide units to the lipid A moiety, but also reacts slightly to the acylation pattern. In contrast, the cytokine production of human immune cells induced by LPS sensitively depends on the type of its aggregate structure. In the case of a hexa-acylated bisphosphorylated lipid A structure, high activity is only observed with cubic inverted aggregates. Furthermore, addition of antimicrobial agents (such as polymyxin B) leads to a nearly complete inhibition of cytokine production, whereas the reduction in the Limulus assay is much lower. These data are important since a reliable determination of endotoxin concentrations, in particular with respect to its ability to elicit severe infections, is of high interest.
Assuntos
Infecções Bacterianas/diagnóstico , Glucosamina/metabolismo , Leucócitos Mononucleares/metabolismo , Teste do Limulus/métodos , Lipídeo A/metabolismo , Animais , Infecções Bacterianas/sangue , Infecções Bacterianas/imunologia , Células Cultivadas , Citocinas/metabolismo , Endotoxinas/sangue , Endotoxinas/química , Glucosamina/análogos & derivados , Glucosamina/química , Caranguejos Ferradura , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/patologia , Lipídeo A/análogos & derivados , Lipídeo A/química , Ativação Linfocitária , Valor Preditivo dos Testes , Multimerização Proteica , Projetos de PesquisaRESUMO
Two types of synthetic peptidoglycan fragments, diaminopimelic acid (DAP)-containing desmuramylpeptides (DMP) and muramyldipeptide (MDP), induced secretion of interleukin (IL)-8 in a dose-dependent manner in human monocytic THP-1 cells, although high concentrations of compounds are required as compared with chemically synthesized Toll-like receptor (TLR) agonists mimicking bacterial components: TLR2 agonistic lipopeptide (Pam3CSSNA), TLR4 agonistic lipid A (LA-15-PP) and TLR9 agonistic bacterial CpG DNA. We found marked synergistic IL-8 secretion induced by MDP or DAP-containing DMP in combination with synthetic TLR agonists in THP-1 cells. Suppression of the mRNA expression of nucleotide-binding oligomerization domain (NOD)1 and NOD2 by RNA interference specifically inhibited the synergistic IL-8 secretion induced by DMP and MDP with these TLR agonists respectively. In accordance with the above results, enhanced IL-8 mRNA expression and the activation of nuclear factor (NF)-kappaB induced by MDP or DMP in combination with synthetic TLR agonists were markedly suppressed in NOD2- and NOD1-silenced cells respectively. These findings indicated that NOD2 and NOD1 are specifically responsible for the synergistic effects of MDP and DMP with TLR agonists, and suggested that in host innate immune responses to invading bacteria, combinatory dual signalling through extracellular TLRs and intracellular NODs might lead to the synergistic activation of host cells.
Assuntos
Acetilmuramil-Alanil-Isoglutamina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Interleucina-8/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Glicoproteínas de Membrana/agonistas , Monócitos/metabolismo , Peptidoglicano/metabolismo , Receptores de Superfície Celular/agonistas , Acetilmuramil-Alanil-Isoglutamina/imunologia , Linhagem Celular Tumoral , Escherichia coli/imunologia , Escherichia coli/metabolismo , Expressão Gênica , Inativação Gênica , Humanos , Interleucina-8/análise , Lipídeo A/análogos & derivados , Lipídeo A/metabolismo , Monócitos/imunologia , NF-kappa B/metabolismo , Proteína Adaptadora de Sinalização NOD1 , Proteína Adaptadora de Sinalização NOD2 , Oligodesoxirribonucleotídeos/síntese química , Oligodesoxirribonucleotídeos/farmacologia , Peptidoglicano/imunologia , Interferência de RNA , RNA Mensageiro/análise , Receptor 2 Toll-Like , Receptor 4 Toll-Like , Receptor Toll-Like 9 , Receptores Toll-LikeRESUMO
Peptidoglycan recognition proteins (PGRPs), a novel family of pattern recognition molecules (PRMs) in innate immunity conserved from insects to mammals, recognize bacterial cell wall peptidoglycan (PGN) and are suggested to act as anti-bacterial factors. In humans, four kinds of PGRPs (PGRP-L, -Ialpha, -Ibeta and -S) have been cloned and all four human PGRPs bind PGN. In this study, we examined the possible regulation of the expression of PGRPs in oral epithelial cells upon stimulation with chemically synthesized pathogen-associated molecular patterns (PAMPs) in bacterial cell surface components: Escherichia coli-type tryacyl lipopeptide (Pam3CSSNA), E. coli-type lipid A (LA-15-PP), diaminopimelic acid containing desmuramyl peptide (gamma-D-glutamyl-meso-DAP; iE-DAP), and muramyldipeptide (MDP). These synthetic PAMPs markedly upregulated the mRNA expression of the four PGRPs and cell surface expression of PGRP-Ialpha and -Ibeta, but did not induce either mRNA expression or secretion of inflammatory cytokines, in oral epithelial cells. Suppression of the expression of Toll-like receptor (TLR)2, TLR4, nucleotide-binding oligomerization domain (NOD)1 and NOD2 by RNA interference specifically inhibited the upregulation of PGRP mRNA expression induced by Pam3CSSNA, LA-15-PP, iE-DAP and MDP respectively. These PAMPs definitely activated nuclear factor (NF)-kappaB in the epithelial cells, and suppression of NF-kappaB activation clearly prevented the induction of PGRP mRNA expression induced by these PAMPs in the cells. These findings suggested that bacterial PAMPs induced the expression of PGRPs, but not proinflammatory cytokines, in oral epithelial cells, and the PGRPs might be involved in host defence against bacterial invasion without accompanying inflammatory responses.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/biossíntese , Células Epiteliais/metabolismo , Escherichia coli/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mucosa Bucal/metabolismo , Oligopeptídeos/farmacologia , Acetilmuramil-Alanil-Isoglutamina/química , Acetilmuramil-Alanil-Isoglutamina/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/agonistas , Linhagem Celular , Citocinas/metabolismo , Diglicerídeos/química , Diglicerídeos/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/microbiologia , Escherichia coli/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/agonistas , Lipídeo A/química , Lipídeo A/farmacologia , Mucosa Bucal/citologia , Mucosa Bucal/microbiologia , Proteína Adaptadora de Sinalização NOD1 , Proteína Adaptadora de Sinalização NOD2 , Oligopeptídeos/química , Peptidoglicano/metabolismo , Ácidos Pimélicos/química , Ácidos Pimélicos/farmacologia , RNA Mensageiro/biossíntese , RNA Interferente Pequeno/genética , Receptor 2 Toll-Like/agonistas , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/agonistas , Receptor 4 Toll-Like/genética , Regulação para CimaRESUMO
The time-averaged solution conformation of a unique bis-sulfated glycolipid (HSO3)2-2,6Manalpha-2Glcalpha-1-sn-2,3-O-alkylglycerol , was studied in terms of the torsional angles of two glycosidic linkages, phi (H1-C1-O-Cx) and psi (C1-O-Cx-Hx), derived from heteronuclear three-bond coupling constants (3JC,H), and inter-residual proton-proton distances from J-HMBC 2D and ROESY experiments, respectively. The dihedral angles of Glcalpha1Gro in glycolipids were determined for the first time. The C1-C4 diagonal line of the alpha-glucose ring makes an angle of approximately 120 degrees with the glycerol backbone, suggesting that the alpha-glucose ring is almost parallel to the membrane surface in contrast with the perpendicular orientation of the beta-isomer. Furthermore, minimum-energy states around the conformation were estimated by Monte Carlo/stochastic dynamics (MCSD) mixed-mode simulations and the energy minimization with assisted model building and energy refinement (AMBER) force field. The Glcalpha1Gro linkage has a single minimum-energy structure. On the other hand, three conformers were observed for the Manalpha2Glc linkage. The flexibility of Manalpha2Glc was further confirmed by the absence of inter-residual hydrogen bonds which were judged from the temperature coefficients of the chemical shifts, ddelta/dT (-10-3 p.p.m. degrees C-1), of hydroxy protons. The conformational flexibility may facilitate interaction of extracellular substances with both sulfate groups.
Assuntos
Glicolipídeos/química , Espectroscopia de Ressonância Magnética/métodos , Simulação por Computador , Glucose/química , Ligação de Hidrogênio , Modelos Químicos , Método de Monte Carlo , Conformação Proteica , TemperaturaRESUMO
Several new para-substituted benzyl- or phenyl-type protecting groups and their application to linkers for solid-phase synthesis are described. p-Acylaminobenzyl groups have higher acid stability than the p-methoxybenzyl (MPM) group, but are readily cleaved with 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ). The p-azidobenzyl (Azb) group also has higher acid stability than the MPM group and can be removed much faster than the MPM group by DDQ oxidation after conversion of the azide group into the corresponding iminophosphorane. The acid stability of the p-azido-m-chlorobenzyl group (Cl-Azb) is higher than that of the Azb group. The former can be readily removed by DDQ oxidation after conversion of the azide group into the iminophosphorane. The p-acylaminophenyl glycoside linker can be readily obtained from p-nitrophenyl glycoside and can be readily cleaved by ammonium cerium(IV) nitrate (CAN) oxidation. This type of linker should be useful not only for the solid-phase synthesis of oligosaccharides but also for general solid-phase synthesis.