RESUMO
Positron emission tomography (PET) imaging studies in laboratory animals are almost always performed under isoflurane anesthesia to ensure that the subject stays still during the image acquisition. Isoflurane is effective, safe, and easy to use, and it is generally assumed to not have an impact on the imaging results. Motivated by marked differences observed in the brain uptake and metabolism of the PET tracer 3-[18F]fluoro-4-aminopyridine [(18F]3F4AP) between human and nonhuman primate studies, this study investigates the possible effect of isoflurane on this process. Mice received [18F]3F4AP injection while awake or under anesthesia and the tracer brain uptake and metabolism was compared between groups. A separate group of mice received the known cytochrome P450 2E1 inhibitor disulfiram prior to tracer administration. Isoflurane was found to largely abolish tracer metabolism in mice (74.8 ± 1.6 vs. 17.7 ± 1.7% plasma parent fraction, % PF) resulting in a 4.0-fold higher brain uptake in anesthetized mice at 35 min post-radiotracer administration. Similar to anesthetized mice, animals that received disulfiram showed reduced metabolism (50.0 ± 6.9% PF) and a 2.2-fold higher brain signal than control mice. The higher brain uptake and lower metabolism of [18F]3F4AP observed in anesthetized mice compared to awake mice are attributed to isoflurane's interference in the CYP2E1-mediated breakdown of the tracer, which was confirmed by reproducing the effect upon treatment with the known CYP2E1 inhibitor disulfiram. These findings underscore the critical need to examine the effect of isoflurane in PET imaging studies before translating tracers to humans that will be scanned without anesthesia.
RESUMO
A novel organomediated cleavage of benzoyl group using ethane-1,2-diamine and acetic acid under neutral condition enables an efficient synthesis of 1-(6-nitropyridin-2-yl)thiourea, which previously has been challenging to prepare by conventional methods. The successful synthesis of 1-(6-nitropyridin-2-yl)thiourea as a synthon permits development of a variety of 18F labeled heterocycles as PET imaging ligands such as N-(pyridin-2-yl)thiazol-2-amine derivatives. The utility of this synthon is demonstrated with the synthesis of a 18F-labeled PET tracer for studying prion disease. In vitro autoradiography using this PET tracer on sagittal rat brain slices showed highest accumulation of radioactivity in the hippocampus, cortex, and striatum, in accordance with reported immunostaining of PrPc in rat brain.
Assuntos
Encéfalo , Tioureia , Animais , Encéfalo/diagnóstico por imagem , Ligantes , Tomografia por Emissão de Pósitrons/métodos , RatosRESUMO
Serine proteases are fundamental components of biology, including innate immunity, which is systematically orchestrated in an orderly, balanced fashion in the healthy host. Such serine proteases are found in two well-recognized pathways of an innate immune network, coagulation and complement. Both pathways, if uncontrolled due to a variety of causes, are pathogenic in numerous diseases, including coagulation disorders and infectious diseases. Previous studies have reported sequence homologies, functional similarities and interplay between these two pathways with some implications in health and disease. The current study newly reveals that complement component factor B (Bf), the second component of the alternative complement pathway, has thrombin-like activity, which is supported by a characteristic homology of the trypsin-like domain of Bf to that of thrombin. Moreover, we newly report that the trypsin-like domain of Bf is closely related to Limulus clotting factor C, the LPS sensitive clotting factor of the innate immune system. We will also discuss potential implications of our findings in diseases.
Assuntos
Fator B do Complemento/genética , Trombina/genética , Tripsina/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Fator B do Complemento/classificação , Fator B do Complemento/metabolismo , Variação Genética , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Filogenia , Homologia de Sequência de Aminoácidos , Trombina/metabolismo , Tripsina/metabolismoRESUMO
Low-tidal volume (Vt) ventilation might protect healthy lungs from volutrauma but lead to inflammation resulting from other mechanisms, namely alveolar derecruitment and the ensuing alveolar collapse and tidal reexpansion. We hypothesized that the different mechanisms of low- and high-volume injury would be reflected in different mechanical properties being associated with development of pulmonary inflammation and mortality: an increase of hysteresis, reflecting progressive alveolar derecruitment, at low Vt; an increase of elastance, as a result of overdistension, at higher Vt. Mice were allocated to "protective" (6 ml/kg) or "injurious" (15-20 ml/kg) Vt groups and ventilated for 16 hours or until death. We measured elastance and hysteresis; pulmonary IL-6, IL-1ß, and MIP-2 (macrophage inflammatory protein 2); wet-to-dry ratio; and blood gases. Survival was greater in the protective group (60%) than in the injurious group (25%). Nonsurvivors showed increased pulmonary cytokines, particularly in the injurious group, with the increase of elastance reflecting IL-6 concentration. Survivors instead showed only modest increases of cytokines, independent of Vt and unrelated to the increase of elastance. No single lung strain threshold could discriminate survivors from nonsurvivors. Hysteresis increased faster in the protective group, but, contrary to our hypothesis, its change was inversely related to the concentration of cytokines. In this model, significant mortality associated with pulmonary inflammation occurred even for strain values as low as about 0.8. Low Vt improved survival. The accompanying increase of hysteresis was not associated with greater inflammation.
Assuntos
Interleucina-6/sangue , Pneumonia/etiologia , Respiração Artificial/métodos , Mecânica Respiratória/fisiologia , Animais , Ensaios Clínicos como Assunto , Citocinas/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Pneumonia/mortalidade , Pneumonia/fisiopatologia , Respiração Artificial/efeitos adversos , Testes de Função RespiratóriaRESUMO
OBJECTIVE: Lynch syndrome (LS), an autosomal-dominant inherited disorder, increases the risk for LS-associated cancers (LS-AC). Molecular LS assessment for all cases is referred to as universal screening (U/S) and is recommended for endometrial cancer (EC) and colorectal cancer. Lynch-like cases (LL) lack LS-pathogenic mutations despite being suspected as LS by U/S, but have been poorly investigated in EC. The aim of this study was to capture the features of LL in EC and to devise LL management in EC. METHODS: U/S, consisting of immunohistochemistry and reflex methylation analysis, was applied to 348 Asian ECs, and sporadic cancer (SC) cases were screened out. Genetic testing was offered to "suspected-LS" cases selected by U/S. The features of the LS, LL, and SC groups were recorded and compared. RESULTS: U/S screened 306 ECs as SC. The recurrence rates of suspected-LS and SC cases were 14.3% (6/42) and 26.5% (81/306), respectively. Of the 42 suspected-LS cases, 10 were identified as LS, 17 were classified as LL, and 15 did not undergo genetic testing. In the LS group, the frequency of personal history (50%) and family history (100%) of LS-AC were prominent. Of note, the prevalence of family history of LS-AC and gastric cancer was significantly higher in the LL group than in the SC group (76.5% vs. 38.6% and 47.1% vs. 25.2%, respectively). CONCLUSIONS: Herein, we report the features of LL classified by LS identification via U/S in Asian EC. LL should be candidates for tailored surveillance based on regionality and family history.
Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/diagnóstico , Neoplasias do Endométrio/diagnóstico , Adulto , Idoso , Povo Asiático/genética , Neoplasias Colorretais Hereditárias sem Polipose/genética , Neoplasias Colorretais Hereditárias sem Polipose/metabolismo , Detecção Precoce de Câncer/métodos , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Proteína 1 Homóloga a MutL/biossíntese , Proteína 1 Homóloga a MutL/genética , Recidiva Local de Neoplasia/diagnóstico , Estudos RetrospectivosRESUMO
Interfering with the assembly of Amyloid ß (Aß) peptides from monomer to oligomeric species and fibrils or promoting their clearance from the brain are targets of anti-Aß-directed therapies in Alzheimer disease. Here we demonstrate that cromolyn sodium (disodium cromoglycate), a Food and Drug Administration-approved drug already in use for the treatment of asthma, efficiently inhibits the aggregation of Aß monomers into higher-order oligomers and fibrils in vitro without affecting Aß production. In vivo, the levels of soluble Aß are decreased by over 50% after only 1 week of daily intraperitoneally administered cromolyn sodium. Additional in vivo microdialysis studies also show that this compound decreases the half-life of soluble Aß in the brain. These data suggest a clear effect of a peripherally administered, Food and Drug Administration-approved medication on Aß economy, supporting further investigation of the potential long-term efficacy of cromolyn sodium in Alzheimer disease.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Cromolina Sódica/farmacologia , Aprovação de Drogas , Fragmentos de Peptídeos/metabolismo , Animais , Células Cultivadas , Cromolina Sódica/química , Modelos Animais de Doenças , Flavonoides/química , Flavonóis , Humanos , Camundongos , Camundongos Transgênicos , Microglia/metabolismo , Microscopia Eletrônica de Transmissão , Estados Unidos , United States Food and Drug AdministrationRESUMO
The complement system is involved in mediation of joint damage in rheumatoid arthritis, with evidence suggesting activation of both the classical and alternative pathway (AP). The AP is both necessary and sufficient to mediate collagen Ab-induced arthritis, an experimental animal model of immune complex-induced joint disease. The AP in mice is dependent on MASP-1/3 cleavage of pro-factor D (pro-FD) into mature factor D (FD). The objectives of the current study were to determine the cells synthesizing MASP-1/3 and pro-FD in synovial tissue. Collagen Ab-induced arthritis was studied in wild-type C57BL/6 mice, and the localization of mRNA and protein for FD and MASP-1/3 in synovial adipose tissue (SAT) and fibroblast-like synoviocytes (FLS) was determined using various techniques, including laser capture microdissection. SAT was the sole source of mRNA for pro-FD. Cultured differentiated 3T3 adipocytes, a surrogate for SAT, produced pro-FD but no mature FD. FLS were the main source of MASP-1/3 mRNA and protein. Using cartilage microparticles (CMPs) coated with anti-collagen mAb and serum from MASP-1/3(-/-) mice as a source of factor B, pro-FD in 3T3 supernatants was cleaved into mature FD by MASP-1/3 in FLS supernatants. The mature FD was eluted from the CMP, and was not present in the supernatants from the incubation with CMP, indicating that cleavage of pro-FD into mature FD by MASP-1 occurred on the CMP. These results demonstrate that pathogenic activation of the AP can occur in the joint through immune complexes adherent to cartilage and the local production of necessary AP proteins by adipocytes and FLS.
Assuntos
Adipócitos/metabolismo , Artrite Experimental/metabolismo , Ativação do Complemento/fisiologia , Via Alternativa do Complemento/fisiologia , Fibroblastos/metabolismo , Adipócitos/imunologia , Animais , Artrite Experimental/imunologia , Artrite Experimental/patologia , Artrite Reumatoide/imunologia , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Western Blotting , Fibroblastos/imunologia , Imuno-Histoquímica , Microdissecção e Captura a Laser , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Membrana Sinovial/imunologia , Membrana Sinovial/metabolismo , Membrana Sinovial/patologiaRESUMO
Endometrial cancer (EC) rates are rising in Japan. Lymph node (LN) metastasis is an important prognostic factor in EC, and its risk is increased with higher tumor grade, deep myometrial invasion, larger tumor size, and lymphovascular space invasion (LVSI). Current methodologies to assess these factors are unreliable. We previously showed the association between C-reactive protein (CRP) 1846C>T (rs1205) polymorphism and LN metastasis in esophageal, non-small cell lung, and breast cancers. The CRP gene is located on chromosome 1q21-q23, and the polymorphism in the noncoding region (1846C>T) of this gene decreases serum CRP levels. We investigated the relationship between CRP 1846C>T genetic polymorphism and LN metastasis or LVSI in 130 EC patients using polymerase chain reaction-restriction fragment length polymorphism. The CRP 1846C/T genotype was C/C in 11 patients, C/T in 58 patients and T/T in 61 patients. The patients were divided into two groups based on their CRP 1846 genotypes: "C/C" and "C/T + T/T". Nine (7%) and 18 (13%) patients, all with the polymorphism, had LN metastasis and moderate or prominent lymphatic invasion, respectively. LN metastasis and/or severe lymphatic invasion were observed in the C/T + T/T group, while patients with the C/C genotype had no LN metastases or severe lymphatic invasion. Univariate and multivariate logistic regression models revealed that the C/T + T/T patients had a significant likelihood of developing LN metastasis and/or severe lymphatic invasion. Our results suggest that CRP genetic polymorphism is a novel risk predictor of LN metastasis and/or lymphatic invasion in EC.
Assuntos
Proteína C-Reativa/genética , Neoplasias do Endométrio/genética , Metástase Linfática/genética , Adulto , Idoso , Neoplasias do Endométrio/patologia , Feminino , Genótipo , Humanos , Metástase Linfática/patologia , Pessoa de Meia-Idade , Invasividade Neoplásica , Razão de Chances , Polimorfismo Genético/genéticaRESUMO
Type 1 diabetes is an autoimmune disease influenced by multiple genetic loci. Although more than 20 insulin-dependent diabetes (Idd) loci have been implicated in the nonobese diabetic (NOD) mouse model, few causal gene variants have been identified. Here we show that RNA interference (RNAi) can be used to probe candidate genes in this disease model. Slc11a1 encodes a phagosomal ion transporter, Nramp1, that affects resistance to intracellular pathogens and influences antigen presentation. This gene is the strongest candidate among the 42 genes in the Idd5.2 region; a naturally occurring mutation in the protective Idd5.2 haplotype results in loss of function of the Nramp1 protein. Using lentiviral transgenesis, we generated NOD mice in which Slc11a1 is silenced by RNAi. Silencing reduced the frequency of type 1 diabetes, mimicking the protective Idd5.2 region. Our results demonstrate a role for Slc11a1 in modifying susceptibility to type 1 diabetes and illustrate that RNAi can be used to study causal genes in a mammalian model organism.
Assuntos
Proteínas de Transporte de Cátions/genética , Diabetes Mellitus Tipo 1/genética , Predisposição Genética para Doença , Interferência de RNA , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos NODRESUMO
Serum antibodies and mannose-binding lectin (MBL) are important host defense factors for host adaptive and innate immunity, respectively. Antibodies and MBL also initiate the classical and lectin complement pathways, respectively, leading to opsonophagocytosis. We have shown previously that Staphylococcus aureus wall teichoic acid (WTA), a cell wall glycopolymer consisting of ribitol phosphate substituted with α- or ß-O-N-acetyl-d-glucosamine (GlcNAc) and d-alanine, is recognized by MBL and serum anti-WTA IgG. However, the exact antigenic determinants to which anti-WTA antibodies or MBL bind have not been determined. To answer this question, several S. aureus mutants, such as α-GlcNAc glycosyltransferase-deficient S. aureus ΔtarM, ß-GlcNAc glycosyltransferase-deficient ΔtarS, and ΔtarMS double mutant cells, were prepared from a laboratory and a community-associated methicillin-resistant S. aureus strain. Here, we describe the unexpected finding that ß-GlcNAc WTA-deficient ΔtarS mutant cells (which have intact α-GlcNAc) escape from anti-WTA antibody-mediated opsonophagocytosis, whereas α-GlcNAc WTA-deficient ΔtarM mutant cells (which have intact ß-GlcNAc) are efficiently engulfed by human leukocytes via anti-WTA IgG. Likewise, MBL binding in S. aureus cells was lost in the ΔtarMS double mutant but not in either single mutant. When we determined the serum concentrations of the anti-α- or anti-ß-GlcNAc-specific WTA IgGs, anti-ß-GlcNAc WTA-IgG was dominant in pooled human IgG fractions and in the intact sera of healthy adults and infants. These data demonstrate the importance of the WTA sugar conformation for human innate and adaptive immunity against S. aureus infection.
Assuntos
Anticorpos Antibacterianos/imunologia , Parede Celular/imunologia , Epitopos/imunologia , Imunoglobulina G/imunologia , Leucócitos/imunologia , Lectina de Ligação a Manose/imunologia , Fagocitose/imunologia , Staphylococcus aureus/química , Ácidos Teicoicos/imunologia , Imunidade Adaptativa/fisiologia , Adulto , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Parede Celular/química , Epitopos/química , Feminino , Humanos , Imunidade Inata/fisiologia , Lactente , Recém-Nascido , Leucócitos/microbiologia , Masculino , Lectina de Ligação a Manose/sangue , Mutação , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/imunologia , Staphylococcus aureus/enzimologia , Staphylococcus aureus/imunologia , Ácidos Teicoicos/químicaRESUMO
The complement system plays a key role in host defense against pneumococcal infection. Three different pathways, the classical, alternative and lectin pathways, mediate complement activation. While there is limited information available on the roles of the classical and the alternative activation pathways of complement in fighting streptococcal infection, little is known about the role of the lectin pathway, mainly due to the lack of appropriate experimental models of lectin pathway deficiency. We have recently established a mouse strain deficient of the lectin pathway effector enzyme mannan-binding lectin associated serine protease-2 (MASP-2) and shown that this mouse strain is unable to form the lectin pathway specific C3 and C5 convertases. Here we report that MASP-2 deficient mice (which can still activate complement via the classical pathway and the alternative pathway) are highly susceptible to pneumococcal infection and fail to opsonize Streptococcus pneumoniae in the none-immune host. This defect in complement opsonisation severely compromises pathogen clearance in the lectin pathway deficient host. Using sera from mice and humans with defined complement deficiencies, we demonstrate that mouse ficolin A, human L-ficolin, and collectin 11 in both species, but not mannan-binding lectin (MBL), are the pattern recognition molecules that drive lectin pathway activation on the surface of S. pneumoniae. We further show that pneumococcal opsonisation via the lectin pathway can proceed in the absence of C4. This study corroborates the essential function of MASP-2 in the lectin pathway and highlights the importance of MBL-independent lectin pathway activation in the host defense against pneumococci.
Assuntos
Ativação do Complemento , Imunidade Inata , Lectinas/imunologia , Serina Proteases Associadas a Proteína de Ligação a Manose/metabolismo , Infecções Pneumocócicas/imunologia , Streptococcus pneumoniae/imunologia , Animais , Colectinas/metabolismo , Complemento C4/imunologia , Humanos , Lectinas/metabolismo , Lectina de Ligação a Manose/metabolismo , Serina Proteases Associadas a Proteína de Ligação a Manose/deficiência , Serina Proteases Associadas a Proteína de Ligação a Manose/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Opsonizantes , Receptores de Reconhecimento de Padrão/metabolismo , FicolinasRESUMO
The pluripotency of embryonic stem (ES) cells is thought to be maintained by a few key transcription factors, including Oct3/4 and Sox2. The function of Oct3/4 in ES cells has been extensively characterized, but that of Sox2 has yet to be determined. Sox2 can act synergistically with Oct3/4 in vitro to activate Oct-Sox enhancers, which regulate the expression of pluripotent stem cell-specific genes, including Nanog, Oct3/4 and Sox2 itself. These findings suggest that Sox2 is required by ES cells for its Oct-Sox enhancer activity. Using inducible Sox2-null mouse ES cells, we show that Sox2 is dispensable for the activation of these Oct-Sox enhancers. In contrast, we demonstrate that Sox2 is necessary for regulating multiple transcription factors that affect Oct3/4 expression and that the forced expression of Oct3/4 rescues the pluripotency of Sox2-null ES cells. These results indicate that the essential function of Sox2 is to stabilize ES cells in a pluripotent state by maintaining the requisite level of Oct3/4 expression.
Assuntos
Diferenciação Celular/fisiologia , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Células-Tronco Pluripotentes/metabolismo , Transativadores/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Proteínas de Ligação a DNA/genética , Desenvolvimento Embrionário/fisiologia , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/genética , Proteínas de Transporte de Cátions Orgânicos/genética , Fatores de Transcrição SOXB1 , Transativadores/genética , Fatores de Transcrição/genética , Ativação Transcricional/genética , Regulação para Cima/genéticaRESUMO
There is mounting evidence indicating an important role for complement in the pathogenesis of cerebral ischemia-reperfusion injury, or ischemic stroke. The role of the alternative complement pathway in ischemic stroke has not been investigated, and there is conflicting data on the role of the terminal pathway. In this study, we show that compared with wild-type mice, mice deficient in the alternative pathway protein factor B or mice treated with the alternative pathway inhibitor CR2-fH have improved outcomes after 60-min middle cerebral artery occlusion and 24-h reperfusion. Factor B-deficient or CR2-fH-treated mice were protected in terms of improved neurologic function and reduced cerebral infarct, demyelination, P-selectin expression, neutrophil infiltration, and microthrombi formation. Mice deficient in both the classical and lectin pathways (C1q/MBL deficient) were also protected from cerebral ischemia-reperfusion injury, and there was no detectable C3d deposition in the ipsilateral brain of these mice. These data demonstrate that the alternative pathway is not alone sufficient to initiate complement activation and indicate that the alternative pathway propagates cerebral injury via amplification of the cascade. Deficiency of C6, a component of the terminal cytolytic membrane attack complex, had no effect on outcome after ischemic stroke, indicating that the membrane attack complex is not involved in mediating injury in this model. We additionally show that the protective effect of factor B deficiency and CR2-fH treatment is sustained in the subacute stage of infarct development, adding to the clinical relevance of these findings.
Assuntos
Isquemia Encefálica/imunologia , Isquemia Encefálica/patologia , Via Alternativa do Complemento/imunologia , Mediadores da Inflamação/fisiologia , Traumatismo por Reperfusão/imunologia , Traumatismo por Reperfusão/patologia , Acidente Vascular Cerebral/imunologia , Acidente Vascular Cerebral/patologia , Animais , Isquemia Encefálica/genética , Antígenos CD59/genética , Complemento C1q/deficiência , Complemento C1q/genética , Complemento C6/deficiência , Complemento C6/genética , Fator B do Complemento/deficiência , Fator B do Complemento/genética , Complexo de Ataque à Membrana do Sistema Complemento/deficiência , Complexo de Ataque à Membrana do Sistema Complemento/genética , Via Alternativa do Complemento/genética , Lectina de Ligação a Manose da Via do Complemento/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Traumatismo por Reperfusão/genética , Acidente Vascular Cerebral/genéticaRESUMO
Bleeding disorders and thrombotic complications constitute a major cause of death and disability worldwide. Although it is known that the complement and coagulation systems interact, no studies have investigated the specific role or mechanisms of lectin-mediated coagulation in vivo. FeCl(3) treatment resulted in intra-arterial occlusive thrombogenesis within 10 min in wild-type (WT) and C2/factor B-null mice. In contrast, mannose-binding lectin (MBL)-null and MBL-associated serine protease (MASP)-1/-3 knockout (KO) mice had significantly decreased FeCl(3)-induced thrombogenesis. Reconstitution with recombinant human (rh) MBL restored FeCl(3)-induced thrombogenesis in MBL-null mice to levels comparable to WT mice, suggesting a significant role of the MBL/MASP complex for in vivo coagulation. Additionally, whole blood aggregation demonstrated increased MBL/MASP complex-dependent platelet aggregation. In vitro, MBL/MASP complexes were captured on mannan-coated plates, and cleavage of a chromogenic thrombin substrate (S2238) was measured. We observed no significant differences in S2238 cleavage between WT, C2/factor B-null, MBL-A(-/-), or MBL-C(-/-) sera; however, MBL-null or MASP-1/-3 KO mouse sera demonstrated significantly decreased S2238 cleavage. rhMBL alone failed to cleave S2238, but cleavage was restored when rMASP-1 was added to either MASP-1/-3 KO sera or rhMBL. Taken together, these findings indicate that MBL/MASP complexes, and specifically MASP-1, play a key role in thrombus formation in vitro and in vivo.
Assuntos
Coagulação Sanguínea , Trombose das Artérias Carótidas/enzimologia , Lectina de Ligação a Manose da Via do Complemento , Serina Proteases Associadas a Proteína de Ligação a Manose/fisiologia , Animais , Coagulação Sanguínea/imunologia , Trombose das Artérias Carótidas/induzido quimicamente , Trombose das Artérias Carótidas/genética , Cloretos/toxicidade , Lectina de Ligação a Manose da Via do Complemento/genética , Modelos Animais de Doenças , Compostos Férricos/toxicidade , Humanos , Imunidade Inata/genética , Lectinas de Ligação a Manose/deficiência , Lectinas de Ligação a Manose/genética , Serina Proteases Associadas a Proteína de Ligação a Manose/efeitos adversos , Serina Proteases Associadas a Proteína de Ligação a Manose/deficiência , Serina Proteases Associadas a Proteína de Ligação a Manose/genética , Camundongos , Trombina/fisiologiaRESUMO
Wall teichoic acid (WTA) of Staphylococcus aureus is a major cell envelope-associated glycopolymer that is a key molecule in promoting colonization during S. aureus infection. The complement system plays a key role in the opsonization and clearance of pathogens. We recently reported that S. aureus WTA functions as a ligand of human serum mannose-binding lectin (MBL), a recognition molecule of the lectin complement pathway. Intriguingly, serum MBL in adults does not bind to WTA because of an inhibitory effect of serum anti-WTA-IgG. In this study, serum anti-WTA-IgG was purified to homogeneity using a purified S. aureus WTA-coupled affinity column to examine the biological function of human anti-WTA-IgG. The purified anti-WTA-IgG contained the IgG2 subclass as a major component and specifically induced C4 and C3 deposition on the S. aureus surface in the anti-WTA-IgG-depleted serum, but not in C1q-deficient serum. Furthermore, the anti-WTA-IgG-dependent C3 deposition induced phagocytosis of S. aureus cells by human polymorphonuclear leukocytes. These results demonstrate that serum anti-WTA-IgG is a real trigger for the induction of classical complement-dependent opsonophagocytosis against S. aureus. Our results also support the fact that a lack of the lectin complement pathway in MBL-deficient adults is compensated by Ag-specific, Ab-mediated adaptive immunity.
Assuntos
Anticorpos Antibacterianos/imunologia , Parede Celular/imunologia , Imunoglobulina G/imunologia , Neutrófilos/imunologia , Fagocitose/imunologia , Staphylococcus aureus/imunologia , Ácidos Teicoicos/imunologia , Adulto , Complexo Antígeno-Anticorpo/imunologia , Complemento C3/imunologia , Complemento C4/imunologia , Via Clássica do Complemento/imunologia , Humanos , Neutrófilos/citologiaRESUMO
The complement system, especially the alternative pathway, plays essential roles in the induction of injury in collagen Ab-induced arthritis (CAIA) in mice. The goal of the current study was to directly compare the roles of receptors for C3a and C5a, as well as the membrane attack complex, as effector mechanisms in the pathogenesis of CAIA. Clinical disease activity in C3aR(-/-), C5aR(-/-), and C6-deficient (C6-def) mice was decreased by 52, 94, and 65%, respectively, as compared with wild-type mice. Decreases in histopathologic injury as well as in IgG and C3 deposition paralleled the clinical disease activity. A decrease in the percentage of synovial neutrophils was observed in C3aR(-/-), C5aR(-/-), and C6-def mice, and a decrease in macrophages was observed in C3aR(-/-) and C5aR(-/-), but not in C6-def, mice. Synovial mRNA obtained by laser capture microdissection exhibited a decrease in TNF-α in C5aR(-/-) mice and in IL-1ß in both C5aR(-/-) and C6-def mice, whereas C3aR(-/-) mice demonstrated no change in either cytokine. Our findings show that absent C3aR-, C5aR-, or membrane attack complex-initiated effector mechanisms each decrease susceptibility to CAIA, with clinical effects most pronounced in C5aR-deficient mice. Although the absence of C3aR, C5aR, or C6 led to differential deficiencies in effector mechanisms, decreased proximal joint IgG and C3 deposition was common to all three genotypes in comparison with wild-type mice. These data suggest the existence of positive-feedback amplification pathways downstream of all three effectors that promote additional IgG deposition and C3 activation in the joint.
Assuntos
Artrite Experimental/etiologia , Complemento C6/deficiência , Receptor da Anafilatoxina C5a/fisiologia , Receptores de Complemento/fisiologia , Animais , Ativação do Complemento , Complemento C3a/imunologia , Complemento C6/fisiologia , Citocinas , Suscetibilidade a Doenças , Imunoglobulina G , Macrófagos/patologia , Camundongos , Camundongos Knockout , Neutrófilos/patologia , Líquido Sinovial/imunologiaRESUMO
The collectins have been shown to have a role in host defense against influenza A virus (IAV) and other significant viral pathogens (e.g., HIV). The ficolins are a related group of innate immune proteins that are present at relatively high concentrations in serum, but also in respiratory secretions; however, there has been little study of the role of ficolins in viral infection. In this study, we demonstrate that purified recombinant human H-ficolin and H-ficolin in human serum and bronchoalveolar lavage fluid bind to IAV and inhibit viral infectivity and hemagglutination activity in vitro. Removal of ficolins from human serum or bronchoalveolar lavage fluid reduces their antiviral activity. Inhibition of IAV did not involve the calcium-dependent lectin activity of H-ficolin. We demonstrate that H-ficolin is sialylated and that removal of sialic acid abrogates IAV inhibition, while addition of the neuraminidase inhibitor oseltamivir potentiates neutralization, hemagglutinin inhibition, and viral aggregation caused by H-ficolin. Pandemic and mouse-adapted strains of IAV are generally not inhibited by the collectins surfactant protein D or mannose binding lectin because of a paucity of glycan attachments on the hemagglutinin of these strains. In contrast, H-ficolin inhibited both the mouse-adapted PR-8 H1N1 strain and a pandemic H1N1 strain from 2009. H-ficolin also fixed complement to a surface coated with IAV. These findings suggest that H-ficolin contributes to host defense against IAV.
Assuntos
Antivirais/farmacologia , Glicoproteínas/fisiologia , Vírus da Influenza A Subtipo H3N2/imunologia , Lectinas/fisiologia , Pandemias , Estações do Ano , Replicação Viral/imunologia , Animais , Linhagem Celular Tumoral , Células Cultivadas , Testes de Fixação de Complemento , Cães , Humanos , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H1N1/patogenicidade , Vírus da Influenza A Subtipo H3N2/patogenicidade , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Camundongos , Testes de Neutralização , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Proteínas da Matriz Viral/antagonistas & inibidoresRESUMO
Collectin kidney 1 (CL-K1) is a recently identified collectin that is synthesized in most organs and circulates in blood. CL-K1 is an innate immune molecule that may play a significant role in host defense. As some collectins also play a role in coagulation, we hypothesized that an effect of CL-K1 may be apparent in disseminated intravascular coagulation (DIC), a gross derangement of the coagulation system that occurs in the setting of profound activation of the innate immune system. DIC is a grave medical condition with a high incidence of multiple organ failure and high mortality and yet there are no reliable biomarkers or risk factors. In our present study, we measured plasma CL-K1 concentration in a total of 659 specimens, including 549 DIC patients, 82 non-DIC patients and 27 healthy volunteers. The median plasma CL-K1 levels in these cohorts were 424, 238 and 245 ng/ml, respectively, with no significant difference in the latter two groups. The incidence of elevated plasma CL-K1 was significantly higher in the DIC patients compared to the non-DIC patients, resulting in an odds ratio of 1.929 (confidence interval 1.041-3.866). Infection, renal diseases, respiratory diseases, and cardiac diseases were more frequently observed in the DIC group than in the non-DIC group. In the DIC group, vascular diseases were associated with elevated plasma CL-K1 levels while age and acute illness had little effect on plasma CL-K1 levels. Independent of DIC, elevated plasma CL-K1 levels were associated with respiratory disease and coagulation disorders. These results suggest that specific diseases may affect CL-K1 synthesis in an organ dependent manner and that elevated plasma CL-K1 levels are associated with the presence of DIC. Further investigations in cohorts of patients are warranted. We propose that elevated plasma CL-K1 may be a new useful risk factor and possibly biomarker for the prediction of developing DIC.
Assuntos
Colectinas/sangue , Coagulação Intravascular Disseminada/sangue , Fatores Etários , Idoso , Biomarcadores/sangue , Transtornos da Coagulação Sanguínea/sangue , Estudos de Coortes , Humanos , Masculino , Pessoa de Meia-Idade , Doenças Respiratórias/sangue , Fatores de RiscoRESUMO
Spinal cord injuries (SCI) often lead to lifelong disability. Among the various types of injuries, incomplete and discomplete injuries, where some axons remain intact, offer potential for recovery. However, demyelination of these spared axons can worsen disability. Demyelination is a reversible phenomenon, and drugs like 4-aminopyridine (4AP), which target K+ channels in demyelinated axons, show that conduction can be restored. Yet, accurately assessing and monitoring demyelination post-SCI remains challenging due to the lack of suitable imaging methods. In this study, we introduce a novel approach utilizing the positron emission tomography (PET) tracer, [ 18 F]3F4AP, specifically targeting K+ channels in demyelinated axons for SCI imaging. Rats with incomplete contusion injuries were imaged up to one month post-injury, revealing [ 18 F]3F4AP's exceptional sensitivity to injury and its ability to detect temporal changes. Further validation through autoradiography and immunohistochemistry confirmed [ 18 F]3F4AP's targeting of demyelinated axons. In a proof-of-concept study involving human subjects, [ 18 F]3F4AP differentiated between a severe and a largely recovered incomplete injury, indicating axonal loss and demyelination, respectively. Moreover, alterations in tracer delivery were evident on dynamic PET images, suggestive of differences in spinal cord blood flow between the injuries. In conclusion, [ 18 F]3F4AP demonstrates efficacy in detecting incomplete SCI in both animal models and humans. The potential for monitoring post-SCI demyelination changes and response to therapy underscores the utility of [ 18 F]3F4AP in advancing our understanding and management of spinal cord injuries.
RESUMO
Innate immunity is the earliest response to invading microbes and acts to contain infection in the first minutes to hours of challenge. Unlike adaptive immunity that relies upon clonal expansion of cells that emerge days after antigenic challenge, the innate immune response is immediate. Soluble mediators, including complement components and the mannose binding lectin (MBL) make an important contribution to innate immune protection and work along with epithelial barriers, cellular defenses such as phagocytosis, and pattern-recognition receptors that trigger pro-inflammatory signaling cascades. These four aspects of the innate immune system act in concert to protect from pathogen invasion. Our work has focused on understanding the protection provided by this complex defense system and, as discussed in this review, the particular contribution of soluble mediators such as MBL and phagocytic cells. Over the past two decades both human epidemiological data and mouse models have indicated that MBL plays a critical role in innate immune protection against a number of pathogens. As demonstrated by our recent in vitro work, we show that MBL and the innate immune signaling triggered by the canonical pattern-recognition receptors (PRRs), the Toll-like receptors (TLRs), are linked by their spatial localization to the phagosome. These observations demonstrated a novel role for MBL as a TLR co-receptor and establishes a new paradigm for the role of opsonins, which we propose to function not only to increase microbial uptake but also to spatially coordinate, amplify, and synchronize innate immune defenses mechanism. In this review we discuss both the attributes of MBL that make it a unique soluble pattern recognition molecule and also highlight its broader role in coordinating innate immune activation.