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1.
Pediatr Dev Pathol ; 22(5): 431-439, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30922166

RESUMO

INTRODUCTION: In pregnancy, the presence of preeclampsia (PEC), systemic lupus erythematosus (SLE), and/or antiphospholipid antibody syndrome (APLS) is characterized by poor obstetric outcomes, with potential adverse effects for both mother and fetus. Although the histopathologic changes observed in these entities have been well established, the pathogenic mediators associated with tissue injury are poorly understood. METHODS: Forty placentas were evaluated, including 10 patients with preeclampsia, 9 with SLE, 11 with APLS, and 10 disease-free controls. Each case was subjected to a panel of immunohistochemical markers including C3b, C4d, Annexin A5, and C5b-9. Staining was graded on intensity and distribution. RESULTS: C4d staining was distinctly different among disease groups and controls. Moreover, 6/10 PEC cases, 3/9 SLE cases, and 4/11 APLS cases showed at least focal staining for C4d. All controls were negative. Annexin A5 (AnxA5) staining showed intrinsic variability in all disease groups, while 10/10 controls showed diffuse, strong staining (2+ or 3+). C3b staining was heterogeneous among groups. DISCUSSION: Previously, antiphospholipid antibody (aPLA)-associated pregnancy complications have been thought to be a consequence of a unique aPLA-mediated pathogenic mechanism. However, the immunohistochemical similarity (increased complement and decreased AnxA5 staining) observed in placentas from patients with APLS, PEC, and SLE suggests that aPLA-associated pregnancy complications may reflect a more general autoimmune mechanism.


Assuntos
Síndrome Antifosfolipídica , Lúpus Eritematoso Sistêmico , Placenta/patologia , Pré-Eclâmpsia , Complicações na Gravidez/imunologia , Anexina A5/análise , Anexina A5/biossíntese , Complemento C3b/análise , Complemento C3b/biossíntese , Complemento C4b/análise , Complemento C4b/biossíntese , Complexo de Ataque à Membrana do Sistema Complemento/análise , Complexo de Ataque à Membrana do Sistema Complemento/biossíntese , Feminino , Humanos , Imuno-Histoquímica , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/biossíntese , Gravidez , Estudos Retrospectivos
2.
Mol Immunol ; 90: 227-238, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28843904

RESUMO

The complement system not only plays a critical role in efficient detection and clearance of bacteria, but also in intestinal immune homeostasis as mice deficient for key complement components display enhanced intestinal inflammation upon experimental colitis. Because underlying molecular mechanisms for this observation are unclear, we investigated the crosstalk between intestinal epithelial cells (IEC), bacteria and the complement system in the course of chronic colitis. Surprisingly, mouse intestinal epithelial cell lines constitutively express high mRNA levels of complement component 3 (C3), Toll-like receptor 2 (Tlr2) and Tlr4. Stimulation of these cells with lipopolysaccharide (LPS), but not with flagellin, LD-muramyldipeptide or peptidoglycan, triggered increased C3 expression, secretion and activation. Stimulation of the C3aR on these cell lines with C3a resulted in an increase of LPS-triggered pro-inflammatory response. Tissue biopsies from C57BL/6J mice revealed higher expression of C3, Tlr1, Tlr2 and Tlr4 in colonic primary IECs (pIECs) compared to ileal pIECs, while in germ-free mice no differences in C3 protein expression was observed. In DSS-induced chronic colitis mouse models, C3 mRNA expression was upregulated in colonic biopsies and ileal pIECs with elevated C3 protein in the lamina propria, IECs and the mucus. Notably, increased C3b opsonization of mucosa-attached bacteria and decreased fecal full-length C3 protein was observed in DSS-treated compared to untreated mice. Of significant interest, non-inflamed and inflamed colonic biopsy samples from CD but not UC patients displayed exacerbated C3 expression compared to controls. These findings suggest that a novel TLR4-C3 axis could control the intestinal immune response during chronic colitis.


Assuntos
Colite Ulcerativa/patologia , Complemento C3a/biossíntese , Complemento C3b/biossíntese , Células Epiteliais/metabolismo , Mucosa Intestinal/patologia , Animais , Bactérias/imunologia , Linhagem Celular , Colite Ulcerativa/induzido quimicamente , Complemento C3a/metabolismo , Complemento C3b/metabolismo , Sulfato de Dextrana/toxicidade , Humanos , Inflamação/patologia , Mucosa Intestinal/imunologia , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/imunologia , Receptor 1 Toll-Like/biossíntese , Receptor 2 Toll-Like/biossíntese , Receptor 4 Toll-Like/biossíntese
3.
J Immunol ; 196(1): 385-94, 2016 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-26608922

RESUMO

The group B Streptococcus (GBS) is a leading cause of neonatal invasive disease. GBS bacteria are surrounded by a thick capsular polysaccharide that is a potent inhibitor of complement deposition via the alternative pathway. Several of its surface molecules can however activate the classical and lectin complement pathways, rendering this species still vulnerable to phagocytic killing. In this study we have identified a novel secreted protein named complement interfering protein (CIP) that downregulates complement activation via the classical and lectin pathways, but not the alternative pathway. The CIP protein showed high affinity toward C4b and inhibited its interaction with C2, presumably preventing the formation of the C4bC2a convertase. Addition of recombinant CIP to GBS cip-negative bacteria resulted in decreased deposition of C3b on their surface and in diminished phagocytic killing in a whole-blood assay. Our data reveal a novel strategy exploited by GBS to counteract innate immunity and could be valuable for the development of anti-infective agents against this important pathogen.


Assuntos
Proteínas de Bactérias/imunologia , Complemento C4b/imunologia , Via Clássica do Complemento/imunologia , Lectina de Ligação a Manose da Via do Complemento/imunologia , Evasão da Resposta Imune/imunologia , Streptococcus agalactiae/imunologia , Adulto , Sequência de Aminoácidos , Ativação do Complemento/imunologia , Complemento C3b/biossíntese , Complemento C3b/imunologia , Via Alternativa do Complemento/imunologia , Via Clássica do Complemento/efeitos dos fármacos , Lectina de Ligação a Manose da Via do Complemento/efeitos dos fármacos , Humanos , Imunidade Inata , Dados de Sequência Molecular , Fagocitose/imunologia , Ligação Proteica/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Streptococcus agalactiae/genética , Streptococcus agalactiae/metabolismo
4.
PLoS One ; 9(7): e101422, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24983375

RESUMO

We have previously reported that in vitro HCV infection of cells of hepatocyte origin attenuates complement system at multiple steps, and attenuation also occurs in chronically HCV infected liver, irrespective of the disease stage. However, none of these regulations alone completely impaired complement pathways. Modulation of the upstream proteins involved in proteolytic processing of the complement cascade prior to convertase formation is critical in promoting the function of the complement system in response to infection. Here, we examined the regulation of C2 complement expression in hepatoma cells infected in vitro with cell culture grown virus, and validated our observations using randomly selected chronically HCV infected patient liver biopsy specimens. C2 mRNA expression was significantly inhibited, and classical C3 convertase (C4b2a) decreased. In separate experiments for C3 convertase function, C3b deposition onto bacterial membrane was reduced using HCV infected patient sera as compared to uninfected control, suggesting impaired C3 convertase. Further, iC3b level, a proteolytically inactive form of C3b, was lower in HCV infected patient sera, reflecting impairment of both C3 convertase and Factor I activity. The expression level of Factor I was significantly reduced in HCV infected liver biopsy specimens, while Factor H level remained unchanged or enhanced. Together, these results suggested that inhibition of C3 convertase activity is an additional cumulative effect for attenuation of complement system adopted by HCV for weakening innate immune response.


Assuntos
Complemento C2/biossíntese , Convertases de Complemento C3-C5/metabolismo , Complemento C3b/biossíntese , Fator H do Complemento/biossíntese , Regulação da Expressão Gênica , Hepacivirus , Hepatite C Crônica/metabolismo , Imunidade Inata , Fígado/metabolismo , Feminino , Hepatite C Crônica/patologia , Humanos , Fígado/patologia , Fígado/virologia , Masculino , RNA Mensageiro/biossíntese
5.
J Immunol ; 191(4): 1827-34, 2013 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-23833239

RESUMO

We examine whether complement factor C3 or C5 is synthesized by human skin-derived mast cells and whether their synthesis is regulated by cytokines. C3 and C5 mRNAs were assessed by RT-PCR, and proteins by flow cytometry, confocal microscopy, Western blotting, and ELISA. C3 and C5 mRNAs were each expressed, and baseline protein levels/10(6) cultured mast cells were 0.9 and 0.8 ng, respectively, and located in the cytoplasm outside of secretory granules. C3 accumulated in mast cell culture medium over time and by 3 d reached a concentration of 9.4 ± 8.0 ng/ml, whereas C5 levels were not detectable (<0.15 ng/ml). Three-day incubations of mast cells with IL-1α, IL-1ß, IL-17, IFN-γ, IL-6, or anti-FcεRI did not affect C3 protein levels in culture medium, whereas incubations with PMA, TNF-α, IL-13, or IL-4 enhanced levels of C3 1.7- to 3.3-fold. In contrast with C3, levels of C5 remained undetectable. Importantly, treatment with TNF-α together with either IL-4 or IL-13 synergistically enhanced C3 (but not C5) production in culture medium by 9.8- or 7.1-fold, respectively. This synergy was blocked by attenuating the TNF-α pathway with neutralizing anti-TNF-α Ab, soluble TNFR, or an inhibitor of NF-κB, or by attenuating the IL-4/13 pathway with Jak family or Erk antagonists. Inhibitors of PI3K, Jnk, and p38 MAPK did not affect this synergy. Thus, human mast cells can produce and secrete C3, whereas ß-tryptase can act on C3 to generate C3a and C3b, raising the likelihood that mast cells engage complement to modulate immunity and inflammation in vivo.


Assuntos
Complemento C3/biossíntese , Complemento C5/biossíntese , Mastócitos/metabolismo , Células Cultivadas , Complemento C3/genética , Complemento C3/metabolismo , Complemento C3a/biossíntese , Complemento C3b/biossíntese , Complemento C5/genética , Meios de Cultivo Condicionados/química , Sinergismo Farmacológico , Humanos , Interleucinas/farmacologia , Pulmão/citologia , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Pele/citologia , Acetato de Tetradecanoilforbol/farmacologia , Fatores de Tempo , Triptases/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/farmacologia , Células U937
6.
J Immunol ; 188(4): 2030-7, 2012 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-22250080

RESUMO

Atypical hemolytic uremic syndrome has been associated with dysregulation of the alternative complement pathway. In this study, a novel heterozygous C3 mutation was identified in a factor B-binding region in exon 41, V1636A (4973 T > C). The mutation was found in three family members affected with late-onset atypical hemolytic uremic syndrome and symptoms of glomerulonephritis. All three patients exhibited increased complement activation detected by decreased C3 levels and glomerular C3 deposits. Platelets from two of the patients had C3 and C9 deposits on the cell surface. Patient sera exhibited more C3 cleavage and higher levels of C3a. The C3 mutation resulted in increased C3 binding to factor B and increased net formation of the C3 convertase, even after decay induced by decay-accelerating factor and factor H, as assayed by surface plasmon resonance. Patient sera incubated with washed human platelets induced more C3 and C9 deposition on the cell surface in comparison with normal sera. More C3a was released into serum over time when washed platelets were exposed to patient sera. Results regarding C3 and C9 deposition on washed platelets were confirmed using purified patient C3 in C3-depleted serum. The results indicated enhanced convertase formation leading to increased complement activation on cell surfaces. Previously described C3 mutations showed loss of function with regard to C3 binding to complement regulators. To our knowledge, this study presents the first known C3 mutation inducing increased formation of the C3 convertase, thus explaining enhanced activation of the alternative pathway of complement.


Assuntos
Convertases de Complemento C3-C5/biossíntese , Complemento C3a/genética , Complemento C3a/metabolismo , Complemento C3b/genética , Complemento C3b/metabolismo , Síndrome Hemolítico-Urêmica/imunologia , Animais , Síndrome Hemolítico-Urêmica Atípica , Células COS , Chlorocebus aethiops , Ativação do Complemento , Convertases de Complemento C3-C5/genética , Convertases de Complemento C3-C5/metabolismo , Complemento C3a/biossíntese , Complemento C3b/biossíntese , Complemento C9/biossíntese , Complemento C9/metabolismo , Fator B do Complemento/metabolismo , Fator H do Complemento/metabolismo , Via Alternativa do Complemento/genética , Glomerulonefrite/genética , Síndrome Hemolítico-Urêmica/genética , Humanos , Masculino , Pessoa de Meia-Idade
7.
Med Princ Pract ; 20(6): 581-3, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21986021

RESUMO

OBJECTIVE: To investigate the activation of different complement pathways in myasthenia gravis (MG) subtypes. SUBJECTS AND METHODS: Levels of complement breakdown products for different complement pathways were measured using ELISA in sera of acetylcholine receptor antibody (AChR-Ab)-positive (n = 21), muscle-specific receptor tyrosine kinase (MuSK)-Ab-positive (n = 23) and seronegative generalized MG patients (n = 21) and healthy controls (n = 22). Levels of factor Bb (FBb), the breakdown product of factor B, and C4d, the breakdown product of C4, were measured to evaluate the activity of the alternative and classical complement pathways, respectively. Serum iC3b levels were analyzed to assess total complement activity. The results were expressed as OD values. RESULTS: MuSK-Ab-positive MG patients had a significantly higher mean concentration of serum FBb (0.638) than other MG subtypes (0.446 for AChR-Ab-positive, 0.537 for seronegative MG patients) and healthy controls (0.434) (p = 0.045). Mean serum iC3b (1.549-1.780) and C4d (0.364-0.395) levels were comparable among the groups. CONCLUSION: Our results suggest that MuSK-Ab-positive MG patients might have a complement-activating serum factor and the alternative complement pathway might be involved in the pathogenesis of the disease.


Assuntos
Ativação do Complemento , Proteínas do Sistema Complemento/biossíntese , Miastenia Gravis/patologia , Receptores Colinérgicos , Adolescente , Adulto , Idoso , Análise de Variância , Reações Antígeno-Anticorpo , Autoanticorpos/biossíntese , Autoanticorpos/imunologia , Autoanticorpos/metabolismo , Estudos de Casos e Controles , Criança , Complemento C3b/biossíntese , Complemento C3b/imunologia , Complemento C3b/metabolismo , Complemento C4a/imunologia , Complemento C4a/metabolismo , Fator B do Complemento/biossíntese , Fator B do Complemento/metabolismo , Proteínas do Sistema Complemento/imunologia , Proteínas do Sistema Complemento/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/imunologia , Músculo Esquelético/metabolismo , Miastenia Gravis/imunologia , Receptores Proteína Tirosina Quinases , Adulto Jovem
8.
Cancer Biol Ther ; 10(3): 232-41, 2010 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-20574151

RESUMO

MicroRNAs (miRNAs), non-coding RNAs that function as post-transcriptional gene regulators, play a pivotal role in cancer development. In the present study, we elucidated the roles of miR-520b and miR-520e in breast cancer cells. We examined the expression levels of miR-520b and miR-520e in the immortalized breast cell line, HBL-100, and in three breast cancer cell lines: MCF-7, LM-MCF-7 and MDA-MB-231. We show the expression levels of miR-520b and miR-520e in the breast cancer cell lines were lower than that in the HBL-100 cells. Furthermore, the breast cancer cell lines showed less sensitivity to complement-dependent cytotoxicity (CDC). We found that overexpression of miR-520b and miR-520e increases the sensitivity of the breast cancer cells to CDC, whereas further suppression of miR-520b and miR-520e decreases the sensitivity of the breast cancer cells to CDC. We then demonstrate that miR-520b and miR-520e are able to directly target the 3'untranslated regions (3'UTR) of the membrane-bound complement regulatory protein CD46; suggesting that miR-520b and miR-520e down-regulate CD46 at post-transcriptional level. Enzyme-linked immunosorbent assay (ELISA) showed that overexpression of miR-520b and miR-520e results in the increased expression of C3b, which is mediated by downregulated CD46. These results suggest that miRNA-520b and miR-520e mediated down-regulation of CD46 induces opsonization of cancer cells via an alternative pathway resulting in complement activation. Thus, we conclude that miR-520b and miR-520e contribute to CDC in breast cancer cells via directly targeting the 3'UTR of CD46.


Assuntos
Regiões 3' não Traduzidas , Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Complemento C3b/imunologia , Proteína Cofatora de Membrana/biossíntese , MicroRNAs/biossíntese , Sequência de Bases , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Complemento C3b/biossíntese , Citotoxicidade Imunológica , Regulação para Baixo , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Proteína Cofatora de Membrana/genética , Proteína Cofatora de Membrana/imunologia , MicroRNAs/genética , MicroRNAs/imunologia , Transfecção
9.
Immunobiology ; 215(12): 949-55, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20334949

RESUMO

Equine recurrent uveitis serves as a spontaneous model for human autoimmune uveitis. Unpredictable relapses and ongoing inflammation in the eyes of diseased horses as well as in humans lead to destruction of the retina and finally result in blindness. However, the molecular mechanisms leading to inflammation and retinal degeneration are not well understood. An initial screening for differentially regulated proteins in sera of uveitic cases compared to healthy controls revealed an increase of the alternative pathway complement component factor B in ERU cases. To determine the activation status of the complement system, sera were subsequently examined for complement split products. We could demonstrate a significant higher concentration of the activation products B/Ba, B/Bb, Bb neoantigen, iC3b and C3d in uveitic condition compared to healthy controls, whereas for C5b-9 no differences were detected. Additionally, we investigated complement activation directly in the retina by immunohistochemistry, since it is the main target organ of this autoimmune disease. Interestingly, infiltrating cells co-expressed activated factor Bb neoantigen, complement split product C3d as well as CD68, a macrophage marker. In this study, we could demonstrate activation of the complement system both systemically as well as in the eye, the target organ of spontaneous recurrent uveitis. Based on these novel findings, we postulate a novel role for macrophages in connection with complement synthesis at the site of inflammation.


Assuntos
Doenças Autoimunes/metabolismo , Fator B do Complemento/biossíntese , Doenças dos Cavalos/metabolismo , Uveíte/metabolismo , Animais , Antígenos CD/biossíntese , Antígenos de Diferenciação Mielomonocítica/biossíntese , Doenças Autoimunes/sangue , Doenças Autoimunes/diagnóstico , Ativação do Complemento , Complemento C3b/biossíntese , Complemento C3d/biossíntese , Complexo de Ataque à Membrana do Sistema Complemento/biossíntese , Modelos Animais de Doenças , Eletroforese em Gel Bidimensional , Feminino , Doenças dos Cavalos/sangue , Doenças dos Cavalos/diagnóstico , Cavalos , Humanos , Imuno-Histoquímica , Macrófagos/metabolismo , Masculino , Retina/imunologia , Retina/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Uveíte/sangue , Uveíte/diagnóstico
10.
Invest Ophthalmol Vis Sci ; 50(3): 1392-9, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19029031

RESUMO

PURPOSE: Studies implicate activation of complement among the processes involved in the pathogenesis of age-related macular degeneration (AMD). Questions pertain to the trigger(s) responsible for the complement-associated events. The authors previously reported that photooxidation products of A2E can activate complement. Here they have further explored these events. METHODS: In vitro assays using human serum as a source of complement were used, and the C3 split product iC3b was measured by enzyme immunoassay. Serum was placed in contact with ARPE-19 cells and polarized human fetal retinal pigment epithelium that had accumulated A2E and were irradiated (430 nm). Serum was also incubated in wells precoated with bisretinoid pigments of lipofuscin and their oxidized forms. iC3b generation in normal human serum (NHS) was compared with that in factor B-depleted and C1q-depleted human serum. RESULTS: iC3b levels were elevated in NHS placed in contact with A2E-laden retinal pigment epithelium that were irradiated to generate A2E photooxidation products. iC3b was also increased in serum incubated in wells precoated with peroxy-A2E, the lipofuscin pigment all-trans-retinal dimer, and oxidized forms of all-trans-retinal dimer. Substitution of NHS with factor B-depleted sera abrogated these increases in iC3b. Complement activation was also suppressed by the addition of C-reactive protein and by a C3 cleavage inhibitor. CONCLUSIONS: The authors suggest that bisretinoid pigments of retinal pigment epithelial lipofuscin, subsequent to photoactivation and cleavage, serve to activate complement. Complement activation by this mechanism is dependent on the alternative pathway and can be modulated by an inhibitor of C3 cleavage. These events in the setting of complement dysregulation could contribute to the chronic inflammation that underlies AMD pathogenesis.


Assuntos
Ativação do Complemento/fisiologia , Complemento C3b/biossíntese , Lipofuscina/metabolismo , Compostos de Piridínio/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Retinoides/metabolismo , Apoptose/efeitos dos fármacos , Proteína C-Reativa/farmacologia , Células Cultivadas , Complemento C3b/antagonistas & inibidores , Proteínas Inativadoras do Complemento C3b/farmacologia , Via Alternativa do Complemento/fisiologia , Etoposídeo/farmacologia , Humanos , Técnicas Imunoenzimáticas , Lipofuscina/química , Oxirredução , Peptídeos Cíclicos/farmacologia , Compostos de Piridínio/química , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/embriologia , Retinoides/química
11.
J Immunol ; 181(12): 8727-34, 2008 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-19050293

RESUMO

Complement activation products are elevated in the cerebrospinal fluid and spinal cord of patients with amyotrophic lateral sclerosis (ALS). In this study, we demonstrate complement system involvement in a rodent model of ALS (human SOD1(G93A) transgenic rats). With end-stage disease, SOD1(G93A) rats displayed marked deposition of C3/C3b, and a significant up-regulation of the C5aR in the lumbar spinal cord. This was associated with increased numbers of C5aR-positive astrocytes. However, expression of C5L2, the alternative receptor for C5a, was highest on motor neurons early in the disease process. To determine the contribution of C5a to the pathology displayed by this model of ALS, rats were administered an orally active, selective C5aR antagonist (PMX205; 1 mg/kg/day, oral). Animals treated with PMX205 displayed a significant extension of survival time and a reduction in end-stage motor scores, as compared with vehicle-treated rats. PMX205-treated animals also displayed reduced levels of astroglial proliferation in the lumbar spinal cord. This study provides the first demonstration of an involvement of C5a in an ALS model and suggests that inhibitors of complement activation could be beneficial in the treatment of this neurodegenerative disease.


Assuntos
Esclerose Lateral Amiotrófica/imunologia , Esclerose Lateral Amiotrófica/patologia , Complemento C5a/fisiologia , Sequência de Aminoácidos , Esclerose Lateral Amiotrófica/tratamento farmacológico , Animais , Complemento C3/antagonistas & inibidores , Complemento C3/biossíntese , Complemento C3b/biossíntese , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Dados de Sequência Molecular , Peptídeos Cíclicos/uso terapêutico , Ratos , Ratos Transgênicos , Receptor da Anafilatoxina C5a/antagonistas & inibidores , Receptor da Anafilatoxina C5a/biossíntese , Medula Espinal/imunologia , Medula Espinal/metabolismo , Medula Espinal/patologia , Superóxido Dismutase/genética , Regulação para Cima/genética , Regulação para Cima/imunologia
12.
Proc Natl Acad Sci U S A ; 103(44): 16182-7, 2006 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-17060630

RESUMO

Recent studies have implicated local inflammation and activation of complement amongst the processes involved in the pathogenesis of age-related macular degeneration (AMD). Several lines of investigation also indicate that bis-retinoid pigments, such as A2E, that accumulate as lipofuscin in retinal pigment epithelial (RPE) cells, contribute to the disease process. In an investigation of a potential trigger for complement activation in AMD, we explored the notion that the complex mixture of products resulting from photooxidation of A2E might include a range of fragments that could be recognized by the complement system as "foreign" and that could serve to activate the complement system, leading to low-grade inflammation. To this end, we established an in vitro assay by using human serum as a source of complement, and we measured products of C3 activation by enzyme immunoassay. Accordingly, we found that the C3 split products inactivated C3b (iC3b) and C3a were elevated in serum, overlying ARPE-19 cells that had accumulated A2E and were irradiated to induce A2E photooxidation. Precoating of microtiter plates with two species of oxidized A2E, peroxy-A2E, and furano-A2E, followed by incubation with serum, also activated complement. We suggest that products of the photooxidation of bis-retinoid lipofuscin pigments in RPE cells could serve as a trigger for the complement system, a trigger than would predispose the macula to disease and that, over time, could contribute to chronic inflammation. These findings link four factors that have been posited as being associated with AMD: inflammation, oxidative damage, drusen, and RPE lipofuscin.


Assuntos
Complemento C3a/biossíntese , Complemento C3b/biossíntese , Lipofuscina/metabolismo , Compostos de Piridínio/metabolismo , Pigmentos da Retina/metabolismo , Retinoides/metabolismo , Linhagem Celular , Complemento C3b/química , Epitélio/química , Epitélio/metabolismo , Furanos/metabolismo , Humanos , Lipofuscina/química , Espectrometria de Massas , Estrutura Molecular , Oxirredução , Fotoquímica , Compostos de Piridínio/química , Pigmentos da Retina/química , Retinoides/química
13.
Vaccine ; 23(3): 329-35, 2004 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-15530677

RESUMO

Complement component C3, which plays an important role in both the innate and adaptative immune response, is present at low level in human infants. We show here that: (i) serum C3 amount is weak also in infant mice, (ii) these young animals fail to upregulate C3 to adult levels following tetanus toxoid immunization, (iii) neonatal macrophages have a limited capacity to synthesize C3 upon LPS exposure, (iv) conjugation of antigen to C3b significantly enhances antibody response elicited in 1-week-old mice--although it does not increase primary IgG response in adult mice. Altogether, this identifies C3 as one of the factors limiting early life antibody response and emphasizes the potential interest of immunization strategies overcoming this limitation.


Assuntos
Complemento C3b/imunologia , Imunoglobulina G/sangue , Ovalbumina/imunologia , Fatores Etários , Animais , Animais Recém-Nascidos , Células Cultivadas , Complemento C3b/biossíntese , Feminino , Humanos , Esquemas de Imunização , Lipopolissacarídeos/farmacologia , Macrófagos Peritoneais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Especificidade da Espécie , Toxoide Tetânico/imunologia , Regulação para Cima
14.
Curr Opin Lipidol ; 14(5): 477-82, 2003 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-14501586

RESUMO

PURPOSE OF REVIEW: Atherosclerosis is characterized by a strong inflammatory component. One factor contributing to inflammation in the arterial intima is the complement system. Here we summarize recent progress in the field of complement research on atherogenesis. RECENT FINDINGS: The complement system is activated in human atherosclerotic lesions and is actively regulated by the local synthesis of complement components and of complement regulatory proteins. Potential triggers of complement activation in the arterial intima include immunocomplexes, C-reactive protein, modified lipoproteins, apoptotic cells, and cholesterol crystals. Complement activation releases anaphylatoxins, and anaphylatoxin receptors have been identified in human atherosclerotic lesions. However, experiments on genetically engineered mice with severe hyperlipidemia have been unable to show a major role for complement in experimental atherogenesis. SUMMARY: In humans there is extensive circumstantial evidence for a role of complement in atherosclerosis, which is somewhat contradictory to recent modest or negative findings in atherosclerosis-prone genetically engineered hyperlipidemic mice.


Assuntos
Arteriosclerose/imunologia , Ativação do Complemento/fisiologia , Animais , Arteriosclerose/patologia , Proteína C-Reativa/metabolismo , Colesterol/metabolismo , Complemento C3b/biossíntese , Complemento C3b/imunologia , Complexo de Ataque à Membrana do Sistema Complemento/biossíntese , Complexo de Ataque à Membrana do Sistema Complemento/imunologia , Proteínas do Sistema Complemento/biossíntese , Humanos , Inflamação/imunologia , Inflamação/patologia , Lipoproteínas/metabolismo , Modelos Animais , Túnica Íntima/imunologia , Túnica Íntima/patologia
15.
J Biol Chem ; 277(51): 49782-90, 2002 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-12388551

RESUMO

Chromosome 13 dementias, familial British dementia (FBD) and familial Danish dementia (FDD), are associated with neurodegeneration and cerebrovascular amyloidosis, with striking neuropathological similarities to Alzheimer's disease (AD). Despite the structural differences among the amyloid subunits (ABri in FBD, ADan in FDD, and Abeta in AD), these disorders are all characterized by the presence of neurofibrillary tangles and parenchymal and vascular amyloid deposits co-localizing with markers of glial activation, suggestive of local inflammation. Proteins of the complement system and their pro-inflammatory activation products are among the inflammation markers associated with AD lesions. Immunohistochemistry of FBD and FDD brain sections demonstrated the presence of complement activation components of the classical and alternative pathways as well as the neo-epitope of the membrane attack complex. Hemolytic experiments and enzyme-linked immunosorbent assays specific for the activation products iC3b, C4d, Bb, and C5b-9 indicated that ABri and ADan are able to fully activate the complement cascade at levels comparable to those generated by Abeta1-42. ABri and ADan specifically bound C1q with high affinity and formed stable complexes in physiological conditions. Activation proceeds approximately 70-75% through the classical pathway while only approximately 25-30% seems to occur through the alternative pathway. The data suggest that the chronic inflammatory response generated by the amyloid peptides in vivo might be a contributing factor for the pathogenesis of FBD and FDD and, in more general terms, to other neurodegenerative conditions.


Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Cromossomos Humanos Par 13 , Ativação do Complemento , Complemento C4b , Demência/genética , Demência/metabolismo , Sequência de Aminoácidos , Amiloide/biossíntese , Amiloide/química , Sítios de Ligação , Western Blotting , Encéfalo/metabolismo , Cromatografia Líquida de Alta Pressão , C3 Convertase da Via Alternativa do Complemento , Complemento C3b/biossíntese , Complemento C4/biossíntese , Complexo de Ataque à Membrana do Sistema Complemento/biossíntese , Demência/diagnóstico , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Epitopos , Hemólise , Humanos , Imuno-Histoquímica , Dados de Sequência Molecular , Fragmentos de Peptídeos/biossíntese , Peptídeos/química , Ligação Proteica , Homologia de Sequência de Aminoácidos
16.
J Immunol Methods ; 258(1-2): 97-109, 2001 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-11684127

RESUMO

CD21 (complement receptor 2, CR2) binds the terminal proteolytic fragments of the third component of complement (C3) that have been covalently attached to immune complexes or other targets during the activation of complement. We used the technique of in vivo biotinylation to create a recombinant multivalent ligand for CD21. A sequence coding for a biotinylation signal peptide was added to the 3' end of the human C3dg cDNA. The modified C3dg was expressed in Escherichia coli and biotinylated intracellularly by the bacterial biotin holoenzyme synthetase (BirA) enzyme. Monomeric C3dg was unable to bind to CD21 as determined by flow cytometry, while biotinylated recombinant C3dg (rC3dg) complexed with fluorochrome-conjugated streptavidin bound tightly. Binding was observed using CD21 positive B cells but not seen on pre-B cells that do not express this complement receptor. Two assays were used to assess the functional capacity of the recombinant C3dg. First, multimeric C3dg caused the phosphorylation of the mitogen-activated kinase, p38, in mature B lymphoma cells. Second, C3dg greatly enhanced the activation of primary B cells in combination with a sub-stimulatory concentration of anti-IgM monoclonal antibody. These results illustrate the utility of the technique of in vivo biotinylation to generate ligands for cell surface receptors that require multimerization for high avidity binding and function.


Assuntos
Complemento C3b/biossíntese , Fragmentos de Peptídeos/biossíntese , Receptores de Complemento 3d/metabolismo , Sequência de Aminoácidos , Linfócitos B/imunologia , Sequência de Bases , Biotina , Linhagem Celular , Complemento C3b/química , Complemento C3b/genética , Complemento C3b/metabolismo , DNA Complementar/genética , Escherichia coli/genética , Humanos , Técnicas In Vitro , Ligantes , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Engenharia de Proteínas , Sinais Direcionadores de Proteínas/genética , Estrutura Quaternária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais
17.
Transplantation ; 67(2): 253-8, 1999 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-10075589

RESUMO

BACKGROUND: OKT3 monoclonal antibody therapy results in an acute clinical syndrome (ACS) associated with the release of tumor necrosis factor and sequestration of neutrophils in the lungs. We have previously shown that inhibition of tumor necrosis factor does not completely eliminate OKT3-ACS, suggesting that other factors also contribute to the ACS. The current studies analyzed complement activation in vivo during the first hour after OKT3 administration. METHODS: Renal (n=4) and lung (n=4) transplant recipients received OKT3 as treatment for rejection and induction therapy, respectively. Complement activation products C4d, Bb, iC3b, and SC5b-9 were measured by ELISA. Hemodynamic parameters were also monitored in the lung transplant recipients. Neutrophil expression of CD11a, CD11b, and CD18 was monitored by flow cytometry. Controls included patients receiving methylprednisolone for rejection (n=4), two adults with adult respiratory distress syndrome who received extracorporeal membrane oxygenation, and normal volunteers (n=5). P values less than 0.05 (*) were considered significant. RESULTS: Increases in the plasma levels of C4d, Bb, iC3b, and SC5b-9 were observed in seven of eight patients after OKT3 administration. Mean values (n=8) at 0, 15, and 60 min (in microg/ml) were as follows-C4d: 1.865, 2.644*, and 2.607*; Bb: 0.245, 0.411, and 0.385; iC3b: 10.881, 17.242*, and 15.145*; and SC5b-9: 0.232, 0.269, and 0.302*. An increase in CD11b and CD18 and a decrease of CD11a on neutrophils in parallel with complement activation was observed. In lung transplant recipients, C3 activation correlated with increases in mean pulmonary and central venous pressures (P<0.05). As compared with extracorporeal membrane oxygenation, which activated classical and alternative pathways, OKT3 predominantly activated complement by the classical pathway. Methylprednisolone pulses did not activate complement. CONCLUSIONS: Complement activation is an early event after OKT3 administration and is associated with the increased expression of adhesion molecules on neutrophils and with pulmonary hemodynamic changes. Effective therapeutic approaches to the control of early monoclonal antibody side effects may require measures that limit complement activation in addition to reducing cytokine activity.


Assuntos
Complemento C3b/biossíntese , Complemento C4/biossíntese , Complemento C4b , Complexo de Ataque à Membrana do Sistema Complemento/biossíntese , Hemodinâmica , Imunossupressores/uso terapêutico , Transplante de Rim/imunologia , Transplante de Pulmão/imunologia , Muromonab-CD3/uso terapêutico , Fragmentos de Peptídeos/biossíntese , Adulto , Idoso , Pressão Sanguínea , Ativação do Complemento , C3 Convertase da Via Alternativa do Complemento , Ensaio de Imunoadsorção Enzimática , Feminino , Frequência Cardíaca , Humanos , Transplante de Rim/fisiologia , Transplante de Pulmão/fisiologia , Masculino , Metilprednisolona/uso terapêutico , Pessoa de Meia-Idade , Modelos Químicos
18.
J Infect Dis ; 178(6): 1597-603, 1998 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-9815210

RESUMO

Human fibroblasts weakly activated the alternative complement pathway, as assessed by C3b deposition, while 4- to 5-fold more C3b was observed 4 days after infection on cytomegalovirus (CMV)-infected fibroblasts when incubated with human serum. CMV-infected fibroblasts activated via the classical complement pathway independent of specific anti-CMV antibody and incubation of CMV-infected fibroblasts with serum deficient in complement components revealed that C1q, but not mannan-binding lectin, was required for complement activation. The enhanced complement activation by CMV-infected cells was observed as early as 4 h after infection and required the active transcription of CMV genes. No difference in the complement activation by CMV-infected cells was observed with the use of CMV-seropositive or -seronegative serum as a complement source, suggesting that CMV infection induces or up-regulates a protein that binds directly to C1q in a complement-activating conformation.


Assuntos
Via Clássica do Complemento/fisiologia , Citomegalovirus/imunologia , Pele/imunologia , Sangue , Células Cultivadas , Complemento C1q/fisiologia , Complemento C3b/análise , Complemento C3b/biossíntese , Via Clássica do Complemento/imunologia , Meios de Cultura , Citomegalovirus/genética , Fibroblastos/imunologia , Fibroblastos/virologia , Regulação Viral da Expressão Gênica , Genes Virais , Humanos , Imunoglobulina G/análise , Imunoglobulina M/análise , Recém-Nascido , Cinética , Masculino , Pele/virologia , Transcrição Gênica
19.
Immunology ; 93(2): 177-83, 1998 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-9616366

RESUMO

We have shown previously that normal B cells share, with Epstein-Barr virus-transformed and malignant B cells, the ability to activate the alternative pathway (AP) of complement in vitro, resulting in the deposition of C3 fragments on the cell surface. Complement receptor type 2 (CR2, CD21) has been implicated directly as the site for formation of an AP convertase, which provides nascent C3b for deposition at secondary sites on the B-cell surface. In the present study, we have examined C3 fragment deposition in vitro in more detail by (1) assessing the importance of locally generated C3b for the deposition process, (2) investigating whether CR2 is the sole requirement for conferring AP activation capacity on a cell, and (3) determining whether CR2's function, as an AP activator, has different structural requirements from ligand binding. Increasing the availability of native C3, by increasing the serum (NHS) concentration, resulted in enhanced C3 fragment deposition on the B cells, whereas use of factor 1-depleted NHS, which showed massive fluid phase C3 conversion during the incubation, diminished the deposition. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting of untreated and hydroxylamine-treated lysates from B cells, after in vitro activation, revealed that the majority of C3 fragments (primarily iC3b and C3dg) had been covalently bound to the cell surface. Transfection of COS cells with wild-type CR2 or a deletion mutant lacking 11 of the molecule's 15 homologous domains, but retaining the ligand-binding site, revealed that expression of intact CR2 conferred a 12-fold increase in AP-activating capacity on these cells, while no increase in AP activity was apparent on cells transfected with the mutant CR2.


Assuntos
Linfócitos B/imunologia , Complemento C3/metabolismo , Via Alternativa do Complemento/imunologia , Receptores de Complemento 3d/imunologia , Western Blotting , Técnicas de Cultura de Células , Complemento C3b/biossíntese , Complemento C3b/metabolismo , Relação Dose-Resposta Imunológica , Eletroforese em Gel de Poliacrilamida , Humanos , Fragmentos de Peptídeos/metabolismo
20.
Exp Neurol ; 146(2): 388-94, 1997 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-9270049

RESUMO

Activation of the classical complement cascade by beta-amyloid peptides has been hypothesized to underlie the neurodegeneration observed in Alzheimer's diseased brains. In this study, various lots of synthetic beta-amyloid peptides, A beta(1-40), A beta(1-42), and A beta(25-35), were tested for their ability to activate both early complement cascade events and formation of the membrane attack complex through terminal pathway activation. Unlike recent reports which did not assess activation of complement terminal pathway, we found that concentrations of beta-amyloid which activated early cascade events, to an extent comparable to aggregated IgG, failed to elicit formation of comparable levels of membrane attack complex.


Assuntos
Peptídeos beta-Amiloides/farmacologia , Via Clássica do Complemento/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Complemento C3b/biossíntese , Ensaio de Atividade Hemolítica de Complemento , Complexo de Ataque à Membrana do Sistema Complemento , Proteínas do Sistema Complemento/biossíntese , Glicoproteínas/biossíntese , Humanos
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