RESUMEN
KEY MESSAGES: Considerable proportion of patients with SpA have been immunized to the subcutaneous anti-TNF drug they are using. Concomitant use of MTX protects from immunization, whereas SASP does not. Patients with SpA using subcutaneous anti-TNF drugs can benefit from monitoring of the drug trough levels. Immunization to biological drugs can lead to decreased efficacy and increased risk of adverse effects. The objective of this cross-sectional study was to assess the extent and significance of immunization to subcutaneous tumor necrosis factor (TNF) inhibitors in axial spondyloarthritis (axSpA) patients in real-life setting. A serum sample was taken 1-2 days before the next drug injection. Drug trough concentrations, anti-drug antibodies (ADAb) and TNF-blocking capacity were measured in 273 patients with axSpA using subcutaneous anti-TNF drugs. The clinical activity of SpA was assessed using the Bath AS Disease Activity Index (BASDAI) and the Maastricht AS Entheses Score (MASES). ADAb were found in 11% of the 273 patients: in 21/99 (21%) of patients who used adalimumab, in 0/83 (0%) of those who used etanercept, in 2/79 (3%) of those who used golimumab and in 6/12 (50%) of those who used certolizumab pegol. Use of methotrexate reduced the risk of formation of ADAb, whereas sulfasalazine did not. Presence of ADAb resulted in decreased drug concentration and reduced TNF-blocking capacity. However, low levels of ADAb had no effect on TNF-blocking capacity and did not correlate with disease activity. The drug trough levels were below the consensus target level in 36% of the patients. High BMI correlated with low drug trough concentration. Patients with low drug trough levels had higher disease activity. The presence of anti-drug antibodies was associated with reduced drug trough levels, and the patients with low drug trough levels had higher disease activity. The drug trough levels were below target level in significant proportion of patients and, thus, measuring the drug concentration and ADAb could help to optimize the treatment in SpA patients.
Asunto(s)
Antirreumáticos , Espondiloartritis , Espondilitis Anquilosante , Anticuerpos Monoclonales Humanizados/uso terapéutico , Antirreumáticos/efectos adversos , Estudios Transversales , Humanos , Metotrexato/uso terapéutico , Espondiloartritis/tratamiento farmacológico , Espondilitis Anquilosante/tratamiento farmacológico , Inhibidores del Factor de Necrosis Tumoral/uso terapéutico , Factor de Necrosis Tumoral alfaRESUMEN
Proteogenomic databases use genomic and transcriptomic information for improved identification of peptides and proteins from mass spectrometry analyses. One application of such databases is in the discovery of variants/mutations. In this study, we created a proteogenomic database that contained sequences with variants derived from Pooled sequencing experiments (137 Group G Streptococcus strains sequenced in 3 pools) and used tandem mass spectrometry (MS/MS) to analyse eight protein samples from randomly selected strains sequenced in the pools. Using the proteogenomic variant database, we identified 385 variant peptides from the eight samples, none of which could be identified from the single genome conventional database utilized, while 71.2% and 93.5% of them were identified from the databases that contained 4 complete genomes and 26 assemblies, respectively. The proteogenomic variant databases exhibited the same properties as the conventional databases in terms of the Andromeda score distributions and the posterior error probability (PEP) values of the identified peptides. SIGNIFICANCE: For bacterial populations, such as Group G Streptococcus (GGS), with substantial intra-species diversity, simultaneous sequencing of large numbers of strains and generation of proteogenomic databases from those aids in improving the discovery of peptides in mass spectrometric analyses. Therefore, generation of proteogenomic variant protein databases from Pooled sequencing experiments can be a cost-effective method to complement conventional databases and discover subtle strain wise differences.
Asunto(s)
Proteínas Bacterianas , Bases de Datos de Proteínas , Genoma Bacteriano , Proteogenómica , Streptococcus , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Streptococcus/genética , Streptococcus/metabolismoRESUMEN
Factor H is an important regulator of complement activation in plasma and on cell surfaces in both humans and mice. If FH function is compromised, inappropriate complement activation on self-surfaces can have disastrous effects as seen in the kidney diseases atypical haemolytic uremic syndrome (aHUS) and C3 glomerulopathy. As FH constructs have been proposed to be used in treatment for these diseases, we studied the distribution of exogenous FH fragments in mice. Full-length mFH, mFH1-5 and mFH18-20 fragments were radiolabelled, and their distribution was examined in WT, FH-/- and FH-/- C3-/- mice in vivo. Whole body scintigraphy revealed accumulation of radioactivity in the abdominal part of the mice, but also to the thyroid gland and urinary bladder. At organ level in WT mice, some full-length FH accumulated in internal organs, but most of it remained in the circulation. Both of the mFH fragments accumulated in the kidneys and were excreted in urine. For mFH1-5, urinary secretion is the likely cause for the accumulation. Concentration of mFH18-20 to kidneys was slower, and at tissue level, mFH18-20 was localized at the proximal tubuli in WT and FH-/- C3-/- mice. No C3-independent binding to glomeruli was detected. In conclusion, these results show that glomerular glycosaminoglycans and sialic acids alone do not collect FH in kidneys. Deposition of C3 fragments is also needed, which implies that in aHUS, the problem is in simultaneous recognition of C3 fragments and glycosaminoglycans or sialic acids by FH, not just the inability of FH to recognize glomerular endothelium as such.
Asunto(s)
Factor H de Complemento/metabolismo , Animales , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fragmentos de Péptidos/metabolismo , Distribución TisularRESUMEN
To cause infections microbes need to evade host defense systems, one of these being the evolutionarily old and important arm of innate immunity, the alternative pathway of complement. It can attack all kinds of targets and is tightly controlled in plasma and on host cells by plasma complement regulator factor H (FH). FH binds simultaneously to host cell surface structures such as heparin or glycosaminoglycans via domain 20 and to the main complement opsonin C3b via domain 19. Many pathogenic microbes protect themselves from complement by recruiting host FH. We analyzed how and why different microbes bind FH via domains 19-20 (FH19-20). We used a selection of FH19-20 point mutants to reveal the binding sites of several microbial proteins and whole microbes (Haemophilus influenzae, Bordetella pertussis, Pseudomonas aeruginosa, Streptococcus pneumonia, Candida albicans, Borrelia burgdorferi, and Borrelia hermsii). We show that all studied microbes use the same binding region located on one side of domain 20. Binding of FH to the microbial proteins was inhibited with heparin showing that the common microbial binding site overlaps with the heparin site needed for efficient binding of FH to host cells. Surprisingly, the microbial proteins enhanced binding of FH19-20 to C3b and down-regulation of complement activation. We show that this is caused by formation of a tripartite complex between the microbial protein, FH, and C3b. In this study we reveal that seven microbes representing different phyla utilize a common binding site on the domain 20 of FH for complement evasion. Binding via this site not only mimics the glycosaminoglycans of the host cells, but also enhances function of FH on the microbial surfaces via the novel mechanism of tripartite complex formation. This is a unique example of convergent evolution resulting in enhanced immune evasion of important pathogens via utilization of a "superevasion site."
Asunto(s)
Bacterias/metabolismo , Candida albicans/metabolismo , Factor H de Complemento/metabolismo , Bacterias/genética , Bacterias/inmunología , Bacterias/patogenicidad , Sitios de Unión , Bordetella pertussis/genética , Bordetella pertussis/inmunología , Bordetella pertussis/metabolismo , Bordetella pertussis/patogenicidad , Borrelia/genética , Borrelia/inmunología , Borrelia/metabolismo , Borrelia/patogenicidad , Candida albicans/genética , Candida albicans/inmunología , Candida albicans/patogenicidad , Membrana Celular/metabolismo , Activación de Complemento , Factor H de Complemento/química , Haemophilus influenzae/genética , Haemophilus influenzae/inmunología , Haemophilus influenzae/metabolismo , Haemophilus influenzae/patogenicidad , Humanos , Unión Proteica , Estructura Terciaria de Proteína , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/inmunología , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidad , Staphylococcus aureus/genética , Staphylococcus aureus/inmunología , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidad , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/inmunología , Streptococcus pneumoniae/metabolismo , Streptococcus pneumoniae/patogenicidadRESUMEN
Atypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy often caused by mutations in complement factor H (CFH), the main regulator of alternative complement pathway. Because CFH is produced mainly by the liver, combined liver-kidney transplantation is a reasonable option in treatment of patients with severe aHUS. We studied complement activation by monitoring activation markers during liver transplantation in two aHUS patients treated extensively with plasma exchange and nine other liver transplantation patients. After the reperfusion, a clear increase in all the activation markers except C4d was observed indicating that the activation occurs mainly through the alternative pathway. Concentration of SC5b-9 was higher in the hepatic than the portal vein indicating complement activation in the graft. Preoperatively and early during the operation, the aHUS patients showed highest C3d concentrations but otherwise their activation markers were similar to the other patients. In the other patients, correlation was found between perioperative SC5b-9 concentration and postoperative alanine aminotransferase and histological changes. This study explains why supply of normal CFH by extensive plasma exchange is beneficial before combined liver-kidney transplantation of aHUS patients. Also the results suggest that perioperative inhibition of the terminal complement cascade might be beneficial if enhanced complement activation is expected.
Asunto(s)
Activación de Complemento , Síndrome Hemolítico-Urémico/cirugía , Trasplante de Hígado , Adolescente , Adulto , Humanos , Persona de Mediana Edad , Adulto JovenRESUMEN
Recently, Plasmodium knowlesi has been recognised as the fifth Plasmodium species causing malaria in humans. Hundreds of human cases infected with this originally simian Plasmodium species have been described in Asian countries and increasing numbers are reported in Europe from travellers. The growing impact of tourism and economic development in South and Southeast Asia are expected to subsequently lead to a further increase in cases both among locals and among travellers. P. knowlesi is easily misidentified in microscopy as P. malariae or P. falciparum. We developed new primers for the rapid and specific detection of this species by low-cost real-time polymerase chain reaction (PCR) and added this method to an already existing panel of primers used for the molecular identification of the other four species in one reaction. Reference laboratories should now be able to identify undisputably and rapidly P. knowlesi, as it is a potentially fatal pathogen.
Asunto(s)
Malaria/diagnóstico , Plasmodium knowlesi/clasificación , Plasmodium knowlesi/genética , Plasmodium/clasificación , Plasmodium/genética , Reacción en Cadena de la Polimerasa/métodos , Animales , Benzotiazoles , Cartilla de ADN , ADN Protozoario/análisis , ADN Protozoario/genética , ADN Protozoario/aislamiento & purificación , Diaminas , Europa (Continente) , Humanos , Malaria/parasitología , Compuestos Orgánicos , Plasmodium/aislamiento & purificación , Plasmodium knowlesi/aislamiento & purificación , Reacción en Cadena de la Polimerasa/economía , Quinolinas , Sensibilidad y Especificidad , Especificidad de la Especie , ViajeRESUMEN
A 12-month-old boy and his 16-year-old aunt became acutely ill 6 months apart and were diagnosed to have atypical hemolytic uremic syndrome (aHUS). Genetic analysis revealed heterozygous R1215Q mutation in complement factor H (CFH) in both patients. The same mutation was found in five healthy adult relatives indicating incomplete penetrance of the disease. The patients developed terminal renal failure and experienced reversible neurological symptoms in spite of plasma exchange (PE) therapy. In both cases, liver-kidney transplantation was successfully performed 6 months after the onset of the disease. To minimize complement activation and prevent thrombotic microangiopathy or overt thrombotic events due to the malfunctioning CFH, extensive PE with fresh frozen plasma was performed pre- and perioperatively and anticoagulation was started a few hours after the operation. No circulatory complications appeared and all four grafts started to function immediately. Also, no recurrence or other major clinical setbacks have appeared during the postoperative follow-up (15 and 9 months) and the grafts show excellent function. While more experience is needed, it seems that liver-kidney transplantation combined with pre- and perioperative PE is a rational option in the management of patients with aHUS caused by CFH mutation.
Asunto(s)
Sustitución de Aminoácidos/genética , Factor H de Complemento/genética , Síndrome Hemolítico-Urémico/genética , Síndrome Hemolítico-Urémico/cirugía , Trasplante de Riñón , Trasplante de Hígado , Adolescente , Femenino , Tamización de Portadores Genéticos , Síndrome Hemolítico-Urémico/terapia , Humanos , Lactante , Masculino , Linaje , Intercambio PlasmáticoRESUMEN
Intravascular Schistosoma mansoni worms seem to take up immunoglobulins from blood by surface Fc-receptors, but the process whereby bound immunoglobulins are processed by the parasite is poorly understood. We here present morphological data suggesting that two distinct main processes are involved: Host immunoglobulins were seen at two distinct locations in the parasite: in the frontal part of the enteric tube, the oesophagus, and as a fine granular staining at the surface and in the subtegumental region. The latter staining pattern corresponds to host immunoglobulin localization in discrete organelle-like aggregates tentatively identified as 'discoid or elongate bodies' at the ultrastructural level using immunogold staining. Immunoglobulin uptake by intravascular worms was also demonstrated in vivo after passive administration of 125I-labelled rabbit and mouse immunoglobulins. Radiolabelled immunoglobulins were taken up by the worms and shown to localize as fine strands running perpendicular to the parasite surface. Our results suggest that intravascular schistosomes take up host immunoglobulins both as part of their enteric digestion and by a surface Fc-receptor-mediated mechanism, involving transport and processing within organelles, 'elongate bodies'. Immunoglobulins taken up by intravascular schistosomes form a distinct organelle-like granules, which seem to be processed within the excretory system of the parasite.
Asunto(s)
Inmunoglobulinas/metabolismo , Schistosoma mansoni/metabolismo , Animales , Autorradiografía/métodos , Inmunoglobulinas/sangre , Inmunoglobulinas/inmunología , Inmunohistoquímica/métodos , Ratones , Ratones Endogámicos C3H , Microscopía Fluorescente/métodos , Microscopía de Polarización/métodos , Schistosoma mansoni/inmunología , Esquistosomiasis mansoni/inmunología , Esquistosomiasis mansoni/metabolismo , Esquistosomiasis mansoni/parasitologíaRESUMEN
Relapsing fever is a rapidly progressive and severe septic disease caused by certain Borrelia spirochetes. The disease is divided into two forms, i.e., epidemic relapsing fever, caused by Borrelia recurrentis and transmitted by lice, and the endemic form, caused by several Borrelia species, such as B. duttonii, and transmitted by soft-bodied ticks. The spirochetes enter the bloodstream by the vector bite and live persistently in plasma even after the development of specific antibodies. This leads to fever relapses and high mortality and clearly indicates that the Borrelia organisms utilize effective immune evasion strategies. In this study, we show that the epidemic relapsing fever pathogen B. recurrentis and an endemic relapsing fever pathogen, B. duttonii, are serum resistant, i.e., resistant to complement in vitro. They acquire the host alternative complement pathway regulator factor H on their surfaces in a similar way to that of the less serum-resistant Lyme disease pathogen, B. burgdorferi sensu stricto. More importantly, the relapsing fever spirochetes specifically bind host C4b-binding protein, a major regulator of the antibody-mediated classical complement pathway. Both complement regulators retained their functional activities when bound to the surfaces of the spirochetes. In conclusion, this is the first report of complement evasion by Borrelia recurrentis and B. duttonii and the first report showing capture of C4b-binding protein by spirochetes.
Asunto(s)
Borrelia/metabolismo , Proteína de Unión al Complemento C4b/metabolismo , Factor H de Complemento/metabolismo , Inactivadores del Complemento/metabolismo , Fiebre Recurrente/metabolismo , Borrelia/inmunología , Borrelia/patogenicidad , Borrelia burgdorferi/inmunología , Humanos , Fiebre Recurrente/inmunologíaRESUMEN
The complement inhibitor Factor H has three distinct binding sites for C3b and for heparin, but in solution uses specifically the most C-terminal domain, i.e. short consensus repeats (SCR) 20 for ligand interaction. Two novel monoclonal antibodies (mABs C14 and C18) that bind to the most C-terminal domain SCR 20 completely blocked interaction of Factor H with the ligands C3b, C3d, heparin and binding to endothelial cells. In contrast, several mAbs that bind to the N-terminus and to the middle regions of the molecule showed no or minor inhibitory effects when assayed by enzyme-linked immunosorbent assay (ELISA) and ligand interaction assays. This paradox between a single functional binding site identified for native Factor H versus multiple interaction sites reported for deletion constructs is explained by a compact conformation of the fluid phase protein with one accessible binding site. On zymosan particles mAbs C14 and C18 blocked alternative pathway activation completely. Thus demonstrating that native Factor H makes the first and initial contact with the C terminus, which is followed by N terminally mediated complement regulation. These results are explained by a conformational hypothetical model: the native Factor H protein has a compact structure and only one binding site accessible. Upon the first contact the protein unfolds and exposes the additional binding sites. This model does explain how Factor H mediates recognition functions during complement control and the clustering of disease associated mutations in patients with haemolytic uraemic syndrome that have been reported in the C-terminal recognition domain of Factor H.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Sitios de Unión de Anticuerpos/inmunología , Complemento C3b/inmunología , Complemento C3d/inmunología , Factor H de Complemento/genética , Factor H de Complemento/inmunología , Vía Alternativa del Complemento/inmunología , Células Endoteliales/inmunología , Epítopos/inmunología , Heparina/inmunología , Humanos , Ligandos , Modelos Biológicos , Mutación , Conformación Proteica , Zimosan/inmunologíaRESUMEN
The most characteristic features of the Lyme disease pathogens, the Borrelia burgdorferi sensu lato (s.l.) group, are their ability to invade tissues and to circumvent the immune defenses of the host for extended periods of time, despite elevated levels of borrelia-specific antibodies in serum and other body fluids. Our aim in the present study was to determine whether B. burgdorferi is able to interfere with complement (C) at the level of C3 by accelerating C3b inactivation and thus to inhibit the amplification of the C cascade. Strains belonging to different genospecies (Borrelia garinii, B. burgdorferi sensu stricto, and Borrelia afzelii) were compared for their sensitivities to normal human serum and abilities to promote factor I-mediated C3b degradation. B. burgdorferi sensu stricto and B. afzelii strains were found to be serum resistant. When the spirochetes were incubated with radiolabeled C3b, factor I-mediated degradation of C3b was observed in the presence of C-resistant B. afzelii (n = 3) and B. burgdorferi sensu stricto (n = 1) strains but not in the presence of C-sensitive B. garinii (n = 7) strains or control bacteria (Escherichia coli, Staphylococcus aureus, and Enterococcus faecalis). Immunoblotting and radioligand binding analyses showed that the C-resistant strains had the capacity to acquire the C inhibitors factor H and factor H-like protein 1 (FHL-1) from growth medium and human serum. A novel surface protein with an apparent molecular mass of 35 kDa was found to preferentially bind to the N terminus region of factor H. Thus, the serum-resistant B. burgdorferi s.l. strains can circumvent C attack by binding the C inhibitors factor H and FHL-1 to their surfaces and promoting factor I-mediated C3b degradation.
Asunto(s)
Grupo Borrelia Burgdorferi/inmunología , Activación de Complemento/inmunología , Complemento C3b/inmunología , Animales , Proteínas Bacterianas/metabolismo , Actividad Bactericida de la Sangre , Proteínas Sanguíneas/metabolismo , Grupo Borrelia Burgdorferi/patogenicidad , Proteínas Inactivadoras del Complemento C3b , Factor H de Complemento/metabolismo , Humanos , Immunoblotting , Enfermedad de Lyme/microbiologíaRESUMEN
Foreign particles and damaged host cells can activate the complement system leading to their destruction by the host defense system. Factor H (fH) plays a vital role in restricting complement activation on host cells through interactions with polyanions such as heparin, while allowing activation to proceed on foreign surfaces. Complement activation by damaged host cells is also down regulated by fH, which is localized to injured areas through interactions with C-reactive protein (CRP). A number of pathogens have developed mechanisms by which they can also bind fH and thus exploit its protective properties. One such organism is Group A Streptococcus (GAS) which mediates fH binding via its surface expressed M-protein. fH consists of 20 conserved short consensus repeat (SCR) units and mutagenesis studies indicate that the seventh repeat is responsible for interactions with heparin, CRP and M-protein. We recently performed molecular modelling of fH SCR 7 and identified a cluster of positively charged residues on one face of the domain. By alanine replacement mutagenesis, we demonstrated that these residues are involved in heparin, CRP and M protein binding, which indicates that there is a common site within fH SCR 7 responsible for multiple ligand recognition.
Asunto(s)
Antígenos Bacterianos , Proteínas de la Membrana Bacteriana Externa , Factor H de Complemento/química , Factor H de Complemento/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/inmunología , Sitios de Unión , Proteína C-Reactiva/inmunología , Proteínas Portadoras/inmunología , Activación de Complemento , Factor H de Complemento/genética , Heparina/química , Humanos , Técnicas In Vitro , Infecciones/etiología , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Ácido N-Acetilneuramínico/inmunología , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Streptococcus pyogenes/inmunologíaRESUMEN
The surface plasmon resonance (SPR) phenomenon is utilized in a number of new real time biosensors. In this study, we have used this technique to study interactions between the central complement component C3b and its multiple ligands by using the Biacore equipment. The SPR technique is particularly suitable for analysis of the alternative complement pathway (AP) because the inherent nature of the latter is to amplify deposition of C3b on various surfaces. C3b was coupled onto the sensor surface and the coupling efficiency was compared under various conditions on both polystyrene and carboxymethylated dextran surfaces. After enzymatic C3b coupling or standard amine C3b coupling, we analyzed and compared the binding of four C3b ligands to the surface: factor B, factor H, C5 and the soluble complement receptor 1 (sCR1, CD35). Binding of each ligand to C3b was detected when C3b had been coupled either enzymatically or using the amine coupling, but the half-lives of the interactions were found to vary depending on the coupling procedure. Factor H binds to C3b via three interaction sites. The target sites are exposed on the C3b, C3c and C3d fragments of C3, respectively. Therefore, we also tested by using the Biacore whether factor B, C5 and sCR1 bind to C3c and/or C3d. It was found that factor B bound to C3d, but not to C3c. On the other hand, both C5 and sCR1 bound to C3c, but not to C3d. In conclusion, this study shows that SPR is a powerful tool in analyzing and mapping the interactions of C3b with its multiple ligands.
Asunto(s)
Complemento C3b/metabolismo , Sitios de Unión , Complemento C3c/metabolismo , Complemento C3d/metabolismo , Complemento C5/metabolismo , Factor B del Complemento/metabolismo , Factor H de Complemento/metabolismo , Vía Alternativa del Complemento , Dextranos , Humanos , Técnicas In Vitro , Ligandos , Poliestirenos , Receptores de Complemento 3b/metabolismo , Resonancia por Plasmón de Superficie , Propiedades de SuperficieRESUMEN
Factor H and the FHL-1/reconectin protein are two human plasma proteins that act as important regulators of the alternative complement pathway. Each protein is encoded by a unique transcript, but both mRNAs are derived from the factor H gene by means of alternative processing. In order to address potential functional differences between the two proteins we analysed their expression in hepatic and non-hepatic cells and studied their regulation by inflammatory mediators. We demonstrate that factor H and FHL-1/reconectin transcripts which are regulated by the same gene promoter and are initiated at the same transcription start site are differently expressed. Expression of the molecules is induced and regulated by the inflammatory mediators interferon-gamma (IFN-gamma) and the anti-inflammatory glucocorticoid dexamethasone. Both factor H and FHL-1/reconectin are expressed and secreted by synovial fibroblasts and are present in synovial fluid derived from patients suffering from rheumatoid or reactive arthritis. The local synthesis in synovial fibroblasts and their induction by IFN-gamma and dexamethasone, but not by tumour necrosis factor-alpha, suggests for each of the two complement regulators a protective role in RA.
Asunto(s)
Empalme Alternativo , Artritis Reumatoide/metabolismo , Enfermedades Autoinmunes/metabolismo , Proteínas Sanguíneas/biosíntesis , Factor H de Complemento/biosíntesis , Dexametasona/farmacología , Regulación de la Expresión Génica , Interferón gamma/farmacología , Artritis Reactiva/metabolismo , Artritis Reumatoide/genética , Enfermedades Autoinmunes/genética , Proteínas Sanguíneas/genética , Western Blotting , Línea Celular , Proteínas Inactivadoras del Complemento C3b , Factor H de Complemento/genética , Fibroblastos/metabolismo , Humanos , Hígado/metabolismo , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes , Líquido Sinovial/metabolismo , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Multiple ovulations are uncommon in humans, cattle and many breeds of sheep. Pituitary gonadotrophins and as yet unidentified ovarian factors precisely regulate follicular development so that, normally, only one follicle is selected to ovulate. The Inverdale (FecXI) sheep, however, carries a naturally occurring X-linked mutation that causes increased ovulation rate and twin and triplet births in heterozygotes (FecXI/FecX+; ref. 1), but primary ovarian failure in homozygotes (FecXI/FecXI; ref. 2). Germ-cell development, formation of the follicle and the earliest stages of follicular growth are normal in FecXI/FecXI sheep, but follicular development beyond the primary stage is impaired. A second family unrelated to the Inverdale sheep also has the same X-linked phenotype (Hanna, FecXH). Crossing FecXI with FecXH animals produces FecXI/FecXH infertile females phenotypically indistinguishable from FecXI/FecXI females. We report here that the FecXI locus maps to an orthologous chromosomal region syntenic to human Xp11.2-11.4, which contains BMP15, encoding bone morphogenetic protein 15 (also known as growth differentiation factor 9B (GDF9B)). Whereas BMP15 is a member of the transforming growth factor beta (TGFbeta) superfamily and is specifically expressed in oocytes, its function is unknown. We show that independent germline point mutations exist in FecXI and FecXH carriers. These findings establish that BMP15 is essential for female fertility and that natural mutations in an ovary-derived factor can cause both increased ovulation rate and infertility phenotypes in a dosage-sensitive manner.
Asunto(s)
Proteínas Morfogenéticas Óseas/genética , Sustancias de Crecimiento/genética , Infertilidad Femenina/genética , Péptidos y Proteínas de Señalización Intercelular , Mutación , Ovulación/fisiología , Cromosoma X , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteína Morfogenética Ósea 15 , Proteínas Morfogenéticas Óseas/química , Mapeo Cromosómico , ADN Complementario , Femenino , Factor 9 de Diferenciación de Crecimiento , Sustancias de Crecimiento/química , Humanos , Masculino , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Oocitos/metabolismo , Linaje , Conformación Proteica , OvinosRESUMEN
Factor H (fH) restricts activation of the alternative pathway of complement at the level of C3, both in the fluid phase and on self-structures, but allows the activation to proceed on foreign structures. To study the interactions between fH and C3b we used surface plasmon resonance analysis (Biacore(R)) and eight recombinantly expressed fH constructs containing fragments of the 20 short consensus repeat domains (SCRs) of fH. We analyzed the binding of these constructs to C3b and its cleavage products C3c and C3d. Three binding sites for C3b were found on fH. Site 1 was localized to the five amino-terminal SCRs (SCR1-5), and its reciprocal binding site on C3b was found to be lost upon the cleavage of C3b to C3c and C3d. Site 2 on fH was localized by exclusion probably within or near SCRs 12-14 (fragment SCR8-20 bound to C3b, C3c, and C3d; SCR8-11 did not bind to C3b at all; and SCR15-20 bound only to the C3d part of C3b). Site 3 on fH for C3b was localized to the carboxyl-terminal SCRs 19-20, and its reciprocal binding site was mapped to the C3d part of C3b. In conclusion, we confirmed and mapped three binding sites on fH for C3b and demonstrated that the three binding sites on fH interact with distinct sites on C3b. Multiple reciprocal interactions between C3b and fH can provide a basis for the different reactivity of the alternative pathway with different target structures.
Asunto(s)
Complemento C3b/química , Complemento C3b/metabolismo , Factor H de Complemento/química , Factor H de Complemento/metabolismo , Sitios de Unión , Técnicas Biosensibles , Clonación Molecular , Secuencia de Consenso , Humanos , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Secuencias Repetitivas de Aminoácido , Resonancia por Plasmón de Superficie/métodosRESUMEN
Of over 20 nucleated cell lines we have examined to date, human H2 glioblastoma cells have turned out to be the most resistant to complement-mediated cytolysis in vitro. H2 cells expressed strongly the membrane attack complex inhibitor protectin (CD59), moderately CD46 (membrane cofactor protein) and CD55 (decay-accelerating factor), but no CD35 (complement receptor 1). When treated with a polyclonal anti-H2 Ab, anti-CD59 mAb, and normal human serum, only 5% of H2 cells became killed. Under the same conditions, 70% of endothelial-like EA.hy 926 cells and 40% of U251 control glioma cells were killed. A combined neutralization of CD46, CD55, and CD59 increased H2 lysis only minimally, demonstrating that these complement regulators are not enough to account for the resistance of H2 cells. After treatment with Abs and serum, less C5b-9 was deposited on H2 than on U251 and EA.hy 926 cell lines. A reason for the exceptional resistance of H2 cells was revealed when RT-PCR and protein biochemical methods showed that the H2 cells, unlike the other cell lines tested, actively produced the soluble complement inhibitors factor H and factor H-like protein 1. H2 cells were also capable of binding human factor H from the fluid phase to their cell surface and promoted the cleavage of C3b to its inactive form iC3b more efficiently than U251 and EA.hy 926 cells. In accordance, anti-factor H mAbs enhanced killing of H2 glioblastoma cells. Taken together, our results show that production and binding of factor H and factor H-like protein 1 is a novel mechanism that these malignant cells utilize to escape complement-mediated killing.
Asunto(s)
Proteínas Sanguíneas/biosíntesis , Factor H de Complemento/biosíntesis , Proteínas del Sistema Complemento/inmunología , Citotoxicidad Inmunológica/inmunología , Glioblastoma/inmunología , Anticuerpos Monoclonales/farmacología , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/fisiología , Membrana Celular/inmunología , Membrana Celular/metabolismo , Activación de Complemento , Complemento C3/inmunología , Complemento C3/metabolismo , Factor H de Complemento/genética , Factor H de Complemento/inmunología , Factor H de Complemento/metabolismo , Proteínas del Sistema Complemento/biosíntesis , Proteínas del Sistema Complemento/metabolismo , Femenino , Glioblastoma/metabolismo , Humanos , Inmunidad Innata , Unión Proteica/inmunología , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
Trichomonas vaginalis is a globally common sexually transmitted human parasite. Many strains of T. vaginalis from around the world have been described to be resistant to the current drug of choice, metronidazole. However, only a few cases of metronidazole resistance have been reported from Europe. The resistant strains cause prolonged infections which are difficult to treat. T. vaginalis infection also increases the risk for human immunodeficiency virus transmission. We present a practical method for determining the resistance of T. vaginalis to 5-nitroimidazoles. The suggested method was developed by determining the MICs and minimal lethal concentrations (MLCs) of metronidazole and ornidazole for T. vaginalis under various aerobic and anaerobic conditions. Using this assay we have found the first three metronidazole-resistant strains from Finland, although the origin of at least one of the strains seems to be Russia. Analysis of the patient-derived and previously characterized isolates showed that metronidazole-resistant strains were also resistant to ornidazole, and MLCs for all strains tested correlated well with the MICs. The suggested MICs of metronidazole for differentiation of sensitive and resistant isolates are >75 microg/ml in an aerobic 24-h assay and >15 microg/ml in an anaerobic 48-h assay.
Asunto(s)
Antitricomonas/farmacología , Metronidazol/farmacología , Trichomonas vaginalis/efectos de los fármacos , Aerobiosis , Anaerobiosis , Animales , Resistencia a Medicamentos , Femenino , Humanos , Ornidazol/farmacología , Oxígeno/farmacología , Vaginitis por Trichomonas/parasitología , Trichomonas vaginalis/crecimiento & desarrolloRESUMEN
A unique monoclonal Ig lambda light chain dimer (protein LOI) was isolated from the serum and urine of a patient with hypocomplementemic membranoproliferative glomerulonephritis. In vitro the lambda light chain dimer efficiently activated the alternative pathway of complement (AP). When added to normal human serum, LOI temporarily enhanced AP hemolytic activity, but during a prolonged incubation the hemolytic activity was depleted. Protein LOI was found to bind to factor H, the main regulator molecule of AP. By binding to the short consensus repeat domain 3 of factor H, the dimer LOI blocked one of three interaction sites between H and C3b and thus inhibited the activity of H and induced an uncontrolled activation of the AP. Structural analysis showed that LOI belonged to the Vlambda3a subgroup of lambda light chains. The variable (V) region of LOI was most closely related to the predicted product of the Vlambda3 germline gene Iglv3s2, although it contained several unique residues that in a tertiary homology model structure form an unusual ring of charged residues around a hydrophobic groove in the putative Ag binding site. This site fitted considerably well with a putative binding site in the molecular model of domain 3 of factor H containing a reciprocal ring of charged amino acids around a hydrophobic area. Apparently, functional blocking of factor H by the Ab fragment-like lambda light chain dimer had initiated the development of a severe form of membranoproliferative glomerulonephritis. Thus, the lambda light chain dimer LOI represents the first described pathogenic miniautoantibody in human disease.
Asunto(s)
Autoanticuerpos/química , Factor H de Complemento/inmunología , Glomerulonefritis Membranoproliferativa/inmunología , Cadenas lambda de Inmunoglobulina/química , Secuencia de Aminoácidos , Autoanticuerpos/metabolismo , Autoanticuerpos/fisiología , Sitios de Unión de Anticuerpos , Factor H de Complemento/antagonistas & inhibidores , Factor H de Complemento/metabolismo , Proteínas Inactivadoras de Complemento/fisiología , Vía Alternativa del Complemento/inmunología , Cristalografía por Rayos X , Dimerización , Femenino , Glomerulonefritis Membranoproliferativa/metabolismo , Humanos , Cadenas lambda de Inmunoglobulina/metabolismo , Cadenas lambda de Inmunoglobulina/fisiología , Persona de Mediana Edad , Modelos Moleculares , Datos de Secuencia Molecular , Relación Estructura-ActividadRESUMEN
C-reactive protein (CRP) is a major acute phase protein whose functions are not totally clear. In this study, we examined the interaction of CRP with factor H (FH), a key regulator of the alternative pathway (AP) of complement. Using the surface plasmon resonance technique and a panel of recombinantly expressed FH constructs, we observed that CRP binds to two closely located regions on short consensus repeat (SCR) domains 7 and 8-11 of FH. Also FH-like protein 1 (FHL-1), an alternatively spliced product of the FH gene, bound to CRP with its most C-terminal domain (SCR 7). The binding reactions were calcium-dependent and partially inhibited by heparin. In accordance with the finding that CRP binding sites on FH were distinct from the C3b binding sites, CRP preserved the ability of FH to promote factor I-mediated cleavage of C3b. We propose that the function of CRP is to target functionally active FH and FHL-1 to injured self tissues. Thereby, CRP could restrict excessive complement attack in tissues while allowing a temporarily enhanced AP activity against invading microbes in blood.