RESUMEN

CD55 is a major regulator of the complement system, a complex network of proteins that cooperate to clear tissue and blood pathogens from the organism. Indeed, overexpression of CD55 is associated with many diseases and is connected to the resistance mechanisms exhibited by several cancers towards immunotherapy approaches. High level of CD55 expression on tumour cells renders it a good target for both imaging and immunotherapy. Indeed, a conceivable approach to tackle disease is to interfere with CD55-mediated complement regulation with the use of CD55-targeting antibodies. However, the large size and poor tissue penetration together with to the high costs of antibodies often limits their widespread therapeutic use. Here, we employed bioinformatic and chemical approaches to design and synthesize molecules of small dimensions able to mimic a CD55 blocking antibody. As a result, a bicyclic peptide, named as miniAB55, proved to bind CD55 with nanomolar affinity. This molecule represents an attracting chemical scaffold for CD55-directed diagnostic tools in diseases associated with CD55 overproduction. To further support the applicative potential of miniAB55, we prove that the miniAB55 binds CD55 on the same region involved in inactivation of the complement C3 and C5 convertases, thus opening promising scenarios for the development of complement-modulating tools.

Asunto(s)

Anticuerpos/farmacología , Antígenos CD55/inmunología , Miniaturización , Péptidos Cíclicos/química , Secuencia de Aminoácidos , Sitios de Unión de Anticuerpos/inmunología , Antígenos CD55/química , Ciclización , Humanos , Cinética , Modelos Moleculares , Simulación del Acoplamiento Molecular

RESUMEN

The rapid spread of the virus in Latin America and the association of the infection with microcephaly in newborns or Guillain-Barré Syndrome in adults prompted the WHO to declare the Zika virus (ZIKV) epidemic to be an international public health emergency in 2016. As the virus was first discovered in monkeys and is spread not only by mosquitos but also from human to human, we investigated the stability to the human complement of ZIKV derived from mosquito (ZIKVInsect), monkey (ZIKVVero), or human cells (ZIKVA549 and ZIKVFibro), respectively. At a low serum concentration (10%), which refers to complement concentrations found on mucosal surfaces, the virus was relatively stable at 37 °C. At higher complement levels (up to 50% serum concentration), ZIKV titers differed significantly depending on the cell line used for the propagation of the virus. While the viral titer of ZIKVInsect decreased about two orders in magnitude, when incubated with human serum, the virus derived from human cells was more resistant to complement-mediated lysis (CML). By virus-capture assay and Western blots, the complement regulator protein CD55 was identified to be incorporated into the viral envelope. Blocking of CD55 by neutralizing Abs significantly increased the sensitivity to human complement. Taken together, these data indicate that the incorporation of CD55 from human cells contributes to the stability of ZIKV against complement-mediated virolysis.

Asunto(s)

Anticuerpos Antivirales/inmunología , Antígenos CD55/inmunología , Proteínas del Sistema Complemento/inmunología , Infección por el Virus Zika/inmunología , Células A549 , Aedes , Animales , Chlorocebus aethiops , Fibroblastos , Humanos , Células Vero , Carga Viral

RESUMEN

The human complement system is an important part of the innate immune system. Its effector pathways largely mediate virus neutralization. Vesicular stomatitis virus (VSV) activates the classical pathway of the complement, leading to virus neutralization by lysis. Two host-derived membrane-associated regulators of complement activation (RCA), CD55 and CD46, which are incorporated into the VSV envelope during egress, confer protection by delaying/resisting complement-mediated neutralization. We showed previously that CD55 is more effective than CD46 in the inhibition of neutralization. In this study, we identified that, at the protein level, VSV infection resulted in the down-regulation of CD46 but not CD55. The mRNA of both the RCAs was significantly down-regulated by VSV, but it was delayed in the case of CD55. The immunoblot analysis of the levels of RCAs in the progeny virion harvested at three specific time intervals, points to an equal ratio of its distribution relative to viral proteins. Besides reconfirming the dominant role of CD55 over CD46 in shielding VSV from complement, our results also highlight the importance of the subtle modulation in the expression pattern of RCAs in a system naturally expressing them.

Asunto(s)

Antígenos CD55/inmunología , Proteínas del Sistema Complemento/inmunología , Estomatitis Vesicular/inmunología , Virus de la Estomatitis Vesicular Indiana/inmunología , Células A549 , Línea Celular Tumoral , Activación de Complemento/inmunología , Células HeLa , Humanos , Proteína Cofactora de Membrana/inmunología , Pruebas de Neutralización/métodos , ARN Mensajero/inmunología , Virión/inmunología

RESUMEN

Decay accelerating factor (DAF), a key complement activation control protein, is a 70 kDa membrane bound glycoprotein which controls extent of formation of the C3 and C5 convertases by accelerating their decay. Using clustered regularly-interspaced short palindromic repeats, (CRISPR)/associated protein 9 (Cas9) genome editing we generated a novel DAF deficient (Daf-/-) rat model. The present study describes the renal and extrarenal phenotype of this model and assesses renal response to complement-dependent injury induced by administration of a complement-fixing antibody (anti-Fx1A) against the glomerular epithelial cell (podocyte). Rats generated were healthy, viable and able to reproduce normally. Complete absence of DAF was documented in renal as well as extra-renal tissues at both protein and mRNA level compared to Daf+/+ rats. Renal histology in Daf-/- rats showed no differences regarding glomerular or tubulointerstitial pathology compared to Daf+/+ rats. Moreover, there was no difference in urine protein excretion (ratio of urine albumin to creatinine) or in serum creatinine and urea levels. In Daf-/- rats, proteinuria was significantly increased following binding of anti-Fx1A antibody to podocytes while increased C3b deposition was observed. The DAF knock-out rat model developed validates the role of this complement cascade regulator in immune-mediated podocyte injury. Given the increasing role of dysregulated complement activation in various forms of kidney disease and the fact that the rat is the preferred animal for renal pathophysiology studies, the rat DAF deficient model may serve as a useful tool to study the role of this complement activation regulator in complement-dependent forms of kidney injury.

Asunto(s)

Lesión Renal Aguda/genética , Antígenos CD55/genética , Activación de Complemento/genética , Podocitos/metabolismo , Lesión Renal Aguda/patología , Albuminuria , Animales , Anticuerpos Antiidiotipos/farmacología , Antígenos CD55/deficiencia , Antígenos CD55/inmunología , Sistemas CRISPR-Cas/genética , Activación de Complemento/inmunología , Convertasas de Complemento C3-C5/genética , Complemento C5/genética , Técnicas de Inactivación de Genes , Complejo Antigénico de Nefritis de Heymann/genética , Complejo Antigénico de Nefritis de Heymann/inmunología , Humanos , Podocitos/patología , Ratas

RESUMEN

Altered glycosylations, which are associated with expression and activities of glycosyltransferases, can dramatically affect the function of glycoproteins and modify the behavior of tumor cells. ST3GAL1 is a sialyltransferase that adds sialic acid to core 1 glycans, thereby terminating glycan chain extension. In breast carcinomas, overexpression of ST3GAL1 promotes tumorigenesis and correlates with increased tumor grade. In pursuing the role of ST3GAL1 in breast cancer using ST3GAL1-siRNA to knockdown ST3GAL1, we identified CD55 to be one of the potential target proteins of ST3GAL1. CD55 is an important complement regulatory protein, preventing cells from complement-mediated cytotoxicity. CD55 had one N-linked glycosylation site in addition to a Ser/Thr-rich domain, which was expected to be heavily O-glycosylated. Detailed analyses of N- and O-linked oligosaccharides of CD55 released from scramble or ST3GAL1 siRNA-treated breast cancer cells by tandem mass spectrometry revealed that the N-glycan profile was not affected by ST3GAL1 silencing. The O-glycan profile of CD55 demonstrated a shift in abundance to nonsialylated core 1 and monosialylated core 2 at the expense of the disialylated core 2 structure after ST3GAL1 silencing. We also demonstrated that O-linked desialylation of CD55 by ST3GAL1 silencing resulted in increased C3 deposition and complement-mediated lysis of breast cancer cells and enhanced sensitivity to antibody-dependent cell-mediated cytotoxicity. These data demonstrated that ST3GAL1-mediated O-linked sialylation of CD55 acts like an immune checkpoint molecule for cancer cells to evade immune attack and that inhibition of ST3GAL1 is a potential strategy to block CD55-mediated immune evasion.

Asunto(s)

Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Neoplasias de la Mama/patología , Antígenos CD55/inmunología , Evasión Inmune/inmunología , Sialiltransferasas/metabolismo , Neoplasias de la Mama/inmunología , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Glicosilación , Humanos , ARN Interferente Pequeño/metabolismo , Sialiltransferasas/genética , Sialiltransferasas/inmunología , beta-Galactosida alfa-2,3-Sialiltransferasa

RESUMEN

Receptor usage that determines cell tropism and drives viral classification closely correlates with the virus structure. Enterovirus B (EV-B) consists of several subgroups according to receptor usage, among which echovirus 30 (E30), a leading causative agent for human aseptic meningitis, utilizes FcRn as an uncoating receptor. However, receptors for many EVs remain unknown. Here we analyzed the atomic structures of E30 mature virion, empty- and A-particles, which reveals serotype-specific epitopes and striking conformational differences between the subgroups within EV-Bs. Of these, the VP1 BC loop markedly distinguishes E30 from other EV-Bs, indicative of a role as a structural marker for EV-B. By obtaining cryo-electron microscopy structures of E30 in complex with its receptor FcRn and CD55 and comparing its homologs, we deciphered the underlying molecular basis for receptor recognition. Together with experimentally derived viral receptor identifications, we developed a structure-based in silico algorithm to inform a rational prediction for EV receptor usage.

Asunto(s)

Complejo Antígeno-Anticuerpo/ultraestructura , Enterovirus Humano B/ultraestructura , Antígenos Virales/ultraestructura , Antígenos CD55/inmunología , Microscopía por Crioelectrón , Enterovirus Humano B/inmunología , Epítopos/ultraestructura , Humanos , Receptores Fc/inmunología , Virión/ultraestructura

RESUMEN

Echovirus 30 (E30), a serotype of Enterovirus B (EV-B), recently emerged as a major causative agent of aseptic meningitis worldwide. E30 is particularly devastating in the neonatal population and currently no vaccine or antiviral therapy is available. Here we characterize two highly potent E30-specific monoclonal antibodies, 6C5 and 4B10, which efficiently block binding of the virus to its attachment receptor CD55 and uncoating receptor FcRn. Combinations of 6C5 and 4B10 augment the sum of their individual anti-viral activities. High-resolution structures of E30-6C5-Fab and E30-4B10-Fab define the location and nature of epitopes targeted by the antibodies. 6C5 and 4B10 engage the capsid loci at the north rim of the canyon and in-canyon, respectively. Notably, these regions exhibit antigenic variability across EV-Bs, highlighting challenges in development of broad-spectrum antibodies. Our structures of these neutralizing antibodies of E30 are instructive for development of vaccines and therapeutics against EV-B infections.

Asunto(s)

Anticuerpos Neutralizantes/ultraestructura , Complejo Antígeno-Anticuerpo/ultraestructura , Proteínas de la Cápside/inmunología , Enterovirus Humano B/inmunología , Animales , Anticuerpos Monoclonales/ultraestructura , Antígenos Virales , Antígenos CD55/inmunología , Microscopía por Crioelectrón , Epítopos/ultraestructura , Humanos , Meningitis Aséptica/virología , Ratones , Receptores Fc/inmunología , Serogrupo

RESUMEN

Understanding molecular mechanisms that dictate B cell diversity is important for targeting B cells as anti-cancer treatment. Through the single-cell dissection of B cell heterogeneity in longitudinal samples of patients with breast cancer before and after neoadjuvant chemotherapy, we revealed that an ICOSL+ B cell subset emerges after chemotherapy. Using three immunocompetent mouse models, we recapitulated the subset switch of human tumor-infiltrating B cells during chemotherapy. By employing B-cell-specific deletion mice, we showed that ICOSL in B cells boosts anti-tumor immunity by enhancing the effector to regulatory T cell ratio. The signature of ICOSL+ B cells is imprinted by complement-CR2 signaling, which is triggered by immunogenic cell death. Moreover, we identified that CD55, a complement inhibitory protein, determines the opposite roles of B cells in chemotherapy. Collectively, we demonstrated a critical role of the B cell subset switch in chemotherapy response, which has implications in designing novel anti-cancer therapies. VIDEO ABSTRACT.

Asunto(s)

Linfocitos B/inmunología , Neoplasias de la Mama/inmunología , Ligando Coestimulador de Linfocitos T Inducibles/metabolismo , Animales , Antineoplásicos/metabolismo , Linfocitos B/metabolismo , Antígenos CD55/inmunología , Antígenos CD55/metabolismo , Línea Celular Tumoral , Proteínas del Sistema Complemento/metabolismo , Modelos Animales de Enfermedad , Femenino , Humanos , Ligando Coestimulador de Linfocitos T Inducibles/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Ratones , Ratones Endogámicos C57BL , Receptores de Complemento 3d/inmunología , Receptores de Complemento 3d/metabolismo , Transducción de Señal/inmunología , Linfocitos T Reguladores/inmunología

RESUMEN

Germinal centers (GC) are sites for extensive B cell proliferation and homeostasis is maintained by programmed cell death. The complement regulatory protein Decay Accelerating Factor (DAF) blocks complement deposition on host cells and therefore also phagocytosis of cells. Here, we show that B cells downregulate DAF upon BCR engagement and that T cell-dependent stimuli preferentially led to activation of DAFlo B cells. Consistent with this, a majority of light and dark zone GC B cells were DAFlo and susceptible to complement-dependent phagocytosis, as compared with DAFhi GC B cells. We could also show that the DAFhi GC B cell subset had increased expression of the plasma cell marker Blimp-1. DAF expression was also modulated during B cell hematopoiesis in the human bone marrow. Collectively, our results reveal a novel role of DAF to pre-prime activated human B cells for phagocytosis prior to apoptosis.

Asunto(s)

Linfocitos B/inmunología , Antígenos CD55/inmunología , Regulación de la Expresión Génica/inmunología , Centro Germinal/inmunología , Activación de Linfocitos , Fagocitosis , Linfocitos B/citología , Centro Germinal/citología , Humanos , Factor 1 de Unión al Dominio 1 de Regulación Positiva/inmunología

RESUMEN

In recent years, efforts to treat cancer by improving the immune function of patients have received a great deal of attention. As part of the immune system, complement is also under such evaluation. Among the many components of the complement system, complement decay accelerating factor (CD55 or DAF) is known to inhibit complement­mediated cell lysis. However, little is known about the role of CD55 in terms of cancer therapy. The present study aimed to demonstrate that increased levels of CD55 are strongly correlated with the progression of colorectal cancer. A novel CD55 chimeric monoclonal antibody was developed that may boost the immune response, thereby suppressing cancer. The CD55 antibody treatment activated complement and therefore suppressed the proliferation, invasion and migration of colorectal cancer cells. This tumoricidal activity is partly explained by the inflammatory response via the activation of proinflammatory cytokines. In addition, the CD55 antibody treatment synergistically enhanced the tumoricidal activity of 5­FU in colorectal cancer cells, suggesting that combined treatment may be a better strategy in colorectal cancer therapy.

Asunto(s)

Anticuerpos Monoclonales/farmacología , Antígenos CD55/genética , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/tratamiento farmacológico , Antígenos CD55/inmunología , Antígenos CD55/farmacología , Movimiento Celular/efectos de los fármacos , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/patología , Proteínas del Sistema Complemento/genética , Citotoxicidad Inmunológica/efectos de los fármacos , Sinergismo Farmacológico , Fluorouracilo/farmacología , Células HT29 , Humanos , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Metástasis de la Neoplasia

RESUMEN

Studies conducted in animal models have suggested that membrane complement regulatory proteins play an important role in the pathophysiology of coronary artery disease (CAD). In this study, a total of 100 individuals, with stable CAD and 100 healthy controls, both groups predominantly male, were recruited. We evaluated the plasma levels of complement regulatory proteins (Cregs) CD35, CD46, CD55, and CD59 and their surface expression on granulocytes, lymphocytes, and monocytes by flow cytometry. The mRNA expression of these Cregs in total leukocytes was determined by quantitative PCR. The soluble forms of Cregs, C3c, Mannose binding protein-associated serine protease 2 (MASP-2), Platelet activating factor-acetyl hydrolase (PAF-AH), and inflammatory cytokines were quantified by ELISA. High plasma levels of C3c, indicative of complement activation, in addition to significantly low levels of Cregs, were observed in CAD patients. A significantly lower expression of CD46 and CD55 on the surface of lymphocytes, monocytes, and granulocytes and higher surface expression of CD35 and CD59 on granulocytes (p < 0.0001) was seen in CAD patients as compared to healthy donors. The high expression of CD59 on granulocytes positively correlated with the severity of disease and may serve as a potential marker of disease progression in CAD.

Asunto(s)

Antígenos CD55/inmunología , Antígenos CD59/inmunología , Proteínas del Sistema Complemento/inmunología , Enfermedad de la Arteria Coronaria/inmunología , Leucocitos/inmunología , Proteína Cofactora de Membrana/inmunología , Receptores de Complemento 3b/inmunología , Biomarcadores/metabolismo , Estudios de Casos y Controles , Activación de Complemento/inmunología , Enfermedad de la Arteria Coronaria/metabolismo , Femenino , Humanos , Leucocitos/metabolismo , Linfocitos/inmunología , Linfocitos/metabolismo , Masculino , Persona de Mediana Edad , Monocitos/inmunología , Monocitos/metabolismo , Neutrófilos/inmunología , Neutrófilos/metabolismo

RESUMEN

Complement defects are associated with an enhanced risk of a broad spectrum of infectious as well as systemic or local inflammatory and thrombotic disorders. Inherited complement deficiencies have been described for virtually all complement components but can be mimicked by autoantibodies, interfering with the activity of specific complement components, convertases or regulators. While being rare, diseases related to complement deficiencies are often severe with a frequent but not exclusive manifestation during childhood. Whereas defects of early components of the classical pathway significantly increase the risk of autoimmune disorders, lack of components of the terminal pathway as well as of properdin are associated with an enhanced susceptibility to meningococcal infections. The impaired synthesis or function of C1 inhibitor results in the development of hereditary angioedema (HAE). Furthermore, complement dysregulation causes renal disorders such as atypical hemolytic uremic syndrome (aHUS) or C3 glomerulopathy (C3G) but also age-related macular degeneration (AMD). While paroxysmal nocturnal hemoglobinuria (PNH) results from the combined deficiency of the regulatory complement proteins CD55 and CD59, which is caused by somatic mutation of a common membrane anchor, isolated CD55 or CD59 deficiency is associated with the CHAPLE syndrome and polyneuropathy, respectively. Here, we provide an overview on clinical disorders related to complement deficiencies or dysregulation and describe diagnostic strategies required for their comprehensive molecular characterization - a prerequisite for informed decisions on the therapeutic management of these disorders.

Asunto(s)

Vía Alternativa del Complemento/inmunología , Proteínas del Sistema Complemento/inmunología , Enfermedades por Deficiencia de Complemento Hereditario/inmunología , Antígenos CD55/inmunología , Antígenos CD59/inmunología , Hemoglobinuria Paroxística/inmunología , Humanos

RESUMEN

The innate immune complement system helps clear invading pathogens by forming membrane attack complexes (MACs) on their surface. Abnormal activation of the complement system may aggravate atopic dermatitis (AD) symptoms in AD patients. Here, we investigated the anti-AD effects of LTAs isolated from Lactobacillus plantarum (pLTA) and Staphylococcus aureus (aLTA) by examination of complement regulatory proteins (CRPs). Combination treatment with pLTA and aLTA increased CD55 and CD59 production in HaCaT cells. The regulation of CD55 and CD59 was mediated by p38 mitogen-activated protein kinase (p38) signaling pathways in pLTA- and aLTA-treated cells. Complement-dependent cytotoxicity (CDC) and bactericidal assays revealed that combination treatment with pLTA and aLTA down-regulated the complement system. In experiments using an irritant contact dermatitis (ICD)-induced mouse model, the levels of MAC and C3 convertase (C3C) were lower in serum collected from pLTA- and aLTA-injected mice than in serum from mice who were untreated or received pLTA or aLTA alone. Combination treatment also inhibited IgE and CCL2 levels in ICD mice. On the other hand, IFN-γ level was significantly increased, indicating that combination treatment switches the Th2 response to a Th1 response. Our results suggest that combination treatment with LTAs could be a good therapeutic approach to alleviate AD by reducing formation of MACs and inducing a Th1 response.

Asunto(s)

Antígenos CD55/inmunología , Antígenos CD59/inmunología , Dermatitis Atópica , Lactobacillus plantarum/química , Lipopolisacáridos , Staphylococcus aureus/química , Ácidos Teicoicos , Regulación hacia Arriba/efectos de los fármacos , Animales , Línea Celular , Dermatitis Atópica/tratamiento farmacológico , Dermatitis Atópica/inmunología , Dermatitis Atópica/patología , Humanos , Lipopolisacáridos/química , Lipopolisacáridos/aislamiento & purificación , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos BALB C , Ácidos Teicoicos/química , Ácidos Teicoicos/aislamiento & purificación , Ácidos Teicoicos/farmacología

RESUMEN

Staphylococcus aureus is frequently isolated from patients with community-acquired pneumonia and acute respiratory distress syndrome (ARDS). ARDS is associated with staphylococcal phosphatidylinositol-specific phospholipase C (PI-PLC); however, the role of PI-PLC in the pathogenesis and progression of ARDS remains unknown. Here, we showed that recombinant staphylococcal PI-PLC possesses enzyme activity that causes shedding of glycosylphosphatidylinositol-anchored CD55 and CD59 from human umbilical vein endothelial cell surfaces and triggers cell lysis via complement activity. Intranasal infection with PI-PLC-positive S. aureus resulted in greater neutrophil infiltration and increased pulmonary oedema compared with a plc-isogenic mutant. Although indistinguishable proinflammatory genes were induced, the wild-type strain activated higher levels of C5a in lung tissue accompanied by elevated albumin instillation and increased lactate dehydrogenase release in bronchoalveolar lavage fluid compared with the plc- mutant. Following treatment with cobra venom factor to deplete complement, the wild-type strain with PI-PLC showed a reduced ability to trigger pulmonary permeability and tissue damage. PI-PLC-positive S. aureus induced the formation of membrane attack complex, mainly on type II pneumocytes, and reduced the level of CD55/CD59, indicating the importance of complement regulation in pulmonary injury. In conclusion, S. aureus PI-PLC sensitised tissue to complement activation leading to more severe tissue damage, increased pulmonary oedema, and ARDS progression.

Asunto(s)

Proteínas Bacterianas/metabolismo , Proteínas del Sistema Complemento/metabolismo , Fosfoinositido Fosfolipasa C/metabolismo , Edema Pulmonar/inmunología , Edema Pulmonar/microbiología , Síndrome de Dificultad Respiratoria/microbiología , Infecciones Estafilocócicas/inmunología , Staphylococcus aureus/enzimología , Células Epiteliales Alveolares/enzimología , Células Epiteliales Alveolares/inmunología , Células Epiteliales Alveolares/microbiología , Animales , Proteínas Bacterianas/genética , Antígenos CD55/inmunología , Antígenos CD59/inmunología , Citocinas/metabolismo , Glicosilfosfatidilinositoles/inmunología , Glicosilfosfatidilinositoles/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ratones , Ratones Endogámicos BALB C , Fosfoinositido Fosfolipasa C/genética , Edema Pulmonar/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Síndrome de Dificultad Respiratoria/inmunología , Síndrome de Dificultad Respiratoria/metabolismo , Infecciones Estafilocócicas/metabolismo , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo

RESUMEN

Recent studies traced inflammatory bowel disease in some patients to deficiency of CD55 [decay-accelerating factor (DAF)], but the mechanism underlying the linkage remained unclear. Herein, we studied the importance of DAF in enabling processes that program tolerance in the gut and the eye, two immune-privileged sites where immunosuppressive responses are continuously elicited. Unlike oral feeding or ocular injection of ovalbumin in wild-type (WT) mice, which induced dominant immune tolerance, identical treatment of DAF-/- mice or DAF-/- to WT bone marrow chimeras did not. While 10% to 30% of mesenteric and submandibular lymph node CD4+ cells became robust T-regulatory cells (Tregs) in WT forkhead box P3 (Foxp3)-green fluorescent protein mice, few in either site became Tregs with little suppressor activity in DAF-/- Foxp3-green fluorescent protein mice. Phenotyping of CD103+ dendritic cells (DCs) from the ovalbumin-fed DAF-/- mice showed impaired expression of inducer of costimulation (ICOS) ligand, programmed death receptor 1-ligand 1 (PD1-L1), CxxxC chemokine receptor 1 (Cx3CR1), CCR7, and CCR9. Analyses of elicited DAF-/- Foxp3+ Tregs showed reduced expression of interferon regulatory factor 8 (IRF-8)/aldehyde dehydrogenase 1 family member A2 (Aldh1a2) and glycoprotein A repetitions predominant/latency-associated protein associated with Treg transforming growth factor-ß production and presentation, as well as integrin ß6/integrin ß8 associated with Treg and CD103+ DC transforming growth factor-ß release. Thus, DAF is required for the properties of CD103+ DCs and their naïve CD4+ cell partners that together program tolerance.

Asunto(s)

Antígenos CD/inmunología , Enfermedades Autoinmunes/inmunología , Antígenos CD55/inmunología , Células Dendríticas/inmunología , Tolerancia Inmunológica , Cadenas alfa de Integrinas/inmunología , Animales , Antígenos CD/genética , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/patología , Enfermedades Autoinmunes/prevención & control , Antígenos CD55/genética , Células Dendríticas/patología , Cadenas alfa de Integrinas/genética , Cadenas beta de Integrinas/genética , Cadenas beta de Integrinas/inmunología , Ratones , Ratones Noqueados , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patología

RESUMEN

Anti-phospholipid syndrome (APS) is characterized by recurrent pathological pregnancy, arterial or venous thrombosis in the presence of anti-phospholipid antibody (aPL). Complement activation is recognized as an intermediate link leading to placental thrombosis and placental inflammation in APS model mice. Decay accelerating factor (DAF, CD55), MAC-inhibitory protein (MAC-IP, CD59) and membrane co-factor protein (MCP, CD46) are important complement inhibitory proteins (CIPs) highly expressed in normal placenta to curb excessive complement activation and its mediated injuries. Anti-ß2 glycoprotein I (anti-ß2GPI) antibody is an important aPL. We found that placental DAF and CD46 decreased in ß2GPI passively immunized APS model mice, accompanied by C3 deposition, neutrophil infiltration and increased proinflammatory cytokine levels detected in its placenta. Progesterone supplement can up-regulate DAF but not CD46 expression, curb C3 activation and decrease proinflammatory cytokines levels to reduce fetal loss frequency. Progesterone receptor antagonist (mifepristone) or knock-down DAF with specific siRNA, above the protective effects of progesterone, were significantly weakened. Another sex hormone, oestrogen, has no significant effect on placental DAF and C3 contents and fetal loss frequency in the APS mice model. This may be an important mechanism by which progesterone induces maternal-fetal immune tolerance. At the same time, it may provide evidence for the use of progesterone in APS abortion patients.

Asunto(s)

Síndrome Antifosfolípido/inmunología , Antígenos CD55/inmunología , Placenta/inmunología , Placenta/lesiones , Complicaciones del Embarazo/inmunología , Progesterona/farmacología , Regulación hacia Arriba/efectos de los fármacos , Animales , Síndrome Antifosfolípido/patología , Femenino , Ratones , Ratones Endogámicos BALB C , Placenta/patología , Embarazo , Complicaciones del Embarazo/tratamiento farmacológico , Complicaciones del Embarazo/patología , Regulación hacia Arriba/inmunología

RESUMEN

Xenotransplantation research has made considerable progress in recent years, largely through the increasing availability of pigs with multiple genetic modifications. We suggest that a pig with nine genetic modifications (ie, currently available) will provide organs (initially kidneys and hearts) that would function for a clinically valuable period of time, for example, >12 months, after transplantation into patients with end-stage organ failure. The national regulatory authorities, however, will likely require evidence, based on in vitro and/or in vivo experimental data, to justify the inclusion of each individual genetic modification in the pig. We provide data both from our own experience and that of others on the advantages of pigs in which (a) all three known carbohydrate xenoantigens have been deleted (triple-knockout pigs), (b) two human complement-regulatory proteins (CD46, CD55) and two human coagulation-regulatory proteins (thrombomodulin, endothelial cell protein C receptor) are expressed, (c) the anti-apoptotic and "anti-inflammatory" molecule, human hemeoxygenase-1 is expressed, and (d) human CD47 is expressed to suppress elements of the macrophage and T-cell responses. Although many alternative genetic modifications could be made to an organ-source pig, we suggest that the genetic manipulations we identify above will all contribute to the success of the initial clinical pig kidney or heart transplants, and that the beneficial contribution of each individual manipulation is supported by considerable experimental evidence.

Asunto(s)

Animales Modificados Genéticamente/genética , Rechazo de Injerto/prevención & control , Porcinos/genética , Trasplante Heterólogo , Animales , Animales Modificados Genéticamente/inmunología , Antígeno CD47/genética , Antígeno CD47/inmunología , Antígenos CD55/genética , Antígenos CD55/inmunología , Receptor de Proteína C Endotelial/genética , Receptor de Proteína C Endotelial/inmunología , Galactosiltransferasas/deficiencia , Galactosiltransferasas/genética , Galactosiltransferasas/inmunología , Técnicas de Sustitución del Gen , Técnicas de Inactivación de Genes , Rechazo de Injerto/inmunología , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/inmunología , Humanos , Proteína Cofactora de Membrana/genética , Proteína Cofactora de Membrana/inmunología , Oxigenasas de Función Mixta/deficiencia , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/inmunología , N-Acetilgalactosaminiltransferasas/deficiencia , N-Acetilgalactosaminiltransferasas/genética , N-Acetilgalactosaminiltransferasas/inmunología , Porcinos/inmunología , Trombomodulina/genética , Trombomodulina/inmunología

RESUMEN

Scintillation proximity assay (SPA) is a type of radioimmunoassay (RIA). We apply ultrasound enhancement to the general SPA. All assay procedures, including the antibody coating and radiolabeled antigen binding are achieved by simply mixing then standing for 5 min in an ultrasound chamber. No additional incubation time is required. To further demonstrate the capability of the UE-SPA, a quantitative measurement of CD55 in various grades of colon tumors was assessed on human tissue slides. The results showed a significant correlation between CD55 expression and tumorigenesis. In conclusion, we confirmed that UE-SPA is a reliable, rapid and alternative to RIA.

Asunto(s)

Antígenos CD55/análisis , Radioinmunoensayo/métodos , Anticuerpos Monoclonales/inmunología , Antígenos CD55/inmunología , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Humanos , Sonicación

RESUMEN

Pig-to-human organ transplantation has drawn attention in recent years due to the potential use of pigs as an alternative source of human donor organs. While GGTA1 knockout (GTKO) can protect xenografts from hyperacute rejection, complement-dependent cytotoxicity might still contribute to this type of rejection. To prolong the xenograft survival, we utilized a T2A-mediated pCMV-hCD55-T2A-hCD59-Neo vector and transfected the plasmid into GTKO Diannan miniature pig fetal fibroblasts. After G418 selection combined with single-cell cloning culture, four colonies were obtained, and three of these were successfully transfected with the hCD55 and hCD59. One of the three colonies was selected as donor cells for somatic cell nuclear transfer (SCNT). Then, the reconstructed embryos were transferred into eight recipient gilts, resulting in four pregnancies. Three of the pregnant gilts delivered, yielding six piglets. Only one piglet carried hCD55 and hCD59 genetic modification. The expression levels of the GGTA1, hCD55, and hCD59 in the tissues and fibroblasts of the piglet were determined by q-PCR, fluorescence microscopy, immunohistochemical staining, and western blotting analyses. The results showed the absence of GGTA1 and the coexpression of the hCD55 and hCD59. However, the mRNA expression levels of hCD55 and hCD59 in the GTKO/hCD55/hCD59 pig fibroblasts were lower than that in human 293T cells, which may be caused by low copy number and/or CMV promoter methylation. Furthermore, we performed human complement-mediated cytolysis assays using human serum solutions from 0 to 60%. The result showed that the fibroblasts of this triple-gene modified piglet had greater survival rates than that of wild-type and GTKO controls. Taken together, these results indicate that T2A-mediated polycistronic vector system combined with SCNT can effectively generate multiplex genetically modified pigs, additional hCD55 and hCD59 expression on top of a GTKO genetic background markedly enhance the protective effect towards human serum-mediated cytolysis than those of GTKO alone. Thus, we suggest that GTKO/hCD55/hCD59 triple-gene-modified Diannan miniature pig will be a more eligible donor for xenotransplantation.

Asunto(s)

Antígenos CD55/genética , Antígenos CD59/genética , Galactosiltransferasas/deficiencia , Vectores Genéticos/genética , Técnicas de Transferencia Nuclear , Péptidos/genética , Animales , Animales Modificados Genéticamente , Antígenos CD55/inmunología , Antígenos CD55/metabolismo , Antígenos CD59/inmunología , Antígenos CD59/metabolismo , Ensayo de Actividad Hemolítica de Complemento , Femenino , Fibroblastos/metabolismo , Galactosiltransferasas/metabolismo , Expresión Génica , Humanos , Péptidos/química , Embarazo , ARN Mensajero/metabolismo , Porcinos , Porcinos Enanos/genética , Distribución Tisular , Trasplante Heterólogo

RESUMEN

The control of complement activation within embryo-endometrium environment is critical for embryo survival. Cell evasion from complement attack requires interaction of complement regulatory proteins (CRPs) with cell adhesion αvß3 integrin. We aim to compare the expression of CRPs in endometria of women with and without endometriosis and to examine the molecular interaction of decay accelerating factor (DAF) with αvß3 integrin. Endometrial expression of Membrane cofactor protein (CD46), Decay accelerating factor (DAF), Membrane attack complex inhibitory factor (CD59) and ß3 integrin subunit were determined through menstrual cycle by immunohistochemistry. DAF protein quantity was determined by Western blot and mRNA levels measured in epithelial cells isolated by laser capture microdissection (LCM). Using in vitro assay, we examined DAF and ß3 integrin expression through paracrine regulation between endometrial compartments. To determine whether ß3 integrin and DAF interacts in vivo, endometrial samples were subjected to immunoprecipitation and colocalization using dual immunofluorescence technique. DAF and ß3 integrin expression were significantly low in samples from women with endometriosis during mid secretory phase. This observation was supported by decreased DAF protein quantity; faint DAF and ß3 integrin interaction and reduced mRNA levels in cells dissected by LCM. Moreover epithelial DAF and ß3 integrin expression through paracrine regulation by progesterone from stromal compartment was disrupted in endometriosis. Endometria from women with endometriosis exhibits aberrant expression of complement proteins. The abnormal DAF expression potentially compromises embryo survival, contributing to understand the implantation failure in women with endometriosis.

Asunto(s)

Antígenos CD55/metabolismo , Proteínas del Sistema Complemento/inmunología , Endometriosis/inmunología , Endometrio/inmunología , Integrina beta3/metabolismo , Adulto , Biopsia , Antígenos CD55/inmunología , Activación de Complemento/inmunología , Proteínas del Sistema Complemento/metabolismo , Implantación del Embrión/inmunología , Endometriosis/patología , Endometrio/patología , Femenino , Humanos , Integrina beta3/inmunología , Ciclo Menstrual/inmunología , Comunicación Paracrina/inmunología , Progesterona/metabolismo