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1.
Bone Marrow Transplant ; 50(7): 924-30, 2015 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-25915810

RESUMEN

Fms-like tyrosine kinase-3 ligand (Flt3L) uniquely binds the Flt3 (CD135) receptor expressed on hematopoietic stem cells (HSCs), early progenitor cells, immature thymocytes and steady-state dendritic cells (DCs) and induces their proliferation, differentiation, development and mobilization in the bone marrow, peripheral blood and lymphoid organs. CDX-301 has an identical amino-acid sequence and comparable biological activity to the previously tested rhuFlt3L, which ceased clinical development over a decade ago. This Phase 1 trial assessed the safety, pharmacokinetic, pharmacodynamic and immunologic profile of CDX-301, explored alternate dosing regimens and examined the impact of rhuFlt3L on key immune cell subsets. Thirty healthy volunteers received CDX-301 (1-75 µg/kg/day) over 5-10 days. One event of Grade 3 community-acquired pneumonia occurred. There were no other infections, dose-limiting toxicities or serious adverse events. CDX-301 resulted in effective peripheral expansion of monocytes, hematopoietic stem and progenitor cells and key subsets of myeloid DCs and plasmacytoid DCs, with no clear effect on regulatory T cells. These data from healthy volunteers support the potential for CDX-301, as monotherapy or in combination with other agents, in various indications including allogeneic HSC transplantation and immunotherapy, but the effects of CDX-301 will need to be investigated in each of these patient populations.


Asunto(s)
Células Dendríticas/inmunología , Trasplante de Células Madre Hematopoyéticas/métodos , Acondicionamiento Pretrasplante/métodos , Tirosina Quinasa 3 Similar a fms/genética , Tirosina Quinasa 3 Similar a fms/metabolismo , Adolescente , Adulto , Femenino , Voluntarios Sanos , Humanos , Masculino , Persona de Mediana Edad , Adulto Joven
2.
Arterioscler Thromb Vasc Biol ; 20(9): 2106-12, 2000 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-10978256

RESUMEN

Using a vaccine approach, we immunized New Zealand White rabbits with a peptide containing a region of cholesteryl ester transfer protein (CETP) known to be required for neutral lipid transfer function. These rabbits had significantly reduced plasma CETP activity and an altered lipoprotein profile. In a cholesterol-fed rabbit model of atherosclerosis, the fraction of plasma cholesterol in HDL was 42% higher and the fraction of plasma cholesterol in LDL was 24% lower in the CETP-vaccinated group than in the control-vaccinated group. Moreover, the percentage of the aorta surface exhibiting atherosclerotic lesion was 39.6% smaller in the CETP-vaccinated rabbits than in controls. The data reported here demonstrate that CETP activity can be reduced in vivo by vaccination with a peptide derived from CETP and support the concept that inhibition of CETP activity in vivo can be antiatherogenic. In addition, these studies suggest that vaccination against a self-antigen is a viable therapeutic strategy for disease management.


Asunto(s)
Aorta/patología , Arteriosclerosis/metabolismo , Proteínas Portadoras/inmunología , Glicoproteínas , Vacunas Sintéticas/inmunología , Animales , Anticuerpos/sangre , Arteriosclerosis/inmunología , Arteriosclerosis/patología , Arteriosclerosis/terapia , Western Blotting , Células CHO , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Transferencia de Ésteres de Colesterol , Colesterol en la Dieta/farmacología , Cricetinae , Modelos Animales de Enfermedad , Humanos , Pruebas de Función Renal , Lipoproteínas/análisis , Conejos , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Vacunas Sintéticas/administración & dosificación
3.
Biotechnol Appl Biochem ; 31(1): 5-13, 2000 02.
Artículo en Inglés | MEDLINE | ID: mdl-10669397

RESUMEN

We sought to produce a complement inhibitory protein possessing oligosaccharides specifically modified to contain the sialyl Lewis x (sLe(x)) moiety. This modified glycoprotein could combine anti-complement activity with the ability to inhibit selectin-mediated interactions and concentrate this activity to sites of activated endothelium where selectins are upregulated. Soluble complement receptor type 1 (sCR1), previously shown to be effective in inhibiting the complement cascade, was produced in a cell line capable of adding fucose to N-linked oligosaccharides in the alpha1-3 linkage, which is necessary for sLe(x) glycosylation. The glycoprotein purified from these cells was designated sCR1sLe(x), and may prove to be more effective than sCR1 in some clinical applications. Detailed analysis and characterization of sCR1sLe(x) was performed to confirm that the N-linked oligosaccharides possessed sLe(x) moieties and also to determine the extent of sLe(x) glycosylation. The glycoproteins were characterized by oligosaccharide profiling, sequencing, linkage analysis and quantified by differential enzymic digestion, using fluorophore-assisted carbohydrate electrophoresis. The major glycans were identified as biantennary oligosaccharides (including sialylated and non-core fucosylated glycans). The linkages of sialic acid and the branched fucose were analysed by digestion with linkage-specific enzymes and subsequent separation by electrophoresis. All data were consistent with the presence of sLe(x) moieties on the N-linked oligosaccharides of sCR1sLe(x). sCR1sLe(x) is a prime example of a recombinant protein expressed with oligosaccharides engineered for a specific biological function, and produced using a commercially viable method.


Asunto(s)
Glicoproteínas/química , Antígeno Lewis X/química , Oligosacáridos/química , Receptores de Complemento/metabolismo , Conformación de Carbohidratos , Secuencia de Carbohidratos , Glicoproteínas/genética , Glicoproteínas/metabolismo , Glicosilación , Antígeno Lewis X/análisis , Antígeno Lewis X/metabolismo , Oligosacáridos/análisis , Oligosacáridos/metabolismo , Receptores de Complemento/química , Receptores de Complemento/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
4.
Br J Pharmacol ; 128(5): 945-52, 1999 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-10556930

RESUMEN

1 This study investigated the effects of soluble complement receptor type 1 (sCR1) or sCR1sLex, agents which function as a complement inhibitor or as a combined complement inhibitor and selectin adhesion molecule antagonist, respectively, on the infarct size and cardiac troponin T (cTnT) release caused by regional myocardial ischaemia and reperfusion in the rat. 2 Eighty-two, male Wistar rats were subjected to 30 min occlusion of the left anterior descending coronary artery (LAD) followed by 2 h of reperfusion. Haemodynamic parameters were continuously recorded and at the end of the experiments infarct size (with p-nitro-blue tetrazolium) and cTnT release were determined. 3 Infusion of sCR1 (1, 5 or 15 mg kg-1, each n=7) or sCR1sLe(x) (1, 5 or 15 mg kg-1, n=7, 13 or 13, respectively) 5 min prior to LAD-reperfusion caused a reduction in infarct size from 59+/-2% (PBS - control, n=12) to 46+/-6%, 25+/-9% and 37+/-6% or 42+/-6%, 35+/-6% and 35+/-4%, respectively. 4 Infusion of sCR1 (15 mg kg-1, n=5) or sCR1sLe(x) (15 mg kg-1, n=5) also reduces the myocardial TnT release from 80+/-20 ng ml-1 (control) to 13+/-7 or 4+/-1 ng ml-1, respectively. 5 Thus, sCR1 or sCRsLe(x) significantly reduce infarct size and cardiac TnT release caused by 30 min of regional myocardial ischaemia and 2 h of reperfusion in the rat. The mechanisms of the cardioprotective effects of sCR1 or sCR1sLe(x) are not entirely clear, but may be due complement inhibition and/or prevention of the adhesion and activation of neutrophils.


Asunto(s)
Fármacos Cardiovasculares/farmacología , Infarto del Miocardio/patología , Oligosacáridos/farmacología , Receptores de Complemento/química , Animales , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/fisiología , Glicosilación , Frecuencia Cardíaca/efectos de los fármacos , Frecuencia Cardíaca/fisiología , Hemodinámica/efectos de los fármacos , Masculino , Monocitos/fisiología , Isquemia Miocárdica/patología , Miocardio/citología , Miocardio/metabolismo , Ratas , Ratas Wistar , Proteínas Recombinantes/farmacología , Antígeno Sialil Lewis X , Troponina T/sangre
5.
J Immunol ; 163(6): 3549-58, 1999 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-10477630

RESUMEN

In vitro complement-mediated lysis of heavily sensitized sheep erythrocytes by C4-deficient (C4D) guinea pig and C2-deficient (C2D) human sera was demonstrated some years ago. It was postulated that these "complement-bypass" pathways resulted from activation of C1 and components of the alternative pathway. We used normal, C2D, and C4D guinea pigs in a Forssman shock model to test the in vivo relevance of the C2- and C4-bypass pathways of complement activation. High concentrations of both anti-Forssman Ab and C2D or C4D guinea pig serum induced efficient lysis of sheep erythrocytes in vitro. The most efficient lysis was observed when IgG Ab and C2D guinea pig serum were used. Blocking either the classical pathway (treatments with EGTA-Mg2+ or soluble recombinant complement receptor type 1 (sCR1)) or the alternative pathway (treatment with heating at 50 degrees C, sCR1, or soluble recombinant CR1 lacking the first of the four long homologous repeat sequences (sCR1[desLHR-A])) inhibited lysis; both pathways were required for lysis of sheep erythrocytes by C2D and C4D guinea pig sera. i.v. injection of anti-Forssman Ab in normal guinea pigs resulted in rapid death from pulmonary shock, whereas C4D guinea pigs had no adverse effect. Surprisingly, C2D guinea pigs either died in a delayed fashion or had a sublethal reaction. sCR1 treatment prevented Forssman shock in both normal and C2D guinea pigs, whereas sCR1[desLHR-A] prevented Forssman shock only in C2D animals. Our results suggest that the C2-bypass pathway occurs in vivo to produce tissue damage. Activation of complement in the absence of C2 appears to be far more efficient than in the absence of C4.


Asunto(s)
Activación de Complemento/inmunología , Complemento C2/deficiencia , Hemólisis/inmunología , Inmunoglobulina G/administración & dosificación , Pulmón/patología , Anafilaxia/inmunología , Anafilaxia/patología , Animales , Anticuerpos Heterófilos/administración & dosificación , Activación de Complemento/genética , Complemento C2/genética , Complemento C4/deficiencia , Complemento C4/genética , Vía Alternativa del Complemento/genética , Vía Alternativa del Complemento/inmunología , Vía Clásica del Complemento/genética , Vía Clásica del Complemento/inmunología , Cobayas , Hemólisis/genética , Humanos , Inyecciones Intravenosas , Pulmón/inmunología , Modelos Inmunológicos , Edema Pulmonar/inmunología , Edema Pulmonar/patología
6.
Science ; 285(5427): 595-9, 1999 Jul 23.
Artículo en Inglés | MEDLINE | ID: mdl-10417391

RESUMEN

Glycoprotein adhesion receptors such as selectins contribute to tissue injury in stroke. Ischemic neurons strongly expressed C1q, which may target them for complement-mediated attack or C1qRp-mediated clearance. A hybrid molecule was used to simultaneously inhibit both complement activation and selectin-mediated adhesion. The extracellular domain of soluble complement receptor-1 (sCR1) was sialyl Lewis x glycosylated (sCR1sLex) to inhibit complement activation and endothelial-platelet-leukocyte interactions. sCR1 and sCR1sLex colocalized to ischemic cerebral microvessels and C1q-expressing neurons, inhibited neutrophil and platelet accumulation, and reduced cerebral infarct volumes. Additional benefit was conferred by sialyl Lewis x glycosylation of the unmodified parent sCR1 molecule.


Asunto(s)
Trastornos Cerebrovasculares/tratamiento farmacológico , Ataque Isquémico Transitorio/tratamiento farmacológico , Fármacos Neuroprotectores/uso terapéutico , Oligosacáridos/uso terapéutico , Receptores de Complemento/uso terapéutico , Animales , Plaquetas/fisiología , Adhesión Celular , Corteza Cerebral/irrigación sanguínea , Corteza Cerebral/inmunología , Corteza Cerebral/metabolismo , Infarto Cerebral/tratamiento farmacológico , Circulación Cerebrovascular , Trastornos Cerebrovasculares/inmunología , Trastornos Cerebrovasculares/fisiopatología , Activación de Complemento , Complemento C1q/metabolismo , Glicosilación , Humanos , Ataque Isquémico Transitorio/inmunología , Ataque Isquémico Transitorio/fisiopatología , Leucocitos/fisiología , Ratones , Neuronas/inmunología , Neuronas/metabolismo , Fármacos Neuroprotectores/administración & dosificación , Fármacos Neuroprotectores/efectos adversos , Fármacos Neuroprotectores/metabolismo , Neutrófilos/fisiología , Oligosacáridos/administración & dosificación , Oligosacáridos/efectos adversos , Oligosacáridos/metabolismo , Adhesividad Plaquetaria , Receptores de Complemento/administración & dosificación , Receptores de Complemento/metabolismo , Daño por Reperfusión/tratamiento farmacológico , Daño por Reperfusión/inmunología , Daño por Reperfusión/metabolismo , Selectinas/metabolismo , Antígeno Sialil Lewis X , Factores de Tiempo
7.
J Biol Chem ; 274(16): 11237-44, 1999 Apr 16.
Artículo en Inglés | MEDLINE | ID: mdl-10196211

RESUMEN

Soluble human complement receptor type 1 (sCR1, TP10) has been expressed in Chinese hamster ovary (CHO) DUKX-B11 cells and shown to inhibit the classical and alternative complement pathways in vitro and in vivo. A truncated version of sCR1 lacking the long homologous repeat-A domain (LHR-A) containing the C4b binding site has similarly been expressed and designated sCR1[desLHR-A]. sCR1[desLHR-A] was shown to be a selective inhibitor of the alternative complement pathway in vitro and to function in vivo. In this study, sCR1 and sCR1[desLHR-A] were expressed in CHO LEC11 cells with an active alpha(1,3)-fucosyltransferase, which makes possible the biosynthesis of the sialyl-Lewisx (sLex) tetrasaccharide (NeuNAcalpha2-3Galbeta1-4(Fucalpha1-3)GlcNAc) during post-translational glycosylation. The resulting glycoproteins, designated sCR1sLex and sCR1[desLHR-A]sLex, respectively, retained the complement regulatory activities of their DUKX B11 counterparts, which lack alpha(1-3)-fucose. Carbohydrate analysis of purified sCR1sLex and sCR1[desLHR-A]sLex indicated an average incorporation of 10 and 8 mol of sLex/mol of glycoprotein, respectively. sLex is a carbohydrate ligand for the selectin adhesion molecules. sCR1sLex was shown to specifically bind CHO cells expressing cell surface E-selectin. sCR1[desLHR-A]sLex inhibited the binding of the monocytic cell line U937 to human aortic endothelial cells, which had been activated with tumor necrosis factor-alpha to up-regulate the expression of E-selectin. sCR1sLex inhibited the binding of U937 cells to surface-adsorbed P-selectin-IgG. sCR1sLex and sCR1[desLHR-A]sLex have thus demonstrated both complement regulatory activity and the capacity to bind selectins and to inhibit selectin-mediated cell adhesion in vitro.


Asunto(s)
Activación de Complemento/efectos de los fármacos , Glicoproteínas/farmacología , Selectinas/metabolismo , Animales , Western Blotting , Células CHO , Adhesión Celular , Cricetinae , Electroforesis/métodos , Citometría de Flujo , Glicoproteínas/química , Humanos , Espectrometría de Masas , Monosacáridos/análisis , Oligosacáridos/análisis , Unión Proteica , Ensayo de Unión Radioligante , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Células U937 , Regulación hacia Arriba
8.
Transplantation ; 66(6): 723-32, 1998 Sep 27.
Artículo en Inglés | MEDLINE | ID: mdl-9771835

RESUMEN

BACKGROUND: Lung dysfunction after transplantation continues to be a significant clinical problem. Soluble complement receptor 1 (sCR1) is a potent inhibitor of complement activation. We evaluated the inhibitory effect of sCR1 on complement activation and reperfusion injury in pig lung allografts. METHODS: In a randomized and blinded study, left lung transplantation was performed in 13 pigs. Donor lungs were flushed and then stored for 30 hr at 4 degrees C. Control pigs (n=7) received saline, and the treatment group (n=6) received 15 mg/kg sCR1 1 hr before reperfusion. One hour after reperfusion, the right pulmonary artery was clamped for 10 min to assess the function of the transplanted lung. Pulmonary function was assessed again on day 3. RESULTS: Complement inhibition was 93% in the sCR1 group and returned to baseline (8% inhibition) after 3 days. There was a trend toward a higher partial pressure of oxygen at 1 hr in the sCR1 group compared with the control group (mean +/- SE: 408+/-42 mmHg vs. 288+/-69 mmHg, P = 0.19). Alveolar ventilation was better in the sCR1 group than in the control group (P = 0.01) at 1 hr. Mixed venous saturation was significantly lower in the control group at both 1 hr (P = 0.02) and 3 days (P = 0.001). The wet/dry weight of the lung tissue was lower in the sCR1 group compared with the control group on day 3 (P < 0.05). Chemiluminescence, an index of phagocyte priming, was lower in the sCR1 group when cells were stimulated with complement opsonized zymosan but not when stimulated with zymosan or phorbol myristate acetate. CONCLUSION: sCR1 improves ventilation, reduces pulmonary edema, and may be beneficial in improving posttransplant lung oxygenation.


Asunto(s)
Proteínas Inactivadoras de Complemento/farmacología , Trasplante de Pulmón , Pulmón/efectos de los fármacos , Pulmón/fisiología , Receptores de Complemento/fisiología , Animales , Células CHO , Complemento C3b/análisis , Cricetinae , Técnica del Anticuerpo Fluorescente , Pulmón/citología , Oxígeno/sangre , Oxígeno/metabolismo , Presión Parcial , Distribución Aleatoria , Receptores de Complemento/sangre , Porcinos
9.
Clin Exp Immunol ; 112(1): 27-33, 1998 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-9566786

RESUMEN

Complement receptor type one (CR1; CD35) binds and processes C3b and C4b opsonized immune complexes and regulates complement activation. We have characterized the epitopes of 13 previously reported and seven new MoAbs to human CR1. The MoAbs formed seven groups based on their reactivity with a panel of deletion forms of CR1. Seventeen of the MoAbs reacted with CR1 at more than one site, a consequence of its repetitive sequence. All five of the MoAbs recognizing epitopes in the nearly identical repeats 3, 10, and 17, as well as one MoAb which reacted with repeats 8 or 1/2 of 9 and 15 or 1/2 of 16, blocked cofactor activity for C3b. Knowledge of the repeats bearing the epitopes for these MoAbs should facilitate the further characterization of CR1.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Mapeo Epitopo , Receptores de Complemento 3b/inmunología , Animales , Células CHO , Cricetinae , Humanos , Epítopos Inmunodominantes/inmunología , Ratones
10.
Transplantation ; 64(9): 1248-55, 1997 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-9371664

RESUMEN

Although agents that inhibit complement activation may be beneficial in discordant xenotransplantation, it is not known whether local complement activation occurs and is deleterious after isogeneic lung transplantation. Lungs were harvested from Lewis rats subjected to 4 degrees C 6-hr preservation followed by transplantation into strain-, gender-, and weight-matched recipients. Transplanted lungs demonstrated increased immunostaining for C5b-9 compared with nontransplanted controls, confirming local complement activation in this isograft model. To investigate the physiologic relevance of complement activation in the transplanted lung, the native pulmonary artery was ligated after transplantation, and pulmonary vascular resistance (mmHg/ml/min), arterial oxygenation (mmHg), graft neutrophil infiltration (myeloperoxidase activity, deltaAbs 460 nm/min), and recipient survival were measured at 30 min. Animals received either saline (control; n=22) or soluble complement receptor type-1 (sCR1, 15 mg/kg; n=19) 2 min before reperfusion. Animals treated with sCR1 showed a marked reduction in serum complement hemolytic activity (CH50; 90% lower than that of control animals, P<0.001). Compared with controls, sCR1-treated animals showed reduced pulmonary vascular resistance (2.9+/-1.1 vs. 8.5+/-1.5 mmHg/ml/min, P<0.05), improved arterial oxygenation (194+/-34 vs. 91+/-17 mmHg, P<0.05), decreased neutrophil infiltration (35% decrease, P<0.005), and improved recipient survival (74% vs. 23%, P<0.005). In parallel with the reduction in complement hemolytic activity in sCR1-treated animals, immunohistology of the transplanted lung revealed decreased C5b-9 deposition compared with controls. Taken together, these data indicate that complement activation occurs after lung preservation and transplantation in an isograft model, and that inhibiting complement activation improves outcome after transplantation.


Asunto(s)
Activación de Complemento/fisiología , Rechazo de Injerto/inmunología , Hipotermia Inducida , Trasplante de Pulmón/inmunología , Pulmón , Preservación de Órganos/métodos , Animales , Masculino , Circulación Pulmonar/fisiología , Ratas , Ratas Endogámicas Lew , Trasplante Heterólogo , Trasplante Homólogo
11.
Transplantation ; 63(6): 900-2, 1997 Mar 27.
Artículo en Inglés | MEDLINE | ID: mdl-9089232

RESUMEN

A single bolus of soluble complement (C) receptor type 1 (sCR1, TP-10) has been shown to delay hyperacute rejection (HAR) of porcine cardiac xenografts (Xgs) by primate recipients. In these recipients, C activity slowly returned and C deposition was noted in the Xgs at rejection. To evaluate the effect of sustained C inhibition using sCR1 on HAR, two additional cynomolgus monkeys received porcine cardiac Xgs and a continuous infusion of sCR1. In the first recipient, Xgs survival was 5 days (120+ hr), whereas in the second, Xg survival was 7 days (168+ hr). Serial biopsies of the Xgs were remarkable for an increasing cellular infiltrate composed predominantly of neutrophils and macrophages, and the development of edema, hemorrhage, and myocyte necrosis. These findings suggest that once C-mediated HAR has been inhibited, infiltration of the Xg by these cells may lead to accelerated acute rejection, which is an additional barrier to successful longer term Xg survival.


Asunto(s)
Proteínas Inactivadoras de Complemento , Rechazo de Injerto/prevención & control , Supervivencia de Injerto/inmunología , Trasplante de Corazón/inmunología , Receptores de Complemento/inmunología , Trasplante Heterólogo/inmunología , Animales , Trasplante de Corazón/métodos , Terapia de Inmunosupresión/métodos , Infusiones Intravenosas , Macaca fascicularis , Reperfusión Miocárdica , Porcinos , Factores de Tiempo , Trasplante Heterólogo/métodos
12.
Eur J Immunol ; 26(8): 1729-35, 1996 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-8765013

RESUMEN

The human complement receptor type 1 (CR1, CD35), is a single-chain glycoprotein consisting of 30 repeating homologous protein domains known as short consensus repeats (SCR) followed by transmembrane and cytoplasmic domains. The SCR themselves, considered in groups of seven, form long homologous repeats (LHR) which have been designated LHR-A, -B, -C, and -D for the most common human allotype of CR1. A soluble deletion mutant of CR1 which lacks the first seven N-terminal SCR (LHR-A) as well as the transmembrane and cytoplasmic domains was produced and characterized. The resulting protein, designated sCR1[desLHR-A], lacks the C4b binding site found in LHR-A, but retains the two C3b binding sites found in LHR-B and -C, respectively. The functional activities of sCR1[desLHR-A] were quantitatively compared in vitro to those of soluble complement receptor type 1 (sCR1) which has been shown to retain all known functions of the native cell surface receptor. sCR1[desLHR-A] and sCR1 competed equally for the binding of dimeric C3b to erythrocyte CR1. sCR1[desLHR-A] and sCR1 were similar in their capacity to serve as a cofactor in the factor I-mediated degradation of the C3b and C4b alpha chains. sCR1[desLHR-A] and sCR1 were comparable in their capacity to inhibit erythrocyte lysis and anaphylatoxin production mediated by the alternative complement pathway. sCR1[desLHR-A], however, was significantly less effective an inhibitor of erythrocyte lysis and anaphylatoxin production than sCR1 under conditions which allow classical pathway activation. These results demonstrate sCR1[desLHR-A] to be a selective inhibitor of the alternative complement pathway in vitro.


Asunto(s)
Complemento C4b/deficiencia , Complemento C4b/genética , Proteínas Inactivadoras de Complemento/farmacología , Vía Alternativa del Complemento/genética , Glicoproteínas , Mutación/inmunología , Receptores de Complemento/deficiencia , Receptores de Complemento/genética , Receptores de Complemento/fisiología , Eliminación de Secuencia/inmunología , Unión Competitiva/inmunología , Complemento C3b/metabolismo , Proteínas Inactivadoras de Complemento/genética , Vía Alternativa del Complemento/efectos de los fármacos , Vía Clásica del Complemento/efectos de los fármacos , Humanos , Unión Proteica/inmunología , Receptores de Complemento/química , Solubilidad
13.
Transplantation ; 62(3): 336-42, 1996 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-8779679

RESUMEN

Hyperacute xenograft rejection may be modified by the activation and depletion of complement (C) using cobra venom factor (CVF). This method of prolonging xenograft survival is toxic and associated with systemic inflammation, which may potentially contribute to the pathologic features of delayed xenograft rejection. Soluble complement receptor type 1 (sCR1) inhibits both the classical and alternative C pathways and thus limits the production of proinflammatory products such as the anaphylatoxins. Hence, we investigated the effects of various sCR1 and CVF regimens, and combinations thereof, in the discordant guinea pig-to-Lewis rat cardiac xenograft model. Mean graft survival time (MST) was significantly prolonged with repetitive dosing (MST=22 hr) or continuous infusion of sCR1 (MST=32 hr) as compared with unmodified controls (MST=15 min). However, sCR1 did not prevent intragraft deposition of C3 or neutrophil infiltration and resulted in only partial inhibition of C-mediated hemolytic activity in vitro. Grafts in rats treated with a single dose of CVF (MST=67 hr) or repetitive doses of CVF (MST=69 hr) survived significantly longer than those treated with sCR1 alone, and lacked C3 deposition or neutrophil accumulation. Sera from these animals were completely depleted of C-mediated hemolytic activity. Animals treated with a single dose of CVF, or sCRI plus a single dose of CVF (MST=64 hr), had similar xenograft survival times. However, immunohistologic studies showed that addition of sCR1 to a single dose of CVF resulted in decreased macrophage activation and reduced levels of cytokines (tumor necrosis factor-alpha and interleukin-1beta) within xenografts as compared with that in recipients treated with CVF alone. Such decreased macrophage activation may result from the binding of C4b by sCR1, since combination therapy was associated with decreased intragraft C4b as compared with either therapy alone. High doses of sCR1 were well tolerated by rats and significantly prolonged discordant xenograft survival (MST=32 hr), although not to the same extent as CVF. The modification of the intragraft immune responses seen with CVF/sCR1 combination therapy may augment further therapeutic manipulations to achieve discordant xenograft survival without the attendant toxicity associated with repeated CVF administration.


Asunto(s)
Venenos Elapídicos/administración & dosificación , Supervivencia de Injerto/efectos de los fármacos , Trasplante de Corazón , Receptores de Complemento/fisiología , Trasplante Heterólogo , Animales , Proteínas del Sistema Complemento/análisis , Venenos Elapídicos/farmacología , Rechazo de Injerto/patología , Cobayas , Masculino , Miocardio/patología , Ratas , Ratas Endogámicas Lew , Proteínas Recombinantes , Solubilidad , Factores de Tiempo
16.
J Med Chem ; 38(9): 1437-45, 1995 Apr 28.
Artículo en Inglés | MEDLINE | ID: mdl-7739002

RESUMEN

The terpenoid 6,7-diformyl-3',4',4a',5',6',7',8',8a'-octahydro-4,6',7'-trihydrox y-2',5',5', 8a'-tetramethylspiro[1'(2'H)-naphthalene-2(3H)-benzofuran] (1a; K-76), a natural product of fungal origin, and its monocarboxylate sodium salt 1c (R = COONa; K-76COONa) inhibit the classical and alternative pathways of complement, and 1c was shown to inhibit the classical pathway at the C5 activation step. In an attempt to elucidate the essential pharmacophore of 1a,c, the natural product was used as a "topographical model" for the design of partial analogs retaining the desired complement inhibiting potency. Therefore, A/C/D-ring analogs have been synthesized, as shown in Scheme 1 using 3-methoxyphenol (3) and limonene chloride (5) as starting materials, which contain functional groups similar to those found on the natural product. The use of (4R)-(+)- and (4S)(-)-limonene chloride (5a,b, respectively) provided two series of compounds differing in the stereochemistry of the C-4 chiral center (limonene moiety numbering). The in vitro assay results of the inhibition of anaphylatoxin production and classical complement-mediated hemolysis revealed that 7-carboxy-2-(R,S)-methyl-2-(1'-methylcyclohexen-(4'R)-yl)-4-met hoxybenzofuran (13a) and 7-carboxy-2-(R,S)-methyl-2-(1'-methylcyclohexen-(4'S)-yl)-4-met hoxybenzofuran (13b) were active in the same range of concentrations as the natural product.


Asunto(s)
Proteínas Inactivadoras de Complemento/síntesis química , Sesquiterpenos/síntesis química , Stachybotrys/química , Animales , División Celular/efectos de los fármacos , Complemento C3a/antagonistas & inhibidores , Complemento C3a/biosíntesis , Complemento C5a/antagonistas & inhibidores , Complemento C5a/biosíntesis , Proteínas Inactivadoras de Complemento/farmacología , Diseño de Fármacos , Cobayas , Hemólisis/efectos de los fármacos , Humanos , Linfocitos/efectos de los fármacos , Ratones , Sesquiterpenos/farmacología
17.
J Biol Chem ; 269(10): 7696-701, 1994 Mar 11.
Artículo en Inglés | MEDLINE | ID: mdl-8125996

RESUMEN

The complement receptor CR1 (CD35) is a transmembrane protein composed in its extracellular portion of short consensus repeats (SCR 1-30) organized into four long homologous repeats (LHR-A, LHR-B, LHR-C, and LHR-D). Each LHR, except LHR-D, contains a binding site for C3b and/or C4b within its first four SCR. The binding reaction between CR1 and soluble dimers of C4b (C4b2) was analyzed using the native receptor on human erythrocytes and full-length recombinant CR1 expressed in stably transfected Chinese hamster ovary (CHO) cells. CR1 mutants expressed similarly were used to determine the SCR of LHR-A required for C4b2 binding and the potential of C3b binding sites in CR1 to bind C4b2. Erythrocyte CR1, CHO cells expressing full-length recombinant CR1 (ABCD), constructs ACD, and SCR(1-4)D each bound C4b2 with similar affinities (Kd, approximately 4 x 10(-7) M). Construct SCR(1-2)D bound C4b2 with lower affinity (Kd, 1.4 x 10(-6) M) indicating that SCR(1-4) are required for a fully functional C4b2 binding site. Construct SCR(15-18)D, which contains a C3b site, also bound C4b2 with lower affinity (Kd 1.2 x 10(-6) M) than its binding to C3b dimers. Constructs SCR(15-16)D and D did not bind C4b2. Each CR1 construct that bound C4b2 functioned as a cofactor for factor I-mediated cleavage to C4d.


Asunto(s)
Proteínas Portadoras/metabolismo , Complemento C4b/metabolismo , Proteínas Inactivadoras de Complemento , Glicoproteínas , Receptores de Complemento 3b/metabolismo , Animales , Unión Competitiva , Células CHO , Cricetinae , Eritrocitos/metabolismo , Humanos , Cinética , Proteínas Recombinantes/metabolismo
18.
Transplantation ; 57(3): 363-70, 1994 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-8108871

RESUMEN

The use of xenografts (Xgs) from distantly related species to relieve the increasing shortage of organs for clinical transplantation is prevented by the occurrence of hyperacute rejection (HAR). This process, in which C activation plays a central role, cannot be inhibited with currently available immunosuppressants. In two clinically relevant xenotransplantation models, this study evaluated the effect of C inhibition using recombinant soluble complement receptor type 1 (sCR1) on HAR. In an ex vivo model in which porcine cardiac Xgs were perfused with human blood, cardiac function ceased within 34 min when the perfusate blood was untreated (n = 3). When the perfusate blood was treated with sCR1 (300 micrograms/ml), cardiac Xg function was maintained for up to 4 hr (n = 3). Immunohistologic examination of these Xgs demonstrated deposition of C3b/iC3b and C3d in Xgs perfused with untreated human blood but only C3d deposition in those Xgs perfused with sCR1-treated human blood. These findings are consistent with the cofactor activity of sCR1 for factor I-mediated degradation of deposited C3b/iC3b to C3d. Treatment with sCR1 also prevented the histopathologic changes of HAR observed when untreated blood was used as the perfusate. In an in vivo pig-to-primate heterotopic cardiac xenotransplantation model, in which porcine Xgs transplanted into untreated cynomolgus monkey recipients underwent HAR in 1 hr or less (n = 3), a single intravenous bolus of sCR1 (15 mg/kg) administered to the recipient immediately before Xg reperfusion markedly inhibited total and alternative pathway serum C activity and prolonged Xg survival to between 48 and 90 hr (n = 5). These studies confirm the important role of C activation in HAR of porcine cardiac Xgs by primates and indicate that sCR1 may be a useful agent for xenotransplantation.


Asunto(s)
Rechazo de Injerto/inmunología , Receptores de Complemento/fisiología , Trasplante Heterólogo/inmunología , Animales , Anticuerpos Heterófilos/sangre , Biopsia , Supervivencia de Injerto , Haplorrinos , Trasplante de Corazón/patología , Trasplante de Corazón/fisiología , Humanos , Masculino , Microscopía Fluorescente , Modelos Biológicos , Porcinos , Trasplante Heterólogo/fisiología
19.
J Immunol Methods ; 164(2): 233-44, 1993 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-8370929

RESUMEN

A soluble, recombinant form of the human T cell receptor (TCR) beta-chain containing the V beta 3.1 sequence has been constructed, expressed in Chinese hamster ovary cells, amplified by dihydrofolate reductase selection, and purified in quantities appropriate for the generation of monoclonal antibodies (mAb). The V beta 3 sequence was chosen because of its reported elevated usage in the synovial T cells of rheumatoid arthritis patients but the approach described should be applicable to other known human V beta gene sequences. By this method, two mAb were prepared which reacted with up to 10% of normal, live peripheral blood T cells but with reactivity varying greatly among individual donors. Both mAb specifically bound to a murine T cell line transfected with a human TCR V beta 3.1 and immunoprecipitated a protein of the expected molecular weight for the TCR beta-chain. Both antibodies were mitogenic for T cells and analysis of peripheral blood lymphocyte cultures stimulated with the mAb suggested that both were specific for the V beta 3.1 subfamily and not D beta or J beta. Clones expressing V beta 3, which were derived from mAb-stimulated peripheral blood lymphocytes of a single individual, preferentially (8/13), but not exclusively, utilized the J beta 2.7 gene segment. The V beta 3.1 usage showed no preference for the CD8+ or CD4+ subpopulations of normal peripheral blood T cells.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Linfocitos T/inmunología , Animales , Secuencia de Bases , Femenino , Humanos , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Receptores de Antígenos de Linfocitos T alfa-beta/química , Proteínas Recombinantes/inmunología , Solubilidad
20.
J Biol Chem ; 267(34): 24754-61, 1992 Dec 05.
Artículo en Inglés | MEDLINE | ID: mdl-1447213

RESUMEN

The C3b/C4b receptor, also known as complement receptor type 1 (CR1, CD35), is a single chain glycoprotein consisting of 30 repeating homologous protein domains known as short consensus repeats (SCR) followed by transmembrane and cytoplasmic domains. A series of recombinant proteins derived from CR1 has been prepared and assessed for the capacity to inhibit complement lysis of the host Chinese hamster ovary (CHO) cells. The full-length recombinant CR1 inhibited human complement-mediated CHO cell lysis, and the efficiency of inhibition was directly proportional to the number of receptors/cell. The SCR 15-18 of CR1, but not SCR 15-16, inhibited complement lysis of the host CHO cell, bound monomeric C3b (Kd,app = 6.5 x 10(-7) M), and dimeric C3b (Kd = 1.8 x 10(-8) M), and served as a cofactor in the proteolysis of C3b by factor I, confirming and extending the observations of Fearon and colleagues (Kalli, K. R., Hsu, P., Bartow, T. J., Ahearn, J. M., Matsumoto, A. K., Klickstein, L. B., and Fearon, D. T. (1991) J. Exp. Med. 174, 1451-1460). The SCR 1-4 of CR1, but not SCR 1-2, also inhibited complement lysis of the host CHO cell, indicating that more than two SCR are necessary and that four SCR are sufficient for optimal C4b binding to CR1. Thus, the structural requirements for C4b binding are analogous to those for C3b binding, namely, four SCR of CR1 form the binding sites for each of these proteins. CR1 has long been recognized to regulate extrinsic complement activation, that is, to bind to and promote the degradation of fluid phase C3b and of C3b attached to immune complex. These results demonstrate that CR1 is also an intrinsic regulator of complement activation in that, under appropriate conditions, CR1 inhibits complement-mediated lysis of the cell on which it is expressed.


Asunto(s)
Proteínas del Sistema Complemento/farmacología , Receptores de Complemento 3b/fisiología , Animales , Secuencia de Bases , Células CHO , Supervivencia Celular/efectos de los fármacos , Cricetinae , Humanos , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligodesoxirribonucleótidos , Plásmidos , Reacción en Cadena de la Polimerasa/métodos , Ensayo de Unión Radioligante , Receptores de Complemento 3b/genética , Receptores de Complemento 3b/metabolismo , Proteínas Recombinantes/metabolismo , Transfección
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