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Cloning and characterization of anti-cathepsin L single chain variable fragment whose expression inhibits procathepsin L secretion in human melanoma cells

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BIOCHEMICAL JOURNAL 2002年第1期367卷 219-227页

作者： Guillaume-Rousselet, N Jean, D Frade, R Hop St Antoine INSERM U354 Ctr INSERM F-75012 Paris France

We previously demonstrated that increase of procathepsin L secretion by human melanoma cells strongly increased their tumourigenicity and switched their phenotype from low to highly metastatic. Thus, we herein analysed whether it was possible to inhibit procathepsin L secretion using anti-cathepsin L ScFv. For this purpose, we produced different forms of fusion cathepsin L in prokaryotic or eukaryotic expression systems. An anticathepsin L monoclonal antibody (mAb), named 3138, was isolated from mice immunized with purified procathepsin L-His. This 3138 mAb interacted with an epitope localized on the 156-197 amino acid sequence of cathepsin L and recognized recombinant or native forms of cathepsin L synthesized by human melanoma cells. An active anti-cathepsin L ScFv was generated and characterized from 3138 mAb heavy and light variable chains. Then, human melanoma cells were transiently co-transfected with 3138 ScFv and cathepsin L cDNAs. Data demonstrated that increase of 3138 ScFv expression in human melanoma cells totally inhibited procathepsin L secretion and induced accumulation of intracellular procathepsin L. Our results constitute the first demonstration that anti-cathepsin L ScFv could be used in human melanoma cells to inhibit procathepsin L secretion. This ScFv represents a new molecular tool to explore cell therapy of human melanomas.

关键词： cell therapy cysteine protease third component of complement

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Nutrition support in hospitalized patients with diabetes mellitus

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MAYO CLINIC PROCEEDINGS 1996年第6期71卷 587-594页

作者： McMahon, MM Rizza, RA MAYO CLIN & MAYO FDN DIV ENDOCRINOL METAB & INTERNAL MEDROCHESTERMN 55905

Many physicians will manage the care of hospitalized patients with diabetes mellitus who require parenteral nutrition or enteral tube feeding, The nutritional assessment, indications for nutrition support, estimate of nutritional needs, and biochemical monitoring guidelines for critically ill patients with diabetes are similar to those for nondiabetic patients, In general, a weight loss of up to 10% of body weight is well tolerated and, in the absence of severe stress, the provision of dextrose-containing crystalloid solutions and electrolytes is adequate for as long as 7 to 10 days, Studies that demonstrate a beneficial influence of nutrition support on clinical outcome administered nutrition for a minimum of 1 week, No data have established that support for a briefer duration is of clinical benefit, An important goal in the care of the hospitalized patient with diabetes is to avoid the extremes of hypoglycemia and hyperglycemia. Herein we provide our approach to achieving glucose control in stressed hospitalized patients with diabetes mellitus who are receiving parenteral and enteral nutrition, Although evidence is increasing that hyperglycemia impairs immune function, well-designed prospective randomized trials are needed to determine the risks, costs, and benefits of achieving glucose control and of providing nutrition support to hospitalized patients with diabetes mellitus.

关键词： C3 third component of complement PN parenteral nutrition

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INFANTILE SYSTEMIC LUPUS-ERYTHEMATOSUS WITH ONSET SIMULATING CONGENITAL NEPHROTIC SYNDROME

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JOURNAL OF PEDIATRICS 1994年第1期124卷 27-31页

作者： MASSENGILL, SF RICHARD, GA DONNELLY, WH UNIV FLORIDA DEPT PATHOL GAINESVILLE FL 32611 USA

Two white female infants were seen with congenital nephrotic syndrome at age 6 weeks and 3 months, respectively. Both had hypocomplementemia, elevated antinuclear antibody and anti-double-stranded DNA titers, and diffuse proliferative glomerulonephritis with positive immunofluorescence in their initial renal biopsy samples. Although uncommon, infantile systemic lupus erythematosus should be considered in the evaluation of congenital nephrotic syndrome.

关键词： ANA Antinuclear antibody Anti-dsDNA anti-double-stranded DNA C3 third component of complement C4 Fourth component of complement CoNS Congenital nephrotic syndrome HLA Human leukocyte antigen IVIg Intravenously administered immuneglobulin SLE Systemic lupus erythematosus

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DETERMINATION OF OPTIMAL INVITRO CONDITIONS FOR CAPRINE ALTERNATIVE complement PATHWAY ASSAY

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VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY 1992年第3-4期32卷 359-364页

作者： VENUGOPAL, G RAM, GC BANSAL, MP INDIAN VET RES INST IMMUNOL SECTIZATNAGAR 243122UTTAR PRADESHINDIA

Optimal in vitro testing conditions for caprine alternative complement pathway assay were determined. Effects of the following variables were tested: heterologous erythrocytes;pH, ionic strength and Mg2+ ion concentration of the complement diluent;incubation time and temperature. Rabbit erythrocytes were the optimal target cells. The optimal buffer conditions were: pH 8.0, ionic strength 0.06 mmol NaCl and 5mmol Mg2+ ion. Optimal incubation time and temperature were 75 min and 30-degrees-C, respectively.

关键词： ACP alternative complement pathway C3 third component of complement GVBS VBS containing 10 mM EDTA plus 0.1% gelatin (pH 7.4) VBS veronal buffered saline VBS-EGTA 2+ VBS containing 10 mM EGTA and 10 mM MgCl 2 (pH 7.4)

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Leucocyte adhesion protein deficiency in Irish setter dogs

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Veterinary Immunology and Immunopathology 1992年第3-4期32卷 261-280页

作者： Trowald-Wigh, Gunilla Håkansson, Lena Johannisson, Anders Norrgren, Leif Hård af Segerstad, Carl Department of Medicine and Surgery Swedish University of Agricultural Sciences Uppsala Sweden Department of Clinical Chemistry University Hospital Uppsala Sweden Department of Pathology Swedish University of Agricultural Sciences Uppsala Sweden Department of Genetics Uppsala University Uppsala Sweden

Investigation of 12 Irish setter puppies from six litters with severe recurrent infections, neutrophilia and low body weight revealed a leucocyte adhesion protein deficiency with a total lack of CD11b and CD18. Their neutrophil function was severely impaired with a totally absent capacity to ingest C3b-opsonized particles, a significantly impaired capacity to ingest IgG-opsonized particles and significantly diminished adherence to nylon wool when compared with neutrophils from healthy control dogs. The chemiluminescence of patient neutrophils activated by C3b-opsonized particles was, consequently, significantly decreased compared with that of control neutrophils, while the respiratory burst assayed by phorbolmyristate acid (PMA) stimulated nitroblue tetrazolium (NBT)-reduction was normal in the patient group. Random migration and chemotactic responses of patient and control neutrophils, were similar. The etiology, pathogenesis and clinical manifestations of the Irish setter leucocyte adhesion deficiency were similar to that of the leucocyte adhesion deficiency in humans. © 1992.

关键词： C3 third component of complement CR3 complement receptor type III FCS fetal calf serum Hb hemoglobin LAD leucocyte adhesion deficiency syndrome NBT nitroblue tetrazolium PBS phosphate-buffered saline PMA phorbolmyristate acid RLU relative light units SEM scanning electron microscope WBC white blood cell

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IMMOBILIZATION OF C3B WITH RETENTION OF FUNCTIONAL-ACTIVITY

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JOURNAL OF IMMUNOLOGICAL METHODS 1990年第1期133卷 67-70页

作者： EDENS, RE LINHARDT, RJ WEILER, JM VET ADM MED CTR IOWA CITY IA 52240 USA UNIV IOWA COLL MED DEPT INTERNAL MED IOWA CITY IA 52242 USA UNIV IOWA COLL PHARM DIV MED & NAT PROD CHEM IOWA CITY IA 52242 USA

We compared eight commercially available, pre-activated affinity chromatography supports for ability to immobilize C3b that would retain functional activity. Pre-activated supports that we studied were: cyanogen bromide activated agarose, N-hydroxysuccinimide activated agarose, Reacti-Gel HW-65, Actigel A aldehyde activated agarose, thiopropyl activated agarose, 1,4-bis(2,3-epoxypropoxy) butane activated agarose, Reacti-Gel GF-2000 and tresyl activated agarose. The amount of C3b immobilized by each support varied from 81% for Actigel A aldehyde activated agarose to only 19% for Reacti-Gel GF-2000. We examined the functional capacity of the C3b immobilized on these various supports to participate in the alternative pathway. Immobilized C3b was mixed with factors D and B of the alternative pathway and examined over time for ability to consume factor B hemolytic activity. C3b immobilized on thiopropyl activated agarose consumed factor B at a rate comparable to unbound fluid phase C3b. C3b immobilized on other supports was less active in participating in factor B consumption. Thus, we have demonstrated the ability to immobilize C3b onto a solid matrix with the immobilized C3b retaining the ability to participate in the alternative pathway. This immobilized C3b can be used to fractionate substances with high C3b binding affinity.

关键词： Affinity chromatography complement C3b Agarose C3 third component of complement C3b cleavage fragment of complement CDI 1,1-carbonyldiimidazole CNBr cyanogen bromide DGVB 2+ dextrose gelatin veronal-buffered saline with calcium and magnesium EAC4b,3b, sheep erythrocytes antibody and complement components C4b and C3b EDTA ethylenediamine tetraacetate EDTA-GVB EDTA solution containing GVB GVB gelatin, veronal buffered saline HSA human serum albumin PBS phosphate-buffered saline Z number of lytic events per cell

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APPEARANCE OF ACCEPTOR-BOUND C3B ON HLA-DR POSITIVE MACROPHAGES AND ON STIMULATED U937 CELLS - INHIBITION OF FC-GAMMA-RECEPTORS BY THE COVALENTLY FIXED C-3 FRAGMENTS

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MOLECULAR IMMUNOLOGY 1988年第3期25卷 295-303页

作者： ERDEI, A BAJTAY, Z FABRY, Z SIM, RB GERGELY, J UNIV L EOTVOS DEPT IMMUNOL JAVORKA S U 14 H-2131 GOD HUNGARY UNIV OXFORD DEPT BIOCHEM MRC IMMUNOCHEM UNIT OXFORD ENGLAND

The appearance and the functional role of acceptor-bound C3b during differentiation of human monocytes into macrophages were studied. Acceptor-bound C3b could be detected by the immune adherence (IA) test parallel to the expression of antigenic determinants specific to mature ***.i.e. on days 4-5 of culture. Consequently, the capacity of these phagocytes to fix C3b covalently via C3b-acceptors (C3Abs) can be considered as one of the signs of their activation/differentiation. All the mature macrophages positive in the IA test were also found to express HLA-DR antigens on their membrane. Using solubilized extracts of stimulated, 35S-cysteine-labelled cells of the human monocytic cell line, U937, we demonstrate that C3 synthesized by these cells can bind to C3bAs of the same cells. Covalently fixed C3 fragments were found to inhibit ***.-receptor-mediated ingestion of immune complexes and also antibody-dependent cellular cytotoxicity of monocyte-derived macrophages.

关键词： ADCC antibody-dependent cellular cytotoxicity BSA bovine serum albumin C3 third component of complement C3b ∗ nascent C3b C3b proteolytic fragments of C3 iC3b proteolytic fragments of C3 C3d proteolytic fragments of C3 C3bA, C3b acceptor structure capable of fixing nascent C3b covalently via the short-living metastable binding site of the complement fragment CR1 receptors for proteolytic fragments of C3 CR2 receptors for proteolytic fragments of C3 CR3 receptors for proteolytic fragments of C3 EA erythrocyte-antibody complex Fc γ R receptor reacting with the Fc-portion of IgG IA immune adherence binding of cells or particles possessing covalently bound C3b to CR1-bearing human red blood cells IAR IA-rosettes IC immune complex Mo monocyte MΦ macrophage PBS phosphate buffered saline PMA phorbol-12-myristate 13-acetate SDS sodium dodecyi sulphate RBC red blood cell Z zymosan

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BIOSYNTHESIS OF THE 3RD component OF complement IN RAT-LIVER EPITHELIAL-CELL LINES AND ITS STIMULATION BY EFFECTOR MOLECULES FROM CULTURED HUMAN MONONUCLEAR-CELLS

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IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY 1987年第12期23卷 821-829页

作者： GUIGUET, M FRIGERE, MFE DETHIEUX, MC BIDAN, Y MACK, G 1. Laboratoire de Biochimie Médicale Unité de Recherche INSERM U208 Faculté de Médecine 7 boulevard Jeanne d'Arc 21033 Dijon Cedex France

Rat liver epithelial cell lines, growing in a serum-supplemented medium, synthesize and secrete into the culture medium the third component of complement (C3). We studied the regulation of C3 production in this system... 详细信息

Rat liver epithelial cell lines, growing in a serum-supplemented medium, synthesize and secrete into the culture medium the third component of complement (C₃). We studied the regulation of C₃ production in this system. We found that human peripheral blood mononuclear leukocytes in culture released one or more soluble factors which stimulated rat liver epithelial cells to produce increased quantitites of C₃. This stimulting effect was strongly enhanced when the mononuclear cell cultures were treated with phytohemagglutinin, a T-lymphocyte mitogen. The factor(s) failed to enhance C₃ biosynthesis by rat dermal fibroblasts, which are known to produce this protein. This reveals a tissue-specific differential response between the fibroblasts and the liver epithelial cells. The physical and chemical characteristics, such as heat sensitivity, 2.8M ammonium sulphate precipitation, and lower activity after digestion by proteases unambiguously indicate that the effector molecules are proteins. When the crude supernatant of mononuclear leukocytes was fractionated by gel filtration, the stimulating factor(s) eluted as two peaks with apparent molecular weight of 25 to 60 and 15 to 20 kdalton, respectively. As to the cellular origin of the C₃-stimulating factor(s), several observations were made: (a) in separate cultures containing either T-cells or monocyte-enriched populations from the same sample of blood mononuclear cells, no activity was detected in the presence or absence of phytohemagglutinin, (b) conditioned media from each of these cultures could not substitute for the corresponding intact cell populations, and (c) the addition of purified T-cells to the monocyte-enriched population in the presence of phytohemagglutinin restored the production of the stimulating activity by the mixed culture. Finally, experiments were c

关键词： rat liver epithelial cell line third component of complement human cytokines

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HEREDITARY PARTIAL DEFICIENCY OF THE 3RD component OF complement ASSOCIATED WITH MINIMAL CHANGE NEPHROTIC SYNDROME

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PEDIATRIC NEPHROLOGY 1987年第4期1卷 608-610页

作者： SPRINGATE, JE MCLEAN, RH WINKELSTEIN, JA FELD, LG CHILDRENS HOSP DEPT PEDIAT219 BRYANT STBUFFALONY 14222

We describe a 10 year old patient admitted to the Children's Hospital of Buffalo with hypocomplementemia associated with steroid responsive minimal change nephrotic syndrome. The sibling also had a low serum C3 concentration and all family members studied hadC 3 slow phenotypes. Factor I levels were at the lower limit of normal in the patient and his brother. Functional assays for CH50, total hemolyticC 3 and serum concentration of C2, C4−C9 and factors B and H were all within normal limits. This case confirms that a depressed serum complement level can occur in minimal change nephrotic syndrome and indicates that this depression could represent a preexisting inherited rather than an acquired deficiency. The findings are consistent with the pressence of a null or hypomorphicC 3 slow allele in hypocomplementemic family members. Additional studies are needed to resolve the association between the inherited partialC 3 deficiency and minimal change nephrotic syndrome.

关键词： Nephrotic syndrome third component of complement

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HIGH-RESOLUTION ELECTROPHORESIS FOR THE ALLOTYPING OF HUMAN C-4 - PROPOSAL OF A RELATIONAL NOMENCLATURE

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JOURNAL OF IMMUNOLOGICAL METHODS 1987年第1期96卷 69-76页

作者： KOLBLE, K RUKAVINA, V KALDEN, JR Institut und Poliklinik für Klinische Immunologie und Rheumatologie Universität Erlangen-Nürnberg Krankenhausstr. 12 D-8520 Erlangen F.R.G.

Among the major histocompatibility complex (MHC)-linked complement genes, the loci for C4A and C4B exhibit the most extensive structural polymorphisms. Therefore the differentiation of variant and complex C4 phenotypes often proves difficult in conventional immunofixation electrophoresis. To improve the available technique of C4 typing a closed horizontal electrophoresis system was combined with poly- and monoclonal alkaline phosphatase immunoprobing on contact diffusion blots. The high resolution and sensitivity of this method not only facilitated C4 allotyping but also revealed additional polymorphic variation. Relative electrophoretic mobilities specific for each C4 allotype were established by computerized remission densitometry and provided the basis for a quantitative denomination of C4 variants. Typing by high resolution electrophoresis and the proposed relational C4 nomenclature could be valuable for further immunogenetical studies of the C4 protein polymorphism.

关键词： C4 polymorphism High resolution electrophoresis Contact diffusion blotting Immunoprobing C4 nomenclature BCIP bromo-chloro-indolyl phosphate BF factor B of the alternative complement pathway C3 third component of complement C4 fourth component of complement EA cells sheep erythrocytes reacted with specific rabbit antibody EDTA ethylenediaminetetraacetic acid HLA human leukocyte antigen ISGN International System for Human Gene Nomenclature MHC major histocompatibility complex MoAb monoclonal antibody M r relative electrophoretic mobility NBT nitro blue tetrazolium SD standard deviation SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis SEM standard error of the mean

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