S.‐E. Svehag

In Vivo and In Vitro Antibacterial Activity of Conglutinin, a Mammalian Plasma Lectin

Conglutinin is a mammalian C‐lype lectin which agglutinates iC3b‐coated erylhrocytes, Ingram [13] found that euglobulin from bovine serum may confer partial protection against experimental infections in mice. We now present evidence that the protective activity in euglobulin against infections of BALB/c mice with Salmonella typhimurium is mediated by conglutinin. Conglutinin also demonstrated antibacterial activity against E. coli and S. typhimurium in vitro. The expression of this activity required the presence of heat‐labile serum factors and peritoneal exudate or spleen cells, but not antibodies to the bacteria. Antibacterial activity was also demonstrated when the bacteria were prelreated with serum at 37°C before incubation with conglutinin and cells. The activity of conglutinin was not observed when factor I‐deficient or EDTA‐treated serum was used instead of normal serum. The active peritoneal exudate or spleen cells showed adherence to plastic.

Serum Amyloid P Component Binds to Influenza A Virus Haemagglutinin and Inhibits the Virus Infection In Vitro

Serum amyloid P component (SAP) is a member of the phylogenetically conserved and structurally related group of proteins called pentraxins. SAP exhibits multispecific calcium‐dependent binding to oligosaccharides with terminal N‐acetyl‐galactosamine, mannose and glucuronic acid. The authors report that SAP can bind to influenza A virus and inhibit agglutination of erythrocytes mediated by the virus subtypes H1N1, H2N2 and H3N2. SAP also inhibits the production of haemagglutinin (HA) and the cytopathogenic effect of influenza A virus in MDCK cells. The binding of SAP to the virus requires physiological calcium concentrations and is blocked by specific SAP antibodies. Denaturated and renaturated SAP retained inhibition of HA. Electron microscopy shows Ca2+‐dependent binding of SAP to spikes on the viral envelope and immunoblotting indicates that SAP binds to a 50–55 kDa peptide corresponding to the mass of the HA1 peptide. Of several monosaccharides tested only D‐mannose interfered with SAPs inhibition of both HA and infectivity. The glycosaminoglycans heparan sulfate and heparin, which bind SAP, reduced SAPs binding to the virus. The results indicate that the inhibition by SAP is due to steric effects when SAP binds to terminal mannose on oligosaccharides localized close to the sialic acid‐binding site of the HA trimer.

Factor I Deficiency and C3 Nephritic Factor: Immunochemical Findings and Association with Neisseria meningitidis Infection in Two Patients

The complement system was examined in two patients with systemic Neisseria meningitidis infections, both of whom had reduced or nondeteclable CH50 as analysed by both pathways. C3 measured by conventional technique revealed 19% anti‐C3c‐reactive protein in the plasma of patient 1 and 3% in patient 2. Patient 1 had circulating C3b but no detectable C3c, C3d, or C4d, whereas patient 2 had normal levels of C3c and C4d and strongly elevated levels of C3d. Factor B analysis revealed no demonstrable native factor B and small amounts of Bb in patient 1 and normal concentration of native factor B plus trace amounts of Bb in patient 2. The depletion of C3 in both patients was due to uncontrolled activation caused by complete factor I deficiency (patient 1) and circulating C3 nephritic factor (patient 2). Both parents of patient 1 had factor I concentrations below (mean‐2 SD) that seen in normal healthy individuals (n= 20). Circulating immune complexes (IC) were demonstrated in patient 1 only, whereas serum from both patients had strongly reduced capacity to solubilize preformed IC.

Binding of complement proteins C1q and C4bp to serum amyloid P component (SAP) in solid contra liquid phase.

Serum amyloid P component (SAP), a member of the conserved pentraxin family of plasma proteins, binds calcium dependently to its ligands. The authors investigated SAPs interaction with the complement proteins C4b binding protein (C4bp) and C1q by ELISA, immunoelectrophoresis and electron microscopy. Binding of these proteins to SAP was demonstrated when SAP was immobilized using F(ab′)2 anti‐SAP, but not when SAP reacted with these proteins in liquid phase; thus the binding to human SAP was markedly phase state dependent. Presaturation of solid phase SAP with heparin, which binds SAP with high affinity, did not interfere with the subsequent binding of C4bp or C1q to SAP. In contrast, collagen I and IV showed partial competition with the binding of C1q to SAP. Using fresh serum, immobilized native SAP bound C4bp whereas binding of C1q/C1 could not be demonstrated. Altogether the results indicate that firm binding of C1q and C4bp to SAP requires that SAP is presented on a solid phase, that C1q and C4bp react with sites distinct from the heparin binding site, and that C1q and collagen I share binding sites on SAP. Immobilized native SAP, aggregated SAP and SAP‐heparan‐sulphate complexes induced no detectable complement activation.

A family with complement factor I deficiency.

A family with inherited factor I deficiency is described. The proband was a 19‐year‐old Caucasian female with one episode of meningococcal meningitis and one episode of suspected septicaemia of unknown cause. Two obligate and two probable heterozyotes with factor I levels below the lower limit of the reference range were identified. None of these exhibited increased susceptibility to infectious diseases. The inheritance was autosomal codominant. In addition, molecular heterogeneity of factor H in plasma from the proband but not from any other family members was demonstrated by crossed Immunoelectrophoresis. The migration of factor H component of fast electrophoretic mobility was retarded by antibodies to C3eand C3d. suggesting the presence of a fluid‐phase complex between factor H and excess C3h generated by the uncontrolled activity of the amplification loop.

Isolation and Characterization of Porcine Mannan-Binding Proteins of Different Size and Ultrastructure

The authors report on the purification and characterization of mannan‐binding proteins (MBP) isolated from porcine serum. The MBPs were purified by use of PEG precipitation, affinity chromatography on mannan‐Sepharose, protein A‐ and anti‐porcine IgM‐Sepharose followed by gel filtration. The MBP proteins were collagenase sensitive and showed γ1‐γ2‐electrophoretic mobility. The MBP designated pMBP‐28 had a molecular mass of 28u2003kDa when analysed on SDS‐PAGE under reducing conditions and eluted corresponding to a molecular mass of approximately 700u2003kDa on gel filtration chromatography. Electron micrographs of pMBP‐28 revealed an oligomeric protein similar to rodent MBP‐A and human MBP but with a predominance of penta‐ and hexameric molecules. Another protein designated pMBP‐27 was composed of peptides of 27u2003kDa and had an Mr of 300–350u2003kDa on gel filtration chromatography. Electron microscopy of pMBP‐27 showed dimer and trimer molecules; the trimers without distinct stalk regions. The N‐terminal 26 (pMBP‐27) and 24 (MBP‐28) amino acid residues showed 54% and 58% identity with human MBP. pMBP‐28 showed a higher degree of sequence similarity to rat and mouse MBP‐A (60% identity) than to mouse and rat MBP‐C (41–45% identity). Both pMBPs exhibited Ca2+‐dependent binding to D‐mannose immobilized on agarose but no significant binding to N‐acetyl‐D‐glucosamine‐ or fucose‐agarose. The results further suggested the presence of a third pMBP which copurified with pMBP‐27 but this protein was not sequenced.

MANNAN-BINDING PROTEIN FORMS COMPLEXES WITH ALPHA -2-MACROGLOBULIN. A PROPOSED MODEL FOR THE INTERACTION

We report that α‐2‐macroglobulin (α2M) can form complexes with a high molecular weight porcine mannan‐binding protein (pMBP‐28). The α2M/pMBP‐28 complexes were isolated by PEG‐precipitation and affinity chromatography on mannan‐Sepharose, protein A‐Sepharose and anti‐IgM Sepharose. The occurrence of α2M/pMBP‐28 complexes was further indicated by crossed immunoelectrophoresis and by use of an anti‐α2M affinity column and chelating Sepharose loaded with Zn2+. The eluates from these affinity columns showed α2M subunits (94 and 180 kDa) and pMBP subunits (28 kDa) in SDS‐PAGE, which reacted with antibodies against α2M and pMBP‐28, respectively, in Western blotting. Furthermore, the α2M/pMBP‐28 complexes were demonstrated by electron microscopy, Fractionation of pMBP‐containing D‐mannose eluate from mannan‐Sepharose on Superose 6 showed two protein peaks which reacted with anti‐C1 s antibodies in ELISA, one of about 650–800 kDa, which in addition contained pMBP‐28 and anti‐α2M reactive material, the other with an Mr of 100–150 kDa. The latter peak revealed rhomboid molecules (7 × 15 nm) in the electron microscope and a 67 kDa band in SDS‐PAGE under reducing conditions. This band was also seen in eluates from the anti‐α2M and chelating Sepharose columns. Based on these observations and previous findings by other investigators of a serine protease with about 67 kDa subunits which copurifies with human MBP we propose a model for the interaction of pMBP‐28 with α2M.

Human‐human hybridoma producing monoclonal antibodies against colorectal cancer‐associated antigens

Lymphocytes from lymph nodes draining the tumor region in patients with colorectal cancer were fused with two different human B‐lymphoblastoid cell lines, LICR‐LON‐HMy‐2 (HMy‐2) and WI‐L2–729‐HF2 (729‐HF2), to generate hybridomas synthesizing antibodies reacting with tumor‐associated antigens. In this way 220 hybridomas were obtained which produce antibody reacting with colon cancer cells. All established clones produced IgM. Four human monoclonal antibodies have been furhter analyzed. The cell lines producing these antibodies are all hybrids based on DNA analysis. Three of the antibodies (G4146, B9165 and D4213) showed binding to differentiation antigens by immunocytochemical analysis on different cancer cell lines and normal human leucocytes and by immunohistochemical analysis on sections of frozen malignant and normal tissues, while the fourth (F11348) showed a reaction with all cells and tissues tested. Western blots of tumor extracts showed binding of G4146 to two components from colon cancer cells with Mr of 59 K. and 61 K, while B9165 bound to a 43 K component and F11348 to several components with Mr from 30 to 200K. D4213 showd no binding in this analysis. The results obtained demonstrate the successful application of hybridoma technology to produce human monoclonals with reactivity to differentiation antigens.

Increased Number of IgG Fc Receptors on Monocyte‐Enriched Peripheral Blood Leucocytes from Patients with Rheumatoid Arthritis

The number of free Fc receptors (FcR) per cell and the association constant (Kass) for the binding of monomeric IgG were determined for monocyte‐enriched peripheral blood mononuclear cells, isolated from 16 patients with active classical rheumatoid arthritis (RA) and from 15 normal healthy donors. The assay system was based on binding under equili brium conditions of 125I‐labelled monomeric rabbit IgG to monocytes purified from peripheral blood on a continuous gradient of Petcoll. Monocytes from 14 untreated RA patients (6 seropositive, 8 seronegative) expressed on the average 4.8±1.3 × 104 FcR/cell. This number was significantly higher (P<0.01) than that found in the control group (3.46±0.7 × 104 FcR/cell). There was also a significant difference between the mean Kass of the RA group and the control group‐2.1±0.7 × 1031/mol and 2.6±1.0 × 103 1/mol, respectively (0.05 >P> 0.01). Two seropositive RA patients receiving systemic treatment with penicillamine expressed the same number of FcR/cell as the mean of the control group (3.6 ± 104). Levels of circulating immune complexes (CIC) and of the complement‐factor C3 split product C3d were also measured. No correlation was found between the number of FcR/cell and the concentration of C3d, but there was a weak correlation between the number of FcR/ccll and the level of CIC.

The third complement factor (C3) and its in vivo cleavage products: Interaction with lectins and precipitation with polyethylene glycol

Five molecular forms of C3 expressing D but not C epitopes were identified following in vivo activation of the complement system. Examination of concanavalin A (Con-A) reactivity in crossed immunoelectrophoresis revealed that native C3, C3c and the beta mobile form 4 of C3d were completely precipitated by 100 micrograms Con A/cm2. The alpha-1 mobile form 1 of C3d did not interact with Con A, whereas the alpha-2 mobile forms 2 and 3 were retarded in electrophoretic migration by Con A. Native C3, C3c, and forms 4 and 5 of C3d were precipitated by 12% (w/v) polyethylene glycol (PEG). Form 1 of C3d was soluble in these PEG concentrations, whereas forms 2 and 3 were partially precipitated.