Mikulska J

Human placentae membrane receptor for IgG—i. Studies on properties and solubilization of the receptor☆

Abstract Characterization of the human placental membrane receptor for human 125 I-IgG is described. The receptor bound specifically both monomers and aggregates of human IgG. Human colostral IgA, bovine, sheep, pig, and horse IgG were not bound. No effect of pH in the range 6.6–7.4, ionic strength in the range 0.1–0.5, and temperature between 4 and 45°C on the binding was found. A water-soluble fraction containing the active receptor (glycoprotein fraction-PGP) was obtained from the placental membranes using lithium diiodosalicylate. The solubilized receptor interacted with IgG better at 4°C than at 20°C or 37°C. The results on replacement of monomeric IgG by aggregated IgG, and vice versa, suggest that both monomers and aggregates of human IgG, were bound to the same receptor sites. The apparent association constant for monomeric human IgG was 0.86 ± 0.2 × 10 7 mole −1 , and 2.0 ± 0.16 × 10 15 IgG molecules were bound per l mg of the membrane protein. Formaldehyde (0.1%), 2-mercaptoethanol (50 m M ), and periodate (4 m M ) showed no effect on the binding properties of the membrane-bound and on the solubilized receptor, as well. Higher concentrations of periodate (10 m M or 20 m M ) decreased the binding of IgG to membranes but showed no effect on the water-soluble receptor. Both the membrane-bound and the solubilized receptor were sensitive to papain. Pronose abolished the receptor activity after prolonged proteolysis only. Neuraminidase did not affect the activity of the receptor. The decrease of the binding activity of the membrane-bound receptor by trypsin and phospholipase C was due to a release of a material containing an active receptor. No effect of trypsin or phospholipase C on the activity of solubilized receptor was observed. The results obtained suggest a protein character of the placental Fc receptor. After electrophoresis of 125 I-labeled solubilized receptor in polyacrylamide gel in the presence of SDS, 2 major protein peaks with molecular weights of 74,000 and 104,000 and 3 minor peaks with molecular weights of 56,000, 144,000, and 163,000 were found.

Human placental membrane receptor for IgG. Purification of the receptor and its subunit structure

The method of purification of the human placental Fc receptor to an active form is described. The FcR was purified from the glycoprotein fraction of the placental membranes by immunoprecipitation and chromatography on DEAE-cellulose. The purified FcR corresponded to 1.5-2% of the protein present in the crude glycoprotein fraction (PGP) and showed the tendency to aggregate. In the presence of 1% SDS, 4 M urea or 5 M guanidine- HCl the placental FcR dissociated into subunits of molecular weight of 60,000-65,000. The 60,000-65,000 dalton glycoprotein subunits regarded as monomers of FcR are composed of two chains of molecular weight 25,000-30,000, linked by disulphide bonds. The subunits, after removal of dissociating agents, displayed IgG binding activity.

A Proline‐Rich Polypeptide Complex (PRP) from Ovine Colostrum. Studies on the Effect of PRP on Nitric Oxide (NO) Production Induced by LPS in THP‐1 Cells

Abstract A proline‐rich polypeptide complex (PRP) isolated from ovine colostrum shows immunoregulatory activity. Similar activity was observed when PRP was replaced with a nonapeptide (NP) isolated from chymotryptic digest of PRP. The polypeptide complex also shows procognitive activity. In the form of orally administered tablets called Colostrinin®, containing 100 µg of PRP, it improves the outcome of Alzheimers disease (AD) patients. The mechanism of action of PRP/Colostrinin® in AD is not yet clarified. Microglial cells involvement in AD has been related to amyloid β (Aβ) internalization, the release of inflammatory cytokines, overproduction of nitrogen oxide (NO) and superoxide anion (O2−), and the development of neuritic plaques. It has been previously found in our laboratory that PRP regulates the secretion of an array of cytokines. It also was shown, in preliminary experiments using human blood cells and murine macrophages, that PRP inhibits production of NO and O2− induced by LPS. In the present work, to study the effect of PRP and NP on the release of NO and O2− induced by LPS we applied THP‐1 cells. The human monocyte/macrophage THP‐1 cell line has been widely used as a model of human microglial cells. The results obtained showed that THP‐1 cells release NO when activated with LPS. However, neither PRP nor NP induced production of NO. Although the nonapeptide, at higher concentration (100 µg/mL), showed an inhibitory activity on the release of NO induced by LPS, no inhibition was observed when PRP was used. THP‐1 cells treated with LPS, PRP or NP did not release O2−.

Analysis of Response Elements Involved in the Regulation of the Human Neonatal Fc Receptor Gene (FCGRT)

Human epithelial, endothelial and PMA-differentiated THP-1 cell lines were used as model systems to study the transcriptional regulation of the human FCGRT gene encoding the alpha chain of hFcRn. The data obtained from site-directed mutagenesis in transient transfection experiments indicate that the Sp1 sites at positions -641, -635, and -313, CF1/YY1 elements at positions -586 and -357, and the AP-1 motif at -276 within the-660/-233 fragment of the human FCGRT promoter (hFCGRT) participate in the regulation of human FCGRT in all selected cell lines. However, their individual contribution to promoter activity is not equivalent. The Sp1 binding site at -313 and the AP-1 site at -276 are critical for the activity of the hFCGRT promoter in epithelial and endothelial cells. Moreover, the CF1/YY1 site at -586 in differentiated THP-1 cells, plays an essential role in the transcriptional activity of the promoter. In addition, the C/EBPbeta binding site at -497 of the hFCGRT promoter in epithelial and endothelial cells, and the C/EBPbeta motif located at -497 and -233 within the hFCGRT promoter in differentiated THP-1 cells may function as positive regulatory sequences in response to LPS or PMA stimulation. EMSA and supershift analyses showed that the functionally identified binding motifs in the hFCGRT promoter were able to specifically interact with their corresponding (Sp1, Sp2, Sp3, c-Fos, c-Jun, YY1, and C/EBPbeta or C/EBPdelta) transcription factors (TFs), suggesting their possible involvement in the regulation of the human FCGRT gene expression.

Synthetic antigens--V. Properties of rabbit antibodies against a synthetic antigen: interpolymer of styrene and maleic acid.

Abstract Properties of rabbit antibodies against a new synthetic antigen: interpolymer of styrene and maleic acid (PSM)‡ are described. Rabbits immunized with PSM produced precipitating and nonprecipitating antibodies of IgG class. Interactions of anti-PSM antibodies with PSM are mainly of an electrostatic character and were inhibited by various low molecular weight compounds resembling the structural unit of PSM. Anti-PSM antibodies belong to low-affinity antibodies and show multispecificity. The apparent affinity constant for PSM was of the order of 10 5 M −1 . Anti-PSM antibodies showed no restriction of heterogeneity in isoeleetric focusing. The results of spectral measurements showed that tyrosine residues may be involved in the interaction of antibodies with PSM. No major change of conformation of anti-PSM antibodies occurred during the interaction with PSM or hapten-like compounds.

Synthetic antigens—VI: Horse ‘natural’ antibodies against an interpolymer of styrene and maleic acid

Abstract -It was found that normal horse sera contained proteins precipitating a synthetic antigen: interpolymer of styrene and maleic acid (PSM). The results presented in this paper showed that the precipitation was caused by the presence of comparatively high concentration of ‘natural’ anti-PSM antibodies in horse sera. The antibodies were of IgG class. Interaction of PSM with horse natural anti-PSM antibodies depended strongly on ionic strength and pH. The antibodies studied belonged to low-affinity antibodies and showed multispecific character. The precipitation was inhibited by low molecular weight compounds resembling and different from the structural unit of PSM. The antibodies showed no restriction of heterogeneity in the isoelectric focusing. Properties of horse natural anti-PSM antibodies showed strong similarity to antibodies produced by rabbits immunized with PSM.