Immunological communications最新文献

Many applications exist and others are envisioned for the chemical coupling of macromolecules to membrane proteins on the surface of mammalian cells. The ability to use antibody as a means to label and subsequently to follow the distribution of cell surface proteins is reported here. A new procedure is outlined for covalently coupling monoclonal antibodies to thiol-containing membrane proteins. The key reagent in the coupling reaction is the commercially available heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP). The coupling proceeds in a simple two-step reaction in aqueous medium under very mild conditions. This results in a very efficient and stable attachment of anti-hapten antibodies to a selected set of cell surface proteins without any loss in cell viability and without denaturing the antibody molecule. The hapten-binding activity of the antibody is exploited to monitor the re-distribution of the antibody-labeled cell surface proteins periodically after the coupling reaction. The hapten binding activity can also be utilized to isolate membrane macromolecules via affinity chromatography.

Model in vivo and in vitro experimental systems have been used to study the efficacy of specific and nonspecific immunization against Candida albicans infection induced in mice. Experiments were designed to compare the extent of resistance in specificity immunized, endotoxin treated and saline treated animals. In vitro phagocytic and postphagocytic killing (cytopepsis) of macrophages or lymphocyte-macrophage combinations from such animals were determined. In the in vitro experiments the macrophage systems destroyed the yeast cells more rapidly than did the lymphocyte-macrophage combinations. Since equivalent numbers of yeast cells were phagocytized, the differences observed were a function of cytopepsis of the organisms.

Dissociation constant (Kd) of antigen-antibody reactions can be obtained from the rates (measured by the progression of the precipitate vs. time) with which antigens diffuse into antibody-containing gels, as a function of antibody-concentration. A bovine serum albumin vs. rabbit anti-bovine serum albumin system was studied with whole antiserum and with its purified IgG fraction. A value was found for Kd of approximately 1.0 x 10-5 moles per liter. It is note- worthy that in monodimensional single diffusion gel precipitation systems of this type, the rate of progression of the precipitate front is significantly faster than the molecular diffusion coefficient of the antigen.

The L1210 enzyme-linked immunoassay (ELIA) for circulating immune complexes, previously developed for use in humans, has been adapted for use in mice. The assay detected elevated levels of immune complexes in 100% of NZB/W female mice (6-10 months old) with age-related immune complex-mediated glomerulonephritis. Application of the assay to the serum of one year old C57Bl/6 male mice failed to demonstrate elevated levels of serum immune complexes in spite of the routine finding of renal deposition of IgG plus proteinuria in aged mice of this strain. Since the L1210 ELIA detects all IgG-containing immune complexes, regardless of size or complement fixing capacity, these results suggest that the renal IgG deposits seen in aged C57Bl/6 mice are not derived from circulating IgG immune complexes.

Pseudomonas aeruginosa produces a factor (PF) which alters the enterobacterial common antigen (ECA). Its effect on the immunogenicity of two types of immunogenic ECA, namely, the ethanol-soluble preparation freed of lipopolysaccharide and the LPS-coupled form from the R-mutant E. coli 014 was investigated. The antibody response following intravenous immunization was determined by means of the hemagglutination test. It is shown that PF abolishes the immunogenicity of the former but not of the latter. PF obtained from a strain of P. maltophilia yielded the same results. Antiserum against Pseudomonas aeruginosa of types 1 and 6 neutralizes PF produced by either type. These results suggest that PF alters the lipid part and not the haptenic determinant of ECA and that this activity is neutralized by P. aeruginosa antiserum of either type 1 or type 6. This interpretation is compatible with the identification of PF as a lipase.

Serum obtained from patients with spontaneous systemic lupus erythematosus or from patients treated at least 3 months with procainamide could specifically inhibit Con A mitogenesis of cultured peripheral blood mononuclear cells obtained from procainamide-treated patients or from normal donors. Transfer of procainamide treated patients to N-acetylprocainamide eliminated the blocking factor from their serum. The blocking factor is not procainamide itself since adding the drug to normal serum and only slight effects on mitogenesis of normal peripheral blood mononuclear cells. These data suggest that systemic lupus erythematosus and procainamide-induced lupus may differ in the relative reversibility of a regulatory defect associated with a Con A responsive population of peripheral blood mononuclear (PBM) cells.

Cultured human myeloid cells (K562) are known to bear Fc receptors that bind with aggregated human IgG (AHG). These cells were used to develop a radiometric assay for detection and quantitation of immune complexes (IC) in human sera. The binding of AHG or in vitro-formed IC between keyhole lympet hemocyanin (KLH) and human anti-KLH to the K562 cells did not require complement. When the K562 radiometric assay was compared to the complement-consumption assay, the K562 radiometric assay could detect IC over a wider range of antibody:antigen ratios. The incidence and mean IC values detected by the K562 radiometric assay in sera from cancer patients and patients with connective tissue diseases (498 +/- 445 and 436 +/- 209 micrograms AHG equ/ml, respectively) were significantly higher than in sera from healthy volunteers (107 +/- 62 micrograms AHG equ/ml). The IC level among sera from cancer patients ranged from 1-3200 micrograms AHG equ/ml. Studies with a limited number of sera from melanoma and sarcoma patients revealed that the mean IC values were significantly higher in patients who had clinically detectable disease than in those with no evidence of disease. Since the K562 cells do not require complement to interact with AHG, the K562 radiometric assay may be potentially useful for detecting IC in pathologic sera which may or may not contain in vivo-bound complement components.

Liposomes bearing human tumor membrane vesicles were effective immunogenic complexes for inducing antibodies in rabbits. Multilamellar liposomes (MLV) at 7:4:1 molar ratios of phosphatidylcholine, cholesterol and phosphatidic acid were prepared with sonicated membrane (MN) isolated from LS174T colon tumor cells. MLV liposomes prepared together with MN (i.e., MN-MLV antigens) or MN added to preformed MLV (i.e., MN+MLV antigens), and MN antigens alone were used as immunogens. Rabbits were immunized i.v. with 100 g protein of each antigen group. Boosters (i.v.) were at days 13 and 29. Binding assays were performed by indirect radioimmunoassay on viable tumor cell targets. The MN+MLV groups were distinguished by earlier reactivity and greater specificity to the colon tumor antigens.

Concentrated rat bronchial washings (BW) were analyzed by gel-filtration and immunochemical methods. BW contained mainly albumin, transferrin and IgG. Free secretory component and secretory IgA were identified in BW; the BW-IgA had the same three sedimentation coefficients, i.e. +/- 11 S, 13 S, 15 S by sucrose density gradient ultracentrifugation, as rat milk and rat bile IgA; the three peaks were secretory IgA. Compared to serum, and relatively to albumin, BW were significantly enriched in IgA, although much less than rat bile. Purified polyclonal rat polymeric 125I-IgA was injected intravenously into normal rats, and into rats with bile duct ligation or partial hepatectomy, which decrease the liver plasma-to-bile transfer of IgA. BW were then collected, one or four hours later, to assess the recovery of the 125I-IgA in BW and to estimate the contribution of serum IgA to BW-IgA. Very little 125I-IgA (less than 0.2%) was recovered in all BW. The specific activity, measured only in the rat with the highest recovery in BW, was 20 times lower in BW than in serum. The data demonstrate that rat serum IgA does not contribute significantly to IgA in BW.

Helix pomatia (HP) receptors are present on approximately 60 percent of neuraminidase (NANAase)-treated rat T lymphocytes. The HP+ spleen cells (SpC) can be separated from HP-, immunoglobulin-bearing (Ig+) cells by affinity chromatography. The HP+ SpC mediate the local graft-versus-host reaction (GVHR). Less than 1% of rat lymphocytes bear both HP and Ig markers. Approximately 25% of Lewis rat bone marrow cells are HP+ (only 1% are HP+ Ig+), suggesting that the HP receptor may be a differentiation marker.