Immunological communications最新文献

Lymphocyte thymidine uptake in leucocytes prepared by NH4Cl induced RBC lysis was compared with that of lymphocytes obtained following Ficoll-Hypaque centrifugation using polynomial regression dose response curves which adjusted for inherent subject variability. Eleven male subjects were studied. Heparinized blood was either layered in Ficoll-Hypaque or treated with 0.87% NH4Cl to induce RBC lysis. Phytohemagglutinin-P (PHA) dose response curves were determined for each subject using triplicate 5 day cultures. For each subject, at each Pha concentration ranging from 12.5 to 167micrograms/ml, triplicate mean cpm of Ficoll-Hypaque treated lymphocytes and NH4Cl treated lymphocytes were compared by the 2 tailed Student's T test. Responses of cultures of NH4Cl treated lymphocytes were significantly different (less than 0.05) from the responses of cultures of Ficoll-Hypaque treated lymphocytes for at least 2 PHA doses in 8 of the 11 subjects. The quadratic dose response curves were also significantly different (p less than 0.05) for 9 of the 11 subjects. A repeated measures quadratic regression was used to determine the best fit dose response curves for each of the experimental conditions. For Ficoll-Hypaque treated lymphocytes, mean cmp=44,977+1400.4 (PHA concentration)-5.90 (PHA concentration)2+ subject effect; s.e.=22,256; for NH4Cl treated lymphocytes, mean cpm=58,039+1022.8(Pha concentration)-5.32 (PHA concentration)2+ subject effect; s.e.=37,966. The p values from the F tests for significance of these quadratic regressions are different, F=1.91, d.f.=30,30; p less than 0.001. The lack of significance of the NH4Cl treated lymphocytes' does response quadratic regression and its larger s.e. indicates that the NH4Cl treated lymphocyte culture dose response curve is less predictable than that of Ficoll-Hypaque treated lymphocytes.

Incubation of human serum with either D- (1-14C) galactose (5 mM), D- (1-14C) glucose (5 mM) or L- (1-14C) fucose (5 mM) in vitro for 7 days under physiological conditions resulted in the accumulation of radioactivity into trichloroacetic acid precipitable material. Separation of the serum proteins by Sephadex G-200 chromatography, revealed the association of radioactivity with the albumin fraction (95%) and to a lesser extent with IgG (4%) and IgM (1%). D-galactose glycosylated purified human IgG at 2 to 3 fold the rate of D-glucose of L-fucose. The rate of glycose incorporation into IgG increased parabolically with increasing pH and temperature of incubation, and followed a first order dependence with either the glycose or the IgG concentration. The post-translational modification of IgG through nonenzymatic glycosylation may affect its immunological properties in clinical conditions associated with increased blood sugar concentrations.

The molecular localization of the full antigenic profile for T-cell recognition of a complex multi-determinant protein antigen has not to date been accomplished. Previously, this laboratory has introduced a comprehensive strategy for the systematic localization of all continuous antigenic sites within a protein. This strategy depends on the synthesis of a series of overlapping peptides that together account for the entire structure of a protein. Such a strategy has been applied, in this report, to the delineation of the continuous sites of T-cell recognition of sperm whale myoglobin. Thirteen peptides, accounting for the entire protein chain, were synthesized and subsequently examined in vitro for their ability to stimulate lymph node cells from myoglobin primed DBA/2 (H-2d) mice, a known high responder. This strategy has enabled for the first time the localization of the full profile of the protein regions which contain the sites of T-cell recognition. Three regions gave a high response (one being immunodominant and coinciding with antigenic, i.e. antibody binding, site 4 of myoglobin). At least three regions appear to coincide with previously known antigenic (antibody binding) sites. Noteworthy is the finding of regions that are recognized by T-cells but to which no detectable antibody response is directed.

Alloimmune antiserum against the L antigen of red cells from sheep of the LK phenotype is known to stimulate by several fold active Na/K transport in LK cells. We have shown that monomeric fragments, Fab1, of anti-L, as well as dimeric fragments, F(ab1)2, stimulate transport to the same extent as intact anti-L Ig. Special care was taken to obtain pure fragments. Two earlier reports on the effect of immunoglobulin fragments were contradictory.

A study was made of the increases in ionic strength (mu) and in pH that would effect: a) the inhibition of association of anti-double stranded (ds) DNA and dsDNA, and b) the dissociation of anti-dsDNA from dsDNA, with respect to high avidity as well as to low avidity human anti-dsDNA antibodies, using Crithidia luciliae kinetoplasts as a source of dsDNA. The results obtained appear to indicate that the electrostatic component of the antigen-antibody bond does not become stronger with time (for times spans less than 1 hr).

Using a technique for combined autoradiography and immunoperoxidase staining, we studied the incorporation of 3H-Uridine and 3H-Leucine into T and B lymphocytes of rats and mice. While in mouse both T and B cells were clearly labeled by incorporation of 3H-Uridine, a portion of rat T cells remained unlabeled. Furthermore, the majority of rat B cells was not labeled. In contrast, mouse T and B cells as well as rat T and B cells could be labeled with 3H-Leucine to a comparable degree.

Sera from three normal dogs were assessed for levels of Clq binding IgG and complement consumption after perfusion over Staphylococcus aureus Cowan I (SAC). Increased levels of Clq binding IgG were detected after perfusion of sera over SAC and were associated with complement consumption. Canine antiserum to human erythrocytes were also perfused over SAC and assessed for Clq binding IgG and hemolytic activity. Increased levels of Clq binding IgG in post-perfusion samples were detected which were associated with a decrease in hemolytic activity. IgG was determined to be present in molecular weight fractions greater than 200,000 M.W. in post-perfusion chromatographically fractionated sera. Moreover, 5% polyethylene glycol (PEG) precipitated IgG from post-perfusion sera was functional in antibody dependent cellular cytotoxicity assays. Putative staphylococcal protein A isolated from post-perfusion sera produced a precipitin band in double diffusion agarose gel studies when reacted with normal human and canine sera. A polypeptide co-migrating with purified protein A could be detected by polyacrylamide gel electrophoresis (PAGE) analysis of the post-perfusion isolated protein A. Addition of purified protein A to canine antiserum resulted in decreased hemolytic activity of the serum which was associated with increased levels of Clq binding IgG.

The acute phase of inflammation is characterized by numerous changes in blood composition, perhaps the most dramatic of these being the elevation of C-reactive protein levels. C-reactive protein (CRP) is known to bind to molecules containing phosphocholine-substituents following reaction with Ca2+ ions. Luminescence energy transfer (LET) has been used effectively to study the Ca2+ and Mg2+ binding properties of many proteins by employing appropriate lanthanides (III). We have used Tb3+ as an isomorphous analogue to study Ca2+ binding to CRP. Energy transfer occurs effectively and demonstrates the importance of aromatic residues (viz., tyrosine and tryptophan) in the binding of Tb3+. The binding of Tb3+ is remarkably dependent on the pH and indicates the requirement of a deprotonated residue in the pH range 6.4 +/- 0.2 for effective Tb3+ binding. A 50-fold molar excess of Ca2+ is sufficient to displace the Tb3+ suggesting that Tb3+ is bound with greater affinity to CRP than the natural analogue Ca2+. We propose that Tb3+ (by inference Ca2+) binding takes place near the CRP subunit disulfide bond, where two histidine residues are present. The pH dependency of Tb3+ binding is best explained by the deprotonation of a histidine residue(s) in CRP.

Immunoglobulins were conjugated to peroxidase by the biotin-avidin method and used in ELISA systems for measuring myelin basic protein (MBP) and anti-MBP antibodies. To measure concentration of MBP, microplate wells were coated with affinity purified rabbit anti-MBP antibodies and incubated with varying concentrations of MBP. Bound antigen was measured by incubating with biotinylated anti-MBP antibodies and avidin-peroxidase. As little as 0.2 ng/ml of MBP could be measured by this assay. To measure anti-MBP antibodies, microplate wells were coated with human MBP and incubated with varying concentrations of affinity purified rabbit anti-human MBP antibodies. Binding was measured by incubating with either peroxidase-conjugated anti-rabbit antibodies or biotinylated anti-rabbit antibodies and avidin peroxidase. The two methods were equally sensitive. The avidin-biotin method for enzyme conjugation promises to be a useful and versatile tool for ELISA systems.

The in vitro blastogenic response of rat splenocytes to concanavalin A stimulation is inhibited by inclusion of asparaginase in the culture medium. The glutaminase-free asparaginase from Vibrio succinogenes is as potent an inhibitor as the Escherichia coli enzyme which has 2% glutaminase activity. The polyethylene glycol-modified forms of both enzymes are also inhibitory. We suggest that previously proposed explanations for the ability of asparaginases to inhibit blastogenesis are not likely to be correct and propose that asparaginase interacts with a mitogenic factor.