Comparison of lysis of bromelain and papain treated red cells in ADCC assays.

Red cells were pretreated with the proteolytic enzymes bromelain or papain prior to use in antibody-dependent cell-mediated cytotoxicity (ADCC) assays with lymphocytes or peripheral blood mononuclear cells (PBMC) as effector cells. At low concentrations of anti-D or anti-A, lysis of papain-treated cells by lymphocytes was greater than that of bromelain-treated cells. Papain digestion resulted in both greater sensitivity to haemolysis by lymphocytes or PBMC and higher agglutination titres of anti-D-sensitised red cells than bromelain. With anti-A, however, although papain also promoted greater haemolysis, it was slightly less effective at red cell agglutination than bromelain.     

Studies on plasminogen activator and other proteases in subcultured human vascular cells

Plasminogen activator activity (PAA) of human vascular cells in culture was quantitated in an assay system in which the conversion of purified plasminogen to plasmin was measured by activity against a soluble low-molecular-weight plasmin substrate. Reliable detection of PAA required cell lysis and use of membrane-disrupting detergents. After such treatment, PAA was found only in the 100,000 g subcellular fractions of human aortic smooth muscle and mixed populations of rabbit aortic cells. No corresponding activity was found in any fraction from human umbilical vein endothelial cells. In contrast, PAA was detected in significant amounts in intact mouse embryo fibroblasts (3T3 cells) used as controls. Very low levels of enzymatic activity against a spectrum of substrates preferentially amidolyzed by serine proteases were also demonstrable in human aortic smooth muscle or 3T3 fibroblast subcultures. Neither whole cell homogenates nor subcellular fractions demonstrated the previously described acid-labile inhibitor of PAA when assayed in this system. The above findings suggest that significant differences in PAA exist between various cell types and vascular segments. In addition, the subcellular localization and quantitatively low levels of PAA and other serine proteases found in human arterial and venous cells may reflect the presence of membrane-associated enzymes whose biological role is restricted to local homeostasis.     

Terasaki-ELISA for murine IgE-antibodies. I. Quality of the detecting antibody: production and specificity testing of antisera specific for IgE

In order to develop an ELISA for the quantitative determination of murine IgE, antisera specific for murine IgE were prepared in the goat and rabbit. As immunogen, monoclonal IgE antibody mixtures of several allotypically different hybridomas were used. Before use, these antibodies were purified employing procedures that allow maximum recovery of binding activity. The goat and rabbit mouse epsilon chain-specific antisera were adsorbed on normal mouse serum. The purified antisera were found to be free of allotypic activity. However, immunoadsorption on NMS could not always remove contaminating anti-idiotypic antibodies. Repeated adsorptions with monoclonal antibodies of different isotypes carrying a similar idiotype were necessary to remove all detectable anti-idiotypic activity. Only after these precautions were the antisera suitable for detecting IgE molecules on nitrocellulose blots as well as for quantitating circulating IgE antibodies (ELISA) and IgE-secreting cells (plaque assay and reverse ELISA plaque assay). The purity and reactivity of several commercially available anti-IgE preparations were tested in similar types of specificity assays. Since the specificity of antibodies used in ELISA determines the monospecificity of the assay, retesting for contaminating cross-reactivities in commercial preparations was shown to be necessary.     

Studies on the specificity of agglutinins against human erythrocytes modified by Newcastle disease virus.

The specificity of agglutinins in human sera against human erythrocytes modified by Newcastle disease virus (NDV-O agglutinins) was studied with agglutination and agglutination inhibition techniques. Virus grown in both embryonic eggs and tissue culture was used in the experiments. The findings were compatible with the existence of four specificities of agglutinins. In some sera, including those from subjects recently inoculated with vaccines prepared in eggs, the agglutinins were directed against a component of the allantoic fluid. In high-titred infectious mononucleosis (IM sera the agglutinins were directed against antigenic groupings present in the Vic strain but not the B1 strain of NDV, whereas in certain other sera they were directed against antigens present in both of them. Finally, in some sera the agglutinins probably reacted with a red cell antigen uncovered by the viral neuraminidase.     

Studies on the specificity of smooth-muscle antibodies.

Purified contractile proteins from smooth and striated muscles have been used to test the specificity of human smooth muscle antibodies (SMA) from patients with chronic liver disease (IgG-SMA) and acute hepatitis (IgM-SMA). The reactions, as detected by indirect immunofluorescence, of IgG-SMA with renal vessel walls, renal glomeruli, peritubular fibrils and the luminal part of the tubular cells could be completely abolished by absorption with either smooth muscle or skeletal muscle F-actin, while absorption with myosin and tropomyosin had no effect. The specificity of IgG-SMA for actin was confirmed by their staining of the actin-rich I-bands of skeletal muscle myofibrils and by the blocking of this reaction by pretreatment of myofibrils and isolated smooth muscle cells with smooth muscle myosin subfragment 1 (S-1). IgM-SMA from patients with acute hepatitis-stained renal vessel walls and some sera also stained renal glomeruli. The IgM-SMA titres decreased after absorption both with myosin and F-actin but not with tropomyosin. The reactivity of some IgM-SMA could be blocked by S-1 while others could not. Thus the specificity of IgM-SMA seemed to be variable, and apparently differed from IgG-SMA in some cases.     

Banding of human chromosomes treated with papain

Differential Giemsa staining techniques have been used to produce discrete banding patterns for Individual human chromosomes. Similar banding patterns have now been observed after pre-treatment with papain.     

Studies on the specificity of antibodies to ovalbumin in normal human serum: technical considerations in the use of ELISA methods.

As part of a study designed to reveal information about molecular features of allergenic food proteins after absorption from the gut the specificity of antibodies in normal human serum to hen's egg ovalbumin was investigated using ELISA techniques. Preliminary investigations with monoclonal antibodies and hyperimmune rabbit antiserum specific for ovalbumin in its native and denatured form established that the molecule underwent an extensive conformational change on adsorption to polyvinyl chloride microtitre plates. The native conformation could be retained by using antibodies to couple the protein to the surface. Serum from 90% of healthy adult human donors contained IgG antibodies to ovalbumin. In nearly all cases the antibodies were specific predominantly for the native molecule and could not be absorbed with denatured ovalbumin or peptides prepared from it by cleavage with cyanogen bromide or trypsin. Antibodies to denatured ovalbumin were detected in most sera but at very low levels and were preferentially absorbed by the homologous antigen; peptides and native ovalbumin showing variable absorptive activity. Thus, although ovalbumin is ingested largely in a denatured form, the serum antibody response is stimulated mainly by topographic epitopes of the native molecule.     

Studies on the substrate specificity of alpha- and beta-amylase of Entamoeba histolytica

From the supernatant fraction of cell homogenates of Entamoeba histolytica alpha-amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) and beta-amylase (1,4-alpha-D-glucan maltohydrolase, EC 3.2.1.2) were separated and purified by gel filtration and isoelectric focusing, followed by DEAE- and CM-chromatography, respectively. Both enzymes catalyzed the degradation of amylose, amylopectin and glycogen. Hydrolysis of polysaccharides by alpha-amylase yielded as reaction products maltose, maltotriose, maltopentaose and maltohexaose, but no free glucose. beta-Amylase produced as main degradation product of glucopolysaccharides maltose and to a minor extend maltotriose, but no glucose. alpha- and beta-amylase were able to hydrolyze 4-nitrophenyl-alpha-D-glucooligosaccharides (Gn-pNP) containing more than two glucose units per molecule. Under identical conditions G6-pNP was cleaved at highest velocity by alpha-amylase, while beta-amylase exhibited the highest activity with G4-pNP as substrate. alpha-Amylase hydrolyzed G4-pNP, G5-pNP and G6-pNP yielding as main reaction product G2-pNP, but also the formation of G1-pNP and G3-pNP from G4-pNP, of G1-pNP, G3-pNP and G4-pNP from G5-pNP, of G1-pNP, G3-pNP, G4-pNP and G5-pNP from G6-pNP was observed. alpha-Amylase as endo-glucohydrolase attacked all glycosidic bonds in G6-pNP, G5-pNP and G4-pNP, while beta-amylase successively removed maltose units from the non-reducing ends of the glycosides.     

Attention modulates the specificity of automatic imitation to human actors

The perception of actions performed by others activates one’s own motor system. Recent studies disagree as to whether this effect is specific to actions performed by other humans, an issue complicated by differences in perceptual salience between human and non-human stimuli. We addressed this issue by examining the automatic imitation of actions stimulated by viewing a virtual, computer-generated, hand. This stimulus was held constant across conditions, but participants’ attention to the virtualness of the hand was manipulated by informing some participants during instructions that they would see a “computer-generated model of a hand,” while making no mention of this to others. In spite of this attentional manipulation, participants in both conditions were generally aware of the virtualness of the hand. Nevertheless, automatic imitation of the virtual hand was significantly reduced––but not eliminated––when participants were told they would see a virtual hand. These results demonstrate that attention modulates the “human bias” of automatic imitation to non-human actors.

Allergic reactions to ampicillin. Studies on the specificity and selectivity in subjects with immediate reactions

Background and Objective Ainpicillin (AMP) is a drug that has been prescribed extensively. Reactions that have been reported include exanthema. desquamative contact eczema, urticaria and anaphylaxis. Experimental evidence indicates that the side chain of AMP is a structure that may induce a selective immune response either at the humoral or lymphocyte T-cell level. With regard to IgE reactions, the selectivity and specificity of the response needs to be studied in humans. Objective To study tbe specificity of tbe IgE response in a group of subjects who had an immediate allergic reaction after the administration of AMP. Methods Subjects developing an immediate response (anapbylaxis or urticaria) after the administration of AMP or an aminopenicillin derivative witb the same side chain as AMP were studied. Skin tests were made to determinants generated from benzyl penicillin (BP): benzyl penicilloyl (BPO) and minor determinant mixture (MDM), as well as amoxicillin (AX) and AMP. Specific IgE antibodies were determined to benzyl penicilloyl polylisine (BPO-PLL), amoxicilloyl-polyllsine (AX-PLL) and ampicilloyl-polylisine (AMPPLL). The specificity of the IgE antibody response was studied by RAST and RAST inbibition. Subjects were classified in three categories: group A: those who were skin test and/or RAST positive to determinants derived from benzylpenicllin, group B: those who were negative to determinants derived from benzylpenicillin but were skin lest and/or RAST positive to determinants derived from AX and AMP and group C: those who were exclusively positive to determinants derived from AMP. Results A total of 48 subjects was included in the study. In group A there were 35 cases, in group B 10 cases, and in group C tbree cases. RAST inhibition studies showed that in some instances tbe side chain of AMP could induce specific responses with a variable degree of crossreactivity between BP and AX. Conclusions Atbough AMP can induce an immediate IgE response in subjects allergic to betalactams and tbe structure of the side chain may contribute to the specificity of the response, our results indicate tbat in most instances crossreactivity with the other penicillins exists and that in the groups studied selective reactions to just AMP derived determinants were uncommon.     

Analysis of the specificity of natural human antibody reactive to Actinomyces

The specificity of a frequently-occurring precipitin response to soluble antigens from cell-walls and culture filtrates of A. viscosus ATCC 19246 was examined. After precipitation with isopropanol (50-75% v/v), antigen fractions of different charge and molecular weight were isolated by ion exchange and gel filtration. When heated in mineral acid or alkali above 0.15 M, each of the purified antigens lost precipitating activity, but now inhibited the precipitin reaction between serum and exogenous unheated antigen. The inhibitor was isolated over Biogel P30 and characterized as a peptide fragment (mol. wt about 2 kd) containing approximately 50 moles of ornithine and 6-12 moles, respectively, of aspartate, serine, threonine, glutamate, glycine, alanine and histidine per 100 moles amino acids. The inhibitor was totally destroyed by heating for 1.0 hr in 2.0 M HCl. Variability in the number of fragments and differences in the non-antigenic portions probably accounted for the complexity of the antigens. Ornithine, putrescine, N-acetyl putrescine and various sugars had little or no effect on the precipitin reaction with intact antigen at high concentrations (200 mM), whereas the fragment inhibited completely at 0.4 mM. This indicates that neither ornithine nor its side-chain amides are exclusively recognized by antibody. However, ornithine may be part of a larger sequence and/or important in forming the configuration recognized by the human antibodies.