Predicting development of sustained unresponsiveness to milk oral immunotherapy using epitope-specific antibody binding profiles

1. Download high-res image (260KB)
2. Download full-size image

     

Comparison of radioimmunoassay and ELISA methods for detection of antibodies to chromatin components

A solid phase radioimmunoassay has been compared with an enzyme-linked immunosorbent assay (ELISA) for efficacy in measuring anti-chromatin antibodies. The low backgrounds achieved with the radioimmunoassay method produced a high signal-to-noise ratio and enabled detection of the human test antiserum at a dilution of 1:102,400. By contrast, the ELISA could detect the same antiserum only at a dilution of 1:3200 and above. The radioimmunoassay was consistently more sensitive than the ELISA for detection of anti-chromatin antibodies in a number of human and mouse sera and ascites fluid containing a monoclonal antibody. Factors affecting sensitivity in both assays are discussed.     

Development of acute autoimmune encephalomyelitis in mice: Factors regulating the effector phase of the disease

The development of acute experimental autoimmune encephalomyelitis (EAE) in mice is potentiated by the use of Bordetella pertussis vaccine as an adjuvant. Histamine sensitizing factor (HSF) extracted from B. pertussis is the active adjuvant agent and causes a mild increase in cerebrovascular permeability. During the development of EAE, there is an additional increase in vascular permeability of the brain and spinal cord. The adjuvant action of B. pertussis HSF does not appear to mimic a generalized beta-adrenergic blockade, since the course of EAE is not potentiated by adrenalectomy. The cerebrovascular permeability changes observed in EAE are probably mediated by vasoactive amines, since the expression of EAE can be blocked by vasoactive amine antagonists.     

Histidine-rich protein genes and their transcripts in Plasmodium falciparum and P. lophurae

The presence of histidine-rich protein (HRP) related genes and gene products in Plasmodium falciparum was demonstrated using a synthetic pentahistidine-encoding oligonucleotide and a cloned HRP cDNA probe prepared from the avian parasite P. lophurae. In Northern blotting experiments, two knobby clones of P. falciparum were found to contain a 3500 nucleotide RNA species that hybridized with the oligonucleotide and HRP cDNA probes. As this component had the expected size for an mRNA encoding an 80-90 kDa protein and was absent from two knobless clones of P. falciparum, we concluded that it represented a 'knob protein' mRNA. Using the restriction enzyme EcoRI, three identical cross-hydribizing HRP gene fragments were found in the DNA of both knobby and knobless clones of P. falciparum. These fragments differed in size from those present in P. lophurae. These results suggest that the absence of knob protein mRNA in knobless clones is not due to loss of the corresponding gene(s).     

The instability of membrane markers expressed by human monocytes and macrophages in culture

Surface markers were tested on freshly isolated human monocytes and following their in vitro maturation to macrophages. The markers tested were HLA-DR antigens, receptors for the Fc of IgG and complement as well as membrane markers defined by monoclonal antibodies. The results revealed a dynamic expression of some of the markers on monocytes which was influenced by several variables. The expression of the markers was modulated by the presence of different sera, by treatment with lymphokines and interferon and following the in vitro maturation of monocytes to macrophages. The most unstable marker was found to be the HLA-DR, which was modulated by all these variables. The 63D3 was affected by different sera and culture supernatant, as well as following the maturation of monocytes to macrophages, but not by lymphokines and interferon. One of the markers, the Mac 120, was found to be relatively stable and did not change significantly following the maturation of monocytes to macrophages. The Fc and complement receptors were also stable in their expression under these conditions, but were probably partially blocked in the presence of human serum. These results indicated that at least some of the heterogeneity related to the monocyte population was probably not due to the occurrence of stable subsets of cells, but rather to reversible changes in marker expression.     

Hydrophobic interactions in Plasmodium falciparum invasion into human erythrocytes

Human glycophorins block in vitro invasion of Plasmodium falciparum merozoites into human erythrocytes. A segment of glycophorin A which appears to be involved in the inhibition, is at, or adjacent to, the membrane-spanning domain of the molecule. To study the role of hydrophobic interactions in the inhibition, a series of proteins were derivatized with lipophilic side groups, and tested for inhibitory activity. Glycophorin A became five times more inhibitory after derivatization with nitrobenzylfurazan groups. Bovine serum albumin was derivatized to different degrees with nitrobenzylfurazan, dinitrobenzyl, trinitrobenzyl, dansyl, disulfonic stilbene, and fluorescein groups. The presence of hydrophobic side groups on the protein rendered it highly inhibitory to invasion, whereas the presence of hydrophilic substitutes such as disulfonic stilbenes did not. Other soluble proteins such as human serum albumin, transferrin, ovalbumin, fetuin and casein derivatized with dinitrobenzyl groups, were also found to block invasion. Inhibition was not a result of toxic effects of the protein derivatives on parasite metabolism or development. A minimum of ten hydrophobic side groups per bovine serum albumin was required in order to elicit appreciable inhibition. The invasion blocking activity was highly correlated with the rate and affinity of binding of the derivatized macromolecules to heptyl-Sepharose. The latter provided a quantitative measure for the capacity of amphiphiles to undergo hydrophobic interactions with insoluble matrices. The results of the present study indicate that hydrophobic interactions may be an essential component in the invasion of P. falciparum merozoites into human erythrocytes.     

An enzyme-linked immunosorbent assay (ELISA) for the measurement of antibodies to different parts of the gram-negative lipopolysaccharide core region

Bovine serum albumin was complexed with the core antigens of either Escherichia coli J5 LPS, Salmonella minnesota R595 LPS or E. coli lipid A. These core-BSA complexes were used for solid-phase coating in ELISAs for anti-core antibodies. Antibodies, binding to various parts of the core region were easily quantified in a single experimental set-up, which was hitherto not possible. The ELISA has only 3 incubation steps and is not costly as only moderate amounts of the core antigens (i.e., 1 microgram per test) were needed for coating. The sensitivity proved to be excellent and the complexes were biologically fully active (compared to native, smooth LPS), which make them suitable for the screening (after fusion) of monoclonal anti-core antibodies. Another possible application is the large-scale screening of blood-bank sera in order to find samples with a high anti-core antibody content.     

Interaction of nanobacteria with cultured mammalian cells

Nanobacteria were recently isolated from human blood and commercial fetal bovine serum (FBS) and were located in the -2 subgroup of proteobacteria based upon their 16S rRNA gene sequence. They can be cultured even in the absence of mammalian cells, and have extraordinary properties, like very slow growth rate and an impermeable cell wall, making their detection difficult by standard microbiological techniques. Since they are present in FBS, and thus in cell cultures, it is essential to clarify their effects on cultured mammalian cells. In this study, we show that four out of six nanobacterial isolates from different sera exerted a cytotoxic effect on 3T6 fibroblasts verified by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] viability assay, lactate dehygrogenase (LDH) release and by direct microscopy. The cytotoxic effect of nanobacteria was attenuated after they had been subcultured several times. The cytotoxic effect was similar with all tested murine and human fibroblastoid cell lines. Differential interference contrast and electron microscopy, and FITC staining with specific monoclonal antibodies indicated selective, possibly receptor-mediated adherence, followed by internalization and cytotoxicity in the 3T6 fibroblasts used as a model in these interaction studies. Thus, nanobacteria have a special way of invading mammalian cells: they trigger cells that are not normally phagocytic to engulf them. These organisms seem to be an important cause for cell vacuolization, poor thriving and unexpected cell lysis, problems frequently encountered in mammalian cell culture.     

Induction of specific anti-actin antibodies and their measurement by ELISA technique.

Using the ELISA technique we have been able to quantify antibodies directed against actin and to follow the kinetics of antibody production. Specific anti-actin antisera have been raised in rabbits by immunization with chemically modified white muscle rabbit actin. Two or three dinitrophenyl groups linked per actin molecule were sufficient to break natural tolerance, while linkage of three phosphorylcholine groups to actin was not.