Antigenic components in the cytoplasmic extract of Candida albicans were examined after fractionation by concanavalin A-Sepharose and DEAE-Sephacel ion-exchange chromatography. Fractions from the DEAE column were tested by fused rocket immunoelectrophoresis for their reactivity with antibodies in the sera of 20 patients with disseminated candidiasis. Three groups of fractions (regions A, B, and C) from the DEAE column were defined by their reactivity with these sera. Immunoblot analysis with 20 human sera identified 18 antigenic components in regions A, B, and C. Region A contained nine antigens, region B contained four antigens, and region C contained five antigens. Region A contained an antigen with an apparent molecular weight of 48,000 that was recognized by 7 of 10 sera from patients with disseminated candidiasis. Immunoprecipitation experiments with labeled proteins from region A and 51 human sera also demonstrated the presence of a major antigen whose apparent molecular weight is 48,000 to 52,000. The 48to 52-kilodalton protein is an abundant protein in region A and is the most frequently recognized protein by antibodies in the sera of patients with disseminated candidiasis. Patients with disseminated candidiasis had significantly higher levels of antibody (immunoglobulin G) (P < 0.001) directed against the 48to 52kilodalton protein than did patients with noninvasive forms of candidiasis, patients with other fungal infections, or normal, healthy persons.
Endotoxin, the lipopolysaccharide (LPS) ~ obtained from the cell wall of gram-negative bacteria, elicits many physiological responses in mammals (1). The large number and diversity of these endotoxic reactions to LPS have made it difficult to analyze the control mechanisms involved. In particular, it has been impossible to determine whether most or all of these diverse responses to LPS are regulated by the expression of common genetic loci. In this paper we compare the genetic control of a number of endotoxic responses which appear to result from the interaction of LPS with B lymphocytes in mice, to responses involving different cell types.The C3H/HeJ mouse strain is unique in its resistance to many of the effects of LPS observed in other mouse strains (2-9). ~ These include resistance to the mitogenic, polyclonal, and adjuvant effects of LPS (4-9), 2 which all appear to result from the interaction of LPS with bone marrow-derived (B) lymphocytes (4, 5), as well as to a variety of other effects such as toxicity (3), resistance to bacterial infectious (10), macrophage activation (10, 11), and increases in levels of various serum components such as colony-stimulating factor (CSF) (12), and the acute phase serum amyloid protein, SAA (13). LPS elicits several different types of responses in murine B lymphocytes. LPS induces the nonproliferative expression of cell surface antigens in more immature B-cell types (14, 15), and a mitogenic response in more mature B-cell types (2-9). 2 B lymphocytes from C3H/HeJ mice are unresponsive in both of these LPS response assays (16). The implication from all of these observations is that the defective gene in C3H/HeJ mice that limits LPS responsiveness in these various assay systems is involved in the expression of a receptor found in many different cell types. 2 However, there are no genetic studies that directly show that the loci responsible for the defective LPS responses of different cell types in C3H/HeJ, are in fact identical.The low responsiveness to LPS of C3H/HeJ mice is determined by a single gene linked to Mup-1 on chromosome 4 in mice.
Recombinant DNA clones containing chick alpha-actin mRNA sequence have been isolated and used as probes to analyze the structure and developmental expression of the chick alpha-actin gene. The full length, 2000 nucleotide alpha-actin mRNA is detected in poly(A) RNA at early and late stages of in vivo leg muscle development. As expected, the alpha-actin mRNA is present at very low levels at early myogenic stages but is a high abundance species in terminally differentiated muscle. However, most of the alpha-actin mRNA from fused leg muscle is shorter than 2000 nucleotides, and occurs in relatively discrete size classes. An alpha-actin-like mRNA can be detected in poly(A) RNA from early embryonic brain, indicating that transcription of the alpha-actin gene may not be strictly muscle-specific at all stages of development. We have identified at least 3, very short (< 100 base pairs) intervening sequences in the alpha-actin gene which was isolated from a chick genomic library. The structure of the chick alpha-actin gene differs, therefore, from the structures of actin genes from yeast and Drosophila, both of which contain a single, relatively long, intervening sequence.
Three monoclonal antibodies, designated A2C7, C2C7, and F19, were produced which recognize proteins from Candida albicans. All are of the immunoglobulin GI heavy chain and kappa light chain class. A2C7 and C2C7 immunoprecipitated three proteins contained in a partially purified fraction (region A) of a mycelial cytoplasmic extract of C. albicans. The apparent molecular weights of these proteins are 120,000 (120K) to 135K, 44K to 52K, and 35K to 38K. Monoclonal antibody F19 was reactive with proteins of 42K, 43K, and 50K in immunoblotting experiments. F19 was also able to form a precipitin band in agarose gel with protein(s) contained in region A. Limited proteolytic digestion of the three proteins immunoprecipitated by A2C7 and C2C7 demonstrated that both monoclonal antibodies recognized the same three Candida proteins and that there exists a significant degree of relatedness in primary structure among the three proteins. Proteins with apparent molecular weights of 120K to 135K, 44K to 52K, and 35K to 38K that were immunoprecipitated by sera from two patients with invasive candidiasis and by the serum from a rabbit immunized against a 48K (44K to 52K) Candida protein were also analyzed by limited proteolysis. Patterns of peptide fragments generated by enzymatic digestion of these proteins showed that the proteins recognized by the monoclonal antibodies are the same proteins recognized by antibodies in the sera of patients during an invasive Candida infection and by antibodies in the serum of the immune rabbit.
Summary.We have generated monoclonal antibodies (MABs) to staphylococcal enterotoxin B (SEB) in BALB/c mice. Five out of 20 clones which produce anti-SEB MABs have been characterised. Among them, three produce IgGl/lc, one produces IgM/A, and one apparently produces both IgGl/A and IgM/iZ MABs. The anti-SEB titres of ascites fluids range from 3200 to > 819200 by ELISA. All of the MABs analysed thus far neutralise the mitogenic response of BALB/c splenocytes to a suboptimal dose of SEB. Also, the induction of suppressor cells by SEB in vitro is reversed by pre-incubating SEB with these MABs. Limited digestion with chymotrypsin, trypsin or Staphylococcus aureus V8 protease yields peptide fragments which have been tested by Western-blot analysis. MABs 1FD7 and 2GD9 are specific for the carboxy-terminal end of SEB, and have a similar, but not identical, binding epitope. MABs 2DA3 and 2HAlO bind to intact SEB but not to cleaved products, and are probably specific for antigenic determinants altered by the cleavage or by the denaturing conditions of the electrophoresis, or by both.
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