The murine monoclonal antibody (MAb) 18B7 [immunoglobulin G1(κ)] is in preclinical development for treatment ofCryptococcus neoformans infections. In anticipation of its use in humans, we defined the serological and biological properties of MAb 18B7 in detail. Structural comparison to the related protective MAb 2H1 revealed conservation of the antigen binding site despite several amino acid differences. MAb 18B7 was shown by immunofluorescence and agglutination studies to bind to all four serotypes of C. neoformans, opsonize C. neoformans serotypes A and D, enhance human and mouse effector cell antifungal activity, and activate the complement pathway leading to deposition of complement component 3 (C3) on the cryptococcal capsule. Administration of MAb 18B7 to mice led to rapid clearance of serum cryptococcal antigen and deposition in the liver and spleen. Immunohistochemical studies revealed that MAb 18B7 bound to capsular glucuronoxylomannan in infected mouse tissues. No reactivity of MAb 18B7 with normal human, rat, or mouse tissues was detected. The results show that both the variable and constant regions of MAb 18B7 are biologically functional and support the use of this MAb in human therapeutic trials.
Antibodies to double-stranded DNA are pathognomonic of systemic lupus erythematosus and deposit in the kidneys of lupus patients to cause glomerulonephritis. Recent data suggest that a significant proportion of anti-DNA antibodies may cross-react with renal antigens and be sequestered in the kidney by virtue of this cross-reactivity. If this is true, antigenic competition for pathogenic antibodies might prevent their deposition in kidneys and the ensuing tissue damage. To generate surrogate antigens that could be used for this purpose, we have used peptide display phage libraries to identify peptides that react with R4A, a pathogenic mouse monoclonal anti-DNA antibody that deposits in glomeruli. We have demonstrated that the peptides bind in or near the double-stranded DNA binding site. Furthermore, the peptides are bound preferentially by the R4A antibody as compared with two closely related antibodies derived from it, one of which deposits in renal tubules and one of which displays no renal pathogenicity. Administration of one of these peptides in a soluble form protects mice from renal deposition of the R4A anti-DNA antibody in vivo. This represents a new therapeutic approach in systemic lupus erythematosus that focuses on protecting target organs from antibody mediated injury.
Monoclonal antibodies (mAbs) to the polysaccharide capsule of Cryptococcus neoformans can prolong survival in mice. However, the properties of antibodies that mediate protection are not fully understood. The IgM mAbs 12A1 and 13F1 originated from the same B cell and differ only by somatic mutations in their variable regions; yet mAb 12A1 protects against serotype D infection, while mAb 13F1 does not. Phage peptide display libraries were used to analyze the fine specificity of these two mAbs. The selection of distinct peptide motifs from identical libraries confirmed that mAbs 12A1 and 13F1 bound to two distinct epitopes. Immunofluorescence and immunoelectron microscopy studies revealed differences in antibody localization within the capsule of serotype D strain; mAb 12A1 bound to the outer rim of the capsule resulting in an annular pattern, whereas mAb 13F1 bound throughout the capsule and had a punctate appearance. The difference in the binding pattern of mAb 12A1 and 13F1 was not observed on serotype A organisms, where both mAbs bound to the capsule with an annular fluorescence pattern. The fluorescence pattern of binding correlated with protective efficacy; mAb 13F1 prolonged survival of mice infected with the J11 serotype A strain (annular fluorescence), but not serotype D strains (punctate pattern). Annular binding, but not punctate binding, was associated with increased opsonic efficacy for phagocytosis of C. neoformans by J774.16 macrophage-like cells. The correlation between capsular binding pattern, opsonic activity, and ability to prolong survival suggests that the efficacy of anticryptococcal antibodies is dependent upon where they bind in the polysaccharide capsule.
Cryptococcus neoformans is a fungus that is pathogenic in humans and that can produce melanin in vitro. Melanization is associated with virulence, but there is no evidence that melanin is made during infection. Melanins are difficult to study because they are amorphous and insoluble. Melanin-binding peptides from a phage display library were used to demonstrate that C. neoformans makes melanin-like compounds in tissue. Melanin-binding peptides were characterized by a high proportion of positively charged and aromatic residues. Two other methods, demonstration of an antibody response to melanin in mice infected with C. neoformans and analysis of yeast cell walls in infected tissue by light microscopy, were used to support these findings. The demonstration that C. neoformans melanizes in tissue has important implications for pathogenesis and drug discovery.Melanins are pigments of biological origin that conform to a unique electron spin resonance pattern (4,19). In contrast to the other great natural pigments, such as the hemoglobins, chlorophylls, flavonoids, and carotenoids, little is known about the structure of naturally occurring melanin (2,4,7,10,11,15,27). It has been difficult to use existing biochemical and biophysical techniques to study melanins because they are insoluble and amorphous. Although the pigments are usually black or brown, blue, green, and red melanins also exist (27). This indicates that melanins are structurally heterogeneous. Some chemical and biological properties of melanin include electron exchange, free radical production and absorbtion, protection from UV light, and drug binding (7). In addition, some biological species utilize melanin for camouflage or sexual display (7). Melanin is also believed to play a significant role in the pathogenesis of malignant melanoma (6) and may interfere with the efficacy of melanoma therapy by modifying the effects of ionizing radiation (17) or by binding and chelating antineoplastic drugs.Cryptococcus neoformans is an encapsulated fungus that causes life-threatening meningoencephalitis in 6 to 8% of patients with AIDS (3). C. neoformans has a laccase that catalyzes the synthesis of melanin in the presence of phenolic compounds, such as L-dopa (14, 28). The ability of C. neoformans to melanize in vitro has been associated with virulence (16, 18), but melanin synthesis in vivo has not been demonstrated. Melanin has been shown to protect C. neoformans against oxidants (25), amphotericin B (23), and macrophages in vitro (22). Some drugs that bind melanin are toxic to eukaryotic cells containing melanin. For example, the melaninbinding compound trifluoperazine has greater fungicidal activity against melanized than nonmelanized cryptococcal cells (26). Establishing whether melanization occurs in vivo is important for understanding the relationship of phenoloxidase activity, pigment production, and virulence. To date, this has not been possible, because stains for melanin are not specific for this compound (9). Melanin forms a shell in the cell wall of C....
The molecular diversity of the luminal endothelial cell surface arising in vivo from local variations in genetic expression and tissue microenvironment may create opportunities for achieving targeted molecular imaging and therapies. Here, we describe a strategy to identify probes and their cognate antigens for targeting vascular endothelia of specific organs in vivo. We differentially screen phage libraries to select organ-targeting antibodies by using luminal endothelial cell plasma membranes isolated directly from tissue and highly enriched in natively expressed proteins exposed to the bloodstream. To obviate liver uptake of intravenously injected phage, we convert the phage-displayed antibodies into scFv-Fc fusion proteins, which then are able to rapidly target select organ(s) in vivo as visualized directly by ␥-scintigraphic whole-body imaging. Mass spectrometry helps identify the antigen targets. This comprehensive strategy provides new promise for harnessing the power of phage display for mapping vascular endothelia natively in tissue and for achieving vascular targeting of specific tissues in vivo.antibody ͉ vascular targeting ͉ endothelial cell ͉ protein expression
Oxidative metabolism of a 3-aroylthiophene, 1, by rat liver microsomes in the presence of mercaptoethanol as a trapping agent led to the isolation of four main compounds, 2-5, which have been isolated and characterized by UV, 1H NMR, and mass spectroscopy. They all derive from two primary metabolites, 2 and 3, which result from the nucleophilic addition of mercaptoethanol to a reactive, very electrophilic intermediate formed by sulfoxidation of the thiophene ring of 1. Further reactions of diastereoisomers 2 and 3 with mercaptoethanol led to compound 4 that is opened at the level of its thiophene ring and, eventually, to a final metabolite 5 resulting formally from the addition of mercaptoethanol on the 4, 5-double bond of the thiophene ring of 1. Compound 5 is very stable even in the presence of a large excess of mercaptoethanol. Similar reactions were observed upon microsomal oxidation of 1 in the presence of another thiol, N-acetylcysteine. Final metabolites 8a and 8b equivalent to 5 except for the replacement of its mercaptoethanol substituent with an N-acetylcysteinyl group were isolated and characterized by UV, 1H NMR, and mass spectroscopy. Interestingly, after treatment of rats with 1, metabolites 8a and 8b could be detected in urine, indicating that the successive reactions, that were observed in vitro after microsomal oxidation of 1 in the presence of a thiol-containing trapping agent, also occur in vivo, glutathione acting as a nucleophile in that case. These data provide clear evidence for the intermediate formation of a reactive. electrophilic thiophene sulfoxide in metabolic oxidation of 1 in vitro and in vivo. They also provide the first data on the complex reactivity of such thiophene sulfoxides, whose chemistry is poorly known, and on their fates in living organisms.
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