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Biomedical subjects

M D Scharff

Publications and source records attributed to M D Scharff.

At least 19 recordsLinked to original sources
PubMed1992

Monoclonal antibody based ELISAs for cryptococcal polysaccharide.

Mouse monoclonal antibody (MAb)-based enzyme-linked immunosorbent assays (ELISAs) have been developed to detect Cryptococcus neoformans capsular polysaccharide from the four serotypes A, B, C and D. The ELISAs avoid the problem of unreliable polysaccharide binding to polystyrene plates by using MAbs to capture and immobilize the polysaccharide antigen. The presence of polysaccharide is detected using MAbs of a different isotype from that of the capture MAb. The capturing MAbs are themselves immobilized on the plates using commercial goat anti-mouse polyclonal sera. The MAbs bind to the glucuronoxylomannan component of cryptococcal polysaccharide. The ELISAs can be used to measure the concentration of polysaccharide in biological fluids and are potentially useful tools for basic research and clinical studies.

Animals↗
PubMed1992

Identification of rare immunoglobulin switch variants using the ELISA spot assay.

We describe here the use of the ELISA spot assay to identify, quantify, and isolate rare hybridoma subclones that have switched to expressing a new class or subclass of Ig. This technique is less labor intensive and time consuming than sib selection and standard ELISA and eliminates the many false positives that complicated those techniques. The use of the ELISA spot assay also allows screening large populations of cells and accurate quantitation of the rate of isotype switching.

Animals↗
PubMed1992

Antibodies elicited by a Cryptococcus neoformans-tetanus toxoid conjugate vaccine have the same specificity as those elicited in infection.

The antibody responses of BALB/c mice to serotype A Cryptococcus neoformans capsular polysaccharide (CNPS) were compared after cryptococcal infection and immunization with a serotype A glucuronoxylomannan-tetanus toxoid conjugate (GXM-TT). Infection rarely resulted in a rise of serum antibody titer to CNPS. In contrast, mice immunized with GXM-TT produced serum IgM and IgG to CNPS. Six IgM and one IgG1 monoclonal antibodies (MAbs) were generated from the spleen of one infected mouse. Nine IgM, 1 IgG3, 16 IgG1, and 7 IgA MAbs were generated from the spleen of one GXM-TT-immunized mouse. All MAbs generated from both mice bound to the GXM fraction of the capsular polysaccharide. For some MAbs, de-O-acetylation of serotype A GXM abolished or greatly reduced MAb binding compared with the native GXM. All MAbs reacted with CNPS from C. neoformans serotypes A-D. MAbs generated from the infected mouse competitively inhibited the binding of MAbs generated from the GXM-TT-immunized mouse. These results indicate that some antibodies elicited by infection with C. neoformans or by immunization with GXM-TT bind to the same antigenic determinant in the GXM.

Animals↗
PubMed1992

Protective murine monoclonal antibodies to Cryptococcus neoformans.

Several murine monoclonal antibodies (MAbs) specific for the capsular glucuronoxylomannan of Cryptococcus neoformans were studied for their capacity to confer protection when passively administered to lethally infected mice. The MAb group studied recognized at least three distinct epitopes and included immunoglobulin M (IgM), IgG3, IgG1, and IgA isotypes. The protection model used A/J and BALB/c mice infected intraperitoneally with 10(8) cryptococci. The MAbs were administered either immediately preceding or, in one experiment, 24 to 48 h prior to infection. Protective efficacy was assessed by the ability of passively administered MAbs to prolong the survival of lethally infected mice. Three IgM MAbs, each of which recognized a distinct epitope, were able to prolong survival of lethally infected mice to different extents. A set of IgM, IgG3, IgG1 and IgA MAbs which utilize the same immunoglobulin gene elements and were derived from the same B-cell clone exhibited significant class differences in protective efficacy with IgA, IgG1 > IgM > IgG3. The results confirm that protective MAbs against C. neoformans capsular polysaccharide exist and strongly suggest that both epitope specificity and isotype are important determinants of protective efficacy.

Animals↗
PubMed1992

Extensive allelic variation in Cryptococcus neoformans.

The orotidine monophosphate pyrophosphorylase (OMPPase) gene locus of the DNA of 13 Cryptococcus neoformans var. neoformans strains, including 10 recent clinical isolates, was studied by using restriction fragment length polymorphisms and nucleotide sequence analysis. The OMPPase locus (URA5) is highly polymorphic, and at least six alleles were identified. The nucleotide sequences of some alleles differed by up to 5%. The majority of the nucleotide polymorphisms in the protein-coding region occurred at the third codon position and were silent. The low frequency of replacement nucleotide substitutions relative to silent nucleotide substitutions implied that there is strong selection against amino acid changes in OMPPase. The allelic variation suggested that there is extensive genomic diversity among C. neoformans clinical isolates from one geographic area. The various alleles are potentially useful markers in the study of the population structure, epidemiology, and pathogenesis of C. neoformans strains.

Alleles↗
PubMed1992

The role of somatic mutation in the pathogenic anti-DNA response.

Anti-DNA antibodies represent a significant autospecificity in systemic lupus erythematosus because they are essentially diagnostic of the disease and they contribute to renal pathology. The molecular genetic characterization of these antibodies from both lupus-prone mice and humans with lupus has shown them to be somatically mutated. In many cases the nature of the mutations suggests that DNA or some structurally homologous molecule is driving the response. In other cases the high replacement-to-silent mutation ratio in framework regions of the antibody suggests selection by idiotype or by some mechanism other than antigen itself. Current studies of immunoglobulin variable region genes encoding anti-DNA antibodies reveal no disease associated polymorphisms. There are also no data suggesting that the nature of the recombination process that forms intact variable region genes or of the process of somatic mutation differs in autoimmune and nonautoimmune strains or kindred. Current data suggest, rather, that a defect in regulation is responsible for auto-antibody production in SLE. The finding that most if not all anti-double stranded DNA antibodies are somatically mutated suggests the defect is in maintenance of peripheral rather than central tolerance.

Animals↗
PubMed1991

The mouse antibody response to infection with Cryptococcus neoformans: VH and VL usage in polysaccharide binding antibodies.

Cryptococcus neoformans is a ubiquitous fungus that can cause serious infections in humans. The fungus has a polysaccharide (C. neoformans capsular polysaccharide; CNPS) capsule that contributes to its pathogenicity and can elicit an antibody response. Nevertheless, only 4 of 60 BALB/c mice chronically infected with C. neoformans had a detectable increase in serum anti-CNPS. The sera of three responder mice contained both IgM and IgG anti-CNPS antibody, and the titers of lambda and kappa anti-CNPS antibody were approximately equal. Eight IgM and one IgG3 monoclonal antibodies (mAbs) were generated from the spleen of one responder mouse, and one IgA was generated from the spleen of another mouse. Seven of the IgMs, the IgG3, and the IgA mAb had lambda light chains and were specific for serotype D CNPS. Molecular analysis confirmed that this was a highly restricted antibody response. All of the D-specific antibodies used VH441, JH3, and either V lambda 2/J lambda 2 or V lambda 1/J lambda 1, and all had the same heavy chain CDR3 amino acid sequence, even though there were differences in the nucleotide sequence of the N/D segment. One IgM mAb reacted with both serotype A and D CNPS, and this mAb used different VH and JH genetic elements and had kappa light chains. All the anti-CNPS mAbs used J proximal VH gene elements that have previously been shown to bind dextran and other polysaccharides. Sequence and Southern blot analysis indicate that the serotype-D CNPS-specific mAbs arose from only a few precursor B cells.

Amino Acid Sequence↗
PubMed1991

Murine adenovirus infection of SCID mice induces hepatic lesions that resemble human Reye syndrome.

Murine adenovirus type 1 (MAV-1) infection of CB-17 SCID mice (which are homozygous for the severe combined immunodeficiency mutation) induces hepatic histopathologic and ultrastructural features that are strikingly similar to human Reye syndrome. Gross pathologic examination of MAV-1-infected mice revealed only pale yellow liver tissue. Histopathologic studies of tissue from MAV-1-infected mice revealed diffuse hepatic injury manifested by microvesicular fatty degenerative changes of hepatocytes and electron microscopic evidence of focal mitochondrial swelling with disruption of cristae and depletion of glycogen. Serum aminotransferase activities increased markedly in the infected animals; however, plasma ammonia levels were not elevated at the times assayed. Although all mice infected with MAV-1 died, neutralizing anti-MAV-1 monoclonal antibodies provided a dose-dependent delay in the appearance of clinical disease and hepatic histopathologic findings. Other findings included rare viral inclusions with only minimal inflammation in spleen, adrenal, and liver of infected mice. Our findings indicate that MAV-1 infection of SCID mice may provide important insights into the pathogenesis of the hepatic lesions of Reye syndrome.

Adenoviridae↗
PubMed1991

The molecular and biochemical characterization of mutant monoclonal antibodies with increased antigen binding.

Mutant mAb with increased Ag binding were generated from a hybridoma cell line, 36-65, that secretes an IgG1,kappa anti-p-azophenylarsonate-(Ars) specific antibody. The mutant antibodies were identified using an Ars-specific ELISA and sib selection so that approximately 10(6) cells could be analyzed. The ELISA used as Ag a low ratio of Ars coupled to BSA and was set up so that only those antibodies that had higher binding than the parent would be detected. Seven mutant producing cell lines were isolated from five independent clones of 36-65. The mutant antibodies bind Ag 20 to more than 200-fold better than the parent and have wild type V region sequences. All have C region mutations that result in an increased avidity. At least five different genetic events are responsible for the C region mutations.

Amino Acid Sequence↗
PubMed1991

A V region mutation in a phosphocholine-binding monoclonal antibody results in loss of antigen binding.

A V region mutant producing an antibody that had lost the ability to bind phosphocholine was isolated from a hybridoma producing a germline encoded T15 antibody. The mutation resulted in a single aspartic acid to asparagine substitution at residue 95 of the H chain V region. This confirms that the aspartic acid at residue 95 plays a major role in Ag binding. The results also suggest that somatic cell genetic techniques can be used to generate mAb with useful changes in Ag binding.

Amino Acids↗
PubMed1991

Characterization of somatically mutated S107 VH11-encoded anti-DNA autoantibodies derived from autoimmune (NZB x NZW)F1 mice.

We have studied 19 S107 heavy chain variable region gene (VH11)-encoded monoclonal antibodies from NZBWF1 mice. These studies show that a single VH gene can encode both antibodies to foreign antigens (anti-phosphorylcholine) and to self antigens (anti-double-stranded DNA) in the same animal. All of the anti-DNA antibodies contain many somatic mutations compared with the relevant germline genes. Since the anti-DNA antibodies were extensively somatically mutated and had undergone isotype switching, the response seems to be T cell dependent. While some of the antibodies appear to be the products of an antigen-driven and antigen-selected response, a number of characteristics of the antibodies suggest that forces other than antigen are contributing to the stimulation and selection of this response.

Amino Acid Sequence↗
PubMed1991

Instability of immunoglobulin genes in S107 cell line.

Somatic mutation occurs frequently in rearranged and expressed immunoglobulin variable region genes in vivo. In contrast, V region hypermutation seldom occurs in antibody-forming cells in culture. The S107 mouse myeloma cell line is one of the few cell lines that has been observed to generate V region mutations frequently and spontaneously in vitro. Detailed examination reveals that both the S107 tumor and the cell line derived from it contain and express a duplicated heavy-chain gene. In culture, only one of the two heavy-chain genes undergoes both V and C region mutation, and variants with complex phenotypes and genotypes arise as a result of mutation and segregation of these duplicated genes.

Animals↗
PubMed1990

Intravascular metabolism of normal and mutant mouse immunoglobulin molecules.

The metabolism of IgG immunoglobulins in the body is tightly regulated in order to maintain their intravascular concentration. Different subclasses may have different intravascular half-lives, and in the mouse, passively administered IgG2b disappears from the circulation more rapidly than IgG2a. We have attempted to localize the sequences in the constant region which are responsible for this difference by examining the intravascular metabolism of mutant immunoglobulins that were generated in tissue culture and have undergone deletions of individual constant region domains or contain different combinations of gamma 2b and gamma 2a CH2 and CH3 domains. Our results suggest that the regulation of intravascular metabolism is complex but indicate that sequences in the CH3 domain are important in determining the different intravascular half-lives of IgG2b and IgG2a antibodies in the mouse.

Animals↗
PubMed1990

Current state of the hybridoma technology.

The first description of the hybridoma technology in 1975 seemed to hold enormous promise for the treatment of a variety of human disease. The ability to produce monoclonal antibodies led to the availability of large amounts of homogeneous and predictable preparations of antibody. The potential to renew indefinitely a particular antibody surmounted many of the technical and regulatory problems that made polyclonal antisera difficult to use as therapeutic agents in man. The hybridoma technology seemed even more valuable as it became clear that it could lead to the generation of pure, highly specific antibodies from impure, poorly characterized antigens. Monoclonal antibodies have been extremely useful in basic investigations and have facilitated the development of new diagnostic tests for serum and tissue components and infectious agents. However, novel approaches are needed in order to provide more useful, less immunogenic antibodies which could be used routinely for passive immunization in the treatment of infections or for tumor targeting.

Animals↗
PubMed1989

The role of somatic hypermutation in the generation of antibody diversity.

The immune system is capable of establishing an enormous repertoire of antibodies before its first contact with antigen. Most antibodies that express germ-line sequences are of relatively low affinity. Once antigen enters the system, it stimulates a somatic mutational mechanism that generates antibodies of higher affinity and selects for the expression of those antibodies to produce a more effective immune response. The details of the mechanism and regulation of somatic hypermutation remain to be elucidated.

Animals↗
PubMed1989

Immunologic and pharmacologic concepts of monoclonal antibodies.

While monoclonal antibodies have solved many of the difficulties of using immunologic reagents for radioimmunodiagnosis and therapy, in the 13 years since their introduction a number of persistent problems remain, most notably a low yield of antibody-producing cells from the fusion process, difficulty in obtaining high-affinity antibodies, and the potential immunogenicity of murine immunoglobulins (Igs). Several solutions are under development, including fusion techniques that enrich for cells producing desired antibodies, production of human-mouse chimeric antibodies by recombinant DNA technology, and the generation of human monoclonal antibodies by promising new approaches. Until these upcoming methodologies are established, and to better direct their development and application, a sound understanding of the pharmacology of presently available native and modified monoclonal antibodies is crucial. Although much has been already determined in this area, a great deal of further clarification remains necessary.

Animals↗
PubMed1989

Significant structural and functional change of an antigen-binding site by a distant amino acid substitution: proposal of a structural mechanism.

To study the molecular basis for antibody diversity and the structural basis for antigen binding, we have characterized the loss of phosphocholine (P-Cho) binding both experimentally and computationally in U10, a somatic mutant of the antibody S107. Nucleotide sequencing of U10 shows a single base change in JH1, substituting Asp-101 with Ala, over 9 A distant from the P-Cho-binding pocket. Probing with antiidiotypic antibodies suggests local, not global, conformational changes. Computational results support a specific structural mechanism for the loss of P-Cho binding. The U10 mutation eliminates the charged interaction between Asp-101 and Arg-94, which allows the Arg-94 side chain to disrupt P-Cho binding sterically and electrostatically by folding into the P-Cho-binding site. These results specifically show the importance of the Arg-94 to Asp-101 side chain salt bridge in the heavy-chain CDR3 conformation and suggest that residues distant from the binding site play an important role in antibody diversity and inducible complementarity.

Animals↗