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Biomedical subjects

M D Scharff

Publications and source records attributed to M D Scharff.

At least 55 records · Page 3Linked to original sources
PubMed1994

Clonal variants of hybridoma cells that switch isotype at a high frequency.

As B cells differentiate under the influence of antigen and T cells, they frequently switch from the expression of IgM antibody to the expression of other isotypes. This is accomplished by rearranging the expressed variable region gene to downstream constant region genes and deleting the intervening sequences. Some B-cell lines that represent early stages in development switch constitutively in culture at frequencies that approach those of lipopolysaccharide- or lymphokine-stimulated normal B cells. Hybridoma cells represent a later stage of development and rarely switch in culture. In contrast to early B-cell lines, hybridomas produce large amounts of immunoglobulin, and single cells can be assayed easily for the expression of new isotypes. We have used the ELISA spot assay and fluctuation analysis to determine the rate of switching of two hybridoma cell lines. By identifying subclones that switched more frequently, we have progressively enriched for cells that switch spontaneously at higher rates. These cells, like normal cells, switch by rearrangement and deletion, and the frequency of switched cells in some of the clones is comparable to that which has been observed in less differentiated B-cell lines and in normal B cells.

Animals↗
PubMed1994

The use of severe combined immunodeficiency mice to study the metabolism of human immunoglobulin G.

BACKGROUND: Although the four human immunoglobulin G (IgG) isotypes are similar in structure, they exhibit significant differences in effector function and catabolic half-life. With advent of structurally engineered antibodies, there is the potential to design antibody constructs with desired half-lives; however, it is first necessary to discover the structures and mechanisms that control immunoglobulin metabolism. METHODS: Radioiodinated chimeric antibodies, consisting of a mouse antidansyl variable region and the four human IgG constant regions, were injected intravenously into Balb/c and severe combined immunodeficiency (SCID) mice, and their half-lives were determined by whole body and whole blood counting. Dependence of the rate of immunoglobulin catabolism on immunoglobulin concentration, a normal regulatory phenomenon specific to IgG, was evaluated by the introduction of large amounts of human gamma-globulin intraperitoneally. RESULTS: Whole body and blood half-lives were statistically indistinguishable. The four IgG isotypes were eliminated from the whole animals in a predominantly single-phasic manner, with the half-life being dependent on the isotype studied. In Balb/c mice, immune elimination frequently occurred after 6 days, although this was not observed in SCID mice. Relevance of the model was confirmed by the demonstration of the presence of the concentration-catabolism phenomenon, a relationship unique to normal IgG regulation. CONCLUSIONS: SCID mice provide an adequate initial animal model for the study of human-mouse chimeric antibodies. Further understanding of the factors governing immunoglobulin catabolism can be probed by study of recombinant human constant regions in this animal system.

Animals↗
PubMed1994

In vitro activation of a nonproductive immunoglobulin allele by a single base pair insertion.

We have shown that one of the alleles in a hybridoma was nonproductive because the sequence in the N region of the heavy chain caused it to be out of frame and to terminate prematurely. This allele became productive, and the gamma 1 heavy chain that it encoded was secreted when an adenosine was inserted to produce an open reading frame. This event occurred at a very low frequency following mutagenesis of the cultured cells. This result suggests that similar sorts of events must occur in vivo when both productive and nonproductive alleles undergo frequent mutations and that new genes may be expressed in hybridomas as they are being subcloned.

Alleles↗
PubMed1994

Therapeutic efficacy of monoclonal antibodies to Cryptococcus neoformans glucuronoxylomannan alone and in combination with amphotericin B.

The therapeutic efficacy of the immunoglobulin G1 (IgG1) monoclonal antibody (MAb) 2H1 to the Cryptococcus neoformans capsular polysaccharide was studied with and without amphotericin B (AmB) in a murine model of intravenous (i.v.) infection. MAb and AmB were administered by intraperitoneal (i.p.) injection after i.v. infection with a C. neoformans serotype D strain. Intraperitoneal administration of MAb 2H1 resulted in rapid distribution to the intravascular compartment, and the half-lives of i.p. and i.v. administered MAb were similar. Administration of MAb 2H1 alone resulted in increased survival, decreased lung fungal burden, and reduced serum glucuronoxylomannan antigen levels when given 2 to 6 h but not 24 h after infection. In vivo, the combination of MAb 2H1 and AmB was more effective at prolonging survival than either agent alone. MAbs of IgM, IgG1, IgG3, and IgA isotypes given 1 day after infection were effective in reducing serum GXM-D levels, with their relative efficacy being IgG1 > IgG3 > IgM > IgA. In vitro, MAb 2H1 was a potent opsonin of C. neoformans and the combination of MAb 2H1 and AmB was more effective than either agent alone in decreasing C. neoformans colony counts in the presence of the murine macrophage cell line J774.16. The results confirm that capsule-binding MAbs can enhance the effect of AmB against C. neoformans and provide support for considering combined therapy in humans.

Amphotericin B↗
PubMed1994

Mouse-human immunoglobulin G1 chimeric antibodies with activities against Cryptococcus neoformans.

Passive antibody administration is a potentially useful approach for the therapy of human Cryptococcus neoformans infections. To evaluate the efficacy of the human immunoglobulin G1 (IgG1) constant region against C. neoformans and to construct murine antibody derivatives with reduced immunogenicities and longer half-lives in humans, two mouse-human IgG1 chimeric antibodies were generated from the protective murine monoclonal antibodies 2D10 (IgM) and 18B7 (IgG1). The 2D10 mouse-human IgG1 chimeric antibody (ch2D10) had significantly lower binding affinity than its parent murine antibody (m2D10), presumably because of a loss of avidity contribution on switching from IgM to IgG. The 18B7 mouse-human IgG1 chimeric antibody (ch18B7) had higher affinity for cryptococcal polysaccharide antigen than its parent murine antibody (m18B7). ch18B7 and ch2D10 promoted phagocytosis of C. neoformans by primary human microglial cells and the murine J774.16 macrophage-like cell line. ch18B7 and m18B7 enhanced fungistatic or fungicidal activity of J774.16 cells and prolonged the survival of lethally infected mice. We conclude that the human IgG1 constant chain can be effective in mediating antifungal activity against C. neoformans. ch18B7 or similar antibodies are potential candidates for passive antibody therapy of human cryptococcosis.

Animals↗
PubMed1994

Monoclonal antibodies to Cryptococcus neoformans capsular polysaccharide modify the course of intravenous infection in mice.

Immunoglobulin G1 (IgG1) monoclonal antibodies (MAbs) to the capsular glucuronoxylomannan (GXM) were studied for their ability to modify the course of intravenous Cryptococcus neoformans infection in mice. A/J mice were given intraperitoneal injection of 1.0 mg of either a GXM-binding IgG1 MAb (2H1 or 2D10 gamma 1) or the irrelevant isotype-matched control MAb 36-65 prior to intravenous infection. Parameters used to study antibody efficacy were lung and brain tissue fungal burden, lung and brain weights, serum GXM levels, and histopathological examination of lung, brain, heart, kidney, and spleen tissues. Mice given GXM-binding MAb had significantly reduced lung tissue fungal burden as measured by CFU. In contrast to the reduction in lung tissue burden, the reduction in brain tissue burden was small and did not achieve statistical significance. Serum GXM levels were reduced in mice receiving GXM-binding MAb. Histopathological examination revealed reduced numbers of granulomas and C. neoformans organisms in the lungs, brains, and kidneys of MAb 2H1-treated mice relative to control mice. The lungs and brains of mice receiving GXM-binding MAb weighed significantly less than those of control animals, consistent with the reduced inflammation noted histologically. Subendocardial inflammation and kidney cortical infarctions were present in control infected mice but not in MAb 2H1-treated mice. Immunocytochemical staining for polysaccharide antigen revealed a marked reduction in the amount of tissue polysaccharide in mice treated with MAb 2H1 relative to control mice. The results support an useful role for passive antibody administration in C. neoformans infections.

Animals↗
PubMed1994

Cryptococcus neoformans infection can elicit protective antibodies in mice.

An IgG1 monoclonal antibody generated from a mouse infected with Cryptococcus neoformans modified the course of intravenous cryptococcal infection in A/J mice by prolonging survival and reducing lung fungal burden, brain mass, and serum polysaccharide levels. The results demonstrate that C. neoformans infection can elicit useful antibodies.

Animals↗
PubMed1993

The use of chemiluminescence and the ELISA spot assay to identify and enumerate rare immunoglobulin switch variants.

The use of chemiluminescence and the ELISA spot assay for identifying rare immunoglobulin switch variants is described. The technique utilizes nitrocellulose membranes and allows rapid screening of a large number of cells. The number of spots can be recorded either manually or automatically by using a commercially available colony counting program. This modification of the ELISA spot assay makes it less labor intensive and time consuming and can be adapted for the search for rare cells secreting small amounts of Ig or other macromolecules.

Animals↗
PubMed1993

Antibody-mediated protection in mice with lethal intracerebral Cryptococcus neoformans infection.

The fungus Cryptococcus neoformans is an important opportunistic pathogen for patients with AIDS. C. neoformans infections frequently involve the brain and are often fatal. In the setting of AIDS C. neoformans infections are incurable and new treatment strategies are urgently needed. Passive administration of antibody is a potential therapeutic option for the prevention and treatment of C. neoformans. The IgG1 murine monoclonal antibody 2H1 to the capsular polysaccharide of C. neoformans was studied for its ability to modify the course of lethal intracerebral cryptococcal infection in mice. Intraperitoneal administration of antibody 2H1 resulted in small, yet significant, prolongations in the average survival of mice given intracerebral infection and reduced the number of C. neoformans colonies in brain tissue. Histopathological examination of brain tissues revealed a diffuse cryptococcal meningitis with fewer organisms in the brains of mice that received antibody 2H1 than in the control group. Thus, systemic administration of a monoclonal antibody can modify the course of lethal intracerebral C. neoformans infection in mice by prolonging survival and decreasing fungal burden in brain tissues.

Animals↗
PubMed1993

Molecular characterization of the humoral responses to Cryptococcus neoformans infection and glucuronoxylomannan-tetanus toxoid conjugate immunization.

The molecular characteristics of the humoral immune response to a serotype A Cryptococcus neoformans infection were compared with the response elicited by a cryptococcal glucuronoxylomannan-tetanus toxoid (GXM-TT) conjugate. Anticryptococcal monoclonal antibodies (mAbs) isolated from both responses have previously been shown to recognize the same antigenic determinant of cryptococcal GXM. Southern blot and sequence analyses indicate that the hybridomas isolated from each response arose from only a few precursor B cells. All the mAbs generated from the infected and GXM-TT conjugate-immunized mice utilize the same VH7183 family member: JH2/JH4, v kappa 5.1, and J kappa 1; mAbs generated by different B cells had complementarity-determining region 3's (CDR3s) composed of seven amino acids with a common sequence motif. Thus, the molecular analysis of these anticryptococcal mAb-producing hybridomas indicated that the response to both cryptococcal infection and conjugate immunization was oligoclonal and highly restricted with regard to immunoglobulin gene utilization. The GXM-TT conjugate primarily stimulated isotype switching and clonal proliferation, and did not result in hybridomas expressing additional immunoglobulin repertoires. The mAbs from both responses had a number of replacement mutations at the 5' end of CDR2 that appear to be the result of antigen-driven selection. Somatic mutation also resulted in altered epitope specificity for one mAb, 13F1. Passive administration of representative mAbs from different clones generated in response to the GXM-TT conjugate prolonged survival of lethally infected mice.

Animals↗
PubMed1993

Mu switch region deletion is associated with both T cell independent and T cell dependent responses.

Isotype switching is a process by which immunoglobulin variable gene regions initially proximal to and expressed with the mu constant region gene (C mu) can rearrange downstream to other constant region genes. Thus the same antigen binding site can be expressed with each of the other constant region isotypes and perform the full panoply of effector functions. Although isotype switching is thought to involve highly reiterated 'switch site' sequences located 5' to constant region genes, the exact role of these switch sites is unknown. It has been reported that prior to switching, the 'donor' switch site 5' of C mu occasionally undergoes deletions, but it is not known whether this is an adventitious event or one which predisposes to or prevents isotype switching. Since T cell independent (TI) immune responses are dominated by IgM while T cell dependent (TD) responses are associated with switching to IgG, we have examined the state of the mu switch site in 51 IgM-producing hybridomas isolated from a variety of TI and TD responses. Although more hybridomas from the TI responses studied exhibited S mu deletions, deletion of S mu also occurred in hybridomas isolated from TD responses. Analysis of a well-characterized clonally related subset of IgM hybridomas also revealed that mu switch region deletion can be associated with the productive allele.

Animals↗
PubMed1993

Variable region gene utilization and mutation in a group of neutralizing murine anti-human immunodeficiency virus type 1 principal neutralizing determinant antibodies.

The heavy (VH) and light (VL) chain variable region nucleotide sequences of four neutralizing anti-human immunodeficiency virus type 1 (HIV-1) murine monoclonal antibodies (mAbs) were determined. These mAbs bind to native gp120, recombinant gp120, and a linear HIV-1 principal neutralizing determinant (PND) peptide that spans amino acid 308-328. Three mAbs that bind to the same linear determinant, 110.3, 110.4, and 110.5, all use the same VL gene elements, a VK21 gene and JK2. These three mAbs also share the same VKJK junctional diversity and specific somatic mutations. They have identical VL immunoglobulin gene rearrangement patterns on Southern blot. Two of the antibodies, 110.4 and 110.5, also use the same VH gene elements, SB32-D-JH4, and have identical VD and DJ junctions and N sequences. Two different anti-HIV-1 PND murine mAbs reported by others, BAT123 and 0.5 beta, also use VK21-JK2, and BAT123 also uses the SB32 VH gene element. Although 110.3 uses the same VL region gene as 110.3 and 110.4, it uses a different VH gene that appears to be a member of the 7183 VH family. 110.6, an mAb that recognizes a discrete, overlapping PND compared to 110.3, 110.4, and 110.5, uses entirely different VH and VL gene elements and has unique immunoglobulin VH and VL rearrangement patterns. Our data, taken together with reports of the BAT123 and 0.5 beta mAb sequences, suggest that the murine antibody response to HIV-1 PND may be restricted to a small subset of VH and VL gene elements.

Amino Acid Sequence↗
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↗