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Biomedical subjects

D M Klinman

Publications and source records attributed to D M Klinman.

At least 91 records · Page 5Linked to original sources
PubMed1995

Increased activation and altered ratio of interferon-gamma: interleukin-4 secreting cells in MRL-lpr/lpr mice.

Cytokine specific ELIspot assays were used to monitor the number of cells secreting IL-4 and IFN gamma in MRL-lpr/lpr mice of different ages. The cytokine repertoire expressed by MRL-lpr/lpr mice was skewed towards the over-production of IFN gamma (and away from IL-4) when compared to that of MRL-+/-/+/- and BALB/c mice. With increasing age and disease severity, the ratio of IFN gamma: IL-4 secreting cells rose in MRL-lpr/lpr mice but decreased in normal animals. This changing ratio of IFN gamma: IL-4 secreting cells correlated with changes in the ratio of IgG2a: IgG1 secreting cells in these mice. Our findings suggest that cytokine secreting CD4+ and CD8+ cells are hyperactivated in young MRL-lpr/lpr mice, and that disease progression is associated with a disruption in the normal ratio of Th1:Th2 production in vivo.

Age Factors↗
PubMed1995

Increased utilization of polyreactive B cells during periods of generalized immune activation.

This work examines the hypothesis that B cells secreting polyreactive antibodies (antibodies capable of binding to more than one self or foreign antigen) are preferentially utilized during periods of generalized immune stimulation. Four conditions characterized by such stimulation were examined: chronic virus infection, mitogen treatment, autoimmune disease and neonatal repertoire development. In normal adult mice, polyreactive IgM secreting lymphocytes constituted 8-9% of the actively expressed repertoire. Under conditions of generalized immune activation, this frequency increased to 13-19% (p. < .01). Polyreactive IgG secreting B cells, which were present at frequencies of < 0.5% in normal adult mice, were found at freqeuncies of 6-10% in mice with autoimmune disease, chronic virus infection or following mitogen treatment (p. < .001). We postulate that polyreactive lymphocytes are preferentially activated when the immune system is confronted with stimuli inadequately controlled by antigen-specific responses.

Animals↗
PubMed1994

Treatment with dextran-conjugated anti-IgD delays the development of autoimmunity in MRL-lpr/lpr mice.

The onset of clinical disease in autoimmune MRL-lpr/lpr mice is preceded by a switch from predominantly IgM production to IgG production. Previous studies have shown that IgG autoantibodies play a central role in the development of life-threatening glomerulonephritis in this strain. Delaying or preventing the switch from IgM to IgG production might therefore be of therapeutic benefit. We previously documented similarities in the B cell repertoire expressed by young MRL-lpr/lpr mice and normal mice treated with the polyclonal activator LPS. Recent in vivo studies indicate that cross-linking membrane IgM or IgD can suppress LPS-dependent IgG production in normal animals. These observations led us to examine whether membrane cross-linking could also lower serum IgG levels in MRL-lpr/lpr mice. Lupus-prone animals were treated with multivalent anti-IgD conjugated to high m.w. dextran. This anti-IgD dextran conjugate was previously shown to reduce IgG production in LPS-stimulated normal animals. Treatment of young lupus-prone MRL-lpr/lpr mice resulted in a significant reduction in the total number of B cells secreting IgG and lower serum titers of IgG anti-DNA and IgG anti-histone autoantibodies. Anti-IgD dextran treatment also delayed the development of glomerulonephritis and improved survival. Thus, anti-IgD dextran interfered with autoantibody-dependent disease progression, perhaps by inhibiting the switch from IgM to IgG autoantibody production.

Age Factors↗
PubMed1994

Cross-reactivity of IgG anti-DNA-secreting B cells in patients with systemic lupus erythematosus.

This study is the first to analyze the cross-reactivity of in vivo activated B cells from patients with systemic lupus erythematosus. A chamber ELIspot assay was used to determine whether lymphocytes secreting antibodies that bound to DNA or 2,4,6-trinitrophenol (TNP)-keyhole limpet-hemocyanin (KLH) could simultaneously bind to the unrelated antigens actin or ovalbumin. IgM anti-DNA-, IgM anti-TNP-KLH- and IgG anti-TNP-KLH-secreting B cells from patients and controls showed similar levels of cross-reactivity (ranging from 6% to 23%, depending upon the antibody isotype and antigen pair examined). In general, IgG-producing cells were less cross-reactive than IgM producers from the same individual (on the average threefold, p < 0.001). In contrast, IgG anti-DNA-secreting B cells from lupus patients (i) showed no decrease in cross-reactivity when compared to IgM anti-DNA-secreting cells and (ii) were significantly more cross-reactive than control IgG anti-DNA-secreting cells and IgG anti-TNP-KLH secreting cells from patients (p < 0.001). The degree of IgG anti-DNA cross-reactivity correlated with disease activity (r = 0.52, p < 0.02). The implications of these findings with respect to repertoire expression and disease pathogenesis are discussed.

Adult↗
PubMed1994

Studies of marrow progenitor abnormalities in lupus-prone mice. II. Further studies of NZB Thy 1(neg)Lin(neg) bone marrow cells.

Bone marrow cells from NZB mice were fractionated and enriched in cells lacking surface markers characteristic of mature lineages, termed Thy 1neg Lineage(neg) cells. These cells represent approximately 1% of all marrow cells and constitute a much greater fraction of the bone marrow than do Thy 1lo Lineage(neg) cells. The NZB Thy 1neg Lineage(neg) cells were able to protect nonautoimmune, histocompatible DBA/2 recipients from lethal doses of irradiation, suggesting that this subpopulation contained progenitor cells. Consistent with this observation, fractioned Lip 6+ Thy 1neg Lineage(neg) cells, representing early B lineage cells, were less effective than Lip 6neg Thy 1neg Lineage(neg) cells in radioprotection. NZB marrow contains a great many more CFU-S than does marrow from nonautoimmune strains. DBA/2 mice transplanted with Thy 1neg Lineage(neg) cells from NZB marrow had substantial numbers of CFU-S, much greater than controls. This CFU-S potential was found primarily in the Lip 6neg Thy 1neg Lineage(neg) fractionated marrow, suggesting that that population contained early progenitor cells that had not yet differentiated into B lineage cells. Both radioprotection and increased CFU-S were transmitted serially by bone marrow from DBA/2 recipients of Thy 1neg Lineage(neg) NZB marrow to secondary and tertiary (irradiated) DBA/2 recipients. Also serially transplanted were precursors of antibody forming cells. These findings suggest that NZB Thy 1neg Lineage(neg) marrow cells play a critical role in the development of the abnormal phenotype of NZB mice. However, because this probably is not a homogeneous population, additional work will be necessary to define the surface and molecular properties of the cell or cells within the NZB Thy 1neg Lineage(neg) marrow population which serve as progenitors of the cells which mediate NZB disease.

Animals↗
PubMed1994

Cross-reactivity of IgM- and IgG-secreting B cells in murine chronic graft-versus-host reaction.

A chamber ELISpot assay was used to measure the frequency with which in vivo activated B cells secreted cross-reactive antibodies during a chronic graft-versus-host reaction in mice. Both normal (C57BL/10 x DBA/2)F1 mice and F1 recipients of syngeneic spleen cells (SynF1) expressed repertoires in which 13-17% of IgM and 3-5% of IgG-secreting cells were cross-reactive. By comparison, 43-55% of IgM and 9-40% of IgG-secreting cells from F1 recipients of parental DBA/2 spleen cells (GVHF1) were cross-reactive (P < 0.01, GVHF1 versus F1 or SynF1). The cross-reactivity of the GVHF1 IgM secreting cells remained elevated for > 2 months following cell transfer. In contrast, the cross-reactivity of IgG-secreting cells, which was significantly high at Week 3 post-transfer, declined to nearly normal levels by Week 9. These findings demonstrate that cross-reactive B cells constitute an abnormally large fraction of the repertoire expressed by mice with chronic GVHR in a period when B cell hyperstimulation was occurring.

Animals↗
PubMed1994

Cross-reactivity of IgG and IgM secreting B cells in normal and autoimmune mice.

Antibodies from animals with lupus react with a diverse array of foreign and self antigens. This has been attributed to the activation of large numbers of mono-specific B cells, although it could result from the stimulation of a smaller population of highly cross-reactive lymphocytes. To examine this issue, a chamber ELIspot assay was used to compare the cross-reactivity of Ig-secreting B cells from autoimmune vs normal mice. The cross-reactivity of IgM-secreting lymphocytes was significantly greater than that of IgG-secreting cells in normal BALB/c and DBA/2 mice. For example, 5-23% of IgM but only 0.1%-0.4% of cells secreting IgG antibodies against DNA or TNP-BSA were cross-reactive. When normal animals were immunized and boosted with TNP-KLH, the resultant IgG anti-TNP response was essentially mono-specific. There was no significant difference in the proportion of lymphocytes producing cross-reactive IgM antibodies in normal vs autoimmune mice. In contrast, the frequency of cross-reactive IgG-secreting B cells in lupus-prone MRL-lpr/lpr and (NZB x NZW)F1 animals was at least 15 times higher than in normal mice. These observations indicate that the repertoire of IgG-secreting cells in active lupus differs qualitatively from that seen spontaneously or following antigenic stimulation in normal animals.

Animals↗
PubMed1994

Individual cells simultaneously produce both IL-4 and IL-6 in vivo.

IL-4 and IL-6 are pleiotropic cytokines that can act independently or synergistically to regulate lymphocyte growth and differentiation. Both of these factors are produced by cultures of Th2 cells but it is uncertain whether they are secreted simultaneously by a single cell. We used a sensitive and specific ELIspot assay to identify individual cells spontaneously producing IL-4 or IL-6 under physiological conditions in vivo. The fraction of cells producing these two lymphokines was regulated concurrently in cloned Th2 cells but varied independently in the lymphoid organs of normal BALB/c mice. A sandwich ELIspot assay was developed to determine whether individual cells simultaneously produced both cytokines. Thirteen per cent of cells in the spleen and 25% of those in the bone marrow of 10-week-old BALB/c mice secreting either IL-4 or IL-6 concurrently produced both of these lymphokines. These observations establish a physiological mechanism by which the concurrent production of synergistic cytokines is regulated in a given micro-environment.

Animals↗
PubMed1994

Lack of correlation between the number of circulating B cells and the concentration of serum antibodies reactive with the HIV-1 envelope glycoprotein.

Cells obtained from the peripheral blood of HIV-infected patients and volunteers immunized with HIV-1 vaccines are commonly used to study anti-viral responses, since lymphocytes from the central lymphoid organs are difficult to obtain. Analyses involving PBMC implicitly assume that circulating B cells provide an accurate reflection of the systemic humoral response induced by the HIV antigens. We examined this assumption by comparing the number of B cells secreting IgG anti-gp160/120 antibodies in the peripheral circulation with serum antibody titers. Results indicate that neither the magnitude nor duration of the serologic response detected in HIV-infected patients or gp160/gp120-immunized volunteers reproducibly correlates with the number of B cells secreting anti-envelope antibodies in the blood.

AIDS Vaccines↗
PubMed1993

VH family utilization by IgG anti-DNA-secreting lymphocytes derived from autoimmune MRL-lpr/lpr mice.

OBJECTIVE: To evaluate the heterogeneity of the IgG anti-DNA autoantibody response of MRL/lpr mice. METHODS: B cell clones were grown on nitrocellulose membranes. Those producing IgG anti-DNA antibodies were identified by a modified enzyme-linked immunospot assay, and their utilization of IgVH genes was determined by hybridization. RESULTS: Four hundred sixty-eight IgG anti-DNA-secreting colonies were derived from 9 autoimmune MRL/lpr mice. Individual VH families contributed to anti-DNA production at a frequency roughly proportional to their representation in the expressed repertoire (except for the more frequent use of VH 7183 and less frequent use of VH 36-60). In individual mice, anti-DNA antibodies were encoded by 2-5 different IgVH families, with no single family constituting more than 27-65% of any animal's anti-DNA response. CONCLUSION: The IgG anti-DNA response of individual MRL/lpr mice is oligoclonal.

Animals↗
PubMed1993

Cross-reactivity of IgM- and IgG-secreting B cells in autoimmune mice.

OBJECTIVE: To determine the frequency with which in vivo-activated B cells secrete cross-reactive antibodies in lupus-prone mice. METHODS: Analysis of freshly isolated splenic lymphocytes by chamber enzyme-linked immunospot assay. RESULTS: Young New Zealand black, New Zealand black x New Zealand white, and MRL/lpr mice expressed repertoires in which 16-20% of IgM-secreting cells and 0-2% of IgG-secreting cells were cross-reactive. By comparison, 21-31% of IgM- and 4-14% of IgG-secreting cells in adult animals with active lupus were cross-reactive (P < 0.01). CONCLUSION: Cross-reactive B cells constitute an abnormally large proportion of the repertoire expressed in mice with active autoimmune disease.

Animals↗
PubMed1993

B-1a and conventional B cells from autoimmune NZB.H-2bm12 mice exhibit similar functional characteristics in vivo.

NZB.H-2bm12 mice develop an autoimmune syndrome characterized by the overproduction of anti-DNA antibodies and the expansion of B-1 B cells. Thus, these animals provide a useful model to examine the antigenic specificity, cross-reactivity and functional capability of B-1 versus conventional lymphocytes. Neither the repertoire expressed by in vivo activated Ly-1+ splenic lymphocytes, nor their cross-reactivity, differed significantly from that of conventional splenic B cells. When Ly-1+ cells were cultured in vitro in the presence of lipopolysaccharide plus interleukin-4 or interferon gamma, they underwent isotype switching at the same frequency as conventional B cells. Of interest, B-1 cells from the peritoneal cavity were significantly less likely to undergo isotype switching than those from the spleen. These findings indicate that in vivo activated B-1a and conventional B cells from mice with lupus manifest similar functional characteristics.

Animals↗
PubMed1993

Administration of a phosphorothioate oligonucleotide antisense to murine endogenous retroviral MCF env causes immune effects in vivo in a sequence-specific manner.

Previous in vitro studies had suggested that a product of the env gene of murine MCF (polytropic)-related sequences plays a role in regulating lymphocyte activation. To determine whether such an effect occurs in vivo, we have studied mice injected with phosphorothioate oligonucleotides antisense to such sequences. Injection of mice with antisense to the initiation region of the env gene resulted in (i) increased spleen cell numbers, primarily due to an increase in splenic B cells, (ii) increased class II MHC expression on B cells, (iii) increased RNA and DNA synthesis, and (iv) increased numbers of Ig producing cells. These results obtained with the antisense to MCF-related env did not occur with two scrambled phosphorothioate oligonucleotides or with antisense oligonucleotides to the initiation region of the env gene of xenotropic or ecotropic retroviral sequences. These data suggest that products of certain endogenous retroviral sequences regulate lymphocyte activation in vivo.

Animals↗
PubMed1993

Immunization with recombinant gp160 prolongs the survival of HIV-1 transgenic mice.

A strain of mouse transgenic for the env gene of the HIV-1 virus was used to study the immunogenicity of a gp160-derived vaccine (the protein encoded by the HIV env gene) and its effect on disease progression. Untreated transgenic mice frequently developed a rapidly progressive renal disease similar to that affecting approximately 10% of HIV-infected humans. When transgenic mice were immunized with recombinant purified gp160, their edema, proteinuria, and serum BUN levels were substantially reduced and their survival prolonged (p < 0.01). The increased longevity of immunized transgenic mice correlated with the production of IgG antibodies reactive with gp160.

AIDS Vaccines↗
PubMed1992

Cross-reactivity of IgM-secreting B cells from normal BALB/c mice.

Cross-reactive antibodies capable of binding to foreign and self Ag are present in the serum of normal newborn and adult animals. In our work, a chamber ELISA assay was used to quantitate the cross-reactivity of B cells actively secreting Ig in BALB/c mice of different ages. Individual lymphocytes were tested for the production of IgM antibodies capable of binding to a series of four unrelated Ag (DNA, TNP, actin, and OVA). Results indicate that nearly one-quarter of IgM secreting lymphocytes from 6-day-old animals were cross-reactive. This frequency was two- to fourfold higher than that found in adult mice. Very old animals, however, showed a selective increase in the cross-reactivity of anti-DNA (but not anti-TNP) secreting lymphocytes. Evidence from Ag inhibition experiments indicated that low concentrations of soluble Ag could block the binding of polyreactive antibodies, and that approximately one-half of "naturally" cross-reactive B cells produced antibodies capable of binding to three or more unrelated Ag.

Actins↗
PubMed1992

Analysis of B lymphocyte cross-reactivity at the single cell level.

A method is described for assaying whether individual in vivo activated B cells produce antibodies which cross-react with two distinct antigens. The technique utilizes a modification of the ELISA spot assay to determine the isotype and antigenic specificity(s) of these antibody secreting cells. To perform the assay, two plastic slides are coated with different antigens and secured together to form chambers capable of holding a monolayer of Ig secreting lymphocytes. The immunoglobulin secreted during culture binds to one or both sides of the antigen-coated chamber (depending upon the specificity and cross-reactivity of the antibody product). This technique was used to evaluate the cross-reactivity of hybridomas and freshly isolated IgM and IgG secreting B cells.

Actins↗