Biomedical subjects

D M Klinman

Publications and source records attributed to D M Klinman.

At least 127 records · Page 7Linked to original sources

PubMed1989

Quantitation of autoantibody-secreting B cells in systemic lupus erythematosus.

An ELISA spot assay was used to quantitate the number of autoantibody-secreting B cells in the peripheral blood of patients with systemic lupus erythematosus. Patients with active disease had 20 fold more anti-DNA, 4 fold more anti-actin and 3 fold more anti-myosin secreting lymphocytes than controls but normal numbers of anti-cardiolipin and anti-transferrin secreting B cells. 60% of SLE patients had increased numbers of B cells reactive with multiple autoantigens. These data suggest that B cell activation in SLE may be influenced by both antigen-specific and antigen-independent factors.

Actins↗

PubMed1988

Acquisition and maturation of expressed B cell repertoires in normal and autoimmune mice.

The expressed B cell repertoires of mice from 1 day to 5 mo of age were examined. The ELISA-spot assay was used to enumerate individual Ig-secreting splenic B cells and determine the proportion of these cells producing antibodies reactive with each of six autoantigens and two conventional Ag. Results indicate that i) neonatal B cells producing antibodies against specific members of the Ag panel arose in a temporally defined sequence, ii) antibodies produced by 1- to 5-wk-old mice appeared to be more cross-reactive than those produced by adult mice, iii) no bias toward autoantibody production was found in the neonatal repertoires of autoimmune-prone mice, and iv) as a whole, the pattern of repertoire development among diverse strains of mice was highly conserved, although individual mice varied considerably in the absolute number of B cells committed to the production of antibodies of a given specificity.

Aging↗

PubMed1988

Autoantibody production is associated with polyclonal B cell activation in autoimmune mice which express the lpr or gld genes.

Systemic autoimmune disease in humans and mice is characterized by hypergammaglobulinemia and abnormally high levels of serum autoantibodies. The presence of certain genes, such as the lpr and gld genes, induces otherwise normal mice to spontaneously develop systemic autoimmunity. To better understand the effect of these genes on the development of hypergammaglobulinemia, we quantitated the absolute number of splenic B cells producing antibodies reactive with each of four autoantigens and two conventional antigens and compared this to the total number of Ig-secreting spleen cells present in these mice. Whereas autoimmune mice had significantly greater numbers of autoantibody-secreting spleen cells than normal mice, they also had significantly greater numbers producing antibodies of conventional specificity. When expressed as a proportion of the total repertoire, no bias towards autoantibody production was present when autoimmune lpr and gld animals were compared to their congenic nonautoimmune C57BL/6 and C3H/HeJ counterparts. We also examined the B cell repertoires of recombinant inbred mice derived by mating autoimmune NZB with normal NFS mice. Some recombinant inbred (RI) lines developed hypergammaglobulinemia and produced large quantities of autoantibody. While evidence for specific (auto)antigenic stimulation was present in some RI lines, hypergammaglobulinemia was commonly associated with polyclonal B cell activation in these autoimmune mice as well.

Animals↗

PubMed1988

Studies on the influence of the internal environment on autoantibody production by B cells.

Purified splenic B cells from autoimmune NZB and nonautoimmune DBA/2 mice were transferred to unmanipulated H-2 compatible xid recipients. The number of autoantibody-secreting clones present in recipient mice was quantitated at varying times after transfer using a splenic fragment assay. We found that NZB and DBA/2 B cells expanded equally well in equivalent xid environments. Cells from either donor expanded significantly better in autoimmune-prone NZB.xid as compared with DBA/2.xid recipients. Moreover, clones producing antibodies reactive with T cell surface antigens, bromelain-treated mouse red cells, or DNA expanded more rapidly than did cells producing antibodies to the nonautoantigen TNP-KLH. Serum autoantibody levels rose in concert with the increased numbers of autoantibody-producing lymphocytes. We conclude that factors present in the internal milieu of autoimmune-prone NZB.xid mice, rather than an intrinsic B cell defect, facilitate the expansion of (auto)antibody-secreting B cells.

Animals↗

PubMed1988

Antibodies from patients and mice with autoimmune diseases react with recombinant hnRNP core protein A1.

Sera from autoimmune patients and normal volunteers were tested for antibodies to the A1 core protein of heteronuclear ribonucleoprotein (hnRNP) particles by ELISA and Western blot assays. The A1 protein used in these studies was produced by recombinant DNA technology. Thirty-seven per cent of patients with systemic lupus erythematosus produced anti-A1 antibodies, compared to 7% of normal controls. Several strains of autoimmune mice were also analysed. They spontaneously made high-titered responses to A1. Normal strains showed very low anti-A1 response unless they were specifically immunized with the antigen. Following immunization, normal mice also made high-titered responses to the A1 protein. These studies demonstrated that the A1 protein itself is an autoantigenic component of hnRNP particles. Our studies uncovered no evidence of linkage between the production of anti-A1 and of anti-DNA antibodies.

Animals↗

PubMed1988

Natural murine autoantibodies and conventional antibodies exhibit similar degrees of antigenic cross-reactivity.

Splenic B cells from normal and autoimmune mice were transferred to MHC-compatible xid recipients. Monoclonal antibodies were secreted by the transferred B cells in splenic fragment cultures. These antibodies were evaluated for reactivity and cross-reactivity against a panel of six autoantigens and two conventional antigens using an ELISA assay. The autoantibodies and conventional antibodies produced in splenic fragment cultures by normal DBA/2 and autoimmune NZB B cells expressed similar degrees of antigenic cross-reactivity. Previous studies have demonstrated that ELISA assays of splenic fragment culture supernatants detect antibodies with affinities of 5 x 10(6) M-1 or greater. We therefore also analyzed the cross-reactivity of monoclonal antibodies derived from hybridomas. This permitted an assessment of antibodies with lower binding affinities. Cross-reactivity was detected more frequently among these hybridomas. Consistent with our earlier observations, hybridoma antibodies specific for conventional antigens exhibited cross-reactivity with a frequency similar to that of antibodies specific for autoantigens.

Animals↗

PubMed1988

NIH conference. Angioimmunoblastic lymphadenopathy with dysproteinemia.

Angioimmunoblastic lymphadenopathy with dysproteinemia is a disorder characterized by a sudden onset of constitutional symptoms and lymphadenopathy. Patients often have hypergammaglobulinemia, autoantibodies, rashes, thrombocytopenia, or hemolytic anemia. Diagnosis requires a lymph node biopsy that shows architectural effacement, absence of germinal centers, arborization of postcapillary venules, and a polymorphous infiltrate that includes immunoblasts. Early in the disease, activated T cells in blood and lymph nodes stimulate B cells to proliferate and produce antibody. However, late in the disease, immune suppression may result from increased suppressor function. Clonal rearrangements, which are seen in all patients with regard to either the T-cell receptor beta-chain gene or immunoglobulin genes, have been followed by malignant transformation and frank lymphoma in some patients. Thus, this disorder stands partway between benign lymphoid proliferation and clonal lymphoid transformation. The prognosis of this disorder is poor; 75% of patients die within 2 years or develop a lymphoid malignancy. The rest usually go into a sustained remission. Current treatment with corticosteroid and immunosuppressive agents is unsatisfactory, especially because of late immunosuppression and predisposition to infections.

Blood Protein Disorders↗

PubMed1988

Pathogenesis of systemic lupus erythematosus.

SLE is a syndrome characterized by diverse clinical signs and symptoms, including rash, serositis, nephritis, central nervous system disease, thrombocytopenia, and leukopenia. These and other manifestations are present in patients with immune abnormalities indicative of B cell hyperactivity: hypergammaglobulinemia, increased amounts of antibodies reactive with self-determinants (characteristically including nuclear antigens), and increased numbers of circulating antibody-producing cells. It is this immune hyperactivity, inclusive of anti-self responses, which groups the patients with diverse clinical manifestations into a single syndrome called lupus.

Animals↗

PubMed1987

Sequence analysis and expression of an X-linked, lymphocyte-regulated gene family (XLR).

The XLR gene family consists of approximately 10 X-linked genes, the expression of which is regulated in lymphocyte development. Certain members of the gene family are closely linked to the murine xid immune deficiency mutation. Sequence analysis of a cDNA clone pM1 derived from the plasmacytoma MOPC167 showed an open reading frame capable of coding for a protein of 208 amino acids and mol wt 24,000. The lack of a signal peptide or transmembrane region indicates a probable cytoplasmic or nuclear localization for the predicted pM1 protein. The predicted protein shares significant homology with lamins A and C and other members of the intermediate filament family of proteins, and shares features important for the coiled-coil structure proposed for these proteins. Analysis of cDNA clones derived from a presecretory lymphoma and from adult thymus indicates that B and T lymphocytes transcribe a common major mRNA identical to pM1, while other rare transcripts were also identified by these studies. A series of clonal T lymphoma lines representing distinct stages of thymic differentiation showed that, as with B lymphoid tumors, XLR expression is correlated with the maturation of the thymomas.

Amino Acid Sequence↗

PubMed1987

Similar in vivo expansion of B cells from normal DBA/2 and autoimmune NZB mice in xid recipients.

B cells from normal DBA/2 and autoimmune NZB mice were transferred into H-2-compatible xid recipients where they engrafted without irradiation or other manipulation of the host. The properties of these cells and their interaction with the host environment were analyzed at the single cell level with a splenic focus assay. When similar numbers of NZB and DBA/2 anti-DNA-producing B cell precursors were transferred, they expanded at similar rates in xid recipients. The rate of expansion varied with the strain of the recipient: it was fastest in autoimmune-prone NZB . xid and slowest in DBA/2 . xid hosts. Cells producing antibodies reactive with the autoantigen DNA proliferated substantially faster than those reactive with the non-autoantigen trinitrophenylated keyhole limpet hemocyanin. These results suggest that 1) B cells from NZB mice do not behave differently from DBA/2 B cells, 2) the internal milieu of the recipient into which the cells are transferred has an important effect on B cell proliferation, and 3) B cells capable of autoantibody production may have a selective growth and/or differentiation advantage relative to other B cells.

Animals↗

PubMed1987

Novel ELISA and ELISA-spot assays used to quantitate B cells and serum antibodies specific for T cell and bromelated mouse red blood cell autoantigens.

The frequency of splenic B cells producing antibodies reactive with bromelain-treated mouse red blood cells (BrMRBC) or T cell surface antigens was examined in autoimmune and normal mice. This was accomplished by fixing target cells to microtiter plates such that their membrane antigens could be detected in ELISA and ELISA-spot assays. This technique was rapid, sensitive, and permitted antibodies of both the IgG and IgM isotypes to be measured independently. Autoimmune NZB, BXSB male and MRL-lpr/lpr mice had 10-100-fold higher levels of serum anti-BrMRBC and anti-T cell antibodies than did control DBA/2 and CBA/J animals. The frequency of splenic B cells producing autoantibodies of these specificities was similarly increased among autoimmune mice. In general, the number of antibody-forming cells (AFC) reactive with BrMRBCs was 2-5 times higher than the number reactive with T cell surface determinants. In NZB mice these cells produced primarily IgM autoantibodies whereas in MRL-lpr/lpr animals they secreted primarily IgG. The concentration of serum autoantibody did not precisely correlate with AFC frequency, indicating that immunoglobulin catabolism and other factors play a role in regulating serum antibody concentration.

Animals↗

PubMed1987

Systemic autoimmune disease arises from polyclonal B cell activation.

The number of B cells producing antibodies reactive with any of seven autoantigens or two conventional antigens was compared at the single-cell level to the total number of Ig-secreting B cells present in the spleens of NZB, MRL lpr/lpr, and BXSB autoimmune mice. The proportion of lymphocytes producing antibodies of each specificity, expressed as a percentage of the total B cell repertoire, was virtually identical among autoimmune and congenic nonautoimmune animals. Furthermore, B cells and serum antibodies reactive with conventional antigens increased commensurately with those reactive with autoantigens. These results indicate that systemic autoimmune diseases arise from polyclonal B cell activation.

Animals↗

PubMed1987

The interaction of the xid and me genes.

The murine "motheaten" (me) mutation has been bred onto the NFS background and combined with the X-linked immunodeficiency (xid) mutation to investigate the effect of the xid-induced B cell maturational block on the widespread immune dysfunction, high levels of autoantibodies, and early mortality found in the motheaten mice. The xid markedly reduced spontaneous IgM secretion by spleen cells, serum IgM, anti-ssDNA antibodies, anti-bromelain-treated-erythrocyte antibodies, and T cell binding (but not thymocytotoxic) antibodies; however, neither phenotype nor mortality was affected, suggesting that other factors are responsible for early death. Marked expansion of the Ly-1+ B cell pool was prevented by xid in the motheaten mouse leaving only a very small population of sIgM-positive B cells. This failure of non-Ly-1+ B cell development in me/me X xid mice suggests that me/me leads to inhibition of non-Ly-1+ B cells and preferential expansion of Ly-1+ B cells in motheaten mice, perhaps as a result of their high levels of maturation and activation factors.

Animals↗

PubMed1987

Preserving primary cDNA libraries.

A technique for the long term storage of primary cDNA libraries in a form such that relevant DNA sequences can be readily identified and retrieved is described. cDNA libraries produced using the lambda gt 10 cloning vector were plated out on host bacteria in 0.7% top agarose supplemented with 30% glycerol. Nitrocellulose lifts of these libraries were made and stored. These lifts could be screened at a later time to permit identification of bacteriophage plaques containing specific cDNA inserts. The plated libraries were then transferred to a -70 degrees C freezer. The combination of freezing and glycerol treatment allowed the bacteriophage in these primary cDNA libraries to remain viable for significantly longer than 1 year.

Bacteriophage lambda↗

PubMed1987

Transfer of anti-DNA-producing B cells from NZB to unmanipulated xid recipients: effects of age, sex, and environment.

NZB mice have been extensively studied in an attempt to understand the pathogenesis of systemic autoimmune disease. In a recent report we demonstrated that NZB B cells would engraft and function when transferred to unmanipulated NZB.xid recipients. Anti-DNA-producing donor B cells expanded dramatically in the NZB.xid recipients. The present study examines the requirements for this expansion. We found that the age and sex of the donor were important: NZB mice less than 2 weeks of age had fewer anti-DNA-secreting precursors than did older mice; male donors had 10-fold fewer precursors than did females of the same age. An effect of the recipient environment on donor cell expansion also was noted: male recipients allowed much less expansion of anti-DNA-producing cells than did female recipients. Castration of the male recipients led to an increase in rate of expansion, suggesting that androgens played a role in regulating lymphocyte proliferation and/or activation. These studies demonstrate that anti-DNA precursors increase with age under the influence of host factors.

Age Factors↗

PubMed1987

Genetic and molecular genetic studies of murine and human lupus.

Mice and humans with systemic lupus erythematosus (SLE) have been studied with regard to cellular, genetic and molecular genetic abnormalities. B cell hyperactivity and autoantibody production are the hallmarks of this illness. In humans with SLE, there is increased stem cell, B cell precursor and B cell proliferation. The same is true of NZB mice. In lpr/lpr and gld/gld mice, marked expansion of a subpopulation of T cells allows extrathymic terminal T cell maturation and secondary B cell hyperactivity. Androgens suppress these processes and polyclonal immune activators accelerate them. Three types of genes are identified: inducing genes, accelerating genes and background genes. These give rise to abnormal expression of various cellular oncogenes, T cell receptor genes and immunoglobulin genes. The data suggest that abnormal immune regulation plays a critical role in the development of SLE, with polyclonal B cell activation being common to both mice and humans with SLE. Different genetic and cellular abnormalities underlie the ultimate syndrome, the common denominator, generalized autoimmunity, that we call SLE.

Animals↗

PubMed1986

Effect of cyclophosphamide therapy on oncogene expression in angioimmunoblastic lymphadenopathy.

The expression of four cellular oncogenes was studied by means of northern blot analysis of messenger RNA in peripheral-blood mononuclear cells from patients with angioimmunoblastic lymphadenopathy (AILD). On average, cells from patients with AILD and from those with systemic lupus erythematosus (SLE) expressed significantly more N-ras and significantly less c-fos mRNA than did cells from healthy controls. Expression of these cellular oncogenes was most abnormal in patients with the most severe disease. In contrast, increased levels of c-myc mRNA were found in patients with SLE but not in those with AILD. Administration of cyclophosphamide to patients with AILD was followed by return to normal of both N-ras and c-fos expression.

Cyclophosphamide↗

PubMed1986

Proliferation of anti-DNA-producing NZB B cells in a non-autoimmune environment.

This study demonstrates that purified NZB B cells, but not other NZB spleen cell populations, are capable of transferring anti-DNA antibody production into unirradiated H-2-compatible xid recipients. The number of autoantibody-producing B cells and the concentration of anti-DNA antibody found in the recipients correlated directly with the number of NZB B cells transferred. In addition, the number of anti-DNA-secreting lymphocytes found in the xid hosts increased exponentially with time post cell transfer. Several lines of evidence suggest that this phenomenon reflected the rapid proliferation of donor NZB B cells in the xid environment. Significantly, such proliferation was characteristic of donor cells that produced autoantibodies, but not of splenic B cells as a whole. These results suggest that stimulated NZB B cells can both induce and perpetuate autoantibody production in a normally non-autoimmune environment and in the absence of autoimmune helper cells.

Animals↗