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T L O'Keefe

Publications and source records attributed to T L O'Keefe.

13 recordsLinked to original sources
PubMed2000

Dysregulated expression of the Cd22 gene as a result of a short interspersed nucleotide element insertion in Cd22a lupus-prone mice.

The Cd22 gene encodes a B cell-specific adhesion molecule that modulates B cell Ag receptor-mediated signal transduction, and is allelic to a lupus-susceptibility locus in New Zealand White (NZW) mice. In this study, we show that, in addition to the wild-type transcripts, NZW (Cd22a) mice synthesize aberrant CD22 mRNAs that contain approximately 20-120 nucleotide insertions upstream of the coding region between exons 2 and 3, and/or approximately 100-190 nucleotide deletions of exon 4. Sequence analysis revealed that these aberrant mRNA species arose by alternative splicing due to the presence in the NZW strain of a 794-bp sequence insertion in the second intron, containing a cluster of short interspersed nucleotide elements. Both the presence of sequence insertion and aberrantly spliced mRNAs were specific to mice bearing the Cd22a and Cd22c alleles. Up-regulation of CD22 expression after LPS activation appeared impaired in Cd22a spleen cells (twice lower than in Cd22b B cells). Furthermore, we show that partial CD22 deficiency, i.e., heterozygous level of CD22 expression, markedly promotes the production of IgG anti-DNA autoantibodies in C57BL/6 (Cd22b) mice bearing the Y chromosome-linked autoimmune acceleration gene, Yaa. Taken together, these results suggest that a lower up-regulation of CD22 on activated B cells (resulting from Cd22 gene anomaly in Cd22a mice or from CD22 heterozygosity in mutants obtained by gene targeting) is implicated in autoantibody production, providing support for Cd22a as a possible candidate allele contributing to lupus susceptibility.

5' Untranslated Regions↗
PubMed2000

Correlation of anti-viral B cell responses and splenic morphology with expression of B cell-specific molecules.

This study attempted to evaluate and compare the role of various B cell-specific markers for anti-viral immune responses in mouse strains lacking molecules belonging to the B cell receptor (BCR) complex (IgM, Ig alpha and C(kappa)), the co-stimulatory molecules (CD19 and CD22), the protein kinases [Bruton's tyrosine kinase (Btk)] or the transcription factors (OBF-1). These mice were tested in two model infections [vesicular stomatitis virus (VSV) and lymphocytic choriomeningitis virus (LCMV)] using T cell-independent (TI) or T cell-dependent (TD) antigens. All mice controlled an LCMV infection indicating that cytotoxic T cell functions were within normal ranges. In contrast, OBF-1(-/-) mice were partially protected and mb-1(delta c/delta c) mice not at all protected against VSV infection, a virus that is controlled virtually exclusively by neutralizing antibodies. Susceptibility to VSV infection was correlated with structural defects in the spleen: absence of mature B cells and follicles with marginal zone macrophages and absence of germinal centers with follicular dendritic cells correlated with lack or substantial reduction of protective IgM and IgG responses respectively. The lack of kappa light chain did not affect the neutralizing response, indicating that it could easily be replaced by the lambda chain. Absence of the co-stimulatory molecules CD19 and CD22 or of the signaling molecule Btk had modulating effects, but did not increase susceptibility to VSV or LCMV. Our findings suggest that there are crucial molecules for B cell activation at the beginning (BCR complex) and the end (transcription) of the signaling cascade, whereas fine-tuning factors modulating the response in between exhibit considerable functional overlap.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1999

Deficiency in CD22, a B cell-specific inhibitory receptor, is sufficient to predispose to development of high affinity autoantibodies.

CD22 is a B cell-specific transmembrane glycoprotein that acts to dampen signals generated through the B cell antigen receptor (BCR): B cells from CD22-deficient mice give increased Ca2+ fluxes on BCR ligation. Here we show that this B cell hyperresponsiveness correlates with the development of autoantibodies. After the age of eight months, CD22-deficient mice developed high titers of serum IgG directed against double-stranded DNA; these antibodies were of multiclonal origin, somatically mutated, and high affinity. Increased titers of antibodies to cardiolipin and myeloperoxidase were also noted. The results demonstrate that a single gene defect exclusive to B lymphocytes is, without additional contrivance, sufficient to trigger autoantibody development in a large proportion of aging animals. Thus, CD22 might have evolved specifically to regulate B cell triggering thresholds for the avoidance of autoimmunity.

Age Factors↗
PubMed1998

Targeted gene disruption reveals a role for natural secretory IgM in the maturation of the primary immune response.

Accelerated development of the secondary immune response may be attributable in part to the rapid delivery of antigen to lymphoid follicles by circulating antibody elicited on primary immunization. Here we provide evidence indicating that the nonspecific IgM present in naive mice (natural antibody) plays a role in the acceleration of the primary response. Targeted deletion of the Ig microseconds polyadenylation site by use of Cre recombinase allowed the creation of mice that, although harboring a normal number of B cells expressing surface IgM, completely lacked serum IgM while retaining the other Ig isotypes. These mice retained a broadly normal B lymphocyte distribution (although containing a somewhat expanded peritoneal B1a subset) but exhibited substantial delays in mounting affinity-matured IgG responses to T cell-dependent antigens. The T cell-independent response, however, was augmented. The data indicate that the IgM present before antigen challenge (as well, possibly, as that elicited immediately after immunization) accelerates maturation of the primary response, presumably by complexing with the antigen and facilitating lymphocyte activation and/or antigen trapping.

Animals↗
PubMed1998

Mice carrying a CD20 gene disruption.

CD20 is a hallmark antigen of B lymphocytes. Its expression is restricted to precursor and mature B cells but it is not expressed on plasma cells. The protein is a membrane-embedded phosphoprotein that appears likely to transverse the membrane four times. Its function is unknown although CD20 has been variously proposed to play a role in B-cell activation, proliferation, and calcium transport. A unique homologue of human CD20 has been described in mouse, which also shows a B-cell-specific pattern of expression. Here we describe the generating of mice carrying a CD20 gene disruption. So far, we have failed to detect any major effect of the gene disruption on the differentiation and function of B lymphocytes as judged by the expression of surface markers, antigen receptor signaling, proliferative responses, or calcium uptake. We did note, however, that the mice homozygous for the gene disruption [generated by intercrossing (129 x C57BL/6)F1 CD20+/- heterozygotes] showed a substantial depletion of the sub-population of peritoneal B cells that lack expression of the B220 (RA3-6B2) isoform of CD45. The loss of the IgM+ 6B2- peritoneal B cells is not, however, attributable to the CD20 gene disruption itself. Rather, it segregates with a polymorphic difference between the 129 and C57BL/6 strains that is linked to the CD20 locus which, intriguingly, is itself close to the CD5 gene. This demonstrates that caution must be exercised when comparing the phenotypes of F2 litter-mates generated from crosses between 129 embryonic stem-cell-derived chimeras and mice of other strains.

Animals↗
PubMed1996

Hyperresponsive B cells in CD22-deficient mice.

CD22 is a surface glycoprotein of B lymphocytes that is rapidly phosphorylated on cytoplasmic tyrosines after antigen receptor cross-linking. Splenic B cells from mice with a disrupted CD22 gene were found to be hyperresponsive to receptor signaling: Heightened calcium fluxes and cell proliferation were obtained at lower ligand concentrations. The mice gave an augmented immune response, had an expanded peritoneal B-1 cell population, and contained increased serum titers of autoantibody. Thus, CD22 is a negative regulator of antigen receptor signaling whose onset of expression at the mature B cell stage may serve to raise the antigen concentration threshold required for B cell triggering.

Animals↗
PubMed1995

The development of anti-CD79 monoclonal antibodies for treatment of B-cell neoplastic disease.

The B-cell antigen receptor (BCR) consists of cell surface IgM associated with the CD79 alpha/beta heterodimer. In this paper we describe a panel of monoclonal antibodies (mAbs) recognising the extracellular regions of human CD79 alpha and beta. FACS analysis demonstrated that the mAbs bind to a range of Burkitt's lymphoma lines, a mouse B-cell line (JO-72) transfected with human CD79 alpha and beta, and tumour biopsies from NHL patients. The specificity of the mAbs was confirmed by immunoprecipitation. The Ka for the binding of IgG from the anti-CD79 alpha mAbs to cell surface CD79 alpha on Ramos cells was 3 x 10(8) M-1, and their maximum level of binding, 1.7-2 x 10(5) molecules/cell, matched that obtained with anti-Fc mu and anti-Fd mu mAbs. All four anti-CD79 beta mAbs were of lower affinity. Interestingly, in growth arrest studies, we found that while all anti-Fc mu mAbs caused profound inhibition of proliferation of Ramos cells, a range of other anti-BCR mAbs, which included the anti-CD79, anti-Fab mu, anti-gamma and anti-idiotype reagents, all performed poorly giving a maximum of 25% inhibition. These differences in performance are believed to relate to the ability of anti-BCR mAbs to cross-link neighbouring surface BCR and suggest that, unlike anti-Fc mu which favours cross-linking, most of these mAbs are binding in a monogamous, non-cross-linking, union with the BCR.

Animals↗
PubMed1992

Cationic residues in pathogenic anti-DNA autoantibodies arise by mutations of a germ-line gene that belongs to a large VH gene subfamily.

The F1 progeny of autoimmune NZB and normal SWR mice uniformly develop severe and accelerated lupus nephritis. The (SWR x NZB)F1 mice produce an oligoclonally expanded population of nephritogenic anti-DNA autoantibodies that share a recurrent cross-reactive idiotype (Id564), use highly homologous VH genes and surprisingly have the CH region allotype of the normal SWR parent. From extensive library analyses, we isolated 15 SWR germ-line genes which are most closely related to the pathogenic autoantibody VH564 gene and which also belong to the NPb subfamily of J558 VH genes. We found that the pathogenic VH genes are probably somatic variants of a SWR germ-line VH gene, SW6-3, and they have several basic amino acid substitutions, in addition to those already present in the SW6-3 germ-line gene. Since these pathogenic autoantibodies are not detectable in the sera of the normal SWR mice despite the presence of the SW6-3 gene, strong selection and expansion of B cells expressing the SW6-3 VH gene is probably occurring in (SWR x NZB)F1 lupus-prone mice. We also isolated eight germ-line genes from the NZB mouse which are homologous to SW6-3. The autoimmune NZB parent that rarely develops nephritis lacks the SW6-3 gene, but has several highly homologous germ-line VH genes that would encode less cationic antibodies. These results establish a correlation between structure and pathogenic potential of VH genes.

Amino Acid Sequence↗
PubMed1991

Numbers of cortical vitreous cells and onset of cataracts in Royal College of surgeons rats.

Royal College of Surgeons (RCS) rats have hereditary retinal degeneration with associated posterior subcapsular opacities. A link between light, retinal degeneration, and cataracts may consist in peroxidation of polyunsaturated fatty acids of rod outer segment lipids to yield water-soluble toxic aldehydes that can traverse the vitreous and react with bow cells and posterior lens fibers. In an immune reaction to the retinal degeneration, macrophages multiply in the retina and in the cortex of the vitreous. In dystrophics, the cortical vitreous separates readily from attachments to retina, ciliary body and lens, and from the vitreous gel. This web-like structure was stained and spread on a counting chamber. Cells were counted at 15-130 postnatal days in pink- and black-eyed RCS dystrophics and in congenic controls to correlate numbers of cells, temporal and geographic patterns of retinal degeneration, and onset of opacities. Rats were reared in cyclic light (10-40 lux inside the cage) and fed a natural ingredient diet (NIH-07). Cortical vitreous cells increased markedly in pink- and black-eyed dystrophics at 50-53 days when slit-lamp detectable opacities occurred in both. The increase was 4.6-fold in pink- and 2.3-fold in black-eyed rats compared with controls. At 50-53 days, the dystrophy affected all quadrants of the retina severely in pink-eyed RCS but only the inferior periphery in black-eyed RCS. Consequently, severe degeneration in one quadrant may suffice to initiate an opacity.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1990

V region sequences of an idiotypically connected family of pathogenic anti-DNA autoantibodies.

The H and L chain V region sequences of nine anti-DNA mAb that are representative of a pathogenic population of autoantibodies produced by the nephritis prone (SWR x NZB)F1 (SNF1) mice, were determined. These nine anti-DNA autoantibodies were idiotypically connected members of a cross-reactive Id family called the Id564 cluster. Moreover, these autoantibodies were all cationic in charge, IgG2b in isotype, and their H chain C regions had the normal SWR parent's allotype. Although derived from two different SNF1 animals, these pathogenic autoantibodies possessed highly homologous Leader-VH sequences that could account for their idiotypic cross-reactivity. Furthermore, the VH region sequences of these anti-DNA antibodies contained numerous basic residues that could impart their cationic charge. The Leader-VH sequences of these autoantibodies were also highly homologous to that of an anti-NP antibody-related germ-line gene of C57BL/6 mice, called VH-23. Among these nine pathogenic autoantibodies, three sets of clonally related anti-DNA antibodies could be identified. Thus the Id564 cluster of cationic anti-DNA autoantibodies of SNF1 mice are encoded by highly related VH genes, and this idiotypically connected population of pathogenic autoantibodies are selected to undergo an oligoclonal expansion in the lupus-prone SNF1 mice.

Amino Acid Sequence↗
PubMed1990

Prevention of cataracts in pink-eyed RCS rats by dark rearing.

Royal College of Surgeons rats have hereditary retinal degeneration and associated posterior subcapsular opacities (PSO) of the lens, detectable by slitlamp at 7-8 postnatal weeks in both pink- and black-eyed rats. The retinal degeneration is intensified by light, especially in pink-eyed rats. A fourth of pink-eyed rats developed mature cataracts by 9-12 months of age, but black-eyed rats whose retinas are protected from light by pigmented irises and pigment epithelium rarely have mature cataracts (3% or less), indicating light may be a factor in cataractogenesis. Prior work had shown that dark rearing reduced the rate of retinal degeneration in pink- but not black-eyed rats, but cataracts were not studied. In the present work, pregnant pink-eyed females were placed in a darkroom 1 week before parturition. Pups were removed over intervals at 20-85 postnatal days for: (a) microscopic study of fresh lenses and of fixed, stained retina and lens, and (b) counts of cells mm-2 of the web-like vitreous cortex after it had been dissected free. The macrophage-like cells are a quantitative index of immune reaction to retinal damage. At 50-53 postnatal days, in pink-eyed cyclic light reared RCS, the mean number of macrophages was 4.6-fold that in congenic controls, but in those that were dark reared it was only 1.4-fold. This was less than the increase in cyclic light reared black-eyed RCS (2.3-fold that in congenic black-eyed controls). Total absence of light reduced retinal degeneration and the number of macrophages, and prevented PSO detectable microscopically.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Selective strain distribution pattern of a germline VH gene for a pathogenic anti-DNA autoantibody family.

Autoimmune NZB mice rarely develop nephritis, but the SNF1, progeny of Crosses between NZB and the normal SWR strain uniformly develop severe lupus nephritis, indicating that the normal SWR strain makes a genetic contribution to the development of nephritis. The SNF1 mice produce a select population of cationic IgG anti-DNA autoantibodies that share a recurrent cross-reactive idiotype called Id564 and these autoantibodies play a prominent role in the development of nephritis. These pathogenic autoantibodies of SNF1 possess the IgCH allotype of the SWR, indicating their origin from the normal parent. The leader-VH sequences of these Id564+ pathogenic anti-DNA autoantibodies are highly homologous and they are also related by 95% homology to a germline gene of normal C57BL/6 mice, called VH-23, that is a member of an anti-NP antibody gene family. Herein, we cloned the flanking and coding regions of the expressed VHDJH genes of the anti-DNA mAb 564, the prototype member of the pathogenic Id564 family. By restriction analysis and partial sequencing, we found that the VH564 gene is related but distinct in its 5' flanking region from all of the known anti-NP VH genes of C57BL/6 and BALB/c mice. Hybridization with four probes complementary to different segments of the flanking and coding regions of the expressed VH564 gene indicated that the germline gene for VH564 is contained in an approximately 5.2 kb EcoRI fragment of SWR genomic DNA. Moreover, high stringency hybridization with oligonucleotide probes complementary to unique CDR2 and 5' flanking sequences of the expressed VH564 gene revealed that the 'approximately 5.2 kb' germline allele for VH564 that is possessed by the normal SWR parental strain is lacking in the NZB parental strain. C57BL/6 mice also lack this allele of the anti-DNA VH564 germline gene, although this strain possesses the highly homologous, anti-NP-related VH-23 germline gene. Thus germline VH genes for certain pathogenic autoantibodies may have a selective strain distribution pattern.

Alleles↗