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T Dörner

Publications and source records attributed to T Dörner.

At least 55 records · Page 3Linked to original sources
PubMed2000

Diagnostic importance of anti-proteasome antibodies.

20S proteasome represents the proteolytic core complex for cytoplasmic protein degradation that is involved in the activation and regulation of the immune response. In this context, proteasome generates antigenic peptides for the MHC class I pathway and activates NF-kappaB. In a recent analysis, we could identify a frequent humoral autoimmune response directed against specific proteasomal subunits in patients with autoimmune myositis, systemic lupus erythematosus and primary Sjögren's syndrome. The outer ring subunit HC9(alpha3) was identified as the predominant target of the anti-proteasome response in these entities. In addition to the reactivity against HC9(alpha3), patients with primary Sjögren's syndrome expressed a more polyspecific recognition pattern of proteasomal subunits involving the active inner ring proteins. In follow-up analysis, anti-proteasome antibody titers revealed a correlation with disease activity in patients with autoimmune myositis and systemic lupus erythematosus. The current review summarizes recent data providing evidence that the 20S proteasome represents an important target of the humoral autoimmune response in systemic autoimmune diseases and extends insight into pathogenic aspects of these diseases.

Adenosine Triphosphatases↗
PubMed2000

Increased serum soluble CD14, ICAM-1 and E-selectin correlate with disease activity and prognosis in systemic lupus erythematosus.

To improve monitoring of immunological and disease activity, we determined soluble markers of activity of the monocyte/macrophage system (sCD14) and the vascular endothelium (sE-selectin, sICAM-1) in patients with systemic lupus erythematosus (SLE) and primary Sjögren's syndrome (pSS) in comparison to patients with infections or sepsis. Concentrations of sCD14, sICAM-1 and sE-selectin (soluble CD14, ICAM-1 and E-selectin, respectively) were measured in serum samples from patients with SLE and pSS, patients with sepsis, different infectious diseases and healthy controls using ELISA systems. Elevated levels of sE-selectin and sICAM-1 were detected in patients with SLE as well as sepsis, in contrast to patients with a localized infection (SLE and sepsis, respectively, versus infection P<0.001; Kruskal-Wallis test). Levels of sCD14 were persistently elevated in sera from patients with SLE, whereas these values decreased rapidly after effective therapy in patients with sepsis or infection. A continuous elevation of all of these three parameters was associated with a fatal outcome in patients with sepsis as well as in patients with SLE. Combined elevation of sCD14, sICAM-1 and sE-selectin correlates with the prognosis in patients with active SLE and indicates a remarkable immune activation involving the monocyte/macrophage system and the endothelium comparable to an activation found only in patients with sepsis.

Adolescent↗
PubMed2000

Enhanced mutational activity and disturbed selection of mutations in V(H) gene rearrangements in a patient with systemic lupus erythematosus.

To determine the impact of somatic hypermutation and selective influences on the V(H) gene repertoire in SLE, the mutational frequency and pattern of mutations in nonproductively and productively rearranged V(H) genes obtained from genomic DNA of individual CD19+ B cells were analyzed in a patient with SLE. The mutational frequencies of nonproductive (6.54 x 10(-2)) as well as of productive (4.38 x 10(-2)) V(H) rearrangements were significantly higher in the SLE patient than in normal controls (3.8 x 10(-2), p<0.001 and 3.3 x 10(-2); p<0.001, respectively). Analysis of nonproductive rearrangements documented only minor abnormalities of the targeting of the mutator in the SLE patient. The majority of "mutational hot spots", although different than in normals, appeared in the CDRs and an increased frequency of mutations in RGYW/WRCY sequences was observed. Moreover, no biases in base pair changes were found in the nonproductive repertoire. In contrast, there was a selection against A and T mutations and towards G mutations within the productive repertoire. Importantly, there were no significant differences in the R/S ratios of mutations within the FRs between the nonproductive and productive repertoire consistent with abnormalities in elimination of B cells expressing V(H) genes with these mutations. The result of this abnormality was a significantly higher R/S ratio of the V(H)genes in the productive repertoire of the SLE patient compared to normals (p<0.05). These data indicate that the mutational machinery was markedly enhanced in this SLE patient but exhibited nearly normal targeting, whereas selective influences were abnormal. These findings suggest that both enhanced mutational activity and disturbances in selection may have played a role in the emergence of autoreactivity in this SLE patient.

Amino Acid Substitution↗
PubMed1999

Ig lambda and heavy chain gene usage in early untreated systemic lupus erythematosus suggests intensive B cell stimulation.

To determine the distribution of Vlambda and Jlambda as well as VH and JH gene usage in a patient with systemic lupus erythematosus (SLE), productive and nonproductive VJ and V(D)J rearrangements were amplified from individual peripheral CD19+ B cells and were analyzed. No differences in the Vlambda and Jlambda or the VH and JH gene usage in the nonproductive gene repertoire of this SLE patient were found compared with the distribution of genes found in normal adults, whereas marked skewing of both Vlambda and VH was noted among the productive rearrangements. The distribution of productive Vlambda rearrangements was skewed, with significantly greater representation of the Jlambda distal cluster C Vlambda genes and the Vlambda distal Jlambda7 element, consistent with the possibility that there was receptor editing of the Vlambda locus in this patient. Significant bias in VH gene usage was also noted with VH3 family members dominating the peripheral B cell repertoire of the SLE patient (83%) compared with that found in normal subjects (55%; p < 0.001). Notably, a clone of B cells employing the VH3-11 gene for the heavy chain and the Vlambda1G segment for the light chain was detected. These data are most consistent with the conclusion that extreme B cell overactivity drives the initial stages of SLE leading to remarkable changes in the peripheral V gene usage that may underlie on fail to prevent the emergence of autoimmunity.

Adult↗
PubMed1999

Molecular mechanisms and selection influence the generation of the human V lambda J lambda repertoire.

To define the lambda light chain repertoire in humans, a single-cell PCR technique using genomic DNA obtained from individual peripheral B cells was employed. Of the 30 known functional V lambda genes, 23 were detected in either the nonproductive or productive repertoires. Specific V lambda genes, including 2A2, 2B2, 1G, and 4B, were overexpressed in the nonproductive repertoire, whereas some V lambda genes, such as 3R, 2A2, 2B2, 1C, 1G, and 1B, were overexpressed in the productive repertoire. Comparison of the nonproductive and productive repertoires indicated that no V lambda genes were positively selected, whereas a number of V lambda genes, including 4C, 1G, 5B, and 4B, were negatively regulated. All four of the functional J lambda segments were found in both repertoires, with J lambda 7 observed most often. Evidence of terminal deoxynucleotidyltransferase activity was noted in nearly 80% of nonproductive V lambda J lambda rearrangements, and exonuclease activity was apparent in the majority. Despite this, the mean CDR3 length was 30 base pairs in both productive and nonproductive repertoires, suggesting that it was tightly regulated at the molecular level. These results have provided new insights into the dimensions of the human V lambda repertoire and the influences that shape it.

Adult↗
PubMed1999

Somatic hypermutation of VkappaJkappa rearrangements: targeting of RGYW motifs on both DNA strands and preferential selection of mutated codons within RGYW motifs.

Productive and nonproductive VkappaJkappa gene rearrangements from individual peripheral blood B cells were analyzed for the pattern and distribution of mutations. The eight RGYW motifs and their inverse repeats, WRCY, were present in germ-line Vkappa genes significantly more often than anticipated by random chance (1.6-fold and 1.4-fold, respectively) and were also mutated in nonproductive rearrangements significantly more often than expected, with a frequency 1.96 fold greater than that of non-RGYW / WRCY motifs. As a result, 50 % of all mutations in nonproductive VkappaJkappa rearrangements occurred in RGYW / WRCY motifs. Each RGYW tetramer and its corresponding WRCY contained mutations at comparable frequencies. Furthermore, mutations of G and C were significantly more frequent in RGYW / WRCY but not in other tetranucleotides. Finally, mutations in codons contained within RGYW / WRCY were significantly more frequent in complementarity-determining regions but not framework regions of productive compared to nonproductive rearrangements and were increased by a factor that was significantly greater than for mutations in other motifs. These results indicate that the mutational machinery targets the overrepresented RGYW motifs in Vkappa genes on both DNA strands and that the resulting replacement mutations are preferentially selected into the productive repertoire.

B-Lymphocytes↗
PubMed1999

Autoantibodies in primary Sjögren's syndrome are directed against proteasomal subunits of the alpha and beta type.

OBJECTIVE: The proteasome subunit HC9 (alpha3) has recently been identified as a major target of the humoral autoimmune response in patients with autoimmune myositis and systemic lupus erythematosus. Since B cell hyperreactivity is a common feature of systemic autoimmune diseases, patients with primary Sjögren's syndrome (SS) and other control groups were investigated to evaluate the significance of autoantibodies against the proteasome. METHODS: Analyses of autoantibodies directed against the 20S proteasome were performed using enzyme-linked immunosorbent assay, immunoblot, and 2-dimensional electrophoresis. Forty-three patients with primary SS, 47 patients with rheumatoid arthritis including 9 with secondary SS, 19 patients with gastrointestinal tumors, and 80 healthy controls were tested for antiproteasome antibodies. RESULTS: Antiproteasome antibodies were detected in 39% of patients (17 of 43) with primary SS. In contrast, only 1 of 47 patients with rheumatoid arthritis showed positive reactivity (P < 0.001). Serum samples from 19 tumor patients (P < 0.003) and 80 healthy controls (P < 0.001) were serologically negative. Moreover, immunoblotting and 2-dimensional analysis of the antiproteasome response revealed a polyspecific recognition pattern in 7 patients with primary SS. Different proteasomal subunits of the alpha and beta type, including subunits that carried the proteolytic active sites, were recognized by the patients' sera. CONCLUSION: The humoral antiproteasome response in primary SS, in contrast to its secondary form, is characterized by an extensive recognition pattern of several subunits, indicating a polyspecific B cell activation against the 20S proteasome. Moreover, proteolytically active beta-type subunits, which are important for the generation of major histocompatibility complex class I-restricted antigens, appear to be targets of the autoimmune response. The data indicate that the proteasome itself may stand on a cross point of pathways that links mechanisms of the immune defense with features of systemic autoimmunity.

Antibodies, Antinuclear↗
PubMed1999

Enhanced mutational activity of Vkappa gene rearrangements in systemic lupus erythematosus.

To determine the differential impact of somatic hypermutation and selective influences on the light chain repertoire in systemic lupus erythematosus (SLE), the frequency and pattern of somatic hypermutations were compared between the productive and nonproductive Vkappa gene repertoire manifested by individual CD19(+) B cells in a patient with SLE. The mutational frequency of nonproductive rearrangements in the SLE patient was significantly (P < 0.001) increased (3.7 x 10(-2)) compared to normals (4.8 x 10(-3)). Similarly, the mutational frequency of the productive Vkappa rearrangements was also significantly increased in the SLE patient (2.8 x 10(-2) vs 1.1 x 10(-2)) (P < 0.001). There were no differences in the R/S ratios of mutations in productive and nonproductive Vkappa rearrangements. Moreover, a variety of mutational "hot spots" were noted, but, unexpectedly, in the FRs. As in normals, mutations were found most frequently in RGYW/WRCY sequences accounting for 39.3% (nonproductive) and 40.1% (productive) of all mutations. Of note, nonproductive Vkappa rearrangements harbored significantly more mutations than productive rearrangements (P < 0.05) indicating that there was overall selection against mutations in the expressed repertoire. This was most apparent in the CDR3. These data are most consistent with the conclusion that, in this SLE patient, the mutational machinery was markedly enhanced compared to normals, but with no subsequent positive selection of mutations. The enhanced mutational activity may play a role in the emergence of autoreactivity in this SLE patient.

Base Pairing↗
PubMed1998

Immunoglobulin kappa chain receptor editing in systemic lupus erythematosus.

To determine whether receptor editing of Vkappa genes was involved in the pathogenesis of systemic lupus erythematosus (SLE), the usage of Vkappa and Jkappa gene elements from individual peripheral CD19(+) B cells obtained from a patient with untreated SLE was examined. No differences in the Vkappa and Jkappa gene usage in the nonproductive gene repertoire of this SLE patient were noted compared with the distribution of genes found in normal adults. However, an increased usage of Jkappa5 segments, and a significant overrepresentation of the Vkappa1 and Vkappa4 families, especially the L15, O14/O4, and B3 genes characterized the productive Vkappa gene repertoire of the SLE patient. Furthermore, Jkappa5-containing Vkappa gene rearrangements in the productive but not the nonproductive repertoire manifested significantly fewer mutations compared with Vkappa genes recombined with Jkappa1-4. These data are consistent with the conclusion that receptor editing of Vkappa is much more apparent in this SLE patient than in normals and suggest that a deficiency in this means to counteract the emergence of autoimmunity is not an essential feature of SLE.

DNA Mutational Analysis↗
PubMed1998

Pairing of variable heavy and variable kappa chains in individual naive and memory B cells.

A functional Ig consists of two heterodimers each of which is composed of a heavy and a light chain. Although there is increasing knowledge about the events that govern the rearrangement of the genes encoding each individual chain, only very limited information is available about the mechanisms governing the pairing of variable heavy (V(H)) and variable light (V(L)) chains. Using a single cell PCR, we were able to obtain V(H) and Vkappa chains from 144 individual human CD19+/IgM+ B cells. Pairing of specific V(H) or Vkappa families was not observed, nor was the length or the amino acid composition of the CDR3s of V(H) and Vkappa chains in individual B cells similar. Comparison of V(H) and Vkappa genes in B cells in which one or both contained evidence of somatic hypermutation with those with no mutations revealed a significant decrease in the mean length of the V(H) CDR3. Moreover, there was a significant correlation between the frequencies of mutations in V(H) and Vkappa gene pairs in individual B cells. These results indicate that Ag-mediated selection as opposed to V(H)DJ(H) recombination or subsequent Ig chain pairing tended to approximate the CDR3 lengths and the frequency of mutations of V(H) and Vkappa in individual B cells.

Adult↗
PubMed1998

Delineation of selective influences shaping the mutated expressed human Ig heavy chain repertoire.

After Ag exposure, somatic hypermutation and subsequent selection play significant roles in shaping the peripheral B cell repertoire. However, the disparate impact of each process has not been completely delineated. To address this, the mutational patterns of a large panel of productive V(H)DJ(H) rearrangements of individual human B cells were analyzed and compared with those of a previously reported panel of nonproductive V(H)DJ(H) rearrangements. The productive V(H) rearrangements exhibited a significantly lower mutational frequency and a significantly smaller number of replacement mutations than the nonproductively rearranged genes, suggesting that structural constraints of the Ig molecule and selective influences both impacted the repertoire, militating against replacement mutations. Positive selection favored a mean of four to six replacements in complementarity-determining region 1 (CDR1) and CDR2, and less than two replacements in the framework regions (FRs). In contrast, the negative impact of replacement mutations generated an increased number of silent mutations within both the CDRs and FRs of the productive repertoire accompanied by a net increase in the ratio of replacement to silent mutations in the CDRs compared with that in the FRs. Moreover, there was a negative influence on the distribution of amino acid changes resulting from mutations of highly mutable codons, such as AGY, TAY, GTA, and GCT, preferentially leading to conservative changes in the expressed Ig repertoire. The results are consistent with the conclusion that the expressed repertoire is limited, compared with the potential generated by the mutational machinery, by the dual requirements of avoiding autoreactivity and satisfying structural constraints of an intact Ig molecule.

Codon↗
PubMed1998

Comparable impact of mutational and selective influences in shaping the expressed repertoire of peripheral IgM+/CD5- and IgM+/CD5+ B cells.

Somatic hypermutation and subsequent selection play a significant role in shaping the peripheral B cell repertoire. This repertoire is composed of CD5+ (5%) and CD5- B cells (95%) which are known to traffic through different lymphoid compartments. Previous studies have shown that V(H) gene usage by CD5+ and CD5- B cells is similar, although mutations are more frequent in the latter. However, the effect of mutation and subsequent selection on the expressed V(H) repertoire of CD5+ and CD5- B cells has not been delineated in detail. This study, therefore, analyzed the mutational pattern of individual IgM+/CD5+ and IgM+/CD5- B cells. In both populations, mutations can occur without heavy chain isotype switching. Despite the differences in mutational frequency, the patterns of mutation and subsequent selection were comparable in CD5+ and CD5- B cells. These results imply that although mutations are more frequent in CD5- B cells, the overall mechanisms governing somatic hypermutation and subsequent positive and negative selection are similar in CD5+ and CD5- B cells.

Adenine↗
PubMed1998

Somatic hypermutation of human immunoglobulin heavy chain genes: targeting of RGYW motifs on both DNA strands.

The impact of the somatic hypermutational machinery was examined by analyzing the frequency and distribution of mutations in nonproductive V(H)DJ(H) rearrangements obtained from individual human peripheral B cells. A strong bias toward nucleotide substitutions within the quadruplet motif RGYW was observed. In addition, there was a comparably increased frequency of mutations of the inverse repeat of RGYW, WRCY. Together, mutations of RGYW/WRCY accounted for 37% of all nucleotide substitutions. No significant strand polarity of the distribution of mutations was evident when nucleotide substitutions of highly mutated quartets and triplets as well as of their inverse repeats were analyzed. Furthermore, detailed analysis of mutations of specific triplets, such as AGC and TAC provided evidence that they were mutated more frequently when they were included within RGYW and WRCY, respectively. Despite being a target of the mutational machinery, neither RGYW nor WRCY was mutated in the absence of a large number of substitutions of other nucleotides in the same sequence. These results indicate that the mutational machinery targets RGYW sequences for mutations on either DNA strand and do not support the contention that the mutational machinery exhibits DNA strand polarity.

Base Sequence↗
PubMed1998

Similar characteristics of the CDR3 of V(H)1-69/DP-10 rearrangements in normal human peripheral blood and chronic lymphocytic leukaemia B cells.

The variable heavy chain (V(H)) gene segment V(H)1-69/DP-10 has been shown to be over-represented in B-cell chronic lymphocytic leukaemia (CLL). Because of certain similar characteristics of their complementarity determining region 3 (CDR3), including preferential utilization of J(H)6 elements and an extended length, it has been suggested that antigenic stimulation might be involved in leukaemogenesis. Utilizing single-cell PCR to amplify and sequence genomic DNA from individual normal human peripheral blood B cells, we have obtained 7/421 productively and 1/69 nonproductively rearranged V(H) genes that used V(H)1-69/DP-10. All productive rearrangements were unmutated, used J(H)6 and had an average CDR3 length similar to that previously found in V(H)1-69/DP-10-expressing CLL cells. These results suggest that CLL may arise from B cells commonly found in the peripheral B-cell repertoire and do not represent expansion of a unique subset of specific antigen-reactive B cells.

Adult↗
PubMed1998

Analysis of the targeting of the hypermutational machinery and the impact of subsequent selection on the distribution of nucleotide changes in human VHDJH rearrangements.

B cells are unique in that they generate and tolerate a high rate of mutations in their antigen receptor genes and employ these mutations as a basis of avidity maturation. The precise role of the mutational machinery versus subsequent selection in determining the frequency and distribution of mutations has not been fully analyzed. To address these issues, the influence of the intrinsic mutational machinery and subsequent selection on the frequency and distribution of mutations in the expressed human immunoglobulin repertoire was analyzed. Analysis of non-productively rearranged VH genes from individual human B cells provided an opportunity to examine the immediate impact of somatic hypermutation without superimposed selective influences. Comparison with the frequency and distribution of mutations in the productively rearranged human VH genes permitted an estimate of the influences of subsequent selection.

B-Lymphocytes↗
PubMed1997

Analysis of the human VH gene repertoire. Differential effects of selection and somatic hypermutation on human peripheral CD5(+)/IgM+ and CD5(-)/IgM+ B cells.

To analyze the immunoglobulin repertoire of human IgM+ B cells and the CD5(+) and CD5(-) subsets, individual CD19(+)/ IgM+/CD5(+) or CD5(-) B cells were sorted and non-productive as well as productive VH gene rearrangements were amplified from genomic DNA and sequenced. In both subsets, the VH3 family was overrepresented largely as a result of preferential usage of a small number of specific individual family members. In the CD5(+) B cell subset, all other VH families were found at a frequency expected from random usage, whereas in the CD5(-) population, VH4 appeared to be overrepresented in the nonproductive repertoire, and also negatively selected since it was found significantly less often in the productive compared to the nonproductive repertoire; the VH1 family was significantly diminished in the productive rearrangements of CD5(-) B cells. 3-23/DP-47 was the most frequently used VH gene segment and was found significantly more often than expected from random usage in productive rearrangements of both CD5(+) and CD5(-) B cells. Evidence for selection based on the D segment and the JH gene usage was noted in CD5(+) B cells. No differences were found between the B cell subsets in CDR3 length, the number of N-nucleotides or evidence of exonuclease activity. Somatically hypermutated VHDJH rearrangements were significantly more frequent and extensive in CD5(-) compared to CD5(+) IgM+ B cells, indicating that IgM+ memory B cells were more frequent in the CD5(-) B cell population. Of note, the frequency of specific VH genes in the mutated population differed from that in the nonmutated population, suggesting that antigen stimulation imposed additional biases on the repertoire of IgM+ B cells. These results indicate that the expressed repertoire of IgM+ B cell subsets is shaped by recombinational bias, as well as selection before and after antigen exposure. Moreover, the influences on the repertoires of CD5(+) and CD5(-) B cells are significantly different, suggesting that human peripheral blood CD5(+) and CD5(-) B cells represent different B cell lineages, with similarities to murine B-1a and B-2 subsets, respectively.

Adult↗