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Thomas Dörner

Publications and source records attributed to Thomas Dörner.

At least 37 records · Page 2Linked to original sources
PubMed2005

Cryopreserved human B cells as an alternative source for single cell mRNA analysis.

Reverse transcription-polymerase chain reaction (RT-PCR) of individual B-lymphocytes has been shown to be a powerful tool for the simultaneous analysis of different mRNA specificities in both malignant and non-malignant B cell subpopulations. However, especially for longitudinal studies, this may also require analyses of cryopreserved cells. Therefore, the current study assessed whether cryopreserved (liquid nitrogen, dimethyl sulfoxide [DMSO]-stored) viable B cells are an alternative source for single cell RT-PCR analysis. Fresh (non-frozen) and post-thawed human peripheral blood B cells were analyzed by fluorescence-activated cell sorting (FACS). As a result, different B cell subpopulations could be reliably stained and separated from both fresh and post-thawed cells by four-color flow cytometry, although slightly diminished fluorescence intensities of some subpopulation markers were observed when analyzing cryopreserved cells. Subsequently, viable individual CD19+CD27+ memory B cells were sorted into single wells and analyzed for the expression of mRNA transcripts of the 'house-keeping gene' glyceraldehyde phosphate dehydrogenase (GAPD), the constitutive B cell homing receptor CXCR4, and immunoglobulin heavy chain variable region (IgVH) genes by nested RT-PCR protocols. Comparing both B cell sources, RT-PCR analysis revealed comparable yields of cells expressing transcripts for the three mRNA specificities tested (GAPD, CXCR4, IgVH) indicating the integrity of the respective mRNAs in cryopreserved B cells. In conclusion, these data indicate that optimally cryopreserved B cells may be an alternative source for single-cell RT-PCR analysis, especially in longitudinal B cell studies. However, the settings for both FACS analysis and RT-PCR should be re-evaluated for each distinct subpopulation and target mRNA of interest when analyzing post-thawed cells.

Antigens, Surface↗
PubMed2005

Effects of lyophilization on the infectivity of enveloped and non-enveloped viruses in bone tissue.

Recently reported qualitative experiments proved that retroviral infectivity is not destroyed by lyophilization performed on systemically infected bone and tendon. The now accomplished quantitative determination of residual infectivity for enveloped and non-enveloped viruses allows a validation of the production process regarding viral safety in freeze-dried bone transplants. The lyophilization effect on the infectivity of two non-enveloped viruses (Maus Elberfeld virus, MEV; Porcine parvovirus, PPV) and one enveloped virus (Vesicular Stomatitis virus, VSV) was examined for virus-spiked bone material in comparison to lyophilized viruses, original virus stock, and air-dried viruses. All experiments were carried out with both cell-free and cell-associated virus. Significant differences were observed regarding the reduction of virus titers (TCID50). Infectivity of VSV was reduced by about 3-4 log10 using lyophilization in presence of bone matrix and of MEV by 6-7 log10, while no substantial reduction in virus titers was observed for PPV. Lyophilization of cell-free or cell-associated virus is not sufficient to inactivate viruses completely. However, lyophilization could have an additive effect in line with other production steps used in the manufacturing process.

Animals↗
PubMed2005

Immunopathogenesis of primary Sjögren's syndrome: implications for disease management and therapy.

PURPOSE OF REVIEW: Recent studies have broadened our understanding of the etiopathogenesis and immunopathology of primary Sjögren's syndrome. This review highlights recent advances in understanding the underlying mechanisms of the disease as well as their implications for clinical handling and therapeutic options. RECENT FINDINGS: It becomes increasingly apparent that certain disturbances of the immune system (i.e. B-cell hyperreactivity and enhanced levels of B-cell-activating factor/B-lymphocyte stimulator) play a central role in this entity. Whether this is a primary abnormality or the result of predisposing factors or infectious, e.g. viral, agents remains uncertain. New insights into the pathogenesis also provide candidates for better diagnosis and classification of disease severity, such as flow cytometric analysis, measurement of soluble cell surface molecules, autoantibodies, cytokines, and ligands (B-cell-activating factor/B-lymphocyte stimulator). Whether B-cell-directed therapies (i.e. blocking B-cell-activating factor/B-lymphocyte stimulator, anti-CD20 therapy) will have an impact on primary Sjögren's syndrome needs to be shown in clinical trials. Alternative therapeutic approaches such as organ-targeted gene transfer are in development but must be carefully evaluated for safety and efficacy in preclinical models that resemble human primary Sjögren's syndrome. SUMMARY: The pathogenesis of primary Sjögren's syndrome is complex and the factors initiating and driving autoimmunity in this entity are largely unknown. Recent studies provide new insights into potential pathogenetic mechanisms of the disease and, thereby, the chance for improved strategies in disease management and therapy.

Animals↗
PubMed2004

Generation of migratory antigen-specific plasma blasts and mobilization of resident plasma cells in a secondary immune response.

Maintenance of protective humoral immunity depends on the generation and survival of antibody-secreting cells. The bone marrow provides niches for long-term survival of plasma cells generated in the course of systemic immune responses in secondary lymphoid organs. Here, we have analyzed migratory human plasma blasts and plasma cells after secondary vaccination with tetanus toxin. On days 6 and 7 after immunization, CD19(+)/CD27(high)/intracellular immunoglobulin G(high) (IgG(high))/HLA-DR(high)/CD38(high)/CD20(-)/CD95(+) tetanus toxin-specific antibody-secreting plasma blasts were released in large numbers from the secondary lymphoid organs into the blood. These cells show chemotactic responsiveness toward ligands for CXCR3 and CXCR4, probably guiding them to the bone marrow or inflamed tissue. At the same time, a population of CD19(+)/CD27(high)/intracellular IgG(high)/HLA-DR(low)/CD38(+)/CD20(-)/CD95(+) cells appeared in the blood in large numbers. These cells, with the phenotype of long-lived plasma cells, secreted antibodies of unknown specificity, not tetanus toxoid. The appearance of these plasma cells in the blood indicates successful competition for survival niches in the bone marrow between newly generated plasma blasts and resident plasma cells as a fundamental mechanism for the establishment of humoral memory and its plasticity.

Adult↗
PubMed2004

Marburg I polymorphism of factor VII-activating protease is associated with idiopathic venous thromboembolism.

The factor VII-activating protease (FSAP) variant Marburg I is known to attenuate the profibrinolytic system in vitro and was recently shown to be a significant predictor for the evolution and progression of carotid stenosis. The objective of this case-control study was to assess FSAP Marburg I's role in the occurrence of venous thromboembolism (VTE). The frequency of FSAP Marburg I was significantly increased in patients with a history of VTE (17 of 213 patients, 8.0%, P = .014) or idiopathic VTE (12 of 103 patients, 11.7%, P = .002) compared to healthy controls (5 of 213 controls, 2.3%). Logistic regression analysis confirmed FSAP Marburg I to be an independent risk factor for VTE (odds ratio, 3.5; 95% confidence interval [CI], 1.2-10.0) and idiopathic VTE (odds ratio, 6.2; 95% CI, 2.0-18.9).

Adolescent↗
PubMed2004

CD38 low IgG-secreting cells are precursors of various CD38 high-expressing plasma cell populations.

Despite the important role immunoglobulin G (IgG)-secreting plasma cells play in memory immune responses, the differentiation and homeostasis of these cells are not completely understood. Here, we studied the differentiation of human IgG-secreting cells ex vivo and in vitro, identifying these cells by the cellular affinity matrix technology. Several subpopulations of IgG-secreting cells were identified among the cells isolated from tonsils and bone marrow, particularly differing in the expression levels of CD9, CD19, and CD38. CD38 low IgG-secreting cells were present exclusively in the tonsils. A major fraction of these cells appeared to be early plasma cell precursors, as upon activation of B cells in vitro, IgG secretion preceded up-regulation of CD38, and on tonsillar sections, IgG-containing, CD38 low cells with a plasmacytoid phenotype were found in follicles, where plasma cell differentiation starts. A unitary phenotype of migratory peripheral blood IgG-secreting cells suggests that all bone marrow plasma cell populations share a common precursor cell. These data are compatible with a multistep model for plasma cell differentiation and imply that a common CD38 low IgG-secreting precursor gives rise to a diverse plasma cell compartment.

ADP-ribosyl Cyclase↗
PubMed2004

Abnormalities in peripheral B cell memory of patients with primary Sjögren's syndrome.

OBJECTIVE: To delineate disturbances in peripheral B cell memory in primary Sjögren's syndrome (SS). METHODS: Isotype-specific immunoglobulin (Ig) heavy-chain transcripts were analyzed in single-sorted CD19+,CD27- naive and CD19+,CD27+ memory B cells from patients with primary SS and normal healthy control subjects. RESULTS: A significantly higher frequency of B cells expressing mu-, alpha-, and/or gamma-chain transcripts were found in patients with primary SS compared with controls (58.0% versus 14.3%; P < 0.0001). Notably, 30.5% of individual B cells (for primary SS, 38.7%; for controls, 12.7% [P < 0.0001]) simultaneously expressed transcripts for different Ig heavy-chain isotypes using identical V(H)-D-J(H) rearrangements. However, these cells lacked surface expression of more than one of the respective Ig heavy-chain isotypes as well as messenger RNA (mRNA) transcripts for 2 germinal center markers, activation-induced cytidine deaminase, and Bcl-6. In contrast with the findings in normal healthy controls, peripheral B cell memory in patients with primary SS was characterized by 1) circulating CD27+ B cells expressing heavily mutated Ig V(H) transcripts (mutational frequency 8.6% versus 4.3%; P < 0.0001), 2) significantly enhanced mutational frequencies of C mu transcripts (9.6% versus 2.5%; P < 0.0001), 3) a high proportion (61.2%) of CD27+ B cells expressing transcripts for multiple Ig heavy-chain isotypes, and 4) a CD27- memory-type B cell subpopulation expressing mutated C mu transcripts. CONCLUSION: Altogether, both B cell hyperactivity and striking abnormalities in peripheral B cell memory are indicated at the single-cell mRNA level in patients with primary SS. Detection of multiple Ig heavy-chain transcripts in peripheral CD19+,CD27+ memory B cells of patients with SS may represent the abnormal retention of pre-switch mRNA transcripts in circulating post-switch B cells.

Adult↗
PubMed2004

Involvement of inducible costimulator in the exaggerated memory B cell and plasma cell generation in systemic lupus erythematosus.

OBJECTIVE: In systemic lupus erythematosus (SLE), the increased generation of memory B cells and plasma cells leads to autoimmune hypergammaglobulinemia and destructive immunoglobulin deposits in the kidneys. We undertook this study to determine the biologic mechanism driving this overactivation of the B cell compartment, which is the central issue in SLE. METHODS: We used flow cytometry to analyze expression of the T cell-specific inducible costimulator (ICOS) and its ligand (ICOS-L) on B cells obtained from the peripheral blood of SLE patients. We correlated ICOS-L expression with the differentiation status of the B cells using a large panel of surface antigens. In addition, SLE kidneys were analyzed by immunohistology. RESULTS: We found an increased expression of ICOS on CD4+ as well as CD8+ T cells in SLE. At the same time, we documented a down-regulation of ICOS-L on a high proportion of peripheral blood memory B cells. Based on in vitro experiments, we inferred that this ICOS-L down-regulation on B cells was a signature of recent interaction with ICOS+ T cells in vivo. In the kidneys of SLE patients, we found clusters of B cells and plasma cells in close contact with ICOS+ T cells. CONCLUSION: Detailed analysis of B cells with down-regulated ICOS-L suggests that ICOS is one of the forces driving the formation of memory B cells and plasma cells in SLE. Furthermore, our identification of plasma cells in areas of T cell-B cell interaction in kidneys suggests that components of a T cell-driven B cell activation process may take place in peripheral tissues in SLE.

Adolescent↗
PubMed2004

Universal leukodepletion of blood components results in a significant reduction of febrile non-hemolytic but not allergic transfusion reactions.

BACKGROUND: Universal leukodepletion of blood components to prevent acute non-hemolytic transfusion reactions (NHTRs) is still a subject of debate. PATIENTS AND METHODS: Transfusion-associated NHTRs observed at our hospital in the last 6 years were retrospectively analyzed. Buffy-coat depleted red blood cells (bc-RBCs), and if indicated, leucodepleted post-storage (ld-RBCs) were initially used. In April 1997, universal leukodepletion was implemented at our hospital, and thereafter only prestorage ld-RBCs were used. All platelet concentrates transfused during this time were prestorage filtered single-donor apheresis platelets (SDAPs). RESULTS: A total of 163,090 blood products were transfused from April 1995 to April 2001 (bc-RBC: n=34,040 units; ld-RBC: n=66,967; SDAP: n=14,516; FFP: n=47,567). The number of post-transfusion febrile NHTRs occurring with each blood product was 65 (0.19%) for bc-RBCs, 8 (0.16%) for post-storage ld-RBCs, 16 (0.03%) for prestorage ld-RBCs, 16 (0.11%) for SDAPs, and 10 (0.02%) for FFP. Allergic reactions (n=116) were most frequently observed after SDAP transfusion (0.32%) and occurred at a similarly low rate after transfusion of all other blood components (0.03-0.08%). CONCLUSION: In conclusion, acute NHTRs rarely occur after the use of leukodepleted blood components. Prestorage appears to be more effective than post-storage leukodepletion in preventing febrile reactions following a blood transfusion.

Blood Component Removal↗
PubMed2004

Rheumatoid factor revisited.

PURPOSE OF REVIEW: Initial studies of the pathogenesis of rheumatoid arthritis focused on the role of rheumatoid factor and immune complex-associated vasculitis and synovitis. Subsequent work has delineated T cell responses, the role of cytokines, chemokines, and the aggressive nature of rheumatoid synovitis. Recent findings underscore the importance of humoral immunity in this entity and are the subject of this review. RECENT FINDINGS: By the discovery of anti-cyclic citrullinated peptide, anti-RA33, and anti-GPI antibodies in the human and mouse systems, respectively, the impact of humoral autoimmunity in rheumatoid arthritis regained remarkable interest. This review summarizes recent insights into humoral autoimmunity in rheumatoid arthritis in the context of the generation of rheumatoid factors, including B cell activation via toll-like receptors and genetic predispositions that can trigger the induction of rheumatoid arthritis. The generation of rheumatoid factors that can also be found during host defense against infectious agents and under pathologic conditions, such as rheumatoid arthritis, Sjögren syndrome, and hepatitis C-associated mixed cryoglobulinemia after hepatitis C infection is likely the result of genetic predispositions and the intensity of the (primary) immune reaction. Models of the role of rheumatoid factors in health and disease, including related lymphomagenesis, will be discussed. SUMMARY: In patients with rheumatoid arthritis, the induction of rheumatoid factors can be taken as an indicator of severe disease with a striking involvement of B cell activation. Very recent clinical trials using B cell depletion support the concept that humoral immunity, as evidenced by the production of rheumatoid factors, plays a significant role in the course of the disease.

Animals↗
PubMed2003

sE-selectin for stratifying outcome in rheumatoid arthritis.

OBJECTIVES: To determine the usefulness of sE-selectin as a marker for early diagnosis and stratification of rheumatoid arthritis. METHODS: We investigated several markers of disease activity, including circulating adhesion molecules and other standard laboratory tests, in a 2-3 year followup analysis of patients with rheumatoid arthritis. RESULTS: The mean +/- SD levels of sE-selectin (91.68 +/- 31.8 ng/ml versus 49.83 +/- 14.76 ng/ml) and rheumatoid factor (375.7 +/- 394.4 U versus 44.66 +/- 37.63 U) were strongly elevated in severe (n = 15) versus mild (n = 7) courses of disease. Statistical calculation of mean and standard deviation revealed that sE-selectin represents a highly significant marker for the presence of persistent and aggressive disease over time, regardless of therapeutic intervention and observation time points (P = 0.0004). Notably, regression analysis identified constant values for all parameters analyzed and, therefore, a stable course of the disease could be predicted from the beginning. CONCLUSION: sE-selectin appears to be a powerful marker to predict the severity of rheumatoid arthritis.

Adult↗
PubMed2003

Semiquantitative and qualitative assessment of B-lymphocyte V H repertoire by a fluorescent multiplex PCR.

We established a new tool to perform semiquantitative and qualitative screening for V(H) gene usage frequency during IgH rearrangements in human B-lymphocytes. In two separate multiplex PCRs, the rearranged VDJ regions were amplified with V(H) family-specific primers labeled with different fluorescent dyes (FAM, HEX, NED, or ROX). The relative amount of each of the particular V(H) family products and their ratios were determined by fragment analysis on a ABI PRISM 377 sequencer. We verified that the fluorescent multiplex PCR (FMPCR) shows high specificity and sensitivity, acceptable reproducibility and reliability. Data obtained were well in agreement with results revealed by sequencing following single-cell PCR. Ten healthy volunteers showed a comparable semiquantitative V(H) family distribution. The FMPCR also correctly detected a monoclonal peak in a CLL patient. Thus, labeling primers with various fluorescent dyes allows for an assessment of V(H) family usage and an immediate determination of the involved V(H) gene family if any clonal peaks are present. This method provides a quick, easy, and reliable tool for V(H) repertoire screening of larger populations of patients suffering from diseases with changes in the V(H) repertoire allowing for selection of cases worth a more detailed and cumbersome sequence analysis later on.

Adult↗
PubMed2003

Correlation between circulating CD27high plasma cells and disease activity in patients with systemic lupus erythematosus.

OBJECTIVE: Disease activity in systemic lupus erythematosus (SLE) is usually assessed with complex disease activity scores comprising a variety of different parameters. In order to determine whether SLE disease activity correlates with abnormal B lymphocyte activity, B cell subsets were analyzed, and their relationship to clinical and humoral measures of disease activity was assessed. METHODS: The distribution of B cell subsets was determined by fluorescence-activated cell sorting analysis and assessed in relation to the autoantibody profile, disease activity measured by the SLE Disease Activity Index (SLEDAI) and the European Consensus Lupus Activity Measure scores, disease duration, and therapy. RESULTS: The number and frequency of CD27(high) plasma cells were significantly correlated with the SLE disease activity indices and with the titer of anti-double-stranded DNA (anti-dsDNA) autoantibodies. Circulating B cell subsets were not influenced by age or sex, but appeared to relate to the duration of disease and the therapeutic regimen, with the number and frequency of CD27(high) plasma cells increasing and those of CD27- naive B cells decreasing over time. Patients were divided into those with a SLEDAI score of 0-8 (low disease activity) and those with SLEDAI score >8 (high disease activity). Patients with high disease activity had an increased frequency of both CD19+ B cells and CD27(high) plasma cells. By using a nonparametric data sieving algorithm, we observed that these B cell abnormalities provided predictive values for nonactive and active disease of 78.0% and 78.9%, respectively. The predictive value of the B cell abnormalities (78.9%) was greater than that of the humoral/clinical data pattern (71.4%), including anti-dsDNA antibody levels, circulating immune complexes, increased erythrocyte sedimentation rate, mucocutaneous involvement, and acute renal involvement. CONCLUSION: Flow cytometric monitoring of B cell subsets in the peripheral blood provides new insights into abnormalities of B cell function in SLE and may also be a diagnostically valuable option for monitoring the activity of this autoimmune disease.

Adult↗
PubMed2003

[B-cell depletion in the treatment of rheumatoid arthritis].

New strategies including the blockade of TNFalpha showed excellent therapeutic effects even in so far refractory rheumatic diseases. Moreover, these approaches provided us with important informations about the pathogenic role of the mediators involved and distinct autoimmune cellular subsets. Here, we discuss recent data of a temporary B cell depletion using a monoclonal anti-CD20 antibody in the therapeutic arsenal of rheumatoid arthritis.

Antibodies, Monoclonal↗
PubMed2003

Acute intravascular hemolysis after transfusion of a chimeric RBC unit.

BACKGROUND: Natural blood cell chimerism rarely occurs in humans. The case of a patient who developed transfusion reaction due to the transfusion of chimeric RBCs is reported. CASE REPORT: A 61-year-old male patient with blood group O received two units of packed and O-grouped RBCs after elective kidney surgery. Immediately after blood transfusion, the patient developed a hemolytic transfusion attack. The serologic re-examination revealed only a mixed-field pattern of agglutination of RBCs in one of the two transfused units. The donor of this unit was an apparently healthy 24-year-old male with a twin sister. Both of them showed an identical mixture of roughly 95 percent group O and 5 percent group B RBCs by gel agglutination technology and flow cytometry. The results were also confirmed by ABO blood group genotyping. CONCLUSIONS: This is the first report of a hemolytic transfusion reaction related to the transfusion of chimeric RBCs.

ABO Blood-Group System↗
PubMed2003

The role of B cells in rheumatoid arthritis: mechanisms and therapeutic targets.

Our understanding of the role of B cells as part of the immune system has been remarkably expanded in the past few years. Autoimmunity, the production of autoantibodies or the activation and expansion of autoimmune T cells, is relatively common, whereas the development of autoimmune diseases with destruction of tissue is much less frequent. In rheumatoid arthritis, the autoantigen(s) involved in tissue damage and their role in disease have not been fully elucidated. Recent data suggest that the impact of B cells in rheumatoid arthritis may be of significance; therefore, a depleting anti-B cell therapy appears to be another therapeutic strategy. This review will focus on recent findings of the role of B cells in rheumatoid arthritis and the implications to target B cells in this disease.

Antirheumatic Agents↗