Analysis of the DNA-binding profile and function of TALE homeoproteins reveals their specialization and specific interactions with Hox genes/proteins.

The interactions of Meis, Prep, and Pbx1 TALE homeoproteins with Hox proteins are essential for development and disease. Although Meis and Prep behave similarly in vitro, their in vivo activities remain largely unexplored. We show that Prep and Meis interact with largely independent sets of genomic sites and select different DNA-binding sequences, Prep associating mostly with promoters and housekeeping genes and Meis with promoter-remote regions and developmental genes. Hox target sequences associate strongly with Meis but not with Prep binding sites, while Pbx1 cooperates with both Prep and Meis. Accordingly, Meis1 shows strong genetic interaction with Pbx1 but not with Prep1. Meis1 and Prep1 nonetheless coregulate a subset of genes, predominantly through opposing effects. Notably, the TALE homeoprotein binding profile subdivides Hox clusters into two domains differentially regulated by Meis1 and Prep1. During evolution, Meis and Prep thus specialized their interactions but maintained significant regulatory coordination.

[Guideline for the joint swelling symptom--primary physician problem management and referral indications. German Society of Rheumatology "Quality Assurance" Committee]. / Leitlinie für das Symptom Gelenkschwellung--Primärärztliches Problemmanagement und Uberweisungsindikationen. Deutsche Gesellschaft für Rheumatologie Kommission "Qualitätssicherung".

The guideline "Joint Swelling" is addressed to primary care physicians--general practitioners, internists or orthopedists without special experience in rheumatology. It provides a framework for interviewing patients, as well as for physical, laboratory and imaging examinations and for selection of treatment appropriate to the level of primary care. Situations which call for urgent evaluation and criteria for referral to rheumatologists are described. The appendix contains comments on signs and symptoms to differentiate arthralgia from joint swelling and on the diagnostic value of a history of joint swelling without confirmation by the physician. Further recommendations for the evaluation of patient history and physical and technical examinations are given in a tabular form. The significance of laboratory and imaging procedures is discussed.

Clonal expansion and decreased occurrence of peripheral blood gamma delta T cells of the V delta 2J delta 3 lineage in multiple sclerosis patients.

gamma delta T cells are implicated in autoimmune diseases but their precise function remains unclear. In multiple sclerosis (MS) brain tissue, gamma delta T cells co-localize with heat shock protein (hsp)65+ oligodendrocytes and are oligoclonally restricted in the V delta 2J delta 3 lineage. To investigate the homing properties and the degree of heterogeneity of V delta 2J delta 3+ gamma delta T cells in MS, peripheral blood lymphocytes (PBL) from 34 MS patients, 42 controls (14 autoimmune control patients, 28 healthy volunteers), and 11 lymphatic tissues of MS patients and controls were studied by RT-PCR and sequencing. V delta 2J delta 3 TCR rearrangement was detected in 27 out of 28 healthy controls, and was significantly less frequent in MS patients (24 out of 34) and autoimmune control patients (seven out of 14). It was present only in five out of 11 tissue specimens, none of them from MS patients. Direct sequencing and cloning/automated sequencing of the V delta 2J delta 3 PCR products indicated heterogeneity in healthy controls and oligoclonality in MS patients, but also in autoimmune control patients. Differences between MS patients and healthy controls were more accentuated in exacerbating hospitalized patients than in clinically stable outpatients participating in a clinical trial. Only one V delta 2J delta 3 sequence of a total of 85 different sequences obtained was shared between two MS patients. Taken together, evidence for clonal expansion of V delta 2J delta 3+ gamma delta T cells was found in PBL of MS patients. This T cell subset, previously shown to be present in 100% of chronic-active MS plaques, might home to the CNS in MS, resulting in its under-representation in the blood.

Kinetics of cytokine secretion by mononuclear cells of the blood from rheumatoid arthritis patients are different from those of healthy controls.

Mononuclear cells from peripheral blood (PBMC) of rheumatoid arthritis (RA) patients and healthy controls were incubated with alpha-CD3. Cytokine secretion from 2 h to 72 h of incubation was measured by ELISA. There were no significant differences in secretion of T cell derived IL-2 and IL-4 in cultures from RA patients and controls. The macrophage-derived cytokines, IL-1 beta and tumour-necrosis factor-alpha (TNF-alpha) were secreted with a steep increase of concentration during the first 16 h of incubation by PBMC from RA patients. PBMC from healthy controls secreted both cytokines at a constantly rising rate with a maximum for TNF-alpha at 48 h and for IL-1 beta at 72 h. Interferon-gamma (IFN-gamma) is secreted in significantly reduced concentrations by PBMC from untreated RA patients compared with controls. Gold-salt treatment led to a slightly delayed and enhanced secretion of TNF-alpha and IL-1 beta, an enhanced secretion of IL-2 and a restored secretion of IFN-gamma.

[Interleukin-1 in the pathogenesis of chronic polyarthritis]. / Interleukin-1 in der Pathogenese der chronischen Polyarthritis.

The cytokine interleukin-1 plays an important role in the production and modulation of the immune response in rheumatoid arthritis. It is produced by macrophages of the inflamed synovial tissue and induces the autocrine production of interleukin-1, amplifies the T-cell dependent immune response and has potent effects on inflammatory reactions of many non-lymphoid cell-systems. By means of a sensitive and specific ELISA interleukin(Il)-1 beta was measured in the peripheral blood and synovial fluid of patients with rheumatoid arthritis in comparison to controls in significantly increased levels (medium values: 280 pg/ml and 325 pg/ml versus less than 20 pg/ml). The Il-1 beta concentrations in the peripheral blood and in the synovial fluid were well correlated, but there was no correlation to other inflammation parameters like erythrocyte sedimentation rate or C-reactive protein, however, a good correlation to the nephelometrically measured rheumatoid factor (r = 0.71). In twelve hour cultures of adherent cells significantly increased spontaneous intracellular Il-1 beta-production was determined in synovial fluid macrophage cultures of rheumatoid arthritis patients compared to peripheral blood monocyte cultures of controls (median values 91.0 ng/10(6) cells versus 31.5 ng/10(6) cells). The secretion into the culture supernatant has to be stimulated by additional lipopolysaccharide. Interferon-gamma inhibits the spontaneous intracellular Il-1 beta-production of synovial fluid macrophages from rheumatoid arthritis patients. These findings may be of importance for the effect of the interferon-gamma therapy in the treatment of rheumatoid arthritis.

Spontaneous and LPS-stimulated production of intracellular IL-1 beta by synovial macrophages in rheumatoid arthritis is inhibited by IFN-gamma.

In the peripheral blood (PB) as well as the synovial fluid (SF) of rheumatoid arthritis (RA) patients significantly elevated levels of interleukin 1 beta (IL-1 beta) were determined compared to controls by means of a sensitive and specific ELISA (median values: 280 pg/ml and 325 pg/ml vs. less than 20 pg/ml). In 12-h cell cultures of adherent cells, significantly increased spontaneous intracellular IL-1 beta production was determined in SF macrophage (SFM phi) cultures of RA patients compared to PB monocyte (PBMo) cultures of controls (median values: 91.0 ng/10(6) cells vs. 31.5 ng/10(6) cells). However, secretion must be elicited by additional stimulation with lipopolysaccharide (LPS). Interferon-gamma (IFN-gamma) significantly inhibited the spontaneous intracellular IL-1 beta production in SFM phi 12-h cultures of RA patients.

Interleukin-2 secretion by synovial fluid lymphocytes in rheumatoid arthritis.

Interleukin-2 (IL-2) receptor bearing cells and soluble IL-2, measured in a bioassay with IL-2 dependent human T-cell blasts, were recognized in synovial fluid, but not in the peripheral blood of patients with rheumatoid arthritis (RA). After stimulation in vitro with appropriate concentrations of the mitogen concanavalin A (Con-A), comparable proportions of IL-2 receptor (IL-2R) bearing cells were seen in cultures of synovial fluid lymphocytes (SFL) and in cultures of peripheral blood lymphocytes (PBL). On the other hand, higher levels of secreted IL-2 were found in SFL cultures compared to corresponding PBL cultures of RA patients and normal donors. Specificity of the IL-2 bioassay was confirmed by blocking the T-cell blast proliferation (induced by SFL culture supernatants), by 83 +/- 4%, after addition of a monoclonal anti-IL-2R antibody. Despite the high levels of soluble IL-2, only a weak proliferative response was observed in the corresponding SFL cultures.

An ELISA for IgA-IgG and IgM-RF measurement. II. RF in several disease and control groups and under gold therapy in RA.

The before introduced solid phase ELISA was employed for the RF determination in the sera of RA patients and controls. The threshold values for positive results (calculated as the 95% distribution percentile of healthy donors) were 8, 3, and 3 U/ml for IgA, IgG, and IgM-RF, respectively. The results confirm the validity of the assay with clear negative results in several negative control groups (healthy donors, patients of the oto-, rhino-, laryngeal ambulance, diabetes mellitus, degenerative arthropathies; n = 111, median IgA, IgG and IgM-RF values of less than or equal to 2, less than or equal to 2 and less than or equal to 1 U/ml, respectively; 25-75% distribution percentiles within the median value) and positive results in the positive control group (seropositive RA; n = 20, median IgA, IgG and IgM-RF values of 324, 479 and 170 U/ml, respectively). 16/24 patients with so-called seronegative RA (negative Latex Fixation Test or Waaler Rose Test) had positive results in the ELISA, two of them had rheumatoid nodules clinically. The IgG-RF activity in the ELISA appears to be a good parameter for the course control of RA under gold therapy. 10 RA patients with clinical improvement of disease (declining ESR, CRP, joint index) after six months of gold therapy (= 0.6 g total gold amount) had a decline of total RF activity of 70% in median, whereas 10 patients with no clear effect on disease activity had only a decline of 20% in median.

An ELISA for IgA, IgG and IgM-RF measurement. I. Parameters of the assay.

A simple and rapid solid phase ELISA for the stepless determination of IgA, IgG and IgM-RF was developed. The ELISA works with Fc parts of human IgG as antigen. Specificity, validity and precision were tested. RF complex dissociation by urea and separation by gel filtration was performed in several samples to obtain information of the transfer of non-RF-IgG by pentameric IgM-RF. The RF-ELISA may give better information on the significance of RF of the three Ig classes in the clinical course of RA.

Superoxide anion production by monocytes and synovial fluid macrophages of patients with chronic inflammatory joint disorders.

Peripheral blood monocytes (PBMo) and synovial fluid macrophages (SFMO) of patients with rheumatoid arthritis (RA), HLA B27-positive reactive oligoarthritis and controls were investigated for their capacity to generate superoxide anions (O2-) upon stimulation with phorbolmyristoacetate (PMA) in a cytochrome c (cyt c) microassay. PBMo of RA patients, patients with reactive arthritis and controls did not reveal any significant differences and also treatment of RA patients with gold salts or immunosuppressive therapy had no effect on the oxidative burst in PBMo. In contrast, in SFMO of RA patients treated only with nonsteroidal anti-inflammatory drugs (NSAID) we found significantly enhanced O2- release, compared with PBMo of the same group. Treatment with gold salts had no effect on this enhanced oxidative response, whereas immunosuppressive therapy with azathioprin or corticosteroids significantly reduced the O2- release of SFMO. In patients suffering from reactive arthritis we did not find significant differences between SFMO and PBMo. The O2- release of SFMO of this group was significantly reduced, when compared to that of SFMO of RA patients, treated with NSAID. These results indicated that SFMO but not PBMo in RA in cyt c microassay produce increased levels of activated oxygen species. In comparison to PBMo, SFMO of patients suffering from reactive arthritis do not show such an increased oxidative burst. These findings suggest that in RA, activated oxygen species have a local destructive effect in inflamed joints. This seems to be caused by activation of catalytic enzymes and complement components, as well as induction of release of interleukins or prostaglandins, contributing to the augmentation of the chronic inflammatory process.

Evidence for enhanced interleukin 2 (IL-2) secretion and IL-2 receptor presentation by synovial fluid lymphocytes in rheumatoid arthritis.

Synovial fluid lymphocytes (SFL) and peripheral blood lymphocytes (PBL) from patients with rheumatoid arthritis (RA) and reactive oligoarthritis were investigated for activated T cells (Ia+SIg-), IL-2 receptor bearing cells (Tac+) and IL-2 production in vivo and in vitro. In contrast to negative results with blood, the synovial fluid of the arthritic joints contains considerable amounts of IL-2 activity (median: 11.8 mu/ml), elevated proportions of Ia+SIg- activated T cells (median: 12.5%) and of IL-2 receptor bearing cells (median: 2.5%). In vitro, after stimulation with several Concanavalin A (Con A) doses, SFL develop proportions of IL-2 receptor cells comparable to PBL. Furthermore, they produce higher values of IL-2 activity than comparable PBL cultures. The proportions of Ia+SIg- activated T cells increase only moderately after Con A stimulation compared to in vivo data, indicating different activated T cell subsets in the synovial fluid (Ia+SIg-, Tac+). The findings are discussed as an expression of an acute hyperactivation of lymphocytes in an inflamed joint.

[Detection of macrophage activity in the blood and synovia of chronic polyarthritis patients using an activated oxygen microassay]. / Nachweis der Makrophagenaktivität in Blut und Synovia von chronisch Polyarthritis-Kranken mittels eines aktivierten Sauerstoff-Mikroassays.

A microassay was developed for the estimation of macrophage (M phi)-activation by released activated oxygen. From peripheral blood or synovia M phi are isolated. O-2 which is released by activated M phi is photometrically detected by cytochrome c reduction. Differences in O-2-production do not exist between monocytes of rheumatoid arthritis patients and controls. In contrast synovial M phi from rheumatoid arthritis patients show increased levels of O-2-production.

[A solid-phase ELISA with specificity for rheumatoid factors of classes IgA, IgG and IgM]. / Ein Festphasen-ELISA mit Spezifität für Rheumafaktoren der Klassen IgA, IgG und IgM.

A solid phase ELISA was developed for the simple and rapid determination of rheumatoid factors (RF) of all major immunoglobulin classes. Micro-ELISA-plates were coated with human Fc-fragments and incubated with various dilutions of serum samples as well as with an international RF reference serum. Rheumatoid factors were quantitatively detected by rabbit antibodies (RaHIgA(alpha), RaHIgM(mu), RaHIgG(Fab] conjugated with alkaline phosphatase. The specificity of the ELISA was proved by means of binding inhibition of all rheumatoid factor classes by heat aggregated human IgG. A comparison of the IgM-RF titres of the latex fixation test with the IgM-RF concentration values of the ELISA yielded high correlation (r = .81).

Enumeration of T cell subsets and functional suppressor cell assays in patients with rheumatoid arthritis and HLA-B27-associated arthritis.

Seronegative rheumatoid arthritis (RA) patients have increased proportions of OKT-4+ (helper) T cells and diminished proportions of OKT-8+ (suppressor/cytotoxic) T cells in the peripheral blood. This phenomenon corresponds to diminished inhibition of B cell activation by peripheral blood lymphocytes (PBL). Seropositive RA patients show a broad range of OKT-8+ T cell proportions (9%-45%) in the peripheral blood, resulting in a mean level comparable to that in controls. Inhibition of T cell activation by suppressor cells in peripheral blood is greater in this group than in controls. HLA-B27-positive arthritis patients show no significant differences from controls with respect to markers and functional suppressor cell assays. In the synovial fluid of all patients both OKT-8+ T cell proportions and functional suppressor cell activity are greatly increased.

Studies on immunoregulatory mechanisms in acute and chronic hepatitis B.

Patients with acute hepatitis B and HBV-induced chronic hepatitis as well as normal control persons participated in the study. Hepatitis patients of both groups have decreased OKT4+/OKT8+T cell ratios due to an percental increase of OKT8+T cells in peripheral blood compared to the data of controls. Lymphocyte cultures of chronic hepatitis patients show reduced DNA synthesis after stimulation by allogeneic non-T cells, PHA, Con A and PWM. PWM-induced immunoglobulin secretion by B cells, determined by means of a reverse haemolytic plaque assay (RHPA) and a solid phase ELISA, showed comparable results in hepatitis B patients and controls. The AMLR, which is thought to reflect an autologous immunoregulatory phenomenon, is slightly impaired in cultures of hepatitis B patients in comparison to controls. Con A-induced suppressor cell activity on T cell reactions is decreased in hepatitis, whereas suppressor cell activity on B cell activation is within the same range as in cultures of controls. It is concluded from these data, that suppressor cell activity on T cell function is impaired in hepatitis B, whereas B cell functions and suppressor cell activity on B cell function are in the normal range. The results with the functional assays and the finding of increased proportions of OKT8+T cells in hepatitis B are considered to reflect properties of different T cell subpopulations, responsible for different immunoregulatory functions.

[Follow-up study on antibody dependent cell-mediated cytotoxicity and cell-mediated cytotoxicity to HBs-antigen conjugated target cells in patients with hepatitis B (author's transl)]. / Antikörperabhängige zellbedingte Zytotoxizität (ADCC) und zellvermittelte Zytotoxizität (CMC) gegenüber HBs-Antigen gekoppelten Targetzellen bei Patienten mit Hepatitis B.

Results of a follow up study on ADCC and CMC to HBs antigen conjugated target cells in patients with hepatitis B are given. The cytotoxic reaction was measured immediately after onset of the disease, three weeks and three months thereafter CMC was increased over the whole observation period. The results in hepatitis B patients were significantly different to those in normal controls. The ADCC in the presence of an antiserum to HBs antigen was in patients with hepatitis B immediately after onset of the disease reduced in comparison to the controls; it increased during the three months to the values of the controls in patients with uncomplicated disease. Experiments with isolated lymphocyte populations showed that the ADCC is mainly dependent on Fc receptor bearing lymphocytes whereas the CMC is mediated by T-cells and Fc-receptor bearing cells.

In vitro studies on lymphocytotoxicity to synovial cells and Chang cells in rheumatoid arthritis patients and healthy controls.

Lymphocytotoxicity to autologous or allogeneic synovial cells and Chang cells was studied in 27 patients with rheumatoid arthritis (RA), in 5 patients with osteoarthrosis of the hip or knee and in 17 healthy controls. Ficoll gradient-separated lymphocytes from the peripheral blood, T cells and non-T cells were used as effector cells. T lymphocytes were isolated as E-rosette forming cells 10 percent of which carried Fc- receptors. The differential counts for T and B cells in the peripheral blood of the RA and osteoarthrosis patients were approximately the same as in the blood of the healthy controls. The counts of Fc-receptor-bearing cells in the RA patients were, however, significantly higher. Cytotoxic reactivity of lymphocytes from RA patients, osteoarthrosis patients or healthy controls to synovial cells of autologous or allogeneic origin could not be demonstrated in our study, in which 125I-iododeoxyuridine labelled target cells were used in the microcytotoxicity test of Cohen et al. However, lymphocytes of the peripheral blood showed an increased cytotoxicity to Chang cells, an effect for which Fc-receptor bearing cells were responsible. Serum did not affect the cytotoxicity of lymphocytes. The results are interpreted as demonstrating an enhanced natural killer (NK) cell activity in RA patients; they do not indicate a specific cell mediated immune reaction to synovial cells.