Monocyte dependence of pokeweed mitogen-induced differentiation of immunoglobulin-secreting cells from human peripheral blood mononuclear cells.

Human peripheral blood mononuclear cells (PBM) lost the capacity to generate immunoglobulin-secreting cells (ISC) in response to pokeweed mitogen (PWM) when depleted of adherent cells (AC). The diminished responsiveness of the nonadherent cells (NAC) could not be ascribed to cell death, altered PWM dose response characteristics, or a change in the length of incubation required to generate a response. Supplementation with autologous or homologous AC, but not 2-mercaptoethanol, restored the capacity of NAC to generate ISC after PWM stimulation. By standard criteria AC were found to contain 85 to 90% monocytes. Furthermore, the monocytes and not the few lymphocytes contaminating the AC were responsible for restoring PWM responsiveness to the NAC. PWM-induced DNA synthesis of NAC also was markedly reduced compared to PBM. Again, supplementation with monocytes restored responsiveness to NAC. The monocyte dependence of PWM-induced proliferation and generation of ISC was most apparent when cultural conditions were employed that limited cell-to-cell interaction.     

Flow cytometric assessment of the signaling status of human B lymphocytes from normal and autoimmune individuals

Abnormalities in lymphocyte signaling cascades are thought to play an important role in the development of autoimmune disease. However, the large amount of cellular material needed for standard biochemical assessment of signaling status has made it difficult to evaluate putative abnormalities completely using primary lymphocytes. The development of technology to employ intracellular staining and flow cytometry to assess the signaling status of individual cells has now made it possible to delineate the perturbations that are present in lymphocytes from patients with autoimmune disease. As an example, human B cells from the Ramos B cell line and the periphery of systemic lupus erythematosus (SLE) patients or normal nonautoimmune controls were assessed for activation of the NF-κB and mitogen activated protein kinase (MAPK) signaling cascades by intracellular multiparameter flow cytometric analysis and biochemical Western blotting. In combination with fluorochrome conjugated antibodies specific for surface proteins that define B cell subsets, antibodies that recognize activated, or phosphorylated inhibitors of κB (IκB) as well as the extracellular regulated kinase (ERK), jun N-terminal kinase (JNK) or p38 MAPKs were used to stain fixed and permeabilized human B cells and analyze them flow cytometrically. Examination of the known signaling pathways following engagement of CD40 on human B cells confirmed that intracellular flow cytometry and Western blotting equivalently assay CD154-induced phosphorylation and degradation of IκB proteins as well as phosphorylation of the MAPKs ERK, JNK and p38. In addition, B cells from the periphery of SLE patients had a more activated status immediately ex vivo as assessed by intracellular flow cytometric analysis of phosphorylated ERK, JNK and p38 when compared with B cells from the periphery of normal, nonautoimmune individuals. Together, these results indicate that multiparameter intracellular flow cytometric analysis of signaling pathways, such as the NF-κB and MAPK cascades, can be used routinely to assess the activation status of a small number of cells and thus delineate abnormalities in signaling molecules expressed in primary lymphocytes from patients with autoimmune disease.     

Morphology agrees with molecular data: phylogenetic affinities of Euptychiina butterflies (Nymphalidae: Satyrinae)

Euptychiina is the most species‐rich subtribe of Neotropical Satyrinae, with over 450 known species in 47 genera (14 monotypic). Here, we use morphological characters to examine the phylogenetic relationships within Euptychiina. Taxonomic sampling included 105 species representing the majority of the genera, as well as five outgroups. A total of 103 characters were obtained: 45 from wing pattern, 48 from genitalia and 10 from wing venation. The data matrix was analysed using maximum parsimony under both equal and extended implied weights. Euptychiina was recovered as monophyletic with ten monophyletic genera, contrasting previous DNA sequence‐based phylogenies that did not recover the monophyly of the group. In agreement with sequence‐based hypotheses, however, three main clades were recognized: the ‘Megisto clade’ with six monophyletic and three polyphyletic genera, the ‘Taygetis clade’ with nine genera of which three were monophyletic, and the ‘Pareuptyhia clade’ with four monophyletic and two polyphyletic genera. This is the first morphology‐based phylogenetic hypothesis for Euptychiina and the results will be used to complement molecular data in a combined analysis and to provide critical synapomorphies for clades and genera in this taxonomically confused group.     

Precursor frequency of human T4 cells responding to stimulation through the CD3 molecular complex: role of various cytokines in promoting growth and IL2 production

The frequency of human T4 cells induced to grow and produce IL2 in response to the anti-CD3 mAb, 64.1, was examined. T4 cells were cultured at limiting dilution and stimulated with either soluble or immobilized 64.1 in the presence of various cytokines and/or irradiated B lymphoblastoid cells as accessory cells (AC). The frequency of responding cells was assessed by examining wells microscopically for visible growth and supernatants for IL2. Immobilized, but not soluble, 64.1 was able to induce T4 cells to grow in the complete absence of AC, but only when exogenous cytokines were present. IL2 was most effective at supporting T4 cell growth in this system, with a mean of 26.0 +/- 3.8% of immobilized 64.1-activated T4 cells generating a colony in cultures supplemented with IL2. IL4 could also support the growth of immobilized 64.1-activated T4 cells, but the frequency of responding cells was much lower (3.7 +/- 0.9%). The combination of IL2 and IL4 was not more effective than IL2 alone. TNF alpha, IL1 beta, and IL6 were unable to support T4 cell growth alone, but each increased the frequency of T4 cells responding in the presence of IL2. AC could support the growth of a small number of 64.1-stimulated T4 cells in the absence of exogenous IL2 and enhanced the frequency of T4 cells responding to immobilized 64.1 in the presence of IL2. The percentage of immobilized 64.1-stimulated T4 cells producing IL2 was also examined. Immobilized 64.1 stimulated less than 1.4 in 1000 T4 cells to produce IL2 in the absence of AC and neither IL4 nor TNF alpha enhanced this response. Fixed AC and IL1 beta, on the other hand, caused a small increase in the frequency of immobilized 64.1-activated T4 cells that secreted IL2. The frequency of T4 cells stimulated to produce IL2 by immobilized 64.1 was greatly enhanced by the addition of AC. The data indicate that in the absence of AC, a stimulatory matrix of immobilized 64.1 is sufficient for some T4 cells to be activated to become IL2 or IL4 responsive and for a smaller percentage to secrete IL2. Additional T4 cells require IL1 beta, TNF alpha, IL6, or AC to become IL2 responsive, whereas only IL1 beta and AC can promote IL2 production. In the presence of AC, the amount of cytokine produced endogenously appears to be sufficient to sustain the growth of some T4 cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression, regulation, and function of B cell-expressed CD154 in germinal centers.

Activated B cells and T cells express CD154/CD40 ligand in vitro. The in vivo expression and function of B cell CD154 remain unclear and therefore were examined. Tonsillar B and T cells expressed CD154 at a similar density both in situ and immediately ex vivo, whereas a significantly higher percentage of the former expressed CD154. CD154-expressing B cells were most frequent in the CD38positiveIgD+ pre-germinal center (GC)/GC founder, CD38positive GC and CD38-IgD- memory populations, and were also found in the CD38-IgD+ naive and CD38brightIgD+ plasmablast subsets, but not in the CD38brightIgD- plasma cell subset. B cell expression of CD154 was induced by engaging surface Ig or CD40 by signals that predominantly involved activation of AP-1/NF-AT and NF-kappaB, respectively. The functional importance of CD154-mediated homotypic B cell interactions in vivo was indicated by the finding that mAb to CD154 inhibited differentiation of CD38positiveIgD- GC B cells to CD38-IgD- memory cells. In addition, mAb to CD154 inhibited proliferation induced by engaging sIg or CD40, indicating the role of up-regulation of this molecule in facilitating B cell responsiveness. Of note, CD154 itself not only functioned as a ligand but also as a direct signaling molecule as anti-CD154-conjugated Sepharose beads costimulated B cell responses induced by engaging surface Ig. These results indicate that CD154 is expressed by human B cells in vivo and plays an important role in mediating B cell responses.     

Chemokine receptor expression and functional effects of chemokines on B cells: implication in the pathogenesis of rheumatoid arthritis

Introduction  

Accumulation of B cells in the rheumatoid arthritis (RA) synovium has been reported, and it has been thought that these cells might contribute to the pathogenesis of RA by antigen presentation, autoantibody production, and/or inflammatory cytokine production. Chemokines could enhance the accumulation of B cells in the synovium. The aims of this study were to determine chemokine receptor expression by B cells both in the peripheral blood of normal donors and subjects with RA, and at the inflammatory site in RA, and the effects of chemokines on B cell activation.     

Challenges and opportunities of publishing supplements at <Emphasis Type="Italic">Arthritis Research &; Therapy</Emphasis>

Supplements are the cause of much debate in the world of journal publishing. Supplements are criticized for the fact that often they are funded by an external source, and journals have been known to shy away from their publication [1]. But is refusing to publish supplements the only answer? At Arthritis Research & Therapy, we feel that - if subjected to the full rigors of peer review - supplements can provide invaluable educational resources, exploring themes in a detailed and focused way that might not always be possible in the main journal.At Arthritis Research & Therapy, we have decided that the potential risk of a loss of objectivity in industry-sponsored supplements can be managed by scrupulous attention to the peer-review process. Therefore, in our experience, the benefit of publishing supplements greatly outweighs any perception of loss of objectivity. We should note that our colleagues in the pharmaceutical industry have embraced our approach with no efforts to circumvent our rules, as far as we know.We consider proceedings, review collections, and meeting abstracts for inclusion as supplements. Examples of recent supplements are easily accessed on the journal website. It will be apparent that the emphasis is on the molecular and cellular basis of immune and inflammatory mechanisms of disease. The underwriting by the sponsor of the cost of peer review and production ensures that the high-quality publication is freely available. Outlined below is the procedure that we follow when considering any potential supplements for the journal. We recognize the potential for competing interests to influence the content of articles where there is industry involvement, but we believe that by adhering to a stringent publication process we negate this risk with our supplement content.So, what is the procedure? First, in a change from the approach taken by many journals, we appoint an ''internal'' Supplement Editor (usually from the journal''s Editorial Board), whose role is to select peer reviewers and assess the suitability of the supplement articles for publication in the journal. This internal editor is selected by the Editors-in-Chief, and the sponsor''s approval is not sought in making this appointment. Before their appointment, internal editors are asked to declare any potential conflicts of interest, and full disclosures are included in both online and print versions of any supplements. In cases in which internal editors receive a stipend, this is paid for by the publisher - not the sponsor. All conflict of interest disclosures associated with supplement publications follow the National Library of Medicine policy for indexing supplement articles in MEDLINE [2].For some supplements, particularly where articles are commissioned externally, there may be an ''external'' Supplement Editor as well as the internal editor. Before their appointment, external supplement editors must be approved by the Editors-in-Chief. Their role is to identify the subject matter and propose suitable authors for the individual supplement articles, with the proviso that all content must be approved by the Editors-in-Chief. The external editor is not involved in the peer review of any of the articles once submitted. They are also asked for full conflict of interest disclosures, which are included in the supplement publication alongside those of the internal editor.One of the common criticisms levied against supplements is that the articles are not peer-reviewed to the journal''s normal standards. At Arthritis Research & Therapy, supplement articles go through the same thorough peer-review process as articles do in the main journal [3]. The Editors-in-Chief have full editorial control, including the ability to ask authors to make extensive revisions, and reserve the right to reject articles that do not meet the journal''s standards. And in accordance with the guidelines of the International Committee for Medical Journal Editors [4], all contracts clearly include information about editorial control and the role of the internal supplement editor.In addition to disclosures from the individual editors, sources of funding for a supplement are prominently displayed on the supplement title page, and funding is also acknowledged in each individual article. We ensure that supplements can be clearly distinguished as separate from the main journal content on the journal homepage. As with all of our articles, individual authors declare their conflicts of interest. This complies with the good publication practice (GPP2) guidelines [5]. We also adhere to the guidelines of the European Medical Writers Association [6] by ensuring that the involvement of any medical writers is disclosed in an article''s acknowledgments section along with their sources of funding.We hope that by ensuring that there is full transparency of disclosures from everyone involved in supplements and following strictly the peer-review procedure described above, we avoid the potential pitfalls of supplement publishing. The guidelines of the International Committee of Medical Journal Editors themselves state that supplements ''serve useful purposes'' and it is our responsibility as editors to ensure that they continue to be useful without allowing ourselves to be subject to industry influence. By publicly disclosing our policy for supplement review here, we hope that other journals will be more open about their peer-review policies for supplements and that they adopt similarly stringent practices in the future.     

IFNGR1 single nucleotide polymorphisms in rheumatoid arthritis

On the basis of their biological function, potential genetic candidates for susceptibility to rheumatoid arthritis can be postulated. IFNGR1, encoding the ligand-binding chain of the receptor for interferon gamma, IFNγR1, is one such gene because interferon gamma is involved in the pathogenesis of the disease. In the coding sequence of IFNGR1, two nucleotide positions have been described to be polymorphic in the Japanese population. We therefore investigated the association of those two IFNGR1 single nucleotide polymorphisms with rheumatoid arthritis in a case-control study in a central European population. Surprisingly, however, neither position was polymorphic in the 364 individuals examined, indicating that IFNGR1 does not contribute to susceptibility to rheumatoid arthritis, at least in Caucasians.