The growing availability of genomic sequence information, together with improvements in analytical methodology, have enabled high throughput, high sensitivity protein identification. Silver staining remains the most sensitive method for visualization of proteins separated by two-dimensional gel electrophoresis (2-D PAGE). Several silver staining protocols have been developed which offer improved compatibility with subsequent mass spectrometric analysis. We describe a modified silver staining method that is available as a commercial kit (Silver Stain PlusOne; Amersham Pharmacia Biotech, Amersham, UK). The 2-D patterns abtained with this modified protocol are comparable to those from other silver staining methods. Omitting the sensitizing reagent allows higher loading without saturation, which facilitates protein identification and quantitation. We show that tryptic digests of proteins visualized by the modified stain afford excellent mass spectra by both matrix-assisted laser desorption/ionization and tandem electrospray ionization. We conclude that the modified silver staining protocol is highly compatible with subsequent mass spectrometric analysis.

ObjectivesTo assess the safety of intra-articular (IA) autologous tolerogenic dendritic cells (tolDC) in patients with inflammatory arthritis and an inflamed knee; to assess the feasibility and acceptability of the approach and to assess potential effects on local and systemic disease activities.MethodsAn unblinded, randomised, controlled, dose escalation Phase I trial. TolDC were differentiated from CD14+ monocytes and loaded with autologous synovial fluid as a source of autoantigens. Cohorts of three participants received 1×106, 3×106 or 10×106 tolDC arthroscopically following saline irrigation of an inflamed (target) knee. Control participants received saline irrigation only. Primary outcome was flare of disease in the target knee within 5 days of treatment. Feasibility was assessed by successful tolDC manufacture and acceptability via patient questionnaire. Potential effects on disease activity were assessed by arthroscopic synovitis score, disease activity score (DAS)28 and Health Assessment Questionnaire (HAQ). Immunomodulatory effects were sought in peripheral blood.ResultsThere were no target knee flares within 5 days of treatment. At day 14, arthroscopic synovitis was present in all participants except for one who received 10×106 tolDC; a further participant in this cohort declined day 14 arthroscopy because symptoms had remitted; both remained stable throughout 91 days of observation. There were no trends in DAS28 or HAQ score or consistent immunomodulatory effects in peripheral blood. 9 of 10 manufactured products met quality control release criteria; acceptability of the protocol by participants was high.ConclusionIA tolDC therapy appears safe, feasible and acceptable. Knee symptoms stabilised in two patients who received 10×106 tolDC but no systemic clinical or immunomodulatory effects were detectable.Trial registration numberNCT01352858.

ObjectivesTolerogenic dendritic cells (tolDCs) constitute a promising experimental treatment for targeting autoreactive T cells in autoimmune diseases, including rheumatoid arthritis (RA). The authors' goal is to bring tolDC therapy for RA to the clinic. Here the authors address key translational issues related to the manufacturing of tolDCs from RA patients with current good manufacturing practice (cGMP)-compliant reagents, the stability of tolDCs, and the selection of suitable quality control markers.MethodsHuman monocyte-derived tolDCs were established from RA patients and healthy controls (HCs) using the immunosuppressive drugs dexamethasone and vitamin D3, and the cGMP-grade immunomodulator, monophosphoryl lipid A, in the cGMP-compliant medium, CellGroDC. The functionality of tolDCs and tolDC-modulated autologous CD4 T cells was determined by flow cytometry, [3H]thymidine incorporation and ELISA.ResultsClinical-grade tolDCs established from patients with RA exhibit a typical tolerogenic phenotype of reduced costimulatory molecules, low production of proinflammatory cytokines and impaired stimulation of autologous antigen-specific T cells, comparable to HC tolDCs. Toll-like receptor 2 (TLR-2) was highly expressed by tolDCs but not mature DCs. Furthermore, tolDCs suppressed mature DC-induced T cell proliferation, interferon γ and interleukin 17 production, and rendered T cells hyporesponsive to further stimulation. Importantly, tolDCs were phenotypically stable in the absence of immunosuppressive drugs and were refractory to further challenge with proinflammatory mediators.ConclusionstolDCs established from patients with RA are comparable to those derived from healthy donors. TLR-2 was identified as an ideal marker for quality control of tolDCs. Potently tolerogenic and highly stable, these tolDCs are a promising cellular therapeutic for tailored immunomodulation in the treatment of RA.

Autoimmune pathologies are caused by a breakdown in self-tolerance. Tolerogenic dendritic cells (tolDC) are a promising immunotherapeutic tool for restoring self-tolerance in an antigen-specific manner. Studies about tolDC have focused largely on generating stable maturation-resistant DC, but few have fully addressed questions about the antigen-presenting and migratory capacities of these cells, prerequisites for successful immunotherapy. Here, we investigated whether human tolDC, generated with dexamethasone and the active form of vitamin D3, maintained their tolerogenic function upon activation with LPS (LPS-tolDC), while acquiring the ability to present exogenous autoantigen and to migrate in response to the CCR7 ligand CCL19. LPS activation led to important changes in the tolDC phenotype and function. LPS-tolDC, but not tolDC, expressed the chemokine receptor CCR7 and migrated in response to CCL19. Furthermore, LPS-tolDC were superior to tolDC in their ability to present type II collagen, a candidate autoantigen in rheumatoid arthritis. tolDC and LPS-tolDC had low stimulatory capacity for allogeneic, naïve T cells and skewed T cell polarization toward an anti-inflammatory phenotype, although LPS-tolDC induced significantly higher levels of IL-10 production by T cells. Our finding that LPS activation is essential for inducing migratory and antigen-presenting activity in tolDC is important for optimizing their therapeutic potential.

Results. Tolerogenic DCs displayed a semimature phenotype, produced low levels of inflammatory cytokines, and exhibited low T cell stimulatory capacity. Upon intravenous injection into arthritic mice, tolerogenic DCs migrated to the spleen, liver, lung, feet, and draining lymph nodes. Treatment of arthritic mice with type II collagen-pulsed tolerogenic DCs, but not unpulsed tolerogenic DCs or mature DCs, significantly inhibited disease severity and progression. This improvement coincided with a significant decrease in the number of Th17 cells and an increase in the number of interleukin-10-producing CD4؉ T cells, whereas tolerogenic DC treatment had no detectable effect on Th1 cells or interleukin-17-producing ␥/␦ T cells.Conclusion. Treatment with type II collagenpulsed tolerogenic DCs decreases the proportion of Th17 cells in arthritic mice and simultaneously reduces the severity and progression of arthritis.

Skeletal muscle plays a major role in whole body protein metabolism, and changes in the rates of synthesis and degradation of proteins are likely to lead to characteristic changes in the amounts of different proteins in muscle under various physiological and pathological conditions. This paper demonstrates the feasibility of a proteomic approach to analyzing the protein composition of skeletal muscle. We report here the initial establishment of two-dimensional gel electrophoresis (2-D PAGE) reference maps for mixed skeletal muscle taken from the abdominal wall of a normal adult rat. We used immobilized pH gradients of 3-10 (non-linear) and 4-7 (linear), and matrix assisted laser desorption/ionization--time of flight mass spectrometry for protein identification by peptide mass fingerprinting. More than 600 protein spots were detected on each gel, of which 100 were excised and characterized. In-gel digestion followed by peptide mass fingerprinting enabled tentative identification of 74 of these, which included a wide range of myofibrillary and sarcoplasmic proteins. This database should provide the nucleus of a valuable resource for investigation of the biochemical basis of skeletal muscle pathologies in general and such specific disorders as alcoholic myopathy and injury.

Apoptotic cells induce dendritic cell-mediated suppression via interferon-c-induced IDO IntroductionDendritic cells (DC) are professional antigen-presenting cells that play a critical role in the induction of immunity and tolerance. Regulation of these outcomes depends on several factors, such as the occurrence of specialized DC subsets and the maturation state of the DC.1-3 Immature DC (iDC) sample their local environment and efficiently capture antigen but are ineffective in T-cell priming because of their low expression of major histocompatibility complex (MHC) class I and II and costimulatory molecules and therefore contribute to the induction of tolerance. Upon receipt of a maturation signal, mature DC (mDC) show a decreased ability to capture antigen, concurrent with an upregulation of MHC and costimulatory molecules, which facilitates T-cell activation and subsequently the ability to induce an immune response.Apoptosis is an active phenomenon regulating cellular homeostasis. In contrast, necrosis, associated with pathological tissue injury, is characterized by rapid,

SummaryTolerogenic dendritic cells (tolDC) are a new immunotherapeutic tool for the treatment of rheumatoid arthritis (RA) and other autoimmune disorders. We have established a method to generate stable tolDC by pharmacological modulation of human monocyte-derived DC. These tolDC exert potent pro-tolerogenic actions on CD4 1 T cells. Lack of interleukin (IL)212p70 production is a key immunoregulatory attribute of tolDC but does not explain their action fully. Here we show that tolDC express transforming growth factor (TGF)-b1 at both mRNA and protein levels, and that expression of this immunoregulatory cytokine is significantly higher in tolDC than in mature monocyte-derived DC. By inhibiting TGF-b1 signalling we demonstrate that tolDC regulate CD4 1 T cell responses in a manner that is at least partly dependent upon this cytokine. Crucially, we also show that while there is no significant difference in expression of TGFbRII on CD4 1 T cells from RA patients and healthy controls, RA patient from RA patients can, nonetheless, be regulated efficiently by tolDC in a TGF-b1-dependent manner. This work is important for the design and development of future studies investigating the potential use of tolDC as a novel immunotherapy for the treatment of RA.