We describe a modification of two-dimensional (2-D) polyacrylamide gel electrophoresis that requires only a single gel to reproducibly detect differences between two protein samples. This was accomplished by fluorescently tagging the two samples with two different dyes, running them on the same 2-D gel, post-run fluorescence imaging of the gel into two images, and superimposing the images. The amine reactive dyes were designed to insure that proteins common to both samples have the same relative mobility regardless of the dye used to tag them. Thus, this technique, called difference gel electrophoresis (DIGE), circumvents the need to compare several 2-D gels. DIGE is reproducible, sensitive, and can detect an exogenous difference between two Drosophila embryo extracts at nanogram levels. Moreover, an inducible protein from E. coli was detected after 15 min of induction and identified using DIGE preparatively.

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Objective. Regulatory T cells play an important role in the prevention of autoimmunity and have been shown to be effective in the treatment of experimental colitis, a T cell-mediated and organ-specific disease. We previously demonstrated that intrinsic CD25؉ regulatory T cells modulate the severity of collagen-induced arthritis (CIA), which, in contrast to colitis, is a systemic antibody-mediated disease and an accepted model of rheumatoid arthritis. We undertook this study to determine whether regulatory T cells have the potential to be used therapeutically in arthritis.Methods. We transferred CD4؉,CD25؉ T cells into mice exhibiting arthritis symptoms, both immunocompetent mice and mice subjected to lethal irradiation and rescued with syngeneic bone marrow transplantation.Results. A single transfer of regulatory T cells markedly slowed disease progression, which could not be attributed to losses of systemic type II collagenspecific T and B cell responses, since these remained unchanged after adoptive transfer. However, regulatory T cells could be found in the inflamed synovium soon after transfer, indicating that regulation may occur locally in the joint. Conclusion.Our data indicate that CD25؉ regulatory T cells can be used for the treatment of systemic, antibody-mediated autoimmune diseases, such as CIA.

Objective. CD4؉,CD25؉ T regulatory cells may offer opportunities to intervene in the course of autoimmune disease. We wished to evaluate their potential for influencing systemic and chronic joint inflammation by investigating their involvement in collagen-induced arthritis (CIA).Methods. We depleted DBA/1 mice of CD25؉ regulatory cells by injection of a depleting monoclonal antibody specific for CD25 14 days before a single immunization with type II collagen (CII) in Freund's complete adjuvant. CD4؉,CD25؉ T cells were adoptively transferred to some groups of mice during immunization. Mice were then scored for signs of arthritis, and blood was taken periodically to measure the amounts of CII-specific antibodies. Splenocytes of treated mice were examined in vitro to determine the effects of depletion on proliferation to CII and control antigens.Results. CD25؉ cell-depleted DBA/1 mice had significantly more severe disease than control mice following collagen immunization. The magnified severity was also accompanied by higher antibody titers against collagen, and in vitro tests showed increased proliferation of collagen-specific T cells. Adoptively transferring CD4؉,CD25؉ T cells into depleted mice was shown to reverse the heightened severity. Control mice, which were depleted and immunized with the neoantigen keyhole limpet hemocyanin (KLH), had neither an increased antibody response toward KLH nor an augmented proliferative response, indicating that CD25؉ cell depletion preferentially affects immunity against self antigen.Conclusion. These results establish a link between CD4؉,CD25؉ regulatory cells and CIA and provide a rationale for investigating CD4؉,CD25؉ T regulatory cells in the treatment and prevention of arthritis.

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ObjectiveProline–glycine–proline (PGP) has been shown to have chemotactic effects on neutrophils via CXCR2 in several lung diseases. PGP is derived from collagen by the combined action of matrix metalloproteinase (MMP) 8 and/or MMP9 and prolyl endopeptidase (PE). We investigated the role of PGP in inflammatory bowel disease (IBD).DesignIn intestinal tissue from patients with IBD and mice with dextran sodium sulfate (DSS)-induced colitis, MMP8, MMP9 and PE were evaluated by ELISA, immunoblot and immunohistochemistry. Peripheral blood polymorphonuclear cell (PMN) supernatants were also analysed accordingly and incubated with collagen to assess PGP generation ex vivo. PGP levels were measured by mass spectrometry, and PGP neutralisation was achieved with a PGP antagonist and PGP antibodies.ResultsIn the intestine of patients with IBD, MMP8 and MMP9 levels were elevated, while PE was expressed at similar levels to control tissue. PGP levels were increased in intestinal tissue of patients with IBD. Similar results were obtained in intestine from DSS-treated mice. PMN supernatants from patients with IBD were far more capable of generating PGP from collagen ex vivo than healthy controls. Furthermore, PGP neutralisation during DSS-induced colitis led to a significant reduction in neutrophil infiltration in the intestine.ConclusionsThe proteolytic cascade that generates PGP from collagen, as well as the tripeptide itself, is present in the intestine of patients with IBD and mice with DSS-induced colitis. PGP neutralisation in DSS-treated mice showed the importance of PGP-guided neutrophilic infiltration in the intestine and indicates a vicious circle in neutrophilic inflammation in IBD.

CD4+CD25+ regulatory T (Treg) cells play an essential role in maintaining tolerance to self and nonself. In several models of T cell-mediated (auto) immunity, Treg cells exert protective effects by the inhibition of pathogenic T cell responses. In addition, Treg cells can modulate T cell-independent inflammation. We now show that CD4+CD25+ Treg cells are able to shed large amounts of TNFRII. This is paralleled by their ability to inhibit the action of TNF-α both in vitro and in vivo. In vivo, Treg cells suppressed IL-6 production in response to LPS injection in mice. In contrast, Treg cells from TNFRII-deficient mice were unable to do so despite their unhampered capacity to suppress T cell proliferation in a conventional in vitro suppression assay. Thus, shedding of TNFRII represents a novel mechanism by which Treg cells can inhibit the action of TNF, a pivotal cytokine driving inflammation.

The effects on gut blood flow velocities of parenteral indomethacin (0-2 mg/kg) given either quickly as a bolus or slowly as an infusion were compared in consecutive studies of two groups of infants with symptomatic patent ductus arteriosus. In the presence of patent ductus arteriosus the range of velocities in the superior mesenteric artery before indomethacin was given was characterised by pronounced abnormalities including absent-or in some cases even retrograde-diastolic flow.In eight subjects the first rapidly given bolus dose of indomethacin (duration 20 seconds or less) caused a pronounced and sustained fail in the velocity of the superior mesenteric artery blood flow (mean peak systolic velocity (cm/second): before 74; after 38; median time to maximum fall 7-4 minutes; median time to recovery 50 minutes). A further 10 subjects received their first dose of indomethacin by slow infusion (duration 30-35 min) and the percentage fall in peak systolic velocity was both substantialiy less (22% compared with 47%) and later (median time to maximum fall 37-3 minutes) than after rapid infusion. Qualitatively similar but smaller changes were seen in the coeliac axis. Return of antegrade end diastolic flow in the superior mesenteric artery within one hour of the first dose of indomethacin was a good predictor of subsequent closure of the ductus.These data suggest that there is a profound disturbance in mid gut perfusion in infants with patent ductus, which is exacerbated by indomethacin given rapidly by intravenous bolus. They may also provide a rational explanation for the well recognised association between necrotising enterocolitis and both patent ductus arteriosus and indomethacin administration. The unwanted effects of the indomethacin are abrogated by slow infusion, without loss of efficacy in closure of the ductus.Neonatal Unit,