Metformin is the first-line drug in the treatment of type 2 diabetes. In addition to its hypoglycemic effect, metformin has an anti-inflammatory function, but the precise mechanism promoting this activity remains unclear. High mobility group box 1 (HMGB1) is an alarmin that is released from necrotic cells and induces inflammatory responses by its cytokine-like activity and is, therefore, a target of anti-inflammatory therapies. Here we identified HMGB1 as a novel metformin-binding protein by affinity purification using a biotinylated metformin analogue. Metformin directly bound to the C-terminal acidic tail of HMGB1. Both in vitro and in vivo, metformin inhibited inflammatory responses induced by full-length HMGB1 but not by HMGB1 lacking the acidic tail. In an acetaminophen-induced acute liver injury model in which HMGB1 released from injured cells exacerbates the initial injury, metformin effectively reduced liver injury and had no additional inhibitory effects when the extracellular HMGB1 was blocked by anti-HMGB1-neutralizing antibody. In summary, we report for the first time that metformin suppresses inflammation by inhibiting the extracellular activity of HMGB1. Because HMGB1 plays a major role in inflammation, our results suggest possible new ways to manage HMGB1-induced inflammation.

BACKGROUND AND PURPOSEUpon stimulation, neutrophils release their nuclear contents called neutrophil extracellular traps (NETs), which contain unfolded chromatin and lysosomal enzymes. NETs have been demonstrated to play a critical role in host defence, although the role of PGE 2 , a bioactive substance generated in inflammatory tissues, in the formation of NETs remains unclear. EXPERIMENTAL APPROACHThe effects of PGE 2 , agonists and antagonists of its receptors, and modulators of the cAMP-PKA pathway on the formation of NETs were examined in vitro in isolated neutrophils and in vivo in a newly established mouse model. KEY RESULTSPGE 2 inhibited PMA-induced NET formation in vitro through EP 2 and EP 4 Gαs-coupled receptors. Incubation with a cell-permeable cAMP analogue, dibutyryl cAMP, or various inhibitors of a cAMP-degrading enzyme, PDE, also suppressed NET formation. In the assay established here, where an agarose gel was s.c. implanted in mice and NET formation was detected on the surface of the gel, the extent of the NET formed was inhibited in agarose gels containing rolipram, a PDE4 inhibitor, and butaprost, an EP 2 receptor agonist. CONCLUSIONS AND IMPLICATIONSPGE 2 inhibits NET formation through the production of cAMP. These findings will contribute to the development of novel treatments for NETosis-related diseases. AbbreviationsBSA, bovine serum albumin;

SummaryThe proliferation of Vd1 + gd T lymphocytes has been described in various infections including human immunodeficiency virus (HIV), cytomegalovirus (CMV) and malaria. However, the antigen specificity and functions of the human Vd1 + T cells remain obscure. We sought to explore the biological role for this T cell subset by investigating the reconstitution of T cell receptor (TCR) repertoires of Vd1 + gd T lymphocytes after human allogeneic haematopoietic stem cell transplantation (HSCT). We observed skewed TCR repertoires of the Vd1

Summary:We have examined the reconstitution of ␥␦ T cell repertoire diversity after human allogeneic hematopoietic cell transplantation using a polymerase chain reaction (PCR)-based complementarity-determining region (CDR) 3 size spectratyping and DNA sequencing. The CDR3 complexity in the variable region of the T cell receptor (TCR)-␦ chain was different amongst the individuals studied. Furthermore, CDR3 size distribution patterns of allogeneic hematopoietic cell transplant recipients were almost completely recovered by a few months after transplantation. In some patients, clonal predominance of the TCRDV1+ T cells became evident during the period after transplantation. In one particular donor/recipient pair, clonal predominance of TCRDV1+ T cells was already present in blood lymphocytes of the donor, and was also observed in the recipient after transplantation. Using this donor/recipient pair, we have questioned whether ␥␦ T cell regeneration occurs via the peripheral expansion of mature T cells in the graft. In the donor lymphocytes, two expanding ␥␦ T cell clones, which were demonstrated by CDR3 sequences of the TCR-␦ chain, were recognized. These two clones were identified in the T cells from the recipient post transplant, but not before transplantation. One of the two clones was still detectable 1. years after the transplant procedure. These results strongly suggest that peripheral expansion of mature T cells in the graft is the principal pathway of ␥␦ T cell regeneration after allogeneic hematopoietic cell transplantation in adults. Bone Marrow Transplantation (2000) 26, 177-185. Keywords: T cell receptor; ␦-chain; diversity; transplantation High-dose chemoradiotherapy depletes T cells in blood and marrow transplant recipients and adequate T cell regeneration is essential for successful transplantation. The repertoire of T cells requires to be sufficiently diverse in order to recognize a wide range of microorganism-derived antigens. ␣␤ T cells are almost exclusively thymus-dependent

Summary. We analysed T-cell receptor a-chain variable region (TCRAV) and T-cell receptor b-chain variable region (TCRBV) repertoires in peripheral blood mononuclear cells (PBMCs) from 34 recipients of allogeneic bone marrow transplantation (allo-BMT), seven of allogeneic peripheral blood stem cell transplantation and 19 of autologous peripheral blood stem cell transplantation using the quantitative microplate hybridization assay. TCR usage skewed at an early period (6±7 weeks) after BMT. The change was more apparent in allogeneic recipients than in autologous recipients. In particular, a predominant increase was detected in the frequency of VA1-4 (26%, 11 of 41 recipients), VA3-1 (32%) and VB24-1 (28%). Interestingly, acidic amino acid residues frequently followed the arginine residue in complementarity-determining region 3 of BV24S1.We further examined the extent of skew using samples obtained at serial time points after transplantation. The normalization of skewed repertoires occurred over a long period of time (. 8 years). There was a significant difference in the rate of normalization of skewed TCR repertoires between adult and child recipients (P , 0´05). The results suggest that these T cells may have expanded in response to allogeneic antigens, such as miHA (minor histocompatibility antigen), and that altered repertoires are eventually normalized by T-cell regeneration via a thymic-dependent pathway in children.