Optimal site-specific PEGylation of mutant TNF-alpha improves its antitumor potency

Recently, we created a lysine-deficient mutant tumor necrosis factor-alpha [mTNF-alpha-Lys(-)] with full bioactivity in vitro compared with wild-type TNF-alpha (wTNF-alpha), and site-specific PEGylation of mTNF-alpha-Lys(-) was found to selectively enhance its in vivo antitumor activity. In this study, we attempted to optimize this PEGylation of mTNF-alpha-Lys(-) to further improve its therapeutic potency. mTNF-alpha-Lys(-) was site-specifically modified at its N-terminus with linear polyethylene glycol (LPEG) or branched PEG (BPEG). While randomly mono-PEGylated wTNF-alpha (ran-LPEG5K-wTNF-alpha) with 5 kDa of LPEG (LPEG5K) had about only 4% in vitro bioactivity of wTNF-alpha, mono-PEGylated mTNF-alpha-Lys(-) [sp-PEG-mTNF-alpha-Lys(-)] with LPEG5K, LPEG20K, BPEG10K, and BPEG40K had 82%, 58%, 93%, and 65% bioactivities of mTNF-alpha-Lys(-), respectively. sp-LPEG-mTNF-alpha-Lys(-) and sp-BPEG10K-mTNF-alpha-Lys(-) had much superior antitumor activity to those of both unmodified TNF-alphas and ran-LPEG5K-wTNF-alpha, though sp-BPEG40K-mTNF-alpha-Lys(-) did not show in vivo antitumor activity. Thus, the molecular shape and weight of PEG may strongly influence the in vivo antitumor activity of sp-PEG-mTNF-alpha-Lys(-).     

Involvement of syntaxin 4 in the transport of membrane-type 1 matrix metalloproteinase to the plasma membrane in human gastric epithelial cells

Membrane-type 1 matrix metalloproteinase (MT1-MMP) localized on the plasma membrane plays a central role in various normal biological responses including tissue remodeling, wound heeling, and angiogenesis and in cancer cell invasion and metastasis, by functioning as a collagenase and activating other matrix metalloproteinases. In order to elucidate the molecular mechanism of the MT1-MMP targeted localization on the plasma membrane, we examined the participation of syntaxin proteins in MT1-MMP intracellular transport to the plasma membrane in human gastric epithelial AGS cells. Western blotting showed that syntaxin 3 and 4 proteins, which are known to function in intracellular transport towards the plasma membrane, were expressed in AGS cells. Immunocytochemistry revealed that transient transfection of AGS cells with dominant-negative mutant syntaxin 4 decreased plasma membrane MT1-MMP expression. In contrast, transient transfection with either dominant-negative mutant syntaxin 3 or 7 did not affect MT1-MMP localization on the plasma membrane. Cell surface biotinylation assay and Matrigel chamber assay demonstrated that stable transfection with dominant-negative mutant syntaxin 4 decreased the amount of MT1-MMP on the plasma membranes and inhibited the cell invasiveness. We suggest that syntaxin 4 is involved in the intracellular transport of MT1-MMP toward the plasma membrane.     

A subpopulation of bone marrow cells depleted by a novel antibody, anti-Liv8, is useful for cell therapy to repair damaged liver

We previously reported a new in vivo model named as "GFP/CCl(4) model" for monitoring the transdifferentiation of green fluorescent protein (GFP) positive bone marrow cell (BMC) into albumin-positive hepatocyte under the specific "niche" made by CCl(4) induced persistent liver damage, but the subpopulation which BMCs transdifferentiate into hepatocytes remains unknown. Here we developed a new monoclonal antibody, anti-Liv8, using mouse E 11.5 fetal liver as an antigen. Anti-Liv8 recognized both hematopoietic progenitor cells in fetal liver at E 11.5 and CD45-positive hematopoietic cells in adult bone marrow. We separated Liv8-positive and Liv8-negative cells and then transplanted these cells into a continuous liver damaged model. At 4 weeks after BMC transplantation, more efficient repopulation and transdifferentiation of BMC into hepatocytes were seen with Liv8-negative cells. These findings suggest that the subpopulation of Liv8-negative cells includes useful cells to perform cell therapy on repair damaged liver.     

Polyphenol (-)-epigallocatechin gallate inhibits apoptosis induced by irradiation in human HaCaT keratinocytes

Green tea is a rich source of polyphenols, and (-)-epigallocatechin-3-gallate (EGCG) is a major constituent of green tea polyphenols. In the present study, we investigated the effect of EGCG on apoptosis induced by irradiation in the human keratinocytic cell line HaCaT. Irradiation by gamma-ray induced apoptosis with concomitant cleavage of caspase-3 and its in vivo substrate poly(ADP-ribose) polymerase. Treatment of cells with EGCG inhibited irradiation-induced apoptosis as detected by Hoechst staining and internucleosomal cleavage of DNA, and prevented the cleavage of these proteins by irradiation. We also found that the treatment of cells with EGCG alone suppressed cell growth and induced apoptosis in these cells. Our results suggest that EGCG inhibits irradiation-induced apoptosis by inactivating the caspase pathway in HaCaT cells. Our study also indicates that EGCG has a dual effect on the survival of these keratinocytes.     

Transendothelial migration of human basophils

During allergic reactions, basophils migrate from the blood compartment to inflammatory sites, where they act as effector cells in concert with eosinophils. Because transendothelial migration (TEM) represents an essential step for extravasation of cells, for the first time we have studied basophil TEM using HUVEC. Treatment of HUVEC with IL-1beta significantly enhanced basophil TEM, which was further potentiated by the presence of a CCR3-specific ligand, eotaxin/CCL11. In addition to CCR3 ligands, MCP-1/CCL2 was also active on basophil TEM. Although stromal cell-derived factor-1/CXCL12, a CXCR4 ligand, failed to induce TEM in freshly isolated basophils, it caused strong TEM in 24-h cultured cells. IL-3 enhanced basophil TEM by increasing the chemokinetic response. Spontaneous TEM across activated HUVEC was inhibited by treatment of cells with anti-CD18 mAb, but not with anti-CD29 mAb, and also by treatment of HUVEC with anti-ICAM-1 mAb. Anti-VCAM-1 mAb alone failed to inhibit TEM, but showed an additive inhibitory effect in combination with anti-ICAM-1 mAb. In contrast, eotaxin- and IL-3-mediated TEM was significantly inhibited by anti-CD29 mAb as well as anti-CD18 mAb. These results indicate that beta2 integrins play the primary role in basophil TEM, but beta1 integrins are also involved, especially in TEM of cytokine/chemokine-stimulated basophils. In conclusion, the regulatory profile of basophil TEM is very similar to that reported for eosinophils. Our results thus support the previous argument for a close relationship between basophils and eosinophils and suggest that the in vivo kinetics of these two cell types are similar.     

Role of gut-associated lymphoreticular tissues in antigen-specific intestinal IgA immunity

This study assessed the roles of the postnatal lymphotoxin-beta receptor (LTbetaR)-mediated signals in the gut-associated lymphoreticular tissues of mice for subsequent regulation of Ag-specific intestinal IgA responses. Blockade of LTbetaR-dependent events by postnatal administration of the fusion protein of LTbetaR and IgG Fc (LTbetaR-Ig) reduced both the size and numbers of Peyer's patches (PP) without influencing the PP microarchitecture. Interestingly, inhibition of LTbetaR-dependent signaling revealed significant reductions in the formation of follicular dendritic cell clusters in mesenteric lymph nodes (MLN). Furthermore, these postnatal signaling events controlled the development of isolated lymphoid follicles (ILF) because treatment with LTbetaR-Ig eliminated the formation of ILF. LTbetaR-Ig-treated mice with altered microarchitecture of MLN and lacking ILF were still able to produce significant Ag-specific mucosal IgA responses after oral immunization; however, the levels were significantly lower than those seen in control mice. These results imply the importance of ILF for Ag-specific intestinal immunity. However, mice treated with both TNFR55-Ig and LTbetaR-Ig in utero, which lack PP and MLN, but retain intact ILF, failed to induce Ag-specific IgA responses after oral immunization. These findings demonstrate that ILF are not essential for induction of intestinal IgA Ab responses to orally administered Ag. Furthermore, the induction of intestinal IgA Ab responses requires the proper maintenance of the MLN microarchitecture, including a follicular dendritic cell network.     

Plasma interleukin (IL)-18 (interferon-gamma-inducing factor) and other inflammatory cytokines in patients with gouty arthritis and monosodium urate monohydrate crystal-induced secretion of IL-18

To determine whether levels of interleukin (IL)-18, together with those of IL-1beta, tumor necrosis factor-alpha, IL-6, and IL-8, are elevated in the plasma of patients with gouty arthritis, the plasma concentrations of those cytokines were measured in 31 males with gouty arthritis. Further, CD14+ cells were obtained from human blood and thioglycolate medium-induced peritoneal cells obtained from caspase 1-deficient mice, and then separately cultured in the presence of monosodium urate monohydrate (MSU) crystals. In addition, in an animal in vivo experiment, MSU crystals were injected into subcutaneous air pouches of IL-18-deficient mice. The plasma concentrations of IL-18, IL-6, and IL-8 were elevated in the presence of gouty arthritis in the gout patients. In the in vitro study, the presence of MSU crystals stimulated CD14+ cells (monocytes) to secrete IL-18 and increased the activity of caspase 1 in CD14+ cells, whereas there was no significant effect on IL-18 messenger RNA in CD14+ cells and only a slight induction of IL-18 secretion from thioglycolate medium-induced caspase 1-deficient peritoneal cells. In the in vivo experiment, MSU crystals injected into the air pouch promoted neutrophil accumulation along with an increase in concentrations of keratinocyte-derived chemokine (KC) and macrophage inflammatory protein (MIP)-1alpha in air-pouch fluids in both IL-18-deficient and wild-type mice. However, there was no increase in the concentration of IL-18 in air-pouch fluids in either mouse strain. Our results suggest that plasma IL-18, IL-6, IL-8, and C-reactive protein (CRP) levels reflect local inflammation associated with gouty arthritis, though IL-18 does not play an important role in neutrophil accumulation. Further, they suggest that MSU crystals accelerate the processing of IL-18 from an inactive to active form via the activation of caspase 1.