Cytokine mRNA expression in unilateral ischemic-reperfused rat lung with salt solution supplemented with low-endotoxin or standard bovine serum albumin

Our aim was to determine whether cytokine mRNA expression is induced by experimental manipulation including artificial perfusate or ischemia-reperfusion (I/R) in an isolated, perfused rat lung model. Constant pulmonary flow [Krebs-Henseleit solution supplemented with low-endotoxin (LE) or standard (ST) bovine serum albumin 4%, 0.04 ml/g body wt] and ventilation were maintained throughout. Right and left pulmonary arteries were isolated, and the left pulmonary artery was occluded for 60 min and then reperfused for 30 min. Analysis of tumor necrosis factor-alpha, IL-1 beta, IL-6, IL-10, and IFN-gamma mRNA expression by RT-PCR and evaluation of vascular permeability by bronchoalveolar lavage (BAL) fluid albumin content were conducted separately in right and left lung. Both LE and ST groups (each 12 rats) showed increases in vascular permeability by I/R (BAL fluid albumin content: 5.53 +/- 1.55 vs. 15.63 +/- 8.87 and 4.76 +/- 2.71 vs. 16.72 +/- 4.85 mg.ml BAL fluid-1.g lung dry wt-1, mean +/- SD; right vs. left lung in LE and ST groups, P < 0.05 between right and left). Cytokine mRNA expression was significantly higher in the I/R lung than in the control lung in the LE group, whereas it was higher in the control lung in the ST group (P < 0.05). mRNAs of not only proinflammatory but also anti-inflammatory cytokines were expressed in I/R lung, which are expected to aggravate I/R injury. The reversed pattern of cytokine mRNA expression in the ST group was possibly due to the longer perfusion of control lung with perfusate containing endotoxin, which caused no lung damage without I/R.     

Intratracheal gene transfer of decorin reduces subpleural fibroproliferation induced by bleomycin

Decorin, a small leucin-rich proteoglycan, is a negative regulator of transforming growth factor-beta, but the antifibrotic effect of decorin gene transfer has not been examined in a mouse model of usual interstitial pneumonia (UIP). We constructed a replication-defective recombinant adenovirus harboring human decorin gene (AdCMV.DC) and administered 1 x l0(9) plaque-forming units of AdCMV.DC intratracheally or intravenously to C57BL/6 mice with intraperitoneal injection of bleomycin, which induces a subpleural fibroproliferation, mimicking UIP, by day 28. Only intratracheal administration of AdCMV.DC increased decorin mRNA expression in the lung and decreased the hydroxyproline content augmented in bleomycin-induced pulmonary fibrosis (1.13 +/- 0.02 to 0.96 +/- 0.02, P = 0.006). In contrast, intravenous administration of AdCMV.DC increased the decorin expression only in the liver, but not in the lung, and without reducing lung fibrosis. These results indicate that adenoviral decorin gene transfer is effective only by direct administration to fibrosing lungs.     

Targeting apoptotic tumor cells to Fc gamma R provides efficient and versatile vaccination against tumors by dendritic cells

Dendritic cells (DCs) loaded with tumor-associated Ags (TAAs) act as potent adjuvant that initiates antitumor immune responses in vivo. However, TAA-based DC vaccination requires prior identification of TAAs. Apoptotic tumor cells (ATCs) can be an excellent source for DC loading because their potential uncharacterized Ags would be efficiently presented to T cells without any prior characterization and isolation of these Ags. However, ATCs alone are considered to be inefficient for activating antitumor immunity, possibly because of their inability to induce DC maturation. In this study, the aim was to enhance antitumor immune response by taking advantage of ATCs that have been opsonized with IgG (ATC-immune complexes, ATC-ICs) so as to target them to FcR for IgG (FcgammaRs) on DCs. It was found that when compared with ATCs, ATC-ICs were efficiently internalized by DCs via FcgammaRs, and this process induced maturation of DCs, which was more efficient than that of ATCs. Importantly, ATC-IC loading was shown to be more efficient than ATCs alone in its capacity for inducing antitumor immunity in vivo, in terms of cytotoxic T cell induction and tumor rejection. These results show that using ATC-ICs may overcome the limitations and may enhance the immune response of current ATC-based DC vaccination therapy.     

Human leukocyte-derived arginine aminopeptidase. The third member of the oxytocinase subfamily of aminopeptidases

In this study we report the cloning and characterization of a novel human aminopeptidase, which we designate leukocyte-derived arginine aminopeptidase (L-RAP). The sequence encodes a 960-amino acid protein with significant homology to placental leucine aminopeptidase and adipocyte-derived leucine aminopeptidase. The predicted L-RAP contains the HEXXH(X)18E zinc-binding motif, which is characteristic of the M1 family of zinc metallopeptidases. Phylogenetic analysis indicates that L-RAP forms a distinct subfamily with placental leucine aminopeptidase and adipocyte-derived leucine aminopeptidase in the M1 family. Immunocytochemical analysis indicates that L-RAP is located in the lumenal side of the endoplasmic reticulum. Among various synthetic substrates tested, L-RAP revealed a preference for arginine, establishing that the enzyme is a novel arginine aminopeptidase with restricted substrate specificity. In addition to natural hormones such as angiotensin III and kallidin, L-RAP cleaved various N-terminal extended precursors to major histocompatibility complex class I-presented antigenic peptides. Like other proteins involved in antigen presentation, L-RAP is induced by interferon-gamma. These results indicate that L-RAP is a novel aminopeptidase that can trim the N-terminal extended precursors to antigenic peptides in the endoplasmic reticulum.     

Coupling between cyclooxygenases and prostaglandin F(2alpha) synthase. Detection of an inducible,glutathione-activated,membrane-bound prostaglandin F(2alpha)-synthetic activity

Distinct functional coupling between cyclooxygenases (COXs) and specific terminal prostanoid synthases leads to phase-specific production of particular prostaglandins (PGs). In this study, we examined the coupling between COX isozymes and PGF synthase (PGFS). Co-transfection of COXs with PGFS-I belonging to the aldo-keto reductase family into HEK293 cells resulted in increased production of PGF(2alpha) only when a high concentration of exogenous arachidonic acid (AA) was supplied. However, this enzyme failed to produce PGF(2alpha) from endogenous AA, even though significant increase in PGF(2alpha) production occurred in cells transfected with COX-2 alone. This poor COX/PGFS-I coupling was likely to arise from their distinct subcellular localization. Measurement of PGF(2alpha)-synthetic enzyme activity in homogenates of several cells revealed another type of PGFS activity that was membrane-bound, glutathione (GSH)-activated, and stimulus-inducible. In vivo, membrane-bound PGFS activity was elevated in the lung of lipopolysaccharide-treated mice. Taken together, our results suggest the presence of a novel, membrane-associated form of PGFS that is stimulus-inducible and is likely to be preferentially coupled with COX-2.     

Analysis of the cellular heterogeneity in the basal layer of mouse ear epidermis: an approach from partial decomposition in vitro and retroviral cell marking in vivo

In the thin epidermis, the existence of epidermal proliferation units was hypothesized. Each unit is supposed to be partitioned into each column of polygonal-shaped cornified plates, estimated to contain a central stem cell in its basal layer. We attempted to verify this hypothesis in vitro by analyzing the partially decomposed fragment of mouse ear epidermis and in vivo using retroviral cell marking. Partially decomposed fragments of the mouse ear epidermis, mostly composed of cytokeratin 14-expressing basal keratinocytes, formed multicellular colonies in vitro. They were composed of heterogeneously shaped cells, morphologically resembling the cells in each single cell-derived colony, including potential stem cells with great proliferative potency in vitro. The estimated frequency of the candidates of stem cells in the fragments was much lower than the prediction from the representative hypothesis. Retroviral cell marking with nuclear localizing LacZ protein in vivo suggested the existence of a large clonal cellular unit for epidermal renewal. From these in vitro and in vivo observations, we propose a new model for the epidermal proliferation unit.     

Human Fc epsilon RIalpha-specific human single-chain Fv (scFv) antibody with antagonistic activity toward IgE/Fc epsilon RIalpha-binding

The alpha-chain of Fc epsilon RI (Fc epsilon RIalpha) plays a critical role in the binding of IgE to Fc epsilon RI. A fully human antibody interfering with this interaction may be useful for the prevention of IgE-mediated allergic diseases. Here, we describe the successful isolation of a human single-chain Fv antibody specific to human Fc epsilon RIalpha using human antibody phage display libraries. Using the non-immune phage antibody libraries constructed from peripheral blood lymphocyte cDNA from 20 healthy subjects, we isolated three phage clones (designated as FcR epsilon 27, FcR epsilon 51, and FcR epsilon 70) through two rounds of biopanning selection. The purified soluble scFv, FcR epsilon 51, inhibited the binding of IgE to recombinant Fc epsilon RIalpha, although both FcR epsilon 27 and FcR epsilon 70 showed fine binding specificity to Fc epsilon RIalpha. Since FcR epsilon 51 was determined to be a monomer by HPLC, BIAcore analysis was performed. The dissociation constant of FcR epsilon 51 to Fc epsilon RIalpha was estimated to be 20 nM, i.e., fortyfold lower than that of IgE binding to Fc epsilon RIalpha (K(d) = 0.5 nM). With these characteristics, FcR epsilon 51 exhibited inhibitory activity on the release of histamine from passively sensitized human peripheral blood mononuclear cells.     

Molecular, biochemical and immunological analyses of canine pancreatic DNase I

The DNase I from canine pancreas was purified 260-fold to electrophoretic homogeneity with a 35% yield using three-step column chromatography. The activity of the purified enzyme was completely inhibited by 20 mM EDTA, an antibody specific to the purified enzyme and G-actin. A 1,373-bp cDNA encoding canine DNase I was constructed from the total canine pancreatic RNA using a rapid amplification of cDNA ends method, followed by sequencing. The mature canine DNase I protein was found to consist of 262 amino acids. A survey of DNase I in 13 different canine tissues revealed the highest levels of both DNase I enzyme activity and gene expression in the pancreas; therefore, the canine DNase I is of the pancreatic type. Phylogenetic and sequence identity analyses, studies of immunological properties and the tissue-distribution patterns of DNase I indicated that the canine enzyme is more closely related to the human DNase I than to other mammalian DNases I. Therefore, canine DNase I is found to be one of the best substitutes in studies of human DNase I.