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The development of chronic graft-versus-host disease (GVHD), which is induced by the transfer of DBA/2 spleen cells into (C57BL/6 × DBA/2)F1 (BDF1) mice, is closely related to diminished donor anti-host CTL activity and host B cell hyperactivation. Therefore, an approach which activates donor CD8+ T cells or suppresses donor CD4+ T cell-host B cell interaction may have clinical utility in the treatment of chronic GVHD. We have previously demonstrated that IL-18 induces the development of naive CD8+ T cells into type I effector cells in DBA/2 anti-BDF1 MLC. In this paper we examined the effect of IL-18 administration on the development of chronic GVHD in mice. The treatment was started before or after the onset of clinical evidence of the disease. Regardless of the treatment schedule, IL-18 significantly decreased immunological parameters indicative of chronic GVHD, such as elevated serum IgG antinuclear Abs, IgG1, and IgE levels, and host B cell numbers and their activation. Importantly, IL-18-treated mice did not show the same acute GVHD-like symptoms reported for IL-12 treatment, because there was no weight loss, death, or severe immunodeficiency as indicated by a decrease in IL-2 and IFN-γ production by Con A-stimulated spleen cells. In contrast, IL-18 treatment partially but significantly restored the production of these cytokines. Data further suggested that these IL-18-mediated therapeutic effects may be due to the induction of donor CD8+ CTL, the decrease in donor CD4+ T cell numbers, and a down-regulation of host B cell MHC class II expression. Thus, our results suggest that IL-18 has beneficial effects in the prevention and treatment of chronic GVHD.

The gene (bdb) for protein thiol-disulfide oxidoreductase cloned from Bacillus brevis was found to encode a polypeptide consisting of 117 amino acid residues with a signal peptide of 27 residues. Bdb contains a well-conserved motif, Cys-X-X-Cys, which functions as the active center of disulfide oxidoreductases such as DsbA, protein disulfide isomerase, and thioredoxin. The deduced amino acid sequence showed significant homology with those of several bacterial thioredoxins. The bdb gene complemented the Escherichia coli dsbA mutation, restoring motility by means of flagellar and alkaline phosphatase activity. The Bdb protein overproduced in B. brevis was enzymatically active in both reduction and oxidization of disulfide bonds in vitro. Immunoblotting indicated that Bdb could function at the periphery of the cell.Disulfide bonds play an important role in producing the three-dimensional structures responsible for the specific properties of proteins. Although disulfide bonds in a protein can form spontaneously in vitro, the process is much slower and less effective than that in vivo (20), suggesting the existence of proteins which catalyze native disulfide bond formation in vivo. Disulfide oxidoreductases such as thioredoxin and protein disulfide isomerase (PDI) contain the Cys-X-X-Cys motif as the catalytic active site (16) and facilitate disulfide exchange in vitro (10).The dsbA gene in Escherichia coli has been discovered independently by two groups (3, 12). DsbA is a periplasmic protein with the sequence motif Cys-Pro-His-Cys, which resembles the active site of thioredoxin and PDI. The dsbA mutation causes a defect in the disulfide bond formation of several periplasmic proteins such as alkaline phosphatase, the OmpA protein, ␤-lactamase (3), and the P-ring protein of the flagellar basal body (7). DsbA appears to be required for the formation of disulfide bonds. DsbA homologs are also found in bacteria such as Vibrio cholerae (19) and Haemophilus influenzae (26). They are thought to form a new family of bacterial disulfide oxidoreductase.All bacterial disulfide oxidoreductases have been isolated only from gram-negative bacteria, while no data have been obtained on those of gram-positive bacteria. In gram-positive bacteria, secretory nascent polypeptides are transported to the external medium and must fold into precise shapes to produce functional proteins outside or on the surface of the cell. It is of interest to examine how extracellular proteins acquire disulfide bonds in the protein secretory pathway. We describe here the cloning and characterization of a gene termed bdb (for Bacillus disulfide bond formation), which encodes the protein thioldisulfide oxidoreductase of Bacillus brevis, a gram-positive bacterium used as a host for heterologous protein production (31). MATERIALS AND METHODSBacterial strains, media, plasmids, and transformation. The bacterial strains used were B. brevis HPD31 (24) and E. coli XL1-Blue (Stratagene), JCB502 ( D69 lacZ::Tn10 tetS by fusaric acid) (3), JCB572 (JCB502 dsbA::kan-1) (...

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