Hirotake Sakuraba

Vaccination with Nontoxic Mutant Toxic Shock Syndrome Toxin 1 Protects against Staphylococcus aureus Infection

To investigate whether vaccination with nontoxic mutant toxic shock syndrome toxin 1 (mTSST-1) can protect against Staphylococcus aureus infection, mice were vaccinated with mTSST-1 and challenged with viable S. aureus. Survival in the mTSST-1-vaccinated group was higher, and bacterial counts in organs were significantly lower than those of control mice. Passive transfer of mTSST-1-specific antibodies also provided protection against S. aureus-induced septic death. Interferon (IFN)-gamma production in the serum samples and spleens from vaccinated mice was significantly decreased compared with that in controls, whereas interleukin-10 titers were significantly higher in vaccinated mice. IFN-gamma and tumor necrosis factor-alpha production in vitro were significantly inhibited by serum samples from mTSST-1-immunized mice but not from control mice. These results suggest that vaccination with mTSST-1 devoid of superantigenic properties provides protection against S. aureus infection and that the protection might be mediated by TSST-1-neutralizing antibodies as well as by the down-regulation of IFN-gamma production.

Cyclosporine regulates intestinal epithelial apoptosis via TGF-β-related signaling

Cyclosporine is a potent immunomodulator and has a beneficial effect in the treatment of ulcerative colitis (UC). We analyzed the mechanism of the effects of cyclosporine on the regulation of epithelial apoptosis via TGF-beta-related signaling, because the balance between the apoptosis and regeneration of epithelial cells seems to be a key factor to maintain the intestinal homeostasis. For this purpose, colitis was induced by treatment of 4% dextran sulfate sodium (DSS), and the effect of treatment with cyclosporine and anti-TGF-beta antibody was assessed. Treatment with cyclosporine ameliorated body weight loss, mucosal destruction, and epithelial apoptosis in DSS-induced colitis. Cyclosporine was shown to upregulate the expression of TGF-beta in the colonic tissue, enhance the expression of p-Smad2 and cFLIP in epithelial cells, and inhibit caspase-8 activity but not caspase-1 or -9. Upregulation of cFLIP in the colonic epithelial cells, amelioration of body weight loss, and mucosal destruction by cyclosporine were attenuated by anti-TGF-beta antibody treatment. These results indicated that cyclosporine could have a protective role against epithelial apoptosis associated with upregulation of TGF-beta-related signaling.

Retinoic acid-inducible gene-I is constitutively expressed and involved in IFN-γ-stimulated CXCL9-11 production in intestinal epithelial cells

Retinoic acid-inducible gene-I (RIG-I) is a member of the DExH/D family proteins, and plays an important role in antiviral response via interferon-stimulated genes (ISGs) and type 1 IFN. In this study, the roles of RIG-I in the epithelial cells in the cross-talk between type 2 IFN and inducible chemokines production are high-lighted. The results showed that RIG-I was constitutively expressed in normal surface epithelia lining the colonic mucosa. RIG-I was constitutively expressed in the epithelial cell lines HT-29, and IFN-gamma and TNF-alpha enhanced the RIG-I expression in a dose-dependent manner. IFN-gamma was shown to stimulate CXCL9-11 production, and RNA interference against RIG-I resulted in significant decrease of IFN-gamma-induced CXCL9-11 productions. These results suggest that RIG-I play an important role in the cross-talk between inflammatory cytokines and immune cell trafficking. In conclusion, RIG-I might regulate the gut barrier function in homeostatic and inflammatory conditions.

Interferon-alpha2b induces p21cip1/waf1 degradation and cell proliferation in HeLa cells.

Type I interferons (IFNs) are a family of cytokines that exhibit various biological activities. Besides their roles in immune response, IFNs have been known to modulate cell proliferation and to induce apoptosis. Thus, IFNs are used as an anti-tumor agent against certain types of cancer, but it is unclear why many other cancers are not influenced by IFNs. Here, we found that IFN-a2b, a subfamily of IFN-a, enhanced proliferation of HeLa cells, a cell line derived from human cervical cancer. IFN-a2b was rather inhibitory on the growth of other types of cervical cancer cells including those positive for HPV. Among the proliferation- and the apoptosis-related genes, p21cip1/waf1 (p21) was upregulated by IFN-a2b, whereas p53, p27 or BCL-2 associated X protein (BAX) was not affected. IFN-a2b did not alter promoter activities of p21 but did prolong the decay of p21 mRNA. In contrast, the level of p21 protein was lowered by IFN-a2b, and half-life analysis of p21 protein revealed that IFN-a2b enhances p21 protein instability in HeLa cells. Pretreatment of the cells with MG132, a proteasome inhibitor, abolished the IFN-a2b-mediated p21 degradation, suggesting that IFN-a2b accelerated the ubiquitin-proteasome dependent degradation of p21. Consistent with these results, IFN-a2b increased S-phase cell cycle distribution in HeLa cells. In addition, IFN-a2b liberated the cells from G1-phase arrest by 5-fluorouracil (5-FU) and from G2-phase arrest by paclitaxel. These results provide a novel role of Type I IFNs in cell cycle regulation and may define an importance of individualized IFN-based therapy against specific types of cancer.

The Presentation of Haptenated Proteins and Activation of T Cells in the Mesenteric Lymph Nodes by Dendritic Cells in the TNBS Colitis Rat

Abstract: We described the role of dendritic cells (DCs) in aspects of T cell activation at the mesenteric lymph nodes (MLN) in trinitrobenzene sulfonic acid (TNBS)‐induced colitis. An antigenic‐immune response is induced at the MLN, and dendritic cells are the affected cell type. Cross‐linking and GRO/CINC‐1 have synergistic effects for DC maturation.

Transforming growth factor-β regulates susceptibility of epithelial apoptosis in murine model of colitis

Abstract: Transforming growth factor (TGF)‐beta has a key role in intestinal homeostasis. Our present data suggest that TGF‐beta, which was constitutively expressed by lamina propria mononuclear cells and epithelium, affected epithelial cells. Abnormal suppression of TGF‐beta could enhance the sensitivity of epithelial cells to apoptosis associated with interferon‐gamma in DSS‐induced colitis.

Macrophage Migration Inhibitory Factor and Activator Protein-1 in Ulcerative Colitis

Abstract: Macrophage migration inhibitory factor (MIF) is a cytokine that has potent antisteroid effects. We determined that MIF is involved in the glucocorticoid‐resistant inflammatory process of ulcerative colitis (UC), and the altered AP‐1 signal is a potent therapeutic target for refractory UC.

Clinical and structural remission rates increased annually and radiographic progression was continuously inhibited during a 3-year administration of tocilizumab in patients with rheumatoid arthritis: A multi-center, prospective cohort study by the Michinoku Tocilizumab Study Group

Abstract Objective: To evaluate the clinical and structural efficacy of tocilizumab (TCZ) during its long-term administration in patients with rheumatoid arthritis (RA). Methods: In total, 693 patients with RA who started TCZ therapy were followed for 3 years. Clinical efficacy was evaluated by DAS28-ESR and Boolean remission rates in 544 patients. Joint damage was assessed by calculating the modified total Sharp score (mTSS) in 50 patients. Results: When the reason for discontinuation was limited to inadequate response or adverse events, the 1-, 2-, and 3-year continuation rates were 84.0%, 76.8%, and 72.2%, respectively. The mean DAS28-ESR was initially 5.1 and decreased to 2.5 at 6 months and to 2.2 at 36 months. The Boolean remission rate was initially 0.9% and increased to 21.7% at 6 months and to 32.2% at 36 months. The structural remission rates (ΔmTSS/year ≤ 0.5) were 68.8%, 78.6%, and 88.9% within the first, second, and third years, respectively. The structural remission rate at 3 years (ΔmTSS ≤ 1.5) was 66.0%, and earlier achievement of swollen joint count (SJC) of 1 or less resulted in better outcomes. Conclusions: TCZ was highly efficacious, and bone destruction was strongly prevented. SJC was an easy-to-use indicator of joint destruction.

Existence of Variant γδT Cells in Crohn’s Disease

Background and Objectives: γδT cell populations are well-known for their unique distribution (e.g. intra-epithelial lymphocytes). Though their ligands play a major role in the immune response they have remained largely obscure. To shed light on this issue, we have analyzed in this study the complementarity determining region (CDR) 3 of the T cell receptor (TCR) Vδ2 chains. This provides an insight into the antigenic immune response in the intestinal mucosa in sickness and health. Methodology: Total RNA was extracted from surgically resected intestinal mucosa of patients with Crohn’s disease (CD) and controls. TCR Vδ2 cDNA was PCR-amplified using Vδ2 sense primer and Cδ antisense primer. The PCR products were then subcloned in pUC18 plasmid. From each sample, 20 subclones were randomly selected and subjected to nucleotide sequence analysis. Results: Sequence analysis revealed that the CDR3 sequences of the TCR Vδ2 chains were unique to each individual. The evidence also showed a significant restriction of the junctional diversity of the TCR Vδ2 chains while no such restriction was found for CD. Conclusions: The marked complexity of the TCR Vδ2 junctional sequences and the oligoclonality of the TCR Vδ2 genes in the control subjects are indicative of a positive selection and expansion of specific T cells in the normal, healthy condition. In CD patients, however, the expression of distinct, non-overlapping TCR Vδ2 clonotypes can be found, suggesting polyclonal activation of γδT cells in the diseased colon of CD patients. These findings have led us to conclude that accumulation of multiple γδT cell clonotypes may be involved in the pathogenesis of CD.

NUDT15 codon 139 is the best pharmacogenetic marker for predicting thiopurine-induced severe adverse events in Japanese patients with inflammatory bowel disease: a multicenter study

BackgroundDespite NUDT15 variants showing significant association with thiopurine-induced adverse events (AEs) in Asians, it remains unclear which variants of NUDT15 or whether additional genetic variants should be tested to predict AEs. To clarify the best pharmacogenetic test to be used clinically, we performed association studies of NUDT15 variants and haplotypes with AEs, genome-wide association study (GWAS) to discover additional variants, and ROC analysis to select the model to predict severe AEs.MethodsOverall, 2630 patients with inflammatory bowel disease (IBD) were enrolled and genotyped for NUDT15 codon 139; 1291 patients were treated with thiopurines. diplotypes were analyzed in 970 patients, and GWASs of AEs were performed with 1221 patients using population-optimized genotyping array and imputation.ResultsWe confirmed the association of NUDT15 p.Arg139Cys with leukopenia and alopecia (p = 2.20E−63, 1.32E−69, OR = 6.59, 12.1, respectively), and found a novel association with digestive symptoms (p = 6.39E−04, OR = 1.89). Time to leukopenia was significantly shorter, and when leukopenia was diagnosed, thiopurine doses were significantly lower in Arg/Cys and Cys/Cys than in Arg/Arg. In GWASs, no additional variants were found to be associated with thiopurine-induced AEs. Despite strong correlation of leukopenia frequency with estimated enzyme activities based on the diplotypes (r2 = 0.926, p = 0.0087), there were no significant differences in the AUCs of diplotypes from those of codon 139 to predict severe AEs (AUC = 0.916, 0.921, for acute severe leukopenia, AUC = 0.990, 0.991, for severe alopecia, respectively).ConclusionsGenotyping of NUDT15 codon 139 was sufficient to predict acute severe leukopenia and alopecia in Japanese patients with IBD.