Fuminobu Kuroda

Inhibitory Effects of Polyphenols on Gastric Injury by Helicobacter pylori VacA Toxin

Background.  Helicobacter pylori induces gastric damage and may be involved in the pathogenesis of gastric cancer. H. pylori‐vacuolating cytotoxin, VacA, is one of the important virulence factors, and is responsible for H. pylori‐induced gastritis and ulceration. The aim of this study is to assess whether several naturally occurring polyphenols inhibit VacA activities in vitro and in vivo.

Regulation of brefeldin A-inhibited guanine nucleotide-exchange protein 1 (BIG1) and BIG2 activity via PKA and protein phosphatase 1γ

Brefeldin A-inhibited guanine nucleotide-exchange proteins (GEPs) BIG1 and BIG2 activate ADP-ribosylation factor (ARF) GTPases, which are required for vesicular trafficking. Both molecules contain one or more sites for binding protein kinase A, i.e., A kinase-anchoring protein (AKAP) sequences. Elevation of cell cAMP caused PKA-catalyzed phosphorylation and nuclear accumulation of BIG1 but not BIG2. We then asked whether BIG1 phosphorylation altered its GEP activity. Incubation of BIG1 or BIG2 with PKA catalytic subunits and ATP resulted in retardation of their electrophoretic migration, consistent with PKA phosphorylation. Okadaic acid inhibits many protein phosphatases, including protein phosphatase 1 (PP1) and PP2A, that can reverse PKA-catalyzed phosphorylation. Incubation of HepG2 cells with okadaic acid caused concentration-dependent accumulation of presumably phosphorylated BIG1 and BIG2 with decreased mobility, which was increased by subsequent incubation in vitro with specific recombinant phosphatases, PP1γ > PP2A ≫ PP1α. For assays of GEP activity, BIG1 and BIG2 were immunoprecipitated from cells that had been depleted, respectively, of BIG2 and BIG1 by using specific siRNA. GEP activity of each was significantly decreased after incubation with recombinant PKA plus ATP and restored by incubation with PP1γ. In agreement with a role for PP1γ in regulation of BIG, endogenous PP1γ, but not PP1α or β, was immunoprecipitated with BIG1 or BIG2 from microsomal fractions. All observations are consistent with the effects of BIG1 and BIG2 phosphorylation on vesicular trafficking, via alterations in ARF activation and regulatory roles for cAMP, PKA, and PP1γ in ARF activation by BIG1 and BIG2.

Attenuation of lung inflammation and fibrosis in CD69-deficient mice after intratracheal bleomycin

BackgroundCluster of differentiation 69 (CD69), an early activation marker antigen on T and B cells, is also expressed on activated macrophages and neutrophils, suggesting that CD69 may play a role in inflammatory diseases. To determine the effect of CD69 deficiency on bleomycin(BLM)-induced lung injury, we evaluated the inflammatory response following intratracheal BLM administration and the subsequent fibrotic changes in wild type (WT) and CD69-deficient (CD69-/-) mice.MethodsThe mice received a single dose of 3 mg/kg body weight of BLM and were sacrificed at 7 or 14 days post-instillation (dpi). Lung inflammation in the acute phase (7 dpi) was investigated by differential cell counts and cytokine array analyses of bronchoalveolar lavage fluid. In addition, lung fibrotic changes were evaluated at 14 dpi by histopathology and collagen assays. We also used reverse transcription polymerase chain reaction to measure the mRNA expression level of transforming growth factor β1 (TGF-β1) in the lungs of BLM-treated mice.ResultsCD69-/- mice exhibited less lung damage than WT mice, as shown by reductions in the following indices: (1) loss of body weight, (2) wet/dry ratio of lung, (3) cytokine levels in BALF, (4) histological evidence of lung injury, (5) lung collagen deposition, and (6) TGF-β1 mRNA expression in the lung.ConclusionThe present study clearly demonstrates that CD69 plays an important role in the progression of lung injury induced by BLM.

Immunogenicity of a monovalent pandemic influenza A H1N1 vaccine in health‐care workers of a university hospital in Japan

A phase III observational study evaluating a single‐dose of an inactivated, split‐virus, unadjuvanted AH1pdm vaccine in HCW was conducted. A safe and effective vaccine was needed after the emergence of AH1pdm in April 2009. We analyzed the immunogenicity and safety of the vaccine. A total of 409 subjects were enrolled and given 15 μg hemagglutinin antigen by s.c. injection. Antibody titers were measured using hemagglutination‐inhibition antibody assays before vaccination and 28 days after. The co‐primary immunogenicity end‐points were the proportion of subjects with antibody titers of 1:40 or more, the proportion of subjects with either seroconversion or a significant increase in antibody titer, and the factor increase in geometric mean titer. We collected 389 pair samples. Antibody titers of 1:40 or more were observed in 148 of 389 subjects (38.0%, 95% CI: 33.2–42.9). The immunogenicity was also confirmed in other end‐points, but was not sufficient and was lower than in previous reports. A total of 96 of adverse events was reported: 51 local events and 57 systemic events. There were 12 subjects with both local and systemic events. Nearly all events were mild to moderate except in four subjects. A single 15‐μg dose of AH1pdm vaccine did not induce sufficient immunogenicity in HCW, with mild‐to‐moderate vaccine‐associated adverse events. We need to consider further improvement of the AH1pdm vaccine program in HCW for the prevention of nosocomial infection, as well as for the benefit of HCW.

Role of CD69 in acute lung injury

AIMS CD69 is an early activation marker in lymphocytes and an important signal transmitter in inflammatory processes. However, its role in acute lung injury (ALI) is still unknown. We used a lipopolysaccharide (LPS)-induced mouse model of ALI to study the role of macrophage-surface CD69 in this condition. MAIN METHODS We investigated bronchoalveolar lavage fluid (BALF) cell subpopulations, myeloperoxidase levels in lung homogenates, lung pathology, and lung oedema in CD69-deficient (CD69(-/-)) mice 24h after LPS instillation. We also determined cytokine/chemokine expression levels in BALF and macrophage culture supernatant from CD69(-/-) and wild type (WT) mice. Also, we investigated CD69, keratinocyte-derived chemokine (KC) and macrophage inflammatory protein (MIP)-2 localization in the lungs after LPS administration. Furthermore, we examined the effect of anti-CD69 antibody on LPS-induced cytokine/chemokine release from cultured macrophages. KEY FINDINGS Our study shows that intratracheal instillation of LPS-induced neutrophilic infiltration, histopathological changes, myeloperoxidase positivity, and oedema in the lung to a lower degree in CD69(-/-) mice than in WT mice. The immunoreactivities for CD69, KC and MIP2 were induced in the lung of WT mice instilled with LPS and were predominantly localized to the macrophages. Moreover, the cytokine/chemokine expression profile between the two genotypes of cultured macrophages in response to LPS was similar to that observed in the BALF. In addition, anti-CD69 antibody inhibited the LPS-induced cytokine/chemokine expression. SIGNIFICANCE These results suggest that CD69 on macrophages plays a crucial role in the progression of LPS-induced ALI and may be a potentially useful target in the therapy for ALI.

Cigarette smoke-induced pulmonary inflammation is attenuated in CD69-deficient mice

Cluster of differentiation 69 (CD69) has been identified as a lymphocyte early activation marker, and recent studies have indicated that CD69 mediates intracellular signals and plays an important role in various inflammatory diseases. Cigarette smoke (CS) is a strong proinflammatory stimulus that induces the release of proinflammatory mediators by recruiting macrophages and neutrophils into the lung tissue, and is one of the main risk factors for a number of chronic diseases. However, the potential role of CD69 in CS-induced pulmonary inflammation has not been determined. To address to this question, CD69-deficient (KO) and wild-type (WT) mice were subjected to CS-induced acute pulmonary inflammation. After the exposure with CS, the expression of CD69 in the lung of WT mice was significantly induced, it was predominantly observed in macrophages. In conjunction with this phenomenon, neutrophil and macrophage cell counts, and expression of several cytokines were significantly higher in the bronchoalveolar lavage fluid (BALF) of CS-exposed WT mice compared with air-exposed WT mice. Likewise, the CS-induced accumulation of inflammatory cells and cytokines expression were significantly lower in CD69-KO mice than in WT mice. These results suggest that CD69 on macrophages is involved in CS-induced acute pulmonary inflammation.