Junji Washizu

Expression of Toll-Like Receptor 2 on γδ T Cells Bearing Invariant Vγ6/Vδ1 Induced by Escherichia coli Infection in Mice

We recently reported that the number of γδ T cells was increased after infection with Escherichia coli in C3H/HeN mice. We here showed that an i.p. injection with native lipid A derived from E. coli induced an increase of γδ T cells in the peritoneal cavity of LPS-responsive C3H/HeN mice and, albeit to a lesser degree, also in LPS-hyporesponsive C3H/HeJ mice. The purified γδ T cells from C3H/HeN and C3H/HeJ mice expressed a canonical TCR repertoire encoded by Vγ6-Jγ1/Vδ1-Dδ2-Jδ2 gene segments and proliferated in response to the native lipid A derived from E. coli in a TCR-independent manner. The lipid A-reactive γδ T cells bearing canonical Vγ6/Vδ1 expressed Toll-like receptor (TLR) 2 mRNA, while TLR4 mRNA was undetectable. Treatment with a TLR2 anti-sense oligonucleotide resulted in hyporesponsiveness of the γδ T cells to the native lipid A. TLR2-deficient mice showed an impaired increase of the γδ T cells following injection of native lipid A. These results suggest that TLR2 is involved in the activation of canonical Vγ6/Vδ1 T cells by native E. coli lipid A.

The NF-κB binding site is essential for transcriptional activation of the IL-15 gene

Abstractu2003We cloned the 5’ upstream region of IL-15 genomic DNA and examined promoter activity in macrophages stimulated with lipopolysaccharide(LPS). The 1.2 kilobase (kb) fragment of the 5’ upstream region contained binding elements for LPS-inducible transcription factors such as NFIL-6 or NF-κB. Determined by luciferase assay following transient transfection in the J774A.1 macrophage cell line, the 1.2 kb of the 5’ upstream region exhibited high promoter activity in response to LPS, while promoter activity was significantly reduced by the 5’ deletion of 313 base pairs containing the NF-κB binding motif. Nuclear protein prepared from LPS-stimulated macrophages formed a complex with the NF-κB binding sequence of the IL-15 promoter. Taken together, the binding of nuclear protein to the NF-κB binding site is required for transcriptional activation of the IL-15 gene in LPS-stimulated macrophages.

Effect of flow on the detoxification function of rat hepatocytes in a bioartificial liver reactor.

Ethoxyresorufin-o-deethylation (EROD) can be used as a sensitive measure of hepatic detoxification function. In this study, we employed a fluorescence assay based on EROD to study the effect of varying Peclet number (or flow) on hepatic function in a microchannel flat-plate bioartificial liver (BAL) reactor containing a coculture of hepatocytes and fibroblasts. Static culture and reactor flow experiments established that: 1) a pseudo-steady-state detoxification rate could be attained at each Peclet number, 2) the steady-state detoxification rate increased nonlinearly with Peclet number (ranging from 167 to 2500), 3) the uptake rate of substrate was a linear function of cell surface substrate concentration (<1 μM), and 4) a shear stress of 10 dyne/cm2 did not adversely affect hepatic function for at least 12 h. A convection–diffusion–reaction model supports the conclusion that increased convective mass transfer of substrate to the cell surface is the primary cause of the observed increase in EROD rate with Peclet number. Our results suggest that detoxification rates can be enhanced by an order of magnitude by choosing an appropriate Peclet number. For our bioreactor configuration, this optimum corresponds to a Peclet number range of 1000–2000 at a Damkohler number of 0.55. The usefulness of the mathematical model is discussed in the context of scale-up to a clinical BAL reactor for human application.

Amino acid supplementation improves cell-specific functions of the rat hepatocytes exposed to human plasma.

Maintaining hepatocyte function during plasma exposure is critical for the successful development of hepatocyte-based bioartificial liver assist systems. Past attempts to culture hepatocytes in plasma yielded discouraging results. Using a stable culture model based on sandwiching hepatocytes between two layers of collagen gel, we investigated the effect of hormone and amino acid supplementation during exposure of rat hepatocytes to heparin-treated human plasma for 1 week. Morphology and hepatocyte-specific functions were evaluated for hepatocytes cultured in Dulbeccos Modified Eagle medium (DMEM), nonsupplemented plasma, plasma supplemented with hormones, or with hormones plus amino acids. Amino acids were supplemented at four-fold concentration of Basal Medium Eagle with 4 mM glutamine, whereas hormones included 7.5 microg/mL of hydrocortisone and 50 microU/mL of insulin. Cuboidal structure and bile canaliculi formation were observed throughout the 1-week exposure period for control hepatocytes in DMEM and for hepatocytes cultured in hormone supplemented plasma. Albumin and urea synthesis rates of hepatocytes in hormone plus amino acid supplemented plasma during the last day of plasma exposure were 60.4 +/- 13.7 and 75.6 +/- 6.5 (microg/day per 1 x 10(6) cells, mean +/- SD), respectively, comparable to cultures in standard culture medium. On the other hand, hepatocytes exposed to nonsupplemented plasma suffered significant morphological and functional damage. The results of this study indicate that hormone plus amino acid supplementation help to restore function in hepatocytes exposed to plasma.

Long-Term Maintenance of Cytochrome P450 Activities by Rat Hepatocyte/3T3 Cell Co-cultures in Heparinized Human Plasma

Little information on the effect of plasma on hepatocyte cytochrome P450 (CYP) activities is currently available. We characterized the effect of plasma on CYPs of hepatocyte-mesenchymal cell co-cultures, which exhibit stable liver specific functions and may be potentially useful for bioartificial liver design. Rat hepatocyte-mouse 3T3-J2 cell co-cultures were maintained for 6 days in medium, and then switched to heparinized human plasma containing 3-methylcholanthrene (3MC; 2 microM), phenobarbital (PB; 1 mM), or no inducer for up to 7 days. CYP activities were measured in situ based on the o-dealkylation of ethoxy- (EROD), methoxy- (MROD), pentoxy- (PROD), or benzyloxy- (BROD) resorufin. Plasma alone increased PROD/BROD but not EROD/MROD. The endogenous inducer was in the high molecular weight fraction (>5 kD) of plasma and inhibited by >5 nM okadaic acid and >10 microM dibutyryl cyclic AMP, two inhibitors of PB-inducible CYPs. Furthermore, plasma increased CYP1A1 and CYP2B1/2 mRNA levels. In plasma, 3MC induced EROD/MROD to about 60% of the level induced in culture medium while PB induced PROD/BROD that were three- to 10-fold above levels induced in medium. CYP activities decreased between days 2 and 7 of plasma exposure, but were enhanced by plasma supplementation with amino acids, insulin, glucagon, and hydrocortisone.

Interleukin-15 production at the early stage after oral infection with Listeria monocytogenes in mice.

We previously reported that exogenous interleukin‐15 (IL‐15) induces proliferation and activation of intestinal intraepithelial lymphocytes (i‐IEL) in naive mice. To investigate the ability of endogenous IL‐15 to stimulate i‐IEL in vivo, we monitored i‐IEL and intestinal epithelial cells (i‐EC) in mice after an oral infection with Listeria monocytogenes. Although the populations of αβ and γδ i‐IEL were not significantly changed after the oral infection, the expression level of interferon‐γ (IFN‐γ) was increased both at transcriptional and protein levels, and a conversely marked decrease in interleukin‐4 (IL‐4) was detected in the i‐IEL on day 1 after infection as compared with before infection. The T helper 1 (Th1)‐biased response of i‐IEL coincided with a peak response of IL‐15 production in the i‐EC after oral infection. These results suggested that IL‐15 produced from i‐EC may be at least partly involved in the stimulation of i‐IEL to produce IFN‐γ after oral infection with L. monocytogenes.

Computed Tomographic Demonstration of a Fish Bone in Abdominal Actinomycosis: Report of a Case

A 53-year-old man who had the habit of consuming fish bones was referred to our clinic because of a suspected malignant abdominal wall tumor. Computed tomography (CT) showed a mass (10 × 5u2009cm) in continuity with the transverse abdominal muscle, containing a small calcification. A laparotomy was performed with a preoperative diagnosis of an inflammatory mass due to fish bone penetration from the sigmoid colon. A fish bone, measuring 2.3u2009cm in length, was detected within the tumor by specimen radiography. The pathological findings demonstrated actinomycotic colonies. We herein present the first case of a CT demonstration showing a fish bone in an abdominal mass which was pathologically confirmed to be actinomycosis. Evidence of the presence of a foreign body is valuable for diagnosing inflammatory nodules such as actinomycosis and differentiation from malignancies.