Tomoharu Shimizu

Tissue-specific expression of estrogen receptors and their role in the regulation of neutrophil infiltration in various organs following trauma-hemorrhage

Although 17β‐estradiol (E2) administration after trauma‐hemorrhage (T‐H) reduces tissue neutrophil sequestration in male rodents, it remains unknown which of the estrogen receptor (ER) subtypes mediates this effect and whether the same ER subtype is involved in all the tissues. We hypothesized that the salutary effects of E2 on attenuation of neutrophil accumulation following T‐H are tissue and receptor subtype‐specific. Male Sprague‐Dawley rats underwent sham operation or T‐H (mean blood pressure, 40 mmHg for 90 min and then resuscitation). E2 (50 μg/kg), ER‐α agonist propyl pyrazole triol (PPT; 5 μg/kg), ER‐β agonist diarylpropiolnitrile (DPN; 5 μg/kg), or vehicle (10% dimethyl sulfoxide) was administered subcutaneously during resuscitation. Twenty‐four hours thereafter, tissue myeloperoxidase (MPO) activity (a marker of neutrophil sequestration), cytokine‐induced neutrophil chemoattractant (CINC)‐1, CINC‐3, and intercellular adhesion molecule (ICAM)‐1 levels in the liver, intestine, and lung were measured (n=6 rats/group). ER‐α and ER‐β mRNA levels in sham‐operated rats were also determined. T‐H increased MPO activity, CINC‐1, CINC‐3, and ICAM‐1 levels in the liver, intestine, and lung. These parameters were improved significantly in rats receiving E2 after T‐H. Administration of the ER‐α agonist PPT but not the ER‐β agonist DPN improved the measured parameters in the liver. In contrast, DPN but not PPT significantly improved these parameters in the lung. In the intestine, ER subtype specificity was not observed. ER‐α mRNA expression was highest in the liver, whereas ER‐β mRNA expression was greatest in the lung. Thus, the salutary effects of E2 administration on tissue neutrophil sequestration following T‐H are receptor subtype and tissue‐specific.

Epithelial expression of interleukin-37b in inflammatory bowel disease

Interleukin (IL)‐37 is a member of the IL‐1 cytokine family. We investigated IL‐37b expression in the inflamed mucosa of inflammatory bowel disease (IBD) patients. Furthermore, we analysed IL‐37b expression in human colonic epithelial cells. The human colonic epithelial cell line T84 and human colonic subepithelial myofibroblasts (SEMFs) were used. IL‐37b expression in the IBD mucosa was evaluated by immunohistochemistry. IL‐37b mRNA and protein expression were determined by real time‐polymerase chain reaction (PCR) and Western blotting, respectively. IL‐37b was not detected in the normal colonic mucosa. In the inflamed mucosa of IBD patients, epithelial IL‐37b expression was increased markedly. In ulcerative colitis (UC) and Crohns disease (CD) patients, IL‐37b expression was enhanced in the affected mucosa. In the intestinal epithelial cell line T84, the expression of IL‐37b mRNA and protein was enhanced by tumour necrosis factor (TNF)‐α. This IL‐37b induction by TNF‐α was mediated by nuclear factor (NF)‐κB and activator protein (AP)‐1 activation. Furthermore, IL‐37b inhibited TNF‐α‐induced interferon‐γ‐inducible protein (IP)‐10 expression significantly in human colonic SEMFs. Epithelial IL‐37b expression was increased in IBD patients, especially UC patients. IL‐37b may be involved in the pathophysiology of IBD as an anti‐inflammatory cytokine and an inhibitor of both innate and acquired immune responses.

Inhibition of cardiac PGC-1α expression abolishes ERβ agonist-mediated cardioprotection following trauma-hemorrhage

PGC‐1α (peroxisome proliferator‐acti‐vated receptor [PPARγ] coactivator‐1α) activates PPARα and mitochondrial transcription factor A (Tfam), which regulate proteins, fatty acid and ATP metabolism (i.e., FAT/CD36, MCAD, and COX I). Recently we found that the salutary effects of estradiol (E2) on cardiac function following trauma‐hemorrhage (T‐H) are mediated via estrogen receptor (ER)β. In this study we tested the hypothesis that ERβ‐mediated cardioprotection is induced via up‐regulation of PGC‐1α through PPARα or Tfam‐dependent pathway. Male rats underwent T‐H and received ERα agonist propylpyra‐zole‐triol (PPT), ERβ agonist diarylpropionitrile (DPN), E2, or vehicle. Another group was treated with antisense PGC‐1α oligonucleotides prior to administration of DPN. E2 and DPN treatments attenuated the decrease in cardiac mitochondrial ATP, abrogated the T‐H‐induced lipid accumulation, and normalized PGC‐1α, PPARα, FAT/CD36, MCAD, Tfam, and COX I after T‐H. In contrast, PPT administration did not abrogate lipid accumulation. Moreover, in PPT‐treated animals mitochondrial ATP remained significantly lower than those observed in DPN‐ or E2‐treated animals. Prior administration of antisense PGC‐1α prevented DPN‐mediated cardioprotection and increase in ATP levels and Tfam but not in PPARα following T‐H. These findings suggest that the salutary effects of E2 on cardiac function following T‐H are mediated via ERβ up‐regulation of PGC‐1 α through Tfam‐dependent pathway.—Hsieh, Y.‐C., Choudhry, M. A., Yu, H.‐P., Shimizu, T., Yang, S., Suzuki, T., Chen, J., Bland, K. I., Chaudry, I. H. Inhibition of cardiac PGC‐1 α expression abolishes ERβ agonist‐mediated cardioprotection following trauma‐hemorrhage. FASEB J. 20, 1109–1117 (2006)

MR-guided microwave ablation for malignancies

Since we first successfully performed magnetic resonance (MR)-guided microwave coagulation therapy for liver tumors in January 2000, we have developed new MR-compatible instruments, laparoscopy and thoracoscopy, which have enabled us to approach liver tumors located just below the diaphragm and in contact with other organs. We have customized software for an MR gradient-based tracking system for the easy detection of the location and orientation of treatment area and for the real-time display of MR temperature maps with a scale bar. Navigation software was customized to enable real-time image navigation. The reformatted images in the two perpendicular planes complemented the limitations of real-time MR imaging. Evaluation software, “FootPrint,” was useful for distinguishing treated areas from untreated areas and improved the evaluation of treatment accuracy. These newly developed MR-guided systems that utilize microwave have played important roles in more accurate, safer, and easier treatment for liver tumors. We have treated 184 patients using these new techniques without major complications.

Indocyanine green fluorescence imaging system for sentinel lymph node biopsies in early breast cancer patients.

PurposeThis study presents a new method that enables the detection of sentinel lymph nodes (SLN) with high sensitivity using indocyanine green (ICG) fluorescence imaging.MethodsThis study enrolled 128 patients with clinically node-negative breast cancer. Fluorescence imaging was obtained after ICG was injected into the areola. Subcutaneous lymphatic channels were immediately visible.ResultsLymphatic channels and SLN were successfully visualized in all patients. One lymphatic channel was 60%, two channels were 24%, and three channels were 16%. The number of fluorescence SLN ranged from 1 to 6, and blue-dyed SLN ranged from 0 to 3. In the latter, SLN were not identified in 44 patients. Nineteen patients had pathologically identified lymph node metastases. All of them were recognized by fluorescence imaging, but 8 patients had lymph nodes with metastases were not identified by dye method.ConclusionThis ICG fluorescence imaging technique is feasible and safe for detecting SLN in a less invasive manner than conventional mapping, with real-time observations.

Adiponectin deficiency is associated with severe polymicrobial sepsis, high inflammatory cytokine levels, and high mortality

BACKGROUND Adiponectin, a key substance in metabolic syndrome, is known to have anti-inflammatory properties. The relationship between adiponectin and sepsis in vivo is unclear. In this study, the possible involvement of adiponectin in polymicrobial sepsis was investigated using adiponectin-knockout (APN-KO) mice that underwent cecal ligation and puncture (CLP) and received the peroxisome proliferator-activated receptor gamma (PPAR-gamma) that increases the plasma adiponectin concentration. METHODS APN-KO and wild-type (WT) mice underwent either CLP or a sham operation. The plasma adiponectin, endotoxin, tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6) levels were determined before and at 2, 4, 6, 8, 12, 16, and 24 hours after the procedures, and the survival rates were compared. Mice were injected with rosiglitazone, a selective PPAR-gamma agonist, and compared survival rates after CLP with those without rosiglitazone. RESULTS After CLP, APN-KO mice had a significantly higher mortality than WT mice. The plasma endotoxin, TNF-alpha, and IL-6 levels in APN-KO mice were significantly higher than those in WT mice 24 hours after CLP. Within 4 hours after CLP, the plasma adiponectin level in WT mice decreased to half of the initial levels. Pre-CLP treatment with PPAR-gamma was shown to increase the plasma adiponectin level and to improve significantly mortality of WT mice during sepsis; mortality among APN-KO mice did not improve. CONCLUSION These results suggest that adiponectin deficiency may cause the high mortality and the high inflammatory cytokine levels in mice with polymicrobial sepsis.

Flutamide attenuates pro-inflammatory cytokine production and hepatic injury following trauma-hemorrhage via estrogen receptor-related pathway.

Objective:To determine the mechanism by which flutamide administration following trauma-hemorrhage (T-H) decreases cytokine production and hepatic injury under those conditions. Summary Background Data:Although studies have demonstrated that flutamide administration following T-H improves hepatic and immune functions, the mechanism by which flutamide produces the salutary effects remains unknown. Methods:Male Sprague-Dawley rats underwent a 5-cm laparotomy and hemorrhagic shock (40 mm Hg for ∼90 minutes), followed by resuscitation with 4 times the shed blood volume in the form of Ringers lactate. Flutamide (25 mg/kg body weight, sc) was administered at the middle of resuscitation and animals were killed 2 hours thereafter. To block estrogen receptor (ER), ER antagonist ICI 182,780 was administrated with flutamide. Results:Hepatic injury, myeloperoxidase activity, nuclear factor-kappaB (NF-κB) DNA binding activity and protein expression of intercellular adhesion molecule-1, and cytokine-induced neutrophil chemoattractant (CINC-1 and CINC-3) markedly increased following T-H. Hepatic mRNA and plasma IL-6 levels were also elevated following T-H. The alterations in these parameters induced by T-H were significantly attenuated by flutamide administration. The decreased plasma estradiol levels following T-H were restored to sham levels in the flutamide-treated T-H animals. Coadministration of ICI 182,780 prevented those salutary effects of flutamide administration on pro-inflammatory responses and hepatic injury following T-H. Conclusion:These findings suggest that the reduction in the production of pro-inflammatory mediators and hepatic injury produced by flutamide administration following T-H is likely due to the down-regulation in hepatic NF-kappaB DNA binding activity. Moreover, the salutary effects of flutamide administration appear to be mediated at least in part via ER-related pathway.

Mechanism of the salutary effects of flutamide on intestinal myeloperoxidase activity following trauma-hemorrhage: up-regulation of estrogen receptor-β-dependent HO-1

Hemeoxygenase (HO)‐1 induction following adverse circulatory conditions is known to be protective, and precastrated males have less intestinal damage than sham‐operated males following trauma‐hemorrhage (T‐H). Previous studies have also shown that administration of flutamide up‐regulated estrogen receptor (ER) expression in males following T‐H. We hypothesized that flutamide administration in males following T‐H up‐regulates HO‐1 via an ER‐dependent pathway and protects against intestinal injury. Male Sprague‐Dawley rats underwent T‐H [mean blood pressure (MBP) 40 mmHg for 90 min and then resuscitation]. A single dose of flutamide (25 mg/kg body weight), with or without an ER antagonist (ICI 182,780), a HO enzyme inhibitor [chromium‐mesoporphyrin (CrMP)], or vehicle, was administered subcutaneously during resuscitation. At 2 h after T‐H or sham operation, intestinal myeloperoxidase (MPO) activity, intercellular adhesion molecule (ICAM)‐1, cytokine‐induced neutrophil chemoattractant (CINC)‐1, and CINC‐3 levels were measured. Intestinal ER‐α, ER‐β, androgen receptor, and HO‐1 mRNA/protein levels were also determined. Results showed that T‐H increased intestinal MPO activity, ICAM‐1, CINC‐1, and CINC‐3 levels. These parameters were improved significantly in the flutamide‐treated rats subjected to T‐H. Flutamide treatment increased intestinal HO‐1 and ER‐β mRNA/protein levels as compared with vehicle‐treated T‐H rats. Administration of the ER antagonist ICI 182,780 or the HO inhibitor CrMP prevented the flutamide‐induced attenuation of shock‐induced intestinal damage. Thus, the salutary effects of flutamide administration on attenuation of intestinal injury following T‐H are mediated via up‐regulation of ER‐β‐dependent HO‐1 expression.

Elevation of plasma peptidoglycan and peripheral blood neutrophil activation during hemorrhagic shock: plasma peptidoglycan reflects bacterial translocation and may affect neutrophil activation.

Objective To investigate the relations among bacterial translocation, plasma peptidoglycan elevation, and peripheral blood neutrophil activation during hemorrhagic shock. Design Prospective, randomized, unblinded animal study. Setting Surgical research laboratories of Shiga University of Medical Science. Subjects Male, specific pathogen–free Sprague-Dawley rats. Interventions The rats were randomly divided into three groups: a conventional group with normal intestinal flora (NF), an antibiotic (streptomycin and penicillin G) decontaminated group (AD), and a sham shock group with normal intestinal flora. The NF and AD groups were subjected to hemorrhagic shock (mean arterial pressure 30 mm Hg, for 30 to 90 mins). Rats were killed at 30, 60, and 90 mins after shock induction. Systemic blood and mesenteric lymph nodes (MLNs) were cultured for the determination of bacterial translocation (BT). Systemic plasma peptidoglycan and endotoxin concentrations were measured. To evaluate peripheral blood neutrophil activation, phagocytosis and hydrogen peroxide generation were assayed by flow cytometry. Measurements and Main Results In the NF group, BT to MLNs was significantly increased from 30 mins after shock induction. Blood culture and plasma endotoxin were positive at 90 mins but there were no significant differences. Assayed plasma peptidoglycan was significantly increased at 90 mins. Phagocytosis and hydrogen peroxide generation were significantly increased. Assayed plasma peptidoglycan concentrations showed significant positive correlations with the magnitude of BT to MLNs (r2 = .54) and hydrogen peroxide generation (r2 = .22) in individual animals. Furthermore, BT and these parameters were significantly suppressed in the AD group. Conclusions First, we concluded that assayed plasma peptidoglycan reflects BT induced by hemorrhage because the increase in assayed plasma peptidoglycan was suppressed, as was BT, by antibiotic decontamination. Second, peripheral blood neutrophil activation was also suppressed when BT was prevented. We concluded BT to be involved in neutrophil activation. Our findings suggest hydrogen peroxide generation by neutrophils to be involved in plasma peptidoglycan elevation.

The role of bacterial translocation on neutrophil activation during hemorrhagic shock in rats

Some biological responses to hemorrhage have been reported to be associated with bacterial translocation (BT). While the relationship between peripheral blood neutrophils and BT in the late phase of hemorrhagic shock or burn injury has been reported, this relationship in the early phase has not been fully elucidated. We investigate the role of BT in neutrophil activation and priming during hemorrhagic shock. The experimental rats were divided into three groups: a group with normal intestinal flora (NF group), an antibiotic-decontaminated group (AD group), and a sham shock group with normal intestinal flora (sham group). Hemorrhagic shock was induced in the NF and AD groups (MAP 30 mm Hg for 30-90 min). The rats were sacrificed at 30, 60, or 90 min following the shock induction. Cultures were taken from the liver, spleen, mesenteric lymph nodes (MLNs), and systemic blood to assess the occurrence of BT. Hydrogen peroxide generation and CD11b/c expression were assayed by flow cytometry to evaluate peripheral blood neutrophil activation and priming, respectively. In the NF group, significant BT to the MLNs and spleen was noted from 30 min after the shock induction, and significant hydrogen peroxide generation was also noted from 30 min. The expression of CD11b/c on neutrophils was significantly up-regulated at 90 min after the shock induction. Furthermore, BT, as also the aforementioned parameters of neutrophil function, was significantly suppressed in the AD group. We, therefore, concluded that neutrophil activation and priming during hemorrhagic shock might be closely related to BT, and that infectious factors possibly influence the host responses starting from the early phase of damage, even in noninfectious stress-inducing conditions.