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Bronchial asthma is an inflammatory disease of the airways. The Sec14l3 gene, encoding a 45-kDa secretory protein, is specifically expressed in airway epithelium. Here, we report on the kinetics of Sec14l3 expression following allergic inflammation of the lung. Brown Norway rats were sensitized by intraperitoneal injection of ovalbumin, followed by challenge with aerosolized ovalbumin after a 3-week interval. This animal model showed many features similar to human allergic asthma: an increase in inflammatory cells such as eosinophils, lymphocytes and neutrophils in bronchoalveolar lavage (BAL) fluid and histopathological alteration of lung tissue, exhibiting infiltration of these inflammatory cells and degeneration and necrosis of alveolar epithelium. These parameters reached their maximal level 24h after allergen challenge. In contrast, quantitative polymerase chain reaction analyses demonstrated a rapid and significant reduction of Sec14l3 mRNA in lung tissue and maximum reduction (to 1.4% of the control) was observed at 24h. Pretreatment with dexamethasone significantly suppressed both the Sec14I3 mRNA reduction and all of the inflammatory changes. The 45-kDa secretory protein was identified in the supernatant of BAL fluids. Two-dimensional gel images of the supernatant proteome also revealed down-regulation of the protein following inflammation (to approximately 30% of the control at 24h). Thus, Sec14l3 expression is highly and inversely associated with the progression of airway inflammation. Sec14l3 mRNA and protein may function in the homeostasis of airway epithelial cells under normal conditions.

To date, few cases of extraintestinal infection with Clostridium difficile have been reported. We describe a case of surgical-site infection with C. difficile following a colonic operation. Administration of metronidazole was considered to be effective for treatment of the infection. The isolate was a toxin A-nonproducing and toxin B-producing strain.

Background/aims: Phosphodiesterase type 4 (PDE4) has been previously shown to regulate colonic contractile activity in vitro. In this study, the effects of PDE4 inhibition were assessed in a model of stress-induced defecation previously demonstrated to be due to increased colonic transit/evacuation., Methods: Rats were individually placed in a mild restraint cage and placed into a 12 degrees C environment (cold-restraint stress) for 60 min. Mice received restraint (only) stress at room temperature for 30 min. Loperamide (positive control compound) or two different PDE4 inhibitors (rolipram and roflumilast) were administered orally at several doses to the rodents 1 h before stress began. Vehicle alone was administered for comparison. The number of fecal pellets expelled during stress (fecal pellet output), total fecal pellet wet weight and total fecal water content were measured., Results: Loperamide produced a dose-related decrease (ID(50)s in mg/kg) in fecal pellet output (rat = 7.4, mouse = 0.7) and significantly decreased fecal wet weight (72.9%) and decreased fecal percent water content (9.4%). The two PDE4 inhibitors produced a similar dose-related inhibition of fecal pellet output. Rolipram exhibited ID(50)s in rat and mouse of 14.1 and 27.1, respectively. Rolipram significantly decreased fecal wet weight (58.8%) but increased fecal percent water content (15.0%). For roflumilast, ID(50)s were 24.2 mg/kg and 12.4 in the rat and mouse, respectively. Although roflumilast also significantly (p < 0.05) decreased fecal wet weight (47.2%), it did not significantly increase fecal percent water content., Conclusions: These data indicate that PDE4 inhibition is effective in reducing rodent stress-induced defecation, provides the first functional data on a potential role for PDE4 activity in the colonic evacuation response to stress, and indicates the potential utility of PDE4 inhibitors in functional bowel disease such as irritable bowel syndrome requires further evaluation., ((c) 2008 S. Karger AG, Basel.)

Bacillus anthracis causes anthrax, a lethal disease affecting humans, which has attracted attention due to its bioterrorism potential. gamma-Phage specifically infects B. anthracis, and is used for its detection. gamma-Phage lysin, PlyG, specifically lyses B. anthracis. Mutational analysis of PlyGB (PlyG binding domain; residues 156-233) indicated that positions 190-199 are necessary for binding to B. anthracis. This region is the central part of PlyGB and is predicted to form a beta-sheet. The amino acid residues of this region are also conserved in other lysins specific for B. anthracis. Alanine substitution at position 190 or 199 within this region resulted in significantly reduced binding, suggesting that L190 and Q199 play key roles in binding of PlyGB to B. anthracis. Our observations provide new insight into the mechanism of specific binding of lysin to B. anthracis, and may be useful in establishing new methods for detection of B. anthracis.

T cells play an important role in initiating autoimmune responses and maintaining synovial inflammation in rheumatoid arthritis. Although, anti-type II collagen antibody-induced arthritis (CAIA) is generally believed to be a T cell- and B cell-independent model, the detailed pathogenesis of CAIA remains unclear. In the present study, to elucidate the contribution of T cells to the pathogenesis of CAIA, we evaluated the effects of CTLA4 Ig and cyclosporin (CsA). Arthritis was induced in mice by intravenous injection of anti-type II collagen antibody followed by intraperitoneal injection of lipopolysaccharide. CTLA4 Ig was intraperitoneally administered and CsA was subcutaneously administered; then the severity of arthritis was evaluated by scoring the edema and erythema of paws and by measuring hind paw thickness. Paw samples were collected 12 days after the antibody injection, and the mRNA expression levels were analyzed by real-time quantitative polymerase chain reaction. Administration of CTLA4 Ig ameliorated the increases in arthritic score and paw thickness in the later phase, but not in the early phase of arthritis. CsA suppressed the increases in arthritic score and paw thickness in both the early and later phases of arthritis. CTLA4 Ig and CsA suppressed mRNA up-regulation of T-cell markers, CD3 and CD25, and immune response-related mediators, IFN-gamma and IL-12. They also suppressed the up-regulation of macrophage marker, F4/80, and proinflammatory cytokines, TNF-alpha, IL-1beta and IL-6. The results provide direct evidence that arthritis in this model is T-cell activation dependent.

Activin receptor-like kinase 5 (ALK5) is a type I receptor of transforming growth factor (TGF)-beta. ALK5 inhibition has been reported to attenuate the tissue fibrosis including pulmonary fibrosis, renal fibrosis and liver fibrosis. To elucidate the inhibitory mechanism of ALK5 inhibitor on pulmonary fibrosis in vivo, we performed the histopathological assessment, gene expression analysis of extracellular matrix (ECM) genes and immunohistochemistry including receptor-activated Smads (R-Smads; Smad2/3), CTGF, myofibroblast marker (alpha-smooth muscle actin; aSMA) and type I collagen deposition in the lung using Bleomycin (BLM)-induced pulmonary fibrosis model. ALK5 inhibitor, SB-525334 (10 mg/kg or 30 mg/kg) was orally administered at twice a day. Lungs were isolated 5, 7, 9 and 14 days after BLM treatment. BLM treatment led to significant pulmonary fibrotic changes accompanied by significant upregulation of ECM mRNA expressions, Smad2/3 nuclear translocation, CTGF expression, myofibroblast proliferation and type I collagen deposition. SB-525334 treatment attenuated the histopathological alterations in the lung, and significantly decreased the type I and III procollagen and fibronectin mRNA expression. Immunohistochemistry revealed that SB-525334 treatment showed significant attenuation in Smad2/3 nuclear translocation, decrease in CTGF-expressing cells, myofibroblast proliferation and type I collagen deposition. These results suggest that ALK5 inhibition attenuates R-Smads activation thereby attenuates pulmonary fibrosis.

Cathepsin K is a cysteine proteinase that is highly expressed by osteoclasts and is being pursued as a potential drug target for the treatment of osteoporosis. We have reported that microcomputed tomography (micro-CT) analysis of bone microarchitecture may serve as a valuable tool for evaluating both antiresorptive and anabolic agents in ovariectomized (OVX) mice. The purpose of this study was to evaluate the effect of SB-553484, a novel cathepsin K inhibitor (human Ki,app=0.14 nM, mouse Ki,app=26 nM), on the OVX mice by micro-CT bone morphometric analysis. Seven weeks female BALB/c mice were OVX or sham-operated. OVX animals were treated with SB-553484 (30 mg/kg, sc) or Rolipram (10 mg/kg, po), a phosphodiesterase 4 inhibitor used as a positive bone anabolic agent, twice a day for 2 weeks. Both SB-553484 and Rolipram significantly prevented the decrease of trabecular bone volume as well as the deterioration of trabecular architecture in OVX mice. Interestingly, SB-553484 demonstrated a more pronounced effect in improvement of trabecular separation, number and connectivity, and a weaker effect in improvement of trabecular thickness compared to that of Rolipram. These differences indicate that SB-553484 mainly acted as an antiresorptive agent in OVX-induced loss of trabecular bone. On the other hand, SB-553484 significantly increased cortical bone volume and cortical thickness as well as Rolipram in OVX mice indicating an unexpected stimulatory effect of SB-553484 on cortical bone formation. These data suggest that targeting cathepsin K may prove therapeutically beneficial in the treatment of diseases with accelerated bone loss such as postmenopausal osteoporosis not only by inhibiting bone resorption but also by potentially stimulating cortical bone formation.

Background: Recent advances in fibrosis biology have identified transforming growth factor (TGF)-beta type I receptor-mediated activation of Smads as playing a central part in the development of fibrosis. However, to date, there have been few studies that examined the localisation and distribution of receptor-activated Smads protein (R-Smads: Smad2 and 3) during the fibrosis progression., Aims: To histopathologically assess the time-course change of the localisation and distribution of the Smads protein in pulmonary fibrosis., Methods: Pulmonary fibrosis was induced by intranasal injection of bleomycin (0.3 U/mouse). Lungs were isolated 2, 5, 7, 9 and 14 days after bleomycin treatment. Histological changes in the lungs were evaluated by haematoxylin-eosin stain or Masson's trichrome stain, and scored. TGF-beta1, Smad3 and phosphorylated Smad2 localisations in lung tissues were determined by immunohistochemistry., Results: The bleomycin treatment led to considerable pulmonary fibrotic changes accompanied by marked increase in TGF-beta1 expression in infiltrating macrophages. With the progression in fibrosis (day 7-14), marked increases in Smad3-positive and pSmad2-positive cells were observed. There were intense Smad3-positive and pSmad2-positive signals localised to the nuclei of the infiltrating macrophages and to type II epithelial cells, and less intense signals in fibroblasts and hyperplastic alveolar/bronchiolar epithelial cells., Conclusions: The time-course data of TGF-beta1 and R-Smads indicate that progressive enhancement of TGF-beta1 signalling via R-Smad is activated in the process of fibrosis progression.

In Japan, many clinical laboratories may not have recognized toxin A-nonproducing/toxin B-producing (A-/B+) Clostridium difficile, because rapid diagnostic kits detecting toxin B of C. difficile have not been available in the laboratories. Therefore, we examined the prevalence of A-/B+ strains in the Tsukuba-Tsuchiura district, Japan. Fecal specimens submitted for C. difficile toxin tests in four tertiary hospitals in the district were collected for 6 months. Several C. difficile A-/B+ strains, isolated in two nosocomial outbreaks that had occurred in geographically distant areas in Japan, were also simultaneously analyzed as controls. C. difficile was isolated from 159 of 332 specimens collected. Ten (6.3%) of the 159 C. difficile strains were A-/B+ strains. Nine A-/B+ strains, isolated in one hospital, had an identical genomic pattern by polymerase chain reaction (PCR) ribotyping and macrorestriction analysis with pulsed-field gel electrophoresis (PFGE). The A-/B+ strain isolated in another hospital and some of those in the geographically distant hospitals were indistinguishable from the nine strains by PCR ribotyping but were distinguishable with PFGE analysis. We concluded that A-/B+ strains are not epidemic in this district and that PFGE analysis may be preferable to PCR ribotyping for the genotyping of C. difficile A-/B+ strains. The reason why most of the A-/B+ strains were detected in the one hospital was unclear.

Objectives: The purposes of this study were to develop an osteoporosis model in a short period of 2 weeks after ovariectomy in mice and to investigate whether analysis of microcomputed tomography (muCT) 3-dimensional bone parameters could provide useful information on the mechanism of action of antiosteoporotic agents., Materials and Methods: Mice were ovariectomized (OVX) or sham-operated, and the OVX mice were treated daily with 17beta-estradiol (E2), parathyroid hormone (PTH[1-34]), raloxifene, rolipram, or vehicle for 2 weeks. On day 14 post-OVX, the left femur bones were removed and then the distal metaphyseal bone was analyzed by both muCT and histomorphometry., Results: The trabecular bone volume, thickness, number, and connectivity significantly decreased and the number of osteoclasts increased in OVX mice. Treatment of OVX animals with each of the 4 antiosteoporotic agents significantly increased the bone volume and improved the bone architecture. However, the improvement of trabecular thickness in the rolipram-treated group and that of cortical thickness in the PTH(1-34)-treated group were the most marked, whereas the improvement of connectivity in the rolipram-treated group was the least among the drug-treated groups. These different improving effects of agents on the bone parameters reflect the differential effects of these agents on bone formation and bone resorption., Conclusions: This study demonstrated the feasibility of evaluating the effect of the antiosteoporotic agents within 2 weeks after ovariectomy in mice. The muCT analysis may serve as a valuable tool, specifically in a high-throughput pharmacological screening test, offering useful information regarding the effects of test compounds on both bone resorption and formation.

Transdermal nicotine patch (TNP) contains approximately 16.6 and 24.9 mg of nicotine per 20 and 30 cm2 (TNP-20 and TNP-30). The aims of the study are to investigate linearity of nicotine pharmacokinetics after single application of different strengths of TNP and to directly compare plasma nicotine concentrations with those during cigarette smoking. Twelve healthy Japanese male smokers were randomly allocated to 1 of 2 cohorts consisting of 6 subjects each. Cohort 1 subjects received 1 sheet of TNP-20 (TNP-20x1) in period 1, and 2 sheets of TNP-20 (TNP-20x2) in period 3. Cohort 2 subjects were received 1 sheet of TNP-30 (TNP-30x1) in period 2, and smoked a total of 12 cigarettes at 1 h intervals in period 4. Each TNP was applied to the upper arm for 16 h. After TNP-20x1 or TNP-20x2 treatment in cohort 1, the amount of nicotine delivered from TNP (Dose) was proportional to surface area of TNP. Cmax and AUC of nicotine increased with the surface area (Dose), and tmax, t(1/2), CL/F and percentage of dose excreted in urine were almost the same between both treatments. These suggest the linear pharmacokinetics of nicotine in proportion to the surface area and Dose following single application of TNP in identical subjects. In cohort 2, the plasma nicotine concentrations after TNP-30x1 treatment were approximately half those just before each smoking.

A 48-year-old man underwent subtotal esophagectomy for pStage III (pT 3 pN 3) thoracic esophageal carcinoma on June 20, 2002, in combination with chemotherapy (5-FU 500 mg/day day 1-14, CDDP 10 mg/day day 1-14, VDS 3 mg on days 1 and 8) before and after the operation. Recurrence was seen 7 months after the operation in right pleura and lower mediastinum. Chemo (same regimen)-radiotherapy (50 Gy) was then performed but without effect. Thereafter, lung and upper mediastinal metastases were found, and weekly administration of paclitaxel (70 mg/m2, day 1, 8, 15, q 4w) was initiated in combination with radiotherapy (40 Gy). Two cycles of treatment resulted in PR, and CR was achieved after the 8th cycle was completed. Although treatment was terminated after the 12 th cycle due to development of peripheral neuropathy (grade 2), CR was still maintained 8 months after the completion of treatment. These results suggested the effectiveness of the treatment in cases that show resistance to conventional 5-FU-based chemotherapy.

Background/aims: To prevent an anastomotic failure due to impaired blood supply, several trials have been performed such as preoperative ischemic conditioning by transarterial embolization of the left gastric, right gastric and splenic arteries or microvascular anastomosis. We assess the significance of an automatic anastomotic coupling device for vessel anastomosis, which we have continuously utilized, to simplify the task and shorten the anastomotic time since March 1999., Methodology: 8 patients who underwent venous anastomosis by an automatic anastomotic coupling device were evaluated for the time of anastomosis, total ischemic time and outcomes., Results: Venous anastomosis was completed within 5 minutes on average. Microscopic arterial anastomosis by hand took 35 minutes on average. For gastric tube reconstruction, venous anastomosis by an automatic coupling device took only 5 minutes. The top of the gastric tube showed congestion before venous anastomosis, but rapidly recovered from it after anastomosis. Postoperative endoscopic observation of the mucosal color of the replaced intestine or gastric tube was started 3 days after surgery and revealed no ischemia or congestion. The postoperative course was uneventful except one case suffering from pneumonia but leakage was not observed in any case., Conclusion: An automatic anastomotic coupling device can perform an easy and reliable vascular anastomosis for patients who undergo esophageal reconstruction. The device may shorten the operating time and consequently the ischemic time of the gastric tube or jejunal or colonic graft, which in turn may lead to a decrease of complications.

Anti-HER2 antibody can induce antitumor responses, and can be used in delivering drugs to HER2-overexpressing cancer. Previously, we produced hyperthermia using magnetite nanoparticles that generate heat in an alternating magnetic field. In the present study, we constructed anti-HER2 immunoliposomes containing magnetite nanoparticles, which act as tumor-targeting vehicles, combining anti-HER2 antibody therapy with hyperthermia. The magnetite nanoparticle-loaded anti-HER2 immunoliposomes exerted HER2-mediated antiproliferative effects on SKBr3 breast cancer cells in vitro. Moreover, 60% of magnetite nanoparticles were incorporated into SKBr3, and the cells were then heated at 42.5 degrees C under an alternating magnetic field, resulting in strong cytotoxic effects. These results suggest that this novel therapeutic tool is applicable to treatment of HER2-overexpressing cancer.

A novel series of 1-pyridylisoquinoline and 1-pyridyldihydroisoquinoline derivatives has been prepared. These compounds showed potent PDE4 inhibitory activities and a broad margin between the K(i) value of the rolipram binding affinity and the IC(50) value of PDE4 inhibition. They also exhibited potent inhibitory activities toward LPS-induced TNF-alpha production in mice.

Background: The regulatory mechanisms of IL-5 gene transcription in human peripheral T cells are unclear because the transfection efficiency of plasmid constructs into nontransformed T cells is very low., Methods: Concanavalin A (ConA)-stimulated blastocytes derived from peripheral blood lymphocytes of asthmatic subjects were transiently transfected with the human IL-5 gene promoter/enhancer-luciferase gene construct, pIL-5 (-511)Luc, and cultured with THP-1 cells (human monocytoid cells) and anti-CD3 monoclonal antibody (mAb). IL-5 level in the culture medium was determined by an enzyme-linked immunosorbent assay. Transcriptional activity of the IL-5 gene was measured by luciferase reporter analysis., Results: ConA-blast lymphocytes of asthmatic patients produced a significant amount of IL-5 upon combined stimulation with anti-CD3 mAb and THP-1 cells, but not with anti-CD3 mAb alone. Costimulation with anti-CD28 mAb also enhanced the anti-CD3 mAb- induced IL-5 production. Accordingly, luciferase activity induced by anti-CD3 mAb stimulation in pIL-5(-511)Luc-transfected ConA-blast lymphocytes was increased 1.9- and 3.4-fold by the addition of anti-CD28 mAb and THP-1 cells, respectively. Serial 5' deletion analysis of the reporter gene demonstrated that the cis-regulatory element located at -119 to -80 is critical for anti-CD3 mAb-induced IL-5 gene transcription., Conclusions: Our present findings provide a useful model reflecting IL-5 gene transcription in human peripheral T cells in vivo, and clearly demonstrate that an interaction with monocytes enhances IL-5 gene transcription., (Copyright 2003 S. Karger AG, Basel)

Duodenum-preserving resection of the pancreatic head is a risk factor for pancreatic fistula because of the wide surface of the transected pancreas. We report on a 72-year-old woman undergoing this procedure for cystadenoma in the pancreatic head using the ultrasonic coagulating shears. The soft pancreatic parenchyma was extensively transected around the cyst with the coagulating shears. Suture of the cut stump was not necessary due to absence of bleeding. The distal pancreatic duct was well preserved for pancreatojejunostomy. We oversewed the possible opening of the main pancreatic duct on the remaining pancreas attached to the duodenum after confirming the location by intrabiliary dye injection. The postoperative course was uneventful. The draining fluid amylase level was low and there were no viscous materials from the drains. We compared histologic changes of a porcine pancreas transected with the coagulating shears or electrocautery to evaluate the sealing effect of the transected surface. The cut stump was covered by a continuing layer of thick protein coagulum in cases of coagulating shears, but by a disrupted layer of coagulating necrosis with charring in cases of electrocautery. The coagulating shears are useful for pancreatic transection in duodenum-preserving resection of the pancreatic head to prevent bleeding and pancreatic fistula from the cut surface.

A 78-year-old woman was admitted to our hospital after computed tomography (CT) had revealed in her the presence of a giant ascending aorta-arch aneurysm. This aneurysm was about 8 cm in diameter and associated with DeBakey type II dissection. Aortography showed the same condition as the CT view with the entry on the ascending aorta. The ascending aortaarch was replaced with a Hemashield 24 mm, by using deep-hypothermic selective cerebral perfusion and the open distal method. There were no complications during her peripostoperative state and no evidence of leakage and remnant dissection on CT and aortography. This is a rare case in which thoracic aortic aneurysm coexisted with dissection. In this case of severe atherosclerosis, deep-hypothermic selective cerebral perfusion and the open distal method provided effective treatment.

Bacterial translocation is believed to occur during cardiopulmonary bypass (CPB) because serum endotoxin concentrations rise. Intestinal ischemia during CPB, however, has never been proven directly. The condition of the intestinal mucosa during CPB was studied by measuring serum diamine oxidase (DAO) activity, an index of intestinal ischemia. Serum DAO activity, blood lactate concentration, and the arterial ketone body ratio (AKBR) were measured intraoperatively in four successive patients who underwent aortic arch replacement by the open distal anastomosis method. DAO activity rose after restoration of blood flow to the lower half of the body, and continued to rise throughout CPB. The lactate concentration also rose, mirroring the change in DAO activity, and returned to nearly normal 12 h after the operation. The AKBR decreased during CPB, with a mean minimum vale of 0.16-0.07 immediately after the restoration of blood flow to the lower half of the body. The parallel rise in DAO activity and serum lactate concentration once blood flow to the lower half of the body was restored implies that ischemic injury to the mucosa of the small intestine occurs during CPB. The continued rise in these parameters throughout CPB is consistent with ongoing injury due to splanchnic hypoperfusion, as reflected in the decrease in the AKBR during the same period.

A 54-year-old man suffering from Borrmann type 4 advanced gastric cancer with pancreatic invasion and paraaortic lymph node metastases underwent a total gastrectomy, which was a radical C operation. From postoperative month 4, he visited our hospital with multiple liver metastases and increased lymph node metastases. After chemotherapy with CDDP and 5-FU, CDDP and UFT was administered on an outpatient basis. The effect of this therapy was PD, therefore, docetaxel and 5'-DFUR combination chemotherapy was performed as second line therapy. After 2 courses of this therapy, the size of liver and lymph node metastases was reduced and the effect of this therapy was PR. The patient has undergone 4 courses of this therapy and is maintaining a clinical PR. It is conceivable that docetaxel and 5'-DFUR combination chemotherapy is useful for patients with advanced and recurrent gastric cancer, even if they had been treated with 5-FU administration as first line therapy.

Cancer chemotherapy targeted to angiogenic vessels is expected to cause indirect tumor regression through the damage of the neovasculature without the induction of drug resistance. To develop a tool for neovasculature-specific drug delivery, we isolated novel peptides homing to angiogenic vessels formed by a dorsal air sac method from a phage-displayed peptide library. Three distinct phage clones that markedly accumulated in murine tumor xenografts presented PRPGAPLAGSWPGTS-, DRWRPALPVVLFPLH- or ASSSYPLIHWRPWAR-peptide respectively. After the determination of the epitope sequences of these peptides, we modified liposomes with epitope penta-peptides. Liposome modified with APRPG-peptide showed high accumulation in murine tumor xenografts, and APRPG-modified liposome encapsulating adriamycin effectively suppressed experimental tumor growth. Finally, specific binding of APRPG-modified liposome to human umbilical endothelial cells, and that of PRP-containing peptide to angiogenic vessels in human tumors, i.e., islet cell tumor and glioblastoma, were demonstrated. The present study indicates the usefulness of APRPG-peptide as a tool for anti-neovascular therapy, a novel modality of cancer treatment.

The activity and toxicity of a weekly infusion of low-dose paclitaxel was studied. Twelve patients with metastatic or advanced breast cancer received paclitaxel (80 mg/m2 over 1 h) every week. Administration was continued for 6 weeks with two weeks rest until disease progression or limiting toxicity. Dexamethasone 20 mg, diphenhydramine 50 mg, and ranitidine 50 mg were given prior to each dose of paclitaxel. Six patients had received prior standard CMF therapy, and four patients had received CMF and docetaxel therapy. Two patients had not received prior therapy. The overall response rate was 58% with 17% complete responses and 42% partial responses. Responses were observed in both patients without prior therapy, and in five of 10 (50%) with prior therapy. Grade 3/4 neutropenia occurred in one patient; febrile neutropenia was not observed. There was no neuropathy or hypersensitivity. Weekly paclitaxel is active and well tolerated in patients with metastatic or advanced breast cancer. This schedule allows a high cumulative dose of paclitaxel without major myelo- or neurotoxicity. This weekly regimen deserves further exploration.

For borderline malignant diseases of the pancreas such as cystic adenoma, partial pancreatectomy or pancreatoduodenectomy including pylorus-preserving pancreaticoduodenectomy have been performed depending on tumor location under large median laparotomy. To investigate the feasibility of a technique with minimal skin incision, while retaining safety equivalent to conventional resection of the pancreatic tail, by making use of the advantages of laparoscopic procedure, we performed a minimally invasive laparoscopic resection of the pancreatic tail with preservation of the spleen. A 69-year-old woman underwent surgery for a diagnostic therapy for a cystic lesion of the pancreatic tail. The procedure was performed as follows: All procedures were performed completely laparoscopically under CO2 insufflation. After dissection of the omentum, laparoscopic ultrasound was performed to identify the location of the tumor and splenic vessels. The splenic hilus was dissected with preservation of the splenocolic ligament to maintain the lower blood supply to the spleen. The left gastroepiploic artery and the short gastric arteries and veins could be preserved. After division of the splenic hilus, the splenic artery and vein were identified from behind the pancreas by being held up and dissected individually by intracorporeal ligation by 3-0 Nylon. Then, pancreatic transection was performed 1 cm proximal to the tumor with the Endo-GIAII. The duration of operation was 4.5 hours. Intraoperative blood loss was under 50 mL. Histological examination revealed mucinous cytadenoma. She could walk the day after surgery and was discharged from the hospital uneventfully. CT prior to discharge from the hospital revealed sufficient blood flow in the spleen. Thus, it may be feasible to select laparoscopic spleen-preserving distal pancreatectomy as a first choice for diagnostic therapy for cystic lesions of the pancreatic tail.

To clarify the function of integrin alpha(v)beta3 in the early stage of liver metastasis, we investigated the interactions of metastatic cells with their target organ under the actual blood flow by using positron emission tomography (PET). The cells used were CHO-K1 cells and their transfectants bearing human integrin alpha(v)beta3 cDNA (alpha(v)beta3-CHO-K1 cells). The liver accumulation of alpha(v)beta3-CHO-K1 cells was significantly higher than that of CHO-K1 cells after injection via the portal vein, whereas no significant difference was observed in the lung accumulation after tail vein injection, suggesting a specific interaction of alpha(v)beta3-CHO-K1 cells with the hepatic sinusoids. Furthermore, to clarify the precise location of each cell in the liver, i.e., to determine whether individual cells were intravascularly localized or had extravasated, we performed intravital fluorescence microscopy (IVM) on the liver by using stable transfectants bearing the green fluorescent protein (GFP) gene, namely, GFP-CHO-K1 and GFP-alpha(v)beta3-CHO-K1 cells. Both types of cells remained in the hepatic blood vessels 1 h after injection via the portal vein. On the other hand, expression of integrin alpha(v)beta3 promoted the cells to reach the extravascular region after 24 h. These results suggest the possibility that the specific accumulation of alpha(v)beta3-CHO-K1 cells in the liver is followed by migration of the cells into the extravascular region. Interestingly, the adhesion of the two types of cells to hepatic sinusoidal endothelial cells in vitro did not correspond to in vivo accumulation of these cells. Therefore, integrin alpha(v)beta3 may function to promote extravasation of integrin alpha(v)beta3-expressing tumor cells in liver through a process possibly mediated by vitronectin produced by this organ.

Bronchial asthma is characterized by chronic eosinophilic inflammation of the bronchial mucosa in which Th2 cells play crucial roles. Ovalbumin-reactive Th2 clones were labeled with a fluorescent-probe then infused into unprimed mice to elucidate the dynamics of antigen-specific T cells involved in allergic inflammation. Infiltration of not only labeled antigen-specific T cells, but also unlabeled nonspecific CD4(+) T cells into the bronchial mucosa following inhaled antigen challenge was detectable under confocal microscopy and flow cytometry. Accordingly, labeled T cells in the spleen were decreased, whereas those in hilar lymph nodes were increased upon antigen challenge. Approximately 45% of antigen-specific T cells that migrated into the lungs bore CD25, while another early activation marker, CD69, was expressed on 80% of the migrated T cells. Accordingly, antigen challenge to the mice induced in situ proliferation of antigen-specific T cells as well as bronchial epithelial cells in the lungs. Expression of vascular cell adhesion molecule (VCAM)-1, but not intercellular adhesion molecule (ICAM)-1, on the vascular endothelium in the lungs was enhanced following antigen challenge. Nevertheless, treatment with anti-VCAM-1 antibody, and also anti-ICAM-1 antibody strongly suppressed the accumulation of T cells, suggesting that both VCAM-1 and ICAM-1 are essential for antigen-stimulated T cell mobilization into peripheral tissues. Our current study visualized the kinetics and the mechanism of antigen-specific T cell migration in response to local challenge with a protein antigen.

Objective: To assess the feasibility of cure of early gastric cancer by the endoscopic procedure done through a temporary gastrostomy and to use it to treat patients with various severe complications., Design: Prospective study., Setting: University hospital, Japan., Subjects: Four patients who required gastric mucosal resection for early gastric cancer., Main Outcome Measures: Ease of the procedure, cure rate, safety under epidural anaesthesia, and outcome., Results: All patients had the procedure under epidural anaesthesia without pain or complaints during operation. Mean operation time was 120 minutes (60-200). Histological examination showed that the surgical margin was free of tumour in 3 of the cases. One patient who had microscopic invasion of lymph nodes subsequently underwent second-look conventional gastrectomy with lymphadenectomy. Postoperative recovery was rapid, and all patients were discharged from the hospital uneventfully., Conclusion: This organ-preserving procedure enables good visualisation of the tumour and is useful even for high-risk patients.

We measured pyrimidine nucleoside phosphorylase (PyNPase), a known angiogenetic factor, and dihydropyrimidine dehydrogenase (DPD) in advanced gastric cancers. PyNPase was expressed in cytoplasm of the cancer cells and surrounding interstitial cells. The levels of PyNPase and DPD were significantly higher in cancer tissue. With respect to tumor factors, the level of PyNPase was significantly higher in cases positive for venous invasion. We divided patients into two groups, with high and low activities of IAP and MMP-9. The level of PyNPase was significantly higher in the high IAP activity group. A correlation was suggested between the level of PyNPase and the activity of IAP. 5'-Deoxy-5-fluorouridine (5'-DFUR) is transformed into 5-FU by PyNPase and manifests antitumor effects. DPD is a rate-limiting enzyme in the process of degradation of 5-FU. In the present study, the level of PyNPase/DPD was significantly higher in cancer tissue. PyNPase/DPD suggests not only the malignant potential of the tumor but also the efficiency of chemotherapy using 5-FU, especially 5'-DFUR.

We report a rare case of carcinoma of the cystic duct (CCD) associated with pancreaticobiliary maljunction (PBM). A 63-year-old man had presented with relapsing cholecystitis of 4 months, duration. Computed tomography showed a distended gallbladder: however, small mass in the cystic duct was overlooked. Endoscopic retrograde cholangiopancreatography demonstrated a long common channel (20-mm-long) and fusiform dilatation of the common bile duct, findings, which were consistent with PBM. At laparotomy, we found a papillary tumor, 20 mm in diameter, that obstructed the cystic duct. The patient underwent resection of the gallbladder and the common bile duct, lymph node dissection in the hepatoduodenal ligament, and hepaticojejunostomy. Histologic study revealed a papillary adenocarcinoma confined within the subserosal space. There was no lymphatic or perineural invasion of cancer cells. The surrounding cystic ductal mucosa showed dysplasia and hyperplasia, and the gallbladder and common bile duct showed severe inflammation. The patient has been doing well for 16 months after surgery, without tumor recurrence. This case suggests a relationship between CCD and chronic biliary inflammation caused by PBM, as in cases of gallbladder carcinoma.

Combined liver-intestine transplantation is an evolving procedure, and auxiliary liver transplantation has several advantages over standard orthotopic liver transplantation. We present a new model of combined intestine-auxiliary liver transplantation in rats. Total small bowel and 60% liver were harvested en bloc. An aortic segment that contained the celiac axis and superior mesenteric artery ensured blood supply to the graft. Venous drainage of the grafted intestine was achieved via the intact portal vein of the graft. The infrahepatic vena cava was cut at different levels during the modification period and at the oblique level of the left renal vein in consecutive series. Revascularization was accomplished by end-to-side anastomosis of the aorta and of the infrahepatic vena cava. The recipient small bowel was resected and the intestine continuity restored by anastomosis. Total operation time averaged 130 min. The overall survival rate of 3 months in the consecutive series was 80% (16/20). Exploratory laparatomy and histologic study in 3 rats on 90 days after transplantation revealed normal and viable grafts. Liver function was normal and both grafted liver and intestine showed normal histologic architectures in 5 rats observed for 12 months after transplantation. The present model is reproducible and allows preclinical research on several aspects of experimental combined intestine-auxiliary liver transplantation.

Background: IL-5 plays a central role in allergic diseases associated with eosinophilic inflammation. We have previously reported that IL-5 production by peripheral blood mononuclear cells (PBMC) is greatly enhanced in both atopic and nonatopic asthmatics compared to control subjects., Method: Concanavalin A (Con A) blast lymphocytes were derived from PBMC of allergic and control subjects. Transcriptional regulation of the IL-5 gene was investigated by transient transfection assay., Results: A significant amount of IL-5 was produced by Con A blast lymphocytes derived from allergic subjects upon stimulation with phorbol ester and Ca(2+) ionophore, whereas the cells derived from control subjects did not produce a detectable amount of IL-5. Production of IL-2 and IL-4 was not significantly different between the two groups. A luciferase reporter plasmid containing the human IL-5 promoter/enhancer region was transcribed by Con A blast lymphocytes derived from allergic subjects, but not by the cells from control subjects, upon activation., Conclusion: IL-5 synthesis by nontransformed T cells of allergic subjects is enhanced at the level of gene transcription. Elucidation of the molecular mechanism of IL-5 gene transcription by allergic T cells may delineate the pathogenesis of allergic disease for the future therapeutic intervention., (Copyright 2000 S. Karger AG, Basel.)

The relationship among the real-time trafficking of lung metastatic B16BL6 cells, metastatic potential, and the injected number of the cells was examined, since the smaller the number of tumor cells injected, the more clearly the immune defense may be observed. When 1x10(6) or 1x10(5) B16BL6 cells were injected into mice via the tail vein, both numbers of cells accumulated in the lung at a similar rate: there was an approximately 10-fold difference in the number of accumulated cells between the two doses. Elimination from the lung was not dependent on the cell number but on the proportion of accumulated cells. However, the injection of 1x10(4) cells resulted in lung accumulation less than one-tenth of that obtained with 1x10(5) cell injection. Metastasis was observed when 1x10(5) or 1x10(6) B16BL6 cells were injected, but not after injection of 1x10(4) cells. To clarify the roles of the immune defense system at the initial phase of metastasis, we challenged macrophage-depleted mice with 1x10(4) tumor cells. Treatment of mice with 2-chloroadenosine prior to the tumor cell challenge cancelled the suppression of not only metastasis but also the lung accumulation. Furthermore, the administration of 2-chloroadenosine following the tumor cell challenge had little effect on the metastatic potential. These results suggest that the immune surveillance whose action was obvious at the low dose of challenged tumor cells functions strongly at the initial phase but not at the advanced stages of the metastatic process, and that macrophages play an important role in the suppression of metastasis.

We examined the subtype-selective binding site of the beta-adrenergic receptors (betaARs). The beta(1)/beta(2)-chimeric receptors showed the importance of the second and seventh transmembrane domains (TM2 and TM7) of the beta(2)AR for the binding of the beta(2)-selective agonists such as formoterol and procaterol. Alanine-substituted mutants of TM7 of the beta(2)AR showed that Tyr(308,) located at the top of TM7, mainly contributed to beta(2) selectivity. However, Tyr(308) interacted with formoterol and procaterol in two different ways. The results of Ala- and Phe-substituted mutants indicated that the phenyl group of Tyr(308) interacted with the phenyl group in the N-substituent of formoterol (hydrophobic interaction), and the hydroxyl group of Tyr(308) interacted with the protonated amine of procaterol (hydrophilic interaction). In contrast to beta(2)AR, TM2 is a major determinant that beta(1)-selective agonists such as denopamine and T-0509 bound the beta(1)AR with high affinity. Three amino acids (Leu(110), Thr(117), and Val(120)) in TM2 of the beta(1)AR were identified as major determinants for beta(1)-selective binding of these agonists. Three-dimensional models built on the basis of the predicted structure of rhodopsin showed that Tyr(308) of the beta(2)AR covered the binding pocket formed by TM2 and TM7 from the upper side, and Thr(117) of the beta(1)AR located in the middle of the binding pocket to provide a hydrogen bonding for the beta(1)-selective agonists. These data indicate that TM2 and TM7 of the betaAR formed the binding pocket that binds the betaAR subtype-selective agonists with high affinity.

1. To delineate the mechanism by which cyclic AMP (cAMP) suppresses interleukin (IL)-5 synthesis, the effects of prostaglandin (PG) E2, forskolin, dibutyryl (db)-cAMP and the Ca2+ ionophore, ionomycin on cytokine synthesis, proliferation and CD25 expression of human T cells were investigated. Further studies were performed by measurement of the intracellular concentrations of cyclic AMP ([cAMP]i) and Ca2+ ([Ca2+]i) and by electrophoretic mobility shift analysis (EMSA). 2. PGE2, forskolin and db-cAMP suppressed IL-5 production by human T cell line following T cell receptor (TCR)-stimulation. PGE2 suppressed TCR-induced messenger RNA (mRNA) expression of IL-2, IL-4 and IL-5, as well as proliferation and CD25 expression. 3. Cyclic AMP-mediated suppression of cytokine synthesis, proliferation and CD25 expression in human T cells were attenuated by ionomycin. 4. [cAMP]i was increased by PGE2 and forskolin. PGE2 suppressed the TCR-induced biphasic increase in [Ca2+]i. EMSA revealed that four specific protein-DNA binding complexes related to NF-AT were detected at the IL-5 promoter sequence located from -119 to -90 relative to the transcription initiation site. The slowest migrating complex induced by TCR stimulation was enhanced by PGE2 and further upregulated by ionomycin. Another binding which did not compete with cold AP-1 oligonucleotides, was constitutively present and was unaffected by PGE2 but enhanced by ionomycin. 5. The suppressive effect of cyclic AMP on human IL-5 synthesis is mediated by interference with intracellular Ca2+ mobilization but distinct from the NF-AT-related pathway.

Background: The essential role of Th cells and T cell cytokines in eosinophilic inflammation has been established., Methods: To determine whether Th cells are sufficient for the development of airway eosinophilic inflammation, ovalbumin-reactive murine Th clones were established and infused into unprimed mice., Results: Eosinophilic infiltration into the lung was induced upon antigen inhalation in parallel with the rise in bronchoalveolar lavage fluid (BALF) eosinophil peroxidase activity. Neither IgG, IgA, nor IgE antibodies were present in this model. Pathological examination showed swelling and desquamation of epithelial cells, mucous plugs, and goblet cell hyperplasia, all of which well resemble human asthma. Fluorescent probe labeled Th clones migrated into the lung prior to the eosinophil accumulation. Bronchial hyperresponsiveness (BHR) was clearly induced upon antigen inhalation. Anti-IL-5 monoclonal antibody abrogated the responses. Dexamethasone and cyclosporin A suppressed cytokine production by Th cells both in vitro and in vivo, BALF eosinophilia, and BHR. The number of eosinophils recovered in the BALF correlated with the intensity of BHR., Conclusion: The results clearly indicated that monoclonal Th cells are sufficient for the development of both airway eosinophilia and BHR. Agents capable of downregulating IL-5 production seem promising for the treatment of bronchial asthma.

Background: We have recently demonstrated that allergic eosinophilic inflammation is transferred to unprimed mice by infusing IL-5-producing CD4+ T cells. The contribution of mast cells to the development of eosinophilic inflammation is controversial., Methods: To clarify the possible different roles of CD4+ T cells and mast cells in eosinophilic inflammation, we compared antigen-induced airway eosinophilia between mast-cell-deficient mice (WBB6F1-W/W(v)) and their congenic normal littermates (WBB6F1-+/+)., Results: The time course study indicated that equivalent numbers of eosinophils were recruited into the airway of both +/+ and W/W(v) mice 6, 24, 96, and 216 h after antigen challenge, whereas the number of eosinophils 48 h after antigen challenge was significantly lower in W/W(v) compared to +/+ mice. Administration of either anti-CD4 or anti-IL-5 monoclonal antibody almost completely inhibited antigen-induced eosinophil recruitment in W/W(v) mice 48 h after antigen challenge. In contrast, the inhibitory effect of these antibodies in +/+ mice were partial (approximately 50% inhibition). Anti-CD4 and anti-IL-5 antibodies equally suppressed airway eosinophilia in both +/+ and W/W(v) mice 96 h after antigen challenge., Conclusion: Our study indicates that CD4+ T cells are crucially involved in the development of allergic eosinophilic inflammation, while mast cells may play a supplemental role depending on the kinetics of the response.

To elucidate the early events of blood-borne metastasis under actual blood flow, real-time trafficking of RAW117 large cell lymphoma cells, namely parental RAW117-P and liver-metastatic RAW117-H10 cells, was investigated using positron emission tomography (PET). Both types of cells accumulated in the liver immediately after injection via the portal vein, and were eliminated from the liver time-dependently. The elimination rate of RAW117-H10 cells, however, was slower than that of RAW117-P cells, suggesting that RAW117-H10 cells interact more strongly with hepatic sinusoidal endothelium than the parental cells. This result correlated with the metastatic potential of these cells: RAW117-H10 cells metastasized in the liver to a greater extent than RAW117-P cells after injection via this route. To investigate the role of sialylglycoconjugates in the interaction of RAW117-H10 cells with the hepatic endothelium after injection via the portal vein, the trafficking of RAW117-H10 cells was examined after the cells had been treated with sialidase. The elimination rate of RAW117-H10 cells from liver was observed to be greatly accelerated by sialidase treatment. To elucidate what kind of sialylglycoconjugates is related to this phenomenon, we analyzed the distribution of sialyl Lewis A and sialyl Lewis X antigens of both sublines of RAW117 by using flow cytometry. RAW117-H10 cells were found to express a much higher level of sialyl Lewis A than RAW117-P cells, whereas the amount of sialyl Lewis X did not differ significantly. These findings suggest that some sialylglycoconjugates, perhaps sialyl Lewis A in particular, play an important role in the initial interaction of RAW117-H10 cells with the hepatic endothelium, leading to metastasis.

We have recently demonstrated that airway eosinophilic inflammation can be transferred to unprimed mice by infusion of IL-5-producing T cell clones. In this study, we investigated the effects of dexamethasone and cyclosporin A on the airway eosinophilic inflammation in mice transferred with T cell clones. An ovalbumin-reactive T cell clone, KW29, produced IL-5 as well as IL-2 and IL-4 upon stimulation with relevant antigen. Dexamethasone and cyclosporin A dose-dependently suppressed the production of these cytokines in vitro. The number of eosinophils recovered in the bronchoalveolar lavage fluid and the airway responsiveness to acetylcholine were increased in KW29-transferred mice after antigen provocation. Both responses were dose-dependently suppressed by the administration of dexamethasone or cyclosporin A in vivo. We concluded that airway eosinophilic inflammation can be controlled by agents capable of downregulating IL-5 production in T cells.

1. We have recently demonstrated that airway eosinophilic inflammation can be transferred to unprimed mice by infusing interleukin (IL)-5-producing T cell clones. Using that murine model, we performed this study to delineate the mechanism of cyclosporin A and dexamethasone to inhibit allergic airway eosinophilic inflammation. 2. The ovalbumin-reactive murine T cell clones, FJ17, produced IL-2, IL-4 and IL-5 upon stimulation with relevant antigen. In FJ17-transferred mice, messenger RNA (mRNA) of IL-2 and IL-5 expressed in the lungs, the number of eosinophils in bronchoalveolar lavage fluid (BALF) was increased and the bronchial responsiveness to acetylcholine was enhanced after antigen provocation. 3. Cyclosporin A (10, 100 ng ml(-1)) and dexamethasone (10, 100 ng ml(-1) suppressed the production of IL-5 as well as IL-2 and IL-4 by FJ17 in vitro. 4. Subcutaneously administered cyclosporin A (30 mg kg(-1)) and dexamethasone (10 mg kg(-1)) inhibited antigen-induced mRNA expression of IL-2 and IL-5, increase of BALF eosinophils and bronchial hyperresponsiveness of FJ17-transferred mice in vivo. The number of BALF eosinophils was correlated with the bronchial responsiveness to acetylcholine (r=0.672). 5. The results clearly indicated that the suppression of IL-5 synthesis by T cells is involved in the effects of cyclosporin A and dexamethasone to inhibit allergic airway eosinophilic inflammation.

We investigated the effects of dexamethasone and cyclosporin A on Sephadex bead (Sephadex G-200, Sephadex)-induced airway hyperresponsiveness (AHR) and inflammation in rats. Sephadex (0.5 mg/animal) was intravenously administered on days 0, 2 and 5. Bronchoalveolar lavage, histological study and measurement of AHR were performed on day 7. Dexamethasone (0.1, 1 and 10 mg/kg, p.o. x 3) and cyclosporin A (0.1, 1 and 10 mg/kg, s.c. x 3) clearly inhibited the increase in eosinophils in bronchoalveolar lavage fluid after Sephadex injection. On histological study, pulmonary eosinophilia, granulomatous arteritis with horseshoe-shaped multinuclear giant cell formation and goblet hyperplasia were observed after Sephadex injection. Both dexamethasone (10 mg/kg x 3) and cyclosporin A (10 mg/kg x 3) inhibited these findings and an increase in eosinophil peroxidase in the lung. Dexamethasone dose-dependently inhibited AHR induced by Sephadex, and completely suppressed it at a dose of 1 mg/kg (x 3). Cyclosporin A, however, did not inhibit AHR even at a dose of 10 mg/kg ( x 3). These results show that there is a difference between dexamethasone and cyclosporin A in the inhibitory effect on Sephadex-induced AHR, and they suggest that eosinophils are not directly associated with the development of AHR after Sephadex injection.

Background: The Lewis rat among highly inbred strains exhibits significant airway hyperresponsiveness (AHR) following intravenous administration of Sephadex G-200 (Sephadex). The aim of this study was to investigate the association of Sephadex-induced AHR with changes in airway inflammation in Lewis rats., Methods: A suspension (0.5 mg/ml/rat) of Sephadex was intravenously administered to male Lewis rats on days 0, 2 and 5. Measurement of airway responsiveness to serotonin, bronchoalveolar lavage (BAL) and histological study were performed on day 2-11., Results: Significant AHR induced by Sephadex was recognized on day2 (p < 0.05), and AHR reached a maximum on day 7 (p < 0.001). In the BAL study, eosinophils increased on day2 (p < 0.01) with a peak on day 5 (p < 0.05). In the histological study, we found Sephadex beads trapped in small arteries of the lung and granulomatous arteritis on day 2 or later. Pulmonary granulomas, horseshoe-shaped multinuclear giant cells, eosinophils and goblet cell hyperplasia were observed on day 2, and the degree became intense on day 5-7. GCC-AP0341 (10 mg/kg, i.p. x 3) inhibited the recruitment of eosinophils in BAL fluid and in lung tissue, but it did not inhibit AHR. The compound also inhibited pulmonary granulomas and goblet cell hyperplasia., Conclusion: The mechanism of Sephadex-induced AHR may not be directly associated with inflammatory changes such as recruitment of eosinophils, pulmonary granulomas and hyperplasia of goblet cells in rats.

The effect of a novel anti-allergic drug, betotastine besilate (betotastine) on interleukin (IL)-5 production by human peripheral blood mononuclear cells (PBMC) was investigated. PBMC of Dermatophagoides farinae extract (Df)-sensitive donors produced IL-5 and showed a proliferative response upon stimulation with relevant antigen (10 microg/ml). Df-induced IL-5 production by PBMC was significantly inhibited by betotastine at 10 and 100 microM. Betotastine also suppressed proliferation of PBMC with less potency. The effect of betotastine on IL-5 production was enhanced and significant even at 0.1 microM when the drug was added 120 min before antigen stimulation. Ketotifen and cetirizine also inhibited IL-5 production, but the effects of these drugs were significant only at 100 microM. These findings indicate that the suppression of IL-5 production may be involved in the anti-allergic effect of betotastine.

To determine the structural basis for binding subtype selective agonists in the beta-adrenergic receptor (beta AR), we examined the interaction of the mutant beta 2AR and chimeric beta 1/beta 2AR with a selective beta 2AR agonist, TA-2005 (8-hydroxy-5-[(1R)-1-hydroxy-2-[N-[(1R)-2-(p-methoxyphenyl)-1-methyle thy l] amino]ethyl] carbostyril hydrochloride). The beta 2AR mutant with Ala substituted for Ser204 (S204A) significantly decreased the affinities for TA-2005, des-8-hydroxy-TA-2005 derivative (compound I), and isoproterenol. In contrast, a S207A mutation slightly decreased the affinities for TA-2005 and compound I, although the affinity for isoproterenol was decreased dramatically. The EC50 values of TA-2005 to activate adenylyl cyclase were not changed in either the S204A- or S207A-beta 2AR. In contrast with TA-2005, the EC50 values of compound I were reduced in the S204A-beta 2AR but not in the S207A-beta 2AR. These results suggest that Ser204 is important for high affinity binding but not necessary to activate adenylyl cyclase. Although TA-2005 was highly selective at the beta 2AR, the compounds lacking p-methoxyphenyl-ethyl (compound II) or p-methoxyphenyl-methylethyl groups (compound III) on the amine portion of TA-2005 lost beta 2AR subtype selectivity. When the second and seventh transmembrane (TM) region but not the TM1 region of the beta 2AR were replaced with the corresponding regions of the beta 1AR, the affinities of the chimeras for TA-2005 decreased compared with those of the wild type beta 2AR. Furthermore, substitution of the TM7 region of the beta 1AR with the corresponding region of the beta 2AR significantly increased the affinities for TA-2005. The affinities for isoproterenol and compounds II and III were not affected in the chimeras. These data suggest that the TM7 region of the beta 2AR plays an important role in beta 2-selective agonist binding. To determine the specific amino acid which confers this high affinity binding of TA-2005 to the beta 2AR, an alanine-scanning mutagenesis approach was employed. All amino acids that were different from those of the beta 1AR were individually changed to alanine. One mutant receptor (Y308A-beta 2AR) out of 10 point-mutated beta 2ARs showed a dramatically reduced affinity for TA-2005. These results indicate that Tyr308 is an essential amino acid for high affinity binding of the beta 2-selective agonist TA-2005.

Effect of a selective type 4 phosphodiesterase (PDE4) inhibitor, T-440, on intracellular cyclic AMP (cAMP) level and interleukin (IL)-2 production of Jurkat cells was investigated. T-440 suppressed both cAMP-PDE activities in cytosolic and membrane fractions of Jurkat cells. Intracellular cAMP level in Jurkat cells was elevated by PGE2 and forskolin but not by T-440. T-440, however, significantly enhanced the increase of cAMP by PGE2. PGE2 and forskolin inhibited IL-2 production of Jurkat cells stimulated with concanavalin A. T-440 by itself did not affect IL-2 production, but significantly enhanced the effect of PGE2 on IL-2 production. The increase of intracellular cAMP by T-440, PGE2, forskolin and T-440 plus PGE2 was well correlated with the inhibition of IL-2 production. These results indicate that IL-2 production of T cells is regulated by cAMP-PDE activity. Immunomodulatory effects of PDE4 inhibitors like T-440 should further be explored in vivo.

We studied the binding region of several beta1 and beta2 selective agonists by using chimeric beta1 and beta2ARs, and point-mutated beta2 adrenergic receptors (ARs). By replacing a single transmembrane domain (TMD) of beta1AR (or beta2AR) with the corresponding region of beta2AR (or beta1AR), we found that beta2 or beta1 selectivities were determined by TMD2 and TMD7 of beta2AR or by TMD2 of beta1AR, respectively. Alanine-substituted beta2AR mutants showed that tyrosine at position 308 in TMD7 played an important role in binding of beta2 selective agonists with high affinity. These data also suggested that the substituent on the amine portion was important for subtype selective agonist binding.

We investigated the selectivity of T-440 for the inhibition of phosphodiesterases (PDEs) in vitro and for bronchial anti-spasmogenic effects in vivo. Using a fast protein liquid chromatography system, five PDE isozymes, PDE 1, PDE 2, PDE 3, PDE 4 and PDE 5 were prepared from guinea pig and dog tissues. T-440 selectively inhibited PDE 4 with an IC50 of 0.071 microM and 0.13 microM for guinea pig lung and dog trachea, respectively. The IC50 values for all other PDE isozymes were over 20 microM. In contrast, theophylline nonselectively inhibited all the tested PDE isozymes, and the inhibition did not exceed 50%, even at 100 microM. T-440 inhibited the histamine-induced bronchoconstriction of anesthetized dogs in a dose-dependent manner with an ED50 of 0.029 mg/kg, indicating that T-440 is 600 times more potent than theophylline. Both T-440 and theophylline increased LV dp/dt/P (LVP; left ventricular pressure) in anesthetized dogs with ED50 values of 3.6 mg/kg and 4.4 mg/kg, respectively. This potency was 1/125 times the bronchial anti-spasmogenic effects for T-440 and 4.2 times that of theophylline. Rolipram, a PDE 4 inhibitor, also showed selective bronchial anti-spasmogenic effects in anesthetized dogs. These results suggest that T-440, which specifically inhibits PDE 4 activity, has potent and selective bronchial anti-spasmogenic effects.

To delineate the critical role of T cells on asthma, we tested whether eosinophilic inflammation of the bronchial mucosa is induced by transfer of IL-5-producing T cell clones, in the absence of antigen-specific immunoglobulins (IgE, A and G). Ovalbumin-specific T cell clone, FI5, that produced IL-5 upon challenge with relevant antigen was established. Eosinophilic inflammation of the lung occurred when unprimed mice were transferred with FI5 and challenged by the inhaled antigen. Eosinophil infiltration was completely suppressed by the administration of anti-IL-5 neutralizing antibody, indicating the essential role of IL-5. We concluded that the existence of IL-5-producing helper T cells is sufficient for the development of airway eosinophilic inflammation.

It has been proven that increasing cyclic adenosine 3',5'-monophosphate (cAMP) in human helper T cells results in decreased production of interleukin (IL)-2. As we have recently found that IL-2 stimulates IL-5 production, the effects of cAMP on IL-5 synthesis of T cells was investigated in this study. Prostaglandin E2 and forskolin raised intracellular cAMP level of Dermatophagoides farinae extract-reactive human T cell line and inhibited T cell receptor-stimulated IL-5 production. The cAMP analog, dibutyryl-cAMP, also inhibited IL-5 production, whereas the protein kinase A inhibitor, H-89, enhanced IL-5 production. The IL-5 production was completely suppressed by anti-IL-2 neutralizing antibody. Recombinant human IL-2 itself induced IL-5 production, suggesting that IL-5 production stimulated through T cell receptor is dependent on the autocrine production of IL-2. Prostaglandin E2, forskolin and dibutyryl-cAMP enhanced but H-89 suppressed recombinant human IL-2-induced IL-5 production. Prostaglandin E2 suppressed T cell receptor-stimulated mRNA expression of IL-2 as well as IL-5 in the T cell line, whereas it potentiated IL-5 mRNA expression stimulated by recombinant human IL-2. These results suggest that the inhibitory effect of cAMP on IL-5 production is mediated by the suppression of IL-2 production. On the contrary, IL-2-induced IL-5 synthesis is enhanced by increasing cAMP. Our study clearly indicated that cAMP regulates IL-5 production of human T cells by two differential effects.

Objective: The aim of this study was to compare Sephadex G-200 (Sephadex)-induced airway hyperresponsiveness (AHR) and pulmonary eosinophilia among some rat strains., Materials: Sprague-Dawley (SD), Brown-Norway (BN), Fischer 344 (Fischer), Lewis and Wistar-Kyoto (WKY) rats were used., Methods: Sephadex (0.5 mg/animal) was intravenously administered on days 0, 2 and 5. Measurement of AHR using serotonin and bronchoalveolar lavage (BAL) was performed on day 7., Results: The Lewis strain exhibited significant AHR to Sephadex but the BN, Fischer and WKY strains did not. Additionally, the degree of AHR in Lewis rats was smaller than in SD rats throughout the study. Eosinophils in BAL fluid, however, increased in each strain, and the magnitude of the response was BN > Lewis > WKY > Fischer. Both the AHR and pulmonary eosinophilia in Lewis rats were inhibited by dexamethasone (0.1 mg/kg, p.o. x3)., Conclusion: These results indicate that the Lewis rat is a useful strain for analysis of the mechanism of Sephadex-induced AHR and airway inflammation.

We investigated the effects of phosphodiesterase (PDE) inhibitors on ozone-induced airway hyperresponsiveness (AHR) in guinea pigs. Theophylline (10-100 mg/kg), rolipram (1-100 mg/kg) and T-440 (1-100 mg/kg) were orally administered 30 min before ozone exposure (3 ppm, for 30 min). Ozone exposure caused an increase in airway responsiveness to methacholine aerosol, and log PC(10) (log-transformed methacholine concentration causing a 10 cm H(2)O increase in pulmonary inflation pressure) in the control and ozone-exposed group was 4.43±0.05 (n=6) and 3.26±0.15 (n=12), respectively. Theophylline at 100 mg/kg, 10 mg/kg rolipram and 10 mg/kg T-440 significantly inhibited AHR with log PC(10) value of 4.73±0.16 (n=7), 3.74±0.11 (n=7), 3.82±0.15 (n=6), respectively. On histological examination, epithelial damage in the trachea and peripheral airways was recognized after ozone exposure. At 100 mg/kg, rolipram, T-440 and theophylline caused complete inhibition of AHR, and prevented epithelial damage of the trachea and peripheral airways. These results indicate that PDE inhibitors prevent not only ozone-induced AHR but also airway epithelial injury by ozone.