Kan Terawaki

Absence of Leukotriene B4 Receptor 1 Confers Resistance to Airway Hyperresponsiveness and Th2-Type Immune Responses

Bronchial asthma is an increasingly common disorder that remains poorly understood and difficult to manage. The disease is characterized by airway hyperresponsiveness, chronic inflammation, and mucus overproduction. Based on the finding that leukotriene B4 receptor 1 (BLT1) is expressed highly in Th2 lymphocytes, we analyzed the roles of BLT1 using an OVA-induced bronchial asthma model. BLT1-null mice did not develop airway hyperresponsiveness, eosinophilic inflammation, and hyperplasia of goblet cells. Attenuated symptoms were accompanied by reduced IgE production, and accumulation of IL-5 and IL-13 in bronchoalveolar lavage fluid, suggesting attenuated Th2-type immune response in BLT1-null mice. Peribronchial lymph node cells of sensitized BLT1-null mice showed much attenuated proliferation and production of Th2 cytokines upon re-stimulation with Ag in vitro. Thus, LTB4-BLT1 axis is required for the development of Th2-type immune response, and blockade of LTB4 functions through BLT1 would be novel and useful in the effort to ameliorate bronchial asthma and related Th2-biased immune disorders.

Helix 8 of the Leukotriene B4 Receptor Is Required for the Conformational Change to the Low Affinity State after G-protein Activation

Recent studies have revealed that G-protein-coupled receptors contain a putative cytoplasmic helical domain, helix 8. Leukotriene B4 (LTB4) receptor 1 derivatives with truncated or mutated helix 8 showed much higher LTB4 binding than wild-type (WT) receptors. Similar to the WT receptor, LTB4 promoted guanosine 5′-3-O-(thio)triphosphate (GTPγS) binding in these mutants. Unlike the WT receptor, however, the addition of GTPγS did not inhibit LTB4 binding to the mutant receptors. Scatchard analyses revealed that mutants maintained high affinity for LTB4, even in the presence of excess GTPγS. Consistently, mutant receptors showed a more prolonged Ca2+ mobilization and cellular metabolic activation than the WT receptor. From mutational studies and three-dimensional modeling based on the structure of bovine rhodopsin, we conclude that the helix 8 of LTB4 receptor 1 plays an important role in the conformational change of the receptor to the low affinity state after G-protein activation, possibly by sensing the status of coupling Gα subunits as GTP-bound.

Identification of the intracellular region of the leukotriene B4 receptor type 1 that is specifically involved in Gi activation.

Many G-protein-coupled receptors can activate more than one G-protein subfamily member. Leukotriene B4 receptor type 1 (BLT1) is a high affinity G-protein-coupled receptors for leukotriene B4 functioning in host defense, inflammation, and immunity. Previous studies have shown that BLT1 utilizes different G-proteins (the Gi family and G16 G-proteins) in mediating diverse cellular events and that truncation of the cytoplasmic tail of BLT1 does not impair activation of Gi and G16 proteins. To determine responsive regions of BLT1 for G-protein coupling, we performed an extensive mutagenesis study of its intracellular loops. Three intracellular loops (i1, i2, and i3) of BLT1 were found to be important for both Gi and G16 coupling, as judged by Gi-dependent guanosine 5′-(γ-thio) triphosphate (GTPγS) binding and G16-dependent inositol phosphate accumulation assays. The i3-1 mutant, with a mutation at the i3 amino terminus, exhibited greatly reduced GTPγS binding but intact inositol phosphate accumulation triggered by leukotriene B4 stimulation. These results suggest that the i3-1 region is required only for Gi activation. Moreover, in the i3-1 mutant, the deficiency in Gi activation was accompanied by a loss of the high affinity leukotriene B4 binding state seen with the wild type receptor. A three-dimensional model of BLT1 constructed based on the structure of bovine rhodopsin suggests that the i3-1 region may consist of the cytoplasmic end of the transmembrane helix V, which protrudes the helix into the cytoplasm. From mutational studies and three-dimensional modeling, we propose that the extended cytoplasmic helix connected to the transmembrane helix V of BLT1 might be a key region for selective activation of Gi proteins.

Attenuated Th1 induction by dendritic cells from mice deficient in the leukotriene B4 receptor 1

Dendritic cells (DCs) are important antigen-presenting cells that control Th1- and Th2-type immunological reactions by releasing cytokines and interacting directly with T cells. Leukotriene B4 (LTB4), a classical proinflammatory lipid mediator for phagocytes, was recently identified as an important attractant for effector CD4(+) and CD8(+) T cells. However, little information is available on the roles of LTB4 and its receptor BLT1 in DCs. Here we show that functional BLT1 expressed in mouse bone marrow-derived DCs (BMDCs) plays important role in initiating Th1-type immune response. Detailed analyses using BMDCs revealed that BLT1-deficient DCs produced less IL-12p70 than WT DCs, leading to attenuated IFN-gamma production in an allogeneic mixed lymphocyte reaction. Adoptive transfer of antigen-loaded BLT1-deficient DCs into naïve WT mice induced a weakened Th1- and enhanced Th2-response in vivo compared to WT DCs. BLT1-deficient mice consistently showed much attenuated delayed-type hypersensitivity (DTH), in which Th1-type cellular responses play a key role, and popliteal lymph node cells of BLT1-deficient mice showed reduced production of Th1 cytokines after DTH induction compared to cells from WT mice. Thus, in addition to its role in inflammation, the LTB4-BLT1 axis is important in initiating Th1-type immunological reactions mediated by DCs.

Long-Term Survival of a Baby with Body Stalk Anomaly: Report of a Case

Body stalk anomaly is characterized by severe scoliosis, severe pulmonary hypoplasia, and giant omphalocele. The prognosis of the disease is poor and most obstetricians consider it fatal. Very few patients with body stalk anomaly survive. We report the case of a baby diagnosed with body stalk anomaly in fetal life, who was saved by intensive care after birth. We closed the giant omphalocele successfully by placing karaya gum sheets over it, which created a humidified environment and promoted natural skin epithelization over the skin defect.

A Pilot Study of Laparoscopic Gastric Pull-Up by Using the Natural Orifice Translumenal Endoscopic Surgery Technique: A Novel Procedure for Treating Long-Gap Esophageal Atresia (Type A)

AIM This study aimed to develop a novel procedure for treating long-gap pure esophageal atresia. This procedure, which entails the combined use of laparoscopy and natural orifice translumenal endoscopic surgery (NOTES), would enable primary repair without cervical and thoracic incisions and prevent postoperative gastroesophageal reflux disease (GERD). METHODS Nonsurvival experiments were conducted in 9 pigs to study the technical feasibility. The procedure comprised the following: (1) creation of the disease model by laparoscopic resection of the lower esophagus; (2) laparoscopic fundoplication, complete mobilization of the stomach, and enlargement of the esophageal hiatus; (3) formation of a peroral transesophageal entry site into either the postmediastinum or the right thoracic cavity followed by fashioning a tunnel to the peritoneal cavity; (4) gastric pull-up by using both laparoscopy and NOTES; (5) esophagoesophageal anastomosis using BraceBar™, a prototype of the double T-bar suturing device (Olympus Medical Systems Co., Tokyo, Japan). RESULTS Laparoscopic procedures were performed without complications. The postmediastinal tunnel was successfully created three times with a complication of pleural injury. However, gastric pull-up via this route could not be completed due to porcine anatomical reasons. Gastric pull-up through the right thoracic route was achieved five times in six attempts. Two disorientations and a hemorrhagic death occurred during the procedures. CONCLUSIONS This study showed that combined use of laparoscopy and NOTES enabled gastric pull-up without cervical and thoracic incisions. Our method has the potential of lowering the incidence of GERD and enabling primary repair of this disease.

Tracheoplasty with cartilage-engineered esophagus environments

PURPOSE Our objective was to investigate the feasibility of engineering cartilage on the esophagus layer and outside the esophagus. Moreover, we investigated the feasibility of tracheoplasty with cartilage engineered on the esophagus in rabbits. METHODS Chondrocytes were isolated from auricular cartilages. 1. Engineered cartilage formation by histological findings on/into the esophageal layer was compared with that of injectable scaffold and preformed scaffold with chondrocytes. 2. Chondrocytes adhered to gelatin+vicryl mesh™ and b-FGF, were implanted on the outer esophageal surface. Four weeks after seeding, we found that cartilage was implanted in the midposterior portion of the cervical trachea (n=5), and it was retrieved 8weeks after seeding. RESULTS 1. A gelatin sponge incorporating β-TCP with vicryl mesh™ showed the best performance for fabricating engineered cartilage on the outer side of the esophagus. 2. Two of 5 rabbits died due to obstructed esophagus. Cartilage engineered outside the esophagus by a composite scaffold as the main material in the gelatin sponge, maintained the airway structure for up to 1month after implantation. Tracheal epithelial regeneration occurred in the internal lumen of this engineered cartilage. CONCLUSION Tracheoplasty with cartilage engineered outside the esophagus may be useful for reconstructing airways.

Slow release of basic fibroblast growth factor (b-FGF) promotes growth of tracheal cartilage ☆

PURPOSE Tracheomalacia is a major cause of morbidity in conditions such as oesophageal atresia. However, symptoms usually improve with age. A more rapid growth of tracheal cartilage can be induced by basic-Fibroblast Growth Factor (b-FGF). This study aimed to investigate whether slow-release b-FGF could act as a novel treatment for tracheomalacia. METHODS Biodegradable gelatin hydrogel sheets incorporating 0.5, 5, or 50 μg/20 μl of b-FGF solution were inserted between the cervical trachea and esophagus of rats. No intervention was performed in rats in a control group. All animals were sacrificed 4 weeks later, and the luminal area of the cervical trachea and the thickness of the cartilage were measured. RESULTS The mean luminal areas in the control group and in the b-FGF groups were 3.1, 3.2, 3.8, and 2.6mm(2), respectively, and showed a peak area at 5 μg of b-FGF. A significant difference was seen only between the control group and the b-FGF 5 μg group (p<0.05). The mean thickness of the tracheal cartilage was 0.12, 0.13, 0.19, and 0.32 mm in the control and the b-FGF groups, respectively, and showed a dose-dependent increase, which was statistically significant between the b-FGF 5 μg or 50 μg groups and the control group (p<0.01). CONCLUSION This study showed that slow-release b-FGF enlarges the tracheal lumen and thickens the cartilage in a dose-dependent fashion.

Translumenal Esophageal Anastomosis for Natural Orifice Translumenal Endoscopic Surgery: An Ex Vivo Feasibility Study

AIM This study aimed to develop a novel procedure for esophagoesophageal anastomosis for natural orifice translumenal endoscopic surgery (NOTES). MATERIALS AND METHODS An ex vivo feasibility study was performed in eight porcine models. The procedure was as follows: (1) A BraceBar™ (Olympus Medical Systems Corp., Tokyo, Japan), a double T-bar suturing device, was placed endoscopically at the blind end of the upper esophagus (UE). (2) The blind end was incised, and the scope was advanced out of the esophagus. (3) A balloon catheter was inserted into the lower esophagus (LE). (4) The catheter and a thread on the BraceBar were withdrawn so that the end of the UE was inverted, and the LE was pulled into the UE. (5) After the catheter was removed, a short tube was placed inside the duplicated part of the esophagus via the transgastric route. (6) A double ligature was performed using a ligating device over the tube. A liquid leak test was performed after the procedure. RESULTS All steps in this procedure were technically successful under the endoscopic visualization without any assistance from outside of the esophagus. The median time of this procedure was 31 (23-66) minutes. The median internal pressure of the UE was 122 (82-142) mm Hg when the anastomosed esophagus was separated into two specimens during the leak test. CONCLUSIONS Translumenal esophagoesophageal anastomosis was feasible. The duration of the procedure was short, and the anastomoses appear to have sufficient strength for use in clinical practice. An in vivo survival study is needed to confirm the safety and reliability of this NOTES procedure.

Tumor cell Dynamics and Metastasis in Advanced Neuroblastoma

This study deals with the advancement process of neuroblastoma through clinical observations and circulating tumor cell exploration. Clinical feature, tumor biology, and circulating tumor cell detected by the previously described polymerase chain reaction(PCR) method were analyzed in 31 patients with advanced neuroblastoma treated in our department since 1991 through 2004. Treatment was completed in 28 patients, of whom 17 are alive without the disease and 11 died. The primary lesion was not confirmed in 2 patients with disseminated metastasis, both of whom showed positive circulating tumor cell. Circulating tumor cell was positive in 6 of 9 examined at their first appearance at the hospital, all had stage 4 disease, and 4 of the 6 (66.7%) died of systemic spread of the disease. N-myc was amplified in 15 patients, of whom only 2 (13.3%) died of systemic metastasis. N-myc amplification did not correlate with positive circulating tumor cell. A certain population of neuroblastoma may provide circulating tumor cells from the early period of the disease to form metastatic lesions independently of the primary lesion, which must be regulated by factors other than N-myc. Circulating tumor cells may suggest higher risk for systemic dissemination and poor prognosis.