Improving the Stability of Protein-Protein Interaction Assay FlimPIA Using a Thermostabilized Firefly Luciferase.

The protein-protein interaction assay is a key technology in various fields, being applicable in drug screening as well as in diagnosis and inspection, wherein the stability of assays is important. In a previous study, we developed a unique protein-protein interaction assay "FlimPIA" based on the functional complementation of mutant firefly luciferases (Fluc). The catalytic step of Fluc was divided into two half steps: D-luciferin was adenylated in the first step, while adenylated luciferin was oxidized in the second step. We constructed two mutants of Fluc from Photinus pyralis (Ppy); one mutant named Donor is defective in the second half reaction, while the other mutant named Acceptor exhibited low activity in the first half reaction. To date, Ppy has been used in the system; however, its thermostability is low. In this study, to improve the stability of the system, we applied Fluc from thermostabilized Luciola lateralis to FlimPIA. We screened suitable mutants as probes for FlimPIA and obtained Acceptor and Donor candidates. We detected the interaction of FKBP12-FRB with FlimPIA using these candidates. Furthermore, after the incubation of the probes at 37°C for 1 h, the luminescence signal of the new system was 2.4-fold higher than that of the previous system, showing significant improvement in the stability of the assay.

Th17 cells reflect colon submucosal pathologic changes in active eosinophilic granulomatosis with polyangiitis.

BACKGROUND: Chronic eosinophilic pneumonia (CEP) or eosinophilic gastroenteritis (EG), or both, with asthma precede the onset of eosinophilic granulomatosis with polyangiitis (EGPA) in half of all EGPA patients. It is not known what determines whether patients with CEP or with EG following asthma will develop EGPA. METHODS: We studied 17 EGPA patients and 12 patients with CEP but without EGPA. We assayed serum ICAM-1, VCAM-1, and VEGF, and the percentage of peripheral blood CD4(+) T cells producing IL-17 (Th17 cells), at both onset and remission. We also examined the numbers of submucosal eosinophils and the basement membrane-to-crypt and crypt-to-crypt distance to evaluate edema in the colon submucosa at onset and remission in EGPA and at onset in CEP. RESULTS: Nine of 12 (75.0%) CEP patients had symptoms or endoscopic findings. Colonic submucosal eosinophil counts and edema in EGPA at onset were greater than at remission or in CEP at onset. Th17 cells (%) and serum ICAM-1 levels at onset were greater in EGPA than in CEP. In EGPA, peripheral blood Th17 cells (%) were significantly correlated with serum ICAM-1 level, colonic submucosal eosinophil count, and degree of edematous change; inversely correlated with serum VEGF level; but not correlated with VCAM-1 level. CONCLUSIONS: Eosinophilia and colonic submucosal edematous change were greater in EGPA than in CEP. The mechanism of vasculitis in EGPA appears related to increases in serum Th17 cell numbers and ICAM-1 levels and decreases in VEGF levels.

Advanced gastric cancer showing long-term complete remission in response to S-1 monotherapy: two case reports.

We herein report two cases showing long-term complete remission (CR) in response to S-1 monotherapy. Case 1 was a 65-year-old male diagnosed with an advanced poorly differentiated adenocarcinoma of the stomach with paraaortic lymph node metastases, which disappeared after S-1 monotherapy. Subsequently a total gastrectomy was performed, and histological CR was evident. His progress is presently uneventful without recurrence 50 months after surgery. Case 2 was a 59-year-old female who underwent a total gastrectomy with a jejunal pouch. The resected tumor was a medullary type poorly differentiated adenocarcinoma infiltrating the serosa and involving the regional lymph nodes. One year after surgery, endoscopy revealed a recurrent tumor in the jejunal pouch. After the administration of S-1, this recurrent tumor completely disappeared, and she has since maintained CR for 39 months. These cases suggest that a subgroup of patients with advanced gastric cancer may attain CR with S-1 monotherapy.

Optimal dose-finding study of bi-weekly paclitaxel in unresectable advanced or recurrent gastric cancer.

BACKGROUND: Tri-weekly and weekly regimens of paclitaxel have been reported to be effective in unresectable advanced or recurrent gastric cancer. The present study was conducted to determine the optimal dose of bi-weekly paclitaxel and evaluate safety and antitumor effect. PATIENTS AND METHODS: The study included patients with a histologically confirmed diagnosis of advanced or recurrent gastric cancer. Paclitaxel was intravenously infused for 1 h bi-weekly. One cycle consisted of four weeks and at least two cycles were performed. Dose levels 1, 2, 3, 4 and 5 were 100, 120, 140, 160 and 180 mg/m2, respectively. RESULTS: Of the 21 patients enrolled, 18 patients (85.7%) had received prior treatment. The maximum tolerated dose was 160 mg/m2 (level 4) and dose-limiting toxicities included grade 3 fatigue, anorexia and neuropathy and grade 4 neutropenia and allergic reaction. The recommended dose was designated as to be 140 mg/m2 (level 3). At this dose level, only one patient experienced a dose-limiting toxicity of neutropenia. No patient had grade 3 or higher non-hematological toxicity. The response rate was 12.5% (2/16) and 14 patients (87.5%) had partial response or stable disease. The median number of treatment courses was four (up to 13 courses). The median survival time was 222 days and the time to progression was 76 days. CONCLUSION: In advanced or recurrent gastric cancer, bi-weekly paclitaxel may be safely administered at a recommended dose of 140 mg/m2. A phase II study is now underway.

[A case of long-term survival of 3-years 4 months after combination chemotherapy of MTX, 5-FU and low-dose CDDP (MFP) for type 4 gastric cancer with pleuritis, peritoneal dissemination and Krukenberg tumor].

A 41-year-old woman presented to the Department of Obstetrics and Gynecology of our hospital because of abdominal distension and irregular genital bleeding. Computed tomography and ultrasonography of the abdomen revealed bilateral ovarian tumors, massive ascites, and bilateral pleural effusion. Type IV advanced gastric cancer was diagnosed on upper gastrointestinal endoscopy. The patient was admitted to our department. She received 3 courses of combination chemotherapy with methotrexate, 5-fluorouracil, and low-dose cisplatin. Pleural effusion and ascites disappeared. Surgery (total gastrectomy, resection of the tail of the pancreas, lymph-node dissection, total hysterectomy, and adnexectomy) was performed, and the patient was discharged. Chemotherapy was repeated after surgery. Lymph-node metastasis recurred 1 year 8 months after the start of chemotherapy. Treatment was switched to irinotecan plus cisplatin, and the lymph nodes shrank. After 9 months, 3 courses of TS-1 were administered. Two years 10 months after starting chemotherapy, abdominal and low back pain developed. Bone scintigraphy revealed bone metastasis. Lymph node swelling was present. The patient responded to radiotherapy with chemotherapy (cisplatin plus 5-fluorouracil). Subsequently, abdominal computed tomography showed lymph-node swelling, multiple metastases to the liver, ascites, and a right pleural effusion. She was readmitted to the hospital and received intraperitoneal chemotherapy with cisplatin. Her condition deteriorated, and she died. The patient survived for about 3 years 4 months after the start of treatment. Chemotherapy with methotrexate, 5-fluorouracil, and low-dose cisplatin may thus be an effective therapeutic option in patients who have advanced gastric cancer with peritoneal dissemination.

[Clinical development of S-1 (TS-1) for advanced gastric cancer].

The 5-FU plus cisplatin containing regimen like FP, ECF and DCF, is considered to be the most effective treatment for advanced gastric cancer in the United States, Europe, and Korea. In Japan, oral fluoropyrimidine S-1 (TS-1) is currently considered to be the first candidate as the standard drug for advanced gastric cancer. S-1 based combination therapies with other promising drugs like cisplatin, irinotecan and taxanes, are expected to yield good results. Above all, S-1+CDDP therapy showed a high efficacy and expected to be a standard therapy for advanced gastric cancer. Two large phase III studies, JCOG 9912 5-FU vs S-1 vs CPT-11 +CDDP and S-1 vs S-1+CDDP, are now on going to establish an acceptable frontline standard for patients with AGC. We therefore need to develop new agents and combination chemotherapy regimens to achieve a greater survival benefit in AGC.

[A gastric stromal tumor (GIST) with a prolonged partial response to a reduced dose of imatinib mesilate].

A 78-year-old woman was admitted to our hospital because of tarry stools. A gastric stromal tumor with liver metastasis was diagnosed. Treatment with imatinib mesilate was begun in a dose of 400 mg daily. After 1 month, the primary tumor showed a partial response; the response of the liver metastasis was stable disease. However, grade 2 edema, leukocytopenia, and anemia developed, and the dose of imatinib mesilate was reduced to 200 mg daily. The adverse reactions resolved promptly, and a partial response of both the primary tumor and liver metastasis to imatinib mesilate has been maintained for 28 months. Strategies for lowering the dose of imatinib mesilate are reviewed.

Small-cell carcinoma of the esophagus associated with a paraneoplastic neurological syndrome: a case report documenting a complete response.

We describe a case of small-cell carcinoma of the esophagus associated with a paraneoplastic neurological syndrome. Sensorimotor neuropathy had developed 3 years earlier, and neurological symptoms had slowly worsened. Small-cell carcinoma of the esophagus was incidentally diagnosed while investigating the cause of the neurological symptoms. A paraneoplastic neurological syndrome was diagnosed on the basis of cancer and exclusion of other known causes of neurological symptoms. The patient was given combination chemoradiotherapy. There was a complete response to three courses of chemoradiotherapy, with no evidence of disease recurrence 6 years after the diagnosis. There was no progression of paraneoplastic neurological symptoms after the complete response.

[Combination chemotherapy of TS-1 +cisplatin for inoperable gastric cancer].

There is no chemotherapy considered to be standard treatment for advanced gastric cancer worldwide, and there is no consensus as to whether combination or single agent therapy is preferred. In the phase I portion, a dose-escalation study of cisplatin (CDDP) combined with TS-1, new oral dihydropyrimidine dehydrogenase inhibitory fluoropyrimidine, was performed to determine the maximum-tolerated dose (MTD), recommended dose (RD), dose-limiting toxicities (DLTs), and objective response rate (RR) in advanced gastric cancer (AGC). TS-1 was given orally at 40 mg/m2 bid for 21 consecutive days following a 2-week rest. CDDP was planned to be given intravenously on day 8, at a dose of 60, 70, or 80 mg/m2, depending on the DLT. Treatment was repeated every 5 weeks, unless disease progression was observed. In the phase I portion, the MTD of CDDP was presumed to be 70 mg/m2, because 33.3% of patients (2/6) developed DLTs; mainly neutropenia. Therefore, the RD of CDDP was estimated as 60 mg/m2. In the phase II portion, 19 patients including 6 patients of the RD phase I portion were evaluated. The median administered courses was 4 (range: 1-8). The incidence of haematological and non-haematological toxicities (> or = grade 3) was 15.8 and 26.3%, respectively, but all were manageable. The RR was 74% (14/19, 95%) confidence interval: 54.9 (90.6%), and the median survival days were 383. This regimen is considered to be active against AGC with acceptable toxicity. In addition, currently, a randomized phase III study (JCOG 9912) for AGC patients not treated previously with chemotherapy is underway in Japan. It compares three arms: 5-FU alone, TS-1 alone and CPT-11 with CDDP therapy. We also initiated a randomized phase III study comparing TS-1 alone, and with CDDP for AGC. From those two phase III studies, we may be able to evaluate the clinical benefit of TS-1 in combination with CDDP versus TS-1 single, or 5-FU combined with CDDP therapy in terms of survival benefits and improving the QOL for AGC patients.