Potential curing and beneficial effects of Ooitabi (Ficus pumila L.) on hypertension and dyslipidaemia in Okinawa.

BACKGROUND: Over 30% of the population of Okinawa Prefecture have a high body mass index. The incidence of hypertension and dyslipidaemia has also increased in recent years. We found that Ooitabi (Ficus pumila L.), a plant native to Okinawa, was useful for hypertension. During ancient times, the extracts of Ooitabi leaves were used for making Ishimaki tea in some areas of Okinawa Prefecture. The plants in Okinawa are rich in antioxidants, and four flavonoid glycosides, including rutin, have been identified in Ooitabi. METHODS: In the present study, we conducted clinical verification tests on the effects of drinking Ishimaki tea on outpatients with hypertension and dyslipidaemia. Of 3814 Japanese patients who underwent medical check-ups in Okinawa, 38 individuals with high blood pressure, dyslipidaemia, liver dysfunction and gout visited our hospital as outpatients and were asked to drink Ishimaki tea. RESULTS: After 3 months, there were significant reductions in body mass index, systolic and diastolic blood pressure, total cholesterol, low-density lipoprotein cholesterol, γ-glutamyltrans peptidase, uric acid and ratio of blood vessel insulin resistance. CONCLUSIONS: Ooitabi extract can lower blood pressure and improve lipid abnormalities and has likely contributed to the well-known health and longevity of the population in Okinawa.

Regeneration of transformed verbena (Verbena x hybrida) by Agrobacterium tumefaciens.

Verbena (Verbena x hybrida), an important floricultural species, was successfully regenerated from stem segments on Murashige and Skoog's basal medium supplemented with thidiazuron and indole-3-acetic acid. A transformation system was developed using cvs. Temari Scarlet, Temari Sakura, Tapien Rose and TP-P2. Agrobacterium tumefaciens strain Agl0 harboring the sGFP gene was infected into stem segments. Transformation efficiency was improved by evaluating and manipulating the age of the plant material, the concentration of kanamycin in the medium during selection, and the length of the culture period in the dark. After 2-3 months of culture on the selection medium, GFP-positive shoots were obtained in all four of the cultivars tested. These shoots were successfully acclimated and set flowers within 2-3 months in a greenhouse. GFP was expressed in all of the organs including the floral parts. Stable genomic transformation was confirmed by Southern blot analysis. No morphological differences were observed between the transformed plants and their host plants.

[A patient with esophageal cancer with subcutaneal abscess and esophago-tracheal fistula who survived more than 2 years following treatment by drainage and chemoradiation therapy].

A 48-year-old man presented at the hospital because of neck swelling and pain. A diagnosis of esophageal cancer with subcutaneous abscess was made based on examination and biopsy results. The cancer was Ce T4NxMx Stage III-IVa. Curative surgery was considered impossible, so chemoradiation therapy was performed (5-FU 500 mg + CDDP 5 mg/day + 2 Gy/day x 31 days) after drainage. During the therapy, an esophago-tracheal fistula was observed, but it later vanished. After chemoradiation therapy, the abscess and tumor vanished. No serious adverse reactions were observed. Now, 2 years after therapy, no recurrence has been found. The patient is now in good health with no symptoms and undergoes regular check-ups. Chemoradiation therapy is effective for inoperable advanced esophageal cancer.

[Diagnosis of the postoperative local recurrence of rectal cancer by FDG-PET--a case report].

Positron emission tomography (PET) using 18F-fluorodeoxyglucose (FDG) as the tracer of glucose metabolism was performed to identify a postoperative recurrent lesion of rectal cancer. A 66-year-old-man underwent trans-sacral local resection of the rectum for rectal cancer in 1992. A local recurrent mass was discovered, and abdomino-perineal resection of the rectum was performed in 1999. The serum CEA level increased gradually August in 2000, but there was no sign of recurrence on CT or MRI. FDG-PET was performed to reveal a presacral recurrent lesion. Total pelvic evisceration combined with resection of the sacrum, and a bilateral ureterostomy were performed in April 2001. The beneficial role of FDG-PET in the diagnosis of the postoperative local recurrence of rectal cancer is emphasized.

[A case of recurring breast cancer causing pericardial metastasis showing a good response following local treatment with methotrexate].

A 52-year-old woman was admitted with a chief complaint of dyspnea. She had undergone right mastectomy for Stage IIB breast cancer 2 years and five months earlier. Chest roentgenogram revealed cardiomegaly and bilateral pleural effusion, and a cardiac echogram showed marked retention of pericardial effusion. A diagnosis of cardiac tamponade was made and pericardiocentesis for continuous drainage was carried out cytologically, the effusion was class V and showed evidence of pericardial metastasis of breast cancer. Pericardiocentesis followed by methotrexate instillation 6 times in a dose of 110 mg successfully controlled the cardiac tamponade, after which the catheter could be removed from the pericardial space. Systemic chemotherapy (CEF) was started at the same time. The patient was discharged very much improved after these treatments, but she died of brain metastasis after 9 months. This case suggests that intrapericardial application of methotrexate may be very useful in the management of carcinomatous cardiac tamponade without any serious side effects.

[Sigmoid colon cancer with multiple liver metastasis (H3) effectively treated with CPT-11 and DSM (degradable starch microsphere) HAI therapy and intensive high dosage 5-FU HAI therapy--a case report].

A weekly HAI therapy (CPT-11 80 mg, MMC 4 mg, degradable starch microsphere (DSM) 600 mg) was given to a patient with sigmoid colon cancer and multiple liver metastasis (H3) who had been taking tegafur 300 mg/day and 5-FU 750 mg HAI/week, which resulted in PD. This therapy was carried out on an outpatient basis with minimum side effects (< grade 2). After 8 weeks, the tumor marker dropped to one tenth and the liver metastasis decreased in size (PR). The time courses of the concentrations of CPT-11, SN-38 and SN-38G were determined by drawing blood after HAI with or without DSM. The Cmax and AUC inf. of SN-38 at HAI without DSM were 17 ng/ml and 90.55 ng/h/ml, respectively, which was comparable to that at i.v. administration. The Cmax and AUC inf. of SN-38 at HAI with DSM were 12 ng/ml and 129.19 ng/h/ml, respectively, implying that DSM might have an enhancing effect on CPT-11 due to stasis of the hepatic artery that slows the conversion of CPT-11 to SN-38 resulting in a longer existence of SN-38.

[A case of locally advanced breast cancer successfully resected after selective intra-arterial chemotherapy].

A 25-year-old female with a large tumor on her left breast was examined at our hospital from August, 1999. Ipsilateral supraclavicular, infraclavicular and axillary lymph nodes were swollen. She was diagnosed as having locally advanced breast cancer of stage IIIb by fine needle aspiration cytology. After the administration of docetaxel (60 mg/m2/3 weeks x 3) failed to improve her condition, we changed the treatment to selective intra-arterial chemotherapy with THP-ADR (60 mg/body/day, day 1 & 8, 41 & 48) by Seldinger's method. The target vessels were the internal thoracic, lateral thoracic, thoracodorsal and deep cervical arteries. We also combined 5-FU 500 mg/body div and CPA 500 mg/body i.v. on the same days with intra-arterial chemotherapy. As a result, the main tumor and metastatic lymph node swelling was remarkably reduced (down-staging was obtained). No recurrence was found for 5 months after curative resection.

[Repetitive chemo-embolization with degradable starch microspheres (DSM) to each left and replaced right hepatic artery in a patient with multiple liver metastases of colon cancer].

In a patient with a right hepatic artery arising from the superior mesenteric artery bearing multiple liver metastases from colon cancer, hepatic arterial chemo-embolization was performed in combination with degradable starch microspheres (DSM) administered independently to the left and replaced right hepatic artery via a percutaneal approach. As the first line chemotherapy from hepatic artery with DSM 300 mg, 5-FU 500 mg and MMC 10 mg resulted in PD. DSM 300 mg, epirubicin (EPI) 50 mg, MMC 4 mg was administered with the RHA:LHA ratio of 3:1 as a second line. Four weeks later it was evaluated as NC by angiography and by tumor-marker dropped extremely. The same regimen was repeated every four weeks, and the NC status remained for 20 weeks in total. Each time, the left and replaced right hepatic artery got perfect re-perfusion and DSM enabled an effective whole liver distribution of anti-cancer drugs and repetitive administrations of them. This regimen could be an alternative choice for patients with a replaced right hepatic artery who have liver metastasis of colon cancer.

[A case of locally advanced rectal carcinoma with liver metastasis treated with a combination of CRT, HAI, and systemic chemotherapy].

A 55-year-old man with locally advanced rectal carcinoma and liver metastasis was treated with a combination of chemo-radiotherapy (5-FU suppository 100 mg/day and 63 Gy of RT), hepatic arterial infusion chemotherapy (5-FU 1,000 mg/3 h, biweekly), and systemic chemotherapy (5'-DFUR 800 mg/day + cimetidine 800 mg/day). His rectal tumor was reduced and his symptoms such as pain and bleeding had markedly decreased. The river metastasis did not change during the entire course. HAI and administration of 5-FU suppository, 5'-DFUR, and cimetidine were continued. As of 18 months after the onset of the combination therapy, NC has been maintained, and the general condition of the patient is favorable.

Pulmonary hypoplasia induced by liquid paraffin injection into fetal thoracic cavity with special reference to renal development in rats.

The present study was designed to clarify lung-kidney interrelation in fetal rats. On fetal day 20, liquid paraffin (LP) was injected into fetal thoracic cavity to produce pulmonary hypoplasia. No significant difference in body and renal weights were noted between the LP injected and control fetuses. The weight of lung, however, was significantly lower in the LP injected fetuses than in the control ones. Histological examinations on the lung and kidney of the LP injected fetuses revealed that the lung was hypoplastic characterized by rich interstitium and reduced air spaces. In the kidney, mature types of glomeruli and profiles of proximal tubules near them were increased in number. Furthermore, strong expression of EGF immunoreactivity was noted in the apical cytoplasm of epithelium of the proximal tubules in the LP injected fetuses. These findings indicate that lung-kidney interrelation exists in fetal rats during late gestational days, and suggest that interruption of the lung development induces accelerated growth of the kidney in fetal rats.

[Evaluation of intermittent hepatic arterial infusion chemotherapy for multiple liver metastasis of colorectal cancer].

Five patients with synchronous multiple hepatic metastasis of colorectal cancer were treated with hepatic arterial infusion chemotherapy. All cases received intermittent 5-FU infusion (5-FU 250-1,000 mg/2-3 hrs/1-2 weeks) on an outpatient basis. In the evaluation of 5 cases, 3 PR and 1 NC were observed. One case administered arterial infusion for adjuvant chemotherapy has no recurrence in liver. In two patients, extra-hepatic metastases were found. In conclusion, this therapy was effective and useful for hepatic metastasis. Moreover, other forms of treatment for extra-hepatic metastasis must be used.

[Intraperitoneal cisplatinum chemotherapy in patients with carcinomatous peritonitis due to colorectal scirrhous cancer--case report].

Two patients received intraperitoneal cisplatinum chemotherapy for carcinomatous ascites due to colorectal cancer recurrence. The patients were a 47-year-old man who had rectal cancer and 51-year-old woman who had colon cancer. They had received the operation and adjuvant chemoradiation therapy and chemotherapy respectively. However, five months and two years after resection, respectively, they presented massive ascites due to carcinomatous peritonitis and were given cisplatin injection intraperitoneally. The amount of ascites was significantly diminished. One patient had been discharged and been able to stay at home, and the other patient underwent gastrostomy for ileus. The results suggested that intraperitoneal cisplatinum chemotherapy may be useful for the patient with carcinomatous ascites due to colorectal cancer.

[Palliative chemo-radiotherapy for abdominal recurrence of esophageal carcinoma--case report].

A 71-year-old man with stage IV esophageal carcinoma was treated by chemo-radiotherapy (5-FU 500 mg/day + CDDP 10 mg/day for 4 weeks and 67.6 Gy of RT). The esophageal tumor showed a complete response to the treatment. Six months later, he had obstructive jaundice due to an abdominal recurrent mass. A secondary (palliative) CRT was performed (5-FU 500 mg/day + CDDP 10 mg/day for 3 weeks and 45 Gy of RT). The abdominal tumor became remarkably smaller and jaundice disappeared. Though the patient died from pulmonary carcinomatous lymphangitis, the primary lesion showed CR. CRT was very effective for local treatment and for palliative therapy.

[A case of multiple bone metastases from advanced breast cancer effectively treated with pamidronate].

A 57-year-old woman, with bone, lymph node and skin metastases underwent mastectomy and extirpation of skin tumors. Chemoenderine-therapy was performed from the 15th day after operation, with a toremifene and CEF regimen consisting of cyclophosphamide, epirubicin and 5-fluorouracil. She had nausea and neurological symptoms from hypercalcemia (21.5 mg/dl) on the 28th day after operation. Her serum PTHrP level was found to be high at 214 pmol/l. We administered pamidronate in a dose of 45 mg biweekly, and she improved. The CEF regimen and pamidronate therapy was continued for 6 cycles and the regions of bone metastases were reduced on the bone scintography. Thereafter she has been administered pamidronate 30 mg/4 weeks as an outpatient with no further symptoms, and serum Ca and PTHrP have remained normal. In conclusion, pamidronate combined with chemotherapy can be a therapeutic option for not only hypercalcemia but also bone metastases of breast cancer.

Augumentation of splenic antitumor immunity by local immunotherapy in gastric cancer patients.

We previously reported that the antitumor effect of OK-432, a streptococcal preparation, was markedly augmented when this agent was injected into tumors together with fibrinogen. In order to elucidate the effect of this treatment on the spleen, we assessed splenic function in gastric cancer patients receiving preoperative local immunotherapy with OK-432 and fibrinogen. Immunohistochemical studies of the spleen at 7 days after intratumoral injection therapy revealed numerous macrophages phagocytizing OK-432 in the splenic sinuses. Phenotypic analysis of splenocytes by flow cytometry revealed an increase in the CD4/CD8 ratio and in the expression of HLA-DR, CD25, and Leu M3 by splenic T cells of the patients treated with OK-432 plus fibrinogen when compared to patients treated with OK-432 alone or untreated patients. Splenic T cells from patients treated with OK-432 plus fibrinogen showed significantly higher cytotoxicity against Daudi and K562 cells than T cells from control patients (p < 0.05), and culture of these splenic T cells with recombinant IL-2 induced the expansion of lymphokine-activated killer cells. These results demonstrate that local immunotherapy with a mixture of OK-432 and fibrinogen effectively augumented splenic antitumor immunity in gastric cancer patients.

Analysis of cytotoxic activity of the CD4+ T lymphocytes generated by local immunotherapy.

We previously reported that the anti-tumour effect of OK-432 is considerably enhanced by its intratumoral injection together with fibrinogen. In the present study, we generated killer T cells by culturing tumour-infiltrating lymphocytes from thyroid cancer patients who had received this local immunotherapy. Phenotypic analysis revealed that the T cells were positive for CD3+, CD4+, Leu8-, CD45RO+ and T-cell receptor (TCR)alpha beta+, as well as showing strong surface expression of HLA-DR, CD25, LFA-1 and ICAM-1. The generated CD4+ T cells secreted interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha, TNF-beta, and interleukin (IL)-6 (but not IL-4), and exhibited a high level of cytolytic activity against several tumour cell lines. The cytolytic activity of these T cells for Daudi cells was inhibited by preincubation with an anti-intercellular adhesion molecule (ICAM)-1 antibody, but not by preincubation with anti-TCR alpha beta, anti-CD2, or anti-LFA-1 antibodies. Pretreatment with anti-ICAM-1 antibody inhibited T-cell cytolytic activity, but not conjugation with target cells. In addition, incubation with immobilised anti-ICAM-1 enhanced the secretion of IFN-gamma by T cells. We conclude that ICAM-1 expressed on the effector cytotoxic CD4+ T lymphocytes delivers regulatory signals that enhance IFN-gamma secretion.

A new family of zinc finger proteins in petunia: structure, DNA sequence recognition, and floral organ-specific expression.

We have previously cloned a gene for a zinc finger protein (EPF1) that is expressed specifically in petals and interacts with the promoter region of the 5-enolpyruvylshikimate-3-phosphate synthase gene in petunia. In an attempt to isolate genes encoding additional factors that interact with this promoter, we cloned four novel genes encoding zinc finger proteins (EPF2-5a, EPF2-5b, EPF2-4, and EPF2-7). Sequence analyses revealed that overall similarity between the EPF1 and the EPF2 protein family, except in the zinc finger motifs and the basic amino acid cluster, was very low, suggesting that the two groups belong to different subfamilies. DNA binding specificities of EPF1, EPF2-5, and EPF2-4 were very similar, as expected from the conserved zinc finger motifs. However, EPF2-7 showed no binding to the probes tested in spite of having the conserved motifs. DNA binding studies using a series of spacing mutant probes suggested a binding mechanism in which the EPF proteins recognize spacings in target DNA. RNA gel blot analyses and histochemical analyses with a promoter and beta-glucuronidase fusion revealed that expression of the EPF2-5 gene (EPF2-5) was petal and stamen specific. Expression of the EPF2-7 gene (EPF2-7) was sepal and petal specific and localized in vascular tissues. The preferential expression in two adjacent floral organs raises the possibility that these genes are downstream transcription factors of floral homeotic genes.

The effect of local immunotherapy for breast cancer using a mixture of OK-432 and fibrinogen supplemented with activated macrophages.

OK-432 is an immunomodulatory agent prepared from a strain of Streptococcus pyogenes. We have previously reported that intratumoral injection of a mixture of OK-432 and fibrinogen (hereinafter referred to as OK/fbg) is very effective in the local immunotherapy for colorectal cancer. However, we found that the intratumoral injection of OK/fbg into tumor tissues of breast cancers did not always induce a strong antitumor effect. With conventional OK/fbg treatment, tumor necrosis observed in breast cancer tumors was significantly less than that in colorectal cancer tumors; the formation of fibrin meshwork and macrophage infiltration, in particular, were poor. In this study, the OK/fbg mixture was supplemented with activated macrophages for local immunotherapy of breast cancers. Macrophages were prepared from peripheral blood of breast cancer patients and activated with 0.05 mg/ml of OK-432. Between 2-7 days before operation, a single intratumoral injection of the above mixtures was done. The addition of activated macrophages to the OK/fbg mixture resulted in marked degrees of fibrin meshwork formation, macrophage infiltration and cancer cell necrosis. These findings suggest that the recruitment of macrophages in tumor stroma and their activation are necessary for sufficient induction of antitumor immunity, and supplementation of activated macrophages at the site of immune reaction may be an alternative method for reinforcement of the antitumor effect of local immunotherapy.

Augmentation of antitumor immunity in regional lymph nodes by local immunotherapy.

We have previously reported that the antitumor effect of OK-432, a streptococcal preparation, is markedly augmented when injected intratumorally together with fibrinogen (OK-432/fbg) [1]. In order to elucidate the effects of this immunotherapy on regional lymph nodes (RLN), we carried out both morphological and functional analyses of the RLN from colonic cancer patients treated with OK-432/fbg. Computer-aided morphometry revealed that the maximal cross-sectional areas and the broadest diameters of the RLN were significantly greater (p < 0.01) in patients who had undergone local immunotherapy than in patients who had not. The component structures of RLN, such as sinus, follicle and paracortex, were all enlarged in the OK-432/fbg-treated patients, and necrosis of metastatic tumors was observed. RLN lymphocytes recovered from OK-432/fbg treated patients showed elevated reactivity to phytohemagglutinin (PHA) and the stimulation index was clearly higher than that of control patients. Flow cytometric analysis revealed a predominance of T-cells, especially CD4 subsets, and higher positivity for both CD25 and HLA-DR. Furthermore, RLN lymphocytes killed more effectively K562 and Daudi cells in the patients who had had immunotherapy. These results suggest that the effect of local immunotherapy with OK-432/fbg is not restricted to the site of injection but extends to the lymph nodes, and contributes to tumor regression through the augmentation of cellular immunity.

[Effect of intratumoral injection of the mixture of OK-432 and fibrinogen (OK/fbg) on gastric cancer].

We have reported the intratumoral injection of OK/fbg is a very effective immunotherapy for colorectal cancer. In this study, we injected OK/fbg into gastric cancer. Histopathological examinations revealed the formation of fibrin fibers, marked infiltration of inflammatory cells and regression of tumor tissue. Seven days after injection, cytotoxicity of splenic lymphocytes was significantly high and HLA-DR, CD25+ splenic lymphocytes were increased. But there was no significant change in peripheral blood and lymph node lymphocytes. On the contrary, several hours after injection, cytotoxicity and surface markers were changed in peripheral blood and lymph node lymphocytes. However, there was no change in splenic lymphocytes. We suppose that activation of splenic lymphocytes was caused by activation of peripheral blood and regional lymph nodes.