Cepharanthine induces the proliferation of human dermal papilla cells and stimulates vascular endothelial growth factor expression through increased intracellular calcium mobilization and hypoxia-inducible factor activation.

BACKGROUND: Cepharanthine (CEP), a compound extracted from the vine Stephania cephalantha, is commonly prescribed to treat alopecia areata; however, the scientific evidence for its efficacy is limited. AIM: To investigate the effect of CEP and its structural analogues on human hair growth in vitro. METHODS: The effects of CEP and three of its structural analogues on the proliferation of human dermal papilla cells (hDPCs) and human outer root sheath cells (hORSCs) were investigated. Their effects on vascular endothelial growth factor (VEGF) expression were also assessed by real-time PCR. Activation of pathways leading to VEGF expression, such as intracellular Ca2+ mobilization and hypoxia-inducible factor (HIF) expression, was also characterized. RESULTS: CEP and two of its structural analogues significantly stimulated the growth of hDPCs but not hORSCs. Moreover, CEP and all three structural analogues significantly induced the expression of VEGF in hDPCs. CEP increased the intracellular Ca2+ concentration in hDPCs. CEP also increased the expression of HIF-1α and HIF-2α and induced the expression of HIF-responsive genes in hDPCs, even under normoxia. CONCLUSIONS: These results suggest that CEP and its structural analogues have the potential to restore hair growth by promoting the proliferation of hDPCs and increasing their expression of VEGF.

Gene delivery to periodontal tissue using Bubble liposomes and ultrasound.

BACKGROUND AND OBJECTIVE: Periodontitis is the most common inflammatory disease caused by oral biofilm infection. For efficient periodontal treatment, it is important to enhance the outcome of existing regenerative therapies. The physical action of an ultrasound may be able to deliver a therapeutic gene or drugs into the local area of the periodontium being treated for periodontal regeneration. Previously, we developed "Bubble liposomes" as a useful carrier for gene or drug delivery, and reported that delivery efficiency was increased with high-frequency ultrasound in vitro and in vivo. Hence, the aim of the present study was to examine the possibility of delivering genes into gingival tissues using Bubble liposomes and ultrasound. MATERIAL AND METHODS: We attempted to deliver naked plasmid DNA encoding luciferase or enhanced green fluorescent protein (EGFP) into the lower labial gingiva of Wistar rats using Bubble liposomes, with or without ultrasound exposure. Ultrasound parameters were optimized for intensity (0-4.0 W/cm(2) ) and exposure time (0-120 s) to establish the most efficient conditions for exposure. The efficacy and duration of gene expression in the gingiva were investigated using a luciferase assay and fluorescence microscopy. RESULTS: The strongest relative luciferase activity was observed when rats were treated under the following ultrasound conditions: 2.0 W/cm(2) intensity and 30 s of exposure time. Relative luciferase activity, 1 d after gene delivery, was significantly higher in gingiva treated using Bubble liposomes and ultrasound than in gingiva of the other treatment groups. Histological analysis also showed that distinct EGFP-expressing cells were observed in transfected gingiva when rats were treated under optimized conditions. CONCLUSION: From these results, the combination of Bubble liposomes and ultrasound provides an efficient technique for delivering plasmid DNA into the gingiva. This technique can be applied for the delivery of a variety of therapeutic molecules into target tissue, and may serve as a useful treatment strategy for periodontitis.

"Obama system:" a new easy, safe, and economical technique for laparoscopic single-port surgery.

INTRODUCTION: Various approaches to laparoscopic single-site surgery (LESS) have been developed to reduce pain and other complications, promote recovery, and improve cosmetic outcomes, particularly relative to conventional open or laparoscopic surgery. Three-port procedures for LESS have been reported to be superior to single-port access, but they usually require expensive, technically sophisticated instruments. To avoid these problems, we have developed a modified procedure for performing LESS with a single port, referred to as the "Obama system." METHODS: From January 2009 through December 2010, we performed laparoscopic cholecystectomy (LC) in 61 patients. Conventional LC with three ports was performed in 39 patients, LESS with a SILS Port was performed in 4 patients, and modified LESS was performed using the Obama system in 18 patients. The operative results were compared. RESULTS: LC was successfully completed in all 61 patients, with no postoperative complications. The mean operating time was 102.3 min (C-reactive protein [CRP] ≤ 2) and 160.1 min (CRP > 2) in the 39 patients who underwent conventional LC, 108.3 min (CRP ≤ 2) in the 4 patients who underwent LESS with a SILS Port, and 116.5 min (CRP ≤ 2) and 186.5 min (CRP > 2) in the 18 patients who underwent LESS using the Obama system. No morbidity or mortality was associated with any technique. CONCLUSION: The Obama system is easier to use and more efficient and reliable than any other technique currently available for LESS. This system is expected to greatly contribute to the further development and wider acceptance of LESS.

MR and CT findings of leiomyomatosis peritonealis disseminata with emphasis on assisted reproductive technology as a risk factor.

Leiomyomatosis peritonealis disseminata (LPD) is a rare benign smooth muscle tumour located in the peritoneal cavity. Increased oestrogen exposure appears to be an aetiological factor for LPD. We report two cases of LPD after leiomyomectomy and assisted reproductive technology pregnancy, which can cause a high serum concentration of oestrogen. CT and MR scanning demonstrate many intraperitoneal well-demarcated nodules of varying size that mimic widespread intraperitoneal malignancy.

Methylenetetrahydrofolate reductase C677T is not associated with expression of pyrimidine metabolic enzyme genes in colorectal cancer.

Methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism may influence the chemosensitivity of colorectal cancers to fluorouracil (5-FU) by increasing intracellular 5,10-methylenetetrahydrofolate. The effect of this polymorphism on the expression of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), orotate phosphoribosyl transferase (OPRT) and thymidine phosphorylase (TP) in colorectal cancer was investigated. The MTHFR C677T polymorphism was analysed and TS, DPD, OPRT and TP mRNA expression was measured in tumour and adjacent normal mucosal tissue. In all patients, the genotypes of the tumour and normal tissues were identical. No differences were found in the expression of TS, DPD or TP mRNA by genotype in either tumour or normal tissue. Although the OPRT mRNA level in tumour tissue was not associated with the genotype, normal mucosa with the TT genotype showed a significantly higher OPRT mRNA level than mucosa with other genotypes. The MTHFR C667T polymorphism is not associated with intratumoural expression of TS, DPD, OPRT or TP.

[Collision cancer of squamous cell carcinoma and small cell carcinoma of the lung; report of a case].

We report a rare case of a primary collision cancer in the lung consisting of squamous cell carcinoma and small cell carcinoma. A 65-year-old man with an abnormal shadow in the right S6 was diagnosed as squamous cell carcinoma by transbronchial lung biopsy. A right lower lobectomy with mediastinal lymph node dissection was performed. The pathological stage of squamous cell carcinoma was IIIA (T2N2M0). The other element diagnosed by pathological examination was small cell carcinoma of which pathological stage was IA (T1N0M0). Each element was clearly distinguished and touched each other. Following the operation, the patient received systemic chemotherapy against small cell carcinoma with cisplatin and irinotecan hydrochloride for 1 course, and cisplatin and etoposide for 3 courses. Since the prognosis of collision cancer is generally reported to be influenced by more advanced element of cancer, the prognosis of the present case is suspected to be dependent on the squamous cell carcinoma.

[Pleomorphic lung cancer; a clinicopathologic study].

Pulmonary pleomorphic carcinoma is a comparatively rare histologic type of lung carcinoma, and the incidence among all lung carcinomas has been reported to be 0.4%. We reported our experience with 8 patients who had been diagnosed as pulmonary pleomorphic carcinoma, and discussed clinicopathologically the preoperative diagnosis and treatment. In 2 of 8 patients, preoperative transbronchial lung biopsy revealed spindle cell component, highly suggesting pulmonary pleomorphic carcinoma. All patients underwent surgical treatment and 2 of then had incomplete resections because of intrathoracic disseminations or carcinomatous pericarditis. Pathological findings showed invasions into the surrounding thoracic organs such as the chest wall, pericardium, adjacent pulmonary lobe or mediastinal pleura in 5 cases, intrapulmonary metastasis of the same lobe in 3 and lymph node involvement in 3. Recurrence occurred in 6 patients immediately after the operation. Although the preoperative diagnosis of biphasic tumor such as pulmonary pleomorphic carcinoma is difficult, it is possible to suspect the diagnosis when sarcomatous components were detected by preoperative biopsy. The efficacy of chemotherapy and radiotherapy have not been established yet, and thus we would like to emphasize that surgery might be the treatment of choice.

[Surgical resection of T3 and T4 lung cancer].

Lung cancer invading neighboring anatomical structures such as the chest wall, pericardium, diaphragm, and left atrium are categorized as T3 or T4, which is regarded as locally advanced lung cancer. The purpose of this study was to evaluate results of surgical treatment of T3-4N0-2M0 non-small cell lung cancer according to involved organs. From 1981 to April 2005, 148 patients with lung cancer invading neighboring organs were surgically treated in our hospital. The 5-year survival was 41.4% in all cases. According to 5-year survival of clinical characteristics, the chest wall (parietal pleura) group (45.5%) had a significantly better prognosis compared with the left atrium (0%, p = 0.03) and diaphragm (0%, p = 0.04) groups. T3N0 (50.3%), IIB (55.4%), IIIA (44.6%), and complete resection groups (49.0%) showed a significantly better prognosis compared with T3N2 (27.9%, p = 0.01), III B (0%, p < 0.0001), and incomplete resection groups (13.9%, p < 0.0001), respectively. These results indicate that the prognosis of patients with N2 disease or incomplete resection remains poor in regardless with the type of involved organs.

[Perioperative management for lung cancer with interstitial pneumonia].

The purpose of this study was to review perioperative managements from the clinical features and the postoperative course of lung cancer patients with interstitial pneumonia (IP). Twenty-two patients with IP were divided into 2 groups: the acute exacerbation (AE) group (6 patients) and the non-acute exacerbation (NAE) group (16 patients). There was no significant difference in the patient background between the 2 groups. In hematological examination, KL-6 levels were significantly higher in the AE group than in the NAE group. There was no significant difference in the respiratory function tests in the both groups, and the heart rate after 2 flights test was significantly higher in the AE group than in the NAE group. There was no significant difference in operation-related factors, tumor-related factors and the postoperative course in the both groups. No postoperative death occurred in our 22 patients probably due to adequate treatments of IP which was managed by our detailed manual. Long-term follow-up for lung cancer patients with IP undergoing surgical intervention is needed to prevent AE.

Flavopiridol as a radio-sensitizer for esophageal cancer cell lines.

Flavopiridol is a synthetic flavone that has shown an antitumor effect against several cancers. Here, we investigated the in vitro effect of flavopiridol alone and the combined effect of low-dose flavopiridol plus radiation on esophageal squamous cell carcinoma cell lines. Esophageal squamous cell carcinoma cell lines (TE8, TE9 and KE4) were exposed to flavopiridol (0.05-400 nmol/L) for 48 h. Growth inhibition was evaluated by MTT assay, cell cycle distribution was determined by flow cytometry, and cyclin D1, Bcl-2 and Rb protein expression was detected by Western blotting. The effect of 0.05 nmol/L flavopiridol as a radio-sensitizer was determined by clonogenic assay. The IC50 was approximately 110-250 nmol/L. Exposure to 0.05 nmol/L flavopiridol for 48 h increased the G2/M population, while 300 nmol/L increased the G1 population. At a concentration of 300 nmol/L, nuclear fragmentation and chromatin condensation were observed in all three cell lines. Exposure to 300 nmol/L flavopiridol decreased the levels of cyclin D1 and Rb protein in all three cell lines and Bcl-2 protein was also decreased in TE8 and KE4 cells. Moreover, exposure to 0.05 nmol/L flavopiridol slightly decreased the levels of cyclin D1, Rb and Bcl-2 protein in KE4 cells. Flavopiridol treatment (0.05 nmol/L) enhanced the radio-sensitivity in all three cell lines. Low-dose flavopiridol augmented the response of esophageal squamous cell carcinoma cell lines to radiation. Administration of a low dose of flavopiridol could be a potent new therapeutic approach for improving the efficacy of radiotherapy against esophageal cancer.

[The clinicopathological features of peripheral small-sized (2 cm or less) squamous cell carcinoma of the lung].

Recently the diagnosis of peripheral small-sized lung cancers has increased with the development of computed tomography. The vast majority of them are adenocarcinoma, whereas squamous cell carcinoma is rare. From 1981 to 2002, 1,054 patients underwent pulmonary resection for primary lung cancer in National Nishigunma Hospital. Among of them, 17 patients with peripheral small-sized (2 cm or less) squamous cell carcinoma underwent lobectomy and systemic nodal dissection were retrospectively reviewed. These were 15 men and 2 women, with a mean age of 68 years (range, 56-75). Regarding the pathologic stage, 15 patients were classified in stage IA, 1 in IIA, and 1 in IIIA. Among of them, only 1 patient with n 2 disease died of cancer at 17 months after surgery. Overall 5-year and 10-year survival rates of this disease were 84.4% and 73.8%, respectively. Based on the present data, we conclude that mediastinal nodal dissection would be unnecessary in the patients with peripheral small-sized squamous cell carcinoma of the lung.

[Clinical study of synchronous multiple primary lung cancers; problems in diagnosis and treatment].

We evaluated retrospectively 33 patients with synchronous multiple primary lung cancers. These were 20 men and 13 women, with a mean age of 67 years (range, 51-79 years). In 27 cases, the tumors were located in the ipsilateral lung, and in 6 cases, they were in the bilateral lung. In patients with synchronous multiple primary lung cancers, combinations of adenocarcinoma and adenocarcinoma (12 cases, 36.4%), adenocarcinoma and others (6 cases, 18.2%) were most commonly observed histologically. Lobectomy was performed in 18, bi-lobectomy in 3, pneumonectomy in 4, lobectomy with partial resection in 6, and lobectomy with laser therapy or irradiation in 2 patients. Overall 5-year survival rate of this disease was 78.3%. Eight patients died within 1 year after surgical resection, and 2 of them died of treatment-related accident. Although optimal treatment of choice for synchronous multiple primary lung cancers remains an unresolved problem, we think that careful planning of the treatment for this disease including selection of surgical methods is much important.

The next generation of liposome delivery systems: recent experience with tumor-targeted, sterically-stabilized immunoliposomes and active-loading gradients.

Three topics are discussed. Enhanced anti-tumor efficacy of targeted doxorubicin-containing sterically-stabilized liposomes using an anti-beta1 integrin Fab' ligand. Use of tumor targeting with an internalizing ligand to improve the efficacy of a non-leaky cisplatin-containing sterically-stabilized liposome formulation. Formulation variables (remote-loading with dextran ammonium sulfate, rigid lipid bilayer) used to optimize in vivo performance of a liposomal camptothecin analog.

Sesamin, a sesame lignan, decreases fatty acid synthesis in rat liver accompanying the down-regulation of sterol regulatory element binding protein-1.

The effect of sesamin, one of the most abundant lignans in sesame seed, on hepatic fatty acid synthesis was examined in rats. Rats were fed experimental diets containing varying amounts (0, 0.1 and 0.2% for Exp. 1 and 0, 0.2 and 0.4% for Exp. 2, respectively) of sesamin for 15 days. The activity and gene expression of enzymes involved in fatty acid synthesis including acetyl-CoA carboxylase, fatty acid synthase, ATP-citrate lyase and glucose-6-phosphate dehydrogenase decreased as the dietary level of sesamin increased in Exp. 1 and in rats fed the 0.2% sesamin diet they were approximately one-half those in animals fed a sesamin-free diet. In Exp. 2, the 0.2% sesamin diet lowered these parameters to one-half the level for a sesamin-free diet, but no further reduction was seen in animals fed the 0.4% sesamin diet. Dietary sesamin dose-dependently decreased the sterol regulatory element binding protein-1 (SREBP-1) mRNA level, and the value in rats fed a 0.4% sesamin diet was approximately one-half that in those fed a sesamin-free diet. The protein content of the membrane-bound precursor form of SREBP-1 decreased as dietary sesamin increased and was 37% lower in rats fed the 0.4% sesamin diet than in those fed a sesamin-free diet. Dietary sesamin exerted a more marked influence on the protein content of the mature nuclear form of SREBP-1. Diets containing 0.2 and 0.4% sesamin lowered the amount of mature SREBP-1 protein to less than one-fifth of that in the animals fed a sesamin-free diet. It was suggested that the dietary sesamin-dependent decrease in lipogenic enzyme gene expression is due to the suppression of the gene expression of SREBP-1 as well as the proteolysis of the membrane-bound precursor form of this transcriptional factor to generate the mature form.

Dietary gamma-linolenic acid dose-dependently modifies fatty acid composition and immune parameters in rats.

gamma-linolenic acid (GLA) has been reported to improve several inflammatory disorders through regulation of eicosanoid production. However, since GLA is a precursor of arachidonic acid, it may bring about increasing tissue arachidonic acid levels with subsequent pro-inflammatory events. To explore this possibility, we examined the effect of high-dose GLA acid on the fatty acid profile of immune cells, leukotriene B4 production by peritoneal exudate cells and immunoglobulin productivity of mesenteric lymph node lymphocytes of Sprague-Dawley rats. Male rats were fed 10% fat diets containing graded levels, 0, 20, 40 and 60% of GLA for 3 weeks. The results showed the distinction in activity of metabolizing GLA between immune cells and liver. Thus, in immune cells such as mesenteric lymph node and spleen lymphocytes and peritoneal exudate cells, more dihomo-gamma-linolenic acid was found than in the liver. Leukotriene B4 production by peritoneal exudate cells was significantly suppressed when fed the highest level of GLA suggesting a lower risk of allergic reaction. Moreover, immunoglobulin productivity in mesenteric lymph node lymphocytes was promoted by dietary GLA. The present study indicates that a high dose of GLA may exert anti-inflammatory effects through suppression of leukotriene B4 release and strengthening of gut immune system, thus ameliorating allergic reaction.

Mechanisms of phytosterolemia in stroke-prone spontaneously hypertensive and WKY rats.

Analysis of sterol composition in serum, liver, adipose tissue, adrenals, and abdominal aorta demonstrated that the contents of plant sterols, campesterol and sitosterol, were evidently higher in WKY and stroke-prone spontaneously hypertensive (SHRSP) rats than in Wistar and WKA rats fed a diet containing a 0.5% plant sterol mixture. Lymphatic 24-hour recovery of 3H-sitosterol was about 2-fold higher in the WKY and SHRSP rats than in the WKA rats. Lymphatic absorption of 14C-cholesterol was also higher in WKY and SHRSP rats compared with WKA rats, but the difference was smaller than in the case of sitosterol. The remarkable increase of sitosterol absorption in WKY and SHRSP rats was observed between 9 and 24 hours after the administration. In SHRSP rats, lymphatic absorption of sitosterol between 0 and 3 hours was also higher than those in the other rat strains. Markedly less esterified 3H-sitosterol was detected in lymph than 14C-cholesterol in all strains, and in WKY and SHRSP rats, only a small increase in the esterified forms of sitosterol and cholesterol was observed. Although the incorporation of micellar 3H-sitosterol and 14C-cholesterol into intestinal brush border membranes was higher in SHRSP rats than in WKA rats, no difference was observed between WKY and WKA rats. These observations suggest that the incorporation into the brush border membranes and the esterification of sterols are not the major determinants for the hyperabsorption of sitosterol and cholesterol in SHRSP and WKY rats. Secretion of sitosterol and cholesterol in the bile of rats fed a plant sterol mixture was lower in SHRSP than in WKA rats. These results suggest that WKY and SHRSP strains deposit plant sterols in the body by enhancing the absorption and lowering the excretion of plant sterols. These strains of rats may be suitable models for studying mechanisms of differential absorption of various sterols.

Human dendritic cells pulsed with autologous Epstein-Barr virus transformed B-cell lymphoblastoid cell (BCL) lysate elicit a BCL specific MHC-class II restricted T-cell response.

Epstein Barr Virus (EBV) associated lymphoproliferative disorders (LPD) express EBV latent antigens that are also expressed on normal B-cells transformed with EBV. This could potentially be exploited to develop immunotherapeutic strategies for LPD and other EBV associated malignancies. To this end we investigated the capacity of human monocyte derived dendritic cells (DC) pulsed with lysate from autologous EBV transformed B-cell lymphoblastoid cell (BCL) lysate to elicit an in vitro antitumor response. BCL lysate pulsed DC generate BCL specific cytotoxic lymphocytes, as lymphocytes primed with such DCs induce cytolysis of autologous (>60%) but not allogeneic BCL (<5%). In addition, lymphocytes primed with BCL lysate pulsed DC secrete gamma-IFN (3176 pg/ml). Whereas gamma-IFN production was markedly reduced (>99%) when BCL specific T-cells were stimulated by BCL lysate pulsed DC in the presence of blocking antibodies to HLA-DR, DP and DQ, use of antibodies to MHC class-I resulted in only a minimal reduction in gamma-IFN production (17%). These studies demonstrate that BCL lysate pulsed DC elicit a predominantly BCL specific, MHC class-Il restricted T cell response. This suggests that vaccination with autologous BCL lysate pulsed DC may represent a viable immunotherapeutic approach for the treatment of LPD.

[A case of IgA2-lambda type M-protein that IgA concentration differs from the values of M-protein by serum protein electrophoresis].

We report an IgA-lambda type M-protein in which the IgA concentration differed from the values of M-protein by serum protein electrophoresis found in a 53-year-old man with multiple myeloma. The M-protein value as determined by serum protein electrophoresis was 6,170 mg/dl. However, the serum IgA concentration was 3,052 mg/dl by turbidimetric immunoassay. Immuno-fixation electrophoresis using IgA subclass antisera revealed that this M-protein was the IgA2-lambda type. Western blotting analysis showed that the IgA2 molecules were composed of two approximately 68 kDa alpha 2 chains and two 28 kDa lambda chains. In addition the free lambda chain band was detected at the position of 28 kDa without 2-mercaptoethanol(2-ME) even though the patient IgA was purified. Since it is known that IgA2m(1) allotype easily release light chains from the IgA molecules in SDS-PAGE without 2-ME, we speculated that in this patient the IgA was the IgA2m(1) allotype. After peripheral blood stem cell transplantation(PBSCT), immunofixation electrophoresis of the patient serum revealed not only the bands of IgA2-lambda type M-protein, but also three bands of IgG1-kappa type M-protein in the gamma region.