Satoshi Okada

Prevalence of Endometriosis in Ovarian Cancer

Endometriosis may be the precursor of clear cell or endometrioid ovarian cancer. In this review, we focus on the prevalence of endometriosis in ovarian cancer and related clinical and epidemiological issues. According to 15 published reports, the rank order of the prevalence of endometriosis in each histologic type was clear cell (39.2%) > endometrioid (21.2%) > serous (3.3%) > mucinous type (3.0%). The high prevalence of endometriosis in clear cell and endometrioid types is a consistent finding in Japan and western countries. However, the incidence of the clear cell type is much higher (15–20% vs. 7–8%), and that of the endometrioid type is lower (7–16% vs. 18–26%), in Japan compared with western countries. This review is also concerned with the relationship between the presence of ovarian endometriosis and clinical features such as age, parity, menopausal status, clinical stage, and survival in ovarian cancer patients.

High incidence of silent venous thromboembolism before treatment in ovarian cancer

Venous thromboembolism (VTE) such as deep-vein thrombosis (DVT) and pulmonary thromboembolism (PTE) often occurs after surgery and rarely occurs even before surgery in patients with ovarian cancer. It is well known that levels of plasma D-dimer (DD) before treatment in most ovarian cancer patients are increased. This study therefore examined whether increased levels of DD are associated with presence of VTE before treatment of ovarian cancer. Between November 2004 and March 2007, DD levels prior to initial treatment were measured in 72 consecutive patients with presumed epithelial ovarian cancer (final diagnosis: epithelial ovarian cancer, n=60; and epithelial ovarian borderline malignancy, n=12). Venous ultrasound imaging (VUI) of the lower extremity was conducted for all patients except for two patients in whom DVT was detected by pelvic computed tomography (CT). When DVT was found, pulmonary scintigraphy was subsequently performed to ascertain presence of PTE. D-dimer levels were above the cut-off value (0.5 μg ml−1) in 65 of 72 patients (90.2%). Venous ultrasound imaging or CT revealed DVT in 18 of 72 patients (25.0%) and pulmonary scintigraphy found PTE in 8 patients (11.1%). All patients with VTE were asymptomatic when VTE was found. D-dimer levels were associated with incidence of VTE (0–1.4 μg ml−1; 0 of 26 (0%), 1.5–7.4 μg ml−1; 9 of 30 (30%) and ⩾7.5 μg ml−1; 9 of 16 (56.3%), P for trend=0.0003). However, even if 1.5 μg ml−1 was used as a cut-off value, this had low specificity and positive predictive value (47.2, 38.3%), though it had high sensitivity and negative predictive value (100, 100%). Therefore, ovarian cancer patients with DD level ⩾1.5 μg ml−1 should be examined using VUI to detect silent DVT. Patients with VTE underwent preventive managements including anticoagulant therapy before initial treatment, chemotherapy or surgery, and after surgery. There was no clinical onset of postoperative VTE in all 72 patients. Measurement of DD levels and subsequent ultrasonography revealed that silent or subclinical VTE frequently occurs before surgery in ovarian cancer. The usefulness of preoperative assessment of VTE needs further confirmation in randomised controlled trials.

Safety and immunogenicity of a peptide containing the cross-neutralization epitope of HPV16 L2 administered nasally in healthy volunteers

Amino acid (aa) 108-120 of L2 protein of human papillomavirus (HPV) type 16 contains a cross-neutralization epitope against genital HPV. We designed a placebo-controlled trial in healthy adults to evaluate the safety and immunogenicity of a synthetic peptide consisting of the aa 108-120 of HPV16 L2 (L2-108/120) region. A total of 13 volunteers were given nasal inoculations with 0.1 (n=5) or 0.5mg (n=5) doses of the peptides or placebo (n=3) without adjuvant at weeks 0, 4, and 12. Sera were collected before inoculation and at 6, 16 and 36 weeks. The inoculation caused no serious local and systemic complications. The inoculation generated anti-L2 antibodies binding to both HPV16 and 52 L1/L2-capsids in four of the five recipients in the 0.5mg group. Sera of the four recipients showed neutralizing activities against HPV16 and 52. Serological responses to the peptides were not found in the 0.1mg group and the placebo group recipients. This study suggests the L2-108/120 peptide is tolerable in humans and has the potential as a broad-spectrum prophylactic vaccine against genital HPV.

Ethanol-induced aggregation and fusion of small phosphatidylcholine liposome: participation of interdigitated membrane formation in their processes.

The mechanism for the ethanol-induced aggregation/fusion of uniform-sized small liposomes comprised of dipalmitoyl (DPPC) or egg yolk (eggPC) phosphatidylcholines was studied by measuring the average size using a photon correlation spectroscopy, by observing directly the states in the liposomal solutions using freeze-fracture electron microscopy and by attempting resonance energy transfer using flurophore-labeled phospholipids. Abrupt increases in the apparent size of DPPC liposomes were observed in the presence of above 44 mg/ml ethanol, where microscopically plateau membranes form interdigitated structure, in which the acyl chains fully interpenetrate the hydrocarbon chains of the apposing monolayer. On the contrary, in the eggPC liposome, where the membranes cannot form interdigitated structures even in the presence of high concentration of ethanol, such intense aggregation and fusion were not observed, suggesting their intimate relation to the interdigitated structure formation. The formation of interdigitated structures in the adhering region leads to an increase in the interfacial area and an exposure of hydrophobic acyl chain terminal on the surface area, and enhances hydrophobic interactions between two interdigitated bilayers. Thus, the resultant interdigitated structure makes the aggregated state stable and partially initiates the bilayer mixing between the two apposed membranes, leading to fusion.

A phase I trial of weekly gemcitabine and concurrent radiotherapy in patients with locally advanced pancreatic cancer

This study investigated the maximum-tolerated dose of gemcitabine based on the frequency of dose-limiting toxicities of weekly gemcitabine treatment with concurrent radiotherapy in patients with locally advanced pancreatic cancer. Fifteen patients with locally advanced pancreatic cancer that was histologically confirmed as adenocarcinoma were enrolled in this phase I trial of weekly gemcitabine (150–350 mg m−2) with concurrent radiotherapy (50.4 Gy in 28 fractions). Gemcitabine was administered weekly as an intravenous 30-min infusion before radiotherapy for 6 weeks. Three of six patients at the dose of 350 mg m−2 of gemicitabine demonstrated dose-limiting toxicities involving neutropenia/ leukocytopenia and elevated transaminase, while nine patients at doses of 150 mg m−2 and 250 mg m−2 did not demonstrate any sign of dose-limiting toxicity. Of all 15 enrolled patients, six patients (40.0%) showed a partial response. More than 50% reduction of serum carbohydrate antigen 19-9 level was observed in 13 (92.9%) of 14 patients who had pretreatment carbohydrate antigen 19-9 levels of 100 U ml−1 or greater. The maximum-tolerated dose of weekly gemcitabine with concurrent radiotherapy was 250 mg m−2, and this regimen may have substantial antitumour activity for patients with locally advanced pancreatic cancer. A phase II trial of weekly gemcitabine at the dose of 250 mg m−2 with concurrent radiation in patients with locally advanced pancreatic cancer is now underway.

Silent venous thromboembolism before treatment in endometrial cancer and the risk factors

Venous thromboembolism (VTE) often occurs after surgery and can even occur before surgery in patients with gynaecological malignancies. We investigated the incidence of VTE before treatment of endometrial cancer and associated risk factors. Plasma D-dimer (DD) levels before initial treatment were examined in 171 consecutive patients with endometrial cancer. Venous ultrasound imaging (VUI) of the lower extremities was performed in patients with DD ⩾1.5 μg ml−1, as the negative predictive value of DD for VTE is extremely high. For patients with deep vein thrombosis (DVT), pulmonary scintigraphy was performed to ascertain the presence of pulmonary thromboembolism (PTE). Risk factors for VTE were analysed using univariate and multivariate analyses for 171 patients. Of these, 37 patients (21.6%) showed DD ⩾1.5 μg ml−1, 17 (9.9%) displayed DVT by VUI and 8 (4.7%) showed PTE on pulmonary scintigraphy. All patients with VTE were asymptomatic. Univariate analysis for various risk factors revealed older age, non-endometrioid histology and several variables of advanced disease as significantly associated with VTE before treatment. Obesity, smoking and diabetes mellitus were not risk factors. Multivariate analysis confirmed extrauterine spread and non-endometrioid histology as independently and significantly associated with risk of VTE. These data suggest that silent or subclinical VTE occurs before treatment in at least around 10% of patients with endometrial cancer. Risk factors for VTE before treatment might not be identical to those after starting treatment.

Increased permeability of phase-separated liposomal membranes with mixtures of ethanol-induced interdigitated and non-interdigitated structures

It has been suggested by many workers using model membranes that the interdigitated structure formation, in which the acyl chains fully interpenetrate the hydrocarbon chains of the opposing monolayer, plays an important role in regulating many functions of biomembranes. In the present study the control of permeability was focused on as one of the biomembrane functions, and the effects of ethanol on the permeability of large unilamellar vesicles made by the extrusion technique (LUVET) (average diameter: about 250 nm), composed of dipalmitoyl or egg yolk phosphatidylcholines, were studied by monitoring the leakage of fluorescent dye, calcein, entrapped in the inner aqueous phase of the LUVET. The permeability was estimated from the apparent rate constant of calcein leakage at 25 degrees C. Large permeabilities were observed in the region of 0.6 M to 1.3 M ethanol, with a concentration dependence. In this range of ethanol concentrations the normal bilayer and interdigitated structure coexist and the membrane is in a phase-separated state. The large permeability is due to the instability of the boundary regions, the interdigitated membrane being characterized by a thinner structure and more rigid hydrocarbon regions in the layer than its non-interdigitated counter part. These results suggest the possibility of biomembrane-permeability regulation by interdigitated membrane formation.

Effects of the acyl chain composition of phosphatidylcholines on the stability of freeze-dried small liposomes in the presence of maltose

The effects of the acyl chain composition of phosphatidylcholines (PCs) on the stability of small unilamellar vesicles during freeze-drying and rehydration in the presence of maltose were studied by monitoring the retention of a trapped marker, calcein, in the internal liposome compartment. In dipalmitoyl PC, beta-oleoyl-gamma-palmitoyl-PC and egg yolk PC liposomes, good or fair retentions (>50%) were observed in the presence of maltose, but maltose was ineffective in preserving retention in the dioleoyl PC (DOPC) liposomes (<10%). The extremely low retention in the DOPC liposome was ascribed to neither a formation of the inverted hexagonal phase of the liposomal membrane nor the fusion/aggregation of the liposomes in the drying-rehydration process. Differential scanning calorimetry measurements suggested that interactions of maltose with PC headgroups were essential to stabilizing the dry liposomes. These interactions were significant in the saturated or mixed chain liposomes but were markedly reduced in the DOPC liposomes.

EFFECTS OF ETHANOL ON PERMEABILITY OF PHOSPHATIDYLCHOLINE/CHOLESTEROL MIXED LIPOSOMAL MEMBRANES

Abstract Effects of ethanol on permeability of large unilamellar vesicles (about 170 nm in diameter), composed of a dipalmitoyl phosphatidylcholine (DPPC)/cholesterol binary mixture, were studied by monitoring leakage of a fluorescent dye, calcein, entrapped in the inner aqueous phase of the vesicles. In the presence of 25 mol% cholesterol, the leakage was dramatically increased, and the permeability at 1.0 M ethanol was about eight times larger than that without ethanol and cholesterol. This effect of ethanol on the membrane permeability was similar to our previous findings concerning mixed membranes of dilauroyl phosphatidylethanolamine and DPPC (H. Komatsu and S. Okada (1996) Biochim. Biophys. Acta 1283, 73–79). It was suggested that the presence of ethanol can lead to high permeability even if the membranes are stable and have a low permeability in its absence.

Photocontrolled Compound Release System Using Caged Antimicrobial Peptide

A novel photocontrolled compound release system using liposomes and a caged antimicrobial peptide was developed. The caged antimicrobial peptide was activated by UV irradiation, resulting in the formation of pores on the liposome surface to release the contained fluorophores. The compound release could be observed using fluorescence measurements and time-lapse fluorescence microscopy. UV irradiation resulted in a quick release of the inclusion compounds (within 1 min in most cases) under simulated physiological conditions. The proposed system is expected to be applicable in a wide range of fields from cell biology to clinical sciences.