Usefulness of immunocytochemistry using a Breast Marker antibody cocktail targeting P63/cytokeratin7/18/cytokeratin5/14 for fine needle aspiration of the breast: a retrospective cohort study of 139 cases.

OBJECTIVE: The Breast Marker Cocktail from Biocare Medical comprises five antibodies recognising p63, and cytokeratins (CKs) 7, 18, 5 and 14. Immunohistochemistry using this cocktail is useful for diagnosing proliferative intraductal breast lesions. However, cytology using the cocktail has not been reported. METHODS: We report 139 cases of mammary samples collected by fine needle aspiration (FNA) for which histological diagnoses were available. After cell transfer, immunocytochemistry was performed using the cocktail, and clusters of cells were classified. A cluster with no or limited CK5/14 expression (<20% of cells) was classified as a monotonous cluster. One with more than 20% of cells showing CK5/14 expression was defined as a mosaic cluster. When at least one p63-positive cell was present, we defined it as a cluster with p63. We also evaluated background p63-positive myoepithelial cell densities. RESULTS: The diagnostic sensitivity and specificity for carcinomas were 97.8% (89/91) and 91.7% (11/12), respectively, using the criterion of two or more monotonous clusters lacking p63. Two false-negative cases were triple-negative cancers; one false-positive was an apocrine papilloma. The numbers of monotonous clusters with p63 differed significantly between benign lesions, ductal carcinoma in situ (DCIS)/lobular carcinoma in situ (LCIS) and invasive carcinomas (P < 0.001). The background myoepithelial cell density was significantly higher in fibroepithelial tumours than in other lesions (P < 0.001). CONCLUSIONS: Immunocytochemistry using this antibody cocktail showed good sensitivity and specificity for diagnosing breast cancers. Thus, this method is useful for mammary cytology using FNA.

Method for screening ecdysone-inducible stable cell lines.

Ecdysone-inducible systems are useful tools to study the function of various genes in different types of mammalian cells. However, it is technically difficult to establish stable cell lines. This is partly because the conditional expression system requires the expression of two or more components driven by different kinds of promoters. In this paper, we describe the use of a luciferase reporter gene system for rapid screening of cell lines that express the ecdysone and retinoid X receptors. Using this system, two human stable colon cancer cell lines, SW480/VgRXR and HCT116/VgRXR, were successfully generated. The expression of these receptors remained high after six months of continuous culturing. A tight regulation of gene induction in SW480/VgRXR was observed using 2 microM Ponasterone A. The gene induction was rapid and persistent. Our results demonstrated the advantage of establishing cell lines that continuously express high levels of ecdysone receptor proteins.

A mammalian two-hybrid system for adenomatous polyposis coli-mutated colon cancer therapeutics.

Colon cancer cells frequently lose expression of the tumor suppressor adenomatous polyposis coli (APC). As result, beta-catenin accumulates and activates transcription of Tcf-responsive genes. Here we describe a novel mammalian two-hybrid system that selectively kills APC-mutated cells. This system consists of GAL4/beta-catenin, VP16/Tcf4, and a gene that is transcribed when GAL4 and VP16 associate. In APC-mutated human colon cancer cells, such as SW480, GAL4/beta-catenin accumulates, and in the presence of VP16/Tcf4, induces high levels of expression of the reporter gene. Expression of wild-type APC reduced GAL4/beta-catenin and intact beta-catenin levels and inhibited reporter gene expression. In colon cancer cells such as SW48 that have wild-type APC, GAL4/beta-catenin was degraded, and expression levels of the output gene were low. Replacement of the reporter gene with a suicide gene resulted in selective killing of SW480 cells. This system may be applicable for broader use of gene therapy by targeting diseases that involve protein degradation.

Inhaled flow and handling of fluticasone diskhaler by asthmatic patients.

We investigated the inhaled flow and handling of a Fluticasone Diskhaler (FDH) by patients familiar with the beclomethasone dipropionate inhaler (BDI), a metered dose inhaler. Before the FDH was introduced in our hospital, 174 patients were instructed in the use of the Diskhaler and measured the flow of Diskhaler inhalation. Three months after the introduction of the FDH, approximately 95 patients were using them. During their regular visit to the hospital, we checked the patients' handling of FDH and the flow of FDH inhalation (n=81). It was found that only 22% of the patients correctly handled the FDH. The major erros concerned breath-holding and disk rotation after use, but 9.9% of the patients handled the inhaler with serious error, e.g., incomplete puncturing of the blister. The mean FDH flow was 86.5 L/min, which was significantly higher than that recorded at the first FDH trial (69.1 L/min). In 9.9% of the patients, the inhaled flow was inappropriately low (<50 L/min), in 29.6% of the patients, it was unnecessarily high (>100 L/min). In conclusion, handling the FDH is easy for patients who are already familiar with the BDI. However, in 40% of the patients, the inhaled flow rate was not sufficient.

Simplification for measuring input function of FDG PET: investigation of 1-point blood sampling method.

UNLABELLED: The current method for quantitative FDG PET study requires application of multiple arterial blood sampling for measuring the input function, but the procedure is invasive and complicated. The purpose of this study was to establish a 1-point blood sampling technique that gives data comparable with the data of more elaborate serial arterial sampling. METHODS: We established a time point for 1-point arterial sampling that exhibited the highest correlation between plasma radioactivity at the time point and the real integrated value (IV) of the measured input function obtained by multiple arterial sampling in 120 patients and the smallest coefficient of variation of the real IV divided by plasma radioactivity at the time point in 120 patients. Scaling factors for estimation at each sampling point were determined, and a reference table was established to make the supposed input function. RESULTS: The optimal time for 1-point arterial sampling was 12 min after FDG injection. A good correlation was observed between the real IVs and those estimated from 1-point arterial blood sampling at 12 min using the supposed input function (n = 120; P < 0.001). The time point at which the difference between values of arterial and venous blood disappeared was 40 min after FDG injection. The percentage errors of IV estimation by 1-point sampling were 1.70% (n = 120) for arterial blood at 12 min and 3.64% (n = 10) for venous blood at 40 min. CONCLUSION: We conclude that the simplified 1-point sample method works in a manner that is comparable with serial arterial sampling and should be useful for clinical PET.

Margin clearance and HPV infection do not influence the cure rates of early neoplasia of the uterine cervix by laser conization.

A lesion existing in the endocervical and/or ectocervical conized margin and HPV-DNA existing in a conized specimen are reported to be at risk of persistence or recurrence of early neoplasia of the cervix when treated by conization. The aim of this study was to investigate whether margin clearance and HPV infection influenced the outcome in our series of laser conization. Excisional conization with the KTP/YAG Surgical Laser System or Nd-YAG laser was performed in this study. Eighty patients with cervical neoplasias were included: 47 with dysplasia, 25 with carcinoma in situ (CIS) and eight with microinvasive carcinoma. The endocervical and ectocervical conized margins were examined microscopically. HPV-DNA was analyzed with the primer for types 16, 18, 31, 33, 35, 52b and 58 amplified by the PCR method. The margins of the conized specimens were confirmed histopathologically to be clear in 58 cases (73%), whereas in 22 cases (27%) they were involved by neoplasia. HPV-DNA was positive in 38% of dysplasias, 40% of CISs and 50% of microinvasive carcinomas. The overall rate of the initial cure at 10 weeks after treatment appeared to be 100% in all 80 cases. Primary cure rates were 100% for 47 cases with dysplasia, 96% for 24 cases with CIS and 100% for four cases with microinvasive carcinoma regardless of margin positivity and HPV-DNA status. Involved margins and HPV infection did not influence the cure of early neoplasia of the uterine cervix achieved by our laser conization procedure. The favorable results may be due to the procedure of vaporizing the cut surface forming a dome-shaped tissue defect.

Synthesis and Properties of an Overcrowded Silabenzene Stable at Ambient Temperature.

Solid-state, room-temperature stability for silabenzene 1 is granted by the bulky silane rendering kinetic inertness. Silabenzene 1 is synthesized in good yield from the crowded chlorosilane 2 with tert-butyllithium. The delocalized, aromatic nature of 1 was confirmed by NMR spectroscopy and the (1)J(Si-C) value, as well as calculations on an analogous silabenzene.

Functional characterization of structural alterations in the sequence of the vasodilatory peptide maxadilan yields a pituitary adenylate cyclase-activating peptide type 1 receptor-specific antagonist.

Maxadilan is a vasodilatory peptide derived from sand flies that is an agonist at the pituitary adenylate cyclase-activating peptide (PACAP) type 1 receptor. Surprisingly, maxadilan does not share significant sequence homology with PACAP. To examine the relationship between structure and activity of maxadilan, several amino acid substitutions and deletions were made in the peptide. These peptides were examined in vitro for binding to crude membranes derived from rabbit brain, a tissue that expresses PACAP type 1 receptors; and induction of cAMP was determined in PC12 cells, a line that expresses these receptors. The peptides were examined in vivo for their ability to induce erythema in rabbit skin. Substitution of the individual cysteines at positions 1 and 5 or deletion of this ring structure had little effect on activity. Substitution of either cysteine at position 14 or 51 eliminated activity. Deletion of the 19 amino acids between positions 24 and 42 resulted in a peptide with binding, but no functional activity. The capacity of this deletion mutant to interact with COS cells transfected with the PACAP type 1 receptor revealed that this peptide was a specific antagonist to the PACAP type 1 receptor.

[Prolonged antinociceptive effect of poly (DL-lactic acid)-fentanyl composites after their intrathecal injection in rats].

We synthesized poly (DL-lactic acid)-fentanyl composites and compared the duration of analgesia after the administration of a single intrathecal dose of these agents in rats. The drug was injected with an intrathecal catheter into the intrathecal space. Fentanyl composites or plain fentanyl in doses of 2.5 or 25 micrograms were administered, respectively. Animals were then tested for analgesia using the tail-flick test. The release rate of fentanyl from fentanyl composites in vitro was also evaluated. The antinociceptive effect of fentanyl composites (25 micrograms) was significantly longer than that of plain fentanyl. Administration of poly (DL-lactic acid) alone did not induce the antinociceptive effect. Four of 7 animals given plain fentanyl (25 micrograms) exhibited temporary respiratory depression, but none of the animals given fentanyl composites showed this response. In vitro experiments demonstrated a slow release of fentanyl from the fentanyl composites. We conclude that the antinociceptive effect of fentanyl can be prolonged when administered as a poly (DL-lactic acid)-fentanyl composite in the intrathecal space with decreased systemic side effects compared with the plain formulation.

New Treatment Strategy for Bone Metastases from Breast Cancer.

Breast cancer patients frequently develop bone metastasis. Parathyroid hormone-related protein, an osteoclast activating factor, might be necessary for tumorto erode bone and grow at skeletal site. Bisphosphonates have an affinity for bone and are potent inhibitors of osteoclastic bone resorption. In light of this,53 patients with bone metastasis from breast cancer were treated with chemoendocrine(mainly high-dose medroxyprogesterone acetate as the endocrine therapy) therapy + bisphosphonate (pamidronate, Aredia (R)). During the previous 6 years (median 27 months), 53 breast cancer patients with bone metastasis were treated with pamidronate + chemoendocrine therapy. The regimen consisting of pamidronate + chemoendocrine agent was administered to 27 patients as a post relapse first-line regimen and to the remaining 26 cases, which failed first- or second-line treatment as a second or third line regimen. As a result of the combination therapy, sclerotic changes were observed in the osteolytic lesions in 31 of the 53 patients (59%). The effect on the osteolytic lesions did not correlate with the duration of disease free interval, estrogen receptor (ER) status, presence/absence of previous therapy or number of " hot spot(s) ] on bone scintigraphy. Lessening of pain from the bone metastasis was achieved in 83% of the patients after 3 months of pamidronate administration. Pamidronate + chemoendocrine therapy seems highly promising.

[Anesthetic management of a patient with dilated cardiomyopathy using olprinone].

A 51-year-old man with dilated cardiomyopathy, who had been treated with medication for five years, was scheduled for abdomioperineal resection of the rectum. Preoperative echocardiography demonstrated left ventricular dilation and hypertrophy, with an ejection fraction of 0.34. Anesthesia was induced with ketamine 40 mg and fentanyl 0.5 mg intravenously. Endotracheal intubation was facilitated by administration of vecuronium 10 mg. Anesthesia was maintained with nitrous oxide-oxygen-sevoflurane and fentanyl. In order to regulate myocardial contractility and after-load, use of a phosphodiesterase III inhibitor was considered, although phosphodiesterase III inhibitors are known to induce arrhythmias, which should be avoided in dilated cardiomyopathy patients. We chose olprinone, because its inotropic action is not associated with arrhythmogenecity. Before infusing olprinone, cardiac output was 4.5 l.min-1 and systemic vascular resistance was 1306 dynes.sec.cm-5. When olprinone was continuously infused for one hour, cardiac output increased to 5.2 l.min-1 and systemic vascular resistance decreased to 958 dynes.sec.cm-5. Some premature ventricular contractions occurred, but they were easily controlled by administration of 50 mg lidocaine. These clinical data demonstrate that olprinone enhanced myocardial contractility, and decreased after-load and arrhythmogenecity in a dilated cardiomyopathy patients. In conclusion, olprinone is useful in the perioperative cardiovascular management of surgical patients with dilated cardiomyopathy.

[Fluorine-18 labeled 6-fluoro-L-dopa: systematization and evaluation of its usefulness].

6-[18F]Fluoro-L-dopa (L-3,4-dihydroxy-6-[18F]fluorophenylalanine; 6-[18F]FDPA) is useful to assess presynaptic dopamine metabolism in central nervous system. In this paper, we report on the usefulness of the 6-[18F]FDOPA synthesis system developed for the routine synthesis. This system consists of the 6-[18F]FOPA synthesis and the separation units in conjunction with controller using a personal computer. The synthesis time of 6-[18F]FDOPA was 73 minutes. The typical yield and specific activity were 1.4-2.4 GBq and 244-270 MBq/mumol at the end of synthesis, respectively, under the irradiation condition of 50 microA for 130 minutes. The radiochemical yields of 6-[18F]FDOPA were 31.3-38.7% based on the [18F]acetylhypofluorite, and the results were affected with the condition of potassium acetate (AcOK) to produce gaseous [18F]acetylhypofluorite. This system is useful for the routine production of 6-[18F]FDOPA because of its high yield and high specific activity while maintaining AcOK in good condition, and decreasing the radiation exposure for chemist.

[Prolonged antinociceptive effect after epidural injection of polyethylene glycol-morphine composites in rats].

Epidurally administered morphine is useful in the management of postoperative or cancer pain, and a reliable method which can produce prolongation of analgesia with a single dose may be very useful. We synthesized a polyethylene glycol-morphine (PEG-morphine) composites and examined the duration of analgesia after a single epidural administration dose of this agent in the rat. The molecular weight of PEG was functionally evaluated. PEG-morphine was injected surgically along the epidural space. Morphine in doses of 2.5, 5.0 and 7.5 mg and PEG only were administered. A second group of animals received intramuscular injections of PEG-morphine (5.0mg). Animals were then tested for analgesia using the tail-flick test. The antinociceptive effect of 7.5mg was significantly longer than that of 2.5mg or 5.0mg. Neither PEG alone nor intramuscular administration of PEG-morphine induced antinociceptive effect. Sensory blockade was reversible and the animal appeared to have normal sensory perception. We conclude that the antinociceptive effect of morphine is dose-dependent and its duration can be prolonged when administered as a PEG-morphine composite in the epidural space.

Radioiodinated diacylglycerol analogue: a potential imaging agent for single-photon emission tomographic investigations of cerebral ischaemia.

Phospholipid metabolism is closely related to membrane perturbation in cerebral ischaemia. We investigated in vivo topographical lipid metabolism using an iodine-123-labelled diacylglycerol analogue, (1-(15-(4-iodine-123-iodophenyl)-pentadecanoyl)-2-stearoyl-rac-gly cerol) (123I-labelled DAG), in a middle cerebral artery (MCA) occlusion model with the aim of positive imaging of ischaemic insult. Sprague-Dawley rats underwent coagulation of the MCA to induce permanent occlusion. MCA occlusion times prior to injection of 123I-labelled DAG ranged from 15 min to 14 days. Each rat was injected with 11-37 MBq of 123I-labelled DAG via a tail vein. After 30 min, in vivo autoradiographs were reconstructed. Scanning of the living rat brain in this MCA occlusion model was performed using a gamma camera with a pinhole collimator. Cerebral infarctions were recognized in the frontal cortex, the parietal cortex and the lateral portion of the caudate-putamen by 2,3,5-triphenyltetrazolium hydrochloride staining. In infarcted regions (region 1), 123I-labelled DAG incorporation showed a slight decrease up to 12 h; it then increased up to 6 days and decreased thereafter. In peri-infarcted regions (region 2), the incorporation showed almost no change up to 12 h, then increased up to 5-6 days and decreased thereafter. In other regions (region 3), the incorporation showed no change. Lipid analysis showed that 123I-labelled DAG was metabolized to 15-(4-iodine-123-iodophenyl)-pentadecanoic acid by DAG lipase and to 123I-labelled phosphatidylcholine. Scanning of the ischaemic region showed higher accumulation than on the non-lesioned side. We established a method to visualize ischaemic foci as positive images. The early changes in 123I-labelled DAG incorporation were closely related to DAG lipase, which degraded the accumulated intrinsic DAG, and increased 123I-labelled DAG incorporation in the chronic stage involves several aspects of neural destruction in the process of autolysis. It is concluded that the reported method could have a clinical future.

Decreased expression of the DCC gene in human breast carcinoma.

Inactivation of the "deleted in colon cancer" (DCC) gene on chromosome 18 is known to be associated with the tumorigenesis and metastasis of colorectal cancer. In the present study, we investigated the expression of DCC and the c-erbB-2 product in surgical specimens from 45 patients with breast cancer by immunohistochemical staining, and found the expression of DCC to be decreased in 23 (51%) tumors. In 8 years of follow-up, 11 of 22 (50%) patients with DCC-positive staining tumors, and 17 of 23 (74%) patients with DCC-negative tumors developed recurrence. The stratified analysis, according to the status of axillary lymph node metastasis, showed the same tendency. Overexpression of erbB-2 was detected in 13 (29%) of the 45 breast cancer specimens, but there were no differences in the relapse rate between patients with erbB-2 positive and those with erbB-2 negative tumors. Although the individual alteration of DCC or erbB-2 did not possess independent prognostic significance for the prediction of recurrence, patients with tumors having the double alteration of DCC-negative and erbB-2-positive showed adverse relapse-free survival (0.025 < P < 0.05). These findings suggest that a decrease in DCC expression and erbB-2 overexpression may influence the progression of breast carcinoma.

[Carbon-11 labeled diacylglycerol for signal transduction imaging by positron CT: evaluation of the quality and safety for clinical use].

To elucidate the synaptic transmission in the neural system, we have been developing fundamental studies for intracellular signaling. For clinical application of carbon-11 labeled diacylglycerol (1-[1-11C]butyryl-2-palmitoyl-rac-glycerol: 11C-DAG) using positron emission computed tomography (PET), we evaluated the quality and the safety of 11C-DAG as the solution for injection. As a result, 11C-DAG was synthesized within 50 minutes, including the preparation step for injection. The half life time and energy spectrum of 11C-DAG were the same as the physical character of carbon-11, and other radioisotopes were not detected. In the quality control, 11C-DAG solution was negative in the examination of bacterial contamination and the pyrogen test in three successive synthesis procedures. In the acute toxicity test by administration of 11C-DAG and 100 mumol/kg of non-radioactive DAG to the rat intravenously, the systemic condition of the rat was not changed and no abnormalities were found in any organ 24 hours after administration. These findings indicated the safety of 11C-DAG solution. Clinical application of 11C-DAG using positron emission tomography may be useful to elucidate the dysfunction of intracellular signaling in disorders of higher cortical function such as Alzheimer disease.

[Carbon-11 labeled diacylglycerol for signal transduction imaging: effect of the solubilizer on the distribution and radiation dosimetry].

Carbon-11 labeled diacylglycerol (11C-DAG) has been developed as a signal transduction imaging agent for the CNS, and it can visualize the second messenger. For clinical application by positron CT (PET), the 11C-DAG solution must be prepared for intravenous injection. However, the 11C-DAG does not dissolve in water because of its lipophilicity and requires a solubilizer such as human serum albumin (HSA) and Tween 80 (TW-80). We examined the influence of these solubilizers on the tissue distribution of 11C-DAG, and estimated the radiation dosimetry. In the brain, uptake of 11C-DAG dissolved with HSA was 1.3-1.8 times higher than that of dissolved with TW-80. On the other hand, the lung and spleen showed a higher uptake of 11C-DAG using TW-80 than when using HSA. Especially, the lungs showed 20-40 times higher uptake than when using HSA. Also, the washout of radioactivity from tissue was slower, and the dose of radiation exposure was estimated to be higher, with TW-80 than with HSA. Therefore, between TW-80 and HSA with different solubilizing mechanisms, the later was suggested to be a better solubilizer of 11C-DAG.

[Anaphylactic shock by ophthalmic wash solution containing chlorhexidine].

A 58 year-old male patient developed anaphylactic shock, possibly due to the use of chlorhexidine as ophthalmic wash solution. He was successfully resuscitated without any sequelae. The patient had increased levels of both histamine and tryptase. The skin test for allergy resulted in strong positive to chlorhexidine. There have been many reports regarding severe adverse reactions associated with use of chlorhexidine. It is necessary to pay attention to anaphylactic shock due to chlorhexidine.

[A clinical study on the complications associated with laser therapy for cervical neoplasia].

Six hundred and fifty-nine patients with cervical neoplasia were treated by either vaporization (395 patients) or excisional conization (264 patients) with various Lasers. The complications associated with the therapies were examined with reference to the Laser sources and in the local anesthesia and non anesthesia groups. Six hundred and thirty-nine patients were treated on an outpatient basis without local anesthesia (37.7%) or with local anesthesia (62.3%). During the procedures, the patients complained of pain and a hot sensation in the lower abdomen in 64.6% in the vaporization (vapor) group and 60.7% in the conization (cone) group. In the local anesthesia group, 2.9% of the vapor and 4.0% of cone group complained of nothing. Uncontrollable bleeding during the procedures was most common in 12.8% and 32.3% in the CO2 vapor and cone groups, respectively. Misirradiation of the vaginal wall and vulva occurred in 3.3% in all vapor groups. On the other hand, the incidence of burns to both areas in all cone groups was 7.2%. The patients who were given an analgesic were 1.3% in all vapor groups and 2.5% in all cone groups. Delayed bleeding in those who received some treatment to stop it occurred in 14.7% in all vapor groups and also in 11.0% in all cone groups. Cervical stenosis was seen in 3.8% in all vapor groups and in 8.6% in all cone groups. No infection of the lasered site was observed in the vapor groups, but was observed in 0.8% in the cone groups. The culture test showed both E. coli positive.