Impact of modifiable lifestyle factors on outcomes after breast cancer diagnosis: the Setouchi Breast Cancer Cohort Study.

The primary purpose of this large cohort study is to investigate the effects on breast cancer outcomes of modifiable lifestyle factors after breast cancer diagnosis. These factors include physical activity, smoking, alcohol consumption, obesity and weight gain after diagnosis, alternative medicine and dietary factors. Women diagnosed with Stage 0 to III breast cancer are eligible for participation to this study. Lifestyle, use of alternative medicine, psychosocial factors, reproductive factors and health-related quality of life will be assessed using a questionnaire at the time of breast cancer diagnosis (baseline), and 1, 2, 3 and 5 years after diagnosis. Clinical information and breast cancer outcomes will be obtained from a breast cancer database. The primary endpoint will be disease-free survival. Secondary endpoints are overall survival, health-related quality of life, breast cancer-related symptoms and adverse events. Patient recruitment commenced in February 2013. Enrollment of 2000 breast cancer patients is planned during the 5-year recruitment period. The concept of the study is described in this article.

Concentrated ambient particles alter myocardial blood flow during acute ischemia in conscious canines.

BACKGROUND: Experimental and observational studies have demonstrated that short-term exposure to ambient particulate matter (PM) exacerbates myocardial ischemia. OBJECTIVES: We conducted this study to investigate the effects of concentrated ambient particles (CAPs) on myocardial blood flow during myocardial ischemia in chronically instrumented conscious canines. METHODS: Eleven canines were instrumented with a balloon occluder around the left anterior descending coronary artery and catheters for determination of myocardial blood flow using fluorescent microspheres. Telemetric electrocardiographic and blood pressure monitoring was available for four of these animals. After recovery, we exposed animals by inhalation to 5 hr of either filtered air or CAPs (mean concentration+/-SD, 349.0+/-282.6 microg/m3) in a crossover protocol. We determined myocardial blood flow during a 5-min coronary artery occlusion immediately after each exposure. Data were analyzed using mixed models for repeated measures. The primary analysis was based on four canines that completed the protocol. RESULTS: CAPs exposure decreased total myocardial blood flow during coronary artery occlusion by 0.12 mL/min/g (p<0.001) and was accompanied by a 13% (p<0.001) increase in coronary vascular resistance. Rate-pressure product, an index of myocardial oxygen demand, did not differ by exposure (p=0.90). CAPs effects on myocardial blood flow were significantly more pronounced in myocardium within or near the ischemic zone versus more remote myocardium (p interaction<0.001). CONCLUSIONS: These results suggest that PM exacerbates myocardial ischemia by increased coronary vascular resistance and decreased myocardial perfusion. Further studies are needed to elucidate the mechanism of these effects.

Mechanisms of inhaled fine particulate air pollution-induced arterial blood pressure changes.

BACKGROUND: Epidemiologic studies suggest a positive association between fine particulate matter and arterial blood pressure, but the results have been inconsistent. OBJECTIVES: We investigated the effect of ambient particles on systemic hemodynamics during a 5-hr exposure to concentrated ambient air particles (CAPs) or filtered air (FA) in conscious canines. METHODS: Thirteen dogs were repeatedly exposed via permanent tracheostomy to CAPs (358.1+/-306.7 microg/m3, mean+/-SD) or FA in a crossover protocol (55 CAPs days, 63 FA days). Femoral artery blood pressure was monitored continuously via implanted telemetry devices. We measured baroreceptor reflex sensitivity before and after exposure in a subset of these experiments (n=10 dogs, 19 CAPs days, 20 FA days). In additional experiments, we administered alpha-adrenergic blockade before exposure (n=8 dogs, 16 CAPs days, 15 FA days). Blood pressure, heart rate, rate-pressure product, and baroreceptor reflex sensitivity responses were compared using linear mixed-effects models. RESULTS: CAPs exposure increased systolic blood pressure (2.7+/-1.0 mmHg, p=0.006), diastolic blood pressure (4.1+/-0.8 mmHg; p<0.001), mean arterial pressure (3.7+/-0.8 mmHg; p<0.001), heart rate (1.6+/-0.5 bpm; p<0.001), and rate-pressure product (539+/-110 bpm x mmHg; p<0.001), and decreased pulse pressure (-1.7+/-0.7 mmHg, p=0.02). These changes were accompanied by a 20+/-6 msec/mmHg (p=0.005) increase in baroreceptor reflex sensitivity after CAPs versus FA. After alpha-adrenergic blockade, responses to CAPs and FA no longer differed significantly. CONCLUSIONS: Controlled exposure to ambient particles elevates arterial blood pressure. Increased peripheral vascular resistance may mediate these changes, whereas increased baroreceptor reflex sensitivity may compensate for particle-induced alterations in blood pressure.

Permanent tracheostomy for long-term respiratory studies.

BACKGROUND: We describe a modified surgical technique for permanent, anterior tracheal-wall stoma for chronic, repeat respiratory studies in trained, conscious dogs. These cannula-free tracheostomies require minimal daily maintenance, permit repeat intubation with endotracheal tubes modified for airflow respiratory measurement, and facilitate up to 6 h continuous administration of aerosol agents during long-term or repeat respiratory studies. METHODS: In 20 dogs, during a 30 to 40 min procedure, portions of tracheal rings 2-4 were removed to create an oval stoma, approximately 2 x 1 cm. The dermis was secured to the transected cartilage and tracheal mucosa in such a manner that skin covered the sternohyoid muscles and grew-in flush with the tracheal mucosa at the stomal opening. Stomas were cleaned daily, and fur was clipped weekly around the stomal site. No other maintenance procedures or environmental modifications were needed. Animals breathed through both the stoma and the upper airway and barked normally. RESULTS: Stomas remained viable in long-term animals (n = 4) ongoing for 70.3 +/- 32.2 mo (mean +/- SEM), with an ongoing maximum of 126 mo. Postmortem examinations were performed on shorter-term animals (n = 16) sacrificed at 16.7 +/- 7.3 mo. Thirteen showed no appreciable tracheal stenosis and three showed <10% stenosis at the level of the stoma. Histopathological examination of the stomal opening and surrounding tissue revealed minimal chronic inflammation and no evidence of necrosis or infection. CONCLUSIONS: During long-term respiratory studies, this practical and dependable tracheal stoma provides a means for examining acute and chronic effects of environmental and pathophysiological influences on the respiratory system of conscious dogs.

Repeat microsphere delivery for serial measurement of regional blood perfusion in the chronically instrumented, conscious canine.

INTRODUCTION: For chronic, repeat hemodynamic studies in conscious dogs, we designed and tested a chronically instrumented canine microsphere delivery model. The goals of this study were (1) to investigate the accuracy of repeated estimations of blood perfusion using fluorescent-labeled microspheres and (2) to develop and validate a chronic preparation that permits consecutive estimations in the same conscious animal over an extended protocol. METHODS: Via thoracotomy, nine dogs were instrumented with left atrial appendage and aortic vascular access catheters connected to subcutaneous vascular access ports. Four animals received seven serial injections of 1.6 million 15 microm microspheres (total: 11.2 million), and five animals received 8 serial injections of 2.25 million microspheres (total: 18 million) over the course of 11 or 18 wk. RESULTS: All catheters have remained bidirectionally patent during protocol for 14.9 +/- 0.8 (mean +/- SEM) wk. Sphere accumulation did not significantly alter global myocardial (P = 0.69, P = 0.25), renal (P = 0.92, P = 0.12), hepatic (P = 0.84, P = 0.32), or splenic (P = 0.33, P = 0.70) blood perfusion in either set of animals. CONCLUSIONS: Catheters remained bidirectionally patent for months, did not interfere with the hemodynamic responses of the preparation, and allowed repeat percutaneous injection of microspheres and withdrawal of reference arterial blood from within conscious canines. Eight serial injections totaling 18 million microspheres over 18 weeks did not alter regional myocardial, hepatic, renal, or splenic blood flow. This dependable, chronic, percutaneous arterial access preparation provides a means for examining acute and long-term effects of pathophysiological, pharmaceutical, and environmental influences on regional arterial blood flow in conscious, large animals.

Long-term pericardial catheterization is associated with minimum foreign-body response.

OBJECTIVES: The goals of this study were to assess the feasibility and to characterize the foreign-body response of a long-term catheter in the pericardium. BACKGROUND: Long-term access to the normal pericardial space provides opportunities for diagnostic sampling and therapeutic intervention. METHODS: After thoracotomy, in 7 anesthetized canines, the pericardium was opened and a 5 French silicone vascular access catheter was advanced 10 cm into the pericardial sac toward the apex of the heart. A hydraulic coronary balloon occluder was implanted (N=6). Pericardium was sealed with Prolene suture. Catheters were tunneled to the nape of the neck, attached to a subcutaneous vascular access port, and buried in the fascia. Animals underwent multiple experimental coronary artery occlusions across months. At sacrifice, we assessed the histopathological response of pericardium and epicardium to chronically indwelling silicone catheters. RESULTS: Post-mortem examinations were performed at 213 days post-operatively (mean, range=96-413, N=6), with one animal maintained for longer-term study. At sacrifice, all catheters were bidirectionally patent and completely mobile in the pericardium without evidence of tissue overgrowth around the intrapericardial segment. Adhesion tissue was found only at the site of catheter entry through the pericardium. Microscopic histopathological examination at catheter entry site, surrounding pericardium, and myocardium revealed minimum chronic inflammation. CONCLUSIONS: This subcutaneous system provides dependable, chronic access to the normal pericardial space for drug delivery and sampling. The presence of a chronic silicone catheter in the pericardium does not precipitate clinically significant pathologic changes even after repeated ischemic events.

Technique for implantation of chronic indwelling aortic access catheters.

INTRODUCTION: For chronic, repeated cardiovascular studies in trained, conscious dogs, we describe a technique for implantation of an arterial vascular access catheter in the aorta. In comparison to previous techniques, our technique enables arterial catheter implantation without interrupting the systemic circulation or compromising peripheral arterial flow, requires only a single penetration of the aortic wall, and results in a catheter facing downstream in the aorta. The catheter is usable for both arterial blood sampling and intra-arterial injection of pharmacologic agents. METHODS: After thoracotomy, two purse-string sutures were set in the aortic wall at the catheter entry-site. A Debakey-Satinsky vena cava clamp was used to partially clamp and isolate a 3-cm portion of aorta surrounding the pursestring sutures. The catheter was prepared by inserting a 0.35 mm guide wire, curve tip end first, into a 7 French silicone Access Technologies catheter. This caused a slight bend at the tip of the catheter, which later facilitated entry into the aorta and prevented damage to the intima of the aortic wall. Catheters were tunneled from the thorax to the nape of the neck, attached to a subcutaneous vascular access port (VAP), and buried in the fascia. Catheters were locked with 2 mL heparinized saline (20 IU/mL). With this technique, we implanted 16 aortic vascular access catheters in 16 dogs. RESULTS: Of the 16 animals, 8 are being maintained for long-term study, 7 were sacrificed for histopathological examination, and 1 died due to improper catheter implantation without the aid of the curved-tip guide-wire technique. VAPs have remained bidirectionally patent in all long-term animals (n = 8) ongoing for 7.5 +/- 1.4 months (mean +/- SEM), with an ongoing maximum of 14 months. In these long-term animals, VAPs were used 5.88 +/- 1.34 times. Postmortem examinations were performed on short-term animals (n = 8) sacrificed at 2.85 +/- 1.29 months. The catheters have remained bidirectionally patent in all but the one animal that died. In the short-term animals, the 7 patent VAPs were used 12.71 +/- 1.64 times. Histopathological examination of hematoxylin and eosin (H&E) slides from the catheter entry site revealed only minimal chronic inflammation. No evidence of tissue overgrowth around any of the intravascular segments of these silicone catheters was noted in any animal. CONCLUSIONS: Thus, this dependable subcutaneous arterial access system provides a means for examining acute and long-term effects of environmental and pathophysiological influences in conscious dogs. These catheters have remained bidirectionally patent ongoing for more than 1 year and allowed infusion of agents and withdrawal of central arterial blood samples.

An organic-inorganic hybrid scaffold for the culture of HepG2 cells in a bioreactor.

Much interest has recently been shown in the potential utility of bioartificial liver (BAL) as a bridge support for patients and as a module for experimental purposes. A radial-flow bioreactor (RFB), one of the perfused bed/scaffold-type bioreactors, enables a highly functional three-dimensional culture as BAL. The functional capacity of bioreactors depends not only on their mechanistic structures but also on scaffolds packed in them. In the present study, we examined the possible utility of a new porous organic-inorganic-hybrid scaffold in an RFB. The scaffold was made from tetraethoxysilane (TEOS) and polydimethylsiloxane (PDMS) by a sol-gel method using sieved sucrose particles as a porogen. In the porous TEOS-PDMS hybrid scaffold, human hepatocellular carcinoma cells (HepG2) proliferated actively and formed cell clusters more efficiently than they did in a polyvinyl-alcohol scaffold. When cultivated in PDMS-TEOS, HepG2 cells secreted a approximately three-fold greater amount of albumin than that secreted in a monolayer culture. For potential application of BAL to pharmacological studies and future clinical use, it is essential to develop a method to propagate liver cells that maintain highly specific functions. The present results indicate that PDMS-TEOS may be a promising scaffold for developing such functional culture methods.

Propagation of adult rat bone marrow-derived hepatocyte-like cells by serial passages in vitro.

Previously, we found that hepatocyte growth factor receptor (c-Met)-and alpha-fetoprotein (AFP)-expressing cells were present in adult rat bone marrow, and that these cells also expressed hematopoietic stem cell markers, such as CD34, Thy-1, and c-Kit. When bone marrow cells were cultured in a hepatocyte growth medium (HGM) with HGF and EGF, colonies composed of polygonal cells resembling mature hepatocytes appeared by 2 weeks and grew very slowly because of overgrowth of stromal cells. At days 34-41, 2-mm2 sheets of hepatocyte-like cells were cut out of their colonies by scratching with an injection needle under observation with a phase contrast microscope, transferred into wells of 24-well plates, and cultured in the HGM medium in the presence or absence of HGF and EGF. When cells reached confluence, cells were detached with trypsin and EDTA and transferred step by step into bigger culture vessels. Thus, hepatocyte-like cells were expanded 1000-fold during less than 4 months. These cells were immunocytochemically stained for albumin and also for AFP and the hematopoietic stem cell markers described above, showing characteristics of oval cells. By RT-PCR, we detected mRNAs of tryptophan-2,3-dioxygenase and tyrosine aminotransferase, markers of hepatocytes at a terminal differentiation stage. The present culture system may be useful for supply of hepatocyte resources for cell transplantation therapy.

Expression of CYP3A4 by an immortalized human hepatocyte line in a three-dimensional culture using a radial-flow bioreactor.

Cytochrome P450 (CYP) 3A is responsible for about 50% of drug metabolizing activity in the liver. The present study was undertaken to establish a CYP3A4-active model for in vitro analysis of human drug metabolism. The cells used were immortalized normal human fetal hepatocytes (OUMS-29) and its HNF4alpha-introduced subline (OUMS-29/H-11). The cells were cultivated under high-density three-dimensional conditions in a radial-flow bioreactor (RFB). The number of OUMS-29 cells increased 15-fold over 49 days and their apical surfaces were covered with abundant microvilli, a characteristic of hepatocytes in vivo. The amount of albumin secreted by OUMS-29 cells in the three-dimensional RFB culture was 6-fold higher than those in a monolayer culture. CYP3A4 protein and an intermediate metabolite of testosterone by CYP3A4 were detected in OUMS-29/H11 cells cultivated in RFB >29 days. These results indicate that the RFB culture of OUMS-29/H-11 cells is useful for screening and developing new drugs.

Involvement of interferon regulatory factor 1 and S100C/A11 in growth inhibition by transforming growth factor beta 1 in human hepatocellular carcinoma cells.

Growth inhibition by transforming growth factor (TGF)-beta 1 has been attributed to the induction of cyclin-dependent kinase inhibitors, among which p21/Waf1 plays a major role in many biological contexts. In the present study, two new intracellular mediators for the induction of p21/Waf1 by TGF-beta 1 were identified in a human hepatocellular carcinoma cell line (JHH-5) expressing mutant-type p53. After addition of TGF-beta 1 to JHH-5 cells, a marked increase of the p21/Waf1 expression preceded the inhibition of DNA synthesis. Expression of IFN regulatory factor (IRF)-1, a known transacting factor for p21/Waf1 promoter, was elevated just before or in parallel with the increase of p21/Waf1. Transduction of antisense IRF-1 inhibited the increase in p21/Waf1 in JHH-5 cells treated with TGF-beta 1 and partially released the cells from the growth arrest by TGF-beta 1. Expression of S100C/A11, a member of the Ca(2+)-binding S100 protein family, also markedly increased after addition of TGF-beta 1. S100C/A11 protein was translocated to and accumulated in nuclei of TGF-beta 1-treated JHH-5 cells, where p21/Waf1 was concomitantly accumulated. When a recombinant S100C/A11 protein was introduced into nuclei of JHH-5 cells, DNA synthesis was markedly inhibited in a dose-dependent manner in the absence of TGF-beta 1. Prior transfection of p21/Waf1-targeted small interfering RNA efficiently blocked decrease of DNA synthesis in JHH-5 cells caused by TAT-S100C/A11 or TGF-beta 1 and markedly inhibited expression of p21/Waf1 protein in the cells. These results indicate that IRF-1 and S100C/A11 mediate growth inhibition by TGF-beta 1 via induction of p21/Waf1.

Improved conditions to induce hepatocytes from rat bone marrow cells in culture.

Recent studies have revealed that bone marrow cells can develop into hepatocytes by in vivo transplantation under certain circumstances. However, little is known about the mechanism of bone marrow cell differentiation into hepatocytes. It is important to determine suitable culture conditions in which bone marrow cells will be differentiated into hepatocytes not only for understanding differentiation mechanisms but also for efficient amplification of hepatocyte-progenitor cells of bone marrow origin, this being a prerequisite for potential therapeutic use. In the present study, we found that hepatocyte growth factor (HGF) receptor (c-Met)- and alpha-fetoprotein-expressing cells were present in adult rat bone marrow. We also found that these cells also express hematopoietic stem cell markers, such as CD34, Thy-1, and c-Kit. Using an HGM medium with HGF and EGF, we succeeded in propagating hepatocyte-like cells induced from adult rat bone marrow in culture. These cells were immunocytochemically stained for albumin. By RT-PCR analysis of cultures containing the hepatocyte-like cells, we detected mRNAs of tryptophan-2,3-dioxygenase and tyrosine aminotransferase, markers of hepatocytes at a terminal differentiation stage. The present culture therefore can be a useful resource for cell transplantation therapy for liver diseases.