Kazuyuki Yazawa

Bifidobacterium longum as a delivery system for cancer gene therapy: selective localization and growth in hypoxic tumors.

A fundamental obstacle in gene therapy for cancer is the specific delivery of an anticancer gene product to a solid tumor, and yet no systemic delivery system that specifically targets solid tumors currently exists. A strain of domestic bacteria, Bifidobacterium longum, which is nonpathogenic and anaerobic, selectively localized and proliferated in several types of mouse solid tumors after systemic application. In this report, we further describe a novel approach to cancer gene therapy in which genetically engineered Bifidobacterium is used as a tumor-specific vector. Similarly to wild-type B. longum, genetically engineered B. longum could be detected in tumor tissue only and was not found in a large survey of normal mouse tissues after intravenous injection. This finding strongly suggests that obligate anaerobic bacteria such as Bifidobacterium can be used as highly specific gene delivery vectors for cancer gene therapy.

Bifidobacterium longum as a delivery system for gene therapy of chemically induced rat mammary tumors.

A fundamental obstacle in cancer gene therapy is the specific targeting of therapy directly to a solid tumor, and no systemic delivery system yet exists. A strain of domestic bacteria, Bifidobacteriumlongum, which is nonpathogenic and anaerobic, selectively localized to and proliferated in 7,12‐dimethylbenz[a]anthracene‐induced rat mammary tumors after systemic application. We further ascertained the tumor specificity of genetically engineered, as well as wild‐type, Bifidobacterium longum. This is the first demonstration that Bifidobacterium longum can be utilized as a specific gene delivery vector for gene therapy on solid breast tumors.

Current Progress in Suicide Gene Therapy for Cancer

Standard chemotherapeutic agents and ionizing radiation destroy dividing cells. Because tumor cells divide more rapidly than normal cells, there is a therapeutic index in which damage to the cancer cells is maximized while keeping the toxicity to the normal host cells acceptable. Suicide gene therapy strives to deliver genes to the cancer cells, which convert nontoxic prodrugs into active chemotherapeutic agents. With this strategy, the systemically administered prodrug is converted to the active chemotherapeutic agent only in cancer cells, thereby allowing a maximal therapeutic effect while limiting systemic toxicity. A literature search was conducted using the MEDLINE database from 1990 to 2001 to identify articles related to suicide gene therapy for cancer. A number of suicide gene systems have been identified, including the herpes simplex virus thymidine kinase gene, the cytosine deaminase gene, the varicella-zoster virus thymidine kinase gene, the nitroreductase gene, the Escherichia coli gpt gene, and the E. coli Deo gene. Various vectors, including liposomes, retroviruses, and adenoviruses, have been used to transfer these suicide genes to tumor cells. These strategies have been effective in cell culture experiments, laboratory animals, and some early clinical trials. Advances in tissue- and cell-specific delivery of suicide genes using specific promoters will improve the clinical utility of suicide gene therapy.

Cloned cytosine deaminase gene expression of Bifidobacterium longum and application to enzyme/pro-drug therapy of hypoxic solid tumors.

Bifidobacterium longum is a nonpathogenic anaerobic bacterium among normal bacterial flora. Recently, it was reported that B. longum accumulated in hypoxic solid tumors. The gene of interest was expressed in transfected B. longum by the shuttle vector pBLES100 in solid tumors. In this report, we constructed pBLES100-S-eCD, which included the cytosine deaminase gene. We confirmed by western blotting that transfected B. longum produced cytosine deaminase. In addition, transfected B. longum produced cytosine deaminase that converted 5-fluorocytosine into 5-fluorouracil. B. longum could be useful for enzyme/pro-drug therapy of hypoxic solid tumors.

Synchronous gastric tumors associated with esophageal cancer: A retrospective study of twenty-four patients

OBJECTIVES Synchronous gastric tumors (including benign and secondary tumors) associated with esophageal cancer present diagnostic and therapeutic issues. We investigated this synchronous association, and retrospectively determined the frequency of the gastric tumors and the clinical characteristics. METHODS In a series of 208 patients with esophageal cancer, we investigated the synchronous gastric tumors, as well as the frequency of association, clinicopathological characteristics, diagnosis, treatment, and the clinical outcome after surgery. RESULTS Twenty-eight gastric tumors were found in 24 patients. Adenocarcinoma was most frequent. Most of these tumors were located at the upper or middle third of the stomach. Eight gastric tumors in six patients could not be detected preoperatively. Six of these tumors including a gastric remnant cancer were detected in the resected stomach, and two leiomyomas were detected during the operation. In one patient in which an endoscope could not pass through the esophagus, a leiomyoma was detected in the resected stomach. For the gastric cancers, total gastrectomy or proximal gastrectomy with lymph node dissections was performed. For the benign tumors, partial resection of the stomach was performed, and endoscopic resection was performed preoperatively for an adenoma. In both the postoperative hospital mortality rate and the survival rate after surgery, there were no significant differences between the patients with and without gastric tumors. CONCLUSIONS Synchronous gastric tumors associated with esophageal cancer are not rare. When an endoscope cannot pass through the esophagus before surgery, other techniques must be performed to explore the stomach. For these patients, surgical treatment should be adapted positively.

Oesophageal cancer associated with other primary cancers: A study of 31 patients

The aim of this study was to investigate the characteristics of oesophageal cancer associated with other primary cancers and the survival rate after surgery for the patients with these cancers. Of 202 patients with oesophageal cancer treated in the Second Department of Surgery, Shinshu University School of Medicine between 1981 and 1995, 31 patients (15.3%) had oesophageal cancer associated with other primary cancers. Twenty‐one synchronous and 10 metachronous associated cancers were found and 25 of them were resected. Early‐stage oesophageal cancer was much more frequent in the associated cases than in the non‐associated cases. The stomach was the most frequently associated organ. The numbers of cases with triple and quadruple cancers were three and one, respectively. Three of these cases had intervals of over 6 years between tumours. Three cases with other primary cancers which had intervals of over 7 years after oesophagectomy were found, and two were carcinomas of the reconstructed gastric tube. In the outcome after surgery for oesophageal cancer, there was no difference between the associated and the non‐associated cases, and also no difference between the synchronous and metachronous associated cases. Regarding the five‐year and 10‐year survival rates after surgery for the first cancers, the synchronous cases had a poorer outcome than did the metachronous cases. In conclusion, oesophageal cancer with other primary cancers is not always rare, and its outcome is not poor compared with that of the non‐associated cases. These patients may achieve survival by early detection of both lesions and positive treatment. It is important to consider the risk of other primary cancers after oesophagectomy, and the success of the reconstructed gastric tube should be followed by endoscopy.

Specific gene expression and therapy for pancreatic cancer using the cytosine deaminase gene directed by the rat insulin promoter

Suicide gene therapy has been shown to be an effective means of destroying pancreatic cancer cells, but cell-specific delivery of the gene is required to limit host toxicity. The objective of this study is to determine whether the rat insulin promoter (RIP) will permit cell-specific gene delivery and subsequent cell death in human pancreatic cancer cells. The RIP DNA was amplified using polymerase chain reaction (PCR), and the purified fragment was inserted into pCR-Blunt II-TOPO plasmid at the SpeI site, which contains the coding sequence of yeast cytosine deaminase (CD). Transfection assays were carried out using both RIP-lacZ and RIP-CD DNA constructs in two human pancreatic cancer cell lines, PANC-1 and MIA PaCa-2. Reporter assays using X-gal staining were performed, and the in vitro cytotoxicity was examined in RIP-CD-transfected cells treated with 5-.ucytosine for 5 days. The expression levels of CD protein in the transfected cells were determined 2 days after transfection by Western blot analysis. The expression levels of insulin promoter factor (IPF-1/PDX-1) in these human pancreatic cell lines, as well as in freshly isolated human pancreatic cancer specimens, were determined using in situ immunohistochemistry analysis. After transfection with RIP-lacZ, only PANC-1 cells, but not MIA PaCa-2 cells, were positive for RIP-lacZ expression, indicating that RIP-directed reporter gene expression occurred only in PANC-1 cells. After transfection with RIP-CD and treatment with 5-flucytosine, PANC-1 cells had a significantly increased cell death rate compared with that of MIA PaCa-2 cells, suggesting that RIP-directed suicide gene expression occurred only in PANC-1 cells. Western blot analysis demonstrated that only PANC-1 cells were able to express the CD protein and that significantly increased levels of PDX-1 were found in PANC-1 but not in Mia PaCa-2 cells. In situ immunohistochemical analysis of both cell lines showed that PDX-1 was only expressed in the nuclei of PANC-1 cells and not in MIA PaCa-2 cells. Furthermore, two freshly isolated human pancreatic cancer specimens had significantly increased levels of PDX-1. The RIP is activated in PANC-1 cells, but not in Mia PaCa-2 cells, and the mechanism of activation is via PDX-1. Pancreatic cancer-specific cytotoxicity can be achieved with the use of RIP-CD and 5-flucytosine treatment in vitro. Significantly increased levels of PDX-1 have been found in human pancreatic cancer specimens. These results suggest that RIP could be used for cell-specific suicide gene therapy to target human pancreatic tumors.

Medical care for a mass gathering: the Suwa Onbashira Festival.

INTRODUCTION The Suwa Onbashira Festival is held every six years and draws approximately one million spectators from across Japan. Men ride the Onbashira pillars (logs) down steep slopes. At each festival, several people are crushed under the heavy log. During the 2004 festival, for the first time, a medical care system that coordinated a medical team, an emergency medical service, related agencies, and local hospitals was constructed. OBJECTIVE The aims of this study were to characterize the spectrum of injuries and illness and to evaluate the medical care system of this festival. METHODS The festival was held 02 April-10 May 2004. The medical records of all of the patients who presented to an on-site medical tent or who were treated at the scene and transported to hospitals over a 12-day period were reviewed. The following items were evaluated: (1) the emergency medical system at the festival; (2) the environmental circumstances; and (3) patient data. RESULTS All medical usage rates are reported as patients per 10,000 attendees (PPTT). A total 1.8 million spectators attended the festival during the 12-day study period; a total of 237 patients presented to the medical tent (1.32 PPTT), and 63 (27%) were transferred to hospitals (0.35 PPTT). Of the total, 135 (57%) suffered from trauma--two were severely injured with pelvic and cervical spine fractures; and 102 (43%) had medical problems including heat-related illness. CONCLUSIONS Comprehensive medical care is essential for similar mass gatherings. The appropriate triage of patients can lead to efficient medical coverage.

Intravenous Delivery of Liposome-mediated Nonviral DNA Is Less Toxic than Intraperitoneal Delivery in Mice

Suicide gene therapy has been shown to be an effective means of destroying pancreatic cancer cells. Liposomes have been described as having better efficacy in gene delivery, and an advantage of using liposomes as gene carriers is that they can be used repeatedly in vivo. The objective of this study is to compare the effect of gene delivery routes and to determine whether systemic delivery of the rat insulin promoter (RIP)–directed suicide gene construct would permit cell-specific gene delivery in vivo. Severe combined immunodeficient (SCID) mice were injected with liposome-RIP-TK (thymidine kinase) complex by either the intraperitoneal or the intravenous route. Twenty-four hours post gene delivery, mice received ganciclovir (GCV) treatment twice daily for 14 days. Mice were sacrificed at various time points. Complete necropsy and serum chemistry analysis were performed. Islet morphology was determined using hematoxylin and eosin (H&E) staining. Serum glucose and insulin levels were also determined. To determine the toxic effect on pancreatic islet cells, immunostaining of insulin-producing and glucagon-producing cells was carried out at each time point. H&E staining indicated that both intravenous and intraperitoneal liposome-RIP-TK gene expression had no effect in normal endocrine islet cells. Both gene-delivery routes in mice resulted in normal glycemia and serum insulin levels. The endocrine islets were intact, with a normal distribution pattern of insulin-producing beta cells and glucagon-secreting alpha cells. However, serum chemistry analysis revealed significantly elevated levels of liver enzymes; suggesting that possible liver damage had occurred with the intraperitoneal gene delivery of liposome-pRIP-TK. Intravenous liposome-mediated gene delivery had no effect on liver enzyme levels. Liposome-mediated gene delivery via intravenous injection was less toxic than intraperitoneal delivery. This gene-delivery route requires fewer liposome-DNA complexes and maintains normal liver function. Thus, intravenous delivery of gene therapy would be superior to intraperitoneal administration of gene therapy in mice.

Can Sedation Reduce the Cardiac Stress During Gastrointestinal Endoscopy? A Study With Non-invasive Automated Cardiac Flow Measurement by Color Doppler Echocardiography

Background: Upper gastrointestinal endoscopy (UGIE) may cause some cardiac stress. The effect of sedation on hemodynamics during UGIE has not been fully studied, and therefore the aim of this study was to clarify whether or not sedation can reduce cardiac stress during UGIE. Methods: Eight normal male volunteers undergoing UGIE with sedation (0.1 mg/kg of midazolam) and without it (two endoscopies per volunteer in random order) were monitored throughout the procedure by means of electrocardiogram, blood pressure and peripheral oxygen saturation (SpO 2 ). Cardiac output was measured at six points before, during and after endoscopy from automated cardiac flow measurement by color Doppler echocardiography. Serum norepinephrine, epinephrine, dopamine and ACTH concentrations were measured before and after the examination. Results: No significant differences in heart rate, systolic blood pressure, rate-pressure product, cardiac output and left ventricular work index were observed between the sedated and non-sedated groups. SpO 2 hardly changed during endoscopy in the non-sedated group, but decreased slightly in the sedated group ( P = 0.075). Although all serum catecholamine concentration changes were within normal limits in both groups, after endoscopy only epinephrine concentration was significantly lower in the sedated group than in the non-sedated group ( P = 0.0027). Conclusions: Conscious sedation with midazolam does not reduce the cardiac stress during UGIE.