Hygiene risk of waterborne pathogenic viruses in rural communities using onsite sanitation systems and shallow dug wells.

We evaluated the hygienic influence of onsite sanitation systems (OSSs) on drinking water wells in rural Sri Lanka by determining the safe setback distance between wells and the management of OSSs. Although previous studies have used bacterial indicators such as E. coli to evaluate the OSS impact, these parameters cannot assess the hygiene risk for waterborne pathogenic viruses (e.g. rotaviruses). Therefore, pepper mild mottle virus was selected as an indicator of human-specific faecal virus contamination. From a viral perspective, not only can the horizontal distance between a well and the nearest OSS reasonably represent hygiene safety, but the OSS sludge management can mitigate the contamination of wells even at short distances from the OSSs. Quantitative microbial risk assessment suggests that the infection risk of rotavirus was extremely high compared to the international standard. As proper management of OSSs would be key to reducing viral risk, it is necessary to reach out to the residents who are unaware of the importance and necessity of such management.

Partial analysis of hepatitis B virus DNA from hepatocellular carcinoma showing negative hepatitis B virus surface antigen: an analysis of two Japanese cases.

It has been reported that hepatitis B virus (HBV) DNA is detected in serum and/or liver in patients with hepatocellular carcinoma (HCC) without HBsAg. To adress this issue, we analyzed HBV genome in 2 HCC cases without HBsAg. The DNA from serum from patients with HCC was amplified with a nested PCR, and 'a' determinant of S region, core promoter region and precore region were sequenced. The first case, a 50 years-old male, was negative for HBsAg and HBeAg, and positive for anti-HBs, anti-HBe and anti-HBc. Viral load of HBV in serum was 4.0 log genome equivalent/ml by TMA assay, and was 1.1 X 105 copy/ml by real-time PCR system. A nucleotide analysis of the common 'a' determinant of S gene showed that the 5 first amino acids of 'a' determinant, CTIPA, were changed to CKTCTTPA. The second case, a 76 years-old male, was positive for anti-HBe, but negative for HBsAg, anti-HBs, HBeAg and anti-HBc. No missense or nonsense mutations were seen in 'a' determinant of S region. Viral load of serum HBV was < 3.7 log genome equivalent/ml by TMA assay, but was 2.4X103 copy/ml by real-time PCR system. The results of the present study suggest that the mechanisms of HBsAg loss are diverse among HCC patients without HBsAg, and that an analysis of HBV genome is a useful tool to dissolve molecular mechanisms losing HBs antigenicity.

[Esophagogastric variceal bleeding in a case of liver cirrhosis associated with thalassemia].

A 58-year-old man was admitted to our hospital because of hematemesis. Laboratory tests indicated microcytic hypochromic anemia and iron overload. We performed urgent endoscopic examination, and diagnosed bleeding from esophagogastric varices. Abdominal CT showed liver cirrhosis and marked splenomegaly, but no evidence of gastrorenal shunt. The varices were treated by Hassab's operation and splenectomy. Pathologic examination revealed hepatocytes in the cirrhotic nodules filled with iron pigment. The cause of the liver cirrhosis was considered to be due to iron overload resulting from thalassemia. We diagnosed the cause of the microcytic anemia as thalassemia by gene analysis, which revealed heterozygosity of a deletion (deltabeta thalassemia Jpn-type II) and one point mutation (-31A-->G).

[A case of portal vein thrombosis associated with congenital protein S deficiency].

A 25-year-old man was admitted to our hospital because of abdominal pain, nausea and low-grade fever. An abdominal CT showed remarkable thickening of the wall of the small intestine and extensive thrombosis of the mesenteric, portal and splenic veins. Because neither intestinal infarction nor peritonitis was seen, anticoagulation therapy was chosen. Heparin was administered intravenously and was used alternatively with warfarin later. The patient's symptoms and clinical data improved gradually. Concerning the etiological factors of the thrombosis, only protein S activity definitely decreased. Genetic analysis indicated a variant of protein S, protein S Tokushima.

Serum human telomerase reverse transcriptase messenger RNA as a novel tumor marker for hepatocellular carcinoma.

PURPOSE: We previously reported the usefulness of a qualified highly sensitive detection method for human telomerase reverse transcriptase (hTERT) mRNA in serum with 89.7% sensitivity for hepatocellular carcinoma (HCC). In this study, we developed a quantitative detection method for serum hTERT mRNA and examined the clinical significance in HCC diagnosis. EXPERIMENTAL BACKGROUND: In 64 patients with HCC, 20 with liver cirrhosis, 20 with chronic hepatitis, and 50 healthy individuals, we measured serum hTERT mRNA by using the newly developed real-time quantitative reverse transcription-PCR with SYBR Green I. We examined its sensitivity and specificity in HCC diagnosis, clinical significance in comparison with other tumor markers, and its correlations with the clinical variables by using multivariate analyses. RESULTS: Serum hTERT mRNA showed higher values in patients with HCC than those with chronic liver diseases. hTERT mRNA expression was shown to be independently correlated with clinical variables such as tumor size, number, and degree of differentiation (P < 0.001, each). The sensitivity/specificity of hTERT mRNA and alpha-fetoprotein (AFP) mRNA in HCC diagnosis were 88.2%/70.0% for hTERT and 71.6%/67.5% for AFP, respectively. hTERT mRNA proved to be superior to AFP mRNA, AFP, and des-gamma-carboxy prothrombin in HCC diagnosis. Furthermore, hTERT mRNA in serum was associated with that in HCC tissue. CONCLUSIONS: The usefulness of hTERT mRNA expression in HCC diagnosis and its superiority to conventional tumor markers were shown. Therefore, serum hTERT mRNA is a novel and available marker for HCC diagnosis.

[A case of gastrointestinal stromal tumor with liver and bone metastases effectively treated with radiofrequency ablation and imatinib mesylate].

A 58-year-old man was admitted because of perforation of the small intestine by a gastrointestinal stromal tumor (GIST). First, the small intestine including a GIST was resected; and then 2 month later, a part of the liver (S5) conforming to metastatic lesion was surgically removed. Twelve months later, another liver metastases was found, and surgical treatment was recommended; but the patient requested non-surgical therapy, so a radiofrequency ablation (RFA) was successfully performed. After that, recurrence of liver metastasis was not observed, but another metastasis was observed on the fifth lumbar vertebra; so administration of imatinib mesylate was started. 28 months after the initial administration the metastatic liver lesion was still invisible, and the bone metastatic lesion had not grown. The patient is alive with good performance status. This report shows that multi-modality therapy by surgery, RFA and imatinib mesylate was effective for liver and bone metastases of GIST.

Synergistic effects of (-)-epigallocatechin gallate with sulindac against colon carcinogenesis of rats treated with azoxymethane.

(-)-Epigallocatechin gallate (EGCG), a major constituent of green tea, has been shown to exhibit anti-cancer activity. Sulindac is also well known as a cancer-preventive agent against colon cancer, but its usage is restricted because of its adverse effects, as exemplified by gastrointestinal bleeding. In the present study, we examined whether a combination of EGCG and sulindac shows synergistic effects for cancer-preventive activity for rat colon carcinogenesis induced by azoxymethane (AOM); we examined the number of aberrant crypt foci (ACF) representing preneoplastic lesions, the argyrophilic nucleolar organizer region (AgNOR) as an indicator of cell proliferation, and the incidence of apoptosis. The AOM treatment induced an average of 46.2+/-4.9 ACF/colon, and sulindac and EGCG significantly reduced the incidence of ACF/colon to 21.4+/-3.4 and 19.5+/-5.8, respectively (P<0.01). The co-treatment with EGCG and sulindac resulted in significantly reduced ACF formation (10.0+/-3.2; P<0.01). The results of the AgNOR analysis indicated that the treatment with EGCG and/or sulindac suppressed AOM-induced cell proliferation. The present results also revealed that the combination of EGCG and sulindac synergistically enhanced apoptosis significantly (P<0.01). Thus, our findings suggest that EGCG with sulindac synergistically suppresses ACF formation by enhancing apoptosis and, therefore, that EGCG is a suitable candidate for use in combination with cancer-preventive agents, such as sulindac, to reduce their adverse effects.

Effects of cyclooxygenase-2 inhibitor NS-398 on APC and c-myc expression in rat colon carcinogenesis induced by azoxymethane.

BACKGROUND: Nonsteroidal anti-inflammatory drugs (NSAIDs) have been reported to protect against the development of colon cancer; however, the mechanism(s) by which NSAIDs exert their effects is still unclear. The aim of this study was to examine the effects of NSAIDs on the expression of the tumor suppressor APC gene and the c-myc oncogene in the colons of rats treated with a colon-specific carcinogen, azoxymethane (AOM). METHODS: Gene expression levels were estimated by a reverse transcription (RT)-competitive polymerase chain reaction (PCR) method. RESULTS: The group of rats simultaneously administered AOM and NS-398, a cyclooxygenase (COX)-2 inhibitor, showed a significant reduction in the number of preneoplastic lesions of colon cancer compared with that in the group of rats treated with AOM alone. Furthermore, the APC expression level in the group of rats treated with both AOM and NS-398 was significantly greater than that in the group of rats treated with AOM alone; this result for APC gene expression was reconfirmed by the immunohistochemical staining of APC protein. In addition, c-myc mRNA expression was clearly decreased in the group of rats treated with both AOM and NS-398 compared with the level in the group of rats treated with AOM alone. CONCLUSIONS: Our data thus indicate that NS-398 causes an increase in APC expression and a decrease in c-myc expression.

A new animal model of continuous catheterization for investigating mechanisms of arteritis associated with chemotherapy.

Although superselective continuous intra-arterial infusion has advantages for cancer therapy, intra-arterial chemotherapy is often interrupted by arterial damage due to arteritis. Therefore, an animal model must be developed to elucidate the mechanism of arteritis associated with continuous anti-cancer drug infusion. We developed a new rat model with which to investigate the causal mechanism(s) of vascular damage associated with continuous catheterization chemotherapy. Chemotherapeutic agents (fluorouracil (5-FU) or peplomycin (PEP)) were continuously administered for 7 days into the abdominal aorta of male Sprague-Dawley rats through a catheter fixed in situ. We found that the incidence of apoptotic endothelial cells of the aorta was higher nearer the tip of the catheter. The incidence of apoptosis was higher in the group treated with 5-FU than with PEP. This animal model will be useful to improve arterial damage among patients undergoing chemotherapy using continuous catheterization.

Clinical usefulness of serum telomerase reverse transcriptase (hTERT) mRNA and epidermal growth factor receptor (EGFR) mRNA as a novel tumor marker for lung cancer.

Using a newly developed assay of telomerase reverse transcriptase (hTERT) mRNA in serum by real-time RT-PCR, we previously reported this assay to be superior to other tumor markers for hepatoma. In this study, we aimed to clarify its clinical significance as a biomarker for lung cancer. In 112 patients with lung tumor and 80 individuals without cancer, we measured serum hTERT mRNA and epidermal growth factor receptor (EGFR) mRNA levels, using a quantitative one-step real-time RT-PCR assay. We examined its sensitivity and specificity in lung cancer diagnosis, its clinical significance in comparison with other tumor markers, and its correlation with the clinical parameters using multivariate analyses and correlation relative tests. The copy number of serum hTERT mRNA was independently correlated with tumor size, tumor number, presence of metastasis and recurrence, and smoking (all P < 0.05). EGFR mRNA correlated with tumor number and clinical stage (both P < 0.05). The sensitivity and specificity in lung cancer diagnosis were 89.0% and 72.7% for hTERT mRNA, and 71.3% and 80.0% for EGFR mRNA, respectively. hTERT mRNA was superior to other tumor markers in lung cancer diagnosis. For both mRNAs, serum levels were significantly correlated with levels in lung cancer tissues (both P < 0.05). The copy number of hTERT mRNA significantly decreased after the surgical treatment. The data suggest that hTERT mRNA, especially when combined with EGFR mRNA, is a novel and excellent biomarker for pulmonary malignancies to diagnose and assess the clinical stage.

Trapidil enhances the slowly activating delayed rectifier potassium current and suppresses the transient inward current induced by catecholamine in Guinea pig ventricular myocytes.

We examined the electrophysiological effects of trapidil on the ionic currents influencing the repolarization and on the transient inward current (ITi) that can cause triggered arrhythmia using the whole-cell patch-clamp technique in guinea pig ventricular myocytes. Trapidil shortened the action potential duration (APD) and increased the delayed rectifier potassium current (IK) in a concentration-dependent manner. The effect of trapidil on the rapidly and slowly activating components of IK (IKr and IKs, respectively) was studied by the envelope of tails test. Trapidil failed to affect IKr and selectively enhanced IKs. Trapidil increased the amplitude of the L-type Ca2+ current (ICa,L), with an acceleration of its inactivation, whereas isoproterenol, a beta-adrenoceptor agonist, increased the amplitude of the ICa,L in a different manner. Isoproterenol activated ITi; however, trapidil not only failed to facilitate ITi but also suppressed isoproterenol-induced ITi. The inhibitory effect of trapidil on isoproterenol-induced ITi is at least partly via a reduction of Ca2+ overload through an acceleration of ICa,L inactivation and/or a sarcoplasmic reticulum (SR) Ca channel modulation. These results suggest that trapidil does not prolong the QT interval and has an antiarrhythmic effect on arrhythmias elicited by triggered activity secondary to Ca2+ overload at much higher concentrations than clinical concentration.

Ischemia of rat stomach mobilizes ECL cell histamine.

Microdialysis was used to study how ischemia-evoked gastric mucosal injury affects rat stomach histamine, which resides in enterochromaffin-like (ECL) cells and mast cells. A microdialysis probe was inserted into the gastric submucosa, and the celiac artery was clamped (30 min), followed by removal of the clamp. Microdialysate histamine was determined by enzyme-linked immunosorbent assay. In addition, we studied the long-term effects of ischemia on the oxyntic mucosal histidine decarboxylase activity in omeprazole-treated rats. Gastric mucosal lesions induced by the ischemia were enlarged on removal of the clamp. The microdialysate histamine concentration increased immediately on clamping (50-fold rise within 30 min) and declined promptly after the clamp was removed. In contrast, histidine decarboxylase activity of the ECL cells was lowered by the ischemia and returned to preischemic values 9 days later. Mast cell-deficient rats responded to ischemia-reperfusion much like wild-type rats with respect to histamine mobilization. Pretreatment with the irreversible inhibitor of histidine decarboxylase, alpha-fluoromethylhistidine, which is known to eliminate histamine from ECL cells, prevented the rise in microdialysate histamine. Pharmacological blockade of acid secretion (cimetidine or omeprazole) prevented the lesions induced by ischemia-reperfusion insult but not the mobilization of histamine. In conclusion, ischemia of the celiac artery mobilizes large amounts of histamine from ECL cells, which occurs independently of the gross mucosal lesions. The prompt reduction of the mucosal histidine decarboxylase activity in response to ischemia probably reflects ECL cell damage. The lesions develop not because of mobilization of histamine per se but because of ischemia plus reperfusion plus gastric acid.

Effects of Sho-Saiko-to on hepatocarcinogenesis and 8-hydroxy-2'-deoxyguanosine formation.

Oxidative stress plays an important role in hepatocarcinogenesis. Although Sho-saiko-to (TJ-9), a Japanese herbal medicine which has been recently administered to patients with chronic liver disease in Japan, prevents hepatocarcinogenesis, the mechanism by which TJ-9 protects against cancer development is not fully understood. 8-Hydroxy-2'-deoxyguanosine (8-OHdG), a DNA adduct by reactive oxygen species, is known as a parameter of genetic risk for hepatocarcinogenesis. To clarify whether the preventive effect on hepatocarcinogenesis by TJ-9 is dependent on 8-OHdG, the effect on 8-OHdG levels by TJ-9 was examined by using high-performance liquid chromatography-mass spectrometry (LC-MS) in a diethylnitrosamine (DEN)-induced hepatocarcinogenesis model of male Fisher rats. TJ-9 reduced the number of preneoplastic cells, detected as the glutathione S transferase P (GST-P)-positive hepatocytes, and inhibited the development of liver tumors. TJ-9 also significantly decreased the formation of 8-OHdG, as indicated by LC-MS and immunohistochemical analysis. In addition, ornithine decarboxylase (ODC) activity and the number of proliferating cell nuclear antigen (PCNA)-positive cells were not altered. An electron paramagnetic resonance spin-trapping technique showed that TJ-9 scavenges hydroxyl radicals in a dose-dependent manner. In conclusion, the results of the present study suggest that TJ-9 prevents hepatocarcinogenesis in association with inhibition of 8-OHdG formation.