Long-term outcomes of hemostatic therapy for variceal bleeding and the challenge pending in the post-direct-acting antivirals era.

Background and study aims: This study evaluated the longterm outcomes of mainly endoscopic hemostatic therapy for gastrointestinal variceal bleeding and of the transition of hemostatic therapy. Patients and methods: Among 1,163 patients treated for gastrointestinal varices between April 2006 and June 2020, a total of 125 patients who underwent emergency hemostatic therapy were enrolled. Survival rates and secondary evaluation points were analyzed. Additionally, patients were classified into two groups: the previous and latter term. Patients' background, therapeutic method, and treatment results were compared between the groups. Results: 94.4% had cirrhosis. The average Child-Pugh score was 8.90. Successful primary hemostasis rate was 98.4%, and 5.6% died within 2 weeks, all with a Child-Pugh score ≥9. The respective 1- and 5-year survival rates for Child-Pugh grade A/B were 81.3% and 55.4%, while those for Child-Pugh grade C were 58.1% and 17.8%. Child-Pugh grade C or hepatocellular carcinoma was significantly associated with poor prognosis. In total, 21.6% experienced variceal re-bleeding; 62.9% of these cases were triggered by continued alcohol consumption. There was no significant difference in survival between patients with and without variceal re-bleeding and in post-treatment survival between the previous and latter terms. In the latter term, the number of cases caused by continued alcohol consumption significantly increased. Conclusions: Multidisciplinary treatment and continuation of proper management after hemostatic therapy for variceal bleeding are crucial. Continued alcohol consumption leads to variceal bleeding and re-bleeding; its proper management, including alcohol abstinence, is one of the major challenges left in the post-directacting antivirals era.

Interobserver agreement in endoscopic evaluation of reflux esophagitis using a modified Los Angeles classification incorporating grades N and M: a validation study in a cohort of Japanese endoscopists.

The Los Angeles classification system is the most widely employed criteria associated with the greatest interobserver agreement among endoscopists. In Japan, the Los Angeles classification system has been modified (modified LA system) to include minimal changes as a distinct grade of reflux esophagitis, rather than as auxiliary findings. This adds a further grading M defined as minimal changes to the mucosa, such as erythema and/or whitish turbidity. The modified LA system has come to be used widely in Japan. However, there have been few reports to date that have evaluated the interobserver agreement in diagnosis when using the modified LA classification system incorporating these minimal changes as an additional grade. A total of 100 endoscopists from university hospitals and community hospitals, as well as private practices in the Osaka-Kobe area participated in the study. A total of 30 video clips of 30-40 seconds duration, mostly showing the esophagocardiac junction, were created and shown to 100 endoscopists using a video projector. The participating endoscopists completed a questionnaire regarding their clinical experience and rated the reflux esophagitis as shown in the video clips using the modified LA classification system. Agreement was assessed employing kappa (kappa) statistics for multiple raters. The kappa-value for all 91 endoscopists was 0.094, with a standard error of 0.002, indicating poor interobserver agreement. The endoscopists showed the best agreement on diagnosing grade A esophagitis (0.167), and the poorest agreement when diagnosing grade M esophagitis (0.033). The kappa-values for the diagnoses of grades N, M, and A esophagitis on identical video pairs were 0.275-0.315, with a standard error of 0.083-0.091, indicating fair intraobserver reproducibility among the endoscopists. The study results consistently indicate poor agreement regarding diagnoses as well as fair reproducibility of these diagnoses by endoscopists using the modified LA classification system, regardless of age, type of practice, past endoscopic experience, or current workload. However, grade M reflux esophagitis may not necessarily be irrelevant, as it may suggest an early form of reflux disease or an entirely new form of reflux esophagitis. Further research is required to elucidate the pathophysiological basis of minimal change esophagitis.

Toxicity of a trivalent organic arsenic compound, dimethylarsinous glutathione in a rat liver cell line (TRL 1215).

BACKGROUND AND PURPOSE: Although inorganic arsenite (As(III)) is toxic in humans, it has recently emerged as an effective chemotherapeutic agent for acute promyelocytic leukemia (APL). In humans and most animals, As(III) is enzymatically methylated in the liver to weakly toxic dimethylarsinic acid (DMAs(V)) that is a major pentavalent methylarsenic metabolite. Recent reports have indicated that trivalent methylarsenicals are produced through methylation of As(III) and participate in arsenic poisoning. Trivalent methylarsenicals may be generated as arsenical-glutathione conjugates, such as dimethylarsinous glutathione (DMAs(III)G), during the methylation process. However, less information is available on the cytotoxicity of DMAs(III)G. EXPERIMENTAL APPROACH: We synthesized and purified DMAs(III)G using high performance TLC (HPTLC) methods and measured its cytotoxicity in rat liver cell line (TRL 1215 cells). KEY RESULTS: DMAs(III)G was highly cytotoxic in TRL 1215 cells with a LC(50) of 160 nM. We also found that DMAs(III)G molecule itself was not transported efficiently into the cells and was not cytotoxic; however it readily became strongly cytotoxic by dissociating into trivalent dimethylarsenicals and glutathione (GSH). The addition of GSH in micromolar physiological concentrations prevented the breakdown of DMAs(III)G, and the DMAs(III)G-induced cytotoxicity. Physiological concentrations of normal human serum (HS), human serum albumin (HSA), and human red blood cells (HRBC) also reduced both the cytotoxicity and cellular arsenic uptake induced by exposure to DMAs(III)G. CONCLUSIONS AND IMPLICATIONS: These findings suggest that the significant cytotoxicity induced by DMAs(III)G may not be seen in healthy humans, even if DMAs(III)G is formed in the body from As(III).

Induction of hepatic metallothionein synthesis by endoplasmic reticulum stress in mice.

Metallothionein (MT) is a small sulfhydryl-rich protein whose levels are elevated by various inducers of organelle stresses, such as nuclear stress (cisplatin), mitochondrial stress (antimycin A, 2,4-dinitrophenol) and lysosomal stress (paraquat). Although abnormal folding of protein in the endoplasmic reticulum (ER) causes ER stress, induction of MT synthesis by ER stress has never been investigated. In this study, we examined the induction of MT by an inducer of ER stress, tunicamycin (Tun), which induces ER stress by inhibiting N-linked glycosylation of protein in the ER. Administration of Tun (0.5-1.5 mg/kg, sc) increased hepatic MT levels in C57BL/6J mice (3.1-fold). The maximal increase in hepatic MT was observed 48-96 h after the administration of Tun (1.0 mg/kg). Expressions of MT-I, II and glucose-regulated protein 78 (Bip/GRP78), which is a molecular chaperone induced by ER stress, mRNA were also detected by administration of Tun. Thapsigargin (Thap), a generator of ER stress by inhibiting ER Ca(2+)-ATPase, also increased both hepatic MT levels and expression of MT-I and -II mRNA. The level of expression of Bip/GRP78 mRNA induced by Tun administration in MT-null mice was greater than that in wild-type mice. Taken together, these findings suggest that inhibitors of ER are potent inducers of MT.

Capillary electrophoretic determination of phosphate based on the formation of a Keggin-type [PMo12O40]3- complex.

Based on the formation of a Keggin-type [PMo12O40]3- complex, a sensitive capillary electrophoresis (CE) method was developed for the determination of P(V) with direct UV detection at 220 nm. A mixture of alpha- and beta-Keggin-type [PMo12O40]3- complexes was readily formed in a sample solution consisting of a trace amount of P(V), 2.5 mM Mo(VI), 0.050 M p-C6H3(CH3)-2-SO3H (XSA), and 60% v/v CH3CN. When a 0.05 M HCl and 60% v/v CH3CN solution was used as a migration electrolyte, the Keggin complexes exhibited a sharp and well-defined peak in the electropherogram. The peak area was linearly dependent on the P(V) concentration in the range of 5 x 10(-7)-5 x 10(-5) M; a detection limit of 1 x 10(-7) M was achieved. In comparison with indirect UV detection, the direct UV detection is about ten times more sensitive, because the Keggin complexes possess high molar absorptivities. The developed CE method was applied to the determination of P(V) in river water, and the results were in good agreement with those obtained by ion chromatography (IC) and colorimetry (COL) based on the formation of mixed-valence heteropoly blue species.

Heat moisture treatment of high amylose cornstarch increases its resistant starch content but not its physiologic effects in rats.

To examine whether the physiologic effects of high amylose cornstarch (HACS) are affected by gelatinization or heat moisture treatment, male rats were fed for 21 d a fiber-free purified diet containing 40 g/100 g gelatinized normal cornstarch (G-CS), HACS, gelatinized high amylose cornstarch (G-HACS) or heat moisture-treated HACS (HMCS). Dietary fiber (DF) content in G-HACS was 87% lower than that in HACS. The apparent starch and protein digestibilities were higher in the G-HACS group than in the HACS group. Fecal wet weight and fecal bile acid excretion were lower in the G-HACS group than in the HACS group. The cecal tissue weight, cecal surface area, cecal content weight and cecal pH were lower in the G-HACS group than in the HACS group. The cecal n-butyric acid and succinic acid concentrations were higher and lower, respectively, in the G-HACS group than in the HACS group. The plasma cholesterol and triacylglycerol concentrations did not differ between the G-HACS group and the HACS group. On the other hand, the DF content in HMCS was 330% higher than that in HACS, but the HMCS and HACS groups generally did not differ except in cecal surface area. Dietary starch did not affect fecal moisture, fecal neutral sterol (cholesterol + coprostanol) excretion, liver cholesterol level, total short-chain fatty acid (SCFA) concentration or apparent Ca, Fe, Mg and Zn absorptions. These results show that the heat moisture treatment of HACS for the most part does not alter its physiologic effects despite the greater DF content.

Accumulation of p53 in esophageal squamous cell carcinoma.

p53 is one of the most important tumor suppressor genes. Mutation of the p53 gene can be detected immunohistochemically as over-expression of its protein in the nucleus. The p53 gene product is known to regulate cell growth and proliferation. Genetic alterations related to the carcinogenesis or progression of esophageal cancer are poorly understood. We examined accumulation of p53 protein in esophageal squamous cell carcinomas including early-stage cancers, and its clinicopathological significance. p53 immunoreactivity in the cancer tissues was found in 61 (79.2%) of 77 esophageal squamous cell carcinomas. Over-expression of p53 protein (diffusely and focally positive staining) was seen in 70.1% (54/77). p53 over-expression was detected not only in the cases of in situ or intramucosal carcinomas, but also in invasive carcinomas. No significant correlations were found between p53 over-expression and clinicopathological features such as depth of tumor invasion, lymph node metastasis or venous/lymphatic invasion. These results suggested that p53 mutations may be closely associated with the early-stage of pre-invasive esophageal carcinoma, and p53 gene mutations may play an important role in the carcinogenesis of human esophageal cancer.

Efficacy of oral UFT plus leucovorin therapy for colon cancer with ovarian and multiple liver metastases: report of two cases.

Case 1: a patient was diagnosed as having ascending colon cancer with right ovarian metastasis, and underwent palliative right hemicolectomy plus oophorectomy. The tumor was a well-differentiated adenocarcinoma with right ovarian metastasis, and the disease was classified as stage IV. Oral chemotherapy with UFT plus LV was performed for about 3 years, and the patient is still being followed up with no recurrence at 5 years postoperatively. Case 2: a patient was diagnosed as having incomplete large bowel obstruction caused by ascending colon cancer, and underwent curative right hemicolectomy. The tumor was a moderately differentiated adenocarcinoma, and the disease was classified as stage II. Since multiple liver metastases developed at 3 months postoperatively, oral chemotherapy with UFT plus LV was started. Imaging studies showed the complete elimination of liver metastases after 2 months. Subsequently, liver metastasis recurred about 10 months later. The patient died of unrelated cerebral infarction at 2 years and 6 months postoperatively.

Effects of selenium deficiency on expression of selenoproteins in bovine arterial endothelial cells.

Damage to the vascular endothelium by reactive oxygen species causes many cardiovascular diseases including atherosclerosis. Such damage can be prevented by selenium (Se), which is thought to exert its actions mainly through the expression of selenoproteins. Se deficiency increased the susceptibility to tert-butylhydroperoxide (t-BuOOH) and enhanced lipid peroxidation in bovine arterial endothelial cells (BAEC). We investigated the effects of Se deficiency on the expression of the selenoproteins in BAEC. 75Se metabolic labeling analysis and RT-PCR analysis revealed that BAEC expressed two glutathione peroxidase (GPx) isozymes, cytosolic GPx (cGPx) and phospholipid hydroperoxide GPx (PHGPx), three thioredoxin reductase (TrxR) isozymes, TrxR1, TrxR2 and TrxR3, and selenoprotein P (SelP). Se deficiency reduced both enzyme activity and mRNA level of cGPx, but did not affect those of PHGPx. SelP mRNA level was also reduced by Se deficiency, although the extent of reduction was much smaller than that of cGPx mRNA. We further found that TrxR activity was also decreased by Se deficiency but none of the mRNA levels of TrxR isozymes were reduced. These results indicate that vascular endothelial cells express several selenoproteins including cGPx, PHGPx, TrxR isozymes and SelP which might play important roles in the defense system against oxidative stresses and that the expressions of these selenoproteins are differently regulated by Se status.

Glutathione content is correlated with the sensitivity of lines of PC12 cells to cisplatin without a corresponding change in the accumulation of platinum.

A study of the involvement of glutathione (GSH) in cellular resistance to cisplatin was performed using methylmercury-resistant sublines (PC12/TM series) of the PC12 line of rat pheochromocytoma cells. The seven clonal sublines of PC12 cells (PC12/TM, PC12/TM2, PC12/TM5, PC12/TM11, PC12/TM15, PC12/TM23, PC12/TM26) used in the study had intracellular levels of GSH that ranged from 8.7-39.9 nmol/mg protein. The intracellular level of GSH was significantly correlated (p < 0.01, r = 0.87) with the sensitivity to cisplatin of PC12 cells and the seven sublines. Among the seven sublines, PC12/TM cells contained the highest concentration of GSH and were the most resistant to cisplatin. Treatment of PC12/TM cells with L-buthionine-SR-sulfoximine, which reduced the level of GSH to that in the parental PC12 cells, significantly reduced the resistance of the cells to cisplatin. The amount of platinum accumulated by resistant PC12/TM cells after treatment with cisplatin was higher than that by sensitive PC12 cells. These results suggest that the intracellular level of GSH might be directly involved in the resistance to cisplatin of these cell lines. However, a high intracellular concentration of GSH does not appear to contribute to a decrease in the accumulation of cisplatin in these cells.

A novel approach to endoscopic colorectal mucosal resection using a three-channel outer tube and multiple forceps: an experimental assessment.

BACKGROUND AND STUDY AIMS: Colorectal endoscopic mucosal resection (EMR) has limitations both anatomically and technically when it is done using the conventional snare wire method. The aim of this study was to develop a new method and instrument for colorectal EMR. METHODS: A total of 21 EMR procedures were done using ten surgical specimens. Saline was injected into the normal submucosa of freshly resected colorectal specimens to prepare a pseudotumor. EMR was performed experimentally by employing a three-channel outer tube with three forceps and a colonoscope with a needle-type precutting knife. This method was assessed in terms of safety and the size of the resected specimens. RESULTS: Perforation occurred only twice in the initial stage of this study. The size of the specimens resected by EMR was 28-39 mm (long diameter 34.8+/-3.11), by 22-28 mm (short diameter 25.8+/-2.07). CONCLUSION: This method can achieve safety and en bloc mucosal resection to the submucosal layer. This novel approach may be promising for clinical application as a new form of endoscopic surgery.

Quintuple carcinomas with metachronous triple cancer of the esophagus, kidney, and colonic conduit following synchronous double cancer of the stomach and duodenum.

A patient who had undergone radical gastrectomy for synchronous gastric cancer (T(1)N(0)M(0), stage I) and duodenal cancer (Tis, stage 0) in November 1987 was found to have esophageal cancer in November 1994, and underwent radical thoracolaparotomy at our hospital (T(1)N(0)M(0), stage I). After follow-up for about 3.5 years, renal cancer was detected in April 1998, and radical nephrectomy was performed (T(1)N(0)M(0), stage I). Two years later, in April 2000, the patient was found to have a polypoid lesion in the colonic conduit used for reconstruction after esophagectomy, and endoscopic mucosal resection was performed (Tis, stage 0). The patient remains under careful follow-up, including observation of the colonic conduit and the residual large intestine.

Simultaneous electrophoretic determination of vanadium(V) and vanadium(IV) based on the complex formation with a Mo(VI)-P(V) reagent.

A capillary electrophoretic (CE) method was developed for the sensitive determination of V(V) and V(IV). A Mo(VI)-P(V) reagent reacted with a mixture of trace amounts of V(V) and V(IV) to form the [P(V(V)Mo(11))O40]4- and [P(V(IV)Mo11)O40]5- complexes in 0.1 M monochloroacetate buffer (pH 2.2). Since the V-substituted Keggin anions possessed high molar absorptivities in the UV region and the peaks due to their migrations were well separated in the electropherogram, the pre-column complex formation reaction was applied to the simultaneous CE determination of V(V) and V(IV) with direct UV detection at 220 nm. The calibration curves were linear over two orders of magnitude with detection limits of 5 x 10(-7) M for V(V) and 2 x 10(-7) M for V(IV).

Enhancement of androgen-dependent transcription and cell proliferation by tributyltin and triphenyltin in human prostate cancer cells.

Tributyltin (TBT) and triphenyltin (TPT) are known to cause imposex, the superimposing of male genitals on female ones, in some species of gastropods. However, the molecular mechanism of the trialkyltin-induced endocrine dysfunction remains to be elucidated. To clarify the effects of organotin compounds on the activation of androgen receptor (AR)-mediated responses in mammals, a LA16 clone that stably expresses androgen-responsive luciferase reporter gene and proliferates in response to androgen was established from human prostate cancer cell line LNCaP. Stimulation of LA16 cells with 100 nM TBT or 1 nM TPT enhanced both AR-dependent transcription of luciferase gene and cell growth to the same extent as those by 1 nM dihydrotestosterone (DHT). TBT or TPT also enhanced the DNA synthesis and expression of endogenous AR target genes such as prostate specific antigen, but not the expression of AR itself. However, an androgen antagonist, flutamide, did not inhibit the TBT- or TPT-induced AR activation. On the other hand, simultaneous treatment of LA16 cells with DHT and TBT or TPT caused highly enhanced effects on AR activation. These results indicate that trialkyltin compounds have an ability to activate AR-mediated transcription in mammalian cells and suggest that a novel target site other than the ligand-binding site of AR is involved in this activation.

Frequent expression of sialyl Le(a) in human esophageal squamous cell carcinoma.

Sialyl Le(a) antigen (CA19-9), a member of a family of high molecular weight glycoproteins, was originally described as a gastrointestinal- and pancreatic-specific tumor marker. Recent studies have demonstrated that sialyl Le(a) is a ligand for E-selectin and may play an important role in tumor metastasis. However, expression patterns of sialyl Le(a) have not yet been established in human esophageal carcinomas. In this study, we examined sialyl Le(a) expression and its histopathological localization in human esophageal squamous cell carcinoma. Sialyl Le(a) immunoreactivity was detected in 28 (51.9%) of the 54 esophageal squamous cell carcinomas, regardless of the depth of tumor invasion, vascular invasion or lymph nodal status. In 13 cases (29.5%), significant sialyl Le(a) expression was detected not only in the intramucosal carcinoma components, but also in the invasive carcinoma components. These observation suggested that sialyl Le(a) expression is associated with early-stage cancer progression.

Activation of the beta-catenin gene by interstitial deletions involving exon 3 as an early event in colorectal tumorigenesis.

beta-Catenin has been identified as an oncogene in several tumors including colorectal cancers. beta-Catenin gene is activated by interstitial deletions involving exon 3 in colorectal carcinomas of Japanese population, in contrast to amino acid substitutions detected among Caucasian population. The aim of this study was to examine the type and frequency of beta-catenin gene mutation during early stages of colorectal tumorigenesis. We screened 100 colorectal adenomas for somatic mutations in the beta-catenin gene by single-strand conformation polymorphism method, as well as polymerase chain reaction amplification. In cases with mutations, sequencing analyses and immunohistochemical staining were also performed. Somatic interstitial deletions of 272-413 bp, each of which included all parts of exon 3, were detected in three tumors. However, no adenoma carried missense mutations. We confirmed accumulation of aberrant beta-catenin protein in cytoplasm and nuclei of adenoma cells by immunohistochemical analysis. Our results suggested that activation of the beta-catenin gene by interstitial deletions involving exon 3 might be less frequent compared with frequent alterations of adenomatous polyposis coli (APC) gene, but could be an early event in colorectal tumorigenesis equivalent to APC gene alterations in the Japanese population.

HeLa cell transformants overproducing mouse metallothionein show in vivo resistance to cis-platinum in nude mice.

Plasmid pSV2MT-I encoding mouse metallothionein-I (MT-I) designed to be expressed under the control of an SV40 promoter was introduced into human HeLa S3 cells. Several transformants (HeLa/MTH) carrying multi-copies of mouse MT-I cDNA in their genomes were isolated. These transformants produced 4 to 20-fold larger amounts of MT than their parent cells. The MT levels in HeLa/MTH were well correlated with the extent of resistance to cadmium, but not with that to cis-platinum (cis-DDP) in vitro. To study the role of MT in resistance to cis-DDP in vivo, nude mice were inoculated subcutaneously with two independent HeLa/MTH clones. MT levels in these tumors were about 3-fold higher than those in the parental cells. The growth of tumors derived from either HeLa/MTH clone was not inhibited in the presence of 15 micromol/kg of cis-DDP, which completely inhibited the growth of tumors derived from the parental HeLa cells. These data strongly suggest that the elevated level of MT confers resistance to cis-DDP in vivo but not in vitro. Thus, the results of this study indicate that in vitro determinations of the influence of MT on cis-DDP resistance may underestimate its importance in in vivo situations.

Changes in serum hepatic fibrosis markers in biochemical responders to interferon therapy for chronic hepatitis C.

Serum hepatic fibrosis markers (7s domain of type IV collagen, N-terminal peptide of type III procollagen, and hyaluronate) were determined during and after a 6-month interferon treatment of patients with chronic hepatitis C. Changes in these markers were compared among the patients who showed a sustained normalization of serum alanine transaminase (ALT) levels with and without eradication of serum hepatitis C virus RNA (complete responders and biochemical responders) and nonresponders. In the case of complete responders, the serum 7s domain of type IV collagen and the N-terminal peptide of type III procollagen levels decreased at the end and 24 weeks after the end of the treatment. Hyaluronate levels were significantly decreased 24 weeks after the end of the treatment, as compared with those prior to the treatment. During and after interferon treatment, changes in these markers in the case of biochemical responders were nearly the same as those in the complete responders. These results suggest that serum hepatic fibrosis markers decrease in patients with chronic hepatitis C who show a sustained normalization of ALT after interferon treatment, even if serum hepatitis C virus RNA fails to be eradicated.

Suppression of a high-affinity transport system for manganese in cadmium-resistant metallothionein-null cells.

Cadmium is a hazardous heavy metal existing ubiquitously in the environment, but the mechanism of cadmium transport into mammalian cells has been poorly understood. Recently, we have established a cadmium-resistant cell line (Cd-rB5) from immortalized metallothionein-null mouse cells, and found that Cd-rB5 cells exhibited a marked decrease in cadmium uptake. To investigate the mechanism of altered uptake of cadmium in Cd-rB5 cells, incorporation of various metals was determined simultaneously using a multitracer technique. Cd-rB5 cells exhibited a marked decrease in manganese incorporation as well as that of cadmium. However, the reduced uptake of manganese was observed only at low concentrations, suggesting that a high-affinity component of the Mn(2+) transport system was suppressed in Cd-rB5 cells. Competition experiments and kinetic analyses revealed that low concentrations of Cd(2+) and Mn(2+) share the same high-affinity pathway for their entry into cells. The mutual competition of Cd(2+) and Mn(2+) uptake was also observed in HeLa, PC12, and Caco-2 cells. The highest uptake of Cd(2+) and Mn(2+) by parental cells occurred at neutral pH, suggesting that this pathway is different from a divalent metal transporter 1 that can transport various divalent metals including Cd(2+) and Mn(2+) under acidic conditions. These results suggest that a high-affinity Mn(2+) transport system is used for mammalian cellular cadmium uptake, and that the suppression of this pathway caused a marked decrease in cadmium accumulation in cadmium-resistant metallothionein-null cells.