High incidence of autoimmune gastritis in patients misdiagnosed with two or more failures of H. pylori eradication.

BACKGROUND: Although autoimmune gastritis (AIG) is generally considered relatively rare, we frequently encounter AIG among patients at to our hospital who have experienced at least two episodes of Helicobacter pylori eradication failure. AIMS: We investigated the incidence of AIG in consecutive patients who consulted our department for H. pylori eradication with reference to eradication history. METHODS: A total of 404 consecutive patients who visited the H. pylori-specific out-patient unit of our hospital from June 2015 to June 2017 were enrolled. Of these, 137 were treatment-naive, 47 had failed treatment once (single failure), and 220 had failed treatment twice or more (multiple failures) by 13 C-UBT. Gastroscopy was performed in all patients. Culture tests of gastric mucosal samples were performed for H. pylori and other bacteria positive for urease activity. Anti-parietal cell antibody (APCA) was measured. Patients with severe atrophy in the gastric corpus and positivity for APCA were diagnosed as having AIG. RESULTS: A total of 43 patients were diagnosed as having AIG, of whom two were treatment-naive (1.5%, 2/137), 1 failed eradication once (2.1% 1/47), and 40 failed treatment at least twice (18.2%, 40/220). The incidence of AIG was significantly higher in the multiple failure group than in the single failure or treatment-naive groups. Urease-positive bacteria, such as Klebsiella pneumoniae and alpha-streptococcus, were identified in 33 of the 35 AIG patients who underwent culture testing. CONCLUSION: AIG patients were often misdiagnosed as refractory to eradication therapy, probably because achlorhydria in AIG might allow urease-positive bacteria other than H. pylori to colonise the stomach, causing positive 13 C-UBT results.

Potent acid inhibition by vonoprazan in comparison with esomeprazole, with reference to CYP2C19 genotype.

BACKGROUND: Acid inhibitory effects of proton pump inhibitors (PPIs) are influenced by CYP2C19 genotype. In contrast, the potent acid inhibition of vonoprazan is not influenced by CYP2C19 genotype. AIM: To compare the acid inhibitory effects of vonoprazan and esomeprazole in relation to CYP2C19 genotype. METHODS: Twenty-eight healthy Japanese volunteers [7 CYP2C19 poor metabolisers (PMs), 11 intermediate metabolisers (IMs) and 10 rapid metabolisers (RMs)] received four different regimens in a randomised crossover manner: (i) vonoprazan 20 mg twice daily (b.d.), (ii) vonoprazan 20 mg daily, (iii) esomeprazole 20 mg b.d. and (iv) esomeprazole 20 mg daily. The timing of each dosing was 1 h before a meal. Twenty-four-hour intragastric pH monitoring was performed on day 7 on each regimen. RESULTS: In the overall genotype group, pH ≥4 holding time ratios (pH 4 HTRs) with vonoprazan b.d., vonoprazan daily, esomeprazole b.d. and esomeprazole daily were 100%, 95%, 91%, and 68% respectively. pH 5 HTRs were 99%, 91%, 84% and 54% respectively. Vonoprazan b.d. potently suppressed acid for 24 h, and was significantly superior to other regimens irrespective of CYP2C19 genotype. Vonoprazan daily was equivalent to esomeprazole b.d. in IMs and PMs, but superior in RMs. CYP2C19 genotype-dependent differences were observed in esomeprazole daily but not in vonoprazan b.d. or daily. CONCLUSION: Vonoprazan 20 mg b.d. inhibits acid irrespective of CYP2C19 genotype, more potently than esomeprazole 20 mg b.d., pH 4 and 5 holding time ratios reached 100% and 99%, respectively.

Twice-daily dosing of esomeprazole effectively inhibits acid secretion in CYP2C19 rapid metabolisers compared with twice-daily omeprazole, rabeprazole or lansoprazole.

BACKGROUND: Twice-daily dosing of proton pump inhibitors (PPIs) is used to treat Helicobacter pylori or acid-related diseases, such as gastro-oesophageal reflux disease (GERD) refractory to standard dose of a PPI. Genetic polymorphisms of CYP2C19 are involved to different extents in the metabolism of four kinds of PPIs (omeprazole, lansoprazole, rabeprazole and esomeprazole) available in Japan. AIM: To compare acid-inhibitory effects of the four PPIs dosed twice daily in relation to CYP2C19 genotype. METHODS: We performed 24-h pH monitoring studies on Day 7 of PPI treatment for 40 Japanese H. pylori-negative volunteers [15 CYP2C19 rapid metabolisers (RMs), 15 intermediate metabolisers (IMs) and 10 poor metabolisers (PMs)] using a randomised four-way crossover design: omeprazole 20 mg, esomeprazole 20 mg, lansoprazole 30 mg and rabeprazole 10 mg twice daily. RESULTS: Although median pH values with esomeprazole, omeprazole, lansoprazole and rabeprazole were 5.7 (3.5-7.2), 5.5 (2.4-7.2), 5.5 (3.7-7.3) and 5.2 (2.5-7.3), respectively (no statistically significant differences), CYP2C19 genotype-dependent differences were smaller for esomeprazole and rabeprazole compared with values for omeprazole and lansoprazole. In CYP2C19 RMs, the median pH with esomeprazole [5.4 (3.5-6.8)] was significantly higher than those with omeprazole [5.0 (2.4-5.9), P = 0.018], lansoprazole [4.7 (3.7-5.5), P = 0.017] or rabeprazole [4.8 (2.5-6.4), P = 0.002]. In IMs and PMs, the median pH was >5.0 independent of the PPI. CONCLUSIONS: In intermediate and rapid metabolisers of CYP2C19, PPIs dosed twice daily could attain sufficient acid suppression, while in CYP2C19 RMs, esomeprazole 20 mg twice daily caused the strongest inhibition of the four PPIs. Therefore, esomeprazole may be effective in Japanese population when dosed twice daily.

A novel tumor-derived SGOL1 variant causes abnormal mitosis and unstable chromatid cohesion.

Mitosis is the most conspicuous cell cycle phase, because it is the phase in which the dynamic physical distributions of cellular components into the two daughter cells occur. The separation of sister chromatids is especially important during mitosis, because of the extreme accuracy required for distribution to the next generation of cells. Shugoshin-like 1 (SGOL1) is a key protein in protecting sister chromatids from precocious separation. We have reported finding that chromosome instability is more likely in SGOL1-downregulated colorectal cancers, but it is still unknown whether there is an association between cancer and SGOL1 transcript variation. Here, we identified a novel SGOL1 variant, SGOL1-P1, in human colon cancer. The SGOL1-P1 transcript contains an exon-skip of exon 3 that results in a stop codon occurring within exon 4. Overexpression of SGOL1-P1 in HCT116 cells resulted in an increased number of cells with aberrant chromosome alignment, precociously separated chromatids and delayed mitotic progression, occasionally followed by inaccurate distribution of the chromosomes. These phenotypes, observed when SGOL1-P1 was present, were also observed very frequently in SGOL1-knockdown cells. Furthermore, the overexpression of SGOL1-P1 inhibited the localization of endogenous SGOL1 and cohesin subunit RAD21/SCC1 to the centromere. These results suggest that SGOL1-P1 may function as a negative factor to native SGOL1, and that abundant expression of SGOL1-P1 may be responsible for chromosomal instability.

Human Sgo1 downregulation leads to chromosomal instability in colorectal cancer.

BACKGROUND AND AIMS: Chromosomal instability (CIN) is recognised as a hallmark of cancer and is caused by a spindle assembly checkpoint disorder or chromosome mis-segregation during mitosis. Although the recent identification of human shugoshin (hSgo1), an important player in proper chromosome segregation, has suggested the involvement of hSgo1 in colorectal tumourigenesis, little is known about how it is involved. The aim of this study was to obtain information about the status of hSgo1 in human colorectal cancer. METHOD AND RESULTS: Among the 46 colorectal cancer cases, hSgo1 mRNA expression was decreased in the tumour tissue in comparison with the corresponding normal tissue (p = 0.032). Human Sgo1-downregulated tumours (tumour to normal mucosa ratio<0.5) had preferential location on the left side large bowel rather than on the right side (p = 0.012), and a higher variation of centromere numbers revealed by fluorescence in situ hybridisation (FISH). To assess the effects of hSgo1 downregulation, hSgo1 knockdown was performed by transfecting the diploid HCT116 cell line with a short hairpin RNA expression vector. hSgo1 knockdown cells proliferated slowly because of both G(2)/M arrest and apoptosis (p<0.001), and markers of CIN in the form of aneuploidy (p<0.001) and micronuclei (p<0.005) were later observed in hSgo1 knockdown cells. Increased centrosome amplification (p<0.05), the presence of binucleated cells and mitotic catastrophes were also noted in hSgo1 knockdown cells. CONCLUSIONS: These findings suggest that hSgo1-downregulated colorectal cancers have a clinicopathological character of CIN, and hSgo1 downregulation leads to CIN in colorectal cancer cells.

Induction of centrosome amplification and chromosome instability in p53-deficient lung cancer cells exposed to benzo[a]pyrene diol epoxide (B[a]PDE).

Benzo[a]pyrene diol epoxide (B[a]PDE), the ultimate carcinogenic metabolite of benzo[a] pyrene, has been implicated in the mutagenesis of the p53 gene involved in smoking-associated lung cancer. To further understand the role of B[a]PDE in lung tumour progression, we investigated its effect on the numerical integrity of centrosomes and chromosome stability in lung cancer cells lacking p53. Exposure of p53-deficient H1299 lung cancer cells to B[a]PDE resulted in S-phase arrest, leading to abnormal centrosome amplification. Analysis of H1299 cells stably expressing fluorescence-tagged centrin (a known centriolar marker) revealed that the centrosome amplification was primarily attributable to excessive centrosome duplication rather than to centriole splitting. Forced expression of POLK DNA polymerase, which has the ability to bypass B[a]PDE-guanine lesions in an error-free manner, suppressed the B[a]PDE-induced centrosome amplification. Fluorescence in situ hybridization analyses with probes specific for chromosomes 2, 3, and 16 revealed that B[a]PDE exposure also led to chromosome instability, which was likely to have resulted from centrosome amplification. We extended these findings to primary lung carcinomas containing non-functional p53, and found a strong association between centrosome amplification and a high level of B[a]PDE-DNA accumulation. Therefore B[a]PDE contributes to neoplasia by inducing centrosome amplification and consequent chromosome destabilization as well as its mutagenic activity.

Pharmacogenomics-based tailored versus standard therapeutic regimen for eradication of H. pylori.

Helicobacter pylori eradication rates by triple therapy with a proton pump inhibitor, amoxicillin, and clarithromycin at standard doses depend on bacterial susceptibility to clarithromycin and patient CYP2C19 genotypes. We examined the usefulness of a personalized therapy for H. pylori infection based on these factors as determined by genetic testing. First, optimal lansoprazole dosing schedules that would achieve sufficient acid inhibition to allow H. pylori eradication therapy in each of different CYP2C19 genotype groups were determined by a 24-h intragastric pH monitoring. Next, 300 H. pylori-positive patients were randomly assigned to the standard regimen group (lansoprazole 30 mg twice daily (b.i.d.)), clarithromycin 400 mg b.i.d., and amoxicillin 750 mg b.i.d. for 1 week) or the tailored regimen group based on CYP2C19 status and bacterial susceptibility to clarithromycin assessed by genetic testing. Patients with failure of eradication underwent the second-line regimen. The per-patient cost required for successful eradication was calculated for each of the groups. In the first-line therapy, the intention-to-treat eradication rate in the tailored regimen group was 96.0% (95% CI=91.5-98.2%, 144/150), significantly higher than that in the standard regimen group (70.0%: 95% CI=62.2-77.2%, 105/150) (P<0.001). Final costs per successful eradication in the tailored and standard regimen groups were $669 and $657, respectively. In conclusion, the pharmacogenomics-based tailored treatment for H. pylori infection allowed a higher eradication rate by the initial treatment without an increase of the final per-patient cost for successful eradication. However, the precise cost-effectiveness of this strategy remains to be determined.

Some features in prostaglandin synthesis of the cancer cells which metastasized into liver from intestinal cancer lesions.

In order to study the mechanism of cancer metastasis, AH100B cells, an ascitic hepatoma cell line, were transplanted into the small intestine of male Donryu rats. Each metastatic nodule in the liver was collected with the respective intestinal lesion. Each sample thus obtained was injected into the peritoneal cavity of male Donryu rats to make free cancer cells. Then, the cancer cells, having an intact cell surface, of the metastatic and primary intestinal lesion were collected respectively. After washing in Dolbecco's PBS (Ca2+ and Mg(2+)-free, pH 7.2), the definite numbers of cancer cells of the metastatic and primary intestinal lesion were incubated in the PBS containing [1-14C]-AA at 25 degrees C for 30 min, respectively. AA metabolites formed during the incubation period were extracted and subjected to TLC, followed by autoradiography. Each radioactive part was scraped off the plate and measured for its radioactivity. The pattern of the ability to synthesize PGs was different between the cancer cells which metastasized to the liver and those of the primary lesion, that is, percentage values of PGE2 and PGF2 alpha were higher (p < 0.01) in the cancer cells which metastasized to liver as compared with those of the primary intestinal lesion. These results suggest that PGs produced by hepatic metastatic cancer cells might play an important role in cancer metastasis.

Some features of prostaglandin synthesis of the cancer cells metastasized into liver from gastric cancer lesions.

In order to study the mechanism of cancer metastasis, AH100B rat hepatoma cells were transplanted to the stomach of male Donryu rats. Each hepatic metastatic nodule was collected with the respective primary gastric lesions. Each sample thus obtained was injected separately into the peritoneal cavity of male Donryu rats to make free cancer cells; then, intact cancer cells of the hepatic metastatic and primary gastric lesions were collected. After washing in Dulbecco's phosphate-buffered saline (Ca2+ and Mg(2+)-free, pH 7.2), the definite number of the metastatic and primary gastric cancer cells were incubated in the phosphate-buffered saline containing [1-14C] arachidonic acid at 25 degrees C for 30 min. Arachidonic acid metabolites formed during the incubation period were extracted and subjected to thin-layer chromatography, followed by autoradiography. Each radioactive spot was scraped off the plate and measured for its radioactivity. The pattern of the ability to produce PGs was different between the cancer cells which metastasized to the liver and those of the primary lesions, that is, percentage of PGF2 alpha was higher (p < 0.05) and that of PGE2 was quite higher (p < 0.01) in the hepatic metastatic cancer cells as compared with those of the primary gastric lesion. These results suggest that PGs produced by hepatic metastatic cancer cells might play an important role in hepatic metastatic formation.

A difference in prostaglandin-producing ability between cancer cells metastasized into liver and kidney.

In order to study the mechanism of cancer metastasis AH100B cells, a rat hepatoma cell line, were injected into the left carotid artery of male Donryu rats to form metastatic lesions. Each metastatic nodule in the liver and kidney was collected and injected into the peritoneal cavity of normal rats. About 3 weeks later, intact metastatic cancer cells were collected from each ascites that was not bloody. After washing in Dulbecco's phosphate-buffered saline (PBS, Ca2+ and Mg(2+)-free, pH 7.2), 1 x 10(6) cancer cells were incubated in the PBS containing [1-14C]-arachidonic acid (AA) at 24 degrees C for 5 min. AA metabolites formed during the incubation period were extracted and subjected to thin layer chromatography, followed by autoradiography. Each radioactive spot was scraped off the plate and its radioactivity was measured. In the cancer cells which metastasized to the liver, the ability to produce prostaglandin (PG) E2 was higher (p less than 0.05) but those to produce PGF2 alpha and 6-keto-PGF1 alpha were lower (p less than 0.01) than in the cancer cells which metastasized to the kidney. These results suggest that cancer cells metastasizing to the liver and the kidney are different from each other in the ability to produce PG.

Electron spin resonance studies of wild-type and mutant cytochromes P-450d: effects of mutations at proximal, aromatic and distal sites on g values.

Low-temperature (6-40 K) electron spin resonance (ESR) spectra of cytochrome P-450d (P-450d) and its 17 mutants have been measured. The spectra of the wild-type and all mutant P-450ds showed signals at around g = 8, 3.7 and 1.7, while they didn't show any signal at around g = 2 up to 40 K. It was thus suggested that all of these P-450ds essentially take the ferric high-spin form. The g values of the proximal mutants were closer to those of the wild-type than those of the distal and aromatic mutants, suggesting that mutations at the distal and aromatic sites influence the electronic state of the heme more profoundly than those of the proximal site. The distal multiple mutants whose distal sequences are the same as those of the low-spin type P-450s such as rat P-450c, mouse P1-450 and P3-450 showed only high-spin ESR signals. Thus the spin state of P-450ds (the wild-type and all mutants) may not be solely due to specific characteristics of the distal site, but to the unique nature of the whole heme environment of P-450d. It is also suggested that the amino acids at the distal region of P-450d may be located close to the heme, so that the water molecule cannot bind to the heme, thus taking the high-spin state. Both the aromatic mutants showed rather large deviations of the g values from those of wild-type P-450d, suggesting that the aromatic region somehow interacts with the heme.

Some features of the metastatic cancer cells in prostaglandin production.

In order to establish metastatic lesions, 2.5 x 10(6) AH100B cells were injected into the left carotid artery of male Donryu rats. Each metastatic nodule in the liver or kidney, 1 mm or less in diameter, thus obtained was then injected into the peritoneal cavity in which these metastatic cells come to free. About 3 weeks later, each ascites was collected from the rats, while not bloody. Then, cancer cells obtained from each ascites were suspended in Dulbecco's PBS without Ca2+ and Mg2+ (pH 7.2) after washing. Then, 10(6) metastasized or control cancer cells were incubated in 0.1 ml of PBS mentioned above together with 0.1 microCi of (1-14C)-AA at 24 degrees C for 3 min, respectively. After the extraction procedure, AA metabolites formed were separated by means of TLC, and each TLC plate was subjected to autoradiography. In the metastasized cells, PG production ability was generally accelerated and especially in that of PGF2 alpha as compared with that of the control.

A role of the cancer cell membrane fluidity in the cancer metastases: an ESR study.

AH66F or Yoshida sarcoma (YS) cells were transplanted intraperitoneally into male Donryu rats. Cancer cells obtained from ascites were suspended in saline solution (10(7) cells/ml) after washing. Then, 0.1 ml of each suspension obtained from both strains was injected into the tail vein of 5 rats, respectively. Each metastatic nodule, 1 mm or less in a diameter, thus obtained was then injected into the peritoneal cavity in which these metastatic cells come to free. After 10 days, cancer cells obtained from each ascites were suspended in phosphate buffered saline (Ca2+ and Mg2+ free, pH 7.2) after washing. Each suspension (10(7) cells/ml) was violently vibrated with a definite amount of 5-doxyl stearic acid and spin labeling of cancer cell membrane was done. Furthermore, each specimen thus obtained was subjected to the electron spin resonance (ESR) measurement and the order parameter was determined from the spectra. In both YS and AH66F strains, the cell membrane fluidity of the metastatic cancer cell was increased at each temperature measured from 5 degrees C through 35 degrees C. The results obtained here suggest that the change of the cell membrane fluidity of cancer cell is closely related with the cancer metastases.

EPR studies of copper(II) and cobalt(II) complexes of adriamycin.

Cu2+ and Co2+ complexes of adriamycin (ADM) in aqueous solutions have been examined using EPR spectroscopy. An appreciable amount of Cu2+ and Co2+ complexes formed in the solutions were found to be in the EPR silent associated form, where the metal ions are antiferromagnetically coupled. The associated form of the Cu2+ complex may be neither a simple dimer nor coordination polymer but aggregates of a stacked type. Formation of a complex having Cu2+-ADM stoichiometry of 1:2 was observed for the solutions containing excess of ADM as an EPR observable species. The complex having Cu2+-ADM stoichiometry of 1:1 was not observed directly by EPR, but the presence of the complex is undeniable, especially at low pH range so far as large excessive ADM is not present. The Co2+ complex of ADM observed by EPR is in the high-spin (S = 3/2) state and may have a coordination structure of tetragonal symmetry. The EPR spectra of these complexes apparently show that the Cu2+ and Co2+ ions are bound at the carbonyl and phenolate oxygen in the 1,4-dihydroxyanthraquinone moiety and the amino nitrogen in the sugar part does not seem to participate in the coordination to the metal ions.

Electron paramagnetic resonance studies of highly anisotropic low-spin states of ferrimyoglobin derivatives.

The effects of addition of nitrogenous bases, which gave low-spin ferric porphyrin complexes with highly anisotropic g values, were investigated for ferrimyoglobin by low-temperature EPR measurements. Concomitant denaturation of myoglobin upon addition of the exogenous bases was also of interest. By addition of pyridine-type bases under regulated pH, Mb(Fe3+) complexes showing EPR spectra with highly anisotropic g values were formed. These complexes have the electronic states close to the spin-crossover point but not so close as that of the ferric porphyrin highly anisotropic low-spin (HALS) complexes previously reported. Several types of low-spin species, LSi, LSa and LSb, were produced by the denaturation of myoglobin caused by addition of some exogenous ligands. The LSi was assigned to a complex with histidine-E7 coordinated on the sixth position and LSa to the one with OH- and histidine-F8[Im0].

A correlation between cancer metastases and the fluidity of cancer cell membrane.

Male Donryu rats weighing from 100 to 120 g were transplanted with AH100B, AH66F or Yoshida sarcoma (YS) cells intraperitoneally. After collecting ascites, while not bloody, the cancer cells were washed three times with Dulbecco's phosphate buffered saline (PBS) without Ca++ and Mg++ (pH 7.2). Then, each sediment was suspended in PBS mentioned above. Each suspension in which 10 million of cancer cells were contained was violently vibrated with a definite amount of 5-doxyl stearic acid and spin labeling of cancer cell membrane was done. Furthermore, each specimen thus obtained was subjected to measurements of ESR spectra and the order parameter was determined from the spectra. The order parameter of AH100B was the highest and that of YS the lowest at each temperature measured from 5 degrees C to 35 degrees C. YS cells have been reported to be found in blood stream within 24 hr, AH66F cells in a day and AH100B cells in 10 days or more after intraperitoneal inoculation. The results obtained here suggest that the accelerated fluidity of cancer cell membrane is related with the cancer metastasis.

Formation of free radicals from carcinogenic benz[c]acridines in the presence of proteins.

Formation of a free radical from carcinogenic and noncarcinogenic benz[c]acridine derivatives in the presence of proteins was examined. When aqueous mixture of benz[c]acridine and protein was stirred for a long period, shielded from light, benz[c]acridines were converted into free radicals. Albumin had the greatest effect in accelerating the free radical formation, and the effect was smaller in globulin, histone, and deoxyribonuclease. The g-value of the free radicals thus obtained was 2.005. Intensity of the electron spin resonance (ESR) signals of the free radical from carcinogenic derivatives was higher than those of the free radical from noncarcinogenic derivatives. There was a corresponding correlation among the ESR signal intensity of the free radical formed from the mixed system of benz[c]acridine and protein, charge of the K-region or ring nitrogen of the compound, and carcinogenicity of benz[c]acridines.

Electron spin resonance study on the mode of generation of free radicals of daunomycin, adriamycin, and carboquone in NAD(P)H-microsome system.

The mode of generation of free radicals of daunomycin, adriamycin, and carboquone in the NADPH-rat liver microsome system was studied at room temperature by electron spin resonance (ESR) spectroscopy. ESR signals of all these quinoid anticancer chemicals were detected when dissolved oxygen in the reaction mixture was consumed since the radicals are easilyaut oxidizable. All the radicals had an appreciable lifetime under anaerobic conditions. However, there were differences in the mode of their generation between daunomycin and adriamycin, on the one hand, and carboquone, on the other, with respect to the lag time and the effect of the amount of chemicals, pH of the medium, kind of electron donors, NADPH and NADH, and the presence of excess of DNA. Especially, ESR signal reappeared after the first signal had decreased considerably, in the case of daunomycin and adriamycin but not in carboquone. Intact Ehrlich ascites tumor cells also gave rise to an ESR signal of adriamycin and carboquone, but the former signal was prevented from appearing in the presence of glucose.