[The Usefulness of Strain Elastography for Determining Liver Fibrosis Stage in Chronic Viral Hepatitis: An Analysis Using Tissue Doppler Imaging with Manual Compression].

BACKGROUND/AIM: Accurate determination of the stage of liver fibrosis is an essential component in choice of treatment and assessment of cancer risk in patients with chronic viral hepatitis. The aim of this study was to evaluate the usefulness of strain elastography based on tissue Doppler imaging for liver fibrosis in chronic viral hepatitis. METHODS: A total of 37 patients with chronic viral hepatitis and 8 healthy volunteers were enrolled. Strain value was measured by using a conventional ultrasound machine that included strain imaging technolo- gy. Strain elastography was performed at the right subcostal area with manual compression. Liver fibrosis stages were assessed by using liver biopsy and compared with strain values. Diagnostic performance of the strain value for fibrosis stage 4, cirrhosis, was determined by performing a receiver-operating characteristics (ROC) curve analysis. RESULTS: Twenty-seven patients were positive for HCV RNA, 9 were positive for HBs antigen, and 1 was positive for both (Fibrosis stage F1, n=11; F2, n=7; F3, n=15; F4, n=4). The strain value of F3 and F4 was 0.066±0.02 and 0.042±0.011, respectively. These strain Values were significantly lower compared to those of healthy volunteers (0.112 ±0.018) (P< 0.05). Using a cutoff value of 0.042, the area under ROC curve was 0.88 for the diagnosis of F4. The sensitivity, specificity, positive predictive value, and negative predictive value were 75%, 92%, 50%, and 94%, respectively. CONCLUSIONS: Strain elastography based on tissue Doppler imaging with manual compression appears to be a useful tool to diagnose cirrhosis in patients with chronic viral hepatitis. [Original].

Association between Temperament and Stress-related Gene Expression in Day-old Chickens.

Stress in day-old chickens from commercial hatcheries is associated with problematic behavior in adult animals. Recently, we developed a new behavioral handling test for day-old chickens and demonstrated that it assessed temperament differences between seven breeds of native Japanese and Western chickens. In this study, we used 2-day-old male chicks from five of the above breeds to investigate the relationship between temperament and mRNA levels of three stress-related genes (nuclear receptor subfamily 3 group C member 1 (NR3C1), cytochrome P450 family 11 subfamily A member 1, and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1) involved in the hypothalamic-pituitary-adrenal axis. Principal component analysis of 10 behavioral traits for the handling test revealed that the Fayoumi breed and Hiroshima line of the Chabo breed, both of which exhibited boisterous temperament, clustered separately from the other breeds. Only NR3C1 expression showed a significant positive correlation with two behavioral traits (general vocalization and approaching the wall), and a negative correlation with movement. These results suggest that the complex temperament of day-old chickens is regulated, in part, by stress-related genes along the hypothalamic-pituitary-adrenal axis.

New Behavioral Handling Test Reveals Temperament Differences in Native Japanese Chickens.

It is well known in the poultry industry that fear and stress experienced during the handling of day-old chicks in commercial hatcheries can have long-lasting effects on their behavior later in life. These hatchery-related stresses are more intense and complex than those encountered in traditional behavioral tests. Consequently, a single behavioral test may not be sufficient to measure hatchery stresses and chicken temperament. In this study, we developed a new behavioral handling test for day-old chickens, which incorporated concepts from established behavioral tests used with both young and adult birds. The new test assessed 10 behavioral traits, including vocalization frequency and responses to human interaction. It was conducted on 96 two-day-old chicks from seven breeds of native Japanese and Western chickens. The results of the principal component analysis classified chicken temperaments into three distinct categories: bustle, aggression, and timidity. Using these categories, the seven breeds were classified into five groups, each with distinct temperaments. This study highlights the reliability and value of the new handling test in characterizing the temperaments of various chicken breeds and provides insights into the complex behaviors of chickens.

Antibody level dynamics until after the third dose of COVID-19 vaccination.

The antibody titers of volunteers, including elderly people, were investigated after the second dose of the BNT162b2 vaccine (Pfizer-BioNTech) as an mRNA vaccine against the coronavirus disease 2019 (COVID-19). Serum samples were collected from 105 volunteers (44 healthcare workers and 61 elderly people) 7-14 days after the second vaccine dose, and antibody titers were measured. The antibody titers of study participants in their 20s were significantly higher than those of other age groups. Furthermore, the antibody titers of participants aged <60 years were significantly higher than those of participants aged ≥60 years. Serum samples were repeatedly collected from 44 healthcare workers until after the third vaccine dose. Eight months after the second round of vaccination, the antibody titer levels decreased to the same level as that before the second vaccine dose. After the third booster vaccination, the antibody titer recovered to the same level as that after the second dose. Neutralizing activities were also investigated at four time points before and after the second vaccine dose. The antibody titers and neutralizing activity were positively correlated. Therefore, neutralizing activity can be predicted by measuring the antibody titer. In conclusion, the antibody titers in the elderly population were significantly lower than those in the younger population. Although the antibody titers increased following vaccination, their levels showed a decline after several months, returning to the same level as that after a single dose of mRNA vaccination. The antibody titer levels recovered after the third dose of vaccination, which had already been administered in Japan. Routine administration of vaccine should be considered in the future.

Is a small-caliber or large-caliber endoscope more suitable for colonic self-expandable metallic stent placement? A randomized controlled study.

OBJECTIVES: The colonic self-expandable metallic stent (C-SEMS) with a 9-French (Fr) delivery system allows for a small-caliber endoscope (SCE) to be used to treat malignant colonic obstruction. Despite the lack of evidence, the SCE has become popular because it is considered easier to insert than the large-caliber endoscope (LCE). We aimed to determine whether the SCE is more suitable than the LCE for C-SEMS placement. METHODS: Between July 2018 and November 2019, 50 consecutive patients who were scheduled to undergo C-SEMS for colon obstruction were recruited in this study. Patients were randomized to the SCE or LCE group. The SCE and LCE were used with 9-Fr and 10-Fr delivery systems, respectively. The primary outcome was the total procedure time. Secondary outcomes were the technical success rate, complication rate, clinical success rate, insertion time, guidewire-passage time, stent-deployment time, and colonic obstruction-scoring-system score. RESULTS: Forty-five patients (SCE group, n = 22; LCE group, n = 23) were analyzed. The procedure time in the LCE group (median, 20.5 min) was significantly (p = 0.024) shorter than that in the SCE group (median, 25.1 min). The insertion time in the LCE group (median, 2.0 min) was significantly (p = 0.0049) shorter than that in the SCE group (median, 6.0 min). A sub-analysis of the procedure difficulties showed that the insertion time in the LCE group (median, 5.0 min) was significantly shorter than that in the SCE group (median, 8.5 min). CONCLUSION: Both LCE and SCE can be used for C-SEMS; however, LCE is more suitable than SCE as it achieved a faster and equally efficacious C-SEMS placement as that of SCE. CLINICAL TRIAL REGISTRATION NUMBER: University Hospital Medical Information Network Clinical Trials Registry (UMIN 32748).

Efficacy of hybrid endoscopic submucosal dissection with SOUTEN in gastric lesions: An ex vivo porcine model basic study.

BACKGROUND: Hybrid endoscopic submucosal dissection (ESD) that comprises mucosal incision and partial submucosal dissection followed by snaring in a planned manner, has been developed for endoscopic resection of gastrointestinal neoplasms to overcome the technical barrier of ESD. Although the superiority of hybrid ESD with SOUTEN, a single multifunctional device, over conventional ESD has been indicated, the actual effect of snaring itself remains unclear since SOUTEN could be applied to hybrid ESD group, but not to the conventional ESD group, due to ethical issue in clinical practice. AIM: To determine whether and how hybrid ESD was superior to conventional ESD in the endoscopic treatment of gastric lesions in an ex vivo porcine model basic study. METHODS: Sixteen endoscopists participated in this basic study in August 2020 at Kyushu University, performing 32 procedures each for hybrid ESD and conventional ESD. Mock lesions (10-15 mm, diameter) were created in the porcine stomach. The primary outcome was total procedure time and secondary outcomes were en bloc or complete resection, perforation, procedure time/speed for both, mucosal incision, and submucosal dissection. Factors associated with difficulty in ESD including longer procedure time, incomplete resection, and perforation, were also investigated. Categorical and continuous data were analyzed using the chi-square test or Fisher's exact test and the Mann-Whitney U test, respectively. RESULTS: The median total procedure time of hybrid ESD was significantly shorter than that of conventional ESD (median: 8.3 min vs 16.2 min, P < 0.001). Time, speed, and the amount of hyaluronic acid during submucosal dissection were more favorable in hybrid ESD than conventional ESD (time, 5.2 min vs 10.4 min, P < 0.001; speed, 43.7 mm2/min vs 23.8 mm2/min, P < 0.00; injection volume, 1.5 mL vs 3.0 mL, P < 0.001), although no significant differences in those factors were observed between both groups during mucosal incision. There was also no significant difference between both groups in the en bloc/complete resection rate and perforation rate (complete resection, 93.8% vs 87.5%, P = 0.67; perforation, 0% vs 3.1%, P = 1). Selection of conventional ESD as the treatment method was significantly associated with difficulties during ESD (odds ratio = 10.2; highest among factors). CONCLUSION: Hybrid ESD with SOUTEN improves the treatment outcomes of gastric lesions. It also has the potential to reduce medical costs since SOUTEN is a single multifunctional device that is inexpensive.

ZAK Inhibitor PLX4720 Promotes Extrusion of Transformed Cells via Cell Competition.

Previous studies have revealed that, at the initial step of carcinogenesis, transformed cells are often eliminated from epithelia via cell competition with the surrounding normal cells. In this study, we performed cell competition-based high-throughput screening for chemical compounds using cultured epithelial cells and confocal microscopy. PLX4720 was identified as a hit compound that promoted apical extrusion of RasV12-transformed cells surrounded by normal epithelial cells. Knockdown/knockout of ZAK, a target of PLX4720, substantially enhanced the apical elimination of RasV12 cells in vitro and in vivo. ZAK negatively modulated the accumulation or activation of multiple cell competition regulators. Moreover, PLX4720 treatment promoted apical elimination of RasV12-transformed cells in vivo and suppressed the formation of potentially precancerous tumors. This is the first report demonstrating that a cell competition-promoting chemical drug facilitates apical elimination of transformed cells in vivo, providing a new dimension in cancer preventive medicine.

Hypoxia-independent overexpression of hypoxia-inducible factor 1alpha as an early change in mouse hepatocarcinogenesis.

Hypoxia-inducible factor 1 (HIF-1) is involved in tumor progression/metastasis and activated in various cancers. Here we show that HIF-1alpha, which plays a major role in HIF-1 activation, is overexpressed in preneoplastic hepatocytic lesions from a very early stage during hepatocarcinogenesis in mice and man. Transcriptional targets of HIF-1, such as vascular endothelial growth factor, glut-1, c-met, and insulin-like growth factor II (IGF-II), were also overexpressed in mouse lesions. Oxygen tension within the lesions was not different from that of the normal hepatic tissues, indicating that HIF-1alpha expression was independent of hypoxia. On the other hand, Akt, the pathway of which can up-regulate HIF-1alpha expression, was activated in the mouse lesions, whereas HIF-1alpha was markedly down-regulated in the mouse hepatocellular carcinoma (HCC) cell lines after treatment with a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, indicating that HIF-1alpha expression is dependent on PI3K/Akt signaling. Conversely, HIF-1alpha knockdown by short interfering RNA in the HCC cell line resulted in decreased expression of activated Akt together with the HIF-1 target genes, indicating that Akt activation is reversely dependent on HIF-1 activation. Treating the HCC cells with IGF-II or epidermal growth factor (EGF) up-regulated both phospho-Akt and HIF-1alpha, whereas inhibition of IGF-II or EGF signaling down-regulated them both, suggesting that IGF-II and EGF can, at least in part, mediate the activation of Akt and HIF-1alpha. However, Akt was not activated by IGF-II or EGF in the HIF-1alpha knockdown cells, indicating that expression of the HIF-1 target genes is necessary for the Akt activation. These findings suggest that the reciprocal activation of PI3K/Akt signaling and HIF-1alpha may be important in the progression of hepatocarcinogenesis.

Low selection of preneoplastic hepatocytes after treatment with the 2-acetylaminofluorene diet-partial hepatectomy regimen in the liver of hepatocarcinogenesis-resistant DRH strain rats.

In hepatocarcinogenesis-resistant DRH rats, preneoplastic hepatocytic lesions are smaller than those of usual rats during carcinogenesis. When preneoplastic hepatocytes from DRH and Donryu (original strain of DRH) were reciprocally transplanted into the livers of DRH and Donryu treated with 2-acetylaminofluorene (2-AAF) diet/two-thirds hepatectomy (PH), the Donryu cells formed small colonies within the DRH liver, whereas the DRH cells formed large colonies within the Donryu liver. The DRH liver showed less degree of oval cell proliferation after treatment with 2-AAF and PH, and DRH hepatocytes were more resistant to the growth-inhibitory effect of 2-AAF after PH. Furthermore, DRH hepatocytes were generally resistant to cytotoxicity of hepatotoxins. The tissue environment of the DRH liver, therefore, is less effective for selective growth of preneoplastic hepatocytes during the carcinogen treatment, which is probably a major cause of the hepatocarcinogenesis-resistance in DRH rats.

Molecular characterization and functional expression of equine interleukin-1 type I and type II receptor cDNAs.

cDNA generated from lipopolysaccharide-stimulated equine peripheral blood mononuclear cells was used to amplify and clone type I and type II equine interleukin-1 receptors (IL-1RI and IL-1RII) using primers derived from semi-conserved regions between human and mouse IL-1RI and IL-1RII sequences, respectively. 5' and 3' terminal sequences of equine IL-1RI and IL-1RII were amplified by 5' and 3' rapid amplification of cDNA ends. The deduced amino acid sequence of equine IL-1RI demonstrated 77, 64 and 63% similarity with human, mouse and rat sequences, respectively. The predicted amino acid sequence of equine IL-1RII demonstrated 70, 60 and 58% similarity with human, mouse and rat sequences, respectively. Recombinant equine soluble IL-1RI and IL-1RII produced in insect cells bound recombinant equine IL-1alpha and IL-1beta. Furthermore, both receptors suppressed the growth inhibitory activities of equine IL-1alpha and IL-1beta toward A375 cells in a dose-dependent manner, indicating that the present equine IL-1RI and IL-1RII cDNA encodes biologically active proteins.

Unique Properties of Hepatocarcinogenesis-Resistant DRH Rat Hepatocytes Linked or Not Linked to the Drh1 Locus on Rat Chromosome 1.

Hepatocarcinogenesis-resistant DRH rats exhibit few and small preneoplastic hepatocytic lesions during hepatocarcinogenesis, of which traits have been assigned to two major chromosomal regions, Drh1 and Drh2. In this study, hepatocytes from DRH.F344-Drh1, a congenic strain in which the Drh1 chromosomal region was replaced with that of F344 rats, were compared to hepatocytes from Donryu (original strain), DRH, and F344 rats. Although DRH hepatocytes exhibited low proliferation and p38 dephosphorylation after lead nitrate (LN) treatment despite cytokine and Cox2 activation, DRH.F344-Drh1 hepatocytes exhibited high responses, as did Donryu and F344 hepatocytes. Moreover, although DRH hepatocytes were resistant to hepatotoxins, DRH.F344-Drh1 hepatocytes were as sensitive to hepatotoxins as Donryu and F344 hepatocytes. However, DRH.F344-Drh1 hepatocytes like DRH hepatocytes proliferated at lower rates in vitro and contained smaller nuclei than Donryu and F344 hepatocytes. Thus, low responses to LN and resistance to hepatotoxins in DRH hepatocytes were linked to the Drh1 locus, while low proliferation in vitro and small nuclear size were not linked to the Drh1 locus.

Low p38 MAPK and JNK activation in cultured hepatocytes of DRH rats; a strain highly resistant to hepatocarcinogenesis.

DRH rats are a hepatocarcinogenesis-resistant strain isolated from hepatocarcinogenesis-sensitive Donryu rats, and the liver of DRH shows less histological damage and fewer/smaller neoplastic hepatic lesions by the treatment with hepatocarcinogens. To investigate the mechanism of the resistance, the properties of hepatocytes of DRH and Donryu were compared. In primary culture, DRH hepatocytes exhibited higher proliferation and less apoptosis than Donryu hepatocytes in the presence of EGF and insulin. However, such difference was not correlated to the degree of DNA damage associated with cell culture or cell cycle checkpoint function. Although the mitogen-activated protein kinases [EGF receptor (EGFR) and extracellular signal regulating kinases (ERK1/2)] were activated to the same degree, the stress-activated protein kinases [p38 mitogen-activated protein kinase (p38) and c-jun N-terminal kinase (JNK)] were activated to a lesser degree in the DRH hepatocytes. Treatment with 2-acetylaminofluorene (2-AAF) in vivo also resulted in less JNK and p38 activation in the DRH livers. Furthermore, apoptosis signal-regulating kinase 1 (ASK1) was inhibited by the lysate from the DRH but not by the Donryu hepatocytes. The low activation of the stress-activated protein kinases may be linked to the resistance to cellular stress, which may underlie the hepatocarcinogenesis-resistance in DRH rats.