Clinicopathological and genomic features of superficial esophageal squamous cell carcinomas in nondrinker, nonsmoker females.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is sometimes detected in non-drinker and non-smoker females who are considered to have very low risk of ESCC development in daily practice. This study examined the clinicopathological and genomic characteristics of ESCCs in females with no history of drinking and smoking. METHODS: The sample comprised 118 ESCC lesions occurring in 95 female patients who underwent endoscopic submucosal dissection at our department between January 2008 and December 2019. The patients were categorized into two groups: 51 lesions in 49 patients with no history of drinking and smoking (nondrinker/nonsmoker [NDNS] group) and 69 lesions in 45 patients with a history of drinking or smoking (drinker/smoker [DS] group). We analyzed the differences in clinicopathological and cancerous genomic characteristics between the groups. Significant genomic alterations were validated using immunohistochemistry. RESULTS: Multiple logistic regression revealed that older age, fewer multiple Lugol-voiding lesions (LVLs), and reflux esophagitis (RE) were independently associated with the occurrence of ESCCs in the NDNS group. ESCC lesions in the NDNS group were predominantly located in the mid-thoracic esophagus, posterior wall side, with 0-IIa, the aspect ratio of the lesion >2 (vertical/horizontal), and endoscopic keratinization. Genetic analysis showed that CDKN2A driver alterations were significantly more frequent and KMT2D alterations were significantly less frequent in the NDNS group than in the DS group. KMT2D alterations were strongly correlated with immunostaining. CONCLUSION: Older nondrinker, nonsmoker females with RE and fewer multiple LVLs may develop longitudinal 0-IIa ESCC with keratinization of the posterior wall of the mid-thoracic esophagus. ESCCs in nondrinker, nonsmoker females had fewer KMT2D alterations and more CDKN2A alterations, which may be a biomarker for treatment.

Clinical factors associated with stable treatment of chronic constipation in Japanese patients.

BACKGROUND/AIMS: Chronic constipation (CC) is one of the most common gastrointestinal disorders in the general population. Although there are many treatment options, achieving a stable treatment for CC remains one of the challenges in clinical practice. This study aimed to evaluate the clinical factors associated with stable treatment for CC in Japanese patients. METHODS: A retrospective, cross-sectional, and multicenter study was carried out. Patients were eligible for inclusion if they fulfilled the Rome IV criteria for diagnosing CC and had been treated for at least one and a half years. Patients with up to two prescription modifications for CC in one year were defined as the stable treatment group, whereas those with three or more prescription changes were defined as the unstable treatment group. Univariate and multivariate analyses were carried out to identify factors associated with CC. RESULTS: A total of 114 patients have been recruited. There were 82 patients (77.0%) in the stable treatment group and 32 patients (23.0%) in the unstable treatment group. Based on multivariate likelihood analysis, only using acid-suppressive drugs contributed to stability treatment in CC patients (odds ratio: 2.81, 95% confidence interval: 1.12-7.08, p = 0.03). CONCLUSION: Administration of acid-suppressive drugs was the only factor related to the stability of CC treatment. Further studies are needed to validate the results as well as clarify the causes.

Predictive factors for esophageal stenosis in patients receiving prophylactic steroid therapy after endoscopic submucosal dissection for esophageal squamous cell carcinoma.

BACKGROUND: Methods to prevent esophageal stenosis (ES) after endoscopic submucosal dissection (ESD) for superficial esophageal squamous cell carcinoma (ESCC) have received increasing attention. Although steroid administration is a prophylactic treatment, the risk factors for ES during prophylactic steroid therapy remain unknown. Therefore, this study aimed to retrospectively evaluate the risk factors for refractory ES in patients administered prophylactic steroids after ESD for ESCC. METHODS: Among 795 patients with ESCC (854 lesions), 180 patients (211 lesions) administered local triamcinolone acetonide (TrA) and/or oral prednisolone were recruited for this study. We compared the total number of endoscopic balloon dilatation (EBD) procedures performed for post-ESD ES and clinical findings (tumor size, ESD history or chemoradiation therapy [CRT], entire circumferential resection, muscle layer damage, supplemental oral prednisolone administration, EBD with TrA injection, and additional CRT) between patients with refractory and non-refractory ES. EBD was continued until dysphagia resolved. We categorized cases requiring ≥ 8 EBD procedures as refractory postoperative stenosis and divided the lesions into two groups. RESULTS: Multivariate logistic regression analysis revealed that factors such as ESD history, CRT history, tumor size, and entire circumferential resection were independently associated with the development of refractory ES. The withdrawal rates of EBD at 3 years were 96.1% (52/53) and 58.5% (39/59) in the non-refractory and refractory groups, respectively. CONCLUSIONS: Our data suggest that entire circumferential resection and CRT history are risk factors for refractory post-ESD ES in ESCC, even with prophylactic steroid administration.

Endoscopic findings suggestive of a high risk of non-radical cure after definitive chemoradiotherapy for cT1bN0M0 esophageal squamous cell carcinoma.

BACKGROUND: Definitive chemoradiotherapy (DCRT) is a curative treatment option for cT1bN0M0 esophageal squamous cell carcinoma (ESCC); however, local residual disease and recurrence after complete remission may occur. We aimed to identify endoscopic findings associated with the risk of non-radical cure (local remnant or recurrence) after DCRT for cT1bN0M0 ESCC. METHODS: We retrospectively analyzed 40 consecutive patients with cT1bN0M0 ESCC who had undergone DCRT between January 2007 and December 2017. We examined the endoscopic findings in patients with residual or recurrent (RR) disease (RR group) and those without RR disease [non-RR (NRR) group] after DCRT. We also evaluated outcomes after DCRT for each endoscopic finding. RESULTS: There were 10 patients in the RR group and 30 patients in the NRR group. The RR group had a significantly larger tumor size and a higher proportion of lesions with type 0-I. The 5-year relapse-free survival rate was significantly lower in type 0-I and in the presence of B3 vessels. Endoscopic findings in 15 patients with cT1bN0M0 ESCC, type 0-I, who underwent DCRT revealed significantly more reddish lesions in the RR group compared to the NRR group. CONCLUSIONS: cT1bN0M0 ESCC large size, with B3 vessels, and type 0-I has a high risk of non-radical cure after DCRT, especially the reddish type 0-I, which may need to be considered for treatment similar to advanced cancer, including surgery with preoperative DCRT.

The diversity of bovine MHC class II DRB3 and DQA1 alleles in different herds of Japanese Black and Holstein cattle in Japan.

In cattle, bovine leukocyte antigens (BoLAs) have been extensively used as markers for bovine diseases and immunological traits. In this study, we sequenced alleles of the BoLA class II loci, BoLA-DRB3 and BoLA-DQA1, from 650 Japanese cattle from six herds [three herds (507 animals) of Japanese Black cattle and three herds (143 animals) of Holstein cattle] using polymerase chain reaction-sequence-based typing (PCR-SBT) methods. We identified 26 previously reported distinct DRB3 alleles in the two populations: 22 in Japanese Black and 17 in Holstein. The number of DRB3 alleles detected in each herd ranged from 9 to 20. Next, we identified 15 previously reported distinct DQA1 alleles: 13 in Japanese Black and 10 in Holstein. The number of alleles in each herd ranged from 6 to 10. Thus, allelic divergence is significantly greater for DRB3 than for DQA1. A population tree on the basis of the frequencies of the DRB3 and DQA1 alleles showed that, although the genetic distance differed significantly between the two cattle breeds, it was closely related within the three herds of each breed. In addition, Wu-Kabat variability analysis indicated that the DRB3 gene was more polymorphic than the DQA1 gene in both breeds and in all herds, and that the majority of the hypervariable positions within both loci corresponded to pocket-forming residues. The DRB3 and DQA1 heterozygosity for both breeds within each herd were calculated based on the Hardy-Weinberg equilibrium. Only one Japanese Black herd showed a significant difference between the expected and observed heterozygosity at both loci. This is the first report presenting a detailed study of the allelic distribution of BoLA-DRB3 and -DQA1 genes in Japanese Black and Holstein cattle from different farms in Japan. These results may help to develop improved livestock breeding strategies in the future.

Elastin gene polymorphisms in neovascular age-related macular degeneration and polypoidal choroidal vasculopathy.

PURPOSE: To study and reveal genetic variation in the elastin gene (ELN) that may be associated with neovascular age-related macular degeneration (AMD) and/or polypoidal choroidal vasculopathy (PCV). Eyes with neovascular AMD and PCV exhibit substantially different structural alterations of the elastic layer in the Bruch's membrane. The hypothesis for the present study was that ELN polymorphisms may play a role in the development of neovascular AMD and PCV and that genetic differences in ELN between these two phenotypes may be a reason for the histopathologic differences. To test these hypotheses, ELN was screened for genetic variation in a Japanese case-control dataset. METHODS: Two hundred eighty-five subjects were enrolled: 78 with neovascular AMD, 103 with PCV, and 104 control. We genotyped five tagged single nucleotide polymorphisms (SNPs) in ELN, and allele, genotype, and haplotype frequency distributions among neovascular AMD, PCV, and control subjects were compared by chi(2) tests. RESULTS: A common ELN variant was significantly associated with susceptibility to PCV. The age- and sex-adjusted odds ratio was 7.56 for individuals homozygous for the risk allele compared with those carrying no more than one copy of the risk allele. Significantly different distributions were found in allele and haplotype frequencies between neovascular AMD and PCV in this region, but no particular ELN SNPs or haplotypes were significantly associated with neovascular AMD. CONCLUSIONS: The findings implicate ELN as a susceptibility gene for PCV, and suggest that a different pathogenic process may be involved in the phenotypic expression of neovascular AMD and PCV.

The upregulation of angiogenic gene expression in cultured retinal pigment epithelial cells grown on type I collagen.

PURPOSE: Increases in matrix proteins, such as type I collagen and fibronectin, are observed with aging in the retinal pigment epithelium (RPE) basement membrane. However, little is known about altered gene expression profiles of RPE associated with increases in matrix proteins. We investigated changes in gene expression profiles of a human RPE cell line (ARPE-19) cultured on type I collagen. METHODS: Visually confluent ARPE-19 cells were grown on either Matrigel (M group) or type I collagen (C group) without serum over 3 days. Total RNA was extracted and reverse transcribed. Gene expression profiles in both groups were compared using microarray analyses. Several angiogenic genes including integrin alpha V, integrin alpha 2, integrin beta 1, integrin beta 3, VEGF-A, VEGF-B, and VEGF-C were subjected to quantitative analyses using real-time PCR. RESULTS: Out of 192 genes examined, angiogenesis-related genes (17.7%) and extracellular matrix-related genes (30.2%) were expressed highly (with more than 1.5-fold difference) in the C group when compared with the M group. In real-time PCR analyses, all VEGF and integrin family genes examined were expressed more in the C group than in the M group. CONCLUSIONS: Type I collagen likely causes an upregulation in a number of angiogenic gene expression patterns as seen in RPE in vitro experiments.

LOC387715/HTRA1 variants in polypoidal choroidal vasculopathy and age-related macular degeneration in a Japanese population.

PURPOSE: To investigate whether variants in the LOC387715 locus and the HtrA serine peptidase 1 (HTRA1) gene within the 10q26 locus are associated with polypoidal choroidal vasculopathy (PCV) and wet age-related macular degeneration (AMD) in a Japanese population, and whether genetic diversity exists between PCV and wet AMD in this locus. DESIGN: Cross-sectional study. METHODS: We genotyped 243 Japanese individuals, including 76 PCV cases, 73 wet AMD cases, and 94 controls using two single nucleotide polymorphisms (SNPs) that are located in the LOC387715 locus (rs10490924) or the HTRA1 gene (rs11200638). Genotyping was performed using TaqMan assays (Applied Biosystems, Foster City, California, USA). RESULTS: Two SNPs generated highly significant allelic associations with PCV (rs10490924, P = 5.7 x 10(-6); rs11200638, P = 5.2 x 10(-6)) and AMD (rs10490924, P = 1.4 x 10(-6); rs11200638, P = 3.4 x 10(-7)). The odds ratios and population attributable risks were higher for the AMD cases than for the PCV cases. Homozygotes for the risk allele at rs11200638 had a 6.33-fold increased risk of PCV and a 13.77-fold increased risk of wet AMD when compared with homozygotes for the wild-type allele. There were no significant differences in either allelic or genotypic frequencies between PCV and AMD cases. CONCLUSIONS: The LOC387715/HTRA1 variants are associated with PCV and wet AMD in the Japanese population. The associations are stronger in AMD than in PCV. PCV and AMD share common genetic factors, which suggests that PCV and wet AMD are similar in some pathophysiologic aspects.

The expression of advanced glycation endproduct receptors in rpe cells associated with basal deposits in human maculas.

Basal deposits within Bruch's membrane are associated with aging and age-related macular degeneration (AMD) although the factors causing their formation are incompletely understood. Advanced glycation endproducts (AGEs) accumulate in Bruch's membrane including basal deposits and drusen with aging. One mechanism by which AGEs alter a cell's phenotype is via AGE receptors. The purpose of this study was to immunolocalize and quantify the expression of AGE receptors by RPE cells associated with basal deposits or normal Bruch's membrane that were microdissected from human maculas. Postmortem eyes from 14 aged control donors and five donors with non-neovascular AMD were cryopreserved. RPE cells associated with normal Bruch's membrane or basal deposits were laser capture microdissected. The RNA was extracted and used for RT-qPCR to quantify the expression of RAGE, AGE R1, AGE R2, and AGE R3. Streptavidin alkaline phosphatase immunohistochemistry for these receptors was also performed and sections were bleached from 14 normal and nine AMD donors. RT-qPCR showed significant upregulation of RAGE, AGE R1, and AGE R3 in RPE cells overlying basal deposits compared to cells attached to morphologically normal Bruch's membrane. Immunohistochemical analysis for RAGE, AGER1, R2, and R3 showed diffuse, light staining of RPE cells and strong choriocapillaris staining in areas of normal Bruch's membrane. In areas of basal deposits, the RPE had more intense staining for RAGE and AGER1 compared to regions of normal Bruch's membrane. These results suggest that AGE receptors could influence the formation of basal deposits during aging and AMD.

Advanced glycation endproduct-induced aging of the retinal pigment epithelium and choroid: a comprehensive transcriptional response.

Advanced glycation endproduct (AGE) formation is a trigger for the onset of age-related disease. To evaluate AGE-induced change in the ocular fundus, 5-mo-old C57BL/6 mice were given low-dose D-galactose (D-gal) for 8 wk and evaluated by AGE fluorescence, electroretinography (ERG), electron microscopy, and microarray analysis for 20 wk. Although AGE fluorescence was increased in D-gal-treated retinal pigment epithelium (RPE)-choroid compared with controls at all time points, ERG showed no AGE-induced functional toxicity. Progressive ultrastructural aging in the RPE-choroid was associated temporally with a transcriptional response of early inflammation, matrix expansion, and aberrant lipid processing and, later, down-regulation of energy metabolism genes, up-regulation of crystallin genes, and altered expression of cell structure genes. The overall transcriptome is similar to the generalized aging response of unrelated cell types. A subset of transcriptional changes is similar to early atherosclerosis, a chronic inflammatory disease characterized by matrix expansion and lipid deposition. These changes suggest an important contribution of a single environmental stimulus to the complex aging response.

Different effects of angiopoietin-2 in different vascular beds: new vessels are most sensitive.

In this study, we used double transgenic mice with inducible expression of angiopoietin-2 (Ang2) to investigate the role of Ang2 in the retinal and choroidal circulations and in three models of ocular neovascularization (NV). Mice with induced expression of Ang2 ubiquitously, or specifically in the retina, survived and appeared grossly normal. They also had normal-appearing retinal and choroidal circulations, demonstrating that high levels of Ang2 did not induce regression of mature retinal or choroidal vessels. When Ang2 expression was induced soon after birth, there was increased density of the deep capillary bed on postnatal day (P) 11 that returned to normal by P18, the time that retinal vascular development is usually completed. In mice with ischemic retinopathy, induction of Ang2 during the ischemic period resulted in a significant increase in retinal NV, but induction of Ang2 at a later time point when ischemia (and vascular endothelial growth factor [VEGF]) was less, hastened regression of NV. In triple transgenic mice that coexpressed VEGF and Ang2, the increased expression of Ang2 inhibited VEGF-induced NV in the retina. Increased expression of Ang2 also resulted in regression of choroidal neovascularization. These data suggest that ocular neovascularization, but not mature retinal or choroidal vessels, is sensitive to Ang2; a high Ang2/VEGF ratio promotes regression, while high Ang2 in the setting of hypoxia and/or concomitantly high Ang2 and VEGF stimulate neovascularization.

Similarity of mRNA phenotypes of morphologically normal macular and peripheral retinal pigment epithelial cells in older human eyes.

PURPOSE: To determine the expression profiles of morphologically normal human retinal pigment epithelial (RPE) cells that originate from the macula and periphery. METHODS: Morphologically normal RPE cells from 15 human globes from donors aged 52 to 82 years old were laser capture microdissected. Total RNA from 5000 cells was SMART amplified, [33]P-labeled, and hybridized to a cDNA array containing 4325 known genes. Expression profiles were analyzed by hierarchical cluster analysis, Prediction Analysis of Microarrays (PAM), and Significance Analysis for Microarrays (SAM). Differentially expressed genes were evaluated further by real time RT-PCR. RESULTS: The overall expression profiles of RPE cells from the macula and periphery were similar. Unsupervised and supervised hierarchical cluster analysis showed that patient genotype was a stronger separating factor than topographical location. SAM analysis identified 11 genes that were underexpressed by macular RPE cells. The expression patterns of these 11 genes were confirmed by real time RT-PCR, with 5 genes reaching statistical significance. CONCLUSIONS: Whereas the overall expression profiles were similar between cells from the macula and periphery, subtle differential expression of five genes could contribute to RPE phenotypic differences based on topographic location.

Ultrastructural aging of the RPE-Bruch's membrane-choriocapillaris complex in the D-galactose-treated mouse.

PURPOSE: Low-dose D-galactose treatment in mice induces accelerated aging due to advanced glycation endproduct (AGEs) formation. The purpose of this study was to identify ultrastructural aging in the retinal pigment epithelium (RPE)-Bruch's membrane-choriocapillaris. METHODS: Five-month-old C57Bl6 mice were injected daily with D-galactose or control buffer for 8 weeks. Eighteen-month-old mice were also treated with control buffer for 8 weeks. Eyes were prepared for electron microscopy and AGE-specific fluorescence at ex = 370 nm/em = 440 nm and ex = 330 nm/ex = 390 nm. RESULTS: D-Galactose treatment induced AGE-specific fluorescence in lens and RPE/choroid compared to buffer-treated controls. In D-galactose-treated animals, the RPE had dilated and fewer basolateral infoldings. Bruch's membrane had alterations that included significant thickening, sub-RPE and prominent outer collagenous layer deposits, and choriocapillaris basement membrane duplication/splitting and thickening. The choriocapillaris endothelium displayed fenestration loss. CONCLUSIONS: Ultrastructural aging to the RPE-Bruch's membrane-choriocapillaris developed in mice treated with low-dose D-galactose. These changes could contribute to age-related changes that promote early age-related disease.

The expression of native and cultured RPE grown on different matrices.

The purpose of this work was to determine the expression profiles of retinal pigment epithelial (RPE) cells grown on different matrices and to assess the degree of culture-induced artifact by comparing the profiles to native RPE. Visually confluent ARPE-19 cells were grown on plastic, Matrigel, collagen I, collagen IV, laminin, and fibronectin for 1 wk, and serum was withdrawn for 3 days. Morphologically normal, macular RPE cells were laser-capture microdissected from three human eye globes. Total RNA was extracted from 5,000 cells and reverse transcribed, and radiolabeled cDNA probes were hybridized to an array containing 4,325 known genes. Arrays were assessed by cluster analysis and significance analysis of microarrays (SAM). Real-time RT-PCR was used to validate differentially expressed genes. Despite similar morphology, ARPE-19 demonstrated different expression profiles when grown on different matrices. Cluster analysis showed that cells grown on collagen IV, laminin, and fibronectin had similar profiles that were distinct from cells grown on collagen I. Cells grown on plastic clustered closest to native RPE. This expression pattern was confirmed with supervised cluster analyses. The number of differentially expressed genes, function of differentially expressed genes, and profile of expressed and unexpressed genes suggest that the overall expression profile of cultured cells is significantly different from native RPE. RPE cells grown on collagen IV, laminin, and fibronectin have profiles more similar than cells grown on plastic, Matrigel, or collagen I. The overall mRNA phenotype, however, is different from morphologically normal, native macular RPE.

Differences in Ibaraki virus RNA segment 3 sequences from three epidemics.

Phylogenetic tree and partial nucleotide sequence analysis of RNA segment 3 were conducted to compare the Ibaraki virus (IBAV) strains from three epidemics in Japan, and serotype 2 epizootic hemorrhagic disease virus strains isolated in Australia, Taiwan, and Canada. Each strain was classified relative to the Ibaraki disease (IBAD) epidemics, which occurred in 1959-1960, 1987, or 1997-1998. In particular, major variation of the gene was identified in the strains isolated after 1997 when a new type of IBAD with the abnormal birth was confirmed. Ibaraki viruses isolated in Japan were more closely related to Taiwanese and Australian strains based on genetics, while the Canadian strain was more distantly related.

Evaluation of the effect of aging on retinal nerve fiber layer thickness measured by optical coherence tomography.

PURPOSE: To evaluate the relationship between age and retinal nerve fiber layer (RNFL) thickness in normal subjects, as measured by optical coherence tomography (OCT). METHODS: One hundred and forty-four normal subjects (144 eyes), ranging from 16 to 84 years of age, were enrolled in this cross-sectional study. The RNFL thickness was determined using OCT with three circle scans 3.4 mm in diameter. RESULTS: The average RNFL thickness was inversely correlated with age (r = -0.348, p < 0.001). Analyzing the quadrants as a parameter, RNFL thickness in the superior, temporal and inferior quadrants also decreased with age. Using 30-degree segments, there were significant correlations between age and the RNFL thickness of temporal segments (7-11 o'clock). The average RNFL thickness had the highest correlation among all parameters (r = -0.348, p < 0.001). Regarding nasal quadrant thickness, RNFL ratios (average, superior, temporal and inferior RNFL thickness relative to the nasal quadrant thickness) were not significantly correlated with age. The refractive error did not affect RNFL thickness (r = 0.091, p = 0.276). CONCLUSION: Our study revealed that RNFL thickness, in particular in the temporal quadrant, measured by OCT significantly decreased with age. Age has to be taken into consideration when we compare RNFL thickness between normal and glaucomatous eyes.

Analysis of cytokine mRNAs in murine herpes simplex virus type 1 retinitis.

PURPOSE: To investigate cytokine mRNA expression during the inflammatory process induced in the contralateral eyes by uniocular inoculation of herpes simplex virus type 1 (HSV-1) via the anterior chamber. METHODS: BALB/c mice were inoculated in the anterior chamber with 5 x 10(4) plaque-forming units of HSV-1 (KOS). mRNA was extracted from the inflamed posterior segments of the uninoculated eyes at 0 (control), 9, 11, 14, and 21 days postinoculation (p.i.). Reverse transcription-polymerase chain reaction was performed for semiquantitative analysis of mRNA expression of interleukin (IL)-1beta, IL-2, IL-4, IL-10, IL-12p35, IL-12p40, interferon (IFN)gamma, tumor necrosis factor (TNF)alpha, transforming growth factor (TGF)beta2 and induced nitric oxide synthase (iNOS). RESULTS: Peak mRNA expression of iNOS was observed at day 14 p.i. The time profiles of mRNA expression for IL-1beta, IL-2, IL-4, IL-10, IFN-gamma, TNFalpha were similar to that of iNOS, while TGFbeta2, IL-12p35, and IL-12p40 demonstrated a reverse pattern. CONCLUSIONS: The kinetics of the analyzed cytokines synchronized with the clinicopathological activity of the experimental murine HSV-1 retinitis. The immunosuppressive cytokines TGFbeta2 and IL-10 demonstrated different peaks of mRNA expressions suggesting that the down-regulation phase of the inflammatory process was controlled by several factors working at different phases.