UbiA prenyltransferase domain-containing protein 1 (UBIAD1) variant c.695 A > G identified in a multigenerational Japanese family with Schnyder corneal dystrophy.

PURPOSE: We aimed to identify pathogenic variations in the UbiA prenyltransferase domain-containing protein 1 (UBIAD1) gene in a Japanese family with Schnyder corneal dystrophy (SCD). STUDY DESIGN: Clinical study METHODS: Three clinically diagnosed SCD patients from a single pedigree participated. Patients 1 and 2 were 69- and 65-year-old sisters, and patient 3 was the 42-year-old daughter of patient 1. Blood samples from the patients were obtained for genetic analysis. Mutation screening of the two UBIAD1 exons was performed using polymerase chain reaction (PCR)-based DNA sequencing. RESULTS: All participants were found to be heterozygous for the pathogenic missense variation c.695 A > G (p.Asn232Ser) in exon 2 of UBIAD1. CONCLUSION: This is the first report on the pathogenic UBIAD1 variation c.695 A > G (p.Asn232Ser) in a Japanese population. SCD is a rare corneal dystrophy, and further research on additional cases will aid in the elucidation of disease mechanisms and development of therapeutic strategies.

Regular and irregular astigmatism of bullous keratopathy using Fourier harmonic analysis with anterior segment optical coherence tomography.

Bullous keratopathy (BK) is known to present with corneal edema and Descemet's folds, which can cause corneal astigmatism. However, no report quantitatively evaluated BK astigmatism by separating it into regular and irregular astigmatism. This study investigated the regular and irregular astigmatism of the anterior and posterior corneal surface with Fourier harmonic analysis and anterior segment optical coherence tomography. Preoperative data from 43 eyes of 41 BK patients who received corneal endothelial transplantation were compared with the data from 43 eyes of 43 subjects without corneal disease. Anterior and posterior cylinder power, central corneal thickness (CCT) and thinnest corneal thickness were significantly greater in BK. With Fourier harmonic analysis, BK eyes were found to have significantly larger anterior and posterior regular astigmatism, asymmetry component and higher-order irregularity. Asymmetry component and higher-order irregularity that accounted for the posterior irregular astigmatism increased as CCT increased in BK. Higher-order irregularity in the posterior cornea also positively correlated with worsening best corrected visual acuity. Subgroup analysis found significant correlations between CCT and posterior higher-order irregularity for intraocular surgery and laser iridotomy, but not Fuchs endothelial corneal dystrophy. This study has significance in that it revealed the characteristics of the corneal posterior irregular astigmatism of BK.

Impaired psychomotor vigilance associated with sleep-disordered breathing in women working in the care of older adults in Japan.

PURPOSE: With a rapidly aging society, there is increasing interest in the health of female workers in the field of care services for older adults due to increasing demands to maintain 24-h care and to support older adults without errors or accidents. Therefore, the purpose of this cross-sectional study was to examine the association between sleep-disordered breathing (SDB) and sustained attention in women caring for older adults in Japan. METHODS: The study was conducted in women aged 18-67 years old working in care service facilities for older adults in Japan. The sustained attention of participants was measured by the 10-min psychomotor vigilance task (PVT). SDB was assessed based on the respiratory disturbance index (RDI), which was measured using an ambulatory airflow monitor with a polyvinylidene fluoride (PVDF) film sensor to monitor the respiratory airflow of nasal and oral breathing. The participants wore the monitor to record the breathing status while asleep at home. The severity of SDB was categorized as follows: normal, RDI < 5 events/h; mild SDB, RDI 5-10 events/h; and moderate-to-severe SDB, RDI ≥ 10 events/h. RESULTS: Of 688 women enrolled, medians of age, body mass index (BMI), sleep duration, and prevalence of hypertension tended to be higher with increasing RDI. No significant association was found between RDI and PVT parameters. However, when we limited the analysis to women with BMI ≥ 22 kg/m2, those with moderate-to-severe SDB had significantly higher odds of having the slowest 10% reaction times compared to those without SDB (OR = 2.03; 95% CI = 1.17-3.53). The association did not decrease after adjusting to account for sleep duration, alcohol drinking habits, and history of hypertension (OR = 1.97; 95% CI = 1.10-3.52). A significant increasing trend was also found between RDI and the slowest 10% of reaction times (p for trend = 0.03). CONCLUSIONS: Our findings suggest that SDB is associated with reduced sustained attention in participants with BMI ≥ 22 kg/m2, although the number of assessments of SDB and PVT was only once per participant due to the nature of the cross-sectional study.

Toxicity profiles of fixed-combination eye drops for glaucoma therapy using cultivated human corneal epithelial sheets.

PURPOSE: We aimed to investigate the toxicity of 6 fixed-combination drugs for glaucoma therapy using human corneal epithelial sheets (HCES). STUDY DESIGN: Experimental. MATERIALS AND METHODS: We used 6 kinds of commercially available fixed-combination drugs: latanoprost/carteolol (LAT/CAR), latanoprost/timolol (LAT/TIM), tafluprost/timolol (TAF/TIM), travoprost/timolol (TRA/TIM), brinzolamide/timolol (BRZ/TIM), and dorzolamide/timolol (DRZ/TIM) including different preservatives. The cell viability and barrier function of the HCES after exposure to the eye drops for 10 or 30 minutes were assessed using the WST-1 assay and transepithelial electrical resistance (TEER) measurements, respectively. The HCES were also evaluated using hematoxylin and eosin (HE) staining and transmission electron microscopy. RESULTS: The cell viability significantly decreased in the HCES treated with LAT/TIM or DRZ/TIM after 10 and 30 minutes and in those treated with BRZ/TIM after 30 minutes. The barrier function increased significantly in the HCES treated with LAT/CAR. Histologically, the HCES were damaged after treatment with LAT/TIM, BRZ/TIM, or DRZ/TIM for 30 minutes. Transmission electron microscopy indicated narrow intercellular spaces and multiple intercellular junctions in the HCES treated with LAT/CAR, TAF/TIM, or TRA/TIM. The HCES treated with DRZ/TIM, BRZ/TIM, or LAT/TIM contained cytoplasmic vacuoles and collapsed cellular structures. CONCLUSION: Glaucoma fixed-combination eye drops demonstrated a different toxic effect on the cell viability, barrier function, and morphologic changes of HCES.

Prevalence of sleep-disordered breathing among women working in the aged care services in Japan.

OBJECTIVES: The aim of this study was to examine the prevalence of sleep-disordered breathing (SDB) in women working in the field of aged care in Japan. METHODS: A cross-sectional study was conducted for female employees aged 18-60 years in aged care facilities in Japan. The analyzed set consisted of 712 participants with complete data. SDB was determined by respiratory disturbance index (RDI), measured using an ambulatory airflow monitor with a polyvinylidene fluoride film sensor to detect nasal and oral airflow overnight at home. Based on the findings of previous studies, RDI 10 was considered equivalent to apnea-hypopnea index (AHI) 15. RESULTS: The prevalence of moderate-to-severe SDB (RDI ≥ 10) was 22.8%. The mean age was 38.1 years, and mean sleeping time of participants was 6.1 h. The median body mass index (BMI) was 22.0. Women with moderate-to-severe SDB (RDI ≥ 10) had a higher age, neck circumference, neck-height ratio, BMI, systolic blood pressure, and increased prevalence of hypertension, short sleepers (amount of sleep on the SDB testing day < 6 h), and habitual snorers, compared to women with no or mild SDB (RDI < 10). CONCLUSIONS: Our study found that women working in the aged care services in Japan were heavily burdened by SDB even though they were relatively young and slim. To prevent CVD from developing in the future, programs through which workplaces can help their employees improve their lifestyle, and early diagnosis and treatment of SDB are highly recommended.

High Permeability and Intercellular Space Widening With Brimonidine Tartrate Eye Drops in Cultured Stratified Human Corneal Epithelial Sheets.

PURPOSE: To investigate the toxicity of topical glaucoma medications using cultured stratified human corneal epithelial sheets (HCES). METHODS: HCES were exposed for 30 minutes to the following glaucoma medications: 0.1% brimonidine with sodium chlorite as the preservative, 0.005% latanoprost with 0.02% benzalkonium chloride (BAC) as the preservative, and 0.5% timolol with 0.005% BAC as the preservative. Then, cell viability and barrier function were tested by the WST-1 assay and carboxyfluorescein permeability assay, respectively. After exposure to glaucoma medications, HCES were evaluated by hematoxylin and eosin staining, periodic acid-Schiff staining, scanning electron microscopy, and transmission electron microscopy. RESULTS: HCES exposed to brimonidine showed higher viability and better preservation of cell morphology and microvilli compared with cell sheets exposed to latanoprost or timolol. The carboxyfluorescein permeability assay demonstrated that the barrier function was preserved after HCES were exposed to timolol, but not after exposure to brimonidine or latanoprost. Transmission electron microscopy revealed widening of intercellular junctions with prominent deposits of glycogen or mucopolysaccharide (periodic acid-Schiff positive) after exposure of HCES to brimonidine. CONCLUSIONS: The toxicity of 0.1% brimonidine containing sodium chlorite for HCES was lower than that of ophthalmic preparations containing BAC. Reduction of the barrier function occurred after HCES were exposed to brimonidine because of widening of intercellular junctions.

High expression of organic anion transporter 2 and organic cation transporter 2 is an independent predictor of good outcomes in patients with metastatic colorectal cancer treated with FOLFOX-based chemotherapy.

Although metastatic colorectal cancer (mCRC) is commonly treated with 5-fluorouracil (5-FU)/leucovorin/oxaliplatin (FOLFOX), their response to FOLFOX varies, and no biomarkers predictive of treatment outcome have been validated. Organic anion transporter 2 (OAT2) and organic cation transporter 2 (OCT2) are critical determinants in uptake of 5-FU and oxaliplatin, respectively. In this study, we evaluated whether OAT2 and OCT2 levels can predict effectiveness of FOLFOX-based therapy. We retrospectively assessed 90 patients with mCRC who were treated with first-line FOLFOX with or without bevacizumab. We immunohistochemically determined OAT2 and OCT2 expression levels at invasion fronts of their tumors and correlated the levels to clinicopathological parameters, including objective tumor response (OTR) and progression-free survival (PFS). High expression of OAT2 (OAT2(High)) and OCT2 (OCT2(High)) were detected in 36% and 60% of the tumors, respectively. OCT2(High) was significantly associated with invasion depth (P=0.03), whereas OAT2(High) was not associated with any clinicopathological parameters. In univariate analysis, OAT2(High) was significantly correlated with good OTR (P=0.02), and OCT2(High) with long PFS (P=0.03). Multivariate analyses showed that OAT2(High) and OCT2(High), respectively, were the sole independent predictors of good OTR (P=0.02) and long PFS (P=0.03). We found that patients with OAT2(High)/OCT2(High) showed the best treatment outcomes (good OTR and long PFS) with significantly higher frequency than patients with other expression patterns (P=0.003). OAT2(High)/OCT2(High) status was also the only independent predictive factor in multivariate analysis. This study suggests that OAT2(High) and OCT2(High) are important independent predictors of good outcomes in FOLFOX-treated mCRC.

Expression of organic anion-transporting polypeptide 1A2 and organic cation transporter 6 as a predictor of pathologic response to neoadjuvant chemotherapy in triple negative breast cancer.

Organic anion-transporting polypeptide 1A2 (OATP1A2) and organic cation transporter 6 (OCT6) are involved in the uptake of taxanes and anthracyclines, respectively. The aim of this study was to evaluate expression levels of OATP1A2 and OCT6 as a predictor of response to neoadjuvant chemotherapy (NAC) in breast cancer. A total of 124 patients who received anthracycline/taxane-based NAC were included. Expression levels of OATP1A2 and OCT6 were immunohistochemically assessed in core needle biopsies obtained prior to NAC. A pathologic good response (pGR) and a pathologic complete response (pCR) were achieved in 24 and 10 % of patients, respectively. In univariate analysis of the entire cohort, negative hormone receptor (HR) status (pGR and pCR, P < 0.001), high Ki-67 level (pGR, P = 0.03; pCR, P = 0.02), triple negative (TN) subtype (pGR, P = 0.001; pCR, P < 0.001), and high OCT6 (pGR, P = 0.003) were associated with the response. In combined analysis, high OATP1A2/high OCT6 level was also a significant factor for pGR (P = 0.001) and pCR (P = 0.001). Two separate multivariate analyses showed that HR status, TN subtype and combined high OATP1A2/high OCT6 level were significant independent predictors. When TN and non-TN tumors were assessed separately in univariate analysis, high Ki-67 level (P = 0.04) were associated with pGR and combined high OATP1A2/high OCT6 level was associated with both pGR (P = 0.005) and pCR (P = 0.03) in the TN group. Multivariate analysis identified the combined high OATP1A2/high OCT6 level as the sole independent predictor of pGR. In the non-TN group, negative HR status (P = 0.03) and positive HER2 status (P = 0.005) were associated with pGR, but HER2 status was the sole independent predictor of pGR. These results suggest that response-associated predictors may differ between the TN and non-TN tumors. Combined high OATP1A2/high OCT6 may be a potential predictor of response to anthracycline/taxane-based chemotherapy in breast cancer, especially in TN tumors.

Organic cation transporter 2 and tumor budding as independent prognostic factors in metastatic colorectal cancer patients treated with oxaliplatin-based chemotherapy.

Oxaliplatin is currently approved for patients with metastatic colorectal cancer (mCRC). Its uptake and consequent cytotoxicity is determined by the levels of organic cation transporter 2 (OCT2). In addition, tumor budding (TB) is associated with high malignant potential. However, the impact of the levels of OCT2 and TB on clinicopathological findings and the prognosis of mCRC patients treated with oxaliplatin-based chemotherapy remains unclear. Here, 80 mCRC patients were retrospectively assessed. Immunohistochemistry was performed to determine the levels of OCT2 and TB. High levels of OCT2 (47/80, 59%) were detected at the invasion front and were associated with depth of invasion (P=0.03), whereas high levels of TB (40/80, 50%) were associated with extensive lymphatic invasion (P=0.03). In univariate analysis, high OCT2 levels were significantly correlated with longer progression-free survival (PFS) (P=0.02) whereas high TB levels were associated with shorter PFS (P=0.01). In combined analysis, patients with 2 favorable factors (high OCT2/low TB) had longer PFS than those with 1 (P=0.03) or 0 (P<0.001) favorable factors. Multivariate analysis confirmed that the OCT2 level (P=0.007), TB level (P=0.004), and combined OCT2/TB status (P=0.001) were independent predictors for PFS. These results suggest that high levels of OCT2 indicate severe invasion, but also better prognosis in mCRC patients treated with oxaliplatin-based chemotherapy, possibly because of its role in oxaliplatin susceptibility. Combined analysis of OCT2 and TB status may guide the selection of patients for successful oxaliplatin-based chemotherapy.

Prognostic value of organic anion transporting polypeptide 1B3 and copper transporter 1 expression in endometrial cancer patients treated with paclitaxel and carboplatin.

Paclitaxel and carboplatin (TC) chemotherapy is an effective and well-tolerated regimen against advanced endometrial cancer. Organic anion transporting polypeptide 1B3 (OATP1B3) and copper transporter 1 (CTR1) are critical for the uptake of paclitaxel and carboplatin, respectively. This study aimed to address the prognostic impact of OATP1B3 and CTR1 in endometrial cancer patients treated with adjuvant TC chemotherapy. We immunohistochemically evaluated the expressions of OATP1B3 and CTR1 in 47 stage III endometrial cancers. The high expression levels of OATP1B3 were significantly correlated with type I tumor (P = 0.0005). In univariate analysis, high expression levels of OATP1B3 (P = 0.047) and CTR1 (P = 0.009) were significantly associated with longer disease-free survival (DFS) and longer overall survival (OS), respectively. The patients with tumors showing high expression levels of at least one of OATP1B3 and CTR1 had potentially longer DFS (P = 0.058) and significantly longer OS (P = 0.003) sin the univariate analysis. Combined OATP1B3/CTR1 expression was the sole independent prognostic factor for longer OS in the multivariate analysis (P = 0.013). Our findings suggest that combined OATP1B3/CTR1 expression is a possible predictive/prognostic factor for a good outcome in stage III endometrial cancer patients treated with adjuvant TC chemotherapy.

Auxiliary role for D-alanylated wall teichoic acid in Toll-like receptor 2-mediated survival of Staphylococcus aureus in macrophages.

We previously reported that Staphylococcus aureus avoids killing within macrophages by exploiting the action of Toll-like receptor 2 (TLR2), which leads to the c-Jun N-terminal kinase (JNK)-mediated inhibition of superoxide production. To search for bacterial components responsible for this event, a series of S. aureus mutants, in which the synthesis of the cell wall was interrupted, were screened for the level of JNK activation in macrophages. In addition to a mutant lacking the lipoproteins that have been suggested to act as a TLR2 ligand, two mutant strains were found to activate the phosphorylation of JNK to a lesser extent than the parental strain, and this defect was recovered by acquisition of the corresponding wild-type genes. Macrophages that had phagocytosed the mutant strains produced more superoxide than those engulfing the parental strain, and the mutant bacteria were more efficiently killed in macrophages than the parent. The genes mutated, dltA and tagO, encoded proteins involved in the synthesis of D-alanylated wall teichoic acid. Unlike a cell wall fraction rich in lipoproteins, D-alanine-bound wall teichoic acid purified from the parent strain by itself did not activate JNK phosphorylation in macrophages. These results suggest that the d-alanylated wall teichoic acid of S. aureus modulates the cell wall milieu for lipoproteins so that they effectively serve as a ligand for TLR2.

Pretaporter, a Drosophila protein serving as a ligand for Draper in the phagocytosis of apoptotic cells.

Phagocytic removal of cells undergoing apoptosis is necessary for animal development and tissue homeostasis. Draper, a homologue of the Caenorhabditis elegans phagocytosis receptor CED-1, is responsible for the phagocytosis of apoptotic cells in Drosophila, but its ligand presumably present on apoptotic cells remains unknown. An endoplasmic reticulum protein that binds to the extracellular region of Draper was isolated. Loss of this protein, which we name Pretaporter, led to a reduced level of apoptotic cell clearance in embryos, and the overexpression of pretaporter in the mutant flies rescued this defect. Results from genetic analyses suggested that Pretaporter functionally interacts with Draper and the corresponding signal mediators. Pretaporter was exposed at the cell surface after the induction of apoptosis, and cells artificially expressing Pretaporter at their surface became susceptible to Draper-mediated phagocytosis. Finally, the incubation with Pretaporter augmented the tyrosine-phosphorylation of Draper in phagocytic cells. These results collectively suggest that Pretaporter relocates from the endoplasmic reticulum to the cell surface during apoptosis to serve as a ligand for Draper in the phagocytosis of apoptotic cells.

Identification of lipoteichoic acid as a ligand for draper in the phagocytosis of Staphylococcus aureus by Drosophila hemocytes.

Phagocytosis is central to cellular immunity against bacterial infections. As in mammals, both opsonin-dependent and -independent mechanisms of phagocytosis seemingly exist in Drosophila. Although candidate Drosophila receptors for phagocytosis have been reported, how they recognize bacteria, either directly or indirectly, remains to be elucidated. We searched for the Staphylococcus aureus genes required for phagocytosis by Drosophila hemocytes in a screening of mutant strains with defects in the structure of the cell wall. The genes identified included ltaS, which encodes an enzyme responsible for the synthesis of lipoteichoic acid. ltaS-dependent phagocytosis of S. aureus required the receptor Draper but not Eater or Nimrod C1, and Draper-lacking flies showed reduced resistance to a septic infection of S. aureus without a change in a humoral immune response. Finally, lipoteichoic acid bound to the extracellular region of Draper. We propose that lipoteichoic acid serves as a ligand for Draper in the phagocytosis of S. aureus by Drosophila hemocytes and that the phagocytic elimination of invading bacteria is required for flies to survive the infection.

The tryptophan pathway is involved in the defense responses of rice against pathogenic infection via serotonin production.

The upregulation of the tryptophan (Trp) pathway in rice leaves infected by Bipolaris oryzae was indicated by: (i) enhanced enzyme activity of anthranilate synthase (AS), which regulates metabolic flux in the Trp pathway; (ii) elevated levels of the AS (OASA2, OASB1, and OASB2) transcripts; and (iii) increases in the contents of anthranilate, indole, and Trp. The measurement of the contents of Trp-derived metabolites by high-performance liquid chromatography coupled with tandem mass spectrometry revealed that serotonin and its hydroxycinnamic acid amides were accumulated in infected leaves. Serotonin accumulation was preceded by a transient increase in the tryptamine content and by marked activation of Trp decarboxylase, indicating that enhanced Trp production is linked to the formation of serotonin from Trp via tryptamine. Feeding of radiolabeled serotonin to inoculated leaves demonstrated that serotonin is incorporated into the cell walls of lesion tissue. The leaves of a propagating-type lesion mimic mutant (sl, Sekiguchi lesion) lacked both serotonin production and deposition of unextractable brown material at the infection sites, and showed increased susceptibility to B. oryzae infection. Treating the mutant with serotonin restored deposition of brown material at the lesion site. In addition, the serotonin treatment suppressed the growth of fungal hyphae in the leaf tissues of the sl mutant. These findings indicated that the activation of the Trp pathway is involved in the establishment of effective physical defenses by producing serotonin in rice leaves.

Induction of serotonin accumulation by feeding of rice striped stem borer in rice leaves.

Tryptophan (Trp)-related secondary metabolism has been implicated in the defense against pathogen infection and insect feeding in various gramineous species. Recently, we also reported that rice plant accumulated serotonin and tryptamine as well as their amide compounds coupled with phenolic acids in response to the infection by fungal pathogen. These compounds were likely to play an important role in the formation of physical barrier to the invading pathogens. To extend our study to elucidate the defensive role of Trp-derived secondary metabolism in gramineous plants, we examined in this study whether it is activated in response to herbivore attack as well. Third leaves of rice plant were fed on by third instar larvae of rice striped stem borer for 24 h or 48 h. The analysis of four Trp-derived metabolites including tryptamine, serotonin feruloyltryptamine (FerTry) and p-coumaroylserotonin (CouSer) by liquid chromatography coupled with tandem mass spectrometry revealed that their contents clearly increased in response to the larvae feeding. The respective amounts of tryptamine, serotonin, FerTry and CouSer in the larvae-fed leaves were 12-, 3.5-, 33- and 140-fold larger than those in control leaves 48 h after the start of feeding.

Evidence for phagocytosis of influenza virus-infected, apoptotic cells by neutrophils and macrophages in mice.

Influenza virus-infected cells undergo apoptosis and become susceptible to phagocytosis by macrophages in vitro, and this leads to the propagation of the virus being inhibited. We previously showed that inhibitors of phagocytosis increased the rate of mortality among influenza virus-infected mice. However, the mode of the phagocytosis of influenza virus-infected cells in vivo has not been investigated. We, in this study, assessed this issue by histochemically analyzing bronchoalveolar lavage cells and lung tissue obtained from C57BL/6 mice infected with influenza A/WSN (H1N1) virus. Both neutrophils and macrophages accumulated in the lung soon after the viral challenge, and either type of cell was capable of phagocytosing influenza virus-infected, apoptotic cells. Changes in the level of phagocytosis and the amount of virus in lung tissue roughly correlated with each other. Furthermore, alveolar macrophages prepared from influenza virus-infected mice showed greater phagocytic activity than those from uninfected mice. The phagocytic activity of macrophages was stimulated in vitro by a heat-labile substance(s) released from influenza virus-infected cells undergoing apoptosis. These results suggested that the level of phagocytosis is augmented both quantitatively and qualitatively in the lung of influenza virus-infected animals so that infected cells are effectively eliminated. Finally, lack of TLR4 caused an increase in the rate of mortality among influenza virus-challenged mice and a decrease in the level of phagocytosis of apoptotic cells in the lung. TLR4 could thus play an important role in the host defense against influenza by positively regulating the phagocytic elimination of infected cells.

Augmentation of fatality of influenza in mice by inhibition of phagocytosis.

Influenza virus-infected cells undergo apoptosis and become susceptible to phagocytosis by macrophages, and this leads to the inhibition of virus propagation in vitro. To assess if this were also true in vivo, mice infected with influenza A/WSN (H1N1) virus were administered with phagocytosis inhibitors and examined for the progress of influenza. Administration of the inhibitors caused a decrease in the level of phagocytosis observed with bronchoalveolar lavage cells. We found that both the lethality in mice and the extent of inflammation in the lung were augmented in those mice. These results suggest that phagocytosis of virus-infected cells helps suppress the progress of influenza in mice.