A case of anti-aquaporin-4 antibody-positive optic neuritis treated by selective immunoadsorption.

Anti-aquaporin-4 (AQP4) antibody-positive optic neuritis is a condition in which a patient testing positive for anti-AQP4 antibody presents with optic neuritis only. The disease is classified as a neuromyelitis optica spectrum disorder (NMOSD) and is a steroid-resistant refractory optic neuritis. Patients are treated by oral administration of steroids, steroid pulse therapy, and apheresis therapy. The patient in our case was a 48-year-old female who was diagnosed with anti-AQP4 antibody-positive optic neuritis by her ophthalmologist, and was referred to our hospital. Selective plasma exchange (SePE) was initially started, but she strongly preferred treatment as an outpatient due to family circumstances. Therefore, selective immunoadsorption (SeIA) was used from the second session to minimize loss of coagulation factors. The RRs were 16.5-33.3% for anti-AQP4 antibody, 7.48-18.57% for fibrinogen, and 0.8-4.57% for factor XIII. After the 7th SeIA session, the patient was followed up with a maintenance dose of 10 mg/day oral prednisolone as an outpatient at our Department of Ophthalmology. This is the first report to investigate the removal rate (RR) of anti-AQ4 antibody using SeIA. In our case, the anti-AQP4 antibody level before the last SeIA session was still not completely negative, but there was clinical improvement in vision. SeIA was highly effective in maintaining coagulation factor levels. Therefore, our results suggest that SeIA is a safe treatment that can be performed in an outpatient setting.

Discovery of an NRF1-specific inducer from a large-scale chemical library using a direct NRF1-protein monitoring system.

NRF1 (NF-E2-p45-related factor 1) plays an important role in the regulation of genes encoding proteasome subunits, a cystine transporter, and lipid-metabolizing enzymes. Global and tissue-specific disruptions of the Nrf1 gene in mice result in embryonic lethality and spontaneous development of severe tissue defects, respectively, suggesting NRF1 plays a critical role in vivo. Mechanistically, the continuous degradation of the NRF1 protein by the proteasome is regarded as a major regulatory nexus of NRF1 activity. To develop NRF1-specific inducers that act to overcome the phenotypes related to the lack of NRF1 activity, we constructed a novel NRF1ΔC-Luc fusion protein reporter and developed cell lines that stably express the reporter in Hepa1c1c7 cells for use in high-throughput screening. In screening of a chemical library with this reporter system, we identified two hit compounds that significantly induced luciferase activity. Through an examination of a series of derivatives of one of the hit compounds, we identified T1-20, which induced a 70-fold increase in luciferase activity. T1-20 significantly increased the level of NRF1 protein in the mouse liver, indicating that the compound is also functional in vivo. Thus, these results show the successful identification of the first small chemical compounds which specifically and significantly induce NRF1.

Nrf2 induces fibroblast growth factor 21 in diabetic mice.

Transcription factor Nrf2 (nuclear factor E2-related factor 2) is a master regulator of cellular defense system against oxidative and electrophilic stresses and is negatively regulated by an adaptor protein Keap1 (Kelch-like ECH-associated protein 1). Nrf2 also plays a pivotal role in metabolic homeostasis, such as lipid metabolism and energy expenditure as well as redox homeostasis. FGF21 (fibroblast growth factor 21) is known as a key mediator of glucose and lipid metabolism. Here, we found that Nrf2 is involved in FGF21 regulation in diabetic model mice. Nrf2 induction by genetic knockdown of Keap1 increased plasma FGF21 level and hepatic Fgf21 expression in diabetic db/db mice and high-calorie-diet-induced obesity model mice. Administration of CDDO-Im (oleanolic triterpenoid 1-[2-cyano-3,12-dioxooleane-1, 9(11)-dien-28-oyl] imidazole), a potent Nrf2 inducer, up-regulated plasma FGF21 level and hepatic Fgf21 expression in db/db mice, whereas CDDO-Im did not induce FGF21 in db/db mice with Nrf2 knockout background. Furthermore, in Keap1-knockdown db/db mice, Nrf2 enhanced expression of glucose- and lipid-metabolism-related genes in adipose tissues, which improved plasma lipid profiles. These results show that Nrf2 positively regulates FGF21 expression in diabetic mice. We propose that FGF21 is a potential efficacy biomarker that mediates metabolic regulation by the Keap1-Nrf2 system.

Evaluation of Dry Eye With Videokeratographer Using a Newly Developed Indicator.

PURPOSE: To analyze the effectiveness of a newly developed indicator that quantitatively assesses disturbance in Meyer-ring (MR) images obtained via videokeratographer and assess its usefulness for the clinical evaluation of dry eye (DE). DESIGN: Cross-sectional study. METHODS: This study involved 79 eyes of 79 DE patients (10 males and 69 females; mean age: 62.7 years). After MR images were obtained via videokeratographer, the degree of blur was quantified at multiple points on the ring, with the total value across the cornea being defined as the disturbance value (DV). Correlations between total DV (TDV; the sum of DV for 5 seconds after eye opening) and 12 DE symptoms, Dry Eye-Related Quality of Life Score (DEQS), tear meniscus radius (mm), tear film (TF) lipid-layer spread grade (SG; grades 1-5, 1 = best), TF noninvasive breakup time (NIBUT, seconds), fluorescein breakup time (FBUT, seconds), corneal epithelial damage score (CEDS; maximum: 15 points), conjunctival epithelial damage score (CjEDS; maximum: 6 points), and Schirmer 1 test value (mm) were analyzed via univariate and multivariate analyses. RESULTS: No significant correlations were found between TDV and each DE symptom or DEQS, yet significant correlations were found between TDV and SG, NIBUT, FBUT, CEDS, and CjEDS (r = 0.56, -0.45, -0.45, 0.72, and 0.62, respectively, all P < .01). TDV was found to be described as 2334 + (412.1 × CEDS) - (302.0 × FBUT) (R2 = 0.593, P < .0001). CONCLUSIONS: Our newly developed indicator, DV, reflecting TF dynamics and stability and corneoconjunctival epithelial damage, may be useful for quantitatively assessing DE ocular-surface abnormalities.

Successful Detection of the Characteristics of Tear Film Breakup Appearing Immediately after Eye Opening by Videokeratography with a Newly-Developed Indicator.

Spot break (SB), a tear film breakup (TFBU) subtype seen in decreased wettability dry eye (DE), is characterized by a spot-like TFBU that appears immediately after eye opening. It is sometimes difficult to detect using currently available devices for evaluating non-invasive TFBU. The purpose of this study was to investigate the effectiveness of using a newly developed videokeratography indicator for detecting SB. The study involved 44 eyes of 44 DE patients (21 eyes with SB (SB group) and 23 eyes with random break in which fluorescein breakup time was ≤ 5 s (s) (RB group)). All eyes were examined using videokeratography, with digital Meyer-ring images being obtained. By calculation of the degree of luminance blur on the cornea in the Meyer-ring images, termed 'disturbance value' (DV), DVs at 0 s (DV(0)]), 2 s (DV(2)), and 5 s (DV(5)) after eye opening, and the changes of DV between each time, were compared between the SB and RB groups. Results: No significant differences in DV(2) and DV(5) and the rate of change between DV(2) and DV(5) were found between the two groups. However, DV(0) and rate of change between DV(0) and DV(2) in the SB group were significantly greater (p < 0.001) than those in the RB group. SB characteristics were successfully detected by videokeratography using a new videokeratography DV indicator.

Dry Eye Subtype Classification Using Videokeratography and Deep Learning.

We previously reported on 'Tear Film Oriented Diagnosis' (TFOD), a method for the dry eye (DE) subtype classification using fluorescein staining and an examination of fluorescein breakup patterns via slit-lamp biomicroscopy. Here, we report 'AI-supported TFOD', a novel non-invasive method for DE subtype classification using videokeratography (VK) and "Blur Value" (BV), a new VK indicator of the extent of blur in Meyer-ring images and deep learning (DL). This study involved 243 eyes of 243 DE cases (23 males and 220 females; mean age: 64.4 ± 13.9 (SD) years)-i.e., 31 severe aqueous-deficient DE (sADDE) cases, 73 mild-to-moderate ADDE (m/mADDE) cases, 84 decreased wettability DE (DWDE) cases, and 55 increased evaporation DE (IEDE) cases diagnosed via the fluorescein-supported TFOD pathway. For DL, a 3D convolutional neural network classification model was used (i.e., the original image and BV data of eyes kept open for 7 s were randomly divided into training data (146 cases) and the test data (97 cases), with the training data increased via data augmentation and corresponding to 2628 cases). Overall, the DE classification accuracy was 78.40%, and the accuracies for the subtypes sADDE, m/mADDE, DWDE, and IEDE were 92.3%, 79.3%, 75.8%, and 72.7%, respectively. 'AI-supported TFOD' may become a useful tool for DE subtype classification.

Characteristics of Pre-Lens Tear Film Behavior in Eyes Wearing Delefilcon A Silicone Hydrogel Water Gradient Contact Lenses.

The pre-lens tear film (PLTF) over (i) delefilcon A silicone hydrogel water gradient (WG; 33-80% from core to surface) contact lenses (CLs) (SHWG-CLs) and (ii) subjects' own non-WG soft CLs (SCLs) (SO-SCLs) was studied in 30 eyes of 30 subjects to assess the hypothesized PLTF stabilization over SHWG-CLs. In both eyes, delefilcon A SHWG-CLs (DAILIES TOTAL1®; Alcon, Fort Worth, TX, USA) or SO-SCLs were worn. After 15 min of wearing each lens, the tear meniscus radius (TMR, mm), lipid-layer interference grade (IG) and spread grade (SG), and non-invasive breakup time (NIBUT, seconds) were evaluated and compared between the SHWG-CLs and the SO-SCLs. The comparison between the SHWG-CL and SO-SCL groups (SHWG-CL and SO-SCL, mean ± SD) revealed that TMRs temporarily decreased and reached a plateau value after 15 min (0.21 ± 0.06; 0.21 ± 0.06) compared to the value prior to CL insertion (0.24 ± 0.08; 0.25 ± 0.08), with no significant difference between the two groups. The NIBUT, IG, and SG values after 15 min of wearing the CLs were (9.7 ± 3.7; 4.7 ± 4.2), (1.0 ± 0.2; 1.8 ± 1.0), and (1.1 ± 0.4; 1.9 ± 1.5), respectively, and all values were significantly better in the SHWG-CL group (p < 0.0001, p = 0.0039, and p < 0.0001, respectively). We found that compared to the SO-SCLs, the maintenance of the PLTF on the SHWG-CLs was supported by the thicker and more stable PLTF.

JAK inhibition ameliorates bone destruction by simultaneously targeting mature osteoclasts and their precursors.

BACKGROUND: Rheumatoid arthritis (RA) is characterized by chronic inflammation and resultant cartilage/bone destruction because of aberrantly activated osteoclasts. Recently, novel treatments with several Janus kinase (JAK) inhibitors have been shown to successfully ameliorate arthritis-related inflammation and bone erosion, although their mechanisms of action for limiting bone destruction remain unclear. Here, we examined the effects of a JAK inhibitor on mature osteoclasts and their precursors by intravital multiphoton imaging. METHODS: Inflammatory bone destruction was induced by local injection of lipopolysaccharides into transgenic mice carrying reporters for mature osteoclasts or their precursors. Mice were treated with the JAK inhibitor, ABT-317, which selectively inhibits the activation of JAK1, and then subjected to intravital imaging with multiphoton microscopy. We also used RNA sequencing (RNA-Seq) analysis to investigate the molecular mechanism underlying the effects of the JAK inhibitor on osteoclasts. RESULTS: The JAK inhibitor, ABT-317, suppressed bone resorption by blocking the function of mature osteoclasts and by targeting the migratory behaviors of osteoclast precursors to the bone surface. Further exhaustive RNA-Seq analysis demonstrated that Ccr1 expression on osteoclast precursors was suppressed in the JAK inhibitor-treated mice; the CCR1 antagonist, J-113863, altered the migratory behaviors of osteoclast precursors, which led to the inhibition of bone destruction under inflammatory conditions. CONCLUSIONS: This is the first study to determine the pharmacological actions by which a JAK inhibitor blocks bone destruction under inflammatory conditions; this inhibition is beneficial because of its dual effects on both mature osteoclasts and immature osteoclast precursors.

Characterizations of Three Major Cysteine Sensors of Keap1 in Stress Response.

The Keap1-Nrf2 system plays a central role in cytoprotection against electrophilic/oxidative stresses. Although Cys151, Cys273, and Cys288 of Keap1 are major sensor cysteine residues for detecting these stresses, it has not been technically feasible to evaluate the functionality of Cys273 or Cys288, since Keap1 mutants that harbor substitutions in these residues and maintain the ability to repress Nrf2 accumulation do not exist. To overcome this problem, we systematically introduced amino acid substitutions into Cys273/Cys288 and finally identified Cys273Trp and Cys288Glu mutations that do not affect Keap1's ability to repress Nrf2 accumulation. Utilizing these Keap1 mutants, we generated stable murine embryonic fibroblast (MEF) cell lines and knock-in mouse lines. Our analyses with the MEFs and peritoneal macrophages from the knock-in mice revealed that three major cysteine residues, Cys151, Cys273, and Cys288, individually and/or redundantly act as sensors. Based on the functional necessity of these three cysteine residues, we categorized chemical inducers of Nrf2 into four classes. Class I and II utilizes Cys151 and Cys288, respectively, while class III requires all three residues (Cys151/Cys273/Cys288), while class IV inducers function independently of all three of these cysteine residues. This study thus demonstrates that Keap1 utilizes multiple cysteine residues specifically and/or collaboratively as sensors for the detection of a wide range of environmental stresses.

Positron emission tomography to elucidate pharmacokinetic differences of regioisomeric retinoid x receptor agonists.

RXR partial agonist NEt-4IB (2a, 6-[ethyl-(4-isobutoxy-3-isopropylphenyl)amino]pyridine-3-carboxylic acid: EC50 = 169 nM, E max = 55%) showed a blood concentration higher than its E max after single oral administration at 30 mg/kg to mice, and repeated oral administration at 10 mg/kg/day to KK-A(y) mice afforded antitype 2 diabetes activity without the side effects caused by RXR full agonists. However, RXR full agonist NEt-3IB (1a), in which the isobutoxy and isopropyl groups of 2a are interchanged, gave a much lower blood concentration than 2a. Here we used positron emission tomography (PET) with tracers [(11)C]1a, [(11)C]2a and fluorinated derivatives [(18)F]1b, [(18)F]2b, which have longer half-lives, to examine the reason why 1a and 2a exhibited significantly different blood concentrations. As a result, the reason for the high blood concentration of 2a after oral administration was found to be linked to higher intestinal absorbability together with lower biliary excretion, compared with 1a.

RXR partial agonist produced by side chain repositioning of alkoxy RXR full agonist retains antitype 2 diabetes activity without the adverse effects.

We previously reported RXR partial agonist CBt-PMN (1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)-1H-benzotriazole-5-carboxylic acid: 5, EC50 = 143 nM, Emax = 75%), which showed a potent glucose-lowering effect without causing serious adverse effects. However, it remains important to elucidate the structural requirements for RXR efficacy and the glucose-lowering effect because RXR-permissive heterodimers such as PPAR/RXR or LXR/RXR are reported to be activated differently depending upon the chemical structure of RXR agonists. In this work, we show that an RXR partial agonist, NEt-4IB (6-[ethyl-(4-isobutoxy-3-isopropylphenyl)amino]pyridine-3-carboxylic acid: 8b, EC50 = 169 nM, Emax = 55%), can be obtained simply by repositioning the side chains (interchanging the isobutoxy and isopropoxy groups) at the hydrophobic moiety of the RXR full agonist NEt-3IB (6-[ethyl-(3-isobutoxy-4-isopropylphenyl)amino]pyridine-3-carboxylic acid: 7b, EC50 = 19 nM). NEt-4IB (8b) showed antitype 2 diabetes activity without the above side effects upon repeated oral administration to mice at 10 mg/kg/day, similarly to 5.

The Keap1-Nrf2 system prevents onset of diabetes mellitus.

Transcription factor Nrf2 (NF-E2-related factor 2) regulates a broad cytoprotective response to environmental stresses. Keap1 (Kelch-like ECH-associated protein 1) is an adaptor protein for cullin3-based ubiquitin E3 ligase and negatively regulates Nrf2. Whereas the Keap1-Nrf2 system plays important roles in oxidative stress response and metabolism, the roles Nrf2 plays in the prevention of diabetes mellitus remain elusive. Here we show that genetic activation of Nrf2 signaling by Keap1 gene hypomorphic knockdown (Keap1flox/-) markedly suppresses the onset of diabetes. When Keap1flox/- mice were crossed with diabetic db/db mice, blood glucose levels became lower through improvement of both insulin secretion and insulin resistance. Keap1flox/- also prevented high-calorie-diet-induced diabetes. Oral administration of the Nrf2 inducer CDDO-Im {oleanolic acid 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl] imidazole} also attenuated diabetes in db/db mice. Nrf2 induction altered antioxidant-, energy consumption-, and gluconeogenesis-related gene expression in metabolic tissues. Thus, the Keap1-Nrf2 system is a critical target for preventing the onset of diabetes mellitus.

The 24-bp consensus sequence responsible for regulation of the BphS1T1 two-component system in a hybrid promoter.

Rhodococcus jostii RHA1 degrades polychlorinated biphenyls (PCBs) by cometabolism with biphenyl. The bphS1T1-coding two-component system, which is composed of a sensor kinase, BphS1, and a response regulator, BphT1, activates the transcription of biphenyl/PCB degradation genes from the five promoters of bphAa, etbAa1, etbAa2, etbAd, and etbD1 in the presence of aromatics, such as biphenyl and ethylbenzene. The transcription start sites of etbAd and etbD1 were determined and the results indicated that the 18-bp consensus sequence is shared by all five promoters at the equivalent position from their transcriptional start sites. To investigate the involvement of the 18-bp consensus sequence in the regulation of BphS1T1, a hybrid promoter was constructed by connecting the 18-bp consensus sequence of bphAa promoter to a portion of the benzoate dioxygenase gene promoter, which is not under the control of BphS1T1. The ethylbenzene-dependent induction of the hybrid promoter by BphS1T1 was not observed. Recently, a 24-bp consensus sequence that included the 18-bp consensus sequence of the bphAa promoter was identified in the regions conserved among RHA1 and other rhodococcal degraders. When the 24-bp consensus sequence was employed instead, both BphS1T1-dependent basal activation and ethylbenzene-dependent induction of the hybrid promoter were observed. Mutations in the six extra residues outside the 18-bp sequence in the 24-bp consensus sequence, affected not only ethylbenzene-dependent induction but also BphS1T1-dependent basal activation. The outstanding conservation of the 24-bp consensus sequence was confirmed by multiple sequence alignment. These results indicate that the 24-bp consensus sequence is really responsible for the regulation of BphS1T1.