LKB1 Regulates Inflammation of Fibroblast-like Synoviocytes from Patients with Rheumatoid Arthritis via AMPK-Dependent SLC7A11-NOX4-ROS Signaling.

Fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA) patients have increased reactive oxygen species (ROS) levels and an impaired redox balance compared with FLS from control patients. Liver kinase B1 (LKB1) plays a key role in ROS scavenging and cellular metabolism in various cancers. Here, we aimed to determine the specific mechanism of LKB1 in RA pathogenesis. FLS were obtained from RA patients (n = 10). siRNA-induced LKB1 deficiency in RA FLS increased ROS levels via NADPH oxidase 4 (NOX4) upregulation. RA FLS migration and expression of inflammatory factors, including interleukin (IL)-1ß, IL-6, IL-8, tumor necrosis factor-alpha (TNF-α), and vascular endothelial growth factor (VEGF), were enhanced by LKB1 deficiency. LKB1-deficient RA FLS showed increased sensitivity to oxidative stress damage caused by hydrogen peroxidase exposure. siRNA-induced solute carrier family 7 member 11 (SLC7A11) deficiency in RA FLS enhanced NOX4 and ROS expression and increased cell migration. When LKB1-deficient RA FLS were stimulated with an AMP-activated protein kinase (AMPK) activator, the LKB1-inhibition-induced cell migration significantly decreased through the restoration of SLC7A11/NOX4 expression. LKB1 regulates the AMPK-mediated SLC7A11-NOX4-ROS pathway to control cell migration and inflammation. Our data indicate that LKB1 is a key regulator of redox homeostasis in RA FLS.

Immediate Vitrectomy for Acute Endophthalmitis in Patients with a Visual Acuity of Hand Motion or Better.

PURPOSE: To determine the efficacy of immediate pars plana vitrectomy as the primary treatment for acute endophthalmitis in patients with a visual acuity (VA) of hand motion (HM) or better. METHODS: A total of 149 patients who were referred to a single center for acute endophthalmitis after cataract surgery over the 13-year study period were retrospectively analyzed. Only patients presenting with a VA of at least HM were included. Patients were initially treated with either primary vitrectomy or intravitreal antibiotic injection alone, and their visual outcomes and reintervention rates after initial treatment were compared. RESULTS: There was no significant difference in the proportion of good (final VA ≥20 / 40) and poor (VA ≤ counting finger) visual outcomes between the groups. However, subgroup analysis of patients with a VA of HM (92 eyes) showed that the incidence of reintervention (14 of 72 eyes [19.4%] vs. 9 of 20 eyes [45.0%]) and poor visual outcomes (10 of 72 eyes [13.9%] vs. 8 of 20 eyes [40.0%]) were lower after prompt vitrectomy than after intravitreal antibiotic injection alone (p = 0.019 and p = 0.022, respectively). For those with a VA of at least counting finger, no significant difference was observed between the groups. CONCLUSIONS: For patients with endophthalmitis presenting with a VA of HM, performing a prompt vitrectomy reduced the incidence of reintervention and poor visual outcomes than the administration of intravitreal antibiotics alone. Our results suggest that primary vitrectomy for patients with endophthalmitis presenting with a VA of HM could be more beneficial than intravitreal antibiotic injection alone.

Hypoxia-Inducible Factor-2 Alpha Regulates the Migration of Fibroblast-like Synoviocytes via Oxidative Stress-Induced CD70 Expression in Patients with Rheumatoid Arthritis.

This study aimed to examine the role of CD70, which is highly expressed on fibroblast-like synoviocytes (FLS), in rheumatoid arthritis (RA) patients. FLS isolated from RA (n = 14) and osteoarthritis (OA, n = 4) patients were stimulated with recombinant interleukin-17 (IL-17; 5 ng/mL) and tumor necrosis factor alpha (TNF-α; 5 ng/mL) for 24 h. Expression of CD70, CD27/soluble CD27 (sCD27), and hypoxia-inducible factor-2 alpha (HIF-2α) was analyzed by RT-qPCR, flow cytometry, and ELISA assays, respectively. Reactive oxygen species (ROS) expression and cell migration were also examined. The HIF-2α inhibitor PT-2385 and CD70 inhibitor BU69 were used to specifically suppress these pathways. Stimulation with IL-17 and TNF-α significantly induced CD70 expression in RA FLS. Although the synovial fluids from patients with RA contained high levels of sCD27, surface expression of CD27, a ligand of CD70, was rarely detected in RA FLS. Cytokine-induced CD70 expression was significantly decreased following antioxidant treatment. Following HIF-2α inhibition, RA FLS had decreased expression of CD70 and ROS levels. Migration of RA FLS was also inhibited by inhibition of CD70 or HIF-2α. The surface expression of CD70 is regulated by HIF-2α and ROS levels and is a key contributor to cytokine-enhanced migration in RA FLS.

Cytoplasmic zinc promotes IL-1ß production by monocytes and macrophages through mTORC1-induced glycolysis in rheumatoid arthritis.

The essential micronutrient zinc regulates immune responses by affecting signaling pathways. In activated monocytes and macrophages, signaling networks mediate the metabolic reprogramming that meets the demands of participation in immune responses. Here, we demonstrated that cytoplasmic, bioavailable zinc was essential for promoting IL-1ß production in activated human monocytes and macrophages downstream of glycolysis induced by the kinase-containing multiprotein complex mTORC1. The concentration of cytoplasmic zinc was determined by that of extracellular zinc, which was brought into cells through the zinc-specific importer Zip8. The abundance of Zip8 was increased in monocytes from patients with rheumatoid arthritis (RA), as well as in LPS-stimulated monocytes and macrophages from healthy individuals. The mTORC1-mediated phosphorylation of S6 kinase (S6K) was enhanced by zinc-mediated inhibition of PP2A, a phosphatase that targets S6K. As a result, IL-1ß production was increased due to the activation of mTORC1-induced glycolysis. In monocytes of patients with RA, the expression of Zip8 and the zinc-inducible metallothionein isoform MT2A and the phosphorylation of S6K were enhanced compared with those of healthy controls. Furthermore, Zip8 expression correlated with more severe RA clinical parameters, suggesting that Zip8-mediated zinc influx is related to inflammatory conditions. These results provide insight into the role of cytoplasmic, bioavailable zinc in the metabolic reprogramming of human monocytes and macrophages in inflammatory responses.

CD14+ monocytes and soluble CD14 of synovial fluid are associated with osteoarthritis progression.

Objectives: This study aims to investigate the role of cluster of differentiation 14 (CD14) expressed monocytes and soluble CD14-mediated pathway in the synovial inflammation of knee osteoarthritis (OA). Patients and methods: Between May 2012 and July 2013, a total of 35 patients with knee OA (9 males, 26 females; mean age: 66.3±8.8 years; range, 52 to 79 years) were included in this cross-sectional study. Synovial fluid was obtained from knee joints of 35 OA patients. The CD14+ monocytes from synovial fluid mononuclear cells (SFMCs) were isolated using the MACS. The fibroblast-like synoviocytes (FLSs) isolated from knee joint tissue were incubated with recombinant CD14 and lipopolysaccharide (LPS) for 24 h. Cytokine profiling was performed with the Luminex® Performance Assay or magnetic bead panel kit. The expression of CD14 and CD16 was analyzed by immunohistochemistry and flow cytometry. Results: The concentration of sCD14 in synovial fluid was correlated with the interleukin-6 (IL-6) level (n=35) (ρ=0.654, p<0.001). The culture supernatants of CD14+ monocytes isolated from SFMC (n=15) showed a correlation between sCD14 and IL-6 (ρ=0.784, p=0.001), along with complement component 3 (ρ=0.756, p=0.010), IL-1b (ρ=0.652, p=0.012), and tumor necrosis factor-alpha (ρ=0.806, p=0.001). Following recombinant CD14 and LPS treatment, OA FLS synergistically enhanced the secretion of IL-6, IL-8, and matrix metalloproteinase 3 (n=3, p<0.05). In five paired-samples from identical patients, the proportions of CD14+ monocytes were significantly elevated in recurred synovial fluid compared to those in initial synovial fluid (p=0.043). When monocyte subsets were analyzed in SFMC (n=26), CD14+CD16+monocytes were abundant (p=0.019) and had higher toll-like receptor 4 expression than CD14+CD16- (p<0.001). Conclusion: Our study results suggest that CD14+ monocytes and the sCD14-mediated pathway play an important role in OA aggravation through inflammatory cytokine secretion.

The effect of nicotinamide adenine dinucleotide phosphate oxidase 4 on migration and invasion of fibroblast-like synoviocytes in rheumatoid arthritis.

BACKGROUND: Reactive oxygen species (ROS) regulate the migration and invasion of fibroblast-like synoviocytes (FLS), which are key effector cells in rheumatoid arthritis (RA) pathogenesis. Nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) induces ROS generation and, consequently, enhances cell migration. Despite the important interrelationship between RA, FLS, and ROS, the effect of NOX4 on RA pathogenesis remains unclear. METHODS: FLS isolated from RA (n = 5) and osteoarthritis (OA, n = 5) patients were stimulated with recombinant interleukin 17 (IL-17; 10 ng/ml) and tumor necrosis factor alpha (TNF-α; 10 ng/ml) for 1 h. Cell migration, invasion, adhesion molecule expression, vascular endothelial growth factor (VEGF) secretion, and ROS expression were examined. The mRNA and protein levels of NOX4 were analyzed by RT-qPCR and western blotting, respectively. The NOX4 inhibitor GLX351322 and NOX4 siRNA were used to inhibit NOX4 to probe the effect of NOX4 on these cellular processes. RESULTS: Migration of RA FLS was increased 2.48-fold after stimulation with IL-17 and TNF-α, while no difference was observed for OA FLS. ROS expression increased in parallel with invasiveness of FLS following cytokine stimulation. When the expression of NOX was examined, NOX4 was significantly increased by 9.73-fold in RA FLS compared to unstimulated FLS. Following NOX4 inhibition, cytokine-induced vascular cell adhesion molecule 1 (VCAM1), VEGF, and migration and invasion capacity of RA FLS were markedly decreased to unstimulated levels. CONCLUSION: NOX4 is a key contributor to cytokine-enhanced migration and invasion via modulation of ROS, VCAM1, and VEGF in RA FLS.

Intravitreal aflibercept for submacular hemorrhage secondary to neovascular age-related macular degeneration and polypoidal choroidal vasculopathy.

PURPOSE: To evaluate the efficacy of intravitreal aflibercept monotherapy for submacular hemorrhage secondary to neovascular age-related macular degeneration (AMD) and polypoidal choroidal vasculopathy (PCV). METHODS: This prospective, phase 4 clinical trial included 29 patients diagnosed with fovea-involving submacular hemorrhage secondary to neovascular AMD (7 patients) or PCV (22 patients). Patients were initially administered 3 monthly aflibercept injections, followed by 1 injection every 2 months. The primary outcome measure was changes in Early Treatment Diabetic Retinopathy Study (ETDRS) best-corrected visual acuity (BCVA) during the 56-week study period. Other key outcome measures were the proportion of patients who exhibited changes in BCVA of ≥ 15 ETDRS letters from baseline and changes in central retinal thickness (CRT). RESULTS: The mean size of hemorrhage was 6.2 ± 4.8-disc-diameter area. The mean BCVA significantly improved from 52.9 ± 17.8 ETDRS letters at week 0 (baseline) to 71.8 ± 16.1 letters at week 56 (P < 0.001). At week 56, improvement in BCVA of ≥ 15 letters was noted in 16 patients (55.2%), whereas none of the patients experienced a loss of ≥ 15 letters. The mean CRT significantly decreased from 498.9 ± 194.2 µm at week 0 to 248.3 ± 45.0 µm at week 56 (P < 0.001). During the study period, retinal break developed in one patient. CONCLUSIONS: Intravitreal aflibercept administered every 2 months after the 3 initial monthly doses was found to be an effective and safe treatment method for submacular hemorrhage secondary to neovascular AMD.

Activated Platelets Convert CD14+CD16- Into CD14+CD16+ Monocytes With Enhanced FcγR-Mediated Phagocytosis and Skewed M2 Polarization.

Monocytes are important cellular effectors of innate immune defense. Human monocytes are heterogeneous and can be classified into three distinct subsets based on CD14 and CD16 expression. The expansion of intermediate CD14+CD16+ monocytes has been reported in chronic inflammatory diseases including rheumatoid arthritis (RA). However, the mechanism underlying induction of CD16 and its role in monocytes remains poorly understood. Here, we demonstrate that activated platelets are important for induction of CD16 on classical CD14+CD16- monocytes by soluble factors such as cytokines. Cytokine neutralization and signaling inhibition assays reveal that sequential involvement of platelet-derived TGF-ß and monocyte-derived IL-6 contribute to CD16 induction on CD14+CD16- monocytes. Activated platelet-induced CD16 on monocytes participates in antibody-dependent cellular phagocytosis (ADCP) and its level is positively correlated with phagocytic activity. CD14+CD16- monocytes treated with activated platelets preferentially differentiate into M2 macrophages, likely the M2c subset expressing CD163 and MerTK. Lastly, the amount of sCD62P, a marker of activated platelets, is significantly elevated in plasma of RA patients and positively correlates with clinical parameters of RA. Our findings suggest an important role of activated platelets in modulating phenotypical and functional features of human monocytes. This knowledge increases understanding of the immunological role of CD14+CD16+ cells in chronic inflammatory diseases.

Development of conditional cell lysis mutants of Saccharomyces cerevisiae as production hosts by modulating OCH1 and CHS3 expression.

The traditional yeast Saccharomyces cerevisiae has been widely used as a host for the production of recombinant proteins and metabolites with industrial potential. However, its thick and rigid cell wall presents problems for the effective recovery of products. In this study, we modulated the expression of ScOCH1, encoding the α-1,6-mannosyltransferase responsible for outer chain biosynthesis of N-glycans, and ScCHS3, encoding the chitin synthase III required for synthesis of the majority of cell wall chitin, by exploiting the repressible ScMET3 promoter. The conditional single mutants PMET3-OCH1 and PMET3-CHS3 and the double mutant PMET3-OCH1/PMET3-CHS3 showed comparable growth to the wild-type strain under normal conditions but exhibited increased sensitivity to temperature and cell wall-disturbing agents in the presence of methionine. Such conditional growth defects were fully recovered by supplementation with 1 M sorbitol. The osmotic lysis of the conditional mutants cultivated with methionine was sufficient to release the intracellularly expressed recombinant protein, nodavirus capsid protein, with up to 60% efficiency, compared to lysis by glass bead breakage. These mutant strains also showed approximately three-fold-enhanced secretion of a recombinant extracellular glycoprotein, Saccharomycopsis fibuligera ß-glucosidase, with markedly reduced hypermannosylation, particularly in the PMET3-OCH1 mutants. Furthermore, a substantial increase of extracellular glutathione production, up to four-fold, was achieved with the conditional mutant yeast cells. Together, our data support that the conditional cell wall lysis mutants constructed based on the modulation of ScOCH1 and ScCHS3 expression would likely be useful hosts for the improved recovery of proteins and metabolites with industrial application.

Clinical and Laboratory Characteristics and Mortality in Korean Patients with Systemic Sclerosis: A Nationwide Multicenter Retrospective Cohort Study.

OBJECTIVE: We aimed to investigate demographic and clinical features and predictors of mortality in Korean patients with systemic sclerosis (SSc). METHODS: We performed a retrospective multicenter medical chart review in Korean patients diagnosed with SSc from 1986 to 2016 at 11 university hospitals representing each geographic area of Korea. SSc patients were defined according to the American College of Rheumatology preliminary classification criteria and subtyped as limited cutaneous (lcSSc) or diffuse cutaneous (dcSSc) SSc. RESULTS: We enrolled 751 patients (female, 86.7%; mean age at diagnosis, 48.9 yrs). The most common organ involvement was interstitial lung disease (52.7%), followed by gastroesophageal reflux disease (32.9%) and pulmonary arterial hypertension (13.6%). Patients with lcSSc were more common than those with dcSSc (64.8 vs 35.2%), whereas anti-Scl-70 and anticentromere antibody positivity were identified in 302 (42.5%) and 175 (25.5%) patients, respectively. In the 46 (6.1%) patients who developed a malignancy, lung cancer (23.9%) was the most common diagnosis, followed by gastric (13%) and breast cancer (13%). During the study period, 57 (7.6%) patients died, and the 5- and 10-year survival rates were 94% and 87%, respectively. Increased age at diagnosis, cardiovascular involvement, and anti-Scl-70 antibody positivity were significant predictors of death. CONCLUSION: Clinical manifestations and survival rates in Korean SSc patients are similar to those of other populations. However, the prevalence of anti-Scl-70 antibody is higher in Korean SSc patients compared with whites, while the prevalence of anticentromere antibody is lower.

A Novel Mitochondrial Serine O-Acetyltransferase, OpSAT1, Plays a Critical Role in Sulfur Metabolism in the Thermotolerant Methylotrophic Yeast Ogataea parapolymorpha.

In most bacteria and plants, direct biosynthesis of cysteine from sulfide via O-acetylserine (OAS) is essential to produce sulfur amino acids from inorganic sulfur. Here, we report the functional analysis of a novel mitochondrial serine O-acetyltransferase (SAT), responsible for converting serine into OAS, in the thermotolerant methylotrophic yeast Ogataea parapolymorpha. Domain analysis of O. parapolymorpha SAT (OpSat1p) and other fungal SATs revealed that these proteins possess a mitochondrial targeting sequence (MTS) at the N-terminus and an α/ß hydrolase 1 domain at the C-terminal region, which is quite different from the classical SATs of bacteria and plants. Noticeably, OpSat1p is functionally interchangeable with Escherichia coli SAT, CysE, despite that it displays much less enzymatic activity, with marginal feedback inhibition by cysteine, compared to CysE. The Opsat1Δ-null mutant showed remarkably reduced intracellular levels of cysteine and glutathione, implying OAS generation defect. The MTS of OpSat1p directs the mitochondrial targeting of a reporter protein, thus, supporting the localization of OpSat1p in the mitochondria. Intriguingly, the OpSat1p variant lacking MTS restores the OAS auxotrophy, but not the cysteine auxotrophy of the Opsat1Δ mutant strain. This is the first study on a mitochondrial SAT with critical function in sulfur assimilatory metabolism in fungal species.

TNFα and IL-1ß in the synovial fluid facilitate mucosal-associated invariant T (MAIT) cell migration.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that primarily affect the joints and inflammatory cell migration into inflamed articular sites contribute to this disease. Among the inflammatory cells, human mucosal-associated invariant T (MAIT) cells were recently recognized as critical cellular component with a pathological role in RA. However, their migratory characteristics are poorly understood. The aim of this study was to determine whether human MAIT cells preferentially traffick to inflamed synovial sites in rheumatoid arthritis patients and to elucidate the underlying mechanism. First, we found that TNFα and IL-1ß were elevated in synovial fluid (SF) of RA patients, which resulted in increased expression of E-selectin, ICAM-1 and V-CAM-1 on blood vessel endothelial cells. To understand whether TNFα and IL-1ß in the SF facilitated MAIT cell migration, we analyzed CD161+ TCRα7.2+ MAIT and other CD3+ T cells for differences in migratory capacity. Collectively, our results demonstrate that TNFα and IL-1ß in the SF facilitated MAIT cell migration dependent on expression of selectin ligand, sialyl LewisX (sLeX) and CCR6 on MAIT cells. We also showed that MAIT cells in the SF from RA patients equipped upregulated sLeX compared to the peripheral blood of RA patients and healthy persons, which suggest that TNFα and IL-1ß mediated expression of E-selectin preferentially attract sLeX mediated MAIT cell migration into the SF of RA patients.

Natural Short-term Course of Recurrent Macular Edema Following Intravitreal Bevacizumab Therapy in Branch Retinal Vein Occlusion.

PURPOSE: To evaluate the 3-month natural course of recurrent macular edema secondary to branch retinal vein occlusion (BRVO) treated with intravitreal bevacizumab. METHODS: This retrospective, observational study included 36 eyes with macular edema secondary to BRVO. All patients were initially treated with intravitreal bevacizumab for macular edema. Recurrence of macular edema was either not treated (untreated group) or treated with a single intravitreal bevacizumab injection (treated group). Central foveal thickness (CFT) and best-corrected visual acuity (BCVA) were compared at the time of recurrence and 3 months later. RESULTS: At the time of recurrence, the mean CFT and logarithm of the minimum angle of resolution BCVA were 484.9 ± 124.1 µm and 0.58 ± 0.26 in the untreated group (n = 19) and 456.3 ± 126.8 µm and 0.51 ± 0.21 in the treated group (n = 17), respectively. Three months later, the mean CFT and BCVA had changed to 493.7 ± 123.9 µm and 0.62 ± 0.29 in the untreated group and 294.7 ± 104.4 µm and 0.40 ± 0.24 in the treated group, respectively. The differences in CFT and BCVA between the two time points were not significant in the untreated group (p = 0.106 and p = 0.687, respectively), whereas statistically significant differences were noted in the treated group (p = 0.002 and p < 0.001, respectively). CONCLUSIONS: Unlike the first episode of macular edema following BRVO, recurrent macular edema following intravitreal bevacizumab therapy did not spontaneously resolve, suggesting the potential benefit of prompt treatment.

Association of Single Nucleotide Polymorphisms of PADI4 and HLA-DRB1 Alleles with Susceptibility to Rheumatoid Arthritis-Related Lung Diseases.

OBJECTIVE: Lung diseases (LD) are common extra-articular manifestations in rheumatoid arthritis (RA). However, little is known about factors associated with susceptibility to rheumatoid arthritis-related lung diseases (RA-LD). The aim of the present study was to investigate whether the single nucleotide polymorphisms (SNPs) of PADI4 and HLA-DRB1 alleles were associated with RA-LD. METHODS: Blood samples and clinical data were collected from 116 consecutive RA patients who satisfied the 1987 American College of Rheumatology classification criteria. RA-LD was diagnosed using high-resolution computed tomography of the chest. All patients were genotyped for SNPs of PADI4 and HLA-DRB1 alleles and analyzed for full amino acid sequence of the HLA protein corresponding to a 4-digit HLA typing. Data were analyzed by independent t test (or Mann-Whitney test) for continuous variables, Chi-square test (or Fisher's exact test) and trend test for categorical variables, and logistic regression analysis. RESULTS: Ninety-four (81.0 %) RA patients had LD, of which eight (6.9 %) had interstitial lung disease (ILD) and 92 (79.3 %) had airway abnormalities in which 64 (55.2 %) showed bronchiectasis and 47 (40.5 %) revealed bronchial wall thickening. The recessive genotype of padi4_92 was susceptible to airway abnormalities (OR = 2.22, 95 % CI = 1.05-4.49, p = 0.034). Tryptophan at position 9 of HLA-DRB1 sequence was associated with the susceptibility to RA-ILD (OR = 22.89, 95 % CI = 1.20-432.56, p = 0.037). CONCLUSION: PADI4 polymorphisms and HLA-DRB1 alleles could attribute differently to the development of airway abnormalities and ILD, respectively, in RA.

Fellow-eye neovascularization in unilateral retinal angiomatous proliferation in a Korean population.

PURPOSE: To evaluate the incidence of fellow-eye neovascularization in retinal angiomatous proliferation (RAP) in a Korean population and associated risk factors. METHOD: This retrospective, observational study included 81 eyes (81 patients) diagnosed with unilateral RAP who were followed up for ≥12 months. The RAP diagnosis was based on an indocyanine green angiography reviewed by two retinal specialists. In fellow eyes experiencing neovascularization, the period between RAP diagnosis and neovascularization was compared between eyes with and without reticular pseudodrusen. RESULTS: The mean age (±standard deviation) of the 81 patients was 74.7 ± 6.1 years. The mean follow-up period was 27.8 ± 12.4 months. Fellow-eye neovascularization was noted in 31 patients (38.3%), and 24 of these (77.4%) was a RAP subtype. Fellow-eye involvement was noted within 12 months in 13 eyes (16.0%). The period between diagnosis and fellow-eye neovascularization was significantly shorter in eyes with reticular pseudodrusen (mean 13.8 ± 8.5 months) than in eyes without reticular pseudodrusen (mean 21.2 ± 9.1 months; p = 0.031). CONCLUSION: In our cohort of unilateral RAP patients, fellow-eye neovascularization was noted in 38.3% in 27.8 months. The presence of reticular pseudodrusen in the fellow eye was closely associated with relatively early onset.

Use of the cysteine-repressible HpMET3 promoter as a novel tool to regulate gene expression in Hansenula polymorpha.

OBJECTIVES: The promoter of HpMET3, encoding an ATP sulfurylase, was evaluated for its potential as a repressible promoter to downregulate the expression of target genes in the thermotolerant, methylotrophic yeast Hansenula polymorpha. RESULTS: The expression of lacZ under the control of the 0.6 kb HpMET3 promoter was efficiently downregulated by cysteine, but not by methionine or sulfate. The HpMET3 promoter was used to generate a conditional mutant of the HpPMT2 gene encoding an O-mannosyltransferase, which is involved in post-translational protein modification. The addition of 0.5 mM cysteine adversely affected the growth of the conditional HpMET3(p)-Hppmt2 mutant strain by downregulating transcription of HpPMT2 to approx. 40 % of the normal levels, indicating that the HpPMT2 gene is essential for cell viability. However, the HpMET3 promoter was neither induced nor repressed in the heterologous host Saccharomyces cerevisiae. CONCLUSION: Our results reveal that the cysteine-repressible HpMET3 promoter is a useful tool that downregulates the expression of various genes in H. polymorpha.

Bucillamine prevents cisplatin-induced ototoxicity through induction of glutathione and antioxidant genes.

Bucillamine is used for the treatment of rheumatoid arthritis. This study investigated the protective effects of bucillamine against cisplatin-induced damage in auditory cells, the organ of Corti from postnatal rats (P2) and adult Balb/C mice. Cisplatin increases the catalytic activity of caspase-3 and caspase-8 proteases and the production of free radicals, which were significantly suppressed by pretreatment with bucillamine. Bucillamine induces the intranuclear translocation of Nrf2 and thereby increases the expression of γ-glutamylcysteine synthetase (γ-GCS) and glutathione synthetase (GSS), which further induces intracellular antioxidant glutathione (GSH), heme oxygenase 1 (HO-1) and superoxide dismutase 2 (SOD2). However, knockdown studies of HO-1 and SOD2 suggest that the protective effect of bucillamine against cisplatin is independent of the enzymatic activity of HO-1 and SOD. Furthermore, pretreatment with bucillamine protects sensory hair cells on organ of Corti explants from cisplatin-induced cytotoxicity concomitantly with inhibition of caspase-3 activation. The auditory-brainstem-evoked response of cisplatin-injected mice shows marked increases in hearing threshold shifts, which was markedly suppressed by pretreatment with bucillamine in vivo. Taken together, bucillamine protects sensory hair cells from cisplatin through a scavenging effect on itself, as well as the induction of intracellular GSH.

Intravitreal anti-vascular endothelial growth factor for typical exudative age-related macular degeneration in eyes with good baseline visual acuity.

PURPOSE: To investigate 12-month treatment outcomes of anti-vascular endothelial growth factor therapy in eyes with typical exudative age-related macular degeneration with good baseline visual acuity. METHODS: This retrospective observational case series included 18 eyes (18 patients) with typical exudative age-related macular degeneration with a baseline best-corrected visual acuity of 20 / 25 or better. Patients were treated with anti-vascular endothelial growth factor monotherapy during the 12-month follow-up period. Baseline visual acuity and central foveal thickness were compared to the values at 12 months. RESULTS: Patients received an average of 4.4 ± 1.3 intravitreal anti-vascular endothelial growth factor injections. The mean logarithm of minimum angle of resolution visual acuity was 0.08 ± 0.04, 0.08 ± 0.07, 0.12 ± 0.09, and 0.16 ± 0.11 at baseline, three months, six months, and 12 months, respectively. Visual acuity at 12 months was significantly worse than the baseline value at diagnosis (p = 0.017), and the mean central foveal thickness at the defined time points was 270.2 ± 55.6, 204.4 ± 25.4, 230.1 ± 56.3, and 216.8 ± 48.7 µm, respectively. The central foveal thickness at 12 months was significantly less than the baseline value at diagnosis (p = 0.042). CONCLUSIONS: Deterioration in visual acuity was noted in eyes with typical exudative age-related macular degeneration with good baseline visual acuity, suggesting the need for close patient monitoring and prompt treatment even in patients with good baseline visual acuity.

Surgical outcomes of 23-gauge vitrectomy for the management of lens fragments dropped into the vitreous cavity during cataract surgery.

PURPOSE: To assess the clinical features and surgical outcomes of 23-Gauge (G) vitrectomy for lens fragments dropped into the vitreous during cataract surgery. METHODS: A retrospective, non-comparative, interventional case series at a single medical center. The medical records of 45 eyes from 45 consecutive patients who were referred to our hospital for surgical retrieval of phacoemulsification dropped lens fragments and who underwent 23-G vitrectomy were retrospectively reviewed. Data pertaining to patient demographics, pre- and post-operative Snellen visual acuity, and postoperative complications were recorded. Factors associated with dropped lens fragments were also examined. RESULTS: Mean patient age was 68.18 ± 11.47 years. The preoperative and postoperative mean logarithm of minimum angle of resolution (logMAR) visual acuity was 1.91 ± 0.59 (Snellen equivalent 0.06 ± 0.15) and 0.42 ± 0.51 (Snellen equivalent 0.54 ± 0.31), respectively. Forty-two eyes (93.3%) had dislocated lens fragments <50% of the total lens size. Two eyes (4.4%) had a large and hard lens nucleus, which necessitated the use of a 20-G fragmatome to efficiently and completely remove the lens material. At the final examination, 30 eyes (66.6%) had a visual acuity better than 20/40. Post-vitrectomy complications included elevated IOP for at least 3 months (n = 5 eyes, 11.1%), intraocular lens dislocation (n = 2 eyes, 4.4%), and cystoid macular edema (n = 1 eye, 2.2%). No cases of postoperative endophthalmitis or retinal detachment were observed. CONCLUSIONS: A 23-G vitrectomy is safe and efficient for the surgical management of dropped lens fragments following cataract surgery.

Functional phenotype of synovial monocytes modulating inflammatory T-cell responses in rheumatoid arthritis (RA).

Monocytes function as crucial innate effectors in the pathogenesis of chronic inflammatory diseases, including autoimmunity, as well as in the inflammatory response against infectious pathogens. Human monocytes are heterogeneous and can be classified into three distinct subsets based on CD14 and CD16 expression. Although accumulating evidence suggests distinct functions of monocyte subsets in inflammatory conditions, their pathogenic roles in autoimmune diseases remain unclear. Thus, we investigated the phenotypic and functional characteristics of monocytes derived from synovial fluid and peripheral blood in RA patients in order to explore the pathogenic roles of these cells. In RA patients, CD14+CD16+, but not CD14dimCD16+, monocytes are predominantly expanded in synovial fluid and, to a lesser degree, in peripheral blood. Expression of co-signaling molecules of the B7 family, specifically CD80 and CD276, was markedly elevated on synovial monocytes, while peripheral monocytes of RA and healthy controls did not express these molecules without stimulation. To explore how synovial monocytes might gain these unique properties in the inflammatory milieu of the synovial fluid, peripheral monocytes were exposed to various stimuli. CD16 expression on CD14+ monocytes was clearly induced by TGF-ß, although co-treatment with IL-1ß, TNF-α, or IL-6 did not result in any additive effects. In contrast, TLR stimulation with LPS or zymosan significantly downregulated CD16 expression such that the CD14+CD16+ monocyte subset could not be identified. Furthermore, treatment of monocytes with IFN-γ resulted in the induction of CD80 and HLA-DR expression even in the presence of TGF-ß. An in vitro assay clearly showed that synovial monocytes possess the unique capability to promote Th1 as well as Th17 responses of autologous peripheral CD4 memory T cells. Our findings suggest that the cytokine milieu of the synovial fluid shapes the unique features of synovial monocytes as well as their cardinal role in shaping inflammatory T-cell responses in RA.