Amplified fluorogenic immunoassay for early diagnosis and monitoring of Alzheimer's disease from tear fluid.

Accurate diagnosis of Alzheimer's disease (AD) in its earliest stage can prevent the disease and delay the symptoms. Therefore, more sensitive, non-invasive, and simple screening tools are required for the early diagnosis and monitoring of AD. Here, we design a self-assembled nanoparticle-mediated amplified fluorogenic immunoassay (SNAFIA) consisting of magnetic and fluorophore-loaded polymeric nanoparticles. Using a discovery cohort of 21 subjects, proteomic analysis identifies adenylyl cyclase-associated protein 1 (CAP1) as a potential tear biomarker. The SNAFIA demonstrates a low detection limit (236 aM), good reliability (R2 = 0.991), and a wide analytical range (0.320-1000 fM) for CAP1 in tear fluid. Crucially, in the verification phase with 39 subjects, SNAFIA discriminates AD patients from healthy controls with 90% sensitivity and 100% specificity in under an hour. Utilizing tear fluid as a liquid biopsy, SNAFIA could potentially aid in long-term care planning, improve clinical trial efficiency, and accelerate therapeutic development for AD.

Integrated Analysis of Transcriptome and Proteome of the Human Cornea and Aqueous Humor Reveal Novel Biomarkers for Corneal Endothelial Cell Dysfunction.

Earlier studies have reported that elevated protein levels in the aqueous humor (AH) are associated with corneal endothelial cell dysfunction (CECD), but the details of the underlying mechanism as well as specific biomarkers for CECD remain elusive. In the present study, we aimed to identify protein markers in AH directly associated with changes to corneal endothelial cells (CECs), as AH can be easily obtained for analysis. We carried out an in-depth proteomic analysis of patient-derived AH as well as transcriptomic analysis of CECs from the same patients with bullous keratopathy (BK) resulting from CECD. We first determined differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) from CECs and AH in CECD, respectively. By combining transcriptomic and proteomic analyses, 13 shared upregulated markers and 22 shared downregulated markers were observed between DEGs and DEPs. Among these 35 candidates from biomarker profiling, three upregulated markers were finally verified via data-independent acquisition (DIA) proteomic analysis using additional individual AH samples, namely metallopeptidase inhibitor 1 (TIMP1), Fc fragment of IgG binding protein (FCGBP), and angiopoietin-related protein 7 (ANGPTL7). Furthermore, we confirmed these AH biomarkers for CECD using individual immunoassay validation. Conclusively, our findings may provide valuable insights into the disease process and identify biofluid markers for the assessment of CEC function during BK development.

The Protective Role of Exosome-Derived MicroRNAs and Proteins from Human Breast Milk against Infectious Agents.

Human breast milk (HBM)-derived exosomes contain various biological and immunological components. However, comprehensive immune-related and antimicrobial factor analysis requires transcriptomic, proteomic, and multiple databases for functional analyses, and has yet to be conducted. Therefore, we isolated and confirmed HBM-derived exosomes by detecting specific markers and examining their morphology using western blot and transmission electron microscopy. Moreover, we implemented small RNA sequencing and liquid chromatography-mass spectrometry to investigate substances within the HBM-derived exosomes and their roles in combating pathogenic effects, identifying 208 miRNAs and 377 proteins associated with immunological pathways and diseases. Integrated omics analyses identified a connection between the exosomal substances and microbial infections. In addition, gene ontology and the Kyoto Encyclopedia of Genes and Genomes pathway analyses demonstrated that HBM-derived exosomal miRNA and proteins influence immune-related functions and pathogenic infections. Finally, protein-protein interaction analysis identified three primary proteins (ICAM1, TLR2, and FN1) associated with microbial infections mediating pro-inflammation, controlling infection, and facilitating microbial elimination. Our findings determine that HBM-derived exosomes modulate the immune system and could offer therapeutic strategies for regulating pathogenic microbial infection.

Retinal Proteome Analysis Reveals a Region-Specific Change in the Rabbit Myopia Model.

Uncovering region-specific changes in the myopic retina can provide clues to the pathogenesis of myopia progression. After imposing form deprivation myopia in the right eye of 6-week-old rabbits, we investigated the proteome profile of each retinal region (central, mid-periphery, and far-periphery retina), using accurate high-resolution mass spectrometry. Protein expression was analyzed using gene ontology and network analysis compared with that of the control, the left eyes. Among 2065 proteins detected from whole retinal samples, 249 differentially expressed proteins (DEPs) were identified: 164 DEPs in the far-periphery, 39 in the mid-periphery, and 83 in the central retina. In network analysis, the far-periphery retina showed the most significant connectivity between DEPs. The regulation of coagulation was the most significant biological process in upregulated DEPs in the far-periphery retina. Proteasome was the most significant Kyoto Encyclopedia of Genes and Genomes pathway in downregulated DEPs in the central retina. Antithrombin-III, fibrinogen gamma chain, and fibrinogen beta chain were identified as hub proteins for myopia progression, which were upregulated in the far-periphery retina. Proteomic analysis in this study suggested that oxidative stress can be the primary pathogenesis of myopia progression and that the far-periphery retina plays a role as the key responder.

Real-time and label-free biosensing using moiré pattern generated by bioresponsive hydrogel.

Bioresponsive hydrogels are smart materials that respond to various external stimuli and exhibit great potential as biosensors owing to their capability of real-time and label-free detection. Here, we propose a sensing platform based on bioresponsive hydrogels, employing the concept of moiré patterns. Two sets of line patterns with different pitch sizes are prepared; a hydrogel grating whose pitch size changes according to external stimuli and a reference grating with constant pitch size. The volume changes of the hydrogel caused by external stimuli changes the pitch size of the hydrogel grating, and subsequently, the pitch sizes of the moiré patterns (moiré signal), whose values can be obtained in a real-time and label-free manner through customized moiré microscopy and signal processing. After confirming that the pH-induced swelling of hydrogel could be monitored using moiré patterns, we performed moiré pattern-based detection of specific proteins using protein-responsive hydrogel that underwent shrinking via interaction with target proteins. Brain-derived neurotrophic factor and platelet-derived growth factor were selected as the model proteins, and our proposed system successfully detected both proteins at nanomolar levels. In both cases, the pitch size change of hydrogel grating was monitored much more sensitively using moiré patterns than through direct measurements. The changes in the moiré signals caused by target proteins were detected in ex-vivo environments using a custom-made intraocular lens incorporating the hydrogel grating, demonstrating the capability of the proposed system to detect various markers in intraocular aqueous humor, when implanted in the eye.

Long-Term Follow-Up of Corneal Endothelial Cell Changes After Iris-Fixated Phakic Intraocular Lens Explantation.

PURPOSE: The purpose of this study was to evaluate long-term corneal endothelial cell changes and visual outcomes after iris-fixated phakic intraocular lens (pIOL) explantation in patients with endothelial damage and to investigate potential predictors of endothelial injury. METHODS: Consecutive patients undergoing pIOL explantation with corneal endothelial cell density (ECD) <2000 cells/mm 2 at the time of the procedure were retrospectively reviewed in a single tertiary center. All patients were treated between April 2016 and October 2020 at a high-volume referral-based tertiary hospital. The primary outcome was the change in corneal endothelial parameters, including ECD, over long-term follow-up. Secondary outcomes included changes in corrected distance visual acuity and analysis of prognostic factors. RESULTS: This study included 44 eyes from 28 patients with an average age of 42.5 ± 7.8 years (range: 27-63). Mean ECD before explantation was 1375.4 ± 468.2 cells/mm 2 (range: 622-1996), and the average duration of follow-up after explantation was 20.5 months (6-58.2). Two years after explantation, ECD had significantly decreased by more than 25% to 1019.6 ± 368.6 (608-1689; P < 0.01). However, there was no significant change in corrected distance visual acuity (20/23-20/22, P = 0.59). Longer operation duration (odds ratio, 1.004; P = 0.04) was the only significant factor weakly associated with postoperative decreases in ECD. CONCLUSIONS: Although ECD continuously decreased despite pIOL explantation on a long-term follow-up, patients did not experience any discomfort or showed decreases in visual acuity. Therefore, a careful follow-up is required for possible endothelial injury after pIOL explantation.

Comparison of Corneal Higher-Order Aberrations Following Topography-Guided LASIK and SMILE for Myopic Correction: A Propensity Score Matching Analysis.

Ocular aberrations, particularly corneal higher-order aberrations (HOAs), which impair visual quality, should be minimized or corrected during any laser vision correction. We compared changes in visual outcomes, including HOAs, in patients who underwent Topography-Guided laser-assisted in situ keratomileusis (TG-LASIK) or small-incision lenticule extraction (SMILE) after propensity score matching (PSM) to reduce selection bias. Of 2749 patients who underwent SMILE or TG-LASIK for myopia, 152 eyes underwent complete ophthalmic examination preoperatively and over six months postoperatively. Visual outcomes were comparatively analyzed after PSM. As a result, 45 eyes were included in each group after PSM. There was a comparable improvement in visual acuity (VA) and refractive parameters postoperatively, with no difference between the two PSM-groups. However, 6.6% in the SMILE PSM-group lost two or more lines of Snellen VA at the six-month follow-up, while none in the TG-LASIK PSM-group did. Specifically, the SMILE PSM-group showed a significant increase in corneal HOAs, including spherical aberration, coma, and total HOAs (0.0736 ± 0.162 µm; 0.181 ± 0.233 µm; and 0.151 ± 0.178 µm, respectively), whereas TG-LASIK PSM-group did not. Furthermore, SMILE PSM-group had greater postoperative corneal HOAs than those in TG-LASIK PSM-group. Collectively, TG-LASIK induces fewer corneal HOAs even after facilitating between-group comparability using PSM analysis. TG-LASIK provides better visual quality than SMILE for myopia.

De Novo L509P Mutation of the TGFBI Gene Associated with Slit-Lamp Findings of Lattice Corneal Dystrophy Type IIIA.

Background: Mutations of the transforming growth factor-ß-induced (TGFBI) gene produce various types of corneal dystrophy. Here, we report a novel de novo L509P mutation not located in a known hot spot of the transforming growth factor-ß-induced (TGFBI) gene and its clinical phenotype, which resembles that of lattice corneal dystrophy type IIIA (LCD IIIA). Case presentation: A 36-year-old man (proband) visited our clinic due to decreased visual acuity with intermittent ocular irritation in conjunction with painful recurrent erosions in both eyes for 10 years. Molecular genetic analyses revealed a TGFBI L509P mutation (c.1526T>C) in the proband and one of his sons. Interestingly, neither TGFBI mutations nor corneal abnormalities were detected in either of the proband's biological parents, indicating the occurrence of a de novo L509P mutation. Clinical examinations, including slit-lamp retro-illumination and Fourier-domain anterior segment optical coherence tomography (FD-OCT), revealed gray deposits in the anterior stroma and deeper refractile lines extending from limbus to limbus in both corneas of the proband, consistent with a diagnosis of LCD IIIA. Superficial diffuse haze and surface irregularity were observed in conjunction with corneal erosions and visual impairment, necessitating phototherapeutic keratectomy (PTK). A 60 µm PTK of the Bowman layer and anterior stroma of the proband's left eye was performed following the removal of the epithelium in order to remove superficial corneal opacities. His BCVA improved from 20/400 to 20/50 at postoperative week 8 and was maintained for 45 months. Pinhole-corrected VA was 20/20 at the last visit, and corneal opacities had not recurred. Conclusions: An inheritable de novo mutation of L509P in the TGFBI gene can produce severe LCD IIIA, which can be successfully treated with OCT-guided PRK.

Correlation Between Salivary Microbiome of Parotid Glands and Clinical Features in Primary Sjögren's Syndrome and Non-Sjögren's Sicca Subjects.

Recent studies have demonstrated that the oral microbiome in patients with Sjögren's syndrome (SS) is significantly different from that in healthy individuals. However, the potential role of the oral microbiome in SS pathogenesis has not been determined. In this study, stimulated intraductal saliva samples were collected from the parotid glands (PGs) of 23 SS and nine non-SS subjects through PG lavage and subjected to 16S ribosomal RNA amplicon sequencing. The correlation between the oral microbiome and clinical features, such as biological markers, clinical manifestations, and functional and radiological characteristics was investigated. The salivary microbial composition was examined using bioinformatic analysis to identify potential diagnostic biomarkers for SS. Oral microbial composition was significantly different between the anti-SSA-positive and SSA-negative groups. The microbial diversity in SS subjects was lower than that in non-SS sicca subjects. Furthermore, SS subjects with sialectasis exhibited decreased microbial diversity and Firmicutes abundance. The abundance of Bacteroidetes was positively correlated with the salivary flow rate. Bioinformatics analysis revealed several potential microbial biomarkers for SS at the genus level, such as decreased Lactobacillus abundance or increased Streptococcus abundance. These results suggest that microbiota composition is correlated with the clinical features of SS, especially the ductal structures and salivary flow, and that the oral microbiome is a potential diagnostic biomarker for SS.

Tissue extracellular matrix hydrogels as alternatives to Matrigel for culturing gastrointestinal organoids.

Matrigel, a mouse tumor extracellular matrix protein mixture, is an indispensable component of most organoid tissue culture. However, it has limited the utility of organoids for drug development and regenerative medicine due to its tumor-derived origin, batch-to-batch variation, high cost, and safety issues. Here, we demonstrate that gastrointestinal tissue-derived extracellular matrix hydrogels are suitable substitutes for Matrigel in gastrointestinal organoid culture. We found that the development and function of gastric or intestinal organoids grown in tissue extracellular matrix hydrogels are comparable or often superior to those in Matrigel. In addition, gastrointestinal extracellular matrix hydrogels enabled long-term subculture and transplantation of organoids by providing gastrointestinal tissue-mimetic microenvironments. Tissue-specific and age-related extracellular matrix profiles that affect organoid development were also elucidated through proteomic analysis. Together, our results suggest that extracellular matrix hydrogels derived from decellularized gastrointestinal tissues are effective alternatives to the current gold standard, Matrigel, and produce organoids suitable for gastrointestinal disease modeling, drug development, and tissue regeneration.

Evaluation of Meibum Lipid Composition According to Tear Interferometric Patterns: RRH: Meibum Composition According to Interferometric Patterns.

PURPOSE: To determine the relationship between tear film interferometric patterns and properties of lipid, including rheological properties. DESIGN: Prospective, cross-sectional laboratory investigation. METHOD: This study included 105 subjects (94 dry eye patients and 11 normal participants). The subjects were divided into 3 categories (group 1, normal; group 2, thin; and group 3, irregular) according to interferometric patterns. According to tear interferometric patterns, ultra-performance liquid chromatography (LC) quadrupole-linear ion trap/mass spectrometry (MS)-based analysis was used to investigate lipid profiling of meibum. Rheological properties were examined by using a Langmuir-Blodgett trough with saline solution. RESULTS: Normal subjects showed Pearl-like patterns, and dry eye patients showed either irregular or thin patterns. Group 2 tended to be the evaporative type, and group 3 tended to be the aqueous-deficient type. Lipid profiling using LC-MS identified 280 lipid species of 25 lipid classes. In the meibum of the patient groups, the content of cholesteryl esters and nonpolar lipids was lower than that in the normal group. However, the content of polar lipids such as sphingolipids and phospholipids in the patient groups was higher than that in the normal group. Rheological properties showed that the lift-off areas were comparable among the 3 groups and the surface tension was the highest in group 1, followed by group 3 and group 2. CONCLUSIONS: The findings of this study suggest that tear interferometric patterns are associated with lipid profiling of meibum and its rheological properties. These results may contribute toward the development of new treatment modalities.

How Long to Continue Eyelid Hygiene to Treat Meibomian Gland Dysfunction.

To determine the efficacy duration of eyelid hygiene for meibomian gland dysfunction (MGD) treatment, a total of 1015 participants with primary MGD, followed for at least 6 months, were enrolled. The participants were classified into the eyelid hygiene group and the control group. The participants who had stopped eyelid hygiene at any point in the observation period after the initial 2 months were classified into the withdrawal group. Analysis was conducted with a generalized linear mixed model. Treatment group, age, sex, ocular surface inflammation, anti-inflammatory treatments, and baseline MGD subtype were considered as fixed effects, and the individual factor was considered as a random effect. The MGD stage decreased significantly for the observational period in the eyelid hygiene group (p < 0.001). Approximately 40.1% of the participants continuously maintained eyelid hygiene throughout the observational period. The MGD stage in the eyelid hygiene group continued to decrease for 6 months and was maintained thereafter. After 4 months of stopping eyelid hygiene, the MGD stage in the withdrawal group was worse than in the eyelid hygiene group (p < 0.001) and similar to that in the control group (p = 0.762). Maintaining eyelid hygiene was significantly effective in MGD treatment. Efficacy increased with treatment for 6 months, and the efficacy duration was maintained for 4 months even after stopping eyelid hygiene. Therefore, we recommend that patients with MGD maintain eyelid hygiene, and compliance should be checked continuously.

Effects of meibomian gland dysfunction and aqueous deficiency on friction-related disease.

PURPOSE: To evaluate the effects of meibomian gland dysfunction (MGD) and aqueous deficiency (AD) on friction-related disease (FRD). METHODS: Cross-sectional comparative study. This study included 550 eyes (550 patients) with dry eye disease (DED). The DED subtype and dynamic tear-film parameters by automated assessments were investigated for the analysis of FRD (superior limbic keratoconjunctivitis, conjunctivochalasis, and lid wiper epitheliopathy). RESULTS: Patients with FRD had a higher proportion of moderate-to-severe MGD and AD (p < 0.001 and p < 0.001, respectively). The dropout rate of the meibomian gland was higher (30.5 ± 31.8 and 14.1 ± 25.0%, p < 0.001), tear meniscus height (TMH) was lower (227.8 ± 60.4 and 241.7 ± 55.6 µm, p = 0.008), and he first non-invasive keratographic tear break-up time (NIKBUT-1) was shorter (5.9 ± 3.5 and 7.3 ± 3.7 s, p < 0.001) in patients with FRD than in those without FRD. In the logistic regression analysis with clinical manifestation, both moderate-to-severe MGD and AD were associated with FRD (odds ratios [OR] 12.27, 95% confidence interval [CI] 7.72-19.50, and 2.31, 95% CI 1.43-3.71], p < 0.001 and p < 0.001, respectively). The dropout rate was positively associated with FRD (OR 1.017, 95% CI 1.010-1.023, p < 0.001). TMH and NIKBUT-1 were negatively associated with FRD (OR 0.995, 95% CI 0.991-0.999, and 0.90, 95% CI 0.85-0.95, p = 0.039 and p < 0.001, respectively). CONCLUSIONS: This study showed that FRD was highly associated with MGD and meibomian gland dropout rate, suggesting that FRD may be mainly affected by lipid components. AD and TMH also had a good but relatively lower association with FRD, compared to MGD and meibomian gland dropout rate.

Comparison of Treatment Modalities for Dry Eye in Primary Sjögren's Syndrome.

PURPOSE: To evaluate the effectiveness of different treatment modalities for dry eye in primary Sjögren's syndrome with their potential overlapping influences. METHODS: This study included 199 patients with newly diagnosed primary Sjögren's syndrome from 2005 to 2020. Various treatment modalities for primary Sjögren's syndrome were compared. Improvement of corneal staining based on Sjögren's International Collaborative Clinical Alliance (SICCA) scores was the primary outcome. RESULTS: The average follow-up period was 5.4 ± 3.1 (range, 2.0-14.1) years. Analysis of the individual treatments showed that punctal plug insertions in the lower and upper eyelids were strongly associated with improvement of SICCA scores (ß = 2.70 and 1.80, p < 0.001 and <0.001, respectively). With ocular surface inflammation, corneal staining scores improved significantly with steroid eye drops. Prednisolone (1%) had the strongest association with improvement of corneal staining scores (ß = 1.48, p < 0.001); this was based on the frequency of administration. Without ocular surface inflammation, diquafosol (3%), carbomer gel, and lanolin ointment were effective (ß = 1.37, 1.06, and 1.17; p = 0.003, 0.003, and <0.001, respectively). CONCLUSIONS: Punctal plug insertion, primarily targeting aqueous deficiency, is the mainstay of the treatment for dry eye in primary Sjögren's syndrome even in the presence of ocular surface inflammation. Furthermore, the effectiveness of treatment modalities for dry eye in primary Sjögren's syndrome was dependent on the presence of ocular surface inflammation.

Proteome alterations in the aqueous humor reflect structural and functional phenotypes in patients with advanced normal-tension glaucoma.

Previous reports have shown possible association between altered protein levels in aqueous humor (AH) and normal-tension glaucoma (NTG), but the underlying pathogenetic mechanism as well as specific molecular biomarkers for NTG remains still elusive. Here, we aimed to identify novel biomarkers for advanced NTG by analyzing the proteome of patient-derived AH and their correlation with various functional and structural parameters from the visual field test (VF), optical coherence tomography (OCT), and OCT angiography (OCTA). We determined differentially expressed proteins (DEPs) of the AH of patients with advanced NTG (n = 20) using label-free quantitative (LFQ) proteomics with pooled samples and data-independent acquisition (DIA) analysis with individual samples, and the roles of AH DEPs in biological pathways were evaluated using bioinformatics. We identified 603 proteins in the AH of patients with advanced NTG, and 61 of them were selected as DEPs via global proteome LFQ profiling. Individual DIA analyses identified a total of 12 DEPs as biomarker candidates, seven of which were upregulated, and five were downregulated. Gene ontology enrichment analysis revealed that those DEPs were mainly involved in the immune response. Moreover, IGFBP2, ENO1, C7, B2M, AMBP, DSP, and DCD showed a significant correlation with the mean deviation of VF and with peripapillary and macular parameters from OCT and OCTA. The present study provides possible molecular biomarkers for the diagnosis of advanced NTG.

Evaluation of dry eye subtypes and characteristics using conventional assessments and dynamic tear interferometry.

BACKGROUND/AIMS: To evaluate subtypes and characteristics of dry eye (DE) using conventional tests and dynamic tear interferometry, and to investigate determinants of disease severity in each DE subtype. METHODS: 309 patients diagnosed with DE and 69 healthy controls were prospectively enrolled. All eyes were evaluated using Ocular Surface Disease Index (OSDI), Schirmer's test I (ST1) and Meibomian gland dysfunction (MGD) grade were analysed. The tear interferometric pattern and lipid layer thickness were determined using DR-1α and LipiView II, respectively. RESULTS: Dynamic interferometric analysis revealed 56.6% of patients with DE exhibited Jupiter patterns, indicative of aqueous-deficiency, while 43.4% exhibited crystal patterns, indicative of lipid deficiency. These findings were in accordance with classification based on ST1 scores and MGD grade. Conventional assessment indicated 286 patients exhibited evidence of evaporative DE (EDE) due to MGD, while only 11 exhibited signs of pure aqueous-deficient DE (pure ADDE, only ST1 ≤5 mm). Interestingly, of 286 patients with EDE, 144 were categorised into the mixed-ADDE/EDE group, in which ST1 was identified as a strong negative determinant of OSDI. In contrast, 72.2% of patients with mixed-ADDE/EDE exhibited Jupiter patterns (Jupiter mixed), while 27.8% exhibited crystal patterns (crystal mixed). OSDI values were significantly higher in the crystal-mixed group than in the Jupiter mixed, in which OSDI scores were independently associated with ST1 values only. CONCLUSIONS: Our findings indicate that majority of EDE patients also exhibit aqueous deficiency, which can aggravate symptoms even in patients with lipid-deficient mixed-ADDE/EDE. Conventional assessments should be combined with interferometric tear analysis to determine the most appropriate treatment for each DE patient.

The Dopaminergic Neuronal System Regulates the Inflammatory Status of Mouse Lacrimal Glands in Dry Eye Disease.

Purpose: Comparison of the parasympathetic and sympathetic neurons, including the dopaminergic neural system, in dry eye (DE)-induced pathophysiology has not been elucidated well. This study investigated the presence of dopamine receptors (DRs) and their functional roles in the lacrimal glands (LGs) of DE-induced mice. Methods: After DE was induced in B6 mice for 2 weeks, the expression of tyrosine hydroxylase (TH), dopamine, and DRs (DR1, DR2, etc.) in the LGs and corneas were measured by quantitative RT-PCR, immunoblot, and ELISA. Using flow cytometry and ELISA, immune cell infiltration and inflammatory cytokine expression were determined in DE-induced LGs with or without DR blockers, SCH-23390 (DR1i), or melperone (DR2i). Corneal erosion scores were also investigated. Results: The mRNA and protein levels of TH significantly increased in DE-induced LGs. The dopamine concentration of LGs was 9.51 pmol in DE (versus naive: 1.39 pmol; P < 0.001). Both DR1 and DR2 mRNA expression were significantly enhanced in desiccating stress compared with those in naive (3.7- and 2.1-fold, P < 0.001). Interestingly, DR1 and DR2 immunostaining patterns stained independently in DE-induced LGs. CD3+ and CD19+ cell infiltration was significantly increased by DR2i (P < 0.001) but not by DR1i. Furthermore, IFN-γ, IL-17, and TNF-α were significantly upregulated by DR2i compared with the blow-only condition. The severity of corneal erosion and inflammation was also aggravated by DR2i. Conclusions: Upregulation of DR1 and DR2 was observed in DE-induced mouse LGs. As the inflammatory conditions are aggravated by the inhibition of DRs, especially DR2, their activity may be an important factor preserving ocular surface homeostasis.

Compound heterozygous mutations in TGFBI cause a severe phenotype of granular corneal dystrophy type 2.

We investigated the clinical and genetic features of patients with severe phenotype of granular corneal dystrophy type 2 (GCD2) associated with compound heterozygosity in the transforming growth factor-ß-induced (TGFBI) gene. Patients with severe GCD2 underwent ophthalmic examination (best-corrected visual acuity test, intraocular pressure measurement, slit-lamp examination, and slit-lamp photograph analysis) and direct Sanger sequencing of whole-TGFBI. The patient's family was tested to determine the pedigrees. Five novel mutations (p.(His174Asp), p.(Ile247Asn), p.(Tyr88Cys), p.(Arg257Pro), and p.(Tyr468*)) and two known mutations (p.(Asn544Ser) and p.(Arg179*)) in TGFBI were identified, along with p.(Arg124His), in the patients. Trans-phase of TGFBI second mutations was confirmed by pedigree analysis. Multiple, extensive discoid granular, and increased linear deposits were observed in the probands carrying p.(Arg124His) and other nonsense mutations. Some patients who had undergone phototherapeutic keratectomy experienced rapid recurrence (p.(Ile247Asn) and p.(Asn544Ser)); however, the cornea was well-maintained in a patient who underwent deep anterior lamellar keratoplasty (p.(Ile247Asn)). Thus, compound heterozygosity of TGFBI is associated with the phenotypic variability of TGFBI corneal dystrophies, suggesting that identifying TGFBI second mutations may be vital in patients with extraordinarily severe phenotypes. Our findings indicate the necessity for a more precise observation of genotype-phenotype correlation and additional care when treating TGFBI corneal dystrophies.

The correction of conjunctivochalasis using high-frequency radiowave electrosurgery improves dry eye disease.

We aimed to determine the clinical impact of conjunctivochalasis (CCh) and its correction using high-frequency radiowave electrosurgery (HFR-ES), for signs and symptoms of dry eye disease (DED). Forty patients diagnosed with symptomatic CCh were prospectively enrolled. As a result, patients with CCh had moderate to severe DED and most of them exhibited meibomian gland dysfunction (MGD). Corneo-conjunctival fluorescein staining score (CFS) and all lid-parallel-conjunctival-folds scores (LIPCOFs) were positively correlated. Nasal LIPCOF significantly correlated with symptoms and tear volume. Central, temporal, and total LIPCOF significantly correlated with MG loss, MGD stage, and lipid layer thickness. Independent significant factors associated with total LIPCOF included CFS, tear break-up time, and MGD stage. One month following HFR-ES, CCh was completely resolved in all cases. Patient age and preoperative nasal LIPCOF were determinants of outcomes associated with postoperative improvements in symptoms. Ocular surface parameters significantly improved, but MGD-related signs did not. Collectively, CCh associated with MGD severity deteriorates not only tear film stability and reservoir capacity, leading to DED exacerbation. Therefore, CCh should be corrected in patients with DED and MGD. Younger patients with nasal CCh are likely to experience more symptomatic relief after HFR-ES. Particularly, management for MGD should be maintained after CCh correction.

Immunostimulatory Effects of Live Lactobacillus sakei K040706 on the CYP-Induced Immunosuppression Mouse Model.

Our previous studies have shown that heat-killed Lactobacillus sakei K040706 exerts immunostimulatory and anti-inflammatory activities in macrophages, cyclophosphamide (CYP)-treated mice, and dextran sulfate sodium-induced colitis mice. However, the immunostimulatory effects of live Lactobacillus sakei K040706 (live K040706) against CYP-induced immunosuppression and its underlying molecular mechanisms remain unknown. Therefore, we investigated the immunostimulatory effects of live K040706 (108 or 109 colony forming unit (CFU)/day, p.o.) in CYP-induced immunosuppressed mice. Oral administration of live K040706 prevented the CYP-induced decreases in body weight, thymus index, natural killer (NK) cell activity, T and B cell proliferation, and cytokine (interferon (IFN)-γ, interleukin (IL)-2, and IL-12) production. The administration of live K040706 also exerted positive effects on the gut microbiota of CYP-induced mice, resulting in a microbiota composition similar to that of normal mice. Moreover, live K040706 significantly enhanced IL-6 and granulocyte-macrophage colony-stimulating factor (GM-CSF) production in the splenocytes and Peyer's patch (PP) cells of mice and increased bone marrow (BM) cell proliferation. Taken together, our data indicate that live K040706 may effectively accelerate recovery from CYP-induced immunosuppression, leading to activation of the immune system. Therefore, live K040706 may serve as a potential immunomodulatory agent against immunosuppression.