Molecular Vision最新文献

Purpose: To collectively investigate the carbohydrate sulfotransferase 6 (CHST6) mutation spectrum and corneal morphological alterations of macular corneal dystrophy (MCD) patients using in vivo confocal microscopy (IVCM), histochemistry, immunohistochemistry, and further ascertaining the immunophenotype using an enzyme-linked immunosorbent assay (ELISA).

Methods: Sanger sequencing-based CHST6 gene screening was performed for 112 study participants (MCD patients, n = 68; family members, n = 44). Twenty-seven MCD patients underwent IVCM analyses, and corneal buttons were analyzed with histochemistry Alcian blue (AB) staining and immunohistochemistry anti-keratan sulfate (KS) monoclonal antibody, 5D4MoAb. An ELISA was used to determine serum KS levels. Quantitative analysis of the central corneal thickness (CCT), epithelial cell thickness, epithelial cell count, and stromal keratocyte cell count was performed using a one-way ANOVA.

Results: Eighteen distinct CHST6 mutations, including one novel (p.L129V), were identified. MCD patients with predominant immunophenotype IA (n = 15) harboring major p.Q182Rfs199 deletion, p.194_R196delinsRC (delins), and open reading frame (ORF) mutations displayed AB positivity corresponding to loss of Bowman's layer, interlamellar glycosaminoglycan (GAG) depositions, and faint KS expression (5D4-MoAb) only in stromal keratocytes. Notably, IVCM imaging revealed BL loss due to confluent clumps of hyper-reflective, granular deposits together with scar tissue seen only in this group. Eight patients (with missense mutations) displayed immunophenotype I with positive GAG deposits and negative KS expression. Patients with immunophenotype II (n = 4) with no mutations showed both positive GAG deposits and KS expression. A quantitative analysis revealed a statistically significant decrease in CCT (p-value < 0.001), epithelial cell thickness, epithelial cell count, and stromal keratocyte cell count among the patients with truncation mutations compared to the control group.

Conclusions: In this current study, the combinational findings of MCD-related corneal morphological alterations, immunophenotypes, and mutation spectrum are presented first, which indicated a severe phenotype in patients identified with truncation (deletion, delins, and deletion of ORF) mutations. However, additional studies with a larger number of patients would help highlight these findings and reinforce the possible correlation between genotypes and immunophenotypes in MCD pathogenesis.

Purpose: The mutation of R124H in TGFBIp causes Avellino corneal dystrophy (ACD, GCD II). However, the molecular mechanisms of ACD caused by the p. R124H mutation are not well understood. In our research, we aimed to explain the molecular mechanisms of ACD caused by the R124H mutation.

Methods: The whole blood of a three-generation family having ACD was studied with the whole exome sequencing. Sanger sequencing was used to identify the mutation gene. The mutant structure of R124H TGFBIp was visualized in Pymol, using the PISA server, Coot and the HDOCK automated docking program. The TGFBIp was expressed in mammalian expression system. And size exclusion chromatography (SEC) was used to identify the aggregate state of TGFBIp.

Results: The whole exome sequencing results showed that there was a c.371G>A mutation in the TGFBI gene in one family, including three patients. In biochemical assays, the purified soluble wild-type TGFBIp and R124H TGFBIp formed a homodimer through a novel interface distinct from the previously proposed FAS1-1: FAS1-4 dimer (interface I). R124H TGFBIp is likely to have formed more severe cross-links and aggregation. Therefore, R124H TGFBIp causes homozygous patients to have more serious symptom than heterozygous patients.

Conclusions: In our study, one family having ACD harboring the mutation of R124H TGFBIp was identified. A new homodimerization interface was determined for wild-type TGFBIp and R124H TGFBIp. Besides, we provided a possible molecular explanation for why the symptom of homozygous patients was more severe than those of heterozygous patients. The possible molecular explanation can provide a new insight into the treatment of ACD.

Purpose: This study aimed to characterize the inflammatory mediators present in the tear film of patients with keratoconus (KC). It also aimed to investigate the gene expression of these mediators in corneal epithelial cells and their immune activity in conjunctival epithelial cells in patients with KC compared to a control group.

Methods: This transversal study included 30 patients with KC and 23 control group participants. Tear samples were collected by washing the ocular surface with 60 μL of sterile buffered saline solution. The levels of interleukin IL-5, IL-13, IL-2, IL-6, IL-10, interferon-gamma, tumor necrosis factor-alpha, and IL-4 were measured using a LEGEND plex HU Th1/Th2 panel kit and analyzed using flow cytometry. Corneal epithelial samples were obtained via manual keratectomy from KC patients scheduled for corneal crosslinking and from individuals scheduled for photorefractive keratectomy (control group). These samples were immediately stored at -70 °C for mRNA extraction and subsequent reverse transcription polymerase chain reaction analysis to measure IL-5 and IL-6 gene expression. Conjunctival epithelium samples were collected using impression cytology and analyzed using immunohistochemistry and confocal microscopy to detect IL-5 and IL-6 immunoreactions.

Results: Our study found no statistically significant differences in the tear film cytokine concentrations between the two groups. In addition, the gene expression of IL-5 and IL-6 in the corneal epithelium was higher in the KC group than in the control group, with IL-5 showing a 50% increase and IL-6 showing a 20% increase. Immunohistochemical analysis revealed a greater immunostaining of IL-5 and IL-6 in the conjunctival epithelium of patients with KC compared to the control group.

Conclusions: In this study, despite higher levels of IL-5 and IL-6 in the tear film of patients with KC, there was no statistically significant difference compared to the control group. However, there was heightened immune activity in the corneal and conjunctival epithelial cells of patients with KC based on IL-5 and IL-6 gene expression and their immunodetection, respectively.

[This retracts the article on p. 92 in vol. 30, PMID: 38601014.].

Purpose: Ranibizumab is a frequently used inhibitor of vascular endothelial growth factor (VEGF) in the treatment of macular edema following central retinal vein occlusion (CRVO). Studying proteins that mediate the beneficial effects of ranibizumab in CRVO can potentially lead to the improved management of macular edema.

Methods: In 14 Danish Landrace pigs, experimental CRVO was induced in the right eyes and treated with either intravitreal ranibizumab (n = 6) or an intravitreal sodium chloride 9 mg/mL solution as a sham injection (n = 8). Successful CRVO was confirmed by fluorescein angiography. Retinal samples were collected 15 days after induced CRVO and analyzed with label-free, quantification, nano-liquid chromatography-tandem mass spectrometry. Validation was performed with western blotting and immunohistochemistry.

Results: CRVO was successfully induced and confirmed by fluorescein angiography. A total of 28 proteins were upregulated, and 31 proteins were downregulated following ranibizumab treatment. A high concentration of the ranibizumab component immunoglobulin kappa chain C region was observed in retinas treated with ranibizumab. Complement C3, the Ig lambda chain C region, and nucleobindin-2 were downregulated following ranibizumab intervention. The downregulation of complement C3 was confirmed by western blotting. Modest changes were observed in the remaining significantly regulated proteins.

Conclusions: Retinal complement C3 was downregulated following ranibizumab intervention in CRVO. The decrease in complement C3 may potentially downregulate the inflammatory response in CRVO. A high retinal concentration of ranibizumab was reached 15 days after injection of the compound.

While the high-dose streptozotocin (STZ; 100 mg/kg) rodent model is the gold standard in modeling Type I diabetes, models for Type II diabetes are needed for this more common form of diabetes. We investigated the retinal, cognitive, and metabolic alterations in a Type II diabetic model induced by high-fat diet (HFD) and low-dose STZ (30 mg/kg). Long Evans rats were assigned to naïve control, HFD, or HFD+STZ groups. Diabetic rats were further stratified into Type I and Type II based on metabolic assessments. Optomotor response (OMR, visual function), electroretinograms (retinal function), and Y-maze (cognitive function) were tested. Serum was analyzed for 12 metabolic markers using a multiplex panel. Type I rats showed severe increases in blood glucose accompanied by impairments in insulin and glucose tolerance, reduced bodyweight, and low insulin levels. In contrast, Type II rats showed moderate changes in blood glucose and insulin and glucose tolerance with weights and insulin levels similar to naïve controls. Type I and II rats showed OMR deficits (p<0.05) and electroretinogram changes (p<0.05). No cognitive deficits were observed. Type I rats displayed reduced serum levels of brain-derived neurotrophic factor (BDNF), C-Peptide, and leptin (p<0.05), and alterations in C-Peptide, PYY, and glucagon levels correlated with retinal function changes (p<0.05). Type II rats exhibited a moderate diabetic state while still developing retinal and visual deficits, which recapitulates phenotypes reported in patients.

Purpose: Congenital cataract is an important cause of visual impairment in childhood. Our previous study reported that the c.110G>C (p.R36P) mutation in the γD-crystallin gene (CRYGD) was associated with congenital cataract in a Chinese family. This study aimed to investigate the potential underlying mechanism through which the p.R36P mutation leads to congenital cataract.

Methods: Plasmids encoding wide-type human γD-crystallin and the mutant R36P γD-crystallin were transfected into HEK293T and SRA01/04 cells. Protein expression levels, including total, soluble, and insoluble fractions, were quantified by Western blotting. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to assess the mRNA expression of other crystallin genes. Cell viability and apoptosis were evaluated using the CCK-8 assay and flow cytometry, respectively.

Results: The total protein, especially the soluble fraction, was significantly reduced in the R36P mutant, while the insoluble part remained unaffected. The decrease of soluble R36P γD-crystallin could not be rescued by the proteinase inhibitor MG132. The mRNA expression of the R36P mutation was lower, but other crystallin RNAs were unchanged. Cell viability was slightly decreased (11%, p<0.05), and cell apoptosis was not significantly increased (12%, p=0.31).

Conclusions: The significant decrease in soluble R36P γD-crystallin may represent a novel mechanism underlying congenital cataract caused by CRYGD gene mutation.

Purpose: To investigate the differences in anterior scleral thickness (AST) among the refractive statuses of Chinese adults aged 18-35.

Methods: This study recruited 170 Chinese participants (mean age, 24.06 ± 2.78 years), including myopes (spherical equivalent refraction [SER] -1.00 to -12.75 diopters [D]; n = 134), emmetropes (SER ± 0.75 D; n = 36), and AST (superior, inferior, nasal, and temporal), which were investigated via swept-source optical coherence tomography. Semiautomated custom-designed software measured the scleral thickness from the scleral spur to 5 mm along four meridians.

Results: The mean axial length and spherical equivalent refractive error were 25.12 ± 1.44 mm and -3.93 ± 3.09 D, respectively. The anterior sclera was thickest in the inferior region and thinnest in the superior region (753.9 ± 88.7 μm versus 613.6 ± 58.4; p < 0.001). The AST in the temporal meridian was significantly thicker than that in the nasal meridian (727.5 ± 60.8, 690.9 ± 55 μm; p < 0.001). There were no significant variations in AST in the myopes and emmetropes along the five latitude lines. AST along the inferior meridian at the 4-mm (r 2 = 0.0992; p < 0.001) and 5-mm (r 2 = 0.0888; p < 0.001) locations decreased significantly with increasing myopia.

Conclusion: With increased myopia, AST at the 4-mm and 5-mm locations showed significant thinning in the inferior meridian. The results indicate that AST, especially along the inferior meridian, may act as a biologic marker to monitor the progression of myopia.

Purpose: We screened 28 female cynomolgus monkeys (CMs) and 25 female rhesus monkeys (RMs) for white dots (WDs) in the macula and detected several animals with WDs in colonies at the Shin Nippon Biomedical Laboratories, Ltd., Drug Safety Research Laboratories (SNBL) facility. To determine the functional and morphological characteristics of WDs, we conducted ophthalmological and pathological examinations on these animals.

Methods: Fundus examination, optical coherence tomography (OCT), and focal electroretinogram (f-ERG) were conducted for all animals. Histopathology and transmission electron microscopy were conducted for one representative adult CM with WDs.

Results: In both CMs and RMs, individual differences were observed in the number of WDs in the macula (ranging from approximately 10 to 500 per eye). Hyperreflective granules were observed between the ellipsoid zone (EZ) and the retinal pigment epithelium (RPE) in OCT. Both CMs and RMs exhibited a significant increase in the thicknesses of the RPE and choroid in WD animals compared to their normal counterparts. In the f-ERG, significant decreases and/or tendencies toward decreases in amplitudes and increases in implicit times of both a- and b-waves were observed in animals with WDs. In pathology, diffuse vacuolization of the RPE cells with tiny granules was observed in the macula.

Conclusions: Based on the results of OCT and pathological examinations, it was suggested that animals with WDs can develop macular degeneration in the future. To assess their suitability as a model for precursor lesions of age-related macular degeneration, it is imperative to continue monitoring the animals used in the present study until they reach a more advanced age of approximately another 5-10 years.