Detection of Novel BEST1 Variations in Autosomal Recessive Bestrophinopathy Using Third-generation Sequencing.

OBJECTIVE: Autosomal recessive bestrophinopathy (ARB), a retinal degenerative disease, is characterized by central visual loss, yellowish multifocal diffuse subretinal deposits, and a dramatic decrease in the light peak on electrooculogram. The potential pathogenic mechanism involves mutations in the BEST1 gene, which encodes Ca2+-activated Cl- channels in the retinal pigment epithelium (RPE), resulting in degeneration of RPE and photoreceptor. In this study, the complete clinical characteristics of two Chinese ARB families were summarized. METHODS: Pacific Biosciences (PacBio) single-molecule real-time (SMRT) sequencing was performed on the probands to screen for disease-causing gene mutations, and Sanger sequencing was applied to validate variants in the patients and their family members. RESULTS: Two novel mutations, c.202T>C (chr11:61722628, p.Y68H) and c.867+97G>A, in the BEST1 gene were identified in the two Chinese ARB families. The novel missense mutation BEST1 c.202T>C (p.Y68H) resulted in the substitution of tyrosine with histidine in the N-terminal region of transmembrane domain 2 of bestrophin-1. Another novel variant, BEST1 c.867+97G>A (chr11:61725867), located in intron 7, might be considered a regulatory variant that changes allele-specific binding affinity based on motifs of important transcriptional regulators. CONCLUSION: Our findings represent the first use of third-generation sequencing (TGS) to identify novel BEST1 mutations in patients with ARB, indicating that TGS can be a more accurate and efficient tool for identifying mutations in specific genes. The novel variants identified further broaden the mutation spectrum of BEST1 in the Chinese population.

Binding properties of chemosensory protein 4 in Riptortus pedestris to aggregation pheromones.

In insects, chemosensory proteins (CSPs) play an important role in the perception of the external environment and have been widely used for protein-binding characterization. Riptortus pedestris has received increased attention as a potential cause of soybean staygreen syndrome in recent years. In this study, we found that RpedCSP4 expression in the antennae of adult R. pedestris increased with age, with no significant difference in expression level observed between males and females, as determined through quantitative real-time polymerase chain reaction (qRT-PCR). Subsequently, we investigated the ability of RpedCSP4 to bind various ligands (five aggregated pheromone components and 13 soybean volatiles) using a prokaryotic expression system and fluorescence competitive binding assays. We found that RpedCSP4 binds to three aggregated pheromone components of R. pedestris, namely, ((E)-2-hexenyl (Z)-3-hexenoate (E2Z3), (E)-2-hexenyl (E)-2-hexenoate (E2E2), and (E)-2-hexenyl hexenoate (E2HH)), and that its binding capacities are most stable under acidic condition. Finally, the structure and protein-ligand interactions of RpedCSP4 were further analyzed via homology modeling, molecular docking, and targeted mutagenesis experiments. The L29A mutant exhibited a loss of binding ability to these three aggregated pheromone components. Our results show that the olfactory function of RpedCSP4 provides new insights into the binding mechanism of RpedCSPs to aggregation pheromones and contributes to discover new target candidates that will provide a theoretical basis for future population control of R. pedestris.

Artificial Intelligence Model for Antiinterference Cataract Automatic Diagnosis: A Diagnostic Accuracy Study.

Background: Cataract is the leading cause of blindness worldwide. In order to achieve large-scale cataract screening and remarkable performance, several studies have applied artificial intelligence (AI) to cataract detection based on fundus images. However, the fundus images they used are original from normal optical circumstances, which is less impractical due to the existence of poor-quality fundus images for inappropriate optical conditions in actual scenarios. Furthermore, these poor-quality images are easily mistaken as cataracts because both show fuzzy imaging characteristics, which may decline the performance of cataract detection. Therefore, we aimed to develop and validate an antiinterference AI model for rapid and efficient diagnosis based on fundus images. Materials and Methods: The datasets (including both cataract and noncataract labels) were derived from the Chinese PLA general hospital. The antiinterference AI model consisted of two AI submodules, a quality recognition model for cataract labeling and a convolutional neural networks-based model for cataract classification. The quality recognition model was performed to distinguish poor-quality images from normal-quality images and further generate the pseudo labels related to image quality for noncataract. Through this, the original binary-class label (cataract and noncataract) was adjusted to three categories (cataract, noncataract with normal-quality images, and noncataract with poor-quality images), which could be used to guide the model to distinguish cataract from suspected cataract fundus images. In the cataract classification stage, the convolutional-neural-network-based model was proposed to classify cataracts based on the label of the previous stage. The performance of the model was internally validated and externally tested in real-world settings, and the evaluation indicators included area under the receiver operating curve (AUC), accuracy (ACC), sensitivity (SEN), and specificity (SPE). Results: In the internal and external validation, the antiinterference AI model showed robust performance in cataract diagnosis (three classifications with AUCs >91%, ACCs >84%, SENs >71%, and SPEs >89%). Compared with the model that was trained on the binary-class label, the antiinterference cataract model improved its performance by 10%. Conclusion: We proposed an efficient antiinterference AI model for cataract diagnosis, which could achieve accurate cataract screening even with the interference of poor-quality images and help the government formulate a more accurate aid policy.

Outcomes of chronic angle-closure glaucoma treated by phacoemulsification and endocyclophotocoagulation with or without endoscopically goniosynechialysis.

AIM: To investigate the surgical outcomes of patients with chronic angle-closure glaucoma (CACG) treated with phacoemulsification (phaco)/endocyclophotocoagulation (ECP) with and without endoscopic goniosynechialysis (E-GSL). METHODS: A retrospective, nonrandomized, comparative case series was conducted. Patients with CACG who underwent phaco in combination with either ECP alone (ECP group) or GSL with ECP (E-GSL group) from 2018 to 2019 were followed for 12mo and reviewed. Clinical features and outcomes were identified and analyzed. The ECP and E-GSL groups were matched in age and baseline intraocular pressure (IOP). Changes in IOP, mean of visual acuity (VA), peripheral anterior synechiae (PAS) formation, and the number of glaucoma medications was examined. RESULTS: The ECP group included 32 eyes of 27 patients, and the E-GSL group included 32 eyes of 26 patients. The preoperative baseline IOP was 22.18±6.48 mm Hg in the ECP group and 22.95±6.71 mm Hg in the E-GSL group (P=0.644). The mean IOP reduction was 26.2% in the ECP group and 41.6% in the E-GSL group at 12mo. The mean postoperative VA (logMAR units) at 12mo was 0.47 in the ECP group and 0.36 in the E-GSL group. The reduction in PAS formation and the number of glaucoma medications was also higher in the ECP group than E-GSL group at 12mo. CONCLUSION: The phaco/ECP and phaco/E-GSL groups both achieve a significant reduction in IOP without complications associated with traditional glaucoma filtration surgeries.

A novel mutation of FOXC1 in a Chinese family with Axenfeld-Rieger syndrome.

Axenfeld-Rieger syndrome (ARS) is a disorder affecting the anterior segment of the eye and causing systemic malformations, and follows an autosomal-dominant inheritance pattern. The aim of the present study was to identify the underlying cause of ARS in a Chinese family. Genomic DNA was extracted from the peripheral blood of the subjects from a family with ARS. The pathogenic variant was identified by targeted next-generation sequencing and confirmed by Sanger sequencing. A novel heterozygous mutation of the forkhead box (FOX)C1 gene (c.1494delG, p.G499Afs*20) was detected in all affected members of the family, while no mutation was identified in the unaffected members or in the 150 normal controls. The affected members exhibited typical ocular and craniofacial anomalies. The results of the present study demonstrated that a novel deletion in exon 1 of the FOXC1 gene caused ARS in this Chinese family.

Haplotype analysis of association of the MYOC gene with primary angle-closure glaucoma in a Han Chinese population.

PURPOSE: The aim of this study is to examine whether or not myocilin (MYOC) genetic variations are associated with susceptibility to primary angle-closure glaucoma (PACG) in the Han Chinese population. METHODS: Four single-nucleotide polymorphisms (SNPs)-rs235913, rs183532, rs12076134, and rs235875-in the MYOC gene were genotyped in 212 adult patients with PACG and 255 age-, sex-, and ethnic-matched healthy controls by using a polymerase chain reaction-restriction fragment length polymorphism assay. Data were analyzed by chi-square analysis. RESULTS: The four SNPs in the MYOC gene were in the Hardy-Weinberg equilibrium in all the subjects. The frequencies of A allele rs183532 were significantly different between the PACG patients and the controls (0.238 vs. 0.169, p=0.008; OR=1.541; 95% CI: 1.117-2.127). The frequencies of the AA genotype and A allele of rs235913 were increased in PACG patients compared with controls, but the difference was not significant (p=0.037, p=0.017, respectively). A comparison of the distributions of the genotypes and alleles of rs12076134 and rs235875 showed no statistically significant differences between the PACG patients and the controls (p>0.05). Haplotype analysis indicated that the frequency of the AATG and AATA haplotypes was significantly higher for PACG patients than for control subjects (both p<0.001). However, the frequency of CGGA and CGTG haplotypes was lower for PACG patients than for control subjects (p<0.001). CONCLUSIONS: Our study suggests that rs183532 is associated with an increased risk of PACG in the Chinese Han population.

Evaluation of corneal graft survival in mice model.

AIM: To investigate the characteristics and criterion of graft rejection in mice model. METHODS: C57BL/6 or BALB/c mice corneal grafts were grafted onto BALB/c hosts. Each group was divided into two subgroups according to the corneal opacity scores 12d after transplantation. The characteristics of opacity and neovascularization were observed. Mice of the 12(th), 50(th) day after transplantation, the grafts biopsy of mice in allogeneic group 1, which opacity score exceed 3, were prepared for histological observation and those restore transparent were endothelial stained. RESULTS: There was no difference of corneal opacity score on the 7(th) and 12(th) day after operation; the histological results had no disparity between syngeneic group and allogeneic group. On the 12(th) day after surgery, the turbidity curve was apparent in grafts with opacity score < 2. Mononuclear cells were shown in grafts with opacity score reached 3 in allogeneic group 1. Different rejection performance was observed in tissue sections on the 50(th) day after surgery. CONCLUSION: Grafts, opacity score exceeds 3 from the 7(th) to the 12(th) day after operation could not be judged as a rejection. We should pay more attention to the variation of grafts opacity since 12d after corneal transplantation.

[Glaucoma and Moscow Eye Microsurgery complex (Moroz) keratoprosthesis].

OBJECTIVE: To investigate the relationship between glaucoma and Moscow Eye Microsurgery Complex (Moroz) keratoprosthesis. METHODS: Analysis and retrospective review of consecutive clinical case series. A total of 90 patients had a Moroz keratoprosthesis implantation between Apr, 2000 and Jun, 2011 at our hospital.Fifteen eyes of 15 patients were included in this research. Twelve eyes were identified glaucoma before Moroz keratoprosthesis surgery, and 3 eyes developed glaucoma afterward.Intraocular pressure (IOP), best-corrected visual acuity (BCVA), and treatment of glaucoma were recorded at 1 to 3 months after the Moroz keratoprosthesis implantation. RESULTS: Moroz keratoprosthesis surgery improved vision dramatically in the majority of patients in our study. The most common preoperative corneal diagnosis was alkali burn (8 eyes, 53.3%), which maybe the risk factor of secondary glaucoma after keratoprosthesis surgery.Five of these 15 patients received cyclocryotherapy after the surgery to control elevated IOP. BCVA decreased from 0.8 to 0.2, 0.25 to light perception, 0.3 to 0.1, 0.2 to hand movement, and 0.25 to counting fingers, respectively. CONCLUSIONS: Moroz keratoprosthesis caused glaucoma in only a small number of patients.Visual field, optic nerve appearance and IOP were the main diagnostic modalities after Moroz keratoprosthesis implantation.

Changes in objective vault and effect on vision outcomes after implantable Collamer lens implantation: 1-year follow-up.

PURPOSE: To investigate the changes in vault and the effect on visual outcomes 1 year after implantable Collamer lens (ICL) implantation. METHODS: In this retrospective study, 127 eyes of 66 patients undergoing ICL implantation were examined both before and up to 1 year after the surgery. The examination contents included white-to-white (WTW) diameter, central vault of the ICL (distance between posterior surface of ICL and anterior surface of crystalline lens), refractive error, and wavefront high-order aberration (HOA). All statistical analyses were performed using SPSS 13.0 software. RESULTS: A significant decrease in vault was noted up to 1 month, after which the value stabilized (p=0.001). The moderate vault decreased significantly after the first 3 months postsurgery (paired-samples t test, p<0.05). Low vault showed a tendency to increase and high vault showed a tendency to decrease, but not significantly, over time. There was no statistically significant correlation between the amount of vault and the refractive error (Pearson correlation coefficient R=0.111, p=0.473) and there was a statistically significant correlation between the vault and HOAs (R=0.304, p=0.024). CONCLUSIONS: Implantable Collamer lens vault over the crystalline lens had the tendency toward a slight decrease with time and did not significantly affect the vision outcome 1 year after surgery.

[The observation of the effects of a micromolecular compound J2 on rat corneal allograft using atomic force microscopy].

OBJECTIVE: To study the cell membrane of corneal endothelium with a micromolecular compound J2 in corneal allograft of rat using atomic force microscopy (AFM). METHODS: Cohort study. Subjects were divided into two groups: group A (n = 15): experimental group; group B (n = 15): placebo control group. At the fifth, tenth, fifteen, twentieth, twenty-fifth day after penetrating keratoplasty, the donor implant was separated from receipt bed, one part of which was stained by HE and the others fixed into AFM sample. Amplitude and height images were obtained in the tapping mode with a scan rate of 2 Hz and an integral gain of 0.3 to 0.5. Statistical analysis was performed using single-factor analysis of variance and P value was calculated. RESULTS: The average transplant survival time in group A was (33.12 ± 6.80) d, and those in group B was (18.87 ± 4.19) d. There were significant difference between two group (F = 47.7449, P = 0.00). There were obvious differences on ultrastructure measured by AFM between two groups. At the fifth day after penetrating keratoplasty, regular hexagonal structure of corneal endothelium was observed by AFM in both two group. The diameter of corneal endothelium was about 15 µm, uneven microstructure of cell could be found. The time being, different changes were arose in two group: a clear microstructure could be found in group A, however the microstructure of cell could not be recognized in group B. One way analysis of variance showed that significant differences on parameters (Ra, Rp and Rv) were found between two groups (P < 0.05). At the fifth day after penetrating keratoplasty, group A: Ra (97.64 ± 31.58) nm, Rp (297.79 ± 25.19) nm, Rv (545.55 ± 25.83) nm; group B: Ra (112.61 ± 34.29) nm, Rp (265.06 ± 24.17) nm, Rv (544.41 ± 21.78) nm (Fa = 30.9416, P = 0.0000; Fp = 263.6018, P = 0.0000; Pv = 1.2013, P = 0.2735). At the tenth day after penetrating keratoplasty, group A: Ra (102.98 ± 32.98) nm, Rp (711.38 ± 21.94) nm, Rv (639.89 ± 22.58) nm; group B: Ra (222.85 ± 31.28) nm, Rp (111.22 ± 20.35) nm, Rv (746.49 ± 23.17) nm (Fa = 2086.4535, P = 0.0000; Fp = 53768.4676, P = 0.0000; Pv = 3257.3178, P = 0.0000). At the fifteenth day after penetrating keratoplasty, group A: Ra (87.44 ± 34.97) nm, Rp (344.18 ± 21.09) nm, Rv (482.61 ± 22.27) nm; group B: Ra (197.64 ± 35.72) nm, Rp (510.76 ± 24.98) nm, Rv (545.62 ± 23.17) nm (Fa = 1458.1057, P = 0.0000; Fp = 7788.6963, P = 0.0000; Pv = 1153.2860, P = 0.0000). At the twentieth day after penetrating keratoplasty, group A: Ra (85.85 ± 32.53) nm, Rp (348.69 ± 21.26) nm, Rv (367.65 ± 23.12) nm; group B: Ra (201.36 ± 34.12) nm, Rp (788.58 ± 20.34) nm, Rv (563.33 ± 21.01) nm (Fa = 1801.1215, P = 0.0000; Fp = 67 057.9516, P = 0.0000; Fv = 11 770.2195, P = 0.0000). At the twenty-fifth day after penetrating keratoplasty, group A: Ra (104.97 ± 32.47) nm, Rp (395.05 ± 20.38) nm, Rv (396.17 ± 21.59) nm; group B: Ra (43.85 ± 31.28) nm, Rp (249.88 ± 20.79) nm, Rv (154.88 ± 22.37) nm (Fa = 551.4134, P = 0.0000; Fp = 7458.9255, P = 0.0000; Pv = 18 070.5189, P = 0.0000). CONCLUSIONS: The morphology and ultrastructure of corneal endothelium in group A with J2 were different from group B by observation with AFM. J2 was an effect micromolecular in prevention of corneal allograft rejection.

[Construction of lentiviral expression vector containing Foxp3 gene and its expression in DC2.4 cell line].

AIM: To construct a Foxp3 lentiviral vector and transfer it into DC2.4 cells, which provides Foxp3+DC cells for further study on its immune modulation. METHODS: We cloned mouse Foxp3 gene into lentiviral vector(pGC-FU) and acquired the plasmid pGC-FU-Foxp3. PCR and sequencing analysis were made for verifying the positive clones. The virus packaging plasmids(pGC-FU-Foxp3, pHelper1.0 and pHelper2.0) were contransfected into 293T cells, and the Lentivirus-Foxp3 was harvested from 293T cells. The Lentivirus-Foxp3 was used to infect DC2.4 cells in vitro and the expression of Foxp3 in infected DC2.4 cells was detected with flow cytometry(FCM). RESULTS: PCR and sequencing revealed that the pGC-FU-Foxp3 plasmid was correctly constructed. The Lentivirus-Foxp3 with a titer of 2×10(8); TU/mL was successfully packaged. Foxp3 expression in DC2.4 cells infected with the Lentivirus-Foxp3 was increased significantly compared with negative control lentivirus. CONCLUSION: The pGC-FU-Foxp3 plasmid has been successfully constructed and the Lentivirus-Foxp3 has been successfully packaged. Foxp3 can be enhanced in DC cells infected with the Lentivirus-Foxp3.

Experimental study of the mechanism of tolerance induction in dexamethasone-treated dendritic cells.

BACKGROUND: The aim of this study was to investigate the mechanisms underlying tolerance induction of dexamethasone (Dex)-treated dendritic cells (DCs). MATERIAL/METHODS: Well-grown DC2.4 cells were randomly assigned to receive control, 50 µg/L, 100 µg/L, or 200 µg/L of dexamethasone and then were cultured for 6 days. The expressions of CD80, CD86, galectin-9, and PD-L1 on the surface of DC2.4 cells were analyzed with flow cytometry and the level of IL-12 secreted by DC2.4 cells was determined by ELISA. The stimulating activity of DC2.4 cells on allogeneic T cells was assessed with mixed lymphocyte reaction. Dexamethasone-treated DC2.4 cells were co-cultured with allogeneic splenic lymphocytes and the Foxp3 expression in naive T lymphocytes was determined with flow cytometry. RESULTS: Compared with the control group, the expressions of CD80, CD86, galectin-9, and PD-L1 on the surface of DC2.4 cells exposed to different doses of dexamethasone showed no significant changes; however, dexamethasone treatment significantly reduced IL-12 secretion and inhibited DC2.4's stimulation on the proliferation of allogeneic T lymphocytes. Moreover, dexamethasone-treated DC2.4 cells effectively promoted FOXP3 expression in naive T lymphocytes. CONCLUSIONS: DC2.4 is a stable cell line with high expressions of CD80, CD86, and PD-L1. Dexamethasone does not significantly change the cell phenotype of DC2.4 cells, but inhibits the secretion of IL-12 cytokine and attenuates DC2.4's stimulation of the proliferation of allogeneic T cells. Dexamethasone-treated DC2.4 cells also effectively promote FOXP3 expression in naive T lymphocytes.

[Outcome after treatment of myopia with implantable Collamer lens].

OBJECTIVE: To evaluate the efficacy, predictability, stability, and safety of the surgical correction myopia using implantable Collamer lenses (ICL) in phakic eyes. METHODS: A prospective study was performed to analyze 91 eyes of 48 patients who had the implantation of ICL for the treatment of myopia from July 2008 to February 2010. Patients were examined preoperatively and followed at 1 week, 1, 3, 6, and 12 months postoperatively. The examinations included the uncorrected visual acuity (UCVA), best corrected visual acuity (BCVA), refraction, contrast sensitivity, wavefront aberration, intraocular pressure, space between crystal lens and intraocular lens (vault), endothelial cell density (ECD), anterior chamber depth (ACD), surgical complication, etc. RESULTS: Successful implantation was achieved in all patients. The mean follow-up time was (9.54 ± 4.12) months (SD)(range 1 to 12 months). The mean preoperative SE was -12.38 diopters (D) (range -5.0D to -23.0D). Postoperatively, UCVA was maintained or improved in all eyes. UCVA achieved 1.0 in 58 eyes (64%) and BCVA gained more than 1 line in 69 eyes (75.9%). The glare and no glare contrast sensitivity were improved at 3cd, 12cd and 18cd, with the exception of 6cd. The aberration decreased in RMS, spherical and coma. Post operative ACD (1 week after operation) diminished 8.92% as compared with the preoperative ACD. The mean vaulting was (452 ± 216.38) µm (6 months) and ranged 130 - 1080 µm at different follow-up periods. There was no statistically significant difference in vaulting between postoperative data at different periods (t = 0.200, P > 0.05). The mean postoperative ECD decreased but had no statistically difference compared with the preoperative ECD. ACD decreased 31% after surgery in 2 eyes (2.1%). Transient high IOP was observed in 13 eyes (2.1%) one week after the operation. CONCLUSION: These results indicate that ICL implantation in the treatment of myopia is efficient, predictable, safe, and reliable.

[Initial study on mechanism of a small non-peptidic CD4 inhibitor J2 in prevention corneal allograft rejection].

OBJECTIVE: To discuss the mechanism of J2 on preventing mice's corneal rejection. METHODS: In experimental study, Balb/c mice had been divided into A, B, C, D, E groups randomly. There were 13 mice in each of Group A, C and E, while 7 in both Group B and D. Group A did no management; Group B, autograft control; Group C, D and E, allograft groups (C57BL/6 were used as donors); Group B and Group C were given placebo; Group D and Group E were treated with orally cyclosporine A (CsA) (10 mg per kilogram of body weight) and J2 (15 mg per kilogram of body weight), respectively. To observe the variation of lymphocyte subgroup of peripheral blood mononuclear cells in different groups by flow cytometer analysis every week after corneal transplantation. DTH assay had been done at week 3, cytokines including interleukin-2 (IL-2), IL-10, interferon-gamma (IFN-gamma), secreting level of mouse spleen cells detected by ELLISPOT assay on day 18 after corneal transplantation. To apply single factor analysis of variance, single factor analysis of variance for repeat measured data and so on to analyze the data. RESULTS: The results of flow cytometer analysis showed: lymphocyte subgroup of peripheral blood mononuclear cells had not proliferated specifically in Group E, compared to Group B and Group D (for CD3(+)CD4(+)T lymphocyte, E-B, P = 0.776, E-D, P = 0.606; for CD3(+)CD8(+)T lymphocyte, E-B, P = 0.941, E-D, P = 0.482). DTH assay showed low reaction to both donor's and third strain mouse spleen cells in Group E (F = 1.00, P = 0.422). The results of ELLISPOT assay indicated: the spot level of IL-2, IFN-gamma in Group E raised slightly compared to Group A, but compared to Group B, there was no statistically significant difference (for IL-2, P = 0.832;for IFN-gamma, P = 0.356). The spot level of IL-10 between all groups had no statistically significant difference (F = 2.911, P = 0.240). CONCLUSION: J2 could block up the process of antigen presentation by inhibiting CD4/major histocompatibility complex-II (MHC-II) moleculeonania, while secreting IL-2, IL-10 and IFN-gamma did not have specificity change.

[Prevention of mouse corneal allograft rejection by micromolecular compound J2].

OBJECTIVE: To investigate the effects of J2 on the prevention of corneal allograft rejection in mice. METHODS: Randomized control design was applied. One hundred C57BL/6 and thirty eight BALB/c mice were used as donors and recipients, respectively, to establish a corneal allograft model. These mice were randomly divided into A, B, C and D groups. There were 25 mice in each group. Group A, autograft control; Group B, allograft control (placebo was used in this group); Group C, allograft and treated with intraperitoneal ciclosporin A (CsA, 10 mg * kg(-1) * d(-1)) and Group D, allograft and treated with intraperitoneal J2 (15 mg * kg(-1) * d(-1)). All medications were applied on the day of transplantation and were continuously administrated for 12 days. Graft survival index, paraffin sections with HE staining and immunohistochemical staining of frozen sections were observed in each group 1, 2, 3 and 4 weeks after operation. IL-2 and IL-10 expression of cornea was detected by RT-PCR. One-Way ANOVO analysis was used to compare transplant survival time of the corneal allograft of each group. RESULTS: The average transplant survival time in the corneal allograft controls was (18.88 +/- 4.19) d. Treatment with J2 resulted in a significant increase of grafts' survival time (33.62 +/- 6.80) d (q = 0.85, P < 0.01). There was no significant difference between J2 and CsA groups. Histological examination showed that numerous lymphocytes infiltrated into the grafts 21 days after the operation in the control groups. Mild lymphocytes infiltration was found in both CsA and J2 groups. Immunohistochemical studies showed that large amount of CD4(+) and CD8(+) T-lymphocytes infiltrated into the grafts of the placebo group at 21 days. J2 and CsA groups only showed a small number of CD4(+) and CD8(+) T-lymphocytes in the grafts. RT-PCR showed that few IL-2 and IL-10 genes were expressed in the autologous transplantation group in the first week, and the expression of these two cytokines showed no significant increase during following three weeks. Both genes were expressed from the first week in the allogeneic transplantation group and significantly increased in the third week. Both genes were expressed from the first week in CsA and J2 group, expression was reduced in the second and third weeks. Expression of these cytokines in the fourth week was stronger than those in previous three weeks. CONCLUSION: J2 can inhibit corneal rejection in allograft mice model and its effect is similar to that of CsA.

Effects of prior freezing or drying on the swelling behaviour of the bovine cornea.

BACKGROUND: Frozen or dried corneal grafts are commonly used for stromal transplantation such as lamellar keratoplasty (full or partial thickness), keratophakia, epikeratophakia. Structural properties are important for the final optical results of these surgeries but the effects of freezing/thawing and drying/rehydration on the properties of the stroma are known little compared with the corneal endothelium, mainly because of lack of non-invasive technique to evaluate the stromal structure. This study aimed to investigate the swelling and structural properties of the bovine corneal stroma following freezing or drying by X-ray diffraction which was a non-invasive technique and could give ultra-structural information in hydrated tissues. METHODS: Bovine corneas were either frozen at -40 degrees C or dried to constant weight in a dessicator over silica gel. Swelling was carried out by placing the corneas into dialysis tubing and equilibrating them against various concentrations of polyethylene glycol (PEG) to obtain a range of tissue hydrations. This method minimises the loss of soluble tissue components during the swelling process. Synchrotron X-ray diffraction was used to measure the average intermolecular spacing, the interfibrillar spacing and the fibril diameter as a function of hydration. Changes in light scattering were detected using a microdensitometer. RESULTS: Freezing and thawing of the cornea caused an increase in light scattering by 63.9% at tissue hydration (H) = 3.4, and by 50.0% at H = 4.9. Repeated freezing and thawing causes further increased by 38.9% at the second time and another 36.0% at the third time (P < 0.05). There was a tendency for both the frozen and the dried corneas to lose some swelling ability, achieving hydrations respectively of 10% and 18% below those of fresh corneas at 0 PEG. There were no changes in the fibril diameters, interfibrillar or intermolecular spacings as measured by X-ray diffraction in the equilibrated fresh, pre-frozen and pre-dried corneas. CONCLUSIONS: The increase in light scattering and the loss of swelling ability after freezing and thawing probably results from structural changes following the close association of the collagen molecules and fibrils whilst the tissue is in the dry or frozen state. Some unknown changes in the extracellular matrix between the collagen fibrils may also play a role in the light scattering. The equilibration technique may improve the quality of rehydrated corneal graft or lenticules used for corneal surgeries.

[Clinical application of keratoprosthesis for corneal opacity caused by chemical burn unsuitable for keratoplasty].

OBJECTIVE: To evaluate the results of keratoprosthesis for eyes with complicated corneal opacities and unsuitable for keratoplasty due to the chemical burn. METHODS: Twenty-eight keratoprosthesis were implanted in 28 patients with bilateral blindness. Preoperative visual acuity (VA) was light perception in all operated eyes. The corneal opacities were caused by severe alkali burn (20 eyes) and, sulfate acid injury (8 eyes). The keratoprosthesis (MICOF.) were made by Moscow Eye Microsurgery Complex in Russia. Russia1 surgical techniques consisted of two stages: first stage, inserting a supporting titanium frame into the lamellar pocket and then, implanting an optical part 3 months later. RESULTS: Follow-up time ranged from 3 to 65 months (22.6 months on average). In 21 of 28 eyes (75%), postoperative VA ranged from 0.05 to 1.0 without correction. Corrected postoperative VA was: 11 eyes (39%) with VA from 0.6 to 1.2; 1 eye (4 %) from 0.3 to 0.5; 5 eyes (18%) from 0.05 to 0.25; 3 eyes (11%) with VA hand movement; 3 eyes (11%) with VA light perception and 1 eye (4%) with VA no light perception. CONCLUSION: Keratoprosthesis improves vision of corneal opacity patients with complicated conditions result caused by chemical burn, such as dense neovascularization and severe ocular surface disorders.