Involvement of Angiopoietin 2 and vascular endothelial growth factor in uveitis.

PURPOSE: Angiopoietin (Ang) 2 is released from vascular endothelial cells by the stimulation of vascular endothelial growth factor (VEGF)A. Ang2 increases the expression of leukocyte adhesion molecules on endothelial cells via nuclear factor κB. The aim of this study was to evaluate the effects of Ang2 and VEGFA on ocular autoimmune inflammation. METHODS: We measured the concentrations of Ang2 and VEGFA in vitreous samples among patients with uveitis. Vitreous samples were collected from 16 patients with idiopathic uveitis (uveitis group) and 16 patients with non-inflammatory eye disease (control group). Experimental autoimmune uveoretinitis (EAU) was induced in B10.BR mice with a human interphotoreceptor retinoid-binding protein-derived peptide. The retinochoroidal tissues of the EAU mice were removed, and the mRNA levels of Ang2 and VEGFA were examined. EAU mice treated with anti-Ang2, anti-VEGFA, a combination of anti-Ang2 and anti-VEGFA, anti-Ang2/VEGFA bispecific, or IgG control antibodies were clinically and histopathologically evaluated. RESULTS: The protein levels of Ang2 and VEGFA were significantly higher in the vitreous samples of patients with uveitis than in controls (P<0.05). The retinochoroidal mRNA levels of Ang2 and VEGFA were significantly upregulated in EAU mice compared to controls (n = 6, P<0.05). Although there was no significant difference, treatment with anti-VEGFA antibody reduced the clinical and histopathological scores. However, treatment with anti-Ang2 antibody reduced the clinical and histopathological scores (n = 18-20, P<0.05). Furthermore, these scores were further decreased when treated by inhibiting both Ang2 and VEGFA. CONCLUSIONS: Based on these results, VEGFA and Ang2 were shown to be upregulated locally in the eye of both uveitis patients and models of uveitis. Dual inhibition of Ang2 and VEGFA is suggested to be a new therapeutic strategy for uveitis.

A case of tubulointerstitial nephritis and uveitis syndrome accompanied by subclinical choroiditis.

BACKGROUND: Tubulointerstitial nephritis and uveitis (TINU) syndrome is an uveits characterized by complications of idiopathic acute tubulointerstitial nephritis, and most cases present only anterior uveitis. We report a case of TINU syndrome in which the presence of choroiditis was revealed by multimodal imaging. CASE PRESENTATION: A 12-year-old male visited our hospital with a 6-day history of ocular pain and hyperemia. Conjunctival and ciliary injections, 1 + flare and 3 + cells of anterior chamber inflammation with mutton fat keratic precipitates were observed in both eyes (OU), together with redness and swelling of the optic disc OU. Laboratory tests showed slightly high levels of soluble IL-2R and serum ß2 microglobulin and markedly high levels of urinary ß2 microglobulin. The diagnosis of probable TINU syndrome was established on the basis of bilateral uveitis and urinalysis results in accordance with a clinical criteria of tubulointerstitial nephritis. With treatment with oral prednisolone (PSL) at 20 mg/day, ocular findings improved, and the dose of PSL was gradually reduced and withdrawn 6 months later. However, 1 month later from the withdrawal, ocular inflammation recurred with the presence of retinal exudates and snowball vitreous opacities in the peripheral retina OU. Fluorescein angiography showed leakages from peripheral retinal vessels and staining corresponding to retinal exudates. Indocyanine green angiography showed hypofluorescent dots scattered over the ocular fundus. Optical coherence tomography revealed the presence of choroidal thickening. Laser speckle flowgraphy color map showed a relatively cooler color. Findings from these multimodal images indicated the presence of subclinical choroiditis; therefore, oral PSL was administered again, and ocular inflammatory findings were improved. CONCLUSIONS: TINU syndrome can exhibit subclinical choroiditis detected with multimodal imaging. Further studies are necessary to determine the frequency of subclinical choroiditis in TINU syndrome.

A case of APMPPE-like panuveitis presenting with extensive outer retinal layer impairment following COVID-19 vaccination.

BACKGROUND: Vaccination against the worldwide pandemic coronavirus disease 2019 (COVID-19) is underway; however, some cases of new onset uveitis after vaccination have been reported. We report a case of bilateral acute posterior multifocal placoid pigment epitheliopathy-like (AMPPE-like) panuveitis after COVID-19 vaccination in which the patient's pathological condition was evaluated using multimodal imaging. CASE PRESENTATION: A 31-year-old woman experienced bilateral hyperemia and blurred vision starting 6 days after her second inoculation of the COVID-19 vaccination. At her first visit, her visual acuity was decreased bilaterally, and severe bilateral anterior chamber inflammation and bilateral scattering of cream-white placoid lesions on the fundus were detected. Optical coherence tomography (OCT) showed serous retinal detachment (SRD) and choroidal thickening in both eyes (OU). Fluorescein angiography (FA) revealed hypofluorescence in the early phase and hyperfluorescence in the late phase corresponding to the placoid legions. Indocyanine green angiography (ICGA) showed sharply marginated hypofluorescent dots of various sizes throughout the mid-venous and late phases OU. The patient was diagnosed with APMPPE and was observed without any medications. Three days later, her SRD disappeared spontaneously. However, her anterior chamber inflammation continued, and oral prednisolone (PSL) was given to her. Seven days after the patient's first visit, the hyperfluorescent lesions on FA and hypofluorescent dots on ICGA partially improved; however, the patient's best corrected visual acuity (BCVA) recovered only to 0.7 OD and 0.6 OS, and the impairment of the outer retinal layer was broadly detected as hyperautofluorescent lesions on fundus autofluorescence (FAF) examination and as irregularity in or disappearance of the ellipsoid and interdigitation zones on OCT, which were quite atypical for the findings of APMPPE. Steroid pulse therapy was performed. Five days later, the hyperfluorescence on FAF had disappeared, and the outer retinal layer improved on OCT. Moreover, the patient's BCVA recovered to 1.0 OU. Twelve months after the end of treatment, the patient did not show any recurrences. CONCLUSIONS: We observed a case of APMPPE-like panuveitis after COVID-19 vaccination featuring some atypical findings for APMPPE. COVID-19 vaccination may induce not only known uveitis but also atypical uveitis, and appropriate treatment is required for each case.

The Steroid-Sparing Effect of Adalimumab in the Treatment for the Recurrent Phase of Vogt-Koyanagi-Harada Disease.

PURPOSE: To reveal the steroid-sparing effect of adalimumab (ADA) in the treatment for the chronic recurrent phase of Vogt-Koyanagi-Harada (VKH) disease. CASES AND METHODS: Thirty-six eyes from 18 cases of the recurrent phase of VKH disease treated with ADA over 12 months were examined retrospectively. Before the introduction of ADA, 4 cases received prednisolone (PSL) monotherapy and other 14 cases received PSL and cyclosporine A (CYA) combination therapy. RESULTS: In cases treated with PSL and CYA, CYA was discontinued when ADA was introduced. The minimum dose of PSL to control intraocular inflammation (min dose of PSL) could be reduced in all cases after the introduction of ADA (from 16.9 ± 7.9 mg to 6.3 ± 3.1 mg). No serious adverse events were observed in the observational periods. CONCLUSION: By comparing the min dose of PSL before and after the introduction of ADA, the steroid-sparing effect of ADA was confirmed.

Clinical significance of serum and vitreous soluble interleukin-2 receptor in patients with intraocular lymphoma.

BACKGROUND: Intraocular lymphoma (IOL) is a masquerade syndrome that mimics uveitis, making diagnosis difficult. The serum soluble interleukin-2 receptor (sIL-2R), which is cleaved by matrix metalloproteinase (MMP) -2 and MMP-9, has been recognized as a tumor-related biomarker of malignant lymphomas. The aim of this study was to review the reliability of serum and vitreous sIL-2R for distinguishing IOL from uveitis. METHODS: Patients who underwent diagnostic vitrectomy for marked vitreous haze at Hokkaido University Hospital between April 2014 and June 2019 were enrolled. The patients were divided into an IOL group and a uveitis group, according to the pathology of their vitreous samples. The IOL group was further divided at the time of vitrectomy into patients who already had extraocular involvement (IOL with extraocular involvement group) and patients with no evidence of having extraocular involvement (IOL without extraocular involvement group). Serum sIL-2R, and intravitreal sIL-2R, MMP-2, and MMP-9 levels were assessed. RESULTS: Twenty-five eyes of 25 patients, and 15 eyes of 15 patients were included in the IOL group and uveitis group, respectively. The serum sIL-2R levels were significantly lower in the IOL group than in the uveitis group (P < 0.05), and 20.0% and 66.7% in the IOL and the uveitis group showed high sIL-2R value above the normal range. Vitreous sIL-2R tended to be higher in the IOL group than in the uveitis group (P = 0.80). Serum sIL-2R was significantly lower in the IOL without extraocular involvement group than in the IOL with extraocular involvement group (P < 0.05); 5.9% in the IOL without extraocular involvement group and 50.0% in the IOL with extraocular involvement group showed high sIL-2R value above the normal range. Vitreous sIL-2R, MMP-2, and MMP-9 tended to be higher in the IOL with extraocular involvement group than in the IOL without extraocular involvement group (P = 0.30, < 0.05, 0.16). CONCLUSIONS: Serum sIL-2R is often within the normal range in IOL patients. Even if it is within the normal range, the possibility of IOL should be considered. Serum sIL-2R is not a reliable biomarker for IOL, whereas vitreous sIL-2R may be useful for the diagnosis of IOL.

Fundus autofluorescence imaging in acute posterior multifocal placoid pigment epitheliopathy.

Purpose: Acute posterior multifocal placoid pigment epitheliopathy (APMPPE) is a disease characterized by multiple yellowish-white placoid lesions. Although most lesions resolve spontaneously, some turn into scars and lead to permanent visual dysfunction. In this report, we found suggestive findings in fundus autofluorescence (FAF) that may be useful for distinguishing severe lesions requiring treatment in APMPPE. Observation: Case 1: A 29-year-old woman was referred to our hospital with multiple yellowish-white placoid lesions on the fundi of both eyes (OU). FAF showed hyperautofluorescence in some of these placoid lesions. Based on the findings of fluorescein angiography, a diagnosis of APMPPE was established, and oral prednisolone (PSL) was initiated, given that some lesions were located in the macula. One week later, exacerbation occurred with the newly developed hyperautofluorescent lesions. Some lesions in the right eye (OD) that were hyperautofluorescent at the first visit became hypoautofluorescent. Afterward, although all hypoautofluorescent lesions persisted, most of the hyperautofluorescent lesions disappeared, so oral PSL could be stopped. Two months later, however, the recurrence occurred along with multiple new placoid lesions. Some lesions located at the macula were hyperautofluorescent on FAF OU, indicating the possibility of becoming scar lesions with hypoautofluorescence. Accordingly, oral PSL was given again. Case 2: A 47-year-old woman noticed decreased vision OD, and she was referred to us. Multiple yellowish-white placoid lesions were seen in the fundi OU. FAF showed hyperautofluorescence both with and without corresponding hypoautofluorescence in the placoid lesions OU. A diagnosis of APMPPE was established, and oral PSL was initiated. Four months later, some lesions that were hyperautofluorescent at the first visit had turned isoautofluorescent, and some lesions OU became hypoautofluorescent. However, all hypoautofluorescent lesions remained hypoautofluorescent OU. Only some hyperautofluorescent lesions recovered to isoautofluorescence without scars. Conclusions and Importance: In APMPPE, lesions showing hyperautofluorescence on FAF may change into hypoautofluorescence indicating scar formation. Therefore, the presence of hyperautofluorescent lesions in the macula may be a good indicator of the need for intensive corticosteroid treatments to avoid leaving hypoautofluorescent scars that are related to irreversible visual dysfunction.

Alteration of oral flora in Mongolian patients with Behçet's disease: a multicentre study.

OBJECTIVES: Behçet's disease (BD) is characterised by repeated acute inflammatory attacks with aphthous ulcers of the oral mucosa, uveitis of the eyes, skin symptoms, and genital ulcers. Although its aetiology is still unknown, there is evidence of the involvement of oral bacteria in systemic diseases. Various types of oral bacteria may be involved in the development and progression of BD. The present study investigated alterations in the oral flora of patients with BD in Mongolia. We collected saliva samples from the Mongolian BD group and healthy control (HC) group, and the oral flora were analysed using next-generation sequencer (NGS). METHODS: DNA was extracted from the unstimulated saliva samples from the 47 BD and 48 HC subjects. The DNA was amplified from the V3-V4 region of 16S rRNA using PCR, and the data were acquired using NGS. Based on the obtained data, we analysed the alpha diversity, beta diversity, and bacterial taxonomy of the salivary flora. RESULTS: Beta diversity differed significantly between the BD and HC flora, but no significant differences were observed in alpha diversity. We found that the proportions of three genera - an S24-7 family unknown species, a mitochondria family unknown species, and Akkermansia species associated with IL-10 production - were significantly lower in the BD than in the HC group. CONCLUSIONS: The reduced proportions of the S24-7 family and symbiotic Akkermansia species may be key phenomena in the oral flora of patients with BD.

A variant in the RP1L1 gene in a family with occult macular dystrophy in a predicted intrinsically disordered region.

SIGNIFICANCE: The responsible genetic variants for occult macular dystrophy (OMD) were found at the predicted intrinsically disordered region (IDR) of the RP1L1 gene. PURPOSE: We examined the phenotypes and genotypes of family members from OMD. In addition, the genetic characteristics of the RP1L1 gene in OMD were investigated. METHODS: Whole-exome sequencing was applied on two affected family members, and Sanger sequencing was performed on three members. The structural property of RP1L1 and pathogenic variants was analyzed using predictor of natural disordered regions (PONDR). RESULTS: Two affected members showed moderate visual impairment and relative central scotoma. The spectral domain optical coherence tomography (SD-OCT) images showed an absence of the interdigitation zone (IZ) and ellipsoid zone (EZ) in one case, and an obscure EZ line in the other case. A RP1L1 variant (c.3593 C > T, p.Ser1198Phe) was identified in two affected members but not in the unaffected member. The PONDR analysis showed that the region from p.1189 to p.1248 could be predicted to be an IDR in the RP1L1 molecule. And the p. Ser1198Phe variant showed significant reduction of PONDR score. CONCLUSIONS: Although, the major pathogenic variant of OMD is p.Arg45Trp, multiple reports indicate that the region between p.1194 and p.1201 is another hot spot of OMD. The PONDR analysis predicted that the RP1L1 molecule is one of the intrinsically disordered proteins. It is speculated that the region around p.1200 is essential for the normal function of the RP1L1 molecule, and the missense variants of that area cause the development of OMD.

Esterification of side-chain oxysterols by lysosomal phospholipase A2.

Side-chain oxysterols produced from cholesterol either enzymatically or non-enzymatically show various bioactivities. Lecithin-cholesterol acyltransferase (LCAT) esterifies the C3-hydroxyl group of these sterols as well as cholesterol. Lysosomal phospholipase A2 (LPLA2) is related to LCAT but does not catalyze esterification of cholesterol. First, esterification of side-chain oxysterols by LPLA2 was investigated using recombinant mouse LPLA2 and dioleoyl-PC/sulfatide/oxysterol liposomes under acidic conditions. TLC and LC-MS/MS showed that the C3 and C27-hydroxyl groups of 27-hydroxycholesterol could be individually esterified by LPLA2 to form a monoester with the C27-hydroxyl preference. Cholesterol did not inhibit this reaction. Also, LPLA2 esterified other side-chain oxysterols. Their esterifications by mouse serum containing LCAT supported the idea that their esterifications by LPLA2 occur at the C3-hydroxyl group. N-acetylsphingosine (NAS) acting as an acyl acceptor in LPLA2 transacylation inhibited the side-chain oxysterol esterification by LPLA2. This suggests a competition between hydroxycholesterol and NAS on the acyl-LPLA2 intermediate formed during the reaction. Raising cationic amphiphilic drug concentration or ionic strength in the reaction mixture evoked a reduction of the side-chain oxysterol esterification by LPLA2. This indicates that the esterification could progress via an interfacial interaction of LPLA2 with the lipid membrane surface through an electrostatic interaction. The docking model of acyl-LPLA2 intermediate and side-chain oxysterol provided new insight to elucidate the transacylation mechanism of sterols by LPLA2. Finally, exogenous 25-hydroxycholesterol esterification within alveolar macrophages prepared from wild-type mice was significantly higher than that from LPLA2 deficient mice. This suggests that there is an esterification pathway of side-chain oxysterols via LPLA2.

Genetic control of CCL24, POR, and IL23R contributes to the pathogenesis of sarcoidosis.

Sarcoidosis is a genetically complex systemic inflammatory disease that affects multiple organs. We present a GWAS of a Japanese cohort (700 sarcoidosis cases and 886 controls) with replication in independent samples from Japan (931 cases and 1,042 controls) and the Czech Republic (265 cases and 264 controls). We identified three loci outside the HLA complex, CCL24, STYXL1-SRRM3, and C1orf141-IL23R, which showed genome-wide significant associations (P < 5.0 × 10-8) with sarcoidosis; CCL24 and STYXL1-SRRM3 were novel. The disease-risk alleles in CCL24 and IL23R were associated with reduced CCL24 and IL23R expression, respectively. The disease-risk allele in STYXL1-SRRM3 was associated with elevated POR expression. These results suggest that genetic control of CCL24, POR, and IL23R expression contribute to the pathogenesis of sarcoidosis. We speculate that the CCL24 risk allele might be involved in a polarized Th1 response in sarcoidosis, and that POR and IL23R risk alleles may lead to diminished host defense against sarcoidosis pathogens.

An Increase of Oxidized Phospholipids and the Role of Macrophages in Intraocular Inflammation.

Purpose: The present study was conducted to examine the profile of oxidized phospholipids (OxPLs) in uveitis using rat model and clinical specimens, and to elucidate the role of macrophages in the metabolism of OxPLs. Methods: Lewis rats were immunized with a bovine interphotoreceptor retinoid- binding protein (bIRBP) peptide with complete Freund's adjuvant (CFA) to induce experimental autoimmune uveitis (EAU). The aqueous humor (AH) was collected 2 weeks after immunization. Fifty-four human AH specimens, among which 21 eyes had a history of chronic uveitis, were collected during their cataract surgery. The profile of OxPLs in the AH specimens were analyzed by liquid-chromatography tandem mass spectrometry (LC-MS/MS). In addition, the involvement of macrophages in the viability of cells treated by OxPLs was investigated through a WST-1 assay using ARPE-19 cells and C57BL/6 mouse alveolar macrophages (AMs). The influence of macrophages in the trend of OxPLs was traced by thin layer chromatography (TLC) using AMs. Results: Six species of OxPLs were detected in the AHs of rats and humans. The content of each OxPL was higher in the uveitis group. Four kinds of OxPLs found in AHs showed cytotoxicity to ARPE-19 cells in a dose-dependent manner. The cytotoxicity was reduced by pretreatment of OxPLs with AMs. When the OxPLs were applied on AMs, a marked reduction of OxPLs in the medium was observed. Conclusions: The OxPLs formed by intraocular inflammation could induce cytotoxicity. The present findings suggest that the phagocytic macrophages emerging in the inflammation site eliminate OxPLs, and prevent intraocular tissue damage following uveitis.

Severe visual impairment due to an optic neuropathy and central retinal vein occlusion in a sarcoidosis patient.

BACKGROUND: The ophthalmic manifestation of neurosarcoidosis is varied. The complication of optic neuropathy and central retinal vein occlusion (CRVO) is rare in sarcoidosis. CASE REPORT: The patient was a 55-year-old female with systemic sarcoidosis suffering from visual loss as hand motion in her left eye. A fundus examination showed severe optic disc head edema and hyperemia, and a central retinal vein occlusion phenotype including engorgement of all branches of the central retinal vein, dot, and flame-shaped hemorrhages. Brain magnetic resonance imaging (MRI) revealed irregular hypertrophy of the left retrobulbar optic nerve. She received several sets of pulse therapy with intravenous methylprednisolone. Although fundus findings of her left eye and the legion around the left retrobulbar optic nerve showed improvement, the final visual outcome was light perception due to optic nerve atrophy. CONCLUSIONS: Our findings suggest neurosarcoidosis of the unilateral retrobulbar optic nerve can cause compressive optic disc edema and resembles the central retinal vein occlusion (CRVO) phenotype.

Intraocular infiltration of Philadelphia chromosome-positive acute lymphoblastic leukemia diagnosed by polymerase chain reaction from the aqueous humor: A case report.

RATIONALE: Intraocular manifestation of hematopoietic tumors is rare and often difficult to distinguish from inflammation. We report a patient with acute lymphoblastic leukemia (ALL) who developed intraocular infiltration during the remission period. PATIENT CONCERNS: A 40-year-old man presented with hypopyon in his right eye. Three months later, extensive subretinal infiltration and the elevation of intraocular pressure were observed. Fourteen months prior to this, he had been diagnosed with Philadelphia chromosome-positive ALL, and had received chemotherapy and bone marrow transplantation that resulted in complete remission. DIAGNOSIS: The breakpoint cluster region-Ableson (BCR/ABL) chimera was detected by polymerase chain reaction (PCR) analysis in the patient's aqueous humor. Additionally, a high expression of WT1 (Wilms tumor gene) mRNA in the aqueous humor was discovered. A bone marrow examination yielded a high expression of BCR/ABL fusion gene, and it was determined the patient had experienced a relapse of ALL. INTERVENTIONS: The dasatinib was administered orally to the patient. OUTCOMES: The intraocular infiltration disappeared, and intraocular pressure was normalized. LESSONS: Intraocular infiltration in leukemia patients may be an indication of relapse regardless of systemic conditions. Analyzing mRNA expression of BCR/ABL and WT1 of ocular fluid in patients with hypopyon is beneficial in diagnosing topical relapses in leukemia.

Epidemiologic and Clinical Characteristics of Optic Neuritis in Japan.

PURPOSE: To elucidate the clinical and epidemiologic characteristics of optic neuritis in Japan. DESIGN: Multicenter cross-sectional, observational cohort study. PARTICIPANTS: A total of 531 cases of unilateral or bilateral noninfectious optic neuritis identified in 33 institutions nationwide in Japan. METHODS: Serum samples from patients with optic neuritis were tested for anti-aquaporin-4 antibodies (AQP4-Abs) and anti-myelin oligodendrocyte glycoprotein antibodies (MOG-Abs) using a cell-based assay and were correlated with the clinical findings. MAIN OUTCOME MEASURES: Antibody positivity, clinical and radiologic characteristics, and visual outcome. RESULTS: Among 531 cases of optic neuritis, 12% were AQP4-Ab positive, 10% were MOG-Ab positive, 77% were negative for both antibodies (double-negative), and 1 case was positive for both antibodies. Pretreatment visual acuity (VA) worsened to more than a median 1.0 logarithm of the minimum angle of resolution (logMAR) in all groups. After steroid pulse therapy (combined with plasmapheresis in 32% of patients in AQP4-Ab-positive group), median VA improved to 0.4 logMAR in the AQP4-Ab-positive group, 0 logMAR in the MOG-Ab-positive group, and 0.1 logMAR in the double-negative group. The AQP4-Ab-positive group showed a high proportion of females, exhibited diverse visual field abnormalities, and demonstrated concurrent spinal cord lesions on magnetic resonance imaging (MRI) in 22% of the patients. In the MOG-Ab-positive group, although posttreatment visual outcome was good, the rates of optic disc swelling and pain with eye movement were significantly higher than those in the AQP4-Ab-positive and double-negative groups. However, most cases showed isolated optic neuritis lesions on MRI. In the double-negative group, 4% of the patients had multiple sclerosis. Multivariate logistic regression analysis of all participants identified age and presence of antibodies (MOG-Ab and AQP4-Ab) as significant factors affecting visual outcome. CONCLUSIONS: The present large-scale cohort study revealed the clinicoepidemiologic features of noninfectious optic neuritis in Japan. Anti-aquaporin-4 antibody-positive optic neuritis has poor visual outcome. In contrast, MOG-Ab positive cases manifested severe clinical findings of optic neuritis before treatment, but few showed concurrent lesions in sites other than the optic nerve and generally showed good treatment response with favorable visual outcome. These findings indicate that autoantibody measurement is useful for prompt diagnosis and proper management of optic neuritis that tends to become refractory.

Visual Function in Mice Lacking GM3 Synthase.

Purpose: Most complex gangliosides in vertebrates are formed from ganglioside GM3. GM3 deficiency in humans can result in epilepsy and visual impairment. To investigate whether a deficiency of GM3 is involved in visual function, ST3GAL5-/- mice with mutations in the ST3GAL5 gene-coded GM3 synthase were employed. Materials and Methods: Sixty mice were employed in this study. The glycosphingolipids of mice retinas were analyzed through high performance thin layer chromatography. The morphology of the optic nerves and retinas were evaluated by hematoxylin and eosin staining and immunohistochemical analysis using an anti-glial fibrillary acidic protein (GFAP) antibody. An electroretinogram (ERG) was applied on the eyes of 4, 9, 12, and 14-month-old mice. Also, visual evoked potential (VEP) was applied on 13-month-old mice. Results: The GM3 in the retinas was detected in ST3GAL5+/+ mice but not ST3GAL5-/- mice. Also, GM1b and GD1α expressions and lactosylceramide accumulation were found in the ST3GAL5-/- mouse retinas. There was no significant difference in GFAP expression in the retinas or optic discs between ST3GAL5+/+ and ST3GAL5-/- mice. Furthermore, the outcome of ERG and VEP analysis showed no disparity between the two strains in 13 and 14-month-old mice. Conclusion: In the eye, neither histopathological abnormalities nor abnormal functions of the retina were found in GM3-deficient mice. Differing from the situation in patients with GM3 deficiency, the lack of GM3 in mice did not lead to optic nerve atrophy.

A fluorometric assay for lysosomal phospholipase A2 activity using fluorescence-labeled truncated oxidized phospholipid.

Lysosomal phospholipase A2 (LPLA2) is a key enzyme involved in the homeostasis of cellular phospholipids. Recently, LPLA2 was reported to preferentially degrade some truncated oxidized phospholipids at the sn-1 position. A commercially available, truncated oxidized phospholipid conjugated with a fluorescent dye, 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphoethanolamine-N-[4-(dipyrrometheneboron difluoride) butanoyl] (PGPE-BODIPY), was used to develop a specific assay for this enzyme. When recombinant mouse LPLA2 was incubated with liposomes consisting of 1,2-O-octadecyl-sn-glycero-3-phosphocholine/PGPE-BODIPY under acidic conditions, PGPE-BODIPY was converted to palmitic acid and a polar BODIPY-product. After phase partitioning by chloroform/methanol, the polar BODIPY-product was recovered in the aqueous phase and identified as 1-lyso-PGPE-BODIPY. The formation of 1-lyso-PGPE-BODIPY was quantitatively determined by fluorescent measurements. The Km and Vmax values of the recombinant LPLA2 for PGPE-BODIPY were 5.64 µM and 20.7 µmol/min/mg protein, respectively. Detectable activity against PGPE-BODIPY was present in LPLA2 deficient mouse sera, but the deacylase activity was completely suppressed by treatment with 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF). AEBSF had no effect on LPLA2 activity. The LPLA2 activity of mouse serum pre-treated with AEBSF was specifically and quantitatively determined by this assay method. The PGPE-BODIPY and AEBSF based LPLA2 assay is convenient and can be used to measure LPLA2 activity in a variety of biological specimens.

Visual impairment by multiple vascular embolization with hydroxyapatite particles.

We demonstrate a case of ocular impairment caused by a hydroxyapatite filler injection and review the prior literature on clinical presentations. A healthy woman, who received a hydroxyapatite filler injection into the glabella for nose augmentation suddenly had symptoms of nausea, diplopia, visual loss in the left eye, and impaired consciousness. Her left eye showed paresis of the inferior branch of the oculomotor nerve, conjunctival injection, cell infiltration in the anterior chamber, and multiple white spots in the nasal fundus. Purpura was detected in the area from the glabella to the left forehead. An orbital computed tomography (CT) scan demonstrated high-density deposits along vessels in the left medial orbit and forehead. Although her consciousness stabilized after a few days, the vision in her left eye deteriorated due to corneal edema and both hypopyon and hyphema in the anterior chamber, and the skin from the glabella to the left forehead developed necrosis. Multiple plaques were observed within the conjunctival and scleral vessels. After 2 months, diplopia and visual loss issues were mostly resolved. A histological examination of the conjunctiva specimen showed multiple foreign bodies plugged vessels that could be dissolved by decalcification. Recently, the number of complications by cosmetic filler injections has increased. The migrated hydroxyapatite particles in vessels cause multiple vascular emboli that can lead to various symptoms.

Preferential hydrolysis of truncated oxidized glycerophospholipids by lysosomal phospholipase A2.

Truncated oxidized glycerophospholipids (ox-PLs) are bioactive lipids resulting from oxidative stress. The catabolic pathways for truncated ox-PLs are not fully understood. Lysosomal phospholipase A2 (LPLA2) with phospholipase A and transacylase activities is a key enzyme in phospholipid homeostasis. The present study assessed whether LPLA2 could hydrolyze truncated ox-PLs. Incubation of LPLA2 with liposomes consisting of 1,2-O-octadecenyl-sn-glycero-3-phosphocholine (DODPC)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or truncated oxidized phosphatidylcholine (ox-PC)/N-acetylsphingosine (NAS) under acidic conditions resulted in the preferential deacylation at the sn-1 position of the truncated ox-PCs. Additionally, the release of free fatty acid from the truncated ox-PCs preferentially occurred compared with the NAS-acylation. Incubation of LPLA2 with the liposomes consisting of DODPC/DOPC/truncated ox-PC/NAS resulted in the same preferential fatty acid release from the truncated ox-PC. The cationic amphiphilic drug, amiodarone, did not inhibit such fatty acid release, indicating that truncated ox-PCs partition from the lipid membrane into the aqueous phase and react with free LPLA2. Consistent with this mechanism, the hydrolysis of some truncated ox-PCs, but not DOPC, by LPLA2 was detected at neutral pH. Additionally, LPLA2-overexpressed Chinese hamster ovary cells efficiently catabolized truncated ox-PC and were protected from growth inhibition. These findings support the existence of a novel catabolic pathway for truncated ox-PLs via LPLA2.

Prolonged Ocular Inflammation in Endotoxin-Induced Uveitis in Lysosomal Phospholipase A2-Deficient Mice.

PURPOSE: The goal of present study was to elucidate the pathophysiological roles of lysosomal phospholipase A2 (LPLA2) in intraocular pressure (IOP) levels and ocular inflammation. METHODS: C57BL/6 (wild-type) and LPLA2-deficient mice with C57BL/6 background were employed. The IOPs were compared between wild-type and LPLA2-deficient mice during their aging, after topical administration of antiglaucoma medications such as travoprost, dorzolamide, or timolol maleate, or after induction of endotoxin-induced uveitis (EIU) using lipopolysaccharide (LPS). Concerning the EIU, ocular inflammation was also evaluated by immunohistochemical analysis by the anti-glial fibrillary acidic protein (GFAP) antibody. RESULTS: The LPLA2-deficient mice showed higher IOP levels than the wild-type mice until 2 months of age (P = 1.60E-06); in older mice there was no difference between the two groups. Significant differences in the IOP changes between groups in young mice were seen after administration of 0.5% timolol (P < 0.05). Upon induction of EIU by LPS, compared with wild-type mice (P < 0.05), IOPs were significantly elevated in LPLA2-deficient mice at maximum levels of the ocular inflammation (48 h). Immunohistochemical analysis indicated that LPLA2-deficient mice showed more prolonged expression of GFAP at the inner plexiform layer and inner nuclear layer by EIU than that found in the wild-type mice (P < 0.05). CONCLUSIONS: These results confirm that LPLA2 plays a significant role in the control of IOP during mouse ocular development or with ocular inflammation by facilitating the digestion of intraocular insoluble materials.

Fluctuation of lysosomal phospholipase A2 in experimental autoimmune uveitis in rats.

Intraocular inflammation leads to oxidative stress and may generate lipid oxidation products. The present study was conducted to elucidate the pathophysiological roles of the lysosomal phospholipase A2 (LPLA2), a phospholipid-degrading enzyme, and the production of oxidized phospholipids (oxPLs) in autoimmune uveitis using a rat model. Lewis rats were immunized with a bovine interphotoreceptor retinoid-binding protein (bIRBP) peptide with complete Freund's adjuvant (CFA) to induce experimental autoimmune uveitis (EAU). The aqueous humor (AH) and serum were collected every week for 4 weeks from the immunized rats. The LPLA2 activity of the AH and serum was detected using liposomes consisting of 1,2-dioleoylphosphatidylglycerol/N-acetylsphingosine as the substrate under acidic conditions. Immunohistochemical analysis was performed using antibodies against LPLA2 and oxPLs. The ocular inflammation was exacerbated at 2 weeks after immunization. The LPLA2 activity in the rat AH was increased by EAU induction, and was concomitant with the extent of inflammation in the anterior chamber (AC). In contrast, the LPLA2 activity in the rat serum was not influenced by EAU induction. At 2 weeks after immunization, immunoreactivity of LPLA2 was observed in infiltrated macrophages in the AC and vitreous cavity of the EAU rats. Furthermore, immunoreactivity of oxPLs was observed in the infiltrated macrophages of EAU rat eyes. These results demonstrated that the LPLA2 activity of the AH is augmented with the inflammation in the AC. The high expression of LPLA2 and production of oxPLs are found in the infiltrated macrophages in the acute inflammation of EAU rats. The present findings suggest the connection between LPLA2 activity and oxPL metabolism in the inflammation sites in the eye.