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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 in the left eye (OS). Moderate anterior segment inflammation was found, without any inflammatory findings in the posterior segment OS. A few days later, ocular inflammatory findings appeared including in the right eye (OD). Conjunctival and ciliary injections, 1+ flare and 3+ cells of anterior chamber inflammation, and mutton fat keratic precipitates were observed in both eyes (OU). In addition, redness and swelling of the optic disc were observed OU. Laboratory tests showed slightly high levels of soluble IL-2R and serum β2 microglobulin and markedly high levels of urinary β2 microglobulin. The diagnosis of probable TINU syndrome was established on the basis of presenting bilateral anterior uveitis and in accordance with a clinical criteria of tubulointerstitial nephritis. Oral prednisolone (PSL) at 20 mg/day was given, and ocular findings improved. The dose of PSL was gradually reduced and withdrawn, but ocular inflammation recurred OU. Retinal exudates and snowball vitreous opacities were observed in the peripheral retina. 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 multimodal imaging caused suspicion of a wide range of choroiditis; therefore, oral PSL was administered again. PSL was gradually tapered to 3 mg/day, and no anterior segment inflammation has been observed, but slight inflammatory findings in the posterior segment have persisted. Conclusions: We describe a case of TINU syndrome complicated by subclinical choroiditis detected with multimodal imaging. Further studies are necessary to determine the frequency of subclinical choroiditis in TINU syndrome.

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.

Concretions of the lacrimal drainage system can cause dacryocystitis. In the present study, our patient developed dacryocystisis with lacrimal concretions of a white soft mass. Rebamipide ophthalmic suspension had been applied for treatment of dry eye. To evaluate the pathogenic mechanism of the case, histological and chemical studies were performed. Our case was woman in her seventies. She had a medical history of rheumatoid arthritis. She was referred to our hospital for dacryocystitis after showing a poor response to treatment with antibiotic agents. A head computed tomography (CT) scan showed ductal high-density deposits along the lacrimal sacs. During dacryocystotomy, the hypertrophy of the lacrimal sacs was found replete with pus and cottage cheese-like white substances. The extracts were surgically removed, and histological and chemical analysis was performed. The histological examination showed granulation tissues and acellular amorphous material with crystal-like structures. Positive staining by Alcian blue and Kossa was found in crystal-like legions. The extract obtained from the concretions showed the same fluorescence band and UV absorption spectrum in thin layer chromatography (TLC) and spectrometry, respectively, as rebamipide. Our findings suggest that the obstruction by lacrimal concretions containing rebamipide resulted in dacryocystitis in this case. In addition, it is speculated that in patients who have impaired tear secretion, refractory dacryocystitis may be evoked following the accumulation of rebamipide in the lacrimal sacs.

The responsible genetic variants for occult macular dystrophy (OMD) were found at the predicted intrinsically disordered region (IDR) of the RP1L1 gene. We examined the phenotypes and genotypes of ...

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.

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).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.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.The reduced proportions of the S24-7 family and symbiotic Akkermansia species may be key phenomena in the oral flora of patients with BD.

Backgroud: Concretions of the lacrimal drainage system can cause dacryocystitis. In the present study, our patient developed dacryocystisis with lacrimal concretions of a white soft mass. Rebamipide ophthalmic suspension had been applied for treatment of dry eye. To evaluate the pathogenic mechanism of the case, histological and chemical studies were performed.Case presentation: Our case was woman in her seventies. She had a medical history of rheumatoid arthritis. She was referred to our hospital for dacryocystitis after showing a poor response to treatment with antibiotic agents. A head computed tomography (CT) scan showed ductal high-density deposits along the lacrimal sacs. During dacryocystotomy, the hypertrophy of the lacrimal sacs was found replete with pus and cottage cheese-like white substances. The extracts were surgically removed and histological and chemical analysis was performed. The histological examination showed granulation tissues and acellular amorphous material with crystal-like structures. Positive staining by Alcian blue and Kossa was found in crystal-like legions. The extract obtained from the concretions showed the same fluorescence band and UV absorption spectrum in thin layer chromatography (TLC) and spectrometry, respectively, as rebamipide.Conclusion: Our findings suggest that the obstruction by lacrimal concretions containing rebamipide resulted in dacryocystitis in this case. In addition, it is speculated that in patients who have impaired tear secretion, refractory dacryocystitis may be evoked following the accumulation of rebamipide in the lacrimal sacs.

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.

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, Akira Meguro et al. report a genome-wide association study for sarcoidosis—a systemic inflammatory disease—in the Japanese population. They identify 3 non-HLA loci with genome-wide significance, 2 of which have not been previously associated with sarcoidosis in any population.

Acute retinal necrosis (ARN) is a rapidly progressive and severe retinitis resulting in a poor visual outcome. Infections caused by herpes viruses such as herpes simplex virus (HSV) types 1 and 2 or the varicella zoster virus (VZV) are known to be implicated in the development of ARN. In the present study, an 80-year-old female with ARN was examined. She had been affected with rheumatoid arthritis and had taken methotrexate for over 10 years. Her right eye showed clinical features of ARN, and her left eye showed mild retinitis. The genomic DNA in the aqueous humor and vitreous fluid from her right eye were analyzed by a comprehensive polymerase chain reaction (PCR) assay to screen infectious pathogens including viruses. The Epstein-Barr virus (EBV) was detected from both specimens, but neither HSV or VZV nor cytomegalovirus was detected. She underwent intraocular surgery following systemic corticosteroid and acyclovir applications. However, although the retinitis of her right eye was extinguished, the final visual outcome was blindness due to optic nerve atrophy. There are few reports indicating that EBV is associated with ARN development. The present findings suggest that EBV alone can be the causative agent of ARN.

In the present study, two female patients with unilateral scleritis without systemic complications were examined. Both patients were suffering from ocular pain and received corticosteroid therapy. The first patient, a 45-year-old woman, was diagnosed with scleritis and iritis in her right eye. Topical corticosteroid treatment could eradicate the iritis but not the scleritis. Oral corticosteroid administration and corticosteroid pulse therapy were applied with little effect. The application of systemic cyclosporine had a satisfactory effect in controlling the scleritis. The other patient, a 60-year-old woman, was suffering from scleritis and lid swelling in her right eye. Not only did topical and systemic corticosteroid therapy prove insufficient, they also resulted in the elevation of her intraocular pressure. After termination of corticosteroid therapy, the systemic cyclosporine was applied orally. Subsequently, the patient's scleritis improved without any severe side effects. Scleritis is a painful and destructive inflammatory disease of the sclera that causes congestion of the scleral venous plexus. In this study, we have established a new grading system for assessing activity in scleritis that can score the extent of ocular pain and the area of congestion. This system provides a practical method for following the clinical course and monitoring the outcome of therapy. We report two cases of unilateral scleritis that were resistant to corticosteroid therapy but responsive to systemic administration of cyclosporine. Findings from these cases indicate that cyclosporine is an effective drug for controlling severe scleritis.

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.

To understand the role of N-glycosylation of lysosomal phospholipase A2 (LPLA2), four potential N-glycosylation sites in human LPLA2 (hLPLA2) were individually modified replacing asparagine (Asn) with alanine by site-direct mutagenesis. COS-7 cells transiently transfected with wild-type (WT) hLPLA2 gene produced catalytically active LPLA2. A single mutation at 273-, 289-, or 398-Asn partially reduced production of active LPLA2. A single mutation at 99-Asn and quadruple mutations at all four Asn sites resulted in a marked reduction of active LPLA2 and loss of active LPLA2, respectively. Western blot analysis using anti-hLPLA2 antibody showed that the LPLA2 expression level was similar between all transfectants. N-glycosidase F digestion revealed that multiple forms of LPLA2 found in individual transfectants are due to different N-glycans linked to the core protein. The LPLA2 activity in individual transfectants was mostly recovered in the soluble fraction and correlated to the quantity of LPLA2 detected in the soluble fraction. LPLA2 mutated at 99-Asn was mostly retained in the membrane fraction. The WT transfectants treated with tunicamycin markedly lost LPLA2 activity. These data indicate that the 99-Asn is the most critical N-glycosylation site for formation of native hLPLA2 in vivo and that the N-glycosylation of LPLA2 is crucial for biosynthesis of catalytically active hLPLA2.

Macrophages have long been known to secrete a Phospholipase A2 with an acidic pH optimum in response to phagocytic stimuli. However, the enzyme or enzymes responsible for this activity have not been identified. We report that mouse alveolar macrophages release lysosomal phospholipase A2 (LPLA2) into the medium of cultured cells following stimulation with zymosan. The release of the enzyme was detected by enzymatic activity assays as well as by Western blotting using an Ab against mouse LPLA2. LPLA2 is a high mannose type glycoprotein found in lysosomes, suggesting that the released enzyme might be reincorporated into alveolar macrophages via a mannose or mannose phosphate receptor. Recombinant glycosylated mouse LPLA2 produced by HEK293 cells was applied to LPLA2-deficient (LPLA2−/−) mouse alveolar macrophages. The uptake of exogenous LPLA2 into LPLA2−/− alveolar macrophages occurred in a concentration-dependent manner. The LPLA2 taken into the alveolar macrophages colocalized with the lysosomal marker, Lamp-1. This uptake was significantly suppressed in the presence of α-methyl-mannoside but not in the presence of mannose 6-phosphate. Thus, the predominant pathway for uptake of exogenous LPLA2 is via the mannose receptor, with subsequent translocation into acidic, Lamp-1-associated compartments. LPLA2−/− alveolar macrophages are characterized by marked accumulation of phosphatidylcholine and phosphatidylethanolamine. Treatment with the recombinant LPLA2 rescued the LPLA2−/− alveolar macrophages by markedly decreasing the phospholipid accumulation. The application of a catalytically inactive LPLA2 revealed that the enzymatic activity of LPLA2 was required for the phospholipid reduction. These studies identify LPLA2 as a high m.w.-secreted Phospholipase A2.

Ceramide-1-phosphate (C1P) is a novel bioactive sphingolipid formed by the phosphorylation of ceramide catalyzed by ceramide kinase (CERK). In this study, we evaluated the mechanism by which increased C1P during phagocytosis enhances phagocytosis and phagolysosome formation in COS-1 cells expressing hCERK. Stable transfectants of COS-1 cells expressing FcgammaRIIA or both FcgammaRIIA/hCERK expression vectors were created. Cell fractionation studies demonstrated that hCERK and the transient receptor potential channel (TRPC-1) were enriched in caveolae fractions. Our data establish that both CERK and TRPC-1 localize to the caveolar microdomains during phagocytosis and that CERK also colocalizes with EIgG in FcgammaRIIA/hCERK-bearing COS-1 cells. Using high-speed fluorescence microscopy, FcgammaRIIA/hCERK transfected cells displayed Ca2+ sparks around the phagosome. In contrast, cells expressing FcgammaRIIA under identical conditions displayed little periphagosomal Ca2+ signaling. The enhanced Ca2+ signals were accompanied by enhanced phagolysosome formation. However, the addition of pharmacological reagents that inhibit store-operated channels (SOCs) reduced the phagocytic index and phagolysosomal fusion in hCERK transfected cells. The higher Ca2+ signal observed in hCERK transfected cells as well as the fact that CERK colocalized with EIgG during phagocytosis support our hypothesis that Ca2+ signaling is an important factor for increasing phagocytosis and is regulated by CERK in a manner that involves SOCs/TRPCs.

Obesity is a serious problem that is growing in developed countries and many children as short-sleepers have been reported to be obese. However, no studies have been conducted on the relationship between BMI shown by mothers of children aged 1-6 years and their own circadian typology. This study aims to clarify this relationship in Japanese mothers. An integrated questionnaire on diurnal rhythms (including the Diurnal Type Scale constructed by Torsval & Akerstedt), sleep habits, meal habits, lighting conditions at night, weight and height was administered to mothers and their children aged 1-6 years. Sixty-two percent of 863 mothers answered the questionnaire for themselves and their children. All the obese mothers (BMI of 28 or more) had MEQ scores of less than 18 and were more evening-typed than non-obese mothers (BMI of less than 28) (p=0.02). A higher ratio of obese mothers watched TV after 11:00 p.m. (p=0.049) and used fluorescent lights while watching TV after 11:00 p.m. (p=0.023) compared to non-obese mothers. Obese mothers showed a higher frequency of depression (p=0.044) and tended to have higher frequency of becoming irritated (p=0.09) than non-obese mothers. The children of obese mothers were significantly more evening-typed (Mean±SD=18.5±3.6) than those of the other mothers (21.1±3.2) (p=0.018). Evening-typed life of mothers with watching mid-night TV would be related to obesity of mothers and children. This study concludes that obese mothers of children aged 1-6 years are evening-typed and the children of obese mothers are also more evening-typed than those of non-obese mothers

Lysosomal phospholipase A(2) (Lpla2) is highly expressed in alveolar macrophages and may mediate the phospholipid metabolism of surfactant. Studies on the properties of this phospholipase are consistent with the presence of both phospholipase A(1) and phospholipase A(2) activities. These activities were studied through the production of O-acyl compounds, produced by the transacylase activity of Lpla2. Liposomes containing POPC and N-acetylsphingosine (NAS) were incubated with the soluble fraction obtained from MDCK cells stably transfected with the mouse Lpla2 gene. Two 1-O-acyl-NASs, 1-O-palmitoyl-NAS and 1-O-oleoyl-NAS, were produced by Lpla2. The formation rate of 1-O-oleoyl-NAS was 2.5-fold that of 1-O-palmitoyl-NAS. When 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (OPPC) was used, the formation rate of 1-O-oleoyl-NAS was 5-fold higher than that of 1-O-palmitoyl-NAS. Thus, Lpla2 can act on acyl groups at both sn-1 and sn-2 positions of POPC and OPPC. When 1-palmitoyl-2-unsaturated acyl-sn-glycero-3-phosphocholines were used as acyl donors, the transacylation of the acyl group from the sn-2 position to NAS was preferred to that of the palmitoyl group from the sn-1 position. An exception was observed for 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC), for which the formation rate of 1-O-palmitoyl-NAS from PAPC was 4-fold greater than that of 1-O-arachidonoyl-NAS. Thus, Lpla2 has broad positional specificity for the sn-1 and sn-2 acyl groups in phosphatidylcholine and phosphatidylethanolamine.

The agonist-stimulated metabolism of membrane lipids produces potent second messengers that regulate phagocytosis. We studied whether human ceramide kinase (hCERK) activity and ceramide 1-phosphate formation could lead to enhanced phagocytosis through a mechanism involving modulation of the membrane-structural order parameter. hCERK was stably transfected into COS-1 cells that were stably transfected with the FcgammaRIIA receptor. hCERK-transfected cells displayed a significant increase in phagocytic index in association with increased ceramide kinase activation and translocation to lipid rafts after activation with opsonized erythrocytes. When challenged with opsonized erythrocytes, hCERK-transfected cells increased phagocytosis by 1.5-fold compared with vector control and simultaneously increased ceramide 1-phosphate levels 2-fold compared with vector and unstimulated control cells. Control and hCERK-transfected cells were subjected to cellular fractionation. Utilizing an antibody against hCERK, we observed that CERK translocates during activation from the cytosol to a lipid raft fraction. The plasma membrane-structural order parameter of the transfectants was measured by labeling cells with Laurdan. Cells transfected with hCERK showed a higher liquid crystalline order than control cells with stimulation, conditions that are favorable for the promotion of membrane fusion at the sites of phagocytosis. The change in the structural order parameter of the lipid rafts probably contributes to phagocytosis by promoting phagosome formation.

Lung surfactant is the surface-active agent comprised of phospholipids and proteins that lines pulmonary alveoli. Surfactant stabilizes the alveolar volume by reducing surface tension. Previously, we identified a lysosomal phospholipase A2, termed LPLA2, with specificity toward phosphatidylcholine and phosphatidylethanolamine. The phospholipase is localized to lysosomes, is calcium-independent, has an acidic pH optimum, and transacylates ceramide. Here, we demonstrate that LPLA2 is selectively expressed in alveolar macrophages but not in peritoneal macrophages, peripheral blood monocytes, or other tissues. Other macrophage-associated phospholipase A2s do not show a comparable distribution. LPLA2 is of high specific activity and recognizes disaturated phosphatidylcholine as a substrate. The lysosomal phospholipase A2 activity is six times lower in alveolar macrophages from mice with a targeted deletion of the granulocyte macrophage colony-stimulating factor (GM-CSF), a model of impaired surfactant catabolism, compared with those from wild-type mice. However, LPLA2 activity and protein levels are measured in GM-CSF null mice in which GM-CSF is expressed as a transgene under the control of the surfactant protein C promoter. Thus LPLA2 may be a major enzyme of pulmonary surfactant phospholipid degradation by alveolar macrophages and may be deficient in disorders of surfactant metabolism.

An acidic phospholipase A(2) (LPLA(2)) was recently purified and cloned. THP-1 cells were used to characterize the gene induction of LPLA(2). THP-1 cells were stimulated with several differentiation agents. The LPLA(2) mRNA and activity increased in cells treated with phorbol ester but not with vitamin D3, interferon-gamma, or granulocyte macrophage colony-stimulating factor. All-trans-retinoic acid enhanced mRNA expression and enzyme activity in a dose- and time-dependent manner. The natural 9-cis and 13-cis isomers of retinoic acid enhanced transcription and activity. Two classes of nuclear receptors, the retinoic acid receptor (RAR) and the retinoid X receptor (RXR), mediate retinoic acid signaling. Specific RAR and RXR agonists were used to identify the nuclear receptor responsible for LPLA(2) induction by retinoic acid. Treatment with the RAR agonist 4-[E-2-tetrahydro-5,5,8,8-tetra-methyl-2-naphthalenyl]1-propenyl benzoic acid (TTNPB) resulted in a small and statistically significant increase of the mRNA expression and activity of LPLA(2). The RXR agonist methoprene acid worked as well as all-trans-retinoic acid at increasing both mRNA and enzyme activity. The methoprene acid and TTNPB effects were not synergistic. The peroxisome proliferator-activated receptor gamma agonists 15-deoxy-Delta(12,14)-prostaglandin J(2) and troglitazone failed to induce LPLA(2) activity and mRNA. Thus, an RXR-dependent pathway controls LPLA(2) gene activation by retinoic acid in THP-1 cells.

Recently, a novel enzyme, 1-O-acylceramide synthase (ACS), was purified and characterized from bovine brain. This enzyme has both calcium-independent phospholipase A(2) and transacylase activities. The discovery of this enzyme led us to propose a new pathway for ceramide metabolism in which the sn-2-acyl group of either phosphatidylethanolamine or phosphatidylcholine is transferred to the 1-hydroxyl group of ceramide. In this study, the partial amino acid sequences from the purified enzyme revealed that the enzyme contains amino acid sequences identical to those of human lecithin:cholesterol acyltransferase-like lysophospholipase (LLPL). The coding sequences of the mouse, bovine, and human genes were obtained from the respective kidney cDNAs by PCR. The open reading frames of LLPL were cloned into pcDNA3 to generate carboxyl-terminally tagged proteins. The expression of mouse LLPL in COS-7 cells demonstrated that transfected cells had higher transacylase and phospholipase A(2) activities than did non-transfected cells. Immunoprecipitation confirmed that LLPL had ACS activity. There were no significant lecithin:cholesterol acyltransferase and lysophospholipase activities in the mouse LLPL-transfected cells under either acidic or neutral conditions. Amino acid sequences from cDNAs of mouse, human, and bovine LLPLs demonstrated a signal peptide cleavage site, one lipase motif (AXSXG), and several N-linked glycosylation sites in each LLPL molecule. The replacement of serine with alanine in the lipase motif of mouse LLPL resulted in elimination of enzyme activity, indicating that the serine residue is part of the catalytic site. Deglycosylation of mouse, human, and bovine LLPLs yielded core proteins with a molecular mass of 42 kDa without change in enzyme activities. LLPL was post-translationally modified by signal peptide cleavage and N-linked glycosylation, and each mature LLPL had the same size core protein. Subcellular fractionation demonstrated that ACS activity co-localized with N-acetylglucosaminidase. Therefore, LLPL encodes a novel lysosomal enzyme, ACS.

PURPOSE The purpose of the present study is to evaluate the effect of selective IKK-β inhibition by IMD-0354 on inflammation, apoptosis, and angiogenesis in diabetic retinopathy (DR). METHODS Six weeks after administration of a streptozotocin (STZ) injection, before diabetic retinopathy (DR) was evident, one group of STZ-induced diabetic mice was systemically administered with IMD-0354 (30 mg/kg) daily for another 6 weeks. Ten weeks after the STZ injection, with DR already present, another group of STZ-induced diabetic mice was administered IMD-0354 for 2 weeks. As controls, nondiabetic mice of the same age were treated with IMD-0354 for 6 weeks, and diabetic mice were treated with 10 μL of dimethyl sulfoxide (DMSO) for 6 weeks. Using these groups of mice, the following effects of IMD-0354 were analyzed: (1) inhibition of nuclear factor-κB (NF-κB) activation, (2) retinal morphology, (3) apoptotic signaling by cleaved caspase-3, (4) retinal vascular permeability, (5) angiogenesis of the retina, and (6) retinal production of VEGF. RESULTS Systemic administration of IMD-0354 for 6 weeks to week-6 diabetic mice caused significant reduction in the loss of retinal ganglion cells and apoptotic signaling, with preservation of retinal vascular integrity and suppression of retinal VEGF expression. When inhibition of NF-κB activation treatment started after the onset of STZ-induced DR (week 10), IMD-0354 was still effective in preventing further DR progression while the vascular integrity was preserved. CONCLUSIONS The present data indicate that NF-κB activation is the key step in the development of DR. Its suppression by IMD-0354 may present a promising therapeutic strategy for DR, especially in the early stages of the disease.

Reversible posterior leukoencephalopathy syndrome (RPLS) is induced by acute cerebral edema. Its symptoms include seizures, headache, altered mental status, and visual disturbances. The clinical and radiological findings are usually transient. This report describes a case of RPLS resulting in bilateral total blindness. A 40-year-old man presented with lethargy and bilateral visual loss. He had a 20-year history of hypertension, but had never been treated. On presentation, the left eye was able to perceive light, but the right eye was not. Radiological examination showed diffuse edema in the brain, and ocular fundus examination revealed severe bilateral hypertensive retinopathy. Antihypertensive therapy improved the patient's general condition, including blood pressure. Radiological findings 5 months later showed resolution of most of the abnormal signal areas. However, total blindness had developed in both eyes by day 15, and two courses of pulsed corticosteroid therapy failed to restore the visual loss.

X-Linked retinitis pigmentosa is a most severe and heterogeneous disorder of the retina. Recently, genes (RP2 and RPGR) from two X-linked loci have been positionally cloned and mutations have been identified in many families. To further evaluate allelic and non-allelic heterogeneity and the genotype — phenotype relationships, and to determine the prevalence of mutations in the gene, we have analyzed one previously unreported X-linked retinitis pigmentosa family, using a combination of haplotype analysis and DNA sequencing. Our extensive analysis of the RP2 gene failed to detect any disease — causing or polymorphic mutations. In the case of the RP3 gene, the alleles of the dinucleotide repeat marker did not segregate with the disease. Although we cannot completely exclude the possibility of the RP2 and RP3 genes as candidate genes, the above results suggest that structural and functional changes associated with the RP2 gene are not responsible for the phenotype in the family analyzed. Further identification of the X-linked genes may facilitate the elucidation of the molecular basis of the disorder in the family analyzed.

A lysosomal phospholipase A2, LPLA2, was recently characterized and shown to have substrate specificity for phosphatidylcholine and phosphatidylethanolamine. LPLA2 is ubiquitously expressed but is most highly expressed in alveolar macrophages. Double conditional gene targeting was employed to elucidate the function of LPLA2. LPLA2-deficient mice (Lpla2-/-) were generated by the systemic deletion of exon 5 of the Lpla2 gene, which encodes the lipase motif essential for the phospholipase A2 activity. The survival of the Lpla2-/- mice was normal. Lpla2-/- mouse mating pairs yielded normal litter sizes, indicating that the gene deficiency did not impair fertility or fecundity. Alveolar macrophages from wild-type but not Lpla2-/- mice readily degraded radiolabeled phosphatidylcholine. A marked accumulation of phospholipids, in particular phosphatidylethanolamine and phosphatidylcholine, was found in the alveolar macrophages, the peritoneal macrophages, and the spleens of Lpla2-/- mice. By 1 year of age, Lpla2-/- mice demonstrated marked splenomegaly and increased lung surfactant phospholipid levels. Ultrastructural examination of Lpla2-/- mouse alveolar and peritoneal macrophages revealed the appearance of foam cells with lamellar inclusion bodies, a hallmark of cellular phospholipidosis. Thus, a deficiency of lysosomal phospholipase A2 results in foam cell formation, surfactant lipid accumulation, splenomegaly, and phospholipidosis in mice.

Lysosomal phospholipase A2 (LPLA2) is an acidic phospholipase that is highly expressed in alveolar macrophages and that may play a role in the catabolism of pulmonary surfactant. The primary structure found in LCAT is conserved in LPLA2, including three amino acid residues potentially required for catalytic activity and four cysteine residues. LPLA2 activity was measured in COS-7 cells transfected with c-myc-conjugated mouse LPLA2 (mLPLA2) or mutated LPLA2. Single alanine substitutions in the catalytic triad resulted in the elimination of LPLA2 activity. Four cysteine residues (C65, C89, C330, and C371), conserved between LPLA2 and LCAT, were replaced with alanine. Quadruple mutations at C65, C89, C330, and C371, double mutations at C65 and C89, and a single mutation at C65 or C89 resulted in the elimination of activity. Double mutations at C330 and C371 and a single mutation at C330 or C371 resulted in a partial reduction of activity. Thus, the presence of a disulfide bond between C330 and C371 is not required for LPLA2 activity. We propose that one disulfide bond between C65 and C89 and free cysteine residues at C330 and C371 and the triad, serine-198, aspartic acid-360, and histidine-392, are required for the full expression of mLPLA2 activity.

Retinopathy of prematurity (ROP) is a leading cause of blindness in premature children. It is a multifactorial disorder which causes fibrovascular tissue changes that affect the retina in low birth-weight and short gestational age infants. To determine the prevalence of Norrie disease (ND) gene mutations, clinical examination and molecular genetic analyses were performed in 100 pre-term babies of different ethnic backgrounds who developed advanced ROP. The leukocyte DNA was extracted, amplified by the polymerase chain reaction (PCR), and analyzed by single-strand conformation polymorphism (SSCP), G/T and C/A scanning, and by DNA sequencing. All three exons, including splice sites and the 3′-untranslated region, were screened. Of the 100 patients analyzed, 2 patients with advanced ROP showed a mobility shift in the DNA. In 1 patient, this mobility shift was caused by the insertion of an additional 12-bp CT repeat in exon 1, and in the second patient, there was a 14-bp deletion in the same exon of the ND gene, as evidenced by direct sequencing of the amplified products. Similar analyses of exons 2 and 3 and the 3′-untranslated region failed to detect additional mutations in the gene. None of the 130 normal, unrelated controls revealed similar changes. Taking into account the above results, as well as those of other studies, it appears that the ND gene mutations can account for 3% of cases of advanced ROP. Although the ND gene is not frequently involved in advanced ROP, the present large-scale study further supports the hypothesis that genetic influences may play an important role in the development of severe ROP in some premature infants.

Juvenile retinoschisis (RS) and Norrie disease (ND) are X-linked recessive retinal disorders. Both disorders, in the majority of cases, are monogenic and are caused by mutations in the RS and ND genes, respectively. Here we report the identification of a family in which mutations in both the RS and ND genes are segregating with RS pathology. Although the mutations identified in this report were not functionally characterized with regard to their pathogenicity, it is likely that both of them are involved in RS pathology in the family analyzed. This suggests the complexity and digenic nature of monogenic human disorders in some cases. If this proves to be a widespread problem, it will complicate the strategies used to identify the genes involved in diseases and to develop methods for intervention.

Norrie disease (ND) is a rare X-linked recessive disorder characterized by congenital blindness, which is often associated with sensorineural hearing loss and mental retardation. X-linked familial exudative vitreoretinopathy (FEVR) is a hereditary disorder characterized by an abnormality of the peripheral retina and is not associated with systemic diseases. X-linked recessive disorders generally do not affect females. Here we show that female carriers can be associated with manifestation of an X-linked disorder.A four-generation family with an affected female, and a history of congenital blindness and hearing loss, was identified through the pro-band. A second family, with a full-term female infant, was evaluated through ophthalmic examinations and found to exhibit ocular features, such as retinal folds, retinal detachment and peripheral exudates. Peripheral blood specimens were collected from several affected and unaffected family members. DNA was extracted and analyzed by single-strand conformation polymorphism (SSCP) following polymerase chain reaction (PCR) amplification of the exons of the Norrie disease gene. The amplified products were sequenced by the dideoxy chain termination method.In an X-linked four-generation family, a novel missense (A118D) mutation in the third exon of the Norrie disease gene, was identified. The mutation was transmitted through three generations and cosegregated with the disease. The affected maternal grandmother and the unaffected mother carried the same mutation in one of their alleles. In an unrelated sporadic family, a heterozygous missense mutation (C96Y) was identified in the third exon of the Norrie disease gene in an affected individual. Analysis of exon-1 and 2 of the Norrie disease gene did not reveal any additional sequence alterations in these families. The mutations were not detected in the unaffected family members and the 116 normal unrelated controls, suggesting that they are likely to be the pathogenic mutations.The results further strengthen the proposal that X-linked disorders can occur in female carriers, due likely to an unfavorable X-inactivation.