A Complex-Type N-Glycan-Specific Lectin Isolated from Green Alga Halimeda borneensis Exhibits Potent Anti-Influenza Virus Activity.

Marine algal lectins specific for high-mannose N-glycans have attracted attention because they strongly inhibit the entry of enveloped viruses, including influenza viruses and SARS-CoV-2, into host cells by binding to high-mannose-type N-glycans on viral surfaces. Here, we report a novel anti-influenza virus lectin (named HBL40), specific for complex-type N-glycans, which was isolated from a marine green alga, Halimeda borneensis. The hemagglutination activity of HBL40 was inhibited with both complex-type N-glycan and O-glycan-linked glycoproteins but not with high-mannose-type N-glycan-linked glycoproteins or any of the monosaccharides examined. In the oligosaccharide-binding experiment using 26 pyridylaminated oligosaccharides, HBL40 only bound to complex-type N-glycans with bi- and triantennary-branched sugar chains. The sialylation, core fucosylation, and the increased number of branched antennae of the N-glycans lowered the binding activity with HBL40. Interestingly, the lectin potently inhibited the infection of influenza virus (A/H3N2/Udorn/72) into NCI-H292 cells at IC50 of 8.02 nM by binding to glycosylated viral hemagglutinin (KD of 1.21 × 10-6 M). HBL40 consisted of two isolectins with slightly different molecular masses to each other that could be separated by reverse-phase HPLC. Both isolectins shared the same 16 N-terminal amino acid sequences. Thus, HBL40 could be useful as an antivirus lectin specific for complex-type N-glycans.

Auto-Lectin Dotcoding by Two Octopuses: Rapid Analysis of Fluorescence-Labeled Glycoproteins by an 8-channel Fully-Automatic Bead Array Scanner with a Rolling-Circle Detector.

Protein glycosylation is a crucial factor that must be evaluated in biological pharmaceuticals. The glycoform profile of a protein can vary depending on the conditions of the cultivation, purification process, and the selection of a host cell. Lectin microarrays are reliable bioanalytical methods used in the early phases of bioprocesses for the detection of glycosylation. The concept of a fully automated glycan detection with a bead array has been previously reported; however, no simple system has been constructed on fluorescence-based detection using a microarray. Here, we present a fully automated detection system equipped with a novel fluorescence detector for a 13-lectin bead array with a single tip. The lattice-like arrangement of a set of fibers proximate to the tip of the light emitting diode and photomultiplier tube detector minimized the noise caused by the reflection of incident light on the plastic capillary tip and bead. A unique rolling-circle fiber unit with quadruple lattices stacked in two layers realizes the 8-parallel automeasurement with a drastic reduction in scanning time and machine size. The 8-glycan profiles obtained automatically within 25 min were identical with those obtained with the conventional lectin microarray after overnight incubation. The signals obtained were represented as lectin dotcodes. Therefore, autolectin dotcoding assisted by the twin 8 legs named as "detection and irradiation octopuses" may be a rapid glyco-evaluation system during the production and development of biopharmaceuticals.

Capture and neutralization of SARS-CoV-2 and influenza virus by algae-derived lectins with high-mannose and core fucose specificities.

We first investigated the interactions between several algae-derived lectins and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). We created lectin columns using high-mannose (HM)-type glycan-specific lectins OAA and KAA-1 or core fucose-specific lectin hypninA-2 and conducted binding experiments with SARS-CoV-2. The results showed that these lectins were capable of binding to the virus. Furthermore, when examining the neutralization ability of nine different lectins, it was found that KAA-1, ESA-2, and hypninA-2 were effective in neutralizing SARS-CoV-2. In competitive inhibition experiments with glycoproteins, neutralization was confirmed to occur through HM-type or core fucose-type glycans. However, neutralization was not observed with other lectins, such as OAA. This trend of KAA-1 and ESA-2 having the neutralizing ability and OAA not having it was also similar to influenza viruses. Electron microscopy observations revealed that KAA-1 and hypninA-2 strongly aggregated SARS-CoV-2 particles, while OAA showed a low degree of aggregation. It is believed that the neutralization of SARS-CoV-2 involves multiple factors, such as glycan attachment sites on the S protein, the size of lectins, and their propensity to aggregate, which cause inhibition of receptor binding or aggregation of virus particles. This study demonstrated that several algae-derived lectins could neutralize SARS-CoV-2 and that lectin columns can effectively recover and concentrate the virus.

A marine sponge-derived lectin reveals hidden pathway for thrombopoietin receptor activation.

N-glycan-mediated activation of the thrombopoietin receptor (MPL) under pathological conditions has been implicated in myeloproliferative neoplasms induced by mutant calreticulin, which forms an endogenous receptor-agonist complex that traffics to the cell surface and constitutively activates the receptor. However, the molecular basis for this mechanism is elusive because oncogenic activation occurs only in the cell-intrinsic complex and is thus cannot be replicated with external agonists. Here, we describe the structure and function of a marine sponge-derived MPL agonist, thrombocorticin (ThC), a homodimerized lectin with calcium-dependent fucose-binding properties. In-depth characterization of lectin-induced activation showed that, similar to oncogenic activation, sugar chain-mediated activation persists due to limited receptor internalization. The strong synergy between ThC and thrombopoietin suggests that ThC catalyzes the formation of receptor dimers on the cell surface. Overall, the existence of sugar-mediated MPL activation, in which the mode of activation is different from the original ligand, suggests that receptor activation is unpredictably diverse in living organisms.

The OAAH Family: Anti-Influenza Virus Lectins.

High mannose (HM)-binding Oscillatoria agardhii agglutinin homologue (OAAH) lectin family is an important class of anti-viral proteins. The OAAH family lectins show potent anti-influenza virus activity with EC50 of nanomolar levels by binding to HM glycans of the envelope glycoprotein hemagglutinin (HA), thereby inhibiting the viral entry into host cells. No broadly effective neutralizing vaccines for influenza virus are available due to the frequent antigenic drift caused by rapid mutations. Alternatives for vaccines need to be developed to prepare for a possible risk of future emergence of a highly virulent virus. Possible use of antiviral lectins is a simple and useful strategy to prevent viral infection by interfering with the interaction between viral HA and the host sialic acid-containing receptor. High-density glycans of surface HA are primary targets for the lectins to inhibit viral entry. In general, the anti-influenza virus potency of lectins is evaluated by a series of inhibitory assays for infection, such as neutral red dye uptake assay to determine the extent of viral cytopathic effect, and immunofluorescence microscopy to detect the expression of viral proteins in infected cells. Direct interaction between lectins and HA could be evaluated by enzyme-linked immunosorbent assay or surface plasmon resonance analysis.

Application of high-mannose-type glycan-specific lectin from Oscillatoria Agardhii for affinity isolation of tumor-derived extracellular vesicles.

Tumor cells secrete membrane vesicles of various sizes, termed extracellular vesicles (EVs), which have gained increasing attention as potential tumor diagnostic markers. Tumor-derived EVs are enriched with high-mannose-type glycans. Here, we report the affinity isolation of EVs from human melanoma A375 cells by using high-mannose-type glycan-specific agglutinin from Oscillatoria Agardhii (OAA). Glycan analysis of melanoma EVs revealed the presence of high-mannose-type glycans with structural units preferred by OAA. We showed that in solution, OAA binds to melanoma EVs in a high-mannose-type glycan-dependent manner. Furthermore, OAA-immobilized beads were found to capture 60% of the particles and most proteinous components from melanoma EVs. Major EV glycoproteins that potentially interact with OAA were identified to be cluster of differentiation 109 (CD109), integrin α6 and a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10). In addition to melanoma EVs, OAA captured EVs from human lung cancer, glioblastoma and colon cancer cells, but not those from endothelial cells and fibroblasts. These results indicate that OAA-immobilized beads may serve as a novel platform for affinity-capture of tumor-derived EVs.

Critical Functionality Effects from Storage Temperature on Human Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium Cell Suspensions.

Human induced pluripotent stem cell (hiPSC)-derived retinal pigment epithelium (hiPSC-RPE) cells suspension have the potential for regenerative treatment. However, practical regenerative applications with hiPSC-RPE cells require the development of simple and cost-effective non-freezing preservation methods. We investigated the effect of non-freezing temperatures on suspended hiPSC-RPE cells in various conditions and analysed mechanisms of cell death, anoikis, Rho GTPases, hypoxia, microtubule destruction, and cell metabolism. Cells stored at 37 °C had the lowest viability due to hypoxia from high cell metabolism and cell deposits, and cells preserved at 4 °C were damaged via microtubule fragility. Cell suspensions at 16 °C were optimal with drastically reduced apoptosis and negligible necrosis. Moreover, surviving cells proliferated and secreted key proteins normally, compared to cells without preservation. hiPSC-RPE cell suspensions were optimally preserved at 16 °C. Temperatures above or below the optimal temperature decreased cell viability significantly yet differentially by mechanisms of cell death, cellular metabolism, microtubule destruction, and oxygen tension, all relevant to cell conditions. Surviving cells are expected to function as grafts where high cell death is often reported. This study provides new insight into various non-freezing temperature effects on hiPSC-RPE cells that are highly relevant to clinical applications and may improve cooperation between laboratories and hospitals.

A simple and static preservation system for shipping retinal pigment epithelium cell sheets.

The ability to move cells and tissues from bench to bedside is an essential aspect of regenerative medicine. In this study, we propose a simple and static shipping system to deliver tissue-engineered cell sheets. Notably, this system is electronic-device-free and simplified to minimize the number of packing and opening steps involved. Shipping conditions were optimized, and application and verification of the system were performed using human iPS cell-derived or fetal retinal pigment epithelium (RPE) cell sheets. The temperature of the compartments within the insulated container was stable at various conditions, and filling up the cell vessel with medium effectively prevented turbulence-induced mechanical damage to the RPE cell sheets. Furthermore, no abnormal changes were observed in RPE morphology, transepithelial electrical resistance, or mRNA expression after transit by train and car. Taken together, our simple shipping system has the potential to minimize the costs and human error associated with bench to bedside tissue transfer. This specially designed regenerative tissue shipping system, validated for use in this field, can be used without any special training. This study provides a procedure for easily sharing engineered tissues with the goal of promoting collaboration between laboratories and hospitals and enhancing patient care.

A Novel High-Mannose Specific Lectin from the Green Alga Halimeda renschii Exhibits a Potent Anti-Influenza Virus Activity through High-Affinity Binding to the Viral Hemagglutinin.

We have isolated a novel lectin, named HRL40 from the green alga Halimeda renschii. In hemagglutination-inhibition test and oligosaccharide-binding experiment with 29 pyridylaminated oligosaccharides, HRL40 exhibited a strict binding specificity for high-mannose N-glycans having an exposed (α1-3) mannose residue in the D2 arm of branched mannosides, and did not have an affinity for monosaccharides and other oligosaccharides examined, including complex N-glycans, an N-glycan core pentasaccharide, and oligosaccharides from glycolipids. The carbohydrate binding profile of HRL40 resembled those of Type I high-mannose specific antiviral algal lectins, or the Oscillatoria agardhii agglutinin (OAA) family, which were previously isolated from red algae and a blue-green alga (cyanobacterium). HRL40 potently inhibited the infection of influenza virus (A/H3N2/Udorn/72) into NCI-H292 cells with half-maximal effective dose (ED50) of 2.45 nM through high-affinity binding to a viral envelope hemagglutinin (KD, 3.69 × 10-11 M). HRL40 consisted of two isolectins (HRL40-1 and HRL40-2), which could be separated by reverse-phase HPLC. Both isolectins had the same molecular weight of 46,564 Da and were a disulfide -linked tetrameric protein of a 11,641 Da polypeptide containing at least 13 half-cystines. Thus, HRL40, which is the first Type I high-mannose specific antiviral lectin from the green alga, had the same carbohydrate binding specificity as the OAA family, but a molecular structure distinct from the family.

High-Mannose Specific Lectin and Its Recombinants from a Carrageenophyta Kappaphycus alvarezii Represent a Potent Anti-HIV Activity Through High-Affinity Binding to the Viral Envelope Glycoprotein gp120.

We previously reported that a high-mannose binding lectin KAA-2 from the red alga Kappaphycus alvarezii, which is an economically important species and widely cultivated as a source of carrageenans, had a potent anti-influenza virus activity. In this study, the full-length sequences of two KAA isoforms, KAA-1 and KAA-2, were elucidated by a combination of peptide mapping and complementary DNA (cDNA) cloning. They consisted of four internal tandem-repeated domains, which are conserved in high-mannose specific lectins from lower organisms, including a cyanobacterium Oscillatoria agardhii and a red alga Eucheuma serra. Using an Escherichia coli expression system, an active recombinant form of KAA-1 (His-tagged rKAA-1) was successfully generated in the yield of 115 mg per liter of culture. In a detailed oligosaccharide binding analysis by a centrifugal ultrafiltration-HPLC method with 27 pyridylaminated oligosaccharides, His-tagged rKAA-1 and rKAA-1 specifically bound to high-mannose N-glycans with an exposed α1-3 mannose in the D2 arm as the native lectin did. Predicted from oligosaccharide binding specificity, a surface plasmon resonance analysis revealed that the recombinants exhibit strong interaction with gp120, a heavily glycosylated envelope glycoprotein of HIV with high association constants (1.48 - 1.61 × 10(9) M(-1)). Native KAAs and the recombinants inhibited the HIV-1 entry at IC50s of low nanomolar levels (7.3-12.9 nM). Thus, the recombinant proteins would be useful as antiviral reagents targeting the viral surface glycoproteins with high-mannose N-glycans, and the cultivated alga K. alvarezii could also be a good source of not only carrageenans but also this functional lectin(s).

High-Mannose Specific Lectin and Its Recombinants from a Carrageenophyta Kappaphycus alvarezii Represent a Potent Anti-HIV Activity Through High-Affinity Binding to the Viral Envelope Glycoprotein gp120.

We previously reported that a high-mannose binding lectin KAA-2 from the red alga Kappaphycus alvarezii, which is an economically important species and widely cultivated as a source of carrageenans, had a potent anti-influenza virus activity. In this study, the full-length sequences of two KAA isoforms, KAA-1 and KAA-2, were elucidated by a combination of peptide mapping and cDNA cloning. They consisted of four internal tandem-repeated domains, which are conserved in high-mannose specific lectins from lower organisms, including a cyanobacterium Oscillatoria agardhii and a red alga Eucheuma serra. Using an Escherichia coli expression system, an active recombinant form of KAA-1 (His-tagged rKAA-1) was successfully generated in the yield of 115 mg per a litter of culture. In a detailed oligosaccharide binding analysis by a centrifugal ultrafiltration-HPLC method with 27 pyridylaminated oligosaccharides, His-tagged rKAA-1 and rKAA-1 specifically bound to high-mannose N-glycans with an exposed α1-3 mannose in the D2 arm as the native lectin did. Predicted from oligosaccharide-binding specificity, a surface plasmon resonance analysis revealed that the recombinants exhibit strong interaction with gp120, a heavily glycosylated envelope glycoprotein of HIV with high association constants (1.48-1.61 × 10(9) M(-1)). Native KAAs and the recombinants inhibited the HIV-1 entry at IC50s of low nanomolar levels (7.3-12.9 nM). Thus, the recombinant proteins would be useful as antiviral reagents targeting the viral surface glycoproteins with high-mannose N-glycans, and the cultivated alga K. alvarezii could also be a good source of not only carrageenans but also this functional lectin(s).

Roles of Epithelial Cell-Derived Type 2-Initiating Cytokines in Experimental Allergic Conjunctivitis.

PURPOSE: To clarify the possible involvement of the type 2-initiating cytokines interleukin (IL)-25, IL-33, and thymic stromal lymphopoietin (TSLP) in the pathophysiology of allergic conjunctivitis, we evaluated ragweed (RW)-induced experimental allergic conjunctivitis (EAC) models by using IL-25 knockout (KO), IL-33 KO, and TSLP receptor (TSLPR) KO mice. METHODS: Interleukin-25 KO, IL-33 KO, TSLPR KO, and BALB/c wild-type mice were sensitized twice with RW in alum and then challenged with RW in eye drops. Clinical scores and eosinophil infiltration were evaluated. Expression levels of serum immunoglobulin E (IgE) and cytokines in the conjunctival tissues were quantified and immunohistochemical analysis was carried out. RESULTS: Significant reductions in clinical scores and numbers of infiltrating eosinophils were observed in the RW-EAC model using IL-33 KO mice. There were no significant differences in clinical scores and numbers of infiltrating eosinophils among IL-25KO, TSLPR KO, and wild-type mice. Serum IgE concentration was upregulated after RW challenges, and there were no differences among the mouse genotypes. Expression levels of of il4, il5, il13, and ccl5 mRNA were diminished in the conjunctivae of the RW-EAC model using IL-33 KO mice compared to those in wild-type mice. Interleukin-33 expression was upregulated as early as 1 hour after RW eye-drop challenge. The number of infiltrating basophils in the conjunctivae of the RW-EAC model using IL-33 KO mice was diminished compared to that in wild-type mice. CONCLUSIONS: Among the type 2-initiating cytokines, IL-33 may play a major role in conjunctival inflammation in an RW-EAC model.

Entry Inhibition of Influenza Viruses with High Mannose Binding Lectin ESA-2 from the Red Alga Eucheuma serra through the Recognition of Viral Hemagglutinin.

Lectin sensitivity of the recent pandemic influenza A virus (H1N1-2009) was screened for 12 lectins with various carbohydrate specificity by a neutral red dye uptake assay with MDCK cells. Among them, a high mannose (HM)-binding anti-HIV lectin, ESA-2 from the red alga Eucheuma serra, showed the highest inhibition against infection with an EC50 of 12.4 nM. Moreover, ESA-2 exhibited a wide range of antiviral spectrum against various influenza strains with EC50s of pico molar to low nanomolar levels. Besides ESA-2, HM-binding plant lectin ConA, fucose-binding lectins such as fungal AOL from Aspergillus oryzae and AAL from Aleuria aurantia were active against H1N1-2009, but the potency of inhibition was of less magnitude compared with ESA-2. Direct interaction between ESA-2 and a viral envelope glycoprotein, hemagglutinin (HA), was demonstrated by ELISA assay. This interaction was effectively suppressed by glycoproteins bearing HM-glycans, indicating that ESA-2 binds to the HA of influenza virus through HM-glycans. Upon treatment with ESA-2, no viral antigens were detected in the host cells, indicating that ESA-2 inhibited the initial steps of virus entry into the cells. ESA-2 would thus be useful as a novel microbicide to prevent penetration of viruses such as HIV and influenza viruses to the host cells.

Purification, primary structure, and biological activity of the high-mannose N-glycan-specific lectin from cultivated Eucheuma denticulatum.

Three isolectins from cultivated Eucheuma denticulatum were isolated. They were commonly monomeric proteins of about 28 kDa with a range of averaged molecular weights from 27,834 to 27,868 Da among the isolectins and shared almost the same 20 N-terminal amino acid sequences. Complementary DNA (cDNA) cloning based on the rapid amplification cDNA ends (RACE) methods elucidated the full-length sequence of EDA-2 which encodes 269 amino acids, including initiating methionine, with four tandemly repeated domains of about 67 amino acids. The primary structure of EDA-2 is highly similar to those of the high-mannose N-glycan specific lectins including Oscillatoria agardhii (OAA) and Burkholderia oklahomensis EO147 (BOA) from cyanobacteria, Myxococcus xanthus (MBHA) and Pseudomonas fluorescens Pf0-1 (PFL) from bacteria, and ESA-2 from a macro red alga. The hemagglutination activities were commonly inhibited by the glycoproteins bearing high-mannose N-glycans, but not by monosaccharides examined, including mannose. In a direct binding experiment with pyridylaminated oligosaccharides, an isolectin EDA-2 exclusively bound to high-mannose type N-glycans, but not to other glycans that include complex types and a core pentasaccharide of N-glycans, indicating that it recognized the branched oligomannoside moiety. Its binding activity was subtly different among the oligomannoside structures examined, showing that the lectin has preference affinity for high-mannose type N-glycans with an exposed (α1-3) mannose residue in the D2 arm. Interestingly, EDAs, the mixture of three isolectins inhibited the growth of shrimp pathogenic bacterium, Vibrio alginolyticus, although it did not affect the growth of V. parahaemolyticus and V. harveyi. Growth inhibition of V. alginolyticus with EDAs was not observed in the presence of yeast mannan bearing high-mannose N-glycans, suggesting that EDAs caused the activity through binding to the target receptor(s) on the surface of V. alginolyticus. These results indicate that cultivated carrageenophyte E. denticulatum is a good source of a lectin(s) that may be useful as a carbohydrate probe and an antibacterial reagent.

Centrifugal ultrafiltration-HPLC method for interaction analysis between lectins and sugars.

The centrifugal ultrafiltration-HPLC method is a simple and rapid method for analyzing the binding interaction between lectins and sugars (oligosaccharides). In this method, a lectin is mixed with a fluorescent-labeled oligosaccharide in buffer and the unbound oligosaccharide recovered by centrifugal ultrafiltration is isolated and quantified by high-performance liquid chromatography. The binding activity is defined as a ratio (percentage) of the amount of bound oligosaccharide to that added, where the former is obtained by subtracting the amount of unbound oligosaccharide from the latter. The oligosaccharide-binding specificity of a lectin can be determined by comparing the binding activities with a variety of fluorescent-labeled oligosaccharides. The association constant and the optimum pH and temperature of the binding interaction between lectins and fluorescent-labeled oligosaccharides can be easily analyzed by this method.

Interaction between conjunctival epithelial cells and mast cells induces CCL2 expression and piecemeal degranulation in mast cells.

PURPOSE: Intraepithelial mast cells are observed in giant papillae tissue samples obtained from patients with atopic keratoconjunctivitis (AKC)/vernal keratoconjunctivitis (VKC). We examined the roles of interaction between the conjunctival epithelial cells and mast cells. METHODS: The interaction between human mast cells and conjunctival epithelial cells (HCjE) was investigated using a coculture model. Protein array analysis, ELISA, and real-time PCR were performed to test the interaction. Tissue samples (n = 6) from giant papillae were resected for therapeutic purposes, and subjected to immunohistological analysis of CCL2 expression. Recombinant CCL2 (10 ng/mL) was reacted with the cultured human mast cells and ultrastructural analysis was performed. A ragweed (RW)-induced mouse experimental allergic conjunctivitis model was used to examine ccl2 mRNA expression and mast cell morphology. RESULTS: Protein array and real-time PCR analyses showed that CCL2 protein/mRNA expression was induced by mast cell-HCjE coculture. Upregulation of CCL2 mRNA was observed in mast cells, whereas in situ CCL2 expression was observed at the conjunctival epithelium of the giant papillae by immunohistochemistry. Ultrastructural analysis showed that recombinant CCL2 treatment induced piecemeal degranulation (PMD) in the mast cells. Ultrastructural analysis of tissues from the giant papillae showed PMD of mast cells within the conjunctival epithelial cells. The RW-induced experimental allergic conjunctivitis model showed increased ccl2 mRNA expression and PMD morphology in the conjunctivae. CONCLUSIONS: Mast cell-conjunctival epithelial cell interaction induces CCL2 expression and subsequent PMD.

Purification, characterization, and cDNA cloning of a novel lectin from the green alga, Codium barbatum.

A novel lectin (CBA) was isolated from the green alga, Codium barbatum, by conventional chromatographic methods. The hemagglutination-inhibition profile with sugars and glycoproteins indicated that CBA had preferential affinity for complex type N-glycans but not for monosaccharides, unlike the other known Codium lectins specific for N-acetylgalactosamine. CBA consisted of an SS-linked homodimer of a 9257-Da polypeptide containing seven cysteine residues, all of which were involved in disulfide linkages. The cDNA of the CBA subunit coded a polypeptide (105 amino acids) including the signal peptide of 17 residues. The calculated molecular mass from the deduced sequence was 9705 Da, implying that the four C-terminal amino acids of the CBA proprotein subunit were post-translationally truncated to afford the mature subunit (84 amino acids). No significantly similar sequences were found during an in silico search, indicating CBA to be a novel protein. CBA is the first Codium lectin whose primary structure has been elucidated.

Involvement of plasminogen activator inhibitor-1 in the pathogenesis of atopic cataracts.

PURPOSE: Further to our previous report of a genetic association between interferon-gamma (IFN-γ) receptor 1 gene and atopic cataract, we investigated the roles of plasminogen activator inhibitor-1 (PAI-1), a fibrosis-related, IFN-γ downstream molecule, in the pathogenesis of atopic cataracts. METHODS: Cultured lens epithelial cells (LECs) were stimulated by IFN-γ and quantified by PAI-1 mRNA/protein expression. PAI-1 and TGF-ß mRNA expression was quantified using cDNA samples obtained from the lens epithelium of atopic cataract patients (n = 7) and of senile cataract patients (n = 8). The anterior capsules obtained from atopic cataracts (n = 9) were immunostained with anti-PAI-1 and anti-alpha smooth muscle actin (α-SMA) antibodies. PAI-1 gene expression was knocked down by PAI-1 siRNA, and α-SMA expression was examined under TGF-ß1 stimulation. Expression of α-SMA was examined as a pathological hallmark of anterior subcapsular cataracts, commonly observed in atopic cataracts. RESULTS: The IFN-γ stimulation induced PAI-1 mRNA/protein expression in the LECs from 24 to 48 hours after stimulation. The expression of PAI-1 mRNA and TGF-ß1 mRNA was significantly higher in the cDNA samples obtained from the atopic cataracts than those obtained from the senile cataracts. PAI-1-positive immunostaining was observed at the fibrotic lesion of the atopic cataracts, and α-SMA-positive myofibroblasts were observed at the vicinity of the PAI-1-positive lesion in all nine samples examined. PAI-1 gene knockdown resulted in reduced α-SMA expression in the LECs. CONCLUSIONS: The findings of this study suggest that the IFN-γ, PAI-1, and TGF-ß1 are involved in the pathophysiology of atopic cataracts.