Injection of Cultured Cells with a ROCK Inhibitor for Bullous Keratopathy.

BACKGROUND: Corneal endothelial cell (CEC) disorders, such as Fuchs's endothelial corneal dystrophy, induce abnormal corneal hydration and result in corneal haziness and vision loss known as bullous keratopathy. We investigated whether injection of cultured human CECs supplemented with a rho-associated protein kinase (ROCK) inhibitor into the anterior chamber could increase CEC density. METHODS: We performed an uncontrolled, single-group study involving 11 persons who had received a diagnosis of bullous keratopathy and had no detectable CECs. Human CECs were cultured from a donor cornea; a total of 1×106 passaged cells were supplemented with a ROCK inhibitor (final volume, 300 µl) and injected into the anterior chamber of the eye that was selected for treatment. After the procedure, patients were placed in a prone position for 3 hours. The primary outcome was restoration of corneal transparency, with a CEC density of more than 500 cells per square millimeter at the central cornea at 24 weeks after cell injection. Secondary outcomes were a corneal thickness of less than 630 µm and an improvement in best corrected visual acuity equivalent to two lines or more on a Landolt C eye chart at 24 weeks after cell injection. RESULTS: At 24 weeks after cell injection, we recorded a CEC density of more than 500 cells per square millimeter (range, 947 to 2833) in 11 of the 11 treated eyes (100%; 95% confidence interval [CI], 72 to 100), of which 10 had a CEC density exceeding 1000 cells per square millimeter. A corneal thickness of less than 630 µm (range, 489 to 640) was attained in 10 of the 11 treated eyes (91%; 95% CI, 59 to 100), and an improvement in best corrected visual acuity of two lines or more was recorded in 9 of the 11 treated eyes (82%; 95% CI, 48 to 98). CONCLUSIONS: Injection of human CECs supplemented with a ROCK inhibitor was followed by an increase in CEC density after 24 weeks in 11 persons with bullous keratopathy. (Funded by the Japan Agency for Medical Research and Development and others; UMIN number, UMIN000012534 .).

Negative regulation of Toll-like receptor-4 signaling through the binding of glycosylphosphatidylinositol-anchored glycoprotein, CD14, with the sialic acid-binding lectin, CD33.

When monocyte-derived immature dendritic cells (imDCs) were stimulated with LPS in the presence of anti-CD33/Siglec-3 mAb, the production of IL-12 and phosphorylation of NF-κB decreased significantly. The cell surface proteins of imDCs were chemically cross-linked, and CD33-linked proteins were analyzed by SDS-PAGE and immunoblotting. It was CD14 that was found to be cross-linked with CD33. A proximity ligation assay also indicated that CD33 was colocalized with CD14 on the cell surface of imDCs. Sialic acid-dependent binding of CD33 to CD14 was confirmed by a plate assay using recombinant CD33 and CD14. Three types of cells (HEK293T cells expressing the LPS receptor complex (Toll-like receptor (TLR) cells), and the LPS receptor complex plus either wild-type CD33 (TLR/CD33WT cells) or mutated CD33 without sialic acid-binding activity (TLR/CD33RA cells)) were prepared, and then the binding and uptake of LPS were investigated. Although the level of LPS bound on the cell surface was similar among these cells, the uptake of LPS was reduced in TLR/CD33WT cells. A higher level of CD14-bound LPS and a lower level of TLR4-bound LPS were detected in TLR/CD33WT cells compared with the other two cell types, probably due to reduced presentation of LPS from CD14 to TLR4. Phosphorylation of NF-κB after stimulation with LPS was also compared. Wild-type CD33 but not mutated CD33 significantly reduced the phosphorylation of NF-κB. These results suggest that CD14 is an endogenous ligand for CD33 and that ligation of CD33 with CD14 modulates with the presentation of LPS from CD14 to TLR4, leading to down-regulation of TLR4-mediated signaling.

MUC1 protein induces urokinase-type plasminogen activator (uPA) by forming a complex with NF-κB p65 transcription factor and binding to the uPA promoter, leading to enhanced invasiveness of cancer cells.

Mucin 1 (MUC1) is overexpressed in various human malignant tumors and its expression is correlated with a poor prognosis. MUC1 engages in signal transduction by interacting with receptors for growth and differentiation factors, which contributes to the growth and survival of cancer cells. However, the mechanism by which MUC1 promotes cancer cell invasion remains unclear. Microarray analysis revealed that expression of urokinase-type plasminogen activator (uPA) was elevated in MUC1-overexpressing cells. Furthermore, up- and down-modulation of MUC1 expression was clearly correlated with the change of uPA expression. An immunochemical study showed that the distribution of uPA coincided with that of MUC1 in various human cancer tissues. The MUC1 C-terminal domain (MUC1-CD) was associated with nuclear factor-κB (NF-κB) p65 in MUC1-expressing cells. Chromatin immunoprecipitation (ChIP) assays demonstrated that MUC1-CD existed with NF-κB p65 on the uPA promoter. Luciferase assays indicated that the uPA transcriptional activity was correlated with the level of MUC1 expression and that this MUC1-enhancing effect on the uPA transcription was abolished by introduction of mutations into the NF-κB binding sites on the uPA promoter. These results indicate that formation of the MUC1-CD and NF-κB p65 complex enhanced nuclear translocation of NF-κB p65 and subsequent occupancy of NF-κB binding region on the uPA promoter, leading to elevated transcription of uPA. We also demonstrated that uPA induced by MUC1 enhanced the matrix metalloproteinase (MMP)-2 and -9 activities, and consequently promoted cancer cell invasion. Thus, a MUC1 co-operating NF-κB signaling pathway plays a critical role in cancer cell invasion in MUC1-expressing cells.

Binding of the sialic acid-binding lectin, Siglec-9, to the membrane mucin, MUC1, induces recruitment of ß-catenin and subsequent cell growth.

Because MUC1 carries a variety of sialoglycans that are possibly recognized by the siglec family, we examined MUC1-binding siglecs and found that Siglec-9 prominently bound to MUC1. An immunochemical study showed that Siglec-9-positive immune cells were associated with MUC1-positive cells in human colon, pancreas, and breast tumor tissues. We investigated whether or not this interaction has any functional implications for MUC1-expressing cells. When mouse 3T3 fibroblast cells and a human colon cancer cell line, HCT116, stably transfected with MUC1cDNA were ligated with recombinant soluble Siglec-9, ß-catenin was recruited to the MUC1 C-terminal domain, which was enhanced on stimulation with soluble Siglec-9 in dose- and time-dependent manners. A co-culture model of MUC1-expressing cells and Siglec-9-expressing cells mimicking the interaction between MUC1-expressing malignant cells, and Siglec-9-expressing immune cells in a tumor microenvironment was designed. Brief co-incubation of Siglec-9-expressing HEK293 cells, but not mock HEK293 cells, with MUC1-expressing cells similarly enhanced the recruitment of ß-catenin to the MUC1 C-terminal domain. In addition, treatment of MUC1-expressing cells with neuraminidase almost completely abolished the effect of Siglec-9 on MUC1-mediated signaling. The recruited ß-catenin was thereafter transported to the nucleus, leading to cell growth. These findings suggest that Siglec-9 expressed on immune cells may play a role as a potential counterreceptor for MUC1 and that this signaling may be another MUC1-mediated pathway and function in parallel with a growth factor-dependent pathway.

Donor Corneal Endothelial Cell Maturity and Its Impact on Graft Survival in Glaucoma Patients Undergoing Corneal Transplantation.

PURPOSE: To examine corneal graft survival via corneal endothelial cell density (ECD) and corneal endothelial cell loss (ECL) at 5 years post-transplantation in the eyes of patients with and without a history of undergoing glaucoma surgery according to the maturity of the donor corneal endothelial cells. DESIGN: Prospective cohort study. METHODS: This prospective cohort study included 17 patients with glaucoma and 51 patients without glaucoma who underwent Descemet's stripping automated endothelial keratoplasty or penetrating keratoplasty at the Baptist Eye Institute, Kyoto, Japan, between October 2014 and October 2016. Human corneal endothelial cells were cultured from residual peripheral donor cornea tissue, and the maturity of the cells was evaluated by cell surface markers (ie, CD166+, CD44-/dull, CD24-, and CD105-) using fluorescence-activated cell sorting. Kaplan-Meier analysis or the chi-square test was used to assess the rate of successful corneal graft survival post-transplantation. RESULTS: At 36 months postoperatively, the mean ECD and ECL in the glaucoma-bleb eyes were 1197 ± 352 cells/mm2 and 55.5% ± 13.9% in the high-maturity group and 853 ± 430 cells/mm2 and 67.7% ± 18.1% in the low-maturity group, respectively. Kaplan-Meier analysis revealed that at 5 years postoperatively, the overall rate of survival was 45%, that is, 100% in the high-maturity group and 25% in the low-maturity group (P < .05). CONCLUSIONS: The findings in this prospective cohort study revealed that the use of donor corneal grafts containing mature-differentiated corneal endothelial cells could maintain the survival of the transplanted graft for a long-term period, even in patients with a history of undergoing glaucoma surgery.

Prohibitins function as endogenous ligands for Siglec-9 and negatively regulate TCR signaling upon ligation.

Previously we demonstrated that prohibitin-1 and -2 (prohibitins) were expressed on the surface of T cell leukemia cell lines and activated T lymphocytes. In the present study, we found that prohibitins play a role as counter receptors for Siglec-9 expressed on macrophages and dendritic cells. Siglec-9 bound to prohibitins in a sialic acid-independent manner. Mutated Siglec-9 with Arg(120) changed to Ala lost the binding activity, suggesting a specific ionic peptide-peptide interaction. Phosphorylation of ERK1/2 in Jurkat cells on treatment with anti-CD3 antibody immobilized beads was markedly diminished on treatment with anti-CD3 antibody and Siglec-9 co-immobilized beads, indicating that engagement of prohibitins with Siglec-9 inhibits ERK1/2 phosphorylation. Phosphorylation of c-Raf was also reduced, maybe due to inhibition of the c-Raf-prohibitin interaction by Siglec-9 ligation. In parallel with inhibition of the ERK cascade, IL-2 production was markedly decreased in Jurkat cells. Thus, this interaction may be a useful immunotherapeutic target.

The Biologic Character of Donor Corneal Endothelial Cells Influences Endothelial Cell Density Post Successful Corneal Transplantation.

Purpose: Corneal endothelial cell density (ECD) gradually decreases after corneal transplantation by unknown biologic, biophysical, or immunologic mechanism. Our purpose was to assess the association between donor corneal endothelial cell (CEC) maturity in culture and postoperative endothelial cell loss (ECL) after successful corneal transplantation. Design: Prospective cohort study. Participants: This cohort study was conducted at Baptist Eye Institute, Kyoto, Japan, between October 2014 and October 2016. It included 68 patients with a 36-month follow-up period who had undergone successful Descemet stripping automated endothelial keratoplasty (DSAEK) or penetrating keratoplasty. Methods: Human CECs (HCECs) from remaining peripheral donor corneas were cultured and evaluated for maturity by surface markers (CD166+, CD44-/dull, CD24-, and CD105-) using fluorescence-activated cell sorting. Postoperative ECD was assessed according to the mature-differentiated HCEC contents: high-maturity group: > 70%, middle-maturity group: 10% to 70%, low-maturity group: < 10%. The successful rate of ECD maintained at 1500 cells/mm2 at 36 months postoperative was analyzed using the log-rank test. Main Outcome Measures: Endothelial cell density and ECL at 36 months postoperative. Results: The 68 included patients (mean [standard deviation] age 68.1 [13.6] years, 47.1% women, 52.9% DSAEK). The high, middle, and low-maturity groups included 17, 32, and 19 eyes, respectively. At 36 months postoperative, the mean (standard deviation) ECD significantly decreased to 911 (388) cells/mm2 by 66% in the low-maturity group, compared with 1604 (436) by 40% and 1424 (613) cells/mm2 by 50% in the high and middle-maturity groups (P < 0.001 and P = 0.007, respectively) and the low-maturity group significantly failed to maintain ECD at 1500 cells/mm2 at 36 months postoperative (P < 0.001). Additional ECD analysis for patients who underwent DSAEK alone displayed a significant failure to maintain ECD at 1500 cells/mm2 at 36 months postoperative (P < 0.001). Conclusions: The high content of mature-differentiated HCECs expressed in culture by the donor peripheral cornea was coincident with low ECL, suggesting that a high-maturity CEC content predicts long-term graft survival. Understanding the molecular mechanism for maintaining HCEC maturity could elucidate the mechanism of ECL after corneal transplantation and aid in developing effective interventions. Financial Disclosures: Proprietary or commercial disclosure may be found after the references.

Expression of prohibitins on the surface of activated T cells.

Prohibitins (prohibitin-1 and -2) comprise a family of highly conserved proteins that are mainly localized to mitochondria. Recent studies showed that prohibitins are up-regulated upon T cell activation and play an essential role in maintaining mitochondrial homeostasis. In the present study, we found that a considerable proportion of prohibitin-1 and -2 induced in response to T cell activation was expressed on the surface of activated T cells. When mouse and human T cells were stimulated with PMA and ionomycin, prohibitins expressed on the cell surface were increased significantly, peaking at 48 h after stimulation. Stimulation of mouse T cells with anti-CD3 and anti-CD28 antibodies also remarkably induced the cell surface expression of prohibitins. Their expression on the cell surface was also detected in T cell leukemia cells such as Jurkat cells. In Jurkat cells, prohibitin-1 and -2 were co-localized with CD3 on the cell surface, and anti-CD3 antibody-induced signaling, the MAP kinase cascade, was inhibited on treatment with protein A magnetic beads co-conjugated with anti-CD3 antibody and anti-prohibitin-1 or anti-prohibitin-2 antibody. These results suggest that prohibitins expressed on the surface of activated T cells are involved in the T cell receptor-mediated signaling cascade.

Different levels of sialyl-Tn antigen expressed on MUC16 in patients with endometriosis and ovarian cancer.

OBJECTIVE: Although CA125 antigen is a useful marker for ovarian cancer, its expression is also elevated in endometriosis. The purpose of this study was to develop an assay method for evaluating differentially glycosylated MUC16 (CA125 core protein) in patients with endometriosis and ovarian cancer. MATERIALS AND METHODS: We prepared MUC16-enriched fractions from peritoneal fluid of patients with endometriosis and conditioned medium of ovarian carcinoma-3 cells by gel filtration, and evaluated the expression of sialyl-Le, Tn, and sialyl-Tn antigens by dot blot analysis. A sandwich enzyme-linked immunosorbent assay was developed to measure the level of sialyl-Tn antigen expressed on MUC16 (sTn/MUC16). The level of sTn/MUC16 was compared between patients with endometriosis (n = 21) and ovarian cancer (n = 36) and in ovarian cancers with different clinical diagnostic criteria. Furthermore, distribution of MUC16 and sialyl-Tn antigen in ovarian cancer tissues was observed immunohistochemically. RESULTS: Sialyl-Tn antigen was markedly detectable in the MUC16-enriched fractions from conditioned medium of ovarian carcinoma-3 cells but negligible in those from the peritoneal fluid of the patients with endometriosis. The level of sTn/MUC16 determined by a sandwich enzyme-linked immunosorbent assay was significantly higher in the patients with ovarian cancer than that in the patients with endometriosis (P < 0.001). An elevated level of sTn/MUC16 was detected in 44% of the patients with ovarian cancer but not all the patients with endometriosis. This level increased more prominently in the patients with ovarian cancer than that of MUC16 as both the clinical stage and cytological grade advanced. An elevated level of sTn/MUC16 was frequently found in the patients with serous and endometrioid carcinomas. Consistent with this, sialyl-Tn antigen was colocalized with MUC16 in serous and endometrioid ovarian cancer tissues. CONCLUSIONS: Estimation of the sTn/MUC16 level may be useful for discriminating endometriosis from ovarian cancer and for evaluating the clinical stage, cytological grade, and histological type of ovarian cancer.

Quiescent innate and adaptive immune responses maintain the long-term integrity of corneal endothelium reconstituted through allogeneic cell injection therapy.

This study aims to clarify the immunogenicity in acquired and innate immune responses of cultured human corneal endothelial cells (hCECs) applied for cell injection therapy, a newly established modality for corneal endothelium failures. Thirty-four patients with corneal endothelial failure received injection of allogeneic hCEC suspension into anterior chamber. No sign of immunological rejection was observed in all 34 patients during the 5-8 years postoperative follow-up period. Cell injection therapy was successful in 2 patients treated for endothelial failure after penetrating keratoplasty and one patient with Descemet membrane stripping automated endothelial keratoplasty failure. ELISPOT assays performed in allo-mixed lymphocyte reaction to the alloantigen identical to that on the injected hCECs, elicited sparse IFN-γ-specific spots in the peripheral blood mononuclear cells of patients who received hCEC injection. The therapy generated simple and smooth graft-host junctions without wound stress. The injection of C57BL/6 CECs into the anterior chamber of BALB/c mice, which rejected C57BL/6 corneas 6 weeks ago, induced no sign of inflammatory reactions after the second challenge of alloantigen. Collectively, injection of the hCEC cell suspension in the aqueous humor induces immune tolerance that contributes to the survival of the reconstituted endothelium.

Repressed miR-34a Expression Dictates the Cell Fate to Corneal Endothelium Failure.

Purpose: To reveal the mechanism triggering the functional disparity between degenerated and non-degenerated corneal endothelium cells in the water efflux from corneal stroma to the anterior chamber. Methods: The varied levels of the microRNA (miR)-34, miR-378, and miR-146 family in human corneal endothelium and cultured cells thereof were investigated using 3D-Gene Human miRNA Oligo Chips. Concomitantly, CD44, p53, c-Myc, matrix metalloprotease (MMP)-2 expression, and Ras homolog gene family member A (Rho A) activity was correlated to the expression intensities of these microRNAs, partly complemented with their altered expression levels with the transfection of the corresponding mimics and inhibitors. The levels of miRs were further associated with intracellular pH (pHi) and mitochondrial energy homeostasis. Results: P53-inducible miR-34a/b repressed CD44 expression, and CD44 was repressed with the elevated c-Myc. The repressed miR-34a activated the CD44 downstream factors Rho A and MMP-2. MiR-34a mimics downregulated pHi, inducing the skewing of mitochondrial respiration to oxidative phosphorylation. The oxidative stress (H2O2) induced on human corneal endothelial cells, which repressed miR-34a/b expression, may account for the impaired signaling cascade to mitochondrial metabolic homeostasis necessary for an efficient water efflux from the corneal stroma. Conclusions: The upregulated expression of CD44, through repressed miR-34a/b by reactive oxygen species and elevated c-Myc by oxidative stress, may impair mitochondrial metabolic homeostasis, leading to human corneal endothelial failure.

Cellular Interplay Through Extracellular Vesicle miR-184 Alleviates Corneal Endothelium Degeneration.

Objective: The objective of the study was to reveal the presence of cellular interplay through extracellular vesicle (EV) microRNAs (miRs), to dampen the vicious cycle to degenerate human corneal endothelium (HCE) tissues. Design: Prospective, comparative, observational study. Methods: The miR levels in neonate-derived corneal tissues, in the aqueous humor (AqH) of bullous keratoplasty and cataract patients, as well as in the culture supernatant (CS) and EV of cultured human corneal endothelial cells (hCECs), were determined using 3D-Gene human miR chips and then validated using the real-time polymerase chain reaction. The extracellularly released miRs were profiled after the forced downregulation of cellular miR-34a, either by an miR-34a inhibitor or exposure to H2O2. The senescence-associated secretory phenotypes and mitochondrial membrane potential (MMP) were assessed to determine the functional features of the released miRs. Main Outcome Measures: Identification of functional miRs attenuating HCE degeneration. Results: The miRs in AqH were classified into 2 groups: expression in 1 group was significantly reduced in neonate-derived tissues, whereas that in the other group remained almost constant, independent of aging. The miR-34a and -29 families were typical in the former group, whereas miR-184 and -24-3p were typical in the latter. Additionally, a larger amount of the latter miRs was detected in AqH compared with those of the former miRs. There was also a greater abundance of miR-184 and -24-3p in hCECs, EV, and CS in fully mature CD44-/dull hCEC, leading to sufficient clinical tissue regenerative capacity in cell injection therapy. The repression of cellular miR-34a, either due to miR-34a inhibitors or exposure to oxidative stress, unexpectedly resulted in the elevated release of miR-184 and -24-3p. Secretions of VEGF, interleukin 6, monocyte chemotactic protein-1, and MMP were all repressed in both mature CD44-/dull and degenerated CD44+++ hCEC, transfected with an miR-184 mimic. Conclusions: The elevated release of miR-184 into AqH may constitute cellular interplay that prevents the aggravation of HCE degeneration induced by oxidative stress, thereby sustaining tissue homeostasis in HCE.

Superiority of Mature Differentiated Cultured Human Corneal Endothelial Cell Injection Therapy for Corneal Endothelial Failure.

PURPOSE: To investigate the safety and efficacy of cultured human corneal endothelial cell (hCEC) injection therapy with mature differentiated (mature) cell subpopulations (SPs) for corneal endothelial failure (CEF). DESIGN: Comparative, interventional case series. METHODS: This study involved 18 eyes with CEF that underwent cultured hCEC injection therapy, categorized into 2 groups: (1) 11 eyes administered a relatively lower proportion (0.1 to 76.3%) of mature cell SPs (group 1 [Gr1]), and (2) 7 eyes administered a relatively higher proportion (>90%) of mature cell SPs (group 2 [Gr2]). From 1 week to 3 years postoperation, corneal endothelial cell (CEC) density (CECD), central corneal thickness (CCT), and best-corrected visual acuity (BCVA) were recorded, and the CEC parameter's "spring constant" was calculated. The proportion of mature SPs was evaluated by fluorescence-activated cell sorting analysis based on cell-surface markers. RESULTS: At 3 years postoperation, corneal restoration with improved BCVA was attained in 10 of the 11 Gr1 eyes and all Gr2 eyes, the median CECD in Gr2 (3083 cells/mm2; range, 2182-4417 cells/mm2) was higher than that in Gr1 (1349 cells/mm2; range, 746-2104 cells/mm2) (P < .001), and the spring constant verified the superiority of the mature cultured hCECs. From 24 weeks through 3 years postoperation, the median percentage of CECD decrease was 3.2% in Gr2 and 23.6% in Gr1 (P < .005). CCT recovery was prompt and constant in Gr2, while diverse in Gr1. No adverse events were observed. CONCLUSION: Our findings showed that mature cell SPs for hCEC injection therapy provide rapid recovery of CCT, better CECD, and low CECD attrition over 3 years postsurgery.

Immunomodulation of monocyte-derived dendritic cells through ligation of tumor-produced mucins to Siglec-9.

Dendritic cells (DCs) play an essential role in the induction and maintenance of an effective immune response and express multiple siglecs. In the present study, we investigated whether or not the ligation of tumor-produced mucins with Siglec-9 expressed on immature DCs is related to escape from immunosurveillance in the tumor-bearing state. Expression of Siglec-9 was up-regulated on the development of monocytes into immature DCs and was decreased in mature DCs. Binding of various mucins and artificial glycopolymers carrying poly (NeuAc α2,6 LacNAc) or poly (NeuAc α2,3 LacNAc) to Siglec-9 was demonstrated by means of a plate assay. These mucins also bound to the surface of immature DCs. When immature DCs were treated with LPS in the presence of these mucins or artificial glycopolymers, the production of IL-12 was significantly reduced, but that of IL-10 was not. Furthermore, IL-12 production was decreased to a similar level on treatment with anti-Siglec-9 mAb. Mucins prepared from serum of cancer patients actually could bind to Siglec-9. These results suggest that Siglec-9 expressed on DCs is involved in immunoregulation through ligation with mucins in an epithelial cancer patient.

Ligation of tumour-produced mucins to CD22 dramatically impairs splenic marginal zone B-cells.

CD22 [Siglec-2 (sialic acid-binding, immunoglobulin-like lectin-2)], a negative regulator of B-cell signalling, binds to alpha2,6- sialic acid-linked glycoconjugates, including a sialyl-Tn antigen that is one of the typical tumour-associated carbohydrate antigens expressed on various mucins. Many epithelial tumours secrete mucins into tissues and/or the bloodstream. Mouse mammary adenocarcinoma cells, TA3-Ha, produce a mucin named epiglycanin, but a subline of them, TA3-St, does not. Epiglycanin binds to CD22 and inhibits B-cell signalling in vitro. The in vivo effect of mucins in the tumour-bearing state was investigated using these cell lines. It should be noted that splenic MZ (marginal zone) B-cells were dramatically reduced in the mice bearing TA3-Ha cells but not in those bearing TA3-St cells, this being consistent with the finding that the thymus-independent response was reduced in these mice. When the mucins were administered to normal mice, a portion of them was detected in the splenic MZ associated with the MZ B-cells. Furthermore, administration of mucins to normal mice clearly reduced the splenic MZ B-cells, similar to tumour-bearing mice. These results indicate that mucins in the bloodstream interacted with CD22, which led to impairment of the splenic MZ B-cells in the tumour-bearing state.

Metabolites Interrogation in Cell Fate Decision of Cultured Human Corneal Endothelial Cells.

Purpose: Aiming to clarify the metabolic interrogation in cell fate decision of cultured human corneal endothelial cells (cHCECs). Methods: To analyze the metabolites in the culture supernatants (CS), 34 metabolome measurements were carried out for mature differentiated and a variety of cHCECs with cell state transition through a facility service. Integrated proteomics research for cell lysates by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed for 3 aliquots of each high-quality or low-quality cHCEC subpopulations (SP). The investigations for the focused genes involved in cHCEC metabolism were performed by using DAVID and its options "KEGG_PATHWAY." Results: The clusters of metabolites coincided well with the distinct content of CD44-/+ SPs. Both secreted pyruvic acid and lactic acid in the CS were negatively correlated with the content of high-quality SPs. Lactic acid and pyruvic acid in the CS exhibited the positive correlation with that of Ile, Leu, and Ser, whereas the negative correlation was with glutamine. Platelet-derived growth factor-ßß in the CS negatively correlated with lactic acid in CS, indicating indirectly the positive correlation with the content of CD44-/+ SPs. Upregulated glycolytic enzymes and influx of glutamine to the tricarboxylic acid cycle may be linked with a metabolic rewiring converting oxidative metabolism in mature differentiated CD44-/+SPs into a glycolytic flux-dependent state in immature SPs with cell state transition. Conclusions: The findings suggest that the cell fate decision of cHCECs may be dictated at least partly through metabolic rewiring.

In Vivo Fluorescence Visualization of Anterior Chamber Injected Human Corneal Endothelial Cells Labeled With Quantum Dots.

Purpose: The injection of cultured human corneal endothelial cells (cHCECs) into the anterior chamber (AC) is a newly developed modality for the successful treatment of corneal endothelium dysfunction. Here, we investigated whether or not cHCECs could be labeled using quantum dots (QDs) composed of semiconductor nanoparticle octa-arginine (R8) to trace injected cHCECs and examined the utility of in vivo fluorescence imaging to analyze the dynamics and accumulation of QD-labeled injected cHCECs in a corneal endothelial dysfunction mouse model. Methods: The cHCECs, either of high quality or with cell-state transition, were labeled by adding a mixture of QDs655 and R8. The labeling efficiency and the unchanging of the cell phenotypes by the labeling was confirmed by flow cytometry. The labeled cHCECs were injected into the AC of either healthy mice or mice with corneal endothelium damaged by cryogenic treatment. The kinetics of the injected cHCECs was traced quantitatively via multiphoton confocal laser microscopy. Results: QD labeling induced no morphologic change in the cHCECs or in the expression of the functional markers of cHCECs (i.e., Na+/K+-ATPase and zonula occludens-1). The injected cHCECs-QDs were quantitatively detected, and the retention of cHCECs-QDs was evident, from 3 to 48 hours post cell injection on the posterior surface in the cryogenically injured corneal endothelium mouse model eyes, yet not in the noninjured healthy control eyes. Conclusions: The findings of this study show that in the field of regenerative medicine, QD labeling of cells presents a convenient and sensitive method of finely monitoring the fate of injected cells in vivo.

A physical biomarker of the quality of cultured corneal endothelial cells and of the long-term prognosis of corneal restoration in patients.

Dysfunction of the corneal endothelium reduces the transparency of the cornea and can cause blindness. Because corneal endothelial cells have an extremely limited proliferative ability in vivo, treatment for corneal endothelial dysfunction involves the transplantation of donor corneal tissue. Corneal endothelium can also be restored via intraocular injection of endothelial cells in suspension after their expansion in vitro. Yet, because quality assessment during the expansion of the cells is a destructive process, a substantial number of the cultured cells are lost. Here, we show that the 'spring constant' of the effective interaction potential between endothelial cells in a confluent monolayer serves as a biomarker of the quality of corneal endothelial cells in vitro and of the long-term prognosis of corneal restoration in patients treated with culture-expanded endothelial cells or with transplanted corneas. The biomarker can be measured from phase contrast imaging in vitro and from specular microscopy in vivo, and may enable a shift from passive monitoring to pre-emptive intervention in patients with severe corneal disorders.

Mucin-induced apoptosis of monocyte-derived dendritic cells during maturation.

Many tumors arising from epithelial tissues produce mucins, which readily come into contact with infiltrating cells in cancer tissues. MUC2 mucins were purified from the conditioned medium of a colorectal cancer cell line, LS180 cells. It is known that in cancer patients, the number of dendritic cells (DCs) is reduced and their function is impaired. Mature DCs were generated from human peripheral blood monocytes through successive treatments with GM-CSF and IL-4, and then with proinflammatory mediators. When monocytes were cultured in the presence of MUC2 mucins in addition to GM-CSF and IL-4 at an early stage of development, mature DCs expressing CD83 decreased and apoptotic cells increased in a dose-dependent manner. During the development of DCs, sialic acid-binding Ig-like lectin (Siglec)-3 was constantly expressed. We prepared recombinant soluble Siglec-3 corresponding to the ectodomain of Siglec-3 and confirmed the binding of soluble Siglec-3 to the MUC2 mucins, probably through alpha2,6-sialic acid-containing O-glycans including a sialyl Tn antigen, which is known to bind to Siglec-3. Apoptosis was partially inhibited by anti-Siglec-3 mAb or recombinant soluble Siglec-3. These results suggest that apoptosis was partially induced through the ligation of the MUC2 mucins with Siglec-3.

Down-modulation of B cell signal transduction by ligation of mucins to CD22.

Epithelial cancer cells secrete mucins carrying carbohydrate antigens such as a sialyl-Tn antigen into cancer tissues and/or the bloodstream, in which mucins may interact with CD22 (Siglec-2). Mucins isolated from colon cancer cells and bovine submaxillary mucins bound to CD22 cDNA transfectants and a human B cell line, Daudi cell, and the binding of soluble recombinant CD22 to the mucins was confirmed by means of a plate assay. The binding specificity was demonstrated by the fact that the mucins bound to the recombinant CD22 with an intact ectodomain but not to that with a mutated ectodomain. Daudi cells were stimulated with anti-IgM F(ab')(2) in the presence or absence of mucins. Ligation of mucins to CD22 decreased the phosphorylation of CD22 and SHP-1 recruitment, and the phosphorylation of ERK-1/2 prominently. The in vivo effect of mucins on splenic B cells in the tumor-bearing state was investigated using mucin-producing (TA3-Ha) and non-producing (TA3-St) mammary adenocarcinoma-bearing mice. When fluorescence-labeled epiglycanins were administered to normal mice, a portion of them was taken up by the spleen and became associated with splenic B cells. We found that splenic B cells were reduced in TA3-Ha-bearing mice but not in TA3-St-bearing ones. These results suggest that in the tumor-bearing state a portion of the mucins in the bloodstream was taken up by the spleen and ligated to CD22 expressed on splenic B cells, which may have led to down-regulation of signal transduction.