Influence of Vitreoretinal Surgery on Ocular Surface Dynamics Using Keratograph 5M.

PURPOSE: To evaluate changes of ocular surface dynamics using Keratograph 5M for 3 months after vitreoretinal surgery. METHODS: Eighty-three patients were divided into three groups: phacoemulsification group, vitrectomy group, and combined group. Keratograph 5M was performed for all patients at 1 week, 1 month, and 3 months after the surgery. Ocular surface dynamics parameters measured by Keratograph 5M, including noninvasive keratograph first tear film breakup time (NifBUT), noninvasive keratograph average tear film breakup time (NiaBUT), and tear meniscus height (TMH) were compared among the three groups over time. RESULTS: The mean age of all patients (46 men and 37 women) was 62.2 ± 8.4 years. NifBUT and NiaBUT were significantly decreased at 1 week after surgery compared to those at baseline in all three groups (all p < 0.001). NifBUT and NiaBUT in the phacoemulsification group almost recovered to the preoperative level, while those in the vitrectomy group and the combined group were still significantly less than those at baseline. NifBUT and NiaBUT in the phacoemulsification group were significantly longer than those in the vitrectomy group and the combined group at 3 months. After 1 week, TMHs were significantly higher in the vitrectomy group (p = 0.001) and the combined group (p = 0.022) than in the phacoemulsification group, while TMHs were significantly less in the vitrectomy group (p = 0.010) and the combined group (p < 0.001) than in the phacoemulsification group at 3 months after surgery. CONCLUSIONS: These results suggest that vitreoretinal surgery could induce alteration of ocular surface dynamics for 3 months. The vitrectomy group and the combined group showed tear film instability compared to the cataract surgery alone group. Patients who underwent vitreoretinal surgery experienced more severe dry eye syndrome symptoms than those who underwent cataract surgery. Thus, managing dry eye syndrome after vitreoretinal surgery should be considered important for patients.

B16 melanoma control by anti-PD-L1 requires CD8+ T cells and NK cells: application of anti-PD-L1 Abs and Trp2 peptide vaccines.

Anti-programmed death ligand 1 (PD-L1) therapy has been beneficial in treating patients with certain cancers. Here, we tested whether anti-PD-L1 therapy is effective for controlling different types of tumors using animal models of TC-1, MC38 and B16. We found that, despite PD-L1 expression, anti-PD-L1 therapy showed little and some antitumor activity in the TC-1 and MC38 models. However, anti-PD-L1 therapy exhibited a more dramatic antitumor effect in the B16 model. This difference in antitumor responses was likely associated with the CD8 + T cell infiltration status of tumor tissues. In the B16 model, CD8 + T cells and to a lesser degree NK cells were found to be responsible for the antitumor response of anti-PD-L1 therapy, as determined by immune cell subset depletion. In particular, CD8 + T cells from B16-bearing mice produced an IFN-γ in response to B16 cells and citrate phosphate buffer-treated B16 cell peptide elutes but not to an immunodominant class I epitope, Trp2180-188, suggesting that CD8 + T cells that recognize neoantigens were induced in B16 tumor-bearing mice and then reactivated by anti-PD-L1 for tumor control. When B16 tumor-bearing mice were treated with anti-PD-L1 in combination with Trp2180-188 peptide vaccines, they displayed significantly more tumor control than either single therapy. Taken together, these studies show that B16 melanomas are more effectively controlled through reactivation of tumor-infiltrating lymphocytes by anti-PD-L1 therapy. Moreover, combined therapy using anti-PD-L1 and Trp2 peptide vaccines is more beneficial for controlling B16 melanomas through reactivation of neoantigen-specific CD8 + T cells and induction of Trp2-specific CD8 + T cells.

BLI-Based Functional Assay in Phage Display Benefits the Development of a PD-L1-Targeting Therapeutic Antibody.

The therapeutic functionality of the antibodies from phage display is verified after an initial screening. Several immunological assays such as ELISA, flow cytometry, the western blot, and surface plasmon resonance (SPR) assay are commonly used; the IgG-format antibody is usually preferred to verify the functionality of antibodies, which need elaborative mammalian expression and purification work. Here, we describe a biolayer interferometry (BLI)-based assay that can evaluate the inhibitory functions of antibodies at an earlier stage of screening. To develop a PD-L1-targeting antibody from phage display, we applied the BLI assay to the initial scFv antibody screening, in addition to common ELISA and fluorescence-activated cell sorting (FACS) assays, which showed high advantages and relevance with the in vitro cell-based PD-1/PD-L1 inhibition assay. The same assays for IgG-format antibodies showed high efficiency of the BLI assay in the functional characterization of antibodies, and one candidate selected from the BLI assay resulted in highly efficacious antitumor activity in an in vivo syngeneic mouse study. The BLI assay was also beneficial when searching for antibodies with diverse epitopes. These results demonstrated that the BLI-based inhibition assay is an excellent technique for high-throughput scFv antibody screening in earlier stages and can make phage-display antibody screening more efficient to develop therapeutic candidates.

Novel Antibodies Targeting MUC1-C Showed Anti-Metastasis and Growth-Inhibitory Effects on Human Breast Cancer Cells.

Mucin1 (MUC1) is aberrantly glycosylated and overexpressed in various cancers, and it plays a crucial role in cancerogenesis. MUC1 is a type I membranous protein composed of α and ß subunits. MUC1-α can be cleaved in cancers, exposing MUC1-ß (MUC1-C). MUC1-C is involved with multiple cancer cellular functions, which makes it an attractive target for cancer treatment. However, its multifunctional mechanisms have not been fully elucidated and there has not been a successful therapeutic development against MUC1-C. Through a phage display process, we isolated the specific antibodies for the extracellular domain of MUC1-C. The relevant full IgG antibodies were produced successfully from mammalian cells and validated for their MUC1-C specificities through ELISA, dual FACS analysis, BLI assay, and confocal image analysis. In the comparison with reference antibody, elected antibodies showed characteristic bindings on target antigens. In the functionality assessment of high-ranking antibodies, SKM1-02, -13, and -20 antibodies highly inhibited invasion by triple-negative breast cancer (TNBC) cells and the SKM1-02 showed strong growth inhibition of cancer cells. Our results showed that these MUC1-C specific antibodies will be important tools for the understanding of MUC1 oncogenesis and are also highly effective therapeutic candidates against human breast cancers, especially TNBC cells.

Generation, Diversity Determination, and Application to Antibody Selection of a Human Naïve Fab Library.

We constructed a large naïve human Fab library (3 × 1010 colonies) from the lymphocytes of 809 human donors, assessed available diversities of the heavy-chain variable (VH) and κ light-chain variable (VK) domain repertoires, and validated the library by selecting Fabs against 10 therapeutically relevant antigens by phage display. We obtained a database of unique 7,373 VH and 41,804 VK sequences by 454 pyrosequencing, and analyzed the repertoires. The distribution of VH and VK subfamilies and germline genes in our antibody repertoires slightly differed from those in earlier published natural antibody libraries. The frequency of somatic hypermutations (SHMs) in heavy-chain complementarity determining region (HCDR)1 and HCDR2 are higher compared with the natural IgM repertoire. Analysis of position-specific SHMs in CDRs indicates that asparagine, threonine, arginine, aspartate and phenylalanine are the most frequent non-germline residues on the antibody-antigen interface and are converted mostly from the germline residues, which are highly represented in germline SHM hotspots. The amino acid composition and length-dependent changes in amino acid frequencies of HCDR3 are similar to those in previous reports, except that frequencies of aspartate and phenylalanine are a little higher in our repertoire. Taken together, the results show that this antibody library shares common features of natural antibody repertoires and also has unique features. The antibody library will be useful in the generation of human antibodies against diverse antigens, and the information about the diversity of natural antibody repertoires will be valuable in the future design of synthetic human antibody libraries with high functional diversity.

Differences in interpalpebral fissure measurement in patients with unilateral enophthalmos resulting from orbital wall fractures.

PURPOSE: To determine the correlation between the degree of enophthalmos and interpalpebral fissure (IPF) measurements in a group of patients with unilateral orbital wall fractures. MATERIALS AND METHODS: The medical charts of 45 patients diagnosed with unilateral enophthalmos resulting from an orbital wall fracture were reviewed. Demographic characteristics were investigated, including patient age, sex, medical history, and type of orbital wall fracture. The correlation between the degree of enophthalmos and IPF was determined, adjusting for confounding demographic factors. RESULTS: In the group with orbital wall fractures, the correlation between the degree of enophthalmos and the IPF measurements was positive and significant (R = 0.299, p = 0.046, Pearson's correlation). The correlation coefficient increased after adjusting for age, sex, medical history, and type of orbital wall fracture (R = 0.316, p = 0.044). CONCLUSION: The patient group with more severe enophthalmos tended to have lower IPF values.

Generation, characterization and preclinical studies of a human anti-L1CAM monoclonal antibody that cross-reacts with rodent L1CAM.

L1 cell adhesion molecule (L1CAM) is aberrantly expressed in malignant tumors and plays important roles in tumor progression. Thus, L1CAM could serve as a therapeutic target and anti-L1CAM antibodies may have potential as anticancer agents. However, L1CAM is expressed in neural cells and the druggability of anti-L1AM antibody must be validated at the earliest stages of preclinical study. Here, we generated a human monoclonal antibody that is cross-reactive with mouse L1CAM and evaluated its pharmacokinetic properties and anti-tumor efficacy in rodent models. First, we selected an antibody (Ab4) that binds human and mouse L1CAM from the human naïve Fab library using phage display, then increased its affinity 45-fold through mutation of 3 residues in the complementarity-determining regions (CDRs) to generate Ab4M. Next, the affinity of Ab4M was increased 1.8-fold by yeast display of single-chain variable fragment containing randomly mutated light chain CDR3 to generate Ab417. The affinities (KD) of Ab417 for human and mouse L1CAM were 0.24 nM and 79.16 pM, respectively. Ab417 specifically bound the Ig5 domain of L1CAM and did not exhibit off-target activity, but bound to the peripheral nerves embedded in normal human tissues as expected in immunohistochemical analysis. In a pharmacokinetics study, the mean half-life of Ab417 was 114.49 h when a single dose (10 mg/kg) was intravenously injected into SD rats. Ab417 significantly inhibited tumor growth in a human cholangiocarcinoma xenograft nude mouse model and did not induce any adverse effect in in vivo studies. Thus, Ab417 may have potential as an anticancer agent.

Expression of Aquaporin-6 in Rat Retinal Ganglion Cells.

Several aquaporins (AQPs) have been identified to be present in the eyes, and it has been suggested that they are involved in the movement of water and small solutes. AQP6, which has low water permeability and transports mainly anions, was recently discovered in the eyes. In the present study, we investigate the localization of AQP6 in the rat retina and show that AQP6 is selectively localized to the ganglion cell layer and the outer plexiform layer. Along with the gradual decrease in retinal ganglion cells after a crushing injury of optic nerve, immunofluorescence signals of AQP6 gradually decreased. Confocal microscope images confirmed AQP6 expression in retinal ganglion cells and Müller cells in vitro. Therefore, AQP6 might participate in water and anion transport in these cells.

A chimeric antibody to L1 cell adhesion molecule shows therapeutic effect in an intrahepatic cholangiocarcinoma model.

Intrahepatic cholangiocarcinoma (ICC), a malignant tumor derived from the intrahepatic bile duct epithelium, has a poor prognosis and is refractory to conventional chemotherapy and radiation therapy. Thus, there is an urgent need to develop new effective therapeutic strategies for this disease. We previously found that L1 cell adhesion molecule (L1CAM) plays an important role in tumor progression of ICC, and we generated a murine mAb, A10-A3 (IgG1), that binds to the Ig1 domain of L1CAM. In the present study, we further characterized A10-A3, constructed a chimeric A10-A3 antibody (cA10-A3) containing the constant regions of human IgG1, and evaluated the therapeutic potential in a human ICC xenograft nude mice model. The affinities (KD) of A10-A3 and cA10-A3 for soluble L1CAM were 1.8 nM and 1.9 nM, respectively, as determined by competition ELISA. A10-A3 inhibited L1CAM homophilic binding and was slowly internalized into the tumor cells, but it did not significantly inhibit proliferation of ICC cells in vitro. cA10-A3 mediated antibody- dependent cell-mediated cytotoxicity in vitro and displayed anti-tumor activity in the ICC animal model. These results suggest that the humanized A10-A3 antibody may have potential as an anticancer agent for the treatment of ICC.

Structural mechanism of the antigen recognition by the L1 cell adhesion molecule antibody A10-A3.

The L1CAM antibody A10-A3 efficiently reduces tumor growth in a nude mouse model. Here, we describe the crystal structure of the Fab fragment of A10-A3 determined at 2.0 angstrom resolution. The A10-A3 antibody H3 loop reveals a characteristic arrangement of exposed aromatic residues that may play an important role in antigen binding. A structure model of the complex between L1CAM Ig1-4 and A10-A3 Fab indicates that the Fab binds to three small loops outside Ig1 and a residue between Ig1 and Ig2, consistent with an epitope mapping result. The data presented here should contribute to the design of high-affinity antibody for therapeutic purposes as well as to the understanding of neural cell remodeling and cancer progression mechanism mediated by L1CAM.

A case of intramuscular hemangioma presenting with large-angle hypertropia.

PURPOSE: To report the case of a patient with large-angle hypertropia of an intramuscular hemangioma of the right superior rectus muscle (SR). METHODS: A 63-year-old man with progressive vertical deviation of the right eye for the past 6 months visited our strabismus department; his condition was not painful. An examination indicated that he had 60PD of right hypertropia at distance and near in primary gaze. Additionally, a significant limitation of his downgaze was noted. The right eye appeared mildly proptotic, and the upper and lower eyelids were slightly edematous. Corrected vision was 20/20 in both eyes. RESULTS: Orbital magnetic resonance imaging (MRI) studies revealed fusiform enlargement of the right superior rectus muscle, with prominent but irregular enhancement following gadolinium administration. Incisional biopsy revealed an intramuscular hemangioma in the superior rectus muscle with cavernous-type vessels. CONCLUSIONS: This case demonstrates that intramuscular hemangioma should be considered in the differential diagnosis of isolated extraocular muscle enlargement and unusual strabismus.

[A case of primary small cell neuroendocrine carcinoma of the liver].

Small cell neuroendocrine carcinoma is a type of undifferentiated, malignant neuroendocrine tumor. Most of neuroendocrine tumors exhibit well-differentiated features and are classified as carcinoid tumors. However, carcinomas of the liver with anaplastic characters, which are classified as small-cell carcinomas are extremely rare and only few cases have been reported in the literature. We report an unusual case of primary small cell neuroendocrine carcinoma of the liver in a 67-year-old man. The patient was found to have a palpable mass on right upper quadrant of abdomen on physical examination. The diagnosis was made by immunohistochemical stains of biopsied specimen from the liver. Other possible primary site was excluded by radiologic and endoscopic evaluations. The tumor was composed of small monotonous and hyperchromatic poorly differentiated cells with higher nuclear to cytoplasmic ratio, and were positive for neuroendocrine tissue markers such as synaptophysin, c-kit, and CD56.