Background: and Purpose Anti-agrin antibodies (agrin Abs) have recently been identified in patients with myasthenia gravis (MG), sometimes in conjunction with antibodies (Abs) to the acetylcholine receptor (AChR), muscle-specific tyrosine kinase (MuSK), or low-density lipoprotein receptor-related protein 4. This study aimed to develop an in-house cell-based assay (CBA) for detecting agrin Abs, and to test its application to serum samples collected from individuals diagnosed with MG. Methods: Agrin complementary DNA as cloned into a pCMV6-AC-GFP vector, which was subsequently transfected into human embryonic kidney 293T (HEK293T) cells. Transfected HEK293T cells were incubated with patient serum and antihuman immunoglobulin G Ab conjugated with a red fluorescent dye. Agrin Ab levels were measured using the CBA in 389 serum samples: 340 from patients with MG, 36 from patients with other neuromuscular diseases, and 13 from healthy controls. The presence of agrin Ab was determined based on the fluorescence intensity and colocalization using fluorescence microscopy. Results: The expression levels of agrin mRNA and protein in transfected HEK293T cells were confirmed using the reverse-transcription polymerase chain reaction and Western blotting, respectively. Agrin expression in cells was further confirmed by immunocytochemistry.Two (0.6%) of the 340 patients with MG tested positive for agrin Ab: 1 of 191 AChR-positive patients and 1 of 54 MuSK-positive patients. Conclusions We have developed and validated a novel CBA for detecting agrin Abs. This CBA was successfully applied to detect agrin Abs in serum samples obtained from individuals with MG.

Background: and Purpose Anti-agrin antibodies (agrin Abs) have recently been identified in patients with myasthenia gravis (MG), sometimes in conjunction with antibodies (Abs) to the acetylcholine receptor (AChR), muscle-specific tyrosine kinase (MuSK), or low-density lipoprotein receptor-related protein 4. This study aimed to develop an in-house cell-based assay (CBA) for detecting agrin Abs, and to test its application to serum samples collected from individuals diagnosed with MG. Methods: Agrin complementary DNA as cloned into a pCMV6-AC-GFP vector, which was subsequently transfected into human embryonic kidney 293T (HEK293T) cells. Transfected HEK293T cells were incubated with patient serum and antihuman immunoglobulin G Ab conjugated with a red fluorescent dye. Agrin Ab levels were measured using the CBA in 389 serum samples: 340 from patients with MG, 36 from patients with other neuromuscular diseases, and 13 from healthy controls. The presence of agrin Ab was determined based on the fluorescence intensity and colocalization using fluorescence microscopy. Results: The expression levels of agrin mRNA and protein in transfected HEK293T cells were confirmed using the reverse-transcription polymerase chain reaction and Western blotting, respectively. Agrin expression in cells was further confirmed by immunocytochemistry.Two (0.6%) of the 340 patients with MG tested positive for agrin Ab: 1 of 191 AChR-positive patients and 1 of 54 MuSK-positive patients. Conclusions We have developed and validated a novel CBA for detecting agrin Abs. This CBA was successfully applied to detect agrin Abs in serum samples obtained from individuals with MG.

Background: and Purpose Anti-agrin antibodies (agrin Abs) have recently been identified in patients with myasthenia gravis (MG), sometimes in conjunction with antibodies (Abs) to the acetylcholine receptor (AChR), muscle-specific tyrosine kinase (MuSK), or low-density lipoprotein receptor-related protein 4. This study aimed to develop an in-house cell-based assay (CBA) for detecting agrin Abs, and to test its application to serum samples collected from individuals diagnosed with MG. Methods: Agrin complementary DNA as cloned into a pCMV6-AC-GFP vector, which was subsequently transfected into human embryonic kidney 293T (HEK293T) cells. Transfected HEK293T cells were incubated with patient serum and antihuman immunoglobulin G Ab conjugated with a red fluorescent dye. Agrin Ab levels were measured using the CBA in 389 serum samples: 340 from patients with MG, 36 from patients with other neuromuscular diseases, and 13 from healthy controls. The presence of agrin Ab was determined based on the fluorescence intensity and colocalization using fluorescence microscopy. Results: The expression levels of agrin mRNA and protein in transfected HEK293T cells were confirmed using the reverse-transcription polymerase chain reaction and Western blotting, respectively. Agrin expression in cells was further confirmed by immunocytochemistry.Two (0.6%) of the 340 patients with MG tested positive for agrin Ab: 1 of 191 AChR-positive patients and 1 of 54 MuSK-positive patients. Conclusions We have developed and validated a novel CBA for detecting agrin Abs. This CBA was successfully applied to detect agrin Abs in serum samples obtained from individuals with MG.

Purpose: To evaluate 24-month outcomes of anti-vascular endothelial growth factor (VEGF) treatment in polypoidal choroidal vasculopathy (PCV). Methods: Retrospective review of medical records was performed for 34 patients (34 eyes) who were diagnosed with PCV. The best-corrected visual acuity (BCVA) was measured before the injection and at 6, 12, and 24 months after the first injection. The value measured before treatment was compared with those measured after treatment. Results: The patients received mean 6.5 ± 2.1 intravitreal anti-VEGF injections during the 24-month follow-up period. The logarithm of minimal angle of resolution (logMAR) BCVA before the injection and at 6, 12, and 24 months after the first injections were 0.57 ± 0.32, 0.43 ± 0.29, 0.46 ± 0.33, and 0.62 ± 0.39, respectively. The BCVA was significantly improved at 6 and 12 months (p = 0.001 and p = 0.037, respectively). However, there was no significant difference between BCVA before the injection and 24 months (p = 1.000) after the first injection. At the 24-month follow-up, stable (< 2 logMAR lines of change) or improved (≥ 2 logMAR lines of improvement) BCVA was noted in 13 (38.2%) and 10 eyes (29.4%), respectively. Conclusions Anti-VEGF therapy was beneficial in maintaining long-term visual acuity in PCV. However, significant improvement in visual acuity was not noted.

Viral load and the duration of viral shedding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are important determinants of the transmission of coronavirus disease 2019.In this study, we examined the effects of viral doses on the lung and spleen of K18-hACE2 transgenic mice by temporal histological and transcriptional analyses. Approximately, 1×105 plaque-forming units (PFU) of SARS-CoV-2 induced strong host responses in the lungs from 2 days post inoculation (dpi) which did not recover until the mice died, whereas responses to the virus were obvious at 5 days, recovering to the basal state by 14 dpi at 1×102 PFU. Further, flow cytometry showed that number of CD8+ T cells continuously increased in 1×102 PFU-virusinfected lungs from 2 dpi, but not in 1×105 PFU-virus-infected lungs. In spleens, responses to the virus were prominent from 2 dpi, and number of B cells was significantly decreased at 1×105PFU; however, 1×102 PFU of virus induced very weak responses from 2 dpi which recovered by 10 dpi. Although the defense responses returned to normal and the mice survived, lung histology showed evidence of fibrosis, suggesting sequelae of SARS-CoV-2 infection. Our findings indicate that specific effectors of the immune response in the lung and spleen were either increased or depleted in response to doses of SARS-CoV-2. This study demonstrated that the response of local and systemic immune effectors to a viral infection varies with viral dose, which either exacerbates the severity of the infection or accelerates its elimination.

Background: As the number of large-scale studies involving multiple organizations producing data has steadily increased, an integrated system for a common interoperable format is needed. In response to the coronavirus disease 2019 (COVID-19) pandemic, a number of global efforts are underway to develop vaccines and therapeutics. We are therefore observing an explosion in the proliferation of COVID-19 data, and interoperability is highly requested in multiple institutions participating simultaneously in COVID-19 pandemic research. Results: In this study, a laboratory information management system (LIMS) approach has been adopted to systemically manage various COVID-19 non-clinical trial data, including mortality, clinical signs, body weight, body temperature, organ weights, viral titer (viral replication and viral RNA), and multiorgan histopathology, from multiple institutions based on a web interface. The main aim of the implemented system is to integrate, standardize, and organize data collected from laboratories in multiple institutes for COVID-19 non-clinical efficacy testings. Six animal biosafety level 3 institutions proved the feasibility of our system. Substantial benefits were shown by maximizing collaborative high-quality non-clinical research. Conclusions This LIMS platform can be used for future outbreaks, leading to accelerated medical product development through the systematic management of extensive data from non-clinical animal studies.

Purpose: To evaluate long-term visual prognosis and changes in the retinal microstructure in patients with macular telangiectasia type 2. Methods: Retrospective analysis of medical records were performed for 61 eyes (32 patients) diagnosed with macular telangiectasia type 2. The visual acuity at diagnosis was compared with that at the final visit. In addition, cases were classified into three grades based on optical coherence tomography (OCT) findings: grade 1, inner retinal cavities only; grade 2, outer retinal cavities with ellipsoid zone disruption; and grade 3, ellipsoid zone disruption with proliferation of retinal pigment epithelium. Change in the grade during the follow-up was verified. In addition, visual acuities were compared between eyes with and without a change in grade. Results: The mean follow-up period was 41.5 ± 33.2 months. Visual acuity had deteriorated significantly from the mean logarithm of the minimal angle of resolution 0.23 ± 0.26 at diagnosis to a mean of 0.31 ± 0.31 at the final visit (p < 0.001). When classified using OCT, 65.6%, 22.9%, and 11.5% were grades 1, 2, and 3, respectively, at diagnosis and 47.5%, 36.1%, and 16.4% at the final visit. Progression of grade was noted in 22.9%. A higher degree of visual deterioration was noted in eyes showing progression than in eyes without (p = 0.002). Conclusions During the long-term follow-up period, visual deterioration was noted in patients with macular telangiectasia type 2. However, the degree of deterioration was not high. Progression in the disruption of the retinal microstructure was the likely cause of visual deterioration.

Purpose: To evaluate the proportion of bevacizumab and the reason for its usage in wet age-related macular degeneration (AMD). Methods: Retrospective analysis of medical records was performed for 1,541 patients who received ranibizumab, aflibercept, or bevacizumab injection to treat wet AMD. The proportion of bevacizumab among the entire set of injections was identified. The reason for selecting bevacizumab was additionally identified. Results: During the study period, a total of 2,929 anti-vascular endothelial growth factor (anti-VEGF) injections were performed; 2,236 (76.3%) were ranibizumab or aflibercept injections and 693 (23.7%) were bevacizumab injections. The most common reason for bevacizumab usage was ‘having a 0.1 or worse best-corrected visual acuity or being unable to assure reimbursement due to the development of extensive scarring or geographic atrophy’ (297 bevacizumab injections, 42.9%). The second most common reason was ‘the inability to assure reimbursement such as extrafoveal choroidal neovascularization (CNV) or early CNV without definite fluid in the foveal region’ (201 bevacizumab injections, 29.0%). Conclusions Bevacizumab was used in 23.7% of the anti-VEGF injections to treat wet AMD. When analyzing patients’ treatment burden and financial impact, the results of the present study may provide useful information. Further multi-center studies are required to evaluate more precisely the usage of anti-VEGF drugs.

Purpose: To evaluate the proportion and reasons for ineligibility to re-register for extended health insurance at 5 years in patients diagnosed with neovascular age-related macular degeneration (AMD) and registered for extended health insurance. Methods: This retrospective study was performed in patients diagnosed with neovascular AMD and registered for extended health insurance with follow-up for at least 5 years. The criteria for re-registration for extended health insurance were determined along with the ineligibility for re-registration. Results: In total, 263 patients were included in the analysis. Of these, 148 (56.3%) did not satisfy the criteria for re-registration. No active treatment was performed in 98 patients during the last 6 months of the study period (no recurrence, 51 patients; additional treatment was not considered beneficial due to retinal damage even without disciform scar formation, 44 patients). Macular disciform scar formation was noted in 50 patients (33.8%). Older age (p = 0.013), poor visual acuity (p = 0.004), and retinal angiomatous proliferation (p < 0.001) were associated with ineligibility for re-registration due to severe retinal damage. Conclusions Among the patients who were initially registered for extended health insurance, 56.3% failed to satisfy the re-registration criteria. The primary reason was advanced retinal damage. The results of this study provide useful information for patient education and to establish long-term treatment strategies.