Investigating the Influence of Temperature and Supplementation Timing on Antifungal Efficacy in Storage Medium for Corneal Transplantation.

INTRODUCTION: Although antifungal supplementation reduces the fungal load in the corneal storage medium, consensus is lacking on the influence of dosing and temperature. The study aims to evaluate the impact of eye bank warming protocol and timing of antifungal supplements on efficacy in Optisol-GS and tissue. METHODS: Corneoscleral rims contaminated with Candida albicans (C. albicans) were incubated in Optisol-GS, either without antifungal agents or with the addition of amphotericin B or voriconazole at various concentrations (2 ×, 5 ×, 10 ×, and 20 × MIC), at different time points, and under various preservation temperatures (2-8 °C versus 2 h-room temperature exposure). Antifungal efficacy was evaluated by counting viable yeast colonies cultured from Optisol-GS samples. Tissue sterility was determined through direct tissue culture and histological examination of the contaminated rims after a 14-day incubation period. RESULTS: Room temperature exposure did not increase colony growth at the same multiple MIC of antifungal agents. Although antifungal addition reduced C. albicans growth in a concentration-dependent manner, yeast growth was still observed in all Optisol-GS samples with a single supplementation after a 14-day incubation. Only groups with additional antifungal supplementation on either day 2 or day 6 showed a 99% or greater reduction of C. albicans growth in Optisol-GS samples and yielded negative results in direct tissue culture. CONCLUSIONS: The eye bank warming protocol did not compromise antifungal efficacy. To sustain the required concentration and effectively reduce C. albicans growth in Optisol-GS and contaminated tissue, additional antifungal supplementation on either day 2 or day 6 was necessary during a 2-week preservation period.

Comparative Proteomics Reveals Prolonged Corneal Preservation Impaired Ocular Surface Immunity Accompanied by Fibrosis in Human Stroma.

Cornea transplantation is one of the most commonly performed allotransplantations worldwide. Prolonged storage of donor corneas leads to decreased endothelial cell viability, severe stromal edema, and opacification, significantly compromising the success rate of corneal transplantation. Corneal stroma, which constitutes the majority of the cornea, plays a crucial role in maintaining its shape and transparency. In this study, we conducted proteomic analysis of corneal stroma preserved in Optisol-GS medium at 4 °C for 7 or 14 days to investigate molecular changes during storage. Among 1923 identified proteins, 1634 were quantifiable and 387 were significantly regulated with longer preservation. Compared to stroma preserved for 7 days, proteins involved in ocular surface immunomodulation were largely downregulated while proteins associated with extracellular matrix reorganization and fibrosis were upregulated in those preserved for 14 days. The increase in extracellular matrix structural proteins together with upregulation of growth factor signaling implies the occurrence of stromal fibrosis, which may compromise tissue clarity and cause vision impairments. This study is the first to provide insights into how storage duration affects corneal stroma from a proteomic perspective. Our findings may contribute to future research efforts aimed at developing long-term preservation techniques and improving the quality of preserved corneas, thus maximizing their clinical application.

Single-Cell Transcriptomics Reveals Cellular Heterogeneity and Complex Cell-Cell Communication Networks in the Mouse Cornea.

Purpose: To generate a single-cell RNA-sequencing (scRNA-seq) map and construct cell-cell communication networks of mouse corneas. Methods: C57BL/6 mouse corneas were dissociated to single cells and subjected to scRNA-seq. Cell populations were clustered and annotated for bioinformatic analysis using the R package "Seurat." Differential expression patterns were validated and spatially mapped with whole-mount immunofluorescence staining. Global intercellular signaling networks were constructed using CellChat. Results: Unbiased clustering of scRNA-seq transcriptomes of 14,732 cells from 40 corneas revealed 17 cell clusters of six major cell types: nine epithelial cell, three keratocyte, two corneal endothelial cell, and one each of immune cell, vascular endothelial cell, and fibroblast clusters. The nine epithelial cell subtypes included quiescent limbal stem cells, transit-amplifying cells, and differentiated cells from corneas and two minor conjunctival epithelial clusters. CellChat analysis provided an atlas of the complex intercellular signaling communications among all cell types. Conclusions: We constructed a complete single-cell transcriptomic map and the complex signaling cross-talk among all cell types of the cornea, which can be used as a foundation atlas for further research on the cornea. This study also deepens the understanding of the cellular heterogeneity and heterotypic cell-cell interaction within corneas.

The versatile roles of lumican in eye diseases: A review.

Lumican is a keratan sulfate proteoglycan that belongs to the small leucine-rich proteoglycan family. Research has lifted the veil on the versatile roles of lumican in the pathogenesis of eye diseases. Lumican has pivotal roles in the maintenance of physiological tissue homogenesis and is often upregulated in pathological conditions, e.g., fibrosis, scar tissue formation in injured tissues, persistent inflammatory responses and immune anomaly, etc. Herein, we will review literature regarding the role of lumican in pathogenesis of inherited congenital and acquired eye diseases, e.g., cornea dystrophy, cataract, glaucoma and chorioretinal diseases, etc.

Association between parental autoimmune disease and childhood atopic dermatitis varied by sex: a nationwide case-control study.

Atopic dermatitis (AD) is a common inflammatory skin disorder induced by dysfunction of immune suppression sharing similar pathogenesis to autoimmune diseases. To explore the association between autoimmune diseases and AD in children, we linked the birth data from National Birth Registry with National Health Insurance Research Database. There were 1,174,941 children obtained from 2006 to 2012 birth cohort. A total of 312,329 children diagnosed with AD before 5 years old were compared to 862,612 children without AD in the control group. Conditional logistic regression was utilized to calculate adjusted odds ratio (OR) and Bonferroni-corrected confidence interval (CI) for overall significance level of 0.05. In 2006-2012 birth cohort, the prevalence rate of AD was 26.6% (95% CI 26.5, 26.7) before 5 years of age. Having parental autoimmune disease (including rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, ankylosing spondylitis, and psoriasis) was associated with a significant higher risk of children AD development. The other associated factors were maternal obstetric complications (including gestational diabetes mellitus and cervical incompetence), parental systemic diseases (including anemia, hypertension, diabetes mellitus, chronic obstructive pulmonary disease, hyperthyroidism, and obstructive sleep apnea), and parental allergic disease (including asthma and AD). The subgroup analysis showed similar results between children's sexes. Moreover, maternal autoimmune disease had higher impact on the risk of developing AD in the child compared with paternal autoimmune disease. In conclusion, parental autoimmune diseases were found to be related to their children's AD before 5 years old.

Experimental Melanoma Immunotherapy Model Using Tumor Vaccination with a Hematopoietic Cytokine.

Fms-like tyrosine kinase 3 ligand (Flt3L) is a hematopoietic cytokine that promotes the survival and differentiation of dendritic cells (DCs). It has been used in tumor vaccines to activate innate immunity and enhance antitumor responses. This protocol demonstrates a therapeutic model using cell-based tumor vaccine consisting of Flt3L-expressing B16-F10 melanoma cells along with phenotypic and functional analysis of immune cells in the tumor microenvironment (TME). Procedures for cultured tumor cell preparation, tumor implantation, cell irradiation, tumor size measurement, intratumoral immune cell isolation, and flow cytometry analysis are described. The overall goal of this protocol is to provide a preclinical solid tumor immunotherapy model, and a research platform to study the relationship between tumor cells and infiltrating immune cells. The immunotherapy protocol described here can be combined with other therapeutic modalities, such as immune checkpoint blockade (anti-CTLA-4, anti-PD-1, anti-PD-L1 antibodies) or chemotherapy in order to improve the cancer therapeutic effect of melanoma.

Keratoplasty Registries: Lessons Learned.

ABSTRACT: Clinical registries have been developed for decades in the field of ophthalmology, and they are especially well-suited to the study of keratoplasty practices. A comprehensive donor/recipient registry system can provide insight into donor, recipient, and surgical factors associated with immediate and long-term outcomes and adverse reactions. Furthermore, linkage with demographic databases can elucidate relationships with social determinants of health and potentially shape public policy. The vast sample size and multicenter nature of registries enable researchers to conduct sophisticated multivariate or multilayered analyses. In this review, we aim to emphasize the importance of registry data for keratoplasty practice and 1) summarize the structure of current keratoplasty registries; 2) examine the features and scientific contributions of the registries from Australia, the United Kingdom, Singapore, the Netherlands, Sweden, Eye Bank Association of America, and European Cornea and Cell Transplant registries; 3) compare registry-based studies with large single-site clinical studies; 4) compare registry-based studies with randomized control studies; and 5) make recommendations for future development of keratoplasty registries. Keratoplasty registries have increased our knowledge of corneal transplant practices and their outcomes. Future keratoplasty registry-based studies may be further strengthened by record linkage, data sharing, and international collaboration.

Extending the utility of anterior corneal buttons through refrigeration and glycerol cryopreservation: utility rate and outcome analysis.

BACKGROUND/AIMS: To evaluate the utility rate, indication, outcome, and cost of refrigeration and glycerol cryopreservation for storing anterior corneal buttons during endothelial keratoplasty for subsequent use in tectonic lamellar patch grafting. METHOD: Anterior corneal buttons collected after precutting or prestripping during endothelial keratoplasty from January 2014 to December 2019 were preserved using the following protocol: (1) refrigeration for up to 4 weeks at 4°C in Optisol-GS and (2) glycerol cryopreservation for up to 2 years. The utility rate, outcome and cost of these cryopreserved anterior corneal buttons were retrospectively examined. RESULTS: During the 6-year study period, 26 anterior corneal buttons were refrigerated and 49 were cryopreserved for extended use. The utility rates for the refrigerated and cryopreserved anterior corneal buttons were 69.2% and 73.5%, respectively. Their average preservation periods were 0.53±0.05 and 12.76±0.94 months, respectively. Noninfection-related perforation was the leading indication for using the extendedly preserved anterior corneal buttons. The average postoperative follow-up periods were 10.03±2.91 and 14.35±2.17 months for refrigerated and cryopreserved anterior corneal buttons. Secondary keratoplasty was required by 7 of 18 (38.9%) and 6 of 36 (16.7%) patients receiving refrigerated and cryopreserved anterior corneal buttons, respectively. None of our patients developed graft infection from donor tissues. CONCLUSION: Cryopreservation can safely extend the utility of anterior corneal buttons. This method not only reduced the wastage of the limited donor tissue but also was cost-effective.

Differential moderation effects of ApoE and 5-HTTLPR genotypes on social vulnerability in predicting mortality among community-dwelling middle-aged and older adults: a nationwide population-based study.

Aging is a dynamic complex process involving social vulnerability over time. The social vulnerability index (SVI) was developed that predicted adverse health outcomes. This study examined effects between SVI status and two genotypes, apolipoprotein E (ApoE) and Serotonin transporter genotyping (5-HTTLPR), on all-cause mortality. Data from the Social Environment and Biomarkers of Aging Study (SEBAS) were obtained, and SVI was constructed using 32 self-reported items of social determinants. Data from 985 participants (age: 65.73 ± 9.47 years, 54.62% males) were obtained for analysis, and the median SVI was 0.35 (IQR 0.29-0.42) with a near normal distribution. Participants with a higher SVI were more likely to be women and have poor cognitive function, more depressive symptoms and poor physical function. Adjusted for age and sex, each incremental deficit in SVI was associated with a 12% increase in mortality risk (HR: 1.12, 95% CI: 1.04-1.20, p = 0.002). An interaction was found between ApoE and SVI but not 5-HTTLPR. The strata-specific hazard ratio confirmed that associations between SVI and mortality was only in non-ε4 carriers (HR: 1.15, 95% CI: 1.07-1.24, p < 0.001), and SVI did not significantly predict mortality among ε4 carriers (HR: 0.84, 95% CI: 0.65-1.10). Differential SVI effects on mortality among middle-age and older adults were identified. In conclusion, a higher SVI was associated with all-cause mortality among middle-aged and older adults, and the association was moderated by ApoE genotypes but not 5-HTTLPR. Further study is needed to evaluate the clinical efficacy of healthy aging intervention programs considering gene-environment interactions and social vulnerability.

Leveraging the Treg-intrinsic CTLA4-PKCη signaling pathway for cancer immunotherapy.

BACKGROUND: Our previous studies revealed a critical role of a novel CTLA4-protein kinase C-eta (PKCη) signaling axis in mediating the suppressive activity of regulatory T cells (Tregs) in antitumor immunity. These studies have employed adoptive transfer of germline PKCη-deficient (Prkch-/-) Tregs into Prkch+/+ mice prior to tumor implantation. Here, we extended these findings into a biologically and clinically more relevant context. METHODS: We have analyzed the role of PKCη in antitumor immunity and the tumor microenvironment (TME) in intact tumor-bearing mice with Treg-specific or CD8+ T cell-specific Prkch deletion, including in a therapeutic model of combinatorial treatment. In addition to measuring tumor growth, we analyzed the phenotype and functional attributes of tumor-infiltrating immune cells, particularly Tregs and dendritic cells (DCs). RESULTS: Using two models of mouse transplantable cancer and a genetically engineered autochthonous hepatocellular carcinoma (HCC) model, we found, first, that mice with Treg-specific Prkch deletion displayed a significantly reduced growth of B16-F10 melanoma and TRAMP-C1 adenocarcinoma tumors. Tumor growth reduction was associated with a less immunosuppressive TME, indicated by increased numbers and function of tumor-infiltrating CD8+ effector T cells and elevated expression of the costimulatory ligand CD86 on intratumoral DCs. In contrast, CD8+ T cell-specific Prkch deletion had no effect on tumor growth or the abundance and functionality of CD8+ effector T cells, consistent with findings that Prkch-/- CD8+ T cells proliferated normally in response to in vitro polyclonal or specific antigen stimulation. Similar beneficial antitumor effects were found in mice with germline or Treg-specific Prkch deletion that were induced to develop an autochthonous HCC. Lastly, using a therapeutic model, we found that monotherapies consisting of Treg-specific Prkch deletion or vaccination with irradiated Fms-like tyrosine kinase 3 ligand (Flt3L)-expressing B16-F10 tumor cells post-tumor implantation significantly delayed tumor growth. This effect was more pronounced in mice receiving a combination of the two immunotherapies. CONCLUSION: These findings demonstrate the potential utility of PKCη inhibition as a viable clinical approach to treat patients with cancer, especially when combined with adjuvant therapies.

Determinants of improved quality of life among older adults with multimorbidity receiving integrated outpatient services: A hospital-based retrospective cohort study.

BACKGROUND: Older adults with multiple complex care needs tend to receive fragmented care that may jeopardize their quality of life (QoL) and health outcomes. This study evaluated the determinants of improved QoL among integrated outpatient service recipients with multimorbidity. METHODS: We conducted a retrospective cohort study of integrated geriatric outpatient services (IGOS) at a tertiary medical center in Taiwan. Data from 2018 to 2019 were retrieved. All patients underwent comprehensive geriatric assessment, which included demographic information, serial functional assessments, and assessment for QoL. QoL was reassessed through a telephone survey 6 months after the patients' first visit to IGOS. Factors associated with the interval changes in QoL were identified using multivariate logistic regression. RESULTS: Data from 995 patients receiving IGOS (mean age: 82.21 ± 7.96 years, 54.5% males) were analyzed. An overall mean improvement in QoL was noted (EQ-5D index: +0.055±0.26, p <0.001) while 747 recipients reported maintained or improved QoL. The results of the multivariate logistic regression showed that poorer nutritional status (OR = 1.56, 95% CI: 1.07-2.28), depressive symptoms (OR = 1.99, 95% CI: 1.38-2.86), and frailty (OR = 1.66, 95% CI: 1.10-2.52) were independent risk factors for poorer QoL after adjustment for baseline QoL. CONCLUSIONS: Integrated outpatient services improved the quality of life of older adults with multimorbidity. Those with poorer nutritional status, depressive symptoms and frailty were less likely to show improvement in their QoL.

Review, analysis, and education of antiseptic related ocular injury in the surgical settings.

Preoperative skin antiseptic preparation is the gold standard for prevention of surgical infection. However, improper use of antiseptics may lead to severe ocular damage. Currently, the most common surgical antiseptics can be divided into aqueous-based and alcohol-based disinfectants, with chlorhexidine and iodine/iodophors being the two major components. Chlorhexidine has a persistent antimicrobial effect and is resistant to neutralization by blood or organic products in surgical wounds. Nevertheless, due to its toxicity to the ears, meninges, and eyes, application of chlorhexidine should be prohibited in these surgical fields. Iodine/iodophor is better tolerated by the ocular surface and is the recommended antiseptic for ophthalmic or head and neck surgeries close to the periocular area. Alcohol is less pricey and has a rapid antiseptic effect, though its desiccating effect and flammability restrict the use in mucosal or laser surgeries. The single or combined use of these antiseptics may inadvertently induce severe ocular damage, especially during time-consuming head and neck surgeries with prone, hyperextension, or lateral tilt positions, or surgeries under general anesthesia. Apart from the choice of antiseptics, appropriate selection and attachment of bio-occlusive dressings are key to avoiding antiseptic-related ocular injuries. In this review, we provided a comprehensive summary of the characteristics of antiseptics used in surgical settings and the possible mechanisms and outcomes of antiseptic-related ocular injuries. The prevention, diagnosis, and acute management of these complications were also discussed.

Keratoconus Screening Based on Deep Learning Approach of Corneal Topography.

Purpose: To develop and compare deep learning (DL) algorithms to detect keratoconus on the basis of corneal topography and validate with visualization methods. Methods: We retrospectively collected corneal topographies of the study group with clinically manifested keratoconus and the control group with regular astigmatism. All images were divided into training and test datasets. We adopted three convolutional neural network (CNN) models for learning. The test dataset was applied to analyze the performance of the three models. In addition, for better discrimination and understanding, we displayed the pixel-wise discriminative features and class-discriminative heat map of diopter images for visualization. Results: Overall, 170 keratoconus, 28 subclinical keratoconus and 156 normal topographic pictures were collected. The convergence of accuracy and loss for the training and test datasets after training revealed no overfitting in all three CNN models. The sensitivity and specificity of all CNN models were over 0.90, and the area under the receiver operating characteristic curve reached 0.995 in the ResNet152 model. The pixel-wise discriminative features and the heat map of the prediction layer in the VGG16 model both revealed it focused on the largest gradient difference of topographic maps, which was corresponding to the diagnostic clues of ophthalmologists. The subclinical keratoconus was positively predicted with our model and also correlated with topographic indexes. Conclusions: The DL models had fair accuracy for keratoconus screening based on corneal topographic images. The visualization mentioned in the current study revealed that the model focused on the appropriate region for diagnosis and rendered clinical explainability of deep learning more acceptable. Translational Relevance: These high accuracy CNN models can aid ophthalmologists in keratoconus screening with color-coded corneal topography maps.

Use of white light in vivo confocal microscopy for the detection of spatial changes in the corneal nerves in cases of early-stage Acanthamoeba keratitis with radial keratoneuritis.

Purpose: Radial keratoneuritis (RK) is a common feature of Acanthamoeba keratitis (AK). In vivo confocal microscopy (IVCM) is noninvasive and provides real-time images for the diagnosis of corneal diseases by allowing the visualization of corneal structures and morphologies of living organisms at the cellular level. Images of AK with RK obtained using commercial white light IVCM devices have not been frequently evaluated. In the present study, a white light IVCM device was used to evaluate the corneal findings and describe spatial changes in the corneal nerves at different depths in cases of early-stage AK with RK. Methods: In this retrospective, observational study, white light IVCM images focused on RK were evaluated for Acanthamoeba cysts/trophozoites, corneal deposits, and altered corneal nerves, with special emphasis on three-dimensional spatial changes in the corneal nerves at different depths. Results: Seventeen eyes of 17 patients exhibiting early-stage AK with RK were included in the study. Acanthamoeba cysts/trophozoites were observed in the corneal epithelium of 13 eyes and stroma of 7 eyes. Alterations in the corneal nerve morphology and density were observed from the basal epithelial layer to the stromal layer in 12 eyes. Acanthamoeba trophozoites were attached to the corneal stromal nerves in five eyes. Conclusion: These findings suggest that white light IVCM can identify consistent corneal findings, particularly spatial changes in the corneal nerves, in cases of early-stage AK with RK.

Liposomal TLR9 Agonist Combined with TLR2 Agonist-Fused Antigen Can Modulate Tumor Microenvironment through Dendritic Cells.

Dendritic cells (DCs) are antigen-presenting cells involved in T cell activation and differentiation to regulate immune responses. Lipoimmunogens can be developed as pharmaceutical lipoproteins for cancer immunotherapy to target DCs via toll-like receptor 2 (TLR2) signaling. Previously, we constructed a lipoimmunogen, a lipidated human papillomavirus (HPV) E7 inactive mutant (rlipoE7m), to inhibit the growth of HPV16 E7-expressing tumor cells in a murine model. Moreover, this antitumor effect could be enhanced by a combinatory treatment with CpG oligodeoxynucleotides (ODN). To improve safety, we developed a rlipoE7m plus DOTAP liposome-encapsulated native phosphodiester CpG (POCpG/DOTAP) treatment to target DCs to enhance antitumor immunity. We optimized the formulation of rlipoE7m and POCpG/DOTAP liposomes to promote conventional DC and plasmacytoid DC maturation in vitro and in vivo. Combination of rlipoE7m plus POCpG/DOTAP could activate conventional DCs and plasmacytoid DCs to augment IL-12 production to promote antitumor responses by intravenous injection. In addition, the combination of rlipoE7m plus POCpG/DOTAP could elicit robust cytotoxic T lymphocytes (CTLs) by intravenous immunization. Interestingly, the combination of rlipoE7m plus POCpG/DOTAP could efficiently inhibit tumor growth via intravenous immunization. Moreover, rlipoE7m plus POCpG/DOTAP combined reduced the number of tumor-infiltrating regulatory T cells dramatically due to downregulation of IL-10 production by DCs. These results showed that the combination of rlipoE7m plus POCpG/DOTAP could target DCs via intravenous delivery to enhance antitumor immunity and reduce the number of immunosuppressive cells in the tumor microenvironment.

Local Irradiation Modulates the Pharmacokinetics of Metabolites in 5-Fluorouracil-Radiotherapy-Pharmacokinetics Phenomenon.

BACKGROUND: The effects of radiotherapy (RT) on the pharmacokinetics (PK) of 5-FU and 5-fluoro-5,6-dihydro-uracil (5-FDHU) were investigated by animal experiments. METHODS: Whole-pelvis RT with 0.5 and 2 Gy was delivered to Sprague-Dawley rats. 5-FU at 100 mg/kg was intravenously infused 24 h after radiation. The pharmacokinetics of 5-FU and 5-FDHU in the plasma and bile system were calculated. RESULTS: The areas under the concentration versus time curve (AUC) of 5-FU in the plasma were reduced by local irradiation by 23.7% at 0.5 Gy (P < 0.001) and 35.3% at 2 Gy (P < 0.001). The AUCs of 5-FDHU were also reduced by 21.4% at 0.5 Gy (P < 0.001) and 51.5% at 2 Gy (P < 0.001). Irradiation significantly increased the clearance values (CLs) of 5-FU by 30.6% at 0.5 Gy and 50.1% at 2 Gy, respectively. The CLs of 5-FDHU were increased by 27.2% at 0.5 Gy and 106% at 2 Gy. The AUCs of 5-FU in the bile were increased by 36.7% at 0.5 Gy (P < 0.001) and 68.6% at 2 Gy (P = 0.005). The AUCs of 5-FDHU in the bile were increased by 40.3% at 0.5 Gy (P < 0.001) and 248.1% at 2 Gy (P < 0.001). The CLs of 5-FU in the bile were increased by 31.8% at 0.5 Gy and 11.2% at 2 Gy. However, the CLs of 5-FDHU in the bile were decreased by 29.1% at 0.5 Gy and 71.0% at 2 Gy. CONCLUSION: Both conventional and low-dose irradiation can affect the pharmacokinetics of 5-FU and its metabolite, 5-FDHU. RT plus 5-FU could cause more adverse events than 5-FU alone by increasing the AUC ratio of 5-FU/5-FDHU. Irradiation decreases the AUC of 5-FU in the plasma, which may cause poor clinical outcomes.

Protein Kinase C-η Deficiency Does Not Impair Antiviral Immunity and CD8+ T Cell Activation.

We reported that protein kinase C-η (PKCη) forms a novel (to our knowledge) signaling complex with the checkpoint inhibitory protein CTLA-4 in regulatory T cells (Tregs). This complex is required for the contact-dependent suppressive activity of Tregs, including suppression of antitumor immunity. However, the importance of PKCη in protective immunity mediated by T effector cells remains unclear. We used mice with germline or conditional Treg-specific deletion of Prkch, the PKCη-encoding gene, to explore CD8+ T cell-dependent antiviral immunity using the lymphocytic choriomeningitis virus Armstrong strain acute infection model as well as the in vitro activation of murine or human CD8+ T cells. Five days following infection, germline Prkch -/- mice displayed enhanced viral clearance compared with control mice. Similarly, Prkch Treg-specific conditional knockout mice also showed improved viral clearance and displayed enhanced expression of granzyme B and IFN-γ by both virus-specific and total CD8+ T cells, demonstrating that enhanced viral clearance in germline Prkch -/- mice is caused by PKCη deficiency in Tregs and the resulting functional defect of Prkch -/- Tregs. In addition, purified Prkch -/- mouse CD8+ T cells as well as PRKCH knockdown human CD8+ T cells displayed intact, or even enhanced, T cell activation in vitro as measured by proliferation and expression of granzyme B and IFN-γ. Thus, global PKCη deletion does not impair overall CD8+ T cell-mediated immunity, including antiviral immunity, implying that selective pharmacological PKCη inhibition could be safely used in vivo to inhibit undesired contact-dependent suppression by Tregs and, thus, enhance tumor-specific and, likely, virus-specific immunity.

Clinical features and outcomes of Acanthamoeba keratitis in a tertiary hospital over 20- year period.

PURPOSE: To analyze the outcomes of Acanthamoeba keratitis (AK) in terms of different clinical presentations in a tertiary hospital in Taiwan over a 20- year period. METHODS: This is a retrospective case series. Patients with AK diagnosed at the National Taiwan University Hospital between January 1996 and December 2015 were identified. A diagnosis of AK was made on the basis of positive Acanthamoeba smear/cultures or pathological identification of Acanthamoeba cysts on keratoplasty specimens. Patient demographics, clinical presentations, treatment courses, and final visual outcomes were collected and analyzed. Visual acuity, postoperative complications and graft survivals were measured as outcomes. RESULTS: Of the 62 patients with AK in our study, 64.5% were medically treated while 35.5% underwent surgical treatment. In those with ring infiltrate, 52.4% patients could be successfully treated with medications. In eyes receiving penetrating keratoplasty, postoperative complications were more common in therapeutic penetrating keratoplasty (TPK) than those in optical penetrating keratoplasty (OPK) group (82.4% versus 40%, p = 0.04). The graft size was larger in TPK than that in OPK group (8.56 versus 7.83 mm, p = 0.002). Furthermore, post-operative glaucoma, which was the major complication, was found to be associated with larger graft size (p = 0.02) and dilated pupil/iris atrophy (p = 0.01). CONCLUSION: Even in advanced cases with ring infiltrate, eradication of infection with anti-amoebic drugs is possible. In those requiring keratoplasty, the surgical timing should be made meticulously considering graft size and signs of dilated pupil/iris atrophy in terms of post-operative glaucoma.

Microbial Keratitis in Taiwan: A 20-Year Update.

PURPOSE: To investigate the demographics, risk factors, microbiology, and resistance pattern at a tertiary hospital and to detect the shifting trend over 2 decades. DESIGN: A retrospective observational case series. METHODS: We reviewed all records of patients with microbial keratitis (MK) that were hospitalized in National Taiwan University Hospital between 2007 and 2016. Demographics, predisposing factors, pathogens, and clinical courses were compared to our previous study conducted from 1992 to 2001. Antibiotic susceptibility was compared with those conducted from 1994 to 2005. RESULTS: The percentage of patients 60 years and older in the MK population was increasing (P = 2.1E-21). The proportion of trauma-related MK declined while MK related to chronic ocular or systemic disorders rose. The prevalence of nontuberculous mycobacteria (NTM) showed a decreasing trend (P = .0032), whereas Microsporidia has been increasingly detected. The 2 most common bacterial isolates were Pseudomonas aeruginosa (35.2%) and Staphylococcus species (13.2%). Management of these infection did not differ in common pathogens between the 2 decades. The susceptibility of Staphylococcus species to oxacillin reduced significantly (P = .002) and there was an increase in methicillin-resistant Staphylococcus aureus keratitis. CONCLUSIONS: Contact lens wear remained the most common predisposing factor, with Pseudomonas species as the major pathogen. However, chronic disorder-related MK was on the rise along with an increasing trend of oxacillin resistance in Staphylococcus species. We found a decreasing trend in NTM keratitis while Microsporidia keratitis was considered as an emerging ocular disease. Though gram-negative isolates remained susceptible to all antibiotics tested, antibiotic resistance was more common in gram-positive isolates.