Automatic measurement of mouse visual acuity based on optomotor response: SKY optomotry.

In the field of visual science study using rodents, several assessment methods have been developed for measuring visual function. However, methods such as electroretinograms tests, visual evoked potentials tests and maze tests have limitations in that they measure function of only a specific type of cells, are difficult to quantify or require sufficient training time. The method which uses an optokinetic reflex and optomotor response, a compensatory eye and head movement in response to changes in the visual scene, became the most widely used method. However, this method requires highly trained experimenters and is time consuming. We showed that measured visual acuity values are significantly different between beginner and expert. Here we suggest an automated optometry program, 'SKY optomotry', which automatically tracks rodents' optomotor response to overcome subjectivity and the lengthy scoring procedure of the existing method. To evaluate the performance of SKY optomotry using 8-12-week-old C57BL/6 mice we compared the binomial decision of SKY optomotry with a skilled expert, and the area under the curve of SKY optomotry was 0.845. Comparing the final visual acuity, the intraclass correlation coefficient value between SKY optomotry and an expert was 0.860 (95% confidence interval (CI) 0.709-0.928), whereas that between an expert and a beginner was 0.642 (95% CI 0.292-0.811). SKY optomotry showed an excellent level of performance with good inter-rater agreements based on the visual acuity measured by an expert. With the use of our application, researchers will be able to test an experimental animal's eyesight more accurately while saving time on specialized training.

Effectiveness and safety of injectable human papilloma virus vaccine administered as eyedrops.

Mucosal vaccines have the advantages of ease of administration and the induction of strong mucosal immunity and a systemic immune response. Recently, the eye mucosa has been shown to be an effective and safe alternative vaccination route against influenza, Toxoplasma gondii infection, and hemolytic uremic syndrome in mice. In this study, we showed that the commercially available human papilloma virus (HPV) vaccine, Cervarix, induced significant immune reactions in terms of anti-HPV antigen (Ag)-specific immunoglobulin G (IgG) and IgA antibody production following eyedrop (ED) vaccination in mice. The HPV ED vaccines (EDV) provoked no signs of inflammation within 24 h, as indicated by the inflammatory cytokine mRNA levels and infiltration of mononuclear cells in inoculation sites. Moreover, the morphology of the cornea and retina and intraocular pressure of mice did not change after the HPV EDV. The functions of photoreceptor cells, including rod and cone cells, were normal following the HPV EDV inoculation in mice. These results suggest that Cervarix EDV could be a potent, safe, and effective mucosal vaccine against HPV-associated cancers.

Eyedrop vaccination: an immunization route with promises for effective responses to pandemics.

INTRODUCTION: Mucosal vaccines have several advantages over parenteral vaccines. They induce both systemic and mucosal antigen-specific immune responses, allow easy administration, and bypass the need for trained medical personnel. AREAS COVERED: Eye mucosa is a novel route of mucosal vaccine administration. Eyedrop vaccination induces systemic and mucosal immune responses similar to other forms of mucosal vaccines such as oral and intranasal vaccines. EXPERT OPINION: Eyedrop vaccines are free of serious adverse side effects like the infiltration of CNS by pathogens. Studies over the years have shown promising results for eye drop vaccines against infectious agents like the influenza virus, Salmonella typhi, and Escherichia coli in animal models. Such efficacy and safety of eyedrop vaccination enable the application of eyedrop vaccines against other infectious diseases as well as chronic diseases. In this review of published literature, we examine the mechanism, efficacy, and safety of eyedrop vaccines and contemplate their role in times of a pandemic.

Granulocyte Macrophage-Colony Stimulating Factor Produces a Splenic Subset of Monocyte-Derived Dendritic Cells That Efficiently Polarize T Helper Type 2 Cells in Response to Blood-Borne Antigen.

Dendritic cells (DCs) are key antigen-presenting cells that prime naive T cells and initiate adaptive immunity. Although the genetic deficiency and transgenic overexpression of granulocyte macrophage-colony stimulating factor (GM-CSF) signaling were reported to influence the homeostasis of DCs, the in vivo development of DC subsets following injection of GM-CSF has not been analyzed in detail. Among the treatment of mice with different hematopoietic cytokines, only GM-CSF generates a distinct subset of XCR1-33D1- DCs which make up the majority of DCs in the spleen after three daily injections. These GM-CSF-induced DCs (GMiDCs) are distinguished from classical DCs (cDCs) in the spleen by their expression of CD115 and CD301b and by their superior ability to present blood-borne antigen and thus to stimulate CD4+ T cells. Unlike cDCs in the spleen, GMiDCs are exceptionally effective to polarize and expand T helper type 2 (Th2) cells and able to induce allergic sensitization in response to blood-borne antigen. Single-cell RNA sequencing analysis and adoptive cell transfer assay reveal the sequential differentiation of classical monocytes into pre-GMiDCs and GMiDCs. Interestingly, mixed bone marrow chimeric mice of Csf2rb+/+ and Csf2rb-/- demonstrate that the generation of GMiDCs necessitates the cis expression of GM-CSF receptor. Besides the spleen, GMiDCs are generated in the CCR7-independent resident DCs of the LNs and in some peripheral tissues with GM-CSF treatment. Also, small but significant numbers of GMiDCs are generated in the spleen and other tissues during chronic allergic inflammation. Collectively, our present study identifies a splenic subset of CD115hiCD301b+ GMiDCs that possess a strong capacity to promote Th2 polarization and allergic sensitization against blood-borne antigen.

Immunogenicity of Exosomes from Dendritic Cells Stimulated with Toxoplasma gondii Lysates in Ocularly Immunized Mice.

Immunogenicity of dendritic cell-derived exosomes stimulated with Toxoplasma gondii lysates (TLA exo), mixed with cholera toxin as an adjuvant, was investigated in mice immunized via 2 mucosal routes (ocular vs intranasal). BALB/c mice were injected 3 times with TLA exo vaccine at 2 week interval, and the levels of IgG in serum and IgA in tear, saliva, feces, and vaginal wash were measured. To observe the expression of T. gondii-specific B1 gene, mice infected with ME49 T. gondii cysts were immunized with TLA exo or PBS exo (not stimulated with TLA), and their brain tissues were examined. The mice vaccinated via intranasal route elicited significantly higher humoral and mucosal immune responses compared with mice treated with PBS alone. Also, mice immunized via ocular route (by eyedrop) induced significantly higher T. gondii-specific IgG in serum and IgA in tear and feces in comparison with PBS controls. B1 gene expression was significantly lower in TLA exo vaccinated mice than in PBS or PBS exo vaccinated mice. These results demonstrated that ocular immunization of mice with TLA exo vaccine has the potential to stimulate systemic or local antibody responses. This study also highlighted an advantage of an eyedrop vaccine as an alternative for T. gondii intranasal vaccines.

Eyedrop Vaccination Induced Systemic and Mucosal Immunity against Influenza Virus in Ferrets.

We investigated eyedrop vaccination (EDV) in pre-clinical development for immunological protection against influenza and for potential side effects involving ocular inflammation and the central nervous system (CNS). Live attenuated influenza EDV, CA07 (H1N1), PZ-4 (H1N2) and Uruguay (H3N2), induced both systemic and mucosal virus-specific antibody responses in ferrets. In addition, EDV resulted in a clinically significant protection against viral challenge, and suppression of viral replication in nasal secretion and lung tissue. Regarding safety, we found that administered EDV flow through the tear duct to reach the base of nasal cavity, and thus do not contact the olfactory bulb. All analyses for potential adverse effects due to EDV, including histological and functional examinations, did not reveal significant side effects. On the basis of these findings, we propose that EDV as effective, while being a safe administration route with minimum local side effects, CNS invasion, or visual function disturbance.

Inactivated Eyedrop Influenza Vaccine Adjuvanted with Poly(I:C) Is Safe and Effective for Inducing Protective Systemic and Mucosal Immunity.

The eye route has been evaluated as an efficient vaccine delivery routes. However, in order to induce sufficient antibody production with inactivated vaccine, testing of the safety and efficacy of the use of inactivated antigen plus adjuvant is needed. Here, we assessed various types of adjuvants in eyedrop as an anti-influenza serum and mucosal Ab production-enhancer in BALB/c mice. Among the adjuvants, poly (I:C) showed as much enhancement in antigen-specific serum IgG and mucosal IgA antibody production as cholera toxin (CT) after vaccinations with trivalent hemagglutinin-subunits or split H1N1 vaccine antigen in mice. Vaccination with split H1N1 eyedrop vaccine antigen plus poly(I:C) showed a similar or slightly lower efficacy in inducing antibody production than intranasal vaccination; the eyedrop vaccine-induced immunity was enough to protect mice from lethal homologous influenza A/California/04/09 (H1N1) virus challenge. Additionally, ocular inoculation with poly(I:C) plus vaccine antigen generated no signs of inflammation within 24 hours: no increases in the mRNA expression levels of inflammatory cytokines nor in the infiltration of mononuclear cells to administration sites. In contrast, CT administration induced increased expression of IL-6 cytokine mRNA and mononuclear cell infiltration in the conjunctiva within 24 hours of vaccination. Moreover, inoculated visualizing materials by eyedrop did not contaminate the surface of the olfactory bulb in mice; meanwhile, intranasally administered materials defiled the surface of the brain. On the basis of these findings, we propose that the use of eyedrop inactivated influenza vaccine plus poly(I:C) is a safe and effective mucosal vaccine strategy for inducing protective anti-influenza immunity.

Protection from hemolytic uremic syndrome by eyedrop vaccination with modified enterohemorrhagic E. coli outer membrane vesicles.

We investigated whether eyedrop vaccination using modified outer membrane vesicles (mOMVs) is effective for protecting against hemolytic uremic syndrome (HUS) caused by enterohemorrhagic E. coli (EHEC) O157:H7 infection. Modified OMVs and waaJ-mOMVs were prepared from cultures of MsbB- and Shiga toxin A subunit (STxA)-deficient EHEC O157:H7 bacteria with or without an additional waaJ mutation. BALB/c mice were immunized by eyedrop mOMVs, waaJ-mOMVs, and mOMVs plus polymyxin B (PMB). Mice were boosted at 2 weeks, and challenged peritoneally with wild-type OMVs (wtOMVs) at 4 weeks. As parameters for evaluation of the OMV-mediated immune protection, serum and mucosal immunoglobulins, body weight change and blood urea nitrogen (BUN)/Creatinin (Cr) were tested, as well as histopathology of renal tissue. In order to confirm the safety of mOMVs for eyedrop use, body weight and ocular histopathological changes were monitored in mice. Modified OMVs having penta-acylated lipid A moiety did not contain STxA subunit proteins but retained non-toxic Shiga toxin B (STxB) subunit. Removal of the polymeric O-antigen of O157 LPS was confirmed in waaJ-mOMVs. The mice group vaccinated with mOMVs elicited greater humoral and mucosal immune responses than did the waaJ-mOMVs and PBS-treated groups. Eyedrop vaccination of mOMVs plus PMB reduced the level of humoral and mucosal immune responses, suggesting that intact O157 LPS antigen can be a critical component for enhancing the immunogenicity of the mOMVs. After challenge, mice vaccinated with mOMVs were protected from a lethal dose of wtOMVs administered intraperitoneally, conversely mice in the PBS control group were not. Collectively, for the first time, EHEC O157-derived mOMV eyedrop vaccine was experimentally evaluated as an efficient and safe means of vaccine development against EHEC O157:H7 infection-associated HUS.

Effect of voriconazole and ultraviolet-A combination therapy compared to voriconazole single treatment on Fusarium solani fungal keratitis.

PURPOSE: To demonstrate that ultraviolet-A (UV-A) and voriconazole combination therapy is more effective than voriconazole single treatment for fungal keratitis. METHODS: The in vitro UV-A (375 nm) fungicidal effect was evaluated on Fusarium solani solutions. Each fungal solution was irradiated with different UV-A irradiation doses. Also, a fungal solution containing voriconazole was also irradiated with UV-A. The in vivo therapeutic effect of UV-A and voriconazole treatment was studied in a rabbit keratitis model. Fungi were injected intrastromally into the cornea of 16 rabbits. Each treatment was initiated 3 days after fungal injection and continued up to 8 days for the following groups: Group 1, control; Group 2, treated with UV-A once a day; Group 3, treated with voriconazole 3 times a day; Group 4, treated with voriconazole 3 times a day and UV-A once a day. On the last day, the sclera-cornea buttons were extracted and microbiological and histological evaluations were performed. RESULTS: The colony-forming units (CFUs) of fungal solutions in culture significantly decreased with UV-A irradiation. The CFUs of fungal solutions containing voriconazole also decreased with UV-A irradiation. In vivo, clinical scores of Group 3 (P=0.03) and Group 4 (P=0.02) 5 days after treatment were significantly lower compared to that of Group 1. The clinical score of Group 4 (P=0.03) 5 days after treatment was significantly lower compared to that of Group 3. The histopathological scores 5 days after treatment were significantly lower in Group 4 compared to those of Group 1 (P<0.01) and Group 3 (P=0.02). Based on our CFU analysis, only Group 4 showed significantly lower CFUs compared to Group 1 (P=0.04). CONCLUSIONS: UV-A and voriconazole combination treatment could be a safe and effective alternative to voriconazole single treatment for fungal keratitis.

Pathological effect of IL-17A-producing TCRγδ(+) T cells in mouse genital mucosa against HSV-2 infection.

Interleukin (IL)-17A is a cytokine that plays an important role in infectious, autoimmune, and inflammatory diseases. In this study, we found that TCRγδ(+)CD4(-)CD8(-) T cells, but not TCRαß(+)CD4(+) T cells, are the primary producers of IL-17A in the genital tract of female mice in the steady-state condition. High mRNA levels of IL-17A and RORγt were determined in TCRγδ(+) T cells isolated from mouse genital tract but lacked detectable expression of IFNγ, T-bet, and FoxP3. IL-17A production by genital TCRγδ(+) T cells was maintained after intravaginal vaccination with cholera toxin or avirulent herpes simplex virus type (HSV)-2 186 syn ΔTK strain. Of note, the deaths of IL-17A(-/-) mice were significantly delayed after intravaginal HSV-2 infection compared with wild-type mice. Further, genital TCRγδ(+) T cells continued to produce comparable amounts of IL-17A after antibiotic treatment. These results imply that genital IL-17A-producing TCRγδ(+) T cells constitutively exist at steady state and that they play a pathogenic effect against HSV-2 infection and are not affected by microflora, unlike conventional Th17 cells.

Mucosa-associated epithelial chemokine/CCL28 expression in the uterus attracts CCR10+ IgA plasma cells following mucosal vaccination via estrogen control.

Previous studies demonstrated cross talk between mucosal and reproductive organs during secretory IgA Ab induction. In this study, we aimed to clarify the underlying mechanisms of this cross talk. We found significantly higher titers of Ag-specific secretory IgA Ab in the vaginal wash after mucosal vaccination by both the intranasal (i.n.) and the intravaginal routes but not by the s.c. route. Interestingly, Ag-specific IgA Ab-secreting cells (ASCs) were found mainly in the uterus but not in the cervix and vaginal canal after i.n. vaccination. The fact that most Ag-specific IgA ASCs isolated from the uteri of vaccinated mice migrated toward mucosa-associated epithelial chemokine (MEC)/CCL28 suggests dominant expression of CCR10 on the IgA ASCs. Further, IgA ASCs in the uteri of vaccinated mice were reduced drastically in mice treated with neutralizing anti-MEC/CCL28 Ab. Most intriguingly, the female sex hormone estrogen directly regulated MEC/CCL28 expression and was augmented by i.n. vaccination with cholera toxin or stimulators for innate immunity. Further, blockage of estrogen function in the uterus by oral administration of the estrogen antagonist raloxifene significantly inhibited migration of Ag-specific IgA ASCs after i.n. vaccination with OVA plus cholera toxin. Taken together, these data strongly suggest that CCR10(+) IgA ASCs induced by mucosal vaccination via the i.n. route migrate into the uterus in a MEC/CCL28-dependent manner and that estrogen might have a crucial role in the protection against genital infection by regulating MEC/CCL28 expression in the uterus.

Gly-Ala-Gly-Val-Gly-Tyr, a novel synthetic peptide, improves glucose transport and exerts beneficial lipid metabolic effects in 3T3-L1 adipoctyes.

Recently, it has been demonstrated that fibroin and fibroin-derived peptides enhances insulin sensitivity and glucose metabolism in adipocytes. Here, we show that a synthetic hexapeptide Gly-Ala-Gly-Val-Gly-Tyr (GAGVGY) derived from repetitive amino acid sequence of fibroin improves glucose transport and exerts beneficial lipid metabolic effects in 3T3-L1 adipocytes. GAGVGY increases both basal and insulin-stimulated glucose uptake through enhancement of GLUT1 expression and PI 3-K-dependent GLUT4 translocation, respectively. GAGVGY treatment also led to a significant reduction in the expression of lipogenic genes including sterol regulatory element binding protein-1c (SREBP1c), peroxisome proliferator-activated receptor-γ (PPARγ), and fatty acid synthase (FAS) in mature 3T3-L1 adipocytes, which was corroborated with decreased lipid accumulation by GAGVGY treatment. Additionally, in cells undergoing differentiation, mRNA levels of adipogenic genes including PPARγ and CCAAT/enhancer binding protein α (C/EBPα), stearoyl-CoA desaturase 1 (SCD1), and FAS were suppressed by GAGVGY. Furthermore, GAGVGY increased AMP-activated protein kinase (AMPK) phosphorylation and adiponectin secretion in 3T3-L1 adipocytes. The latter effect was supported with evidence showing increased AMPK activation in C2C12 myocytes treated with 3T3-L1-adipocyte-conditioned medium. Together, our data suggest that GAGVGY has multiple beneficial effects on glucose and lipid metabolism, and would control hyperglycemia without the adverse effect of weight gain.

Adipose-specific deletion of stearoyl-CoA desaturase 1 up-regulates the glucose transporter GLUT1 in adipose tissue.

Stearoyl-CoA desaturase 1 (SCD1) deficiency protects mice from diet-induced obesity and insulin resistance. To understand the tissue-specific role of SCD1 in energy homeostasis, we have generated mice with an adipose-specific knockout of Scd1 (AKO), and report here that SCD1 deficiency increases GLUT1 expression in adipose tissue of AKO mice, but not global SCD1 knockout (GKO) mice. In 3T3-L1 adipocytes treated with an SCD inhibitor, basal glucose uptake and the cellular expression of GLUT1 were significantly increased while GLUT4 expression remained unchanged. Consistently, adipose-specific SCD1 knockout (AKO) mice had significantly elevated GLUT1 expression, but not GLUT4, in white adipose tissue compared to Lox counterparts. Concurrently, adiponectin expression was significantly diminished, whereas TNF-alpha expression was elevated. In contrast, in adipose tissue of GKO mice, GLUT4 and adiponectin expression were significantly elevated with lowered TNF-alpha expression and little change in GLUT1 expression, suggesting a differential responsiveness of adipose tissue to global- or adipose-specific SCD1 deletion. Taken together, these results indicate that adipose-specific deletion of SCD1 induces GLUT1 up-regulation in adipose tissue, associated with decreased adiponectin and increased TNF-alpha production, and suggest that GLUT1 may play a critical role in controlling glucose homeostasis of adipose tissue in adipose-specific SCD1-deficient conditions.

Fibroin-derived peptides stimulate glucose transport in normal and insulin-resistant 3T3-L1 adipocytes.

Fibroin, the protein of silk, and hydrolyzed fibroin have recently been described to enhance insulin sensitivity and glucose metabolism in 3T3-L1 adipocytes. Here, we report that a series of synthetic peptides derived from the fibroin sequence have enhancing effects on glucose transport in normal and insulin-resistant 3T3-L1 cells. We observed that, among several enzymatic hydrolysates of fibroin, the chymotryptic and peptic hydrolysates were significantly more effective than others in augmenting insulin-stimulated glucose uptake in both cells. We synthesized several peptides of repetitive sequences in fibroin. Treatment with synthesized hexapeptides enhanced insulin-stimulated glucose uptake more than tri-, tetra- or pentapeptides. Among those, the effect of Gly-Ala-Gly-Ala-Gly-Tyr (GAGAGY) was most robust, and especially its activity of blocking off the chronic-insulin-induced loss of insulin-stimulated uptake was remarkable. Data reveal that the residues of tyrosine situated at the ends of the peptides play a critical role for exerting their activities. We demonstrate that the insulin-sensitizing effect of GAGAGY is due to enhancement of phosphoinositide 3-kinase (PI 3-K) signaling pathway. The GAGAGY-induced insulin-stimulated glucose uptake was sensitive to inhibition of PI 3-K by wortmannin. Phosphorylation of Akt was also elevated in GAGAGY-treated cells. Furthermore, GAGAGY significantly increased insulin-induced glucose transporter 4 (GLUT4) translocation without affecting the synthesis of GLUT4. Our findings suggest that fibroin-derived peptides such as GAGAGY could be considered as novel insulin-sensitizing agents with an activity of blocking the development of insulin resistance.

Berberine activates GLUT1-mediated glucose uptake in 3T3-L1 adipocytes.

It has recently been known that berberine, an alkaloid of medicinal plants, has anti-hyperglycemic effects. To explore the mechanism underlying this effect, we used 3T3-L1 adipocytes for analyzing the signaling pathways that contribute to glucose transport. Treatment of berberine to 3T3-L1 adipocytes for 6 h enhanced basal glucose uptake both in normal and in insulin-resistant state, but the insulin-stimulated glucose uptake was not augmented significantly. Inhibition of phosphatidylinositol 3-kinase (PI 3-K) by wortmannin did not affect the berberine effect on basal glucose uptake. Berberine did not augment tyrosine phosphorylation of insulin receptor (IR) and insulin receptor substrate (IRS)-1. Further, berberine had no effect on the activity of the insulin-sensitive downstream kinase, atypical protein kinase C (PKCzeta/lambda). However, interestingly, extracellular signal-regulated kinases (ERKs), which have been known to be responsible for the expression of glucose transporter (GLUT)1, were significantly activated in berberine-treated 3T3-L1 cells. As expected, the level of GLUT1 protein was increased both in normal and insulin-resistant cells in response to berberine. But berberine affected the expression of GLUT4 neither in normal nor in insulin-resistant cells. In addition, berberine treatment increased AMP-activated protein kinase (AMPK) activity in 3T3-L1 cells, which has been reported to be associated with GLUT1-mediated glucose uptake. Together, we concluded that berberine increases glucose transport activity of 3T3-L1 adipocytes by enhancing GLUT1 expression and also stimulates the GLUT1-mediated glucose uptake by activating GLUT1, a result of AMPK stimulation.