Oral Porphyromonas gingivalis infection affects intestinal microbiota and promotes atherosclerosis.

AIM: The link between periodontitis and intestinal dysbiosis, two factors that contribute to atherosclerosis, has not been clearly defined. We investigated the integrative effects of oral infection with Porphyromonas gingivalis (PG), the major pathogen for periodontitis, on intestinal microbiota and atherosclerosis. MATERIALS AND METHODS: ApoE-/- mice were fed a normal chow diet (NC), a Western diet (WD) or a WD with oral PG infection (PG). The PG infection was investigated by placing a total of 109 CFUs of live PG into the oral cavity of each mouse using a feeding needle five times a week for 3 weeks. Atherosclerotic lesions of the aortae were measured, and blood lipoproteins and the expression of molecules related to lipid metabolism in the liver were analysed. We also performed 16S RNA sequencing and a microbiome analysis using faeces. RESULTS: En face bloc preparation of the aortae showed that the PG group had a 1.7-fold increase in atherosclerotic lesions compared with the WD group (p < .01). Serum analyses showed that oral PG infection induced a significant decrease in high-density lipoprotein (HDL) and triglyceride. Western blots of hepatic tissue lysates revealed that PG infection reduced the expression of scavenger receptor class B type 1 (SR-B1) in the liver by 50%. Faecal microbiota analysis revealed that species richness estimates (Chao1, ACE) decreased immediately after PG infection. PG infection also induced a significant decrease in Shannon diversity and an increase in Simpson's indices in the WD-fed mice. PG infection significantly increased the phyla Actinobacteria and Deferribacteres, along with the species Mucispirillum schaedleri and Lactobacillus gasseri, in the mice. The functional study showed that PG infection increased the expression of proteins that function in carbohydrate and glucose metabolism, including phosphotransferase system (PTS) proteins and the GntR family transcriptional regulator. CONCLUSIONS: Oral PG infection promotes atherosclerosis and induces significant metabolic changes, including reduced serum HDL and reduced hepatic SR-B1 and ABCA1 expression, as well as changes in intestinal microbiota. Our study suggests that intestinal dysbiosis accompanies periodontitis and could play a role in atherosclerosis.

TH17 cells mediate inflammation in a novel model of spontaneous experimental autoimmune lacrimal keratoconjunctivitis with neural damage.

BACKGROUND: Dry eye disease (DED) affects one third of the population worldwide. In prior studies, experimental autoimmune lacrimal keratoconjunctivitis (EALK) induced by desiccating stress in mice has been used as a model of DED. This model is complicated by a requirement for exogenous epithelial cell injury and administration of anticholinergic agents with broad immunologic effects. OBJECTIVE: We sought to develop a novel mouse model of EALK and to demonstrate the responsible pathogenic mechanisms. METHODS: CD4+CD45RBhigh naive T cells with and without CD4+CD45RBlow regulatory T cells were adoptively transferred to C57BL/10 recombination-activating gene 2 (Rag2)-/- mice. The eyes, draining lymph nodes, lacrimal glands, and surrounding tissues of mice with and without spontaneous keratoconjunctivitis were evaluated for histopathologic changes, cellular infiltration, and cytokine production in tissues and isolated cells. Furthermore, the integrity of the corneal nerves was evaluated using whole-tissue immunofluorescence imaging. Gene-deficient naive T cells or RAG2-deficient hosts were evaluated to assess the roles of IFN-γ, IL-17A, and IL-23 in disease pathogenesis. Finally, cytokine levels were determined in the tears of patients with DED. RESULTS: EALK developed spontaneously in C57BL/10 Rag2-/- mice after adoptive transfer of CD4+CD45RBhigh naive T cells and was characterized by infiltration of CD4+ T cells, macrophages, and neutrophils. In addition to lacrimal keratoconjunctivitis, mice had damage to the corneal nerve, which connects components of the lacrimal functional unit. Pathogenic T-cell differentiation was dependent on IL-23p40 and controlled by cotransferred CD4+CD45RBlow regulatory T cells. TH17 rather than TH1 CD4+ cells were primarily responsible for EALK, even though levels of both IL-17 and IFN-γ were increased in inflammatory tissues, likely because of their ability to drive expression of CXC chemokines within the cornea and the subsequent influx of myeloid cells. Consistent with the findings of this model, the tears of patients with DED had increased levels of inflammatory cytokines, including IL-17A and TNF-α. CONCLUSION: We describe a novel model of spontaneous EALK that supports a role for TH17 cells in disease pathogenesis and that will contribute to our understanding of autoimmune lacrimal keratoconjunctivitis in many human eye diseases, including DED.

Meibomian gland dysfunction and tear cytokines after cataract surgery according to preoperative meibomian gland status.

BACKGROUND: To investigate the possible mechanisms by which cataract surgery aggravates meibomian gland dysfunction (MGD), we evaluated the changes in tear cytokines and ocular surface parameters after cataract surgery according to the preoperative MGD grade. DESIGN: Prospective, observational case series. PARTICIPANTS: A total of 50 eyes from 50 patients who underwent cataract surgery were included. METHODS: Patients were classified into two groups: Group I had no or minimal MGD, and group II had grades 2-4 MGD. Ocular surface parameters were measured, including tear film break-up time, Schirmer I test, ocular surface staining and Ocular Surface Disease Index, and tear cytokine levels were measured. MAIN OUTCOME MEASURES: The main outcomes were changes in ocular surface parameters and inflammatory tear cytokine concentrations. RESULTS: In group II, preoperative MGD grade, ocular surface staining, tear film break-up time and Ocular Surface Disease Index were worse, and mean interleukin (IL)-2, IL-6 and TNF-α levels were higher than those of group I. MGD and ocular surface parameters were worsened to a greater degree after surgery in group II than in group I (P < 0.050). In group II, IL-6 and TNF-α levels significantly increased at postoperative 1 month, and there were significant correlations between changes in ocular surface parameters and tear cytokines (IL-2, IL-6 and TNF-α; P < 0.050). CONCLUSIONS: The extent to which the MGD grade was aggravated following cataract surgery differed based on preoperative MGD grade. Preoperative MGD and ocular surface status should be carefully evaluated.

Tear Cytokines as Biomarkers for Chronic Graft-versus-Host Disease.

We investigated the tear cytokine profiles in patients who underwent stem cell transplantation (SCT) and attempted to evaluate whether tear cytokines are associated with the presence of systemic chronic graft-versus-host disease (GVHD), regardless of ocular GVHD status. We also tested tear cytokines as biomarkers for chronic ocular GVHD severity. Forty-four patients who underwent SCT were enrolled and their diagnosis of chronic GVHD was confirmed. Ocular surface parameters and tear cytokine profiles were evaluated and the correlations between concentrations of cytokines and ocular surface parameters or several chronic ocular GVHD severity scales were evaluated. Tear interleukin (IL)-2, IL-10, IL-17α, interferon (IFN)-γ, IL-6, and tumor necrosis factor (TNF)-α were elevated in patients with chronic systemic GVHD compared with patients without chronic systemic GVHD. Receiver-operating characteristic curve analysis revealed that area under the curve (AUC) values for tear IL-10 (AUC = .795), IL-17α (AUC = .821), IL-6 (AUC = .912), and TNF-α (AUC = .910) were significantly correlated with the presence of chronic GVHD (all P < .001). Tear IL-10, IL-6, and TNF-α showed a stronger correlation with ocular surface parameters than other cytokines and these cytokines also correlated with several chronic ocular GVHD severity scales (all P < .05). Our data suggest the tear cytokines are useful biomarkers for the diagnosis of chronic GVHD after SCT and chronic ocular GVHD severity.

Decision and Practice of End-of-Life Care in Lung Disease Patients with Physicians Orders for Life Sustaining Treatment.

Purpose: The purpose of this study was to analyze end-of-life care practices in lung disease patients with physician orders for life-sustaining treatment (POLSTs). Methods: We retrospectively analyzed data from medical records regarding the end-of-life care practices of POLST decisions for patients with lung disease hospitalized at a tertiary hospital in Seoul, South Korea. Data were collected from January 1 to June 30, 2021. Results: Of 300 total patients, 198 had lung cancer (66.0%) and 102 had non-malignant lung diseases (34.0%). A POLST was written for 187 patients (62.3%), and an advance directive was written for 20 patients (6.7%). Subsequent treatments were hemodialysis in 13 patients (4.3%), surgery in 3 patients (1.0%), and cardiopulmonary cerebral resuscitation in 1 patient (0.3%). Among cancer patients, chemotherapy was performed in 11 patients (3.7%), targeted therapy in 11 patients (3.7%), immunotherapy in 6 patients (2.0%), and radiation therapy in 13 patients (4.3%). Depending on the type of lung disease, types of treatment differed, including hemodialysis, ventilators, bilevel positive airway pressure, high-flow nasal cannulas, nebulizers, enteral nutrition, central line, inotropic agents, and opioids. Conclusion: Although the goals of hospice care are the same whether a patient has lung cancer or a non-malignant lung disease, because the characteristics of the respective diseases differ, end-of-life care practices and hospice approaches must be considered differently.

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.

Eye mucosa: an efficient vaccine delivery route for inducing protective immunity.

The external part of the eye shares mucosa-associated common characteristics and is an obvious entry site for foreign Ags. We assessed the potential of eyedrop vaccination for effective delivery of vaccines against viral or bacterial infection in mice. Both OVA-specific IgG Ab in serum and IgA Ab in mucosal compartments were induced by eyedrops of OVA with cholera toxin (CT). Eyedrop vaccination of influenza A/PR/8 virus (H1N1) induced both influenza virus-specific systemic and mucosal Ab responses and protected mice completely against respiratory infection with influenza A/PR/8 virus. In addition, eyedrop vaccination of attenuated Salmonella vaccine strains induced LPS-specific Ab and complete protection against oral challenge of virulent Salmonella. Unlike with the intranasal route, eyedrop vaccinations did not redirect administered Ag into the CNS in the presence of CT. When mice were vaccinated by eyedrop, even after the occlusion of tear drainage from eye to nose, Ag-specific systemic IgG and mucosal IgA Abs could be induced effectively. Of note, eyedrops with OVA plus CT induced organogenesis of conjunctiva-associated lymphoid tissue and increased microfold cell-like cells on the conjunctiva-associated lymphoid tissue in the nictitating membrane on conjunctiva, the mucosal side of the external eye. On the basis of these findings, we propose that the eyedrop route is an alternative to mucosal routes for administering vaccines.

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.

Ethylene emitted by viral pathogen-infected pepper (Capsicum annuum L.) plants is a volatile chemical cue that attracts aphid vectors.

Plant viruses are obligate intracellular pathogens, and most depend on insect vectors for transmission between plants. Viral infection causes various physiological and metabolic changes in host traits, which subsequently influence the behavior and fitness of the insect vectors. Cucumber mosaic virus (CMV), one of the most widespread pathogens in pepper (Capsicum annuum L.), is transmitted by aphid vectors in a non-persistent manner. Here, we examined whether CMV infection in pepper affects the behavior of aphid vectors (Myzus persicae and Aphis glycines) in pepper. Aphid preference test revealed that significantly more aphids were attracted to CMV-infected pepper plants than to healthy plants. Comparative transcriptome analysis revealed a significant activation of the ethylene biosynthesis pathway in CMV-infected pepper plants. Indeed, gas chromatography analysis demonstrated that ethylene emission was significantly increased by CMV infection in pepper plants. Elevated ethylene emission in ethephon-treated healthy pepper increased their attractiveness to aphids. In contrast, aphid preference decreased after chemical inhibition of ethylene biosynthesis in CMV-infected pepper plants. Our results suggest that the ethylene emitted by CMV infection is a volatile cue that regulates the attractiveness of pepper plants to M. persicae and A. glycines.

Viral Strain-Specific Activation of Pathogen-Associated Molecular Pattern-Triggered Immunity Enhances Symptom Severity in Broad Bean Wilt Virus 2 Infection.

Broad bean wilt virus 2 (BBWV2) is an emerging virus in various economically important crops, especially pepper (Capsicum annuum L.), worldwide. Recently, the emergence of various BBWV2 strains that induce severe symptoms has increased damage to pepper crops. While the symptomatic variations among virus strains should be associated with differences in the transcriptomic reprogramming of host plants upon infection, underlying molecular mechanisms and associated genes are largely unknown. In the present study, we employed transcriptome analysis to identify responsible host factors for symptom enhancement in the BBWV2-pepper pathosystem using two distinct BBWV2 strains, PAP1 (a severe strain) and RP1 (a mild strain). Comparative analysis of the differentially expressed genes (DEGs) revealed that various genes associated with pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and ethylene signaling were significantly upregulated upon infection with the severe PAP1 strain, but not with the mild RP1 strain. Indeed, hormone analysis revealed that ethylene emission was significantly increased in pepper plants infected with PAP1. These observations imply that the activation of the PTI-associated defense responses reinforce symptom formation during BBWV2 infection in a virus strain-specific manner.

Anti-atherosclerotic vaccination against Porphyromonas gingivalis as a potential comparator of statin in mice.

BACKGROUND: Porphyromonas gingivalis (Pg) is an oral anaerobe which damages teeth and periodontal tissues. Its body infection is known to cause chronic inflammation, thereby inducing an early stage of atherosclerosis through humoral immune actions. Hence, vaccination by immunizing the proteins of P. gingivalis (Pg) post sonication with heating may prevent atherosclerosis. This study aimed to compare the effect of its vaccination with statin, which effectively prevents atherosclerosis by lowering lipids. METHODS: The vaccine was produced by sonicating P. gingivalis through heating, and a total of 32 male APOE-/-mice (8-week old) were subjected Western diet for 8 weeks, in order to induce atherosclerosis in a physiological manner. Then, the mice were grouped to undergo four treatment conditions (i.e., no treatment, pitavastatin, vaccine, or pitavastatin with vaccine). Vaccination was conducted through nasal immunization and confirmed by a Pg-specific humoral immune reaction. Then, half of the mice in each group were orally injected with P. gingivalis for the next 5 weeks while the other half remained uninfected, generating a total of eight groups (n = 4/group). The mice were sacrificed at 3 weeks after the last injection. After harvesting the aorta, Oil Red O staining of en face was conducted with imaging and image analysis, and plaque formation was quantitatively determined. RESULTS: Compared to no treatment, the vaccination through nasal immunization significantly reduced the atherosclerotic plaque sizes in APOE -/- mice under Western diet to the comparable level of statin group. When both vaccine and statin were used, no clear synergistic effect was observed as opposed to expectation. CONCLUSIONS: This study revealed that nasal immunization of heat shock P. gingivalis has a significant impact on the prevention of arteriosclerosis and acts as a potential comparator of statin.

Evaluation of factors related to Anaesthesia-induced Lens opacity in experimental mice.

To investigate conditions that cause temporal lens opacity, we tested chemical and physical factors, such as anaesthesia dose, ocular surface dryness, and infrared (IR) light exposure in anaesthetised C57BL/6 N mice. Mice were anaesthetised with a low (80%; tiletamine/zolazepam 32 mg/kg and xylazine 8 mg/kg, intraperitoneal injection) or high (120%; 48 mg/kg and 12 mg/kg) dose of anaesthetic and examined every 5 min from 10 to 30 min after anaesthesia was induced. Lens opacity levels were assessed and graded (1-6) using the standard classification system. Regardless of the anaesthetic dose, lens opacity grade was 1-2 in moisturised eyes with application of 0.5% carboxymethylcellulose, and 5-6 in dry ocular surface conditions. Lens opacity in mice with high-dose anaesthetic in the dry ocular surface condition was not different from that of mice with low-dose anaesthetic. Lens opacity grade 1-2 was noted in eyes in the wet ocular surface condition, regardless of IR light exposure. During IR light exposure in eyes in the dry ocular surface condition, lens opacity (grade 6) in mice with high-dose anaesthetic was not different from that (grade 6) in mice with low-dose anaesthetic. We demonstrated that ocular surface dryness might be a relevant factor for the formation and progression of lens opacity in anesthetized C57BL/6 N mice. Anaesthesia dose and IR light exposure did not strongly influence lens opacity formation. Furthermore, eyes with corneal dryness-induced lens opacity recovered to normal status without additional intervention.

Comparison of ophthalmic toxicity of light-emitting diode and organic light-emitting diode light sources.

The use of organic light-emitting diodes (OLEDs) has rapidly increased in recent years. However, the effect of OLEDs on human health has not been studied yet. We investigated morphologic and functional changes after OLEDs exposure of human ocular cells, including corneal, conjunctival, lens, and retinal pigment epithelial cells, and mouse eyes. In corneal and conjunctival epithelial cells, the levels of reactive oxygen species production and interleukin-8 expression after white light-emitting diodes (LED) exposure were significantly greater than those after OLED exposure. Although no gross morphologic changes of the eyelid or cornea were found in LED- or OLED-exposed mice, oxidative stress on ocular surface was significantly increased, and the outer nuclear layer (ONL) was significantly shorter in both light-treated groups than the control group. Moreover, ONL thickness was significantly lower in the LED group than the OLED group. The electroretinography response was significantly lower in light exposure group, and there was significant difference between LED- and OLED-treated mice. Although OLED exhibits certain ocular toxicity, it can be less toxic to eyes than LED. The higher blue-wavelength energy of LED light might be the reason for its higher toxicity relative to OLED.

Newly emerged resistance-breaking variants of cucumber mosaic virus represent ongoing host-interactive evolution of an RNA virus.

Understanding the evolutionary history of a virus and the mechanisms influencing the direction of its evolution is essential for the development of more durable strategies to control the virus in crop fields. While the deployment of host resistance in crops is the most efficient means to control various viruses, host resistance itself can act as strong selective pressure and thus play a critical role in the evolution of virus virulence. Cucumber mosaic virus (CMV), a plant RNA virus with high evolutionary capacity, has caused endemic disease in various crops worldwide, including pepper (Capsicum annuum L.), because of frequent emergence of resistance-breaking variants. In this study, we examined the molecular and evolutionary characteristics of recently emerged, resistance-breaking CMV variants infecting pepper. Our population genetics analysis revealed that the high divergence capacity of CMV RNA1 might have played an essential role in the host-interactive evolution of CMV and in shaping the CMV population structure in pepper. We also demonstrated that nonsynonymous mutations in RNA1 encoding the 1a protein enabled CMV to overcome the deployed resistance in pepper. Our findings suggest that resistance-driven selective pressures on RNA1 might have contributed in shaping the unique evolutionary pattern of CMV in pepper. Therefore, deployment of a single resistance gene may reduce resistance durability against CMV and more integrated approaches are warranted for successful control of CMV in pepper.

Meibum Expressibility Improvement as a Therapeutic Target of Intense Pulsed Light Treatment in Meibomian Gland Dysfunction and Its Association with Tear Inflammatory Cytokines.

Many recent studies have demonstrated the efficacy of intense pulsed light (IPL) for the treatment of meibomian gland dysfunction (MGD); however, its effective treatment targets have not yet been elucidated. This study aimed to investigate the baseline characteristics associated with an improvement in symptoms after IPL treatment; to examine the course of change in inflammatory tear cytokines, meibomian gland function, and tear stability; and to investigate the correlation between cytokines and ocular surface parameters. Thirty participants underwent three sessions of IPL treatment. During each examination, tear film lipid layer interferometry, meibography, tear meniscus height measurement, tear sampling, and slit-lamp examination were performed, and the Ocular Surface Disease Index (OSDI) questionnaire was administered. Meibum quality, meibum expressibility, lid margin abnormality, tear film break-up time (TBUT), ocular surface staining, and the OSDI significantly improved after treatment. Poor meibum expressibility and short TBUT were associated with greater recovery in the OSDI after IPL. Tear levels of IL-4, IL-6, IL-10, IL-17A, and TNF-α decreased after IPL, and IL-6, and TNF-α were correlated with the improvement in meibum expressibility. Therefore, IPL treatment improved meibomian gland function, stabilized the tear film, and decreased ocular surface inflammation. Patients with obstructive MGD and tear instability were more likely to experience an improvement in ocular discomfort after IPL treatment.

Molecular dissection of distinct symptoms induced by tomato chlorosis virus and tomato yellow leaf curl virus based on comparative transcriptome analysis.

The viral infection of plants may cause various physiological symptoms associated with the reprogramming of plant gene expression. However, the molecular mechanisms and associated genes underlying disease symptom development in plants infected with viruses are largely unknown. In this study, we employed RNA sequencing for in-depth molecular characterization of the transcriptional changes associated with the development of distinct symptoms induced by tomato chlorosis virus (ToCV) and tomato yellow leaf curl virus (TYLCV) in tomato. Comparative analysis of differentially expressed genes revealed that ToCV and TYLCV induced distinct transcriptional changes in tomato and resulted in the identification of important genes responsible for the development of symptoms of ToCV (i.e., chlorosis and anthocyanin accumulation) and TYLCV (i.e., yellowing, stunted growth, and leaf curl). Our comprehensive transcriptome analysis can provide molecular strategies to reduce the severity of disease symptoms as well as new insights for the development of virus-resistant crops.

Movement protein of broad bean wilt virus 2 serves as a determinant of symptom severity in pepper.

Broad bean wilt virus 2 (BBWV2, genus Fabavirus, family Secoviridae) has a wide host range and infects many economically important crops. Various isolates of BBWV2 have been identified from diverse host plants, and their molecular and biological characteristics have been investigated. In our previous study, we demonstrated that BBWV2 RNA2 contains a symptom determinant(s) capable of enhancing symptom severity by utilizing infectious full-length cDNA clones of two distinct strains of BBWV2, pBBWV2-PAP1 (a severe strain) and pBBWV2-RP1 (a mild strain). In the present study, to identify the symptom determinant(s) of BBWV2, we exploited disease responses of pBBWV2-PAP1- and pBBWV2-RP1-derived chimeric viruses and amino acid substitution mutant viruses in Nicotiana benthamiana and pepper (Capsicum annuum Quarri) and demonstrated that the movement protein (MP) encoded in BBWV RNA2 is the determinant of disease symptom severity in both plants. A single amino acid substitution in the MP was sufficient for changing symptom severity of BBWV2. Our finding provides a role for the MP as a symptom determinant in BBWV2 and increases the understanding of the basis of molecular interactions between host plants and BBWV2.

Development of Allergic Conjunctivitis Induced by House Dust Mite Extract From Dermatophagoides pteronyssinus.

PURPOSE: The purpose of this study was to develop a murine model of allergic conjunctivitis induced by house dust mite (HDM) extract from Dermatophagoides pteronyssinus, a major allergen in humans. METHODS: Forty BALB/c mice were divided into five groups, immunized with placebo, ovalbumin (10 µg), or HDM extract following a schedule. Twenty minutes after topical challenge, mice were examined clinically. Material collected from mice was used for measuring total and specific IgE, antigen-specific lymphocyte proliferation, and supernatant cytokine levels and for conjunctival histopathology and flow cytometric analysis of conjunctival cells. RESULTS: This murine model showed similar clinical signs and laboratory findings to human allergy and the ovalbumin-induced allergic conjunctivitis model. Total IgE levels and conjunctival infiltration of mast cells and eosinophils in immunized mice were significantly higher than in the control group. Cervical lymphocyte proliferation was increased in antigen-stimulated cultures in immunized mice, concomitant with significantly higher IL-4 and IL-5 levels in the culture supernatant. The proportion of conjunctival CD4+ T cells expressing the ST2 receptor was increased, and conjunctival CD4+ST2+ T cells exhibited an increase in intracellular IL-5. CONCLUSIONS: House dust mite extract successfully induced allergic conjunctivitis in BALB/c mice. Ten micrograms of HDM extract was the optimal dose for systemic immunization in this model. This murine model is suitable for further studies on HDM-induced allergic conjunctivitis, and the data show that conjunctival CD4+ T cells expressing ST2 may play an important role in IL-5 secretion, recruiting eosinophils into conjunctiva on ocular allergen challenge.

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.