Global Transcriptomic Profiling of Innate and Adaptive Immunity During Aspergillus flavus Endophthalmitis in a Murine Model.

Purpose: Fungal endophthalmitis is characterized by chronic inflammation leading to the partial or complete vision loss. Herein, we analyzed the transcriptomic landscape of Aspergillus flavus (A. flavus) endophthalmitis in C57BL/6 mice to understand the host-pathogen interactions. Methods: Endophthalmitis was induced by intravitreal injection of A. flavus spores in C57BL/6 mice and monitored for disease progression up to 72 hours. The enucleated eyeballs were subjected to histopathological analysis and mRNA sequencing using the Illumina Nextseq 2000. Pathway enrichment analysis was performed to further annotate the functions of differentially expressed genes (DEGs) and validation of cytokines was performed in vitreous of patients with fungal endophthalmitis using multiplex ELISA. Results: Transcriptomic landscape of A. flavus endophthalmitis revealed upregulated T-cell receptor signaling, PI3K-AKT, MAPK, NF-κB, JAK-STAT, and NOD like receptor signaling pathways. We observed significant increase in the T-cells during infection especially at 72 hours infection along with elevated expression levels of IL-6, IL-10, IL-12, IL-18, IL-19, IL-23, CCR3, and CCR7. Furthermore, host-immune response associated genes, such as T-cell interacting activating receptor, TNF receptor-associated factor 1, TLR1, TLR9, and bradykinin receptor beta 1, were enriched. Histopathological assessment validated the significant increase in inflammatory cells, especially T-cells at 72 hours post-infection along with increased disruption in the retinal architecture. Additionally, IL-6, IL-8, IL-17, TNF-α, and IL-1ß were also significantly elevated, whereas IL-10 was downregulated in vitreous of patients with Aspergillus endophthalmitis. Conclusions: Regulating T-cell influx could be a potential strategy to modulate the excessive inflammation in the retina and potentially aid in better vision recovery in fungal endophthalmitis.

Immune Inhibitor A Metalloproteases Contribute to Virulence in Bacillus Endophthalmitis.

Endophthalmitis is a devastating infection that can cause blindness. Over half of Bacillus endophthalmitis cases result in significant loss of useful vision. Bacillus produces many virulence factors that may contribute to retinal damage and robust inflammation. We analyzed Bacillus immune inhibitor A (InhA) metalloproteases in the context of this disease, hypothesizing that InhAs contribute to Bacillus intraocular virulence and inflammation. We analyzed phenotypes and infectivity of wild-type (WT), InhA1-deficient (ΔinhA1), InhA2-deficient (ΔinhA2), or InhA1, A2, and A3-deficient (ΔinhA1-3) Bacillus thuringiensis. In vitro analysis of growth, proteolysis, and cytotoxicity were compared. WT and InhA mutants were similarly cytotoxic to retinal cells. The ΔinhA1 and ΔinhA2 mutants entered log-phase growth earlier than WT B. thuringiensis. Proteolysis by the ΔinhA1-3 mutant was decreased, but this strain grew similar to WT in vitro. Experimental endophthalmitis was initiated by intravitreally infecting C57BL/6J mice with 200 CFU of WT B. thuringiensis or InhA mutants. Eyes were analyzed for intraocular Bacillus and myeloperoxidase concentrations, retinal function loss, and gross histological changes. Eyes infected with the ΔinhA1 or ΔinhA2 mutant strains contained greater numbers of bacteria than eyes infected with WT throughout the infection course. Eyes infected with single mutants had inflammation and retinal function loss similar to eyes infected with the WT strain. Eyes infected with the ΔinhA1-3 mutant cleared the infection. Quantitative real-time PCR (qRT-PCR) results suggested that there may be compensatory expression of the other InhAs in the single InhA mutant. These results indicate that together, the InhA metalloproteases contribute to the severity of infection and inflammation in Bacillus endophthalmitis.

Transcriptomic and Histological Analysis of Exacerbated Immune Response in Multidrug-Resistant Pseudomonas aeruginosa in a Murine Model of Endophthalmitis.

Multidrug-resistant (MDR) endophthalmitis is a serious threat to the whole spectrum of therapeutic procedures associated with the risk of managing and preventing vision loss. We have earlier shown the interplay of immune mediators in patients with MDR Pseudomonas aeruginosa (PA) endophthalmitis leading to worse outcome. Expanding on these findings, a murine model of endophthalmitis was developed to explore the effects of drug resistance on the pathogenesis by analyzing the temporal changes in retinal morphology along with its transcriptomic signatures. Clinical isolates of susceptible (S-PA) and multidrug-resistant PA (MDR-PA) were injected intravitreally in C57BL/6 mice followed by enucleation at 6 and 24 h time points postinfection. Disease progression and retinal changes were monitored by clinical and histological assessment and transcriptome analysis in a pair-wise manner. Histological assessment of MDR-PA eyeball revealed higher disease severity (p < 0.05), CD45+ cells (p = 0.007), MPO+ cells (p = 0.01), GFAP+ (p = 0.02), along with higher retinal cell death in mice infected with MDR-PA (p = 0.008). Temporal transcriptome analysis revealed differential expression of nearly 923 genes at 6 h p.i. and 2,220 genes at 24 h p.i. (FC ≥2, adjusted p-value <0.05). Pathway enrichment analysis identified differential regulation of chemokine- and cytokine-mediated, MAPK, and NF-Ðºß signaling pathways. In conclusion, rapid deterioration of retinal architecture and immune exacerbation was significantly associated with the MDR endophthalmitis, suggesting the need for immunomodulatory agents to strengthen host cell functions and support antibiotics to save the retinal structure from inevitable deterioration and restoration of the vision.

Innate Immune Interference Attenuates Inflammation In Bacillus Endophthalmitis.

Purpose: To explore the consequences of innate interference on intraocular inflammatory responses during Bacillus endophthalmitis. Methods: Bacillus endophthalmitis was induced in mice. Innate immune pathway activation was interfered by injecting S layer protein-deficient (∆slpA) B. thuringiensis or by treating wild-type (WT)-infected mice with a TLR2/4 inhibitor (WT+OxPAPC). At 10 hours postinfection, eyes were harvested and RNA was purified. A NanoString murine inflammation panel was used to compare gene expression in WT-infected, WT+OxPAPC, ∆slpA-infected, and uninfected eyes. Results: In WT-infected eyes, 56% of genes were significantly upregulated compared to uninfected controls. Compared to WT-infected eyes, the expression of 27% and 50% of genes were significantly reduced in WT+OxPAPC and ∆slpA-infected eyes, respectively. Expression of 61 genes that were upregulated in WT-infected eyes was decreased in WT+OxPAPC and ∆slpA-infected eyes. Innate interference resulted in blunted expression of complement factors (C3, Cfb, and C6) and several innate pathway genes (TLRs 2, 4, 6, and 8, MyD88, Nod2, Nlrp3, NF-κB, STAT3, RelA, RelB, and Ptgs2). Innate interference also reduced the expression of several inflammatory cytokines (CSF2, CSF3, IL-6, IL-1ß, IL-1α, TNFα, IL-23α, TGFß1, and IL-12ß) and chemokines (CCL2, CCL3, and CXCLs 1, 2, 3, 5, 9, and 10). All of the aforementioned genes were significantly upregulated in WT-infected eyes. Conclusions: These results suggest that interfering with innate activation significantly reduced the intraocular inflammatory response in Bacillus endophthalmitis. This positive clinical outcome could be a strategy for anti-inflammatory therapy of an infection typically refractory to corticosteroid treatment.

Evaluation of Susceptibility and Innate Immune Response in C57BL/6 and BALB/c Mice During Candida albicans Endophthalmitis.

Purpose: Candida remains the leading cause of fungal endophthalmitis. However, the pathobiology and innate immune responses in this disease are not well characterized. Here, we developed two murine models of candida endophthalmitis and evaluated their disease susceptibility and differential immune response. Methods: Endophthalmitis was induced in C57BL/6 (B6) and BALB/c mice by intravitreal injection of Candida albicans (CA). Disease progression was monitored by slit-lamp examination and clinical scoring, followed by retinal function assessment using electroretinography (ERG). Enucleated eyes were used to estimate fungal burden and retinal tissue damage by hematoxylin and eosin and TUNEL staining. The level of inflammatory mediators were determined by quantitative Polymerase Chain Reaction (qPCR) and enzyme-linked immunosorbent assay, whereas neutrophil infiltration was assessed by flow cytometry and immunostaining. Results: Intravitreal injection of CA at 6500 colony-forming units resulted in sustained (non-resolving) ocular inflammation in both B6 and BALB/c mice as evidenced by increased levels of inflammatory cytokines (tumor necrosis factor-α, interleukin-1ß, and interleukin-6) and chemokine (CXCL2/MIP-2). In both mouse strains, fungal burden peaked at 24 to 48 hours post-infection (hpi) and decreased by 72 to 96 hpi. CA-infected eyes exhibited increased polymorphonuclear neutrophils (PMN) infiltration and retinal tissue damage. Overall retinal function declined rapidly, with a significant reduction in ERG response at 12 hpi and near-total loss by 24 hpi. Differential analyses revealed increased pathology in BALB/c versus B6 mice. Conclusions: C. albicans was able to cause endophthalmitis in mice. Although BALB/c mice were found to be more susceptible to CA endophthalmitis, both BALB/c and B6 models could be used to study fungal endophthalmitis and test therapeutic modalities.

Toll-like receptor 2 (TLR2) engages endoplasmic reticulum stress sensor IRE1α to regulate retinal innate responses in Staphylococcus aureus endophthalmitis.

Endoplasmic reticulum (ER) stress response has been implicated in a variety of pathophysiological conditions, including infectious and inflammatory diseases. However, its contribution in ocular bacterial infections, such as endophthalmitis, which often cause blindness is not known. Here, using a mouse model of Staphylococcus (S.) aureus endophthalmitis, our study demonstrates the induction of inositol-requiring enzyme 1α (IRE1α) and splicing of X-box binding protein-1 (Xbp1) branch of the ER-stress pathway, but not the other classical ER stress sensors. Interestingly, S aureus-induced ER stress response was found to be dependent on Toll-like receptor 2 (TLR2), as evident by reduced expression of IRE1α and Xbp1 mRNA splicing in TLR2 knockout mouse retina. Pharmacological inhibition of IRE1α using 4µ8C or experiments utilizing IRE1α-/- macrophages revealed that IRE1α positively regulates S aureus-induced inflammatory responses. Moreover, IRE1α inhibition attenuated S aureus-triggered NF-κB, p38, and ERK pathways activation and cells treated with these pathway-specific inhibitors reduced Xbp1 splicing, suggesting a positive feedback inhibition. In vivo, inhibition of IRE1α diminished the intraocular inflammation and reduced PMN infiltration in mouse eyes, but, increased the bacterial burden and caused more retinal tissue damage. These results revealed a critical role of the IRE1α/XBP1 pathway as a regulator of TLR2-mediated protective innate immune responses in S aureus-induced endophthalmitis.

Glycolytic inhibitor 2-deoxyglucose suppresses inflammatory response in innate immune cells and experimental staphylococcal endophthalmitis.

Previously, we have shown that Staphylococcus (S) aureus induces a glycolytic response in retinal residential (microglia) and infiltrated cells (neutrophils and macrophages) during endophthalmitis. In this study, we sought to investigate the physiological role of glycolysis in bacterial endophthalmitis using a glycolytic inhibitor, 2-deoxyglucose (2DG). Our data showed that 2DG treatment attenuated the inflammatory responses of mouse bone marrow-derived macrophages (BMDM) and neutrophils (BMDN) when challenged with either live or heat-killed S. aureus (HKSA). Among the inflammatory mediators, 2DG caused a significant reduction in levels of cytokines (TNF-α, IL-1ß, IL-6) and chemokines (CXCL1 and CXCL2). Western blot analysis of 2DG treated cells showed downregulation of bacterial-induced MEK/ERK pathways. In vivo, intravitreal administration of 2DG both pre- and post-bacterial infection resulted in a significant reduction in intraocular inflammation in C57BL/6 mouse eyes and downregulation of ERK phosphorylation in retinal tissue. Collectively, our study demonstrates that 2DG attenuates inflammatory response in bacterial endophthalmitis and cultured innate immune cells via inhibition of ERK signaling. Thus glycolytic inhibitors in combination with antibiotics could mitigate inflammation-mediated tissue damage in ocular infections.

Bacillus S-Layer-Mediated Innate Interactions During Endophthalmitis.

Bacillus endophthalmitis is a severe intraocular infection. Hallmarks of Bacillus endophthalmitis include robust inflammation and rapid loss of vision. We reported that the absence of Bacillus surface layer protein (SLP) significantly blunted endophthalmitis severity. Here, we further investigated SLP in the context of Bacillus-retinal cell interactions and innate immune pathways to explore the mechanisms by which SLP contributes to intraocular inflammation. We compared phenotypes of Wild-type (WT) and SLP deficient (ΔslpA) Bacillus thuringiensis by analyzing bacterial adherence to and phagocytosis by human retinal Muller cells and phagocytosis by mouse neutrophils. Innate immune receptor activation by the Bacillus envelope and purified SLP was analyzed using TLR2/4 reporter cell lines. A synthetic TLR2/4 inhibitor was used as a control for this receptor activation. To induce endophthalmitis, mouse eyes were injected intravitreally with 100 CFU WT or ΔslpA B. thuringiensis. A group of WT infected mice was treated intravitreally with a TLR2/4 inhibitor at 4 h postinfection. At 10 h postinfection, infected eyes were analyzed for viable bacteria, inflammation, and retinal function. We observed that B. thuringiensis SLPs contributed to retinal Muller cell adherence, and protected this pathogen from Muller cell- and neutrophil-mediated phagocytosis. We found that B. thuringiensis envelope activated TLR2 and, surprisingly, TLR4, suggesting the presence of a surface-associated TLR4 agonist in Bacillus. Further investigation showed that purified SLP from B. thuringiensis activated TLR4, as well as TLR2 in vitro. Growth of WT B. thuringiensis was significantly higher and caused greater inflammation in untreated eyes than in eyes treated with the TLR2/4 inhibitor. Retinal function analysis also showed greater retention of A-wave and B-wave function in infected eyes treated with the TLR2/4 inhibitor. The TLR2/4 inhibitor was not antibacterial in vitro, and did not cause inflammation when injected into uninfected eyes. Taken together, these results suggest a potential role for Bacillus SLP in host-bacterial interactions, as well as in endophthalmitis pathogenesis via TLR2- and TLR4-mediated pathways.

Interplay between IDO1 and iNOS in human retinal pigment epithelial cells.

Human retinal pigment epithelial (hRPE) cells form a selectively permeable monolayer between the neural retina and the highly permeable choroidal vessels. Thus, hRPE cells bear important regulatory functions and are potential targets of pathogens in vivo. Endogenous bacterial endophthalmitis (EBE) is frequently caused by infections with the Gram-positive bacterium Staphylococcus aureus (S. aureus). Upon microbial infection, interferon gamma (IFN-γ), a major cytokine of the adaptive immune response, induces a broad spectrum of effector molecules, such as the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase-1 (IDO1). We stimulated human RPE (hRPE) cells in vitro with proinflammatory cytokines and analyzed the expression levels and enzymatic activities of IDO1 and inducible nitric oxide synthase (iNOS), another antimicrobial effector molecule. The antimicrobial capacity was analyzed in infection experiments using S. aureus and Toxoplasma gondii (T. gondii). Our aim was to characterize the particular importance of IDO1 and iNOS during EBE. We found that an IFN-γ stimulation of hPRE cells induced the expression of IDO1, which inhibited the growth of T. gondii and S. aureus. A co-stimulation with IFN-γ, interleukin-1 beta, and tumor necrosis factor alpha induced a strong expression of iNOS. The iNOS-derived nitric oxide production was dependent on cell-culture conditions; however, it could not cause antimicrobial effects. iNOS did not act synergistically with IDO1. Instead, iNOS activity inhibited IDO1-mediated tryptophan degradation and bacteriostasis. This effect was reversible by the addition of the iNOS inhibitor NG-monomethyl-L-arginine. In conclusion, iNOS mediates anti-inflammatory effects in hRPE cells stimulated with high amounts of IFN-γ together with tumor necrosis factor alpha and Interleukin-1 beta and prevents potential IDO1-dependent tissue damage.

Targets of immunomodulation in bacterial endophthalmitis.

Bacterial infection of the posterior segment of the eye (endophthalmitis) leads to a robust host response that often results in irreversible damage to the layers of the retina, significant vision loss, and in some patients, enucleation of the globe. While a great deal of effort has gone into understanding the role of bacterial virulence factors in disease initiation and propagation, it is becoming increasingly clear that the host response to infection plays a major role in causing the damage associated with endophthalmitis. Researchers have identified the host receptors which detect infecting organisms and initiate the cascade of events that result in inflammation. This inflammation may damage nonregenerative tissues of the eye while attempting to clear the infection. Both Gram-positive and Gram-negative bacteria can cause endophthalmitis. These organisms initiate an immune response by activating toll-like receptor (TLR) pathways. Once an inflammatory response is initiated, the expression of immunomodulators, such as proinflammatory chemokines and cytokines, affect the recruitment of PMNs and other inflammatory cells into the eye. We and others have reported that knockout mice that do not express specific inflammatory pathways and molecules have an attenuated response to infection and retain significant retinal function. These findings suggest that host immune mediators are important components of the response to infections in the posterior segment of the eye, and the timing and level of their production may be related to the severity of the damage and the ultimate visual outcome. If that is the case, a better understanding of the complex and often redundant role of these pathways and inflammatory mediators may identify host molecules as potential anti-inflammatory therapeutic targets. This review highlights potential anti-inflammatory targets during acute inflammation in endophthalmitis, compares and contrasts those with findings in other models of ocular inflammation, and translates current immunomodulatory strategies for other types of infection and inflammation to this blinding disease. Given the poor visual outcomes seen in patients treated with antibiotics alone or in combination with corticosteroids, immunomodulation in addition to antibiotic therapy might be more effective in preserving vision than current regimens.

Bilateral endophthalmitis and symmetrical peripheral gangrene in a patient with chronic kidney disease on maintenance hemodialysis.

Dialysis patients have greater number of complications due to multiple comor-bidity and access-related infections as well as nosocomial infections due to reduced immunity and more frequent hospitalizations. Endogenous endophthalmitis is a potentially blinding ocular infection occurring in chronically debilitated patients and the use of invasive procedures. Symmetric peripheral gangrene (SPG) is defined as symmetrical distal ischemic damage in two or more sites in the absence of a major vascular occlusive disease. It carries a high mortality rate with a very high frequency of multiple limb amputations in the survivors. However, only a few case reports have described endogenous endophthalmitis in dialysis patients. Concomitant endophthalmitis and disseminated intravascular coagulation (DIC), presenting as SPG, is extremely rare and no such case was found in the literature survey. Herein, we report a very rare association of bilateral endophthalmitis with DIC and SPG in a patient with chronic kidney disease on maintenance hemodialysis.

Elevated cytokine levels in vitreous as biomarkers of disease severity in infectious endophthalmitis.

PURPOSE: To investigate the immunopathogenesis of endophthalmitis, and determine if cytokine profiles could serve as biomarkers of disease severity in infectious endophthalmitis. MATERIALS AND METHODS: Vitreous samples of 46 patients clinically diagnosed as endophthalmitis (of which 25 were culture positive) and 20 non-infectious controls from patients with Retinal Detachment (RD) or diabetic retinopathy were included in the study. The cytokine and chemokine expression patterns of 40 immune mediators including 6 antiinflammatory cytokines, 15 proinflammatory cytokines, 9 Growth factors and 10 proinflammatory chemokines in the vitreous were were analyzed by multiplex cytokine immunoassay. In addition, significant immune mediators were correlated with initial and final visual acuity (VA). RESULTS: Our results demonstrated elevated expression of 16 mediators such as GCSF, GRO, IFN-γ, IL-1α, IL-1ß, IL-1 RA, IL-6, IL-8, IP-10, MCP-1, MCP-3, MIP-1α, IL-1ß, TGF-α, TNF-α in patients with culture positive endophthalmitis. Cytokine profile expression significantly differed between patients with proven endophthalmitis and the non-infectious controls in heat map analysis. PCoA plot indicated five mediators (IL-1RA, IL-6, IL-8, GRO, G-CSF) as biomarkers that could be Independent Predictors of Disease especially in culture negative cases. Correlation of cytokines with VA revealed strong association between the initial VA and intraocular levels of TGF-α, IL-1ß and IL-8 but there was no correlation with the severity or visual outcome of infection. CONCLUSION: In comparison to non-infectious ocular conditions, the pathogenesis of infectious endophthalmitis correlates with increased expression levels of IL-1RA, IL-6, IL-8, GRO, G-CSF. Understanding cytokine profiles in culture negative endophthalmitis patients could aid in therapy in non-responders to empirical antibiotic therapy.

[New Therapeutic Approaches in Inflammatory Diseases of the Eye - Targeting Lymphangiogenesis and Cellular Immunity: Research Unit FOR 2240 Presents Itself]. / Neue Therapieansätze bei entzündlichen Augenerkrankungen durch Modulation von Lymphangiogenese und zellulärer Immunität: Die DFG-Forschergruppe 2240 stellt sich vor.

Background Ophthalmology, principally, is a very successful subdiscipline in medicine. Nonetheless, there are still unmet medical needs which necessitate translational research. Methods The funding instrument of a Research Unit (RU) of the German Research Foundation (DFG) is presented as exemplified by the RU 2240 at the Department of Ophthalmology at the University of Cologne. Results The Research Unit integrates different research groups working on pathologic ocular inflammation, macrophages/microglia and (lymph)angiogenesis to collaborate in a synergistic way. Rotation positions allow young clinicians to rotate into research labs for a defined period of time. A Research Unit is also a powerful strategic tool to strengthen clinical and experimental ophthalmology at individual medical faculties. Conclusions The funding instrument of a Research Unit is highly suitable for fostering translational research in a medical subdiscipline such as ophthalmology, supporting the next generation of (clinician) scientists in ophthalmology and finding new cures for our patients.

[Determination of cytomegalovirus IgG synthesis by the albumin correction in the aqueous humor of posner-schlossmann syndrome].

Objective: To evaluate the usefulness of albumin correction in determination of cytomegalovirus IgG in the aqueous humor of Posner-Schlossman syndrome (PSS) patients. Methods: Cases series studies. Forty-two patients (26 men and 16 women) who were diagnosed as PSS were enrolled from Oct. 2009 to Oct. 2015 at the Eye and ENT Hospital. During the same period, 20 patients with primary open-angle glaucoma (POAG) and 30 patients with bacterial endophthalmitis or retinal necrosis were enrolled as negative control group and inflammatory disease control group, respectively. Aqueous humor and serum samples were assayed to detect CMV IgG by enzyme-linked immunosorbent assay (ELISA), and albumin by scattering immunonephelometry. CMV DNA in aqueous humor was assayed by polymerase chain reaction (PCR). The ratio which was calculated as the (aqueous humor CMV IgG/serum CMV IgG)/(aqueous humor concentration of albumin/serum albumin concentration) over 0.6 was considered as intraocular antibody formation. Performance of differentiating control eyes from eyes with CMV-positive PSS was evaluated by the receiver operating characteristic curve. The ANOVA test, Mann-Whitney test and Chi-square test were performed to compare the differences among groups. Results: The detectable rate of CMV IgG antibody in the aqueous humor was 76.2%, 100.0% and 10.0% in PSS, inflammatory disease control and POAG groups, respectively. The levels of CMV IgG antibody in the PSS groups were significantly higher than that of POAG groups (Z=4.23, P<0.001).The positive rate corrected by the albumin was 71.4%, 3.3% and 0.0%.The corrected positive rate in PSS groups was significantly higher than that of the inflammatory disease control and POAG groups (χ(2)=30.38, P<0.01; χ(2)= 24.89, P<0.01), with a sensitivity of 75.0% and a specificity of 98.0%. The area under the curve for calibrated ratio was 0.942 (95%CI: 0.859 to 0.984) which was higher than that of CMV IgG (Z=6.19, P<0.001).The corrected positive rate of CMV IgG antibody (71.4%) was higher than that of CMV DNA (47.6%, χ(2)=4.003, P=0.045). Conclusions: CMV IgG antibody ratio which was corrected by aqueous humor and serum albumin could effectively improve aqueous antibody specificity in PSS patients. Furthermore, CMV IgG antibody ratio combined with PCR could improve the sensitivity of CMV detection. All of which help clarify the CMV infection in PSS in CMV DNA negative eyes. (Chin J Ophthalmol, 2017, 53: 104-108).

Endophthalmitis related to lemon allergy in a heroin addict

BACKGROUND: Heroin and its contaminants may be an important source of allergens in young people. We present a case of severe endophthalmitis in a patient that also suffered from anaphylactoid symptoms (hypotension, urticaria, glottic oedema) whenever he ingested lemon. METHODS: Prick tests with a battery of 42 aeroallergens including fruits and citrus fruits (orange, mandarin, grapefruit and lemon) and specific IgE to these allergens were carried out. Immunodetection was performed using the patient's serum and the following allergens: lemon,Candida, Aspergillus, Penicillium and Alternaria recombinant Alt 1 (Laboratories Diater). RESULTS: Skin tests were negative or Candida, Penicillium, Aspergillus and Cladosporium (ALK-Spain) as were specific IgE antibodies for CAP (Thermofisher, Sweden) and positive only for lemon and, doubtfully, to Candida. Specific IgE tests to pollen, arthropods, fungi, dander and foods were positive only for lemon (0.49kU/L). Serological study of fungi ruled out fungal infection at that time. The immunodetection showed that the patient's serum recognised a protein of approximately 25kDa of lemon peel, one of approximately 12-13kDa of Penicillium, and perfectly recognised Alt a 1. CONCLUSIONS: Lemon surface can be contaminated by Candida and other fungi. In heroin addicts with positive skin tests for lemon, the possibility of these serious complications should be taken into account

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CXCL1, but not IL-6, significantly impacts intraocular inflammation during infection.

During intraocular bacterial infections, the primary innate responders are neutrophils, which may cause bystander damage to the retina or perturb the clarity of the visual axis. We hypothesized that cytokine IL-6 and chemokine CXCL1 contributed to rapid neutrophil recruitment during Bacillus cereus endophthalmitis, a severe form of intraocular infection that is characterized by explosive inflammation and retinal damage that often leads to rapid vision loss. To test this hypothesis, we compared endophthalmitis pathogenesis in C57BL/6J, IL-6-/-, and CXCL1-/- mice. Bacterial growth in eyes of CXCL1-/-, IL-6-/-, and C67BL/6J mice was similar. Retinal function retention was greater in eyes of IL-6-/- and CXCL1-/- mice compared with that of C57BL/6J, despite these eyes having similar bacterial burdens. Neutrophil influx into eyes of CXCL1-/- mice was reduced to a greater degree compared with that of eyes of IL6-/- mice. Histology confirmed significantly less inflammation in eyes of CXCL1-/- mice, but similar degrees of inflammation in IL6-/- and C57BL/6J eyes. Because inflammation was reduced in eyes of infected CXCL1-/- mice, we tested the efficacy of anti-CXCL1 in B. cereus endophthalmitis. Retinal function was retained to a greater degree and there was less overall inflammation in eyes treated with anti-CXCL1, which suggested that anti-CXCL1 may have therapeutic efficacy in limiting inflammation during B. cereus endophthalmitis. Taken together, our results indicate that absence of IL-6 did not affect overall pathogenesis of endophthalmitis. In contrast, absence of CXCL1, in CXCL1-/- mice or after anti-CXCL1 treatment, led to an improved clinical outcome. Our findings suggest a potential benefit in targeting CXCL1 to control inflammation during B. cereus and perhaps other types of intraocular infections.

5-Aminoimidazole-4-carboxamide ribonucleoside-mediated adenosine monophosphate-activated protein kinase activation induces protective innate responses in bacterial endophthalmitis.

The retina is considered to be the most metabolically active tissue in the body. However, the link between energy metabolism and retinal inflammation, as incited by microbial infection such as endophthalmitis, remains unexplored. In this study, using a mouse model of Staphylococcus aureus (SA) endophthalmitis, we demonstrate that the activity (phosphorylation) of 5' adenosine monophosphate-activated protein kinase alpha (AMPKα), a cellular energy sensor and its endogenous substrate; acetyl-CoA carboxylase is down-regulated in the SA-infected retina. Intravitreal administration of an AMPK activator, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), restored AMPKα and acetyl-CoA carboxylase phosphorylation. AICAR treatment reduced both the bacterial burden and intraocular inflammation in SA-infected eyes by inhibiting NF-kB and MAP kinases (p38 and JNK) signalling. The anti-inflammatory effects of AICAR were diminished in eyes pretreated with AMPK inhibitor, Compound C. The bioenergetics (Seahorse) analysis of SA-infected microglia and bone marrow-derived macrophages revealed an increase in glycolysis, which was reinstated by AICAR treatment. AICAR also reduced the expression of SA-induced glycolytic genes, including hexokinase 2 and glucose transporter 1 in microglia, bone marrow-derived macrophages and the mouse retina. Interestingly, AICAR treatment enhanced the bacterial phagocytic and intracellular killing activities of cultured microglia, macrophages and neutrophils. Furthermore, AMPKα1 global knockout mice exhibited increased susceptibility towards SA endophthalmitis, as evidenced by increased inflammatory mediators and bacterial burden and reduced retinal function. Together, these findings provide the first evidence that AMPK activation promotes retinal innate defence in endophthalmitis by modulating energy metabolism and that it can be targeted therapeutically to treat ocular infections.

Autoimmune disease: Conceptual history and contributions of ocular immunology.

Medical historians identify the mid-20th century as the time when the scientific and medical communities acknowledged the existence of autoimmune disease. Several conditions including sympathetic ophthalmia and endophthalmitis phacoanaphylactica, however, were proposed as autoimmune disorders much earlier. During the first half of the century, autoimmune disease was viewed as biologically implausible. Paul Ehrlich coined the term horror autotoxicus to emphasize that autoimmunity would contradict nature's aversion to self-injury. The discoveries of allergy and anaphylaxis were the first clues that the immune system was capable of self-harm. A major obstacle to comprehending the pathogenesis of autoimmunity was how the immune system distinguishes foreign from self, a process eventually understood in the context of immune tolerance. Investigators of sympathetic ophthalmia and endophthalmitis phacoanaphylactica were positioned to invalidate horror autotoxicus but lacked sufficiently convincing experimental and clinical evidence to accomplish the task. Seminal studies of chronic thyroiditis and a series of clinical laboratory breakthroughs led to the general acceptance of autoimmune disease in the 1950s. The travails encountered by ophthalmic investigators offer insights into the how medical ideas take shape. We review the contributions of ocular immunology to the conceptual development of autoimmune disease and explore the reasons why the concept caught on slowly.

Temporal retinal transcriptome and systems biology analysis identifies key pathways and hub genes in Staphylococcus aureus endophthalmitis.

Bacterial endophthalmitis remains a devastating inflammatory condition associated with permanent vision loss. Hence, assessing the host response in this disease may provide new targets for intervention. Using a mouse model of Staphylococcus aureus (SA) endophthalmitis and performing retinal transcriptome analysis, we discovered progressive changes in the expression of 1,234 genes. Gene ontology (GO) and pathway analyses revealed the major pathways impacted in endophthalmitis includes: metabolism, inflammatory/immune, antimicrobial, cell trafficking, and lipid biosynthesis. Among the immune/inflammation pathways, JAK/Stat and IL-17A signaling were the most significantly affected. Interactive network-based analyses identified 13 focus hub genes (IL-6, IL-1ß, CXCL2, STAT3, NUPR1, Jun, CSF1, CYR61, CEBPB, IGF-1, EGFR1, SPP1, and TGM2) within these important pathways. The expression of hub genes confirmed by qRT-PCR, ELISA (IL-6, IL-1ß, and CXCL2), and Western blot or immunostaining (CEBP, STAT3, NUPR1, and IGF1) showed strong correlation with transcriptome data. Since TLR2 plays an important role in SA endophthalmitis, counter regulation analysis of TLR2 ligand pretreated retina or the use of retinas from TLR2 knockout mice showed the down-regulation of inflammatory regulatory genes. Collectively, our study provides, for the first time, a comprehensive analysis of the transcriptomic response and identifies key pathways regulating retinal innate responses in staphylococcal endophthalmitis.