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1.
FASEB J ; 34(10): 13826-13838, 2020 10.
Article in English | MEDLINE | ID: mdl-32813318

ABSTRACT

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.


Subject(s)
Endophthalmitis/immunology , Endoribonucleases/metabolism , Immunity, Innate , Protein Serine-Threonine Kinases/metabolism , Toll-Like Receptor 2/metabolism , Animals , Cell Line , Cells, Cultured , Endophthalmitis/genetics , Endoribonucleases/genetics , Female , MAP Kinase Signaling System , Male , Mice , Mice, Inbred C57BL , NF-kappa B/metabolism , Protein Serine-Threonine Kinases/genetics , Retina/immunology , Retina/microbiology , Staphylococcus aureus/pathogenicity , Toll-Like Receptor 2/genetics , X-Box Binding Protein 1/genetics , X-Box Binding Protein 1/metabolism , p38 Mitogen-Activated Protein Kinases
2.
Infect Immun ; 83(10): 3926-36, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26195555

ABSTRACT

Inflammation caused by infection with Gram-positive bacteria is typically initiated by interactions with Toll-like receptor 2 (TLR2). Endophthalmitis, an infection and inflammation of the posterior segment of the eye, can lead to vision loss when initiated by a virulent microbial pathogen. Endophthalmitis caused by Bacillus cereus develops as acute inflammation with infiltrating neutrophils, and vision loss is potentially catastrophic. Residual inflammation observed during B. cereus endophthalmitis in TLR2(-/-) mice led us to investigate additional innate pathways that may trigger intraocular inflammation. We first hypothesized that intraocular inflammation during B. cereus endophthalmitis would be controlled by MyD88- and TRIF-mediated signaling, since MyD88 and TRIF are the major adaptor molecules for all bacterial TLRs. In MyD88(-/-) and TRIF(-/-) mice, we observed significantly less intraocular inflammation than in eyes from infected C57BL/6J mice, suggesting an important role for these TLR adaptors in B. cereus endophthalmitis. These results led to a second hypothesis, that TLR4, the only TLR that signals through both MyD88 and TRIF signaling pathways, contributed to inflammation during B. cereus endophthalmitis. Surprisingly, B. cereus-infected TLR4(-/-) eyes also had significantly less intraocular inflammation than infected C57BL/6J eyes, indicating an important role for TLR4 in B. cereus endophthalmitis. Taken together, our results suggest that TLR4, TRIF, and MyD88 are important components of the intraocular inflammatory response observed in experimental B. cereus endophthalmitis, identifying a novel innate immune interaction for B. cereus and for this disease.


Subject(s)
Adaptor Proteins, Vesicular Transport/immunology , Bacillus cereus/physiology , Endophthalmitis/immunology , Gram-Positive Bacterial Infections/immunology , Toll-Like Receptor 4/immunology , Adaptor Proteins, Vesicular Transport/genetics , Animals , Bacillus cereus/immunology , Endophthalmitis/genetics , Endophthalmitis/microbiology , Female , Gram-Positive Bacterial Infections/genetics , Gram-Positive Bacterial Infections/microbiology , Humans , Male , Mice , Mice, Inbred C57BL , Myeloid Differentiation Factor 88/genetics , Myeloid Differentiation Factor 88/immunology , Toll-Like Receptor 2/genetics , Toll-Like Receptor 2/immunology , Toll-Like Receptor 4/genetics
3.
J Biol Chem ; 288(50): 35626-35, 2013 Dec 13.
Article in English | MEDLINE | ID: mdl-24142690

ABSTRACT

Vimentin, a type III intermediate filament (IF) protein, is phosphorylated predominantly in mitosis. The expression of a phosphorylation-compromised vimentin mutant in T24 cultured cells leads to cytokinetic failure, resulting in binucleation (multinucleation). The physiological significance of intermediate filament phosphorylation during mitosis for organogenesis and tissue homeostasis was uncertain. Here, we generated knock-in mice expressing vimentin that have had the serine sites phosphorylated during mitosis substituted by alanine residues. Homozygotic mice (VIM(SA/SA)) presented with microophthalmia and cataracts in the lens, whereas heterozygotic mice (VIM(WT/SA)) were indistinguishable from WT (VIM(WT/WT)) mice. In VIM(SA/SA) mice, lens epithelial cell number was not only reduced but the cells also exhibited chromosomal instability, including binucleation and aneuploidy. Electron microscopy revealed fiber membranes that were disorganized in the lenses of VIM(SA/SA), reminiscent of similar characteristic changes seen in age-related cataracts. Because the mRNA level of the senescence (aging)-related gene was significantly elevated in samples from VIM(SA/SA), the lens phenotype suggests a possible causal relationship between chromosomal instability and premature aging.


Subject(s)
Aneuploidy , Cataract/etiology , Cataract/metabolism , Cellular Senescence , Endophthalmitis/etiology , Endophthalmitis/metabolism , Epithelial Cells/pathology , Mitosis , Vimentin/metabolism , Alleles , Amino Acid Sequence , Amino Acid Substitution , Animals , Cataract/genetics , Cataract/pathology , Cell Nucleus/pathology , Endophthalmitis/genetics , Endophthalmitis/pathology , Epithelial Cells/metabolism , Gene Knock-In Techniques , Lens, Crystalline/pathology , Mice , Molecular Sequence Data , Phosphorylation , Vimentin/chemistry , Vimentin/genetics
4.
Invest Ophthalmol Vis Sci ; 65(4): 44, 2024 Apr 01.
Article in English | MEDLINE | ID: mdl-38687493

ABSTRACT

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.


Subject(s)
Adaptive Immunity , Aspergillosis , Aspergillus flavus , Cytokines , Disease Models, Animal , Endophthalmitis , Eye Infections, Fungal , Gene Expression Profiling , Immunity, Innate , Mice, Inbred C57BL , Animals , Aspergillus flavus/genetics , Mice , Eye Infections, Fungal/microbiology , Eye Infections, Fungal/genetics , Eye Infections, Fungal/immunology , Endophthalmitis/microbiology , Endophthalmitis/immunology , Endophthalmitis/genetics , Aspergillosis/microbiology , Aspergillosis/genetics , Aspergillosis/immunology , Adaptive Immunity/genetics , Immunity, Innate/genetics , Cytokines/metabolism , Cytokines/genetics , Transcriptome , Enzyme-Linked Immunosorbent Assay , Vitreous Body/microbiology
5.
Indian J Med Res ; 138(5): 609-19, 2013 Nov.
Article in English | MEDLINE | ID: mdl-24434316

ABSTRACT

Toll-like receptors (TLRs) play a key role in the innate immune response to invading pathogens. Thus, their discovery has opened up a wide range of therapeutic possibilities for various infectious and inflammatory diseases. In the last several years, extensive research efforts have provided a considerable wealth of information on the expression and function of TLRs in the eye, with significant implications for better understanding of pathogenesis of infectious eye diseases affecting the cornea, uvea, and the retina. In this review, by using bacterial keratitis and endophthalmitis as examples, we discuss the possibilities of targeting TLR signaling for the prevention or treatment of ocular infectious diseases.


Subject(s)
Eye Infections/genetics , Inflammation/genetics , Molecular Targeted Therapy , Toll-Like Receptors/genetics , Cornea/pathology , Endophthalmitis/genetics , Endophthalmitis/microbiology , Endophthalmitis/therapy , Eye Infections/microbiology , Eye Infections/pathology , Eye Infections/therapy , Humans , Immunity, Innate/genetics , Inflammation/pathology , Inflammation/therapy , Keratitis/genetics , Keratitis/microbiology , Keratitis/therapy , NF-kappa B/genetics , NF-kappa B/metabolism , Signal Transduction/genetics , Toll-Like Receptors/antagonists & inhibitors
6.
J Clin Microbiol ; 50(4): 1487-90, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22259203

ABSTRACT

We present three unrelated post-cataract surgery endophthalmitis cases caused by Rhizobium radiobacter, hospitalized in three different hospitals. Early diagnosis was obtained in two cases by bacterial DNA detection in vitreous samples. All patients recovered from infection, but pars plana vitrectomy was needed in two patients due to rapid clinical deterioration.


Subject(s)
Agrobacterium tumefaciens , Cataract Extraction/adverse effects , Endophthalmitis/diagnosis , Gram-Negative Bacterial Infections/diagnosis , Aged , Aged, 80 and over , Endophthalmitis/genetics , Endophthalmitis/microbiology , Female , Gram-Negative Bacterial Infections/microbiology , Humans , Male , Molecular Diagnostic Techniques , Molecular Sequence Data , RNA, Bacterial/genetics , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA
7.
Curr Eye Res ; 47(11): 1559-1566, 2022 11.
Article in English | MEDLINE | ID: mdl-36094002

ABSTRACT

PURPOSE: Increasing incidence of multidrug-resistant Pseudomonas aeruginosa (MDR-PA) causing endophthalmitis challenges our ability to manage this vision-threatening condition. In this study, temporal dynamics of immune response in a mouse model of MDR-PA endophthalmitis was investigated by whole transcriptome analysis. METHODS: C57BL/6 mice were infected with MDR-PA and antibiotic susceptible (S-PA) clinical strains and disease severity were monitored at 6 and 24-h postinfection (p.i), following which eyeballs were enucleated. Microarray analysis was performed using SuperPrint G3 Mouse Gene Expression v2 chip and the differential gene expression analysis was performed with limma package in R (v4.0.0.)/Bioconductor (v3.11). RESULTS: Histopathological analysis revealed a significant difference in retinal architecture and vitreous infiltrates at 6 and 24 h. In comparison to S-PA, MDR-PA revealed altered expression of 923 genes at 6 h and 2220 genes at 24 h. Further, 23 and 76% of these altered genes and its downstream interacting proteins showed time-specific expression (6 and 24 h respectively), indicating their association with disease progression. At 24 hours, MDR-PA induced endophthalmitis showed aberrant immune response with the enrichment inflammasome signalling, dysregulated ubiquitination, complement cascade, MMPs NF-κß and IL-1 signalling. CONCLUSION: The rapid development of transcriptional differences between the two-time points reveals that distinct genes contribute to disease severity. The results from this study highlighted a link between innate and adaptive immune responses and provided novel insights in the pathogenesis of MDR-PA endophthalmitis by extending the number of molecular determinants and functional pathways that underpin host-associated damage.


Subject(s)
Endophthalmitis , Pseudomonas Infections , Animals , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Disease Models, Animal , Drug Resistance, Multiple, Bacterial , Endophthalmitis/drug therapy , Endophthalmitis/genetics , Gene Expression Profiling , Inflammasomes/metabolism , Interleukin-1/genetics , Mice , Mice, Inbred C57BL , Pseudomonas Infections/drug therapy , Pseudomonas Infections/genetics , Pseudomonas aeruginosa
8.
Ophthalmology ; 118(4): 772-7, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21055814

ABSTRACT

OBJECTIVE: To analyze the clinical profiles, histopathologic features, and Mycobacterium tuberculosis polymerase chain reaction testing in patients with ocular tuberculosis. DESIGN: Retrospective case series. PARTICIPANTS: Forty-two patients. METHODS: This retrospective study was approved by the Armed Forces Institute of Pathology (AFIP) Institutional Review Board. The AFIP data banks were screened for cases with diagnosis of ocular tuberculosis using key words such as mycobacterium; tuberculosis; and acid-fast bacilli. Files and slides stained with hematoxylin-eosin and acid-fast staining were reviewed by the Division of Ocular Pathology and by the Infectious Diseases and Parasitic Diseases Pathology Branches. When available; blocks and unstained slides were sent to the Doheny Eye Institute; Los Angeles; California; for quantitative polymerase chain reaction (qPCR) analysis to detect Mycobacterium tuberculosis-specific DNA. MAIN OUTCOME MEASURES: Tuberculin skin test (TST) results, as well as the chest radiograph results, were recorded. When acid-fast bacilli were identified in tissue, their locations-ocular or extraocular sites-were recorded. Emphasis was placed on lymph node involvement and any systemic diseases. RESULTS: In the histopathologic specimens, microscopy revealed a paucity of organisms, and often there were only 1 or 2 organisms associated with or near a giant cell or near an area of necrosis. The qPCR analysis was performed on 6 biopsy specimens. These specimens showed necrotizing granulomatous inflammation from 6 different patients; 3 had positive qPCR results. In 2 of the 3 cases with positive qPCR results, acid-fast bacilli were not found in the tissue sections. In 17 patients, TST results were available; 10 had positive results (60%) and 7 had negative results (40%). Fourteen chest radiograph results were submitted, and 8 (57%) of 14 patients had normal chest films. CONCLUSIONS: This study suggests that in dealing with those populations at increased risk of tuberculosis (e.g., immigrants from endemic areas and human immunodeficiency virus-infected patients) or patients receiving biologic therapy, the ophthalmologist should endeavor to entertain this diagnosis and to rely on the support of infectious disease specialists and pulmonologists to help solidify the diagnosis, because the current methods for the diagnosis have limited sensitivity.


Subject(s)
DNA, Bacterial/analysis , Mycobacterium tuberculosis/genetics , Tuberculosis, Ocular/genetics , Tuberculosis, Ocular/pathology , Adolescent , Adult , Aged , Child , Child, Preschool , Databases, Factual , Endophthalmitis/genetics , Endophthalmitis/pathology , Female , Humans , Infant , Male , Mass Chest X-Ray , Middle Aged , Molecular Biology , Polymerase Chain Reaction , Retrospective Studies , Tuberculin Test , Uveitis/genetics , Uveitis/pathology , Young Adult
9.
Invest Ophthalmol Vis Sci ; 61(14): 5, 2020 12 01.
Article in English | MEDLINE | ID: mdl-33263715

ABSTRACT

Purpose: Age, sex, and genetics are important biological variables in determining an individual's susceptibility or response to infectious agents; however, their role has not been evaluated in intraocular infections. In this study, we comprehensively examined the impact of these host biological factors in the pathogenesis of experimental bacterial endophthalmitis. Methods: Endophthalmitis was induced by intravitreal injection of bacteria (Staphylococcus aureus) in the eyes of male and female C57BL/6 mice of different ages: group I (young, 6-8 weeks), group II (mid-age, 18-20 weeks), and group III (old, 1 year). Highly heterogeneous outbred J:DO mice were used for genetic diversity analysis. Eyes were subjected to clinical examination, retinal function testing using electroretinography (ERG), histopathological analysis (hematoxylin and eosin staining), and bacterial burden estimation. The levels of inflammatory mediators were measured using qPCR and ELISA, and the infiltration of neutrophils was determined by flow cytometry. Results: Both inbred C57BL/6 and diversity outbred (J:DO) mice were equally susceptible to S. aureus endophthalmitis, as evidenced by a time-dependent increase in clinical scores, bacterial burden, intraocular inflammation, and retinal tissue damage, in addition to decreased retinal function. However, no significant differences were observed in disease severity and innate responses in male versus female mice. Older mice (group III) exhibited higher clinical scores coinciding with increased bacterial proliferation and intraocular inflammation, resulting in enhanced disease severity. Moreover, bone-marrow-derived macrophages from old mice exhibited reduced phagocytic activity but increased inflammatory response toward S. aureus challenge. Conclusions: Age, but not sex, is an important biological variable in bacterial endophthalmitis. Identification of pathways underlying altered innate immunity and impaired bacterial clearance in aging eyes could provide new insights into the pathobiology of intraocular infections in elderly patients.


Subject(s)
Endophthalmitis/pathology , Age Factors , Animals , Electroretinography , Endophthalmitis/genetics , Enzyme-Linked Immunosorbent Assay , Female , Flow Cytometry , Genetic Variation/genetics , Male , Mice , Mice, Inbred C57BL , Real-Time Polymerase Chain Reaction , Sex Factors , Staphylococcal Infections/genetics , Staphylococcal Infections/pathology
10.
Sci Rep ; 10(1): 14234, 2020 08 28.
Article in English | MEDLINE | ID: mdl-32859978

ABSTRACT

Staphylococcus epidermidis (S. epidermidis) is one of the primary pathogens in postoperative endophthalmitis, which is a devastating complication of cataract surgery and often results in irreversible visual loss and even blindness. Meanwhile, it is the most frequently isolated commensal bacterium in the healthy conjunctiva. In this study, we investigated the differentially expressed genes (DEGs) of S. epidermidis isolated from the patients with postoperative endophthalmitis and the healthy conjunctiva to predict their functions and pathways by Illumina high-throughput RNA sequencing. Using genome-wide transcriptional analysis, 281 genes (142 upregulated and 139 downregulated genes) were found to be differentially expressed (fold change ≥ 2, p ≤ 0.05) in the strains from endophthalmitis. Ten randomly selected DEGs were further validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR). GO enrichment analysis suggested that more DEGs were associated with the thioredoxin system and iron ion metabolism. KEGG pathway analysis revealed that more DEGs were associated with the pathways of the two-component system and pyruvate metabolism. Moreover, the gene SE1634 code for staphylococcal toxin was significantly upregulated in S. epidermidis strains of the endophthalmitis, which might be directly responsible for the pathogenesis of endophthalmitis. In conclusion, this research is helpful for further investigations on genes or pathways related with the pathogenesis and therapeutic targets of S. epidermidis endophthalmitis.


Subject(s)
Conjunctiva/microbiology , Endophthalmitis/genetics , Staphylococcus epidermidis/genetics , Cataract Extraction/methods , China , DNA, Bacterial/analysis , DNA, Bacterial/genetics , Endophthalmitis/microbiology , Eye Infections, Bacterial/genetics , Eye Infections, Bacterial/microbiology , Female , Gene Expression Profiling/methods , Humans , Male , Middle Aged , Postoperative Complications/microbiology , Sequence Analysis, RNA/methods , Staphylococcal Infections/microbiology , Staphylococcus epidermidis/isolation & purification , Staphylococcus epidermidis/pathogenicity
11.
Am J Ophthalmol ; 217: 325-334, 2020 09.
Article in English | MEDLINE | ID: mdl-32217118

ABSTRACT

PURPOSE: To associate detection of potential pathogen DNA in endophthalmitis with clinical outcomes. DESIGN: Prospective cohort study. METHODS: Patients in whom endophthalmitis was diagnosed following an intraocular procedure were recruited. Clinical outcome data from baseline, week-1, month-1, and month-3 visits were collected. Intraocular biopsy samples were cultured by standard methods. Quantitative polymerase chain reaction (qPCR) was performed for specific pathogens and whole-genome sequencing (WGS). RESULTS: A total of 50 patients (mean age 72 years old; 52% male) were enrolled. Twenty-four cases were culture-positive and 26 were culture-negative. WGS identified the cultured organism in 76% of culture-positive cases and identified potential pathogens in 33% of culture-negative cases. Month-1 and -3 visual acuities did not vary by pathogen-positive versus pathogen-negative cases as detected by either culture or WGS. Visual outcomes of Staphylococcus epidermidis endophthalmitis were no different than those of pathogen-negative cases, whereas the patients infected with other pathogens showed worse outcome. Higher baseline bacterial DNA loads of bacteria other than those of S epidermidis detected by WGS were associated with worse month-1 and -3 visual acuity, whereas the S epidermidis loads did not appear to influence outcomes. Torque teno virus (TTV) and Merkel cell polyomavirus (MCV) were detected by qPCR in 49% and 19% of cases, respectively. Presence of TTV at presentation was associated with a higher rate of secondary pars plana vitrectomy (P = .009) and retinal detachment (P = .022). CONCLUSIONS: The presence and higher load of bacteria other than S epidermidis detected by WGS or DNA from TTV by qPCR in ocular fluids is associated with worse outcomes in post-procedure endophthalmitis.


Subject(s)
Bacteria/genetics , DNA, Bacterial/analysis , Endophthalmitis/microbiology , Eye Infections, Bacterial/microbiology , Genome-Wide Association Study/methods , Vitreous Body/microbiology , Adult , Aged , Aged, 80 and over , Endophthalmitis/diagnosis , Endophthalmitis/genetics , Eye Infections, Bacterial/diagnosis , Eye Infections, Bacterial/genetics , Female , Follow-Up Studies , Humans , Male , Middle Aged , Polymerase Chain Reaction , Prognosis , Prospective Studies , Visual Acuity , Vitreous Body/diagnostic imaging
12.
Hum Gene Ther ; 31(1-2): 80-89, 2020 01.
Article in English | MEDLINE | ID: mdl-31544533

ABSTRACT

Both subretinal dosing and intravitreal (IVT) dosing of adeno-associated virus (AAV) in higher species induce mild and transient inflammatory responses that increase with dose. Foreign protein and foreign DNA are known inducers of inflammation, which is also true in the immune-privileged ocular environment. We explored which component(s) of AAV vectors, viral capsid, or viral DNA drive inflammatory responses. Recombinant AAV with three tyrosine to phenylalanine substitutions in the capsid of AAV serotype 2 (rAAV2tYF), and with a generic ubiquitous promoter (cytomegalovirus [CMV]) controlling the expression of humanized green fluorescent protein (hGFP), was processed to enrich for AAV capsids containing genome (full capsids), capsids without genome (empty capsids), and residual material. Nonhuman primate eyes were injected by IVT in both eyes. During in-life, ocular inflammation and development of neutralizing antibodies (NAb) were measured. Following termination, lymph node immunophenotyping was performed, vitreous was processed for cytokine and RNAseq analyses, and ocular sections were assessed for transgene expression (by in situ hybridization) and histopathology. IVT dosing of AAV vectors transiently raised cellular inflammation in the aqueous and induced a more sustained inflammation in the vitreous. Lowering the total capsid dose by removing empty AAV capsids reduced inflammation and improved viral transduction. IVT dosing of AAV induced systemic NAb to AAV irrespective of the vector preparation. Similarly, lymph node immunophenotyping revealed identical profiles irrespective of viral preparation used for dosing. Immune cells in the vitreous were identified based on RNAseq analysis. Three months postdose, cytokine levels were low, indicative of minimal levels of inflammation in agreement with histopathological assessment of the retina.


Subject(s)
Dependovirus/genetics , Genetic Therapy , Genetic Vectors/genetics , Animals , Biomarkers , Capsid Proteins/genetics , Cytokines/metabolism , Disease Models, Animal , Endophthalmitis/diagnosis , Endophthalmitis/genetics , Endophthalmitis/therapy , Gene Expression Regulation , Gene Transfer Techniques , Genes, Reporter , Genetic Therapy/adverse effects , Genetic Therapy/methods , Genetic Vectors/administration & dosage , Genome, Viral , Humans , Immunohistochemistry , Mice , Transduction, Genetic , Transgenes
13.
DNA Cell Biol ; 38(8): 887-894, 2019 Aug.
Article in English | MEDLINE | ID: mdl-31295021

ABSTRACT

Circular RNAs (circRNAs), as with other noncoding RNAs, have emerged as novel molecules of interest in gene regulation and in the development of many diseases. However, the expression and function of circRNAs in inflammation-induced lymphangiogenesis (LG) are still unknown. Microarray profiling in inflamed human lymphatic endothelial cells identified 82 differentially expressed circRNAs, including 6 downregulated and 76 upregulated circRNAs. One of the top 10 upregulated circRNAs, cZNF609, was selected for subsequent quantitative real-time PCR validation, and was found to be significantly upregulated in inflamed corneas from both mouse and human eyes. The expression of miR-184 was significantly lower in inflamed corneas than in control ones, which suggested that cZNF609 might serve as a sponge for miR-184. The expression of heparanase, a potential target gene of miR-184, was significantly increased in inflamed corneas. Therefore, circRNAs may serve as potential regulators of corneal LG. These findings lay a foundation for functional research on circRNAs in corneal LG pathogenesis.


Subject(s)
Cornea/physiology , Endophthalmitis/genetics , Lymphangiogenesis/genetics , RNA , Animals , Cells, Cultured , Cornea/pathology , Endophthalmitis/etiology , Endothelial Cells , Humans , Male , Mice, Inbred C57BL , MicroRNAs , Oligonucleotide Array Sequence Analysis , RNA, Circular
14.
Sci Rep ; 8(1): 4126, 2018 03 07.
Article in English | MEDLINE | ID: mdl-29515160

ABSTRACT

Advances in genomics have the potential to revolutionize clinical diagnostics. Here, we examine the microbiome of vitreous (intraocular body fluid) from patients who developed endophthalmitis following cataract surgery or intravitreal injection. Endophthalmitis is an inflammation of the intraocular cavity and can lead to a permanent loss of vision. As controls, we included vitreous from endophthalmitis-negative patients, balanced salt solution used during vitrectomy and DNA extraction blanks. We compared two DNA isolation procedures and found that an ultraclean production of reagents appeared to reduce background DNA in these low microbial biomass samples. We created a curated microbial genome database (>5700 genomes) and designed a metagenomics workflow with filtering steps to reduce DNA sequences originating from: (i) human hosts, (ii) ambiguousness/contaminants in public microbial reference genomes and (iii) the environment. Our metagenomic read classification revealed in nearly all cases the same microorganism that was determined in cultivation- and mass spectrometry-based analyses. For some patients, we identified the sequence type of the microorganism and antibiotic resistance genes through analyses of whole genome sequence (WGS) assemblies of isolates and metagenomic assemblies. Together, we conclude that genomics-based analyses of human ocular body fluid specimens can provide actionable information relevant to infectious disease management.


Subject(s)
Aqueous Humor/microbiology , Bacteria , Bacterial Typing Techniques/methods , DNA, Bacterial/genetics , Endophthalmitis , Genomics/methods , Postoperative Complications , Bacteria/classification , Bacteria/genetics , Cataract Extraction/adverse effects , Endophthalmitis/etiology , Endophthalmitis/genetics , Endophthalmitis/microbiology , Female , Humans , Intravitreal Injections/adverse effects , Male , Middle Aged , Postoperative Complications/genetics , Postoperative Complications/microbiology
15.
Curr Eye Res ; 43(4): 553-565, 2018 04.
Article in English | MEDLINE | ID: mdl-29199855

ABSTRACT

PURPOSE: The concept of tissue-dependent cytokine hierarchy has been demonstrated in a number of diseases, but it has not been investigated in ophthalmic diseases. Here, we evaluated the functional hierarchy of interleukin-1ß (IL-1ß), IL-6, IL-17A, and tumor necrosis factor (TNF) in the induction of ocular inflammation. MATERIALS AND METHODS: We delivered adeno-associated virus (AAV) vectors expressing IL-1ß, IL-6, IL-17A, or TNF intravitreally in naïve C57/BL6 mice and compared and contrasted the inflammatory effects in the eye 5 weeks after AAV-mediated gene transfer. We also used an in vitro human system to test the effect of cytokines on barrier function. RESULTS: We found that IL-1ß had the highest ability to initiate ocular inflammation. The continuous overexpression of IL-1ß resulted in a significant upregulation of additional proinflammatory mediators in the eye. Using scanning laser ophthalmoscope and optical coherence tomography imaging techniques, we showed that a low dose of AAVIL-1ß was sufficient and was as pathogenic as a high dose of TNF in inducing vascular leakage, retinal degeneration, and cellular infiltration. Furthermore, only a marginal increase in IL-1ß was enough to cause cellular infiltration, thus confirming the highly pathogenic nature of IL-1ß in the eye. Contrary to our expectation, IL-6 or IL-17A had minimal or no effect in the eye. To examine the clinical relevance of our findings, we used an impedance assay to show that IL-1ß alone or TNF alone was able to cause primary human retinal endothelial cell barrier dysfunction in vitro. Again, IL-6 alone or IL-17A alone had no effect on barrier function; however, in the presence of IL-1ß or TNF, IL-17A but not IL-6 may provide additive proinflammatory effects. CONCLUSIONS: Our studies demonstrate the existence of a functional hierarchy of proinflammatory cytokines in the eye, and we show that IL-1ß is the most pathogenic when it is continuously expressed in the eye.


Subject(s)
Cytokines/genetics , Endophthalmitis/genetics , Gene Expression Regulation , RNA/genetics , Animals , Cytokines/biosynthesis , Disease Models, Animal , Endophthalmitis/metabolism , Endophthalmitis/pathology , Enzyme-Linked Immunosorbent Assay , Female , Inflammation/genetics , Inflammation/metabolism , Male , Mice , Mice, Inbred C57BL , Real-Time Polymerase Chain Reaction , Retinal Artery/metabolism , Retinal Artery/pathology , Tomography, Optical Coherence
16.
BMJ Case Rep ; 20162016 Mar 03.
Article in English | MEDLINE | ID: mdl-26941346

ABSTRACT

A 25-year-old woman presented with unilateral red eye and visual blur, and was found to have panuveitis with an inflammatory white mass at the macula, initially presumed to be Toxoplasma retinitis. After failure to respond, she underwent vitrectomy, which produced Candida albicans. Despite intraocular and systemic antifungal treatment, she lost all vision in that eye. Two years later, she developed unilateral hip osteomyelitis leading to total hip replacement and also revealing Candida infection. By clinical exome sequencing, she was then found to have caspase recruitment domain 9 (CARD9) deficiency, an autosomal recessive disorder that causes a specific susceptibility to candidal infections. She remains otherwise well but on lifelong fluconazole prophylaxis.


Subject(s)
CARD Signaling Adaptor Proteins/deficiency , Candidiasis/diagnosis , Endophthalmitis/microbiology , Osteomyelitis/microbiology , Adult , Antifungal Agents/therapeutic use , CARD Signaling Adaptor Proteins/genetics , Candida albicans/isolation & purification , Candidiasis/complications , Candidiasis/drug therapy , Candidiasis/genetics , Endophthalmitis/drug therapy , Endophthalmitis/genetics , Exome , Female , Fluconazole/therapeutic use , Humans , Osteomyelitis/genetics , Osteomyelitis/surgery , Sequence Analysis, DNA
17.
Sci Rep ; 6: 21502, 2016 Feb 11.
Article in English | MEDLINE | ID: mdl-26865111

ABSTRACT

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.


Subject(s)
Endophthalmitis/genetics , Gene Regulatory Networks , Host-Pathogen Interactions , Staphylococcal Infections/genetics , Toll-Like Receptor 2/genetics , Transcriptome , Animals , CCAAT-Enhancer-Binding Proteins/genetics , CCAAT-Enhancer-Binding Proteins/immunology , Cytokines/genetics , Cytokines/immunology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/immunology , Disease Models, Animal , Endophthalmitis/immunology , Endophthalmitis/microbiology , Endophthalmitis/pathology , Female , Gene Expression Profiling , Gene Expression Regulation , Gene Ontology , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Molecular Sequence Annotation , Neoplasm Proteins/genetics , Neoplasm Proteins/immunology , Principal Component Analysis , Retina/immunology , Retina/microbiology , Retina/pathology , Signal Transduction , Staphylococcal Infections/immunology , Staphylococcal Infections/microbiology , Staphylococcal Infections/pathology , Staphylococcus aureus/pathogenicity , Staphylococcus aureus/physiology , Systems Biology , Toll-Like Receptor 2/deficiency , Toll-Like Receptor 2/immunology
18.
Invest Ophthalmol Vis Sci ; 56(3): 1719-32, 2015 Feb 12.
Article in English | MEDLINE | ID: mdl-25678692

ABSTRACT

PURPOSE: The purpose of this study was to investigate the protective mechanisms evoked by TLR2 and MyD88 signaling in bacterial endophthalmitis in vivo. METHODS: Endophthalmitis was induced in wild-type (WT), TLR2(-/-), MyD88(-/-), and Cnlp(-/-) mice by intravitreal injections of a laboratory strain (RN6390) and two endophthalmitis isolates of Staphylococcus aureus. Disease progression was monitored by assessing corneal and vitreous haze, bacterial burden, and retinal tissue damage. Levels of inflammatory cytokines/chemokines were determined using quantitative RT-PCR (qRT-PCR) and ELISA. Flow cytometry was used to assess neutrophil infiltration. Cathelicidin-related antimicrobial peptide (CRAMP) expression was determined by immunostaining and dot blot. RESULTS: Eyes infected with either laboratory or clinical isolates exhibited higher levels of inflammatory mediators at the early stages of infection (≤24 hours) in WT mice than in TLR2(-/-) or MyD88(-/-) mice. However, their levels surpassed that of WT mice at the later stages of infection (>48 hours), coinciding with increased bacterial burden and retinal damage. Both TLR2(-/-) and MyD88(-/-) retinas produced reduced levels of CRAMP, and its deficiency (Cnlp(-/-)) rendered the mice susceptible to increased bacterial burden and retinal tissue damage as early as 1 day post infection. Analyses of inflammatory mediators and neutrophil levels in WT versus Cnlp(-/-) mice showed a trend similar to that observed in TLR2 and MyD88 KO mice. Furthermore, we observed that even a 10-fold lower infective dose of S. aureus was sufficient to cause endophthalmitis in TLR2(-/-) and MyD88(-/-) mice. CONCLUSIONS: TLR2 and MyD88 signaling plays an important role in protecting the retina from staphylococcal endophthalmitis by production of the antimicrobial peptide CRAMP.


Subject(s)
Endophthalmitis/genetics , Endophthalmitis/immunology , Immunity, Innate/genetics , Immunity, Innate/immunology , Myeloid Differentiation Factor 88/genetics , Signal Transduction/genetics , Staphylococcal Infections/genetics , Staphylococcal Infections/immunology , Toll-Like Receptor 2/genetics , Animals , Antimicrobial Cationic Peptides , Cathelicidins/metabolism , Chemokines/metabolism , Cytokines/metabolism , Endophthalmitis/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Retina/immunology , Retina/pathology , Staphylococcal Infections/pathology
19.
J Nutr Biochem ; 26(11): 1357-67, 2015 Nov.
Article in English | MEDLINE | ID: mdl-26362107

ABSTRACT

Green tea extract (GTE) exerts antioxidative activities in ocular tissues of rats, but high levels of (-)-epigallocatechin gallate (EGCG) can induce oxidative stress. In this study, pharmacokinetics, diurnal variation of oxidative status, antioxidation and transcription factors changes in ocular tissues of rats were investigated. Rats were fed intragastrically with GTE and catechin mixtures containing different amounts of EGCG. Plasma and various ocular tissues were taken for pharmacokinetic analysis, oxidation marker testings and gene expression assays. Effects of EGCG on ocular oxidation status were assessed by 8-isoprostane level and reduced/oxidized glutathione ratio. Oxidation, inflammation and apoptosis regulations in retina were evaluated by real-time polymerase chain reaction. Epicatechin, epigallocatechin and EGCG were dominant in various ocular tissues except vitreous humor, where gallocatechin was predominant. Diurnal variation of oxidative status was found in some compartments. GTE caused oxidative stress increase in the plasma, aqueous humor, vitreous humor, cornea and retina but decrease in the lens and choroid-sclera. Catechins mixture containing half dose of EGCG lowered 8-isoprostane in the retina and lens. GTE treatment induced superoxide dismutase 1 and glutathione peroxidase-3 expressions but suppressed catalase in the retina. Our results reveal pro-oxidation of GTE with high EGCG content to the ocular tissues. Optimal EGCG level is needed for protection.


Subject(s)
Apoptosis/genetics , Catechin/analogs & derivatives , Endophthalmitis/genetics , Tea/chemistry , Animals , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Antioxidants/pharmacology , Apoptosis/drug effects , Catechin/blood , Catechin/pharmacokinetics , Catechin/pharmacology , Dose-Response Relationship, Drug , Endophthalmitis/diet therapy , Eye/drug effects , Eye/physiopathology , Female , Gene Expression Regulation/drug effects , Oxidative Stress/drug effects , Plant Extracts/chemistry , Rats, Sprague-Dawley , Vitreous Body/drug effects
20.
Infect Genet Evol ; 1(3): 237-42, 2002 May.
Article in English | MEDLINE | ID: mdl-12798020

ABSTRACT

Genome sequence-based fluorescent amplified-fragment length polymorphism (FAFLP) analysis was investigated for fingerprinting and subtyping 42 multiple drug resistant (MDR) isolates of Pseudomonas aeruginosa from post-surgical endophthalmitis. The FAFLP profiles derived from EcoRI/MseI restricted fragments differentiated clinical isolates and were found to be extremely reproducible. Seventeen different amplification patterns (amplitypes) were observed for all the 42 isolates from 11 different patients. Also, we were able to genotype the isolates based on the differential amplification of a total of 31 FAFLP markers representing genomic fragments from the P. aeruginosa chromosome. This data appears to provide clues to the genetic diversity of endopthalmitis associated strains and could be converted into digitised fingerprints suitable for electronic manipulations and archiving.


Subject(s)
Drug Resistance, Multiple/genetics , Endophthalmitis/microbiology , Pseudomonas aeruginosa/genetics , DNA Fingerprinting , Endophthalmitis/genetics , Humans , India , Phylogeny , Pseudomonas aeruginosa/metabolism
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