Search

The ideal ophthalmic anti-infective exhibits broad-spectrum activity against gram-positive, gram-negative, and atypical bacterial species. These pathogens can cause potentially blinding infections such as keratitis and endophthalmitis, both of which are associated with ophthalmic surgery or traumatic injury. These infections often require aggressive antibacterial therapy, preferably with newer generations of antibiotics. In this study, minimal inhibitory concentration (MIC) values for gatifloxacin and moxifloxacin were determined in vitro against bacterial strains that were isolated from suspected cases of bacterial keratitis and endophthalmitis. The ocular isolates included 7 gram-positive, 4 gram-negative, and 3 atypical bacterial species. Gatifloxacin and moxifloxacin exhibited similar activity against 6 gram-positive organisms: Staphylococcus epidermidis, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Bacillus cereus, and Enterococcus faecalis. MIC90 values for the drugs against these isolates ranged from 0.08 mg/mL to 0.57 mg/mL and were comparable to previously published values against isolates from patients with systemic infections. The MIC90 for gatifloxacin against Streptococcus viridans was 0.22 mg/mL compared with 0.73 mg/mL for moxifloxacin (P = .011). Among the gram-negative isolates, the mean MIC90 for gatifloxacin against Pseudomonas aeruginosa was 1.28 mg/mL compared with 2.60 mg/ mL for moxifloxacin (P = .023). MIC90 values for gatifloxacin against Klebsiella pneumoniae and Enterobacter aerogenes were one fourth to one fifth the values for moxifloxacin. For the atypicals, the MIC90 values for gatifloxacin against Nocardia asteroides and Mycobacterium chelonae were one fourth the corresponding values for moxifloxacin. Gatifloxacin demonstrated a broad spectrum of activity against several key ocular pathogens tested in this study and was at least as effective as moxifloxacin against these pathogens. [ABSTRACT FROM AUTHOR]

Purpose: To test the hypothesis that (C-C motif) ligand 2 (CCL2) and CCL3 impact retinal function decline and inflammation during Staphylococcus aureus endophthalmitis., Methods: Experimental endophthalmitis was initiated by intravitreal injection of 5000 colony-forming units of S. aureus into the eyes of C57BL/6J, CCL2-/-, or CCL3-/- mice. At 12 and 24 hours post-infection, retinal function, bacterial load, and myeloperoxidase levels were quantified., Results: During S. aureus endophthalmitis, we observed a significant improvement in retinal function in CCL2-/- mice relative to C57BL/6J mice at 12 hours but not at 24 hours. In CCL3-/- mice, retinal function was significantly improved relative to C57BL/6J mice at 12 and 24 hours. The absence of CCL2 did not alter intraocular S. aureus intraocular concentrations. However, CCL3-/- mice had significantly lower intraocular S. aureus at 12 hours but not at 24 hours. No difference in myeloperoxidase levels was observed between C57BL/6J and CCL2-/- mice at 12 hours. CCL3-/- mice had almost no myeloperoxidase at 12 hours. At 24 hours, increased myeloperoxidase was observed in CCL2-/- and CCL3-/- mice relative to C57BL/6J mice., Conclusions: Although the absence of CCL2 resulted in improved retinal function retention at 12 hours, CCL3 deficiency resulted in improved retinal function at 12 and 24 hours. CCL3 deficiency, but not CCL2 deficiency, resulted in almost no inflammation at 12 hours. However, at 24 hours, the absence of CCL2 or CCL3 resulted in significantly increased inflammation. These results suggest that, although both CCL2 and CCL3 impact intraocular infection outcomes, CCL3 may have a more significant impact in S. aureus endophthalmitis.

Background: Bacillus cereus (Bc) can cause self-limiting gastrointestinal infections, but when infecting the eye, can cause rapid and irreversible blindness. This study investigated whether clinical ocular and gastrointestinal Bc isolates differed in terms of virulence-related genotypes and endophthalmitis virulence., Methods: Twenty-eight Bc ocular, gastrointestinal, and laboratory reference isolates were evaluated. Hemolysis assays were performed to assess potential differences in hemolytic activity. The presence of twenty Bc virulence-related genes was assessed by PCR. A subset of ocular and gastrointestinal isolates differing in PCR positivity for 5 virulence genes was compared to strain ATCC14579 in an experimental murine model of endophthalmitis. At 8 hours post infection, retinal function was evaluated by electroretinography, and intraocular bacterial concentrations were determined by plate counts., Results: Gastrointestinal Bc isolates were more hemolytic than the Bc ocular isolates and ATCC14579 (p < 0.0001). Bc ocular isolates were more frequently PCR-positive for capK, cytK, hblA, hblC , and plcR compared to the gastrointestinal isolates (p ≤ 0.0002). In the endophthalmitis model, mean A-wave retention did not differ significantly between eyes infected with ATCC14579 and eyes infected with the selected ocular or gastrointestinal isolates (p ≥ 0.3528). Similar results were observed for mean B-wave retention (p ≥ 0.0640). Only one diarrheal isolate showed significantly greater B-wave retention when compared to ATCC14579 (p = 0.0303). No significant differences in mean A-wave (p ≥ 0.1535) or B-wave (p ≥ 0.0727) retention between the selected ocular and gastrointestinal isolates were observed. Intraocular concentrations of ATCC14579 were significantly higher than the selected ocular isolate and 3 of the gastrointestinal isolates (p ≤ 0.0303). Intraocular concentrations of the selected ocular isolate were not significantly different from the gastrointestinal isolates (p ≥ 0.1923)., Conclusions: Among the subset of virulence-related genes assessed, 5 were significantly enriched among the ocular isolates compared to gastrointestinal isolates. While hemolytic activity was higher among gastrointestinal isolates, retinal function retention and intraocular growth was not significantly different between the selected ocular and gastrointestinal isolates. These results suggest that Bc strains causing gastrointestinal infections, while differing from ocular isolates in hemolytic activity and virulence-related gene profile, are similarly virulent in endophthalmitis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declare that this study received funding from Research to Prevent Blindness, Inc. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication., (Copyright © 2023 Coburn, Miller, LaGrow, Mursalin, Gregory, Parrott, Astley and Callegan.)

Aging is a complex biological process that is characterized by low-grade inflammation, called inflammaging. Aging affects multiple organs including eye and lacrimal gland. Tumor necrosis factor (TNF) is a pleiotropic cytokine that participates in inflammation, activation of proteases such as cathepsin S, and formation of ectopic lymphoid organs. Using genetic and pharmacological approaches, we investigated the role of TNF in age-related dry eye disease, emphasizing the ocular surface and lacrimal gland inflammation. Our results show the increased protein and mRNA levels of TNF in aged lacrimal glands, accompanied by increased TNF, IL1β, IL-18, CCL5, CXCL1, IL-2, IL-2 receptor alpha (CD25), IFN-γ, IL-12p40, IL-17, and IL-10 proteins in tears of aged mice. Moreover, genetic loss of the Tnf -/- in mice decreased goblet cell loss and the development of ectopic lymphoid structures in the lacrimal gland compared to wild-type mice. This was accompanied by a decrease in cytokine production. Treatment of mice at an early stage of aging (12-14-month-old) with TNF inhibitor tanfanercept eye drops for eight consecutive weeks decreased cytokine levels in tears, improved goblet cell density, and decreased the marginal zone B cell frequency in the lacrimal gland compared to vehicle-treated animals. Our studies indicate that modulation of TNF during aging could be a novel strategy for age-related dry eye disease., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Bacteriophage lytic enzymes (i.e., phage lysins) are a trending alternative for general antibiotics to combat growing antimicrobial resistance. Gram-positive Bacillus cereus causes one of the most severe forms of intraocular infection, often resulting in complete vision loss. It is an inherently β-lactamase-resistant organism that is highly inflammogenic in the eye, and antibiotics are not often beneficial as the sole therapeutic option for these blinding infections. The use of phage lysins as a treatment for B. cereus ocular infection has never been tested or reported. In this study, the phage lysin PlyB was tested in vitro , demonstrating rapid killing of vegetative B. cereus but not its spores. PlyB was also highly group specific and effectively killed the bacteria in various bacterial growth conditions, including ex vivo rabbit vitreous (Vit). Furthermore, PlyB demonstrated no cytotoxic or hemolytic activity toward human retinal cells or erythrocytes and did not trigger innate activation. In in vivo therapeutic experiments, PlyB was effective in killing B. cereus when administered intravitreally in an experimental endophthalmitis model and topically in an experimental keratitis model. In both models of ocular infection, the effective bactericidal property of PlyB prevented pathological damage to ocular tissues. Thus, PlyB was found to be safe and effective in killing B. cereus in the eye, greatly improving an otherwise devastating outcome. Overall, this study demonstrates that PlyB is a promising therapeutic option for B. cereus eye infections.IMPORTANCEEye infections from antibiotic-resistant Bacillus cereus are devastating and can result in blindness with few available treatment options. Bacteriophage lysins are an alternative to conventional antibiotics with the potential to control antibiotic-resistant bacteria. This study demonstrates that a lysin called PlyB can effectively kill B. cereus in two models of B. cereus eye infections, thus treating and preventing the blinding effects of these infections., Competing Interests: The authors declare no conflict of interest.

Ocular infections can be medical emergencies that result in permanent visual impairment or blindness and loss of quality of life. Bacteria are a major cause of ocular infections. Effective treatment of ocular infections requires knowledge of which bacteria are the likely cause of the infection. This survey of ocular bacterial isolates and review of ocular pathogens is based on a survey of a collection of isolates banked over a ten-year span at the Dean McGee Eye Institute in Oklahoma. These findings illustrate the diversity of bacteria isolated from the eye, ranging from common species to rare and unique species. At all sampled sites, staphylococci were the predominant bacteria isolated. Pseudomonads were the most common Gram-negative bacterial isolate, except in vitreous, where Serratia was the most common Gram-negative bacterial isolate. Here, we discuss the range of ocular infections that these species have been documented to cause and treatment options for these infections. Although a highly diverse spectrum of species has been isolated from the eye, the majority of infections are caused by Gram-positive species, and in most infections, empiric treatments are effective.

Purpose: To test the hypothesis that the C-X-C chemokines CXCL1, CXCL2, and CXCL10 contribute to inflammation during Staphylococcus aureus endophthalmitis., Methods: S. aureus endophthalmitis was induced by intravitreal injection of 5000 colony forming units of S. aureus into the eyes of C57BL/6J, CXCL1-/-, CXCL2-/-, or CXCL10-/- mice. At 12, 24, and 36 hours postinfection, bacterial counts, intraocular inflammation, and retinal function were assessed. Based on these results, the effectiveness of intravitreal administration of anti-CXCL1 in reducing inflammation and improving retinal function was evaluated in S. aureus-infected C57BL/6J mice., Results: We observed significant attenuation of inflammation and improvement in retinal function in CXCL1-/- mice relative to C57BL/6J at 12 hours but not at 24 or 36 hours postinfection with S. aureus. Co-administration of anti-CXCL1 antibodies with S. aureus, however, did not improve retinal function or reduce inflammation at 12 hours postinfection. In CXCL2-/- and CXCL10-/- mice, retinal function and intraocular inflammation were not significantly different from those of C57BL/6J mice at 12 and 24 hours postinfection. At 12, 24, or 36 hours, an absence of CXCL1, CXCL2, or CXCL10 did not alter intraocular S. aureus concentrations., Conclusions: CXCL1 appears to contribute to the early host innate response to S. aureus endophthalmitis, but treatment with anti-CXCL1 did not effectively limit inflammation in this infection. CXCL2 and CXCL10 did not seem to play an integral role in inflammation during the early stages of S. aureus endophthalmitis.

Bacillus cereus (B. cereus) endophthalmitis is a vision-threatening bacterial infection. Uncontrolled inflammatory responses are the hallmark of this disease which cause irreversible damage to the retina. We recently reported C-X-C chemokines as a vital modulators which impacted the pathogenesis of this disease. Here, we investigated the impact of two highly upregulated C-C chemokines, CCL2 and CCL3, on intraocular inflammation this disease. B. cereus was injected into the eyes of C57BL/6J (WT), CCL2 -/- , and CCL3 -/- mice to induce endophthalmitis. Infected eyes were examined for bacterial growth, retinal function, and inflammation. Bacterial growth in CCL2 -/- and CCL3 -/- mice were similar, but retained retinal function was greater in CCL2 -/- and CCL3 -/- eyes compared to that of C57BL/6J eyes. The retinal architecture of infected eyes of CCL2 -/- mice were conserved for a longer period of time than in infected CCL3 -/- eyes. Infected CCL2 -/- and CCL3 -/- eyes had less inflammation than did infected C57BL/6J eyes. Based on these results, we assessed the efficacies of intravitreal anti-CCL2 or anti-CCL3 with or without the antibiotic gatifloxacin. Compared to infected untreated eyes, there was significantly less inflammation and greater retention of retinal function in eyes treated with anti-CCL2 or anti-CCL3 with gatifloxacin. This study showed that B. cereus endophthalmitis in CCL2 -/- mice had a better clinical outcome than in CCL3 -/- mice. Intravitreal administration of anti-CCL2 and anti-CCL3 with gatifloxacin significantly reduced inflammation and provided protection of retinal function. These results suggest that CCL2 and CCL3 are prospective anti-inflammatory targets that should be tested along with other antibiotics for treating Bacillus and perhaps other forms of endophthalmitis., (Copyright © 2022. Published by Elsevier Ltd.)

Pseudomonas aeruginosa is a significant opportunistic pathogen responsible for numerous human infections. Its high pathogenicity resides in a diverse array of virulence factors and an ability to adapt to hostile environments. We report that these factors are tied to the activity of condensins, SMC and MksBEF, which primarily function in structural chromosome maintenance. This study revealed that both proteins are required for P. aeruginosa virulence during corneal infection. The reduction in virulence was traced to broad changes in gene expression. Transcriptional signatures of smc and mksB mutants were largely dissimilar and non-additive, with the double mutant displaying a distinct gene expression profile. Affected regulons included those responsible for lifestyle control, primary metabolism, surface adhesion and biofilm growth, iron and sulfur assimilation, and numerous virulence factors, including type 3 and type 6 secretion systems. The in vitro phenotypes of condensin mutants mirrored their transcriptional profiles and included impaired production and secretion of multiple virulence factors, growth deficiencies under nutrient limiting conditions, and altered c-di-GMP signaling. Notably, c-di-GMP mediated some but not all transcriptional responses of the mutants. Thus, condensins are integrated into the control of multiple genetic programs related to epigenetic and virulent behavior of P. aeruginosa., (© 2022 John Wiley & Sons Ltd.)

Purpose: The purpose of this study was to explore the C-X-C chemokines CXCL2 and CXCL10 as potential anti-inflammatory targets for Bacillus endophthalmitis., Methods: Bacillus endophthalmitis was induced in C57BL/6J, CXCL2-/-, and CXCL10-/- mice. At specific times postinfection, eyes were analyzed for Bacillus, retinal function, and inflammation. The efficacies of intravitreal anti-CXCL2 and anti-CXCL10 with or without gatifloxacin in B. cereus endophthalmitis were also assessed using the same techniques., Results: Despite similar Bacillus growth in eyes of C57BL/6J, CXCL2-/-, and CXCL10-/- mice, retinal function retention was greater in eyes of CXCL2-/- and CXCL10-/- mice compared to that of C57BL/6J mice. Neutrophil migration into eyes of CXCL2-/- and CXCL10-/- mice was reduced to a greater degree compared to that of eyes of C57BL/6J mice. Infected CXCL2-/- and CXCL10-/- mouse eyes had significantly less inflammation compared to that of C57BL/6J eyes. Retinal structures in infected eyes of CXCL2-/- mice were preserved for a longer time than in CXCL10-/- eyes. Compared to untreated eyes, there was less inflammation and significant retention of retinal function in eyes treated with anti-CXCL2 and anti-CXCL10 with or without gatifloxacin., Conclusions: For Bacillus endophthalmitis, the absence of CXCL2 or CXCL10 in mice resulted in retained retinal function and less inflammation. The absence of CXCL2 led to a better clinical outcome than the absence of CXCL10. The use of anti-CXCL2 and anti-CXCL10 limited inflammation during B. cereus endophthalmitis. These results highlight the utility of CXCL2 and CXCL10 as potential targets for anti-inflammatory therapy that can be tested in conjunction with antibiotics for improving treating 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 (Δ inhA1 ), InhA2-deficient (Δ inhA2 ), or InhA1, A2, and A3-deficient (Δ inhA1-3 ) Bacillus thuringiensis. In vitro analysis of growth, proteolysis, and cytotoxicity were compared. WT and InhA mutants were similarly cytotoxic to retinal cells. The Δ inhA1 and Δ inhA2 mutants entered log-phase growth earlier than WT B. thuringiensis. Proteolysis by the Δ inhA1-3 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 Δ inhA1 or Δ inhA2 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 Δ inhA1-3 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.

Bacillus cereus is recognized as a causative agent of gastrointestinal syndromes, but can also cause a devastating form of intraocular infection known as endophthalmitis. We have previously reported that the PlcR/PapR master virulence factor regulator system regulates intraocular virulence, and that the S-layer protein (SlpA) contributes to the severity of B. cereus endophthalmitis. To better understand the role of other B. cereus virulence genes in endophthalmitis, expression of a subset of factors was measured at the midpoint of disease progression in a murine model of endophthalmitis by RNA-Seq. Several cytolytic toxins were expressed at significantly higher levels in vivo than in BHI. The virulence regulators codY , gntR , and nprR were also expressed in vivo . However, at this timepoint, plcR / papR was not detectable, although we previously reported that a B. cereus mutant deficient in PlcR was attenuated in the eye. The motility-related genes fla , fliF , and motB , and the chemotaxis-related gene cheA were detected during infection. We have shown previously that motility and chemotaxis phenotypes are important in B. cereus endophthalmitis. The sodA2 variant of manganese superoxide dismutase was the most highly expressed gene in vivo . Expression of the surface layer protein gene, slpA , an activator of Toll-like receptors (TLR)-2 and -4, was also detected during infection, albeit at low levels. Genes expressed in a mouse model of Bacillus endophthalmitis might play crucial roles in the unique virulence of B. cereus endophthalmitis, and serve as candidates for novel therapies designed to attenuate the severity of this often blinding infection.

Intraocular bacterial infections are a danger to the vision. Researchers use animal models to investigate the host and bacterial factors and immune response pathways associated with infection to identify viable therapeutic targets and to test drugs to prevent blindness. The intravitreal injection technique is used to inject organisms, drugs, or other substances directly into the vitreous cavity in the posterior segment of the eye. Here, we demonstrated this injection technique to initiate infection in the mouse eye and the technique of quantifying intraocular bacteria. Bacillus cereus was grown in brain heart infusion liquid media for 18 hours and resuspended to a concentration 100 colony forming units (CFU)/0.5 µL. A C57BL/6J mouse was anesthetized using a combination of ketamine and xylazine. Using a picoliter microinjector and glass capillary needles, 0.5 µL of the Bacillus suspension was injected into the mid vitreous of the mouse eye. The contralateral control eye was either injected with sterile media (surgical control) or was not injected (absolute control). At 10 hours post infection, mice were euthanized, and eyes were harvested using sterile surgical tweezers and placed into a tube containing 400 µL sterile PBS and 1 mm sterile glass beads. For ELISAs or myeloperoxidase assays, proteinase inhibitor was added to the tubes. For RNA extraction, the appropriate lysis buffer was added. Eyes were homogenized in a tissue homogenizer for 1-2 minutes. Homogenates were serially diluted 10-fold in PBS and track diluted onto agar plates. The remainder of the homogenates were stored at -80 °C for additional assays. Plates were incubated for 24 hours and CFU per eye was quantified. These techniques result in reproducible infections in mouse eyes and facilitate quantitation of viable bacteria, the host immune response, and omics of host and bacterial gene expression.

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-1β, IL-1α, TNFα, IL-23α, TGFβ1, and IL-12β) 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.

Mutations in nucleotide-binding oligomerization domain-containing protein 2 (NOD2) cause Blau syndrome, an inflammatory disorder characterized by uveitis. The antimicrobial functions of Nod2 are well-established, yet the cellular mechanisms by which dysregulated Nod2 causes uveitis remain unknown. Here, we report a non-conventional, T cell-intrinsic function for Nod2 in suppression of Th17 immunity and experimental uveitis. Reconstitution of lymphopenic hosts with Nod2 -/- CD4 + T cells or retina-specific autoreactive CD4 + T cells lacking Nod2 reveals a T cell-autonomous, Rip2-independent mechanism for Nod2 in uveitis. In naive animals, Nod2 operates downstream of TCR ligation to suppress activation of memory CD4 + T cells that associate with an autoreactive-like profile involving IL-17 and Ccr7. Interestingly, CD4 + T cells from two Blau syndrome patients show elevated IL-17 and increased CCR7. Our data define Nod2 as a T cell-intrinsic rheostat of Th17 immunity, and open new avenues for T cell-based therapies for Nod2-associated disorders such as Blau syndrome.

Experimental autoimmune uveoretinitis (EAU) is a mouse model of human autoimmune uveitis. EAU spontaneously resolves and is marked by ocular autoantigen-specific regulatory immunity in the spleen. Kallikrein binding protein (KBP) or kallistatin is a serine proteinase inhibitor that inhibits angiogenesis and inflammation, but its role in autoimmune uveitis has not been explored. We report that T cells activation is inhibited and EAU is attenuated in human KBP (HKBP) mice with no significant difference in the Treg population that we previously identified both before and after recovery from EAU. Moreover, following EAU immunization HKBP mice have potent ocular autoantigen specific regulatory immunity that is functionally suppressive., (Copyright © 2020 Muhammad, Avalos, Mursalin, Ma, Callegan and Lee.)

Bacillus cereus produces many factors linked to pathogenesis and is recognized for causing gastrointestinal toxemia and infections. B. cereus also causes a fulminant and often blinding intraocular infection called endophthalmitis. We reported that the PlcR/PapR system regulates intraocular virulence, but the specific factors that contribute to B. cereus virulence in the eye remain elusive. Here, we compared gene expression in ex vivo vitreous humor with expression in Luria Bertani (LB) and Brain Heart Infusion (BHI) broth by RNA-Seq. The expression of several cytolytic toxins in vitreous was less than or similar to levels observed in BHI or LB. Regulators of virulence genes, including PlcR/PapR, were expressed in vitreous. PlcR/PapR was expressed at low levels, though we reported that PlcR-deficient B. cereus was attenuated in the eye. Chemotaxis and motility genes were expressed at similar levels in LB and BHI, but at low to undetectable levels in vitreous, although motility is an important phenotype for B. cereus in the eye. Superoxide dismutase, a potential inhibitor of neutrophil activity in the eye during infection, was the most highly expressed gene in vitreous. Genes previously reported to be important to intraocular virulence were expressed at low levels in vitreous under these conditions, possibly because in vivo cues are required for higher level expression. Genes expressed in vitreous may contribute to the unique virulence of B. cereus endophthalmitis, and future analysis of the B. cereus virulome in the eye will identify those expressed in vivo, which could potentially be targeted to arrest virulence.

Bacillus cereus (B. cereus) endophthalmitis is a devastating intraocular infection primarily associated with post-traumatic injuries. The majority of these infections result in substantial vision loss, if not loss of the eye itself, within 12-48 h. Multifactorial mechanisms that lead to the innate intraocular inflammatory response during this disease include the combination of robust bacterial replication, migration of the organism throughout the eye, and toxin production by the organism. Therefore, the window of therapeutic intervention in B. cereus endophthalmitis is quite narrow compared to that of other pathogens which cause this disease. Understanding the interaction of bacterial and host factors is critical in understanding the disease and formulating more rational therapeutics for salvaging vision. In this review, we will discuss clinical and research findings related to B. cereus endophthalmitis in terms of the organism's virulence and inflammogenic potential, and strategies for improving of current therapeutic regimens for this blinding disease., Competing Interests: Declaration of competing interest The authors report no proprietary or commercial interest in any product mentioned or concept discussed in this article., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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 ( Δ slpA ) 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 Δ slpA 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., (Copyright © 2020 Mursalin, Coburn, Livingston, Miller, Astley, Flores-Mireles and Callegan.)

Some tissues of the eye are susceptible to damage due to their exposure to the outside environment and inability to regenerate. Immune privilege, although beneficial to the eye in terms of homeostasis and protection, can be harmful when breached or when an aberrant response occurs in the face of challenge. In this review, we highlight the role of the PMN (polymorphonuclear leukocyte) in different bacterial ocular infections that invade the immune privileged eye at the anterior and posterior segments: keratitis, conjunctivitis, uveitis, and endophthalmitis. Interestingly, the PMN response from the host seems to be necessary for pathogen clearance in ocular disease, but the inflammatory response can also be detrimental to vision retention. This "Pyrrhic Victory" scenario is explored in each type of ocular infection, with details on PMN recruitment and response at the site of ocular infection. In addition, we emphasize the differences in PMN responses between each ocular disease and its most common corresponding bacterial pathogen. The in vitro and animal models used to identify PMN responses, such as recruitment, phagocytosis, degranulation, and NETosis, are also outlined in each ocular infection. This detailed study of the ocular acute immune response to infection could provide novel therapeutic strategies for blinding diseases, provide more general information on ocular PMN responses, and reveal areas of bacterial ocular infection research that lack PMN response studies.

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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Purpose: Bacillus causes a sight-threating infection of the posterior segment of the eye. The robust intraocular inflammatory response in this disease is likely activated via host innate receptor interactions with components of the Bacillus cell envelope. S-layer proteins (SLPs) of some Gram-positive pathogens contribute to the pathogenesis of certain infections. The potential contributions of SLPs in eye infection pathogenesis have not been considered. Here, we explored the role of a Bacillus SLP (SlpA) in endophthalmitis pathogenesis., Methods: The phenotypes and infectivity of wild-type (WT) and S-layer deficient (ΔslpA) Bacillus thuringiensis were compared. Experimental endophthalmitis was induced in C57BL/6J mice by intravitreally injecting 100-CFU WT or ΔslpA B. thuringiensis. Infected eyes were analyzed by bacterial counts, retinal function analysis, histology, and inflammatory cell influx. SLP-induced inflammation was also analyzed in vitro. Muller cells (MIO-M1) were treated with purified SLP. Nuclear factor-κB (NF-κB) DNA binding was measured by ELISA and expression of proinflammatory mediators from Muller cells was measured by RT-qPCR., Results: Tested phenotypes of WT and ΔslpA B. thuringiensis were similar, with the exception of absence of the S-layer in the ΔslpA mutant. Intraocular growth of WT and ΔslpA B. thuringiensis was also similar. However, eyes infected with the ΔslpA mutant had significantly reduced inflammatory cell influx, less inflammatory damage to the eyes, and significant retention of retinal function compared with WT-infected eyes. SLP was also a potent stimulator of the NF-κB pathway and induced the expression of proinflammatory mediators (IL6, TNFα, CCL2, and CXCL-1) in human retinal Muller cells., Conclusions: Taken together, our results suggest that SlpA contributes to the pathogenesis of Bacillus endophthalmitis, potentially by triggering innate inflammatory pathways in the retina.

Ocular glands play a critical role in eye health through the secretion of factors directly onto the ocular surface. The cornea is a normally transparent tissue necessary for visual acuity located in the anterior segment of the eye. Corneal damage can occur during microbial infection of the cornea, resulting in potentially permanent visual deficits. The involvement of ocular glands during corneal infection has been only briefly described. We hypothesized that ocular glands contribute to resistance as an arm of the eye-associated lymphoid tissue and may also be susceptible to infection secondary to microbial keratitis. Utilizing a mouse model of herpes simplex virus 1 (HSV-1) keratitis, we found that infection of corneas resulted in subsequent infection of ocular glands, including harderian glands (HGs) and extraorbital glands. Similarly, infection of corneas with Pseudomonas aeruginosa resulted in secondary infection of ocular glands. A robust immune response, characterized by increased numbers of immune cells and inflammatory mediators, occurred within ocular glands following HSV-1 keratitis. Removal of HGs altered corneal resistance to HSV-1, as measured by increased viral load, decreased corneal edema, and decreased inflammatory cell infiltration. These novel findings suggest that ocular glands are involved in microbial keratitis through their susceptibility to secondary infection and contribution to corneal resistance. IMPORTANCE Microbial keratitis accounts for up to 700,000 clinical visits annually in the United States. The involvement of ocular glands during microbial keratitis is not readily appreciated, and treatment options do not address the consequences of ocular gland dysfunction. The present study shows that ocular glands are susceptible to direct infection by common ocular pathogens, including HSV-1 and Pseudomonas aeruginosa , subsequent to microbial keratitis. Additionally, ocular glands contribute soluble factors that play a role in corneal resistance to HSV-1 and alter viral load, corneal edema, and immune cell infiltration. Further studies are needed to elucidate the mechanisms by which this occurs., (Copyright © 2019 American Society for Microbiology.)

Staphylococcus aureus (S. aureus) is a common pathogen of the eye, capable of infecting external tissues such as the tear duct, conjunctiva, and the cornea, as well the inner and more delicate anterior and posterior chambers. S. aureus produces numerous toxins and enzymes capable of causing profound damage to tissues and organs, as well as modulating the immune response to these infections. Unfortunately, in the context of ocular infections, this can mean blindness for the patient. The role of α-toxin in corneal infection (keratitis) and infection of the interior of the eye (endophthalmitis) has been well established by comparing virulence in animal models and α-toxin-deficient isogenic mutants with their wild-type parental strains. The importance of other toxins, such as β-toxin, γ-toxin, and Panton-Valentine leukocidin (PVL), have been analyzed to a lesser degree and their roles in eye infections are less clear. Other toxins such as the phenol-soluble modulins have yet to be examined in any animal models for their contributions to virulence in eye infections. This review discusses the state of current knowledge of the roles of S. aureus toxins in eye infections and the controversies existing as a result of the use of different infection models. The strengths and limitations of these ocular infection models are discussed, as well as the need for physiological relevance in the study of staphylococcal toxins in these models.

Intraocular infections are prevalent after traumatic injuries or after common ocular surgeries. Infections cause inflammation that can damage the retina and architecture of the eye, often resulting in poor visual outcomes. Severe cases may result in blindness or require enucleation of the eye. Treatments for intraocular infections include intravitreal antibiotics and corticosteroids or surgical vitrectomy in serious cases. The increase in multidrug-resistant infections calls for novel treatment options. In the present study, a biomimetic erythrocyte-derived nanosponge was tested for the ability to neutralize pore-forming toxins from the most frequent Gram-positive bacterial causes of intraocular infections ( Staphylococcus aureus , Enterococcus faecalis , Streptococcus pneumoniae , and Bacillus cereus ). Nanosponge pretreatment of supernatants reduced hemolytic activity in vitro. In a murine sterile endophthalmitis model, nanosponge pretreatment of injected supernatants resulted in greater retinal function and less ocular pathology compared to that in eyes injected with untreated supernatants from all pathogens except methicillin-resistant S. aureus In a murine bacterial endophthalmitis model, treatment with gatifloxacin and gatifloxacin-nanosponges reduced intraocular bacterial burdens, except in the case of methicillin-sensitive S. aureus For all pathogens, eyes in both treatment groups showed decreased ocular pathology and inflammation. Overall, reductions in retinal function loss afforded by gatifloxacin-nanosponge treatment were significant for E. faecalis , S. pneumoniae , and methicillin-resistant S. aureus but not for B. cereus and methicillin-sensitive S. aureus These results suggest that clinical improvements in intraocular infections following nanosponge treatment were dependent on the complexity and types of toxins produced. Nanosponges might serve as an adjunctive therapy for the treatment of ocular infections. IMPORTANCE Endophthalmitis is a blinding consequence of bacterial invasion of the interior of the eye. Because of increases in the numbers of ocular surgeries and intraocular injections, the incidence of endophthalmitis is steadily increasing. Staphylococcus aureus , Enterococcus faecalis , Streptococcus pneumoniae , and Bacillus cereus are leading causes of infection following ocular procedures and trauma and are increasingly more difficult to treat due to multidrug resistance. Each of these pathogens produces pore-forming toxins that contribute to the pathogenesis of endophthalmitis. Treatment of these infections with antibiotics alone is insufficient to prevent damage to the retina and vision loss. Therefore, novel therapeutics are needed that include agents that neutralize bacterial pore-forming toxins. Here, we demonstrate that biomimetic nanosponges neutralize pore-forming toxins from these ocular pathogens and aid in preserving retinal function. Nanosponges may represent a new form of adjunct antitoxin therapy for serious potentially blinding intraocular infections., (Copyright © 2019 Coburn et al.)

Background: Endophthalmitis is a serious intraocular infection that frequently results in significant inflammation and vision loss. Because current therapeutics are often unsuccessful in mitigating damaging inflammation during endophthalmitis, more rational targets are needed. Toll-like receptors (TLRs) recognize specific motifs on invading pathogens and initiate the innate inflammatory response. We reported that TLR4 contributes to the robust inflammation which is a hallmark of Bacillus cereus endophthalmitis. To identify novel, targetable host inflammatory factors in this disease, we performed microarray analysis to detect TLR4-dependent changes to the retinal transcriptome during B. cereus endophthalmitis., Results: C57BL/6 J and TLR4 -/- mouse eyes were infected with B. cereus and retinas were harvested at 4 h postinfection, a time representing the earliest onset of neutrophil infiltration. Genes related to acute inflammation and inflammatory cell recruitment including CXCL1 (KC), CXCL2 (MIP2-α), CXCL10 (IP-10), CCL2 (MCP1), and CCL3 (MIP1-α)) were significantly upregulated 5-fold or greater in C57BL/6 J retinas. The immune modulator IL-6, intercellular adhesion molecule ICAM1, and the inhibitor of cytokine signal transduction SOCS3 were upregulated 25-, 11-, and 10-fold, respectively, in these retinas. LIF, which is crucial for photoreceptor cell survival, was increased 6-fold. PTGS2/COX-2, which converts arachidonic acid to prostaglandin endoperoxide H2, was upregulated 9-fold. PTX3, typically produced in response to TLR engagement, was induced 15-fold. None of the aforementioned genes were upregulated in TLR4 -/- retinas following B. cereus infection., Conclusions: Our results have identified a cohort of mediators driven by TLR4 that may be important in regulating pro-inflammatory and protective pathways in the retina in response to B. cereus intraocular infection. This supports the prospect that blocking the activation of TLR-based pathways might serve as alternative targets for Gram-positive and Gram-negative endophthalmitis therapies in general.

Intraocular infections are a potentially blinding complication of common ocular surgeries and traumatic eye injuries. Bacterial toxins synthesized in the eye can damage intraocular tissue, often resulting in poor visual outcomes. Enteroccocus faecalis causes blinding infections and is responsible for 8 to 17% of postoperative endophthalmitis cases. These infections are increasingly difficult to treat due to the emergence of multidrug-resistant strains. Virulent E. faecalis isolates secrete a pore-forming bicomponent cytolysin that contributes to retinal tissue damage during endophthalmitis. We hypothesized that a biomimetic nanosponge, which mimics erythrocytes, might adsorb subunits of the cytolysin and reduce retinal damage, protecting vision. To test the efficacy of nanosponges in neutralizing the cytolysin in vitro , hemoglobin release assays were performed on culture supernatants from cytolysin-producing E. faecalis with and without preincubation with nanosponges. Treatment with nanosponges for 30 min reduced hemolytic activity by ~70%. To determine whether nanosponges could neutralize the cytolysin in vivo , electroretinography was performed on mice 24 h after intravitreal injection with cytolysin-containing supernatants treated with nanosponges. Pretreatment of cytolysin-containing supernatants with nanosponges increased the A-wave retention from 12.2% to 65.5% and increased the B-wave retention from 21.0% to 77.0%. Histology revealed that in nanosponge-treated eyes, retinas remained intact and attached, with little to no damage. Rabbit nanosponges were also nontoxic and noninflammatory when injected into mouse eyes. In an experimental murine model of E. faecalis endophthalmitis, injection of nanosponges into the vitreous 6 h after infection with a wild-type cytolysin-producing strain increased A-wave retention from 5.9% to 31% and increased B-wave retention from 12.6% to 27.8%. Together, these results demonstrated that biomimetic nanosponges neutralized cytolysin activity and protected the retinas from damage. These results suggest that this novel strategy might also protect eyes from the activities of pore-forming toxins of other virulent ocular bacterial pathogens. IMPORTANCE Endophthalmitis is a serious, potentially blinding infection that can result in vision loss, leaving a patient with only the ability to count fingers, or it may require enucleation of the globe. The incidence of postoperative endophthalmitis has markedly increased over the past 2 decades, paralleling the rise in ocular surgeries and intravitreal therapies. E. faecalis is a leading cause of infection following ocular procedures, and such infections are increasingly difficult to treat due to multidrug resistance. Cytolysin is the primary virulence factor responsible for retinal tissue damage in E. faecalis eye infections. Treatment of these infections with antibiotics alone does not impede ocular damage and loss of visual function. Pore-forming toxins (PFTs) have been established as major virulence factors in endophthalmitis caused by several bacterial species. These facts establish a critical need for a novel therapy to neutralize bacterial PFTs such as cytolysin. Here, we demonstrate that biomimetic nanosponges neutralize cytolysin, protect the retina, preserve vision, and may provide an adjunct detoxification therapy for bacterial infections.

Bacterial endophthalmitis is a potentially blinding intraocular infection. The bacterium Bacillus cereus causes a devastating form of this disease which progresses rapidly, resulting in significant inflammation and loss of vision within a few days. The outer surface of B. cereus incites the intraocular inflammatory response, likely through interactions with innate immune receptors such as TLRs. This study analyzed the role of B. cereus pili, adhesion appendages located on the bacterial surface, in experimental endophthalmitis. To test the hypothesis that the presence of pili contributed to intraocular inflammation and virulence, we analyzed the progress of experimental endophthalmitis in mouse eyes infected with wild type B. cereus (ATCC 14579) or its isogenic pilus-deficient mutant (ΔbcpA-srtD-bcpB or ΔPil). One hundred CFU were injected into the mid-vitreous of one eye of each mouse. Infections were analyzed by quantifying intraocular bacilli and retinal function loss, and by histology from 0 to 12 h postinfection. In vitro growth and hemolytic phenotypes of the infecting strains were also compared. There was no difference in hemolytic activity (1:8 titer), motility, or in vitro growth (p > 0.05, every 2 h, 0-18 h) between wild type B. cereus and the ΔPil mutant. However, infected eyes contained greater numbers of wild type B. cereus than ΔPil during the infection course (p ≤ 0.05, 3-12 h). Eyes infected with wild type B. cereus experienced greater losses in retinal function than eyes infected with the ΔPil mutant, but the differences were not always significant. Eyes infected with ΔPil or wild type B. cereus achieved similar degrees of severe inflammation. The results indicated that the intraocular growth of pilus-deficient B. cereus may have been better controlled, leading to a trend of greater retinal function in eyes infected with the pilus-deficient strain. Although this difference was not enough to significantly alter the severity of the inflammatory response, these results suggest a potential role for pili in protecting B. cereus from clearance during the early stages of endophthalmitis, which is a newly described virulence mechanism for this organism and this infection., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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., (© Society for Leukocyte Biology.)

Bacterial endophthalmitis is an infection and inflammation of the posterior segment of the eye which can result in significant loss of visual acuity. Even with prompt antibiotic, anti-inflammatory and surgical intervention, vision and even the eye itself may be lost. For the past century, experimental animal models have been used to examine various aspects of the pathogenesis and pathophysiology of bacterial endophthalmitis, to further the development of anti-inflammatory treatment strategies, and to evaluate the pharmacokinetics and efficacies of antibiotics. Experimental models allow independent control of many parameters of infection and facilitate systematic examination of infection outcomes. While no single animal model perfectly reproduces the human pathology of bacterial endophthalmitis, investigators have successfully used these models to understand the infectious process and the host response, and have provided new information regarding therapeutic options for the treatment of bacterial endophthalmitis. This review highlights experimental animal models of endophthalmitis and correlates this information with the clinical setting. The goal is to identify knowledge gaps that may be addressed in future experimental and clinical studies focused on improvements in the therapeutic preservation of vision during and after this disease., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The blood-retinal barrier (BRB) functions to maintain the immune privilege of the eye, which is necessary for normal vision. The outer BRB is formed by tightly-associated retinal pigment epithelial (RPE) cells which limit transport within the retinal environment, maintaining retinal function and viability. Retinal microvascular complications and RPE dysfunction resulting from diabetes and diabetic retinopathy cause permeability changes in the BRB that compromise barrier function. Diabetes is the major predisposing condition underlying endogenous bacterial endophthalmitis (EBE), a blinding intraocular infection resulting from bacterial invasion of the eye from the bloodstream. However, significant numbers of EBE cases occur in non-diabetics. In this work, we hypothesized that dysfunction of the outer BRB may be associated with EBE development. To disrupt the RPE component of the outer BRB in vivo, sodium iodate (NaIO3) was administered to C57BL/6J mice. NaIO3-treated and untreated mice were intravenously injected with 108 colony forming units (cfu) of Staphylococcus aureus or Klebsiella pneumoniae. At 4 and 6 days postinfection, EBE was observed in NaIO3-treated mice after infection with K. pneumoniae and S. aureus, although the incidence was higher following S. aureus infection. Invasion of the eye was observed in control mice following S. aureus infection, but not in control mice following K. pneumoniae infection. Immunohistochemistry and FITC-dextran conjugate transmigration assays of human RPE barriers after infection with an exoprotein-deficient agr/sar mutant of S. aureus suggested that S. aureus exoproteins may be required for the loss of the tight junction protein, ZO-1, and for permeability of this in vitro barrier. Our results support the clinical findings that for both pathogens, complications which result in BRB permeability increase the likelihood of bacterial transmigration from the bloodstream into the eye. For S. aureus, however, BRB permeability is not required for the development of EBE, but toxin production may facilitate EBE pathogenesis.

Purpose: To test the hypothesis that blood-retinal barrier compromise is associated with the development of endogenous Staphylococcus aureus endophthalmitis., Methods: To compromise the blood-retinal barrier in vivo, streptozotocin-induced diabetes was induced in C57BL/6J mice for 1, 3, or 5 months. Diabetic and age-matched nondiabetic mice were intravenously injected with 108 colony-forming units (cfu) of S. aureus, a common cause of endogenous endophthalmitis in diabetics. After 4 days post infection, electroretinography, histology, and bacterial counts were performed. Staphylococcus aureus-induced alterations in in vitro retinal pigment epithelial (RPE) cell barrier structure and function were assessed by anti-ZO-1 immunohistochemistry, FITC-dextran conjugate diffusion, and bacterial transmigration assays., Results: We observed one bilateral infection in a control, nondiabetic animal (mean = 1.54 × 103 ± 1.78 × 10² cfu/eye, 7% incidence). Among the 1-month diabetic mice, we observed culture-confirmed unilateral infections in two animals (mean = 5.54 × 10² ± 7.09 × 10² cfu/eye, 12% incidence). Among the 3-month diabetic mice, infections were observed in 11 animals, three with bilateral infections (mean = 2.67 × 10² ± 2.49 × 10² cfu/eye, 58% incidence). Among the 5-month diabetic mice, we observed infections in five animals (mean = 7.88 × 10² ± 1.08 × 10³ cfu/eye, 33% incidence). In vitro, S. aureus infection reduced ZO-1 immunostaining and disrupted the barrier function of cultured RPE cells, resulting in diffusion of fluorophore-conjugated dextrans and transmigration of live bacteria across a permeabilized RPE barrier., Conclusions: Taken together, these results indicated that S. aureus is capable of inducing blood-retinal barrier permeability and causing endogenous bacterial endophthalmitis in normal and diabetic animals.

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., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Purpose: Caveolin-1 (Cav-1), the signature protein of caveolae, modulates inflammatory responses, and innate immunity. However, Cav-1's role in retinal inflammation has not been rigorously tested. In this study, we examined the effect of Cav-1 ablation on the sensitivity of the retina to inflammation., Methods: Cav-1 knockout (KO) mice were challenged by intravitreal injection of lipopolysaccharide (LPS) and inflammatory cell recruitment was assessed by flow cytometry and immunohistochemistry. Leukostasis was assessed in retinal flatmounts after perfusion with FITC-labeled Concanavalin A (FITC-ConA). Chemoattractants were measured by multiplex immunoassays. Blood-retinal barrier (BRB) breakdown was assessed quantitatively by a FITC-dextran permeability assay. The ratio of extravascular to total immune cells was determined by CD45 immunohistochemistry of retinal flatmounts., Results: Inflammatory challenge resulted in significant blunting of proinflammatory cytokine (monocyte chemoattractant protein-1 [MCP-1/CCL2], CXCL1/KC, IL-6, and IL-1β) responses as well as reduced inflammatory BRB breakdown in Cav-1 KO retinas. Paradoxically, Cav-1 deficiency resulted in significantly increased recruitment of immune cells compared with controls as well as increased leukostasis. A similar ratio of extravascular/total leukocytes were found in Cav-1 KO and wild-type (WT) retinas suggesting that Cav-1 deficient leukocytes were as competent to extravasate as those from WT mice. We found increased levels of circulating immune cells in naïve (not challenged with LPS) Cav-1 KO mice compared with controls., Conclusions: Caveolin-1 paradoxically modulates inflammatory signaling and leukocyte infiltration through distinct mechanisms. We hypothesize that Cav-1 expression may enhance inflammatory signaling while at the same time supporting the physical properties of the BRB., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

Purpose/aim: Klebsiella pneumoniae causes a blinding infection called endogenous endophthalmitis. The role of innate immune recognition of K. pneumoniae in the eye during infection is not known. We hypothesized that intraocular recognition of K. pneumoniae was mediated by Toll-like receptor (TLR)-4 and may be dependent on MagA-regulated hypermucoviscosity., Materials and Methods: Experimental endophthalmitis was induced in C57BL/6J or TLR4(-/-) mice by intravitreal injection of 100 CFU of wild type or ΔmagA K. pneumoniae. Infection and inflammation were quantified by determining viable K. pneumoniae per eye, retinal responses via electroretinography, myeloperoxidase activity of infiltrating neutrophils and the proinflammatory cytokine and chemokine response., Results: C57BL/6J and TLR4(-/-) mice could not control intraocular wild-type K. pneumoniae growth. TLR4(-/-) mice were less able than C57BL/6J to control the intraocular growth of ΔmagA K. pneumoniae. Retinal function testing suggested that infection with ΔmagA K. pneumoniae resulted in less retinal function loss. There was a TLR4-dependent delay in initial neutrophil recruitment, regardless of the infecting organism. The proinflammatory cytokine/chemokine data supported these results. These findings were not due to an inability of TLR4(-/-) neutrophils to recognize or kill K. pneumoniae., Conclusions: These studies suggest that TLR4 is important in the early intraocular recognition and host response to K. pneumoniae. However, the role of MagA in TLR4-mediated intraocular recognition and subsequent inflammation is less clear.

B. cereus possesses flagella which allow the organism to migrate within the eye during a blinding form of intraocular infection called endophthalmitis. Because flagella is a ligand for Toll-like receptor 5 (TLR5), we hypothesized that TLR5 contributed to endophthalmitis pathogenesis. Endophthalmitis was induced in C57BL/6J and TLR5-/- mice by injecting 100 CFU of B. cereus into the mid-vitreous. Eyes were analyzed for intraocular bacterial growth, retinal function, and inflammation by published methods. Purified B. cereus flagellin was also injected into the mid-vitreous of wild type C57BL/6J mice and inflammation was analyzed. TLR5 activation by B. cereus flagellin was also analyzed in vitro. B. cereus grew rapidly and at similar rates in infected eyes of C57BL/6J and TLR5-/- mice. A significant loss in retinal function in both groups of mice was observed at 8 and 12 hours postinfection. Retinal architecture disruption and acute inflammation (neutrophil infiltration and proinflammatory cytokine concentrations) increased and were significant at 8 and 12 hours postinfection. Acute inflammation was comparable in TLR5-/- and C57BL/6J mice. Physiological concentrations of purified B. cereus flagellin caused significant inflammation in C57BL/6J mouse eyes, but not to the extent of that observed during active infection. Purified B. cereus flagellin was a weak agonist for TLR5 in vitro. These results demonstrated that the absence of TLR5 did not have a significant effect on the evolution of B. cereus endophthalmitis. This disparity may be due to sequence differences in important TLR5 binding domains in B. cereus flagellin or the lack of flagellin monomers in the eye to activate TLR5 during infection. Taken together, these results suggest a limited role for flagellin/TLR5 interactions in B. cereus endophthalmitis. Based on this and previous data, the importance of flagella in this disease lies in its contribution to the motility of the organism within the eye during infection.

Purpose: To determine the bacterial contamination rate of a 27-gauge needle bore during conjunctival penetration in donor eye bank eyes and the effect of short-term use of topical 0.3% gatifloxacin and 0.5% moxifloxacin., Methods: One hundred consecutive human donors had 10 conjunctival penetrations per 10 syringes per eye before antibiotic placement; this was repeated 15 min after antibiotic use. Samples were cultured by expressing 0.3 mL of saline through the needle. Positive cultures were speciated., Results: There were 1,033 positive cultures (25.8% of all cultures); 568 (28.4%) pre-antibiotics, 249 (24.9%) after gatifloxacin (P=0.04, compared to the pre-antibiotic rate), and 216 (21.6%) after moxifloxacin (P<0.001). The most common organism was Staphylococcus epidermidis [334 positive cultures (8.4%)]. No antibiotic effect was seen on this or other organisms except S. aureus [4.6% pre-antibiotic, 2.8% after gatifloxacin (P=0.02), and 1.8% after moxifloxacin (P<0.001)] and other Staphylococcus species [5.3% pre-antibiotic, 3.6% after gatifloxacin (P=0.04), and 3.2% after moxifloxacin (P=0.01)]., Conclusions: Transconjunctival penetration often results in needle bore contamination; bacteria are included in an injected solution. Fifteen minutes of exposure to 2 topical antibiotics had a minimal effect on bacterial contamination and no significant effect on many common pathogens.

Purpose: We tested the hypothesis that changes in the diabetic ocular environment facilitate the development of endogenous bacterial endophthalmitis (EBE)., Methods: C57BL/6J mice were rendered diabetic with streptozotocin (STZ) for 1, 3, or 5 months' duration. Diabetic and age-matched nondiabetic mice were tail vein-injected with 10(8) CFU of Klebsiella pneumoniae, a common cause of EBE in diabetics. After either 2 or 4 days postinfection, the EBE incidence was assessed by electroretinography, histology, bacterial counts, and myeloperoxidase ELISAs. Blood-retinal barrier (BRB) permeability in uninfected diabetic mice also was determined., Results: No cases of EBE were observed among the 1-month diabetic group. Extending the time from diabetes induction to 3 months resulted in a 23.8% EBE incidence after 2 days, and a 22% incidence after 4 days. The incidence of EBE increased to 27% in the 5-month diabetic group. Infected eyes had an average 8.01 × 10(2) and 6.19 × 10(4) CFU/eye for the 3- and 5-month diabetic groups, respectively. There was no significant difference in BRB permeability between control and 1-month uninfected diabetic mice. However, 3- and 5-month diabetic mice had significantly greater BRB permeability than control mice. These results suggested that increasing the time from STZ diabetes induction to 3 and 5 months resulted in an ocular environment more conducive to the development of EBE., Conclusions: These results demonstrated a correlation between an increase in BRB permeability and an increase in EBE incidence, supporting the hypothesis that diabetic ocular changes contribute to the development of EBE.

Purpose: To evaluate the efficacy of vitrectomy with vancomycin for the treatment of experimental Bacillus cereus endophthalmitis., Methods: Endophthalmitis was initiated in rabbits via intravitreal injection of 100 colony-forming unit B. cereus. Treatment groups included 25-gauge transconjunctival sutureless vitrectomy with intravitreal vancomycin (1 mg) or vancomycin alone. Groups were treated at 4, 5, or 6 hours after infection. At 48 hours (for 4-hour and 5-hour groups) or 36 hours (for the 6-hour group) after infection, eyes were analyzed by electroretinography, histology, and inflammatory cell counts., Results: Treatment with vitrectomy/vancomycin at 4 hours resulted in significantly greater retinal function compared with that of vancomycin alone. Intraocular inflammation after treatment at 4 hours was minimal for both the treatment groups. Treatment with vitrectomy/vancomycin or vancomycin alone at 5 hours or 6 hours after infection resulted in similar levels of retinal function loss (i.e., >90%) and significant intraocular inflammation., Conclusion: These results demonstrate that vitrectomy may be of therapeutic benefit in the treatment of B. cereus endophthalmitis but only during the early stages of infection.

Purpose: Endogenous endophthalmitis secondary to Klebsiella pneumoniae liver abscess is a blinding infection that is being reported more frequently in the literature. The K1 capsule and magA contribute to virulence of systemic infection in mice; however, little is known about the role of magA in secondary ocular infections., Methods: To assess the role of K. pneumoniae capsule in endophthalmitis, the authors induced experimental endophthalmitis by direct inoculation of 100 colony-forming unit wild-type, magA-deficient, or magA-complemented K. pneumoniae into the posterior segments of mouse eyes. Eyes were analyzed by quantitation of viable bacteria, retinal function, and inflammatory cell influx as well as by histology., Results: Wild-type K1 K. pneumoniae caused significant ocular disease. At the end point of 24 hours postinfection, eyes infected with wild-type K. pneumoniae retained significantly less retinal A-wave function than eyes infected with an isogenic magA-mutant strain. B-wave function retention was also greater in eyes infected with the magA mutant than with wild-type K. pneumoniae. Additionally, intraocular growth of the magA-deficient strain was less than it was in the wild-type strain. The amount of myeloperoxidase elicited was also significantly higher for wild-type-infected eyes at 24 hours., Conclusions: These results indicate that in the eye, the K1 capsule of invasive K. pneumoniae significantly contributes to the ability of the bacteria to disrupt retinal function, to grow to high density, and to persist despite immune cell recruitment.

Bacillus cereus causes a uniquely rapid and blinding intraocular infection, endophthalmitis. B. cereus replicates in the eye, synthesizes numerous toxins, and incites explosive intraocular inflammation. The mechanisms involved in the rapid and explosive intraocular immune response have not been addressed. Because Toll-like receptors (TLRs) are integral to the initial recognition of organisms during infection, we hypothesized that the uniquely explosive immune response observed during B. cereus endophthalmitis is directly influenced by the presence of TLR2, a known gram-positive pathogen recognition receptor. To address this hypothesis, we compared the courses of experimental B. cereus endophthalmitis in wild type C57BL/6J mice to that of age-matched homozygous TLR2(-/-) mice. Output parameters included analysis of bacterial growth, inflammatory cell (PMN) infiltration, cytokine/chemokine kinetics, retinal function testing, and histology, with N≥4 eyes/assay/time point/mouse strain. B. cereus grew at similar rates to10(8) CFU/eye by 12 h, regardless of the mouse strain. Retinal function was preserved to a greater degree in infected TLR2(-/-) eyes compared to that of infected wild type eyes, but infected eyes of both mouse strains lost significant function. Retinal architecture was preserved in infected TLR2(-/-) eyes, with limited retinal and vitreal cellular infiltration compared to that of infected wild type eyes. Ocular myeloperoxidase activities corroborated these results. In general, TNFα, IFNγ, IL6, and KC were detected in greater concentrations in infected wild type eyes than in infected TLR2(-/-) eyes. The absence of TLR2 resulted in decreased intraocular proinflammatory cytokine/chemokine levels and altered recruitment of inflammatory cells into the eye, resulting in less intraocular inflammation and preservation of retinal architecture, and a slightly greater degree of retinal function. These results demonstrate TLR2 is an important component of the initial ocular response to B. cereus endophthalmitis.

Endophthalmitis is an infection and inflammation of the interior of the eye that can result in significant vision loss. This infection occurs as a result of the seeding of organisms into the interior of the eye following surgery (postoperative), trauma (post-traumatic) or an infection in another site in the body (endogenous). The general rate of endophthalmitis has remained steady over the past several years. However, the increased use of intraocular injections to treat various degenerative and inflammatory ocular diseases, in addition to the already large and growing number of invasive ocular surgeries, may increase the opportunities in which organisms can gain access to the eye. In most cases of endophthalmitis, useful vision can be retained if proper treatment is instituted. However, in severe cases of bacterial endophthalmitis, blindness often occurs despite treatment. This article summarizes information on endophthalmitis epidemiology, treatment issues and current regimens, and recent experimental and clinical efforts to improve the outcome of severe and blinding forms of bacterial endophthalmitis.

Purpose: The purpose of this study was to determine to what extent blood-retinal barrier (BRB) permeability occurred during experimental Bacillus cereus endophthalmitis and whether tight junction alterations were involved in permeability., Methods: Mice were intravitreally injected with 100 colony-forming units of B. cereus, and eyes were analyzed at specific times after infection for permeability to fibrin and albumin, quantitation of intraocular plasma constituent leakage, production of inflammatory cytokines, and alterations in tight junction protein localization and expression at the level of the retinal pigment epithelium., Results: B. cereus induced the leakage of albumin and fibrin into the aqueous and vitreous humor by 8 hours after infection. BRB permeability occurred as early as 4 hours and increased 13.30-fold compared with uninfected controls by 8 hours. Production of proinflammatory cytokines IL-6, MIP-1alpha, IL-1beta, and KC increased over the course of infection. In the retina, ZO-1 disruption began by 4 hours and was followed by decreasing occludin and ZO-1 expression at 4 and 8 hours, respectively. Tubulin condensation and RPE65 degradation occurred by 12 hours. A quorum-sensing mutant B. cereus strain caused BRB permeability comparable to that of wild-type B. cereus. Wild-type and mutant B. cereus sterile supernatants induced blood-ocular barrier permeability similarly to that of wild-type infection., Conclusions: These results indicate that BRB permeability occurs during the early stages of experimental B. cereus endophthalmitis, beginning as early as 4 hours after infection. Disruption of tight junctions at the level of the retinal pigment epithelium may contribute to barrier breakdown. Quorum-sensing dependent factors may not significantly contribute to BRB permeability.

Introduction: Antibacterial activity of ophthalmic fourth-generation fluoroquinolones has traditionally been evaluated by comparing only their active ingredients, gatifloxacin and moxifloxacin. However, ophthalmic formulations of fourth-generation fluoroquinolones differ in terms of the inclusion of preservatives. While gatifloxacin ophthalmic solution 0.3% (Zymar; Allergan, Inc., Irvine, CA, USA) contains 0.005% benzalkonium chloride (BAK), moxifloxacin ophthalmic solution 0.5% (Vigamox; Alcon Laboratories, Inc., Fort Worth, TX, USA) is preservative-free. Recent studies have demonstrated that the presence of BAK dramatically affects the antibacterial activity of the ophthalmic formulation of gatifloxacin. This study was designed to compare the kill rates of ophthalmic solutions of fourth-generation fluoroquinolones against isolates of common ocular bacterial pathogens., Methods: Approximately 5.6 log(10) colony-forming units (CFU)/mL of Haemophilus influenzae (n=1), Streptococcus pneumoniae (n=1), Staphylococcus aureus (n=2), methicillin-resistant Staphylococcus aureus (MRSA) (n=4), methicillinresistant Staphylococcus epidermidis (MRSE) (n=4), and fluoroquinolone-resistant S. epidermidis (n=1) were incubated with ophthalmic solutions of either gatifloxacin or moxifloxacin. Viable bacteria were quantified at specific time points up to 60 minutes., Results: Gatifloxacin 0.3% completely eradicated H. influenzae and Strep. pneumoniae in 5 minutes, one of two S. aureus isolates in 15 minutes, and the other S. aureus isolate in 60 minutes. Gatifloxacin 0.3% completely killed all MRSA, MRSE, and fluoroquinolone-resistant S. epidermidis isolates in 15 minutes. Moxifloxacin 0.5% completely eradicated Strep. pneumoniae and one of four MRSA isolates in 60 minutes. All other isolates incubated with moxifloxacin 0.5% retained viable bacteria ranging from 1.8 to 4.4 log(10) CFU/mL., Conclusions: The ophthalmic solution of gatifloxacin 0.3% eradicated bacteria that frequently cause postoperative ocular infections substantially faster than did the ophthalmic solution of moxifloxacin 0.5%.

Purpose: Klebsiella pneumoniae is a common cause of endogenous bacterial endophthalmitis, a disease that frequently results in a poor visual outcome. Hypermucoviscosity has been identified as a virulence factor among clinical bacteremia isolates of K. pneumoniae. In this study, an experimental murine model of K. pneumoniae endophthalmitis was established, and the role of hypermucoviscosity in its pathogenesis was analyzed., Methods: C57BL/6J mice were intravitreously injected with 100 CFU of hypermucoviscous (HMV+) or nonhypermucoviscous (HMV-) K. pneumoniae. Intraocular bacterial growth, retinal function, gross pathology, and inflammatory responses were monitored every 3 hours until the eyes lost significant (>90%) retinal function, or the infection appeared to clear., Results: The HMV+ strain grew logarithmically in eyes until approximately 15 hours postinfection (PI), reaching a stationary phase of growth at approximately 8.0 log(10) CFU/eye. The HMV- strain grew logarithmically to approximately 7.6 log(10) by 18 hours, but bacterial count declined to approximately 6.4 log(10) CFU/eye by 21 hours PI. Eyes infected with the HMV+ strain retained approximately 35% a-wave and <10% b-wave function by 18 hours PI. These eyes also had a cumulative clinical score of 14+ by 18 hours and underwent phthisis between 21 and 24 hours. Eyes infected with the HMV- strain had a cumulative clinical score of <6 and retained >60% a-wave and >50% b-wave function throughout 21 hours. Five of 7 eyes had <100 CFU HMV- K. pneumoniae at 27 hours PI., Conclusions: The findings demonstrate the site-threatening consequences of K. pneumoniae endophthalmitis and the importance of the hypermucoviscosity phenotype in the pathogenesis of experimental K. pneumoniae endophthalmitis.

Purpose: To determine the contribution of tumor necrosis factor-alpha (TNFalpha) in the pathogenesis of experimental Bacillus cereus endophthalmitis., Methods: Experimental B. cereus endophthalmitis was induced in wild-type control (B6.129F1) and age-matched homozygous TNFalpha knockout mice (TNFalpha(-/-), B6.129S6-Tnf(tm1Gk1)/J). At various times after infection, eyes were analyzed by electroretinography and were harvested for quantitation of bacteria, myeloperoxidase, proinflammatory cytokines and chemokines, and histologic analysis., Results: B. cereus replicated more rapidly in the eyes of TNFalpha(-/-) mice than in the eyes of B6.129F1 mice. Retinal function decreased more rapidly in TNFalpha(-/-) mice than in B6.129F1 mice. Retinal layers were not as structurally intact at 6 and 12 hours after infection in TNFalpha(-/-) eyes as in B6.129F1 eyes. Histologic analysis suggested less polymorphonuclear leukocyte (PMN) infiltration into the vitreous of TNFalpha(-/-) mice than of B6.129F1 mice. B6.129F1 eyes also had greater myeloperoxidase concentrations than did eyes of TNFalpha(-/-) mice. In general, concentrations of proinflammatory cytokines and chemokines (IL-1beta, KC, IL-6, and MIP-1alpha) were greater in eyes of TNFalpha(-/-) mice than of B6.129F1 mice., Conclusions: TNFalpha is important to intraocular pathogen containment by PMNs during experimental B. cereus endophthalmitis. In the absence of TNFalpha, fewer PMNs migrated into the eye, facilitating faster bacterial replication and retinal function loss. Although greater concentrations of proinflammatory cytokines were synthesized in the absence of TNFalpha, the resultant inflammation was diminished, and an equally devastating course of infection occurred.