A rise in the frequency of lasR mutant Pseudomonas aeruginosa among keratitis isolates between 1993 and 2021.

Introduction: Pseudomonas aeruginosa causes vision threatening keratitis. The LasR transcription factor regulates virulence factors in response to the quorum sensing molecule N-3-oxo-dodecanoyl-L-homoserine lactone. P. aeruginosa isolates with lasR mutations are characterized by an iridescent high sheen phenotype caused by a build-up of 2-heptyl-4-quinolone. A previous study demonstrated 22% (n=101) of P. aeruginosa keratitis isolates from India between 2010 and 2016 were sheen positive lasR mutants, and the sheen phenotype correlated with worse clinical outcomes for patients. In this study, a longitudinal collection of P. aeruginosa keratitis isolates from Eastern North America were screened for lasR mutations by the sheen phenotype and sequencing of the lasR gene. Methods: Keratitis isolates (n=399) were classified by sheen phenotype. The lasR gene was cloned from a subset of isolates, sequenced, and tested for loss of function or dominant-negative status based on an azocasein protease assay. A retrospective chart review compared outcomes of keratitis patients infected by sheen positive and negative isolates. Results: A significant increase in sheen positive isolates was observed between 1993 and 2021. Extracellular protease activity was reduced among the sheen positive isolates and a defined lasR mutant. Cloned lasR alleles from the sheen positive isolates were loss of function or dominant negative and differed in sequence from previously reported ocular lasR mutant alleles. Retrospective analysis of patient information suggested significantly better visual outcomes for patients infected by sheen positive isolates. Discussion: These results indicate an increase in lasR mutations among keratitis isolates in the United States and suggest that endemic lasR mutants can cause keratitis.

Rise in frequency of lasR mutant Pseudomonas aeruginosa among keratitis isolates between 1993 and 2021.

Pseudomonas aeruginosa causes severe vision threatening keratitis. LasR is a transcription factor that regulates virulence associated genes in response to the quorum sensing molecule N-3-oxo-dodecanoyl-L-homoserine lactone. P. aeruginosa isolates with lasR mutations are characterized by an iridescent high sheen phenotype caused by a build-up of 2-heptyl-4-quinolone. A previous study indicated a high proportion (22 out of 101) of P. aeruginosa keratitis isolates from India between 2010 and 2016 were sheen positive and had mutations in the lasR gene, and the sheen phenotype correlated with worse clinical outcomes for patients. In this study, a longitudinal collection of P. aeruginosa keratitis isolates from Eastern North America were screened for lasR mutations by the sheen phenotype and sequencing of the lasR gene. A significant increase in the frequency of isolates with the sheen positive phenotype was observed in isolates between 1993 and 2021. Extracellular protease activity was lower among the sheen positive isolates and a defined lasR mutant. Cloned lasR alleles from the sheen positive isolates were loss of function or dominant negative and differed in sequence from previously reported ocular lasR mutant alleles. Insertion elements were present in a subset of independent isolates and may represent an endemic source from some of the isolates. Retrospective analysis of patient information suggested significantly better visual outcomes for patients with infected by sheen positive isolates. Together, these results indicate an increasing trend towards lasR mutations among keratitis isolates at a tertiary eye care hospital in the United States.

Rate of fractional change in corneal tomography parameters in keratoconus using a combination of predictive parameters.

BACKGROUND: To compare the rate of fractional change for multiple corneal tomographic factors in progressive keratoconus (KC). METHODS: In this retrospective case series, 40 eyes (40 patients) with progressive KC (increase in central keratometry of 1.00 D or maximum keratometry of 1.50 D on two visits at least six months apart) were included. Cases with previous history of ocular surgery, poor scans, corneal scars, severe dry eyes, post-excimer ectasia, pellucid marginal degeneration were excluded. Medical records, corneal tomography and anterior corneal wavefront (8 mm) (Scheimpflug tomography, Pentacam, Oculus, Germany) were analyzed. Rate of fractional change (Rx = (x1 - x0)/(|x0|tm)); where, x1 = value at follow-up, x0 = value at initial visit and tm = time in months, was measured. RESULTS: The mean age of the patients was 30.0 ± 8.4 years. The mean follow-up duration was 8.9 ± 4.2 months. Coma (0.076 ± 1.4) had the largest rate of fractional change (P = 1.7 × 10-14, Kruskal-Wallis test). The rate of fractional change was higher for aberrometric parameters (anterior corneal higher-order aberrations root mean square and anterior coma) compared to pachymetric and keratometric parameters (P values ranging from 1.4 × 10-4 to 7.4 × 10-10, Mann-Whitney test, effect size ranging from 0.4-0.7). The rate of fractional change was comparable between pachymetric and keratometric factors (P > 0.05 for all comparisons, Mann-Whitney test). CONCLUSIONS: Anterior corneal wavefront, especially anterior coma, were noted to have higher rate of fractional change compared to single point keratometric and pachymetric indices in progressive KC. This information can be used for decision-making when monitoring patients with KC.

Double Trouble: An Unusual Keratitis Case of Herpes Simplex Virus and Varicella-Zoster Virus Co-infection.

PURPOSE: The aim of this study was to describe a case of herpes simplex virus (HSV) and varicella-zoster virus (VZV) corneal co-infection in a patient with systemic immunosuppression. METHODS: A 77-year-old White man who was recently administered pembrolizumab present with reduction in visual acuity in his left eye from 20/25 to 20/50. There was a known history of ocular HSV keratitis. Slit-lamp examination showed superficial dendritic lesions suggestive of VZV. RESULTS: Viral polymerase chain reaction testing was positive for both HSV and VZV, confirming clinical diagnosis of VZV keratitis in the setting of recurrent HSV keratitis. The infection responded to treatment with topical trifluridine. Two months later, he had another episode of keratitis based on his symptoms reported through telephone encounter which resolved with trifluridine. Unfortunately, the patient committed suicide 4 months after onset. CONCLUSIONS: This is the first case of keratitis with HSV and VZV co-infection likely related to systemic immunosuppression. Clinicians should have a high suspicion for viral co-infections in the setting of systemic immunosuppression.

Bilateral, chronic, bacterial conjunctivitis in giant fornix syndrome.

Giant fornix syndrome (GFS) results in chronic, relapsing conjunctivitis in elderly patients with enophthalmos and enlarged fornices, in which infectious material collects and perpetuates inflammation. A 98-year-old woman presented with persistent, bilateral, purulent conjunctivitis; corneal epithelial defects and progressive blepharospasm that did not respond to artificial tears, topical antibiotics and steroids and amniotic membrane grafts. Additional findings of deep-set orbits with enlarged upper fornices were diagnostic of GFS. Over the next 2 months, she responded to a combination of topical and systemic antibiotics, autologous serum eye drops, povidone-iodine forniceal rinses, and hypochlorous acid treatment of the eyelashes. GFS is an important diagnostic consideration in elderly patients with chronic conjunctivitis and deep-set orbits.

The Antibacterial Comparison of 5% and 2.5% Povidone Iodine to 0.01% Hypochlorous Acid Using Corneoscleral Tissue as a Solid-Phase Medium.

PURPOSE: Prophylactic topical antiseptics used to eliminate bacteria on the ocular surface prior to ocular surgery should be both effective and non-irritating. Five percent povidone iodine (PI) is an accepted antiseptic used for prophylaxis. Dilute 2.5% PI and 0.01% hypochlorous acid (HOCl) may be more patient comfortable and equally effective. PI at 5% and 2.5% were compared to HOCl against a battery of bacterial endophthalmitis isolates using corneoscleral tissue as a solid-phase medium to determine antiseptic efficacy. METHODS: Bacteria from 20 cases of endophthalmitis were tested for the elimination of growth against topical 5% PI, 2.5% PI, HOCl, and no antiseptic using donor corneoscleral tissue. The tissue was inoculated with 103 colony forming units of bacteria prior to a 3-minute contact time with the antiseptics, placed in liquid growth medium, and monitored for growth at three days. No growth indicated antiseptic treatment success. Differences were analyzed using Chi square (χ2). RESULTS: For 20 isolates, 5% PI was comparable to 2.5% PI for preventing bacteria growth (p=0.71), and both were more effective than HOCl (p=0.004). Estimated weighted comparison over a 27-year period indicated that for all bacterial groups, except Streptococcus viridans, 5% PI was equally effective to 2.5% PI for preventing bacterial growth (p=1.0). For Streptococcus viridans, 5% PI was more effective than 2.5% PI (p=0.0001). Both concentrations of PI were more effective than HOCl (p=0.00001). CONCLUSION: Five percent PI appears to be optimal as a prophylaxis prior to ocular surgery.

Comparison of Clinical Features and Treatment Outcomes of Pseudomonas aeruginosa Keratitis in Contact Lens and Non-Contact Lens Wearers.

PURPOSE: To compare the outcomes of Pseudomonas aeruginosa keratitis (PAK) in contact lens wearers (CLWs) and non-contact lens wearers (non-CLWs) and identify risk factors for poor visual acuity (VA) outcomes in each group. DESIGN: Retrospective cohort study METHODS: Two hundred fourteen consecutive cases of PAK were included between January 2006 and December 2019. Clinical features, microbiologic results, and treatment course were compared between CLW and non-CLW groups. Analyses of clinical features predicting poor final VA were performed. RESULTS: This study identified 214 infected eyes in 207 patients with PAK, including 163 eyes (76.2%) in CLWs and 51 eyes (23.8%) in non-CLWs. The average age was 39.2 years in CLWs and 71.9 years in non-CLWs (P < .0001). The average logMAR visual acuity (VA) at presentation was 1.39 in CLWs and 2.17 in non-CLWs (P < .0001); average final VA was 0.76 in CLWs and 1.82 in non-CLWs (P < .0001). Stromal necrosis required a procedural or surgical intervention in 13.5% of CLWs and 49.0% of non-CLWs (P < .0001). A machine learning-based analysis yielded a list of clinical features that most strongly predict a poor VA outcome (worse than 20/40), including worse initial VA, older age, larger size of infiltrate or epithelial defect at presentation, and greater maximal depth of stromal necrosis. CONCLUSIONS: Non-CLWs have significantly worse VA outcomes and required a higher rate of surgical intervention, compared with CLWs. Our study elucidates risk factors for poor visual outcomes in non-CLWs with PAK.

Methicillin-Resistant Staphylococcus aureus Keratitis: Initial Treatment, Risk Factors, Clinical Features, and Treatment Outcomes.

PURPOSE: To analyze the clinical characteristics, management choices, and outcomes of cases of methicillin-resistant Staphylococcus aureus (MRSA) keratitis. DESIGN: Retrospective interventional case series. METHODS: Fifty-two culture-proven (52 eyes) cases of MRSA keratitis diagnosed and treated at the University of Pittsburgh Medical Center were identified and reviewed. RESULTS: The mean age was 66.6 ± 19.2 years with a median follow-up time of 147 days. The most prevalent risk factors included a history of ocular surgery (62.5%), topical corticosteroid use (35.4%), and dry eye syndrome (37.5%). There was a high burden of systemic disease (95.8%). The average presenting logarithm of minimal angle of resolution visual acuity was 1.7 ± 0.8 and the average final logarithm of minimal angle of resolution visual acuity was 1.2 + 1.0. Initial antibiotic treatment varied, with 20.8% receiving moxifloxacin alone, 20.8% receiving fortified cefazolin and fortified tobramycin together, and 12.5% receiving fortified vancomycin and fortified tobramycin, although other antibiotics were used during treatment if warranted. Surgical management was often required as 17.3% of eyes perforated: 13.5% required tarsorrhaphy, 5.8% required penetrating keratoplasty, and 1 eye was enucleated. When patients treated with fourth-generation fluoroquinolones were compared with those treated with fortified vancomycin, no difference in final visual acuity, treatment duration, or need for surgery was found. CONCLUSION: MRSA causes fulminant keratitis often requiring surgical management with poor visual acuity outcomes. Poor ocular surface, topical corticosteroid use, previous ocular surgery, and/or a high burden of systemic disease were identified as common risk factors. Patients treated with fluoroquinolones in our study had comparable outcomes to those treated with fortified vancomycin; however, those treated with fortified vancomycin tended to have more severe ulcers at presentation.

The Prevalence of Bacteria, Fungi, Viruses, and Acanthamoeba From 3,004 Cases of Keratitis, Endophthalmitis, and Conjunctivitis.

PURPOSE: The definitive identification of ocular pathogens optimizes effective treatment. Although the types of ocular pathogens are known; there is less definitive information on the prevalence of causative infections including viruses, fungi, and protozoa, which is the focus of this retrospective laboratory review. METHODS: Data used for laboratory certification were reviewed for the detection of bacteria, viruses, fungi, and protozoa, from patients with infectious keratitis, endophthalmitis, and conjunctivitis. The main outcome parameter was laboratory-positive ocular infection. RESULTS: The distribution of infectious agents for keratitis (n=1,387) (2004-2018) was bacteria 72.1% (Staphylococcus aureus 20.3%, Pseudomonas aeruginosa 18%, Streptococcus spp. 8.5%, other gram-positives 12.4%, and other gram-negatives 12.9%), Herpes simplex virus 16%, fungi 6.7%, and Acanthamoeba 5.2%. For endophthalmitis, (n=770) (1993-2018), the bacterial distribution was coagulase-negative Staphylococcus 54%, Streptococcus spp. 21%, S. aureus 10%, other gram-positives 8%, and gram-negatives 7%. The distribution for conjunctivitis (n=847) (2004-2018) was Adenovirus 34%, S. aureus 25.5%, Streptococcus pneumoniae 9%, Haemophilus 9%, other gram-negatives 8.8%, other gram-positives 6%, coagulase-negative Staphylococcus 4.5% and Chlamydia 3.2%. CONCLUSION: An updated monitoring of ocular pathogens creates an awareness of the different infectious etiologies and the importance of laboratory studies. This information can determine treatment needs for infectious ocular diseases.

Topical Vancomycin 5% Is More Efficacious Than 2.5% and 1.25% for Reducing Viable Methicillin-Resistant Staphylococcus aureus in Infectious Keratitis.

PURPOSE: Topical vancomycin 5% (50 mg/mL) has been used for the treatment of methicillin-resistant Staphylococcus aureus (MRSA) keratitis, but patient comfort has many clinicians using lower concentrations. We compared the efficacy of different concentrations of vancomycin in the treatment of experimental MRSA keratitis. METHODS: The corneas of 45 rabbits were infected with 2000 colony-forming units (CFUs) of MRSA. Corneal epithelium was abraded in the left eyes to mimic corneal ulceration. After 4 hours, the corneal CFUs were determined at the onset of treatment. The remaining rabbits were divided into 4 treatment groups (n = 9): 1) vancomycin 5%, 2) vancomycin 2.5%, 3) vancomycin 1.25%, and 4) saline. The rabbits were treated topically in both eyes every 15 minutes for 5 hours. One hour after treatment, the rabbits were clinically examined and euthanized, corneas were removed, and CFUs were determined to analyze vancomycin penetration, treatment efficacy, and bactericidal effect. RESULTS: Ocular toxicity was concentration dependent from mild to moderate. For the abraded corneas, the CFUs of the vancomycin 5% group were lower than 2.5% and 1.25%, and all vancomycin groups were lower than saline. The CFUs of 2.5% were lower but similar to 1.25%. The vancomycin 5% group demonstrated a bactericidal effect and the best penetration. The CFUs of the abraded corneas treated with saline were lower than those of the intact corneas, indicating a possible antibacterial effect from the ocular surface. CONCLUSIONS: Vancomycin 5% was most potent for treating experimental MRSA keratitis. The clinician may need to reassess treatment regarding antibacterial efficacy and patient comfort.

Postsurgical Cataract Prophylaxis With Intravitreal "Triamcinolone-Moxifloxacin" May Not Be Optimal For Preventing Endophthalmitis.

PURPOSE: Intravitreal injections of antibiotics and anti-inflammatories are used by some cataract surgeons for surgical prophylaxis. To support this prophylaxis, intravitreal triamcinolone-moxifloxacin (TM) and triamcinolone-moxifloxacin-vancomycin (TMV) were tested for preventing Staphylococcus aureus (SA) endophthalmitis in rabbits. METHODS: Trademark formulations of TM (15/1 mg/mL) and TMV (15/1/10 mg/mL) were intravitreally injected into seven groups of eight rabbits each (A-G). Before intravitreal injection, the vitreous was first challenged with clinical SA endophthalmitis isolates (5,000 colony-forming unit) with varying minimum inhibitory concentrations (MICs in µg/mL) to moxifloxacin (denoted by the MIC at the end of each group listed): A) TMV-10, B) TM-10, C) Saline-10, D) TM-2, E) Saline-2, F) TM-0.032, and G) Saline-0.032. After 24 hr, the rabbit eyes were graded for clinical endophthalmitis and cultured for viable SA. RESULTS: Rabbits treated with TMV and challenged by SA with a moxifloxacin MIC of 10 µg/mL did not present with endophthalmitis (0/8, no eyes with endophthalmitis). For SA with moxifloxacin MICs of 10.0 and 2.0 µg/mL, TM did not prevent endophthalmitis (16/16, 100% of eyes with endophthalmitis). For SA with a moxifloxacin MIC of 0.032 µg/mL, endophthalmitis was prevented with TM (0/8, no eyes with endophthalmitis). All saline-treated eyes developed endophthalmitis (23/23, 100% of eyes with endophthalmitis). CONCLUSIONS: Intravitreal monotherapy with TM did not provide consistent prevention of SA endophthalmitis, whereas intravitreal TMV successfully prevented endophthalmitis because of SA with elevated MIC values to moxifloxacin. Cataract surgeons need to be aware that vancomycin seems to be essential for intravitreal prophylaxis to cover moxifloxacin resistance.

AmpliVue Is a Practical and Timely Test for the Detection of HSV From Keratitis Specimens.

OBJECTIVE: The laboratory diagnostic detection of herpes simplex virus (HSV) from eye samples must be practical, timely, and definitive for appropriate therapy. Although polymerase chain reaction (PCR) and/or cell culture can be definitive, HSV results can be delayed. Enzyme Linked Virus Inducible System (ELVIS) is a test that can provide results within 24 to 48 hr. We evaluated "AmpliVue HSV 1+2 Assay" as a molecular colorimetric test that can detect HSV (1 or 2) DNA within 1 hr. METHODS: Cornea/conjunctival samples were tested retrospectively with AmpliVue against 53 true-positive and 20 true-negative specimens collected in chlamydial transport medium. All clinical specimens were tested by cell culture isolation, PCR, and ELVIS for routine patient care. RESULTS: The sensitivity of AmpliVue against ocular samples that were both culture-positive and PCR-positive was 84%. The specificity of AmpliVue was 100%. Only one clinical sample was HSV-2 positive, whereas all others tested positive for HSV-1. Based on PCR-positive and cell culture-negative samples, AmpliVue (11 of 17) tested more positive than ELVIS (0 of 17) (P=0.003, Fisher Exact). CONCLUSIONS: AmpliVue is moderately sensitive and highly specific as a practical and timely diagnostic test for detecting ocular HSV. Expertise is readily achieved and the test is straightforward with easy interpretation. Negative AmpliVue testing must be confirmed with PCR. AmpliVue has potential as an office-based diagnostic test.

Development of retrocorneal membrane following pig-to-monkey penetrating keratoplasty.

Recent reports of long-term survival after wild-type (WT) pig-to-monkey corneal xenotransplantation are encouraging. We experienced the rapid development of retrocorneal membranes, a rare complication after corneal allotransplantation (although seen in infants and young children). The original specific aim of the study was to determine the factors associated with successful (young) pig corneal transplantation in monkeys. However, when it was obvious that retrocorneal membranes rapidly developed, our aims became to determine the factors involved in its development after both WT and Genetically engineered (GE ) pig corneal xenotransplantation and to investigate the characteristics of the retrocorneal membrane. Rhesus monkeys were recipients of penetrating keratoplasty using WT and GE pigs (n=2, respectively, 1-3 months old). Local/systemic steroids were administered for 3 months. Grafts were evaluated by slit lamp for corneal transparency, edema, and neovascularization. Hematoxylin and eosin, Masson trichrome staining, and immunohistochemical analysis were performed. Gal staining was also carried out to distinguish the origin of the membrane. All penetrating keratoplasty recipients developed fibrous retrocorneal membranes in the early post-transplantation period, regardless of whether the graft was from a WT or GE pig. There were no features of rejection, with no cell infiltrate in the graft or anterior chamber during the three-month follow-up. There was no difference in the clinical course between the two groups (WT or GE corneas). Immunohistochemistry indicated that the retrocorneal membranes were CK negative, α-SMA positive, and vimentin positive, suggesting that they were of fibrous (keratocytic) origin. Also, the membrane was Gal positive, suggesting that it is derived from pig cornea. Following pig-to-monkey corneal xenotransplantation, we report that retrocorneal membranes are derived from donor pig keratocytes. Prevention of retrocorneal membranes will be necessary to achieve successful corneal xenotransplantation.

Endophthalmitis Prophylaxis Using a Single Drop of Thermoresponsive Controlled-Release Microspheres Loaded with Moxifloxacin in a Rabbit Model.

PURPOSE: Postsurgical endophthalmitis is a sight-threatening problem. We introduce a simple approach by using a single application of thermoresponsive controlled-release microspheres, loaded with moxifloxacin, to prevent bacterial endophthalmitis in a rabbit endophthalmitis prevention model. METHODS: We separated 24 rabbits into 3 treatment groups in which topical drop treatment was placed onto the conjunctival cul-de-sac: (1) a single drop of controlled-release microspheres containing moxifloxacin, (2) a single drop of controlled-release microspheres without moxifloxacin, and (3) multiple topical treatment with moxifloxacin alone every 15 minutes for 1 hour. All rabbits were challenged, 1 hour after microspheres drop placement and immediately after the fifth topical dose of moxifloxacin, with anterior chamber injections of Staphylococcus aureus. Rabbits in the topical moxifloxacin group also were treated after challenge and four additional times over the next 24 hours. After 24 hours, the rabbits were clinically evaluated for endophthalmitis and the animals were euthanized to culture for intraocular S. aureus. The treatment groups were compared statistically for bacterial endophthalmitis. RESULTS: No eyes had endophthalmitis, based on clinical presentation and/or positive culture, in the groups with controlled-release microspheres loaded with moxifloxacin (0/8, 0%) or multiple drops of topical moxifloxacin (0/8, 0%). In contrast, 8 of 8 eyes (100%; P = 0.0001), had endophthalmitis among eyes treated with controlled-release microspheres drops without moxifloxacin. CONCLUSION: A single drop of controlled-release microspheres loaded with moxifloxacin was successful in preventing endophthalmitis. Further clinical studies will be required to confirm the full potential of controlled-release anti-infective loaded microspheres to prevent endophthalmitis. TRANSLATIONAL RELEVANCE: This study presents a simple method of prophylaxis to prevent postsurgical endophthalmitis.

Comparison of Validated Polymerase Chain Reaction and Culture Isolation for the Routine Detection of Acanthamoeba From Ocular Samples.

PURPOSE: Acanthamoeba keratitis should be definitively diagnosed for appropriate therapy. Our institution has validated polymerase chain reaction (PCR) as a routine diagnostic test to detect Acanthamoeba DNA from ocular samples. We compared PCR with culture isolation for detecting Acanthamoeba from ocular samples. METHODS: The microbiology records of patients that had specimens submitted (May 2012 to January 2014) for laboratory testing for Acanthamoeba keratitis were reviewed for (1) Acanthamoeba culture isolation, (2) Acanthamoeba DNA detection by PCR, and (3) non-Acanthamoeba culture results. For Acanthamoeba isolation, corneal samples were planted on nonnutrient agar overlaid with Enterobacter aerogenes. Validated PCR (May 2012) for Acanthamoeba DNA was processed at the Division of Molecular Diagnostics, UPMC, Pittsburgh, PA. Additional cultures were obtained for bacteria, fungus, and virus (i.e., herpes simplex virus) using standard techniques. RESULTS: Culture isolation and PCR were processed on 125 patients with a differential diagnosis of Acanthamoeba keratitis. Of these, 104 (83.2%) were culture negative, PCR negative; 14 (11.2%) were culture positive, PCR positive; 4 (3.2%) were culture negative, PCR positive; and, 3 (2.4%) were culture positive, PCR negative. Culture and PCR were statistically equivalent for detecting Acanthamoeba from ocular samples (P=1.0, McNemar's test). Nineteen of the culture-negative, PCR-negative corneal samples (18.3%) were positive for other pathogens such as bacteria, fungus, and virus. CONCLUSIONS: There is no clear advantage of PCR over culture isolation for detecting Acanthamoeba in ocular specimens. Other pathogens such as bacteria, fungus, and virus must still be considered in severe persistent keratitis. Polymerase chain reaction seems to be a complementary test for the clinical support of Acanthamoeba keratitis.

Phosphoinositide and Erk signaling pathways mediate activity-driven rodent olfactory sensory neuronal survival and stress mitigation.

Olfactory sensory neurons (OSNs) are the initial site for olfactory signal transduction. Therefore, their survival is essential to olfactory function. In the current study, we demonstrated that while odorant stimulation promoted rodent OSN survival, it induced generation of reactive oxygen species in a dose- and time-dependent manner as well as loss of membrane potential and fragmentation of mitochondria. The MEK-Erk pathway played a critical role in mediating these events, as its inhibition decreased odorant stimulation-dependent OSN survival and exacerbated intracellular stress measured by reactive oxygen species generation and heat-shock protein 70 expression. The phosphoinositide pathway, rather than the cyclic AMP pathway, mediated the odorant-induced activation of the MEK-Erk pathway. These findings provide important insights into the mechanisms of activity-driven OSN survival, the role of the phosphoinositide pathway in odorant signaling, and demonstrate that odorant detection and odorant stimulation-mediated survival proceed via independent signaling pathways. This mechanism, which permits independent regulation of odorant detection from survival signaling, may be advantageous if not diminished by repeated or prolonged odor exposure. We investigated the role of odorant stimulation in generating cellular stress and the molecular mechanisms mitigating such stress and promoting neuronal survival. Odorant stimulation promoted olfactory sensory neuron (OSN) survival and also induced intracellular oxidative stress, which was exacerbated when MEK/Erks pathway was inhibited. Sensory stimulation simultaneously activated at least two parallel pathways, the AC/cAMP cascade responsible for odorant detection, and phosphoinositide hydrolysis to promote odorant stimulation-dependent neuronal survival odorants may activate parallel signaling cascades to mediate sensory detection and sensory stimulation-dependent survival. AC, adenylyl cyclase; cAMP, cyclic adenosine monophosphate; Erk, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase.

Corneal blindness and xenotransplantation.

Approximately 39 million people are blind worldwide, with an estimated 285 million visually impaired. The developing world shoulders 90% of the world's blindness, with 80% of causative diseases being preventable or treatable. Blindness has a major detrimental impact on the patient, community, and healthcare spending. Corneal diseases are significant causes of blindness, affecting at least 4 million people worldwide. The prevalence of corneal disease varies between parts of the world. Trachoma, for instance, is the second leading cause of blindness in Africa, after cataracts, but is rarely found today in developed nations. When preventive strategies have failed, corneal transplantation is the most effective treatment for advanced corneal disease. The major surgical techniques for corneal transplantation include penetrating keratoplasty (PK), anterior lamellar keratoplasty, and endothelial keratoplasty (EK). Indications for corneal transplantation vary between countries, with Fuchs' dystrophy being the leading indication in the USA and keratoconus in Australia. With the exception of the USA, where EK will soon overtake PK as the most common surgical procedure, PK is the overwhelming procedure of choice. Success using corneal grafts in developing nations, such as Nepal, demonstrates the feasibility of corneal transplantation on a global scale. The number of suitable corneas from deceased human donors that becomes available will never be sufficient, and so research into various alternatives, for example stem cells, amniotic membrane transplantation, synthetic and biosynthetic corneas, and xenotransplantation, is progressing. While each of these has potential, we suggest that xenotransplantation holds the greatest potential for a corneal replacement. With the increasing availability of genetically engineered pigs, pig corneas may alleviate the global shortage of corneas in the near future.

The vomeronasal organ and adjacent glands express components of signaling cascades found in sensory neurons in the main olfactory system.

The vomeronasal organ (VNO) is a sensory organ that influences social and/or reproductive behavior and, in many cases, the survival of an organism. The VNO is believed to mediate responses to pheromones; however, many mechanisms of signal transduction in the VNO remain elusive. Here, we examined the expression of proteins involved in signal transduction that are found in the main olfactory system in the VNO. The localization of many signaling molecules in the VNO is quite different from those in the main olfactory system, suggesting differences in signal transduction mechanisms between these two chemosensory organs. Various signaling molecules are expressed in distinct areas of VNO sensory epithelium. Interestingly, we found the expressions of groups of these signaling molecules in glandular tissues adjacent to VNO, supporting the physiological significance of these glandular tissues. Our finding of high expression of signaling proteins in glandular tissues suggests that neurohumoral factors influence glandular tissues to modulate signaling cascades that in turn alter the responses of the VNO to hormonal status.

Hippocalcin in the olfactory epithelium: a mediator of second messenger signaling.

Intracellular Ca2+ plays an important role in a variety of second messenger cascades. The function of Ca2+ is mediated, in part, by Ca2+-binding proteins such as calmodulin, calretinin, calbindin, neurocalcin, recoverin, and visinin-like proteins (VILIPs). These proteins are highly expressed in rat olfactory receptor neurons (ORNs) and are localized to distinct intracellular regions. In the present study, we have identified another Ca2+-binding protein, hippocalcin, in the rat olfactory epithelium (OE). Olfactory/brain hippocalcin shows high sequence homology with hippocalcins expressed in mice and humans. Hippocalcin was predominantly localized to the olfactory cilia, the site of the initial events of olfactory signal transduction, and was found to regulate the activity of ciliary adenylate cyclases (ACs) and particulate guanylyl cyclases (GCs) in a Ca2+-dependent manner. These data indicate that hippocalcin is expressed in rat ORNs, and is likely to regulate second messenger cascades in a Ca2+-dependent manner.