Phosphoinositide 3-Kinase Binds to TRPV1 and Mediates NGF-stimulated TRPV1 Trafficking to the Plasma Membrane

Sensitization of the pain-transducing ion channel TRPV1 underlies thermal hyperalgesia by proalgesic agents such as nerve growth factor (NGF). The currently accepted model is that the NGF-mediated increase in TRPV1 function during hyperalgesia utilizes activation of phospholipase C (PLC) to cleave PIP2, proposed to tonically inhibit TRPV1. In this study, we tested the PLC model and found two lines of evidence that directly challenge its validity: (1) polylysine, a cationic phosphoinositide sequestering agent, inhibited TRPV1 instead of potentiating it, and (2) direct application of PIP2 to inside-out excised patches dramatically potentiated TRPV1. Furthermore, we show four types of experiments indicating that PI3K is physically and functionally coupled to TRPV1: (1) the p85β subunit of PI3K interacted with the N-terminal region of TRPV1 in yeast 2-hybrid experiments, (2) PI3K-p85β coimmunoprecipitated with TRPV1 from both HEK293 cells and dorsal root ganglia (DRG) neurons, (3) TRPV1 interacted with recombinant PI3K-p85 in vitro, and (4) wortmannin, a specific inhibitor of PI3K, completely abolished NGF-mediated sensitization in acutely dissociated DRG neurons. Finally, simultaneous electrophysiological and total internal reflection fluorescence (TIRF) microscopy recordings demonstrate that NGF increased the number of channels in the plasma membrane. We propose a new model for NGF-mediated hyperalgesia in which physical coupling of TRPV1 and PI3K in a signal transduction complex facilitates trafficking of TRPV1 to the plasma membrane

Cytomegalovirus Retinitis After Intravitreous Triamcinolone Injection in a Patient with Central Retinal Vein Occlusion

To report a case of cytomegalovirus (CMV) retinitis after intravitreal injection of triamcinolone acetonide (IVTA). A 77-year-old woman with macular edema due to central retinal vein occlusion (CRVO) developed peripheral retinitis 4 months after IVTA. A diagnostic anterior chamber paracentesis was performed to obtain DNA for a polymerase chain reaction (PCR) test for viral retinitis. The PCR test was positive for CMV DNA. Other tests for infective uveitis and immune competence were negative. Four months after presentation, gancyclovir was intravitreously injected a total of 5 times, and the retinitis resolved completely. CMV retinitis is a rare complication of local immunosuppression with IVTA. It can be managed with timely injection of intravitreal gancyclovir until recovery from local immunosuppression

Atypical Central Serous Chorioretinopathy Masquerading as Vogt-Koyanagi-Harada Disease: A Case Report

Introduction: We aimed to describe a clinical presentation of central serous retinopathy that poses a diagnostic and management dilemma. Case Presentation: A 30-year-old male patient presented with bilateral vision loss and multifocal serous retinal detachments involving the posterior pole of both eyes. Optical coherence tomography revealed prominent bilateral bacillary layer detachments. The patient complained of recent headaches and tinnitus. However, the clinical exam did not reveal overt inflammation and the patient admitted to being under significant stress. The clinical presentation raised concerns for both central serous retinopathy (CSR) and Vogt-Koyanagi-Harada (VKH). Additional findings, including white fundus spots and focal areas of retinal vascular leakage, were seen in our patient. We highlight these because, while they have been described in CSR, they are not commonly discussed and could add to the diagnostic dilemma. After a conservative approach that avoided steroids, our patient showed marked improvement over the following month, supporting a diagnosis of CSR. Conclusion: CSR can mimic VKH disease. A high level of suspicion is needed to avoid instituting steroid therapy that could induce a severe iatrogenic exacerbation of the disease

Presumed Necrotizing Viral Retinitis after Intravitreal Triamcinolone Injection: Case Report

A 56-year-old man presented with anterior chamber inflammation, increased intraocular pressure, peripheral retinal infiltration, and generalized retinal arterial obstruction suggesting acute retinal necrosis five months after intravitreal triamcinolone acetonide injection (IVTA). He was treated with intravenous antiviral agents and aspirin. Shortly after treatment, retinal infiltrations were resolved, and partial recanalization of the obstructed vessel was observed. Viral retinitis may occur as an opportunistic infection following IVTA due to the local immune modulatory effect of the steroid; hence, close observation following IVTA is necessary

Spontaneous Resolution of Intravitreal Steroid-Induced Bilateral Cytomegalovirus Retinitis

A 73-year-old woman underwent vitrectomy and intravitreal triamcinolone acetonide (IVTA) of the right eye and cataract surgery with IVTA of the left eye, for bilateral diabetic macular edema. The patient presented with visual loss in both eyes three-months postoperatively. The fundoscopic examination revealed white-yellow, necrotic peripheral lesions in the superotemporal quadrant of both eyes. Although bilateral acute retinal necrosis was suspected, azotemia resulting from diabetic nephropathy limited the use of acyclovir. Antiviral treatment was not started. A sample of the aqueous humor for polymerase chain reaction (PCR) analysis was obtained. One week later, the PCR results indicated the presence of cytomegalovirus (CMV). Since the retinal lesions did not progress and did not threaten the macula, the patient was followed without treatment for CMV. The retinal lesions progressively regressed and completely resolved in both eyes by six months of follow-up. Patients with IVTA-induced CMV retinitis may not require systemic treatment with ganciclovir

Novel non-contact retina camera for the rat and its application to dynamic retinal vessel analysis

We present a novel non-invasive and non-contact system for reflex-free retinal imaging and dynamic retinal vessel analysis in the rat. Theoretical analysis was performed prior to development of the new optical design, taking into account the optical properties of the rat eye and its specific illumination and imaging requirements. A novel optical model of the rat eye was developed for use with standard optical design software, facilitating both sequential and non-sequential modes. A retinal camera for the rat was constructed using standard optical and mechanical components. The addition of a customized illumination unit and existing standard software enabled dynamic vessel analysis. Seven-minute in-vivo vessel diameter recordings performed on 9 Brown-Norway rats showed stable readings. On average, the coefficient of variation was (1.1 ± 0.19) % for the arteries and (0.6 ± 0.08) % for the veins. The slope of the linear regression analysis was (0.56 ± 0.26) % for the arteries and (0.15 ± 0.27) % for the veins. In conclusion, the device can be used in basic studies of retinal vessel behavior

Design and Evaluation of Meningococcal Vaccines through Structure-Based Modification of Host and Pathogen Molecules

Neisseria meningitis remains a leading cause of sepsis and meningitis, and vaccines are required to prevent infections by this important human pathogen. Factor H binding protein (fHbp) is a key antigen that elicits protective immunity against the meningococcus and recruits the host complement regulator, fH. As the high affinity interaction between fHbp and fH could impair immune responses, we sought to identify non-functional fHbps that could act as effective immunogens. This was achieved by alanine substitution of fHbps from all three variant groups (V1, V2 and V3 fHbp) of the protein; while some residues affected fH binding in each variant group, the distribution of key amino underlying the interaction with fH differed between the V1, V2 and V3 proteins. The atomic structure of V3 fHbp in complex with fH and of the C-terminal barrel of V2 fHbp provide explanations to the differences in the precise nature of their interactions with fH, and the instability of the V2 protein. To develop transgenic models to assess the efficacy of non-functional fHbps, we determined the structural basis of the low level of interaction between fHbp and murine fH; in addition to changes in amino acids in the fHbp binding site, murine fH has a distinct conformation compared with the human protein that would sterically inhibit binding to fHbp. Non-functional V1 fHbps were further characterised by binding and structural studies, and shown in non-transgenic and transgenic mice (expressing chimeric fH that binds fHbp and precisely regulates complement system) to retain their immunogenicity. Our findings provide a catalogue of non-functional fHbps from all variant groups that can be included in new generation meningococcal vaccines, and establish proof-in-principle for clinical studies to compare their efficacy with wild-type fHbps

IFNγ production in peripheral blood of early Lyme disease patients to hLFAα(L) (aa326-345)

BACKGROUND: It has been proposed that outer surface protein A (OspA) of Borrelia burgdorferi sensu stricto contains a T helper 1 (Th1) cell epitope that could play a role in an autoimmune response to hLFA1. METHODS: We used two peptides, hLFAα(L) (aa326-345) and Borrelia burgdorferi OspAB31 (aa164-183), as stimulating antigens to measure Th1 proinflammatory IFNγ cytokine production in peripheral blood of Lyme disease patients presenting with EM without history of arthritis, as well as in peripheral blood of healthy individuals. RESULTS: IFNγ responses to hLFA1 peptide were observed in 11 of 19 Lyme disease patients and in 3 of 15 healthy controls. In contrast, only 2 of 19 of the Lyme disease patients and none of the controls responded to the homologous OspAB31 peptide. CONCLUSIONS: IFNγ was produced in response to stimulation with peptide hLFAα(L) (aa326-345) in peripheral blood of 58% of patients with early Lyme disease without signs of arthritis, as well as in peripheral blood of 20% of healthy individuals, but not in response to stimulation with the homologous OspAB31 (aa164-183) peptide (p < 0.05). Our results suggest that reactivity to the hLFA1 peptide in peripheral blood may be the result of T cell degeneracy