Adaptation processes that build CRISPR immunity: creative destruction, updated.

Prokaryotes can defend themselves against invading mobile genetic elements (MGEs) by acquiring immune memory against them. The memory is a DNA database located at specific chromosomal sites called CRISPRs (clustered regularly interspaced short palindromic repeats) that store fragments of MGE DNA. These are utilised to target and destroy returning MGEs, preventing re-infection. The effectiveness of CRISPR-based immune defence depends on 'adaptation' reactions that capture and integrate MGE DNA fragments into CRISPRs. This provides the means for immunity to be delivered against MGEs in 'interference' reactions. Adaptation and interference are catalysed by Cas (CRISPR-associated) proteins, aided by enzymes well known for other roles in cells. We survey the molecular biology of CRISPR adaptation, highlighting entirely new developments that may help us to understand how MGE DNA is captured. We focus on processes in Escherichia coli, punctuated with reference to other prokaryotes that illustrate how common requirements for adaptation, DNA capture and integration, can be achieved in different ways. We also comment on how CRISPR adaptation enzymes, and their antecedents, can be utilised for biotechnology.

The immune privileged retina mediates an alternative activation of J774A.1 cells.

PURPOSE: We have previously found that retinal pigment epithelial (RPE) cells suppressed endotoxin-stimulated macrophages; moreover, it induced expression of anti-inflammatory cytokines. We further assessed the possibility that the RPE is alternatively activating macrophages. METHODS: J774A.1 cells were stimulated with endotoxin and treated with the conditioned media (CM) of RPE, or neuroretinal eyecups from healthy mouse eyes. The supernatant was assayed for IL-1 beta, TNF-alpha, IL-6, IL-12(p70), and IL-10, and for nitric-oxide generation. The RPE conditioned media (RPE CM) was absorbed of known soluble factors to identify the factor that augments nitric-oxide generation. RESULTS: We found the RPE CM suppressed all cytokine production except IL-10, and augmented nitric-oxide generation. The augmented nitric-oxide levels were mediated by RPE derived alpha-melanocyte stimulating hormone (alpha-MSH). CONCLUSIONS: Healthy RPE not only suppresses inflammatory activity, it promotes an alternative activation of macrophages that can further promote immune privilege.

Acute retinal necrosis features, management, and outcomes.

OBJECTIVE: To determine the viral diagnosis and factors affecting the visual outcome of eyes with acute retinal necrosis. DESIGN: Nonrandomized, retrospective, interventional, noncomparative series. PARTICIPANTS: A cohort of 22 human immunodeficiency virus-negative patients with acute retinal necrosis (ARN). There were 17 unilateral and 5 bilateral cases. INTERVENTION: Diagnostic vitreous biopsy for polymerase chain reaction (PCR) viral DNA analysis, prophylactic barrier laser posterior to necrotic retina to try to prevent rhegmatogenous retinal detachment (RD), intravenous acyclovir in combination with oral, and vitrectomy for RD repair. MAIN OUTCOME MEASURES: Results of PCR viral DNA analysis, relationship between prophylactic barrier argon laser photocoagulation and occurrence of RD, and visual acuities at presentation and follow-up. RESULTS: Varicella-zoster virus (VZV) was detected in 66.7% (12/18) of eyes (66.7% of patients [10/15]) with vitreous biopsy and herpes simplex virus (HSV) in 22.2% (4/18) of eyes (20% of patients [3/15]). Epstein-Barr virus (EBV) was detected in 16.7% (3/18) of eyes (20% of patients [3/15]), and all the EBV-positive eyes were also positive for VZV. Polymerase chain reaction results were identical in both eyes of bilateral cases (5 patients) and were negative in 11.1% (2/18) of eyes (13.3% of patients [2/15]) biopsied. Systemic corticosteroid treatment given before ARN diagnosis did not appear to increase the risk of developing RD (P = 0.69). Rhegmatogenous RD occurred in 35.3% (6/17) of eyes given prophylactic argon laser treatment and in 80% (8/10) of eyes that could not be lasered prohylactically. Of RDs, 96.3% (13/14) occurred after the third week and up to 5 months from onset of symptoms. The VA after surgical repair of RD improved relative to the presentation acuity in 33.3% (4/12) of eyes. CONCLUSION: Varicella-zoster virus is the leading cause of ARN. We recommend the management of ARN to include prompt diagnosis; prophylactic argon laser retinopexy, preferably within the first 2 weeks to reduce risk of RD; systemic acyclovir; and corticosteroids to control the severe inflammation associated with ARN. Despite the guarded visual prognosis, RD repair may result in improved visual outcomes.

Long-term efficacy of mycophenolate mofetil in the control of severe intraocular inflammation.

PURPOSE: To assess whether the previously demonstrated short-term efficacy of the immunosuppressant mycophenolate mofetil (MMF; CellCept, Roche) is maintained in the long-term management of refractory uveitis. METHODS: The study was an open-label, non-comparative retrospective series of 14 patients with refractory uveitis and treated with MMF for a mean of >33 months. Mycophenolate mofetil was given at a dosage of 1 g (oral) twice daily. Indications included prednisolone reduction, additive agent with cyclosporin, or replacement therapy (azathioprine or methotrexate). The intraocular inflammatory response, side-effects, and toxicity were monitored. RESULTS: Intraocular inflammation remained under control in 10 patients, unchanged in three and deteriorated in one patient. Transient side-effects included tiredness, headache and dizziness (one patient each, lasting less than 2 weeks from the time of MMF introduction). Mycophenolate mofetil was stopped in one patient because of absence of prolonged clinical improvement. Vision improved in 25% (7 eyes), did not change in 50% (14 eyes), but was reduced in 25% (7 eyes). CONCLUSIONS: Mycophenolate mofetil is safe for long-term usage and is recommended for treatment of refractory panuveitis or posterior uveitis with uncontrolled inflammation despite high prednisolone maintenance dosage (>15 mg/day) or toxicity or lack of efficacy of other immuno-suppressive agents. However, MMF is less effective for refractory uveitis unresponsive to azathioprine.