rAAV.sFlt-1 Gene Therapy Achieves Lasting Reversal of Retinal Neovascularization in the Absence of a Strong Immune Response to the Viral Vector

PURPOSE. To determine the efficacy of rAAV.sFlt-1-mediated gene therapy in a transgenic mouse model of retinal neovascularization (trVEGF029) and to assess whether rAAV.sFlt-1 administration generated any deleterious, long-lasting immune response that could affect efficacy. METHODS. trVEGF029 mice were injected subretinally with rAAV.sFlt-1 or phosphate-buffered saline. Fluorescein angiography and electroretinography were used to compare the extent of fluorescein leakage from retinal vessels and retinal function, respectively. A group of eyes was enucleated, and the retinal vasculature and morphology were studied by confocal and light microscopy. Cells were isolated from the posterior eyecups and spleens of a further group, and immune cell subset populations were investigated by flow cytometry. sFlt-1 protein levels in the eyes were evaluated by ELISA. RESULTS. After a single rAAV.sFlt-1 injection, sFlt-1 protein levels were upregulated, and there was a reduction in fluorescein leakage from the retinal vessels and an improvement in retinal function. Confocal microscopy of isolectin-IB4-labeled retinal wholemounts showed more normal-appearing capillary beds in rAAV.sFlt-1-injected than in PBS-injected trVEGF029 mouse eyes. Light microscopy demonstrated retinal morphology preservation, with fewer aberrant vessels invading the outer nuclear layer of rAAV.sFlt-1-injected eyes. Furthermore, the immune response to subretinal injection of rAAV.sFlt-1 was limited to a transient increase in CD45 ϩ leukocytes that disappeared by 4 weeks after injection. This transient increase was localized to the eye and did not affect long-term therapeutic efficacy. CONCLUSIONS. The data support the notion that rAAV.sFlt-1 gene therapy is safe and effective for the long-term inhibition of deleterious blood vessel growth in the eye. (Invest Ophthalmol Vis Sci. 2009;50:4279 -4287

Innate immunity defines the capacity of antiviral T cells to limit persistent infection

Effective immunity requires the coordinated activation of innate and adaptive immune responses. Natural killer (NK) cells are central innate immune effectors, but can also affect the generation of acquired immune responses to viruses and malignancies. How NK cells influence the efficacy of adaptive immunity, however, is poorly understood. Here, we show that NK cells negatively regulate the duration and effectiveness of virus-specific CD4+ and CD8+ T cell responses by limiting exposure of T cells to infected antigen-presenting cells. This impacts the quality of T cell responses and the ability to limit viral persistence. Our studies provide unexpected insights into novel interplays between innate and adaptive immune effectors, and define the critical requirements for efficient control of viral persistence

Ballet injuries: injury incidence and severity over 1 year.

STUDY DESIGN: Prospective, descriptive single-cohort study. OBJECTIVE: To assess the incidence and severity of injuries to a professional ballet company over 1 year. METHODS: Data for an elite-level ballet company of 52 professional dancers were collected by an in-house medical team using a time-loss injury definition. RESULTS: A total of 355 injuries were recorded, with an overall injury incidence of 4.4 injuries per 1000 hours (female, 4.1; male, 4.8; P>.05) and a mean of 6.8 injuries per dancer (female, 6.3; male, 7.3; P>.05). Mean injury severity was 7 days (female, 4; male, 9; P.05); mean severity of injury was 3 days for females and 9 days for males (P<.05). The percentage of traumatic injuries was 32% for females and 40% for males (P<.05); the corresponding severity was 6 and 10 days, respectively (P<.05). CONCLUSION: The relatively high number of injuries reported and the resulting loss of dance time support the need to introduce interventions to reduce the risk of injury in professional dancers

Spatiotemporally separated antigen uptake by alveolar dendritic cells and airway presentation to T cells in the lung

Asthma pathogenesis is focused around conducting airways. The reasons for this focus have been unclear because it has not been possible to track the sites and timing of antigen uptake or subsequent antigen presentation to effector T cells. In this study, we use two-photon microscopy of the lung parenchyma and note accumulation of CD11b(+) dendritic cells (DCs) around the airway after allergen challenge but very limited access of these airway-adjacent DCs to the contents of the airspace. In contrast, we observed prevalent transepithelial uptake of particulate antigens by alveolar DCs. These distinct sites are temporally linked, as early antigen uptake in alveoli gives rise to DC and antigen retention in the airway-adjacent region. Antigen-specific T cells also accumulate in the airway-adjacent region after allergen challenge and are activated by the accumulated DCs. Thus, we propose that later airway hyperreactivity results from selective retention of allergen-presenting DCs and antigen-specific T cells in airway-adjacent interaction zones, not from variation in the abilities of individual DCs to survey the lung

The regulation of peripheral T cell responses in TCR transgenic mice

Transgenic mice expressing a T cell receptor specific for cytochrome C in association with I-Ek were employed to study the mechanisms regulating the decision between tolerance and immunity. Tolerance and immunity to the same peptide antigen were . induced by using different routes of administration. Thus tolerance was induced by the intravenous route and immunity by the subcutaneous route. The results presented in this thesis provide a detailed map of the cellular events that occur during these two distinct responses. Intravenous immunisation induced activation of CD4+ cells expressing the transgenic TCR (CD4+Tg0L+ cells), resulting in CD69 expression within two hours, priming of T cell proliferation and Th1 cytokine production by 24 hours. Clonal expansion peaked 3- 4 days after immunisation. The number of CD4+Tg0t+ cells decreased rapidly after day four, such that only 50% of initial cell numbers remained 7—10 days after immunisation. Intravenous peptide also upregulated CD44 expression, increasing the proportion and number of CD4+Tg0t+CD44hi cells at the peak of the response, but having no net effect at the resolution of the response. When labelled CD4+Tg0t+ cells were adoptively transferred to syngeneic non-transgenic hosts, deletional tolerance to intravenous peptide was still seen, demonstrating that it was not an artefact of the high precursor frequency in TCR transgenic mice. Antigen re-challenge experiments demonstrated that CD4+Tg0t+ cells remaining at the resolution of the primary response to intravenous peptide could not respond as vigorously as naive cells either in vitro and in vivo. Interestingly, intravenous administration of intact cytochrome C also stimulated T cell activation, but failed to induce peripheral deletion, and resulted instead in a minor increase in the final proportion of CD4+Tg0t+CD44hi cells

CpG pretreatment enhances antiviral T-cell immunity against cytomegalovirus.

Major histocompatibility complex class I-restricted T-cell immunity is essential to control infection with cytomegalovirus (CMV), a clinically important virus that causes significant disease in immunocompromised individuals. Cross-presentation is considered the primary mode of antigen presentation to generate protective antiviral CD8(+) T-cell immunity. Herpesviruses, including CMV, encode numerous proteins that interfere with direct antigen presentation, leading to the paradigm that T-cell immunity to these pathogens necessitates cross-presentation. However, the antigen presentation requirements needed to generate a protective T-cell response to CMV remain unknown. Here, we show that a fully functional antiviral CD8(+) T-cell response can be generated in a system where cross-presentation is shut down by pretreatment with CpG. Notably, in this setting, CD8(+) T cells demonstrate accelerated control of infection, and organ pathology is limited. These data indicate that protective antiviral T-cell immunity to CMV is generated by direct presentation and can be enhanced by pretreatment with CpG