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Inherited retinal diseases (IRDs) are a leading cause of blindness in industrialized countries, and gene therapy is quickly becoming a viable option to treat this group of diseases. Gene replacement using a viral vector has been successfully applied and advanced to commercial use for a rare group of diseases. This, and the advances in gene editing, are paving the way for the emergence of a new generation of therapies that use CRISPR–Cas9 to edit mutated genes in situ. These CRISPR-based agents can be delivered to the retina as transgenes in a viral vector, unpackaged transgenes or as proteins or messenger RNA using non-viral vectors. Although the eye is considered to be an immune-privileged organ, studies in animals, as well as evidence from clinics, have concluded that ocular gene therapies elicit an immune response that can under certain circumstances result in inflammation. In this review, we evaluate studies that have reported on pre-existing immunity, and discuss both innate and adaptive immune responses with a specific focus on immune responses to gene editing, both with non-viral and viral delivery in the ocular space. Lastly, we discuss approaches to prevent and manage the immune responses to ensure safe and efficient gene editing in the retina. [ABSTRACT FROM AUTHOR]

Objective: von Frey Filament (vFF) is an aesthesiometer to measure paw withdrawal thresholds. Our aim was to validate the manually von Frey test technique for assessing neuropathic pain behavioral signs in a sciatic nerve ligation model. Materials and Methods: In this experimental study, peripheral neuropathic pain associated with sciatic nerve chronic ligation (SN-CL) was induced. Filaments used against posterior pad mid-plantar region using a simplified up-down method (SUDO). In addition to baseline withdrawal thresholds, the behavioral test was repeated after surgery thrice more with an interval of ten days. vFF (2 to 26 g) were used in ascending order for hyperalgesia assessment. Results: In SN-CL rats, the results validate a loss of pain sensation, resulted in, long-lasting ipsilateral allodynia with the development of contralateral allodynia later and an extraterritorial development of neuropathic signs. Variability for the development of ipsilateral and contralateral allodynia over time was noted in sham (SH) control rats. SN-CL group showed a contralateral hyperalgesia development just at the 16th-day after surgery with an absence of ipsilateral hyperalgesia development at the different days of paw withdrawal thresholds measurements. Conclusion: Manually vFF test technique was successfully used for assessing neuropathic pain behavioral signs in sciatic a nerve ligation model with the absence of ipsilateral hyperalgesia development. [ABSTRACT FROM AUTHOR]

To investigate which cytokines, chemokines and growth factors are involved in the immunopathogenesis of idiopathic uveitis, and whether cytokine profiles are associated with. Serum and aqueous humor (AH) samples of 75 patients with idiopathic uveitis were analyzed by multiplex immunoassay. Infectious controls consisted of 16 patients with ocular toxoplasmosis all confirmed by intraocular fluid analyses. Noninfectious controls consisted of 7 patients with Behçet disease related uveitis and 15 patients with sarcoidosis related uveitis. The control group consisted of AH and serum samples from 47 noninflammatory control patients with age-related cataract. In each sample, 27 immune mediators ± IL-21 and IL-23 were measured. In idiopathic uveitis, 13 of the 29 mediators, including most proinflammatory and vascular mediators such as IL-6, IL-8, IL-12, G-CSF, GM-CSF, MCP-1, IP-10, TNF-α and VEGF, were significantly elevated in the aqueous humor when compared to all controls. Moreover, IL-17, IP-10, and IL-21, were significantly elevated in the serum when compared to all controls. We clustered 4 subgroups of idiopathic uveitis using a statistical analysis of hierarchical unsupervised classification, characterized by the order of magnitude of concentrations of intraocular cytokines. The pathogenesis of idiopathic uveitis is characterized by the presence of predominantly proinflammatory cytokines and chemokines and vascular endothelial growth factor with high expression levels as compared to other causes of uveitis. There are indications for obvious Th-1/ IL21-Th17 pathways but also IL9-Th9 and increased IFN-γ-inducing cytokine (IL12) and IFN-γ-inducible CXC chemokine (IP-10). The combined data suggest that immune mediator expression is different among idiopathic uveitis. This study suggests various clusters among the idiopathic uveitis group rather than one specific uveitis entity. [ABSTRACT FROM AUTHOR]

Background. Accumulating evidence illustrates that peripheral nerve injuries ac-tivate glial components of the peripheral and central cellular circuitry which contributes to neuroinflammation and neuropathic pain. Epidemiological surveys suggest that for women the prevalence of pain increases with age with more frequent pain than men. Recently, several studies have found that pain generation, transmission and modulation are under hormonal regulation. To this end, 17beta estradiol was used to investigate the modulatory actions on nociceptive thresholds in neuroinflammation driven neuropathic pain conditions. Methods. Adult female Wistar rats were divided into four groups: Ovariectomised neuropathic untreated and neuropathic 17 beta estradiol treated groups. sham operated and intact control groups. Results. In treated rats, the results validate an anti-inflammatory action of 17 beta estradiol against chronic constriction injury (CCI) induced neuropathic pain by attenuat-ing complement C3 fractions and anti-neutrophil cytoplasmic auto-antibodies (ANCAs) intensity plasma level, an anti-nociceptive action by attenuating ipsilateral, con-trolateral allodynia and hyperalgesia developed in treated animals after chronic con-striction injury and at the last day of treatment by increasing the tail flick latency at the last day of treatment. Conclusions. We conclude that 17beta estradiol might exhibit beneficial effects on peripheral nerve injury consequences. [ABSTRACT FROM AUTHOR]

Today, there are >500 published studies and 40 clinical trials to treat retinal disorders using gene therapy. The great majority of them rely on the use of adeno-associated virus vectors (AAV) for therapeutic gene delivery. Thus far, AAVs have an excellent safety profile in the clinic. Nevertheless, it is known that AAV-mediated gene delivery leads to toxicity at higher input doses in experimental gene therapy. This study reveals the factors that contribute to retinal toxicity after subretinal administration of AAV vectors in wild-type mice. The study shows that alongside the input dose, the nature of the transgene and the cells mediating the expression determine the extent of toxicity. Importantly, the study shows that AAV vectors encoding green fluorescent protein (GFP) used as controls in experimental gene therapy are toxic at doses as low as 5 × 109 vg, confounding the observed therapeutic effect in gene therapy paradigms. Altogether, the data show the importance of reducing input doses while increasing transgene expression levels via the use of more efficient capsids and promoters in order to avoid side effects in AAV-mediated gene therapy. Furthermore, the toxicity observed with AAV-GFP vectors imply a reinterpretation of previous gene therapy studies where the therapeutic effect was measured in relation to this control. [ABSTRACT FROM AUTHOR]

CD4+CD25+Foxp3+ regulatory T (Treg) cell therapy is a promising approach for the treatment of autoimmune diseases. To be effective, Treg cells should be in an activated state in the target tissue. This can be achieved by systemic administration of Ag-specific Treg cells, which are difficult to produce in conditions that can be translated to the clinic. In this paper, we propose an alternative approach consisting of in situ injection of preactivated polyclonal Treg cells that would exert bystander suppression in the target tissue. We show that polyclonal Treg cells suppressed uveitis in mice as efficiently as Ag-specific Treg cells but only when preactivated and administered in the vitreous. Uveitis control was correlated with an increase of IL-10 and a decrease of reactive oxygen species produced by immune cell infiltrates in the eye. Thus, our results reveal a new mechanism of Treg cell-mediated suppression and a new Treg cell therapy approach. [ABSTRACT FROM AUTHOR]

Gene transfer vectors such as lentiviral vectors offer versatile possibilities to express transgenic antigens for vaccination purposes. However, viral vaccines leading to broad transduction and transgene expression in vivo, are undesirable. Therefore, strategies capable of directing gene transfer only to professional antigen-presenting cells would increase the specific activity and safety of genetic vaccines. A lentiviral vector pseudotype specific for murine major histocompatibilty complex class II (LV-MHCII) was recently developed and the present study aims to characterize the in vivo biodistribution profile and immunization potential of this vector in mice. Whereas the systemic administration of a vector pseudotyped with a ubiquitously-interacting envelope led to prominent detection of vector copies in the liver of animals, the injection of an equivalent amount of LV-MHCII resulted in a more specific biodistribution of vector and transgene. Copies of LV-MHCII were found only in secondary lymphoid organs, essentially in CD11c+ dendritic cells expressing the transgene whereas B cells were not efficiently targeted in vivo, contrary to expectations based on in vitro testing. Upon a single injection of LV-MHCII, naive mice mounted specific effector CD4 and CD8 T cell responses against the intracelllular transgene product with the generation of Th1 cytokines, development of in vivo cytotoxic activity and establishment of T cell immune memory. The targeting of dendritic cells by recombinant viral vaccines must therefore be assessed in vivo but this strategy is feasible, effective for immunization and cross-presentation and constitutes a potentially safe alternative to limit off-target gene expression in gene-based vaccination strategies with integrative vectors. [ABSTRACT FROM AUTHOR]

Coxsackie B viruses (CV–B) are usually transmitted via the fecal-oral route and the virus gains the central nervous system (CNS) via the bloodstream. Nevertheless, other routes of spread of the virus to the CNS cannot be excluded, including the neuronal route. Neuronal cells, as well as non-neuronal cells (fibroblasts), were isolated from mice and inoculated with CV-B4 in the absence and presence of neutralizing serum. In the absence of neutralizing serum, virus titers recorded in neuron cultures and rates of infected neurons were non-significantly different compared to those recorded in fibroblast cultures. Higher cell mortality was noted among neurons than fibroblasts. The addition of neutralizing serum to neurons did not reduce significantly virus titers or rates of infected cells and cell viability was not significantly augmented, while virus titers and rates of infected fibroblasts were significantly reduced and their viability was significantly enhanced as well. Our results demonstrate the ineffectiveness of neutralizing serum to prevent neurons infection with CV-B4 which suggests a trans-synaptic transmission of CV-B4 between neurons. • Coxsackievirus B4 infects and replicates in primary neurons. • Coxsackievirus B4 neutralizing serum does not preserve primary neurons from viral cytopathic effect. • Coxsackievirus B4 neutralizing serum does not enhance the viability of primary neurons. • Coxsackievirus B4 is transmitted between neurons via synapses. [ABSTRACT FROM AUTHOR]

Coxsackie B viruses (CV–B) are associated with several central nervous system (CNS) disorders. These viruses are predominantly transmitted by fecal-oral route but vertical transmission can also occur. This work attempted to study the immune response ensuing vertical transmission of CV-B to the brain, and its eventual implementation in the brain pathogenesis. To this end, pregnant Swiss albino mice were inoculated with CV-B4 E2 or with sterile medium for control animals. At different ages after birth, brains were collected and analyzed for virus infection, histopathological changes and immune response. Infectious particles were detected in offspring's brain which demonstrates vertical transmission of the virus. This infection is persistent since the long lasting detection of viral RNA in offspring's brain. Some pathological signs including meningitis, edema and accumulation of inflammatory cells within and surrounding the inflammatory areas were observed. Immunoflorescence staining unveiled the presence of T lymphocytes and microgliosis in the sites of lesion for a long period after birth. Multiplex cytokines measurement upon supernatants of in vitro mixed brain cells and extracted mononuclear cells from offspring's brain has demonstrated an elevated secretion of the pro-inflammatory cytokines TNFα, IL-6 and IFNα and the chemokines RANTES and MCP-1. Hence, vertical transmission of CV-B4 and its persistence within offspring's brain can lead to pathological features linked to increased and sustained immune response. • Coxsackievirus B4 infects and persists in the brain of mice following its vertical transmission. • This infection gives histopathological abnormalities in the brain of infected offspring. • Mixed brain cells and brain mononuclear cells showed an elevated secretion of pro-inflammatory cytokines. • Signs of inflammation including microgliosis and inflammatory cells infiltration detected in the pathological areas. [ABSTRACT FROM AUTHOR]