Optimized culture of retinal ganglion cells and amacrine cells from adult mice.

Cell culture is widely utilized to study the cellular and molecular biology of different neuronal cell populations. Current techniques to study enriched neurons in vitro are primarily limited to embryonic/neonatal animals and induced pluripotent stem cells (iPSCs). Although the use of these cultures is valuable, the accessibility of purified primary adult neuronal cultures would allow for improved assessment of certain neurological diseases and pathways at the cellular level. Using a modified 7-step immunopanning technique to isolate for retinal ganglion cells (RGCs) and amacrine cells (ACs) from adult mouse retinas, we have successfully developed a model of neuronal culture that maintains for at least one week. Isolations of Thy1.2+ cells are enriched for RGCs, with the isolation cell yield being congruent to the theoretical yield of RGCs in a mouse retina. ACs of two different populations (CD15+ and CD57+) can also be isolated. The populations of these three adult neurons in culture are healthy, with neurite outgrowths in some cases greater than 500µm in length. Optimization of culture conditions for RGCs and CD15+ cells revealed that neuronal survival and the likelihood of neurite outgrowth respond inversely to different culture media. Serially diluted concentrations of puromycin decreased cultured adult RGCs in a dose-dependent manner, demonstrating the potential usefulness of these adult neuronal cultures in screening assays. This novel culture system can be used to model in vivo neuronal behaviors. Studies can now be expanded in conjunction with other methodologies to study the neurobiology of function, aging, and diseases.

A Preclinical Safety Study of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells for Macular Degeneration.

Purpose: Age-related macular degeneration (AMD) is a common disease trending towards epidemic proportions and is a leading cause of irreversible vision loss in people over the age of 65. A pathomechanism of AMD is death and/or dysfunction of retinal pigment epithelial (RPE) cells; RPE loss invariably results in photoreceptor atrophy. Treatment options for AMD are very limited, and include vitamin supplements and lifestyle changes. An exciting potential therapy currently being tested in clinical trials is transplantation of stem cell-derived RPE. Methods: We developed a NIH-registered embryonic stem line (CR-4), and in this study set out to determine if CR4-RPE are tolerated in normal mice and in murine models of retinal degeneration by injecting a bolus of CR4-RPE cells in the subretinal space of immunosuppressed wild-type, Mer mutant (Merkd), and Elovl4 deficient mice. Results: Mice with CR-RPE grafts were monitored daily, were examined routinely using OCT, and histology was prepared and examined at terminal end-points. Based on the parameters of the study, none of the animals with CR-RPE grafts (n=36) experienced any obvious adverse reactions. Conclusions: We conclude that transplanted CR-4 hES-derived RPE cells are well tolerated in immunosuppressed healthy and dystrophic murine retinas.

Transcriptomic profiles of retinal ganglion cells are defined by the magnitude of intraocular pressure elevation in adult mice.

Elevated intraocular pressure (IOP) is the major risk factor for glaucoma, a sight threatening disease of retinal ganglion cells (RGCs) and their axons. Despite the central importance of IOP, details of the impact of IOP elevation on RGC gene expression remain elusive. We developed a 4-step immunopanning protocol to extract adult mouse RGCs with high fidelity and used it to isolate RGCs from wild type mice exposed to 2 weeks of IOP elevation generated by the microbead model. IOP was elevated to 2 distinct levels which were defined as Mild (IOP increase >1 mmHg and <4 mmHg) and Moderate (IOP increase ≥4 mmHg). RNA sequencing was used to compare the transcriptional environment at each IOP level. Differentially expressed genes were markedly different between the 2 groups, and pathway analysis revealed frequently opposed responses between the IOP levels. These results suggest that the magnitude of IOP elevation has a critical impact on RGC transcriptional changes. Furthermore, it is possible that IOP-based set points exist within RGCs to impact the direction of transcriptional change. It is possible that this improved understanding of changes in RGC gene expression can ultimately lead to novel diagnostics and therapeutics for glaucoma.

Suppression of Cocaine-Evoked Hyperactivity by Self-Adjuvanting and Multivalent Peptide Nanofiber Vaccines.

The development of anti-cocaine vaccines that counteract the rewarding effects of the drug are currently being investigated as adjunct therapies for prevention of relapse in abstinent users. However, cocaine is weakly immunogenic and requires conjugation to carrier proteins and coadministration with strong adjuvants, which carry the risk of local reactogenicity and systemic toxicity. Here we report synthetic and multivalent self-assembling peptide nanofibers as adjuvant-free carriers for cocaine vaccines. A novel cocaine hapten modified at the P3 site was conjugated to the N-terminus of an amphipathic self-assembling domain KFE8. In aqueous buffers the cocaine-KFE8 conjugate assembled into ß-sheet rich nanofibers, which raised anti-cocaine antibodies without the need for added adjuvants in mice. Vaccinated mice were treated with cocaine and a significant negative correlation was observed between antibody levels and cocaine-evoked hyperactivity. These totally synthetic and multivalent nanofibers with well-defined chemical composition represent the first generation of adjuvant-free cocaine vaccines.

Peptide nanofiber-CaCO3 composite microparticles as adjuvant-free oral vaccine delivery vehicles.

To combat mucosal pathogens that cause gastrointestinal (GI) infections, local mucosal immunity is required which is best achieved through oral vaccination. Oral delivery of vaccines is also a safe and convenient alternative to injected vaccines due to its non-invasive nature and high compliance rate for all ages. However, the lack of effective and safe mucosal adjuvants, the selective permeability of the mucus barrier, and the harsh GI environment continue to pose a significant challenge for oral vaccine development. Microparticle-based strategies are attractive for oral vaccination due to their ability to efficiently penetrate the mucus barrier and have the added advantage of protecting the antigen in the harsh gastric environment. In this work, self-adjuvanting peptide nanofiber-CaCO3 composite microparticles were prepared and investigated for oral vaccine delivery. Compared to polymeric microparticles, inorganic CaCO3 microparticles have unique advantages due to the biocompatibility of CaCO3 as a natural mineral, mild preparation conditions, and its porous structure that is suitable for loading other materials. Particle size distribution, nanofiber loading efficiency, morphology, and degradation in simulated gastric fluid were characterized. The composite microparticles were efficient at penetrating the mucus barrier and were localized to immune inductive sites and elicited the production of mucosal antibody responses, particularly the protective IgA isotype following oral administration. The magnitude of the mucosal immune response was comparable to the gold-standard adjuvant cholera toxin B (CTB). Our results indicate that OVA-KFE8/CaCO3 composite microparticles are efficient self-adjuvanting oral vaccine delivery vehicles for induction of mucosal antibody responses.

Enhancing the Magnitude of Antibody Responses through Biomaterial Stereochemistry.

d-Amino acid analogs of peptides and proteins are attractive for applications in biotechnology and medicine due to their reduced proteolytic sensitivity. Here, we report that self-assembling peptide nanofibers composed of d-amino acids act as immune adjuvants, and investigate their ability to induce antibody responses in comparison to their l-amino acid counterparts. The model antigenic peptide OVA (chicken egg ovalbumin aa 323-339) from chicken egg ovalbumin, known to elicit antibody responses in mice, was linked to an l- or d-amino acid self-assembling peptide domain to generate enantiomeric nanofibers displaying the same epitope. The chiral nature of the fusion peptides was confirmed by circular dichrosim spectroscopy and transmission electron microscopy studies indicated that OVA-bearing enantiomers self-assembled into nanofibers with similar morphologies. In mice, d-amino acid peptide nanofibers displaying OVA elicited stronger antibody responses, equivalent levels of CD4+ T cell responses, and long-term antigen-presentation in vivo compared to l-amino acid nanofibers. Our findings indicate that self-assembling peptides composed of d-amino acids are strong immune adjuvants and that biomaterial stereochemistry can be used as a design tool to program adaptive immune responses for vaccine development.

NK cells and CD8+ T cells cooperate to improve therapeutic responses in melanoma treated with interleukin-2 (IL-2) and CTLA-4 blockade.

BACKGROUND: Melanoma is one of the few types of cancer with an increasing annual incidence. While a number of immunotherapies for melanoma have been associated with significant clinical benefit, including high-dose IL-2 and cytotoxic T lymphocyte antigen 4 (CTLA-4) blockade, clinical response to either of these single agents has been limited to 11-20% of treated patients. Therefore, in this study, we sought to test the hypothesis that the combination of IL-2 and CTLA-4 blockade could mediate a more profound therapeutic response. METHODS: Here, B6 mice were challenged with poorly immunogenic B16 melanoma on day 0, and treated with CTLA-4 blocking antibody (100 µg/mouse) on days 3, 6, and 9, and IL-2 (100,000 units) twice daily on days 4-8, or both. RESULTS: A highly significant synergistic effect that delayed tumor growth and prolonged survival was demonstrated with the combination immunotherapy compared to either monotherapy alone. The therapeutic effect of combination immunotherapy was dependent on both CD8+ T and NK cells and co-depletion of these subsets (but not either one alone) abrogated the therapeutic effect. CTLA-4 blockade increased immune cell infiltration (including CD8+ T cells and NK cells) in the tumor and IL-2 reduced the proportion of highly differentiated/exhausted tumor-infiltrating NK cells. CONCLUSIONS: These results have implications for the design of clinical trials in patients with metastatic melanoma and provide new insights into how the immune system may be mediating anti-tumor activity with combination IL-2 and CTLA-4 blockade in melanoma.