<p><b>The central nervous system was traditionally considered an immune-privileged site, defined as being immunologically inactive. However, recent studies have elucidated that a number of immune cells traffic into and out of the brain in healthy humans to conduct routine immunosurveillance. A unique immunological interface, the choroid plexus, acts as a gatekeeper for the entry of these immune cells during homeostasis. Although the mechanisms are not well described, the choroid plexus also has the capacity to regulate the responses of migrating leukocytes during inflammation.</b></p>
<p>Multiple sclerosis is a complex neuroinflammatory disease characterized by demyelination in the CNS. Autoreactive immune cells invade the central nervous system and orchestrate an attack against myelin sheathes, the insulation layer that protects neurons. The disease affects nearly 1 in 1,000 New Zealanders, and currently has no cure. The most successful treatments for multiple sclerosis target the initial stages by inhibiting the entry of these cells into the central nervous system, however these are often associated with severe side and life-threatening effects and cannot prevent the progression of the disease.</p>
<p>Heparanase, the ubiquitously expresses heparan sulfate degrading enzyme has been thoroughly implicated in the disease processes of multiple sclerosis, and its animal model, EAE. Autoreactive lymphocytes exploit heparanase activity to degrade the extracellular matrix and destabilize the barriers that maintain the relative immune privileged status of the central nervous system. Exogenous heparan sulfate mimetics have previously been shown to ameliorate symptoms of EAE by interfering with heparanase activity. However, the commercialization and clinical translation of these inhibitors is currently inhibited by the complexity of their synthesis. ‘HS16-35’ is a novel heparan sulfate mimetic developed by the Ferrier Institute, comprised of a dendritic core with four heavily sulfated oligosaccharide arms. The synthesis of this compound is much shorter due to its smaller size; however, it has been shown to act similarly to native heparan sulfate molecules. We proposed that HS16-35 is protective in preventing the migration of autoreactive immune cells across the choroid plexus by inhibiting lymphocyte heparanase.</p>
<p>To investigate the efficacy of HS16-35 in vitro, we first established an experimental transwell model of the choroid plexus. This model incorporated core components of the choroid plexus, including fenestrated capillaries, the stromal matrix and epithelial monolayer. We first showed that the model was capable of mimicking homeostatic trafficking across the choroid plexus epithelium, which formed a selective but permeable barrier. Then, we induced T-cell specific inflammatory migration using Concanavalin A or TH1-type cytokines. This migration was found to be interferon-γ dependent and could be mitigated with anti-interferon-γ treatment.</p>
<p>Once this model was established, we next investigated whether HS16-35 was effective in inhibiting inflammatory migration across this structure. To adapt HS16-35 to an in vitro dose, we performed cell viability assays. This confirmed that the compound was mildly cytotoxic to epithelial choroid plexus cells but not murine splenocytes. Further experiments found that low-dose HS16-35 did not impact monolayer permeability. Transwell migration assays showed that low-dose HS16-35 was effective in reducing ConA and interferon-γ mediated inflammatory T-cell migration to a level comparable to homeostatic trafficking. Finally, we assessed cytokine profiles of leukocytes and epithelial choroid plexus cells treated with HS16 35 and found that HS16-35 reduced the expression of key cytokines involved in MS pathogenesis.</p>
<p>In summary, the work described in this thesis shows how HS16-35 may be protective during EAE by suppressing the inflammatory response of autoreactive T-cells, in addition to regulating the infiltration of immune cells into the CNS through the choroid plexus. In a broader sense, these findings show that HS16 36 may be effective in treating MS by regulating, not inhibiting lymphocyte migration into the CNS, mitigating some of the severe side effects that other migration-inhibitors face.</p>
The Nerves to Conduct a Multiple Sclerosis Crime Investigation
