Peter J. Darlington

Emerging multiple sclerosis disease-modifying therapies

Purpose of reviewWe focus on emerging therapies for the treatment of multiple sclerosis (MS) whose progress through late-phase clinical trials has furthered our understanding of MS pathophysiology. Recent findingsA promising array of potential new therapies for MS is targeting a broadening range of pathophysiologic mechanisms. Essentially all emerging therapies in late-phase clinical trials continue to focus on peripheral immune mechanisms that predominate early in the illness. The success of some of these, with varying mechanisms of action, has contributed to an evolution in our conceptual framework of MS. SummarySeveral of the emerging therapies focusing on immune-mediated disease mechanisms seem to offer stronger efficacy than the currently approved immune modulators for MS, although with potential for serious adverse effects. These therapies have also broadened our understanding of MS pathophysiology by demonstrating that significant decreases in new disease activity can be achieved through targeting of distinct cell types and mechanisms.

Innate Immune-Mediated Neuronal Injury Consequent to Loss of Astrocytes

Neuronal injury and loss are recognized features of neuroinflammatory disorders, including acute and chronic encephalitides and multiple sclerosis; destruction of astrocytes has been demonstrated in cases of Rasmussen encephalitis. Here, we show that innate immune cells (i.e. natural killer [NK] and γδ T cells) cause loss of neurons from primary human neuron-enriched cultures by destroying the supporting astrocytes. Interleukin 2-activated NK cells caused loss of astrocytes within 1 hour, whereas neurons were lost at 4 hours. Time-lapse imaging indicated that delayed neuron loss was due to early destruction of supporting astrocytes. Selective blocking of astrocyte death with anti-NKG2D antibodies reduced neuron loss, as did blocking of CD54 on astrocytes. γδ T cells also induced astrocyte cytotoxicity, leading to subsequent neuronal displacement. In astrocytes, NK cells caused caspase-dependent fragmentation of the intermediate filament proteins glial fibrillary acidic protein and vimentin, whereas anti-CD3-activated T cells produced fragmentation to a lesser extent and without measurable cytotoxicity. Glial fibrillary acidic protein fragmentation was also demonstrated in lysates from chronic multiple sclerosis plaques but not from normal control white matter. These data suggest that non-major histocompatibility complex-restricted immune effector cells may contribute to neuron loss in neuroinflammatory disorders indirectly through injury of glia.

Detecting glycogen in peripheral blood mononuclear cells with periodic acid schiff staining.

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Reciprocal modulation of helper Th1 and Th17 cells by the β2‐adrenergic receptor agonist drug terbutaline

Catecholamine hormones are powerful regulators of the immune system produced by the sympathetic nervous system (SNS). They regulate the adaptive immune system by altering T‐cell differentiation into T helper (Th) 1 and Th2 cell subsets, but the effect on Th17 cells is not known. Th17 cells, defined, in part, by chemokine receptor CCR6 and cytokine interleukin (IL)‐17A, are crucial for mediating certain pathogen‐specific responses and are linked with several autoimmune diseases. We demonstrated that a proportion of human Th17 cells express beta 2‐adrenergic receptor (β2AR), a G protein‐coupled receptor that responds to catecholamines. Activation of peripheral blood mononuclear cells, which were obtained from venous blood drawn from healthy volunteers, with anti‐cluster of differentiation 3 (CD3) and anti‐CD28 and with a β2‐agonist drug, terbutaline (TERB), augmented IL‐17A levels (P < 0.01) in the majority of samples. TERB reduced interferon gamma (IFNγ) indicating that IL‐17A and IFNγ are reciprocally regulated. Similar reciprocal regulation was observed with dbcAMP. Proliferation of Th cells was monitored by carboxyfluorescein diacetate N‐succinimidyl ester labeling and flow cytometry with antibody staining for CD3 and CD4. TERB increased proliferation by a small but significant margin (P < 0.001). Next, Th17 cells (CD4+CXCR3− CCR6+) were purified using an immunomagnetic positive selection kit, which removes all other mononuclear cells. TERB increased IL‐17A from purified Th17 cells, which argues that TERB acts directly on Th17 cells. Thus, hormone signals from the SNS maintain a balance of Th cells subtypes through the β2AR.

Comparative morphology and phagocytic capacity of primary human adult microglia with time-lapse imaging

Microglia provide immune surveillance within the brain and spinal cord. Various microglial morphologies include ramified, amoeboid, and pseudopodic. The link between form and function is not clear, especially for human adult microglia which are limited in availability for study. Here, we examined primary human microglia isolated from normal-appearing white matter. Pseudopodic and amoeboid microglia were effective phagocytes, taking up E. coli bioparticles using ruffled cell membrane sheets and retrograde transport. Pseudopodic and amoeboid microglia were more effective phagocytes as compared to ramified microglia or monocyte-derived dendritic cells. Thus, amoeboid and pseudopodic microglia may both be effective as brain scavengers.

Netrin-1 regulates blood–brain barrier function and CNS inflammation

Objective: To identify a biomarker capable of distinguishing patients who, in spite of a primary progressive MS (PPMS) clinical course, may nonetheless benefit from immune therapy. Methods: Presence or absence of both IgG and IgM oligoclonal bands (OCB) were blindly examined in paired CSF and serum samples from a large cohort of 170 patients with PPMS, and then related to clinical and imaging evidence of focal inflammatory disease activity. Results:Using both cross-sectional samples aswell as serial sampling in a unique subgroup of patients followed prospectively as part of the placebo-controlled OLYMPUS study of rituximab in PPMS, we found that the presence of CSF-restricted IgMOCB (but not of IgGOCB), is associated with an active inflammatory disease phenotype in PPMS patients. Interpretation: Presence of CSF IgMOCBmay be a biomarker for a subset of PPMS patients with more active inflammatory disease, who may benefit from immune-directed treatments. doi:10.1016/j.jneuroim.2014.08.071