Laura Airas

Clinical Relevance of Brain Volume Measures in Multiple Sclerosis

Multiple sclerosis (MS) is a chronic disease with an inflammatory and neurodegenerative pathology. Axonal loss and neurodegeneration occurs early in the disease course and may lead to irreversible neurological impairment. Changes in brain volume, observed from the earliest stage of MS and proceeding throughout the disease course, may be an accurate measure of neurodegeneration and tissue damage. There are a number of magnetic resonance imaging-based methods for determining global or regional brain volume, including cross-sectional (e.g. brain parenchymal fraction) and longitudinal techniques (e.g. SIENA [Structural Image Evaluation using Normalization of Atrophy]). Although these methods are sensitive and reproducible, caution must be exercised when interpreting brain volume data, as numerous factors (e.g. pseudoatrophy) may have a confounding effect on measurements, especially in a disease with complex pathological substrates such as MS. Brain volume loss has been correlated with disability progression and cognitive impairment in MS, with the loss of grey matter volume more closely correlated with clinical measures than loss of white matter volume. Preventing brain volume loss may therefore have important clinical implications affecting treatment decisions, with several clinical trials now demonstrating an effect of disease-modifying treatments (DMTs) on reducing brain volume loss. In clinical practice, it may therefore be important to consider the potential impact of a therapy on reducing the rate of brain volume loss. This article reviews the measurement of brain volume in clinical trials and practice, the effect of DMTs on brain volume change across trials and the clinical relevance of brain volume loss in MS.

CD73 is required for efficient entry of lymphocytes into the central nervous system during experimental autoimmune encephalomyelitis

CD73 is a cell surface enzyme of the purine catabolic pathway that catalyzes the breakdown of AMP to adenosine. Because of the strong immunosuppressive and antiinflammatory properties of adenosine, we predicted that cd73−/− mice would develop severe experimental autoimmune encephalomyelitis (EAE), an animal model for the central nervous system (CNS) inflammatory disease, multiple sclerosis. Surprisingly, cd73−/− mice were resistant to EAE. However, CD4 T cells from cd73−/− mice secreted more proinflammatory cytokines than wild-type (WT) mice and were able to induce EAE when transferred into naïve cd73+/+ T cell-deficient recipients. Therefore, the protection from EAE observed in cd73−/− mice was not caused by a deficiency in T cell responsiveness. Immunohistochemistry showed that cd73−/− mice had fewer infiltrating lymphocytes in their CNS compared with WT mice. Importantly, susceptibility to EAE could be induced in cd73−/− mice after the transfer of WT CD73+CD4+ T cells, suggesting that CD73 must be expressed either on T cells or in the CNS for disease induction. In the search for the source of CD73 in the CNS that might facilitate lymphocyte migration, immunohistochemistry revealed a lack of CD73 expression on brain endothelial cells and high expression in the choroid plexus epithelium which regulates lymphocyte immunosurveillance between the blood and cerebrospinal fluid. Because blockade of adenosine receptor signaling with the A2a adenosine receptor-specific antagonist SCH58261 protected WT mice from EAE induction, we conclude that CD73 expression and adenosine receptor signaling are required for the efficient entry of lymphocytes into the CNS during EAE development.

Expansion of CD56Bright natural killer cells in the peripheral blood of multiple sclerosis patients treated with interferon-beta

We studied how interferon-beta (IFN-β) treatment of relapsing-remitting multiple sclerosis (MS) affects subgroups of natural killer cells (NK cells). Following IFN-β treatment, there was an expansion of CD56Bright NK-cells in the peripheral blood of MS patients, while at the same time the proportion of CD56Dim cells was diminished. In a control group, the proportion of CD56Bright NK-cells was significantly higher in secondary lymphoid tissues compared to the peripheral blood of the same individual. Our findings confirm that CD56Bright NK-cells preferably locate within the secondary lymphoid tissues, where they may interact with T cells and thereby contribute to the control of the disease activity in MS.

Immunoregulatory factors in multiple sclerosis patients during and after pregnancy: relevance of natural killer cells

Multiple sclerosis (MS) ameliorates typically during pregnancy but after the delivery the relapse rate often increases. Our study was conducted to understand the immunoregulatory mechanisms accompanying this phenomenon. MS patients were followed‐up prospectively during pregnancy and 6 months postpartum, with immunological characterization of the peripheral blood. Groups of age‐ and parity‐matched healthy pregnant women, and age‐ and sex‐matched non‐pregnant women and non‐pregnant MS patients were studied as controls. In our patient cohort, the annualized relapse rate was 1·0 ± 1·0 relapses/woman/year (mean ± standard deviation) during the year before pregnancy, but dropped to 0·2 ± 0·9 during the third trimester (P = 0·02). After the delivery the relapse rate increased again to 1·4 ± 1·9 (1–3 months postpartum versus third trimester P = 0·003). While percentages of peripheral blood CD3, CD4, CD8 and CD19 immune cell subsets were unchanged over the observation period, reduced disease activity during the last trimester was associated with a significant increase in the percentage of circulating CD56bright natural killer (NK) cells. Simultaneously, the proportion of circulating CD56dim NK cells was clearly reduced. No alteration was noted in CD4+ CD25high forkhead box P3+ regulatory T cells. Production of interferon‐gamma by peripheral blood lymphocytes was down‐regulated significantly during pregnancy in comparison to the postpartum period, resulting in an increased T helper type 2 (Th2) : Th1 ratio during pregnancy. In conclusion, pregnant state in MS patients is characterized by an increase in the percentage of CD56bright NK cells and by enhanced Th2 type cytokine secretion. Our findings suggest a potential role for CD56bright regulatory NK cells in the control of autoimmune inflammation during pregnancy in MS.

Detection of Microglial Activation in an Acute Model of Neuroinflammation Using PET and Radiotracers 11C-(R)-PK11195 and 18F-GE-180

It remains unclear how different translocator protein (TSPO) ligands reflect the spatial extent of astrocyte or microglial activation in various neuroinflammatory conditions. Here, we use a reproducible lipopolysaccharide (LPS)-induced model of acute central nervous system inflammation to compare the binding performance of a new TSPO ligand 18F-GE-180 with 11C-(R)-PK11195. Using immunohistochemistry, we also explore the ability of the TSPO ligands to detect activated microglial cells and astrocytes. Methods: Lewis rats (n = 30) were microinjected with LPS (1 or 10 μg) or saline (1 μL) into the left striatum. The animals were imaged in vivo at 16 h after the injection using PET radiotracers 18F-GE-180 or 11C-(R)-PK11195 (n = 3 in each group) and were killed afterward for autoradiography of the brain. Immunohistochemical assessment of OX-42 and glial fibrillary acidic protein (GFAP) was performed to identify activated microglial cells and reactive astrocytes. Results: In vivo PET imaging revealed an increase in the ipsilateral TSPO binding, compared with binding in the contralateral hemisphere, after the microinjection of 10 μg of LPS. No increase was observed with vehicle. By autoradiography, the TSPO radiotracer binding potential in the injected hemisphere was increased after striatal injection of 1 or 10 μg of LPS. However, the significant increase was observed only when using 18F-GE-180. The area of CD11b-expressing microglial cells extended beyond that of enhanced GFAP staining and mapped more closely to the extent of 18F-GE-180 binding than to 11C-(R)-PK11195 binding. The signal from either PET ligand was significantly increased in regions of increased GFAP immunoreactivity and OX-42 colocalization, meaning that the presence of both activated microglia and astrocytes in a given area leads to increased binding of the TSPO radiotracers. Conclusion: 18F-GE-180 is able to reveal sites of activated microglia in both gray and white matter. However, the signal is increased by the presence of activated astrocytes. Therefore, 18F-GE-180 is a promising new fluorinated longer-half-life tracer that reveals the presence of activated microglia in a manner that is superior to 11C-(R)-PK11195 due to the higher binding potential observed for this ligand.

IFN-α Induced Adenosine Production on the Endothelium: A Mechanism Mediated by CD73 (Ecto-5′-Nucleotidase) Up-Regulation

CD73 (ecto-5′-nucleotidase; EC 3.1.3.5) participates in lymphocyte binding to endothelial cells and converts extracellular AMP into a potent anti-inflammatory substance adenosine. However, the regulation of expression and function of CD73 has remained largely unknown. In this study, we show that IFN-α produces a time- and dose-dependent long-term up-regulation of CD73 on endothelial cells, but not on lymphocytes both at protein and RNA levels. Moreover, CD73-mediated production of adenosine is increased after IFN-α treatment on endothelial cells, resulting in a decrease in the permeability of these cells. Subsequent to induction with PMA, FMLP, dibutyryl cAMP, thrombin, histamine, IL-1β, TNF-α, and LPS, no marked changes in the level of CD73 expression on endothelial cells are observed. We also show that CD73 is up-regulated in vivo on the vasculature after intravesical treatment of urinary bladder cancers with IFN-α. In conclusion, distinct behavior of lymphocyte and endothelial CD73 subsequent to cytokine treatment further emphasizes the existence of cell type-specific mechanisms in the regulation of CD73 expression and function. Overall, these results suggest that IFN-α is a relevant in vivo regulator of CD73 in the endothelial-leukocyte microenvironment in infections/inflammations, and thus has a fundamental role in controlling the extent of inflammation via CD73-dependent adenosine production.

CD73 engagement promotes lymphocyte binding to endothelial cells via a lymphocyte function-associated antigen-1-dependent mechanism.

CD73 is a GPI-anchored lymphocyte adhesion molecule possessing an ecto-5′-nucleotidase enzyme activity. In this work, we show that engagement of lymphocyte CD73 increases lymphocyte binding to cultured endothelial cells (EC) in an LFA-1-dependent fashion. Engagement of CD73 by an anti-CD73 mAb 4G4 increases the adhesion of lymphocytes to cultured EC by about 80% compared with that of lymphocytes treated with a negative control Ab, and the increased adhesion can be blocked by an anti-CD18 mAb. The CD73-regulated increase in lymphocyte adhesion is not due to a conformational change leading to high-affinity LFA-1 receptors as assayed using mAb 24 against an activation-induced epitope of the molecule. Instead, CD73 engagement induces clustering of LFA-1 that is inhibitable by calpeptin, indicating involvement of Ca2+-dependent activation of a calpain-like enzyme in this process. In conclusion, the results shown here demonstrate that CD73 regulates the avidity of LFA-1 by clustering. This indicates a previously undescribed role for CD73 in controlling the poorly characterized activation step in the multistep cascade of lymphocyte extravasation. Moreover, these results suggest that in physiological conditions the activation step may result in clustering of LFA-1 rather than in an affinity change of the molecule.

Pregnancy outcome in women with multiple sclerosis: results from a prospective nationwide study in Finland:

The majority of individuals obtaining the diagnosis of multiple sclerosis are women of childbearing age. They are naturally concerned as to how multiple sclerosis affects the course of pregnancy and the developing foetus. The objective of this study was to prospectively evaluate the incidence of pregnancy complications and delivery risks, and to follow the natural course of multiple sclerosis during and after pregnancy in a cohort of Finnish patients with multiple sclerosis. Sixty-one patients with multiple sclerosis who became pregnant during the years 2003—2005 were prospectively followed-up from early pregnancy until 6 months postpartum. Multiple sclerosis relapses, Expanded Disability Status Scale rates and obstetric details were recorded. The results were compared with the statistics obtained from Finnish Medical Birth Register from the year 2004. We found that patients with multiple sclerosis were no more likely to experience pregnancy complications than Finnish pregnant women generally, but they had a greater likelihood for a need of artificial insemination (4.9% vs. 0.9%; p = 0.0009). Subjects with multiple sclerosis were more likely to undergo assisted vaginal delivery than the at-large cohort(16.4% vs. 6.5%; p = 0.0017). We conclude that pregnancy does not seem to pose a woman with multiple sclerosis to a greater risk for pregnancy complications when compared with women in general. The potential need for instrumental delivery should, however, be taken into account when planning the delivery of a mother with multiple sclerosis.

Breast-feeding, postpartum and prepregnancy disease activity in multiple sclerosis

It is well known that the risk of multiple sclerosis (MS) relapse is starkly diminished during late pregnancy and increases in the postpartum period.1 These alterations in the disease activity are likely associated with pregnancy-related immunologic events serving the well-being of the fetus. Because disease-modifying drugs (DMDs) are not recommended during pregnancy or lactation, mothers need to decide whether to resume early treatment to minimize risk of postpartum relapses or to breast-feed. Interestingly, a recent study suggested that exclusive breast-feeding and concomitant suppression of menses may reduce the risk of postpartum relapses.2 The objective of our study was to determine the prevalence of breast-feeding among Finnish patients with MS and the relationship between breast-feeding, postpartum and prepregnancy relapse frequency. ### Methods. All patients with MS becoming pregnant during the years 2003-2005 in Finland who were encountered by the investigators and gave informed consent to the study protocol were enrolled in the study (n = 61; mean age 30.5 years [range 23-42 years], mean disease duration 5.7 years [range 0-17.5 years], mean total number of relapses before pregnancy 4.1 [range 1-12]). Prospective follow-up continued from early pregnancy (enrollment at 10 to 12 gestational weeks) until 6 months postpartum as described earlier.3 MS relapses, information on DMDs, and breast-feeding history were recorded. The study was approved by the ethical committee of the Turku University Hospital, Finland. ### Results. The average annual relapse rate (RR) of the cohort was 0.82 ± 0.98 (SD) relapses per woman per year during the year before pregnancy (BP). During the third trimester of pregnancy, the mean annualized RR was diminished to 0.40 ± 1.21, but increased again to 1.40 ± 1.92 during the period of 0 to 3 months postpartum (PP) (BP vs third trimester, p = 0.026, and BP vs 0 to 3 months …

IFN-β regulates CD73 and adenosine expression at the blood-brain barrier

IFN‐β treatment reduces the relapse rate in MS but its mechanism of action remains incompletely understood. Our aim was to clarify the beneficial effect of IFN‐β in the treatment of MS. We assessed the influence of IFN‐β treatment on (i) CD73 expression on the surface of primary cultures of human blood–brain barrier endothelial cells (BBB‐EC) and human astrocytes using immunofluorescence staining and flow cytometry, (ii) transmigration of CD4+ T lymphocytes using an in vitro model of BBB and (iii) CD73 enzyme activity, i.e. ecto‐5′‐nucleotidase activity in the serum of MS patients using a radiochemical assay. IFN‐β increases the expression of ecto‐5′‐nucleotidase both on BBB‐EC and astrocytes. As a consequence, lymphocyte transmigration through BBB‐EC is reduced. Importantly, this reduction can be reversed using α,β‐methyleneadenosine‐5′‐diphosphate, a specific inhibitor of ecto‐5′‐nucleotidase. CD73 is strongly expressed in microvasculature in samples of postmortem MS brain and, moreover, in the majority of MS patients there was a clear upregulation both in the soluble serum ecto‐5′‐nucleotidase activity and skin microvascular CD73 expression after IFN‐β treatment. Upregulation of ecto‐5′‐nucleotidase and a subsequent increase in adenosine production might contribute to the beneficial effects of IFN‐β on MS via enhancing the endothelial barrier function.