Up-Regulation of Soluble Axl and Mer Receptor Tyrosine Kinases Negatively Correlates with Gas6 in Established Multiple Sclerosis Lesions

Multiple sclerosis is a disease that is characterized by inflammation, demyelination, and axonal damage; it ultimately forms gliotic scars and lesions that severely compromise the function of the central nervous system. Evidence has shown previously that altered growth factor receptor signaling contributes to lesion formation, impedes recovery, and plays a role in disease progression. Growth arrest-specific protein 6 (Gas6), the ligand for the TAM receptor tyrosine kinase family, consisting of Tyro3, Axl, and Mer, is important for cell growth, survival, and clearance of debris. In this study, we show that levels of membrane-bound Mer (205 kd), soluble Mer (∼150 kd), and soluble Axl (80 kd) were all significantly elevated in homogenates from established multiple sclerosis lesions comprised of both chronic active and chronic silent lesions. Whereas in normal tissue Gas6 positively correlated with soluble Axl and Mer, there was a negative correlation between Gas6 and soluble Axl and Mer in established multiple sclerosis lesions. In addition, increased levels of soluble Axl and Mer were associated with increased levels of mature ADAM17, mature ADAM10, and Furin, proteins that are associated with Axl and Mer solubilization. Soluble Axl and Mer are both known to act as decoy receptors and block Gas6 binding to membrane-bound receptors. These data suggest that in multiple sclerosis lesions, dysregulation of protective Gas6 receptor signaling may prolong lesion activity.Multiple sclerosis (MS) is a debilitating white matter disease of the central nervous system (CNS). Although much of the evidence from animal models and MS suggests it to be an autoimmune disorder mediated by TH-1 type T cells,¹ other possible causes include genetic and environmental factors, antibody-dependent cytotoxicity, and bacterial and viral infections that may mediate altered protein expression resulting in inflammation, axonal and oligodendrocyte damage, demyelination and CNS scarring.² Growth and survival factors that protect against axonal and oligodendrocyte damage or loss, and dampen the inflammatory response are actively being pursued for MS therapy.^2,3,4,5,6 One growth factor associated with oligodendrocyte maturation, survival and dampening the immune response is growth-arrest specific protein 6 (Gas6). Gas6 is a secreted protein that is widely expressed in the central and peripheral nervous systems by endothelial cells and neurons, and is involved in numerous physiological and pathological functions including cell growth, survival and apoptosis.^{7,8,9,10,11,12} Gas6 binds and activates the TAM family of receptor tyrosine kinases consisting of Tyro3 (Rse/Dtk/Sky), Axl (Ufo), and Mer (Eyk).^{8,11,13,14,15} Many cell types express all three receptors and receptor activation can result from homophilic and heterophilic interactions.^16,17 Axl contains the major and minor Gas6 binding groove. Only the minor groove is conserved in Tyro3 and Mer and as a result, response to Gas6 is mediated in a concentration-dependent manner; Gas6 binding affinity is Axl>Tyro3>Mer.¹⁸We previously reported mRNA expression of Axl, Tyro3, and Mer receptors on human fetal oligodendrocytes and the ability of Gas6 to promote oligodendrocyte survival in vitro by activating Axl, resulting in Axl directly and indirectly recruiting phosphatidylinositol 3 kinase and activating the Akt pathway.^19,20 Moreover, we have shown that Gas6/Axl signaling through the Akt pathway can protect oligodendrocytes from tumor necrosis factor α (TNFα)-induced apoptosis.²¹ Down-regulation or deletion of Axl, even in the presence of Gas6, results in loss of protection against TNFα.²²During the relapse phase of relapsing-remitting MS, serum TNFα levels and TNFα mRNA are elevated.^23,24 TNFα is one of the major cytokines expressed in MS lesions.²⁵ TNFα is cleaved to its mature, soluble, secretable form by the matrix metalloproteinase (MMP) TNFα converting enzyme, also known as ADAM17.^26,27,28 MMPs, including ADAM17 and ADAM10 are involved in normal processes such as wound repair and tissue remodeling and are associated with disease states, including MS.^{29,30,31,32,33,34,35,36} Expression of ADAM17 is observed in acute and chronic active MS lesions, primarily in perivascular cuffs and cells morphologically resembling lymphocytes.³⁷ ADAM17 up-regulation in cerebrospinal fluid of MS patients is associated with inflammation and increased soluble TNFα.^37,38 ADAM10 is constitutively expressed on astrocytes in normal appearing white matter and on astrocytes and perivascular macrophages in MS lesions.^38,39,40,41 ADAM10 cleaves Axl, and ADAM17 cleaves Axl and Mer. Cleaved forms of receptors can result in internalization of the receptor and transport off the membrane for recycling. Cleavage can also result in shortened, soluble forms that act as a decoy to regulate the level of a growth factor at the membrane.^41,42Soluble forms of Axl and Mer can reduce the number of viable receptors for Gas6 binding and act as a decoy by sequestering Gas6 extracellularly; potentially a normal mechanism of receptor activation regulation.^40,41 During inflammation, soluble Mer has been reported to inhibit macrophage clearance of apoptotic cells.^41,43 Also, soluble Axl blocked the protective effect of membrane-bound Axl by inhibiting Gas6 induced tyrosine phosphorylation of Axl.⁴⁴ The binding of Gas6 to TAM receptors acts as an inhibitor of inflammation by inhibiting Toll-like receptor and cytokine receptor cascades.⁴⁴ Up-regulation of Axl and its subsequent interaction with interferon α and β receptors results in the expression of cytokine and Toll-like receptor inhibitors.^44,45 Thus, loss of Gas6 signaling, along with dysregulation of the balance between Gas6, Axl and Mer by increased extracellular levels of soluble Axl and Mer, might detrimentally impact the nervous system, especially in established (chronic active and chronic silent) lesions associated with MS. Chronic active MS lesions are characterized by ongoing demyelination, astrogliosis, macrophage and lymphocyte infiltration, astroglial hypertrophy, and oligodendrocyte hyperplasia.⁴⁶ Chronic silent MS lesions are characterized by the absence of actively infiltrating and inflammatory cells, oligodendrocyte loss and no evidence of ongoing demyelination.^46,47,48In this study, we investigated in chronic active and chronic silent MS lesions whether increased expression of soluble Axl and Mer was associated with increased expression of the MMPs ADAM17 and ADAM10, similar to previous studies that showed an association between increased ADAM17 and ADAM10 with TNFα in the CNS of MS patients.^37,38 We also investigated whether in lesions increased soluble Axl and Mer was associated with decreased Gas6, resulting in loss of the beneficial effects from activating membrane-bound Axl and Mer receptors.     

Antenatal interpersonal sensitivity is more strongly associated than perinatal depressive symptoms with postnatal mother-infant interaction quality

Maternal mental health has enduring effects on children’s life chances and is a substantial cost driver for child health, education and social services. A key linking mechanism is the quality of mother-infant interaction. A body of work associates maternal depressive symptoms across the antenatal and postnatal (perinatal) period with less-than-optimal mother-infant interaction. Our study aims to build on previous research in the field through exploring the association of a maternal personality trait, interpersonal sensitivity, measured in early pregnancy, with subsequent mother-infant interaction quality. We analysed data from the Avon Longitudinal Study of Parents and Children (ALSPAC) to examine the association between antenatal interpersonal sensitivity and postnatal mother-infant interaction quality in the context of perinatal depressive symptoms. Interpersonal sensitivity was measured during early pregnancy and depressive symptoms in the antenatal year and across the first 21 months of the postnatal period. In a subsample of the ALSPAC, mother-infant interaction was measured at 12 months postnatal through a standard observation. For the subsample that had complete data at all time points (n?=?706), hierarchical regression examined the contribution of interpersonal sensitivity to variance in mother-infant interaction quality. Perinatal depressive symptoms predicted little variance in mother-infant interaction. Antenatal interpersonal sensitivity explained a greater proportion of variance in mother-infant interaction quality. The personality trait, interpersonal sensitivity, measured in early pregnancy, is a more robust indicator of subsequent mother-infant-interaction quality than perinatal depressive symptoms, thus affording enhanced opportunity to identify vulnerable mother-infant relationships for targeted early intervention.     

Early transvaginal ultrasound following an accurately dated pregnancy: the importance of finding a yolk sac or fetal heart motion

Our goals were to determine the prognostic value of a yolk sac or fetal heart motion seen during an early accurately dated transvaginal ultrasound (TVU). We reviewed 225 consecutive pregnancies for fetal heart motion data. Furthermore, 63 pregnancies following in-vitro fertilization were reviewed for yolk sac information. The TVU was performed between 5 and 6 weeks following presumed conception (heart motion data) and between 22 and 32 days following in-vitro fertilization (yolk sac data). Pregnancies were followed until an ongoing pregnancy or spontaneous abortion was documented. The presence of a yolk sac between 22 and 32 days from fertilization was associated with the development of fetal heart motion in 94% of cases. The absence of the yolk sac by 32 days after fertilization was always associated with a poor outcome. In women < 36 years of age, the presence of fetal heart motion was associated with a spontaneous abortion in only 4.5% of the cases. However, the incidence of spontaneous abortion following fetal heart motion increased to 10% in women 36-39 years and 29% in women > or = 40 years of age. The presence of heart motion should not be considered a reassuring sign in the older woman. These data have implications regarding early embryology and the counselling of infertility patients.      

Development of skin conductance orienting, habituation, and reorienting from ages 3 to 8 years: a longitudinal latent growth curve analysis

Little is known about the development of the skin conductance orienting response (SCOR) in childhood. This longitudinal study examines the effects of age on initial SCOR, habituation, and reorienting. Skin conductance responses to nonsignal auditory stimuli were recorded from 200 male and female children at five different time points (ages 3, 4, 5, 6, and 8 years). Longitudinal latent growth curve analyses were used to determine the trajectory of each SCOR measure during this period. Results indicated that (a) initial SCOR is present at age 3, increases thereafter to peak at age 6, and then levels off, (b) habituation is absent at age 3, but becomes apparent at age 4 years and increases thereafter with increasing age, (c) SC reorienting is absent from ages 3 to 8, and (d) boys and girls do not exhibit different developmental trajectories. Results suggest that from age 3 to 8 years, the transition from the functionally immature to mature neural network underlying orienting and habituation is a continuous process and may be related to children's cognitive development during this period.     

Upregulation of group 1 CD1 antigen presenting molecules in guinea pigs with experimental autoimmune encephalomyelitis: an immunohistochemical study

In humans, group 1 CD1 glycoproteins present foreign and self lipid and glycolipid antigens to T-cells. Homologues of these molecules are not found in mice or rats but are present in guinea pigs (GPs). We examined CD1 and MHC class II expression in the central nervous system (CNS) of GPs sensitized for experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. In normal GPs and the uninflamed CNS, low-level MHC class II (MHC II) immunoreactivity occurred on vascular elements, meningeal macrophages and parenchymal microglial cells, whereas immunoreactivity for CD1 was absent. In the inflamed CNS, the majority of infiltrating cells were MHC II+ and microglia showed increased expression. CD1 immunoreactivity was detected on astrocytes and subsets of inflammatory cells Including B cells and macrophages. Minimal CD1 and MHC II co-expression was noted on inflammatory cells or glia. We conclude that group 1 CD1 molecules are strongly upregulated in the inflamed CNS on subsets of cells distinct from the majority of MHC II bearing cells. The expression of CD1 proteins in such lesions broadens the potential repertoire of antigens recognized at these sites and highlights the value of the GP as a model for studies of the relevance of CD1 molecules in host defense and autoimmune diseases.     

Clinical and genetic analysis of Korean patients with Marfan syndrome: possible ethnic differences in clinical manifestation

Yoo E‐H, Woo H, Ki C‐S, Lee HJ, Kim D‐K, Kang I‐S, Park P, Sung K, Lee CS, Chung T‐Y, Moon JR, Han H, Lee S‐T, Kim J‐W. Clinical and genetic analysis of Korean patients with Marfan syndrome: possible ethnic differences in clinical manifestation. Marfan syndrome (MFS) is an autosomal dominant disorder of the fibrous connective tissue caused by mutations in the fibrillin‐1 (FBN1) gene. Although clinical and genetic analyses have been performed in various populations, there have been few studies in Korea. The aim of this study was to investigate the clinical characteristics and genetic background of Korean patients with MFS. In 39 Korean patients with MFS who met the Ghent criteria, the most common clinical finding was aortic dilatation and/or dissection (94.9%), whereas only 35.9% of patients had ectopia lentis. The majority of MFS patients had fewer than four of the skeletal findings required to fulfill the major skeletal Ghent criterion for MFS. Only 21% of Korean patients had major skeletal abnormalities and most cases showed only minor skeletal involvement. FBN1 gene mutations were detected in 35 out of 39 patients (89.7%), which is similar to rates presented in the previous reports. These results suggest that some clinical features in Korean patients with MFS differed from those reported in Western MFS patients.     

Heat shock protein 70 associations with myelin basic protein and proteolipid protein in multiple sclerosis brains

Heat shock proteins (hsp) are known to facilitate the generation of specific immune responses by chaperoning proteins and peptides involved in T cell activation. Hsp have been shown to be strikingly elevated in multiple sclerosis (MS) lesions. The unique chaperonin properties of hsp70 have allowed identification of immunogenic proteins bound to it by the ex vivo demonstration of hsp associations with proteins implicated in the immune response. We have investigated the association of hsp70 with myelin basic protein (MBP), myelin proteolipid protein (PLP) and myelin oligodendrocyte protein (MOG) in MS and control brain tissue. In co-immunoprecipitation experiments, in all samples of MS brains examined (n = 3), but not control brain tissue (n = 3), direct association of MBP with hsp70, but not with hsp90, was found. In some MS brain samples, association between PLP and hsp70 was also seen. In similar co-immunoprecipitation experiments on brain tissue obtained from mice with experimental autoimmune encephalomyelitis (n = 5) induced by immunization with PLP peptide, specific association of hsp70 with PLP and MBP was found. Using surface plasmon resonance we demonstrated specific binding of hsp70 with MBP in vitro. Analysis of the amounts of MBP bound to hsp70 yielded a molecular ratio of MBP binding to hsp70 at 6.5:1. MBP complexed with hsp70 was taken up at significantly higher rates by antigen-presenting cells than MBP alone and enhanced MBP-specific immune responses. These results indicate that hsp70 specifically associates with MBP in MS brain tissue. This association might be relevant to the enhanced immune recognition of MBP in MS.     

Influence of inorganic nutrition on growth and PSP toxin production of Alexandrium minutum (Dinophyceae) from Cork Harbour, Ireland.

The physiological response of the PSP toxin producing dinoflagellate Alexandrium minutum isolated from the Irish coast was assessed after modulating the initial concentrations of nitrate and phosphate in batch cultures. The cell growth in cultures of strain CK.A02 was primarily controlled by nitrate availability. In all experiments, only gonyautoxins 2 and 3 (GTX2 and 3) were synthesized along the different growth phases, with GTX3 dominating ( approximately 80%) at all stages, making the GTX2-3 toxin profile a possible population marker of A. minutum in Cork Harbour. The cellular toxin quotas remained low and relatively stable at around 2pgcell(-1), except when high N:P ratios were initially used for culture inoculations; in these conditions PSP toxins accumulated up to 14pgcell(-1). Due to the composition of the toxin profile, the toxicity of strain CK.A02 was generally relatively low (from 1.1 to 1.7pg STX eqcell(-1)) in comparison with strains from other geographic areas except when phosphate limiting culture conditions were applied (maximum of 12.5pg STX eqcell(-1)). Results showed that sufficient soluble protein quotas were necessary to observe the intra-cellular accumulation of PSP toxins in phosphate limiting conditions, highlighting also the requirement of adequate nitrogen supplies. The possible existence of localized toxicity hot spots in the field, linked to the accumulation of PSP toxins within A. minutum cells as a metabolic response to adverse environmental conditions, could potentially increase risks for shellfish farming operations.     

Fibreoptic bronchoscopy without general anaesthetic.

We have used flexible fibreoptic bronchoscopy using sedation and local anaesthesia in 50 children aged 2-19 years (median 10) using an Olympus BFP20 instrument. Indications were opportunistic pneumonias (n = 11), persistent atelectasis (n = 11), recurrent pneumonia (n = 7), miscellaneous lower airway disease (n = 7), recurrent wheezing (n = 3), haemoptysis (3), to diagnose infection or rejection of heart-lung transplants (n = 3), stridor (n = 2), suspected airway compression (n = 1), evaluation of tracheostomy (n = 1), and suspected foreign body (n = 1). In 43 cases (86%) the diagnosis was related to the primary indication. In five (10%) unrelated abnormalities were found, and five (10%) were normal. In 13 (26%) treatment was altered as a result of flexible fibreoptic bronchoscopy. Complications were transient respiratory arrest (n = 2), hypoxia (n = 2), pneumonia (n = 2), and laryngospasm (n = 1). All complications were followed by complete recovery. Our results suggest that flexible fibreoptic bronchoscopy is safe. Advantages over rigid bronchoscopy include greater visual range, fewer complications, and the avoidance of a general anaesthetic. Though invasive it can yield important diagnostic and therapeutic information.