Characteristic Cerebrospinal Fluid Cytokine/Chemokine Profiles in Neuromyelitis Optica,Relapsing Remitting or Primary Progressive Multiple Sclerosis

Background

Differences in cytokine/chemokine profiles among patients with neuromyelitis optica (NMO), relapsing remitting multiple sclerosis (RRMS), and primary progressive MS (PPMS), and the relationships of these profiles with clinical and neuroimaging features are unclear. A greater understanding of these profiles may help in differential diagnosis.

Methods/Principal Findings

We measured 27 cytokines/chemokines and growth factors in CSF collected from 20 patients with NMO, 26 with RRMS, nine with PPMS, and 18 with other non-inflammatory neurological diseases (OND) by multiplexed fluorescent bead-based immunoassay. Interleukin (IL)-17A, IL-6, CXCL8 and CXCL10 levels were significantly higher in NMO patients than in OND and RRMS patients at relapse, while granulocyte-colony stimulating factor (G-CSF) and CCL4 levels were significantly higher in NMO patients than in OND patients. In NMO patients, IL-6 and CXCL8 levels were positively correlated with disability and CSF protein concentration while IL-6, CXCL8, G-CSF, granulocyte-macrophage colony-stimulating factor (GM-CSF) and IFN-γ were positively correlated with CSF neutrophil counts at the time of sample collection. In RRMS patients, IL-6 levels were significantly higher than in OND patients at the relapse phase while CSF cell counts were negatively correlated with the levels of CCL2. Correlation coefficients of cytokines/chemokines in the relapse phase were significantly different in three combinations, IL-6 and GM-CSF, G-CSF and GM-CSF, and GM-CSF and IFN-γ, between RRMS and NMO/NMOSD patients. In PPMS patients, CCL4 and CXCL10 levels were significantly higher than in OND patients.

Conclusions

Our findings suggest distinct cytokine/chemokine alterations in CSF exist among NMO, RRMS and PPMS. In NMO, over-expression of a cluster of Th17- and Th1-related proinflammatory cytokines/chemokines is characteristic, while in PPMS, increased CCL4 and CXCL10 levels may reflect on-going low grade T cell and macrophage/microglia inflammation in the central nervous system. In RRMS, only a mild elevation of proinflammatory cytokines/chemokines was detectable at relapse.     

Interferon-β Produces Synergistic Combinatory Anti-Tumor Effects with Cisplatin or Pemetrexed on Mesothelioma Cells

Interferons (IFNs) have been tested for the therapeutic effects in various types of malignancy, but mechanisms of the anti-tumors effects and the differential biological activities among IFN members are dependent on respective cell types. In this study, we examined growth inhibitory activities of type I and III IFNs on 5 kinds of human mesothelioma cells bearing wild-type p53 gene, and showed that type I IFNs but not type III IFNs decreased the cell viabilities. Moreover, growth inhibitory activities and up-regulated expression levels of the major histocompatibility complexes class I antigens were greater with IFN-β than with IFN-α treatments. Cell cycle analyses demonstrated that type I IFNs increased S- and G2/M-phase populations, and subsequently sub-G1-phase fractions. The cell cycle changes were also greater with IFN-β than IFN-α treatments, and these data collectively showed that IFN-β had stronger biological activities than IFN-α in mesothelioma. Type I IFNs-treated cells increased p53 expression and the phosphorylation levels, and activated apoptotic pathways. A combinatory use of IFN-β and cisplatin or pemetrexed, both of which are the current first-line chemotherapeutic agents for mesothelioma, produced synergistic anti-tumor effects, which were also evidenced by increased sub-G1-phase fractions. These data demonstrated firstly to our knowledge that IFN-β produced synergistic anti-tumor effects with cisplatin or pemetrexed on mesothelioma through up-regulated p53 expression.     

Role of Musclin in the Pathogenesis of Hypertension in Rat

Musclin is a novel skeletal muscle-derived secretory factor found in the signal sequence trap of mouse skeletal muscle cDNAs. Musclin possesses a region homologous to the natriuretic peptide family. Thus, musclin is found to bind with the natriuretic peptide clearance receptors. However, the role of musclin in vascular regulation remains unclear. In this study, we aim to investigate the direct effect of musclin on vascular tone and to analyze its role in hypertension using the spontaneously hypertensive rats (SHR). In aortic strips isolated from SHR, musclin induced contractions in a dose-dependent manner. We found that the musclin-induced vasoconstriction was more marked in SHR than in normal rats (WKY). Moreover, this contraction was reduced by blockade of natriuretic peptide receptor C using the ab14355 antibody. Therefore, mediation of the natriuretic peptide receptor in musclin-induced vasoconstriction can be considered. In addition, similar to the natriuretic peptide receptor, expression of the musclin gene in blood vessels was higher in SHR than in WKY. Injection of musclin markedly increased the blood pressure in rats that can be inhibited by anti-musclin antibodies. Musclin-induced vasoconstriction was more pronounced in SHR than in WKY as in its expression. Taken together, these results suggest that musclin is involved in blood pressure regulation. The higher expression of musclin in hypertension indicates that musclin could be used as a new target for the treatment of hypertension in the future.     

Aberrant Assembly of RNA Recognition Motif 1 Links to Pathogenic Conversion of TAR DNA-binding Protein of 43 kDa (TDP-43)

Aggregation of TAR DNA-binding protein of 43 kDa (TDP-43) is a pathological signature of amyotrophic lateral sclerosis (ALS). Although accumulating evidence suggests the involvement of RNA recognition motifs (RRMs) in TDP-43 proteinopathy, it remains unclear how native TDP-43 is converted to pathogenic forms. To elucidate the role of homeostasis of RRM1 structure in ALS pathogenesis, conformations of RRM1 under high pressure were monitored by NMR. We first found that RRM1 was prone to aggregation and had three regions showing stable chemical shifts during misfolding. Moreover, mass spectrometric analysis of aggregated RRM1 revealed that one of the regions was located on protease-resistant β-strands containing two cysteines (Cys-173 and Cys-175), indicating that this region served as a core assembly interface in RRM1 aggregation. Although a fraction of RRM1 aggregates comprised disulfide-bonded oligomers, the substitution of cysteine(s) to serine(s) (C/S) resulted in unexpected acceleration of amyloid fibrils of RRM1 and disulfide-independent aggregate formation of full-length TDP-43. Notably, TDP-43 aggregates with RRM1-C/S required the C terminus, and replicated cytopathologies of ALS, including mislocalization, impaired RNA splicing, ubiquitination, phosphorylation, and motor neuron toxicity. Furthermore, RRM1-C/S accentuated inclusions of familial ALS-linked TDP-43 mutants in the C terminus. The relevance of RRM1-C/S-induced TDP-43 aggregates in ALS pathogenesis was verified by immunolabeling of inclusions of ALS patients and cultured cells overexpressing the RRM1-C/S TDP-43 with antibody targeting misfolding-relevant regions. Our results indicate that cysteines in RRM1 crucially govern the conformation of TDP-43, and aberrant self-assembly of RRM1 at amyloidogenic regions contributes to pathogenic conversion of TDP-43 in ALS.     

MT-LOOP-dependent Localization of Membrane Type I Matrix Metalloproteinase (MT1-MMP) to the Cell Adhesion Complexes Promotes Cancer Cell Invasion

Localization of membrane type I matrix metalloproteinase (MT1-MMP) to the leading edge is thought to be a crucial step during cancer cell invasion. However, its mechanisms and functional impact on cellular invasion have not been clearly defined. In this report, we have identified the MT-LOOP, a loop region in the catalytic domain of MT1-MMP (¹⁶³PYAYIREG¹⁷⁰), as an essential region for MT1-MMP to promote cellular invasion. Deletion of the MT-LOOP effectively inhibited functions of MT1-MMP on the cell surface, including proMMP-2 activation, degradation of gelatin and collagen films, and cellular invasion into a collagen matrix. This is not due to loss of the catalytic function of MT1-MMP but due to inefficient localization of the enzyme to β1-integrin-rich cell adhesion complexes at the plasma membrane. We also found that an antibody that specifically recognizes the MT-LOOP region of MT1-MMP (LOOP_Ab) inhibited MT1-MMP functions, fully mimicking the phenotype of the MT-LOOP deletion mutant. We therefore propose that the MT-LOOP region is an interface for molecular interactions that mediate enzyme localization to cell adhesion complexes and regulate MT1-MMP functions. Our findings have revealed a novel mechanism regulating MT1-MMP during cellular invasion and have identified the MT-LOOP as a potential exosite target region to develop selective MT1-MMP inhibitors.     

Excitotoxicity-induced immediate surge in hippocampal prostanoid production has latent effects that promote chronic progressive neuronal death

Excitotoxicity is involved in neurodegenerative conditions. We investigated the pathological significance of a surge in prostaglandin production immediately after kainic acid (KA) administration [initial phase], followed by a sustained moderate elevation in prostaglandin level [late phase] in the hippocampus of juvenile rats. Numerous pyknotic hippocampal neurons were observed 72 h after KA treatment; this number remained elevated on days 10 and 30. Gross hippocampal atrophy was observed on days 10 and 30. Pre-treatment with indomethacin ameliorated neuronal death on days 10 and 30, and prevented hippocampal atrophy on day 30. Microglial response was moderated by the indomethacin pre-treatment. Blockade of only late-phase prostaglandin production by post-treatment with indomethacin ameliorated neuronal death on day 30. These findings suggest a role for initial-phase prostaglandin production in chronic progressive neuronal death, which is exacerbated by late-phase prostaglandin production. Blockade of prostaglandin production has therapeutic implications in preventing long-term neurological sequelae following excitotoxic brain damage.     

Production of l-Tryptophan by Azaserine-resistant Mutants of Brevibacterium flavum

Azaserine-resistant mutants derived from a 5-fluorotryptophan-resistant, l-tryptophan-producing mutant of Brevibacterium flavum, accumulated 10.3 g/liter of l-tryptophan at maximum. The production increased to 11.4 g/liter when l-serine was added. In the mutant, only anthranilate synthase among enzymes of the tryptophan-specific bio synthetic pathway increased in activity to a 2-fold higher level than that in the parent strain, No. 187. Sensitivity of anthranilate synthase to the feedback inhibition was not altered by the mutation. Activity of 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase, the first common enzyme for aromatic amino acid biosynthesis, also increased 2.7-fold and was less sensitive to the feedback inhibition by phenylalanine and tyrosine. Tryptophan transport activity in strain A-100 was similar as that in the parent. Azaserine inhibited anthranilate synthase activity by 50% at 0.075 mm. The inhibition was of a mixed type with respect to both the two substrates. Anthranilate synthase of strain A-100 was inhibited in a similar manner to that of the parent.     

Construction of Transducing Phage ρ 11 Containing α-Amylase Structural Gene of Bacillus subtilis

We constructed a reporter system to detect a superoxide-generating methyl viologen using SoxRS of Escherichia coli and GFP of Aequorea victoria. E. coli carrying this plasmid exhibited strong fluorescence when grown in the presence of a superoxide-generating reagent methyl viologen. The fluorescence intensity observed in the stationary phase culture of the transformant increased in response to the methyl viologen concentration in a range of 0.01 μM to 10 μM.