Overexpression of the cholesterol-binding protein MLN64 induces liver damage in the mouse.

AIM: To examine the in vivo phenotype associated with hepatic metastatic lymph node 64 (MLN64) over-expression. METHODS: Recombinant-adenovirus-mediated MLN64 gene transfer was used to overexpress MLN64 in the livers of C57BL/6 mice. We measured the effects of MLN64 overexpression on hepatic cholesterol content, bile flow, biliary lipid secretion and apoptosis markers. For in vitro studies cultured CHO cells with transient MLN64 overexpression were utilized and apoptosis by TUNEL assay was measured. RESULTS: Livers from Ad.MLN64-infected mice exhibited early onset of liver damage and apoptosis. This response correlated with increases in liver cholesterol content and biliary bile acid concentration, and impaired bile flow. We investigated whether liver MLN64 expression could be modulated in a murine model of hepatic injury. We found increased hepatic MLN64 mRNA and protein levels in mice with chenodeoxycholic acid-induced liver damage. In addition, cultured CHO cells with transient MLN64 overexpression showed increased apoptosis. CONCLUSION: In summary, hepatic MLN64 over-expression induced damage and apoptosis in murine livers and altered cholesterol metabolism. Further studies are required to elucidate the relevance of these findings under physiologic and disease conditions

Manifiesto fundacional del Foro sobre Cooperación Internacional para América Latina y el Caribe.

Durante el mes de junio de 1996 tuvo lugar en la ciudad de Buenos Aires el Encuentro sobre Cooperación internacional para América Latina y el Caribe, organizado por la Universidad Nacional de Quilmes y el CINDA. Como resultado de dicha reunión, un grupo de participantes decidió la creación del Foro sobre Cooperación Internacional para América Latina y el Caribe y encargó al Instituto de Estudios Sociales de la Ciencia de la Universidad Nacional de Quilmes la redacción del manifiesto fundacional. A continuación se publica dicho documento

Galectin-8 as an immunosuppressor in experimental autoimmune encephalomyelitis and a target of human early prognostic antibodies in multiple sclerosis.

Galectin-8 (Gal-8) is a member of a glycan-binding protein family that regulates the immune system, among other functions, and is a target of antibodies in autoimmune disorders. However, its role in multiple sclerosis (MS), an autoimmune inflammatory disease of the central nervous system (CNS), remains unknown. We study the consequences of Gal-8 silencing on lymphocyte subpopulations and the development of experimental autoimmune encephalitis (EAE), to then assess the presence and clinical meaning of anti-Gal-8 antibodies in MS patients. Lgals8/Lac-Z knock-in mice lacking Gal-8 expression have higher polarization toward Th17 cells accompanied with decreased CCR6+ and higher CXCR3+ regulatory T cells (Tregs) frequency. These conditions result in exacerbated MOG35-55 peptide-induced EAE. Gal-8 eliminates activated Th17 but not Th1 cells by apoptosis and ameliorates EAE in C57BL/6 wild-type mice. β-gal histochemistry reflecting the activity of the Gal-8 promoter revealed Gal-8 expression in a wide range of CNS regions, including high expression in the choroid-plexus. Accordingly, we detected Gal-8 in human cerebrospinal fluid, suggesting a role in the CNS immune-surveillance circuit. In addition, we show that MS patients generate function-blocking anti-Gal-8 antibodies with pathogenic potential. Such antibodies block cell adhesion and Gal-8-induced Th17 apoptosis. Furthermore, circulating anti-Gal-8 antibodies associate with relapsing-remitting MS (RRMS), and not with progressive MS phenotypes, predicting clinical disability at diagnosis within the first year of follow-up. Our results reveal that Gal-8 has an immunosuppressive protective role against autoimmune CNS inflammation, modulating the balance of Th17 and Th1 polarization and their respective Tregs. Such a role can be counteracted during RRMS by anti-Gal-8 antibodies, worsening disease prognosis. Even though anti-Gal-8 antibodies are not specific for MS, our results suggest that they could be a potential early severity biomarker in RRMS

Gal-8 deficit favors Th17 polarization during MOGp-induced EAE and <i>ex-vivo</i> re-stimulation.

<p>Th17 and Th1 subpopulations in splenocytes from <i>Lgals8</i>^-/- (KO) and <i>Lgals8</i>^+/+ (WT) mice obtained after 10 days of EAE induction were analyzed either immediately or after 72 h of <i>ex vivo</i> MOGp re-stimulation, in the absence or presence of Gal-8. Gal-8 KO mice show higher frequency of Th17 cells both at steady state and after MOGp re-stimulation. Incubation with Gal-8 reduced Th17 cells only in Gal-8 KO. Graph shows frequency +/-SD (*p<0.05; ANOVA; n = 4).</p

Function-blocking activity of anti-Gal-8 autoantibodies.

<p>(A) Anti-Gal-8(+) sera block the adhesion of PBMC to Gal-8-coated coverslips. Graph shows number of adhered cells (Average ± SE of three anti-Gal-8(-) and three anti-Gal-8(+) sera tested in triplicate) (***p<0.001; Student’s <i>t</i>-test). (B) Anti-Gal-8 autoantibodies inhibit Gal-8-induced apoptosis of Th17 cells. <i>In vitro</i> differentiated Th17 cells from IL-17A-GFP reporter mice were purified based on IL-17A expression (GFP+) and incubated with Gal-8 (20 μg/ml) in the presence of lactose, sucrose or anti-Gal-8 antibodies affinity purified from pooled serum of MS patients. The extent of apoptosis was quantified as the frequency of Annexin V+ 7AAD+ cells of the sample relative to the frequency of Annexin V+ 7AAD+ cells of the untreated control. Representative contour plots are shown in upper panels. Quantification of a representative experiment is shown in the lower panel. Values represent mean + SEM of triplicates. Data from a representative from four independent experiments is shown. **, p<0.01; ***, p < .001 by one-way ANOVA followed by Tukey’s post-hoc test.</p

Anti-Gal-8 autoantibodies correlate with worse disability scores in RRMS patients.

<p>(A) RRMS patients with and without autoantibodies were followed during an average of 12 months. Patients (n = 17) with anti-Gal-8(+) sera have worse EDSS at the end of follow-up than patients (n = 19) without anti-Gal-8 autoantibodies (mean EDSS 1.5 vs 0, *p = 0.002, nonparametric Mann-Whitney U test), independent of the treatment received or number of relapses during this period. (B) At the end of follow-up, 5/17 patients with anti-Gal-8 autoantibodies developed confirmed EDSS worsening vs 0/19 of patients without anti-Gal-8 autoantibodies (*p = 0.016 by Fisher test).</p

Detection of Gal-8 autoantibodies in sera and CSF from MS patients.

<p>Immunoblot of sera and CSF from different MS patients (Pn) against Gal-8 indicating its (-) or (+) anti-Gal-8 reactivity compared with a negative control (C) from a healthy individual. In some patients (e.g. P2 and P10) anti-Gal-8 reactivity was detected in both sera and CSF, while in others (e.g. P3) was only detected in CSF. P14 is shown only in CSF but is also positive in serum (analyzed in other immunoblot), while P15 and P17 are negative both in CSF and serum (not shown). In most patients (e.g. P4-6) only sera could be analyzed. P9 was only analyzed in CSF.</p

Clinical parameters of EAE disease progression (day 0–20).

<p>Clinical parameters of EAE disease progression (day 0–20).</p

Gal-8 expression in mouse brain and presence in human CSF.

<p>(A) Histochemistry of β-gal staining reveals Gal-8 expression in several regions of the mouse brain (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177472#pone.0177472.s001" target="_blank">S1 Table</a>). Brain slices depict high Gal-8 expression levels in the choroid plexus (CP) of the lateral ventricle (LV) and the dorsal 3rd ventricle (D3V), as well as in the ventrolateral thalamic nucleus. (B) Immunoblot with rabbit anti-Gal-8 antibody show Gal-8 reactivity in the CSF of individuals without MS. Samples C3-11 correspond to non-inflammatory CSF from individuals studied for diplopia (C3), vertiginous syndrome (C7), cephalea (C8 and C11) and febrile syndrome (C9), whereas C6 is an inflammatory CSF from a patient with meningitis. All samples show anti-Gal-8 reactivity, though with variable intensity. *Bands of unknown origin might include Gal-8 dimers or complexes with other proteins, not separable under SDS-PAGE conditions.</p

Gal-8 deficit favors selective Th17 cell differentiation upon polyclonal activation.

<p>Splenocytes isolated from <i>Lgals8</i>^+/+ (WT) and <i>Lgals8</i>^-/- (KO) mice were analyzed by FACS: (A) Dendritic cells (CD11c⁺), B cells (CD19⁺), CD8⁺ T cells and different CD4⁺ T cells subsets, naïve (CD44^-CD62L⁺), effector (CD44⁺CD62L⁺), memory (CD44⁺CD62L^-) and total cells analyzed in the subset of viable CD4⁺ CD25^- T-cells show no differences between WT and KO mice. Graphics of frequency +/-SD (n = 5). (B) T cell activation by 72 h incubation with anti-CD3 (anti-CD3) and anti-CD28 (anti-CD28) antibodies show Th17 increased frequency in KO mice while Th1 and Th2 cells are similar in WT and KO mice. Graph shows frequency +/-SD (*p<0.05; ANOVA; n = 4).</p