A Blood Test for the Diagnosis of Multiple Sclerosis.

Multiple sclerosis (MS) is an autoimmune chronic disease characterized by inflammation and demyelination of the central nervous system (CNS). Despite numerous studies conducted, valid biomarkers enabling a definitive diagnosis of MS are not yet available. The aim of our study was to identify a marker from a blood sample to ease the diagnosis of MS. In this study, since there is evidence connecting the serotonin pathway to MS, we used an ELISA (Enzyme-Linked Immunosorbent Assay) to detect serum MS-specific auto-antibodies (auto-Ab) against the extracellular loop 1 (ECL-1) of the 5-hydroxytryptamine (5-HT) receptor subtype 2A (5-HT2A). We utilized an ELISA format employing poly-D-lysine as a pre-coating agent. The binding of 208 serum samples from controls, both healthy and pathological, and of 104 serum samples from relapsing-remitting MS (RRMS) patients was tested. We observed that the serum-binding activity in control cohort sera, including those with autoimmune and neurological diseases, was ten times lower compared to the RRMS patient cohort (p = 1.2 × 10-47), with a sensitivity and a specificity of 98% and 100%, respectively. These results show that in the serum of patients with MS there are auto-Ab against the serotonin receptor type 2A which can be successfully used in the diagnosis of MS due to their high sensitivity and specificity.

The cultural origin of saving behavior.

Traditional economic interpretations have not been successful in explaining differences in saving rates across countries. One hypothesis is that savings respond to cultural specific social norms. The accepted view in economics so far is that culture does not have any effect on savings. We revisit this evidence using a novel dataset, which allows us to study the saving behavior of up to three generations of immigrants in the United Kingdom. Against the backdrop of existing evidence, we find that cultural preferences are an important explanation for cross-country differences in saving behavior, and their relevance persists up to three generations.

Traditional agricultural practices and the sex ratio today.

We study the historical origins of cross-country differences in the male-to-female sex ratio. Our analysis focuses on the use of the plough in traditional agriculture. In societies that did not use the plough, women tended to participate in agriculture as actively as men. By contrast, in societies that used the plough, men specialized in agricultural work, due to the physical strength needed to pull the plough or control the animal that pulls it. We hypothesize that this difference caused plough-using societies to value boys more than girls. Today, this belief is reflected in male-biased sex ratios, which arise due to sex-selective abortion or infanticide, or gender-differences in access to family resources, which results in higher mortality rates for girls. Testing this hypothesis, we show that descendants of societies that traditionally practiced plough agriculture today have higher average male-to-female sex ratios. We find that this effect systematically increases in magnitude and statistical significance as one looks at older cohorts. Estimates using instrumental variables confirm our findings from multivariate OLS analysis.

Early and late events induced by polyQ-expanded proteins: identification of a common pathogenic property of polYQ-expanded proteins.

To find a common pathogenetic trait induced by polyQ-expanded proteins, we have used a conditional expression system in PC12 cells to tune the expression of these proteins and analyze the early and late consequences of their expression. We find that expression for 3 h of a polyQ-expanded protein stimulates cellular reactive oxygen species (ROS) levels and significantly reduces the mitochondrial electrochemical gradient. 24-36 h later, ROS induce DNA damage and activation of the checkpoint kinase, ATM. DNA damage signatures are reversible and persist as long as polyQ-expanded proteins are expressed. Transcription of neural and stress response genes is down-regulated in these cells. Selective inhibition of ATM or histone deacetylase rescues transcription and restores the expression of silenced genes. Eventually, after 1 week, the expression of polyQ-expanded protein also induces endoplasmic reticulum stress. As to the primary mechanism responsible for ROS generation, we find that polyQ-expanded proteins, including native Ataxin-2 and Huntingtin, are selectively sequestered in the lipid raft membrane compartment and interact with gp91, the membrane NADPH-oxidase subunit. Selective inhibition of NADPH oxidase or silencing of H-Ras signaling dissolves the aggregates and eliminates DNA damage. We suggest that targeting of the polyQ-expanded proteins to the lipid rafts activates the resident NADPH oxidase. This triggers a signal linking H-Ras, ROS, and ERK1/2 that maintains and propagates the ROS wave to the nucleus. This mechanism may represent the common pathogenetic signature of all polyQ-expanded proteins independently of the specific context or the function of the native wild type protein.

A point mutation (G574A) in the chemokine receptor CXCR4 detected in human cancer cells enhances migration.

The chemokine receptor CXCR4 is widely expressed in human cancers and regulates cell invasion, proliferation and survival. Because mutations in the CXCR4 gene could regulate its function we sequenced the coding region of the CXCR4 gene in 18 human melanoma and 3 human colon carcinoma cell lines. The same somatic point mutation (G574A; V160I) in the fourth transmembrane region of CXCR4 was detected in one colon cancer cell line (PD) and one melanoma cell line (LB). CXCR4 was expressed and functional in both PD and LB cells, PD and LB cells migrated specifically toward the receptor ligand, CXCL12 and P-Erk was specifically induced by CXCL12. To give insight into the function of the mutant CXCR4 receptor, human A431, epidermoid carcinoma cells, were stably transfected with both mutant and wild type CXCR4. In vitro, A431 cells harboring CXCR4(G574A) migrated specifically toward CXCL12 and CXCL12 induced ERK phosphorylation. Interestingly, in vivo studies showed that the growth of A431 tumors harboring CXCR4(G574A) was delayed compared to those harboring WT CXCR4. As expected, treatment with AMD3100, a specific CXCR4 inhibitor, reduced the in vivo growth of CXCR4(G574A) tumor b(G574A) but surprisingly, increased the growth of CXCR4(G574A) A431 cells. This is the first report of a spontaneously occurring, functionally active CXCR4 mutation in human cancer cells. While the mutation impairs cell growth in vivo, the CXCR4 inhibitor, AMD3100, stimulated the growth of cells harboring CXCR4(G574A).

Huntingtin inclusion bodies are iron-dependent centers of oxidative events.

Recently, we reported that the transient expression of huntingtin exon1 polypeptide containing polyglutamine tracts of various sizes (httEx1-polyQ) in cell models of Huntington disease generated an oxidative stress whose intensity was CAG repeat expansion-dependent. Here, we have analyzed the intracellular localization of the oxidative events generated by the httEx1-polyQ polypeptides. Analysis of live COS-7 cells as well as neuronal SK-N-SH and PC12 cells incubated with hydroethidine or dichlorofluorescein diacetate revealed oxidation of these probes at the level of the inclusion bodies formed by httEx1-polyQ polypeptides. The intensity and frequency of the oxidative events among the inclusions were CAG repeat expansion-dependent. Electron microscopic analysis of cell sections revealed the presence of oxidation-dependent morphologic alterations in the vicinity of httEx1-polyQ inclusion bodies. Moreover, a high level of oxidized proteins was recovered in partially purified inclusions. We also report that the iron chelator deferroxamine altered the structure, localization and oxidative potential of httEx1-polyQ inclusion bodies. Hence, despite the fact that the formation of inclusion bodies may represent a defense reaction of the cell to eliminate httEx1 mutant polypeptide, this phenomenon appears inherent to the generation of iron-dependent oxidative events that can be deleterious to the cell.

Human melanoma metastases express functional CXCR4.

PURPOSE: The chemokine receptor CXCR4 was identified as an independent predictor of poor prognosis in primary melanoma. The aim of the study was to investigate the role of CXCR4 in human melanoma metastases. EXPERIMENTAL DESIGN: CXCR4 expression was evaluated in melanoma metastases and in metastatic cell lines through immunohistochemistry, immunoblotting, immunofluorescence, and reverse transcription-PCR. The function of CXCR4 was tested in the presence of the ligand, CXCL12, through induction of extracellular signal-regulated kinase-1 and -2 (Erk-1 and -2) phosphorylation, proliferation, apoptosis, and migration capabilities. RESULTS: CXCR4 expression was detected in 33 out of 63 (52.4%) metastases from cutaneous melanomas. Metastatic melanoma cell lines expressed cell surface CXCR4; PES 43, Alo 40, and COPA cell lines showed the highest levels of CXCR4 (>90% of positive cells); PES 41, Alo 39, PES 47, POAG, and CIMA cell lines showed low to moderate degrees of expression (5-65% of positive cells). Other chemokine receptors, CCR7 and CCR10, were detected on the melanoma cell lines; CXCL12 activated Erk-1 and Erk-2, the whose induction was specifically inhibited by AMD3100 treatment. CXCL12 increased the growth in PES 41, PES 43, and PES 47 cells under suboptimal (1% serum) and serum-free culture conditions; AMD3100 (1 mumol/L) inhibited the spontaneous and CXCL12-induced proliferation. No rescue from apoptosis was shown but PES 41, PES 43, and PES 47 cells migrate toward CXCL12. CONCLUSIONS: These findings indicate that CXCR4 is expressed and active in human melanoma metastases, suggesting that active inhibitors such as AMD3100 may be experienced in human melanoma.

Expression of CXCR4 predicts poor prognosis in patients with malignant melanoma.

PURPOSE: CXCR4 receptor and its unique ligand, the CXCL12 chemokine, have been recently implicated in cancer metastasis. Evidence about the role of CXCR4/CXCL12 axis has been reported in several cancers including melanoma. Our goal was to investigate if CXCR4 expression has a prognostic value in malignant melanoma. EXPERIMENTAL DESIGN: Immunohistochemical expression of CXCR4 was evaluated on 71 specimens of primary cutaneous melanoma with a Breslow tumor thickness of >1 mm after radical resection. Associations between baseline patient features and tumors were analyzed by chi(2) test. The prognostic value of CXCR4 expression was evaluated by univariate and multivariate analyses adjusted by age, sex, Breslow tumor thickness, presence of ulceration, and sentinel lymph node metastases. RESULTS: CXCR4 expression was detected in 31 of 71 (43.6%) primary cutaneous melanomas. Membrane or cytoplasmic staining for CXCR4 protein was absent in 56% of the tumors. The positive cases were divided into three score classes according to their staining: low in 15 cases (21%), moderate in 10 (14%), and high in 6 (8%). After a median follow-up of 38 months, 26 patients progressed (16 of 26 expressed CXCR4) and 19 died (12 of 19 expressed CXCR4). The CXCR4 expression on tumor cells was correlated with an unfavorable prognosis with a median disease-free and overall survival of 22 and 35 months, respectively. The hazard ratios of relapse and death, compared with patients with CXCR4-negative tumors, were 2.5 (95% confidence interval, 1.2-6.1) and 3.1 (95% confidence interval, 1.1-7.2), respectively. Median time-to-event (progression and survival) was not reached in patients with CXCR4-negative tumors. In the multivariate analysis, CXCR4 expression, presence of ulceration, and sentinel lymph node status emerged as independent prognostic factors. CONCLUSIONS: This article provides the first evidence that CXCR4 expression could be an independent and powerful prognostic marker in primary cutaneous malignant melanomas.

DNA damage induced by polyglutamine-expanded proteins.

We have developed stable cell lines expressing green fluorescent protein fusion proteins containing polyglutamine repeats of various lengths under tetracycline control. The expression of the expanded (43Q) repeat protein resulted in aggregate formation in a time-dependent fashion. The accumulation of aggregates did not induce apoptosis, although the survival of these cells was critically dependent on the presence of serum and growth factors. However, the expression of 43Q expanded protein strongly activated the ataxia telangiectasia mutated kinase/ATM and Rad3-related kinase (ATM/ATR)-dependent DNA damage response, as shown by selective phosphorylation of ATM substrates. This activation was dependent on 43 CAG protein expression, reversible and sensitive to caffeine and reducing agents. Similarly, we found phosphorylated ATM substrates in fibroblasts from Huntington's disease or SCA-2 patients. Oxidative stress induced accumulation of ATM/ATR phosphorylated protein in HD and SCA-2 patients, but not in normal controls. Furthermore, a significant phosphorylation of H2AX was shown by fibroblasts from patients. We conclude that polyglutamine induces ATM/ATR-dependent DNA damage response through accumulation of reactive oxygen species. ATM activation can be used to monitor the disease in vivo.

Up-regulation of c-Jun N-terminal kinase pathway in Friedreich's ataxia cells.

The severe reduction in mRNA and protein levels of the mitochondrial protein frataxin, encoded by the X25 gene, causes Friedreich ataxia (FRDA), the most common form of recessive hereditary ataxia. Increasing evidence underlines the pathogenetic role of oxidative stress in this disease. We generated an in vitro cellular model of regulated human frataxin overexpression. We identified, by differential display technique, the mitogen activated protein kinase kinase 4 mRNA down regulation in frataxin overexpressing cells. We studied the stress kinases pathway in this cellular model and in fibroblasts from FRDA patients. Frataxin overexpression reduced c-Jun N-terminal kinase phosphorylation. Furthermore, exposure of FRDA fibroblasts to several forms of environmental stress caused an up regulation of phospho-JNK and phospho-c-Jun. To understand if this susceptibility results in cell death, we have investigated the involvement of caspases. A significantly higher activation of caspase-9 was observed in FRDA versus control fibroblasts after serum-withdrawal. Our findings suggest the presence, in FRDA patient cells, of a 'hyperactive' stress signaling pathway. The role of frataxin in FRDA pathogenesis could be explained, at least in part, by this hyperactivity.