Pleiotrophin regulates microglia-mediated neuroinflammation.

BACKGROUND: Pleiotrophin (PTN) is a cytokine found highly upregulated in the brain in different disorders characterized by overt neuroinflammation such as neurodegenerative diseases, drug addiction, traumatic injury, and ischemia. In the present work, we have explored whether PTN modulates neuroinflammation and if Toll-like receptor 4 (TLR4), crucial in the initiation of an immune response, is involved. METHODS: In immunohistochemistry assays, we studied lipopolysaccharide (LPS, 7.5 mg/kg i.p.)-induced changes in glial fibrillary acidic protein (GFAP, astrocyte marker) and ionized calcium-binding adaptor molecule 1 (Iba1, microglia marker) expression in the prefrontal cortex (PFC) and striatum of mice with transgenic PTN overexpression in the brain (PTN-Tg) and in wild-type (WT) mice. Cytokine protein levels were assessed in the PFC by X-MAP technology. The influence of TLR4 signaling in LPS effects in both genotypes was assessed by pretreatment with the TLR4 antagonist (TAK-242, 3.0 mg/kg i.p.). Murine BV2 microglial cells were treated with PTN (0.5 µg/ml) and LPS (1.0 µg/ml) and assessed for the release of nitric oxide (NO). RESULTS: We found that LPS-induced microglial activation is significantly increased in the PFC of PTN-Tg mice compared to that of WT mice. The levels of TNF-α, IL-6, and MCP-1 in response to LPS were significantly increased in the PFC of PTN-Tg mice compared to that of WT mice. Pretreatment with TAK-242 efficiently blocked increases in cytokine contents in a similar manner in both genotypes. Concomitant incubation of BV2 cells with LPS and PTN significantly potentiated the production of NO compared to cells only treated with LPS. CONCLUSIONS: Our findings identify for the first time that PTN is a novel and potent regulator of neuroinflammation. Pleiotrophin potentiates LPS-stimulated microglia activation. Our results suggest that regulation of the PTN signaling pathways may constitute new therapeutic opportunities particularly in those neurological disorders characterized by increased PTN cerebral levels and neuroinflammation.

Genetic variants associated with rheumatoid arthritis patients and serotypes in European populations.

OBJECTIVES: To replicate the association of rheumatoid arthritis (RA) susceptibility loci in an independent European sample and to assess their specificity with anti-citrullinated protein antibodies (ACPA) status. METHODS: A selection of 64 SNP previously associated with RA have been typed in a cohort of 267 RA patients (169 ACPA-positive and 98 ACPA-negative) and 152 controls from the Rheumatology Units of the University Hospital of Pisa (Italy) and the University of Pécs Medical Center (Hungary). Regression analyses were performed first considering overall RA patients and secondly, taking both serotype subgroups as different disease entities. The results have been adjusted for age, gender and origin of individuals. RESULTS: The well-known CD2, REL, TNFAIP3, IRF5, PTPRC, and CCR6 have been confirmed as RA disease associated loci together with recently discovered BACH2, RASGRP1, and IKZF3 loci, taking all RA patients as a unique phenotype. Results from both serological subgroups separately reflect the specificity of these susceptibility loci and show additional ACPA-positive specific associations for variants at IL6R, IL2RA, BLK, DDX6, IL6, and TLE3 genes. CONCLUSIONS: The results from GAPAID project are consistent with previously established RA disease associations for CD2, PTPRC, REL, CCR6, TNFAIP3, IRF5, BLK, IL2RA, and DDX6 loci. In addition, IL6R, BACH2, RASGRP1, TLE3, and IKZF3 are replicated for the first time in an independent European population and IL6 appears to be a suggestive new RA associated locus. The stratified analysis based on ACPA status provides further support for distinct genetic aetiologies of RA subsets, which might have therapeutic implications.

Chronic Cocaine Use Causes Changes in the Striatal Proteome Depending on the Endogenous Expression of Pleiotrophin.

The neurotrophic factor pleiotrophin (PTN) is upregulated in different brain areas after the administration of different drugs of abuse, including psychostimulants. PTN has been shown to prevent cocaine-induced cytotoxicity in NG108-15 and PC12 cells. We previously demonstrated that specific phosphoproteins related to neurodegeneration processes are differentially regulated in the mouse striatum by a single cocaine (15 mg/kg) administration depending on the endogenous expression of PTN. Since neurodegenerative processes are usually observed in patients exposed to toxicants for longer duration, we have now performed a striatal proteomic study using samples enriched in phosphorylated proteins from PTN knockout (PTN-/-) mice, from mice with transgenic PTN overexpression (PTN-Tg) in the brain, and from wild type (WT) mice after a chronic treatment with cocaine (15 mg/kg/day for 7 days). We have successfully identified 23 proteins significantly affected by chronic cocaine exposure, genotype, or both. Most of these proteins, including peroxiredoxin-6 (PRDX6), triosephosphate isomerase (TPI1), ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1), and annexins A5 (ANXA5) and A7 (ANXA7), may be of significant importance because they were previously identified in proteomic studies in animals treated with psychostimulants and/or because they are related to neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease. The data support a protective role of PTN against chronic cocaine-induced neural alterations.

Influence of MTHFR C677T polymorphism on methotrexate monotherapy discontinuation in rheumatoid arthritis patients: results from the GAPAID European project.

OBJECTIVES: Methotrexate (MTX) is the most widely prescribed drug for rheumatoid arthritis (RA) patients, but 45% of them discontinue therapy within two years, either due to inefficacy or toxicity. Several authors have reported contradictory results related to C677T polymorphism in the MTHFR gene and response to MTX in RA. The purpose of this study was to further explore this genotype-response association in a European RA population. METHODS: This retrospective longitudinal study included a total of 269 RA patients from Italy and Hungary, of whom 73.2% had available data on MTX treatment (197 patients). C677T polymorphism (rs1801133) was genotyped by quantitative PCR using TaqMan assays. Genotype association analysis and Kaplan-Meier method were used for statistical comparisons between patients continuing and patients who abandoned MTX treatment. RESULTS: A total of 85 out of the 197 RA patients (43%) abandoned MTX treatment by the time of analysis. No significant genotype-MTX discontinuation association was found for the overall population, either at the end of the study (p=0.375), or during the follow-up (p=0.324). When the analysis was restricted to the 68 patients on MTX monotherapy, a borderline association (OR 3.15, 95% CI 0.93-10.67, p=0.057) was noted with the recessive genetic model. In agreement with that, a Kaplan-Meier analysis showed a significantly shorter time-to-discontinuation of MTX monotherapy for homozygous carriers of the T-allele (p=0.042). CONCLUSIONS: These results demonstrate that the C677T polymorphism in the MTHFR gene is involved in MTX monotherapy discontinuation in a multicentre European patient cohort, confirming previous results.

Pleiotrophin differentially regulates the rewarding and sedative effects of ethanol.

Pleiotrophin (PTN) is a cytokine with important roles in dopaminergic neurons. We found that an acute ethanol (2.0 g/kg, i.p.) administration causes a significant up-regulation of PTN mRNA and protein levels in the mouse prefrontal cortex, suggesting that endogenous PTN could modulate behavioural responses to ethanol. To test this hypothesis, we studied the behavioural effects of ethanol in PTN knockout (PTN(-/-) ) mice and in mice with cortex- and hippocampus-specific transgenic PTN over-expression (PTN-Tg). Ethanol (1.0 and 2.0 g/kg) induced an enhanced conditioned place preference in PTN(-/-) compared to wild type mice, suggesting that PTN prevents ethanol rewarding effects. Accordingly, the conditioning effects of ethanol were completely abolished in PTN-Tg mice. The ataxic effects induced by ethanol (2.0 g/kg) were not affected by the genotype. However, the sedative effects of ethanol (3.6 g/kg) tested in a loss of righting reflex paradigm were significantly reduced in PTN-Tg mice, suggesting that up-regulation of PTN levels prevents the sedative effects of ethanol. These results indicate that PTN may be a novel genetic factor of importance in alcohol use disorders, and that potentiation of the PTN signalling pathway may be a promising therapeutic strategy in the treatment of these disorders.

Implication of the PTN/RPTPß/ζ Signaling Pathway in Acute Ethanol Neuroinflammation in Both Sexes: A Comparative Study with LPS.

Binge drinking during adolescence increases the risk of alcohol use disorder, possibly by involving alterations of neuroimmune responses. Pleiotrophin (PTN) is a cytokine that inhibits Receptor Protein Tyrosine Phosphatase (RPTP) ß/ζ. PTN and MY10, an RPTPß/ζ pharmacological inhibitor, modulate ethanol behavioral and microglial responses in adult mice. Now, to study the contribution of endogenous PTN and the implication of its receptor RPTPß/ζ in the neuroinflammatory response in the prefrontal cortex (PFC) after acute ethanol exposure in adolescence, we used MY10 (60 mg/kg) treatment and mice with transgenic PTN overexpression in the brain. Cytokine levels by X-MAP technology and gene expression of neuroinflammatory markers were determined 18 h after ethanol administration (6 g/kg) and compared with determinations performed 18 h after LPS administration (5 g/kg). Our data indicate that Ccl2, Il6, and Tnfa play important roles as mediators of PTN modulatory actions on the effects of ethanol in the adolescent PFC. The data suggest PTN and RPTPß/ζ as targets to differentially modulate neuroinflammation in different contexts. In this regard, we identified for the first time important sex differences that affect the ability of the PTN/RPTPß/ζ signaling pathway to modulate ethanol and LPS actions in the adolescent mouse brain.

Metabolomics and biochemical alterations caused by pleiotrophin in the 6-hydroxydopamine mouse model of Parkinson's disease.

Pleiotrophin (PTN) is a cytokine involved in nerve tissue repair processes, neuroinflammation and neuronal survival. PTN expression levels are upregulated in the nigrostriatal pathway of Parkinson's Disease (PD) patients. We aimed to characterize the dopaminergic injury and glial responses in the nigrostriatal pathway of mice with transgenic Ptn overexpression in the brain (Ptn-Tg) after intrastriatal injection of the catecholaminergic toxic 6-hydroxydopamine (6-OHDA) at a low dose (5 µg). Ten days after surgery, the injection of 6-OHDA induced a significant decrease of the number of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra and of the striatal TH contents in Wild type (Wt) mice. In contrast, these effects of 6-OHDA were absent in Ptn-Tg mice. When the striatal Iba1 and GFAP immunoreactivity was studied, no statistical differences were found between vehicle-injected Wt and Ptn-Tg mice. Furthermore, 6-OHDA did not cause robust glial responses neither on Wt or Ptn-Tg mice 10 days after injections. In metabolomics studies, we detected interesting metabolites that significantly discriminate the more injured 6-OHDA-injected Wt striatum and the more protected 6-OHDA-injected Ptn-Tg striatum. Particularly, we detected groups of metabolites, mostly corresponding to phospholipids, whose trends were opposite in both groups. In summary, the data confirm lower 6-OHDA-induced decreases of TH contents in the nigrostriatal pathway of Ptn-Tg mice, suggesting a neuroprotective effect of brain PTN overexpression in this mouse model of PD. New lipid-related PD drug candidates emerge from this study and the data presented here support the increasingly recognized "lipid cascade" in PD.

Role of RPTPß/ζ in neuroinflammation and microglia-neuron communication.

Pleiotrophin (PTN) is a cytokine that is upregulated in different neuroinflammatory disorders. Using mice with transgenic PTN overexpression in the brain (Ptn-Tg), we have found a positive correlation between iNos and Tnfα mRNA and Ptn mRNA levels in the prefrontal cortex (PFC) of LPS-treated mice. PTN is an inhibitor of Receptor Protein Tyrosine Phosphatase (RPTP) ß/ζ, which is mainly expressed in the central nervous system. We aimed to test if RPTPß/ζ is involved in the modulation of neuroinflammatory responses using specific inhibitors of RPTPß/ζ (MY10 and MY33-3). Treatment with MY10 potentiated LPS-induced microglial responses in the mouse PFC. Surprisingly, MY10 caused a decrease in LPS-induced NF-κB p65 expression, suggesting that RPTPß/ζ may be involved in a novel mechanism of potentiation of microglial activation independent of the NF-κB p65 pathway. MY33-3 and MY10 limited LPS-induced nitrites production and iNos increases in BV2 microglial cells. SH-SY5Y neuronal cells were treated with the conditioned media from MY10/LPS-treated BV2 cells. Conditioned media from non-stimulated and from LPS-stimulated BV2 cells increased the viability of SH-SY5Y cultures. RPTPß/ζ inhibition in microglial cells disrupted this neurotrophic effect of microglia, suggesting that RPTPß/ζ plays a role in the neurotrophic phenotype of microglia and in microglia-neuron communication.

Quantum-dot-based suspension microarray for multiplex detection of lung cancer markers: preclinical validation and comparison with the Luminex xMAP® system.

A novel suspension multiplex immunoassay for the simultaneous specific detection of lung cancer markers in bronchoalveolar lavage fluid (BALF) clinical samples based on fluorescent microspheres having different size and spectrally encoded with quantum dots (QDEM) was developed. The designed suspension immunoassay was validated for the quantitative detection of three lung cancer markers in BALF samples from 42 lung cancer patients and 10 control subjects. Tumor markers were detected through simultaneous formation of specific immune complexes consisting of a capture molecule, the target antigen, and biotinylated recognition molecule on the surface of the different QDEM in a mixture. The immune complexes were visualized by fluorescently labeled streptavidin and simultaneously analyzed using a flow cytometer. Preclinical validation of the immunoassay was performed and results were compared with those obtained using an alternative 3-plex immunoassay based on Luminex xMAP® technology, developed on classical organic fluorophores. The comparison showed that the QDEM and xMAP® assays yielded almost identical results, with clear discrimination between control and clinical samples. Thus, developed QDEM technology can become a good alternative to xMAP® assays permitting analysis of multiple protein biomarkers using conventional flow cytometers.

Pleiotrophin overexpression regulates amphetamine-induced reward and striatal dopaminergic denervation without changing the expression of dopamine D1 and D2 receptors: Implications for neuroinflammation.

It was previously shown that mice with genetic deletion of the neurotrophic factor pleiotrophin (PTN-/-) show enhanced amphetamine neurotoxicity and impair extinction of amphetamine conditioned place preference (CPP), suggesting a modulatory role of PTN in amphetamine neurotoxicity and reward. We have now studied the effects of amphetamine (10mg/kg, 4 times, every 2h) in the striatum of mice with transgenic PTN overexpression (PTN-Tg) in the brain and in wild type (WT) mice. Amphetamine caused an enhanced loss of striatal dopaminergic terminals, together with a highly significant aggravation of amphetamine-induced increase in the number of GFAP-positive astrocytes, in the striatum of PTN-Tg mice compared to WT mice. Given the known contribution of D1 and D2 dopamine receptors to the neurotoxic effects of amphetamine, we also performed quantitative receptor autoradiography of both receptors in the brains of PTN-Tg and WT mice. D1 and D2 receptors binding in the striatum and other regions of interest was not altered by genotype or treatment. Finally, we found that amphetamine CPP was significantly reduced in PTN-Tg mice. The data demonstrate that PTN overexpression in the brain blocks the conditioning effects of amphetamine and enhances the characteristic striatal dopaminergic denervation caused by this drug. These results indicate for the first time deleterious effects of PTN in vivo by mechanisms that are probably independent of changes in the expression of D1 and D2 dopamine receptors. The data also suggest that PTN-induced neuroinflammation could be involved in the enhanced neurotoxic effects of amphetamine in the striatum of PTN-Tg mice.

Genetic association study of systemic lupus erythematosus and disease subphenotypes in European populations.

Epidemiological studies suggest a strong contribution of genetic factors in the pathogenesis of systemic lupus erythematosus (SLE). In the last decades, many risk loci have been identified in several genetic association studies following both candidate gene and genome-wide approaches. The present work was conducted by GAPAID (Genes And Proteins for AutoImmunity Diagnostics) consortium with a dual aim: to replicate the association of several previously reported SLE susceptibility loci in an independent European sample and to explore their relation with some disease subphenotypes. A total of 48 single nucleotide polymorphisms (SNP) from 40 associated loci were typed in a cohort of 208 SLE patients and 152 controls from Rheumatology Units of the University Hospital of Pisa (Italy) and University of Pécs Medical Center (Hungary). Regression analyses were performed to detect disease susceptibility loci and to identify genes affecting specific disease manifestations (renal, neurological, or skin involvement; arthritis; secondary Sjögren syndrome; and secondary antiphospholipid syndrome). Association of previously described risk alleles from HLA locus has been replicated, while IRF5, BLK, ITGAM, and IRF8 loci have been found to be consistent with previous published results. In addition, two new subphenotype-specific associations have been detected: SNP rs5754217 (UBE2L3) with skin involvement and rs3093030 (ICAM1-ICAM4-ICAM5) with hematological disorders. Overall, results from GAPAID project are consistent with previously established associations for HLA, IRF5, BLK, ITGAM, and IRF8 SLE susceptibility loci and report for the first time two subphenotype-specific associations.

Serological and Genetic Evidence for Altered Complement System Functionality in Systemic Lupus Erythematosus: Findings of the GAPAID Consortium.

Systemic lupus erythematosus is a chronic autoimmune disease with multifactorial ethiopathogenesis. The complement system is involved in both the early and late stages of disease development and organ damage. To better understand autoantibody mediated complement consumption we examined ex vivo immune complex formation on autoantigen arrays. We recruited patients with SLE (n = 211), with other systemic autoimmune diseases (n = 65) and non-autoimmune control subjects (n = 149). Standard clinical and laboratory data were collected and serum complement levels were determined. The genotype of SNP rs1143679 in the ITGAM gene was also determined. Ex vivo formation of immune complexes, with respect to IgM, IgG, complement C4 and C3 binding, was examined using a functional immunoassay on autoantigen microarray comprising nucleic acids, proteins and lipids. Complement consumption of nucleic acids increased upon binding of IgM and IgG even when serum complement levels were decreased due to consumption in SLE patients. A negative correlation between serum complement levels and ex vivo complement deposition on nucleic acid autoantigens is demonstrated. On the contrary, complement deposition on tested protein and lipid autoantigens showed positive correlation with C4 levels. Genetic analysis revealed that the non-synonymous variant rs1143679 in complement receptor type 3 is associated with an increased production of anti-dsDNA IgG antibodies. Notwithstanding, homozygous carriers of the previously reported susceptible allele (AA) had lower levels of dsDNA specific IgM among SLE patients. Both the non-synonymous variant rs1143679 and the high ratio of nucleic acid specific IgG/IgM were associated with multiple organ involvement. In summary, secondary complement deficiency in SLE does not impair opsonization of nucleic-acid-containing autoantigens but does affect other antigens and potentially other complement dependent processes. Dysfunction of the receptor recognizing complement opsonized immune complexes promotes the development of class-switched autoantibodies targeting nucleic acids.

A new biomarker panel in bronchoalveolar lavage for an improved lung cancer diagnosis.

INTRODUCTION: The enormous biological complexity and high mortality rate of lung cancer highlights the need for new global approaches for the discovery of reliable early diagnostic biomarkers. The study of bronchoalveolar lavage samples by proteomic techniques could identify new lung cancer biomarkers and may provide promising noninvasive diagnostic tools able to enhance the sensitivity of current methods. METHODS: First, an observational prospective study was designed to assess protein expression differences in bronchoalveolar lavages from patients with (n = 139) and without (n = 49) lung cancer, using two-dimensional gel electrophoresis and subsequent protein identification by mass spectrometry. Second, validation of candidate biomarkers was performed by bead-based immunoassays with a different patient cohort (204 patients, 48 controls). RESULTS: Thirty-two differentially expressed proteins were identified in bronchoalveolar lavages, 10 of which were confirmed by immunoassays. The expression levels of APOA1, CO4A, CRP, GSTP1, and SAMP led to a lung cancer diagnostic panel that reached 95% sensitivity and 81% specificity, and the quantification of STMN1 and GSTP1 proteins allowed the two main lung cancer subtypes to be discriminated with 90% sensitivity and 57% specificity. CONCLUSIONS: Bronchoalveolar lavage represents a promising noninvasive source of lung cancer specific protein biomarkers with high diagnostic accuracy. Measurement of APOA1, CO4A, CRP, GSTP1, SAMP, and STMN1 in this fluid may be a useful tool for lung cancer diagnosis, although a further validation in a larger clinical set is required for early stages.