Elucidation of the binding site of antagonists for the human chemokine receptor CCR2b

The present study focused his attention on the binding site of two small molecule antagonists of human CCR2b chemokine receptor. Chemokine receptors belong to the family of G protein-coupled receptors. To date, the chemokine system is made up by 50 ligands and 21 receptors, classified on the basis of the cystein pattern of the chemokines. CCR2 and its endogenous ligand CCL2 have been found to be involved in several diseases such as multiple sclerosis, rheumatoid arthritis, metabolic diseases, cancer and neuropathic pain. In these years different treatments have been proposed from mAb to small molecules. Unfortunately, no cures are available on the market yet. The aim of this project was to elucidate the binding site of two molecule antagonists of hCCR2 chemokine receptor: V-10-5191 and V-10-5299. Two different techniques of transfection in mammalian HEK-293 cells were performed: calcium phosphate and PEI transfection. Comparison between these two techniques leaded to the better expression of the receptors. Receptor expression was detected using 125I-CCL2, [3H]-V-10-5191 and [3H]-V-10-5299 in radioligand binding assays. Only the polycation PEI transfection method yielded CCR2 expression for which 125I-CCL2 binding could be detected. For this reason, the PEI method was chosen as a more efficient method to carry out following transfections using WT hCCR2b and in addition ten mutants of hCCR2b kindly provided by J. Peace. Receptor expression was detected using the radioligand binding assays mentioned previously. The analysis of the effects of these mutants on specific binding added information on the chemical interactions that occurs between the residues involved in the binding site and the three molecules. In conclusion these data could support further studies in the design of new antagonists of chemokine receptor CCR2, improving the knowledge on their binding site and clarify the pharmacological profile of the molecules already known

Parkinson’s Disease and Alpha-Synucleinopathies: from Arising Pathways to Therapeutic Challenge

Parkinson’s Disease (PD) and alpha synucleinopathies are multifactorial disorders, which manifest through motor symptoms and non-motor symptoms involving the Central Nervous System (CNS), the Peripheral Nervous System (PNS) and, recently, also the Enteric Nervous System (ENS). The typical hallmarks of alpha synucleinopathies are proteinaceous inclusions of alpha synuclein (αS). In PD they are known as Lewy Bodies (LBs) and Lewy Neurites (LNs), discovered in dopaminergic neurons of substantia nigra (pars compacta) as well as in other regions of the central and peripheral nervous systems. Despite the clear causes which lead to LBs/LNs are still unknown, according to Braak’s theory, these inclusions appear first in PNS to spread, following neuronal innervation, towards the CNS in a spatio- temporal dissemination described in a staging procedure. In line with these observations, several animal models have been used with the purpose to reproduce PD as well as to propose new therapeutic approaches. Different pathways can cooperate to neurodegeneration in PD such as genetic mutations of αS gene, mitochondrial dysfunctions, neuroinflammation. The present review highlights αS as the key-word for PD pathology and alpha synucleinopathies and a main target in PD research. Several therapeutic approaches can be proposed, however all of them are addressed in advanced stages of the pathology. Our focus will be the alteration of αS physiological pathway, which allows to address therapy in early stages at intracellular or extracellular level, such as the use of anti ER-stress compounds and innovative immunotherapy, which could be promising tools to reduce neuronal degeneration and to halt PD progression

Lisch nodules in Schwannomatosis: a new manifestation

Schwannomatosis is a syndrome characterized by presence of schwannomas in the absence of bilateral vestibular schwannomas and meningiomas. Schwannomas interest frequently peripheral nerves (90%) and spinal nerves (75%). Schwannomatosis are generally sporadic; in 15 - 25% are familiar. The genes involved are SMARCB1 (40-50% of familial) and LZTR1. The reported phenotype continues to expand and evolve. We report the case of a patient with Schwannomatosis and Lisch nodules, typical manifestation of NF1

Velusetrag rescues GI dysfunction, gut inflammation and dysbiosis in a mouse model of Parkinson's disease

: In patients with Parkinson's disease (PD), constipation is common, and it appears in a prodromal stage before the hallmark motor symptoms. The present study aimed to investigate whether Velusetrag, a selective 5‑HT4 receptor agonist, may be a suitable candidate to improve intestinal motility in a mouse model of PD. Five months old PrP human A53T alpha-synuclein transgenic (Tg) mice, which display severe constipation along with decreased colonic cholinergic transmission already at 3 months, were treated daily with the drug for 4 weeks. Velusetrag treatment reduced constipation by significantly stimulating both the longitudinal and circular-driven contractions and improved inflammation by reducing the level of serum and colonic IL1β and TNF-α and by decreasing the number of GFAP-positive glia cells in the colon of treated mice. No significant downregulation of the 5-HT4 receptor was observed but instead Velusetrag seemed to improve axonal degeneration in Tgs as shown by an increase in NF-H and VAChT staining. Ultimately, Velusetrag restored a well-balanced intestinal microbial composition comparable to non-Tg mice. Based on these promising data, we are confident that Velusetrag is potentially eligible for clinical studies to treat constipation in PD patients

Enteric α-synuclein impairs intestinal epithelial barrier through caspase-1-inflammasome signaling in Parkinson's disease before brain pathology

Bowel inflammation, impaired intestinal epithelial barrier (IEB), and gut dysbiosis could represent early events in Parkinson’s disease (PD). This study examined, in a descriptive manner, the correlation among enteric α-synuclein, bowel inflammation, impairments of IEB and alterations of enteric bacteria in a transgenic (Tg) model of PD before brain pathology. Human A53T α-synuclein Tg mice were sacrificed at 3, 6, and 9 months of age to evaluate concomitance of enteric inflammation, IEB impairments, and enteric bacterial metabolite alterations during the early phases of α-synucleinopathy. The molecular mechanisms underlying the interplay between α-synuclein, activation of immune/inflammatory responses and IEB alterations were investigated with in vitro experiments in cell cultures. Tg mice displayed an increase in colonic levels of IL-1β, TNF, caspase-1 activity and enteric glia activation since 3 months of age. Colonic TLR-2 and zonulin-1 expression were altered in Tg mice as compared with controls. Lipopolysaccharide levels were increased in Tg animals at 3 months, while fecal butyrate and propionate levels were decreased. Co-treatment with lipopolysaccharide and α-synuclein promoted IL-1β release in the supernatant of THP-1 cells. When applied to Caco-2 cells, the THP- 1-derived supernatant decreased zonulin-1 and occludin expression. Such an effect was abrogated when THP-1 cells were incubated with YVAD (caspase-1 inhibitor) or when Caco-2 were incubated with anakinra, while butyrate incubation did not prevent such decrease. Taken together, early enteric α-synuclein accumulation contributes to compromise IEB through the direct activation of canonical caspase-1-dependent inflammasome signaling. These changes could contribute both to bowel symptoms as well as central pathology.Bowel inflammation, impaired intestinal epithelial barrier (IEB), and gut dysbiosis could represent early events in Parkinson’s disease (PD). This study examined, in a descriptive manner, the correlation among enteric α-synuclein, bowel inflammation, impairments of IEB and alterations of enteric bacteria in a transgenic (Tg) model of PD before brain pathology. Human A53T α-synuclein Tg mice were sacrificed at 3, 6, and 9 months of age to evaluate concomitance of enteric inflammation, IEB impairments, and enteric bacterial metabolite alterations during the early phases of α-synucleinopathy. The molecular mechanisms underlying the interplay between α-synuclein, activation of immune/inflammatory responses and IEB alterations were investigated with in vitro experiments in cell cultures. Tg mice displayed an increase in colonic levels of IL-1β, TNF, caspase-1 activity and enteric glia activation since 3 months of age. Colonic TLR-2 and zonulin-1 expression were altered in Tg mice as compared with controls. Lipopolysaccharide levels were increased in Tg animals at 3 months, while fecal butyrate and propionate levels were decreased. Co-treatment with lipopolysaccharide and α-synuclein promoted IL-1β release in the supernatant of THP-1 cells. When applied to Caco-2 cells, the THP-1-derived supernatant decreased zonulin-1 and occludin expression. Such an effect was abrogated when THP-1 cells were incubated with YVAD (caspase-1 inhibitor) or when Caco-2 were incubated with anakinra, while butyrate incubation did not prevent such decrease. Taken together, early enteric α-synuclein accumulation contributes to compromise IEB through the direct activation of canonical caspase-1-dependent inflammasome signaling. These changes could contribute both to bowel symptoms as well as central pathology

Use of Leptospira spp. strains isolated in Brazil in the microscopic agglutination test applied to diagnosis of leptospirosis in cattle herds in eight brazilian states

Sarmento A.M.C., Azevedo S.S., Morais Z.M., Souza G.O., Oliveira F.C.S., Goncales A.P., Miraglia F. & Vasconcellos S.A. 2012. [Use of Leptospira spp. strains isolated in Brazil in the microscopic agglutination test applied to diagnosis of leptospirosis in cattle herds in eight brazilian states.] Emprego de estirpes Leptospira spp. isoladas no Brasil na microtecnica de soroaglutinacao microscopica aplicada ao diagnostico da leptospirose em rebanhos bovinos de oito estados brasileiros. Pesquisa Veterinaria Brasileira 32(7);601-606. Universidade de Sao Paulo, Faculdade de Medicina Veterinaria e Zootecnia, Departamento de Medicina Veterinaria Preventiva e Saude Animal, Av. Prof. Dr. Orlando Marques de Paiva 87, Sao Paulo, SP 05508-270, Brazil. E-mail: [email protected] The aim of this study was to investigate the adequacy of the use of autochthonous strains of leptospires isolated in Brazil, added to antigen collection of the microscopic agglutination test (MAT) applied to the diagnosis of bovine leptospirosis. By means of non-probability sampling, 109 farms and 9,820 cattle, females at reproductive age were chosen from 85 municipalities in the states of Goias, Mato Grosso, Mato Grosso do Sul, Minas Gerais, Parana, Rio Grande do Sul, Santa Catarina and Sao Paulo. Among the 9,820 examined animals, 5,806 (59.12%) were reactants at MAT for at least one serovar using the 23 reference serovars. Employing the collection of reference serovars and the ten autochthonous strains, 6,400 (65.24%) reactants and significant difference (p=0.001) was found. The most probable serovars identified by the collection of reference antigens were Hardjo (43.03%), Shermani (20%), Wolfi (9.96%), Grippothyphosa (5.42%) and Pomona (4.28%). With the collection amplified with the ten strains isolated in Brazil, the most probable serovars were Hardjo (31%), Guaricura-M4/84 (22.50%), Shermani (15.43%), Wolffi (4.76%), Grippothyphosa (3.71%) and Autumnalis (3.24%). The serovar Guaricura, strain M4/84, isolated from bovines and buffaloes in the State of Sao Paulo, was ranked as one of the three most probable serovars in the states of Goias, Mato Grosso, Mato Grosso do Sul, Minas Gerais and Sao Paulo. The addition of autochthonous strains to the MAT antigen collection provided the confirmation of the diagnosis of leptospirosis in 594 cattle (6%) which have been classified as non-reactants by the reference collection (p=0.001)

ERK1/2 phosphorylation is an independent predictor of complete remission in newly diagnosed adult acute lymphoblastic leukemia

Abstract Extracellular signal-regulated kinase-1/2 (ERK1/2) is frequently found constitutively activated (p-ERK1/2) in hematopoietic diseases, suggesting a role in leukemogenesis. The aim of this study was to assess the expression and clinical role of p-ERK1/2 in adult acute lymphoblastic leukemia (ALL). In 131 primary samples from adult de novo ALL patients enrolled in the Gruppo Italiano per le Malattie Ematologiche dell'Adulto (GIMEMA) Leucemia Acute Linfoide (LAL) 2000 protocol and evaluated by flow cytometry, constitutive ERK1/2 activation was found in 34.5% of cases; these results were significantly associated with higher white blood cell (WBC) values (P = .013). In a multivariate analysis, p-ERK1/2 expression was an independent predictor of complete remission achievement (P = .027). Effective approaches toward MEK inhibition need to be explored in order to evaluate whether this may represent a new therapeutic strategy for adult ALL patients