Neuropeptide Y inhibits cholangiocarcinoma cell growth and invasion

No information exists on the role of neuropeptide Y (NPY) in cholangiocarcinoma growth. Therefore, we evaluated the expression and secretion of NPY and its subsequent effects on cholangiocarcinoma growth and invasion. Cholangiocarcinoma cell lines and nonmalignant cholangiocytes were used to assess NPY mRNA expression and protein secretion. NPY expression was assessed by immunohistochemistry in human liver biopsies. Cell proliferation and migration were evaluated in vitro by MTS assays and matrigel invasion chambers, respectively, after treatment with NPY or a neutralizing NPY antibody. The effect of NPY or NPY depletion on tumor growth was assessed in vivo after treatment with NPY or the neutralizing NPY antibody in a xenograft model of cholangiocarcinoma. NPY secretion was upregulated in cholangiocarcinoma compared with normal cholangiocytes. Administration of exogenous NPY decreased proliferation and cell invasion in all cholangiocarcinoma cell lines studied and reduced tumor cell growth in vivo. In vitro, the effects of NPY on proliferation were blocked by specific inhibitors for NPY receptor Y2, but not Y1 or Y5, and were associated with an increase in intracellular d-myo-inositol 1,4,5-trisphosphate and PKCα activation. Blocking of NPY activity using a neutralizing antibody promoted cholangiocarcinoma growth in vitro and in vivo and increased the invasiveness of cholangiocarcinoma in vitro. Increased NPY immunoreactivity in human tumor tissue occurred predominantly in the center of the tumor, with less expression toward the invasion front of the tumor. We demonstrated that NPY expression is upregulated in cholangiocarcinoma, which exerts local control on tumor cell proliferation and invasion. Modulation of NPY secretion may be important for the management of cholangiocarcinoma.     

Combinatorial analysis and algorithms for quasispecies reconstruction using next-generation sequencing

Background  

Next-generation sequencing (NGS) offers a unique opportunity for high-throughput genomics and has potential to replace Sanger sequencing in many fields, including de-novo sequencing, re-sequencing, meta-genomics, and characterisation of infectious pathogens, such as viral quasispecies. Although methodologies and software for whole genome assembly and genome variation analysis have been developed and refined for NGS data, reconstructing a viral quasispecies using NGS data remains a challenge. This application would be useful for analysing intra-host evolutionary pathways in relation to immune responses and antiretroviral therapy exposures. Here we introduce a set of formulae for the combinatorial analysis of a quasispecies, given a NGS re-sequencing experiment and an algorithm for quasispecies reconstruction. We require that sequenced fragments are aligned against a reference genome, and that the reference genome is partitioned into a set of sliding windows (amplicons). The reconstruction algorithm is based on combinations of multinomial distributions and is designed to minimise the reconstruction of false variants, called in-silico recombinants.     

A major role for side-chain polyglutamine hydrogen bonding in irreversible ataxin-3 aggregation

The protein ataxin-3 consists of an N-terminal globular Josephin domain (JD) and an unstructured C-terminal region containing a stretch of consecutive glutamines that triggers the neurodegenerative disorder spinocerebellar ataxia type 3, when it is expanded beyond a critical threshold. The disease results from misfolding and aggregation, although the pathway and structure of the aggregation intermediates are not fully understood. In order to provide insight into the mechanism of the process, we monitored the aggregation of a normal (AT3Q24) ataxin-3, an expanded (AT3Q55) ataxin-3, and the JD in isolation. We observed that all of them aggregated, although the latter did so at a much slower rate. Furthermore, the expanded AT3Q55 displayed a substantially different behavior with respect to the two other variants in that at the latest stages of the process it was the only one that did the following: i) lost its reactivity towards an anti-oligomer antibody, ii) generated SDS-insoluble aggregates, iii) gave rise to bundles of elongated fibrils, and iv) displayed two additional bands at 1604 and 1656 cm(-1) in FTIR spectroscopy. Although these were previously observed in other aggregated polyglutamine proteins, no one has assigned them unambiguously, yet. By H/D exchange experiments we show for the first time that they can be ascribed to glutamine side-chain hydrogen bonding, which is therefore the hallmark of irreversibly SDS-insoluble aggregated protein. FTIR spectra also showed that main-chain intermolecular hydrogen bonding preceded that of glutamine side-chains, which suggests that the former favors the latter by reorganizing backbone geometry.     

Immunopathogenesis of primary biliary cirrhosis: an old wives' tale

Primary biliary cirrhosis (PBC) is a cholestatic liver disease characterised by the autoimmune destruction of the small intrahepatic bile ducts. The disease has an unpredictable clinical course, but may progress to fibrosis and cirrhosis. Although medical treatment with urseodeoxycholic acid is largely successful, some patients may progress to liver failure requiring liver transplantation. PBC is characterised by the presence of disease specific anti-mitochondrial (AMA) antibodies, which are pathognomonic for PBC development. The disease demonstrates an overwhelming female preponderance and virtually all women with PBC present in middle age. The reasons for this are unknown; however several environmental and immunological factors may be involved. As the immune systems ages, it become less self tolerant, and mounts a weaker response to pathogens, possibly leading to cross reactivity or molecular mimicry. Some individuals display immunological changes which encourage the development of autoimmune disease. Risk factors implicated in PBC include recurrent urinary tract infection in females, as well as an increased prevalence of reproductive complications. These risk factors may work in concert with and possibly even accelerate, immune system ageing, contributing to PBC development. This review will examine the changes that occur in the immune system with ageing, paying particular attention to those changes which contribute to the development of autoimmune disease with increasing age. The review also discusses risk factors which may account for the increased female predominance of PBC, such as recurrent UTI and oestrogens.     

Identification of New Autoantigens by Protein Array Indicates a Role for IL4 Neutralization in Autoimmune Hepatitis

Autoimmune hepatitis (AIH) is an unresolving inflammation of the liver of unknown cause. Diagnosis requires the exclusion of other conditions and the presence of characteristic features such as specific autoantibodies. Presently, these autoantibodies have relatively low sensitivity and specificity and are identified via immunostaining of cells or tissues; therefore, there is a diagnostic need for better and easy-to-assess markers. To identify new AIH-specific autoantigens, we developed a protein microarray comprising 1626 human recombinant proteins, selected in silico for being secreted or membrane associated. We screened sera from AIH patients on this microarray and compared the reactivity with that of sera from healthy donors and patients with chronic viral hepatitis C. We identified six human proteins that are specifically recognized by AIH sera. Serum reactivity to a combination of four of these autoantigens allows identification of AIH patients with high sensitivity (82%) and specificity (92%). Of the six autoantigens, the interleukin-4 (IL4) receptor fibronectin type III domain of the IL4 receptor (CD124), which is expressed on the surface of both lymphocytes and hepatocytes, showed the highest individual sensitivity and specificity for AIH. Remarkably, patients'' sera inhibited STAT6 phosphorylation induced by IL4 binding to CD124, demonstrating that these autoantibodies are functional and suggesting that IL4 neutralization has a pathogenetic role in AIH.Autoantibodies specific for proteins or nonprotein antigens (dsDNA, snRNP, carbohydrates) are often the serological hallmark of autoimmune diseases. Autoantibodies can be simply an epiphenomenon secondary to a chronic inflammatory milieu (1), but they can also play a direct pathogenetic role, as antithyroglobulin antibodies do in Hashimoto''s thyroiditis (2).Autoimmune hepatitis (AIH)¹ is a chronic necro-inflammatory disease of unknown etiology that affects predominantly women with an incidence of 1 to 2 per 100,000 per year and a prevalence of 10 to 20 out of 100,000 (3, 4). AIH is subdivided into two major types on the basis of autoantibody reactivity (5). Antibodies to nuclei and/or to smooth muscle characterize type 1 AIH, whereas antibodies to a liver-kidney microsomal constituent define patients with type 2 AIH. Because the detection of these autoantibodies is done by means of immunofluorescence on rodent multi-organ sections (liver, kidney, stomach), there are problems with the standardization and interpretation of the immunostaining patterns (6). To overcome these methodological problems, the International Autoimmune Hepatitis Group established an international committee to define guidelines and develop procedures and reference standards for more reliable testing (7, 8). Although ELISA and bead assays with purified or recombinant autoantigens are under development (9), they actually represent a complementary, rather than alternative, approach to traditional immunofluorescence. Moreover, serological overlap is frequently observed between AIH and other non-autoimmune liver diseases such as chronic viral hepatitis (10). Therefore, new, highly specific markers represent an unmet medical need for the more accurate diagnosis and classification of AIH.Besides the potential diagnostic application, the discovery of novel AIH autoantigens could provide insights into the disease pathogenicity mechanism. Although some AIH target-autoantigens have been identified and characterized, little is known about their pathogenetic role, and other autoantigens are probably still unknown. Autoantibodies, to be considered pathogenetic, must have at least two features: (i) the target-autoantigen should be either expressed on the plasma membrane of target cells or secreted by cells (i.e. should be exposed to autoantibodies), and (ii) binding of the autoantibodies to the target antigen should disturb a cellular function directly or indirectly. A possible pathogenetic role in AIH has been put forward for autoantibodies specific for cytochrome P450 2D6 (CYP2D6) or Asialoglycoprotein receptor 1 (AGPR-1), which are both present on the hepatocyte cell membrane (10).Protein microarrays are a powerful technology, as they allow the simultaneous screening of thousands of analytes (11). In the present study, to identify new autoantigens with potential diagnostic and/or pathogenetic roles in AIH, we printed a microarray with 1626 human proteins whose main feature was being either secreted or membrane associated (i.e. potentially exposed to autoantibody recognition). We used this microarray to screen panels of sera from patients with AIH and identified six new protein antigens that are recognized with high sensitivity and specificity. One of these six autoantigens is the interleukin-4 (IL4) receptor fibronectin type III (FNIII) domain of the IL4 receptor (CD124), and, interestingly, patients'' autoantibodies specific for CD124 neutralize IL4 signaling, suggesting a possible pathogenetic role for IL4 neutralization in AIH.     

The role of the central flexible region on the aggregation and conformational properties of human ataxin-3

Aggregation of human ataxin-3 (AT3) into amyloid fibrils is responsible for spinocerebellar ataxia type 3. This protein consists of a folded N-terminal domain (Josephin domain, residues 1-182), a central flexible region (residues 183-291), a poly-glutamine sequence of variable length and a short C-terminal flexible region. Very little is known about the influence of the central flexible region on the conformational and aggregation properties of this protein. The present study aimed to investigate the specific role of this portion of the protein (residues 183-291). Accordingly, protein fragments 1-182 (AT3/182) and 1-291 (AT3/291) were produced and compared by thioflavin-T fluorescence, Fourier transform infrared spectroscopy, CD, intrinsic fluorescence and ESI-MS. It is shown that the central flexible region enhances protein aggregation and can populate conformational states with different degrees of compactness. Both monomeric and dimeric partially-folded forms are identified for both protein fragments under denaturing conditions. Partially-folded monomers and dimers accumulate to a larger extent in AT3/291. These species represent good candidates for early intermediates of the aggregation process under the experimental conditions employed in the present study.     

Electrospray-ionization mass spectrometry as a tool for fast screening of protein structural properties

Since the early 1990s, electrospray-ionization mass spectrometry (ESI-MS) has encountered growing interest as a complementary tool to established biochemical and biophysical methods for investigating protein structure and conformation. Nowadays, applications of ESI-MS to protein investigation span from the area of analytical biochemistry to that of structural biology. This review focuses on applications of this technique to the analysis of protein conformational properties and molecular interactions, underscoring their possible relevance for molecular biotechnology, although representing a still very young field. An introductive section presents the major issues related to theoretical and technical aspects of ESI-MS under non-denaturing conditions. Examples from our work and from the literature illustrate which kind of information can be obtained concerning key issues in biotechnology such as stability and aggregation of proteins under both near-native and challenging conditions, and interactions with other proteins, ligands and cofactors.     

Energy balance,leptin, NEFA and IGF-I plasma concentrations and resumption of post partum ovarian activity in swedish red and white breed cows

In the purpose to provide further information in respect of the relationship between metabolism and post partum (PP) ovarian activity resumption in dairy cows, the aim of the present study was to characterize the energy balance (EB) and leptin, NEFA and IGF-I plasma levels in Swedish Red and White (SRW) cows with and without ovarian activity re-initiation within 7 weeks PP. The study was conducted on 12 primiparous SRW cows fed the same diet as total mixed ration for ad libitum intake. The EB was calculated weekly from parturition until seven weeks PP. Blood samples were collected weekly from one week before until 7 weeks after calving for leptin, NEFA and IGF-I analysis. For progesterone (P4) analysis, blood samples were collected two times per week from parturition until the end of the study. P4 profile was used in addition to the clinical examination to detect cows with and without ovarian activity resumption. The clinical and ultrasonographic examination, coupled with P4 profile analysis showed the resumption of ovarian activity within 7 weeks after calving in 8 (group A) and no ovarian resumption in 4 cows (group B). No significant differences were detected in the whole period of observation in the amount of milk production between the two groups, while the mean milk protein content was significantly lower in group B at the third week PP. The calculated EB was negative in both groups in the first three weeks after calving, but more marked in group B. NEFA and Leptin plasma levels did not show significant differences between the two groups. In conclusion, the results of the present study showed that, when low milk producing primiparous cows are concerned, no significant differences in BW loss, milk yield, EB and leptin and NEFA plasma levels between the cows with and without resumption of ovarian activity within 7 weeks post partum were seen. However, significantly higher IGF-I levels in the first two weeks after calving were found in cows with post partum ovarian activity resumption, highlighting the important role of IGF-I as sensitive signal between metabolism and reproduction.     

Characterization of the antibodies to p62 nucleoporin in primary biliary cirrhosis using human recombinant antigen

Reactivity of sera from patients with primary biliary cirrhosis (PBC) with a 60 kDa component of nuclear pore complexes (NPCs), purified by affinity chromatography on wheat-germ agglutinin (WGA)-Sepharose, was previously detected. Recently, clinical significance of the anti-NPC antibodies in PBC became evident. In the light of recent reports, indicating the correlation of the anti-NPC antibodies with severity and progression of the disease, the characterization of the reactive antigens is becoming essential in the clinical management of patients with PBC. Since accurate autoantibody detection represents one of the fundamental requirements for a reliable testing, we have generated a human recombinant p62 protein and validated an immunoprecipitation assay for the detection of anti-p62. We also demonstrated that the generated human recombinant p62 nucleoporin was modified by N-acetylglucosamine residues. More than 50% of tested PBC sera precipitated (35)S-radioactively labeled p62 recombinant nucleoporin and 40% recognized this recombinant antigen by immunoblotting. We compared the reactivity of PBC sera with rat and human nucleoporin. The incidence of anti-p62 nucleoporin positive PBC sera increased by 15% when human recombinant antigen was used. The titer of autoantibodies in p62-positive PBC samples strongly varied. Preadsorption of the PBC sera with p62 recombinant protein completely abolished their reactivity with the antigen. In conclusion, this study unequivocally proves that autoantibodies reacting with the 60 kDa component of NPCs target p62 nucleoporin and, more importantly, provide a better antigen source for future evaluations of the clinical role of anti-p62 in PBC.