Fasciola hepatica in the rat:immune responses associated with the development of resistance to infection

F. hepatica infections were established in rats and immune responses were monitored during primary and challenge infections. Antibody levels peaked at 3 weeks post-primary infection and at 6 days post-challenge infection. No significant correlation was found between antibody titre and number of flukes recovered at autopsy. Immunoblotting revealed a limited number of immunogenic polypeptides. When antibodies from these reactive bands were eluted and tested by IFA they all gave identical binding patterns: on juvenile fluke sections tegumental syncytium, tegumental cells and gut cells were labelled, while on adult sections the same antibodies labelled gut cells, reproductive tissue, excretory ducts and flame cells. This suggested that these tissues shared a common epitope or range of epitopes. A pronounced eosinophilia was observed throughout the infection period studied and infected liver sections showed massive cellular infiltration. Histochemical and immunocytochemical investigation of infected liver revealed the presence of large numbers of eosinophils, neutrophils, lymphocytes and phagocytes. The implications of these findings, to an understanding of concomitant immunity in the rat are discussed.

Demonstration of antibodies in archival sera from Canadian seals reactive with a European isolate of phocine distemper virus

Sera from seals infected during the 1988 European epizootic of phocine distemper virus and sera from Canadian seals collected since 1972 have been tested for the presence of antibodies to morbillivirus. Approximately one third of the Canadian sera have been shown to contain anti-morbillivirus antibodies; the possibility that these populations of seals provided a source of infection for European seals is discussed.

Human B cell colony growth from pre-B cells in vitro

We describe a simple one-step technique for the growth of human B cell colonies in semi-solid agar in vitro. This method used conditioned medium from the human plasmacytoma cell line LICR-LON-H My 2 as a source of stimulating activity. A linear relationship exists between the number of B cells seeded and the number of colonies formed (r = 0.95). Most colony forming cells, approximately 1 in 500 of B cells seeded, lack surface immunoglobulin, possess Fc receptors and mark with the Leu 12 monoclonal antibody. Cells within developing colonies are found to have cytoplasmic IgM, IgA and IgG depending on the length of time in culture.

Determination of Spontaneous Mutation Frequencies in Measles Virus under Nonselective Conditions

There is a paradox between the remarkable genetic stability of measles virus (MV) in the field and the high mutation rates implied by the frequency of the appearance of monoclonal antibody escape mutants generated when the virus is pressured to revert in vitro (S. J. Schrag, P. A. Rota, and W. J. Bellini, J. Virol. 73: 51-54, 1999). We established a highly sensitive assay to determine frequencies of various categories of mutations in large populations of wild-type and laboratory-adapted MVs using recombinant viruses containing an additional transcription unit (ATU) encoding enhanced green fluorescent protein (EGFP). Single and double mutations were made in the fluorophore of EGFP to ablate fluorescence. The frequencies of reversion mutants in the population were determined by measuring the appearance of fluorescence indicating a revertant virus. This allows mutation rates to be measured under nonselective conditions, as phenotypic reversion to fluorescence requires only either a single-or a double-nucleotide change and amino acid substitution, which does not affect the length of the nonessential reporter protein expressed from the ATU. Mutation rates in MV are the same for wild-type and laboratory-adapted viruses, and they are an order of magnitude lower than the previous measurement assessed under selective conditions. The actual mutation rate for MV is approximately 1.8 x 10(-6) per base per replication event. Copyright © 2013, American Society for Microbiology. All Rights Reserved.

Prior Autoimmune Disease and Risk of Monoclonal Gammopathy of Undetermined Significance and Multiple Myeloma:A Systematic Review

Background: Several observational studies have investigated autoimmune disease and subsequent risk of monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma. Findings have been largely inconsistent and hindered by the rarity and heterogeneity of the autoimmune disorders investigated. A systematic review of the literature was undertaken to evaluate the strength of the evidence linking prior autoimmune disease and risk of MGUS/multiple myeloma.

Methods: A broad search strategy using key terms for MGUS, multiple myeloma, and 50 autoimmune diseases was used to search four electronic databases (PubMed, Medline, Embase, and Web of Science) from inception through November 2011.

Results: A total of 52 studies met the inclusion criteria, of which 32 were suitably comparable to perform a meta-analysis. “Any autoimmune disorder” was associated with an increased risk of both MGUS [n = 760 patients; pooled relative risk (RR) 1.42; 95% confidence interval (CI), 1.14–1.75] and multiple myeloma (n>2,530 patients; RR 1.13, 95% CI, 1.04–1.22). This risk was disease dependent with only pernicious anemia showing an increased risk of both MGUS (RR 1.67; 95% CI, 1.21–2.31) and multiple myeloma (RR 1.50; 95% CI, 1.25–1.80).

Conclusions: Our findings, based on the largest number of autoimmune disorders and patients with MGUS/multiple myeloma reported to date, suggest that autoimmune diseases and/or their treatment may be important in the etiology of MGUS/multiple myeloma. The strong associations observed for pernicious anemia suggest that anemia seen in plasma cell dyscrasias may be of autoimmune origin.

Impact: Underlying mechanisms of autoimmune diseases, general immune dysfunction, and/or treatment of autoimmune diseases may be important in the pathogenesis of MGUS/multiple myeloma. Cancer Epidemiol Biomarkers Prev; 23(2); 332–42. ©2014 AACR.

Mutations in the H, F, or M proteins can facilitate resistance of measles virus to neutralizing human anti-MV sera

Although there is currently no evidence of emerging strains of measles virus (MV) that can resist neutralization by the anti-MV antibodies present in vaccinees, certain mutations in circulating wt MV strains appear to reduce the efficacy of these antibodies. Moreover, it has been hypothesized that resistance to neutralization by such antibodies could allow MV to persist. In this study, we use a novel in vitro system to determine the molecular basis of MV's resistance to neutralization. We find that both wild-type and laboratory strain MV variants that escape neutralization by anti-MV polyclonal sera possess multiple mutations in their H, F, and M proteins. Cytometric analysis of cells expressing viral escape mutants possessing minimal mutations and their plasmid-expressed H, F, and M proteins indicates that immune resistance is due to particular mutations that can occur in any of these three proteins that affect at distance, rather than directly, the native conformation of the MV-H globular head and hence its epitopes. A high percentage of the escape mutants contain mutations found in cases of Subacute Sclerosing Panencephalitis (SSPE) and our results could potentially shed light on the pathogenesis of this rare fatal disease.

StaRProtein: a web server for prediction of stability of repeat proteins

Repeat proteins have become increasingly important due to their capability to bind to almost any proteins and the potential as alternative therapy to monoclonal antibodies. In the past decade repeat proteins have been designed to mediate specific protein-protein interactions. The tetratricopeptide and ankyrin repeat proteins are two classes of helical repeat proteins that form different binding pockets to accommodate various partners. It is important to understand the factors that define folding and stability of repeat proteins in order to prioritize the most stable designed repeat proteins to further explore their potential binding affinities. Here we developed distance-dependant statistical potentials using two classes of alpha-helical repeat proteins, tetratricopeptide and ankyrin repeat proteins respectively, and evaluated their efficiency in predicting the stability of repeat proteins. We demonstrated that the repeat-specific statistical potentials based on these two classes of repeat proteins showed paramount accuracy compared with non-specific statistical potentials in: 1) discriminate correct vs. incorrect models 2) rank the stability of designed repeat proteins. In particular, the statistical scores correlate closely with the equilibrium unfolding free energies of repeat proteins and therefore would serve as a novel tool in quickly prioritizing the designed repeat proteins with high stability. StaRProtein web server was developed for predicting the stability of repeat proteins.

Slug-dependent upregulation of L1CAM is responsible for the increased invasion potential of pancreatic cancer cells following long-term 5-FU treatment

BACKGROUND: Pancreatic adenocarcinoma is a lethal disease with 5-year survival of less than 5%. 5-fluorouracil (5-FU) is a principal first-line therapy, but treatment only extends survival modestly and is seldom curative. Drug resistance and disease recurrence is typical and there is a pressing need to overcome this. To investigate acquired 5-FU resistance in pancreatic adenocarcinoma, we established chemoresistant monoclonal cell lines from the Panc 03.27 cell line by long-term exposure to increasing doses of 5-FU.

RESULTS: 5-FU-resistant cell lines exhibited increased expression of markers associated with multidrug resistance explaining their reduced sensitivity to 5-FU. In addition, 5-FU-resistant cell lines showed alterations typical for an epithelial-to-mesenchymal transition (EMT), including upregulation of mesenchymal markers and increased invasiveness. Microarray analysis revealed the L1CAM pathway as one of the most upregulated pathways in the chemoresistant clones, and a significant upregulation of L1CAM was seen on the RNA and protein level. In pancreatic cancer, expression of L1CAM is associated with a chemoresistant and migratory phenotype. Using esiRNA targeting L1CAM, or by blocking the extracellular part of L1CAM with antibodies, we show that the increased invasiveness observed in the chemoresistant cells functionally depends on L1CAM. Using esiRNA targeting β-catenin and/or Slug, we demonstrate that in the chemoresistant cell lines, L1CAM expression depends on Slug rather than β-catenin.

CONCLUSION: Our findings establish Slug-induced L1CAM expression as a mediator of a chemoresistant and migratory phenotype in pancreatic adenocarcinoma cells.