Diagnostic and prognostic value of antibodies against chimeric fibrin/filaggrin citrullinated synthetic peptides in rheumatoid arthritis

Introduction

Evidence suggests that citrullinated fibrin(ogen) may be a potential in vivo target of anticitrullinated protein/peptide antibodies (ACPA) in rheumatoid arthritis (RA). We compared the diagnostic yield of three enzyme-linked immunosorbent assay (ELISA) tests by using chimeric fibrin/filaggrin citrullinated synthetic peptides (CFFCP1, CFFCP2, CFFCP3) with a commercial CCP2-based test in RA and analyzed their prognostic values in early RA.

Methods

Samples from 307 blood donors and patients with RA (322), psoriatic arthritis (133), systemic lupus erythematosus (119), and hepatitis C infection (84) were assayed by using CFFCP- and CCP2-based tests. Autoantibodies also were analyzed at baseline and during a 2-year follow-up in 98 early RA patients to determine their prognostic value.

Results

With cutoffs giving 98% specificity for RA versus blood donors, the sensitivity was 72.1% for CFFCP1, 78.0% for CFFCP2, 71.4% for CFFCP3, and 73.9% for CCP2, with positive predictive values greater than 97% in all cases. CFFCP sensitivity in RA increased to 80.4% without losing specificity when positivity was considered as any positive anti-CFFCP status. Specificity of the three CFFCP tests versus other rheumatic populations was high (> 90%) and similar to those for the CCP2. In early RA, CFFCP1 best identified patients with a poor radiographic outcome. Radiographic progression was faster in the small subgroup of CCP2-negative and CFFCP1-positive patients than in those negative for both autoantibodies. CFFCP antibodies decreased after 1 year, but without any correlation with changes in disease activity.

Conclusions

CFFCP-based assays are highly sensitive and specific for RA. Early RA patients with anti-CFFCP1 antibodies, including CCP2-negative patients, show greater radiographic progression.     

Genome-Wide Screen of Genes Required for Caffeine Tolerance in Fission Yeast

Background

An excess of caffeine is cytotoxic to all eukaryotic cell types. We aim to study how cells become tolerant to a toxic dose of this drug, and the relationship between caffeine and oxidative stress pathways.

Methodology/Principal Findings

We searched for Schizosaccharomyces pombe mutants with inhibited growth on caffeine-containing plates. We screened a collection of 2,700 haploid mutant cells, of which 98 were sensitive to caffeine. The genes mutated in these sensitive clones were involved in a number of cellular roles including the H₂O₂-induced Pap1 and Sty1 stress pathways, the integrity and calcineurin pathways, cell morphology and chromatin remodeling. We have investigated the role of the oxidative stress pathways in sensing and promoting survival to caffeine. The Pap1 and the Sty1 pathways are both required for normal tolerance to caffeine, but only the Sty1 pathway is activated by the drug. Cells lacking Pap1 are sensitive to caffeine due to the decreased expression of the efflux pump Hba2. Indeed, ?hba2 cells are sensitive to caffeine, and constitutive activation of the Pap1 pathway enhances resistance to caffeine in an Hba2-dependent manner.

Conclusions/Significance

With our caffeine-sensitive, genome-wide screen of an S. pombe deletion collection, we have demonstrated the importance of some oxidative stress pathway components on wild-type tolerance to the drug.     

Ancestral Origin of the ATTCT Repeat Expansion in Spinocerebellar Ataxia Type 10 (SCA10)

Spinocerebellar ataxia type 10 (SCA10) is an autosomal dominant neurodegenerative disease characterized by cerebellar ataxia and seizures. The disease is caused by a large ATTCT repeat expansion in the ATXN10 gene. The first families reported with SCA10 were of Mexican origin, but the disease was soon after described in Brazilian families of mixed Portuguese and Amerindian ancestry. The origin of the SCA10 expansion and a possible founder effect that would account for its geographical distribution have been the source of speculation over the last years. To unravel the mutational origin and spread of the SCA10 expansion, we performed an extensive haplotype study, using closely linked STR markers and intragenic SNPs, in families from Brazil and Mexico. Our results showed (1) a shared disease haplotype for all Brazilian and one of the Mexican families, and (2) closely-related haplotypes for the additional SCA10 Mexican families; (3) little or null genetic distance in small normal alleles of different repeat sizes, from the same SNP lineage, indicating that they are being originated by a single step mechanism; and (4) a shared haplotype for pure and interrupted expanded alleles, pointing to a gene conversion model for its generation. In conclusion, we show evidence for an ancestral common origin for SCA10 in Latin America, which might have arisen in an ancestral Amerindian population and later have been spread into the mixed populations of Mexico and Brazil.     

One More Piece in the VACV Ecological Puzzle: Could Peridomestic Rodents Be the Link between Wildlife and Bovine Vaccinia Outbreaks in Brazil?

Background

Despite the fact that smallpox eradication was declared by the World Health Organization (WHO) in 1980, other poxviruses have emerged and re-emerged, with significant public health and economic impacts. Vaccinia virus (VACV), a poxvirus used during the WHO smallpox vaccination campaign, has been involved in zoonotic infections in Brazilian rural areas (Bovine Vaccinia outbreaks – BV), affecting dairy cattle and milkers. Little is known about VACV''s natural hosts and its epidemiological and ecological characteristics. Although VACV was isolated and/or serologically detected in Brazilian wild animals, the link between wildlife and farms has not yet been elucidated.

Methodology/Principal Findings

In this study, we describe for the first time, to our knowledge, the isolation of a VACV (Mariana virus - MARV) from a mouse during a BV outbreak. Genetic data, in association with biological assays, showed that this isolate was the same etiological agent causing exanthematic lesions observed in the cattle and human inhabitants of a particular BV-affected area. Phylogenetic analysis grouped MARV with other VACV isolated during BV outbreaks.

Conclusion/Significance

These data provide new biological and epidemiological information on VACV and lead to an interesting question: could peridomestic rodents be the link between wildlife and BV outbreaks?     

Rapid Differential Diagnosis between Extrapulmonary Tuberculosis and Focal Complications of Brucellosis Using a Multiplex Real-Time PCR Assay

Background

Arduous to differ clinically, extrapulmonary tuberculosis and focal complications of brucellosis remain important causes of morbidity and mortality in many countries. We developed and applied a multiplex real-time PCR assay (M RT-PCR) for the simultaneous detection of Mycobacterium tuberculosis complex and Brucella spp.

Methodology

Conventional microbiological techniques and M RT-PCR for M. tuberculosis complex and Brucella spp were performed on 45 clinical specimens from patients with focal complications of brucellosis or extrapulmonary tuberculosis and 26 control samples. Fragments of 207 bp and 164 bp from the conserved region of the genes coding for an immunogenic membrane protein of 31 kDa of B. abortus (BCSP31) and the intergenic region SenX3-RegX3 were used for the identification of Brucella and M. tuberculosis complex, respectively.

Conclusions

The detection limit of the M RT-PCR was 2 genomes per reaction for both pathogens and the intra- and inter-assay coefficients of variation were 0.44% and 0.93% for Brucella and 0.58% and 1.12% for Mycobacterium. M RT-PCR correctly identified 42 of the 45 samples from patients with tuberculosis or brucellosis and was negative in all the controls. Thus, the overall sensitivity, specificity, PPV and NPV values of the M RT PCR assay were 93.3%, 100%, 100% and 89.7%, respectively, with an accuracy of 95.8% (95% CI, 91.1%–100%). Since M RT-PCR is highly reproducible and more rapid and sensitive than conventional microbiological tests, this technique could be a promising and practical approach for the differential diagnosis between extrapulmonary tuberculosis and focal complications of brucellosis.     

PSSA-2, a Membrane-Spanning Phosphoprotein of Trypanosoma brucei,Is Required for Efficient Maturation of Infection

The coat of Trypanosoma brucei consists mainly of glycosylphosphatidylinositol-anchored proteins that are present in several million copies and are characteristic of defined stages of the life cycle. While these major components of the coats of bloodstream forms and procyclic (insect midgut) forms are well characterised, very little is known about less abundant stage-regulated surface proteins and their roles in infection and transmission. By creating epitope-tagged versions of procyclic-specific surface antigen 2 (PSSA-2) we demonstrated that it is a membrane-spanning protein that is expressed by several different life cycle stages in tsetse flies, but not by parasites in the mammalian bloodstream. In common with other membrane-spanning proteins in T. brucei, PSSA-2 requires its cytoplasmic domain in order to exit the endoplasmic reticulum. Correct localisation of PSSA-2 requires phosphorylation of a cytoplasmic threonine residue (T₃₀₅), a modification that depends on the presence of TbMAPK4. Mutation of T₃₀₅ to alanine (T₃₀₅A) has no effect on the localisation of the protein in cells that express wild type PSSA-2. In contrast, this protein is largely intracellular when expressed in a null mutant background. A variant with a T₃₀₅D mutation gives strong surface expression in both the wild type and null mutant, but slows growth of the cells, suggesting that it may function as a dominant negative mutant. The PSSA-2 null mutant exhibits no perceptible phenotype in culture and is fully competent at establishing midgut infections in tsetse, but is defective in colonising the salivary glands and the production of infectious metacyclic forms. Given the protein''s structure and the effects of mutation of T₃₀₅ on proliferation and localisation, we postulate that PSSA-2 might sense and transmit signals that contribute to the parasite''s decision to divide, differentiate or migrate.     

Gene Dosage Effects of the Imprinted Delta-Like Homologue 1 (Dlk1/Pref1) in Development: Implications for the Evolution of Imprinting

Genomic imprinting is a normal process that causes genes to be expressed according to parental origin. The selective advantage conferred by imprinting is not understood but is hypothesised to act on dosage-critical genes. Here, we report a unique model in which the consequences of a single, double, and triple dosage of the imprinted Dlk1/Pref1, normally repressed on the maternally inherited chromosome, can be assessed in the growing embryo. BAC-transgenic mice were generated that over-express Dlk1 from endogenous regulators at all sites of embryonic activity. Triple dosage causes lethality associated with major organ abnormalities. Embryos expressing a double dose of Dlk1, recapitulating loss of imprinting, are growth enhanced but fail to thrive in early life, despite the early growth advantage. Thus, any benefit conferred by increased embryonic size is offset by postnatal lethality. We propose a negative correlation between gene dosage and survival that fixes an upper limit on growth promotion by Dlk1, and we hypothesize that trade-off between growth and lethality might have driven imprinting at this locus.     

Effects of population size and forest management on genetic diversity and structure of the tuberous orchid Orchis mascula

Due to societal changes and altered demands for firewood, the traditional forest management of coppicing has been largely abandoned. As a result, many forest herbs that are specifically adapted to regular opening of the canopy, have suffered significant declines in abundance, and the remaining populations of these species often tend to be small and isolated. Reduced population sizes and pronounced spatial isolation may cause loss of within-population genetic diversity and increased between-population differentiation through random genetic drift and inbreeding. In this study, we investigated genetic diversity and genetic structure of 15 populations of the food-deceptive orchid Orchis mascula using AFLP markers. Within-population genetic diversity significantly increased with increasing population size, indicating genetic impoverishment in small populations. Genetic differentiation, on the other hand, was rather low (Φ_ST = 0.083) and there was no significant relationship between genetic and geographic distances, suggesting substantial gene flow within the study area. However, strong differences in levels of within-population diversity and among-population differentiation were found for populations located in forests that have been regularly coppiced and populations found in forests that were neglected for more than 50 years and that were totally overgrown by shrubs. Our data thus indicate that a lack of coppicing leads to decreased genetic diversity and increased differentiation in this orchid species, most likely as a result of genetic drift following demographic bottlenecks. From a conservation point of view, this study combined with previous results on the demography of O. mascula in relation to forest management illustrates the importance of coppicing in maintaining viable populations of forest herbs in the long-term.