Role of environmental factors in autoantibody production - importance of a detailed analysis in a small cohort

In the previous issue of Arthritis Research & Therapy, Muro and colleagues reported a detailed epidemiologic analysis in central Japan on one of the new myositis-specific autoantibodies to MDA-5 (melanoma differentiation-associated gene 5), which is associated with clinically amyopathic dermatomyositis accompanying interstitial lung disease. The increasing prevalence of anti-MDA-5, higher prevalence in small rural towns, and geographical clustering in two areas along the Kiso River suggest a role of environmental factors associated with rural communities or the river/water system or both. A detailed analysis of a small cohort may offer clues, which is ignored in multi-center studies, to the pathogenesis of systemic rheumatic diseases and autoantibody production.     

A New Entodiniomorphid Ciliate,Troglocorys cava n. g., n. sp., from the Wild Eastern Chimpanzee (Pan troglodytes schweinfurthii) from Uganda

ABSTRACT. Troglocorys cava n. g., n. sp. is described from the feces of wild eastern chimpanzee, Pan troglodytes schweinfurthii, in Uganda. This new species has a spherical body with a frontal lobe, a long vestibulum, a cytoproct located at the posterior dorsal side of the body, an ovoid macronucleus, a contractile vacuole near the cytoproct, and a large concavity on the left surface of the body. Buccal ciliature is non‐retractable and consists of three ciliary zones: an adoral zone surrounding the vestibular opening, a dorso‐adoral zone extending transversely at the basis of the frontal lobe, and a vestibular zone longitudinally extending in a gently spiral curve to line the surface of the vestibulum. Two non‐retractable somatic ciliary zones comprise arches over the body surface: a short dorsal ciliary arch extending transversely at the basis of the frontal lobe and a wide C‐shaped left ciliary arch in the left concavity. Because of the presence of three ciliary zones in the non‐retractable buccal ciliature, the present genus might be a member of the family Blepharocorythidae, but the large left concavity and the C‐shaped left ciliary arch are unique, such structures have never been described from other blepharocorythids.     

Strain persistence of invasive Candida albicans in chronic hyperplastic candidosis that underwent malignant change

Objectives: The aim of this study was to assess persistence and tissue invasion of Candida albicans strains isolated from a 65 year‐old patient with chronic hyperplastic candidosis (CHC), that subsequently developed into squamous cell carcinoma (SCC). Materials and Methods: C. albicans (n=7) were recovered from the oral cavity of the patient over seven years. Confirmation of CHC and SCC in this patient was achieved by histopathological examination of incisional biopsy tissue. DNA fingerprinting was performed on the seven isolates from the CHC patient together with a further eight isolates from patients with normal oral mucosa (n=2), chronic atrophic candidosis (n=1), SCC (n=1) and CHC (n=4). Genotyping involved the use of inter‐repeat PCR using the eukaryotic repeat primer 1251. Characterisation of the tissue invasive abilities of the isolates was achieved by infecting a commercially available reconstituted human oral epithelium (RHE; SkinEthic, Nice, France). After 24 h. C. albicans tissue invasion was assessed by histopathological examination. Results: DNA fingerprinting demonstrated strain persistence of C. albicans in the CHC patient over a seven year period despite provision of systemic antifungal therapy. The strain of C. albicans isolated from this patient was categorised as a high invader within the RHE compared to other isolates. Conclusions: Candidal strain persistence was evident in a patient with CHC over seven years. This persistence may be due to incomplete eradication from the oral cavity following antifungal therapy or subsequent recolonisation from other body sites or separate exogenous sources. The demonstration of enhanced in vitro tissue invasion by this particular strain may, in part, explain the progression to carcinoma.     

Immunological detection of NADH-specific enoyl-ACP reductase from rape seed (Brassica napus)--induction, relationship of alpha and beta polypeptides, mRNA translation and interaction with ACP

An antibody has been raised against rape seed enoyl-ACP reductase. This recognizes both the alpha and beta polypeptides of the enzyme. Immunoblotting of fresh seed demonstrates that beta is not present in seed material, and that it is produced by proteolysis during isolation. It is thus deduced that rape seed enoyl reductase is an alpha 4 homotetramer. Leaf material from both rape and Arabidopsis have an enoyl reductase with a similar electrophoretic mobility to the rape seed enzyme when analyzed on SDS-PAGE. Quantitative immunoassay has demonstrated that the enzyme continually increases during lipid deposition, indicating that an increase in this enzyme is required to sustain high levels of lipid biosynthesis. In vitro translation experiments show that the enzyme is nuclear coded and synthesized as a precursor form. Immunogold electron microscopy has demonstrated that enoyl reductase is located in plastids. It is shown that ACP-Sepharose may be used as a matrix in the purification of enoyl-ACP reductase.     

A method for the amidation of recombinant peptides expressed as intein fusion proteins in Escherichia coli

The increasing use of peptides as pharmaceutical agents, especially in the antiviral and anti-infective therapeutic areas, requires cost-effective production on a large scale. Many peptides need carboxy amidation for full activity or prolonged bioavailability. However, this modification is not possible in prokaryotes and must be done using recombinant enzymes or by expression in transgenic milk. Methods employing recombinant enzymes are appropriate for small-scale production, whereas transgenic milk expression is suitable for making complex disulfide-containing peptides required in large quantity. Here we describe a method for making amidated peptides using a modified self-cleaving vacuolar membrane ATPase (VMA) intein expression system. This system is suitable for making amidated peptides at a laboratory scale using readily available constructs and reagents. Further improvements are possible, such as reducing the size of the intein to improve the peptide yields (the VMA intein comprises 454 amino acids) and, if necessary, secreting the fusion protein to ensure correct N-terminal processing to the peptide. With such developments, this method could form the basis of a large-scale cost-effective system for the bulk production of amidated peptides without the use of recombinant enzymes or the need to cleave fusion proteins.     

A novel chimpanzee adenovirus vector with low human seroprevalence: improved systems for vector derivation and comparative immunogenicity

Recombinant adenoviruses are among the most promising tools for vaccine antigen delivery. Recently, the development of new vectors has focused on serotypes to which the human population is less exposed in order to circumvent pre-existing anti vector immunity. This study describes the derivation of a new vaccine vector based on a chimpanzee adenovirus, Y25, together with a comparative assessment of its potential to elicit transgene product specific immune responses in mice. The vector was constructed in a bacterial artificial chromosome to facilitate genetic manipulation of genomic clones. In order to conduct a fair head-to-head immunological comparison of multiple adenoviral vectors, we optimised a method for accurate determination of infectious titre, since this parameter exhibits profound natural variability and can confound immunogenicity studies when doses are based on viral particle estimation. Cellular immunogenicity of recombinant E1 E3-deleted vector ChAdY25 was comparable to that of other species E derived chimpanzee adenovirus vectors including ChAd63, the first simian adenovirus vector to enter clinical trials in humans. Furthermore, the prevalence of virus neutralizing antibodies (titre >1:200) against ChAdY25 in serum samples collected from two human populations in the UK and Gambia was particularly low compared to published data for other chimpanzee adenoviruses. These findings support the continued development of new chimpanzee adenovirus vectors, including ChAdY25, for clinical use.