Anti-osteosarcoma effect of antiserum against cross antigen TPD52 between osteosarcoma and Trichinella spiralis

Background Trichinella spiralis (T. spiralis) is a parasite occurring worldwide that has been proven to have antitumour ability. However, studies on the antitumour effects of cross antigens between the tumour and T. spiralis or antibodies against cross antigens between tumours and T. spiralis are rare. Methods To study the role of cross antigens between osteosarcoma and T. spiralis, we first screened the cDNA expression library of T. spiralis muscle larvae to obtain the cross antigen gene tumour protein D52 (TPD52), and prepared fusion protein TPD52 and its antiserum. The anti-osteosarcoma effect of the anti-TPD52 antiserum was studied using cell proliferation and cytotoxicity assays as well as in vivo animal models; preliminary data on the mechanism were obtained using western blot and immunohistochemistry analyses. Results Our results indicated that TPD52 was mainly localized in the cytoplasm of MG-63 cells. Anti-TPD52 antiserum inhibited the proliferation of MG-63 cells and the growth of osteosarcoma in a dose-dependent manner. The tumour inhibition rate in the 100 μg treatment group was 61.95%. Enzyme-linked immunosorbent assay showed that injection of anti-TPD52 antiserum increased the serum levels of IFN-γ, TNF-α, and IL-12 in nude mice. Haematoxylin and eosin staining showed that anti-TPD52 antiserum did not cause significant pathological damage. Apoptosis of osteosarcoma cells was induced by anti-TPD52 antiserum in vivo and in vitro. Conclusions Anti-TPD52 antiserum exerts an anti-osteosarcoma effect by inducing apoptosis without causing histopathological damage. Graphical abstract

Membrane-associated RING-CH (MARCH) proteins down-regulate cell surface expression of the interleukin-6 receptor alpha chain (IL6Rα)

Interleukin 6 (IL6) is a cytokine that regulates a number of important immune and inflammatory pathways. We used the ability of IL6 to inhibit the clonal proliferation of the mouse M1 myeloid leukemia cell line in agar to positively screen a cDNA expression library for proteins that inhibited IL6 activity. We found three clones completely resistant to IL6 that contained the cDNA for the Membrane-Associated RING-CH E3 ubiquitin ligase MARCH2. MARCH2 is a member of a family of membrane-bound E3 ubiquitin ligases that target cell surface receptors for degradation. MARCH2 overexpressing M1 clones retained responsiveness to the related cytokines leukemia inhibitory factor and oncostatin M and we showed that its inhibitory effect was a result of selective down-regulation of the IL6 receptor alpha chain and not the shared receptor subunit, gp130 or other signalling molecules. This activity of MARCH2 was also shared with related proteins MARCH4, MARCH9 and an isoform of MARCH3. The transmembrane domains and C-terminal domains, as well as a functional RING domain, of MARCH proteins were all required for substrate recognition and down-regulation. Genetic deletion of individual MARCH proteins in mice had no or little effect on IL6Rα levels but combined deletions of MARCH2,3 and 4 displayed elevated steady-state levels of IL6Rα in selected haemopoietic cell subsets including CD8+ and CD4+ T cells. These studies extend the potential immunosuppressive roles of MARCH proteins to include down-regulation of IL6 inflammatory responses.

A member of the HSP90 family from ovineBabesiain China: molecular characterization, phylogenetic analysis and antigenicity

SUMMARYHeat shock protein 90 (HSP90) is a key component of the molecular chaperone complex essential for activating many signalling proteins involved in the development and progression of pathogenic cellular transformation. AHsp90gene (BQHsp90) was cloned and characterized fromBabesiasp. BQ1 (Lintan), an ovineBabesiaisolate belonging toBabesia motasi-like group, by screening a cDNA expression library and performing rapid amplification of cDNA ends. The full-length cDNA ofBQHsp90is 2399 bp with an open reading frame of 2154 bp encoding a predicted 83 kDa polypeptide with 717 amino acid residues. It shows significant homology and similar structural characteristics toHsp90of other apicomplex organisms. Phylogenetic analysis, based on the HSP90 amino acid sequences, showed that theBabesiagenus is clearly separated from other apicomplexa genera. Five Chinese ovineBabesiaisolates were divided into 2 phylogenetic clusters, namelyBabesiasp. Xinjiang (previously designated a new species) cluster andB. motasi-like cluster which could be further divided into 2 subclusters (Babesiasp. BQ1 (Lintan)/Babesiasp. Tianzhu andBabesiasp. BQ1 (Ningxian)/Babesiasp. Hebei). Finally, the antigenicity of rBQHSP90 protein from prokaryotic expression was also evaluated using western blot and enzyme-linked immunosorbent assay (ELISA).