Characterization and partial purification of a macrophage-stimulating factor from Mycoplasma hominis.

PROBLEM: Mycoplasma hominis is one of the most common pathogens of the genital tract and is associated with increased production of proinflammatory cytokines in reproductive tissues during preterm labor. The mechanism by which M. hominis, an organism lacking cell walls, increases the production of proinflammatory cytokines is unknown. METHOD OF STUDY: We characterized and purified a macrophage-activating factor from this organism. RESULTS: Extraction of whole organisms with Triton-X-114 demonstrated that the activity was primarily associated with the detergent phase. Macrophage-stimulating activity (MSA) of detergent extracts of M. hominis was not inhibited by polymyxin B or heating but was completely abrogated by alkaline hydrolysis and partially reduced by proteinase K digestion. Further experiments that utilized Toll-like receptor (TLR)-2- and TLR-4-transfected cells, revealed that the detergent extracts activate TLR-2 but not TLR-4 signal transduction. Purification of the activity using preparative SDS-PAGE and reverse phase chromatography experiments led to the isolation of a 29-kDa protein. CONCLUSIONS: These experiments suggest that the MSA of M. hominis is due to a lipophillic factor that interacts with TLR-2 rather than TLR-4 (as does lipopolysaccharide), to increase tumor necrosis factor (TNF)-alpha by macrophages. It is known that TNF-alpha can cause preterm labor and intrauterine fetal death and that it is upregulated in amniotic fluid samples infected with M. hominis.

Establishment of a chicken monoclonal antibody panel against mammalian prion protein.

A panel of chicken monoclonal antibodies (mAbs) was developed against prion protein (PrP), the sequence of which is a highly conserved molecule among mammals. A portion of the splenocytes from chickens immunized with recombinant mouse PrP was fused with the chicken B cell line, MuH1. The remaining splenocytes were used to generate the recombinant mAbs by phage display. A total of 36 anti-PrP mAbs, 2 from cell fusion and 34 from phage display were established. The specificity of these mAbs was determined by Western blot and ELISA using various PrP antigens including recombinant PrPs, synthetic PrP peptides and PrPs from brains or scrapie-infected neuroblastoma cell line. These mAbs were classified into three main groups, protease K (PK)-sensitive (Group I), PK cleavage site proximal (Group II) and PK-resistant (Group III), based on their abilities to recognize PrP following PK-treatment. Some mAbs were found to selectively recognize different glycoforms of PrP as well as the metabolic fragments of PrP. Furthermore, we found that PrP recognition by chickens differed from that by PrP-knockout mouse. These results indicate that these newly generated PrP antibodies from chickens will help to research the PrP and to establish the diagnosis of prion disease.

Copper chelation delays the onset of prion disease.

The prion protein (PrP) binds copper and under some conditions copper can facilitate its folding into a more protease resistant form. Hence, copper levels may influence the infectivity of the scrapie form of prion protein (PrPSc). To determine the feasibility of copper-targeted therapy for prion disease, we treated mice with a copper chelator, D-(-)-penicillamine (D-PEN), starting immediately following intraperitoneal scrapie inoculation. D-PEN delayed the onset of prion disease in the mice by about 11 days (p = 0.002), and reduced copper levels in brain by 29% (p < 0.01) and in blood by 22% (p = 0.03) compared with control animals. Levels of other metals were not significantly altered in the blood or brain. Modest correlation was observed between incubation period and levels of copper in brain (p = 0.08) or blood (p = 0.04), indicating that copper levels are only one of many factors that influence the rate of progression of prion disease. In vitro, copper dose-dependently enhanced the proteinase K resistance of the prion protein, and this effect was counteracted in a dose-dependent manner by co-incubation with D-PEN. Overall, these findings indicate that copper levels can influence the conformational state of PrP, thereby enhancing its infectivity, and this effect can be attenuated by chelator-based therapy.