Structural details and composition of Trichomonas vaginalis lipophosphoglycan in relevance to the epithelial immune function.

Trichomonas vaginalis causes the most common non-viral sexually transmitted infection linked to increased risk of premature birth, cervical cancer and HIV. This study defines molecular domains of the parasite surface glycoconjugate lipophosphoglycan (LPG) with distinct functions in the host immunoinflammatory response. The ceramide phospho-inositol glycan core (CPI-GC) released by mild acid had Mr of approximately 8,700 Da determined by MALDI-TOF MS. Rha, GlcN, Gal and Xyl and small amounts of GalN and Glc were found in CPI-GC. N-acetyllactosamine repeats were identified by endo-beta-galactosidase treatment followed by MALDI-MS and MS/MS and capLC/ESI-MS/MS analyses. Mild acid hydrolysis led to products rich in internal deoxyhexose residues. The CPI-GC induced chemokine production, NF-kappaB and extracellular signal-regulated kinase (ERK)1/2 activation in human cervicovaginal epithelial cells, but neither the released saccharide components nor the lipid-devoid LPG showed these activities. These results suggest a dominant role for CPI-GC in the pathogenic epithelial response to trichomoniasis.

Improving the efficacy and safety of biologic drugs with tolerogenic nanoparticles.

The development of antidrug antibodies (ADAs) is a common cause for the failure of biotherapeutic treatments and adverse hypersensitivity reactions. Here we demonstrate that poly(lactic-co-glycolic acid) (PLGA) nanoparticles carrying rapamycin, but not free rapamycin, are capable of inducing durable immunological tolerance to co-administered proteins that is characterized by the induction of tolerogenic dendritic cells, an increase in regulatory T cells, a reduction in B cell activation and germinal centre formation, and the inhibition of antigen-specific hypersensitivity reactions. Intravenous co-administration of tolerogenic nanoparticles with pegylated uricase inhibited the formation of ADAs in mice and non-human primates and normalized serum uric acid levels in uricase-deficient mice. Similarly, the subcutaneous co-administration of nanoparticles with adalimumab resulted in the durable inhibition of ADAs, leading to normalized pharmacokinetics of the anti-TNFα antibody and protection against arthritis in TNFα transgenic mice. Adjunct therapy with tolerogenic nanoparticles represents a novel and broadly applicable approach to prevent the formation of ADAs against biologic therapies.

Structure elucidation and biological activity of the oversulfated chondroitin sulfate contaminant in Baxter heparin.

From late December 2007 to February 2008, the number of adverse responses to heparin infusions rose noticeably above baseline levels in North America, ultimately resulting in a widespread recall of all heparin vial products made by Baxter Healthcare. Using various analytical techniques and the de novo synthesis of a fully sulfated chondroitin sulfate (FSCS) derivative, the authors have confirmed the identity of the contaminant as an oversulfated chondroitin sulfate (OSCS) and have also defined the heterogeneity and concentration of this contaminant in various lots of heparin. Using both contaminated heparin products and the synthetically produced derivative, the authors have shown that the OSCS produces a dose-dependent hypotension in both pigs and rats and that the response in rats can be abrogated with bradyzide, a rodent-selective B(2) bradykinin receptor antagonist. The no observed effect level (NOEL) for this contaminant appears to be approximately 1 mg/kg, corresponding to a contamination level in finished lots of heparin of approximately 3%. Using human plasma, the OSCS derivative was shown to activate kallikrein. These data provide insight into the etiology of the adverse events, particularly refractory hypotension, observed in patients who were exposed to heparin contaminated with OSCS.

Expression, purification, and characterization of rat interferon-beta, and preparation of an N-terminally PEGylated form with improved pharmacokinetic parameters.

To identify potential new clinical uses and routes of administration for human interferon-beta-1a (IFN-beta-1a), we have developed an expression and purification procedure for the preparation of highly purified rat interferon-beta (IFN-beta) suitable for testing in rat models of human disease. An expression vector containing the rat IFN-beta signal sequence and structural gene was constructed and transfected into Chinese hamster ovary (CHO) cells. The protein was purified from CHO cell conditioned medium and purified to > 99.5% purity using standard chromatographic techniques. Analytical characterization indicated that the protein was a heavily glycosylated monomeric protein, with two of the four predicted N-glycosylation sites occupied. Analysis of the attached oligosaccharides showed them to be a complex mixture of bi-antennary, tri-antennary, and tetra-antennary structures with a predominance of sialylated tri-antennary and tetra-antennary structures. Peptide mapping, N-terminal sequencing, and mass spectrometry confirmed the identity and integrity of the purified protein. The purified protein had a specific activity of 2.1x10(8)U/mg when assayed on rat RATEC cells, which is similar in magnitude to the potencies observed for murine IFN-beta and human IFN-beta-1a assayed on murine and human cells, respectively. We also prepared an N-terminally PEGylated form of rat IFN-beta in which a 20 kDa methoxy polyethylene glycol (PEG)-propionaldehyde was attached to the N-terminal alpha-amino group of Ile-1. The PEGylated protein, which retained essentially full in vitro antiviral activity, had improved pharmacokinetic parameters in rats as compared to the unmodified protein. Both the unmodified and PEGylated forms of rat IFN-beta will be useful for testing in rat models of human disease.