An LC-MS/MS approach to assess total and free protein target in the serum of cynomolgus monkey.

AIM: Develop LC-MS/MS-based assays to measure total and free complement C5 in cynomolgus monkey serum as a target engagement biomarker for pharmacokinetic/pharmacodynamic correlation study. Materials & methods/results: The C5-specific signature peptide derived from pellet digestion of serum proteins with and without prior immunodepletion of the drug-bound C5 by protein A beads was quantified to assess free and total C5 levels, respectively. Conditions for immunodepletion by protein A were optimized to ensure complete depletion of IgGs (and drug-bound C5). The effect of sample dilution on drug-target dissociation and thus free C5 measurement was evaluated by applying a mathematical simulation. CONCLUSION: The procedure described here allows for the assessment of protein target engagement, aiding in pharmacokinetic/pharmacodynamic correlation analysis and human dose projection.

Pompe disease in a Brazilian series: clinical and molecular analyses with identification of nine new mutations.

Pompe disease (glycogen storage disease type II or acid maltase deficiency) is an inherited autosomal recessive deficiency of acid alpha-glucosidase (GAA), with predominant manifestations of skeletal muscle weakness. A broad range of studies have been published focusing on Pompe patients from different countries, but none from Brazil. We investigated 41 patients with either infantile-onset (21 cases) or late-onset (20 cases) disease by muscle pathology, enzyme activity and GAA gene mutation screening. Molecular analyses identified 71 mutant alleles from the probands, nine of which are novel (five missense mutations c.136T > G, c.650C > T, c.1456G > C, c.1834C > T, and c.1905C > A, a splice-site mutation c.1195-2A > G, two deletions c.18_25del and c.2185delC, and one nonsense mutation c.643G > T). Interestingly, the c.1905C > A variant was detected in four unrelated patients and may represent a common Brazilian Pompe mutation. The c.2560C > T severe mutation was frequent in our population suggesting a high prevalence in Brazil. Also, eight out of the 21 infantile-onset patients have two truncating mutations predicted to abrogate protein expression. Of the ten late-onset patients who do not carry the common late-onset intronic mutation c.-32-13T > G, five (from three separate families) carry the recently described intronic mutation, c.-32-3C > A, and one sibpair carries the novel missense mutation c.1781G > C in combination with known severe mutation c.1941C > G. The association of these variants (c.1781G > C and c.-32-3C > A) with late-onset disease suggests that they allow for some residual activity in these patients. Our findings help to characterize Pompe disease in Brazil and support the need for additional studies to define the wide clinical and pathological spectrum observed in this disease.

UV inactivation of Cryptosporidium hominis as measured in cell culture.

The Cryptosporidium spp. UV disinfection studies conducted to date have used Cryptosporidium parvum oocysts. However, Cryptosporidium hominis predominates in human cryptosporidiosis infections, so there is a critical need to assess the efficacy of UV disinfection of C. hominis. This study utilized cell culture-based methods to demonstrate that C. hominis oocysts displayed similar levels of infectivity and had the same sensitivity to UV light as C. parvum. Therefore, the water industry can be confident about extrapolating C. parvum UV disinfection data to C. hominis oocysts.