Redox active or thiol reactive? Optimization of rapid screens to identify less evident nuisance compounds

Compounds that exhibit assay interference or undesirable mechanisms of bioactivity are routinely encountered in assays at various stages of drug discovery. We observed that assays for the investigation of thiol-reactive and redox-active compounds have not been collected in a comprehensive review. Here, we review these assays and subject them to experimental optimization to improve their reliability. We demonstrate the usefulness of our assay cascade by assaying a library of bioactive compounds, chemical probes, and a set of approved drugs. These high-throughput assays should complement the array of wet-lab and in silico assays during the initial stages of hit discovery campaigns to pursue only hit compounds with tractable mechanisms of action.     

Characterization and phenotypic analysis of differentiating CD34+human bone marrow cells in liquid culture

Abstract: Our current understanding of human haematopoietic stem cell biology is based in part on the characterization of human CD34⁺ bone marrow cell differentiation in vitro. CD34 is highly expressed on early stem cells and haematopoietic progenitor cells with clonogenic potential and is gradually lost during differentiation and commitment. However, CD71 (transferrin receptor) is expressed at low levels on early stem cells and generally increases during haematopoietic progenitor cell proliferation. We reasoned that the combination of these surface markers would provide a useful framework for the simultaneous analysis of multiple lineage differentiation of CD34⁺ haematopoietic progenitor cells in liquid culture. In this report, we identify the phenotype of distinct subpopulations of myeloid, erythroid and lymphoid cells in liquid suspension culture using differential expression of CD34 vs. CD71 in combination with specific lineage markers. Freshly isolated human CD34⁺ bone marrow cells were introduced into suspension culture and monitored over a 6-d period using 3-colour flow cytometry. This is the first demonstration that differential expression of CD34 vs. CD71 can be used to simultaneously monitor differentiation of multiple haematopoietic cell lineages in liquid suspension culture, facilitating the study of cytokine-, drug- or chemical-induced alterations in haematopoietic progenitor cell differentiation in vitro.     

Separation of human basophils into two fractions with different density and histamine content

We designed experiments in this study to test the hypothesis suggested by recent purification data that blood basophils comprise two populations of different density, which circulate in numbers characteristic for each human subject. Basophils were separated into two density bands by single step centrifugation on a discontinuous Percoll gradient. Band 1 cells were at the interface between plasma and Percoll of density 1.070 gm/ml. Band 2 cells were at the Percoll 1.070 to 1.080 interface. When the number of band 1 basophils was expressed as a percentage of the total in bands 1 and 2, this relative amount generally remained in a narrow range for blood obtained from the same donor on 3 successive days but differed markedly in different individuals. In a series of leukapheresis experiments, we demonstrated that the percentage of band 1 basophils in postleukapheresis venous blood was strikingly similar to the preleukapheresis value. If basophils that repopulated the leukapheresis-depleted circulation came from the bone marrow, we can conclude that blood levels of basophils in bands 1 and 2 are under physiologic control and that the two types of basophils are released in amounts characteristic for each human subject. Additional evidence for two distinct blood basophil populations was provided by histamine measurements. The histamine content per basophil was consistently higher in cells from band 1 than from band 2, the mean difference between pairs of values for 30 subjects being 0.3 +/- 0.04 pg or about 27% of the band 1 basophil histamine content of 1.1 pg.     

Genetic and antigenic analyses of enterovirus 71 isolates in Taiwan during 1998–2005

Enterovirus 71 (EV71) infections can lead to devastating clinical outcomes in children, with an increasing number of severe cases worldwide. The genetic and antigenic variability of EV71 strains isolated in Taiwan in 1998-2005 was evaluated using partial nucleotide sequence analysis of the VP1 gene and the neutralisation assay. Phylogenetic analyses revealed that most EV71 isolates from the 1998 epidemic belonged to sub-genogroup C2, with a minority belonging to sub-genogroup B4. Between 1999 and 2003, isolates belonging to sub-genogroup B4 predominated, followed by a change to sub-genogroup C4 in 2004 and 2005. Antibodies raised in rabbits or collected from infected patients were able to neutralise EV71 virus stocks at high dilutions, regardless of the sub-genogroup of the virus being challenged. The presence of phylogenetically distinct yet antigenically similar populations of EV71 in Taiwan is of concern in the context of herd immunity and vaccine development.     

Interlaboratory validation of a CD71-based flow cytometric method (Microflow) for the scoring of micronucleated reticulocytes in mouse peripheral blood

An interlaboratory study was performed to validate an anti-CD71/flow cytometry-based technique for enumerating micronucleated reticulocytes (MN-RETs) in mouse peripheral blood. These experiments were designed to address International Workshop on Genotoxicity Test Procedures validation criteria by evaluating the degree of correspondence between MN-RET measurements generated by flow cytometry (FCM) with those obtained using traditional microscopy-based methods. In addition to these cross-methods data, flow cytometric MN-RET measurements for each blood sample were performed at two separate sites in order to evaluate the reproducibility of data between laboratories. In these studies, groups of male CD-1 mice were treated with vehicle (saline or vegetable oil), a negative control (saline or vegetable oil), or four dose levels of five known genotoxicants (clastogens: cyclophosphamide, benzo[a]pyrene, 5-fluorouracil, methotrexate; aneugen: vincristine sulfate). Exposure occurred on 3 consecutive days via intraperitoneal injection, and blood samples were obtained approximately 24 hr after the final treatment. MN-RET frequencies were determined for each sample based on the analysis of 2,000 (microscopy) and 20,000 (FCM) reticulocytes. Regardless of the method utilized, each genotoxic agent was observed to cause statistically significant increases in the frequency of MN-RETs, and each response occurred in a dose-dependent manner. Spearman's correlation coefficient (rs) for FCM versus microscopy-based MN-RET measurements (nine experiments, 252 paired measurements) was 0.740, indicating a high degree of correspondence between methods. The rs value for all flow cytometric MN-RET measurements performed at the two independent sites was 0.857 (n = 248), suggesting that the automated method is highly transferable between laboratories. Additionally, the flow cytometric system offered advantages relative to microscopy-based scoring, including a greater number of cells analyzed, much faster analysis times, and a greater degree of objectivity. Collectively, data presented in this report suggest that the overall performance of mouse peripheral blood micronucleus tests is enhanced by the use of the flow cytometric scoring procedure.     

Incidence and case-fatality rates resulting from the 1998 enterovirus 71 outbreak in Taiwan

In 1998, an epidemic of hand-foot-and-mouth disease and herpangina caused by enterovirus 71 occurred in Taiwan, leaving many fatalities and severely handicapped survivors in its wake. The reasons this rather common pathogen would cause such a large-scale epidemic remain unknown. A seroepidemiological survey to elucidate the epidemiological characteristics of this outbreak, including its incidence and case-fatality rates was undertaken. Microneutralization tests for antibodies against enterovirus 71 were used to screen four collections of serum samples: 1) 202 specimens taken from individuals > or = 4 years old in 1994; 2) 245 specimens collected from individuals of all ages in 1997; 3) 1,258 specimens collected from individuals of all ages in 1999; and 4) sera samples from a birth cohort of 81 children who had yearly blood samples taken from 1988-98. After the maternal antibody had declined, the seropositive rates began to increase with age. Approximately half of all children aged 6 years or older were enterovirus 71 seropositive. Significantly higher seropositive rates were noted in 1999 than in 1997, in children aged 0.5-3 years. The incidence of enterovirus 71 infection during the epidemic was estimated to be 13-22%, with the higher rates in younger children. The case-fatality rate was highest (96.96 per 100,000) in infants aged 6-11 months, and declined in older children. The results showed that enterovirus 71 is endemic in Taiwan. The apparent lack of large-scale enterovirus 71 activity in the 3 years before 1998 might have been the prelude to the epidemic's appearance in 1998, and might suggest that enterovirus 71 infection will reappear every few years. The lack of a protective antibody in younger children may account for the high incidence and case-fatality rate in this age group.     

RNA interference against enterovirus 71 infection

Enterovirus 71 (EV71) is a highly infectious major causative agent of hand, foot, and mouth disease (HFMD) which could lead to severe neurological complications. There is currently no effective therapy against EV71. In this study, RNA interference (RNAi) is employed as a therapeutic approach for specific viral inhibition. Various regions of the EV71 genome were targeted for inhibition by chemically synthesized siRNAs. Transfection of rhabdomyosarcoma (RD) cells with siRNA targeting the 3'UTR, 2C, 3C, or 3D region significantly alleviated cytopathic effects of EV71. The inhibitory effect was dosage-dependent with a corresponding decrease in viral RNA, viral proteins, and plaque formations by EV71. Viral inhibition of siRNA transfected RD cells was still evident after 48 h. In addition, no significant adverse off-target silencing effects were observed. These results demonstrated the potential and feasibility for the use of siRNA as an antiviral therapy for EV71 infections.