Sensitivity to antifolates and genetic analysis of Plasmodium vivax isolates from Thailand

We investigated the association between the Plasmodium vivax dihydrofolate reductase (Pvdhfrtas) and the P. vivax dihydropteroate synthase (Pvdhps) genotype and in vitro sensitivity to the antifolates pyrimethamine, WR99210, chlorcycloguanil, sulfadoxine, and dapsone. Drug responses of 32 P. vivax isolates were assessed in two in vitro systems: schizont maturation inhibition and a yeast expression system. The geometric mean of 50% inhibition concentration (IC(50)) values for pyrimethamine, chlorcycloguanil, WR99210, sulfadoxine, and dapsone were 85 +/- 88, 784 +/- 662, 95 +/- 87, 2,424 +/- 2,784, and 1,625 +/- 1,801 nM, respectively, for the schizont maturation assay. Five different Pvdhfr alleles and four Pvdhps alleles were observed: 26 of 32 quadruple mutant alleles of Pvdhfr (F57I,L/S58R/T61M/S117T), four triple mutants (S58R/T61M/S117T, K49C/S58R/S117N), and two double mutant isolates (S58R/S117N). All isolates carried Pvdhps 585V. Twenty four isolates carried double mutant Pvdhps (A383G/A553G), six an additional mutation, S382A,C/A383G/A553G, and two a single mutation, A383G. Increasing geometric mean IC(50) values were observed with increased number of Pvdhfr mutations from double to quadruple. Results suggest that quadruple mutant alleles confer decreased sensitivity to pyrimethamine but retain sensitivity to WR99210.     

In vitro cytotoxicity of nelarabine, clofarabine and flavopiridol in paediatric acute lymphoblastic leukaemia

The in vitro efficacies of three new drugs--clofarabine (CLOF), nelarabine (NEL) and flavopiridol (FP) - were assessed in a panel of acute lymphoblastic leukaemia (ALL) cell lines. The 50% inhibitory concentration (IC50) for CLOF across all lines was 188-fold lower than that of NEL. B-lineage, but not T-lineage lines, were >7-fold more sensitive to CLOF than cytosine arabinoside (ARAC). NEL IC50 was 25-fold and 113-fold higher than ARAC in T- and B-lineage, respectively. T-ALL cells were eightfold more sensitive to NEL than B-lineage but there was considerable overlap. FP was more potent in vitro than glucocorticoids and thiopurines and at doses that recent phase I experience predicts will translate into clinical efficacy. Potential cross-resistance of CLOF, NEL and FP was observed with many front-line ALL therapeutics but not methotrexate or thiopurines. Methotrexate sensitivity was inversely related to that of NEL and FP. Whilst NEL was particularly effective in T-ALL, a subset of patients with B-lineage ALL might also be sensitive. CLOF appeared to be marginally more effective in B-lineage than T-ALL and has a distinct resistance profile that may prove useful in combination with other compounds. FP should be widely effective in ALL if sufficient plasma levels can be achieved clinically.     

Cost-Effective Algorithm for Detection and Identification of Vancomycin-Resistant Enterococci in Surveillance Cultures

 A study was undertaken to develop an easy-to-use and cost-effective algorithm for the detection and identification of vancomycin-resistant enterococci (VRE) in surveillance cultures, because the incidence of VRE outbreaks in institutions across Canada has made continuous surveillance a necessity. Enterococcus faecium and Enterococcus faecalis carry transferable resistance genes and hence are a problem for infection control. In laboratory surveillance, however, Enterococcus gallinarum and Enterococcus casseliflavus, which exhibit low-level nontransferable resistance, are also encountered and often create confusion in identification. Included in this study were a total of 218 strains of enterococci and other streptococci isolated from surveillance cultures. Conventional methods were used to determine their biochemical activities, and speciation was attempted in 121 strains using a rapid multiplex polymerase chain reaction (PCR) method that utilized primers for the vanA, B, C1, C2/C3 and ddl genes. The results indicated that by using only a few tests (Gram stain, pyrrolidonyl arylamidase activity, motility, xylose and methyl-α-D-glucopyranoside utilization), Enterococcus faecium/faecalis strains could be accurately differentiated from Enterococcus gallinarum/casseliflavus strains. For the 121 strains on which PCR was performed, there was a 100% correlation with the biochemical identification, with the added advantage that the presence of van genes could be determined at the same time. The cost of identification using minimal biochemical testing and PCR was less than that of identification using automated systems or a battery of conventional biochemical methods. The algorithm presented here may be used in the microbiology laboratory.     

Smac mimetic Birinapant induces apoptosis and enhances TRAIL potency in inflammatory breast cancer cells in an IAP-dependent and TNF-α-independent mechanism

X-linked inhibitor of apoptosis protein (XIAP), the most potent mammalian caspase inhibitor, has been associated with acquired therapeutic resistance in inflammatory breast cancer (IBC), an aggressive subset of breast cancer with an extremely poor survival rate. The second mitochondria-derived activator of caspases (Smac) protein is a potent antagonist of IAP proteins and the basis for the development of Smac mimetic drugs. Here, we report for the first time that bivalent Smac mimetic Birinapant induces cell death as a single agent in TRAIL-insensitive SUM190 (ErbB2-overexpressing) cells and significantly increases potency of TRAIL-induced apoptosis in TRAIL-sensitive SUM149 (triple-negative, EGFR-activated) cells, two patient tumor-derived IBC models. Birinapant has high binding affinity (nM range) for cIAP1/2 and XIAP. Using isogenic SUM149- and SUM190-derived cells with differential XIAP expression (SUM149 wtXIAP, SUM190 shXIAP) and another bivalent Smac mimetic (GT13402) with high cIAP1/2 but low XIAP binding affinity (K _d > 1 μM), we show that XIAP inhibition is necessary for increasing TRAIL potency. In contrast, single agent efficacy of Birinapant is due to pan-IAP antagonism. Birinapant caused rapid cIAP1 degradation, caspase activation, PARP cleavage, and NF-κB activation. A modest increase in TNF-α production was seen in SUM190 cells following Birinapant treatment, but no increase occurred in SUM149 cells. Exogenous TNF-α addition did not increase Birinapant efficacy. Neutralizing antibodies against TNF-α or TNFR1 knockdown did not reverse cell death. However, pan-caspase inhibitor Q-VD-OPh reversed Birinapant-mediated cell death. In addition, Birinapant in combination or as a single agent decreased colony formation and anchorage-independent growth potential of IBC cells. By demonstrating that Birinapant primes cancer cells for death in an IAP-dependent manner, these findings support the development of Smac mimetics for IBC treatment.     

Molecular Characterization of 2-Chlorobiphenyl Degrading Stenotrophomonas maltophilia GS-103

The catabolic potential of transformer oil contaminated soil bacteria in aerobic degradation of polychlorinated biphenyls (PCB) were assessed. Transformer oil contaminated soil sample was subjected to microcosm enrichment experiments (PAS medium/biphenyl as sole carbon source). PCB-degrading activity of the enrichment cultures in PAS medium with the addition of 2-chlorobiphenyl were analysed by GC–MS indicated that, although the isolates differed in PCB-degrading capabilities, all of the enrichment cultures expressed activity toward at least some of the lower chlorinated congeners. Biphenyl-utilizing bacteria isolated from the most active PCB-degrading mixed cultures showed little taxonomic diversity and identified as Stenotrophomonas maltophilia GS-103.     

Natural colonization and adaptation of a mosquito species in Galápagos and its implications for disease threats to endemic wildlife

Emerging infectious diseases of wildlife have been recognized as a major threat to global biodiversity. Endemic species on isolated oceanic islands, such as the Galápagos, are particularly at risk in the face of introduced pathogens and disease vectors. The black salt-marsh mosquito (Aedes taeniorhynchus) is the only mosquito widely distributed across the Galápagos Archipelago. Here we show that this mosquito naturally colonized the Galápagos before the arrival of man, and since then it has evolved to represent a distinct evolutionary unit and has adapted to habitats unusual for its coastal progenitor. We also present evidence that A. taeniorhynchus feeds on reptiles in Galápagos in addition to previously reported mammal and bird hosts, highlighting the important role this mosquito might play as a bridge-vector in the transmission and spread of extant and newly introduced diseases in the Galápagos Islands. These findings are particularly pertinent for West Nile virus, which can cause significant morbidity and mortality in mammals (including humans), birds, and reptiles, and which recently has spread from an introductory focus in New York to much of the North and South American mainland and could soon reach the Galápagos Islands. Unlike Hawaii, there are likely to be no highland refugia free from invading mosquito-borne diseases in Galápagos, suggesting bleak outcomes to possible future pathogen introduction events.