The accumulation of lactic acid and its influence on the growth ofPlasmodium falciparum in synchronized cultures

Summary Synchronization ofPlasmodium falciparum cultured in vitro results in a one-step growth pattern that allows the study of stage-specific metabolic activities of the parasites. Lactic acid (LA) was selected as a metabolic marker, and the concentration of this end product found in spent media was correlated with the different erythrocytic stages of the parasites. When the medium was changed at 12 h intervals, cultures containing predominantly trophozoites produced 3.66±0.55 μmol LA per 12 h per 10⁷ parasitized cells (n=26), an amount of LA that is about 8 to 20 times higher than that found in corresponding cultures containing predominantly ring forms. Depending on the stage of development, parasitized red blood cells produced between 5 and 100 times more LA than uninfected erythrocytes (3.72±0.62 μmol LA per 12 hours per 10⁹ red blood cells) (n=41) when cultured under identical conditions. The intraerythrocytic development of the parasites was not impaired by exposure to extracellular concentrations of LA up to 12 mM over a 12 h period. The growth resulting in such cultures was described as uninhibited and was characterized by a multiplication index of 10 or higher. Above the threshold of 12 mM of LA, progressive inhibition of parasite development occurred. The stage-specific LA production reported can be used to predict the amount of LA that will have accumulated at the end of a subsequent 12 h incubation period during synchronized in vitro growth ofPlasmodium falciparum. Using these values, it is possible to establish an optimal medium exchange schedule, thereby assuring uninhibited growth and a correspondingly high parasite yield. J. W. Z. was supported during part of this study by a long-term fellowship of the European Molecular Biology Organization, Heidelberg, West Germany, followed by a Research Associateship from the National Research Council, Washington, D.C. The project was supported by grants from the Medical Research Council to J. G. S. and by the Naval Research and Development Command, Work Unit No. 3M 162 770 A871 AE 312. The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the U.S. Navy Department or the naval service at large.     

Arsenate-induced renal agenesis in rats

The developmental origin of arsenate-induced renal agenesis was investigated. Pregnant Wistar rats were each injected once ip with 45 mg/kg sodium arsenate at day 10 (sperm day = day 0). Pregnancy was terminated at various times following injection and the embryos recovered and serially sectioned. Renal agenesis resulted when the mesonephric duct failded to give rise to a ureteric bud with subsequent failure of induction of the metanephric blastema. The underlying defect was retardation in growth of the mesonephric duct, first observed 48 hours after arsenate injection. A shortened mesonephric duct also resulted in a failure of the mesonephros to attain normal size and in the male resulted in absence of the ductus deferens, seminal vesicle a variable portion of the epididymis. Due to the intimate association of the mesonephric and growing paramesonephric ducts, a shortened mesonephric duct resulted in a shortened paramesonephric duct with resultant lack of a uterine horn.     

Invariant NKT cells exacerbate type 1 diabetes induced by CD8 T cells

Invariant NKT (iNKT) cells have been implicated in the regulation of autoimmune diseases. In several models of type 1 diabetes, increasing the number of iNKT cells prevents the development of disease. Because CD8 T cells play a crucial role in the pathogenesis of diabetes, we have investigated the influence of iNKT cells on diabetogenic CD8 T cells. In the present study, type 1 diabetes was induced by the transfer of CD8 T cells specific for the influenza virus hemagglutinin into recipient mice expressing the hemagglutinin Ag specifically in their beta pancreatic cells. In contrast to previous reports, high frequency of iNKT cells promoted severe insulitis and exacerbated diabetes. Analysis of diabetogenic CD8 T cells showed that iNKT cells enhance their activation, their expansion, and their differentiation into effector cells producing IFN-gamma. This first analysis of the influence of iNKT cells on diabetogenic CD8 T cells reveals that iNKT cells not only fail to regulate but in fact exacerbate the development of diabetes. Thus, iNKT cells can induce opposing effects dependent on the model of type 1 diabetes that is being studied. This prodiabetogenic capacity of iNKT cells should be taken into consideration when developing therapeutic approaches based on iNKT cell manipulation.     

Nationwide molecular surveillance of pandemic H1N1 influenza A virus genomes: Canada, 2009

Background

In April 2009, a novel triple-reassortant swine influenza A H1N1 virus (“A/H1N1pdm”; also known as SOIV) was detected and spread globally as the first influenza pandemic of the 21^st century. Sequencing has since been conducted at an unprecedented rate globally in order to monitor the diversification of this emergent virus and to track mutations that may affect virus behavior.

Methodology/Principal Findings

By Sanger sequencing, we determined consensus whole-genome sequences for A/H1N1pdm viruses sampled nationwide in Canada over 33 weeks during the 2009 first and second pandemic waves. A total of 235 virus genomes sampled from unique subjects were analyzed, providing insight into the temporal and spatial trajectory of A/H1N1pdm lineages within Canada. Three clades (2, 3, and 7) were identifiable within the first two weeks of A/H1N1pdm appearance, with clades 5 and 6 appearing thereafter; further diversification was not apparent. Only two viral sites displayed evidence of adaptive evolution, located in hemagglutinin (HA) corresponding to D222 in the HA receptor-binding site, and to E374 at HA2-subunit position 47. Among the Canadian sampled viruses, we observed notable genetic diversity (1.47×10⁻³ amino acid substitutions per site) in the gene encoding PB1, particularly within the viral genomic RNA (vRNA)-binding domain (residues 493–757). This genome data set supports the conclusion that A/H1N1pdm is evolving but not excessively relative to other H1N1 influenza A viruses. Entropy analysis was used to investigate whether any mutated A/H1N1pdm protein residues were associated with infection severity; however no virus genotypes were observed to trend with infection severity. One virus that harboured heterozygote coding mutations, including PB2 D567D/G, was attributed to a severe and potentially mixed infection; yet the functional significance of this PB2 mutation remains unknown.

Conclusions/Significance

These findings contribute to enhanced understanding of Influenza A/H1N1pdm viral dynamics.