Expression of cathelicidin mRNA in biopsies from the stomach.

<p>(A) Biopsies from the antrum of 10 German and 27 African patients were analyzed by real-time PCR for cathelicidin mRNA expression. (B) Biopsies from the corpus and fundus of 15 German and 11 African patients were analyzed as described under (A). Levels are normalized to glyceraldehydes-3-phosphate dehydrogenase (GAPDH). Data are expressed as ∼fold change in mRNA transcript levels relative to German subjects. Horizontal bars represent median cathelicidin expression.</p

Expression of cathelicidin mRNA in human duodenal biopsies.

<p>Duodenal biopsies from 9 German and 12 African patients were analyzed by real-time PCR for cathelicidin mRNA expression. Levels are normalized to glyceraldehydes-3-phosphate dehydrogenase (GAPDH). Data are expressed as ∼fold change in mRNA transcript levels relative to German subjects. Horizontal bars represent median cathelicidin expression.</p

Clinical data of patients.

<p>Clinical data of patients.</p

Expression of cathelicidin mRNA, interleukin-8 (IL-8) and beta-defensin 2 (hbd2) in biopsies.

<p>From the gastric antrum (A) and corpus/fundus (B) of African and German patients. Data were correlated with helicobacter pylori (HP) infection status. Levels are normalized to glyceraldehydes-3-phosphate dehydrogenase (GAPDH). Data are expressed as ∼fold change in mRNA transcript levels relative to German subjects. Horizontal bars represent median cathelicidin expression. Cathelicidin and hbd2 levels were significantly increased in HP positive compared with HP negative patients. The inflammatory status measured by IL-8 expression correlated well with the HP infection status.</p

Drug resistance test for <i>T</i>. <i>brucei</i> using sublethal concentrations of ethyl pyruvate and effect of pyruvate on cell proliferation.

<p>(A) Cells were treated with sub-lethal concentration of ethyl pyruvate, 1 mM and 2 mM, respectively, for 30 days in separate cell culture flasks. The medium and the trypanocidal compound were changed every two days. The control cells were left in fresh medium without ethyl pyruvate. On day 31, 2x104 cells/100μl from each flask were suspended a 96-well plate. Each cell group was treated with ethyl pyruvate at increasing concentrations ranging from 1 to 20 mM. Absorbance was recorded at 48 hrs of incubation. (B) The impact of pyruvate (0–20 mM) on cell proliferation was analysed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137353#pone.0137353.g001" target="_blank">Fig 1</a>.</p

Effect of ethyl pyruvate and ethyl lactate on T. brucei cells proliferation.

<p>A cell proliferation/viability assay was conducted in 96-well cell culture plates containing 2x104 cells, medium and ethyl pyruvate in a volume of 200 μl. After 24 hrs of incubation the AlamarBlue cell proliferation reagent (20 μl) was added and the absorbance was read: (A) Ethyl pyruvate; (B) Ethyl lactate; (C) For the time-dependency test 2x105 cells/ml were cultured in 24-well plates in the presence of different concentrations of ethyl pyruvate at 0 mM (■), 1 mM (▲), 2.5 mM (x) and 5 mM (□). After certain time intervals aliquots were removed and the number of vital cells was counted. (D) For cell-recovery test 2x105 cells/ml were cultured in the presence of ethyl pyruvate at 0 mM (■), 1 mM (▲), 2.5 mM (▼), 5 mM (□) for 3 hrs in 24-well plates. Then, the medium was removed and the cells were replenished with fresh medium without the ethyl pyruvate and further cultured for 24 hrs. At the indicated time points aliquots were removed and vital cells were counted. Cells were cultured at 37°C containing 5% CO2 in a 100% humidified environment, * p<0.05.</p

Effect of ethyl pyruvate on the activity of glycolytic enyzmes of <i>T</i>. <i>brucei</i>.

<p>Specific activity of glycolytic enzymes in <i>T</i>. <i>brucei</i> cell extracts in the absence and presence of 10 mM ethyl pyruvate. (A) hexokinase, (B) phosphofructokinase and (C) pyruvate kinase. All cell extracts were pre-treated with ethyl pyruvate for 30 min before activity testing. All experiments were performed in triplicates (n = 3), * p<0.05.</p

Cytotoxicity evaluation of ethyl pyruvate on human erythrocytes.

<p>(A) 6x10⁵ red blood cells were mixed to 2x10⁵<i>T</i>. <i>brucei</i> cells in 5 ml fresh medium in a flask. Cells were first incubated at 37°C in 5% CO₂ for 24 hrs. Out of this 1 ml-aliquots were distributed into 24-well plates and incubated with 100 μl of increasing concentrations of ethyl pyruvate (1–15 mM). Controls contained cells without ethyl pyruvate but 100 μl of fresh medium. The cells were then re-incubated for 3 hrs. Afterwards, the number of trypanosomes and human erythrocytes was counted using a haemocytometer. (B) Effect of ethyl pyruvate (10 mM) on red blood cell haemolysis was determined by measuring the optical density of the medium containing the <i>T</i>. <i>brucei</i> and red blood cells co-incubated for 3 hrs. Before measurement, the cells were spun down by centrifugation. Total haemolysis of an equivalent number of cells (labelled as ‘Control’) was achieved by sonication (70% power, 5x5 sec) using an ultrasonicater. Cells without ethyl pyruvate were used as negative controls (labelled as ‘Blank’).</p

Competitive inhibition of pyruvate kinase activity by ethyl pyruvate.

<p>Enzyme activity was measured by following the production of ATP from the substrates phosphoenolpyruvate (PEP) and ADP as described in Materials and Methods. Enzyme activity expressed as nmole ATP/μl/s (ordinate) was recorded in dependence of increasing substrate concentrations (PEP) in the absence and presence of ethyl pyruvate. Controls were (●) without ethyl pyruvate (K_<i>m</i> = 0.22±0.02 mM); (○) 5 mM ethyl pyruvate (K_<i>m</i> = 0.51±0.03mM); (◊) 10 mM ethyl pyruvate (K_<i>m</i> = 1±0.08mM) and (∆) 15 mM ethyl pyruvate (K_<i>m</i> = 1.4±0.08mM). The pure enzyme from rabbit muscle (200 U/mg) was used for the experimental setup to avoid contamination bias in our cell extract. The K<i>m</i> and K<i>i</i> values were calculated by SIGMAPLOT (Systat Softwarwe Inc.).</p

Anti-proliferative effects of pentamidine and suramin on <i>T</i>. <i>brucei</i>.

<p>Cell proliferation/viability was assayed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137353#pone.0137353.g001" target="_blank">Fig 1</a> with the exception of the use of (A) pentamidine and (B) suramin at variable concentrations. (C) Time dependency of anti-proliferative effects of pentamidine at variable concentrations (12.8 to 128 nM) was performed analogously to the experiments shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137353#pone.0137353.g001" target="_blank">Fig 1</a>. Control samples contained 100 μl of fresh medium instead of a trypanocidal compound. Pentamidine concentrations used were: 0 nM (■), 12.8 nM (▲), 51.2 nM (▼), 64 nM (♦) and 128 nM (●). (D) Recovery test of <i>T</i>. <i>brucei</i> cells exposed to pentamidine was done in 24-well in the presence of pentamidine (12.8 nM to 128 nM). Pentamidine concentrations used were: 0 nM (■), 12.8 nM (▲), 51.2 nM (▼), 64 nM (♦) and 128 nM (●).The results depict the mean of three independent measurements (n = 3), *p<0.05.</p