The role of arginine in intestinal infection.

<p>An overview of the intestinal epithelial lining is given in B, with a villus and a crypt being depicted. Proliferating cells in the crypts migrate towards the tip of the villus, undergoing differentiation, and are shed at the tip of the villus after cell death. <i>Giardia</i> trophozoites sit all along the epithelial surface (with exception of the Paneth cells that are most down in the crypts) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045325#pone.0045325-Aley1" target="_blank">[47]</a>. A trophozoite attached to an IEC is shown in A. Dotted lines indicate molelcules released by the trophozoite. Arginine is actively taken up by the parasite and converted into CO₂, NH₃ and ornithine along the dihydrolase pathway, indicated by the enzymes ADI (arginine deiminase), OCT (ornithine carbamoyl transferase) and CK (carbamate kinase). Ornithine is released via an arginine-ornithine antiporter, blocks the cationic amino acid transporters (CAT) of IECs and thereby uptake of arginine. In addition, arginine is degraded extracellular by ADI and OCT released from <i>Giardia</i> upon interaction. All this leads to reduced arginine availability within the host IEC. As shown within this study, the consequences are reduced polyamine levels in IECs that result in cell cycle arrest via upregulation of cell cycle block genes (GADD45A, BTG3). This reduced cell proliferation in the crypts could lower intestinal cell turnover and reduce cell differentiation along the villus-axis, allowing the parasite to live in a more stable environment. The reduced arginine availability in IECs also leads to decreased production of the antimicrobial agent nitric oxide (NO) via nitric oxide synthases (NOS) in all cells of the intestinal epithelial lining.</p

Amino acid analysis of the medium of the interaction between IECs (Caco2 clone TC7) and <i>Giardia</i> trophozoites (isolate WB).

<p>Arbitrary units are used.</p

Effects of ADI on IEC numbers.

<p>Triplicates of IEC numbers were monitored by MTT assay. In A, IECs (Caco2 and HCT-8 cells), were treated with medium only (medium, white bars), with WB trophozoites (WB, black bars) or with WB trophozoites overexpressing ADI (WB-ADI, grey bars) and measured after 4 d. In B, two silver stained SDS-PAGE gels are shown visualizing the purified ADI and OCT at the expected Mw of 65 kDA and 40 kDA respectively (arrows). In C and D, TC7 and HCT-8 cells were treated with 27.5 U/L ADI from <i>Giardia</i> and measured after 4 d (TC7 cells) and 40 h (HCT-8 cells). ADI induced effects were abolished by addition of arginine to 0.4 mM (ADI +Arg). Corresponding controls are given (buffer; ADIb, which is the boiled protein control; Arg that shows that arginine addition alone does not affect the cells). Cell numbers are expressed as relative to buffer ctrl, P-values are displayed. Note the reduction in cell number upon addition of ADI.</p

mRNA expression of cell cycle regulatory proteins (BTG3, GADD45A) in HCT-8 cells upon parasite interaction.

<p>Expression levels are expressed in arbitrary units.</p

Growth curves of Caco2 cells in arginine-free medium compared to arginine- and citrulline-supplemented medium.

<p>Graphs are given for cells growing in complete medium (+Arg, rectangles), in medium without arginine (-Arg, circles) and arginine-free medium complemented with citrulline (+Citr, triangles). In A, cell numbers for proliferating Caco2 cells were measured by MTT assay; in B differentiated Caco2 cells were used. Samples were set up in triplicates, cell numbers expressed as relative to +Arg (d1). P-values calculated in comparison to complete medium values are shown for –Arg and for +Citr. After 3 d, growth was significantly reduced without arginine and citrulline could partially replace for the omitted arginine. In C, argininosuccinate synthase (ASS) mRNA expression in proliferating Caco2 cells at day 2 is displayed for the 3 different growth conditions, as assessed by qPCR in quadruplicates. Note the increase in ASS expression in the presence of citrulline.</p

DNA content profiles of HCT-8 cells upon <i>Giardia</i>-interaction.

<p>The DNA of HCT-8 cells was stained with propidium iodide and analyzed by flow cytometry. The solid lined histograms represent control cells, whereas the dotted lines show profiles of cells after 24 h (A, B and C) and 36 h (D, E and F) of interaction with WB trophozoites. A and D display controls with only water added; in B and E, arginine was added to 0.4 mM; in C and F, citrulline was added to 0.4 mM. Note the histogram shifts upon parasite addition that could be completely reverted by the addition of citrulline.</p

In vivo treatment of secondary <i>E. multilocularis</i> infected Balb/c mice with BTZ reduces parasite weight.

<p>Balb/c mice were i.p. infected with in vitro-cultured <i>E. multilocularis</i> parasite material. After 6 weeks, treatment was performed for 6 weeks (5 days a week) with 2×5 mice in each treatment group. Controls received honey/CMC p.o. and PBS i.p. ABZ received 200 mg/kg ABZ in honey/CMC and PBS i.p. BTZ received honey/CMC p.o. and 0.7 mg/kg BTZ i.p. once a week for three weeks, then 0.5 mg/kg twice a week for another three weeks. ABZ/BTZ received ABZ p.o. and BTZ i.p. as stated above. After euthanization parasite material was resected (A) and weighed (B). A, macroscopical assessment showed proliferating metacestode masses with many big and small vesicles (see black arrows) in the control group. All treated groups showed less metacestodes, especially less proliferating ones and more white and encapsulated, small cysts (see white arrows). B, parasite weight visualized by boxplot. Statistical analysis of log-transformed data confirmed a highly significant parasite mass reduction with ABZ and ABZ/BTZ treatment.</p

<i>In vivo</i> MMV665807 treatment in the secondary alveolar echinococcosis mouse model.

<p>(A) Balb/c mice were i.p. infected with metacestodes obtained from <i>in vitro</i> cultures. After 6 weeks of infection, mice were randomly allocated into 4 groups of 6 mice and treated p.o. during 4 weeks. The different treatment groups were: control (no treatment), oil (p.o. gavage of corn oil, 5 days per week), albendazole (ABZ, p.o. gavage of 200 mg/kg ABZ in corn oil, 5 days per week), MMV665807 (p.o. gavage of 100 mg/kg MMV665807 in corn oil, 5 days per week). At the endpoint, mice were euthanized and parasite weight determined. The only effective treatment reducing parasite weight significantly was ABZ. (B) in a second experiment, mice were infected accordingly, and i.p. injection treatment started after 2 weeks of infection. The treatment groups of 8 randomly allocated mice were as follows: control (i.p. injection of DMSO three times per week and p.o. gavage of corn oil 5 days per week), ABZ (i.p. injection of DMSO three times per week and p.o. gavage of 200 mg/kg ABZ in corn oil 5 days per week), MMV665807 (i.p. injection of 100 mg/kg MMV665807 three times per week and p.o. gavage of corn oil 5 days per week) and MMV665807/ABZ (i.p. injection of 100 mg/kg MMV665807 three times per week and p.o. gavage of ABZ in corn oil 5 days per week). After euthanasia, parasite weight was determined. Effective were treatments of ABZ and MMV665807/ABZ. The drug MMV665807 did not lead to any reduction in parasite growth. P values shown in (A) and (B) were calculated by the non-parametric Wilcoxon signed-rank test with Bonferroni-adjustment.</p

TEM of <i>E</i>. <i>multilocularis</i> metacestode exposed to 0.8 μM MMV665807 for 5 days.

<p>(A) and (B) are lower magnification views. LL = laminated layer, Te = tegument, uc = stem cell with large nucleus and nucleolus. Note largely translucent mitochondria in drug treated parasites (mi). Bars in (A) and (B) = 3 μm. (C) = higher magnification view of mitochondria lacking any clearly discernible internal structures. Also note the presence of clearly discernible accumulation of small vesicles within the LL in the close vicinity of the microtriches (arrows). The yellow arrows in (B) and (C) point towards the same location. Bar = 0.3 μm.</p

Activity of the most active drugs of the FDA library against <i>E. multilocularis</i> metacestodes in vitro.

<p>The seven most active drugs (more than 50% damage of the control DB1127 at 20 µM after 5 days; BTZ, sorafenib tosylate, crystal violet, candesartan cilexetil, nitazoxanide, amlodipine besylate and axitinib) of the FDA drug library screen were further tested in triplicates by PGI assay at different concentrations (20, 10, 5, 1, 0.1 µM) and for their anti-metacestode activity in vitro. As a positive control, DB1127 was applied at 20 µM and the different drug activities are expressed as percentage of the positive control. DMSO served as a negative control at same dilutions as the drugs and was subtracted from all values. Note the high activity of BTZ down to the concentration of 0.1 µM.</p