Some key facts and points on the vicious cycle of nutrition and infectious disease.

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The effect of IgMs in high-, intermediate- or low-virulent infections.

<p>BALB/c WT (□) and IgM^−/− (▴) mice were infected with (A/B) 5000 parasites of the pleomorphic AnTat 1.1E clone by intra-peritoneal injection, (C/D) non clonal <i>T. brucei</i> AnTaR 1 parasites through exposure to tsetse fly or (E/F) with 5000 parasites of the low virulent field isolate TSW196 by intra-peritoneal injection. Parasitemia and mortality of infected mice was assessed as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000122#ppat-1000122-g001" target="_blank">Fig. 1</a>, using 10 mice per experimental group. M.S. = median survival time.</p

Trypanosome infected IgM^−/− mice produce compensatory IgD titers.

<p>Sera were collected from <i>T. brucei</i> AnTat 1.1E infected WT and IgM^−/− BALB/c mice, and were analyzed as serial dilutions (log 10) in a solid phase VSG coated ELISA, using isotype-specific antibodies for detection. Sera were collected throughout the first peak and clearance phase from 5 individual mice. Values are presented as means.</p

The role of IgM in VSG-specific protection.

<p>(A) BALB/c WT (□), IgM^−/− (▴) and µMT (▪) mice were infected with 5000 pleomorphic AnTat 1.1E parasites. (B) BALB/c WT mice were infected with 5000 monomorphic AnTat 1.1 (•) or MITat 1.4 (▒) parasites. (C) AnTat 1.1E infected BALB/c WT (□) mice were super-infected (S.I.) on day 6 or (D) on day 10 with 5000 parasites of a homologous, monomorphic AnTat 1.1 (•) or a non-homologous monomorphic, MITat 1.4 (▒) strain. (E) AnTat 1.1E infected BALB/c µMT (□) and (F) BALB/c IgM^−/− (□) mice were super-infected with AnTat 1.1 (•) or MITat 1.4 (▒) parasites using the same strategy described above in C–D. (G) BALB/c WT (□) and BALB/c ^nu/nu (▾) mice were infected with 5000 AnTat 1.1E parasites and (H) were super-infected AnTat 1.1 (•) or MITat 1.4 (▒) parasites using the same strategy described above in C–D. All primary and super-infections were done by intra-peritoneal inoculation of 5000 parasites. Mortality was recorded using 10 mice per experimental group and the results were compared to mice that only received the primary infection. One out of 3 representative experiments is shown.</p

Quantitative analysis of VSG switching.

<p>Switching of VSG expression was followed by real time RT-PCR, by amplification of peak stage parasite RNA with primers specific for VSG AnTat 1.1E and the housekeeping genes tubulin-zeta (<i>Tubulin</i>). (A) RNA was isolated from WT derived and (B) µMT derived parasites on the first and second peak, respectively occurring on day 6 and 14. Expression of VSG AnTat 1.1E specific RNA on the second peak was calculated and compared to the first peak, after normalization of the first and second peak VSG AnTat 1.1E specific RNA to tubulin-zeta gene expression.. One representative of two experiments are shown. (C) Similarly, total VSG mRNA was amplified from first and second peak µMT derived parasites, using VSG-Uni primers, and results were normalized using <i>Tubulin</i> gene expression. (D) To assess parasite proliferation in µMT mice, the occurrence of dividing ‘long slender’ parasites versus intermediate and non-dividing ‘short stumpy’ parasites was analyzed by light microscopy on day 4 and compared to the chronic infection stage (up to day 22).</p

Trypanosomiasis-associated anemia is present in µMT mice.

<p>(A) C57BL/6 WT (○) and µMT (•) mice were infected with a pleomorphic <i>T. brucei</i> AnTat 1.1E clone by intra-peritoneal inoculation of 5000 parasites. (B) BALB/c WT (□), µMT (▪) and IgM^−/− (▴) mice were infected by intra-peritoneal inoculation of 5000 with the pleomorphic AnTat 1.1E clone. RBC counts were followed by microscopy analysis of tail-cut blood samples. Values are presented as mean±SD of 10 individual mice per group.</p

Avian influenza virus detection, temporality and co-infection in poultry in Cambodian border provinces, 2017–2018

Avian influenza virus detection, temporality and co-infection in poultry in Cambodian border provinces, 2017–201

Mmp14b is not involved in retinal development.

<p><b>A</b> RT-PCR analysis of Mmp14b-splice MO-injected embryos reveals efficient splice blocking, resulting in a dose-dependent increase in aberrantly spliced <i>mmp14b</i> mRNA. Since the splice MO targets intron 1 of <i>mmp14b</i>, which is 11 kb large, 2 different primer sets were used to visualize the splice blocking. Primer set F-R1 generates a fragment spanning exon 1 to 3 to detect correctly spliced transcripts (329 bp), and primer set F-R2 generates a fragment that spans exon 1 and part of intron 1 to detect aberrantly spliced transcripts (432 bp). The house keeping gene β-actin was used as a loading control. <b>B</b> Mmp14b knockdown using either the Mmp14b-ATG MO (5 ng) or the Mmp14b-splice MO (13 ng) in combination with the p53 MO does not affect normal embryonic morphogenesis at 5 dpf. <b>C–E</b> Quantitative analysis of eye size (<b>C</b>), total body length (<b>D</b>) and tectal area innervated by RGC axons (<b>E</b>) at 5 dpf, shows a normal eye diameter and tectal arborization area in Mmp14b morphant embryos using both the Mmp14b-ATG MO and Mmp14b-splice MO as compared to control embryos (<i>n</i> = 65 from 3 independent experiments)., <b>F</b> Combined knockdown of Mmp14a and Mmp14b (both injected at 2 ng) does not aggravate the eye defects observed after single Mmp14a knockdown in 3 dpf embryos (<i>n</i> = 70 from 3 independent experiments). Data are represented as mean ± SEM (*<i>p</i><0.05, Student's <i>t</i>-test). Dpf, days post fertilization; F, forward; MO, morpholino; R, reverse.</p

<i>In vivo</i> interaction between Mmp14a and Mmp2.

<p><b>A, B</b> Quantitative analysis of eye size (<b>A</b>) and tectal area innervated by RGC axons (<b>B</b>) after single or combined suboptimal knockdown of Mmp14a and Mmp2 in 5 dpf embryos reveals that suboptimal knockdown of Mmp2 does not affect eye size nor OT innervation. However, combined knockdown of Mmp14a and Mmp2 results in embryos with normal eye size but a significantly reduced RGC axon innervation area in the OT, as compared to the Mmp14a suboptimal knockdown embryos (<i>n</i> = 75 from 3 independent experiments). <b>C</b> Western blot for Mmp2 on extracts of control and Mmp14a morphant embryos at 30 hpf shows reduced levels of active Mmp2 protein (64 kDa) in Mmp14a morphant embryos. Total protein coomassie blue staining was used for loading control. Data are represented as mean ± SEM (*<i>p</i><0.05 versus controls using a Student's <i>t</i>-test; #<i>p</i><0.05 between 1 ng injections of Mmp14a-MO and Mmp14a+Mmp2-MO using a multilevel model statistical test (SAS proc mixed)). dpf: days post fertilization; hpf: hours post fertilization; MO: morpholino; OT: optic tectum; RGC: retinal ganglion cells.</p

Mmp14a knockdown results in an impaired retinotectal development.

<p><b>A</b> Graphical representation of the PLM from 15 minutes to 2.5 hours post-BrdU exposure at 31 hpf. The number of PH3⁺/BrdU⁺ retinoblasts is lower in retinas of Mmp14a morphant embryos as compared to control embryos, indicative for an impaired/prolonged transition from S to M phase at all time points examined (<i>n</i> = 10 from 2 independent experiments). Inset shows retinas of control embryos immunostained for BrdU (green) and PH3 staining (red) at 15 min and 2.5 h post-BrdU exposure. S-phase cells that have progressed from S to M phase are PH3⁺/BrdU⁺ (yellow). <b>B</b> Whole mount immunostaining for acetylated-α-Tubulin in 3 dpf embryos reveals a reduced tectal neuropil area (dotted circle), indicative for a reduced innervation of the OT by RGC axons. Images show a dorsolateral view of the left OT. <b>C–D</b> Quantitative analysis of the tectal area innervated by RGC axons reveals a dose-dependent decrease after Mmp14a knockdown using both the Mmp14a-ATG MO (<b>C</b>) and the Mmp14a-splice MO (<b>D</b>), as compared to control embryos at 5 dpf (<i>n</i> = 55 from 3 independent experiments). <b>E</b> Quantitative analysis of the tectal area innervated by RGC axons (in µm²) at different time-points between 3 and 10 dpf shows a significant and persistent reduction in RGC axon arborization area in Mmp14a morphant embryos as compared to controls (<i>n</i> = 55 from 3 independent experiments). <b>F</b> Knockdown of Mmp14a results in thinner optic nerves in 2 dpf transgenic <i>Tg(Ath5:GFP)</i> embryos, as compared to control embryos. Images show a ventral view on the optic chiasm (marked by white arrow). Data are represented as mean ± SEM (*<i>p</i><0.05, Student's <i>t</i>-test for <b>C–D</b>, multilevel model statistical test (SAS proc mixed) for <b>A</b>, <b>E</b>). A, anterior; dpf: days post fertilization; D, dorsal; OT: optic tectum; PLM, percent labeled mitosis; P, posterior; RGC: retinal ganglion cells; V, ventral.</p