Detection of tissue remodelling by Fast Field-Cycling methods

Abstract 57Peer reviewedPublisher PD

Evolution of bacterial load over time after infection by <i>M</i>.<i>ulcerans</i>.

<p>ZN stainings of 5 micrometer thick slices of paraffin embedded footpad of a mouse infected <i>s</i>.<i>c</i>. with <i>M</i>. <i>ulcerans</i> NM20/02 4 weeks (upper), 10 weeks (middle) and 15 weeks (lower) after infection.</p

Influence of mouse strain on the outcome of <i>M</i>.<i>ulcerans</i> infection.

<p>A solution of 6.6x10⁴ CFU of <i>M</i>. <i>ulcerans</i> was injected in the footpad of C57Bl/6 (A) and Balb/cJ mice (B). Footpad pictures are shown at week 8 after infection (upper panel). ZN staining of 5μm thick histology slices of paraffin embedded footpads show infiltration area (blue nuclei—middle panel) and bacterial load (pink rod-shape bacilli—lower panel) 12 week after infection in C57Bl/6 (A) and Balb/c (B) mice.</p

Comparison of macroscopic signs of infection in mice inoculated by various <i>M</i>. <i>ulcerans</i> strains.

<p>Suspensions of <i>M</i>. <i>ulcerans</i> from seven strains from Ghana and Cameroon were injected subcutaneous (<i>s</i>.<i>c</i>.) in the footpad of C57Bl/6 mice. Footpad swelling at week 4 after infection is shown.</p

Infiltration of leukocytes within the footpad infected with <i>M</i>. <i>ulcerans</i>.

<p>Immunohistochemistry was performed on 5 micrometer paraffin embedded sections of footpad of mice infected with a suspension of <i>M</i>. <i>ulcerans</i> (NM20/02) in order to identify the nature of the leukocytic infiltrate present within and around the bacterial foci. B cells were stained with anti-CD45R antibody (A), T cells with anti-CD3 antibody (B), Macrophages with MOMA (C) and neutrophils with anti-Ly6G/Ly6C (D).</p

Lymph node cellularity of Balb/Jc vs C57Bl/6 mice.

<p>H&E staining was performed on 5 micrometer paraffin embedded sections of draining (right) and non-draining lymph nodes.</p

Macroscopic and microscopic signs of infection and inflammation in mice infected with <i>M</i>. <i>ulcerans</i>.

<p>The upper panel shows photograph of a C57Bl/6 mouse footpad 5 weeks after injection with <i>M</i>. <i>ulcerans</i> (NM20/02) (A) and the non-injected control footpad (B). Middle panel shows the corresponding 5micrometer thick paraffin embedded footpad slices stained with Ziehl-Neelsen (ZN) staining. Finally, the lower panel shows the same footpad slices stained with Hematoxylin Eosin (H&E). Arrows show the bacterial foci (ZN) and the inflamed tissue (H&E) of the lesion.</p

Origin and subculture history of <i>M</i>.<i>ulcerans</i> strains.

<p>Origin and subculture history of <i>M</i>.<i>ulcerans</i> strains.</p

Expression of <i>M</i>. <i>ulcerans</i> proteins by VRP-infected cells.

<p>(A) Genome maps of recombinant VSV: The genome of VSV encodes for the nucleoprotein N, phosphoprotein P, matrix protein M, glycoprotein G, and the large RNA polymerase L. In VSV*ΔG, the glycoprotein G coding region was replaced by the coding sequence for eGFP. In VSV*ΔG(MUL22332) and VSV*ΔG(MUL3720), the VSV G gene was replaced by the <i>M</i>. <i>ulcerans</i> genes MUL2232 and MUL3720, respectively. The reporter protein eGFP is encoded by an additional transcription cassette located downstream of the <i>M</i>. <i>ulcerans</i> genes. (B) BHK-21 cells were infected with the indicated replicons and indirect immunofluorescence analysis performed with mAbs specific for MUL2232 (B1) or MUL3720 (B2) (red fluorescence). The infection of cells with VRPs is indicated by eGFP expression (green fluorescence). Scale bar equals 30 μm. (C) Equal numbers of L929 fibroblasts were infected with the indicated VRPs at a MOI of 10. At 6 hours post infection, the cells were harvested and protein lysates of the soluble (s) and insoluble (i) fraction analysed by Western blotting for expression of <i>M</i>. <i>ulcerans</i> antigens using mAbs specific for MUL2232 (C1) and MUL3720 (C2), respectively.</p

Infection of immunized mice with <i>M</i>. <i>ulcerans</i>.

<p>Groups of six immunized, female BALB/c mice were infected by s.c. injection of <i>M</i>. <i>ulcerans</i> suspension (30 μl) into the left hind foot pad. (A) Development of the infection was followed by weekly measurements of the foot pad thickness with a caliper. The mean foot pad thickness (dot) ± standard deviation is shown for each animal group. (B) At day 60 after infection, mice were sacrificed and the number of <i>M</i>. <i>ulcerans</i> bacilli in foot pads determined by classical CFU plating. (C) Quantification of <i>M</i>. <i>ulcerans</i> in foot pad lysates by IS2404 specific qPCR. Genome equivalents are shown. (D) Determination of <i>M</i>. <i>ulcerans</i> genome equivalents in immunized and infected mice. * <i>p</i> ≤ 0.05 (Mann-Whitney test).</p