Semiquantitative estimation of amyloid deposits in splenic squash preparations.

<p>A, grade 1. Only one or two amyloid particles are found in the slide. B, grade 2. Several small amyloid deposits are found. C, grade 3. More widely spread amyloid deposits often with large particles appear. D, grade 4. Extensive amyloid material all over the slide. Amyloid deposits are marked by arrows. Bar = 50 µm.</p

Small drops of aggregated synthetic peptides were dried on microscopical slides, labeled with thioflavin S and examined in a fluorescence microscope.

<p>The figure shows that peptides No. 5, 6, 7, 11 and 12 exhibited affinity for the dye and fluorescence. Peptide No. 10, although fibrillar by electron microscopy, did not show this amyloid-like property. Bar = 24 µm.</p

Amyloid deposits perifollicularly in the spleen.

<p>Typically, this is the first region where systemic AA amyloid is seen in the mouse. Bar = 50 µm.</p

All aggregated peptides, listed in Table 1 were examined in transmission electron microscopy.

<p>Peptides No. 4, 5, 6, 7, 10, 11 and 12 formed fibrillar assemblies while peptides No. 3, 8 and 9 gave rise to paper-like sheets. Bar = 200 nm.</p

Analysis of AEF activity in peripheral blood monocytes isolated from mice with AA-amyloid induced by AEF.

<p>AA-amyloid was induced in nine mice (G1–G9) by an i.v. injection of 0.1 ml AEF with concomitant s.c. injection of 0.2 ml 1% silver nitrate day 1, 7, 14, 21 and 28 and the mice were sacrificed on day 35. The presence of amyloid in the spleen was verified by Congo red staining. Peripheral blood monocytes were isolated, sonicated and re-introduced into the blood circulation of new groups of healthy mice (H1–H8). These mice received inflammatory stimuli day 1, 7 and 14 and were sacrificed day 16. The presence of amyloid was analysed in spleen sections after Congo red staining. Mice in group H9 received sonicated monocytes without subsequent inflammatory stimuli and group H10 received monocytes isolated from untreated mice and subsequent inflammatory stimuli on day 1, 7 and 14 and were sacrificed day 16 (group H10).</p

Description of antibodies used for the study.

<p>Description of antibodies used for the study.</p

Spleen amyloid deposits stained with Congo red.

<p>(A) The amyloid appears pink and is localized to the perifollicular zone. (B) The identical area exhibits green birefringence in polarized light. Amyloid is indicated by arrows.</p

Grading of spleen amyloid and quantification of MZMs during amyloid development.

<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079104#pone-0079104-g001" target="_blank">figure 1</a> for definition of groups. *p<0.05; ** p≤0.01; *** p≤0.001.</p><p>Presence of antibodies specific for ER-TR9.</p><p>ND = not determined, MZ = marginal zone and # = no marginal zones without amyloid.</p

Amyloid deposition was accompanied by changes of splenic macrophages.

<p>Group A untreated control, group F received AEF and AgNO₃ day 0 and was sacrificed day 4, group G received AEF and AgNO₃ day 0, additional injections of AgNO₃ on day 7 and 14, and was sacrificed day 16. In the left panel, a double arrow indicates a distance between RPMs and amyloid in group F, which does not persist in group G. The middle panel depicts MMZMs intermingling with amyloid in groups F (white arrow) and G. In the right panel, arrow indicates an onset loss of MZMs in group F, and complete loss of MZMs in marginal zones with extensive amyloid deposits in group G. Capital letter refers to experimental group, amyloid in red, macrophages in green,* indicate white pulp region, and bar = 100 µm.</p

Intracellular amyloid was detected in RPMs, MMZMs and MZMs.

<p>Frozen sections from group G treated with AEF and AgNO₃ on day 0 and additional injections of AgNO₃ on day 7 and 14, sacrificed on day 16 were immunolabelled with F4/80 to detect RPMs, with MOMA-1 to detect MMZMs or with ERTR-9 to detect MZMs. Amyloid was detected by incubation in Congo red solution B. Amyloid is showed in red, macrophages in green and white dotted line encircles amyloid containing cells. Bar = 20 µm.</p