Analysis of AA/SAA reactivity in peripheral blood monocytes by confocal microscopy showed immunoreactivity in 5% of the monocytes isolated from a mouse with AA-amyloidosis (A).

<p>There was no reactivity present in monocytes recovered from a mouse given one AgNO₃ injection 48 hrs prior to isolation (B) or in monocytes isolated from untreated mice (C). The used rabbit antiserum recognizes both protein AA and SAA and was visualized by goat anti rabbit Alexa488-cojugated IgG. Cell nuclei were labeled with TO-PRO3. Bar 10 um.</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

Analysis of AEF activity in peripheral blood monocytes isolated from mice with AA amyloidosis.

<p>The table presents detailed information on animals in group H 1–8. AA-amyloidosis was induced by i.v. injection of AEF and 0.2 ml 1% silver nitrate injections on day 1, 7, 14, 21 and 28. The animals were sacrificed on day 35. Blood was collected and amyloid was verified in spleen sections after Congo red staining. Isolated peripheral blood monocytes were injected into new animals, group H 1–8, and silver nitrate was given day 1, 7 and 14.The animals were sacrificed day 16 and the presence of amyloid was studied in spleen after Congo red staining.</p

SAA 1, SAA 2 and SAA 3 mRNA expression in peripheral blood monocytes were analysed with PCR.

<p>Cells were isolated from mice that developed AA-amyloid after AEF and AgNO₃ injections or from mice that received AEF or AgNO₃ injections only or from untreated mice. Expression of the amyloid-prone SAA 1 or non-amyloidogenic SAA 2 was absent in all monocyte preparations. SAA 3 mRNA was detected in all cells independent of treatment. Mouse liver cDNA was used as a positive control. The PCR products were separated on a 1.6% agarose gel.</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

PrP^Sc is present both in and around MadCam1-positive HEVs in <i>TNFR1^−/−</i>-Ig mesenteric lymph nodes.

<p>TNFR1^−/− mice inoculated i.p. with 6 log LD₅₀ RML6 and treated weekly with control Ig were sacrificed at 60 d.p.i. Immunofluorescence (A–I) and histoblots (J & K) were then performed on frozen sections from prion-infected TNFR1^−/−-Ig mesenteric lymph nodes. Co-IF with anti-serum (XN) against PrP (green; A) and MadCam1 (red; B) showed points of intense PrP immunoreactivity localized to HEVs (C). Confocal co-IF with the amyloid-binding dye, p-FTAA (green; D) and MadCam1 (red; E,F) revealed some points of PrP^Sc association with HEVs (F); however much of the PrP^Sc was present outside of HEVs (I). Histoblots pre-stained with PNAd antibody and developed with AP (pink; J) also revealed some prion-infected HEVs (black arrows), some non-infected HEVs (white arrow), and some PrP^Sc deposits that were not HEV-associated (yellow arrow). (K) Total numbers of PNAd-positive HEVs in histoblot co-stains were counted and scored as PrP^Sc-positive (PrP^Sc+; black) or PrP^Sc-negative (PrP^Sc+; white), and total PrP^Sc deposits were counted and scored as PNAd-positive (PNAd+; black) or PNAd-negative (PNAd; white). 35% of HEVs were PrP^Sc-positive, and 58% of PrP^Sc deposits were PNAd-positive. Size bars in A–F = 50 µm. Size bars in G–I = 100 µm.</p

Primer sequences used for Real Time PCR analysis.

<p>Primer sequences used for Real Time PCR analysis.</p

PrP^Sc accumulation in <i>TNFR1^−/−</i> lymph nodes requires LTβR signaling independent of <i>Prnp</i> expression.

<p>C57BL/6 (WT) or TNFR1^−/− mice inoculated i.p. with 6 log LD₅₀ RML6 and treated weekly with control Ig or LTβR-Ig were sacrificed at 60 d.p.i. Histoblots were performed on frozen sections from spleens (SPL; A–D) or mesenteric lymph nodes (mLN; E–H) from mice in each treatment group to visualize PrP^Sc deposition. Whole organs are shown in left panels, and corresponding higher resolution images for each treatment group are shown in right panels. Note that lack of TNFR1 signaling can prevent PrP^Sc accumulation in spleen (B) but not lymph node (F). However, blocking LTβR signaling can prevent PrP^Sc accumulation in TNFR1^−/− lymph nodes (compare F and H). Prion infectivity titers in mLN homogenates from individual TNFR1^−/− Ig-treated and LTβR-Ig treated mice were measured using the scrapie cell assay (I). Whereas TNFR1^−/−-Ig mLNs all harbored ≥6.1 log TCI units/g tissue, prion infectivity in TNFR1^−/−-LTβR-Ig mLNs was reduced by at least 2.5 log TCI units/g tissue. Total mRNA was isolated from spleens (J) or mesenteric lymph nodes (mLN; K) of mice from the indicated treatment groups and analyzed for Prnp expression by Real Time PCR (Mean ± S.E.M.: Spleen – WT-Ig = 100.69±6.74, WT-LTβR-Ig = 46.02±4.30, TNFR1^−/−-Ig = 48.94±0.55, and TNFR1^−/−-LTβR-Ig = 50.11±0.55; mLN – WT-Ig = 106.05±22.54, WT-LTβR-Ig = 101.69±12.88, TNFR1^−/−-Ig = 56.43±3.13, and TNFR1^−/−-LTβR-Ig = 72.35±6.65). Whereas spleens from WT-LTβR-Ig, TNFR1^−/−-Ig, and TNFR1^−/−-LTβR-Ig mice all showed decreases in Prnp expression relative to WT-Ig spleens (J), no differences could be found in Prnp expression in mLNs from mice in any treatment group (K).</p

MadCam1 immunoreactivity in lymphoid tissue correlates with prion deposition.

<p>Formalin-fixed cryosections from spleens (SPL; A–D) or mesenteric lymph nodes (mLN; F–I) of C57BL/6 (WT) Ig-treated (A & F), TNFR1^−/− Ig-treated (B & G), C57BL/6 (WT) LTβR-Ig-treated (C & H), or TNFR1^−/− LTβR-Ig-treated (D & I) mice were immunostained with an antibody against the stromal cell marker, mucosal addressin cell adhesion molecule 1 (MadCam1), and visualized with alkaline phosphatase. (E) The total number of MadCam1-positive (MadCam1+; black) or MadCam1-negative (MadCam1-; white) lymphoid follicles were scored for spleens and expressed as a percentage of total follicles in each treatment group. (J) The total number of MadCam1-postive (MadCam1+) structures per mesenteric lymph node (mLN) was counted and averaged for each treatment group. WT-Ig mLNs contained 47.5±14 MadCam1+ structures, WT-LTβR-Ig = 9.5±3, TNFR1^−/−-Ig = 74±19, and TNFR1^−/−-LTβR-Ig = 0. MadCam1 immunoreactivity in WT-Ig spleens was localized to the marginal sinus (A; black arrow) and 55% (E) of germinal centers (A; white arrow). This staining pattern was absent (0%; E) in the spleens of mice from all other treatment groups (B,C & D). In contrast, MadCam1 immunoreactivity in WT-Ig mLNs was largely found in thick vessels (F). MadCam1 immunoreactivity was retained in TNFR1^−/−-Ig mLNs (G) but absent in mLNs from mice treated with LTβR-Ig (H–J). Size bars: Left panels = 200 µm; Right panels = 100 µm.</p

Vessel-associated MadCam1 expression in lymph nodes is preserved in the absence of TNFR1 signaling.

<p>Frozen sections from mesenteric lymph nodes (mLN) of C57BL/6 (WT) Ig-treated (A), TNFR1^−/− Ig-treated (B), C57BL/6 (WT) LTβR-Ig-treated (C), or TNFR1^−/− LTβR-Ig-treated (D) mice were analyzed by immunofluorescence with MadCam1 antibody. In WT-Ig mLNs (A), MadCam1 robustly stained thick vessels (yellow arrow), while germinal centers were weakly MadCam1-positive (white arrow). In TNFR1^−/−-Ig mLNs (B), MadCam1-positive germinal centers were absent, while vessel-associated MadCam1 staining persisted. In contrast, both WT-LTβR-Ig and TNFR1^−/−-LTβR-Ig mLNs were MadCam1-negative (C & D). Size bars = 100 µm. Total mRNA was isolated from spleens (E) or mesenteric lymph nodes (F) of C57BL/6 (WT) Ig-treated, C57BL/6 (WT) LTβR-Ig-treated, TNFR1^−/− Ig-treated, or TNFR1^−/− LTβR-Ig-treated mice and analyzed for MadCam1 expression by Real Time PCR (Mean ± S.E.M.: Spleen – WT-Ig = 104.21±16.56, WT-LTβR-Ig = 29.98±1.28, TNFR1^−/−-Ig = 30.41±2.56, and TNFR1^−/−-LTβR-Ig = 27.95±3.77; mLN – WT-Ig = 100.89±8.06, WT-LTβR-Ig = 45.40±6.75, TNFR1^−/−-Ig = 49.42±0.63, and TNFR1^−/−-LTβR-Ig = 24.79±7.25). MadCam1 expression was reduced in spleens of WT-LTβR-Ig-treated, TNFR1^−/− Ig-treated, and TNFR1^−/− LTβR-Ig-treated mice compared to WT-Ig. MadCam1 expression in WT-LTβR-Ig and TNFR1^−/− Ig mLNs was reduced compared to WT-Ig, and MadCam1 expression in TNFR1^−/− LTβR-Ig mLNs was reduced compared to TNFR1^−/−-Ig.</p