the absorbance measured after development. This cor-rected value was then used for the calculation of endo-toxin concentration. The virus inoculum used in this studywas assayed and shown to be free of endotoxin (
Cytokine/Chemokine and Nitrate Production
Cytokine/chemokine levels in monkey sera/plasma wereassayed using commercially available ELISA kits accord-ing to manufacturer
s directions. Cytokines/chemokinesassayed included monkey interleukin (IL)-2, IL-4, IL-10,IL-12, interferon (IFN)-
, and tumor necrosis factor(TNF)-
(BioSource International, Inc., Camarillo, CA).ELISAs for human proteins known to be compatible withcynomolgus macaques included IL-6, IFN-
, and MIP-1
(BioSource); and human IL-1
, IL-8, IL-18, and MCP-1 (R&D Systems, Minneapolis, MN). Nitratelevels were determined using a colorimetric assay ac-cording to manufacturer
s directions (R&D Systems).
A complete necropsy was performed on all animals. Tis-sue samplesofall majororganswere collectedfromeachmonkey for histopathological, immunohistochemical, and
hybridization examination andwereimmersion-fixedin 10% neutral-buffered formalin. Select tissues for ultra-structural examination were immersion-fixed in 4% formal-dehyde plus 1% glutaraldehyde in 0.1 mol/L Millonig
sphosphate buffer for transmission electron microscopy(TEM) or 2% formaldehyde plus 0.1% glutaraldehyde in theMillonig
s buffer for immunoelectron microscopy.
Formalin-fixed tissues for histology, immunohistochemis-try, and
hybridization were processed and embed-ded in paraffin according to conventional methods.
Histology sections were cut at 5 to 6
m on a rotarymicrotome, mounted on glass slides, and stained withhemotoxylin and eosin. Replicate sections of all tissueswere mounted on positively charged glass slides (Super-frost Plus; Fisher Scientific, Pittsburgh, PA) and immuno-histochemically stained for detection of viral antigen byan immunoperoxidase method according to kit proce-dures (Envision System; DAKO Corporation, Carpinteria,CA), or by a fluorescence-based method.
Sections were deparaffinized and rehydrated througha series of graded ethanols, pretreated with ready-to-useProteinase K (DAKO) for 6 minutes at room temperature,and blocked with Protein Block Serum-Free (DAKO) for20 minutes before exposure to antibody. Tissue sectionswere incubated with primary antibody overnight a 4
Cusing an anti-EBOV rabbit polyclonal (kindly provided byCindy Rossi) (1:500) or an equal mixture of mouse mono-clonal antibodies to EBOV GP and VP40 (1:5000).
Analkaline phosphatase-labeled polymer (DAKO EnvisionSystem, alkaline phosphatase) was added for 30 minutesand color development was achieved by exposing tissueto the substrate 6-bromo-2-hydroxyl-3-naphtholic acid(Histomark Red; Kirkegaard and Perry, Gaithersburg, MD)for 50 minutes in the dark. Sections were counterstainedwith hematoxylin. Negative controls included replicate sec-tions exposed to anti-Marburg virus antibodies and unex-posedcynomolgusmonkeytissue;archivedEBOV-infectedcynomolgus tissue served as positive controls.
Tissue sections were deparaffinized, rehydrated, andincubated in 20
g/ml of proteinase K for 30 minutes atroom temperature. Sections were subsequently rinsed,placedinnormalgoatserumfor20minutesandtransferredto a mixture of the anti-EBOV monoclonal antibodies asdescribed above for 30 minutes at room temperature. Afterincubation, sections were rinsed and stained with goat anti-mouse Alexa 594 (Molecular Probes, Eugene, OR), incu-bated with a pan T-cell marker, CD3 (DAKO) for 30 minutesat room temperature, rinsed, and incubated in goat anti-mouse Alexa 488 (Molecular Probes).For co-localization of macrophage and/or dendritic cellmarkers and EBOV antigens, two different techniqueswere performed. Briefly, deparaffinized tissue sectionswere pretreated with proteinase K (20
g/ml) for 30 min-utes at room temperature and incubated in normal goatserum for 20 minutes. Sections were then incubated ineither a macrophage marker (Ab-1, Oncogene ResearchProducts, San Diego, CA), or a marker for dendritic cells(DC-SIGN, kindly provided by Dr. Vineet KewalRamani,NCI-Frederick), for 30 minutes at room temperature. Afterincubation, sections were placed in goat anti-mouse Al-exa 594 (Molecular Probes) for 30 minutes at room tem-perature and rinsed. Sections were transferred to ablocking solution consisting of a cocktail of mouse IgGsand irrelevant mouse anti-Marburg virus antibodies, andincubated for 20 minutes at room temperature. The ex-cess blocking cocktail was removed and sections wereincubated in a fluorescein isothiocyanate (FITC)-conju-gated EBOV for 30 minutes at room temperature. TheFITC-conjugated anti-EBOV pool was produced by dilut-ing the purified, concentrated anti-EBOV murine mono-clonal antibodies (described above) to 1 mg/ml and dia-lyzing overnight at 4
C in 0.1 mol/L sodium bicarbonatebuffer (pH 8.5). The antibodies were recovered andmixed with FITC on celite (
-Aldrich, St. Louis, MO) andincubated, rotating, for 2 hours at room temperature.FITC-conjugated antibodies were then separated fromunbound FITC by passage over a Sephadex G-25 (Am-ersham Biosciences, Piscataway, NJ) column. To in-crease the sensitivity of the staining, sections were incu-bated in an anti-FITC Alexa 488 (Molecular Probes) for 30minutes at room temperature. After rinsing in PBS, sec-tions were mounted in an aqueous mounting mediumcontaining 4
-diamidino-2-phenylindole (DAPI) (Vector
Ebola Pathogenesis in Cynomolgus Monkeys 2349
AJP December 2003, Vol. 163, No. 6