[PubMed] [Google Scholar] 34. of CD3+-, CD4+-, and CD8+-cell populations (explained Casp-8 below). Scheduled necropsies were performed at 2 (= 1), 4 to 5 (= 3), 8 (= 1), 13 to 14 (= 2), iCRT3 and 25 (= 2) weeks postinfection (wpi). The inoculation and sampling routine is usually detailed in Table ?Table22. TABLE 2 Hematologic and histologic observations for inoculated?macaques thead th rowspan=”1″ colspan=”1″ Animal /th th rowspan=”1″ colspan=”1″ Titer /th th rowspan=”1″ colspan=”1″ Route /th th rowspan=”1″ colspan=”1″ Necropsy (wpi) /th th rowspan=”1″ colspan=”1″ Hematology /th th rowspan=”1″ colspan=”1″ Histopathologya /th /thead 29831106p.o.2LN, moderate LFH; Sp, moderate LFH 30457106p.o.4LN, moderate to moderate LFH; tonsil, marked LFH; Sp, moderate LFH; Duod, moderate lymphocytic-plasmacytic duodenitis 30460106p.o.8LN, moderate to marked LFH; tonsil, marked LFH; Sp, moderate LFH; Duod, moderate lymphocytic-plasmacytic duodenitis 29840106i.v.4Transient leukocytosis (4 wpi): moderate neutrophilia, monocytosis, eosinophilia, moderate lymphocytosisLN, moderate to marked LFH; Sp, moderate to marked LFH, sinus neutrophilia and lymphocytosis; tonsil, tonsilitis, neutrophilic, marked LFH; Jej/Duod, dilated lacteal; Ki, moderate membranoproliferative glomerulopathy 29721104i.v.4LN, moderate LFH; tonsil, tonsilitis, neutrophilic, marked LFH; Sp, sinus neutrophilia; Th, moderate lymphoid depletion; Duod/Jej, moderate LFH; Duod/Il, moderate LFH; Li, moderate centrolobular vacuolar degeneration; Ki, moderate membranoproliferative glomerulopathy; Lu, prominent pulmonary lymphoid tissue, focal, perivascular lymphocytosis 29740104i.v.12Mild monocytosis (2, 9 wpi), moderate eosinophilia (7, 9 wpi)LN, marked LFH, hyalinization of germinal centers; Sp, marked LFH, hyalinization of germinal centers, sinus neutrophilia and lymphocytosis; tonsil, marked LFH; Duod/Jej, marked LFH; Lu, prominent lymphoid tissue, multifocal 29762104i.v.12Transient leukocytosis (5, 9 wpi): moderate neutrophilia, monocytosis, moderate lymphocytosisLN, moderate to noticeable LFH, sinus histiocytosis; Sp, moderate LFH, sinus neutrophilia; tonsil, tonsilitis, neutrophilic, marked LFH; Duod, marked mucosal LFH; Il, marked submucosal LFH; Li, moderate multifocal lymphocytic hepatopathy 29848106i.v.25Transient leukocytosis (8 wpi): moderate lymphocytosis, moderate monocytosis (6, 8, 22 wpi), marked eosinophiliaLN, moderate LFH, sinus histiocytosis; Sp, marked LFH; Duod, moderate LFH; BM, moderate myeloid hyperplasia 29850106i.v.25Persistent leukocytosis (2, 3, 6, 8, 17 wpi): moderate neutrophilia (3, 22, 25 wpi), moderate lymphocytosis (3, 8, 13, 17 wpi), moderate monocytosis (1, 2, 3, 6, 8, 17, 22 wpi), moderate eosinophilia (2, 8 iCRT3 wpi)LN, moderate to marked LFH; tonsil, moderate LFH; Sp, moderate LFH sinus neutrophilia; Duod/Jej, moderate LFH; Duod, enteritis, plasmacytic, diffuse, crypts; Li, moderate centrolobular vacuolar degeneration; Lu, prominent pulmonary lymphoid tissue; BM, moderate myeloid hyperplasia Open in a separate windows aLFH, lymphofollicular hyperplasia; Sp, spleen; Duod, duodenum; Jej, jejunum; Ki, kidney; Th, thymus; Il, ileum; Li, liver; Lu, lung; BM, bone marrow.? Hematologic evaluation and CD4/CD8 T-lymphocyte immunophenotyping. Total blood counts were performed by the Clinical Laboratory at the California Regional Primate Research Center with EDTA-anticoagulated blood. Fifty microliters of whole blood was incubated for 15 to 30 min at room heat with monoclonal antibodies specific for CD3 (10 l, fluorescein isothiocyanate conjugated) (catalogue no. 35694X; Pharmingen, San Diego, Calif.), CD4 (5 l, phycoerythrin conjugated) (catalogue no. 703430; Ortho Diagnostics, Raritan, N.J.), and CD8 (5 l, peridinin chlorophyll protein [PerCP] conjugated) (catalogue no. 347314, Becton Dickinson, Mountain View, Calif.). Samples were processed (Q-Prep; Coulter, Hialeah, Fla.) and analyzed by three-color circulation cytometry with a FACScan (Becton Dickinson). Fluorescence-activated cell sorter analysis was performed by the Optical Biology Laboratory, Department of Medical Pathology, University or college of California, Davis. Necropsy and histology. Animals were culled at defined time points during the study period (observe Table ?Table2).2). Total necropsy examinations were iCRT3 performed. Tissues were utilized for histologic examination and nucleic acid purification. Tissues were fixed in 10% formalin for a period of time not exceeding 5 iCRT3 days prior to paraffin embedding (46). PCR. DNA was purified from necropsy tissues by using a lysis buffer made up of 1% sodium dodecyl sulfate, 20 mM Tris (pH 7.5), 250 mM NaCl, 20 mM EDTA, and 0.5 mg of proteinase K per ml. Tissues were incubated in lysis buffer overnight at 63C, and new lysis buffer was added until the tissues were completely digested. After phenol-chloroform extraction and precipitation with ethanol, DNA concentrations were decided spectrophotometrically. Viral DNA was purified from 200 l of plasma by using the QIAmp blood kit (Qiagen, Valencia, Calif.); DNA was eluted in a volume of 50 l of water. Nested PCR (40 cycles of 1 1 min at 94C, 1 min at 55C, and 1 min at 72C) was performed with.