Ribavirin treatment targets eIF4E activity and localization in patients. (A) Top panels show bone marrow specimens for patients 8 (touch print, due to dry aspirate) and 11 (squash preparation) prior to therapy. Note the abundance of blasts. Immediately below, panels show response after 28 days of therapy. Patient 8 achieved PRi and patient 11, a CR. Both exhibit a restoration of hematopoiesis. This is reflected in the improvement in peripheral blood counts, particularly platelets and neutrophils, as shown in the graphs in the bottom panels. Hydrea levels are shown below the graphs indicating that response did not depend on hydrea. As well, white blood cell (WBC) counts remained normal despite decreasing hydrea doses. Squash preparations of bone marrow aspirates and touch prints of bone marrow biopsies were air-dried and stained with Wright-Giemsa. The Leica DM2 LB2 microscope with the Leica H3X PL Fluotar 100× objective lens 1.3 numeric aperture (Leica) and oil immersion were used to examine the specimens. The Infinity 1-3C (color) camera was used to capture images on the Infinity Analyze image acquisition software (Lumenera Corporation). (B, top panel) Ribavirin treatment lowered blast counts. Plot of percentage of peripheral (blue) or bone marrow (red) blasts versus treatment day for patient 6. (Bottom panel) Ribavirin treatment led to relocalization of eIF4E. Immunohistochemistry was carried out as described where cells were stained for eIF4E and DAPI.^1,13  Cells were immunostained with an eIF4E mAb directly conjugated to FITC (BD Bioscience). Specimens were mounted in Vectashield with DAPI (Vector Laboratories). Channels were recorded separately with no cross talk observed between channels. Micrographs were collected on a laser scanning confocal microscope (LSM510; Carl Zeiss) using a 100× objective with a numeric aperture of 1.4, with 2× digital zoom at room temperature. Images were displayed using Photoshop CS2 (Adobe). Micrographs represent single optical sections through the plane of the cell. (C) eIF4E activity and levels were reduced by ribavirin treatment in patients. Western blot analysis was carried out using the antibodies indicated. Antibodies for immunoblotting were obtained from Cell Signaling unless otherwise mentioned: mAb anti-eIF4E (BD Bioscience); pAb anti-NBS; pAb anti–cyclin D1 (Santa Cruz Biotechnology); pAb anti-Akt; anti–phospho Thr308 or Ser473 Akt; and mAb anti-β-actin (AC-15; Sigma -Aldrich). As expected, similar reductions were seen for either Thr308 or Ser473 phosphorylation of Akt as seen in cell culture.¹¹