1. Autocrine nerve growth factor is essential for cell survival and viral maturation in HHV-8–infected primary effusion lymphoma cells
by Francesca Pica, Antonio Volpi, Annalucia Serafino, Marzia Fraschetti, Ornella Franzese, and Enrico Garaci
Blood
Volume 95(9):
May 1, 2000
©2000 by American Society of Hematology

2. Expression of high-affinity and low-affinity NGF receptors by HHV-8+ and HHV-8− B-lymphoma cell lines.The expression of high-affinity (p140trk-A) and low-affinity (p75NGFR) NGF receptors by B-lymphoma cell lines HHV-8+ (BC-1 and BCBL-1 cells) and HHV-8− (RA...
Expression of high-affinity and low-affinity NGF receptors by HHV-8+ and HHV-8− B-lymphoma cell lines.The expression of high-affinity (p140trk-A) and low-affinity (p75NGFR) NGF receptors by B-lymphoma cell lines HHV-8+ (BC-1 and BCBL-1 cells) and HHV-8− (RAMOS cells) is shown. Western blot analysis was performed, and immunostaining was completed with anti-p140 and anti-p75 antibodies of proteins (100 μg/sample) from untreated cells and BC-1 cells treated with 3 mmol/L sodium butyrate and BCBL-1 cells treated with 20 ng/mL TPA at 48 hours of culture.
Francesca Pica et al. Blood 2000;95:
©2000 by American Society of Hematology

3. Effects of endogenous NGF neutralization on the basal growth of HHV-8+ and HHV-8− B-lymphoma cell lines.The figure depicts untreated cells (•), cells treated with 30 μg/mL anti-NGF Ab at 0 hour (○), and cells treated with 30 μg/mL anti-NGF Ab at 0 hour and ...
Effects of endogenous NGF neutralization on the basal growth of HHV-8+ and HHV-8− B-lymphoma cell lines.The figure depicts untreated cells (•), cells treated with 30 μg/mL anti-NGF Ab at 0 hour (○), and cells treated with 30 μg/mL anti-NGF Ab at 0 hour and 48 hours (▵). Cells were seeded in duplicate at (A, C) 2 × 105/mL or (B) 5 × 105/mL at 0 hour. The number of viable cells in each culture was determined daily using the trypan blue dye exclusion method. The values refer to the mean of each duplicate. In all cases, the SD was less than 10% of the mean. Similar results were obtained in 3 separate experiments.
Francesca Pica et al. Blood 2000;95:
©2000 by American Society of Hematology

4. Effects of endogenous NGF neutralization by means of anti-NGF antibodies or NGF antisense oligonucleotides on3H-thymidine incorporation by BCBL-1 cells.(A) Cells were treated with 30 μg/mL anti-NGF Ab at 0 hour (□) and with 30 μg/mL anti-NGF Ab at 0 and
Effects of endogenous NGF neutralization by means of anti-NGF antibodies or NGF antisense oligonucleotides on3H-thymidine incorporation by BCBL-1 cells.(A) Cells were treated with 30 μg/mL anti-NGF Ab at 0 hour (□) and with 30 μg/mL anti-NGF Ab at 0 and 48 hours (▪). (B) Cells were treated with 2 μmol/L (▵) or 10 μmol/L (▴) NGF antisense oligonucleotides and with 10 μmol/L (○) control random-sequence oligonucleotides. Cells were seeded in quadruplicate at 1 × 105 cells/mL in 96-well plates.3H-thymidine (0.037 MBq/well) was added in the final 12 hours of incubation for each time point. Results were calculated as the mean 3H-thymidine incorporation of quadruplicate cultures and expressed as the percentage of untreated controls. Similar results were obtained in 3 separate experiments.
Francesca Pica et al. Blood 2000;95:
©2000 by American Society of Hematology

5. Confocal fluorescence micrographs of BCBL-1 cells cultured in the absence or presence of anti-NGF antibodies.Untreated cells were observed at (A) 24 hours, (C) 48 hours, and (E) 72 hours.
Confocal fluorescence micrographs of BCBL-1 cells cultured in the absence or presence of anti-NGF antibodies.Untreated cells were observed at (A) 24 hours, (C) 48 hours, and (E) 72 hours. Cells were treated with 30 μg/mL anti-NGF Ab at 0 hour and observed at (B) 24 hours, (D) 48 hours, and (F) 72 hours. (G) Cells were treated with 30 μg/mL anti-NGF Ab at 0 and 48 hours and observed at 72 hours. To observe nuclei, cells were stained with propidium iodide. Apoptosis was evaluated using morphological parameters such as chromatin condensation and nuclear fragmentation (seen in dense black). (Original magnification ×40.)‏
Francesca Pica et al. Blood 2000;95:
©2000 by American Society of Hematology

6. Effects of TPA plus hr-NGF or anti-NGF antibodies on BCBL-1
Effects of TPA plus hr-NGF or anti-NGF antibodies on BCBL-1.(A) Cell growth curves and (B) the percentage of apoptotic cells is depicted.
Effects of TPA plus hr-NGF or anti-NGF antibodies on BCBL-1.(A) Cell growth curves and (B) the percentage of apoptotic cells is depicted. Cells were seeded in duplicate at 2 × 105/mL. The figure depicts untreated cells (•), cells treated with 20 ng/mL TPA at 0 hour (▴), cells treated with TPA plus 5 ng/mL hr-NGF at 0 hour (▵), and cells treated with TPA plus 30 μg/mL anti-NGF Ab at 0 and 24 hours (○). The number of viable cells in each culture was determined daily using the trypan blue dye exclusion method. The values refer to the mean of each duplicate. In all cases, the SD was less than 10% of the mean. Similar results were obtained in 3 separate experiments.
Francesca Pica et al. Blood 2000;95:
©2000 by American Society of Hematology

7. Light microscopy of BCBL-1 cells treated with TPA plus hr-NGF or anti-NGF Ab and observed at 48 hours of culture.Semithin sections of cells were embedded in Spurr epoxy resin and stained with methylene blue as shown: (A, B) untreated cells, (C, D) cells tre...
Light microscopy of BCBL-1 cells treated with TPA plus hr-NGF or anti-NGF Ab and observed at 48 hours of culture.Semithin sections of cells were embedded in Spurr epoxy resin and stained with methylene blue as shown: (A, B) untreated cells, (C, D) cells treated with 20 ng/mL TPA at 0 hour, (E, F) cells treated with TPA plus 5 ng/mL hr-NGF at 0 hour, and (G, H) cells treated with TPA plus 30 μg/mL anti-NGF Ab at 0 and 24 hours. (Original magnification ×40 in A, C, E, and G, and ×100 in B, D, F, and H. (C, D) Numerous cells showing aggregation of dense masses of chromatin beneath the nuclear membrane and nuclear fragmentation were observed. (E, F) Only a few cells showed morphological evidence of apoptosis, and (G, H) a high number of apoptotic cells were observed. Arrowheads indicate apoptotic bodies.
Francesca Pica et al. Blood 2000;95:
©2000 by American Society of Hematology

8. Transmission electron microscopy of HHV-8 virions in TPA-treated BCBL-1 cells at 48 hours of culture.The following cells are depicted: (A) untreated cells, (B) cells treated with 20 ng/mL TPA at 0 hour, (C) cells treated with TPA plus 5 ng/mL hr-NGF at 0 ho...
Transmission electron microscopy of HHV-8 virions in TPA-treated BCBL-1 cells at 48 hours of culture.The following cells are depicted: (A) untreated cells, (B) cells treated with 20 ng/mL TPA at 0 hour, (C) cells treated with TPA plus 5 ng/mL hr-NGF at 0 hour, and (D) cells treated with TPA plus 30 μg/mL anti-NGF Ab at 0 and 24 hours. (A) No HHV-8 viral particles were observed in control cells. (B) Mature enveloped virions originated from the nucleus of a TPA-treated cell. A detail, at higher magnification, is shown in the corner. (C) View of numerous mature HHV-8 virions, approximately 110 nm in diameter, in the nucleus of a cell treated with TPA plus hr-NGF. A detail, at higher magnification, is shown in the corner. (D) Nucleus of an apoptotic cell in a culture treated with TPA plus anti-NGF Ab. The condensed chromatin of the apoptotic cell can be seen along the nuclear membrane. Numerous defective virus particles, with internal electron dense structures possibly representing viral DNA, were observed. A detail of a defective virion can be seen at higher magnification in the corner. The bars in (A, B) equal 1 μm; in (C, D), 0.5 μm; and in the corners, 150 nm.
Francesca Pica et al. Blood 2000;95:
©2000 by American Society of Hematology

9. Transmission electron microscopy of BCBL-1 cells treated with TPA plus hr-NGF after 48 hours of culture.(A) Numerous mature HHV-8 virions (arrows) were observed both in cytoplasm and in dilated cisternae of the endoplasmic reticulum (ER).
Transmission electron microscopy of BCBL-1 cells treated with TPA plus hr-NGF after 48 hours of culture.(A) Numerous mature HHV-8 virions (arrows) were observed both in cytoplasm and in dilated cisternae of the endoplasmic reticulum (ER). The small pictures in the upper right-hand corner detail HHV-8 virions in the (1) cell cytoplasm and (2,3) dilated cisternae of ER. In some cases, (2) the outer membrane of the viral particles was connected to the ER membrane, suggesting a possible budding into ER. (B) Electron dense material and mature virions inside intracytoplasmic vesicles (arrowheads) were observed just beneath the cell membrane; a detail at higher magnification is inserted in *. (C) Morphologically mature viral particles are seen in the extracellular space of BCBL-1 cells. The bars in (A, B) equal 400 nm; in (C), 300 nm; and in (1, 2, 3), 200 nm.
Francesca Pica et al. Blood 2000;95:
©2000 by American Society of Hematology