Journal: PLoS pathogens

Article Title: Exploitation of the complement system by oncogenic Kaposi's sarcoma-associated herpesvirus for cell survival and persistent infection.

doi: 10.1371/journal.ppat.1004412

Figure Lengend Snippet: Figure 7. Activation of complement pathway induces STAT3 phosphorylation to enhance cell survival of latently KSHV-infected endothelial cells. (A) The enhanced cell survival of KSHV-infected endothelial cells by complement was mediated by the STAT3 pathway. TIME- KSHV cells were cultured for 48 h in normal human serum in growth factor-depleted medium with and without JAK or STAT3 inhibitor, and the numbers of dead cells (left panel) and live cells (right panel) were determined. Cells cultured in heat-inactivated human serum were used as controls. Results are means 6 SD from three independent experiments with three repeats. * P,0.05, ** P,0.01 and *** P,0.001 by Student’s t-test. (B) Complement activation induced STAT3 tyrosine phosphorylation in TIME-KSHV cells. STAT3 tyrosine (Y705) and serine (S727) phosphorylation in TIME and TIME-KSHV cells switched from full endothelial cell medium with growth factors and 10% heat-inactivated human serum to endothelial cell medium depleted of growth factors with 10% heat-inactivated or normal human serum for the specified lengths of time. (C) Complement activation was required for the enhanced STAT3 tyrosine phosphorylation of TIME-KSHV cells. STAT3 tyrosine phosphorylation was examined in cells cultured for 24 h in 10% C3-depleted human serum or C3-depleted human serum reconstituted with purified C3 protein in endothelial cell medium depleted of growth factors. (D) Formation of C5b-9 complexes was required for the enhanced STAT3 tyrosine phosphorylation. STAT3 tyrosine phosphorylation was examined in cells cultured for 24 h in 10% C6-depleted human serum or C6-depleted human serum reconstituted with purified C6 protein in endothelial cell medium deprived of growth factors. (E) STAT3 tyrosine phosphorylation was unchanged without the continuous presence of normal human serum and growth factors. STAT3 tyrosine phosphorylation was examined in cells cultured in 10% heat-inactivated or normal human serum for 1 h, and then in endothelial cell medium without any serum and growth factors for additional 23 h. (F) JAK but not Src activation mediated STAT3 tyrosine phosphorylation. STAT3 tyrosine phosphorylations was examined in TIME-KSHV cells cultured in 10% normal human serum in endothelial cell medium depleted of growth factors for 24 h with or without the presence of STAT3, JAK or Src inhibitor. doi:10.1371/journal.ppat.1004412.g007

Article Snippet: C1q-depleted human serum, C3-depleted human serum, C6-depleted human serum, factor B-depleted human serum, purified C3 protein, and purified C6 protein were purchased from Quidel Corporation (San Diego, CA).

Techniques: Activation Assay, Phospho-proteomics, Infection, Cell Culture, Purification

Journal: Leukemia & lymphoma

Article Title: Targeting glucosylceramide synthase synergizes with C6-ceramide nanoliposomes to induce apoptosis in natural killer cell leukemia.

doi: 10.3109/10428194.2012.752485

Figure Lengend Snippet: Figure 2. Cellular apoptosis is induced after combination treatment. (A) Patient leukemic NK cells from six patients were treated with 12.5 μ M C 6 -ceramide and 5, 10 or 15 μ M PPMP for 24 h then stained for annexin-V/7-AAD then assayed for apoptosis by fl ow cytometry. (B) RNK-16 cells were treated with 6.25 μ M C 6 -ceramide nanoliposomes and 5, 10 or 15 μ M of PPMP ( n 3 independent experiments), then apoptosis assay was performed. (C) NKL were treated with 12.5 μ M C 6 -ceramide and 5, 10 or 15 μ M PPMP ( n 3 independent experiments), then apoptosis assay was performed. (D) Normal PBMCs were treated with 12.5 μ M C 6 -ceramide nanoliposomes and 5, 10 or 15 μ M PPMP ( n 8), then apoptosis assay was performed. * p 0.05, * * p 0.005, * * * p 0.0005 indicate signifi cant diff erences of C 6 -ceramide nanoliposomes PPMP versus control treated cells (Student ’ s t -test).

Article Snippet: PPMP and C 6 -ceramide nanoliposome drug studies C 6 -ceramide nanoliposomes were manufactured in our laboratory with the following lipids purchased from Avanti Polar Lipids: 1,2-distearoyl- sn -glycero-3-phosphocholine, 1,2-dioleoyl- sn -glycero-3-phosphoethanolamine, N -hexanoyld -erythro-sphingosine (C 6 ), 1,2-distearoyl- sn -glycero-3phosphoethanolamine- N -[methoxy polyethylene glycol-2000] and N -octanoyl-sphingosine-1-[succinyl (methoxy polyethylene glycol-750)] [PEG(750)-C8], which were combined at a molar ratio of 3.75:1.75:3:0.75:0.75, respectively.

Techniques: Staining, Cytometry, Apoptosis Assay, Control

Journal: Leukemia & lymphoma

Article Title: Targeting glucosylceramide synthase synergizes with C6-ceramide nanoliposomes to induce apoptosis in natural killer cell leukemia.

doi: 10.3109/10428194.2012.752485

Figure Lengend Snippet: Figure 4. Mitochondrial disruption occurs in leukemic NK cells after combination treatment. (A) Patient NK cells (12.5 μ M C 6 -ceramide nanoliposomes PPMP) and (B) RNK-16 cells (6.25 μ M C 6 -ceramide nanoliposomes PPMP) were treated for 24 h then stained with JC-1 and assayed for mitochondrial depolarization via cell fl ow cytometry. * p 0.05, * * p 0.005, * * * p 0.0005 indicate signifi cant diff erences of C 6 - ceramide nanoliposomes PPMP versus control treated cells (Student ’ s t -test).

Article Snippet: PPMP and C 6 -ceramide nanoliposome drug studies C 6 -ceramide nanoliposomes were manufactured in our laboratory with the following lipids purchased from Avanti Polar Lipids: 1,2-distearoyl- sn -glycero-3-phosphocholine, 1,2-dioleoyl- sn -glycero-3-phosphoethanolamine, N -hexanoyld -erythro-sphingosine (C 6 ), 1,2-distearoyl- sn -glycero-3phosphoethanolamine- N -[methoxy polyethylene glycol-2000] and N -octanoyl-sphingosine-1-[succinyl (methoxy polyethylene glycol-750)] [PEG(750)-C8], which were combined at a molar ratio of 3.75:1.75:3:0.75:0.75, respectively.

Techniques: Disruption, Staining, Cytometry, Control

Journal: Leukemia & lymphoma

Article Title: Targeting glucosylceramide synthase synergizes with C6-ceramide nanoliposomes to induce apoptosis in natural killer cell leukemia.

doi: 10.3109/10428194.2012.752485

Figure Lengend Snippet: Figure 3. Sphingolipid levels are altered after combination treatment. RNK-16 (4 10 6 ) cells were treated for 24 h with 6.25 μ M C 6 -ceramide and 5, 10 and 15 μ M PPMP. Cells were harvested and lipids were extracted and analyzed with LC-MS/MS. Lipid levels relative to total cell protein levels were determined for: (A) C 6 -ceramide, (B) C 6 -cerebroside, (C) total natural ceramide, (D) sphingosine. * p 0.05 indicates diff erences of C 6 - ceramide nanoliposomes alone vs. C 6 -ceramide nanoliposomes PPMP combinations (one-way ANOVA).

Article Snippet: PPMP and C 6 -ceramide nanoliposome drug studies C 6 -ceramide nanoliposomes were manufactured in our laboratory with the following lipids purchased from Avanti Polar Lipids: 1,2-distearoyl- sn -glycero-3-phosphocholine, 1,2-dioleoyl- sn -glycero-3-phosphoethanolamine, N -hexanoyld -erythro-sphingosine (C 6 ), 1,2-distearoyl- sn -glycero-3phosphoethanolamine- N -[methoxy polyethylene glycol-2000] and N -octanoyl-sphingosine-1-[succinyl (methoxy polyethylene glycol-750)] [PEG(750)-C8], which were combined at a molar ratio of 3.75:1.75:3:0.75:0.75, respectively.

Techniques: Liquid Chromatography with Mass Spectroscopy

Journal: Leukemia & lymphoma

Article Title: Targeting glucosylceramide synthase synergizes with C6-ceramide nanoliposomes to induce apoptosis in natural killer cell leukemia.

doi: 10.3109/10428194.2012.752485

Figure Lengend Snippet: Figure 6. Mcl-1 and survivin expression is decreased in a dose- dependent manner. Western blot analysis was performed for Mcl-1 and survivin, after 24 h treatment of PBMCs from a patient with chronic NK leukemia with 12.5 μ M C 6 -ceramide nanoliposome and 5, 10 and 15 μ M PPMP ( n 3 independent experiments). β -Actin served as a loading control.

Article Snippet: PPMP and C 6 -ceramide nanoliposome drug studies C 6 -ceramide nanoliposomes were manufactured in our laboratory with the following lipids purchased from Avanti Polar Lipids: 1,2-distearoyl- sn -glycero-3-phosphocholine, 1,2-dioleoyl- sn -glycero-3-phosphoethanolamine, N -hexanoyld -erythro-sphingosine (C 6 ), 1,2-distearoyl- sn -glycero-3phosphoethanolamine- N -[methoxy polyethylene glycol-2000] and N -octanoyl-sphingosine-1-[succinyl (methoxy polyethylene glycol-750)] [PEG(750)-C8], which were combined at a molar ratio of 3.75:1.75:3:0.75:0.75, respectively.

Techniques: Expressing, Western Blot, Control

Journal: Leukemia & lymphoma

Article Title: Targeting glucosylceramide synthase synergizes with C6-ceramide nanoliposomes to induce apoptosis in natural killer cell leukemia.

doi: 10.3109/10428194.2012.752485

Figure Lengend Snippet: Figure 5. Increase in ROS level and decrease of Bcl-2 after combination treatment. (A) RNK-16 cells were treated for 24 h with 6.25 μ M C 6 -ceramide nanoliposomes and 5 or 10 μ M PPMP and then assayed for ROS levels via cell fl ow cytometry. * p 0.05 indicates signifi cant diff erences of C 6 - ceramide nanoliposomes PPMP versus control treated cells (Student ’ s t -test). (B) Bcl-2 production decreases with increasing ROS levels ( n 3 independent experiments).

Article Snippet: PPMP and C 6 -ceramide nanoliposome drug studies C 6 -ceramide nanoliposomes were manufactured in our laboratory with the following lipids purchased from Avanti Polar Lipids: 1,2-distearoyl- sn -glycero-3-phosphocholine, 1,2-dioleoyl- sn -glycero-3-phosphoethanolamine, N -hexanoyld -erythro-sphingosine (C 6 ), 1,2-distearoyl- sn -glycero-3phosphoethanolamine- N -[methoxy polyethylene glycol-2000] and N -octanoyl-sphingosine-1-[succinyl (methoxy polyethylene glycol-750)] [PEG(750)-C8], which were combined at a molar ratio of 3.75:1.75:3:0.75:0.75, respectively.

Techniques: Cytometry, Control

Journal: eBioMedicine

Article Title: Preclinical characterisation of the protective capacity of an anti-nucleoprotein hRSV monoclonal antibody

doi: 10.1016/j.ebiom.2025.106104

Figure Lengend Snippet: The humanised anti-N-hRSV mAbs demonstrate antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity activity in vitro . A. The percentage of antibody-dependent cellular cytotoxicity (ADCC) activity for each clone of the humanised anti-N-hRSV mAbs (clones P1-04H, P1-05D, P2-01A, and P2-01D) was measured using luciferase expression levels in Jurkat NFAT-luc FcγRIII cells incubated with the humanised anti-N-hRSV mAbs against N-hRSV. B. The percentage of complement-dependent cytotoxicity (CDC) activity for each clone was assessed by viability assay of HEp-2 cells infected with hRSV-GFP, treated with the four clones of the humanised anti-N-hRSV mAbs, followed by the addition of mouse purified complement. Three independent experiments were conducted for each ADCC and CDC assay (N = 3).

Article Snippet: After 30 min of antibody incubation, 100 μl of a 1:25 solution of mouse-purified complement (Rockland Immunochemical; cat #R.C201-0005) was added, bringing the final concentration in the well to 1:50, and the plate was incubated for 3 h at 37 °C with 5% CO 2 .

Techniques: Activity Assay, In Vitro, Clone Assay, Luciferase, Expressing, Incubation, Viability Assay, Infection, Purification, CDC Assay