Adoptive immunotherapy with high-dose interleukin-2: kinetics of circulating progenitors correlate with interleukin-6, granulocyte colony-stimulating factor level

Abstract Immunotherapy with interleukin-2 (IL-2) and lymphokine-activated killer (LAK) cells results in significant tumor regression in patients with advanced cancer. We have investigated the kinetics of circulating erythroid (BFU-E) and granulocytic-macrophage (CFU-GM) progenitors after IL-2 therapy in 11 cancer patients, mainly affected by metastatic melanoma and renal cell carcinoma. Administration of IL-2 from day 1 through day 5 constantly induced a dramatic decrease of the number of circulating BFU-E and CFU-GM, which then showed a striking rebound (up to values fourfold and sevenfold higher, respectively, than the pretherapy levels) on discontinuation of IL-2, ie, from day 5 through day 10. A similar kinetic pattern was observed during and after the second cycle of IL-2 administration. 3[H]-thymidine killing experiments showed that the cycling activity of the progenitors was virtually unmodified in the rebound phases. To explore the mechanism(s) underlying this kinetic pattern, we have analyzed the plasma concentration of several hematopoietic growth factors, including IL-1 beta, IL-3, IL-4, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, and erythropoietin (Ep). No modifications in the levels of IL-3, GM-CSF, or IL-1 beta were observed, whereas a pronounced increase of IL-6 and G-CSF concentration was monitored, starting at day 3 and peaking at day 5 of treatment (a parallel, but modest, increase of Ep level was also observed). The elevation of IL-6 and G-CSF concentration is directly correlated with and may, at least in part, underlie the subsequent rebound of circulating hematopoietic progenitors. Furthermore, the increase in IL-4 level observed at day 10 of therapy may mediate the eosinophilia gradually starting at this stage of treatment.</jats:p

Adoptive immunotherapy with high-dose interleukin-2: kinetics of circulating progenitors correlate with interleukin-6, granulocyte colony-stimulating factor level

Immunotherapy with interleukin-2 (IL-2) and lymphokine-activated killer (LAK) cells results in significant tumor regression in patients with advanced cancer. We have investigated the kinetics of circulating erythroid (BFU-E) and granulocytic-macrophage (CFU-GM) progenitors after IL-2 therapy in 11 cancer patients, mainly affected by metastatic melanoma and renal cell carcinoma. Administration of IL-2 from day 1 through day 5 constantly induced a dramatic decrease of the number of circulating BFU-E and CFU-GM, which then showed a striking rebound (up to values fourfold and sevenfold higher, respectively, than the pretherapy levels) on discontinuation of IL-2, ie, from day 5 through day 10. A similar kinetic pattern was observed during and after the second cycle of IL-2 administration. 3[H]-thymidine killing experiments showed that the cycling activity of the progenitors was virtually unmodified in the rebound phases. To explore the mechanism(s) underlying this kinetic pattern, we have analyzed the plasma concentration of several hematopoietic growth factors, including IL-1 beta, IL-3, IL-4, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, and erythropoietin (Ep). No modifications in the levels of IL-3, GM-CSF, or IL-1 beta were observed, whereas a pronounced increase of IL-6 and G-CSF concentration was monitored, starting at day 3 and peaking at day 5 of treatment (a parallel, but modest, increase of Ep level was also observed). The elevation of IL-6 and G-CSF concentration is directly correlated with and may, at least in part, underlie the subsequent rebound of circulating hematopoietic progenitors. Furthermore, the increase in IL-4 level observed at day 10 of therapy may mediate the eosinophilia gradually starting at this stage of treatment.</jats:p

Granulocyte-macrophage colony-stimulating factor reactivates fetal hemoglobin synthesis in erythroblast clones from normal adults

Abstract Reactivation of fetal hemoglobin (HbF, alpha 2 gamma 2) synthesis was previously reported in normal human adult erythroblast colonies (“bursts”) generated by erythroid progenitors (BFU-E) in fetal calf serum-supplemented (FCS+) semisolid cultures stimulated with erythropoietin (Ep). Our studies focused on the reactivation of HbF synthesis in normal adult erythroid bursts generated by peripheral blood mononuclear cells (PBMCs) seeded in FCS+ methylcellulose culture. Reactivation is almost totally suppressed when (a) PBMCs are grown in optimized FCS- culture, or (b) PBMCs are first stringently depleted of monocytes and then plated in FCS+ medium (ie, BFU-E growth in FCS+ Mo- culture). In both experimental conditions, the proliferation of lymphocytes and macrophages interspersed among colonies is drastically reduced, and the cloning efficiency of granulocyte-macrophage (GM) progenitors is sharply diminished. In either case, addition of biosynthetic GM colony-stimulating factor (GM-CSF) induces a dose- related increase of HbF synthesis up to the level in FCS+ culture, with even more elevated values on delayed addition of Ep. A dose-related increase was also observed in erythroblast clones generated by highly purified BFU-E. These results suggest that reactivation of HbF synthesis in normal adults is at least in part mediated by GM-CSF. Furthermore, they imply intriguing hypotheses on the mechanism(s) of perinatal Hb switching. Finally, they raise the possibility of reactivation of HbF synthesis in beta-thalassemia and sickle cell anemia by GM-CSF therapy.</jats:p

p53 Localization at Centrosomes during Mitosis and Postmitotic Checkpoint Are ATM-dependent and Require Serine 15 Phosphorylation

We recently demonstrated that the p53 oncosuppressor associates to centrosomes in mitosis and this association is disrupted by treatments with microtubule-depolymerizing agents. Here, we show that ATM, an upstream activator of p53 after DNA damage, is essential for p53 centrosomal localization and is required for the activation of the postmitotic checkpoint after spindle disruption. In mitosis, p53 failed to associate with centrosomes in two ATM-deficient, ataxiatelangiectasia–derived cell lines. Wild-type ATM gene transfer reestablished the centrosomal localization of p53 in these cells. Furthermore, wild-type p53 protein, but not the p53-S15A mutant, not phosphorylatable by ATM, localized at centrosomes when expressed in p53-null K562 cells. Finally, Ser15 phosphorylation of endogenous p53 was detected at centrosomes upon treatment with phosphatase inhibitors, suggesting that a p53 dephosphorylation step at centrosome contributes to sustain the cell cycle program in cells with normal mitotic spindles. When dissociated from centrosomes by treatments with spindle inhibitors, p53 remained phosphorylated at Ser15. AT cells, which are unable to phosphorylate p53, did not undergo postmitotic proliferation arrest after nocodazole block and release. These data demonstrate that ATM is required for p53 localization at centrosome and support the existence of a surveillance mechanism for inhibiting DNA reduplication downstream of the spindle assembly checkpoin

Granulocyte-macrophage colony-stimulating factor reactivates fetal hemoglobin synthesis in erythroblast clones from normal adults

Reactivation of fetal hemoglobin (HbF, alpha 2 gamma 2) synthesis was previously reported in normal human adult erythroblast colonies (“bursts”) generated by erythroid progenitors (BFU-E) in fetal calf serum-supplemented (FCS+) semisolid cultures stimulated with erythropoietin (Ep). Our studies focused on the reactivation of HbF synthesis in normal adult erythroid bursts generated by peripheral blood mononuclear cells (PBMCs) seeded in FCS+ methylcellulose culture. Reactivation is almost totally suppressed when (a) PBMCs are grown in optimized FCS- culture, or (b) PBMCs are first stringently depleted of monocytes and then plated in FCS+ medium (ie, BFU-E growth in FCS+ Mo- culture). In both experimental conditions, the proliferation of lymphocytes and macrophages interspersed among colonies is drastically reduced, and the cloning efficiency of granulocyte-macrophage (GM) progenitors is sharply diminished. In either case, addition of biosynthetic GM colony-stimulating factor (GM-CSF) induces a dose- related increase of HbF synthesis up to the level in FCS+ culture, with even more elevated values on delayed addition of Ep. A dose-related increase was also observed in erythroblast clones generated by highly purified BFU-E. These results suggest that reactivation of HbF synthesis in normal adults is at least in part mediated by GM-CSF. Furthermore, they imply intriguing hypotheses on the mechanism(s) of perinatal Hb switching. Finally, they raise the possibility of reactivation of HbF synthesis in beta-thalassemia and sickle cell anemia by GM-CSF therapy.</jats:p

HOXB cluster genes in activated natural killer lymphocytes: expression from 3'--&amp;gt;5' cluster side and proliferative function.

Abstract The expression of HOXB cluster genes (i.e., B1 through B9) was evaluated in purified IL-2/IL-1 beta-activated NK lymphocytes from normal adult peripheral blood by RNase protection and reverse transcription-PCR. In quiescent NK cells these genes are essentially not expressed. After IL-2/IL-1 beta addition, we observed a coordinate induction wave in the 3'-5' HOXB cluster direction, i.e., from B1 through B9. As notable exceptions, B8 is silent, while B9 RNA is detected starting from 6 h through day 11. Furthermore, the 3' located B2/B3/B4 are expressed earlier and at higher level than the 5' located B5/B6/B7/B8. In IL-2/IL-1 beta-activated NK cells, treatment with antisense oligonucleotides targeting B2 mRNA causes a significant inhibition of both cell proliferation and expression of activation markers (i.e., IL-2R alpha-chain and transferrin receptor). These studies provide novel evidence of the role of HOX genes in adult NK cell proliferation. Thus, 1) a coordinate activation of HOXB genes from the 3'--&amp;gt;5' cluster side apparently underlies IL-2/IL-1 beta-induced NK cell activation. 2) Since NK cell activation and survival induced by IL-12 and c-kit ligand, respectively, are not associated with cell proliferation of HOXB gene expression, it is apparent that HOXB gene induction is specifically associated with IL-2-induced NK cell proliferation. 3) Studies with antisense oligomer targeting HOXB2 mRNA suggest an important role for 82 in NK cell proliferation, possibly in part via the IL-2R.</jats:p

Immunization of woodchucks with adjuvanted sHDAg (p24): immune response and outcome following challenge

The immunogenicity and the protection induced by an hepatitis delta virus (HDV) vaccine consisting of the small nucleoprotein (HDAg) (p24) and adjuvanted with MF59 or Freund's adjuvant (FA) were evaluated in woodchucks chronically infected with woodchuck hepatitis virus (WHV) and challenged with hepatitis delta virus. Humoral and T-cell-mediated responses to HDAg were measured. Anti-HD antibodies appeared earlier in the FA/p24 animals. After challenge, all MF59/p24 vaccinated animals showed a response to HDAg-derived peptides, compared to two of the five FA/p24 animals and one of the control animals. Serum HDV-RNA peak values and persistence were considerably reduced in immunized animals, in comparison to controls. Furthermore, HDV-RNA was absent in autopsy liver tissues of 50% of the MF59/p24 animals, whereas high levels were present in all of the FA/p24 animals and controls. Histological liver analysis performed before and after challenge revealed the presence of acute hepatitis-like lesions only in the controls. Overall, the results suggest that the MF59/p24 vaccine better controls the infection in terms of viral replication and survival. © 2003 Elsevier Ltd. All rights reserved