A specific Toll-like receptor profile on T lymphocytes and values of monocytes correlate with bacterial, fungal, and cytomegalovirus infections in the early period of allogeneic stem cell transplantation.

BACKGROUND: Bacterial, fungal, and viral infections often affect non-relapse mortality after allogeneic stem cell transplantation (alloSCT). Recovery from infections depends on a balanced integration between innate and adaptive immune responses. In this complex interplay, a key role is played by Toll-like receptors (TLRs), which are sensors of pathogen-associated molecular patterns. To our knowledge, no previous study deals with both expression and function of all human TLRs together, in relation to infections in the setting of alloSCT. METHODS: We prospectively evaluated 9 TLRs by flow cytometry on T lymphocytes and monocytes of 35 patients in relation to infectious events from day +30 to day +120. Tumor necrois factor-alpha, interleukin-4, interferon-gamma, and monocyte chemoattractant protein-1 induction upon TLR activation was assessed by enzyme-linked immunosorbent assay on cell supernatants. RESULTS: In multivariate Cox regression analysis, levels of TLR-9 expression on T lymphocytes (P = 0.01) and values of natural killer cells (P = 0.01) correlated negatively with bacterial infections, whereas cytomegalovirus (CMV) infection resulted as a positive predictor. We observed a trend for negative correlation between TLR-7 levels on T lymphocytes and fungal infections (P = 0.07). Values of monocytes were negatively associated with CMV infection (P = 0.03), whereas levels of TLR-5 on T lymphocytes were positive predictors (P = 0.01). Age (P = 0.03) and bacterial infections (P = 0.006) negatively influenced overall survival. Monocyte values were positive predictors of survival (P = 0.003). CONCLUSIONS: Bacterial, fungal, and CMV infections were associated with a different expression of some TLRs on T lymphocytes. The protective role of TLR-7 and TLR-9 seemed dominant over other TLRs involved in recognizing fungi and bacteria. We also observed an atypical involvement of TLR-5 in CMV infection. The dominant and atypical role of some TLRs could depend on their pleiotropic functions and the changing inflammatory environment of transplanted patients. A specific TLR profile and an adequate count of monocytes could improve survival, promoting an effective control of infections, and balanced immune responses. If our findings will be confirmed by further studies, these immunological variables could be useful as parameters to predict susceptibility to infections.

The immunomodulatory molecule pidotimod induces the expression of the NOD-like receptor NLRP12 and attenuates TLR-induced inflammation.

Pidotimod (3-L-pyroglutamyl-L-thiaziolidine-4-carboxylic acid) (PDT) is a synthetic dipeptide with in vitro and in vivo immunomodulatory properties that is largely used for treatment and prevention of infections in paediatric and disease-prone patients. However, the effects of PDT on cellular immune responses are still poorly characterized and there is little information on the mechanism of action of this compound. It has been speculated that PDT action may be exerted through the interaction with a Pattern Recognition Receptor (PRR). Therefore, to gain a further understanding of the immune pathways involved by PDT, we first decided to investigate whether PDT could modify the immune response triggered by TLR ligands. Monocytic cells were exposed to PDT then stimulated with a panel of TLR agonists. Under these experimental conditions, we observed a significant decrease in the synthesis of key proinflammatory mediators in comparison to the production observed in TLR-stimulated cells that were not treated with PDT. Using RT² Profiler PCR Array we have observed that PDT specifically up-regulates the expression of the NOD-like receptor NLRP12 mRNA in the absence of any further costimulation. Increase of NLRP12 in cells treated with PDT was confirmed using specifically designed real-time quantitative PCR and western blotting assays where a clear increase in the amount of NLRP12 protein was detected. Furthermore, in myeloid/monocytic cells we demonstrated that PDT treatment counteracts the NLRP12 reduction induced by TLR agonists. Finally, the results obtained using NLRP12 silenced cells showed that down-regulation of the proinflammatory function occurring in PDT-treated cells upon interaction with TLRs is associated with the increased levels of NLRP12 induced by PDT. To our knowledge this is the first evidence of an immunomodulatory peptide that upregulates NLRP12 and, through this molecule, antagonizes the TLR-induced inflammatory response. These results pave the way for the development of innovative therapeutic approaches aimed at controlling different pathological settings such as tumorigenesis, systemic inflammatory processes and autoimmunity, where NLRP12 plays a crucial role.

Detection of a functional hybrid receptor gammac/GM-CSFRbeta in human hematopoietic CD34+ cells.

A functional hybrid receptor associating the common gamma chain (gammac) with the granulocyte/macrophage colony-stimulating factor receptor beta (GM-CSFRbeta) chain is found in mobilized human peripheral blood (MPB) CD34+ hematopoietic progenitors, SCF/Flt3-L primed cord blood (CB) precursors (CBPr CD34+/CD56-), and CD34+ myeloid cell lines, but not in normal natural killer (NK) cells, the cytolytic NK-L cell line or nonhematopoietic cells. We demonstrated, using CD34+ TF1beta cells, which express an interleukin (IL)-15Ralpha/beta/gammac receptor, that within the hybrid receptor, the GM-CSFRbeta chain inhibits the IL-15-triggered gammac/JAK3-specific signaling controlling TF1beta cell proliferation. However, the gammac chain is part of a functional GM-CSFR, activating GM-CSF-dependent STAT5 nuclear translocation and the proliferation of TF1beta cells. The hybrid receptor is functional in normal hematopoietic progenitors in which both subunits control STAT5 activation. Finally, the parental TF1 cell line, which lacks the IL-15Rbeta chain, nevertheless expresses both a functional hybrid receptor that controls JAK3 phosphorylation and a novel IL-15alpha/gammac/TRAF2 complex that triggers nuclear factor kappaB activation. The lineage-dependent distribution and function of these receptors suggest that they are involved in hematopoiesis because they modify transduction pathways that play a major role in the differentiation of hematopoietic progenitors.

Effects of granulocyte colony stimulating-factor in a rat model of acute liver injury.

BACKGROUND/AIM: Controversial experimental observations suggest that granulocyte colony stimulating-factor may promote hepatic regeneration after hepatectomy and chemical injury either by directly stimulating adult liver cells or facilitating the mobilization of bone marrow cells and their homing to the liver. We investigated whether different schedules of granulocyte colony stimulating-factor administration protect against experimental acute liver injury. METHODS: Acute liver injury was induced in Sprague-Dawley fed rats by injecting a single intraperitoneal dose of carbon tetrachloride. Recombinant human granulocyte colony stimulating-factor or vehicle was given daily after intoxication (4 days) or before (7 days) and after carbon tetrachloride administration. Liver injury and regeneration were assessed 2 and 4 days after damage. Bone marrow cells mobilization was evaluated by the white blood cell count and the assessment of circulating clonogenic haematopoietic progenitors (colony forming unit-cells). RESULTS: In this experimental model, although granulocyte colony stimulating-factor induced the significant mobilization of colony forming unit-cells, the study cytokine had no effect on liver injury (serum alanine amino transaminase level and necrotic index) and liver regeneration (mitotic index and bromodeoxyuridine incorporation), regardless of the administration schedule. CONCLUSIONS: This study does not support the conclusion that: (1) granulocyte colony stimulating-factor exerts a protective effect against toxic-induced, non-lethal acute liver injury and (2) promotes hepatocyte regeneration.

Stem cell factor (c-kit ligand) enhances the interleukin-9-dependent proliferation of human CD34+ and CD34+CD33-DR- cells.

We have studied the effects of recombinant human interleukin-9 (IL-9), alone and combined with stem cell factor (SCF, c-kit ligand), IL-3, and granulocyte-macrophage colony-stimulating factor (GM-CSF) on the clonogenic proliferation of highly enriched human hematopoietic CD34+ and CD34+CD33-DR- progenitor cells. Colony assays were performed under serum-containing and serum-free conditions. IL-9, as a single agent, did not support colony formation. The addition of erythropoietin (Epo) to IL-9 induced the growth of erythroid progenitors (BFU-E) derived from both CD34+ and CD34+CD33-DR- cells. The IL-9-dependent growth of BFU-E derived from CD34+ cells was increased in an additive manner by SCF and, to a lesser extent, by IL-3, whereas CD34+CD33-DR- erythroid precursors were also responsive to GM-CSF in combination with IL-9. The addition of SCF to IL-9 did stimulate the development of CD34+ and CD34+CD33-DR- macroscopic, multicentered BFU-E and multilineage colonies (CFU-GEMM). When IL-9 was used in serum-free conditions, the growth of CD34+ and CD34+CD33-DR- BFU-E was observed in the presence of Epo. Moreover, a marked synergy on BFU-E colony formation was evident when IL-9 was combined with SCF, and their activity was enhanced by the addition of IL-3. IL-9 showed a negligible proliferative activity on colony-forming units-granulocyte/macrophage (CFU-GM). However, it increased the number of CD34+CD33-DR- CFU-GM responsive to IL-3 (37% of the colonies generated by phytohemagglutinin-stimulated lymphocyte conditioned medium [PHA-LCM]). The effects of IL-9 on CD34+CD33-DR- cells were also studied in a short-term suspension culture system, which evaluates the proliferation of progenitors earlier than day 14 CFU-C (Delta assay). In this system, IL-9 had a minimal activity on its own. In combination with SCF, however, it induced a nine-fold expansion of CD34+CD33-DR- cells, which generated a greater number of CFU-GM than BFU-E in secondary methylcellulose cultures. These experiments indicate that IL-9 induces the proliferation of very primitive human erythroid cells, and this effect is potentiated by SCF and other cytokines. Furthermore, IL-9 synergizes in vitro with the c-kit ligand in expanding the pool of early pluripotent hematopoietic progenitor cells.

Interleukin-11 stimulates the proliferation of human hematopoietic CD34+ and CD34+CD33-DR- cells and synergizes with stem cell factor, interleukin-3, and granulocyte-macrophage colony-stimulating factor.

We have studied the effects of recombinant human interleukin-11 (rhIL-11), alone and combined with stem cell factor (SCF or c-kit ligand), IL-3, and granulocyte-macrophage colony-stimulating factor (GM-CSF) on the proliferation of highly enriched human hematopoietic CD34+ and CD34+CD33-DR- progenitor cells. CD34+ cells were purified using the avidin-biotin immunoabsorption technique and CD33+DR+ cells were subsequently removed by immuno-magnetic separation. The colony assays were performed in the presence and absence of exogenous serum. IL-11, as a single agent, induced the growth of a small number of colony-forming units-granulocyte/macrophage (CFU-GM) derived from purified CD34+ cells and failed to support the colony growth of CD34+CD33-DR- cells. The addition of erythropoietin (Epo) to IL-11 induced the growth of erythroid progenitors (BFU-E) derived from CD34+ cells but not from the same population depleted of CD33+DR+ cells. The combination of IL-11 with SCF, IL-3, or GM-CSF, in the presence of Epo, resulted in a synergistic or additive increase in the number of CFU cells (CFU-C) derived from both cell fractions. Moreover, the addition of SCF to IL-11 stimulated the development of macroscopic erythroid and multilineage colonies (CFU-GEMM) containing more than 10(4) cells. A combination of three factors (IL-11, SCF, and IL-3) resulted in the increase of the number of colonies arising from CD34+ and CD34+CD33-DR- cells (but not of their size) compared to the cultures treated with IL-11 plus SCF or IL-11 plus IL-3. The pattern of proliferative response of primitive hematopoietic progenitor cells to IL-11 in serum-free conditions was very similar to the cultures grown in serum-containing medium. It is noteworthy that IL-11 and SCF yielded colony formation that was comparable to that observed in the presence of serum. The effects of IL-11 on CD34+CD33-DR- cells were also studied in a short-term suspension culture system, which was shown to be specific for evaluating the proliferation of pluripotent hematopoietic precursors (Delta assay). In this system, IL-11 had a minimal effect on its own, whereas IL-11 plus SCF acted synergistically and their proliferative activity was improved by the addition of GM-CSF. These experiments indicate that IL-11 may be considered a "permissive" cytokine, capable of initiating the proliferation of very primitive human hematopoietic cells, which are then able to respond to late-acting CSFs.(ABSTRACT TRUNCATED AT 400 WORDS)

Proliferative response of human marrow myeloid progenitor cells to in vivo treatment with granulocyte colony-stimulating factor alone and in combination with interleukin-3 after autologous bone marrow transplantation.

We have recently reported that the hematologic recovery of patients with non-Hodgkin's lymphoma (NHL) and Hodgkin's disease (HD) undergoing autologous bone marrow transplantation (BMT) is significantly faster when recombinant human interleukin-3 (rhIL-3) is combined with recombinant human granulocyte colony-stimulating factor (rhG-CSF) in comparison with patients receiving G-CSF alone. In this paper, we studied the kinetic response and concentration of BM progenitor cells of 17 patients with lymphoid malignancies submitted to autologous BMT and treated with the G-CSF/IL-3 combination. The results were compared with those of five lymphoma patients receiving the same pretransplant conditioning regimen followed by G-CSF alone. rhG-CSF was administered as a single subcutaneous (sc) injection at the dose of 5 micrograms/kg/d from day 1 after reinfusion of autologous stem cells; rhIL-3 was added from day 6 at the dose of 10 micrograms/kg/d sc (overlapping schedule). In both groups (G-CSF- and G-CSF/IL-3-treated patients), cytokine administration was discontinued when the absolute neutrophil count (ANC) was >0.5 x 10(9)/L of peripheral blood (PB) for 3 consecutive days. After treatment with the CSF combination, the percentage of marrow colony-forming units-granulocyte/macrophage (CFU-GM) and erythroid progenitors (BFU-E) in S phase of the cell cycle increased from 9.3 +/- 2% to 33.3 +/- 12% and from 14.6 +/- 3% to 35 +/- 6%, respectively (p < 0.05). Similarly, we observed an increased number of actively cycling megakaryocyte progenitors (CFU-MK and BFU-MK). Conversely, G-CSF augmented the proliferative rate of CFU-GM (22.6 +/- 0.6% compared to a baseline value of 11.5 +/- 3%; p < 0.05) but not of BFU-E, CFU-MK, or BFU-MK, and the increase of S-phase CFU-GM was significantly lower than that observed in the posttreatment samples of patients receiving IL-3 in addition to G-CSF. The frequency of hematopoietic precursors in the BM, expressed as the number of colonies formed per number of cells plated, was unchanged or slightly decreased in both groups of patients. Because of the increase in marrow cellularity, however, a significant augmentation of the absolute number of both CFU-GM (3605 +/- 712/mL BM vs. 2213 +/- 580/mL; p < 0.05) and BFU-E (4373 +/- 608/mL vs. 3027 +/- 516/mL; p < 0.05) was reported after treatment with G-CSF/IL-3 but not G-CSF alone. Similarly, administration of the cytokine combination resulted in a higher number of CD34+ cells/mL BM, and their concentration was significantly greater than that observed in the posttreatment samples of G-CSF patients. Finally, we investigated the responsiveness to CSFs, in vitro, of highly enriched CD34+ cells, collected after priming with G-CSF in vivo (i.e., after 5 days of G-CSF administration). Our results demonstrated that pretreatment with G-CSF modified the response of BM cells to subsequent stimulation with additional CSFs. The results presented in this paper indicate that in vivo administration of two cytokines increases the proliferative rate and concentration of BM progenitor cells to a greater degree than G-CSF alone. These results support the role of growth factor combinations for accelerating hematopoietic recovery after high-dose chemotherapy.

Pharmacological purging of minimal residual disease from peripheral blood stem cell collections of acute myeloblastic leukemia patients: preclinical studies.

UNLABELLED: In this paper we describe an experimental model for ex vivo purging of contaminating tumor cells from peripheral blood stem cell (PBSC) collections obtained from patients with acute myeloblastic leukemia (AML). We studied the combination of the alkylating agent nitrogen mustard (NM; concentrations ranging from 0.25 to 1.25 microg/mL) and etoposide (VP-16; constant dose of 20 microg/mL), and the conventional cyclophosphamide (Cy)-derivative mafosfamide (concentrations: 20-175 microg/mL). THE AIMS OF OUR STUDY WERE: 1) To compare the toxicity of the purging protocols on bone marrow (BM) and circulating trilineage precursors collected from normal donors after priming with granulocyte colony-stimulating factor (G-CSF) or after complete remission (CR) consolidation chemotherapy and G-CSF (leukemic patients); 2) to demonstrate the survival of very primitive hematopoietic progenitors (LTC-IC) in the peripheral blood (PB) and the BM after pharmacological treatment; and 3) to evaluate the antineoplastic efficacy of purging protocols on PBSC collections using 3 well-established leukemic cell lines. Our results demonstrated that the toxicity on BM and PB progenitor cells could be correlated with the complete killing of committed granulocyte-macrophage colony-forming units (CFU-GMs) and erythroid precursors (BFU-Es), a condition reached at the concentration of 1.5 microg/mL of NM (in addition to 20 microg/mL of VP-16) and 175 microg/mL of mafosfamide. Notably, early and late megakaryocyte progenitor cells (CFU-MKs and BFU-MKs, respectively) showed higher sensitivity to NM/VP-16, but not to mafosfamide, than did CFU-GMs and BFU-Es. The dose of NM capable of inhibiting 95% of CFU-MKs and BFU-MKs (ID95) was 0.75 microg/mL. After incubation with the same dose of NM, the recovery of CFU-GMs and BFU-Es was 20 +/- 8% SD and 25 +/- 10% SD, respectively (p < 0.05). Long-term liquid cultures showed the recovery of primitive hematopoietic cells after incubation with the highest concentrations of NM/VP-16 and mafosfamide, with no significant differences between PB and BM samples. Under the same experimental conditions, we observed a more than 5-log reduction of contaminating leukemic cell lines (i.e., K-562, KG-1, and HL-60). In conclusion, we demonstrated that NM/VP-16 and mafosfamide purging agents are capable of killing leukemic cell lines that contaminate leukapheresis products from patients with AML, whereas an acceptable proportion of primitive LTC-IC is spared. Moreover, despite the different kinetic and functional profile of mobilized and steady-state BM progenitors, we did not observe any difference in toxicity of antineoplastic agents on hematopoietic cells at different levels of differentiation. These data suggest that pharmacological strategies developed for eliminating minimal residual disease (MRD) from BM autografts can be effectively and safely applied to circulating stem cell harvests.

Efficient presentation of tumor idiotype to autologous T cells by CD83(+) dendritic cells derived from highly purified circulating CD14(+) monocytes in multiple myeloma patients.

To generate mature and fully functional CD83(+) dendritic cells derived from circulating CD14(+) cells highly purified from the leukapheresis products of multiple myeloma patients.CD14(+) monocytes were selected by high-gradient magnetic separation and differentiated to immature dendritic cells with granulocyte-macrophage colony-stimulating factor and interleukin-4 for 6-7 days and then induced to terminal maturation by the addition of tumor necrosis factor-alpha or stimulation with CD40 ligand. Dendritic cells were characterized by immunophenotyping, evaluation of soluble antigens uptake, cytokine secretion, capacity of stimulating allogeneic T cells, and ability of presenting nominal antigens, including tumor idiotype, to autologous T lymphocytes. Phenotypic analysis showed that 90% +/- 6% of cells recovered after granulocyte-macrophage colony-stimulating factor and interleukin-4 stimulation expressed all surface markers typical of immature dendritic cells and demonstrated a high capacity of uptaking soluble antigens as shown by the FITC-dextran assay. Subsequent exposure to maturation stimuli induced the downregulation of CD1a and upregulation of CD83, HLA-DR, costimulatory molecules and induced the secretion of large amounts of interleukin-12. Mature CD83(+) cells showed a diminished ability of antigen uptake whereas they proved to be potent stimulators of allogeneic T cells in a mixed lymphocyte reaction. Monocyte-derived dendritic cells, pulsed before the addition of maturation stimuli, were capable of presenting soluble proteins such as keyhole limpet hemocyanin and tetanus toxoid to autologous T cells for primary and secondary immune response, respectively. Conversely, pulsing of mature (CD83(+)) dendritic cells was less efficient for the induction of T-cell proliferation. More importantly, CD14(+) cells-derived dendritic cells stimulated autologous T-cell proliferation in response to a tumor antigen such as the patient-specific idiotype. Moreover, idiotype-pulsed dendritic cells induced the secretion of interleukin-2 and gamma-interferon by purified CD4(+) cells. T-cell activation was better achieved when Fab immunoglobulin fragments were used as compared with the whole protein. When dendritic cells derived from CD14(+) cells from healthy volunteers were analyzed, we did not find any difference with samples from myeloma patients as for cell yield, phenotypic profile, and functional characteristics. These studies demonstrate that mobilized purified CD14(+) cells represent the optimal source for the production of a homogeneous cell population of mature CD83(+) dendritic cells suitable for clinical trials in multiple myeloma.

Transforming growth factor beta3 inhibits chronic myelogenous leukemia hematopoiesis by inducing Fas-independent apoptosis.

OBJECTIVE: Transforming growth factor beta3 (TGF-beta3) is a potent suppressor of human hematopoietic progenitor cells. In this article, we compare the activity of TGF-beta3 on highly purified CD34+ cells and more immature CD34-DR(-) cells from chronic myelogenous leukemia (CML) patients in chronic phase and normal donors. MATERIALS AND METHODS: Primitive hematopoietic progenitors were stimulated in liquid cultures and clonogenic assays by early-acting growth factors such as stem cell factor (SCF) and interleukin 11 (IL-11) and the intermediate-late-acting stimulating factors IL-3, granulocyte-macrophage colony-stimulating factor, and erythropoietin. Molecular analysis of bcr/abl mRNA was performed on single CML colonies by nested reverse transcriptase polymerase chain reaction. Moreover, cell cycle analysis and assessment of apoptosis of normal and leukemic CD34+ cells were performed by propidium iodide (PI) alone and simultaneous staining with annexin V and PI, respectively. RESULTS: The colony-forming efficiency of CML CD34+ cells was generally inhibited by more than 90% regardless of whether the colony-stimulating factors were used alone or combined. When compared to normal CD34+ cells, leukemic cells were significantly more suppressed in 6 of 8 culture conditions. The inhibitory effect of TGF-beta3 on CD34+ cells was exerted within the first 24 hours of incubation as demonstrated by short-term preincubation followed by IL-3-and SCF-stimulated colony assays. Evaluation of bcr/abl transcript on residual CML colonies incubated with TGF-beta3 demonstrated a small subset of neoplastic CD34+ cells unresponsive to the inhibitory effect of the study cytokine. TGF-beta3 demonstrated a greater inhibitory activity on primitive CD34+DR cells than on more mature CD34+ cells. Again, CML CD34+DR(-) cells were significantly more inhibited by TGF-beta3 than their normal counterparts in 3 of 8 culture conditions. Kinetic analysis performed on CD34+ cells showed that TGF-beta induces cell cycle arrest in G(1) phase. However, this mechanism of action is shared by normal and leukemic cells. Conversely, TGF-beta3 preferentially triggered the programmed cell death of CML CD34-cells without increasing the proportion of leukemic cells coexpressing CD95 (Fas receptor), and this effect was not reversed by functional blockade of Fas receptor. Conclusion. We demonstrate that TGF-beta3 exerts a potent suppressive effect on CML cells that is partly mediated by Fas-independent apoptosis.

Thrombopoietin and interleukin 11 have different modulatory effects on cell cycle and programmed cell death in primary acute myeloid leukemia cells.

The c-mpl ligand, thrombopoietin (TPO), is a physiologic regulator of platelet and megakaryocytic production, acting synergistically on thrombopoiesis with the growth factors interleukin 11 (IL-11), stem cell factor, interleukin 3 (IL-3), interleukin 6 (IL-6), and granulocyte-macrophage colony-stimulating factor. Because some of these growth factors, especially TPO and IL-11, are now being evaluated clinically to reduce chemotherapy-associated thrombocytopenia in cancer patients, we evaluated 25 acute myeloid leukemia (AML) samples to test whether TPO, IL-11, and other early-acting megakaryocyte growth factors can affect leukemic cell proliferation, cell cycle activation, and programmed cell death (PCD) protection. TPO induced proliferation in the majority of AML samples from an overall mean proportion of S-phase cells of 7.8% +/-1.5% to 14.5% +/- 2.1% (p = 0.0006). Concurrent G0 cell depletion was found in 47.3% of AML samples. TPO-supported leukemic cell precursor (CFU-L) proliferation was reported in 5 of 17 (29.4%) of the samples with a mean colony number of 21.4 +/- 9.6 x 10(5) cells plated. In 13 of 19 samples, a significant protection from PCD (from an overall mean value of 13% +/-0.7% to 8.8% +/- 1.8%;p = 0.05) was detected after TPO exposure. Conversely, IL-11-induced cell cycle changes (recruitment from G0 to S phase) were detected in only 2 of 14 samples (14.2%). In addition, IL-11 showed little, if any, effect on CFU-L growth (mean colony number = 17.5 9.5) or apoptosis. Combination of TPO with IL-11 resulted in only a slight increase in the number of CFU-L, whereas IL-3 and stem cell factor significantly raised the mean colony numbers up to 119.2 +/- 68.3 and 52.9 +/- 22.1 x 10(5) cells plated, respectively. We conclude that TPO induces cell cycle activation in a significant proportion of cases and generally protects the majority of AML blast cells from PCD. On the other hand, IL-11 has little effect on the cell cycle or PCD. Combination of both TPO and IL-11 is rarely synergistic in stimulating AML clonogenic growth. These findings may be useful for designing clinical studies aimed at reducing chemotherapy-associated thrombocytopenia in AML patients.

Managing chronic myeloid leukaemia in the elderly with intermittent imatinib treatment.

The aim of this study was to investigate the effects of a non-standard, intermittent imatinib treatment in elderly patients with Philadelphia-positive chronic myeloid leukaemia and to answer the question on which dose should be used once a stable optimal response has been achieved. Seventy-six patients aged ⩾65 years in optimal and stable response with ⩾2 years of standard imatinib treatment were enrolled in a study testing a regimen of intermittent imatinib (INTERIM; 1-month on and 1-month off). With a minimum follow-up of 6 years, 16/76 patients (21%) have lost complete cytogenetic response (CCyR) and major molecular response (MMR), and 16 patients (21%) have lost MMR only. All these patients were given imatinib again, the same dose, on the standard schedule and achieved again CCyR and MMR or an even deeper molecular response. The probability of remaining on INTERIM at 6 years was 48% (95% confidence interval 35-59%). Nine patients died in remission. No progressions were recorded. Side effects of continuous treatment were reduced by 50%. In optimal and stable responders, a policy of intermittent imatinib treatment is feasible, is successful in about 50% of patients and is safe, as all the patients who relapsed could be brought back to optimal response.

Differential disappearance of inhibitory natural killer cell receptors during HAART and possible impairment of HIV-1-specific CD8 cytotoxic T lymphocytes.

BACKGROUND: Highly active antiretroviral therapy (HAART) is associated with a decrease in viral replication to undetectable levels and with an increase in CD4 T lymphocytes. Residual HIV-1 replication occurs together with incomplete recovery of cytotoxic CD8 T lymphocyte (CTL) numbers and function. We sought to determine whether expression of HLA class I-specific inhibitory natural killer receptors (iNKR) on the CTL of patients who had been treated successfully with HAART for 24 months could be involved, at least in part, in residual CTL functional inhibition. METHODS: Two-colour cytofluorometry was used to analyse the expression of six different iNKR including p58.1, p58.2, p70, p140, CD94/NKG2A and LIR1/ILT2 on the CD3, CD8 lymphocytes of eight patients with successful long-term suppression of viral replication before and after 3, 6 and 24 months of HAART. Healthy subjects were analysed as controls. HIV-1-specific cytotoxic activity was determined after 24 months of HAART in the presence and absence of iNKR-masking. RESULTS: No significant reduction of iNKR expression on CD8 T cells was observed by 6 months. Expression of p70 and p140 was inversely correlated with the increasing CD4 numbers. After 24 months CD8 T-lymphocytes expressing p58.1, p58.2, p70, p140 and CD94/NKG2A returned to levels indistinguishable from those of the healthy controls. A significantly increased proportion of CD8 CTL still expressed LIR1/ILT2, a receptor with broad HLA-class I specificity. Functional analysis of freshly separated cells revealed that the disruption of the interaction between LIR1/ILT2 and HLA-class I could partly restore HIV-1-specific lysis. CONCLUSIONS: A decrease in CD3CD8iNKR cells is observed beyond 6 months of HAART. In some patients functional impairment due to LIR1/ILT2 expression may persist even after 24 months of successful HAART.

Reconstitution of active catalytic trimer of aspartate transcarbamoylase from proteolytically cleaved polypeptide chains.

Treatment of the catalytic (C) trimer of Escherichia coli aspartate transcarbamoylase (ATCase) with alpha-chymotrypsin by a procedure similar to that used by Chan and Enns (1978, Can. J. Biochem. 56, 654-658) has been shown to yield an intact, active, proteolytically cleaved trimer containing polypeptide fragments of 26,000 and 8,000 MW. Vmax of the proteolytically cleaved trimer (CPC) is 75% that of the wild-type C trimer, whereas Km for aspartate and Kd for the bisubstrate analog, N-(phosphonacetyl)-L-aspartate, are increased about 7- and 15-fold, respectively. CPC trimer is very stable to heat denaturation as shown by differential scanning microcalorimetry. Amino-terminal sequence analyses as well as results from electrospray ionization mass spectrometry indicate that the limited chymotryptic digestion involves the rupture of only a single peptide bond leading to the production of two fragments corresponding to residues 1-240 and 241-310. This cleavage site involving the bond between Tyr 240 and Ala 241 is in a surface loop known to be involved in intersubunit contacts between the upper and lower C trimers in ATCase when it is in the T conformation. Reconstituted holoenzyme comprising two CPC trimers and three wild-type regulatory (R) dimers was shown by enzyme assays to be devoid of the homotropic and heterotropic allosteric properties characteristic of wild-type ATCase. Moreover, sedimentation velocity experiments demonstrate that the holoenzyme reconstituted from CPC trimers is in the R conformation. These results indicate that the intact flexible loop containing Tyr 240 is essential for stabilizing the T conformation of ATCase. Following denaturation of the CPC trimer in 4.7 M urea and dilution of the solution, the separate proteolytic fragments re-associate to form active trimers in about 60% yield. How this refolding of the fragments, docking, and association to form trimers are achieved is not known.

Multiple HLA-class I-specific inhibitory NK receptor expression and IL-4/IL-5 production by CD8+ T-cell clones in HIV-1 infection.

Several mechanisms may contribute to the decline in HIV-1 specific CD8+ cytotoxic T-lymphocyte (CTL) activity that is observed in infected patients, including loss of CD4+ cell help, antigenic shift, impaired clonogenicity and functional impairment due to expression of inhibitory NK receptors (iNKRs). In addition to a decrease in HIV-1-specific cytolytic activity, an increased proportion of CD8+ T-cells producing IL-4 and IL-5 has been recently observed in advanced HIV-1 infection. Remarkably, an impaired HIV-1-specific CTL activity was primarily detected among the TC0/Tc2 CD8+ CTLs. A series of CD3+CD8+ T-cell clones expressing inhibitory NK receptors (iNKRs) isolated from HIV-1 infected patients was analyzed in order to determine their cytokine production pattern and to assess the extent of iNKR expression at the single cell level. Our data indicate that iNKR+CD3+CD8+ clones isolated from infected patients frequently express multiple iNKR and may produce IL-4 and IL-5 to a relevant extent.

Thrombopoietin, interleukin-11, and early-acting megakaryocyte growth factors in human myeloid leukemia cells.

In this study we report our data on effects of early-acting megakaryocyte growth factors, particularly the c-mpl ligand also known as thrombopoietin (TPO) and interleukin-11 (IL-11), on cell proliferation and apoptosis (Apo) of primary acute myeloid leukemia (AML) cells. A proliferative response to TPO was noticed in the majority of AML samples (17/19) with an average increase of S-phase cells from 7.8% +/- 1.5 to 14.5% +/- 2.1 (p=0.0006). Resulting cell cycle activation did not always correlate with expression of the c-mpl receptor, although it was coupled, in the majority of samples, by an average decrease of apoptotic cells from 13% +/- 0.7 to 8.8% +/- 1.8 (p=0.05). Clonogenic cell growth (CFU-L) was confirmed in 5/17 of the samples with a mean colony number of 21.4 +/- 9.6 x 10(5) cells plated. Conversely, effects of IL-11 on AML cells demonstrated that cell cycle changes (recruitment from G0 to S phase) were promoted only in a minority of samples (2/14) and there was little, if any, effect on CFU-L growth (mean colony number=17.5 +/- 9.5) or Apo (from 13% +/- 0.7 to 13.3 +/- 1.9). Combination of TPO with IL-11 induced a slight increase of clonogenic cell growth, while the addition of IL-3 or SCF to the c-mpl ligand significantly raised the mean colony numbers up to 119.2 +/- 68.3 and 52.9 +/- 22.1 x 10(5) cells plated, respectively. In summary, TPO shows activity on AML cells by stimulating their proliferation in a significant proportion of cases and generally protecting the majority of AML blast cells from induction of Apo. Conversely, IL-11 exerts little effect on the cell cycle activation and Apo. These data help to understand regulation of myeloid leukemia cell growth and should be considered in the clinical use of early-acting megakaryocyte growth factors in acute leukemia.

Dendritic cell differentiation from hematopoietic CD34+ progenitor cells.

Dendritic cells (DC) are the most powerful antigen presenting cells (APC) and play a pivotal role in initiating the immune response. In light of their unique properties, DC have been proposed as a tool to enhance immunity against infectious agents and in anticancer vaccine strategies. In the last few years, the development of DC has been extensively investigated. The present paper summarizes the most recent findings on the differentiation of myeloid DC from hematopoietic CD34+ progenitors and methods for DC generation in vitro. A better understanding of DC function has important implications for their use in clinical settings.

Cd 34+ cells and clonogenicity of peripheral blood stem cells during chemotherapy treatment in association with granulocyte colony stimulating factor in osteosarcoma.

The introduction of aggressive chemotherapy in the treatment of osteosarcoma has improved the long-term outcome for these patients. With the increasing aggressiveness of chemotherapy protocols, hematopoietic growth factors have emerged as useful adjuncts involving, in some cases, rescue by peripheral blood stem cell (PBSC) infusion to assist faster recovery and maintain relative dose intensity. To evaluate the number of PBSCs needed, we analyzed the number of CD34+ cells and hematopoietic progenitor cells in the peripheral blood of 16 patients with osteoblastic, condroblastic and fibroblastic osteosarcoma enrolled in an Istituto Ortopedico Rizzoli-Scandinavian Sarcoma Group (IOR-SSG) pilot study, consisting of two cycles of preoperative high dose chemotherapy. The blood samples were studied at different times. The CD34+ cells were analyzed by flow cytometry and the hematopoietic progenitor cells were analyzed by tissue culture clonogenic assay. In comparing the two courses of chemotherapy, we observed that modification of the mean values of WBC, CD34+ and CFU-GM were very similar. The second course of chemotherapy seemed to induce greater hematological toxicity. All three parameters showed good correlation. The results demonstrated that the best time to collect PBSC by means of leukapheresis is post G-CSF used as rescue after ifosfamide treatment. We verified the ability of G-CSF to mobilize PBSCs in patients with osteosarcoma through cytofluorimetric analysis of CD34+ cells and their clonogenic capability. Moreover, during this preoperative treatment, we identified the best time to collect a sufficient number of PBSCs, that is after 9-10 days of G-CSF treatment following the first cycle of ifosfamide.

Interleukin-11 (IL-11) and IL-9 counteract the inhibitory activity of transforming growth factor beta 3 (TGF-beta 3) on human primitive hematopoietic progenitor cells.

BACKGROUND: TGF-beta 3 has been proven to be a potent suppressor of human hematopoietic progenitor cells and its effects on hematopoiesis are only inhibitory. METHODS: In this paper we investigated the antiproliferative activity of TGF-beta 3 on highly purified bone marrow (BM) CD34+ cells and more immature CD34+/4-hydroperoxycyclophosphamide (4-HC) resistant cells. Primitive hematopoietic progenitors were stimulated by early acting stimulatory factors such as SCF, IL-11, IL-9 and the intermediate-late acting growth factors IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF), alone and in combination. RESULTS: The addition of TGF-beta 3 to cultures of CD34+ cells containing IL-11, IL-9 or SCF alone resulted in 86% or more inhibition of total colony formation. Conversely, IL-3 and GM-CSF-stimulated colony growth was inhibited by 57% and 58%, respectively (p < 0.02). IL-11 and IL-9 acted synergistically or additively with IL-3 and GM-CSF on the clonogenic growth of BFU-E derived from CD34+ cells, in both the presence and absence of TGF-beta 3. Co-incubation of CD34+ cells with 2 synergistic factors (e.g. IL-11 and SCF or IL-9 and SCF), with or without TGF-beta 3, resulted in the enhancement of both CFU-GM and BFU-E growth. The percentage of CD34+ cells inhibited by TGF-beta 3 was significantly reduced when IL-11 or IL-9, but not SCF, was added to the other cytokines (e.g. IL-11 and IL-3-stimulated cultures were inhibited by 42%, compared to 57% and 90% for the CSF alone; p < 0.05). Similarly, the addition of IL-11 or IL-9 to SCF decreased the suppressive activity of TGF-beta 3 (e.g. IL-11 and SCF in combination were inhibited by 52.4%, compared to 90% or more when the same cytokines were used separately; p < 0.001). These effects were mainly observed on CD(34+)-derived BFU-E although IL-9 appeared to override TGF beta-3 on both CFU-GM and BFU-E. When tested on CD34+/4-HC resistant progenitors, IL-11, IL-9 and SCF increased the number of clonogenic cells responsive to IL-3 and GM-CSF. However, TGF-beta 3 demonstrated a greater inhibitory activity on earlier cells than on the more mature CD34+ cell fraction, and none of the study cytokines completely abrogated the activity of TGF-beta 3. CONCLUSIONS: These data confirm that TGF-beta 3 exerts its suppressive effect on hematopoietic progenitor cells according to the differentiation state of the target cells and the presence of other cytokines interacting with the cells. The permissive growth factors IL-11 and IL-9 seem to be able to partially counteract the negative regulation of TGF-beta 3.