Expression of human glucocerebrosidase in murine long-term bone marrow cultures after retroviral vector-mediated transfer

A retroviral vector (N2-SV-GC) was constructed by inserting a normal human glucocerebrosidase (GC) cDNA under control of the SV40 early region promoter into the Moloney murine leukemia virus-derived N2 vector. N2-SV-GC produced human GC in murine 3T3 fibroblasts at levels in the range of the endogenous murine GC as determined by enzymatic assay and Western blot analysis. The N2-SV-GC retroviral vector was used for studies of gene transduction of murine hematopoietic progenitor cells (HPC). Infection of bone marrow cultured for 2 to 10 days in medium containing hematopoietic growth factors was significantly more efficient than infection of freshly isolated marrow cells (24% to 32% G418-resistant CFU-GM v 15%, respectively). The marrow infected by N2-SV-GC was maintained in long-term bone marrow culture (LTBMC) and had a stable level of G418-resistant HPC over 2 months of serial assays. The human GC gene of the vector was persistently expressed in the nonadherent cell fraction of the murine LTBMC as determined by Northern blotting, Western blotting, and immunohistochemical staining using a monoclonal antibody specific for human GC. N2-SV-GC also expressed the human GC gene in day 12 CFU-S. LTBMC represents a novel system for retroviral vector-mediated gene transduction of HPC and may accurately predict the activities of vectors in vivo.     

The role of platelet alpha-granular proteins in the regulation of thrombopoietin messenger RNA expression in human bone marrow stromal cells

Thrombopoietin (TPO), the specific cytokine that regulates platelet production, is expressed in human bone marrow (BM), kidney, and liver. There appears to be no regulation of TPO in the kidney and liver, but TPO messenger RNA (mRNA) expression can be modulated in the stromal cells of the BM. In this study, we used primary human BM stromal cells as a model to study the regulation of TPO mRNA expression in response to various platelet alpha-granular proteins. We showed that platelet-derived growth factor (PDGF) BB and fibroblast growth factor (FGF) 2 stimulated TPO mRNA expression in both a dose-dependent and time-dependent manner. The addition of 50 ng/mL of PDGF and 20 ng/mL of FGF resulted in maximal induction of TPO mRNA expression in 4 hours. We also found that platelet factor 4 (PF4), thrombospondin (TSP), and transforming growth factor-beta (TGF-beta) are negative modulators of megakaryocytopoiesis. We observed suppression in TPO mRNA expression with 1 microg/mL of both PF4 and TSP and 50 ng/mL of TGF-beta, with maximal suppression occurring 4 hours after the addition of these proteins. Finally, the addition of whole-platelet lysate produced a dose-dependent inhibition of TPO expression. On the basis of these findings, we propose that the platelet alpha-granular proteins studied may regulate TPO gene expression in BM stromal cells by means of a feedback mechanism.     

Adenoviral-mediated gene transfer into ex vivo expanded human bone marrow mesenchymal progenitor cells

OBJECTIVE: Based on their differentiation properties and facilely of ex vivo expansion, human bone marrow mesenchymal progenitor cells (MPC), are considered as attractive targets to deliver foreign genes to the bone marrow or other mesenchymal tissues. In this study we investigated the feasibility of transduce MPC with adenoviral vectors (Adv). METHODS: MPC were expanded ex vivo and transduced with replication-defective Adv-containing reporter genes (lacZ or GFP) under the control of CMV promoter. Transfection efficiency was assessed by microscopical scoring or by flow cytometry. Expression and involvement of Adv-attachment (CAR) and Adv-internalization (integrins alphav) receptors were evaluated by flow cytometric studies. RESULTS: Transgene expression analysis showed that only 19%+/-3% of cells expressed the transgenes at high levels. MPC express the attachment and internalization receptors required for Adv infection. While integrins alphavbeta3 and alphavbeta5 are expressed by all MPC, CAR is solely expressed by a fraction of low size cells. Antibodies against CAR and alphavbeta5, but not against alphavbeta3, blocked Adv-mediated gene transfer into MPC, showing that CAR and alphavbeta5 are required for infection. Because alphavbeta5, as compared with CAR, is overexpressed in MPC, the results suggest that the efficiency of Adv-mediated gene transfer into MPC depends on the level of CAR expression. CONCLUSION: These findings demonstrate that Adv may be useful to engineer a subpopulation of ex vivo expanded human mesenchymal progenitors, with a high level of transgene expression.     

Gene transfer into human bone marrow hematopoietic cells mediated by adenovirus vectors [see comments]

Human bone marrow mononuclear cells (BMMNCs) and enriched CD34 positive (CD34+) cells were transduced with adenovirus vectors encoding Escherichia coli beta-galactosidase gene. Tranductions were carried out by 24-hour coincubation with adenovirus vectors at different multiplicities of infections (moi). Efficacy of gene transfer into BM cells and expression of the gene product (ie, beta-galactosidase) were studied using X-Gal histochemical staining and flow cytometric analysis. X-Gal staining demonstrated that the percentage of positive cells at mois of 5 to 500 was 3.4% to 34.5% for BMMNCs and 6.0% to 20.0% for enriched CD34+ cells. Similar results (1.5% to 35.7% for BMMNCs and 5.4% to 24.2% for enriched CD34+ cells) were obtained with flow cytometric analysis using fluorescein di-beta-D-galactopyranoside (FDG). Multicolor flow cytometry analysis, which included FDG, demonstrated that BM progenitors (CD34+ or CD34+CD38-), T cells (CD2+), B cells (CD19+), natural killer cells (CD56+), granulocytes, and monocytes all expressed the adenovirus transgene. To ascertain the effects of adenovirus vectors on normal BM progenitors, the numbers of colony forming unit-granulocyte/macrophage (CFU-GM), burst-forming unit- erythrocyte (BFU-E), and high-proliferative potential-colony-forming cells (HPP-CFC) after 24-hour coincubation with adenovirus vectors were determined. When BMMNCs or enriched CD34+ cells were incubated with adenovirus vectors at mois of 5 and 50, no significant differences in the numbers of CFU-GM, BFU-E, and HPP-CFC were observed compared with the uninfected control cells. However, the numbers of CFU-GM were significantly (P < .01) decreased when BMMNCs or enriched CD34+ cells were incubated with adenovirus vectors at a moi of 500, compared with the uninfected control cells. The adenovirus infected cells, purified by cell sorting for FDG expression, were capable of growing in culture and gave rise to various colonies (ie, CFU-GM, BFU-E, and HPP-CFC). These data indicate that recombinant adenovirus vectors can be used to transfer genes to human BM hematopoietic cells with expression of the exogenous gene at a high transduction efficiency.     

In vitro suppression of bone marrow progenitor cell differentiation by human herpesvirus 6 infection.

Suppression of marrow function may be one of the most serious effects of human herpesvirus 6 (HHV-6) infection in marrow transplant patients. In this study, normal bone marrow mononuclear cells were infected in vitro with HHV-6, and a methylcellulose-based colony formation assay was used to evaluate the impact of the infection on marrow cell differentiation and proliferation. Results demonstrated that the outgrowth of colony-forming units of granulocyte and macrophage lineages (cfu-GM) was decreased by approximately 43%, that growth of cfu of granulocyte, erythrocyte, macrophage, and megakaryocyte lineages (cfu-GEMM) was inhibited by an average of 71%, and that the erythroid burst-forming unit (bfu-E) was decreased by approximately 73%. Further, outgrowth of the marrow stromal layer was reduced 74%. Direct infection of bone marrow monocytes was observed, although cell-free virus could not be detected in infected culture supernatants. Addition of a neutralizing monoclonal antibody specific for interferon-alpha to the infected cultures resulted in an almost complete reversal of the viral suppressive effects.     

Thiamphenicol induced bone marrow suppression as a therapy of myeloproliferative diseases

Summary Sixteen patients, suffering from myeloproliferative diseases (9 polycythaemia vera, 7 primary thrombocythaemia) and 20 control subjects were treated for 14 days with 1 g of thiamphenicol per day. The effect of 40 treatment cycles was studied. The regimen resulted in lowering the haemoglobin values by 4.8% in controls (p0.001) and patients (p0.01); the reticulocyte count dropped 43% in the patient group (p0.01) and 32% in the controls (p0.05); the thrombocyte count was decreased 42% (p0.0001) vs 29% (p0.0001) in the control group. The administration of 1 g/day of thiamphenicol for a 14 day period reduced the myelopoietic activity by 40%. The decrease of all values was statistically significant. After discontinuation of thiamphenicol therapy the haematological parameters returned to the initial values within 1–2 months. The myelodepressant activity of thiamphenicol may therefore be applied to the therapy of the myeloproliferative diseases in order to reduce the risk of spontaneous haemorrhages and thrombosis.     

The synergistic effect of thrombopoietin in erythropoiesis and myelopoiesis from human bone marrow cells

We sought to determine whether recombinant human thrombopoietin (TPO) acts synergistically with recombinant human erythropoietin (EPO) and/or recombinant human interleukin-3 (IL-3) on erythroid burst formation and granulocyte-macrophage colony formation from human bone marrow (BM). BM cells were from 5 adults and 15 children who underwent bone marrow examination because of a clinical suspicion of malignancy; their bone marrows as well as the complete blood counts were normal and were cultured in a methylcellulose system. TPO has a synergistic effect with EPO or EPO + IL-3 on erythropoiesis of human BM, as the addition of TPO to EPO significantly gave rise to more erythroid bursts (p = 0.0001) and the addition of TPO to EPO + IL-3 might give rise to more erythroid bursts (p = 0.05). TPO also has a synergistic effect with recombinant human granulocyte colony-stimulating factor (G-CSF) on myelopoiesis of human BM, since the addition of TPO to G-CSF gave rise to significantly more granulocyte-macrophage colonies (p = 0. 0001). Besides its well-known significant role in megakaryopoiesis, TPO also has synergistic effects on erythropoiesis and myelopoiesis.     

Kinetics of circulating selectin levels during bone marrow aplasia

Abstract: We have studied the kinetics of plasma levels of circulating (c)selectins in 8 patients undergoing bone marrow or stem cell transplantation to gain estimates for the distribution and half-life of (c)selectins and to potentially identify an endothelial source of cp -selectin in patients who are deprived of platelets and megakaryocytes. Blood was sampled just before conditioning treatment and immediately after, at 2 occasions under bone marrow aplasia (1 and 2 wk after BMT), on 2 separate days (3–8 d apart) before and at 60 min after platelet transfusion, and 30–72 d after BMT. All (c)selectins showed a strikingly parallel decrease during bone marrow aplasia: cp-selectin decreased by a median of 70% (range: 59–95%), CE-selectin by 63% (range: 27–78%), and cl -selectin by 75% (range: 66–90%) compared to baseline (p = 0.012 for all comparisons). Estimates for plasma half-lives of all (c)selectins were obtained from individual time vs. concentration profiles of the maximal decreases and ranged from 2 to 4 d. cp-selectin increased by 23% (p = 0.003), ce -selectin by 5% (p = 0.041) and cl -selectin by 19% (p = 0.009) 1 h after the platelet transfusions. Based on these results the calculated volume of distribution (Vd) of transfused (c)selectins was 2.5-fold higher than the plasma volume, which supports the concept of the in-vivo expression of binding sites, i.e. ligands, for CE-selectin and CL-selectin. In summary, while our study cannot provide any evidence for endothelial cells as a source of cp-selectin, we have found for the first time evidence for the existence of ligands for soluble CE-selectin and cL-selectin in humans.     

In vivo evidence for apoptosis in the bone marrow in systemic lupus erythematosus

An increase in leucocyte apoptosis and impaired clearance of apoptotic cells has been observed in patients with systemic lupus erythematosus (SLE). Apoptotic cells are likely to be a key source of autoantigens in SLE as they express many of the nuclear autoantigens (in surface blebs and apoptotic bodies) that are relevant to this disease. The clearance of apoptotic cells is usually a rapid process, such that few cells are usually seen in the extracellular environment in vivo. We report a case in which multiple apoptotic bodies were observed in the bone marrow of a patient with SLE that was complicated by an immune-mediated pancytopenia. We have subsequently examined the frequency of apoptotic cells, identified morphologically, and by caspase-3 staining in bone-marrow trephine samples taken from patients with SLE over a 10-year period of follow-up. A high proportion of bone marrows contained apoptotic debris. The novel demonstration of apoptotic bodies in vivo in patients with SLE is unusual and supports the notion that the marrow may be a target organ in the disease. Their abundance is also consistent with the hypothesis that normal clearance mechanisms are defective and/or overwhelmed in SLE.     

腺病毒介导的人骨形态发生蛋白-7基因转染大鼠骨髓基质干细胞及其表达

目的 利用人骨形态发生蛋白-7(hBMP-7)腺病毒表达载体(Ad-hBMP-7)转染大鼠骨髓基质干细胞(BMSC)的方法,观察Ad-hBMP-7转染后对BMSC分化的影响,探讨基冈治疗的可能性.方法 获取Wistar大鼠BMSC,并分为3组:Ad-hBMP-7转染组、Ad-绿色荧光蛋白(Ad-GFP)转染组、未转染组.将Ad-hBMP-7和Ad-GFP的病毒液分别转染体外培养的大鼠BMSC,用RT-PCR法检测hBMP-7 mRNA的表达,7 d后用Western blot法检测hBMP-7的蛋白表达,同时进行碱性磷酸酶(ALP)染色.结果 在Ad-hBMP-7转染组,RT--PCR可检测到hBMP-7 mRNA的表达(470 bp),Western blot可检测到hBMP-7蛋白分泌(相对分子质量为15×103),转染后第10天ALP染色多数细胞-胞浆呈棕黑色阳性着色;而Ad-GFP转染组和未转染组ALP染色阳性细胞少见并且未检测到hBMP-7蛋白分泌.结论 Ad-hBMP-7基因可高效转染BMSC,促进BMSC向成骨细胞转化,为利用hBMP-7开展局部基因治疗的研究奠定了基础.     

Endogenous thrombopoietin levels during the clinical management of acute myeloid leukaemia

Thrombocytopenia represents a major problem in the management of acute myeloid leukaemia (AML). The data regarding the alterations of endogenous thrombopoietin (TPO) regulation during the clinical course of AML are limited. The aim of this study was to investigate endogenous TPO dynamics in association with platelets during the clinical course of AML. We serially measured both TPO and platelets concurrently over the entire treatment period of newly diagnosed patients receiving both remission induction and consolidation chemotherapies. The median concentration of TPO in AML patients at the initial diagnosis was 469.71 pg/ml and increased significantly during the aplastic period due to remission induction chemotherapy (median: 1085.33 pg/ml) but then decreased to a level (median: 45.26 pg/ml) encountered in the healthy control subjects (median: 56.90 pg/ml). In the cytopenic period due to consolidation treatment, TPO level again increased significantly to a high level (median: 891.38 pg/ml) during the platelet nadir, but decreased toward normal (median: 100.75 pg/ml) after the thrombocytopenic period had elapsed. In conclusion, endogenous TPO levels exhibit an inverse fluctuation in relation to platelet counts during the clinical course of AML. Pharmacological stimulation of thrombopoiesis in AML with novel molecules, including the recombinant thrombopoietins and the small peptide agonists, should be based on a critical administration strategy that must consider the endogenous levels of TPO. TPO levels in distinct AML disease states may explain the unsuccessful recombinant TPO trials and could help to design better strategies for 'pharmacological stimulation of thrombopoiesis' in AML.     

Culture of human fetal B-cell precursors on bone marrow stroma maintains highly proliferative CD20dim cells

Growth of human B-cell precursors (BCP) was achieved by plating fetal CD10+ surface-mu (s mu)- cells in liquid medium onto bone marrow- derived fibroblastic stromal cell layers deprived of hematopoietic cells. Proliferation of the fetal BCP was strongly potentiated by the addition of interleukin-7 (IL-7) to the cultures. Cultures included both a stroma-adherent and -nonadherent fraction of lymphoid cells, allowing us to expand the number of input BCP to 13-fold. In the presence of exogenous IL-7, proliferation was dose-dependent relative to the number of stromal cells, demonstrating that soluble IL-7 does not act alone to promote optimal growth. We further showed that the lymphoid cells recovered remain CD10+ sIg- BCP and that most cells expressed the maturation-associated CD20 antigen when IL-7 was added to the cultures. Whereas both freshly isolated CD20- and CD20bright BCP proliferated in the presence of stroma, we observed that high- proliferative capacity CD20dim cells were maintained in the cultures. Finally, CD20dim sorted cells were shown to subsequently acquire high levels of CD20 expression in culture, thus demonstrating a partial maturation sequence. The present culture system thus represents a useful model for studying the regulatory signals in early human B lymphopoiesis.     

Retroviral transfer of the hENT2 nucleoside transporter cDNA confers broad-spectrum antifolate resistance in murine bone marrow cells

Antifolate drugs such as methotrexate are commonly used in cancer chemotherapy. It may be possible to increase the antitumor activity of antifolates by the coadministration of drugs that inhibit nucleoside transport, thereby blocking the capacity of tumor cells to salvage nucleotide precursors. An important limitation of this approach is severe myelosuppression caused by many of these drug combinations. For this reason, we have developed a gene therapy strategy to protect bone marrow cells against combined treatment with antifolates and nitrobenzylmercaptopurine riboside (NBMPR), a potent inhibitor of the es nucleoside transporter. A retroviral vector (MeiIRG) was constructed that expressed the NBMPR-insensitive ei transporter, hypothesizing that transduced bone marrow cells would survive drug treatment because of the preservation of nucleoside salvage pathways. In vitro clonogenic assays confirmed that the MeiIRG vector did protect myeloid progenitors against the toxic effects of 3 different antifolates when each was combined with NBMPR. On testing this system in vivo, decreased myelosuppression was observed in mice transplanted with MeiIRG-transduced bone marrow cells and subsequently treated with trimetrexate and NBMPR-P. In these mice, significant increases were noted in absolute neutrophil count nadirs, reticulocyte indices, and the numbers of myeloid progenitors in the bone marrow. Furthermore, a survival advantage was associated with transfer of the MeiIRG vector, indicating that significant dose intensification was possible with this approach. In summary, the MeiIRG vector can decrease the toxicity associated with the combined use of antifolates and NBMPR-P and thereby may provide a strategy for simultaneously sensitizing tumor cells while protecting hematopoietic cells.     

In vitro and in vivo anticancer activity of mitozolomide and sparsomycin in human tumor xenografts,murine tumors and human bone marrow

Summary The colony formation in agar of human tumor xenografts, of murine tumors and of human bone marrow was used as a test system to determine the in vitro activity of the two novel cytostatic agents, mitozolomide and sparsomycin. Mitozolomide was additionally studied in vivo in nine human tumor xenografts. The comparison of in vitro/in vivo activity allows an assessment of the relevant in vitro dose based on in vivo pharmacological behavior of a compound. Both compounds showed clear dose/response effects in vitro. A dose of 3 g/ml mitozolomide, given by continuous exposure, was active (colony number of test <30% of the control group) in 12/42 (29%) human tumor xenografts as well as in the four murine tumors, P388, L1210, B16 melanoma and colon carcinoma 38, whereas the two human bone marrows showed no significant suppression of the ability to form colonies in culture. The comparison of in vitro with in vivo activity suggests that the in vitro dose of 3 g/ml corresponds best to the activity observed in animal experiments. The highest activity was observed in small-cell cancer of the lung (4/5), followed by melanomas (2/7) and non-small-cell cancer of the lung (2/9). Furthermore, activity was found in a cancer of the large bowel, stomach, breast and in one sarcoma. In the treatment of nine human tumor xenografts growing subcutaneously in nude mice, mitozolomide effected a complete or partial remission in 6 out of 9 tumors. In comparison to standard drugs mitozolomide is one of the most effective compounds in these tumors. These data indicate that mitozolomide possesses potent broad-spectrum activity in human tumor xenografts. Sparsomycin (0.1 g/ml, continuous exposure) was active in 11/46 (24%) human tumor xenografts and in 4/5 of the murine tumors, whereas the colony-forming capacity of four human bone-marrows showed no inhibition, suggesting that this dose level may be the relevant in vitro dose. However, the high in vitro activity in murine tumors is incompatible with the in vivo activity. In mice the only responsive tumor was leukemia P388, whereas the L1210, B16 melanoma and colon carcinoma 38 were resistant. At the dose level of 0.03 g/ml only 3/30 (10%) of the human tumor xenografts were sensitive. In an earlier clinical phase I study the dose-limiting adverse effect was eye toxicity and not bone-marrow suppression. This example illustrates that comparing in vitro with in vivo activity in the same tumor results in a more reliable estimation of the relevant in vitro dose than does comparing in vitro activity with in vitro effects on human bone marrow.Abbreviations Mitozolomide 8-carbamoyl-3-(2-chloroethyl)imidazo(5,1-d)-1,2,3,5-tetrazin-4(3H)-one - NSC 353451 formerly known as azolastone - sparsomycin NSC 59 729 - DTIC 5-(3,3-dimethyltriazen-1-yl)-imidazole-4-carboxamide - MTIC 5-(3-methyltriazen-1-yl)imidazole-4-carboxamide - DMSO dimethylsulfoxide Dedicated to Professor Dr. D. Schmähl, Heidelberg, on the occasion of his 65th birthday     

Retroviral-mediated gene transfer into human bone marrow stromal cells: Studies of efficiency and in vivo survival in SCID mice

Abstract: Retroviral-mediated gene transfer into bone marrow cells is extensively used in gene therapy protocols. Cytokines are needed for stimulation, to achieve a high rate of gene transfer. However, the stromal cell compartment of bone marrow is characterized by rapid proliferation even without cytokines. In this study, human bone marrow stromal cells were isolated and subsequently infected with recombinant retrovirus in a cell-free supernatant. The LN retroviral vector used in this study carries the bacterial neomycin phosphotransferase gene (neoR). Transduction efficiency was significantly enhanced by repeated cycles of infection, with a maximum of 91% transduced cells by four rounds of infection. Presence of the neoR-gene was detected by PCR from all stromal cells selected by G418. After culture in vitro for 3 months, cells were still positive for PCR amplification of the neoR-gene. Transduced stromal cells were also injected into SCID mice to study their homing and survival ability in vivo.     

New insights into the thrombopoietic status of patients on dialysis through the evaluation of megakaryocytopoiesis in bone marrow and of endogenous thrombopoietin levels

The thrombopoietic status of patients with uremia remains unclear. This issue was addressed with particular reference to marrow megakaryocytopoiesis and endogenous thrombopoietin (TPO) levels. A study was conducted in 114 patients on hemodialysis, 43 patients on continuous ambulatory peritoneal dialysis, and 48 age-matched controls. Reticulated platelets, a marker of marrow megakaryocytopoiesis, were measured by flow cytometry. Serum TPO levels, platelet-associated IgG (PAIgG) levels, and hepatitis C virus (HCV) antibody titers were also measured by enzyme-linked immunosorbent assay. Circulating and reticulated platelet counts were significantly lower in the patients on dialysis than in the controls. Thrombocytopenia (less than 150 x 10(9)/L) was most frequent in the HCV-positive hemodialysis patients, who had a higher incidences and higher PAIgG titers. The following results were obtained in the HCV-negative dialysis patients: (1) platelet counts chronologically decreased with years on hemodialysis; (2) platelet counts were associated with the reticulated platelet counts; (3) serum TPO levels were significantly elevated in the dialysis patients, responding to the decrease of reticulated platelets; (4) hematocrits had a positive correlation with serum TPO levels, and serum TPO levels were significantly higher in the patients on hemodialysis who did not require recombinant human erythropoietin therapy than in the other patients. In conclusion, thrombocytopenia is a frequent finding in patients on dialysis. The failure of megakaryocyte production could be the principal cause of the platelet reduction, and the peripheral destruction and sequestration of platelets may be concomitantly involved. Elevation of serum TPO may in part serve as an aid to erythropoiesis in dialysis patients.     

Differential mechanisms in the regulation of endogenous levels of thrombopoietin and interleukin-11 during thrombocytopenia: insight into the regulation of platelet production

The regulation of megakaryocytopoiesis and thrombopoiesis appears to be under the control of an array of hematopoietic growth factors. To determine the relationship of endogenous thrombopoietic cytokine levels and circulating platelet (PLT) counts, we measured the levels of thrombo-poietin (TPO), interleukin-11 (IL-11), and interleukin-6 (IL-6) in patients with significant thrombocytopenia secondary to both marrow hypoplasia and increased PLT destruction. Increased endogenous levels of TPO and IL-11, but not IL-6, were detected in bone marrow transplant patients with thrombocytopenia following myeloablative therapy (BMT/MAT) (TPO: 1,455.5 +/- 87.3 pg/mL, [PLT 39,600 +/- 7,800/microL], P < .001, n = 12; IL-11: 227.9 +/- 35 pg/mL, [PLT 32,900 +/- 57,000/microL], P < .05, n = 19; IL-6: 25.8 +/- 8.4 pg/mL, [PLT 32,800 +/- 5,057/microL], P > .05, n = 4] v normal donors [TPO < 150 pg/mL, n = 8; IL-11 < 50 pg/mL, n = 9; IL-6 < 10 pg/mL, n = 5 [PLT 203,000 +/- 7,500/microL]. There was a significant inverse correlation between endogenous levels of TPO and IL-11, but not IL-6, and PLT counts in the MAT/BMT patients (TPO: r = -0.57, P < .0001, n = 188; IL-11: r = - 0.329, P < .0001, n = 249; IL-6: r = -0.1147, P > .05, n = 62). In patients with immune thrombocytopenia purpura (ITP), with decreased PLT survival, but intact bone marrow megakaryocytopoiesis, endogenous IL-11 levels were significantly increased (328.0 +/- 92.6 pg/mL, [PLT: 20,900 +/- 3,000/microL], P < .05, n = 25). However, endogenous TPO levels remained undetectable (< 150 pg/mL, [PLT 30,500 +/- 5,500/microL], n = 15). These results suggest that there may be differential mechanisms regulating endogenous TPO, IL-11, and IL-6 levels during acute thrombocytopenia and suggest that the absolute number of circulating PLTs may not always be the sole regulator of endogenous TPO levels. Other mpl-expressing cells of the megakaryocyte lineage may contribute to the regulation of circulating TPO levels as well. Our results also suggest IL-11 levels may in part, be regulated by a negative feedback loop based on circulating PLT counts, but also may, in part, be regulated by a variety of inflammatory agonists. Both TPO and IL-11, therefore, appear to be active thrombopoietic cytokines regulating, in part, megakaryocytopoiesis during states of acute thrombocytopenia.