Production Of Hematopoietic Growth Factors Research Articles

Thrombopoietin-Dependent Myelo-Megakaryopoiesis Fuels Thromboinflammation and Worsens Antibody-Mediated Chronic Renal Microvascular Injury.

Kidney-derived thrombopoietin (TPO) increases myeloid cell and platelet production during antibody-mediated chronic kidney disease (AMCKD) in a mouse model, exacerbating chronic thromobinflammation in microvessels. The effect is mirrored in patients with extracapillary glomerulonephritis associated with thromboinflammation, TGF β -dependent glomerulosclerosis, and increased bioavailability of TPO. Neutralization of TPO in mice normalized hematopoiesis, reduced chronic thromboinflammation, and ameliorated renal disease. The findings suggest that TPO is a relevant biomarker and a promising therapeutic target for patients with CKD and other chronic thromboinflammatory diseases.Neutralization of TPO in mice normalized hematopoiesis, reduced chronic thromboinflammation, and ameliorated renal disease. The findings suggest that TPO is a relevant biomarker and a promising therapeutic target for patients with CKD and other chronic thromboinflammatory diseases. Chronic thromboinflammation provokes microvascular alterations and rarefaction, promoting organ dysfunction in individuals with various life-threatening diseases. Hematopoietic growth factors (HGFs) released by the affected organ may sustain emergency hematopoiesis and fuel the thromboinflammatory process. Using a murine model of antibody-mediated chronic kidney disease (AMCKD) and pharmacological interventions, we comprehensively monitored the response to injury in the circulating blood, urine, bone marrow, and kidney. Experimental AMCKD was associated with chronic thromboinflammation and the production of HGFs, especially thrombopoietin (TPO), by the injured kidney, which stimulated and skewed hematopoiesis toward myelo-megakaryopoiesis. AMCKD was characterized by vascular and kidney dysfunction, TGF β -dependent glomerulosclerosis, and microvascular rarefaction. In humans, extracapillary glomerulonephritis is associated with thromboinflammation, TGF β -dependent glomerulosclerosis, and increased bioavailability of TPO. Analysis of albumin, HGF, and inflammatory cytokine levels in sera from patients with extracapillary glomerulonephritis allowed us to identify treatment responders. Strikingly, TPO neutralization in the experimental AMCKD model normalized hematopoiesis, reduced chronic thromboinflammation, and ameliorated renal disease. TPO-skewed hematopoiesis exacerbates chronic thromboinflammation in microvessels and worsens AMCKD. TPO is both a relevant biomarker and a promising therapeutic target in humans with CKD and other chronic thromboinflammatory diseases.

Tumor-Derived Autophagosomes (DRibbles) Activate Human B Cells to Induce Efficient Antigen-Specific Human Memory T-Cell Responses.

We have reported that tumor-derived autophagosomes (DRibbles) were efficient carriers of tumor antigens and DRibbles antigens could be present by DRibbles-activated B cells to stimulate effect and naïve T cells in mice. However, the effect of DRibbles on human B cells remains unclear. Herein, we found that DRibbles can also efficiently induce proliferation and activation of human B cells and lead to the production of chemokines, cytokines and hematopoietic growth factors. We further demonstrated human B cells can effectively phagocytose DRibbles directly and cross-present DRibbles antigens to stimulate antigen-specific memory T cells. Furthermore, we found that membrane-bound high-mobility group B1 (HMGB1) on DRibbles was crucial for inducing human B cells activation. Therefore, these findings provide further evidence to promote the clinical application of B-DRibbles vaccines.

The in vitro effects of hepatoblastoma cells on the growth and differentiation of blasts in transient abnormal myelopoiesis associated with Down syndrome

Transient abnormal myelopoiesis (TAM) in neonates with Down syndrome, which spontaneously resolves within several weeks or months after birth, may represent a special form of leukemia developing in the fetal liver (FL). To explore the role of hepatoblasts, one of the major constituents of the FL hematopoietic microenvironment, in the pathogenesis of TAM, we investigated the influence of a human hepatoblastoma cell line, HUH-6, on the in vitro growth and differentiation of TAM blasts. In a coculture system with membrane filters, which hinders cell-to-cell contact between TAM blasts and HUH-6 cells, the growth and megakaryocytic differentiation of TAM blast progenitors were increased in the presence of HUH-6 cells. The culture supernatant of HUH-6 cells contained hematopoietic growth factors, including stem cell factor (SCF) and thrombopoietin (TPO). The neutralizing antibody against SCF abrogated the growth-stimulating activity of the culture supernatant of HUH-6 cells, demonstrating that, among the growth factors produced by HUH-6 cells, SCF may be the major growth stimulator and that TPO may be involved in megakaryocytic differentiation, rather than growth, of TAM blasts. This suggests that hepatoblasts function in the regulation of the growth and differentiation of TAM blasts in the FL through the production of hematopoietic growth factors, including SCF and TPO, and are involved in the leukemogenesis of TAM.

High‐yield production of hematopoietic growth factors by tobacco BY‐2 cells for ex vivo production of red blood cells

Ex vivo generation of clinically available red blood cells (RBCs) from hematopoietic stem cells (HSCs) represents a promising approach for overcoming the limitations associated with the use of donor’s blood, such as shortage of blood and risk of transfusion‐transmitted infections. Both expansion and differentiation of stem cells are highly reliant on growth and differentiation factors, with the activity and purity of these molecules being critical to successful research and clinical applications. The overarching goal of this project is to develop a novel plant cell‐based bioproduction platform for high‐quality (i.e. animal‐free and endotoxin‐free) erythropoietic growth factors (eGFs) that facilitate mass production of RBCs from HSCs at significantly reduced cost. In addressing the low productivity bottleneck of plant cell culture technology, this proposal aims to leverage a unique, plant‐specific, O‐glycosylation process called “hydroxyproline (Hyp)‐O‐glycosylation” for de novo design and engineering of Hyp‐O‐ glycosylated peptides (HypGPs”) that presumably function as a molecular carrier in boosting secretion of conjoined eGFs from cultured plant cells. This will dramatically increase the secreted yields of plant cell‐derived eGFs and simplify the product purification process. Three key eGFs essential for HSCs expansion and differentiation: erythropoietin (EPO), stem cell factor (SCF) and interleukin 3 (IL‐3), are expressed in tobacco BY‐2 cells as fusion with a strategically designed HypGP tag. The secreted protein yields, molecular structure, and Hyp‐O‐glycosylation of the HypGP‐tagged eGFs are characterized. The bioactivities of individual HypGP‐tagged eGF in stimulating proliferation and differentiation of relevant hematopoietic cell lines, includingTF1 erythroleukemic cell and umbilical cord blood‐derived CD34+ cell, are evaluated to establish a suitable design of HypGP tag (size and orientation) for each eGF. A scalable cell culture system fortified sorely with plant cell‐produced eGFs is developed for production of RBCs from CD34+ HSC cells. This project provides a proof‐of‐concept for ex vivo manufacturing of stem cell‐derived RBCs with plant cell‐produced eGFs.Support or Funding InformationNational Science Foundation Directorate for Engineering [1605564]; National Institutes of Health‐Arkansas INBRE [P20GM103429]

Study evaluating testosterone deficiency as a cause of anemia and reduced responsiveness to erythropoiesis-stimulating agents in men on maintenance hemodialysis

IntroductionChronic kidney disease (CKD) is a worldwide disease that is classified into five stages according to the glomerular filtration rate and presents through a variety of symptoms and signs. Anemia is one of the first signs of kidney dysfunction. The most common causes of anemia in CKD are erythropoietin (EPO) hormone deficiency and iron deficiency. Anemia and hyporesponsiveness to erythropoietin-stimulating agents (ESAs) are commonly observed in CKD patients and are associated with increased morbidity, mortality, and a significant healthcare economic burden. Although testosterone deficiency is a prevalent condition in men with CKD, it has so far received relatively little attention in practice. Testosterone stimulates erythropoiesis through the production of hematopoietic growth factors and possible improvement of iron bioavailability.AimThe aim of this study was to evaluate serum testosterone levels in patients on maintenance hemodialysis (MHD) and correlate its level with anemia and response to ESAs therapy.Patients and methodsThis study included 40 male patients from dialysis units, where they were divided equally into group A, group taking ESAs, and group B, group not taking ESAs (EPO-naive group). Another 20 men were included in group C (control group). All groups were subjected to a full assessment of history, full clinical examination, and laboratory investigations to exclude all possible causes of anemia.ResultsThis study showed that in group A, 75% of the participants were anemic, whereas in group B, 100% of the participants were anemic, with a higher degree of anemia. The testosterone level was slightly higher in group B than group A; despite being within the normal range, it was relatively deficient on the basis of the age of the participants in the control group.ConclusionTestosterone deficiency is a prevalent condition in CKD that starts at an earlier age than the normal population. It is an evident independent cause of anemia in EPO-naive CKD patients and is a possible cause of resistance of ESAs in CKD patients; still, the most important causes of anemia in CKD are EPO and iron deficiency.

Over expression of HIF-1α in human mesenchymal stem cells increases their supportive functions for hematopoietic stem cells in an experimental co-culture model

IntroductionBone marrow transplantation is a critical approach for the treatment of many hematological disorders. Success of this approach is dependent on many factors the most important of which is the number of hematopoietic stem cells along with an efficient stroma. Co-transplantation of efficient mesenchymal stem cells can greatly improve the outcome of transplantations. Current researches assign a critical role for hypoxia inducible factor (HIF)-1α in protection of various cells and tissues probably through induction of cytokines. To make this feature applicable to human bone marrow-derived mesenchymal stem cells, we manipulated these cells to over express HIF-1α gene.Materials and methodsFull-length cDNA of human HIF-1α was inserted into human bone marrow mesenchymal stem cells by pcDNA.3.1 non-viral plasmid vector, and the effect of this over expression on production of some hematopoietic growth factors was explored. Moreover, using a co-culture system, the interactive impact of HIF-1α-overexpressed mesenchymal stem cells on hematopoietic stem cells was evaluated.ResultsOver expression of HIF-1α in mesenchymal stem cells in normoxia increased production of one of the most important hematopoietic growth factors, Stem cell factor (also known as Steel factor or c-kit ligand). HIF-1α overexpression had no effect on production of other hematopoietic growth factors. In co-culture of mesenchymal stem cells-HIF-1α with hematopoietic stem cells, enhancement of colony formation and reduced differentiation of hematopoietic stem cells were observed.ConclusionOver expression of HIF-1α in human bone marrow-derived mesenchymal stem cells can augment the production of some hematopoietic growth factors, and we suggest this response of mesenchymal stem cells could help to improve the outcome of bone marrow transplantation.

Microbial Impact on Hematologic Homeostasis

AbstractAbstract SCI-40During systemic infection and inflammation, immune effector cells are in high demand and are rapidly consumed at sites of need. While adaptive immune cells have high proliferative potential, innate mature immune cells are mostly postmitotic and need to be replenished from bone marrow hematopoietic stem and progenitor cells. Indeed, severe clinical infection, particularly infections challenging the innate immune response, lead to an increase in hematopoietic differentiation and throughput in bone marrow, involving subsequent differentiation stages from hematopoietic stem cells, multipotent progenitors, as well as early-lineage and late-lineage restricted hematopoietic progenitors. A fundamental question is how the increased need is sensed and translated in enhanced production and how adequate levels of response are guided. Recent research has shed light on conserved intracellular and extracellular pathogen recognition receptors, such as Toll-like receptors, that are expressed on nonhematopoietic and hematopoietic effector cells and cause activation upon ligation. This activation results in production of hematopoietic growth, survival, activation, and migration factors operating at site on effector cells, but also at remote primary hematopoietic sites to increase production upon need. Recent research by several groups, including ours, surprisingly revealed that conserved pattern-recognition receptors are also expressed on hematopoietic stem and progenitor cells in bone marrow, implying a direct effect of systemically available ligands on these cellular populations. Indeed, it has been demonstrated that, for example, ligation of Toll-like receptor 4 by its cognate agonist lipopolysaccharide can lead to divisional activation, proliferation, lineage-directed differentiation, and migration of hematopoietic stem and lineage-restricted progenitor cells, all aimed at efficient contribution to immune responses and rapid reestablishment of hematopoietic homeostasis. The relative contribution of pathogen sensing by hematopoietic and diverse nonhematopoietic cells to appropriate hematopoietic responses, as well as the subcellular translation of the signals, is the focus of ongoing research. Also to be discussed will be how chronic infectious and inflammatory processes, which are frequently associated with aging, might impinge on hematopoiesis, potentially fostering hematopoietic stem cell diseases as exhaustion or transformation. Disclosures:No relevant conflicts of interest to declare.

Exercise promotes bone marrow cell survival and recipient reconstitution post-bone marrow transplantation, which is associated with increased survival

Bone marrow transplantation (BMT) is associated with a high risk of mortality, partially because of the harmful effects of the preconditioning myeloablative regimens. We have recently demonstrated increased bone marrow cell survival and proliferation in response to exercise training, which may be attributable to increased quality of the niche. The purpose of the present study was to determine the extent to which exercise preconditioning of recipients could increase the success of BMT. Recipient mice remained sedentary (SED) or were exercise-trained (EX) on a treadmill (3 d/wk for 8 weeks) before reconstitution with green fluorescent protein (GFP)-labeled donor marrow. Recipient survival, both donor-derived and total (donor- and recipient-derived) blood reconstitution were measured by flow cytometry. The first and fourth day after BMT apoptosis, cellularity and donor cell homing were determined in the recipients' bone marrow cavity by flow cytometry. Whereas only 25% of SED mice survived, 82% of EX recipients survived the BMT. Homing of donor-derived marrow cells to the recipients' marrow cavity acutely after BMT was not altered in EX, but EX mice displayed decreased levels (10%; p < 0.05) of activated caspase-3/-7 one day after BMT, leading to a maintenance of marrow cellularity in mice preconditioned with exercise. The acute inhibition of marrow cell apoptosis in EX mice resulted in increased total blood cell reconstitution at 1 and 3.5 months after BMT in EX mice (42% and 43%, respectively; both p < 0.05). Short- and long-term donor-derived engraftment was not different between EX and SED recipients. Exercise training increases recipient survival after BMT with increased total blood cell reconstitution.

Leukemoid reaction in a patient with adenocarcinoma of the lung: a case report

IntroductionLung cancers are characterized by high incidence, prevalence and mortality. They may be associated with numerous paraneoplastic syndromes. Mild leukocytosis is not rare. The case described here, however, is of a female patient with adenocarcinoma of the lung who developed extreme leukocytosis at over 140,000 cells/μL. Descriptions of such leukemic forms of lung cancer are few and far between in the literature. In our case, the complete hematological diagnostic investigation, which included cytological, immunocytological, cytogenetic, histological and molecular genetic tests of the bone marrow (mutation analyses of BCR-ABL and JAK2), was accompanied for the first time by a molecular genetic workup of the primary tumor for epidermal growth factor receptor and K-RAS gene mutations.Case presentationWe present the medical case of a 51-year-old female Caucasian patient, who was diagnosed with a poorly differentiated stage IV (International Union Against Cancer staging) adenocarcinoma of the lung. While undergoing treatment, our patient developed extreme leukocytosis, for which, despite extensive diagnostic tests, no infection-related or hematological cause could be identified. The tumor proved to be highly resistant to treatment. Our patient died only five months after the initial diagnosis.ConclusionA leukemoid course can most likely be interpreted as the paraneoplastic production of hematopoietic growth factors. Despite the absence of a verified primary hematological origin, this possibility should always be investigated in all patients in a comparable situation.

INTESTINAL IMMUNE SYSTEM-MODULATING ACTIVITY THROUGH PEYER'S PATCH OF FLAVONOID GLYCOSIDE PURIFIED FROMCITRUS UNSHIUPEEL

After methanol extract (CUP-2) with intestinal immune system-modulating activity through Peyer's patch was prepared from Citrus unshiu peel, CUP-2C-2–2 was purified by solvent partitioning, silica gel 60 and Sephadex LH-20 chromatographies (Pharmacia Biotech, Uppsala, Sweden). Ultraviolet-visible spectral, 1H/13C-NMR, and fast atom bombardment mass spectrometry analyses suggested that CUP-2C-2–2 was identified to flavonoid glycoside (5,7,3′,4′-tetrahydroxyflavone linked by glucose) with molecular weight of 725 Da. When the active chloroform layer (CUP-2C) fractionated from CUP-2 was orally administered in mice, the potent activity was observed on the fifth day of 2.0 g/kg body weight/day (1.59-fold of the saline control). Granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-6 contents in the culture medium of Peyer's patch cell were also significantly increased (1.80- and 2.20-fold, respectively) compared with the saline control. These results suggested that flavonoid glycoside from C. unshiu peel might stimulate Peyer's patch and produce the multifunctional hematopoietic growth factors such as GM-CSF and IL-6 followed by proliferation of bone marrow cells. PRACTICAL APPLICATIONS C. unshiu peel is widely used as the traditional herbal prescription or tea in East Asia. Since the early 1980s, many researchers have been studying the physiologically active substances from citrus fruit peel for the purpose of exploring their more valuable utilization. Current work is focused on low-molecular-weight constituents with intestinal immune system modulating activity through Peyer's patch from citrus peel. It is clearly shown that the purified active substance (CUP-2C-2–2) is characterized as flavonoid glycoside. When the active fraction (CUP-2C) was orally administered in mice, production of hematopoietic growth factors from Peyer's patch cells was stimulated and proliferation of bone marrow cells was induced by these growth factors influxed via systemic circulation. This conclusion supports the rationality of citrus peels as a potential source of biological response modifier for food supplementation or functional foods.

IL-33 Activates Unprimed Murine Basophils Directly In Vitro and Induces Their In Vivo Expansion Indirectly by Promoting Hematopoietic Growth Factor Production

IL-33, a new member of the IL-1 family, has been described as an important inducer of Th2 cytokines and mediator of inflammatory responses. In this study, we demonstrate that murine basophils sorted directly from the bone marrow, without prior exposure to IL-3 or Fc(epsilon)R cross-linking, respond to IL-33 alone by producing substantial amounts of histamine, IL-4, and IL-6. These cells express ST2 constitutively and generate a cytokine profile that differs from their IL-3-induced counterpart by a preferential production of IL-6. In vivo, IL-33 promotes basophil expansion in the bone marrow (BM) through an indirect mechanism of action depending on signaling through the beta(c) chain shared by receptors for IL-3, GM-CSF, and IL-5. IL-3 can still signal through its specific beta(IL-3) chain in these mutant mice, which implies that it is not the unique growth-promoting mediator in this setup, but requires IL-5 and/or GMCSF. Our results support a major role of the latter growth factor, which is readily generated by total BM cells as well as sorted basophils in response to IL-33 along with low amounts of IL-3. Furthermore, GM-CSF amplifies IL-3-induced differentiation of basophils from BM cells, whereas IL-5 that is also generated in vivo, affects neither their functions nor their growth in vitro or in vivo. In conclusion, our data provide the first evidence that IL-33 not only activates unprimed basophils directly, but also promotes their expansion in vivo through induction of GM-CSF and IL-3.

NOV-002, a chemoprotectant/immunomodulator, added to first-line carboplatin/paclitaxel in advanced non-small cell lung cancer (NSCLC): A randomized Phase 1/2, open-label, controlled study

17021 Background: NOV-002 is a proprietary formulation of oxidized glutathione that modulates production of cytokines and hematopoietic growth factors. A randomized, multi-site advanced NSCLC trial conducted in the Russian Federation showed a one-year survival rate of 63% with NOV-002 + cytotoxic chemotherapy compared to 17% with cytotoxic chemotherapy alone. Methods: Forty-four chemotherapy-naïve, Stage IIIB/IV NSCLC patients (ECOG 0–2, stratified by disease stage) were randomized to one of three groups for six months of treatment: Groups A and B: NOV-002 in combination with carboplatin and paclitaxel (C+P). For each nominal 21-day chemotherapy cycle, these groups received 60 mg of NOV-002 i.v. daily for the first 4 days, then 60 mg i.m. (A) or s.c. (B) weekdays for the next 17 days. Group C: C+P alone. Initial doses of C and P in all three groups were 225 mg/m2 and AUC 6, respectively. Initiation of repeat C+P cycles required an absence of hematologic and other toxicities in excess of pre-defined values. Primary study endpoints included tumor response (scans performed at baseline and then every two months) and safety. Results: An intent-to-treat analysis of the best overall objective tumor response (WHO criteria), showed that 11 out of 16 (69%) Group B patients demonstrated greater than 50% tumor shrinkage versus 5 out of 15 (33%) in the control group (C) (p = 0.044, logistic regression stratified on disease stage). Six out of 13 (46%) patients in Group A demonstrated an objective response. 100% of NOV-002 treated patients in Group B and 85% in Group A were able to complete four cycles of C+P compared to 50% of control patients (Group C) (p = 0.004, chi square). NOV-002 was well-tolerated in this patient population. Conclusions: These data continue to suggest increased efficacy of NOV-002 plus cytotoxic chemotherapy compared to chemotherapy alone in advanced NSCLC. [Table: see text]

The hematological etiology of osteoporosis

In this article we present a new working hypothesis, suggesting that overexertion of the hematopoietic system resulting from constant excessive need for blood cell production, plays an important role in the etiology of osteoporosis. It is generally accepted that the development of osteoporosis in postmenopausal women is due to the reduction of estrogen level. However a most striking observation is the fact that in the male organism, which has never been protected by high levels of estrogen, osteoporosis in senescence is significantly less frequent then in the female organism, which has been protected by estrogens for at least 35 years. Healthy women loose about 70 ml of blood every month, which adds up to some 850 ml per year and approximately 30 l over the 35 years of their reproductive life. Blood loss intensifies hematopoiesis by increasing the level of hematopoietic growth factors while, at the same time, stimulating proliferation of osteogenic progenitor cells. Osteogenic activity has, indeed, been detected in the blood of bled animals. The chain of events that converts continuous requiremental pressure on hematopoiesis into development of osteoporosis may be formulated as follows: on the one hand, blood loss creates developmental pressure on the hematopoietic system, augments production of hematopoietic growth factors with subsequent intensified proliferation of hematopoietic progenitor cells, increases the number of hematopoietic cells including osteoclasts, thus intensifying resorption of bone tissue and extension of hematopoietic territories. On the other hand, blood loss leads to stimulation of bone development, extensive proliferation of osteogenic progenitor cells resulting in increased numbers of osteoblasts, followed by new bone formation and, at the same time, increased production and maturation of osteoclasts which then enter the cycle of bone resorption. The bone resorption process itself is characterized by the release of bone morphogenic proteins that induce proliferation of osteogenic cells and subsequent production of osteoclasts that in their turn enter the cycle of bone resorption. Important evidence supporting the substantial role of hematopoietic insufficiency in the development of osteoporosis comes from the field of clinical hematology: hematological diseases accompanied by chronic anemia, such as beta thalassemia major, sickle cell anemia, chronic hemolytic anemia, pernicious anemia, etc., are also characterized by the concomitant development of osteoporosis. Patients suffering from hemophilia tend to develop osteoporosis as well. The possible role of functional interaction between hematopoietic and bone tissues in the development of age related osteoporosis is also discussed.

Effects of Neopterin on the Hematopoietic Microenvironment of Senescence-Accelerated Mice (SAM)

The pteridine neopterin (NP) is produced by monocytes and is known to be a useful marker of immunological activation, although, it remains elusive whether neopterin itself exhibits biological functions. Recently, we found that NP stimulates hematopoietic cell proliferation and differentiation by activating bone marrow stromal cell function. In order to elucidate the biological effect of NP on stromal cells, its effects on hematopoiesis was determined in the mouse model of age-related stromal impairment, senescence-accelerated mice (SAMs). An intraperitoneal administration of NP increased the number of peripheral leukocytes and CFU-GM in the bone marrow and spleen of young SAMs, however, no increase of CFU-GM in old SAMs (stromal impairment) was observed when compared with young SAMs. NP also increased the CFU-GM colony formation of bone marrow and spleen cells from young SAMs in a soft agar culture system, but it did not enhance CFU-GM colony formation of cells from old SAMs cultured in this system. Treatment with NP induced the production of hematopoietic stimulating factors, including IL-6 and GM-CSF, by bone marrow stromal cells from young SAMs but stromal cells from old SAMs did not respond to NP stimulation. Further studies will be required to clarify the mechanism by which NP stimulates the production of hematopoietic growth factors from stromal cells, the results of this study indicate that NP is a potent hematopoietic regulatory factor by activating stromal cell function(s).

A novel polysaccharide of black soybean promotes myelopoiesis and reconstitutes bone marrow after 5-flurouracil- and irradiation-induced myelosuppression

The aim of this study was to investigate the promotion of myelopoiesis by an active polysaccharide of black soybean (PSBS). Murine spleen cells were collected from ICR mice and conditioned media (SCM) was prepared by incubating these cells without PSBS (normal-SCM) or with PSBS in concentrations ranging from 12.5 to 100 μg/ml (PSBS-SCM). Murine bone marrow cells were treated with PSBS alone or SCM to induce the formation of colonies, including CFU-GM, CFU-GEMM, BFU-E and HPP-CFC. The concentrations of six hematopoietic growth factors contained in SCM were measured using enzyme-linked immunoassay. In the live animal experiment, PSBS was administered orally to total body-irradiated (TBI) and 5-fluorouracil (5-FU)-treated mice to assess the reconstitution of bone marrow after myelosuppression. PSBS-SCM stimulated CFU-GM, CFU-GEMM, BFU-E and HPP-CFC colony formation with 45.0, 5.0, 6.2 and 6.6-fold increases, respectively. However, neither PSBS alone nor normal-SCM had such a colony-stimulating effect. In PSBS-SCM, the levels of IL-6, IL-17, G-CSF and GM-CSF were markedly increased, but not those of IL-3 and SCF. Oral administration of PSBS in mice not only restored the leukocyte counts reduced by TBI and 5-FU treatment but also enhanced CFU-GM colony formation of bone marrow cells without a significant change in body weight. We conclude that PSBS promotes myelopoiesis activity in the bone marrow, stimulates production of various hematopoietic growth factors from spleen cells, and reconstitutes bone marrow that has been myelosuppressed by irradiation and 5-FU.

Direct and Indirect Effects of Androgens on Survival of Hematopoietic Progenitor Cells In Vitro

Androgens remain a common treatment for certain type of anemia, based upon its myelostimulating effects; however, it has not been established whether androgens affect apoptosis of hematopoietic progenitor cells (HPCs). We investigated the effects of the androgens, such as testosterone, 5β-dihydrotestosterone (5-DHT), and oxymetholone, on apoptosis of normal hematopoietic progenitor cells in vitro. Androgens did not rescue normal bone marrow (BM) CD34+ cells and colony-forming cells (CFCs), other than mature erythroid CFCs, from apoptosis induced by serum- and growth factor deprivation. Oxymetholone did not affect growth factor-mediated survival of normal CD34+ cells or its inhibition by interferon-gamma (IFN-γ). In a standard methylcellulose clonogenic assay, low concentrations of oxymetholone and 5-DHT stimulated the clonal growth of colony-forming unit (CFU)-erythroid, but did not affect growth of CFU-granulocyte/macrophage or burst-forming unit-erythroid. Oxymetholone and 5-DHT stimulated the production of stem cell factor in normal bone marrow stromal cells (BMSCs) via transcriptional regulation. In agreement with this, oxymetholone-treated BMSCs better supported the survival of HPCs. These data indicate that survival-enhancing or growth-stimulatory effects of androgens on hematopoietic progenitor cells are minimal and mostly restricted to mature erythroid progenitors, and its myelostimulating effects could be attributed, at least in part, to the stimulation of production of hematopoietic growth factors in BMSCs.

Endogenous interleukin-11 (IL-11) levels in newly diagnosed children with acquired severe aplastic anemia (SAA)

Background: Impaired hematopoietic growth factor production is a hypothetical contributing factor to the development of acquired severe aplastic anemia (SAA). The serum levels of most hematopoietic cytokines in SAA patients are elevated. Objective: To measure interleukin-11 levels in newly diagnosed SAA children and attempt to correlate levels with disease severity and response to therapy. Design/methods: Following enrollment into a clinical study but prior to treatment, serum samples were obtained from 11 newly diagnosed children with acquired SAA. These samples were collected between May 2000 and September 2002. IL-11 levels were quantified utilizing an ELISA technique. Results: Ten of the 11 patients had low or normal levels of IL-11 (<85 pg/mL) and one had an elevated level of 409 pg/mL (normal range 15–200 pg/mL). Conclusions: The production of IL-11 does not increase in response to thrombocytopenia in most children with SAA. The significance of this laboratory observation is not clear at this time. Further studies are warranted to determine what, if any, role this plays in the development of this disorder and if the administration of recombinant human IL-11 might be beneficial in the treatment of acquired SAA.

Ligand-activated natural killer T lymphocytes promptly produce IL-3 and GM-CSF in vivo: relevance to peripheral myeloid recruitment.

Natural killer (NK) T cells are prominent for their prompt IL-4 and IFN-gamma production upon TCR ligation that enables them to influence acquired immune responses. In the present study we provide evidence that the regulatory functions of this particular T cell subset extend to the myeloid compartment of bone marrow and spleen through its production of hematopoietic growth factors. Bone marrow and spleen NKT cells responded to a single injection of their specific ligand alpha-galactosylceramide (alpha-GalCer) by producing both IL-3 and granulocyte-macrophage colony stimulating factor (GM-CSF), whose colony-stimulating activity became detectable in the serum as early as 1 h post treatment. These cytokines were not produced in mice lacking NKT cells (CD1d-/-), whose exclusive involvement in this biological activity was further confirmed by intracellular immuno-staining. Growth factor production was accompanied by significant changes in the myeloid compartment of treated mice, namely mobilization of myeloid progenitors (colony-forming unit cells, CFU-C) and neutrophils from the bone marrow to the periphery. Taken together, our data support the notion that activated NKT cells influence innate immune responses by recruiting myeloid progenitors and granulocytes to the periphery through their production of hematopoietic growth factors.

Regulation of hematopoietic growth factor production by genetically modified human bone marrow stromal cells expressing interleukin-1beta antisense RNA.

Interleukin-1 (IL-1) plays a major role in the regulation of bone marrow stromal cell function and hematopoiesis. It is known to induce secretion of the hematopoietic growth factors granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), IL-6, and IL-8 as well as IL-1 itself in stromal cells. We investigated the role of IL-1beta-mediated growth factor production in the human stromal cell line L88/5. Using liposome-mediated DNA transfer, two stromal cell transfectants that constitutively express IL-1beta antisense (AS) RNA were generated. Expression of IL-1beta AS RNA and IL-1beta RNA was determined by RT-PCR. The stromal cell transfectants were strongly impaired in their endogenous IL-1beta production, and this effect was present even when strong IL-1beta inducers, such as IL-1alpha and tumor necrosis factor-alpha (TNF-alpha), were used. Reduced endogenous IL-1beta levels had no effect on the constitutive production of IL-6, IL-8, and GM-CSF measured by ELISA. In contrast to lipopolysaccharide (LPS) stimulation, IL-1alpha-mediated stimulation of GM-CSF production was significantly reduced in AS transfectants. TNF-alpha induced GM-CSF production was also reduced. IL-6 and IL-8 production was increased in transfectants, suggesting a negative regulatory role of IL-1beta in L88/5. This new approach using AS technology to specifically target constitutive RNA expression will allow further characterization of the bone marrow cytokine network in normal and malignant hematopoiesis.

Adoptive cellular immunotherapy: NK cells and bone marrow transplantation.

Allogeneic bone marrow transplantation (BMT) has been increasingly used for the treatment of both neoplastic and non-neoplastic disorders. However, serious obstacles currently limit the efficacy and thus more extensive use of BMT. These obstacles include: graft-versus-host disease (GVHD), relapse from the original tumor, and susceptibility of patients to opportunistic infections due to the immunosuppressive effects of the conditioning regimen. Overcoming these obstacles is complicated by dual outcome of existing regimens; attempts to reduce GVHD by depleting T cells from the graft, result in increased rates of tumor relapse and failure of engraftment. On the other hand, efforts to increase graft-versus-tumor (GVT) effects of the transplant also promote GVHD. In this review, the use of natural killer (NK) cells to overcome some of these obstacles of allogeneic BMT is evaluated. Adoptive immunotherapy using NK cells after allogeneic BMT has several potential advantages. First, NK cells can promote hematopoiesis and therefore engraftment by production of hematopoietic growth factors. Second, NK cells have been shown to prevent the incidence and severity of GVHD. This has been shown to be at least partially due to TGF-beta, an immunosuppressive cytokine. Third, NK cells have been shown to augment numerous anti-tumor effects in animals after BMT suggesting a vital role of NK cells in mediating GVT effects. Finally, NK cells have been demonstrated to affect B cell recovery and function in mice. Therefore, understanding the mechanisms of beneficial effects of NK cells after BMT may lead to significant increases in the efficacy of this procedure.