The milk thistle (Silybum marianum) compound Silibinin stimulates leukopoiesis from mouse embryonic stem cells.

The milk thistle compound Silibinin (i.e., a 1:1 mixture of Silybin A and Silybin B) stimulates vasculogenesis of mouse embryonic stem (ES) cells. Because vasculogenesis and leukopoiesis are interrelated, the effect of Silibinin on leukopoiesis of ES cells was investigated. Treatment of differentiating ES cells with hydrosoluble Silibinin-C-2',3-dihydrogen succinate dose-dependent increased the number of CD18+ , CD45+ , and CD68+ cells, indicating leukocyte/macrophage differentiation. Silibinin treatment activated phosphoinositide 3-kinase (PI3K), AKT (protein kinase B), signal transducer and activator of transcription 3 (STAT3), stimulated hypoxia-induced factor-1α (HIF-1α), and vascular endothelial growth factor receptor 2 (VEGFR2) expression and raised intracellular nitric oxide (NO). Western blot experiments showed that upon coincubation with either the PI3K inhibitor LY294002, the STAT3 inhibitor Stattic, the AKT antagonist AKT inhibitor VIII, or the NO inhibitor L-NAME, the Silibinin-induced expression of CD18, CD45, and CD68 was abolished. Moreover, the stimulation of HIF-1α and VEGFR2 expression was blunted upon STAT3 and PI3K/AKT inhibition. Treatment of differentiating ES cells with L-NAME abolished the stimulation of VEGFR2 and VE-cadherin expression achieved with Silibinin, indicating that NO is involved in vasculogenesis and leukocyte differentiation pathways. In summary, the data of the present study demonstrate that Silibinin stimulates leukocyte differentiation of ES cells, which is associated to vasculogenesis and regulated by PI3K/AKT-, STAT3-, and NO-mediated signaling.

Effects of a Chinese medical herbs complex on cellular immunity and toxicity-related conditions of breast cancer patients.

Rose geranium (Pelargonium graveolens, Geraniaceae) has anti-cancer and anti-inflammatory properties, and promotes wound healing. Similarly, Ganoderma tsugae (Ganodermataceae), Codonopsis pilosula (Campanulaceae) and Angelica sinensis (Apiaceae) are traditional Chinese herbs associated with immunomodulatory functions. In the present study, a randomised, double-blind, placebo-controlled study was conducted to examine whether the Chinese medicinal herb complex, RG-CMH, which represents a mixture of rose geranium and extracts of G. tsugae, C. pilosula and A. sinensis, can improve the immune cell count of cancer patients receiving chemotherapy and/or radiotherapy to prevent leucopenia and immune impairment that usually occurs during cancer therapy. A total of fifty-eight breast cancer patients who received chemotherapy or radiotherapy were enrolled. Immune cell levels in patient serum were determined before, and following, 6 weeks of cancer treatment for patients receiving either an RG-CMH or a placebo. Administration of RG-CMH was associated with a significant reduction in levels of leucocytes from 31·5 % for the placebo group to 13·4 % for the RG-CMH group. Similarly, levels of neutrophils significantly decreased from 35·6 % for the placebo group to 11·0 % for the RG-CMH group. RG-CMH intervention was also associated with a decrease in levels of T cells, helper T cells, cytotoxic T cells and natural killer cells compared with the placebo group. However, these differences between the two groups were not statistically significant. In conclusion, administration of RG-CMH to patients receiving chemotherapy/radiotherapy may have the capacity to delay, or ease, the reduction in levels of leucocytes and neutrophils that are experienced by patients during cancer treatment.

Deleterious effects of Echinacea purpurea and melatonin on myeloid cells in mouse spleen and bone marrow.

The neurohormone, melatonin, a product of the pineal gland, is a potent immune cell stimulant. Phytochemicals contained in root extracts of the plant species Echinacea purpurea are also potent as immune cell stimulants. Both agents are potent stimulants of T, B, and/or natural killer cells, but little is known of their effect on other hemopoietic cells, specifically granular leukocytes, also participants in a wide variety of disease defense processes. Given their current popularity and availability for amelioration of a) jet lag and sleep disorders (melatonin) and b) virus-mediated respiratory infections (E. purpurea), we investigated the effects of these agents on granular leukocytes and their precursors, myeloid cells. Mice received these agents daily for 7 or 14 days via the diet, thus mimicking human administration, after which spleens and bone marrow were removed and assessed for mature, differentiated granulocytes and their myeloid progenitors. The influence of these agents was directly related to the stage of cell maturity. Administration of both agents together resulted in significantly elevated levels of myeloid progenitor cells in both bone marrow and spleen and significantly reduced levels of mature, functional granulocyte progeny in both organs, suggesting a) increased precursor proliferation, b) antiapoptosis among the progenitors, and/or c) inhibition of precursor maturation-the latter readily explaining the paucity of mature granulocyte progeny. In conclusion, individual administration of either the herbal derivative and melatonin was either without effect (E. purpurea) or even advantageous (melatonin) to cells of this lineage, but when administered together, these agents significantly perturbed myelopoiesis.

Effects of the green algae Chlorella vulgaris on the response of the host hematopoietic system to intraperitoneal ehrlich ascites tumor transplantation in mice.

Chlorella vulgaris extract (CVE) was examined for its effects on the Ehrlich ascites tumor-induced suppression in the numbers of bone marrow and spleen granulocyte-macrophage progenitor cells (CFU-GM) in mice. No effects on bone marrow and spleen CFU-GM, as compared to controls, were observed in normal mice given 50, 100 and 200 mg/kg CVE orally for 5 days. In tumor-bearing mice, myelosuppression concomitant with increased number of spleen CFU-GM were observed. The number of CFU-GM in the bone marrow was restored to control levels after the administration of CVE (50, 100 and 200 mg/kg) to tumor-bearing mice, and a slight reduction in spleen colony formation was observed in these animals. In addition, CVE significantly prolonged the survival of mice inoculated with the Ehrlich ascites tumor. These results suggest a protective antitumor effect of CVE which might be attributable, at least in part, to the stimulation of the production and, possibly, maturation of granulocytes and macrophages.

[Chemotherapy-associated myelosuppression in gynecological oncology]. / Chemotherapieassoziierte Myelosuppression in der gynäkologischen Onkologie.

INTRODUCTION AND OBJECTIVE: A clinically important myelosuppression due to adjuvant chemotherapy is seen more frequently as dosage is intensified and new drugs are used. The assessment of the cytopenia expected is frequently hampered by a lack of directly comparable data. The aim of this study was to compare - in our own patient population - the chemotherapy-associated myelosuppression of four chemotherapeutic regimens used in gynecological oncology. METHODS: 79 patients with primary breast cancer and 26 patients with epithelial ovarian carcinoma underwent cytostatic treatment, and the associated myelosuppression was evaluated by the determination of cytopenia and the need for supportive therapy. The chemotherapy regimens investigated were CMF (cyclophosphamide 600 mg/m(2), methotrexate 40 mg/m(2), 5-fluorouracil 600 mg/m(2), 6xq3w), EC/CMF (epirubicin 90 mg/m(2), cyclophosphamide 600 mg/m(2), 4xq3w, followed by CMF, 3xq3w), DE (docetaxel 75 mg/m(2), epirubicin 90 mg/m(2), 6xq3w) and CC (cyclophosphamide 600 mg/m(2), carboplatin AUC 6, 6xq3w). RESULTS: The EC/CMF and DE regimens were used significantly more frequently for more advanced tumor stages, but there were no differences concerning tumor-dependent prechemotherapeutic myelosuppression. Hemopoiesis was most impaired in the CC group with a mean drop of serum hemoglobin of 1.5 g/dl to the end of the cytostatic treatment; correspondingly, most transfusions of concentrated erythrocytes were needed in this group. The strongest suppression of leukopoiesis was found in the DE group, with a mean drop in leukocyte counts of 6.2 x 10(3)/microliter per cycle; in this group, a mean of 7.6 ready-made syringes with 263 microgram Lenogastrim was used to stimulate leukopoiesis. The severest drop in the mean thrombocyte count, i.e. 171.7 x 10(3)/microliter, was found in the CC group. CONCLUSIONS: The CC regimen impairs thrombo- and erythropoiesis most, whereas the DE regimen causes marked leukopenia. The regimen with the smallest myelosuppression was CMF. No severe cytopenia-associated complications were detected in any of the cases investigated.

The role of the reticulo-epithelial (RE) cell network in the immuno-neuroendocrine regulation of intrathymic lymphopoiesis.

The thyrnus provides an optimal cellular and humoral microenvironment for the development of immunocompetent T lymphocytes. Although yolk sac derived pre-T, committed hematopoietic stem cells enter the thymus using a homing receptor, the immigration process also requires secretion of a peptide, called thymotaxin by the cells of the reticulo-epithelial (RE) network of the thymic cellular microenvironment. The thymic RE cells are functionally specialized based on their location within the thymic microenvironment. Thus, although subcapsular, cortical, and medullary RE cells are derived from a common, endodermal in origin epithelial precursor cell, their unique location within the gland causes their specialization in terms of their immunophenotypical and in situ physiological properties. The subcapsular, endocrine, RE cell layer (giant or nurse cells) is comprised of cells filled with PAS positive granules, which also express A2B5/TE4 cell surface antigens and MHC Class I (HLA A, B, C) molecules. In contrast to the medullary RE cells, these subcapsular nurse cells also produce thymosins beta 3 and beta 4. The thymic nurse cells (TNCs) display a neuroendocrine cell specific immunophenotype (IP): Thy-1+, A2B5+, TT+, TE4+, UJ13/A+, UJ127.11+, UJ167.11+, UJ181.4+, and presence of common leukocyte antigen (CLA+). Medullar RE cells display MHC Class II (HLA-DP, HLA-DQ, HLA- DR) molecule restriction. These cells also contain transforming growth factor (TGF)-beta type II receptors and are involved in the positive selection of T cells. Transmission electronmicroscopic (TEM) observations have defined four, functional subtypes of medullary RE cells: undifferentiated squamous, villous and cystic. All subtypes were connected with desmosomes. The secreted thy nic hormones, thymulin, thymosin-alpha 1 and thymopoietin (its short form, thymopentin or TP5) were detected immunocytochemically to be produced by RE cells. Thymic RE cells also produce numerous cytokines including IL-1, IL-6, G-CSF, M-CSF, and GM-CSF molecules that likely are important in various stages of thymocyte activation and differentiation. The co-existence of pituitary hormone and neuropeptide secretion [growth hormone (GH), prolactin (PRL), adrenocorticotropic hormone (ACTH), thyroid stimulating hormone (TSH), triiodothyronine (T3), somatostatin, oxytocin (OT), follicle stimulating hormone (FSH), luteinizing hormone (LH), arginine vasopressin (AVP), growth hormone releasing hormone (GHRH), corticotropin releasing hormone (CRH), nerve growth factor (NGF), vasoactive intestinal peptide (VIP), pro-enkephalin (pro-enk), and beta-endorphin (beta-end)], as well as production of a number of interleukins and growth factors and expression of receptors for all, by RE cells is an unique molecular biological phenomenon. The thymic RE cell network is most probably comprised of cells organized into sub-networks--functional units composed of RE cells with differing hormone production/hormone receptor expression profiles, involved in the various stages of T lymphocyte maturation. Furthermore, it is quite possible that even on the level of individual RE cells, the numerous projections associated with a single cell, which engulf developing lymphocytes, nurturing and guiding them in their maturation, may differ in their hormone production and/or hormone receptor expression profile, thus allowing a single cell to be involved in distinct, separate steps of the T cell maturation process. Based on our systematic observations of the thymus in humans and other mammalian species, we suggest that the thymic RE cells represent an extremely important cellular and humoral network within the thymic microenvironment and are involved in the homeopathic regulation mechanisms of the multicellular organism, in addition to the presentation of various antigens to developing lymphocytes, and providing growth regulatory signals which may range from stimulatory to apoptotic signaling within the thymus. (ABSTRACT TRUNCA

Prostaglandin E2 receptor antagonist (SC-19220) treatment restores the balance to bone marrow myelopoiesis after burn sepsis.

BACKGROUND: Although prostaglandin E2 (PGE2) has been shown to be immunosuppressive, its role in the development of specific bone marrow myeloid lineages after thermal injury and sepsis has yet to be elucidated. The purpose of this study was to demonstrate that alterations in bone marrow progenitor proliferation favoring monocytopoiesis in burn sepsis can be restored by blocking the cellular interactions of PGE2. METHODS: A murine model of burn sepsis with and without treatment with SC-19220, a PGE2 receptor antagonist, was used to determine peripheral monocyte and neutrophil counts as well as the colony forming potential of colony-stimulating factor responsive bone marrow progenitors. RESULTS: Burn sepsis augmented the growth of the early colony-forming unit granulocyte-macrophage and monocyte progenitors and the number of circulating monocytes, whereas granulocyte progenitors and circulating neutrophils demonstrated an opposite response. Treatment with SC-19220 nearly reversed these alterations. CONCLUSION: These data indicate that abrogating PGE2's actions during burn sepsis can restore the balance in bone marrow granulocyte and monocyte production, further consolidating the pivotal role PGE2 plays in the pathogenesis of burn sepsis.

[Toxocological study of Guiera senegalensis Lam (Combretaceae)]. / Etude toxicologique de Guiera senegalensis Lam (Combretaceae).

The study was based on an aqueous extract derived from a 60 degrees ethanolic tincture containing 0.032 g of dry matter per ml. The leaves of Guiera senegalensis Lam (Combretaceae) were collected in December 1991 at Nguekhokh a village within 20 km from Mbour (Senegal). The extract was administered for six months through daily forced-feeling at 2 g/kg to Wistar male and female rats whose weight at the beginning of the experiment tanged between 140 g and 180 g. The urine was analysed during the study and the animals were weighed every four weeks. At the end of the experiment, the animals were slaughtered and various analyses carried out. Haematological features in relation with erythropoiesis, haemoglobinogenesis and leucopoiesis, were studied in relation with renal and hepatic functions; biochemical features too. Some organs (heart, lungs, kidneys, brain, cerebellum, spleen and liver) were removed and examined in order to detect possible lesions following the experiment. Judging by the results, Guiera senegalensis Lam (Combretaceae), as used in the experiment, did not show any significant toxicity.

PEG-rHuMGDF promotes multilineage hematopoietic recovery in myelosuppressed mice.

PEG-rHuMGDF administered to normal mice is a lineage-specific growth factor for megakaryocytes and platelets as judged by morphologic examination of hematologic cells in marrow and peripheral blood smears. The purpose of this study was to document that PEG-rHuMGDF in myelosuppressed mice promotes multilineage hematopoietic recovery. High-dose 5-fluorouracil (5-FU) in mice results in profound myelosuppression and 0-30% survival. Mice receiving a single dose of PEG-rHuMGDF (1000 microg/kg) 1 day after 5-FU (225 mg/kg) demonstrate an increased survival (76% vs 27% in control mice at 14 days). Compared to surviving controls, PEG-rHuMGDF-treated mice not only show the expected higher platelet counts, but also increased marrow colony-forming unit granulocyte-macrophage, increased multilineage marrow cellularity, and increased neutrophil, monocyte, and lymphocyte counts in peripheral blood. PEG-rHuMGDF- and vehicle-treated mice both develop hepatic abscesses after 5-FU treatment, but the abscesses in the PEG-rHuMGDF-treated mice contain more neutrophils, suggesting that myeloid reconstitution contributes to their survival. Furthermore, survival in 5-FU-treated mice is significantly improved by granulocyte colony-stimulating factor and antibiotics, suggesting that infection rather than thrombocytopenia is the predominant cause of death. PEG-rHuMGDF after 5-FU promotes survival accompanied by accelerated lymphohematopoietic repopulation, suggesting that PEG-rHuMGDF, a lineage-specific thrombopoietic factor in normal mice, promotes multilineage hematopoietic recovery in myelosuppressed mice.

Update on thrombopoietin in preclinical and clinical trials.

Thrombopoietin, also termed the c-Mpl ligand, is a lineage-dominant hematopoietic factor that primarily regulates megakaryopoiesis and thrombopoiesis. Treatment of normal animals with recombinant human megakaryocyte growth and development factor, a truncated molecule of the c-Mpl ligand, which is modified with polyethylene glycol (PEG-rHuMGDF), and glycosylated recombinant thrombopoietin stimulates the expansion of bone marrow megakaryocytes and their progenitors, and greatly enhances the production of morphologically and functionally normal platelets. In contrast, this cytokine has only minimal effects on peripheral leukocyte and erythrocyte counts. In myelosuppressed animals, PEG-rHuMGDF or glycosylated thrombopoietin accelerates multilineage hematopoietic recovery effectively improving thrombocytopenia and, in most models, leukopenia (or neutropenia) and anemia. In addition to daily multiple injections, even a single injection of PEG-rHuMGDF after myelosuppressive treatment is fully effective for hematopoietic recovery. In clinical trials, PEG-rHuMGDF or glycosylated recombinant human thrombopoietin potently stimulates thrombopoiesis in cancer patients before chemotherapy. The administration of PEG-rHuMGDF alone or in combination with recombinant human granulocyte colony-stimulating factor (rhG-CSF) reduces the duration of severe thrombocytopenia and in some cases platelet nadirs in patients with advanced cancers after dose-intensive chemotherapy. The recombinant hormone is well-tolerated with little drug-related toxicity.