Production Of Colony-stimulating Factor Research Articles

Enhancement of Umbilical Cord Blood Cell Hematopoiesis by Maitake Beta-Glucan Is Mediated by Granulocyte Colony-Stimulating Factor Production

Maitake beta-glucan (MBG) is an extract from the fruit body of the Grifola frondosa mushroom that is being widely used to treat cancer in Asia. We have previously reported that MBG enhances mouse bone marrow cell (BMC) hematopoiesis in vitro and protects BMC from doxorubicin (DOX) toxicity. In the current study, we investigated the ability of MBG to enhance hematopoiesis and to reduce the toxic effects of DOX on fresh human umbilical cord blood (CB) cells. MBG treatment significantly enhanced the colony formation unit (CFU) response of granulocytes-macrophages (CFU-GM response) over the whole dose range of 12.5 to 100 microg/ml (P < 0.05). The addition of MBG to DOX-treated CB cells significantly protected granulocyte-macrophage colony formation from the toxicity of DOX, which otherwise produced strong hematopoietic repression. MBG also partially replaced recombinant human granulocyte colony-stimulating factor (rhG-CSF), as shown by a significant augmentation of the CFU-GM response in the absence of rhG-CSF. We found that MBG induces granulocyte colony-stimulating factor (G-CSF) production in CB CD33+ monocytes, as detected by intracellular cytokine flow cytometric assessment. In contrast, we found that adult peripheral blood monocytes did not produce a significant G-CSF response to MBG, whereas both adult and CB monocytes produced G-CSF in response to lipopolysaccharide. These studies provide the first evidence that MBG induces hematopoietic stem cell proliferation and differentiation of CFU-GM in umbilical CB cells and acts directly to induce G-CSF.

CYSTATIN C AS A MARKER OF IMMUNE COMPLEX–ASSOCIATED RENAL IMPAIRMENT IN A SUDANESE POPULATION WITH VISCERAL LEISHMANIASIS

Renal function was studied in visceral leishmaniasis (VL) and post-kala-azar dermal leishmaniasis (PKDL) by means of the specific marker cystatin C and related to circulating immune complexes and cytokine production. Forty patients with VL (23 with sub-acute disease and 17 with acute disease), 17 patients with PKDL, and 22 healthy controls were included. Cystatin C, but not creatinine, was significantly raised in VL (P = 0.004). The highest levels of cystatin C were found in those with acute disease (P < 0.0001). In VL, cystatin C levels were positively correlated to circulating immune complexes and production of granulocyte-macrophage colony stimulating factor (GM-CSF), but negatively correlated to aspartate aminotransferase and lactate dehydrogenase. We conclude that cystatin C is a superior marker of glomerular function in leishmaniasis and that immune complex deposition and GM-CSF are two functions that most likely are causally involved in the mechanisms leading to glomerular dysfunction in leishmaniasis.

The β-glucan receptor dectin-1 functions together with TLR2 to mediate macrophage activation by mycobacteria

AbstractPattern recognition receptors (PRRs) play an essential role in a macrophage's response to mycobacterial infections. However, how these receptors work in concert to promote this macrophage response remains unclear. In this study, we used bone marrow–derived macrophages isolated from mannose receptor (MR), complement receptor 3 (CR3), MyD88, Toll-like receptor 4 (TLR4), and TLR2 knockout mice to examine the significance of these receptors in mediating a macrophage's response to a mycobacterial infection. We determined that mitogen-activated protein kinase (MAPK) activation and tumor necrosis factor-α (TNF-α) production in macrophage infected with Mycobacterium avium or M smegmatis is dependent on myeloid differentiation factor 88 (MyD88) and TLR2 but not TLR4, MR, or CR3. Interestingly, the TLR2-mediated production of TNF-α by macrophages infected with M smegmatis required the β-glucan receptor dectin-1. A similar requirement for dectin-1 in TNF-α production was observed for macrophages infected with M bovis Bacillus Calmette-Guerin (BCG), M phlei, M avium 2151-rough, and M tuberculosis H37Ra. The limited production of TNF-α by virulent M avium 724 and M tuberculosis H37Rv was not dependent on dectin-1. Furthermore, dectin-1 facilitated interleukin-6 (IL-6), RANTES (regulated on activation, normal T expressed and secreted), and granulocyte colony-stimulating factor (G-CSF) production by mycobacteria-infected macrophages. These are the first results to establish a significant role for dectin-1, in cooperation with TLR2, to activate a macrophage's proinflammatory response to a mycobacterial infection.

Neutrophil-activating activity and platelet-activating factor synthesis in cytokine-stimulated endothelial cells: Reduced activity in growth-arrested cells

The reactivity of endothelial cells (ECs) to proinflammatory cytokines is critically important for the pathogenesis of vascular diseases. Here, we studied functional alterations of human ECs during culture under a confluent condition; i.e., the alterations of neutrophil-activating activity, platelet-activating factor (PAF) synthesis, and granulocyte–macrophage colony-stimulating factor (GM-CSF) production in cytokine-stimulated ECs. Human umbilical vein-derived ECs exhibited the increased activity in neutrophil activation, PAF synthesis, and GM-CSF production when stimulated by proinflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). The activity of cytokine-stimulated ECs to stimulate superoxide release in human neutrophils and to produce PAF declined markedly in parallel as ECs became growth-arrested during culture under a confluent condition. By contrast, GM-CSF production induced by cytokine stimulation was modestly increased, and up-regulation of intercellular adhesion molecule-1 (ICAM-1) and activation of mitogen-activated protein kinases were not altered. The neutrophil-activating activity of cytokine-stimulated ECs was dependent on PAF synthesis and GM-CSF production from ECs. These findings indicate that the reduced neutrophil-activating activity in growth-arrested ECs may be, at least in part, ascribed to down-regulation of PAF synthesis.

Murine mammary carcinoma 4T1 induces a leukemoid reaction with splenomegaly: Association with tumor-derived growth factors

A leukemoid reaction with granulocytosis and splenomegaly has been observed in animals and humans with a variety of tumors. We have employed four color flow cytometry to characterize the leukemoid reaction induced by the transplantable mouse mammary carcinoma 4T1 in female BALB/c mice. Gr-1 + myeloid cells with the morphology of granulocytes increased in peripheral blood from < 15% pre-transplant to nearly 80% of total CD45 + leukocytes at four weeks post-transplant. Though the granulocyte:lymphocyte ratio increased markedly, the absolute numbers of CD19 + B lymphocytes, CD4 + and CD8 + T lymphocytes, and the CD4/CD8 ratio in peripheral blood did not change significantly. Femurs from tumor-bearing mice showed myeloid hyperplasia of the fatty marrow. There was a notable increase in cells with a Gr-1 dim/CD11b bright immature granulocyte phenotype, and these cells were also found in peripheral blood and spleen. Spleen weights had increased 8.5-fold by four weeks post-tumor transplant, mainly due to granulocytic hyperplasia. Cultured 4T1 tumor cells constitutively expressed mRNA for the myeloid colony-stimulating factors G-CSF and GM-CSF, and IFN-γ-inducible M-CSF transcripts were also detected. Tumors excised from mice had transcripts for G-CSF and GM-CSF, but only G-CSF protein was found in high levels in serum of tumor-bearing mice. These data demonstrate that 4T1 tumor-bearing mice exhibit a leukemoid reaction that apparently is caused by the production of colony-stimulating factors produced by the tumor. The 4T1 tumor may serve as an excellent model for the study of this reaction.

Effects of chorionic gonadotropin on peripheral monocytes vary with the phases of the menstrual cycle

The qualitative and quantitative effects of physiological concentrations of chorionic gonadotropin (CG) on monocytes of women vary with the phases of the menstrual cycle. During the follicular phase, the hormone inhibits phagocytosis; stimulates the secretion of interleukin (IL) 6 (IL-6), interferon α (IFN-α), and the protein component of apolipoprotein A1 (APO-A1); and activates myeloperoxidase (MPO). During the luteal phase, CG stimulates phagocytosis and APO-A1 secretion, inhibits MPO, and does not shift the levels of IL-6 and INF-α. Regardless of the menstrual phase, the hormone does not modify the release of elastase or the production of granulocytic colony-stimulating growth factor, IL-1α, and tumor necrosis factor α.

Aggressive carcinosarcoma of the uterine cervix associated with high levels of granulocyte colony stimulating factor: Case report and laboratory correlates

Production of granulocyte colony stimulating factor (G-CSF) by solid tumors, including cervix cancers, is reported to be associated with an aggressive clinical course. A 64-year-old female with a locally advanced carcinosarcoma of the cervix presents with a WBC count of 48,000 cells/microl with no infectious causes. Her WBCs returned to normal during primary radiotherapy and then increased again with recurrence. She expired of progressive disease 10 weeks after primary therapy. Serum and pleural fluid levels of G-CSF were 1500 and 6000 pg/ml, respectively. Cancer cells secreted G-CSF in culture and recombinant G-CSF increased cancer cell proliferation. Like other sarcomas, carcinosarcomas can produce high levels of G-CSF and demonstrate an aggressive behavior that may be autocrine stimulation.

Prophylactic administration of Withania somnifera extract increases host resistance in Listeria monocytogenes infected mice

In this study, we demonstrated that Withania somnifera L. extract (WSE) protects mice from a lethal dose of Listeria monocytogenes when administered prophylactically at 100, 250 and 500 mg/kg for 10 days, with survival rates up to 30%. These doses also prevented the myelosuppression and the splenomegaly caused by a sublethal infection with L. monocytogenes, due to increased numbers of granulocyte–macrophage progenitors (CFU-GM) in the bone marrow. Investigation of the production of colony-stimulating factors (CSFs) revealed increased colony-stimulating activity (CSA) in the serum of normal and infected mice pre-treated with WSE. Further studies to investigate the levels of interferon-gamma (INF-γ) and lymphocyte cell proliferation were undertaken. We observed dose-dependent increases in cell proliferation and in the levels of INF-γ in mice infected with L. monocytogenes and treated with WSE. All together, our results suggest that WSE indirectly modulates immune activity and probably disengages Listeria-induced suppression of these responses by inducing a higher reserve of myeloid progenitors in the bone marrow, proliferation of lymphocytes and increased INF-γ levels.

Histamine H1 Receptor-Stimulated Interleukin 8 and Granulocyte Macrophage Colony-Stimulating Factor Production by Bronchial Epithelial Cells Requires Extracellular Signal-Regulated Kinase Signaling via Protein Kinase C

Background: Histamine stimulates the release of several cytokines, such as interleukin (IL)-8 and granulocyte macrophage colony-stimulating factor, from bronchial epithelial cells. However, the functional individual histamine receptor subtype and intracellular signaling in bronchial epithelial cells are poorly defined. Methods: Using human primary epithelial cells and the NCI-H292 cell line, we examined the expression of histamine receptor subtypes and histamine-induced second messenger. We also evaluated the involvements of mitogen-activated protein kinase, protein kinase C (PKC) and epidermal growth factor receptor in cytokine expression caused by histamine. Results: Histamine H₁ receptor (H₁R) was the only subtype expressed in both types of cells. Histamine elevated intracellular calcium ion without affecting cAMP levels. Histamine induced the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. Histamine also phosphorylated PKC and myristoylated alanine-rich C kinase substrate. Ro-31-8220, a PKC inhibitor, and PD98059, a mitogen-activated protein/ERK kinase inhibitor, suppressed the histamine-induced ERK activation and the production of granulocyte macrophage colony-stimulating factor and IL-8. On the contrary, histamine had no effect on the phosphorylation of epidermal growth factor receptor, and its specific inhibitor AG1478 failed to inhibit the histamine-induced ERK activation. Olopatadine, an H₁ antagonist, completely blocked the histamine-related responses, whereas H₂ and H₃ antagonists did not. Histamine also augmented the IL-8 production caused by IL-4 or tumor necrosis factor-α. Conclusions: The H₁R-PKC-ERK pathway may play crucial roles in eliciting cytokine production from bronchial epithelial cells stimulated by histamine, leading to airway inflammation.

Osteoblast Dysfunction in Male Idiopathic Osteoporosis

The etiology of primary osteoporosis in young and middle-aged men is unknown. We have studied osteoblast function in cells derived from men with idiopathic osteoporosis and in control cells from age-matched men with osteoarthrosis. Osteoblasts were isolated from transiliac bone biopsies. Osteoblast function was measured as vitamin D-stimulated osteocalcin production and production of cytokines and factors involved in osteoclast activation and bone formation. Cell proliferation was measured as (3)H-thymidine incorporation. Parathyroid hormone-related peptide (PTHrP) mRNA was measured using reverse-transcriptase polymerase chain reaction. In osteoporotic men, bone mineral density at the femoral neck was correlated to in vitro production of osteocalcin. Osteoblasts from osteoporotic men produced significantly less osteocalcin after vitamin D stimulation but had increased production of macrophage colony-stimulating factor (M-CSF) compared to controls. The osteocalcin response was negatively correlated to production of M-CSF, interleukin-6, and C-terminal propeptide of type I collagen. Basal (3)H-thymidine incorporation was similar in cells from osteoporotic patients and controls. PTHrP (10(-9 )M) significantly increased cell proliferation in control cells but not in osteoporotic cells. Basal PTHrP mRNA levels were significantly higher in osteoporotic cells than in cells from controls. The results are in agreement with previous histomorphologic studies indicating that men with idiopathic osteoporosis have an osteoblast dysfunction with decreased osteocalcin production and increased production of factors stimulating osteoclast activation. This indicates a catabolic cellular metabolic balance leading to negative bone turnover, resulting in osteoporosis. The cause of such cellular dysfunction needs further evaluation.

Inactivation of PU.1 in adult mice leads to the development of myeloid leukemia

Genetically primed adult C57BL mice were deleted of exon 5 of the gene encoding the transcription factor PU.1 by IFN activation of Cre recombinase. After a 13-week delay, conditionally deleted (PU.1(-/-)) mice began dying of myeloid leukemia, and 95% of the mice surviving from early postinduction death developed transplantable myeloid leukemia whose cells were deleted of PU.1 and uniformly Gr-1 positive. The leukemic cells formed autonomous colonies in semisolid culture with varying clonal efficiency, but colony formation was enhanced by IL-3 and sometimes by granulocyte-macrophage colony-stimulating factor. Nine of 13 tumors analyzed had developed a capacity for autocrine IL-3 or granulocyte-macrophage colony-stimulating factor production, and there was evidence of rearrangement of the IL-3 gene. Acquisition of autocrine growth-factor production and autonomous growth appeared to be major events in the transformation of conditionally deleted PU.1(-/-) cells to fully developed myeloid leukemic populations.

Paraneoplastic production of granulocyte colony-stimulating factor in a bladder carcinoma

We report the case of a 51-year-old female with poorly differentiated bladder carcinoma who presented with leukocytosis and hypercalcemia. Granulocyte colony-stimulating factor was noted to be twice the normal level. Paraneoplastic leukocytosis in bladder carcinoma is rare, is often associated with paraneoplastic hypercalcemia, and appears to represent a particularly aggressive form of the disease.

Histone deacetylase 2–mediated deacetylation of the glucocorticoid receptor enables NF-κB suppression

Glucocorticoids are the most effective antiinflammatory agents for the treatment of chronic inflammatory diseases even though some diseases, such as chronic obstructive pulmonary disease (COPD), are relatively glucocorticoid insensitive. However, the molecular mechanism of this glucocorticoid insensitivity remains uncertain. We show that a defect of glucocorticoid receptor (GR) deacetylation caused by impaired histone deacetylase (HDAC) 2 induces glucocorticoid insensitivity toward nuclear factor (NF)-κB–mediated gene expression. Specific knockdown of HDAC2 by RNA interference resulted in reduced sensitivity to dexamethasone suppression of interleukin 1β–induced granulocyte/macrophage colony-stimulating factor production. Loss of HDAC2 did not reduce GR nuclear translocation, GR binding to glucocorticoid response element (GRE) on DNA, or GR-induced DNA or gene induction but inhibited the association between GR and NF-κB. GR becomes acetylated after ligand binding, and HDAC2-mediated GR deacetylation enables GR binding to the NF-κB complex. Site-directed mutagenesis of K494 and K495 reduced GR acetylation, and the ability to repress NF-κB–dependent gene expression becomes insensitive to histone deacetylase inhibition. In conclusion, we show that overexpression of HDAC2 in glucocorticoid-insensitive alveolar macrophages from patients with COPD is able to restore glucocorticoid sensitivity. Thus, reduction of HDAC2 plays a critical role in glucocorticoid insensitivity in repressing NF-κB–mediated, but not GRE-mediated, gene expression.

Intense paraneoplastic neutrophilic leukemoid reaction related to a G‐CSF‐Secreting lung sarcoma

A white blood cell count more than 50 x 10(9)/l, not related to bone marrow involvement, is termed leukemoid reaction. We report on the first case of an undifferentiated sarcoma of the lung associated with an intense paraneoplastic neutrophilic leukemoid reaction related to the production of granulocyte colony-stimulating factor (G-CSF). A radiography and a computed tomography scan of the chest revealed a well-limited voluminous and heterogeneous low-density mass of the left lung. The patient died of multiorgan failure related to uncontrolled progressive tumor growth after admission and two cycles of chemotherapy. The patient's G-CSF serum concentration was dramatically elevated (6,538 pg/ml) compared to serum levels observed in normal controls and patients with elevated leukocytosis (31 and 387 pg/ml, respectively). The G-CSF concentration dramatically increased after the first cycle of chemotherapy and during the subsequent neutropenia, as a result of the tumor lyses as well as of disruption of the physiological negative feedback mechanism. Adjunction of the patient's serum to CD34+ cell cultures induced a 12.3-fold increase in CD15+ cells, demonstrating the serum's capacity to induce myeloid differentiation.

Laryngeal squamous cell carcinoma with ectopic production of granulocyte colony-stimulating factor and parathyroid hormone-related protein

It is known that malignant tumors occasionally produce hormone-like substances. However, they rarely produce two or more kinds of hormonal factors simultaneously. We describe the clinical, biochemical, and immunohistochemical findings in a patient with squamous cell carcinoma of the larynx associated with leukocytosis and hypercalcemia, without bone metastasis. The serum granulocyte colony-stimulating factor (G-CSF) level was 222 pg/ml (normal, < 38 pg/ml), and decreased to 15 pg/ml after surgery. The serum parathyroid hormone-related protein (PTHrP) level was 5.6 pmol/l (normal, < 0.5 pmol/l), and this also decreased, to less than 0.5 pmol/l. G-CSF and PTHrP increased again with tumor recurrence. The production of G-CSF from the tumor was confirmed by immunohistochemical staining with monoclonal antibody against human recombinant G-CSF. The tumor was revealed to produce both G-CSF and PTHrP. In this article, we describe this very rare case of primary squamous cell carcinoma of the head and neck with the simultaneous ectopic production of more than one hormone-like substance.

61. Higher In Vivo Maintenance, Biodistribution and Immunopotency of Dendritic Cell Vaccines Self-Differentiated through Lentiviral Vector Programming

Top of pageAbstract Dendritic cell (DC) vaccines are conventionally produced ex vivo by culturing DC precursors in the presence of recombinant growth factors. Methods to facilitate the generation of DCs, to prolong their viability, to facilitate their migration to lymphoid tissues and to augment their capacity to establish and maintain antigen-specific immune responses are critical for improving their effective therapeutic potential for immunotherapy. To address these issues, we have genetically engineered purified human CD14+ monocytes with lentiviral vectors (LV) for production of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4), which triggered their differentiation into cells with typical characteristics of DCs. We previously demonstrated that these lentivirus programmed DCs (DC/LVs) exhibited more potent immunostimulatory function and prolonged viability in vitro than DCs generated by conventional methods. Using the same strategy, we have now generated murine DC/LVs in order to test the efficacy of this approach in vivo. Third generation self-inactivating LV (5 |[mu]|g/ml p24 equivalent) transduced non-adherent bone marrow (BM) cells harvested from C57BL/6 mice at >90% transduction efficiency. BM cells co-expressing GM-CSF and IL-4 after LV transduction produced these cytokines at concentrations similar to those commonly used for generation of conventionally cultured DCs by addition of exogenous cytokines (> 10ng/ml), resulting in the generation of DC/LVs as evidenced by their morphology and immunophenotype (CD11b+, CD11c high, CD80 high, MHCII high). DC/LVs maintained in culture were metabolically active and viable for 3 weeks, whereas conventional DCs showed a drastic decrease in viability in the first week of culture. In vivo bioluminescence imaging studies were then performed using luciferase as a marking gene to track DCs after s.c. injection into nude syngeneic mice. Persistent and robust bioluminescence signals were detectable from the injected DC/LVs for more than 45 days, whereas signals produced by conv. DCs faded away within the first two weeks. To determine whether the higher viability of DC/LVs correlated with greater potency as vaccines, we next examined their antigen presentation and immunostimulatory functions in vivo. DC/LVs and conv. DCs maintained in culture for 7 days were transduced to express the melanoma associated antigen MART-1 and injected s.c. Splenocytes harvested 2 weeks after vaccination with DC/LVs showed up to 2-fold increase in activated T cells by IFN|[gamma]|- ELISPOT assay (p<0.05), and chromium lysis assays using target B16 melanoma cells showed more efficient killing by DC/LV-activated CTLs in comparison to conv. DCs (p<0.05). We are currently evaluating the immunotherapeutic efficacy of DC/LVs injected into mice developing B16 melanoma tumors. Initial experiments in a prophylactic model indicate that mice vaccinated with DC/LV show enhanced survival compared to the group vaccinated with conv.DCs. Our results therefore indicate that DC/LVs can provide enhanced anti-tumoral therapeutic efficacy and warrant further investigation of the clinical applicability of genetically programmed DCs.

Therapeutic Effects ofLycium barbarumPolysaccharide (LBP) on Irradiation or Chemotherapy-Induced Myelosuppressive Mice

The aim of this study was to investigate the effects of Lycium barbarum polysaccharide (LBP) on irradiation- or chemotherapy-induced myelosuppressive mice and cultured peripheral blood mononuclear cells (PBMCs). In an in vivo experiment, mice were irradiated with a sublethal dose of 550 cGy X-ray or intraperitoneally (i.p.) injected with carboplatin (CB) 125 mg/kg to produce severe myelosuppression. Four to 6 hours after the irradiation or injection, mice were subcutaneously (s.c.) injected with LBP (50, 100, and 200 mg/kg) daily from day 0 to day 6. Blood samples were collected from the tail veins of mice at different time points, and peripheral white blood cells (WBC), red blood cells (RBC), and platelet (PLT) counts were monitored. In an in vitro experiment, human PBMCs were incubated with LBP at different concentrations in combination with phytohemagglutinin (PHA), and the production of granulocyte colony-stimulating factor (G-CSF) was tested. Compared to the control, 50 mg/kg LBP (LBP-L) significantly ameliorated the decrease of peripheral WBC of irradiated myelosuppressive mice on day 13, and 100 mg/kg LBP (LBP-M) did the same on days 17 and 21. All dosages of LBP significantly ameliorated the decrease of peripheral RBC of irradiated myelosuppressive mice on days 17 and 25. Two-hundred mg/kg LBP (LBP-H) and LBP-M significantly enhanced peripheral PLT counts of irradiated myelosuppressive mice on days 10, 13, 17, and 21, as did LBP-L on days 13 and 17. All dosages of LBP increased peripheral WBC counts of chemotherapy-induced myelosuppressive mice to some extent, but there was no statistic difference when compared to the control. LBP-H significantly ameliorated the decrease of peripheral RBC of chemotherapy-induced myelosuppressive mice on days 13, 15, 17, and 20, and LBP-M and LBP-L did the same on days 15 and 17. All dosages of LBP significantly enhanced peripheral PLT counts of chemotherapy-induced myelosuppressive mice on days 7 and 10, as did LBP-H on days 13, 15, and 17, and LBP-M on days 13 and 15. Also, LBP could obviously stimulate human PBMCs to produce G-CSF. LBP promoted the peripheral blood recovery of irradiation or chemotherapy-induced myelosuppressive mice, and the effects may be the result of the stimulation of PBMCs to produce G-CSF.

Uncaria tomentosa extract increases the number of myeloid progenitor cells in the bone marrow of mice infected with Listeria monocytogenes

In this study, we demonstrated that Uncaria tomentosa extract (UTE) protects mice from a lethal dose of Listeria monocytogenes when administered prophylactically at 50, 100, 150 and 200 mg/kg for 7 days, with survival rates up to 35%. These doses also prevented the myelosuppression and the splenomegaly caused by a sublethal infection with L. monocytogenes, due to increased numbers of granulocyte-macrophage progenitors (CFU-GM) in the bone marrow. Non-infected mice treated with 100 mg/kg UTE also presented higher numbers of CFU-GM in the bone marrow than the controls. Investigation of the production of colony-stimulating factors revealed increased colony-stimulating activity (CSA) in the serum of normal and infected mice pre-treated with UTE. Moreover, stimulation of myelopoiesis and CSA occurred in a dose-dependent manner, a plateaux being reached with the dose of 100 mg/kg. Further studies to investigate the levels of factors such as IL-1 and IL-6 were undertaken. We observed increases in the levels of IL-1 and IL-6 in mice infected with L. monocytogenes and treated with 100 mg/kg of UTE. White blood cells (WBC) and differential counting were also performed, and our results demonstrated no significant changes in these data, when infected mice were pre-treated with 100 mg/kg of UTE. All together, our results suggest that UTE indirectly modulates immune activity and probably disengages Listeria-induced supression of these responses by inducing a higher reserve of myeloid progenitors in the bone marrow in consequence of biologically active cytokine release (CSFs, IL-1 and IL-6).

The liquid culture filtrates of Paecilomyces tenuipes (Peck) Samson (= Isaria japonica Yasuda) and Paecilomyces cicadae (Miquel) Samson (= Isaria sinclairii (Berk.) Llond) regulate Th1 and Th2 cytokine response in murine Peyer's patch cells in vitro and ex vivo

The effects of liquid culture filtrates of medicinal entomogenous fungi, Paecilomyces tenuipes (Peck) Samson (= Isaria japonica Yasuda or Isaria tenuipes) (PTCF) and Paecilomyces cicadae (Miquel) Samson (= Isaria sinclairii (Berk.) Llond) (PCCF), on cytokine productions in cultured Peyer's patches (PP) from C57BL/6J mice were investigated in vitro and ex vivo. In an in vitro experiment, PTCF (100 and 10 μg/ml) enhanced the production of T helper 1 (Th1) cytokines, interleukin (IL)-2 and interferon (IFN)-γ, in cultured PP cells stimulated with 5 μg/ml concanavalin A (Con A) but did not influence on the production of T helper 2 (Th2) cytokines, IL-4 and IL-5. PTCF also enhanced the production of granulocyte macrophage colony-stimulating factor (GM-CSF) and IL-10 in the cultured PP cells. While, PCCF enhanced the production of IFN-γ but did not alter the level of IL-2 in the PP cells. In an ex vivo experiment using PP cells removed from the mice after oral treatment of PTCF (10 and 100 mg/kg daily for 7 consecutive days), the production of IL-2 and IFN-γ were increased in response to Con A. On the other hand, orally treated PCCF (10 mg/kg/day) suppressed IL-2 production but did not change the levels of IFN-γ and IL-10 in the isolated PP cells. The flow cytometric analysis revealed that the population of CD3 + cells in the PP cells slightly but significantly increased after oral administration of PCCF. Orally administered PTCF did not change the population of T (CD3 +), B (CD19 +), T cell subset (CD4 +and CD8 +) and Th1 (IFN-γ +) and Th2 (IL-4 +). From PTCF, the fraction rich in proteoglycans was separated as active fraction that stimulates Th1 immune response. These results indicate that the mode of action of PTCF and PCCF on mucosal immune response is different and this is contributed to their metabolites. Taken together, there is a possibility of PTCF and PCCF being therapeutic or preventive agents for immune diseases such as cancer, allergy and parasitic disease through activation of mucosal immune response.

Sulphostin, a Novel Inhibitor of Dipeptidyl Peptidases IV (DPPIV) That Stimulates Hematopoiesis in Mice

CD26, a membrane-bound ectopeptidase, is known as an activated T cell marker with dipeptidyl peptidase IV (DPPIV) activity that has diverse functional roles in the regulation of peptide hormones, neuropeptides, chemokines and growth factors. We recently isolated a novel inhibitor of DPPIV, sulphostin, from culture broth of Streptomyces sp. MK251-43F3. We investigated herein the hematopoietic effect of sulphostin in mice and found that sulphostin induced the production of granulocyte colony-stimulating factor (G-CSF), stimulated myeloblasts in bone marrow, and increased neutrophil numbers in peripheral blood in both normal mice and mice with cyclophosphamide-induced leucopenia. Sulphostin desulfonate, in addition to sulphostin, has a similar inhibitory effect on DPPIV and stimulatory effect on neutrophils. These results suggest that DPPIV/CD26 might be a novel target for hematopoietic stimulation and DPPIV inhibitors including sulphostin and derivatives may be candidates for further development.