Crude Heparin Preparations Unveil the Presence of Structurally Diverse Oversulfated Contaminants

Nowadays, pharmaceutical heparin is purified from porcine and bovine intestinal mucosa. In the past decade there has been an ongoing concern about the safety of heparin, since in 2008, adverse effects associated with the presence of an oversulfated chondroitin sulfate (OSCS) were observed in preparations of pharmaceutical porcine heparin, which led to the death of patients, causing a global public health crisis. However, it has not been clarified whether OSCS has been added to the purified heparin preparation, or whether it has already been introduced during the production of the raw heparin. Using a combination of different analytical methods, we investigate both crude and final heparin products and we are able to demonstrate that the sulfated contaminants are intentionally introduced in the initial steps of heparin preparation. Furthermore, the results show that the oversulfated compounds are not structurally homogeneous. In addition, we show that these contaminants are able to bind to cells in using well known heparin binding sites. Together, the data highlights the importance of heparin quality control even at the initial stages of its production

Modulation by Acanthospermum australe extracts of the tumor induced hematopoietic changes in mice

Previous studies on the Ehrlich ascites tumor (EAT) model indicate that tumor progression is associated with reduced myelopoiesis and increased extramedullar hematopoiesis. In order to investigate the in vivo antitumor activity of Acanthospermum australe, its hydroalcoholic extract was partitioned with different solvents and the resulting extracts were monitored by their effects on bone marrow and spleen hematopoietic progenitor cell proliferation and differentiation in EAT-bearing mice. Oral treatment of tumor-bearing mice with 3 doses of 100, 500 and 1000 mg/kg of the crude hydroalcoholic extract and its chloroformic, butanolic and aqueous fractions significantly stimulated myelepoiesis and brought extramedullar hematopoiesis back to near control values. In normal mice, stimulation of myelopoiesis was only observed with the crude and the butanolic extracts. All the extracts at 500 mg/kg significantly increased survival of tumor-bearing mice, however a clear survival advantage in the group treated with the butanolic extract was observed. These results suggest that A. australe may exert effects on myelopoiesis that may be implicated in antitumor immune responses.24227528

Myelopoietic response in tumour-bearing mice by an aggregated polymer isolated from Aspergillus oryzae

The effects of magnesium ammonium phospholinoleate-palmitoleate anhydride (MAPA), a proteic aggregated polymer isolated from Aspergillus oryzae, on the growth and differentiation of granulocyte-macrophage progenitor cells (colony-forming unit-granulocyte-macrophage [CFU-GM]) in normal and Ehrlich ascites tumour-bearing mice were studied. Myelosuppression concomitant with increased numbers of spleen CFU-GM was observed in tumour-bearing mice. Treatment of these animals with MAPA (0.5 - 10 mg/kg) stimulated marrow myelopoiesis in a dose-dependent manner and reduced spleen colony formation. No changes were observed in total and differential marrow cell counts. The dose of 5.0 mg/kg MAPA, given prior or after tumour inoculation, was the optimal biologically active dose in tumour-bearing mice and this dose schedule also stimulated myelopoiesis in normal mice. MAPA significantly enhanced survival and concurrently reduced tumour growth in the peritoneal cavity. We propose that the modulatory effect of MAPA on the myelopoietic response may be related to its antitumour activity as a possible mechanism for regulation of granulocyte-macrophage production and expression of functional activities. (C) 2000 Elsevier Science B.V. All rights reserved.388321922

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.23111913

Stimulation of myelopoiesis in Listeria monocytogenes-infected mice by an aggregated polymer isolated from Aspergillus oryzae

In this work, we investigated the effects of the proteic aggregated polymer of magnesium ammonium phospholinoleate-palmitoleate anhydride (MAPA) isolated from Aspergillus oryzae on the growth and differentiation of bone marrow granulocyte-macrophage progenitor cells (CFU-GM) in Listeria monocytogenes-infected mice. A significant reduction in the CFU-GM number was observed in the initial phase of infection with a sublethal dose of Listeria. Treatment of mice with 0.5, 2.0 and 5.0 mg/kg MAPA for 7 days prior to infection significantly stimulated myelopoiesis in a dose-dependent manner. Moreover, treatment with 0.5 and 5.0 mg/kg MAPA resulted in 30% and 40% cures of mice lethally infected with Listeria, respectively. MAPA added directly to the culture dishes hardly affected colony formation by bone marrow cells, suggesting an indirect effect of this compound on myelopoiesis in vivo. In summary, the data show that MAPA can modulate the CFU-GM generation and antibacterial resistance in listeriosis. As the ability of hematopoietic tissues to produce phagocytes is of particular significance to mediate resistance to Listeria, the promotion of bone marrow CFU-GM by MAPA may contribute to a rapid restoration of phagocyte numbers in infected sites, thus mitigating the course of infection.201384

Natural killer cell activity, lymphocyte proliferation, and cytokine profile in tumor-bearing mice treated with MAPA, a magnesium aggregated polymer from Aspergillus oryzae

The present study examined the effects of MAPA, an antitumor aggregated polymer of protein magnesium ammonium phospholinoleate-palmitoleate anhydride, isolated from Aspergillus oryzae, on concanavalin A (Con A)-induced spleen cell proliferation, cytokine production and on natural killer (NK) cell activity in Ehrlich ascites tumor-bearing mice. The Ehrlich ascites tumor (EAT) growth led to diminished mitogen-induced expansion of spleen cell populations and total NK activity. This was accompanied by striking spleen enlargement, with a marked increase in total cell counts. Moreover, a substantial enhancement in IL-10 levels, paralleled by a significant decrease in IL-2 was observed, while production of IL-4 and interferon-gamma (IFN-gamma) was not altered. Treatment of mice with 5 mg/kg MAPA for 7 days promoted spleen cell proliferation, IL-2 production and NK cell activity regardless of tumor outgrowth. In addition, MAPA treatment markedly enhanced IFN-gamma levels and reduced IL-10 production relative to EAT mice. A 35% reduction in splenomegaly with normal number of nucleated cells was also found. Altogether, our results suggest that MAPA directly and/or indirectly modulates immune cell activity, and probably disengages tumor-induced suppression of these responses. Clearly, MAPA has an impact and may delay tumor outgrowth through immunotherapeutic mechanisms.25330531

Natural killer cell activity and anti-tumour effects of dehydrocrotonin and its synthetic derivatives

In this work, the anti-tumour properties of dehydrocrotonin and its derivatives were investigated in vitro and in vivo using the Ehrlich ascites tumour model. Treatment of Ehrlich ascites tumour-bearing mice with 20 mg/kg dehydrocrotonin for 4 days significantly increased survival, whereas administration of dehydrocrotonin derivatives was ineffective in affording protection. Compound IV exhibited little activity against Ehrlich tumour cells in vitro. Investigation of the effects of dehydrocrotonin treatment on total natural killer (NK) cell activity of tumour-bearing mice as a possible mechanism of dehydrocrotonin action in vivo revealed that this sesquiterpene lactone significantly improved NK cytotoxicity against YAC-1, a Moloney virus-induced mouse T-cell lymphoma of A/SN origin. As expected, tumour growth in non-treated mice markedly suppressed NK cell cytolysis. No effects on NK functional activity were observed in normal mice receiving dehydrocrotonin. In summary, only the natural compound exhibits anti-tumour efficacy and immunomodulatory actions in vivo, which may be related to its chemical structure. (C) 2004 Elsevier B.V. All rights reserved.48741699475

Myelopoietic response in mice exposed to acute cold/restraint stress: Modulation by Chlorella vulgaris prophylactic treatment

In this study, hematopoietic cells from mice pretreated with CVE and exposed to acute cold/restraint stress were stimulated in the presence of growth factors to form colonies, thus providing accurate information about the modulation of the green algae of the stress-induced changes in the hematopoietic response. Our results demonstrated that exposure to acute stress affected hematopoiesis. Mice exposed for a 2.5-hour time period of cold and restraint presented diminished clonal capacity for CFU-GM content per femur, which was decreased by as much as 50% compared with that in control mice, in spite of the significant increase in serum colony-stimulating activity (CSA). Treatment with 50 mg/kg CVE for 5 days, previously to the stress regimen, attenuates the effects of the stress, since comparable levels of myeloid progenitors were found in the bone marrow of both CVE/stress and control mice. Moreover, the sera from stressed mice pretreated with CVE further increased the CFU-GM formation. On the contrary, the spleen seemed to be less sensitive to acute stress in our experimental conditions. These findings are in line with our previous reports showing that the stress-induced reduction in bone marrow CFU-GM of rats exposed to electric shocks is mediated by activation of the HPA axis and by secretion of opioid agonists.([14,15]) No changes were observed in bone marrow, spleen and thymus total cell counts, and in relative organ weights. However, a 50% reduction in the body weight loss produced by the stress was observed in mice given the extract.26345546

Cytotoxicity of materials used in perforation repair tested using the V79 fibroblast cell line and the granulocyte-macrophage progenitor cells

Aim To compare the cytotoxicity of materials used to repair perforations using permanent V79 fibroblasts and murine granulocyte-macrophage progenitor cells (CFU-GM). Methodology Set specimens from amalgam, glass-ionomer, SuperEBA, N-Rickert, MTA and gutta-percha were eluted with culture medium for 72 h and their cytotoxicities were assessed by incubating the extracts with V79 and bone marrow-derived progenitors for 24 h and 7 days, respectively. Cytotoxicity on V79 cells was judged using the total nucleic acid content (NAC), neutral red uptake (NRU) and reduction of the tetrazolium salt (MTT). The number of bone marrow CFU-GM colonies determined in clonal cultures stimulated with recombinant murine granulocyte-macrophage colony-stimulating factor was used to assess cytotoxicity to progenitor cells. Statistical analyses were conducted using the one-way analysis of variance and Tukey's test where appropriate. Results All materials were cytotoxic in both cell systems; however, CFU-GM was more sensitive to the extracts than V79 cells. A similar rank order of toxicity was observed in V79 cells using the NAC and the MTT assays: glass-ionomer > N-Rickert congruent to SuperEBA > gutta-percha > amalgam congruent to MTA (P < 0.05). In contrast, the NRU test exhibited a lower sensitivity to MTA, gutta-percha and amalgam extracts. In the clonal culture assay, the toxicity was less pronounced in the presence of gutta-percha, SuperEBA and MTA. Similar cellular responses were found by placing the set specimens directly in the clonal culture dishes. Conclusions The sensitivity of toxicity depended on the choice of the endpoint and the cell-culture system. Nevertheless, MTA was ranked as the least cytotoxic cement in both cell systems.391404

Biochemical characterization of a Kunitz type inhibitor similar to dendrotoxins produced by Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) hemocytes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)A novel chymotrypsin inhibitor identified in fat body and hemocyte cDNA libraries of Boophilus microplus was named BmCI (B. microplus Chymotrypsin Inhibitor) (Genbank EU636772). The putative BmCI amino acid sequence presented a 22-residue-signal peptide and 58-residue-mature protein. BmCI amino acid sequence analysis allowed its classification as a Kunitz-BPTI inhibitor with six cysteine residues, a theoretical pI of 7.8, and the presence of Tyr at P1 position in the putative reactive site, suggesting inhibitory activity toward chymotrypsin. In this work, we reported the biochemical characterization of BmCI. The recombinant BmCI expressed in yeast Pichia pastoris was purified by size exclusion and reverse phase chromatographies. rBmCI expression yield was of I mg L(-1) of culture. Purified rBmCI confirmed its chymotrypsin inhibitory activity with a low K(i) (6.2 pM). The BmCI gene expression analysis by semi-quantitative RT-PCR indicated its transcription in the hemocytes, salivary gland and ovary. The cytotoxic activity of purified rBmCI was demonstrated in BALB/c 3T3 mouse fibroblasts. As assessed by the MTT reduction assay, rBMCI induced a dose-dependent decrease in 3T3 fibroblasts viability (IC(50) = 8 mu M). Moreover, flow cytometry analysis revealed that rBmCI is able to induce apoptosis, whereas no effect was observed on cell cycle progression. In conclusion, we demonstrated that rBmCI is cytotoxic against mammalian cells and obtained evidence that this growth inhibition is caused by an apoptosis-inducing activity. (C) 2009 Published by Elsevier B.V.16741731SI279287Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)FAPESP [05/03514-9, 06/52206-8