In vivo assessment of the antimalarial and spleen-protective activities of the Saudi propolis methanolic extract.

Antimalarial drug resistance is the main therapeutic challenge to the control of the disease, making the search for new compounds as alternative treatments of central importance. Propolis has a long history of medicinal use due to its antifungal, antibacterial and antiprotozoal properties. The present study therefore aimed to evaluate the antimalarial activity of the Saudi propolis methanolic extract against Plasmodium chabaudi infection in mice. To this end, albino mice were divided into five groups: the first group was the normal control; the second, third, fourth and fifth groups were infected intraperitoneally with 106 P. chabaudi-parasitized erythrocytes. The last three groups of mice were gavaged with 100 µl of propolis extract (PE) at a dose of 25, 50 and 100 mg PE/kg, respectively, once daily for 7 days. PE significantly suppressed the parasitaemia and showed significant efficacy in ameliorating anaemic conditions in P. chabaudi-infected mice in a dose-dependent manner. Histological investigation of the spleen tissue of treated and untreated mice further supports the antimalarial potential of PE. In addition, our study proved that Saudi PE reduced oxidative damage by decreasing the malondialdehyde (MDA) and increasing the catalase (CAT) activity and the glutathione (GSH) levels. Also, Saudi PE increased the level of some pro-inflammatory cytokines such as IFN-γ, TNF-α, GM-CSF and G-CSF, with the most effective dose being 100 mg PE/kg. In conclusion, PE showed antimalarial and antioxidant activities and provided protection against spleen tissue damage in P. chabaudi-infected mice.

Cloning and expression of recombinant human GMCSF from Pichia pastoris GS115--a progressive strategy for economic production.

Human granulocyte-macrophage colony-stimulating factor (hGMCSF) is a proinflammatory cytokine and hematopoietic growth factor. Recombinant human granulocyte-macrophage colony-stimulating factor (rhGMCSF) serves as a biotherapeutic agent in bone marrow stimulations, vaccine development, gene therapy approaches, and stem cell mobilization. The objective of the present study includes construction of rhGMCSF having N-terminal intein tag, expression of protein both extracellularly and intracellularly from yeast expression system followed by its purification in a single step by affinity chromatography. The soluble and biologically active rhGMCSF was obtained from Pichia pastoris GS115. About 122 g DCW/L of final yield was obtained for both cytosolic and secretory expression of Pichia GS115 strain. Purified intracellular hGMCSF was 420 mg/L with a specific activity of 2.1×108 IU/mg, and the purified extracellular recombinant protein was 360 mg/L with a specific activity of 1.9×108 IU/mg. The data presented here indicate the possibilities of exploring the economic ways of producing the rhGMCSF.

In vivo characterization of fusion protein comprising of A1 subunit of Shiga toxin and human GM-CSF: Assessment of its immunogenicity and toxicity.

BACKGROUND: Most cancer cells become resistant to anti-cancer agents. In the last few years, a new approach for targeted therapy of human cancer has been developed using immunotoxins which comprise both the cell targeting and the cell killing moieties. METHODS: In the present study, the recombinant Shiga toxin A1 subunit fused to human granulocyte-macrophage colony stimulating factor (A1-GM-CSF), previously produced in E. coli, was further characterized. RESULTS: The recombinant protein could cause 50% cytotoxicity and induced apoptosis in cells bearing GM-CSF receptors. The non-specific toxicity of the fusion protein was assessed in C57BL/6 and BALB/c mice. No mortality was observed in either group of mice, with different concentration of fusion protein. CONCLUSION: The lymphocyte proliferation assay, induction of specific IgG response and a mixed (Th1/Th2) response were observed only in BALB/c mice. The mixed response in BALB/c mice (Th1/Th2) could be explained on the basis of the two components of the fusion protein i.e. A1 and GM-CSF.

Assessment of a combined, adenovirus-mediated oncolytic and immunostimulatory tumor therapy.

In this study, we identified murine breast cancer cell lines that support DNA replication of E1-deleted adenovirus vectors and which can be killed by an oncolytic adenovirus expressing adenovirus E1A and tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) in a replication-dependent manner (Ad.IR-E1A/TRAIL). We showed that systemic or intratumoral (i.t.) injection of adenovirus vectors into mice increases plasma levels of proinflammatory cytokines and chemokines, including TNF-alpha, INF-gamma, and MCP-1, which are potent inducers of dendritic cell maturation. Furthermore, we showed that in vivo expression of Flt3L from an adenovirus vector increases the number of CD11b+ and CD11c+ cells (populations that include dendritic cells) in the blood circulation. Based on these findings, we tested whether Ad.IR-E1A/TRAIL induced killing of tumor cells in combination with dendritic cell mobilization by Ad.Flt3L or, for comparison, Ad.GM-CSF would have an additive antitumor effect. As a model, we used immunocompetent C3H mice with syngeneic s.c. tumors derived from C3L5 cells. We found that vaccination of mice with C3L5 cells that underwent viral oncolysis in combination with Flt3L or granulocyte-macrophage colony-stimulating factor (GM-CSF) expression induces a systemic antitumor immune response. I.t. injection of the oncolytic and Flt3L expressing vectors into established tumors delayed tumor growth but did not cause efficient tumor elimination. This study shows the effectiveness of a combined oncolytic/immunostimulatory tumor therapy approach.

Short description of an alternative simplified method for screening recombinant clones within the "AdEasy-System" by Duplex-PCR.

BACKGROUND: Recombinant adenoviral vectors are highly efficient for in vitro and in vivo gene delivery. They can easily be produced in large numbers, transduce a wide variety of cell types and generate high levels of transgene expression. The AdEasy system is a widely used system for generating recombinant adenoviral vectors, which are created with a minimum of enzymatic manipulations and by employing homologous recombination in E. coli. In this paper we describe an alternative simplified method for screening recombinant DNA within the AdEasy system. This Duplex-PCR-method is independent of the transgene or insert and can be used for the complete AdEasy system. It is characterized by a simple standard protocol and the results can be obtained within a few hours. The PCR is run with two different primer sets. The primers KanaFor and KanaRev hybridizise with the Kanamycin resistence gene and AdFor and AdRev detect the adenoviral backbone. In case of recombinant clones, two diagnostic fragments with a size of 384 bp and 768 bp are generated. RESULTS: The practicability of this method was verified with three different transgenes: Cytosin Deaminase (AdCD), p53 (Adp53) and Granulocyte Macrophage Colony Stimulating Factor (AdGM-CSF). Recombinant clones are indicated by two diagnostic fragments and are then suitable for further processing. CONCLUSION: In summary, the presented protocol allows fast detection of recombinants with an easy technique by minimizing the amount of necessary steps for generating a recombinant adenovirus. This method is time sparing and cost-effective.

Endocytic internalization of adenovirus, nonspecific phagocytosis, and cytoskeletal organization are coordinately regulated in alveolar macrophages by GM-CSF and PU.1.

GM-CSF gene-targeted (GM(-/-)) mice have impaired pulmonary clearance of bacterial and fungal pathogens by alveolar macrophages (AMs). Because AMs also clear adenovirus from the lung, the role of GM-CSF in endocytic internalization of adenovirus by AMs was evaluated. Pulmonary clearance of adenovirus was severely impaired in GM(-/-) mice compared to wild-type (GM(+/+)) mice as determined by Southern analysis of viral DNA. Internalization of adenovirus by AMs was deficient in GM(-/-) mice in vivo and in vitro as determined by uptake of fluorescently labeled adenovirus or by PCR quantification of adenoviral DNA internalized within AMs. An AM cell line previously established from GM(-/-) mice (mAM) had impaired internalization of adenovirus and transferrin-coated 100-nm latex beads compared to MH-S, a GM(+/+) AM cell line. Phagocytosis of 4- micro m latex beads was also impaired in mAM cells as determined by confocal and fluorescence microscopy. Retroviral vector-mediated reconstitution of PU.1 expression in cultured GM(-/-) AMs restored phagocytosis of 4- micro m beads, endocytosis of adenovirus, and transferrin-coated 100-nm beads (independent of integrin alpha(V) and transferrin receptors, respectively), and restored normal cytoskeletal organization, filamentous actin distribution, and stimulated formation of filopodia. Interestingly, mRNA for the phosphoinositide 3 kinase p110gamma isoform, important in macrophage phagocytic function, was absent in GM(-/-) AMs and was restored by PU.1 expression. These data show that GM-CSF, via PU.1, regulates endocytosis of small ( approximately 100 nm) pathogens/inert particles and phagocytosis of very large inert particles and suggests regulation of cytoskeletal organization by GM-CSF/PU.1 as the molecular basis of this control.

Assessment of G-CSF and GM-CSF mRNA expression in peripheral blood mononuclear cells from patients with severe congenital neutropenia and in human myeloid leukemic cell lines.

Patients with severe congenital neutropenia (SCN), also called Kostmann syndrome, are unable to generate sufficient peripheral blood granulocytes owing to an arrest of myeloid differentiation at the level of promyelocytes. Similarly, myeloid leukemic cells show a maturation arrest at different stages of myeloid maturation coupled with uncontrolled proliferation. Among other cells, defective production of or defective response to granulocyte/macrophage colony-stimulating factor (GM-CSF) or granulocyte CSF (G-CSF) might be involved in the pathophysiology of these disorders of hematopoiesis. Reverse transcription of messenger RNA and subsequent specific amplification by the polymerase chain reaction (RT-PCR) served as a sensitive technique to detect G-CSF and GM-CSF gene expression. We have tested two alternative assays for the specific quantitation of transcript levels for G-CSF. Applying one assay we could demonstrate that: 1) peripheral blood monocytes from 5 patients with SCN are able to express G-CSF and GM-CSF messenger RNA, suggesting that defective production of these factors is not responsible for the neutropenia in this condition; 2) messenger RNA levels from 5 SCN patients were on average higher than the levels determined for three healthy volunteers; 3) 7 of 9 of the examined myeloid cell lines express GM-CSF and all of them G-CSF mRNA. These results show that quantitative PCR techniques can be used as simple tools to elucidate aspects of the pathophysiology of hematologic disorders concerning the production of CSFs.