Artemisinin as a Chinese medicine, selectively induces apoptosis in pancreatic tumor cell line / 中国结合医学杂志

<p><b>OBJECTIVE</b>To investigate the possible mechanism through which Artemisinin induced apoptosis in pancreatic cell line.</p><p><b>METHODS</b>Column chromatography, thin layer chromatography (TLC) and proton NMR spectroscopy were used to purify Artemisinin. The flowcytometry was employed to detect apoptosis and reactive oxygen species (ROS).</p><p><b>RESULTS</b>The results indicated that 50% inhibiting concentration (IC50 value) for pancreatic cell line (RIN) was 45 μmol/L of Artemisinin. Artemisinin had no cytotoxic effect on the growth of peripheral blood lymphocytes. The mechanism of apoptosis was evaluated by measuring intracellular ROS. It was shown that Artemisinin-induced apoptosis occurred independently of the binding of CD95L to CD95 receptor in the RIN cells. Moreover, Artemisinin, in a dose-dependent manner, could significantly increase the level of ROS.</p><p><b>CONCLUSION</b>Artemisinin can induce apoptosis in the RIN cells via the generation of ROS and triggering the intrinsic pathway of cell death.</p>

Cloning, expression, purification and characterization of recorabinant UreB[229-561] from Helicobacter pylori

Helicobacter pylori is a widely distributed gram negative bacterium that infects the human stomach and duodenum. Some antibiotic regimens are subjected to cure the infection but the cost of drugs, poor patient compliance and emerging of antibiotic-resistant strains are limiting the usefulness of these antibiotic therapies. Therefore, interest in a H. pylori vaccine is growing up rapidly. We selected a fragment of B subunit of H. pylori urease enzyme consist of four important epitopes, involving in elevating host immune responses. This 1070bp fragment was amplified by PCR from genomic DNA isolated from H. pylori 22596 and then cloned into the pET28a expression vector. UreB229-561 was expressed and then affinity-purified by Ni2+-Sepharose resin. The recombinant UreB229-561 was reacted with the serum of/. pylori-infected human and rabbit anti-H. pylori polyclonal antibody in western-blot analysis. Having transformed competent E.coli DH5 alpha with ligation product of digested ureB fragment and pET28a, plasmid extraction from single colonies appeared in LB-agar plate after 18-24 h incubation at 37°C, using plasmid extraction kit [Bioneer, Korea]. Applying both infected human serum and rabbit anti-H. pylori polyclonal antibody, brown strip corresponding to the location of the recombinant protein appeared on PVDF membrane after adding DAB solution, hence confirming the antigenicity of the protein. This recombinant fragment showed urease activity. Our findings confirmed that a prokaryotic expression system of rUreB[229-561] was successfully constructed. The results of SDS-PAGE showed that our constructed prokaryotic expression system pET28 alpha- ureB[229-561]-BL21DE3 efficiently produces target recombinant protein in the form of dissoluble inclusion body. Therefore we can suggest that these epitopes can effectively be a vaccine candidate

Tehranolide could shift the immune response towards Th1 and modulate the intra-tumor infiltrated T regulatory cells

Artemisia diffusa contains a new type of sesquiterpene lactone with an endoperoxide group [Tehranolide]. Due to the existing similarity between the structures of Tehranolide and Artemisinin, it was hypothesized that Tehranolide would have similar effects as Artemisinin. In this study, the immunotherapeutic effectiveness of Tehranolide was investigated by direct intra-tumoral injection. Tehranolide was purified from Artemisia diffusa, and its effect on the tumor volume was investigated. The splenocyte proliferation, shifting of cytokine profile, and the presence of naturally-occurring CD4+CD25+Foxp3+ Treg cells were assessed to describe the anti-tumor immune response. Analysis of immune response showed that, intra-tumoral injection of Tehranolide decreased the rate of tumor growth compared to control group. Furthermore, the proliferative response of mice treated with Tehranolide was enhanced. In comparison with the control group, production of both IL-4 and IFN-gamma was induced [p<0.05]. The results indicated a decrease in tumor CD4+CD25+Foxp3+ T lymphocytes in the Tehranolide-treated group compared to the control group. Treatment of tumors with Tehranolide attenuated CD4+CD25+Foxp3+ Treg cell-mediated immune suppression and elicited a persistent anti-tumor immunity against cancer

Purified aged garlic extract modulates allergic airway inflammation in Balb/c Mice

Garlic is known as a potent spice and a medicinal herb with broad therapeutic properties ranging from antibacterial to anticancer and anticoagulant. Our previous studies have shown some immunoregulatory effects for aged garlic extract, suggesting a key role for 14-kD glycoprotein of garlic in shifting the cytokine pattern to T helper-1. In present study, we investigated the effect of 1, 2, and 3 times intraperitoneal injections of aged garlic extract on an established allergic airway inflammation in murine model [BALB/c mice]. The garlic extract, isolated by biochemical method, includes proteins precipitation by ammonium sulfate. After injection of the aged garlic extract, IFN-gamma, anti allergen specific IgE and IgG1 were measured in lavage and serum by ELISA and histological assessment was performed on the lung tissues. The results indicated that three-time intra peritoneal injections of the aged garlic extract caused a significant decrease in the hallmark criteria of allergic airway inflammation levels which included eosinophil percentage in lavage, peribronchial lung eosinophils, IgG1 level in lavage and serum, mucous producing goblet cells grade and peribronchial and perivascular inflammation. Our findings in the present research suggested that aged garlic extract has the potential of attenuation of inflammatory features of allergic airway inflammation in murine model

Humoral and cellular immunomodulation induced by propoxure in C57-w/6 mice

Propoxure [PPX] is a well-known carbamate insecticide, which has been used for several decades in the world and Iran in agriculture and public health programs. However, there is no clear investigation toward its immunotoxicity as yet. In this study, we examined the effects of subchronic i.p. exposure of PPX on humoral [PFC and HA] and cellular [DTH] responses, and also monitored T-Cell subtypes using FACS technique. Briefly, female C57b1/6 inbred mice were administered PPX [0.2, 2 and 10 mg/kg/day i.p. [5 inj/wk] for 28 days] or positive and negative controls. On the day 28, mice were examined for DTH, PFC and HA responses to SRBC. Splenocyte single cell suspension was used for measuring the spleen CD4/CD8 percentage and absolute number. In vitro lymphocyte proliferation response to non-specific antigen [PHA] was also measured using MTT method. Results showed that PPX at 10 mg/kg/day could suppress DTH response and could increase the spleen CD4-/CD8+ T-cell percentage. On the other hand, PPX at medium dose [2 mg/kg] could increase the antibody formation response against SRBC as determined by PFC and HA. Subchronic PPX at low dose [0.2 mg/kg/day] could not show any significant effects on humoral or cellular responses. It could be concluded that, subchronic PPX at high dose [10 mg/kg], possessed cellular immunosuppressive effect. However, PPX at 2 mg/kg does not change cellular response to antigen but can stimulate humoral responses. It seems that PPX has no adverse effects on mice immune system at low doses as 0.2 mg/kg, which is 10 fold greater than PPX Allowed Daily Intake limit

Effect of immunomodulator pyrimethamine and cimetidine on immunosuppression induced by burn blister fluid

Despite recent advances in burn wound management, sepsis remains the main cause of death in patients resuscitated after major thermal injury. Increased susceptibility to infections has been related to severe suppression of the immune system. The aim of this study was to induce immune suppression with blister fluid injection, and to modulate immune response by use of cimetidine and pyrimethamine in animal model. Male Balb/c mice were injected with blister fluid intrapritoneally [ip]. Fluids were collected from parital-thickness burn blisters and then the delayed type hypersensitivity [DTH] to sheep red blood cell [SRBC] and the effects of different doses of immunomodulators [Cimetidine and Pyrimethamine] on this response were quantitated. A marked suppression of DTH was observed in mice injected with blister fluid. Pyrimethamine and Cimetidine at all three doses caused a significant enhancement of DTH response to SRBC compared with blister fluid injected in control group. This finding represents evidence of a host defense defect within the burn wound and also indicates the blister fluid exhibit immunosuppressor factor that can modulate with immunomadulatory drugs like cimetidine and pyrimethamine

Extraction of the outer membrane proteins of H. pylori and evaluation of their presence in stool of the infected individuals

Helicobacter pylori infections are accompanied with the release of some antigenic material into the stool. Outer membranes proteins [OMP] are among the major antigens of bacteria, which in comparison with other bacterial constituents have less cross-reactivity. In this study, OMP of H. pylori were isolated from 11 clinical isolates and rabbit antiserum against them was used to detect possible antigens of H. pylori, which are released into the stool. We used immunoblotting and affinity chromatography to detect such antigens in fecal antigenic extracts of infected individuals. By immunoblotting, we were able to detect a 26 kDa band under reducing and non-reducing conditions, but many more antigens [at least 5 antigens with molecular weights of about 14, 26, 52, 57.5 and 66 kDa] were isolated by affinity chromatography. The 26 kDa antigen had a higher concentration and is seen in nearly all positive samples. Since the 26 kDa antigen is detectable by these two techniques, we suggest that this antigen is one of the major antigens of H. pylori which is released into the stool and can be considered as a candidate diagnostic antigen to be used in diagnostic kit development