Leukemia Inhibitory Factor Suppresses NKG2D mRNA Expression and Presentation on Human Natural Killer Cells.

Leukemia inhibitory factor (LIF) is a multi-functional cytokine secreted from cells such as lymphocytes and hepatocytes. This study aimed to evaluate the effect of LIF on natural killer group 2 member D (NKG2D) receptors' expression and presentation on natural killer (NK) cells.  For this purpose, peripheral blood mononuclear cells taken from 4 young male healthy blood donors were isolated and the effect of LIF (25 ng/mL) after 12, 24, and 48 hours of incubation, on NKG2D receptors expression and presentation was investigated using flow cytometry and real-time-polymerase chain reaction (PCR). All of the steps of the experiment were performed in duplicate. After periods of 12, 24, and 48 hours, LIF reduced both the expression and presentation of the NKG2D receptor on NK cells. The results suggest that this cytokine has a direct modulating activity on the body's immune response through suppression of NKG2D receptor expression and presentation on NK cells.

A. crassicauda, M. eupeus and H. lepturus scorpion venoms initiate a strong in vivo anticancer immune response in CT26-tumor mice model.

In the present in vivo study the anticancer efficacy of the venoms from Androctonus crassicauda, Messobuthus eupeus and Hemiscorpius lepturus scorpions was investigated. In addition, we attempted to clarify whether the immune system is involved in this activity. Initially, the LD50 of the venoms from these scorpions were determined and their 0.1 and 0.2 LD50 were calculated. The toxicity of 0.1 and 0.2 LD50 was tested on healthy mice by daily SC administration of these venoms for 12 consecutive days. CT26 cells were inoculated by SC route in BALB/c mice to establish a sold tumor, and ten days later, the mice were treated with 0.1 and 0.2 LD50 doses of the venoms on daily basis for 12 consecutive days. The tumor volume was measured every 4 days. At day 13, the tumors from untreated-control and venom-treated groups were removed, weighed, and assessed by histopathological and immunohistochemical techniques. In addition, the levels of mRNA expression of IL-12, IFN-γ and IL-1ß were measured by real-time PCR. All the venoms induced anticancer effects as evidenced by significant inhibition in tumor growth; significant increases in inflammatory and CD+-T cells and expression of mRNA IL-12 and IFN-γ in tumor microenvironment of venom-treated as compared to untreated-control. These findings demonstrated, for the first time, that sub-lethal doses of the venoms from these scorpions induce their in vivo anticancer effects by stimulating the immune system. Further studies, specifically designed to identify these active constituents are recommended.

3-Bromopyruvate potentiates TRAIL-induced apoptosis in human colon cancer cells through a reactive oxygen species- and caspase-dependent mitochondrial pathway.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a promising anticancer cytokine with minimal toxicity towards normal cells. Nevertheless, most primary cancers are often intrinsically TRAIL-resistant or can acquire resistance after TRAIL therapy. This study aimed to investigate the inhibitory effect of co-treatment of 3-bromopyruvate (3-BP) as a potent anticancer agent with TRAIL on colon cancer cells (HT-29). The results of present study indicated that combined treatment with 3-BP and TRAIL inhibited the proliferation of HT-29 cells to a greater extent (88.4%) compared with 3-BP (54%) or TRAIL (11%) treatment alone. In contrast, the combination of 3-BP and TRAIL had no significant inhibitory effect on the proliferation of normal cells (HEK-293) (8.4%). At a cellular mechanistic level, the present study showed that 3-BP sensitized human colon cancer cells to TRAIL-induced apoptosis via reactive oxygen species generation, upregulation of Bax, downregulation of Bcl-2 and survivin, release of cytochrome c into the cytosol, and activation of caspase-3. In normal cells, 3-BP, TRAIL, or combination of both had no significant effect on the reactive oxygen species levels, release of cytochrome c, and caspase-3 activity. Therefore, the combination of 3-BP and TRAIL can be a promising therapeutic strategy for treatment of colon cancer.

Induction of IL-12 from human monocytes after stimulation with Androctonus crassicauda scorpion venom.

The objective of this study was to evaluate the capacity of venom from Androctonus crassicauda to induce expression/production of interleukin (IL)-12 by isolated human monocytes. For this purpose, isolated human monocytes were exposed to different concentrations of the venom (0.16-20 µg/ml) for varying periods (6, 12, and 24 h). Apart from measures of venom cytotoxicity (i.e., lactase dehydrogenase activity [LDH] release), measures of IL-12 p40 mRNA (by Real-time PCR) of IL-12 release (by ELISA) were performed. The results showed that the venom produced significant concentration- and duration of incubation-dependent cytotoxicity. Expression of IL-12 p40 mRNA was significantly increased at all exposure timepoints relative to that in unexposed cells, but was maximal after 6 h of exposure. At that timepoint, the effect from a dose of 2.5 µg venom/ml provided the maximal increase among all doses tested. At the level of the protein itself, IL-12 production remained almost consistently elevated (vs. unexposed control values) across all exposure timepoints, with the greatest formation again occurring after 6 h of incubation at a dose of 2.5 µg venom/ml. The findings from this study demonstrated that venom from the A. crassicauda scorpion contained active constituents that could induce a sustained activation of human monocytes that was manifested, in part, as promotion of the expression/production of IL-12.

Hemiscorpius lepturus venom induces expression and production of interluckin-12 in human monocytes.

The objective of this study was to evaluate the capacity of the venom from Hemiscorpius lepturus to induce expression and production of interleukin-12 (IL-12) on isolated human monocytes. For this purpose, isolated human monocytes (250,000-300,000 cells/ml) were exposed to different concentrations of the venom (0.625, 1.25, 2.5, 5, 10 and 20 µg/ml) in 96-well plates for varying incubation periods (6, 12, and 24 h). The end point of assessment included LDH cytotoxicity assay, measurement of expression of IL-12,p40 mRNA by real-time PCR, and quantification of IL-12 release using sandwich ELISA technique. The results showed that this venom produced concentration- and time of incubation-dependent cytotoxicity. The level of enhancement of expression and production of IL-12 were found significantly higher with lowest concentration and after 6 h of incubation. The findings demonstrated that the venom from this scorpion contains active constituents which can direct the immune system to produce IL-12.

A forward to optimization of antivenom therapy: An in vivo study upon the effectiveness of the antivenom against early and delayed nephrotoxicity induced by the venom of the Iranian scorpion Hemiscorpius lepturus in rat.

The aim of the present in vivo study was to identify the optimal effective dose, the most favorable time and the route of administration of the available polyvalent scorpion antivenom against the toxic effects induced by Hemiscorpius lepturus (H. lepturus) venom in rat. The end point for assessment included measurement of alanin-amino-peptidase (AAP) and N-acetyl-b-d-glucosaminidase (NAG), biochemical urine analysis and histopathological assessment. The results showed that a single subcutaneous 50 µg of the venom produced significant increase in the AAP and NAG enzyme activity, urinary biochemical parameters and induced histopathological structural abnormalities in the renal system. The optimal effective co-administered dose of the antivenom was 0.5 ml, which when administered 1 and 2 h of envenomation by intravenous (IV) and subcutaneous (SC) routes respectively produced significant protection against these toxic effects. Prudently, the significance of these findings need to be assessed in further clinical studies.

Assessment of immunogenic characteristics of Hemiscorpius lepturus venom and its cross-reactivity with venoms from Androctonus crassicauda and Mesobuthus eupeus.

Hemiscorpius lepturus (H. lepturus), one of the most venomous scorpions in tropical and sub-tropical areas, belongs to the Hemiscorpiidae family. Studies of antibodies in sera against the protein component of the venom from this organism can be of great use for the development of engineered variants of proteins for eventual use in the diagnosis/treatment of, and prevention of reactions to, stings. In the present in vitro study, the proteins of H. lepturus venom, which could specifically activate the production of immunoglobulin G (IgG) in victims accidently exposed to the venom from this scorpion, were evaluated and their cross-reactivity with venoms from two other important scorpion species including Androctonus crassicauda and Mesobuthus eupeus assessed. H. lepturus venom was analyzed with respect to its protein composition and its antigenic properties against antibodies found in sera collected from victims exposed to the venom of this scorpion within a previous 2-month period. The cross-reactivity of the H. lepturus venom with those from A. crassicauda and M. eupeus was assessed using ELISA and immunoblotting. Electrophoretic analysis of the venom of H. lepturus revealed several protein bands with weights of 8-116 KDa. The most frequent IgG-reactive bands in the test sera had weights of 34, 50, and 116 kDa. A weak cross-reactivity H. lepturus of venom with venoms from A. crassicauda and M. eupeus was detected. The results of immunoblotting and ELISA experiments revealed that H. lepturus venom activated the host immune response, leading to the production of a high titer of antibodies. Clearly, a determination of the major immunogenic components of H. lepturus venom could be valuable for future studies and ultimately of great importance for the potential production of recombinant or hypo-venom variants of these proteins.

Shallominthe active antimicrobial constituent of persian shallot in treatment of oral herpes: a double-blind randomized clinical trial.

BACKGROUND: Previous studies showed that shallomin, the active antimicrobial constituent of Persian shallot, has a wide range of antibacterial and antifungal properties. OBJECTIVES: The objective of this randomized clinical trial was to evaluate the effectiveness of topical shallomin alcoholic solution in treatment of cold sore. PATIENTS AND METHODS: A total of 60 volunteers who met the inclusion criteria were randomly allocated to two equal groups to hourly apply topical of either 0.5% shallomin alcoholic solution or placebo within the first 24 hours of developing cold sores. All the cases were reassessed at six-hour intervals. RESULTS: The cold sores were cleared within six hours among 30% of cases who received shallomin solution and the remaining of the cases in this group were cleared between 6six to 24 hours of application. In the placebo group, clearance of the sores occurred in four cases between 48 to 72 hours and the remaining of cases were cleared after 72 hours. CONCLUSIONS: The results of this study demonstrated that shallomin is a useful natural remedy in preventing the progression and treatment of cold sores and can significantly reduce the duration of ulceration.

An in vitro comparative study upon the toxic properties of the venoms from Hemiscorpius lepturus, Androctonus crassicauda and Mesobuthus eupeus scorpions.

The aim of the present study was to compare the toxic effects of the venoms from Hemiscorpius lepturus (H. lepturus), Androctonus crassicauda (A. crassicauda) and Mesobuthus eupeus (M. eupeus). For this purpose, three in vitro models were employed to compare the toxic effects of various concentrations of the venoms from these three scorpions, namely: hemolytic potential using human RBCs, phospholipase activity using Saubouraud's dextrose agar (SDA) supplemented with 2% egg yolk and lactate dehydrogenase (LDH) enzyme releasing effect using K562 leukemia cell line. In addition, the neutralizing effectiveness of the antivenom against these toxic properties was assessed. The results showed that, unlike the venoms from A. crassicauda and M. eupeus, the venom from H. lepturus produced dose-dependent lysis of human RBCs and showed phospholipase activity. However, all the tested venoms showed variable degrees of LDH releasing properties. The venom from H. lepturus had highest and the venom from M. eupeus had the lowest LDH releasing effect. The antivenom effectively inhibited all the tested toxicities. In conclusion, these results suggest that the venoms from the studied scorpions have variable toxic properties, which may explain the underlying reason for the differences in their clinical manifestations. In addition, the antivenom was effective in neutralizing all the tested toxic effects.

An In Vivo Toxicological Study Upon Shallomin, the Active Antimicrobial Constitute of Persian Shallot (Allium hirtifolium, Boiss) Extract.

BACKGROUND: Previous studies have shown that shallomin, one of the active constituents of Persian shallot, has a broad range of antimicrobial properties. OBJECTIVES: The safety of shallomin must be established before it can be used in clinical applications. Therefore, the aim of the present study was to evaluate the acute toxic effects of shallomin and to estimate its lethal dose low (LDLo) value. MATERIALS AND METHODS: TWO SERIES OF EXPERIMENTS WERE PERFORMED: In the first series, we used functional testing to assess the acute toxic effects of shallomin on the blood, liver, and kidney and examined histopathological changes in the liver, kidney, lung, and heart, following 7 days of daily intraperitoneal administration of 3 standard doses (10, 20, and 30 µg/g body weight of mice). In the second series, the LDLo value was estimated by determining daily mortality in mice after 7-day administration of escalating doses of shallomin (10 to 240 µg/g body weight of mice). RESULTS: The results showed that shallomin (at the anticipated in vivo doses), unlike the placebo (ethanol), did not produce any adverse effects on the tested organs. The LDLo value was observed to be 160 µg/g body weight; this value is 8- to 32-times the anticipated in vivo dose that produces antimicrobial effects under in vitro conditions against various pathogenic organisms. CONCLUSIONS: In conclusion, the results of the present study show that shallomin is a relatively safe agent, although its use needs to be carefully monitored. Further in vivo chronic toxicity tests need to be performed to establish the therapeutic potential of shallomin as an antimicrobial agent.

An In Vitro Method for Assessing the Efficacy of Antivenom Against Hemiscorpius lepturus Venom.

BACKGROUND: Hemolyis of red blood cells is a serious toxic effect commonly found among patients envenomed by Hemiscorpius lepturus scorpion. OBJECTIVES: The aim of the present study was to evlaute the efficay of the avaible polyvalent antivneom in preventing this phenomena. MATERIALS AND METHODS: Using a red blood cell fragility test, the anti-hemolytic effectiveness of a new antivenom serum against Hemiscorpius lepturus venom was investigated. Hemolysis was measured using spectrophotometry. RESULTS: Addition of venom (2, 10, 20, and 40 µg/ml) to 0.5 ml of 5% washed red blood cell suspension produced concentration-dependent hemolysis. Both the pre-incubation of red blood cell suspensions with various concentrations of antivenom (4%, 10%, and 20% v/v) and the co-administration of antivenom with 20 µg/ml venom resulted in concentration-dependent protection against hemolysis. Both the methods resulted in protection against hemolysis at the antivenom concentration of 20% (v/v). However, the inhibition of hemolysis after 24 h was found to be greater for red blood cell suspensions preincubated with antivenom (75% inhibition) than for red blood cell suspensions that were co-administered with antivenom and venom (50% inhibition). CONCLUSIONS: The results suggest that the antivenom against H. lepturus venom is useful in inhibiting hemolysis produced by the venom, but the duration of protection is relatively short and appropriate measures need to be taken, depending on the patients' clinical progress, to re-administer the antivenom at intervals less than 8 h. This proposed treatment method merits further clinical assessment.

A Comparative Study upon the Therapeutic Indices of Some Natural and Synthetic Anti-inflammatory Agents.

OBJECTIVES: THE AIM OF THE PRESENT STUDY WAS TO COMPARE THE THERAPEUTIC INDICES OF SEVERAL AGENTS USED IN TREATMENT OF INFLAMMATORY CONDITIONS WHICH INCLUDED: Vitamin E, hydro-alcoholic extracts of Glycyrrhiza glabra, Matricaria aurea, dexamethasone, piroxicam and diclofenac using Wehi-164 fibrosarcoma cells. MATERIALS AND METHODS: Cytotoxicity evaluation was based on vital dye exclusion assay. Matrix-metalloproteinases inhibition (MMPI) was assessed by gelatinase zymography method. The collected data were used to estimate the IC50 (50% MMPI concentration), LC50 (50% cytotoxicity concentration) and the therapeutic index (LC50/IC50). RESULTS: Among the natural anti-inflammatory agents used, M. aurea was the least toxic and the most effective inhibitor of MMP. Vitamin E not only increased MMP activity, but also was the most toxic of all the agents tested. Next in terms of toxicity to vitamin E was G. glabra. Diclofenac was more toxic than both piroxicam and dexamethasone. CONCLUSION: Findings from this study suggest that medicinal plants reputed to have anti-inflammatory properties are not equally effective and safe. In order to assess the implications of these findings, further in vitro and in vivo studies are needed.

Enzymatic analysis of Hemiscorpius lepturus scorpion venom using zymography and venom-specific antivenin.

Hemiscorpius lepturus envenomation exhibits various pathological changes in the affected tissues, including skin, blood cells, cardiovascular and central nervous systems. The enzymatic activity and protein component of the venom have not been described previously. In the present study, the electrophoretic profile of H. lepturus venom was determined by SDS-PAGE (12 and 15%), resulting in major protein bands at 3.5-5, 30-35 and 50-60 kDa. The enzymatic activities of the venom was, for the first time, investigated using various zymography techniques, which showed the gelatinolytic, caseinolytic, and hyaluronidase activities mainly at around 50-60 kDa, 30-40 kDa, and 40-50 kDa, respectively. Among these, the proteolytic activities was almost completely disappeared in the presence of a matrix metalloproteinase inhibitor, 1, 10-phenanthroline. Antigen-antibody interactions between the venom and its Iranian antivenin was observed by Western blotting, and it showed several antigenic proteins in the range of 30-160 kDa. This strong antigen-antibody reaction was also demonstrated through an enzyme-linked immunosorbent assay (ELISA). The gelatinase activity of the venom was suppressed by Razi institute polyvalent antivenin, suggesting the inhibitory effect of the antivenin against H. lepturus venom protease activities. Prudently, more extensive clinical studies are necessary for validation of its use in envenomed patients.

In vitro and in vivo studies on some toxic effects of the venom from Hemiscorpious lepturus scorpion.

The aim of this laboratory-based study was to investigate some of the toxic effects induced by the venom from Hemiscorpious lepturus (H. lepturus). For this aim, pharmacological, histological, biochemical methods as well as complete blood cell count were used to assess these toxic actions. In addition, in vitro haemolysis studies on human washed blood suspension and cytotoxicity on cultured fibroblasts were also undertaken. In vitro pharmacological test was made on rat isolated ileal segment. To this end, the effects of the venom on the contractile responsiveness to acetylcholine were recorded using F30 transducer and Darco chart recorder. For assessment of the haemolytic potency, varying concentrations (2, 10, 20 and 40 microg/ml) of the venom were added to 0.5 ml of 5% washed human blood and after 30 min, 2, 4, 8, 12 and 24h of exposure, the degree of lysis (extent of redness developed in the supernatant solution after centrifugation) were measured by ELISA method. Cytotoxicity potential of the venom was assessed by trypan blue exclusion test. The venom (0.1, 1 and 10 microg/ml) was mixed with confluent fibroblast cell culture and the extent cytotoxicity was assessed microscopically. In vivo studies were conducted by a subcutaneous administration of sub-lethal dose (10 microg) of the venom and after 7 days the skin, at the site of injection, and kidney samples were stained by H & E method and examined microscopically. In addition, biochemical assessments including measurement of serum aspartate aminotransferase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP) and amylase levels and urine analysis were made. The results showed that the venom prevented the relaxation phase of the acetylcholine-induced contractions on the isolated ileal segments and finally produced sustained spasmodic contractions. This spasmodic action was abolished by 1 microM atropine. The venom produced haemolysis of red blood cells in a concentration-dependent and duration-of-exposure manner, with 100% of haemolysis produced after 24h following exposure to 40 microg/ml of venom. While cultured fibroblasts cells were more sensitive and disintegrated after 15 min of exposure to 1 microg/ml of the venom. Histological findings showed evidences of excessive inflammatory responses accompanied with signs of necrosis in the skin at the site of injection as well as structural damage in the nephrones. There was a significant rise in the serum enzymes. In addition, the number of the RBCs were reduced. The urine showed positive readings for proteinuria, blood and intact RBCs. The overall results suggest that the venom from H. lepturus primarily is a cytotoxic agent and has haemolytic, nephrotoxic and to some extent hepatotoxic activity.