Monarch-1 activation in murine macrophage cell line [j774 a.1] infected with Iranian strain of leishmania major

Leishmania major is an intracellular parasite transmitted through the bite of the female phlebotomine sand flies. Leishmania major is able to escape the host immune defense and survive within macrophages. Modulation of the NF-kappaB [Nuclear Factor-Kappa B] activation and suppression of the pro-inflammatory cytokines by L. major are the main evasion mechanisms that remain to be explored. This study aims to examine the expression level of the Monarch-1 in L. major-infected macrophages, as a negative regulator of the NF-kappaB activation. Murine macrophage cell line [J774 A.1] was infected by metacyclic form of Leishmania promastigotes at macrophage/parasite ratio of 1:10. After harvesting infected cells at different times, total RNA was extracted and converted to cDNA. Semi-quantitative RT-PCR was performed for Monarch-1 by specific primers. Hypoxanthine Phospho-Ribosyl Transferase [HPRT] was used as an internal control to adjust the amount of mRNA in each sample. Semiquantitive analysis of Monarch-1 mRNA expression level showed a significant expression increase within 6 to 30 hours after L. major infection of macrophages when compared to the control macrophages. Monarch-1 expression level reveals a significant increase in the early phase of macrophage infection with L. major, which in turn may suppress IL-12 production in Leishmania infected macrophages and deeply influence the relationship between host and parasite.

Apoptosis of human lymphocytes after exposure to hydatid fluid

Modulation of the immune response is an important strategy by which establishment and growth of hydatid cyst in the internal organs of human is warranted. Induction of apoptosis in the lymphocytes might be a considerable component. This study was designed to evaluate apoptotic impact of hydatid fluid [HF] on human lymphocytes. Human lymphocytes were treated with hydatid fluid. After 6 hours of exposure, caspase-3 activity, the central enzyme of apoptosis cascade, was measured by fluorometric assay in the HF-treated lymphocytes and control cells. In addition, the expression of Bax [a pro-apoptotic protein] and Bcl-2 [an anti-apoptotic protein] mRNA was assessed by RT-PCR after 12 hours of exposure. Both the ratio of Bax/Bcl-2 mRNA expression and Caspase-3 activity were higher in the HF-treated lymphocytes relative to the control group. Apoptosis could be as a possible mechanism by which Echinococcus granulosus overwhelms host defenses

Effects of insulin and ascorbic acid on inhibition of the apoptosis in hippocampus of STZ-lnduced diabetic rats

The aim of this study was to investigate effects of insulin and ascorbic acid on rate of Caspase - 3 activity and DNA Laddering in hippocampus of STZ-induced diabetic rats. Thirty male Wistar rats in five groups, 6 in each group: one control group [group C] and four diabetic groups [diabetic control [group D], treatment with insulin [group I], with ascorbic acid [group AA] and with insulin plus ascorbic acid [group I+AA]] were used in this study. Diabetes was induced by injection of 60 mg/kg STZ IP. After six weeks, rats in group I were treated with insulin [4-6 U/kg/day Sc.], rats in group AA treated with ascorbic acid [200 mg/kg/day, IP] and rats in group I+AA treated with equal dosage of both insulin and ascorbic acid for two weeks. Rats in group D were treated with saline and considered as the diabetic control group. Two weeks after treatment, animals were anesthetized and hippocampus was dissected from hemispheres. Caspase-3 activity was assessed by Fluorometry, and finally, DNA fragmentation due to apoptosis was determined by DNA laddering Assay. Caspase-3 activity in group D significantly increased compared to group C [6.7 fold], whereas it decreased after treatment with insulin, ascorbic acid or both [2.6, 4.2 and 5.1 fold, respectively]. DNA laddering was observed in group D, but not in three treated groups. Conclusions: From this survey it was concluded that treatment of STZ-induced diabetic rats with insulin and/or L-ascorbic acid could possibly inhibit apoptosis in hippocampal tissues using decrease of Caspase -3 activity and prevention of DNA Laddering

[ study of effects of insulin and ascorbic acid on Bcl-2 family expression in hippocampus of streptozotocin -induced diabetic rats]

Diabetes is a metabolic disorder that has been shown to adversely affect both the central and peripheral nervous system by increasing basal neuronal apoptosis. Since Bcl-2 protein family is considered to play a key role in the regulation of apoptosis, in the present study we have examined the effects of insulin and ascorbic acid on expression of Bcl-2 family members including Bax [pro-apoptotic] and Bcl-2 and Bcl-x[L] [anti-apoptotic] on hippocampus of STZ-induced diabetic rats. Five groups of six Wistar rats including one control group [C] and four diabetic groups [D, I, AA and I+AA] were used in this study. Diabetes was induced by injection of 60 mg/kg STZ [IP]. After six weeks, rats in group I were treated with insulin [4-6 U/kg/day Sc], rats in group AA were treated with ascorbic acid [200 mg/kg/day IP] and rats in group I+AA were treated with equal dosage of both insulin and ascorbic acid for two weeks. Rats in group D were treated with normal saline and considered as diabetic control group. Two weeks after treatment, expression of Bcl-2, Bcl-x[L] and Bax genes were measured at both mRNA and protein levels. In diabetic control rats [group D], Bax increased whereas Bcl-2 and Bcl-x[L] decreased at both mRNA and protein levels compared to group C [P<0.01, P<0.001 respectively]. Interestingly, treatment with insulin [group I], ascorbic acid [group AA] and insulin plus ascorbic acid [group I+AA] could reverse these changes both at mRNA and protein levels [p<0.001 for I and AA+I groups, p<0.05 [Bcl-2] and p<0.01 [Bcl-x[L]] for AA group]. It is concluded that insulin and ascorbic acid alone or together can inhibit apoptosis in STZ-induced diabetic rats' hippocampus through increasing the ratio of Bcl-2/Bax and Bcl-x[L]/Bax expressions. We suggest that inhibition of apoptosis may prevent cognitive dysfunctions induced by hippocampal damage in diabetic patients as well. In addition, further experimental studies will need to be performed to confirm such effects

[Development of an indirect ELISA for detection of specific IgG subclasses for saffron pollen allergens]

Allergic reactions to plant pollens are a common problem. There are some evidences that saffron pollen has allergenic properties. Although IgE-mediated hypersensitivity has been reported, other immunologic mechanisms may be involved based on a variety of clinical manifestations in allergic cases. One of the mechanisms that have been suggested is specific IgG and its subclasses. In this study, an Enzyme-linked Immunosorbent Assay [ELISA] was developed for detection of specific IgG subclasses against saffron pollen extract. Briefly, microtiter plates were coated with crude extract of saffron pollen and then blocked with bovine serum albumin [BSA], and the sera were added to the plates which followed by the addition of anti-human IgG. TMB was used as substrate. Finally, after stopping the reaction with addition of HCl, plates read at 450 nm. The method was verified by testing two groups of samples, first, sera from individuals living near saffron farms in Khorasan and second, sera from individuals living in areas not having any saffron farms. The results indicated significant differences between the two groups

[ prevalence of saffron pollen allergy in saffron workers of Khorasan [Iran] in 2002]

Iran is the main producer of Saffron in the world and a great number of Khorasan [the largest province of Iran] are involved in growing this plant. Recently, saffron pollen has found great importance due to inducing allergic reactions. In this study, the prevalence and clinical symptoms of saffron pollen allergy and specific IgE level in saffron workers have been studied. For this purpose, 167 Saffron workers selected randomly from different regions of Khorasan Saffron growing areas were enrolled into the study.Clinical history, skin prick test results, total IgE and specific IgE were used as inclusion and exclusion criteria. Total IgE and specific IgE assessment carried out on 39 cases who had either positive skin prick test or positive clinical symptoms and 10 cases who had neither positive skin prick test nor clinical symptoms [non-allergic group]. From 167 individuals, 21 cases had positive skin prick test to Saffron pollen extract and 11 cases had Saffron specific IgE in their sera [allergic group]. Forty Saffron workers [24%] showed allergic symptoms in Saffron picking season and 19 individuals [11%] suffered from allergic symptoms in other seasons. Clinical symptoms were respectively sneezing, watery nose, itchy eyes, itchy nose and red eyes. Skin symptoms like urticaria and dry skin were reported rarely. Saffron allergic and non-allergic groups showed significant differences in birth month, allergy to three other pollen extracts and allergy history in their siblings. No significant difference was found between Saffron allergic and non-allergic groups in regard to gender, age, family size, the duration of allergen exposure and smoking. Therefore, Saffron pollen can cause allergic reactions occuring basically in eye, nose and upper respiratory system and according to the obtained results these allergic reactions occur through an IgE-dependent mechanism

Immunoblot analysis of saffron pollen proteins recognized by human IgE antibodies

Pollen is one of the major causes of allergy worldwide. Pollen allergens can cause an immediate hypersensitivity [type 1, IgE-mediated] response in susceptible individuals. Hypersensitivity to saffron pollen is a serious problem, especially among the people who working with Saffron flowers. The aim of this study was to identify protein with IgE binding activity [allergenic protein] in a saffron pollen extract. Proteins of the saffron pollen extract recognized by human IgE antibodies were investigated by SDS-PAGE and Western blotting. Proteins with IgE binding activity were identified with sera from 23 patients who suffered from continues sneezing, rhino rhea, conjunctivitis, throat irritation, shortness of breath during exposure with Saffron flowers. A chemiluminescent immuno-detection method was used to study IgE binding activity'of saffron pollen proteins with patients's sera.Six IgE immimoreactive proteins ranging in molecular weight from 13.5 to 85 KD were identified. From among of these proteins, four with apparent molecular weight of 13.5-14, 19.5-20, 42-43 and 85 KD could be identified with more than 35% of our allergic patient's sera. While two proteins with IgE binding activity [58 and 67 KD] could be demonstrated for only some of our saffron allergic patients.In conclusion, in this study we demonstrated the presence of several IgE binding proteins in the total extract of saffron pollen

[ prevalence of saffron pollen allergy in saffron workers of Khorasan [Iran] in 2002]

Iran is the main producer of Saffron in the world and a great number of Khorasan [The largest province of Iran] are involved in growing this plant. Recently, Saffron pollen has found great importance due to inducing allergic reactions. In this study, the prevalence and clinical symptoms of Saffron pollen allergy and specific IgE level in Saffron workers have been studied. For this purpose, 167 Saffron workers selected randomly from different regions of Khorasan Saffron growing areas were enrolled into the study. Clinical history, skin prick test results, total IgE and specific IgE were used as inclusion and exclusion criteria. Total IgE and specific IgE assessment carried out on 39 cases who had either positive skin prick test or positive clinical symptoms and 10 cases who had neither positive skin prick test nor clinical symptoms [Non-allergic group]. From 167 individuals, 21 cases had positive skin prick test to Saffron pollen extract and 11 cases had Saffron specific IgE in their sera [Allergic group]. 40 Saffron workers [24%] showed allergic symptoms in Saffron picking season and 19 individuals [11%] suffered from allergic symptoms in other seasons. Clinical symptoms were respectively sneezing, watery nose, itchy eyes, itchy nose and red eyes. Skin symptoms like urticaria and dry skin were reported rarely. Saffron allergic and non-allergic groups showed significant differences in birth month, allergy to 3 other pollen extracts and allergy history in their siblings. No significant difference was found between Saffron allergic and non-allergic groups in regard to gender, age, family size, the duration of allergen exposure and smoking. Therefore, Saffron pollen can cause allergic reactions occurring basically in eye, nose and upper respiratory system and according to the obtained results these allergic reactions occur through an IgE-dependent mechanism

[Melon allergy and allergenic cross reactivity of melon with other allergens]

Allergenic reaction to melon, Cucumis melo [belongs to Cucurbitaceae family], has been reported in some allergic patients. Oral allergy syndrome was the most common clinical features associated with melon allergy. This study was aimed to confirm allergenicity of Mashadi melon, identify allergenic protein[s] of melon and allergenic cross reactivity of melon with other allergens. Prick test was performed with the extract of different parts of melon [Peel, pulp and loose layer on pulp] on the 35 patients who suffered from allergic symptoms after the ingestion of melon. Total IgE and specific IgE to melon %ere nieasurcd in 21 sera from patients with positive sicin prick test and 15 healthy controls' sera by means of ELISA. The IgE reactive protein of melon extract was detected by westeni blotting, using the 13 patient's sera [with high levels of IgE]. ELISA inhibition carried out in order to detect cross-reactivity between melon and kiwi, banana, Cynodon dactylon and Poa pratensis. Clinical reactions to melon were oral allergy syndrome 61% [immediate oral itching with or without angioedema of the lips and oral mucosa], rhinitis 38%, itching 19% and gastrointestinal symptoms 4.8%. Twenty one of the 35 patients showed positive skin prick test [SPT] to loose layer on pulp. Three patients also showed reaction to pulp and loose layer. Increased specific IgE levels to melon were observed in 18 patients with positive SPT to melon extract. Inhibition experiments showed a strong cross-reactivity of melon specific IgE with two species of ragweed pollen, especially with Cynodon doctylon, but banana and kiwi extract did not inhibit melon specific IgE in inhibition ELISA method. Immunoblot analyses of aqueous protein extract from melon were showed an IgE-binding protein of - 14.4 kDa with 8 of 12 melon-allergic patients' sera. In conclusion, we confirmed IgE-mediated hypersensitivity to melon with common clinical feature of oral allergy syndrome [OAS] and presence of an IgE-binding protein of - 14.4 kDa in melon extract. These findings suggest that main allergen of melon could be profilin

[Anti-D quantification by an enzyme-linked antiglobulin test: A comparison study of two methods]

The quantification of anti-red blood cell antibodies routinely performed by standard direct antiglobulin test [DAT], but there are some limits to its sensitivity and the end-point evaluation is very subjective in this method. Recently, a modified enzyme-linked immunosorbent assay [ELISA] has been applied to detect anti-red blood cell antibodies. In this method, the end-point is determined by enzyme activity of an enzyme conjugated anti-human antibodies. The enzyme converts a soluble substrate to a soluble colored product, which is proportional to amount of primary antibodies. This technique has been called enzyme linked antiglobulin test [ELAT]. In this study, we tried to evaluate the analytical parameters of this technique and compare with conventional DAT method. Our results showed some modification of the previously described ELAT technique makes it feasible and simpler for the routine applications. This modified method was reproducible with the mean coefficient variation of 9.08 and 5.21 for between days and within day assays and found to be at least sixteen times more sensitive than DAT method. Anti-D measurement showed an acceptable correlation [R 0.88] between ELAT and DAT methods. Therefore, this method could be useful for more precise monitoring of allo-immunized mothers and patients with autoimmune hemolytic anti-D anemia and provide an alternative method for assessing anti-D activity of specific total IgG and IgG subclasses preparation