Comparative assessment of proliferation and immunomodulatory potential of Hypericum perforatum plant and callus extracts on mesenchymal stem cells derived adipose tissue from multiple sclerosis patients.

BACKGROUND: Mesenchymal stem cells-derived adipose tissue (AT-MSCs) are recognized for the treatment of inflammatory diseases including multiple sclerosis (MS). Hypericum perforatum (HP) is an anti-inflammatory pharmaceutical plant with bioactive compounds. Plant tissue culture is a technique to improve desired pharmacological potential. The aim of this study was to compare the anti-inflammatory and proliferative effects of callus with field-growing plant extracts of HP on AT-MSCs derived from MS patients. MATERIALS AND METHODS: AT-MSCs were isolated and characterized. HP callus was prepared and exposure to light spectrum (blue, red, blue-red, and control). Total phenols, flavonoids, and hypericin of HP callus and plant extracts were measured. The effects of HP extracts concentrations on proliferation were evaluated by MTT assay. Co-culture of AT-MSCs: PBMCs were challenged by HP plant and callus extracts, and Tregs percentage was assessed by flow cytometry. RESULTS: Identification of MSCs was performed. Data showed that blue light could stimulate total phenols, flavonoids, and hypericin. MTT test demonstrated that plant extract in concentrations (0.03, 1.2, 2.5 and 10 µg/ml) and HP callus extract in 10 µg/ml significantly increased. Both HP extracts lead to an increase in Tregs percentage in all concentrations. In particular, a comparison between HP plant and callus extracts revealed that Tregs enhanced 3-fold more than control groups in the concentration of 10 µg/ml callus. CONCLUSIONS: High concentrations of HP extracts showed effectiveness on AT-MSCs proliferation and immunomodulatory properties with a certain consequence in callus extract. HP extracts may be considered as supplementary treatments for the patients who receiving MSCs transplantation.

Immunotherapy with a recombinant hybrid molecule alleviates allergic responses more efficiently than an allergenic cocktail or pollen extract in a model of chenopodium album allergy.

BACKGROUND: The aim of this study is to assess the therapeutic potential of a recombinant hybrid molecule (rHM) alongside an allergenic cocktail from recombinant wild-type allergens as well as pollen extract on Chenopodium album allergy, using a BALB/c mouse model. METHODS: The BALB/c mice had already been sensitized to C. album via intraperitoneal injections of alum-adsorbed allergenic cocktail and immunotherapy procedure was followed by subcutaneous injections of the rHM, allergenic cocktail and pollen extract at weekly intervals. Humoral immune responses were determined via measurement of specific antibodies in serum. Splenocytes of immunized mice were stimulated in vitro and then proliferation responses, cytokine secretion and mRNA expression of genes involved in immunotherapy were examined by ELISA and real-time PCR. RESULTS: Sensitized mice were identified with high specific IgE against allergenic cocktail when compared with healthy mice. Immunotherapy with the rHM induced the highest ratio of the IgG2a/IgG1 levels compared to allergenic cocktail or C. album pollen extract. The rHM was able to induce proliferative responses as well as the allergenic cocktail in cultured splenocytes. Immunotherapy with the rHM significantly improved secretion of IFN-γ and IL-10, while secretion of IL-13 rapidly diminished. Interestingly, mRNA expression of GATA3 was strongly decreased in rHM-treated mice whereas mRNA expression of T-bet and Foxp3 was significantly increased. CONCLUSION: Our results prove that immunotherapy with the rHM effectively controlled allergic responses by shifting from a Th2-like immune response to a Th1-dominated immune response.

Constructing a hybrid molecule with low capacity of IgE binding from Chenopodium album pollen allergens.

Allergen specific immunotherapy is the only remedy to prevent the progression of allergic diseases. Nowadays, using of recombinant allergens with reduced IgE-binding capacity is an ideal tool for allergen immunotherapy. Therefore, in this study we focused on a hybrid molecule (HM) production with reduced IgE reactivity from Chenopodium album pollen allergens. By means of genetic engineering, a head to tail structure of the three allergens of the C. album pollen was designed. The resulting DNA construct coding for a 46kDa HM was inserted into an expression vector and expressed as hexahistidine tagged fusion protein in Escherichia coli. IgE reactivity of the HM was evaluated by western blotting, inhibition ELISA and in vivo skin prick test and its immunogenic property was tested by proliferation assay. The recombinant HM was expressed and purified by nickel-affinity chromatography. Comparison of the recombinant HM with a mixture of three recombinant allergens, as well as natural allergens in the whole C. album pollen extract via immunological experiments revealed that it has a much lower potential of IgE reactivity. Furthermore, in vivo skin prick tests showed that it has a significantly lower potency to induce cutaneous reactions than the mixture of recombinant wild type allergens and whole extract while, it had been preserved immunogenic properties. Our results have demonstrated that assembling three allergens of C. album in a hybrid molecule can reduce its IgE reactivity.

Diagnosis of Chenopodium album allergy with a cocktail of recombinant allergens as a tool for component-resolved diagnosis.

Chenopodium album pollen is one of the main sources of pollen allergy in desert and semi-desert areas and contains three identified allergens, so the aim of this study is comparison of the diagnostic potential of C. album recombinant allergens in an allergenic cocktail and C. album pollen extract. Diagnostic potential of the allergenic cocktail was investigated in 32 individuals using skin prick test and obtained results were compared with the acquired results from C. album pollen extract. Specific IgE reactivity against the pollen extract and allergenic cocktail was determined by ELISA and western blotting tests. Inhibition assays were performed for the allergenic cocktail characterization. The exact sensitization profile of all patients was identified which showed that 72, 81 and 46% of allergic patients had IgE reactivity to rChe a 1, rChe a 2 and rChe a 3, respectively. Almost all of C. album allergic patients (30/32) had specific IgE against the allergenic cocktail. In addition, there was a high correlation between IgE levels against the allergenic cocktail and IgE levels against the pollen extract. The allergenic cocktail was able to completely inhibit IgE binding to natural Che a 1, Che a 2 and Che a 3 in C. album extract. In addition, positive skin test reactions were seen in allergic patients that tested by the allergenic cocktail. The reliable results obtained from this study confirmed that the allergenic cocktail with high diagnostic potential could be replaced with natural C. album allergen extracts in skin prick test and serologic tests.