Yaser Dorri

Induction of Oral Tolerance in Experimental Sjögren's Syndrome Autoimmunity

Previous studies have showed that immunization with peptides from Ro 60 results in Sjögrens syndrome (SS)‐like condition in BALB/c mice. We hypothesized that oral feeding with Ro 60 peptide or Ro 60 would prevent the disease. Four groups (each consisting of 10) of BALB/c mice were used. Group I–III were immunized with Ro 274 peptide. Group IV mice were administered adjuvant only. Group II mice were fed orally with Ro 274 peptide and Group III with Ro 60 for 5 days before immunization. There was a significant reduction in the binding of sera from both Group II and Group III mice to most of the Ro multiple antigenic peptides bound by Group I mice. In Group III mice, salivary flow was maintained above that of the Group I mice (average: 117.5 versus 58.6 µl; t = 2.7; P = 0.02). Salivary infiltrates were drastically decreased in the Ro peptide or Ro 60‐fed groups, compared to non‐tolerized group. Two of eight mice in Group II and 3/6 mice in Group III had no infiltrates, whereas all eight mice studied in Group I had a significant number of infiltrates. Thus, epitope spreading was prevented, lymphocytic infiltration was blocked and saliva flow was restored by means of oral feeding of either Ro 274 or Ro 60 in this animal model of SS.

Degree of modification of Ro60 by the lipid peroxidation by-product 4-hydroxy-2-nonenal may differentially induce Sjögren syndrome or systemic lupus erythematosus in BALB/c mice

Our previous work showed that immunization of rabbits with 4-hydroxy-2-nonenal-modified Ro60 (HNE-Ro60) accelerates autoimmunity. We extended this model into mice, hypothesizing that the severity of autoimmunity would be dependent on the degree of HNE modification of Ro60. Five groups of BALB/c mice (10/group) were used. Group I was immunized with Ro60. Groups II to IV were immunized with Ro60 modified with 0.4 mM (low), 2 mM (medium), and 10 mM (high) HNE, respectively. Group V controls received Freunds adjuvant. A rapid abrogation of tolerance to Ro60/La antigens occurred in mice immunized with HNE-modified Ro60, especially in the low and medium HNE-Ro60 groups. Lymphocytic infiltration and significantly high decrement in salivary flow (37%) compared to controls was observed only in the high HNE-Ro60 group, suggesting induction of a Sjögren syndrome-like condition in this group. Anti-dsDNA occurred only in mice immunized with medium HNE-Ro60. This group did not have a significant decrement in salivary flow, suggesting induction of a systemic lupus erythematosus-like manifestation in this group. Significantly high antibodies to Ro60 were found in saliva of mice in the low and medium HNE-Ro60 and the Ro60 groups, as well as anti-HNE Ro60 in the low and medium HNE-Ro60 groups. Understanding the mechanism of this differential induction may help discriminate between these two autoimmune diseases.

Environmentally safe removal/disposal of Coomassie Brilliant Blue from gel destain and used gel stain

Gel destaining following Coomassie Brilliant Blue (CBB) staining involves the use of toxic reagents. Here we demonstrate the efficacy of various paper adsorbents in adsorbing CBB. Kimwipes adsorbed the best, followed by Teri towels, multifold towels, and Whatman numbers 1 and 3 filter papers. Three Kimwipes completely adsorbed the dye released from a CBB-stained mini-gel. Nonradioactive destain solution can, therefore, be recycled for destaining CBB-stained gels. Stain removal with Kimwipes helps in reducing destain use and in reducing organic liquid waste, and it is 7.5-fold cheaper compared with an available method for CBB disposal. Following this, we determined the suitability of this procedure to remove the dye from a used CBB staining solution awaiting proper disposal by our Institutional Safety Office. The dye from a 0.05% CBB staining solution could be removed in 5 to 10 min using 75 Kimwipes. The CBB-adsorbed Kimwipes did not release the stain when squeezed dry even after incubation in various salts over 1week and in water for 5 weeks. The CBB removed allows its easy disposal as solid waste and will not leach out from solid landfills. Thus, stain removal with Kimwipes helps in disposing CBB in an environmentally friendly manner and allows recycling of destaining solution.

Spicy SDS-PAGE gels: curcumin/turmeric as an environment-friendly protein stain.

Gel proteins are commonly stained with calorimetric/fluorescent dyes. Here, we demonstrate that heat-solubilized curcumin can serve as a nontoxic and environment-friendly fluorescent/colorimetric reversible protein stain. Curcumin, the yellow pigment found in the rhizomes of the perennial herb Curcuma longa (turmeric), is insoluble in aqueous solvents. However, heat (100°C) solubilization in water renders 1.5% of curcumin soluble. Curcumin solubilized by ethanol or alkali is ineffective in staining proteins. Heat solubilized curry spice turmeric stains proteins similarly. Staining is achieved in 30 min, with a sensitivity almost equaling that of Coomassie Brilliant Blue (CBB). Destaining is not required, and excess curcumin/turmeric can be discarded into the sink. Binding of proteins by silver inhibits curcumin binding, suggesting similarity of protein binding by silver and curcumin. It costs

Detection of Autoantibodies to Tumour-Associated Antigens in Sera of Patients with Systemic Autoimmunity Using a Novel Protein Microblot Array

1.5-2.0 to stain a mini-gel with curcumin, while turmeric costs less than 0.005 cent. CBB staining/destaining costs about 2 cents. However, CBB is toxic and its use necessitates specialized disposal efforts. Curcumin/turmeric, thus, can serve as an ideal nontoxic protein stain.

Coomassie Brilliant Blue removal/disposal from gel destain and used gel stain in an environment-friendly manner.

It is well known that sera of patients with systemic autoimmunity contain autoantibodies to nuclear antigens. It is also known that patients with systemic autoimmunity have an increased risk for the development of tumours. Interestingly, tumour patients frequently develop autoantibodies and there is a growing list of potential tumour‐associated antigens. It is, however, not known whether or not patients with systemic autoimmunity also develop antibodies to tumour‐associated antigens. Here we describe the development of a novel multiprotein array allowing us to screen for autoantibodies to 30 different tumour‐associated antigens in parallel. Using this novel assay, we found that the frequency of autoantibodies to the selected tumour‐associated antigens is increased between 2‐ and 14‐fold in patients with systemic autoimmunity compared with an age‐matched control group.

Whole and Strip Nitrocellulose Membrane as well as a New-Line-Immunoblotting ofAntigen Using the Chemiluminescence Technique

Toxic reagents are employed to destain Coomassie Brilliant Blue (CBB) stained gels. We tested the efficacy of various paper adsorbents in adsorbing CBB released from gels during destaining. Kimwipes were the most efficient, followed by Teri towels, multifold towels, and Whatman (numbers 1 and 3) filter papers. Three Kimwipes added during destaining of a CBB-stained mini-gel helped adsorb the released dye. Thus, stain removal with Kimwipes helps reduce destain use and organic waste accumulation, enables recycling of nonradioactive destaining solution, and is 7.5-fold cheaper than an available method for CBB disposal. Next, we used Kimwipes to deplete the dye from a used CBB staining solution awaiting proper disposal by our Institutional Safety Office. Seventy-five Kimwipes successfully helped remove the dye from a 0.05% CBB staining solution in 5 to 10 min. The blue-colored Kimwipes did not release the stain even when squeezed dry after incubation in various salts, water, or acid solutions for five weeks. The CBB removed thus can be simply disposed as solid waste and will not leach out from solid landfills. Kimwipes, thus, enables CBB disposal in an environmentally friendly manner and allows recycling of destaining solution.

A Miniaturized Blotting System for Simultaneous Detection of Different Autoantibodies

There are a number of techniques in the scientific world that researchers use to detect specific antigens. One such technique that has provided many advantages over typical immunochemical staining is chemiluminescence. The emission of visible radiation by compounds once exposed to sunlight has been known for centuries and currently is the main principle for chemiluminescence. Here, we introduce three different chemiluminescence techniques that are widely used in immunodetection of antigens: (a) whole membrane chemiluminescence detection, (b) strip membrane chemiluminescence detection, and (c) new line blotting chemiluminescence.

An overview of Western blotting for determining antibody specificities for immunohistochemistry.

Sera of tumor patients frequently contain autoantibodies to tumor associated antigens. Here we describe a miniaturized immunoblot platform allowing us to screen sera of patients for the presence of autoantibodies to ten autoantigens in parallel.