Introduction of new formulas and evaluation of the previous red blood cell indices and formulas in the differentiation between beta thalassemia trait and iron deficiency anemia in the Makkah region.

BACKGROUND: Many formulas from red blood cell (RCB) indices are instructed to differentiate between iron deficiency anemia (IDA) and beta thalassemia trait. None had 100% Youden index. AIM OF THE WORK: To introduce two new formulas and evaluate them in the differentiation between IDA and beta thalassemia trait in adults Saudi (male and female; male; female) in the Makkah region. Furthermore, to evaluate the previous formulas in our population. METHODS: A total of 249 participants, 91 with IDA, 123 with beta thalassemia trait, and 35 healthy persons. All subjected to complete blood count, measurement of iron profile, hemoglobin electrophoresis and hemoglobin A2 by column chromatography. The first new formula equal hemoglobin (Hb) + hematocrit (Hct) + RBC and second equals Hb + Hct + RBC-red cell distribution (RDW). The previous formula used is England and Fraser, Mentzer, Strivastava, Ehsani, Green and King, red cell distribution index, Ricerca, and Shine and Lal Results: In both men and women, the England and Fraser was the best with a Youden's index of 70.4%, followed by Green and King 67.4%. In men, the England and Fraser and our new formula 1 had the highest Youden' index 84.7% and 84.1%, respectively. In women, the England and Fraser and RDW index had the highest Youden' index 74% and 69.2%, respectively. CONCLUSION: The England and Fraser and our new formula 1 are the best formulas in men. The England and Fraser and RDW index are the best formulas in women.

Soluble endothelial protein C receptor and high sensitivity C reactive protein levels as markers of endothelial dysfunction in patients with type 1 and type 2 diabetes mellitus: their role in the prediction of vascular complications.

BACKGROUND AND OBJECTIVE: Endothelial dysfunction in diabetes mellitus (DM) is an important factor in the pathogenesis of micro and macrovascular complications. We aimed to measure soluble endothelial protein C receptor (sEPCR) and high sensitivity C reactive protein (hsCRP) levels as markers of endothelial damage in both types of diabetes mellitus and to determine if they can be used as predictors of vascular complications. METHODS: Fifty patients with DM, 20 with type 1 and 30 with type 2 as well as 30 healthy subjects were included. All were subjected to measurement of sEPCR and hsCRP by enzyme linked immunosorbent assay. RESULTS: sEPCR and hsCRP were significantly increased when compared to the control group in both types of DM. sEPCR was a significant predictor of macrovascular complications and thrombosis in type 1 p=0.02, and p=0.015, respectively. hsCRP was a significant predictor of macrovascular complications in type 2 p=0.04. CONCLUSION: Patients with type 1 and type 2 DM exhibit higher sEPCR and hsCRP levels compared to healthy controls which suggesting endothelial damage. sEPCR could be used as a predictor of macrovascular complications and thrombosis in type 1 DM, whereas, hsCRP might be used as a predictor of macrovascular complications in type 2 DM.

Distinct antigenic features of linear epitopes at the N-terminus and C-terminus of 65 kDa glutamic acid decarboxylase (GAD65): implications for autoantigen modification during pathogenesis.

Autoantibodies to 65 kDa glutamic acid decarboxylase (GAD65) are produced in many patients with autoimmune polyendocrine syndrome type II (APS-II) or stiff-man syndrome (SMS) and are heterogeneous in their epitope specificities, recognizing both conformational and linear determinants. Major linear epitopes of GAD, which are recognized by autoantibodies in a minority of these patients, occur in the N-terminal and C-terminal regions. We have investigated antibody recognition of the N- and C-termini of GAD65 in relation to their structural features as an approach to understanding what modifications to the native GAD structure may occur that facilitate the generation of antibodies specific to linear epitopes in these regions during the autoimmune pathogenesis. A monoclonal antibody specific to the N-terminus of GAD65 bound both native and denatured GAD in ELISA, whereas monoclonal and polyclonal antibodies specific to the C-terminus of GAD bound only denatured GAD. These antibodies were epitope mapped using random peptide phage-display libraries and the epitopes related to a previously proposed structural model of GAD65. This has led us to propose that the alpha-helical secondary structure of the C-terminus of GAD65 must be denatured to generate linear epitopes. In contrast, the N-terminus is both surface exposed and linear in the native structure, but may be masked by membrane interactions, which must be broken to facilitate recognition by B cells.

An immuno-precipitation assay for determining specific interactions between antibodies and phage selected from random peptide expression libraries.

Libraries of random peptides displayed by bacteriophage can be screened to select phage expressing peptides that specifically bind antibodies, so that the peptide sequence motifs expressed by the phage can help to define the epitopes of the antibodies. It is often desirable to screen antibody-selected phage for binding of the selecting antibody in an immunoassay in order to verify the specificity of the interaction. Enzyme-linked immunosorbent assays (ELISAs) are commonly used for this purpose. However, for many antibodies, the best techniques for measuring specific, high affinity interactions are immuno-precipitation assays. Immuno-precipitation was therefore investigated as a means of measuring interactions between antibodies and phage clones selected from random peptide display libraries. Three mouse monoclonal antibodies specific for glutamic acid decarboxylase were used to select peptides as 9-mers on T7 phage, linear 12-mers on pIII of M13 phage, or constrained 15-mers on pVIII of M13 phage. Following the cloning and sequencing of selected phage, mixtures of antibody and phage were incubated in solution and the immune complexes were precipitated with Protein G bound to Sepharose beads. In order to detect and quantitate the phage that had formed immune complexes and been precipitated, advantage was taken of the biological properties of the phage by inducing infection of Escherichia coli by the precipitated phage. The aim was to quantitate the phage precipitated by determining the number of plaques produced, which would therefore be proportional to the degree of interaction between the phage and the antibody in solution. The results presented here indicate that this method of measuring monoclonal antibody interactions with phage selected for expression of peptides recognised by the monoclonal antibody is highly specific and sensitive.

Heterogeneity in the occurrence of a subset of autoantibodies to glutamic acid decarboxylase in autoimmune polyendocrine patients with islet cell antibodies.

Glutamic acid decarboxylase-65 (GAD-65) is a major target for autoantibodies and autoreactive T cells in patients with insulin-dependent diabetes mellitus (IDDM). Autoantibodies to GAD are also found in patients with stiff man syndrome (SMS) or polyendocrine autoimmunity (PE). The epitope specificities of autoantibodies to GAD in IDDM and SMS have been well documented, but the locations of autoantibody epitopes of GAD in PE patients have not been mapped. Thus, the properties of anti-GAD antibodies in PE patients (with or without diabetes) were investigated. The ability of PE serum antibodies to inhibit the binding of the mouse monoclonal antibody, GAD-6, to native GAD in ELISA was determined. For PE patients without diabetes, levels of inhibition of GAD-6 binding ranged from 0% to almost 70% and were unrelated to the level of binding of serum antibodies to GAD (P = 0.351) or to the functional affinities of these antibodies. This suggests differences in the epitope specificities of anti-GAD antibodies in different patients. Levels of inhibition were also unrelated to clinical condition. SMS antibodies showed similar levels of inhibition of GAD-6 binding. Similar analysis was applied to PE patients with diabetes and levels of inhibition of GAD-6 binding to GAD were determined. These ranged from 0% to 80%, and levels of inhibition were similar in samples taken before or after diabetes onset. There was no significant difference between anti-GAD antibodies from PE patients with or without diabetes in the range of abilities to inhibit GAD-6 binding to GAD, although the highest levels of inhibition were given by sera from non-diabetic patients. This raises the possibility of differential expression of subsets of anti-GAD antibodies in progressive versus slow or non-progressive anti-islet autoimmune responses. Serum antibodies of PE and SMS patients did not inhibit the binding of antibodies specific for the extreme C-terminus of GAD, indicating that this is not the site of the epitopes for the patients' antibodies or for GAD-6.