[Detection of Autoantibodies Against the 1-Adrenergic Receptor in the Sera of Patients via the Competitive cell-Based Enzyme Linked Immunosorbent Assay].

OBJECTIVE: This study aimed to assess the level of anti-1-adrenergic receptor autoantibodies in patients with ventricular arrhythmias with no signs of organic heart disease and with presence of cardiovascular pathology in comparison with a group of healthy volunteers. MATERIAL AND METHODS: The study included 44 patients with ventricular arrhythmias with no signs of organic heart disease ("idiopathic"), 34 patients with diagnosed dilated cardiomyopathy (DCM) of inflammatory origin, 35 patients with coronary heart disease and ventricular arrhythmias, 12patients with coronary heart disease with no ventricular arrhythmias, and 19 healthy volunteers (control group). The level of autoantibodies against the 1-adrenergic receptor was determined by the developed competitive cell-based enzyme-linked immunosorbent assay (ELISA) and by the standard ELISA using peptides corresponding to the second extracellular loop of the 1-adrenergic receptor. RESULTS: Elevated level of autoantibodies detected by a competitive cell-based ELISA was observed in 62% of patients with DCM compared to 21% of healthy volunteers (p=0.0006). In patients with "idiopathic" ventricular arrhythmias, the level of 1-adrenergic receptor autoantibodies was lower than in healthy subjects (p=0.003). Coronary heart disease patients with or without ventricular arrhythmias exhibited no differences from the control group. The number of significantly positive signals in peptide-based ELISA did not exceed 10% in any of the groups. No correlation between the data from competitive cell-based ELISA and peptide-based ELISA was found. CONCLUSIONS: This study demonstrated that competitive cell-based ELISA technique can be applied for detection of 1-adrenergic receptor autoantibodies. The results in DCM patients generally correspond to the expected. Decreased level of autoantibodies in patients with "idiopathic" ventricular arrhythmias indicates that this disease is related to changes in the immune system. Such relation is not observed in the case of coronary heart disease patients.

[Phospholipase A2, group IIa, as an earlier unknown immunohistological marker of cardiac myxoma].

Phospholipase A2, group IIA, gene expression has been analyzed in primary heart tumors. High expression has been demonstrated through several ways: reverse-transcriptase chain polymerase chain, Northern blotting hybridization at the RNA level and immunoblotting, immunohistochemical assay at the protein level. Human cardiac myxoma exhibits highly positive phospholipase A2, group IIA, immunophenotype (100% positive cases). The immunophenotype is unique among human primary cardiac tumors. Phospholipase A2, group IIA, can be proposed as a tissue marker for pathological examination after heart tumor resection.

Expression level of lipoprotein lipase and dystrophin genes predict survival in B-cell chronic lymphocytic leukemia.

Mutational status of immunoglobulin variable region genes (VH-genes) is known as the strongest predictor of long term prognosis in B-CLL. However, applications in the routine clinical practice are time consuming, and therefore some other predictions are required. In this study, we have compared prognostic values of real time PCR quantification of the expression levels of four genes previously shown to be differentially expressed in V(H)-unmutated and mutated B-CLL subtypes: ZAP-70, ZBTB20, DMD and LPL. The study included 134 B-CLL patients. Expression levels of LPL and DMD genes were significantly correlated to mutational status, while expression levels of of ZAP-70 gene correlated only in CD19+ selected cases (N = 40). No correlation was observed for ZBTB20 gene. Expression levels of LPL and DMD predicted overall survival in the entire cohort of patients. Prognostic values of LPL gene expression levels were significant even for CLL patients with stage A. Quantitative RT-PCR assays for measuring LPL gene expression are robust enough to be introduced into routine clinical practice.

[Expression patterns of the human and mouse IFGP family genes].

The IFGP family is a recently identified group of human and mouse genes structurally related to the genes of leukocyte Fc-receptors. In this study we compared expression patterns of six human and four mouse IFGP genes. With the exception of mouse IFGP2, the genes of the family were found to be predominantly expressed in haemopoietic cells. Expression of human IFGP1-IFGP5 and mouse IFGP3 was B cell-specific. Mouse IFGP1 transcripts were mainly found in B cells, but this gene may be either expressed by nonlymphoid cells. Expression of the human IFGP6 was detected in CD8+ T cells and NK cells. We further demonstrated that alternative splicing of human IFGP1 and IFGP6 mRNA may generate transcripts coding for the previously unknown isoforms. The novel IFGP1 isoform lacks transmembrane domain, whereas the IFGP6 isoform has altered cytoplasmic tail. The data obtained indicate that the receptors of family may contribute to the regulation of development and/or functions of three effector types of lymphocytes, namely B cells, CD8 T cells and NK cells.

Cloning and characterization of the human FCRL2 gene.

We report cloning and characterization of FCRL2, a novel human gene that belongs to the FcR family. The gene is closely linked and structurally similar to the recently identified FCRL/FREB/FcRX gene. The encoded protein is composed of three Ig-like domains and a C-terminal mucin-like domain containing a conserved alpha-helical motif with dileucine signals. Intraexonic splicing may generate two alternative transcripts, coding for isoforms with the third and fourth domains replaced by entirely different amino acid sequences. Like FCRL, the full-length isoform of FCRL2 is expressed intracellularly in transfected 293T cells. Expression analysis revealed FCRL2 mRNA only in placenta. The gene transcripts were not detected in lymphoid tissues or in the main leukocyte subsets isolated from peripheral blood. However, we found that FCRL2 is differentially expressed by transformed B cell lines. Of interest is also the finding that the gene expression may be up-regulated in the progression of melanocytic tumors.

Development of antibody to human GM3 synthase and immunodetection of the enzyme in human tissues.

Polyclonal antibody was raised to a cloned fragment of human GM3 synthase. Affinity purified R27C1 antibody to the tagged recombinant protein inhibited GM3 synthase activity in human liver and HL-60 cells in a dose-dependent manner. However, the R27C1 antibody did not affect liver sialyltransferase activity towards asialofetuin. We are the first to measure GM3 synthase activity in human liver (194 +/- 60 pmol NeuAc/h per mg protein), which was about 10-fold lower than in phorbol myristate acetate-stimulated HL-60 cells (1353 +/- 573 pmol NeuAc/h per mg protein). On immunoblotting the R27C1 antibody recognized a common protein band in a number of human tissues (liver, brain, atherosclerotic aortic intima, HL-60 cells) with molecular mass of about 60 kD, which is similar to that of the purified GM3 synthase from rat liver. In human liver and aortic intima, the 60-kD band was almost a single band, which makes possible the use of the R27C1 antibody for immunohistochemical studies in these tissues.

[T8-positive lymphocytes in inflammatory infiltrates of the vascular wall in nonspecific aortoarteritis]. / T8-pozitivnye limfotsity v vospalitel'nykh infil'tratakh stenki sosudov pri nespecifitsicheskom aortoarterite.

Artery specimens obtained during surgical manipulations from 11 patients suffering from nonspecific aortoarteritis (NAA) were studied immunohistochemically for T4, T8-positive lymphocytes, LFA-1-positive lymphocytes, basal membrane (BM) proteins: laminin and type IV collagen. T8-positive and LFA-1-positive cells were shown to be predominant in the sites of injury. The results obtained point out to the presence of cytotoxic cells in the affected arteries. The targets for such cytotoxic cells are medial smooth muscle cells rather then intimal cells, whereas intimal cells become hyperplastic, expressing BM proteins, which proves their smooth muscular origin. The above data support the suggestion on the autoimmune etiology of NAA.

[Heterogeneity of basal membranes in human tissues detected by monoclonal antibodies to laminin and entactin]. / Geterogennost' bazal'nykh membran v tkaniakh cheloveka, vyiavliaemaia s pomoshch'iu monoklonal'nykh antitel k lamininu i éntaktinu.

The distribution of basal membrane glycoproteins, laminin and entactin, was studied immunohistochemically by peroxidase-antiperoxidase technique in different adult human organs: kidneys, liver, heart, skin, spleen and ileum. Monoclonal antibody against entactin (ELM2) reacted with all basal membranes. Monoclonal antibody against laminin (LT3.1), however, did not react with basal membranes of arterial smooth muscle cells, or with endothelial basal membranes of renal and hepatic sinusoid endothelium. Thus, LT3.1 antibody has revealed basal membrane heterogeneity by laminin. The possible immunochemical heterogeneity of basal membranes by entactin is also discussed.