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2). in CVID may be caused by regulatory T cell disorder. Keywords:Pax5, CD19, CD40, RT-PCR, common variable immunodeficiency Candesartan cilexetil (Atacand) == INTRODUCTION == The Pax5 gene encodes a Candesartan cilexetil (Atacand) B cell-specific activator protein (BSAP), which has been identified as a transcriptional factor that is expressed at early, but not late, stages of B cell differentiation [1]. Numerous binding sites for Pax5 in the promotors of B cell-specific genes have been recognized, including sites in the promotors of the genes encoding CD19 [2], VpreB [3] and Blk [4], as well as multiple sites within the IgH locus, including a region upstream of S2a and S [5]. Furthermore, Pax5 was identical to S-bp, which binds to two sites upstream of C, as well as to S binding proteins [68]. Over-expression of Pax5 in splenic B cells stimulates proliferation of these B cells [9]. Experiments performed using mice that were Pax5-deficient due to targeted gene disruption revealed that Pax5-deficient mice fail to produce small preB, B and plasma cells, owing to a complete arrest of B cell development at an early precursor stage [10]. The expression and function of the Pax5 gene in mouse cells and tissues have been extensively investigated [1113]. It is assumed that this Pax5 gene is usually specifically expressed in mouse B cell lineage haematopoietic cells. However, the lineage specificity of Pax5 gene expression in human cells remains obscure. Common variable immunodeficiency (CVID) is usually characterized by recurrent contamination and decreased serum immunoglobulin levels [14]. Some cases of CVID are associated with a complete absence of surface IgM+B cells and immunoglobulins of all three Rabbit Polyclonal to XRCC5 major isotypes, as in Pax5-deficient mice [10]. In this study we analyse the expression of the human Pax5 gene in various haematopoietic cell lines and tissues and in CVID peripheral blood lymphocytes (PBL). In human cell lines the Pax5 gene was Candesartan cilexetil (Atacand) specifically expressed in B lineage cells, which expressed CD19. Myeloma cell lines did not express the Pax5 gene. In CVID with a decreased number of mature B cells among PBL, Pax5 gene expression was not detected. Activation with anti-CD40 MoAb and cytokines induced Pax5 expression in some CVID PBL. We discuss the role of Pax5 in human B cell differentiation and proliferation. == MATERIALS AND METHODS == == == == Cell lines and human tissues == Human cell lines were cultured in RPMI medium with 10% fetal calf serum (FCS). The cell lines used in this study are outlined inTable 1. PBL were prepared from heparinized blood using FicollHypaque. Human adult tissues were obtained at autopsy and fetal tissues at abortion before 24 weeks of pregnancy. Informed consent was obtained before the tissues were collected. == Table 1. == Expression of Pax5 gene and CD marker in various haematopoietic cell lines [1628] == Immunophenotypic analysis == Cells were stained with a panel of diagnostic reagents in suspension using the direct Candesartan cilexetil (Atacand) and indirect immunofluorescence technique [15]. Briefly, cells were first incubated with a specific MoAb, in excess, for 30 min. In cases in which cytoplasmic immunoglobulin was stained, the cells were preincubated with ice-cold methanol. After being washed, cells were stained with FITC-conjugated goat anti-mouse F(ab)2isotype-specific antisera (Cappel, CA). Screening for terminal deoxy-transferase (TdT) was carried out on methanol-fixed mononuclear cell smears with rabbit anti-calf thymus TdT antiserum followed by staining with FITC-conjugated goat anti-rabbit IgG (Cappel). == Reverse transcriptase-polymerase chain reaction for detecting mRNA expression == RNA was extracted from cell and tissues using Isogen (Nippon Gene, Toyama, Japan). cDNA was synthesized with MMTV reverse transcriptase using 1 g or 100 ng or 10 ng of RNA and oligo dT-primer. The polymerase chain reaction (PCR) primers were as follows: Pax5 sense 5- AATGACACCGTGCCTAGCGT-3, Pax5 antisense 5-GGTGGTGAAGATGTCTGAGT-3; CD19 sense 5-TAAGTCATTGCTGAGCCTAGA-3, CD19 antisense 5-TCGCTGCTCGGGTTTCCATAA-3; -actin Candesartan cilexetil (Atacand) sense 5-TGACGGGGTCACCCACACTGTGCCCATCTA-3, -actin antisense 5-CTAGAAGCATTTGCGGTGGACGATGGAGGG-3. PCR was performed for 1535 cycles (Fig. 1) in a PCR thermal cycler (Takara, Ootsu, Japan) at a denaturation.