C-reactive protein triggers calcium signalling in human neutrophilic granulocytes via FcγRIIa in an allele-specific way.

C-reactive protein (CRP) binds to Fcγ-receptors, FcγRIIa (CD32) with high affinity and to FcγRIa (CD64) with low affinity. The binding to CD32 has been shown to be allele specific, that is, it binds to R/R131 but not to H/H131. Little is known about the cooperation of CRP and neutrophilic granulocytes (PMNs) in inflammatory reactions. The purpose of the present study was to examine CRP signalling in human PMNs, and whether this signalling is also allele specific. Cytosolic calcium of PMN was measured in a single-cell digital imaging system. Receptor expression and polymorphism were studied by real-time RT-PCR, flow cytometry and standard PCR. C-reactive protein induced cytosolic calcium signals in PMNs from homozygote R/R131 donors, but not in PMNs from heterozygote R/H131 donors. However, after the heterozygote PMNs had been incubated with IFN-γ (100 U/ml) for 2 h, both the proportion of cells responding and the size of the CRP-induced calcium signals increased. IFN-γ increased mRNA expression of CD64 about fivefold and surface protein expression of CD64 about fourfold. The calcium signal elicited by CRP was augmented by PMN adhesion to fibronectin, but almost totally abrogated by sphingosine kinase inhibitors. The signals were partly dependent on calcium influx. In conclusion, calcium signalling instigated by CRP in human PMN is FcγRIIa allele specific, as R/R131 responded to CRP, whereas R/H131 did not. However, increased expression of FcγRIa (CD64), stimulated by IFN-γ, can augment calcium signalling by CRP in low-responders. This suggests that the state of the PMNs, as well as the genetic origin, affect sensitivity for CRP.

Identification and physical mapping of genes expressed in the corpus luteum in cattle.

A representational difference analysis was performed to identify genes expressed in the corpus luteum of cattle. The corpus luteum is an ovarian structure that is essential for the establishment and maintenance of pregnancy. Knowledge of gene expression and function of corpus luteum will be important to improve fertility in humans and domestic animals. Housekeeping genes were removed from the corpus luteum representation (tester) using skeletal muscle as the subtracting agent (driver). A total of 80 clones of the final subtraction product were analysed by sequencing and 11 new bovine gene sequences were identified (pBTCL1-11). The sequences were mapped to segments of 10 different chromosomes using a somatic cell hybrid panel and a radiation hybrid panel. With one exception the locations are in agreement with published comparative maps of cattle and man. Expression in corpus luteum was verified by RT-PCR for all the 11 clones.

Cloning and characterization of ZNF189, a novel human Krüppel-like zinc finger gene localized to chromosome 9q22-q31.

A 3-kb-long cDNA encoding a Krüppel-like human zinc finger protein was isolated and mapped to chromosome 9q22-q31. The ZNF189 gene encodes a protein with 16 zinc fingers at its C-terminus and belongs to the Krüppel-associated box (KRAB)-containing group of zinc finger proteins. Four differently spliced cDNA transcripts, differing at the 5' coding region where a KRAB A repressor domain is encoded, were isolated. In addition, Northern blot analysis indicates the presence of two additional unidentified splice variants. Comparison of cDNA and genomic sequences shows that the ZNF189 gene spans approximately 11 kb and is organized into at least four exons, the large 3'-end exon coding for the complete zinc finger domain and the 3' untranslated region. ZNF189 is expressed in all tissues and cell types currently investigated, at varying levels, but with a tissue- or cell-type-restricted expression pattern for the different splice variants. ZNF189 is conserved in the genome of several mammalian species. Direct sequencing of the ZNF189 gene in microdissected tumor biopsies of sporadic basal cell carcinoma and squamous cell carcinoma reveals no mutations in the coding sequence or at exon/intron boundaries.

[Blocking of carbohydrate adhesion. A new principle for therapy of many conditions?]. / Blokkering av karbohydratadhesjon. Et nytt prinsipp for behandling av mange tilstander?

Cell adhesion is of fundamental biological importance. In this article we describe the therapeutic potential of blocking carbohydrate dependent adhesion. The animal studies are promising, and there is a realistic hope that "anti-adhesion" therapy for acute conditions like reperfusion injury and severe infections will be of clinical value. Treatment of more chronic diseases like rheumatoid arthritis and cancer by blocking carbohydrate mediated cell adhesion is theoretically possible, but probably more difficult.

Characterization of a promoter region supporting transcription of a novel human beta-galactoside alpha-2,6-sialyltransferase transcript in HepG2 cells.

In humans, two transcripts encoding beta-galactoside alpha-2,6-sialytransferase (EC 2.4.99.1.) have previously been described. One of the transcripts is widely expressed, whereas the other is restricted to mature B-cells. In this study we demonstrate the existence of a third transcript in the hepatoma cell-line HepG2. The expression of this transcript is controlled by a promoter region which efficiently supports transcription in HepG2 cells, and which harbours putative binding sites for liver-enriched and acute phase inducible transcription factors.

The B cell antigen CD75 is a cell surface sialytransferase.

In this work we have isolated a cDNA clone encoding the B cell antigen CD75. The amino acid sequence of CD75 is shown to be identical to that of human alpha 2,6 sialyltransferase, believed to be primarily associated with the Golgi complex. This is the first demonstration of cell surface expression of sialytransferase which, in B cells, may play an important role in intercellular adhesion and antigen presentation events.

Inhibition of polyphosphoinositide breakdown and c-myc induction accompanying inhibition of human B-cell activation by two monoclonal antibodies against the leucocyte common antigen (CD45).

In this report we show that the two monoclonal anti-CD45 antibodies, EO-1 and FN-126, potently inhibit G0 to G1 transition and S phase entry in human B cells stimulated with anti-mu and low molecular weight B-cell growth factor. Both antibodies were found to inhibit anti-mu-induced inositol phospholipid breakdown and c-myc mRNA induction. In contrast, EO-1 and FN-126 only partially inhibited the early anti-mu-induced increase in cytoplasmic Ca2+ levels, both in normal and in Ca2(+)-depleted medium. B-cell activation provoked by 12-O-tetradecanoylphorbol 13-acetate (TPA) was not inhibited by these antibodies, except when using high concentrations of EO-1. In addition, both antibodies were found to inhibit G1 entry induced by the anti-CD20 antibody 1F5, which confers an activation of B cells without any detectable increase in [Ca2+]i or in phospholipid metabolism. This indicates that alternative mechanisms in addition to the inhibition of polyphosphoinositide (PI) breakdown are involved in the inhibitory action of these antibodies.

Functional properties of CD19+ B lymphocytes positively selected from buffy coats by immunomagnetic separation.

Here we report that human B lymphocytes can be positively selected directly from buffy coats applying the anti-CD19 antibody AB1 coupled to magnetic beads. This isolation protocol is highly efficient and the isolated cell population is of very high purity and viability. As judged by cell cycle analysis and various parameters for cell activation, the cells are still in a resting state after isolation. Furthermore, different functional assays have shown that the isolation procedure does not interfere with either activation or proliferation/differentiation of CD19 selected cells as compared to negatively isolated cells. As a consequence of cross-linking during the isolation process, the CD19 antigen is temporarily down-regulated as measured by AB1 binding. Despite this decreased expression, monoclonal antibodies to the CD19 antigen nevertheless inhibited anti-mu plus B cell growth factor induced B cell activation as reported also for negatively isolated cells. Taken together, the presented data strongly suggest that B cells isolated through the CD19 antigen can be used in critical functional assays.