Measurement of CD4+ T cells in point-of-care settings with the Sysmex pocH-100i haematological analyser.

The decision to provide antiretroviral therapy to HIV-positive patients mainly depends on the CD4(+) T-cell count, with therapy indicated at a cut-off value of <350-200 CD4(+) T cells/microl blood. Monitoring patients is still a major problem in countries with limited resources where blood samples often have to be transported over long distances to regional referral centres in which the count can be performed on flow cytometers. We have evaluated a newly developed simple and inexpensive method for CD4(+) T-cell quantification. It is a variation of the Invitrogen T4 Quant kit, with manual isolation of nuclei from CD4(+) T cells and subsequent counting on the small haematology analyser pocH-100i, Sysmex. We have demonstrated that this new method is highly reproducible and gives stable and linear results over a wide range of CD4(+) T-cell concentrations. Method comparison to two different flow cytometers showed excellent correlation with concordances of about 93%. Overall, this method is rapid, easy to perform and offers a good reliable alternative to measurement by flow cytometry. The pocH-100i has the additional benefit of providing a complete blood count with a three-part white blood cell differential and software for patient data storage and handling.

Somatostatin inhibits the production of vascular endothelial growth factor in human glioma cells.

In various cell types, the neuro- and endocrine peptide somatostatin induces inhibitory and anti-secretory effects. Since somatostatin receptors, especially of the subtype sst2A, are constantly over-expressed in gliomas, we investigated the influence of somatostatin and the receptor subtype-selective peptide/non-peptide agonists octreotide and L-054,522 on the secretion of the most important angiogenesis factor produced by gliomas, vascular endothelial growth factor (VEGF). Cultivated cells from solid human gliomas of different stages and glioma cell lines secreted variable amounts of VEGF, which could be lowered to 25% to 80% by co-incubation with somatostatin or sst2-selective agonists (octreotide and L-054,522). These effects were dose-dependent at nanomolar concentrations. Stimulation with different growth factors (EGF, bFGF) or hypoxia considerably increased VEGF production over basal levels. Growth factor-induced VEGF synthesis could be suppressed to <50% by co-incubation with somatostatin or an sst2-selective agonist; this was less pronounced in hypoxia-induced VEGF synthesis. The effects were detected at the protein and mRNA levels. These experiments indicate a potent anti-secretory action of somatostatin or sst2 agonists on human glioma cells that may be useful for inhibiting angiogenesis in these tumors.

Influence of the somatostatin receptor sst2 on growth factor signal cascades in human glioma cells.

The somatostatin receptor subtype sst2A is highly expressed, non-mutated and functionally active in gliomas. After stimulation of cultivated human U343 glioma cells with somatostatin, octreotide (sst2-, sst3- and sst5-selective peptide agonist) or the sst2-selective non-peptide agonist L-054,522 multiple signal transduction pathways are induced: elevated cAMP levels are reduced, protein tyrosine phosphatases (especially SHP2) are activated and mitogen-activated protein kinases are inhibited. Stimulation of the phosphatases resulted in dephosphorylation of activated receptors for EGF and PDGF (epidermal and platelet-derived growth factor), and as a consequence the mitogen-activated protein kinases ERK 1 and 2 (p42/p44) were de-phosphorylated in co-stimulation experiments. Furthermore, somatostatin or sst2-selective agonists reduced EGF-stimulated expression of the AP-1 complex (c-jun/c-jun) on the transcriptional and translational level. These experiments show that the interaction of stimulatory and inhibitory receptors are important mechanisms for the regulation of signal cascades and gene expression.

Somatostatin receptors in gliomas.

Gliomas differ from non-malignant glial cells in the overexpression or mutations of genes involved in cell cycle or growth regulation. One example is the overexpression of the somatostatin receptor subtype 2 (sst2), especially of the splice variant sst2A. The reasons for this overexpression are not known. However, the coding sequence and part of the promoter region is not mutated. In accordance to this, the sst2 is functionally active and is internalised upon agonist stimulation. Immunoelectronmicroscopic studies show that the activated sst2 is internalised via caveolin-positive endosomal vesicles and later accumulates in multivesicular bodies and lysosomal compartments. The activated sst2 is found to be co-localised with the inhibitory G-protein Gialpha at the plasma membrane and in early endosomal vesicles. Multiple signal transduction pathways are induced. Stimulation of sst2 lowers cAMP levels elicited by forskolin and activates the protein tyrosine phosphatase SHP-2. In contrast to other sst2-expressing cells a long term antiproliferative effect of somatostatin or sst2-selective agonists are not detected in cultivated glioma cells. However, continuous stimulation of sst2 decreases the expression of genes promoting tumour survival.