Geijn, G.J.M. van de

Erythrocyte-associated apolipoprotein B and its relationship with clinical and subclinical atherosclerosis (Article)

2012-04-01T00:00:00Z

Background Apolipoprotein (apo) B-containing lipoproteins are closely linked to atherogenesis. These lipoproteins are transported in plasma and are also associated with blood leucocytes. Our aim was to investigate whether apoB-containing lipoproteins are also present on the surface of erythrocytes and investigate the relationship with the presence of atherosclerosis in a cross-sectional study. Materials and methods Erythrocyte-bound apoB (ery-apoB) was measured by flowcytometry in subjects with (CAD+) and without coronary artery disease (CAD-), based on coronary angiography or on a history of cardiovascular disease. Intima media thickness (IMT) measurements were carried out using B-mode ultrasound. The relationship between ery-apoB and clinical and subclinical atherosclerosis was evaluated with binary logistic regression. Results A total of 166 subjects were included (40 CAD+ and 126 CAD-). ApoB was detected on freshly isolated erythrocytes (range: 0·1-5·5 au; mean±SEM 0·86±0·09 au) in all but nine subjects (four CAD+ and five CAD-). Ery-apoB was lower in CAD+ (0·62±0·09 au) compared to CAD- (1·18±0·10 au; P<0·001). Higher ery-apoB was associated with a lower risk of CAD (adjusted OR: 0·003 (95% CI: 0·001-0·08; P<0·001), but the protective effect was diminished with increasing age (adjusted OR: 1·10 (95% CI: 1·04-1·16; P<0·001). IMT was increased in CAD+ subjects (0·77±0·13mm) compared to CAD- (0·57±0·14mm; P<0·001). A significant negative association was found between ery-apoB and IMT (β=-0·214: 95% CI -0·284 to -0·145; P<0·001). There was no association between ery-apoB and plasma apoB (Pearson's r=-0·45; P=0·57). Conclusions Human erythrocytes carry apoB-containing lipoproteins. Subjects with atherosclerosis have lower ery-apoB. High ery-apoB may be protective against atherosclerosis and may reflect an alternative blood cell-mediated lipoprotein transport system in the circulation, in which these lipoproteins less likely interact with the endothelium. © 2011 The Authors. European Journal of Clinical Investigation

Leukoflow: Multiparameter extended white blood cell differentiation for routine analysis by flow cytometry (Article)

2011-09-01T00:00:00Z

Differential white blood cell count (dWBC) is a frequently used diagnostic tool. For most patient samples an automated blood counter produces a five-part differential count. If this dWBC does not meet pre-set criteria, microscopic dWBC is performed. Microscopy is labor intensive and requires sustained training of technicians. Inter-observer variation and statistical variation are significant, due to limited numbers of cells counted. Flow cytometry is a candidate reference method for dWBC. Advantages are immunological definitions and large number of measured cells. Our goal was to replace (part of) the microscopic dWBC by a flow cytometric dWBC, that gives additional information on blasts, myeloid precursors, and lymphocyte subsets. We designed a cocktail of antibodies (CD4, CD14, CD34, CD16, CD56, CD19, CD45, CD138, CD3, and CD71) combined with a gating strategy and flow cytometric protocol for easy identification of leukocyte populations. This assay, called Leukoflow, requires low sample volume, has few manual handling steps, and a potential turn-around-time shorter than 2 h. We determine percentages and absolute concentrations of at least 13 different cell populations. For quantification of normoblasts a second flow cytometric staining was designed. We compared microscopic dWBC with that of the automated blood counter and Leukoflow for normal and abnormal blood samples. Leukoflow results correlate well with the automated blood counter for leukocytes, neutrophils, eosinophils, monocytes, and lymphocytes. Correlation with manual dWBC is lower. Blast counts reported by Leukoflow suffer less from inter-observer variation compared to manual dWBC. In addition to microscopic or cytometric dWBC-techniques T-lymphocytes, CD4-T-lymphocytes, B-lymphocytes, NK-cells, myeloid progenitors, plasma cells, and blasts are determined by Leukoflow. These populations give potential useful clinical information and are subject for future studies focusing on the additional clinical value. Leukoflow is a highly interesting and promising technique for clinical laboratories.

Cell-mediated lipoprotein transport: A novel anti-atherogenic concept (Article)

2010-05-27T00:00:00Z

Lipoprotein transport is thought to occur in the plasma compartment of the blood, where lipoproteins are modulated by various enzymatic reactions. Subsequently, lipoproteins can migrate through the endothelial barrier to the subendothelial space or are taken up by the liver. The interaction between pro-atherogenic (apoB-containing) lipoproteins and blood cells (especially monocytes and macrophages) in the subendothelial space is well known. This lipoprotein-inflammatory cell interplay is central in the development of the atherosclerotic plaque. In this review, a novel interaction is described between lipoproteins and both leukocytes and erythrocytes in the blood compartment. This lipoprotein-blood cell interaction may also be related to the process of atherosclerosis by inducing inflammatory changes in the case of leukocytes (pro-atherogenic) and as an anti-atherogenic transport-system by adherence to erythrocytes. Triglyceride rich lipoprotein (TRL)-mediated leukocyte activation can lead to an inflammatory situation with generation of oxidative stress and the production of cytokines, ultimately resulting in acute endothelial dysfunction. Binding of apoB containing lipoproteins to erythrocytes may be a potential anti-atherogenic mechanism protecting the vessel wall from the pro-inflammatory effects of these lipoproteins and also playing a role in the removal of these particles from the circulation. One of the proposed mechanisms of this interaction implies complement activation on the lipoprotein surface and binding to the Complement Receptor 1 (CR1) on erythrocytes and leukocytes, followed by clearance by the liver.

Recent developments in testicular germ cell tumor research (Article)

2009-03-01T00:00:00Z

Testicular germ cell tumors of adolescents and adults (TGCTs; the so-called type II variant) are the most frequent malignancies found in Caucasian males between 20 and 40 years of age. The incidence has increased over the last decades. TGCTs are divided into seminomas and nonseminomas, the latter consisting of the subgroups embryonal carcinoma, yolk-sac tumor, teratoma, and choriocarcinoma. The pathogene-sis starts in utero, involving primordial germ cells/gonocytes that are blocked in their differentiation, and develops via the precursor lesion carcinoma in situ toward invasiveness. TGCTs are totipotent and can be considered as stem cell tumors. The developmental capacity of their cell of origin, the primordial germ cells/gonocyte, is demonstrated by the different tumor histologies of the invasive TGCTs. Seminoma represents the germ cell lineage, and embryonal carcinoma is the undifferentiated component, being the stem cell population of the nonseminomas. Somatic differentiation is seen in the teratomas (all lineages), whereas yolk-sac tumors and choriocarcinoma represent extra-embryonal differentiation. Seminomas are highly sensitive to irradiation and (DNA damaging) chemotherapy, whereas most nonseminomatous elements are less susceptible to radiation, although still sensitive to chemotherapy, with the exception of teratoma. To allow early diagnosis and follow up, appropriate markers are mandatory to discriminate between the different subgroups. In this review, a summary will be given related to several recent developments in TGCT research, especially selected because of their putative clinical impact.

Stem cell factor as a novel diagnostic marker for early malignant germ cells (Article)

2008-09-01T00:00:00Z

Carcinoma in situ (CIS) of the testis is the pre-invasive stage of type II testicular germ cell tumours (TGCTs) of adolescents and adults. These tumours are the most frequently diagnosed cancer in Caucasian adolescents and young adults. In dysgenetic gonads, the precursor of type II GCTs can be either CIS or a lesion known as gonadoblastoma (GB). CIS/GB originates from a primordial germ cell (PGC)/gonocyte, ie an embryonic cell. CIS can be cured by local low-dose irradiation, with limited side effects on hormonal function. Therefore, strategies for early diagnosis of CIS are essential. Various markers are informative to diagnose CIS in adult testis by immunohistochemistry, including c-KIT, PLAP, AP-2γ, NANOG, and POU5F1 (OCT3/4). OCT3/4 is the most informative and consistent in presence and expression level, resulting in intense nuclear staining. In the case of maturational delay of germ cells, frequently present in gonads of individuals at risk for type II (T)GCTs, use of these markers can result in overdiagnosis of malignant germ cells. This demonstrates the need for a more specific diagnostic marker to distinguish malignant germ cells from germ cells showing maturation delay. Here we report the novel finding that immunohistochemical detection of stem cell factor (SCF), the c-KIT ligand, is informative in this context. This was demonstrated in over 400 cases of normal (fetal, neonatal, infantile, and adult) and pathological gonads, as well as TGCT-derived cell lines, specifically in cases of CIS and GB. Both membrane-bound and soluble SCF were expressed, suggestive of an autocrine loop. SCF immunohistochemistry can be a valuable diagnostic tool, in addition to OCT3/4, to screen for precursor lesions of TGCTs, especially in patients with germ cell maturation delay.

Further characterization of the first seminoma cell line TCam-2 (Article)

2008-03-01T00:00:00Z

Testicular germ cell tumors of adolescents and adults (TGCTs) can be classified into seminomatous and nonseminomatous tumors. Various nonseminomatous cell lines, predominantly embryonal carcinoma, have been established and proven to be valuable for pathobiological and clinical studies. So far, no cell lines have been derived from seminoma which constitutes more than 50% of invasive TGCTs. Such a cell line is essential for experimental investigation of biological characteristics of the cell of origin of TGCTs, i.e., carcinoma in situ of the testis, which shows characteristics of a seminoma cell. Before a cell line can be used as model, it must be verified regarding its origin and characteristics. Therefore, a multidisciplinary approach was undertaken on TCam-2 cells, supposedly the first seminoma cell line. Fluorescence in situ hybridization, array comparative genomic hybridization, and spectral karyotyping demonstrated an aneuploid DNA content, with gain of 12p, characteristic for TGCTs. Genome wide mRNA and microRNA expression profiling supported the seminoma origin, in line with the biallelic expression of imprinted genes IGF2/H19 and associated demethylation of the imprinting control region. Moreover, the presence of specific markers, demonstrated by immunohistochemistry, including (wild type) KIT, stem cell factor, placental alkaline phosphatase, OCT3/4 (also demonstrated by a specific Q-PCR) and NANOG, and the absence of CD30, SSX2-4, and SOX2, confirms that TCam-2 is a seminoma cell line. Although mutations in oncogenes and tumor suppressor genes are rather rare in TGCTs, TCam-2 had a mutated BRAF gene (V600E), which likely explains the fact that these cells could be propagated in vitro. In conclusion, TCam-2 is the first well-characterized seminoma-derived cell line, with an exceptional mutation, rarely found in TGCTs.

Suppressors Of Cytokine Signaling in G-CSF-induced neutrophil development (Doctoral Thesis)

2004-06-24T00:00:00Z

G-CSF is the most important growth factor involved in the production of neutrophilic granulocytes. Signaling routes activated upon ligand binding to the G-CSF receptor (G-CSF-R) control survival, proliferation and differentiation of myeloid progenitor cells towards mature neutrophils. Elucidation of the signaling pathways involved in this process is important for understanding normal neutrophil development and for better insights in diseases with perturbed neutrophil production such as neutropenia and myeloid leukemia. In Chapter 1 of this thesis an overview of the current knowledge on the G-CSF-R is given and the signaling pathways activated by G-CSF are introduced. Upon GCSF binding, kinases of the JAK and Src kinase families become active and the four tyrosines of the receptor, located at positions 704, 729, 744 and 764 are phosphorylated. Multiple signaling pathways are subsequently activated via recruitment of intermediates to these phosphorylated tyrosines as well as via tyrosine independent mechanisms. A well known signaling route activated by the G-CSF-R is the JAK-STAT pathway. The most prominent STATs that are activated by G-CSF are STAT3, implicated in mediating a cell cycle arrest, and STAT5, which contributes to proliferation and cell survival.

G-CSF receptor truncations found in SCN/AML relieve SOCS3-controlled inhibition of STAT5 but leave suppression of STAT3 intact (Article)

2004-01-01T00:00:00Z

Truncated granulocyte colony-stimulating factor receptors (G-CSF-Rs) are implicated in severe congenital neutropenia (SCN) and the consecutive development of acute myeloid leukemia (AML). Mice expressing G-CSF-R truncation mutants (gcsfr-d715) show defective receptor internalization, an increased signal transducer and activator of transcription 5 (STAT5)/STAT3 activation ratio, and hyperproliferative responses to G-CSF treatment. We determined whether a lack of negative feedback by suppressor of cytokine signaling (SOCS) proteins contributes to the signaling abnormalities of G-CSF-R-d715. Expression of SOCS3 transcripts in bone marrow cells from G-CSF-treated gcsfr-d715 mice was approximately 60% lower than in wild-type (WT) littermates. SOCS3 efficiently suppressed STAT3 and STAT5 activation by WT G-CSF-R in luciferase reporter assays. In contrast, while SOCS3 still inhibited STAT3 activation by G-CSF-R-d715, STAT5 activation was no longer affected. This was due mainly to loss of the SOCS3 recruitment site Tyr729, with an additional contribution of the internalization defects of G-CSF-R-d715. Because Tyr729 is also a docking site for the Src homology 2-containing protein tyrosine phosphatase-2 (SHP-2), which binds to and inactivates STAT5, we suggest a model in which reduced SOCS3 expression, combined with the loss of recruitment of both SOCS3 and SHP-2 to the activated receptor complex, determine the increased STAT5/STAT3 activation ratio and the resulting signaling abnormalities projected by truncated G-CSF-R mutants.

Signaling mechanisms coupled to tyrosines in the granulocyte colony-stimulating factor receptor orchestrate G-CSF-induced expansion of myeloid progenitor cells (Article)

2003-01-01T00:00:00Z

Granulocyte colony-stimulating factor (G-CSF) is the major regulator of neutrophil production. Studies in cell lines have established that conserved tyrosines Tyr704, Tyr729, Tyr744, Tyr764 within the cytoplasmic domain of G-CSF receptor (G-CSF-R) contribute significantly to G-CSF-induced proliferation, differentiation, and cell survival. However, it is unclear whether these tyrosines are equally important under more physiologic conditions. Here, we investigated how individual G-CSF-R tyrosines affect G-CSF responses of primary myeloid progenitors. We generated G-CSF-R-deficient mice and transduced their bone marrow cells with tyrosine "null" mutant (m0), single tyrosine "add-back" mutants, or wild-type (WT) receptors. G-CSF-induced responses were determined in primary colony assays, serial replatings, and suspension cultures. We show that removal of all tyrosines had no major influence on primary colony growth. However, adding back Tyr764 strongly enhanced proliferative responses, which was reverted by inhibition of ERK activity. Tyr729, which we found to be associated with the suppressor of cytokine signaling, SOCS3, had a negative effect on colony formation. After repetitive replatings, the clonogenic capacities of cells expressing m0 gradually dropped compared with WT. The presence of Tyr729, but also Tyr704 and Tyr744, both involved in activation of signal transducer and activator of transcription 3 (STAT3), further reduced replating efficiencies. Conversely, Tyr764 greatly elevated the clonogenic abilities of myeloid progenitors, resulting in a more than 10(4)-fold increase of colony-forming cells over m0 after the fifth replating. These findings suggest that tyrosines in the cytoplasmic domain of G-CSF-R, although dispensable for G-CSF-induced colony growth, recruit signaling mechanisms that regulate the maintenance and outgrowth of myeloid progenitor cells