Jesper Kastrup

Changes in circulating mesenchymal stem cells, stem cell homing factor, and vascular growth factors in patients with acute ST elevation myocardial infarction treated with primary percutaneous coronary intervention

Objective: To investigate the spontaneous occurrence of circulating mesenchymal stem cells (MSC) and angiogenic factors in patients with ST elevation acute myocardial infarction (STEMI) treated with primary percutaneous coronary intervention (PCI). Design: In 20 patients with STEMI, blood samples were obtained on days 1, 3, 7, 14, 21, and 28 after the acute PCI. Fifteen patients with a normal coronary angiography formed a control group. MSC (CD45−/CD34−), plasma stromal derived factor 1 (SDF-1), vascular endothelial growth factor A (VEGF-A), and fibroblast growth factor 2 (FGF-2) were measured by multiparametric flow cytometry and enzyme linked immunosorbent assay (ELISA). Results: Circulating CD45−/CD34− cells were significantly decreased on day 7 compared with day 3. Cell counts normalised one month after the acute onset of STEMI. The changes were mainly seen in patients with a large infarction. Plasma SDF-1 increased significantly from day 3 to day 28, and VEGF-A and FGF-2 increased significantly from day 7 to day 28. Conclusions: Spontaneous sequential fluctuations in MSC and the increase in vascular growth factor concentrations after STEMI suggest that the optimal time for additional stem cell therapy is three weeks after a myocardial infarction to obtain the maximum effects by stimulating endogenous growth factors on the delivered stem cells.

Leucine-based Receptor Sorting Motifs Are Dependent on the Spacing Relative to the Plasma Membrane

Many integral membrane proteins contain leucine-based motifs within their cytoplasmic domains that mediate internalization and intracellular sorting. Two types of leucine-based motifs have been identified. One type is dependent on phosphorylation, whereas the other type, which includes an acidic amino acid, is constitutively active. In this study, we have investigated how the spacing relative to the plasma membrane affects the function of both types of leucine-based motifs. For phosphorylation-dependent leucine-based motifs, a minimal spacing of 7 residues between the plasma membrane and the phospho-acceptor was required for phosphorylation and thereby activation of the motifs. For constitutively active leucine-based motifs, a minimal spacing of 6 residues between the plasma membrane and the acidic residue was required for optimal activity of the motifs. In addition, we found that the acidic residue of leucine-based motifs must be located amino-terminal to the dileucine sequence for proper function of the motifs and that residues surrounding the motifs affect the activity of the motifs. Thus, our observations suggest that the position, the exact sequence, and surrounding residues are major determinants of the function of leucine-based receptor sorting motifs.

Comparison of different culture conditions for human mesenchymal stromal cells for clinical stem cell therapy

Objective. Mesenchymal stromal cells (MSCs) from adult bone marrow (BM) are considered potential candidates for therapeutic neovascularization in cardiovascular disease. When implementing results from animal trials in clinical treatment, it is essential to isolate and expand the MSCs under conditions following good manufacturing practice (GMP). The aims of the study were first to establish culture conditions following GMP quality demands for human MSC expansion and differentiation for use in clinical trials, and second to compare these MSCs with MSCs derived from culture in four media commonly used for MSC cultivation in animal studies simulating clinical stem cell therapy. Material and methods. Human mononuclear cells (MNCs) were isolated from BM aspirates by density gradient centrifugation and cultivated in a GMP‐accepted medium (EMEA medium) or in one of four other media. Results. FACS analysis showed that the plastic‐adherent MSCs cultured in EMEA medium or in the other four media were identically negative for the haematopoietic surface markers CD45 and CD34 and positive for CD105, CD73, CD90, CD166 and CD13, which in combined expression is characteristic of MSCs. MSC stimulation with vascular endothelial growth factor (VEGF) increased expression of the characteristic endothelial genes KDR and von Willebrand factor; the von Willebrand factor and CD31 at protein level as well as the capacity to develop capillary‐like structures. Conclusions. We established culture conditions with a GMP compliant medium for MSC cultivation, expansion and differentiation. The expanded and differentiated MSCs can be used in autologous mesenchymal stromal cell therapy in patients with ischaemic heart disease.

TCRζ is transported to and retained in the Golgi apparatus independently of other TCR chains: implications for TCR assembly

It is generally assumed that TCR assembly occurs in the endoplasmic reticulum (ER), and ER retention/degradation signals have been identified in several of the TCR chains. These signals are probably responsible for retention of incompletely assembled TCR complexes and free TCR chains in the ER. This study focused on the intracellular localization and transport of partially assembled TCR complexes as determined by confocal microscopy analyses. We found that none of the TCR chains except for TCRζ were allowed to exit the ER in T cell variants in which the hexameric CD3γ ϵTiα βCD3δ ϵ complex was not formed. Interestingly, TCRζ was exported from the ER independently of other TCR chains and was predominantly located in a compartment identified as the Golgi apparatus. Furthermore, in the TCRζ‐negative cell line MA5.8, the hexameric CD3γ ϵTiα βCD3δ ϵ complex was allowed to exit the ER and was also predominantly located in the Golgi apparatus. However, neither hexameric TCR complexes nor TCRζ chains were efficiently expressed at the cell surface without the other. The observations that TCRζ and hexameric TCR complexes are transported from the ER to the Golgi apparatus independently of each other and that these partial TCR complexes are unable to be efficiently expressed at the cell surface suggest that final TCR assembly occurs in the Golgi apparatus.

The phosphorylation state of CD3gamma influences T cell responsiveness and controls T cell receptor cycling.

The T cell receptor (TCR) is internalized following activation of protein kinase C (PKC) via a leucine (Leu)-based motif in CD3γ. Some studies have indicated that the TCR is recycled back to the cell surface following PKC-mediated internalization. The functional state of recycled TCR and the mechanisms involved in the sorting events following PKC-induced internalization are not known. In this study, we demonstrated that following PKC-induced internalization, the TCR is recycled back to the cell surface in a functional state. TCR recycling was dependent on dephosphorylation of CD3γ, probably mediated by the serine/threonine protein phosphatase-2A, but independent on microtubules or actin polymerization. Furthermore, in contrast to ligand-mediated TCR sorting, recycling of the TCR was independent of the tyrosine phosphatase CD45 and the Src tyrosine kinases p56Lckand p59Fyn. Studies of mutated TCR and chimeric CD4-CD3γ molecules demonstrated that CD3γ did not contain a recycling signal in itself. In contrast, the only sorting information in CD3γ was the Leu-based motif that mediated lysosomal sorting of chimeric CD4-CD3γ molecules. Finally, we found a correlation between the phosphorylation state of CD3γ and T cell responsiveness. Based on these observations a physiological role of CD3γ and TCR cycling is proposed.

High numbers of clonal CD19+ cells in the peripheral blood of a patient with multiple myeloma

Recent studies concerning the numbers of circulating clonal B cells in patients with multiple myeloma (MM) have reported conflicting data regarding the exact levels of clonal B cells and the existence of clonal cells in the CD34 compartment. In this report we show that high numbers of clonal cells with a phenotype of late‐stage B cells or pre‐plasma cells were present in the peripheral blood (PB) of a patient with MM. During treatment the initial high level of PB clonal cells was markedly reduced and remained low (<1%) post transplant, even after disease progression. In addition, we found that the MM clone did not include a B‐progenitor population defined by CD34.

Ceramide-induced TCR up-regulation.

The TCR is a constitutively recycling receptor meaning that a constant fraction of TCR from the plasma membrane is transported inside the cell at the same time as a constant fraction of TCR from the intracellular pool is transported to the plasma membrane. TCR recycling is affected by protein kinase C activity. Thus, an increase in protein kinase C activity affects TCR recycling kinetics leading to a new TCR equilibrium with a reduced level of TCR expressed at the T cell surface. Down-regulation of TCR expression compromises T cell activation. Conversely, TCR up-regulation is expected to increase T cell responsiveness. The purpose of this study was to identify and characterize potential pathways for TCR up-regulation. We found that ceramide affected TCR recycling dynamics and induced TCR up-regulation in a concentration- and time-dependent manner. Experiments applying phosphatase inhibitors indicated that ceramide-induced TCR up-regulation was most probably mediated by serine/threonine protein phosphatase 2A. Analyses of T cell variants demonstrated that TCR up-regulation was dependent on the presence of an intact CD3γ L-based motif and thus acted on TCR engaged in the recycling pathway. Finally, we showed that TCR up-regulation probably plays a physiological role by increasing T cell responsiveness. Thus, by affecting the TCR recycling kinetics, T cells have the potential both to up- and down-regulate TCR expression and thereby adjust T cell responsiveness.

T-cell receptor downregulation by ceramide-induced caspase activation and cleavage of the zeta chain.

Regulation of T‐cell receptor (TCR) cell surface expression levels is probably an important mechanism by which T‐cell responsiveness is controlled. Previously, two distinct pathways for TCR downregulation have been described. One is dependent on protein kinase C (PKC) and the leucine‐based receptor‐sorting motif (l‐based motif) of the CD3γ chain but independent of tyrosine kinases, whereas the other is dependent on the tyrosine kinase activation but independent of the PKC and the CD3γl‐based motif. In this study, we describe a new pathway for TCR downregulation distinct from both the PKC/CD3γl‐based motif‐dependent and the tyrosine kinase‐dependent pathways. This pathway is dependent on ceramide‐induced activation of caspases and correlate with caspase‐mediated cleavage of the ζ chain. Thus, a 10–15% downregulation of the TCR was induced following the treatment of the T cells with ceramide for 4 h. A close correlation between TCR downregulation, caspase activation, and cleavage of the ζ chain was found. Furthermore, the caspase inhibitors abolished the cleavage of the ζ chain and TCR downregulation in parallel with the inhibition of the caspase activity.

β2-Adaptin is constitutively de-phosphorylated by serine/threonine protein phosphatase PP2A and phosphorylated by a staurosporine-sensitive kinase

Clathrin-mediated endocytosis includes cycles of assembly and disassembly of the clathrin-coated vesicle constituents. How these cycles are regulated is still not fully known but previous studies have indicated that phosphorylation of coat subunits may play a role. Here we describe that beta2-adaptin undergoes cycles of phosphorylation/de-phosphorylation in intact cells. Thus, beta2-adaptin was constitutively de-phosphorylated by serine/threonine protein phosphatase 2A and phosphorylated by a staurosporine-sensitive kinase in vivo. Confocal laser scanning microscopy demonstrated that phosphorylated AP2 complexes were found more evenly distributed at the plasma membrane compared to non-phosphorylated AP2 complexes which were found in aggregates. Finally, we found that phosphorylation of beta2-adaptin correlated with inhibition of clathrin-mediated endocytosis. Our results support the hypothesis that phosphorylation/de-phosphorylation of coat proteins plays a regulatory role in the assembly/disassembly cycle of clathrin-coated vesicles.

In vitro production and characterization of partly assembled human CD3 complexes.

Pairwise assembly of human CD3 chains takes place in the endoplasmic reticulum of T cells. Subsequently, the CD3 heterodimers form complexes with Tiα and Tiß chains forming hexameric TiαβCD3γεδε complexes. Finally, association with the ζ2 homodimer occurs in Golgi apparatus before the fully assembled T‐cell receptor is transported to the cell surface. To study the structural properties of the human CD3 chains, we have developed new methods to produce and fold the extracellular domains of CD3γ, CD3δ and CD3ε. Proteins were expressed in Escherichia coli as denatured chains and de novo folded in vitro. CD3γ and CD3ε folded as soluble monomers, whereas CD3δ did not yield any soluble proteins. When folding the chains pairwise, soluble CD3γε and CD3δε heterodimers could be isolated, whereas CD3γδ heterodimers were not produced. Using antibodies as structural probes, we identified two different types of antigenic epitopes that were dependent on heterodimerization. Our data indicate that CD3ε undergoes a conformational change after dimerization with CD3γ or CD3δ. Furthermore, we demonstrated that the CD3γε heterodimer could be purified using immunoaffinity chromatography.