Gernot Bruchelt

Adult stem cells as an alternative source of multipotential (pluripotential) cells in regenerative medicine.

Embryonic stem cells are by definition the master cells capable of differentiating into every type of cells either in vitro or in vivo. Several lines of evidence suggest, however, that adult stem cells and even terminally differentiated somatic cells under appropriate microenvironmental cues are able to be reprogrammed and contribute to a much wider spectrum of differentiated progeny than previously anticipated. This has been demonstrated by using tissue- specific stem cells, which like embryonic stem cells do not express CD45 as an exclusive hematopoietic marker (skin, adipose, cord blood and bone marrow- derived stem cells). On the other side, there is a great number of reports which demonstrate that hematopoietic cells (CD45+) from different sources (peripheral blood, cord blood, bone marrow) are also able to cross the tissue boundaries and give rise to the cells of the other germ layers. Herein we discuss the differentiation and reprogramming potential of both hematopoietic and non- hematopoietic stem cells along endodermal, mesodermal and neuroectodermal lineage and their importance for regenerative medicine.

Phagocytic Activity and Oxidative Burst of Granulocytes in Persons with Myeloperoxidase Deficiency

In the present study, phagocytosis and the oxidative metabolism of neutrophil granulocytes from five clinically healthy persons with different degrees of myeloperoxidase deficiency were investigated and compared to those of normal persons. The identification of individuals with myeloperoxidase deficiency was performed with the Bayer/Technicon H3 blood cell counter, which differentiates the leukocytes by measuring the peroxidase activity. Neutrophils of three out of five investigated myeloperoxidase deficient persons showed extremely low peroxidase indices (-53 and lower), but only the neutrophils of one person totally lacked myeloperoxidase. This was demonstrated by comparing myeloperoxidase mass concentration measured with an enzyme immunoassay, lack of HOCl production, and was further confirmed by measuring luminol- and lucigenin-enhanced chemiluminescence. Characteristically, myeloperoxidase deficient granulocytes showed a strikingly decreased luminol-enhanced chemiluminescence while the lucigenin-enhanced chemiluminescence was significantly increased compared to normal granulocytes. Although there is a DNA sequence homology of about 70%, the activity of peroxidase in eosinophils was not affected in any myeloperoxidase deficient person investigated. Moreover, a person with a very rare defect of eosinophil peroxidase had completely normal myeloperoxidase activity. The lack of myeloperoxidase activity is compensated for by an increased phagocytic activity, an increased production of superoxide anion (lucigenin-chemiluminescence) and probably by an alternative metabolism of H2O2; since persons lacking myeloperoxidase activity do not normally suffer from severe infections, H2O2 is obviously metabolized to other reactive oxygen substrates than HOCl, e.g. to OH-radicals.

Uptake of mIBG and catecholamines in noradrenaline- and organic cation transporter-expressing cells: potential use of corticosterone for a preferred uptake in neuroblastoma- and pheochromocytoma cells

For imaging of neuroblastoma and phaeochromocytoma, [(123)I]meta-iodobenzylguanidine ([(123)I]mIBG) is routinely used, whereas [(18)F]6-fluorodopamine ([(18)F]6-FDA) is sporadically applied for positron emission tomography in pheochromocytoma. Both substances are taken up by catecholamine transporters (CATs). In competition, some other cell types are able to take up catecholamines and related compounds probably by organic cation (OCT) [extraneuronal monoamine (EMT)] transporters (OCT1, OCT2, OCT3=EMT). In this study, we investigated the uptake of radioiodine-labeled meta-iodobenzylguanidine (mIBG) as well as [(3)H]dopamine (mimicring 6-fluorodopamine) and [(3)H]noradrenaline. SK-N-SH (neuroblastoma) and PC-12 (phaeochromocytoma) cells were used and compared with HEK-293 cells transfected with OCT1, OCT2 and OCT3, respectively. In order to gain a more selective uptake in CAT expressing tumor cells, different specific inhibitors were measured. Uptake of mIBG into OCT-expressing cells was similar or even better as into both CAT-expressing cell lines, whereas dopamine and noradrenaline uptake was much lower in OCT-expressing cells. In presence of corticosterone (f.c. 10(-4) M], catecholamine and mIBG uptake into SK-N-SH and PC-12 cells was only slightly reduced. In contrast, this process was significantly inhibited in OCT2 and OCT3 transfected HEK-293 as well as in Caki-1 cells, which naturally express OCT3. We conclude that the well-known corticosteroid corticosterone might be used in combination with [(18)F]6-FDA or [(123)I]mIBG to improve specific imaging of neuroblastoma and pheochromocytoma and to reduce irradiation dose to nontarget organs in [(131)I]mIBG treatment.

Influence of Dichloroacetate (DCA) on Lactate Production and Oxygen Consumption in Neuroblastoma Cells: Is DCA a Suitable Drug for Neuroblastoma Therapy?

Many cancer cells metabolize glucose preferentially via pyruvate to lactate instead to CO2 and H2O (oxidative phosphorylation) even in the presence of oxygen (Warburg effect). Dichloroacetate (DCA) is a drug which is able to shift pyruvate metabolism from lactate to acetyl-CoA (tricarboxylic acid cycle) by indirect activation of pyruvate dehydrogenase (PDH). This can subsequently lead to an increased flow of oxygen in the respiratory chain, associated with enhanced generation of reactive oxygen species (ROS) which may cause apoptosis. In order to investigate if DCA may be suitable for neuroblastoma therapy, it was investigated on three human neuroblastoma cell lines whether DCA can reduce lactate production and enhance oxygen consumption. The data show, that DCA (in the low millimolar range) is able to reduce lactate production, but there was only a slight shift to increased oxygen consumption and almost no effect on cell vitality, proliferation and apoptosis of the three cell lines investigated. Therefore, DCA at low millimolar concentrations seems to be only of minor efficacy for neuroblastoma treatment.

Analysis of metabolites of glucose pathways in human erythrocytes by analytical isotachophoresis.

The aim of this study was to establish an isotachophoretic (ITP) method for the determination of the main compounds of glycolysis in human erythrocytes in order to analyze the influence of different glucose concentrations (mimicking the situation in diabetes mellitus) on this pathway. Samples for ITP were prepared by isolation of erythrocytes, lysis of the cells by heating in double-distilled water and subsequent ultrafiltration (Mr cut-off: 5000). All the main compounds of glycolysis were characterized by ITP. The influence of different glucose concentrations on the main compounds of the energy metabolism (ATP, ADP, lactate, pyruvate) and 2,3-diphosphoglycerate were analyzed in short- and long-time incubations.

Potential pitfalls of comparative measurements of reticulocytes with flow cytometry and microscopy in prematures and infants

Abstract Reticulocyte counting by flow cytometry (Bayer H*3, ADVIA 120) in blood of prematures, infants and children > 1 year of age was compared with microscopic counting under research conditions (9000 counted red blood cells per slide). While in children > 1 year a good concordance of both methods was observed, 2.3–2.4-fold higher values were obtained in neonates by microscopy (Brilliant Cresyl Blue stain, 0.5%). However, another laboratory found good agreement between H*3-counting and microscopy in samples also obtained from neonates using the same methods. Despite very similar results for all age groups in comparative flow cytometry measurements in both laboratories, counting of smears from neonates differed, showing an approximately 2.3-fold larger amount of reticulocytes in our laboratory. The reason for these observations was a greater enlargement (1250-fold) used routinely in our laboratory compared with 800-fold in the other one. Thus very mature reticulocytes frequently found in neonates could only be detected using a 1250-fold enlargement. Similarly, the low concentration of the colouring matter used in the H*3 (0.0005% oxazin or 0.001% ADVIA 120) is obviously not sufficient for detection of mature reticulocytes. Therefore, it is important to consider this phenomenon and to standardise microscopic enlargement, especially for comparisons in multicentre studies.

Isotachophoretic analysis of the dihydrofolate reductase reaction in the presence of methotrexate and ascorbic acid

The antifolate methotrexate (MTX) is widely used in cancer chemotherapy. In this study, we show that MTX (MTX‐Glu1) and MTX‐polyglutamates (MTX‐Glu2—5) strongly inhibited the growth of the leukemic cell line MOLT‐4. This effect, however, was mitigated by ascorbic acid. We investigated whether ascorbic acid is able to reduce dihydrofolic acid (DHF) to tetrahydrofolic acid (THF) directly or by circumventing the MTX inhibition of dihydrofolate reductase (DHFR). The inhibition of this NADPH‐dependent reduction of DHF by MTX‐Glun in the absence or presence of ascorbate, was determined by analytical isotachophoresis. Using 0.01 M HCl/histidine, pH 6.0, as a leading electrolyte (L) and 0.005 M 2‐(N‐morpholino)ethanesulfonic acid (MES)/histidine, pH 6.0, as a terminating electrolyte (T), MTX‐Glun derivatives including MTX‐Glu1 could be easily separated, whereas the quantitative estimation of THF was not possible. A quantitative characterization of the DHFR reaction by measuring NADPH, NADP+ and ascorbate was achieved with another system (L: 0.01 M HCl/β‐alanine, pH 3.73; T: 0.01 M caproic acid, pH 3.27). Nanomolar concentrations of MTX‐Glu1—5 inhibited consumption of NADPH and production of NADP+. Ascorbic acid was not able to reduce DHF, neither directly nor after inhibition of DHFR by MTX. However, ascorbic acid seemed to diminish the oxidation of THF and this may account for its capacity to reduce the inhibitory effect of MTX on MOLT‐4 cells.

Nifurtimox reduces N-Myc expression and aerobic glycolysis in neuroblastoma

Neuroblastoma is one of the most common solid tumors in childhood and usually accompanied with poor prognosis and rapid tumor progression when diagnosed with amplification of the proto-oncogene N-Myc. The amplification of N-Myc has major influence on the maintenance of aerobic glycolysis, also known as the Warburg effect. This specific switch in the conversion of pyruvate to lactate instead of the conversion of pyruvate to acetyl-coenzyme A even in the presence of oxygen has important benefits for the tumor, e.g. increased production of enzymes and enzyme substrates that are involved in tumor progression, angiogenesis and inhibition of apoptosis. The antiprotozoal drug nifurtimox, which is generally used for the treatment of infections with the parasitic protozoan Trypanosoma cruzi, has been reported to have cytotoxic properties in the therapy of neuroblastoma. However, its action of mechanism has not been described in detail yet. The presented in vitro study on the neuroblastoma cell lines LA-N-1, IMR-32, LS and SK-N-SH shows an increased production of oxidative stress, a reduced lactate dehydrogenase enzyme activity and reduced lactate production after nifurtimox treatment. Furthermore, nifurtimox leads to reduced mRNA and protein levels of the proto-oncogene protein N-Myc. Thus, the current work gives new insights into the effect of nifurtimox on tumor metabolism revealing a shifted glucose metabolism from production of lactate to oxidative phosphorylation and a reduced expression of the major molecular prognostic factor in neuroblastoma N-Myc, presenting nifurtimox as a possible adjuvant therapeutic agent against (high risk) neuroblastoma.

Low-density lipoprotein modification by normal, myeloperoxidase-deficient and NADPH oxidase-deficient granulocytes and the impact of redox active transition metal ions

Abstract The modification of low-density lipoprotein (LDL) by normal, myeloperoxidase (MPO)-deficient and NADPH oxidase-deficient granulocytes was investigated using the monoclonal antibody (mAb) OB/04, which was originally generated against copper-oxidized LDL. Incubation of LDL with normal granulocytes increased the reactivity of LDL with mAb OB/04. These effects were even more pronounced using MPO-deficient granulocytes. Inhibitors of oxidative reactions (the NADPH oxidase inhibitor diphenyleneiodonium chloride [DPI], catalase, superoxide dismutase [SOD]) did not significantly reduce LDL oxidation by normal granulocytes. Furthermore, granulocytes of a patient with NADPH oxidase deficiency were almost equally effective as normal granulocytes, indicating that oxidative burst-derived reactive oxygen species are of only minor importance in the generation of mAb OB/04-detectable new epitopes on LDL in vitro. In contrast, incubation of LDL with iron and copper prior to and during incubation with normal granulocytes markedly enhanced the generation of OB/04-detectable epitopes. It is supposed that, besides superoxide (in normal and MPO-deficient granulocytes) or instead of superoxide (in NADPH oxidase-deficient granulocytes), lytic enzymes released by activated granulocytes may enhance the availability of transition metals for oxidation of LDL. Our results support the concept that transition-metal-dependent pathways of LDL oxidation in combination with degranulation products of granulocytes are important.

Differences in the accumulation of ascorbic acid in normal, myeloperoxidase deficient and NADPH-oxidase deficient granulocytes.

Granulocytes contain large quantities of ascorbic acid (AA). The uptake mechanism is mainly restricted to the accumulation of the oxidized form, dehydroascorbate (DHA). We investigated the uptake of ascorbic acid and dehydroascorbate of normal, myeloperoxidase (MPO)-deficient, and NADPH-oxidase-deficient granulocytes. The accumulation of ascorbic acid was increased in all types of granulocytes after stimulation with phorbol-myristate-acetate, whereas the NADPH-oxidase-deficient cells showed a decreased uptake compared to normal and MPO-deficient cells. The intracellular concentration of ascorbic acid was further enhanced after incubation of granulocytes with DHA, most prominently in NADPH-oxidase-deficient granulocytes. MPO-deficient granulocytes are not able to produce HOCl after activation. The granulocytes of one individual with total MPO deficiency accumulated ascorbate in higher concentrations than did cells with partial MPO deficiency, indicating that HOCl is of minor importance for the oxidation of ascorbate. Since the ability of MPO-deficient cells to kill microorganisms is pronounced in contrast to NADPH-oxidase-deficient cells, effective mechanisms of compensating for the absence of HOCl must exist. We hypothesize that the enhanced uptake of ascorbic acid combined with an enhanced superoxide anion production may favor the generation of OH radicals via the Fenton reaction.