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Chemicals/CAS: Serum Albumin, Bovine

Adherent cells from mouse bone marrow have been shown to promote the in vitro growth of the AVRij-1 tumour cell line. The experiments presented here suggest that the adherent cells involved in this phenomenon are the progeny of bone marrow derived fibroblastoid colony forming units. The latter were characterized by means of cell density distribution analysis. They had a broad distribution pattern and an average peak cell density of 1.069 g.cm-3. The growth promotion activity exerted by adherent cell layers from the various density fractions on the AVRij-1 tumour cell line coincided with the distribution of fibroblastoid colony forming units. On the other hand, the presence of macrophages in the adherent layers seemed to be non-essential for the in vitro promotion of growth of the AVRij-1 cell line.

Chemicals/CAS: glucose 6 phosphate dehydrogenase, 37259-83-9, 9001-40-5; Glucosephosphate Dehydrogenase, EC 1.1.1.49

CD4+ T cells from young and aged mice were sorted into Mel-14+ cells which are regarded as naive cells and Mel-14- cells which are regarded as memory cells. These subsets were stimulated in short-time cultures with anti-CD3 or anti-CD3/anti-CD28 in order to determine the presence of Th1 and/or Th2 cytokines. Based on the simultaneous production of IL-2, IL-4, IL-10, and IFN-γ upon anti-CD3 stimulation by Mel-14- cells from young and aged mice, it is concluded that this cell population comprises Th1, Th2, and/or Th0 cells. Mel-14+ cells from young mice only secrete substantial amounts of IL-2 in the presence of anti-CD28 as a costimulatory signal and can therefore be regarded as Th precursor cells. By contrast, Mel-14+ cells from aged mice responded to anti-CD3 alone, not only by the production of IL-2 but also by the production of high amounts of IFN-γ and minute amounts of IL-4 and IL-10, suggesting that these 'naive' cells in aged mice are enriched for Th1 cells. This was not due to lack of CD28 triggering since anti-CD28 enhanced IFN-γ as well as IL-4 and IL-10 to a similar extent. Our data therefore indicate that Mel-14 is not exclusively expressed on naive CD4+ T cells.

A method is described for isolation of antibody forming cells with the help of cluster formation. This was achieved by incubating spleen cells from immunized mice with sheep erythrocytes at 37°C. During incubation rabbit anti mouse immunoglobulin serum was present in order to facilitate the formation of clusters. Thereafter the clusters were isolated by velocity sedimentation. In this way suspensions were obtained containing more than 80% clusters. Some of the cells in the clusters still produced antibodies against sheep erythrocytes cultured for two days in vitro.

Precision-cut liver slices are frequently used to study hepatic toxicity and metabolism of xenobiotics in vitro. Successful cryopreservation techniques will enhance an efficient and economic use of scarcely available (human) liver tissue. For primary hepatocytes, slow freezing has been accepted as the best approach towards successful cryopreservation. For slices, however, no agreement exists on the optimal way of cryopreservation and both slow and fast freezing techniques have been reported. The aim of the present study was to determine the applicability of a computer-controlled slow freezing technique for the cryopreservation of (rat) liver slices. Thus far, this technique has not been described in detail. Our studies confirmed that slow freezing was most successful in the cryopreservation of primary rat hepatocytes. Based on this observation, the slow freezing technique was applied to the cryopreservation of rat liver slices. Directly after thawing, slice viability was between 60 and 100% of fresh values, depending on the parameter determined. However, after additional culturing, slice viability was reduced. This decrease in slice viability was more pronounced in comparison to primary hepatocytes. In conclusion, the slow freezing technique was confirmed to be a successful approach for the cryopreservation of primary rat hepatocytes, and was found to be of limited use for the cryopreservation of rat liver slices. Copyright (C) 2000 Elsevier Science Ltd. Chemicals/CAS: Adenosine Triphosphate, 56-65-5; Dinitrochlorobenzene, 97-00-7; Formazans; Glutathione Transferase, EC 2.5.1.18; Glutathione, 70-18-8; Lactate Dehydrogenase, EC 1.1.1.27; MTT formazan, 23305-68-2; Proteins; Testosterone, 57-85-2; Tetrazolium Salts; Urea, 57-13-6

For in vitro evaluation of functional properties of endothelial cells seeded on synthetic vascular prostheses accurate and reproducible quantification of cells is mandatory. Comparison of these properties with those resulting from other studies requires correlation of the functional parameters to reliably counted cell numbers. The accuracy of methods of quantification currently being used is unknown due to the lack of a "gold standard" method to which these methods can be compared. To determine the accuracy and reproducibility of four widely used methods, we have developed a "gold standard" model, using a flow cytometer (FACS). Endothelial cells, attached to collagen-coated Dacron vascular prostheses, were counted by four conventional methods and a new method of quantification after the attached number of cells had been determined with 99% accuracy by FACS. Subsequently, ratios were computed by dividing the cell numbers determined by the methods under investigation by those determined by FACS (×100%). The four conventional methods investigated were (1) removal and subsequent counting of cells from substrata by trypsin (T), (2) digestion of cells by citric acid and counting of crystal violet-stained cell nuclei (CV), (3) light microscopy after hematoxylin staining (LM), and (4) scanning electron microscopy (SEM). The new method consists of the measurement of cell fluorescence after labeling with fluorescein-diacetate (FDA). T and CV had average accuracy ratios of 127 ± 58% and 96 ± 48%, respectively (± standard deviation). The ratios for LM and SEM were 116 ± 101% and 44 ± 10% (respectively). FDA had a ratio of 99 ± 7%. Reproducibility of cell quantification by T and CV was significantly less than that of quantification by LM, SEM, and FDA, as expressed by data on inter- and intraobserver agreement. Our results indicate that the investigated conventional methods of quantification failed to meet criteria of both high accuracy and reproducibility. Light microscopy and scanning microscopy methods were inaccurate but yielded reproducible countings. We conclude that the FACS method can serve as a "gold standard" to compare the accuracy and reproducibility of cell quantification methods. Moreover, the FDA method results in both accurate and reproducible quantification of endothelial cells attached to vascular prosthetic material. © 1998 Academic Press, Inc. Chemicals/CAS: 3',6'-diacetylfluorescein, 596-09-8; Fluoresceins

The transplantable C57BL/KaLwRij mouse 5T2 multiple myeloma (MM) is a new animal model for studies on MM in man. Histological examination of the 5T2 MM cells revealed their morphological heterogeneity. In this study we investigated whether this heterogeneity reflects subpopulations of 5T2 MM cells with different biological properties. 5T2 MM bone marrow cells were separated according to their sedimentation velocity (s.v.). When intravenously injected into syngeneic recipient mice, cells with s.v. of 8 mm h-1 led to the development of detectable 5T2 MM after 6 weeks; in contrast, 18 weeks elapsed before the same result was achieved with cells of s.v. lower than 5 mm h-1. Flow cytometric analysis revealed that 5T2 MM cells had an aneuploid DNA content and that most cycling 5T2 MM cells were larger, their s.v. rate exceeding 9 mm h-1. It was further demonstrated that about half of all aneuploid cells carried on their membrane the 5T2 MM idiotype. The majority of the idiotype-positive cells had s.v. rate exceeding 6.5 mm h-1 (16%-39%) or lower than 3 mm h-1 (16%-19%). The 5T2 MM was shown to contain subpopulations of cells of different size, proliferation capacity and expression of their membrane 5T2 idiotype; this, most likely reflects cells in different stages of differentiation. The mouse 5T2 MM corresponds also in this respect with MM in man. Chemicals/CAS: DNA, Neoplasm

A review is presented of the experiments that resulted in the identification of a specific morphologic entity representing the pluripotential hemopoietic stem cell (HSC) in mouse bone marrow. This entity was subsequently discovered in concentrated HSC preparations from bone marrow of rats, monkeys, and humans. In the mouse, a set of physical paramaters (of the HSC) has been collected which agree with its morphologic description. It was also shown that these physical properties, and a number of cell surface properties, do not enable a distinction between HSC and its immediate descendants, the G/M CFU 1 and the E-BFU. The factors that stimulate proliferation of these three cell types have been isolated from human leukocyte conditioned medium and mouse spleen conditioned medium and were partly purified and characterized. The information at present indicates that the three cell types respond to closely related, if not identical, factors. Direct counts of HSC in electron microscopic preparations of density gradient fractions of different enrichment have been compared with HSC values computed from spleen colony counts and f factors for rat and mouse marrow. A high degree of correlation was found between the two types of observations. The slopes of the regression lines for mouse marrow fractions, for concentrates of normal rat marrow, and for concentrates of cycling rat marrow were the same, namely 0.5. The deviation of this value from the expected value of 1.0 is probably not due to the use of erroneous f values. It is proposed that the observed discrepancy may be due to heterogeneity of spleen colony forming cells, in that a proportion of them may not be pluripotential.

The ability of coumarin to induce UDS in male Sprague-Dawley CD rat hepatocytes in vivo was assessed using the unscheduled DNA synthesis (UDS) assay. From a preliminary toxicity study the oral maximum tolerated dose (MTD) of coumarin was determined to be 320mg/kg body weight. For the UDS studies, rats were treated with 0 (corn oil control), 32 (one-tenth the MTD), 107 (one-third the MTD) and 320 (MTD) mg/kg coumarin via oral gavage. Rats were also treated with 20mg/kg body weight dimethylnitrosamine (DMN) or 50mg/kg body weight 2-acetylaminofluorene (2-AAF) as positive controls for the 2-4hr and 12-16hr expression of UDS, respectively. Hepatocytes were isolated by liver perfusion either 2-4hr or 12-16hr after treatment and cultured in medium containing [methyl-³H]thymidine for 4hr and assessed for UDS by grain counting of autoradiographs. Coumarin treatment at doses of 32-320mg/kg body weight had no statistically significant or dose-related effect on UDS in rat hepatocytes either 2-4hr or 12-16hr after dosing. In contrast, both DMN 2-4hr after dosing and 2-AAF 12-16hr after dosing produced significant increases in UDS assessed as the net nuclear grain count. Both genotoxins also increased the percentage of hepatocyte nuclei with greater than 5 net grains. Treatment with coumarin, DMN and 2-AAF had no statistically significant effect on the proportion of rat hepatocytes undergoing replicative DNA synthesis. In summary, this study demonstrates that coumarin does not induce UDS in hepatocytes of male Sprague-Dawley CD rats after oral administration at doses up to the MTD of 320mg/kg. The responsiveness of the animals used in this study to genotoxic agents was demonstrated by the clear induction of DNA repair after treatment with DMN and 2-AAF. Copyright (C) 2000 Elsevier Science Ltd. Chemicals/CAS: 2-Acetylaminofluorene, 53-96-3; Anticoagulants; Coumarins; Dimethylnitrosamine, 62-75-9; DNA, 9007-49-2; Mutagens

Iron absorption can be measured by the incorporation of stable iron isotopes into erythrocytes, 14 days after isotope administration. The disadvantage of this method is the high dose of isotopes needed to obtain a sufficient enrichment. Therefore, in this study cell fractions rich in young erythroid cells were prepared by using a density separation method. From 10 women blood was taken 4, 5, and 7 days after oral and intravenous administration of 57Fe and 58Fe. In these cell fractions and in whole blood taken 14 days after isotope administration, isotope enrichment was measured and absorption calculated. Absorption calculated from the isotope enrichment in the reticulocyte-rich cell fractions (12.2 ± SEM 3.7%) was not significantly different from absorption based on higher isotope enrichment was found in the cell fractions, the required dose of stable isotopes can be reduced to one-third of the dose used in the traditional method without loss of sensitivity.

The effect of intravenously injected endotoxin on inflammatory cells within solid Meth A tumours was studied and central hyperaemia, necrosis and early collapse were observed macroscopically at 4, 24 and 48 h, respectively. The effects were studied in semithin sections and cytocentrifuge preparations of the tumours. The inflammatory cell reaction evoked by the tumours in untreated animals was relatively slight. It was located predominantly around the lateral margins of the tumours and only a few inflammatory cells were found inside the tumour. Prominent effects of endotoxin included a transient increase of mononuclear inflammatory cells in the centre of the tumour by 4 h and a reduction of the influx of lymphocytes, observed in and around the margin of control tumours, by 48 h. Mast cells formed an important part of the inflammatory cell infiltrate, but no distinct changes in number and appearance were observed with time or following treatment. Total host cell numbers within tumours did not increase significantly upon endotoxin-treatment. Results suggest that a direct cytotoxic action of host cells cannot account for the extensive tumour damage observed. Rather, endotoxin-induced regression seems to be related to decreased lymphocyte numbers. Chemicals/CAS: Endotoxins

Objective - Emerging evidence suggests that human blood contains bone marrow (BM)-derived endothelial progenitor cells that contribute to postnatal neovascularization. Clinical trials demonstrated that administration of BM-cells can enhance neovascularization. Most studies, however, used crude cell populations. Identifying the role of different cell populations is important for developing improved cellular therapies. Methods and Results - Effects of the hematopoietic stem cell-containing CD34+ cell population on migration, proliferation, differentiation, stimulation of, and participation in capillary-like tubule formation were assessed in an in vitro 3-dimensional matrix model using human microvascular endothelial cells. During movement over the endothelial monolayer, CD34+ cells remained stuck at sites of capillary tube formation and time- and dose-dependently formed cell clusters. Immunohistochemistry confirmed homing and proliferation of CD34+ cells in and around capillary sprouts. CD34+ cells were transduced with the LNGFR marker gene to allow tracing. LNGFR gene-transduced CD34 + cells integrated in the tubular structures and stained positive for CD31 and UEA-1. CD34+ cells alone stimulated neovascularization by 17%. Coculture with CD34- cells led to 68% enhancement of neovascularization, whereas CD34- cells displayed a variable response by themselves. Cell-cell contact between CD34+ and CD34- cells facilitated endothelial differentiation of CD34+ cells. Conclusions - Our data suggest that administration of CD34+-enriched cell populations may significantly improve neovascularization and point at an important supportive role for (endogenous or exogenous) CD34- cells. © 2005 American Heart Association, Inc. Chemicals / CAS: nitric oxide, 10102-43-9; Antigens, CD34; Biological Markers

We investigated the lobular localization and molecular level of expression of cholesterol 7α-hydroxylase and sterol 27-hydroxylase, two key enzymes in bile acid synthesis, in isolated periportal and pericentral hepatocytes and by in situ hybridization of rat liver. Enzyme activity, mRNA, and gene transcription of cholesterol 7α-hydroxylase were predominant in pericentral hepatocytes of control rats, being 7.9-, 9.9-, and 4.4-fold higher than in periportal hepatocytes, respectively. Similar localization was found for sterol 27-hydroxylase: 2.9-, 2.5-, and 1.7-fold higher enzyme activity, mRNA, and gene transcription, respectively, was found in pericentral hepatocytes. Interruption of the enterohepatic circulation with colestid resulted in upregulation of these parameters for both enzymes, as a consequence of stimulated gene expression mainly in the periportal zone. In contrast, mRNA levels and gene transcription of 3-hydroxy-3-methylglutaryl CoA reductase showed opposite lobular distribution. Selective periportal expression for the latter was enhanced, but remained local, after colestid treatment. In situ hybridization showed unambiguously that cholesterol 7α-hydroxylase mRNA is localized exclusively in the pericentral zone and that sterol 27-hydroxylase mRNA is expressed preferentially in the pericentral region, though less pronounced. Administration of colestid led to expression of both genes within a larger area of the liver lobulus. In conclusion, we suggest that cholesterol 7α-hydroxylase and sterol 27-hydroxylase are coordinately regulated by the bile acid gradient over the lobulus, resulting in predominant expression in the pericentral zone. Opposite lobular localization of cholesterol and bile acid synthesis provides an alternative view to interregulation of these metabolic pathways. Chemicals/CAS: 3 hydroxy 3 methylglutaryl coenzyme A, 1553-55-5; alanine aminotransferase, 9000-86-6, 9014-30-6; cholesterol 7alpha monooxygenase, 9037-53-0; colestipol, 25085-17-0, 37296-80-3, 50925-79-6; glutamate ammonia ligase, 9023-70-5; glyceraldehyde 3 phosphate dehydrogenase, 9001-50-7; pyruvate kinase, 9001-59-6; sterol 27 hydroxylase, 134712-57-5; Biological Markers; Cholesterol 7-alpha-Hydroxylase, EC 1.14.13.17; Colestipol, 50925-79-6; Cytochrome P-450 Enzyme System, 9035-51-2; cytochrome P-450C27/25, EC 1.14.-; RNA, Messenger; Steroid Hydroxylases, EC 1.14.-

Activated microglia are found in a variety of neuroinflammatory disorders where they have attributed roles as effector as well as antigen-presenting cells (APC). Critical determinants for the multifaceted role of microglia are the differentiation potential of microglia and their mode of activation. In this study, we have investigated the effects of M-CSF and GM-CSF-mediated differentiation of adult primate microglia on their cellular phenotype, antigen presentation, and phagocytic function as well as on Toll-like receptor (TLR)-mediated responses. We show that although cell morphology and expression levels of activation markers were markedly different, differentiation with either factor yielded microglia that phenotypically and functionally resemble macrophages. Both M-CSF and GM-CSF-differentiated microglia were responsive to TLR1/2, 2, 3, 4, 5, 6/2, and 8-mediated activation, but not to TLR7 or 9-mediated activation. Intriguingly, M-CSF-differentiated microglia expressed higher levels of TLR8-encoding mRNA and protein, and produced larger amounts of proinflammatory cytokines in response to TLR8-mediated activation as compared to GM-CSF-differentiated microglia. While differentiation of adult microglia by growth factors that can be produced endogenously in the central nervous system is thus unlikely to change their APC function, it can alter their innate responses to infectious stimuli such as ssRNA viruses. Resident primate microglia may thereby help shape rather than initiate adaptive immune responses. © 2007 Wiley-Liss, Inc.