TdR Incorporation Research Articles

The rate of change of a soil bacterial community after liming as a function of temperature.

The response of a bacterial community to liming of a forest humus soil (pH 4.9 increased to pH 7.5) was studied in the laboratory at three temperatures (5, 20, and 30 degrees C). As a comparison an unlimed soil (pH 4.9) and a soil limed in the field 15 years ago (pH around 6) were also included. The bacterial community tolerance of pH was measured using TdR incorporation. The pH of the bacterial suspensions (bacteria directly extracted from soil) was altered to 3.6 and 8.3 using different buffers before measuring TdR incorporation. The logarithmic ratio between TdR incorporation at 8.3 and 3.6 was then used as an indicator of the community pH tolerance. The rate of changes in the community tolerance to pH after liming was fastest for the soil incubated at 30 degrees C, but only minor differences in rate of change could be seen between samples incubated at 5 and 20 degrees C. Changes in phospholipid fatty acid (PLFA) pattern after increasing the pH were most rapid for the bacterial community in the soil incubated at 30 degrees C followed by the soil incubated at 20 degrees C, whereas no changes could be seen in the PLFA pattern of the soil incubated at 5 degrees C, even after 82 days' incubation. Thus, the changes in the PLFA pattern were considerably slower than the changes in bacterial community tolerance to pH measured using TdR incorporation.

Effects of selective prostaglandin EP 2 and EP 4 receptor agonists on bone resorption and formation in fetal rat organ cultures

Prostaglandin E 2 (PGE 2) can stimulate bone resorption and formation through receptors which activate adenylyl cyclase. We examined the effects of selective EP 2 and EP 4 agonists (EP2A and EP4A) on the release of previously incorporated 45 Ca from fetal rat long bones and the incorporation of [ 3 H ]-proline or [ 3 H ]-thymidine (TdR) in fetal rat calvaria to assess the relative effects of these selective agonists on bone formation and resorption. Only EP4A was effective in increasing 45 Ca release. Both agonists increased [ 3 H ]-TdR incorporation and [ 3 H ]-proline incorporation into calvariae, particularly in the presence of cortisol.

Modulation of the bacterial response to spectral solar radiation by algae and limiting nutrients

SUMMARY 1. The response of bacterial production (measured as [3H]TdR incorporation rate) to spectral solar radiation was quantified experimentally in an oligotrophic high‐mountain lake over 2 years. Bacterial responses were consistent: ultraviolet‐B (UVB) was harmful, whereas ultraviolet‐A (UVA) + photosynthetically active radiation (PAR) and PAR enhanced bacterial activity. Full sunlight exerted a net stimulatory effect on bacterial activity in mid‐summer but a net inhibitory effect towards the end of the ice‐free period.2. Experiments were undertaken to examine whether the bacterial response pattern depended on the presence of algae and/or was modulated by the availability of a limiting inorganic nutrient (phosphorus, P). In the absence of algae, [3H]TdR incorporation rates were significantly lower than when algae were present under all light treatments, and the consistent bacterial response was lost. This suggests that the bacterial response to spectral solar radiation depends on fresh‐C released from algae, which determines the net stimulatory outcome of damage and repair in mid‐summer.3. In the absence of algae, UVB radiation inhibited bacteria when they were strongly P‐deficient (mean values of N : P ratio: 46.1), whereas it exerted no direct effect on bacterial activity when they were not P‐limited.4. P‐enrichment of lake water markedly altered the response of bacteria to spectral solar radiation at the end of ice‐free period, when bacteria were strongly P‐deficient. Phosphorus enrichment suppressed the inhibitory effect of full sunlight that was observed in October, both in whole lake water (i.e. including algae) and in the absence of algae. This indicates that the bacterial P‐deficiency, measured as the cellular N : P ratio, was partly responsible for the net inhibitory effect of full sunlight, implying a high bacterial vulnerability to UVB.

Overexpression of chemokine-like factor 2 promotes the proliferation and survival of C2C12 skeletal muscle cells

Chemokine-like factor 1 (CKLF1) is a novel cytokine first cloned from U937 cells. It contains different splicing forms and has chemotactic effects on a wide spectrum of cells both in vitro and in vivo; it can also stimulate the regeneration of skeletal muscle cells in vivo, but the mechanism remains unclear. To probe the myogenesis function of CKLF2, which is the largest isoform of CKLFs, C2C12 murine myoblasts were stably transfected with human CKLF2 eukaryotic expression vector. Compared with control vector transfected C2C12 cells, CKLF2 overexpression causes accelerated myoblast proliferation as determined by cell counting and [ 3H]TdR incorporation assays. In addition, CKLF2 overexpression also promotes cell differentiation, which was determined by higher expression levels of myogenin, creatine kinase, myosin and the accelerated myoblast fusion. Further analysis also indicates that CKLF2 could activate the transcription activity of the bHLH/MyoD and MEF2 families. Finally, DNA synthesis and myotube formation could also be promoted by growing C2C12 cells in conditioned media from CKLF2-transfected cells. These findings strongly suggest a role for human CKLF2 in regulation of skeletal muscle myogenesis.

Novel assays of multiple lymphocyte functions in whole blood measure: New mechanisms of action of mycophenolate mofetil in vivo

The antiproliferative effects of MMF are believed to be the mechanism of its immunosuppressive action. We further investigated the mechanisms of action by assessing the pharmacodynamics (PD) of MMF in treated animals using whole blood assays not only of lymphocyte proliferation but also of activation. In vitro, different MPA concentrations were added to rat whole blood. In vivo, Lewis rats were treated with single doses of 5, 10 or 20 mg/kg MMF ( n=6 rats/dose group). Blood was obtained before and at different times after drug administration. For both in vitro and in vivo studies, different mitogens with calcium-dependent (TCR) or -independent (co-stimulatory) pathways of lymphocyte activation were added to the blood for stimulation. Proliferation was measured by [ 3H]TdR incorporation and by flow cytometric detection of DNA content. Activation was measured by changes in T cell surface expression of CD25, CD134, CD71, CD11a and CD54. In vitro and in vivo studies showed a dose-dependent inhibition by MPA and MMF, respectively, of lymphocyte proliferation and surface antigen expression. We observed high correlations between MMF PD effects over time with both MMF dose and MPA plasma concentrations in vivo. We show that MMF, apart from its antiproliferative effect, induced a dose-dependent suppression of calcium-dependent and -independent stimulated expression of important lymphocyte cell surface antigens. These data suggest that the ex vivo assessment of immune function in whole blood can uncover new mechanisms of MMF action. Our results demonstrated that the measurement of the PD is a means to assess the functional effects of MMF after its administration in vivo.

Increased generation of dendritic cells from myeloid progenitors in autoimmune-prone nonobese diabetic mice.

Aberrant dendritic cell (DC) development and function may contribute to autoimmune disease susceptibility. To address this hypothesis at the level of myeloid lineage-derived DC we compared the development of DC from bone marrow progenitors in vitro and DC populations in vivo in autoimmune diabetes-prone nonobese diabetic (NOD) mice, recombinant congenic nonobese diabetes-resistant (NOR) mice, and unrelated BALB/c and C57BL/6 (BL/6) mice. In GM-CSF/IL-4-supplemented bone marrow cultures, DC developed in significantly greater numbers from NOD than from NOR, BALB/c, and BL/6 mice. Likewise, DC developed in greater numbers from sorted (lineage(-)IL-7Ralpha(-)SCA-1(-)c-kit(+)) NOD myeloid progenitors in either GM-CSF/IL-4 or GM-CSF/stem cell factor (SCF)/TNF-alpha. [(3)H]TdR incorporation indicated that the increased generation of NOD DC was due to higher levels of myeloid progenitor proliferation. Generation of DC with the early-acting hematopoietic growth factor, flt3 ligand, revealed that while the increased DC-generative capacity of myeloid-committed progenitors was restricted to NOD cells, early lineage-uncommitted progenitors from both NOD and NOR had increased DC-generative capacity relative to BALB/c and BL/6. Consistent with these findings, NOD and NOR mice had increased numbers of DC in blood and thymus and NOD had an increased proportion of the putative myeloid DC (CD11c(+)CD11b(+)) subset within spleen. These findings demonstrate that diabetes-prone NOD mice exhibit a myeloid lineage-specific increase in DC generative capacity relative to diabetes-resistant recombinant congenic NOR mice. We propose that an imbalance favoring development of DC from myeloid-committed progenitors predisposes to autoimmune disease in NOD mice.

Molecular Mechanisms Underlying Differential Contribution of CD28 Versus Non-CD28 Costimulatory Molecules to IL-2 Promoter Activation

T cell costimulation via CD28 and other (non-CD28) costimulatory molecules induces comparable levels of [(3)H]TdR incorporation, but fundamentally differs in the contribution to IL-2 production. In this study, we investigated the molecular basis underlying the difference between CD28 and non-CD28 costimulation for IL-2 gene expression. Resting T cells from a mutant mouse strain generated by replacing the IL-2 gene with a cDNA encoding green fluorescent protein were stimulated with a low dose of anti-CD3 plus anti-CD28 or anti-non-CD28 (CD5 or CD9) mAbs. CD28 and non-CD28 costimulation capable of inducing potent [(3)H]TdR uptake resulted in high and marginal levels of green fluorescent protein expression, respectively, indicating their differential IL-2 promoter activation. CD28 costimulation exhibited a time-dependent increase in the binding of transcription factors to the NF-AT and NF-kappaB binding sites and the CD28-responsive element of the IL-2 promoter, whereas non-CD28 costimulation did not. Particularly, a striking difference was observed for the binding of NF-kappaB to CD28-responsive element and the NF-kappaB binding site. Decreased NF-kappaB activation in non-CD28 costimulation resulted from the failure to translocate a critical NF-kappaB member, c-Rel, to the nuclear compartment due to the lack of IkappaBbeta inactivation. These observations suggest that unlike CD28 costimulation, non-CD28 costimulation fails to sustain IL-2 promoter activation and that such a failure is ascribed largely to the defect in the activation of c-Rel/NF-kappaB.

Inhibition of growth of human breast cancer cells in culture by neutron capture using liposomes containing 10B

Cell destruction in boron neutron capture therapy is effected by nuclear reaction between 10B and thermal neutrons with the release of alpha-particles ( 4He) and lithium-7 ions ( 7Li). 4He kills cells within 10 μm of the site of 4He generation, therefore it is theoretically possible to destroy tumour cells without affecting adjacent healthy tissue, given selective delivery of compounds containing 10B. Liposomes wore prepared by vortex dispersion of solutions containing 10B compounds with dried lipid films and the effects of those compounds on human breast cancer cells in culture were examined after thermal neutral irradiation. 〚 3H〛-TdR incorporation by MRK nu/nu-1 cells treated with 10B-containing liposomes showed 40% suppression compared with liposomes without 10B, at 2 x 10 12 n/cm 2 thermal neutron fluence. Inhibition of tumour cell growth with liposomes prepared with 100 mm 10B-compound was as significant as with those made with 500 ppm 10B solution. The concentration of 10B in liposomes was 76.5 ± 3.4 μg/mL. Boronated liposomes can thus deliver sufficient 10B atoms to this line of breast cancer cells in culture to effect cytotoxicity and suppression of growth after thermal neutron irradiation.

CD2 facilitates differentiation of CD4 Th cells without affecting Th1/Th2 polarization.

The role of CD2 in murine CD4 helper T cell differentiation and polarization was examined using TCR-Cyt-5CC7-I transgenic recombination activating gene-2-/- H-2(a) mice on CD2+/+ or CD2-/- backgrounds. In the absence of CD2, thymic development was abnormal as judged by reduction in the steady state number of total, double-positive, and CD4 single-positive (SP) thymocytes, as well as a defect in their restorative dynamics after peptide-induced negative selection in vivo. In addition, in CD2-/- animals, lymph node CD4 SP T cells manifest a 10- to 100-fold attenuated activation response to cytochrome c (CytC) agonist peptides as judged by induction of CD25 and CD69 cell surface expression or [(3)H]TdR incorporation; differences in the magnitude of responsiveness and requisite molar peptide concentrations were even greater for altered peptide ligands. Although the presence or absence of CD2 did not impact the final Th1 or Th2 polarization outcome, CD2 expression reduced the CytC peptide concentration threshold necessary to facilitate both Th1 and Th2 differentiation. In vivo administration of CytC peptide to CD2-/- animals yielded an impaired CD4 SP T cell effector/memory phenotype compared with similarly treated CD2+/+ mice. Analysis of TCR-Cyt-5CC7-I human CD2 double-transgenic mice similarly failed to reveal a preferential Th1 vs Th2 polarization. Collectively, these results indicate that CD2 is important for the efficient development of CD4 SP thymocytes and TCR-dependent activation of mature CD4 lymph node T cells, but does not direct a particular helper T cell subset polarity.

Diurnal variations in depth profiles of UV-induced DNA damage and inhibition of bacterioplankton production in tropical coastal waters

MEPS Marine Ecology Progress Series Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections MEPS 228:25-33 (2002) - doi:10.3354/meps228025 Diurnal variations in depth profiles of UV-induced DNA damage and inhibition of bacterioplankton production in tropical coastal waters Petra M. Visser1,2,*, Jan Jaap Poos1, Bonnie B. Scheper1, Peter Boelen3, Fleur C. van Duyl1 1Department of Biological Oceanography, Netherlands Institute for Sea Research (NIOZ), PO Box 59, 1790 AB Den Burg, Texel, The Netherlands 2Carmabi Foundation Ecological Institute, PO Box 2090, Curaçao, Netherlands Antilles 3Department of Marine Biology, University of Groningen, PO Box 14, 9750 AA Haren, The Netherlands *Present address: IBED/Aquatic Microbiology, University of Amsterdam, Nieuwe Achtergracht 127, 1018 WS Amsterdam, The Netherlands. E-mail: petra.visser@science.uva.nl ABSTRACT: In this study, diurnal changes in bacterial production and DNA damage in bacterioplankton (measured as cyclobutane pyrimidine dimers, CPDs) incubated in bags at different depths in tropical coastal waters were investigated. The DNA damage and inhibition of the bacterial production was highest at the surface and decreased with depth. Inhibition of both leucine (Leu) and thymidine (TdR) incorporation was detectable to a depth of 10 m, while DNA damage was measurable only until 5 m. In addition, the decrease of the inhibition of Leu and TdR incorporation with depth was less than the decrease in DNA damage. Variations in DNA damage in bacteria and in biodosimeters were mostly explained by UV radiation (UVR) dose received over time and depth. UVB radiation (UVBR) contributed more than UVA radiation (UVAR) to the damage. The attenuation coefficient of DNA damage with depth was comparable to the attenuation coefficients of UVBR suggesting that DNA damage was induced mainly by UVBR. For incubations at the surface, there was a trend of increasing DNA damage at a constant rate during the entire period of daylight, while the inhibition of leucine incorporation showed a trend of a less rapid increase in the afternoon than in the morning. This might have been due to photorepair, which would also explain the lack of correlation between the inhibition of leucine incorporation and UV dose. It was concluded that CPD damage in DNA is not necessarily the predominant factor in the inhibition of bacterial production. It cannot explain the large impact of sunlight on bacterioplankton productivity in tropical waters. KEY WORDS: DNA damage · Bacterial production · UV radiation Full text in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in MEPS Vol. 228. Online publication date: March 06, 2002 Print ISSN: 0171-8630; Online ISSN: 1616-1599 Copyright © 2002 Inter-Research.

Assessment of mechanisms of action of immunosuppressive drugs using novel whole blood assays

WHOLE BLOOD ASSAYS are preferred because they better approximate the complex in vivo relationships among drug concentration, the binding of drug to plasma constituents and cellular targets, and the interaction among immune cells and their products. Additionally, the whole blood technique is more rapid and requires smaller sample volumes than methods that rely on purified lymphocytes. We assessed lymphocyte proliferation by measuring [H]-TdR incorporation and also assessed the expression of the T-lymphocyte surface antigens CD11a, CD25, CD54, and CD71 with flow cytometry analysis. We stimulated whole blood with concanavalin A (Con A) and phorbol 13-myristate 12-acetate (PMA) plus anti-CD28 (CD28) and determined the relative potencies of different immunosuppressive drugs on inhibition of lymphocyte function.

Apoptosis and changes in glucose transport early after treatment of Morris hepatoma with gemcitabine.

Apoptosis has been described as an energy-consuming process. This combined in vivo/in vitro study investigated the effects of the antineoplastic agent gemcitabine on tumour metabolism and on the induction of apoptosis. Dynamic positron emission tomography (PET) measurements of fluorine-18 fluorodeoxyglucose (FDG) uptake were done in rats bearing Morris hepatoma prior to and after therapy with 90 mg gemcitabine/kg b.w. Furthermore, thymidine (TdR) incorporation into the DNA of these tumours was determined. In vitro measurements of FDG and TdR uptake were performed immediately and 24 h after the end of gemcitabine treatment, and the amount of apoptotic cells was determined using the TUNEL reaction. In vivo an increase in FDG transport and phosphorylation occurred early after gemcitabine treatment, although TdR incorporation into the DNA of the tumours declined. In vitro, an enhanced glucose transport, an increase in TdR uptake in the cytoplasm and a decrease in TdR incorporation in the nucleic acid fraction early after treatment occurred. Inhibition of glucose transport caused an increase in the amount of apoptotic cells. The increase in glucose uptake and TdR metabolism early after therapy is interpreted as a stress reaction of the tumour cells, protecting the cells from apoptosis during this early period after exposure to cytotoxic drugs like gemcitabine.

Temperature-driven adaptation of the bacterial community in peat measured by using thymidine and leucine incorporation.

The temperature-driven adaptation of the bacterial community in peat was studied, by altering temperature to simulate self-heating and a subsequent return to mesophilic conditions. The technique used consisted of extracting the bacterial community from peat using homogenization-centrifugation and measuring the rates of thymidine (TdR) or leucine (Leu) incorporation by the extracted bacterial community at different temperatures. Increasing the peat incubation temperature from 25 degrees C to 35, 45, or 55 degrees C resulted in a selection of bacterial communities whose optimum temperatures for activity correlated to the peat incubation temperatures. Although TdR and Leu incorporations were significantly correlated, the Leu/TdR incorporation ratios were affected by temperature. Higher Leu/TdR incorporation ratios were found at higher temperatures of incubation of the extracted bacterial community. Higher Leu/TdR incorporation ratios were also found for bacteria in peat samples incubated at higher temperatures. The reappearance of the mesophilic community and disappearance of the thermophilic community when the incubation temperature of the peat was shifted down were monitored by measuring TdR incorporation at 55 degrees C (thermophilic activity) and 25 degrees C (mesophilic activity). Shifting the peat incubation temperature from 55 to 25 degrees C resulted in a recovery of the mesophilic activity, with a subsequent disappearance of the thermophilic activity. The availability of substrate for bacterial growth varied over time and among different peat samples. To avoid confounding effects of substrate availability, a temperature adaptation index was calculated. This index consisted of the log(10) ratio of TdR incorporation at 55 and 25 degrees C. The temperature index decreased linearly with time, indicating that no thermophilic activity would be detected by the TdR technique 1 month after the temperature downshift. There were no differences between the slopes of the temperature adaptation indices over time for peat samples incubated at 55 degrees C 3 or 11 days before incubation at 25 degrees C. Thus, different levels of bacterial activity did not affect the temperature-driven adaptation of the bacterial community.

Thymidine utilization abnormality in proliferating lymphocytes and hepatocytes of the woodchuck

Effective incorporation of tritiated thymidine ([ 3H]TdR) into proliferating lymphocytes is important because [ 3H]TdR is a standard label to study proliferate T-cell responses. We analyzed the thymidine utilization of woodchuck peripheral blood lymphocytes (PBL) since the [ 3H]TdR incorporation assay was not applicable to measure proliferative immune responses in the woodchuck, a current major virus/host model for human hepatitis B virus infection. Incorporation of [ 3H]TdR into DNA as well as the activity of the salvage pathway enzyme thymidine kinase (TK) of proliferating woodchuck PBL was low compared to human lymphocytes. Furthermore, [ 3H]TdR incorporation of proliferating woodchuck PBL remained residual regardless of the use of methotrexate, an inhibitor of the competitive deoxythymidine monophosphate de novo synthesis pathway. Using a human probe, specific for the proliferation-associated TK1, we proved the genomic presence and transcription of TK1 sequences in various species. TK1 sequences were detected in the genome of human, mouse, woodchuck, and chicken specimens. In contrast to proliferating human PBL and 3T3 mouse fibroblasts, no TK1 transcript was found in proliferating woodchuck PBL and hepatic cells. Transfection experiments with vectors containing the murine or human TK1 and selection assays demonstrated the ability of woodchuck cells to transcribe TK1 and to express functional TK1 proteins. Our study characterizes the unique failure of sufficient [ 3H]TdR incorporation into proliferating woodchuck cells and demonstrates tritiated adenine and serine as alternative labels to monitor PBL proliferation in the woodchuck.

Antiproliferative effect of a lectin- and anti-Thy-1.2 antibody-targeted HPMA copolymer-bound doxorubicin on primary and metastatic human colorectal carcinoma and on human colorectal carcinoma transfected with the mouse Thy-1.2 gene.

The aim of this study was to compare the potential of two plant lectins [peanut agglutinin (PNA) and wheat germ agglutinin (WGA)], monoclonal antibody (anti-Thy-1.2), its F(ab')(2) fragments, and galactosamine as targeting moieties bound to the polymer drug carrier to deliver a xenobiotic, doxorubicin, to selected cancer cell lines. We have used primary (SW 480, HT 29) and metastatic (SW 620) human colorectal cancer cell lines and a transfectant, genetically engineered SW 620 cell line with mouse gene Thy-1.2 (SW 620/T) to test the possibility of marking human cancer with xenogeneic mouse gene and use it for effective site-specific targeting. The targeting moieties and doxorubicin were conjugated to a water-soluble copolymer based on N-(2-hydroxypropyl)methacrylamide (HPMA) acting as a carrier responsible for controlled intracellular release of the targeted drug. FACS analysis showed a strong binding of WGA-FITC to all tested cell lines. Binding of PNA-FITC was considerably weaker. The in vitro antiproliferative effect of lectin-targeted HPMA carrier-bound doxorubicin evaluated as [(3)H]TdR incorporation reflected both the intensity of the binding and the different sensitivity of the tested cancer cells lines to doxorubicin. The antiproliferative effect of conjugates targeted with WGA was comparable to that with the conjugates targeted with the anti-Thy-1.2 monoclonal antibody or their F(ab')(2) fragments. The magnitude of the cytotoxic effect of HPMA-doxorubicin targeted with PNA was lower in all tested cell lines. While the conjugates with WGA were more cytotoxic, the conjugates with PNA were more specific as their binding is limited to cancer cells and to the sites of inflammation. Noncytotoxic conjugates with a very low concentration of doxorubicin and targeted with PNA, anti-Thy-1.2, or their F(ab')(2) fragments exerted in some lines (SW 480, SW 620) low mitogenic activity. The Thy-1.2 gene-transfected SW 620 metastatic colorectal cancer cell line was sensitive to the antiproliferative effect of Thy-1.2-targeted doxorubicin as was shown for the Thy-1. 2(+) EL4 cell line and for Thy-1.2(+) concanavalin A-stimulated mouse T lymphocytes. These results represent the first indication of the suitability of transfection of human cancer cells with selected targeting genes for site-specific therapy of malignancies.

Influence of vehicle upon potency estimation in the local lymph node assay: Further investigations

The original local lymph node assay (LLNA) is based on the use of radioactive labelling to measure cell proliferation. Other endpoints for the assessment of proliferation are also authorized by the OECD Guideline 429 provided there is appropriate scientific support, including full citations and description of the methodology (OECD, 2002. OECD Guideline for the Testing of Chemicals; Skin Sensitization: Local Lymph Node Assay, Guideline 429. Paris, adopted 24th April 2002.). Here, we describe the outcome of the second round of an inter-laboratory validation of alternative endpoints in the LLNA conducted in nine laboratories in Europe. The validation study was managed and supervised by the Swiss Agency for Therapeutic Products (Swissmedic) in Bern. Ear-draining lymph node (LN) weight and cell counts were used to assess LN cell proliferation instead of [3H]TdR incorporation. In addition, the acute inflammatory skin reaction was measured by ear weight determination of circular biopsies of the ears to identify skin irritation properties of the test items. The statistical analysis was performed in the department of statistics at the university of Bern.Similar to the EC3 values defined for the radioactive method, threshold values were calculated for the endpoints measured in this modification of the LLNA. It was concluded that all parameters measured have to be taken into consideration for the categorisation of compounds due to their sensitising potencies. Therefore, an assessment scheme has been developed which turned out to be of great importance to consistently assess sensitisation versus irritancy based on the data of the different parameters. In contrast to the radioactive method, irritants have been picked up by all the laboratories applying this assessment scheme.

Tritiated thymidine ([3H]-TdR) and immunocytochemical tracing of cellular fate within the asexually dividing cestode Mesocestoides vogae (syn. M. corti).

This report documents the presence of an active thymidine kinase (TK) system within Mesocestoides vogae tetrathyridia as quantified by tritiated thymidine ([3H]-TdR) incorporation using liquid scintillation counting. A 100-fold increase in [3H]-TdR incorporation was observed at 37 degrees C when compared with its incorporation at 0 degrees C. Thymidine's competitive analogue, BrdU, competed for sites within newly replicated DNA. Immunohistochemical trials performed here using antibodies against BrdU identified cells that have entered and passed through S-phase. Positively stained nuclei were most numerous at the anterior tip of tetrathyridia especially within the ganglia, lesser numbers of these cells occurred along the growing commissure and amongst surface tegumental cytons suggesting that stem cells do not exist in one region but are found throughout the entire body. As M. vogae has no internal organ systems the major sites for cell proliferation are those exhibiting maximal cell recruitment and undergoing tissue repair. These results show that it is possible to monitor changes in the cell recruitment pattern within this cestode. Thus use of BrdU and immunohistochemistry demonstrates how spatial arrangement and cellular reorganization can be successfully traced within M. vogae.

The pharmacodynamic basis for the increased antileukaemic efficacy of cytosine arabinoside-based treatment regimens in acute myeloid leukaemia with a high proliferative activity.

The current study was initiated to explore the mechanisms underlying the previously demonstrated association between the proliferative activity of leukaemic blasts and the response to cytosine arabinoside (AraC)-based therapy in de novo acute myeloid leukaemia (AML). The activity of key enzymes of AraC metabolism-deoxycytidine kinase (DCK), cytidine deaminase (DCD) and polymerase alpha (PolyA) were determined in blast cells from 33 patients. In addition, formation and retention of intracellular levels of AraC triphosphate (AraCTP) and DNA incorporation of AraC were measured, as was the proliferative activity of leukaemic blasts by [3H]-TdR incorporation before and after stimulation with granulocyte-macrophage colony-stimulating factor (GM-CSF) or granulocyte CSF (G-CSF) for 48 h. AraC incorporation into the DNA (median 0.60 pmol/105 cells) was significantly related to the proliferative activity of AML blasts (r = 0.74, P < 0.001). Similarly, priming with GM-CSF or G-CSF increased both the proliferative activity of AML blasts by a median of 1.84- and 1.64-fold, respectively, and the incorporation of AraC into the DNA (1.29- and 1.40-fold respectively). In contrast, no relationship was found between the endogenous proliferative activity (EPA) and enzyme activities regulating AraC activation (DCK; median 4.70 pmol/min/mg protein), inactivation (DCD; median 2.92 pmol/min/mg protein) or inhibitory effects (PolyA; median 1.50 pmol/min/mg protein), nor the formation or retention of AraCTP (median 306.1 ng/107 cell and 1.6 h respectively). When samples were grouped according to EPA (more than or less than the median), slowly proliferating specimens had a higher response to cytokine priming for proliferative activity and incorporation of AraC into DNA. Clinical data of 15 patients were available. Although all eight patients with a high endogenous proliferative activity reached complete remission, only four out of seven patients with a low proliferative activity responded, whereas the other three patients were non-responders (P = 0.077).

Effects of salvianolic acid-A on NIH/3T3 fibroblast proliferation, collagen synthesis and gene expression.

AIM:To investigate the mechanisms of salvianolic acid A (SA-A) against liver fibrosis in vitro.METHODS:NIH/3T3 fibroblasts were cultured routinely, and incubated with 10(-4) mol/L-10(-7)mol/L SA-A for 22h. The cell viability was assayed by (3)H proline incorporation, cell proliferation by (3)H TdR incorporation, cell collagen synthetic rate was measured with (3)H proline impulse and collagenase digestion method.The total RNA was prepared from the control cells and the drug treated cells respectively, and alpha(1) I pro-collagen mRNA expression was semi-quantitatively analyzed with RT-PCR.RESULTS:10(-4)mol/L SA-A decreased cell viability and exerted some cytotoxiciy,while 10(-5)mol/L -10(-7)mol/L SA-A did not affect cell viability, but inhibited cell proliferation significantly, and 10(-6)mol/L SA-A had the best effect on cell viability among these concentrations of drugs. 10 (-5)mol/L -10(-6)mol/L SA-A inhibited intracellular collagen synthetic rate, but no significant influence on extracellular collagen secretion. Both 10(-5)mol/L and 10(-6)mol/L SA-A could decrease alpha(1)I pro-collagen mRNA expression remarkably.CONCLUSION:SA-A had potent action against liver fibrosis. It inhibited NIH/3T3 fibroblast proliferation, intracellular collagen synthetic rate and type I procollagen gene expression, which may be one of the main mechanisms of the drug.

Responsiveness of peripheral blood B cells to recombinant CD40 ligand in patients with systemic lupus erythematosus.

To investigate the immunopathological significance of CD40/CD40 ligand system for B cell hyperactivation in SLE patients, the expression and the function of CD40 on B cells were compared with those of normal controls. Expression of CD40 was evaluated by flow cytometry. DNA synthesis of B cells were measured by 3H - TdR incorporation. Antibody production was assessed by ELISA. There was no significant difference between SLE and normal controls in CD40 expression on peripheral blood B cells. Recombinant CD40 ligand-leucine zipper fusion protein (CD40L-LZ) significantly enhanced 3H - TdR incorporation by both SLE and normal B cells (P<0.01). 3H - TdR incorporation of SLE B cells without stimuli (P<0.001) and with CD40L-LZ stimulation (P<0.05) were significantly lower in SLE patients compared with normal controls. Active SLE B cells spontaneously produced significantly larger amounts of total IgG than normal B cells (P<0.05). CD40L-LZ significantly increased the production of total IgG by SLE B cells (P<0.05), but not by normal B cells. Active SLE B cells spontaneously produced IgG anti-dsDNA and IgG anti-ssDNA antibodies. CD40L-LZ significantly increased the production of these autoantibodies by SLE B cells (P<0.05). B cells from normal controls do not produce these autoantibodies spontaneously nor in response to CD40L-LZ. These findings indicate that signalling via CD40 plays an important role in B cell proliferation and autoantibody production in human SLE.