Cytokine production of in vitro stimulated peripheral lymphocytes during the course of pregnancy and pseudopregnancy in the rat.

PROBLEM: Does maternal lymphocyte cytokine production after in vitro stimulation vary with the stage of pregnancy in the rat? METHOD OF STUDY: Blood samples were taken during the estrus cycle in rats (n = 11). Thereafter, rats were rendered pregnant (n = 6) or pseudopregnant (n = 5) and blood samples were taken at days 4, 8, 11, 15, and 20 of pregnancy and pseudopregnancy. White blood cell (WBC) count was measured and whole blood was stimulated with phorbol 12-myristate 13-acetate and calcium ionophore; interferon-gamma (IFNgamma) and interleukin-4 (IL-4) production as well as (sub)populations of lymphocytes were measured using flow cytometry. RESULTS: We observed an increase of WBC in the second week of pregnancy and a slowly decreasing percentage of lymphocytes during the course of pregnancy. The percentage IFNgamma producing T-lymphocytes after in vitro stimulation was increased during pregnancy (for Th-lymphocytes only in the second week of pregnancy, for Tc-lymphocytes at all days). This increased IFNgamma production in pregnant T-lymphocytes was accompanied by an increase during pseudopregnancy, and therefore may result from increased sex hormone concentrations. The percentage IFNgamma producing natural killer (NK) cells after in vitro stimulation was decreased on day 20 of pregnancy. No effect of pregnancy or pseudopregnancy was seen on percentage IL-4 producing lymphocytes after in vitro stimulation. CONCLUSION: In the rat the IFNgamma production after in vitro stimulation varies during pregnancy and is increased, rather than decreased, during pregnancy.

Monocyte activation, but not granulocyte activation, is inhibited in the presence of developing ovarian follicles.

This study was set up to evaluate the influence of ovarian factors on the acute phase of the endotoxin-induced glomerular inflammatory reaction. Six groups of rats with permanent jugular vein cannulas were used. This included three groups with increased progesterone and/or 17beta-oestradiol concentrations (day 14 pregnant rats, pseudopregnant rats and lactating rats), one group with the presence of developing ovarian follicles (cyclic rats), and two groups with both increased sex hormone concentrations and the presence of developing ovarian follicles (day 14 pregnant rats treated with FSH and day 21 pregnant rats). Rats were infused for 1h with either saline or endotoxin (1 microg/kg body weight) and sacrificed 4h after the infusion. Kidney sections were snap-frozen and prepared for immunohistochemistry. Endotoxin-induced glomerular granulocyte infiltration was increased only in the groups of rats with increased progesterone and/or 17beta-oestradiol concentrations. This could be due to endotoxin-induced ICAM-1 and/or VCAM-1 expression, which was observed in all endotoxin-treated groups and in all endotoxin-treated groups with increased sex hormone concentrations, respectively. It could also be due to an effect on granulocytes per se, since the number of endotoxin-induced CD11b-positive cells in the glomeruli was increased only in the groups with increased sex hormone concentrations. Endotoxin-induced glomerular monocyte infiltration, however, was seen only in those groups in which developing ovarian follicles were lacking (i.e. day 14 pregnant, pseudopregnant and lactating rats), suggesting that developing ovarian follicles produce anti-inflammatory factors. These factors did not have an effect on endothelial or leukocyte adhesion molecule expression. We hypothesize that the presence of elevated progesterone concentrations increased the endotoxin-induced glomerular granulocyte infiltration, while endotoxin-induced glomerular monocyte infiltration was inhibited in the presence of developing ovarian follicles.

Biotransformation studies using rat proximal tubule cells.

The proximal tubule is the main target for nephrotoxic substances due to its specific properties, including efficient drug transport and biotransformation potential. The availability of a pure population of proximal tubule cells (PT cells) as a model to study a range of biological, pharmacological, and toxicological parameters is, therefore, of great value. A two-step PT cell-isolation procedure, based on density-gradient centrifugation, is described; this procedure can easily be introduced into each laboratory setting. The procedure routinely yields a highly pure PT cell population, comprising 20-40 × 10(6) cells, which can be used for preparation of subcellular fractions or brought into primary culture.

Total white blood cell counts and LPS-induced TNF alpha production by monocytes of pregnant, pseudopregnant and cyclic rats.

Pregnancy in the rat may be associated with an activated innate immune system. Therefore, we investigated monocyte function as well as total white blood cell (WBC) counts during the follicular phase of the ovarian cycle, pregnancy and pseudopregnancy in the rat. Rats were equipped with a permanent jugular vein cannula, and 0.43 ml blood samples were taken from this cannula during the 4 days of the regular oestrus cycle of the rat (n=12). Thereafter, six rats were rendered pregnant, and the other six rats were rendered pseudopregnant according to standard methods. Blood samples were withdrawn from the cannula on days 4, 7 and 11 of pseudopregnancy and on days 4, 7, 11 and 20 of pregnancy. From each blood sample, 0.4 ml was stimulated with lipopolysaccharide (LPS) and monocyte intracellular cytokine production measured using flow cytometry. 30 microl of the blood was used to measure WBC counts and differential WBC counts. The results showed that the number of WBC was significantly increased only on day 11 of pregnancy compared with the follicular phase, and that this was due to the increased numbers of polymorphonuclear (PMN) cells. The percentage of TNF alpha-producing monocytes was increased on all days of pseudopregnancy and on day 11 of pregnancy. The fact that the percentage of monocytes producing TNF alpha upon an LPS stimulus was increased during the post-implantation phase of pregnancy and during pseudopregnancy as compared to the follicular phase may indicate that these conditions are proinflammatory conditions. For the post-implantation phase of pregnancy, this is once more stressed by the increased numbers of WBC and PMN.

Glomerular immunoglobulin deposits induce glomerular inflammation in pregnant but not in non-pregnant rats.

PROBLEM: Does an inflammatory stimulus evoke a more intense inflammatory response in pregnant rats as compared with non-pregnant rats? METHOD OF STUDY: Non-pregnant rats were injected with antibodies against the glomerular basement membrane (GBM), 14 days before pregnancy, to induce a subclinical glomerulonephritis. Part of the rats were rendered pregnant, the others remained non-pregnant throughout the experiment. Two experiments were performed: in experiment 1, pregnant and non-pregnant rats were killed at various intervals after the injection with antibody and parameters characteristic of a glomerular inflammation were evaluated using immunohistology on cryostat kidney sections and liver sections. In experiment 2, 24-hr urinary protein excretion was measured at various days after the injection in pregnant and non-pregnant rats. RESULTS: Experiment 1 revealed that a significant glomerular inflammation, as characterized by increased numbers of monocytes and LFA-1 positive cells per glomerulus, was only observed in pregnant rats with glomerulonephritis. Experiment 2 revealed that only pregnant rats with glomerulonephritis showed increased urinary protein excretion. CONCLUSION: The fact that glomerular inflammation coincides with proteinuria only in pregnant rats with glomerulonephritis, may suggest that these phenomena are causally related and promoted by the pregnant condition.

Management of oxidative stress by heme oxygenase-1 in cisplatin-induced toxicity in renal tubular cells.

Induction of heme oxygenase-1 (HO-1) may serve as an immediate protective response during treatment with the cytostatic drug cisplatin (CDDP). Oxidative pathways participate in the characteristic nephrotoxicity of CDDP. In the present study, cultured tubular cells (LLC-PK1) were used to investigate whether induction of HO provided protection against CDDP by maintaining the cellular redox balance. The antioxidants, alpha-tocopherol (TOCO) and N-acetylcysteine (NAC), were used to demonstrate that elevation of ROS levels contribute to the development of CDDP-induced cytotoxicity. Chemical modulators of HO activity were used to investigate the role of HO herein. Hemin was used to specifically induce HO-1, while exposure of the cells to tin-protoporphyrin (SnPP) was shown to inhibit HO activity. Hemin treatment prior to CDDP-exposure significantly decreased the generation of ROS to control levels, while inhibition of HO increased the ROS levels beyond the levels measured in cells treated with CDDP alone. Furthermore, HO induction protected significantly against the cytotoxicity of CDDP, although this protection was limited. Similar results were obtained when the cells were preincubated with TOCO, suggesting that mechanisms other than impairment of the redox ratio are important in CDDP-induced loss of cell viability in vitro. In addition, SnPP treatment exacerbated the oxidative response and cytotoxicity of CDDP, especially at low CDDP concentrations. We therefore conclude that HO is able to directly limit the CDDP-induced oxidative stress response and thus serves as safeguard of the cellular redox balance.

The role of oxidative stress in the ochratoxin A-mediated toxicity in proximal tubular cells.

Balkan endemic nephropathy (BEN), a disease characterized by progressive renal fibrosis in human patients, has been associated with exposure to ochratoxin A (OTA). This mycotoxin is a frequent contaminant of human and animal food products, and is toxic to all animal species tested. OTA predominantly affects the kidney and is known to accumulate in the proximal tubule (PT). The induction of oxidative stress is implicated in the toxicity of this mycotoxin. In the present study, primary rat PT cells and LLC-PK(1) cells, which express characteristics of the PT, were used to investigate the OTA-mediated oxidative stress response. OTA exposure of these cells resulted in a concentration-dependent elevation of reactive oxygen species (ROS) levels, depletion of cellular glutathione (GSH) levels and an increase in the formation of 8-oxoguanine. The OTA-induced ROS response was significantly reduced following treatment with alpha-tocopherol (TOCO). However, this chain-braking anti-oxidant did not reduce the cytotoxicity of OTA and was unable to prevent the depletion of total GSH levels in OTA-exposed cells. In contrast, pre-incubation of the cell with N-acetyl-L-cysteine (NAC) completely prevented the OTA-induced increase in ROS levels as well as the formation of 8-oxoguanine and completely protected against the cytotoxicity of OTA. In addition, NAC treatment also limited the GSH depletion in OTA-exposed PT- and LLC-PK(1) cells. From these data, we conclude that oxidative stress contributes to the tubular toxicity of OTA. Subsequently, cellular GSH levels play a pivotal role in limiting the short-term toxicity of this mycotoxin in renal tubular cells.

Synthesis of stereopentad analogues of the C14-C22 segment of callystatin A through additions of chiral allenylzinc reagents to stereotriads.

The addition of (P)- and (M)-allenylzinc reagents, prepared in situ through Pd-catalyzed metalation of (R)- and (S)-3-butyn-2-ol mesylates, to diastereomeric stereotriad aldehydes 8, 13, 18, and 23 of syn,syn, syn,anti, anti,anti, and anti,syn stereochemistry was examined. Additions to the former two aldehydes afforded the four anti adducts with high diastereoselectivity and negligible mismatching. Significant mismatching was observed with the latter two aldehydes and the (M)-allenylzinc reagent. An evaluation of possible transition states is presented in consideration of steric and dipolar control elements.

Characterization of biotransformation enzyme activities in primary rat proximal tubular cells.

The proximal tubule is a frequent target for nephrotoxic compounds due to it's ability to transport and accumulate xenobiotics and their metabolites, as well as by the presence of an organ-selective set of biotransformation enzymes. The aim of the present study was to characterize the activities of different biotransformation enzymes during primary culturing of rat proximal tubular cells (PT cells). Specific marker substrates for determining cytochrome P450 (CYP450) activity of primary cultured PT cells include 7-ethoxyresorufin (CYP1A1), caffeine (CYP1A), testosterone (CY2B/C, CYP3A), tolbutamide (CYP2C) and dextromethorphan (CYP2D1). Activities of the CYP450 isoenzymes decreased considerably during culture with the greatest loss in activity within 24 h of culture. In addition, expression of CYP450 apoprotein, including CYP1A, CYP2C, CYP2D, CYP2E and CYP4A, was detected in microsomes from freshly isolated PT cells by immunoblotting using specific antibodies. CYP2B and CYP3A apoprotein could not be detected. Activity of the phase II biotransformation enzymes GST, GGT, beta-lyase and UGT was determined with 1-chloro-2,4-dinitrobenzene, L-glutamic acid gamma-(7-amido-4-methyl-coumarin), S-(1,1,2,2-tetrafluoroethyl)-L-cysteine and 1-naphthol, respectively, as marker substrates. Activity of the phase II enzymes remained more stable and, in contrast to CYP450 activity, significant activity was still expressed after 1 week of PT cell culture. Thus, despite the obvious advantages of PT cells as an in-vitro model for studies of biotransformation mediated toxicity, the strong time dependency of especially phase I and, to a lesser extent, phase II biotransformation activities confers limitations to their application.

In vitro analysis of alpha-amanitin-resistant transcription from the rRNA, procyclic acidic repetitive protein, and variant surface glycoprotein gene promoters in Trypanosoma brucei.

In Trypanosoma brucei, transcription resistant to the mushroom toxin alpha-amanitin is not restricted to the rRNA genes (rDNA), as in higher eukaryotes, but extends to genes encoding the major cell surface proteins variant surface glycoprotein (VSG) and procyclin or procyclic acidic repetitive protein (PARP). Here, we report the development of a homologous cell extract from procyclic T. brucei cells in which rDNA and PARP A and VSG gene promoters drive efficient, accurate, and alpha-amanitin-resistant transcription. A comparative analysis revealed that transcription from the three promoters generally required identical reaction conditions for maximal efficiency. Nevertheless, PARP promoter transcription proved to be exceptional by its high efficiency, its lag phase, a high template DNA concentration optimum, and its tolerance to increasing concentrations of Mn(2+). Mutational analysis for both the PARP and rDNA promoters showed that the proximal and distal core elements were essential for efficient transcription in vitro. Deletion of the upstream control regions (UCRs), however, had a different effect. Whereas PARP UCR deletion reduced transcription efficiency almost 10-fold, deletion of the rDNA UCR had only a minor effect on transcription efficiency.

IGF I induction of p53 requires activation of MAP kinase in cardiac muscle cells.

The aim of this study was to investigate whether IGF I induction of p53 expression and p21 promoter require activation of MAP kinase in cardiac muscle cells. Compared to cardiomyocytes transfected with control vector, activation of MAP kinase by IGF I was decreased by approximately 60-70% in the cells transfected with dominant negative MAP kinase Y185. Transfection with Y185 also resulted in decreased induction of p53 mRNA by IGF I (70% reduction). In the cells transfected with a wildtype p21WAF1/CIP1 promoter construct, activation of luciferase reporter gene by IGF I was decreased in the cells co-transfected with Y185. To further confirm these findings, cells were preincubated with PD98059, a specific MAP kinase kinase inhibitor. As expected, PD98059 inhibited induction of p53 mRNA and p21WAF1/CIP1 promoter by IGF I. These data indicate that transcriptional activation of p53 and p21WAF1/CIP1 by IGF I involves MAP kinase pathway in cardiomyocytes, and thus link MAP kinase to negative modulation of the cell cycle in cardiac muscle cells.