TRANSIENT LIPOPOLYSACCHARIDE-INDUCED CYTOKINE RESPONSES IN THE MATERNAL AND FETAL GUINEA PIG

The aim of this study was to further investigate the role of pro-inflammatory cytokines in the pathogenesis of fetal cererbral white matter injury associated with chorioamnionitis by charaterizing the time course of the cytokine response in the pregnant guinea pig following a maternal inflammatory insult. Chorioamnionitis increases the risk for fetal brain injury. In the guinea pig, a threshold maternal inflammatory response must be reached for significant fetal brain injury to occur. However, a previous study demonstrated that, by seven days after an acute maternal inflammatory insult, cytokine levels in both maternal and fetal compartments are not different from controls. The purpose of this study, therefore, was to test the hypothesis that a significant cytokine response occurs within the first seven days following an acute maternal inflammatory response. Pregnant guinea pigs (n=34) were injected intraperitoneally with 100µg/kg lipopolysaccharide (LPS) at 70% gestation and euthanized at 24 hours, 48 hours or 5 days following endotoxin exposure. Control animals were euthanized at 70% gestation without exposure. Concentrations of interleukin-6, interleukin 1-β and tumour necrosis factor-α (IL-6, IL-1β, TNF-α) were quantified in the maternal serum and amniotic fluid by enzyme-linked immunosorbent assay. IL-6 and IL-1β concentrations were elevated in the maternal serum at 24 hours and returned to control levels by five days. In the amniotic fluid, IL-6 peaked at 48 hours and IL-1β at 24 hours. TNF-α levels were not significantly increased. A single maternal LPS injection produces transient increases in cytokine concentrations in the maternal serum and amniotic fluid. This further implicates the cytokines as potential mediators of fetal white matter damage. Although this response might not be sufficient to produce the brain injury itself, it may initiate harmful pro-inflammatory cytokine cascades, which could even continue to harm the fetus following delivery. A human diagnostic protocol was developed to assess the use of serial serum biomarkers, including IL-6 and TNF-α, in the prediction of histological chorioamnionitis. Preliminary analysis of the pilot study suggests that certain biomarkers might be worthy of further investigation in a larger-scale study.

Retinal Pigment Epithelial Cell DNA is Damaged by Exposure to Benzo[a]pyrene, a Constituent of Cigarette Smoke.

This study examined the effect of exogenous benzo[ a ]pyrene (BaP), an important constituent of cigarette smoke, on cultured bovine retinal pigment epithelial (RPE) cells. Evidence is presented for its metabolic conversion into benzo[ a ]pyrene diol epoxide (BPDE) and the consequent formation of potentially cytotoxic nucleobase adducts in DNA. Cultured RPE cells were treated with BaP at concentrations in the range of 0–100 µm. The presence of BaP was found to cause inhibition of cell growth and replication. BaP induced the expression of a phase I drug metabolizing enzyme which was identified as cytochrome P450 1A1 (CYP 1A1) by RT–PCR and by Western blotting. Coincident with the increased expression of CYP 1A1, covalent adducts between the mutagenic metabolite BPDE and DNA could be detected within RPE cells by immunocytochemical staining. Additional support for their formation was afforded by nuclease P1 enhanced 32P-postlabelling assays on cellular DNA. Single-cell gel electrophoresis (comet) assays showed that exposure of RPE cells to BaP rendered them markedly more susceptible to DNA damage induced by broad band UVB or blue light laser irradiation. In the case of UVB, this is consistent with the photosensitization of DNA cleavage by nucleobase adducts of BPDE. Collectively, these findings imply that BaP has a significant impact on RPE cell pathophysiology and suggest mechanisms whereby exposure to cigarette smoke might cause RPE dysfunction and cell death, thus possibly contributing to degenerative disorders of the retina.

Staurosporine-induced apoptosis and hydrogen peroxide-induced necrosis in two human breast cell lines.

The use of apoptosis-inducing agents in the treatment of malignant cancer is increasingly being considered as a therapeutic approach. In this study, the induction of apoptosis and necrosis was examined in terms of temporal dose responses, comparing a malignant and nonmalignant breast cell line. Staurosporine (SSP)-induced apoptosis and H2O2-induced necrosis were evaluated by two cytotoxicity assays, neutral red (NR) and methyl-thiazolyl tertrazolium (MTT), in comparison with a differential dye uptake assay, using Hoechst33342/propidium iodide (Hoechst/PI). Confirmatory morphological assessment was also performed by routine resin histology and transmission electron microscopy. Cell viability was assessed over a 0.5-48 h time course. In nonmalignant HBL-100 cells, 50 nM SSP induced 100% apoptosis after a 48 h exposure, while the same exposure to SSP caused only 4% apoptosis in metastatic T47D cells. Although complete apoptosis of both cell lines was induced by 50 M SSP, this effect was delayed in T47D (24 h) compared with HBL-100 (4 h). Results also showed that neither MTT or NR can distinguish between the modes of cell death, nor detect the early onset of apoptosis revealed by Hoechst/PI.

Diabetes-induced activation of protein kinase C inhibits store-operated Ca2+ uptake in rat retinal microvascular smooth muscle.

AIMS/HYPOTHESIS: To assess the effects of diabetes-induced activation of protein kinase C (PKC) on voltage-dependent and voltage-independent Ca2+ influx pathways in retinal microvascular smooth muscle cells. METHODS: Cytosolic Ca2+ was estimated in freshly isolated rat retinal arterioles from streptozotocin-induced diabetic and non-diabetic rats using fura-2 microfluorimetry. Voltage-dependent Ca2+ influx was tested by measuring rises in [Ca2+]i with KCl (100 mmol/l) and store-operated Ca2+ influx was assessed by depleting [Ca2+]i stores with Ca2+ free medium containing 5 micromol/l cyclopiazonic acid over 10 min and subsequently measuring the rate of rise in Ca2+ on adding 2 mmol/l or 10 mmol/l Ca2+ solution. RESULTS: Ca2+ entry through voltage-dependent L-type Ca2+ channels was unaffected by diabetes. In contrast, store-operated Ca2+ influx was attenuated. In microvessels from non-diabetic rats 20 mmol/l D-mannitol had no effect on store-operated Ca2+ influx. Diabetic rats injected daily with insulin had store-operated Ca2+ influx rates similar to non-diabetic control rats. The reduced Ca2+ entry in diabetic microvessels was reversed by 2-h exposure to 100 nmol/l staurosporine, a non-specific PKC antagonist and was mimicked in microvessels from non-diabetic rats by 10-min exposure to the PKC activator phorbol myristate acetate (100 nmol/l). The specific PKCbeta antagonist LY379196 (100 nmol/l) also reversed the poor Ca2+ influx although its action was less efficacious than staurosporine. CONCLUSION/INTERPRETATION: These results show that store-operated Ca2+ influx is inhibited in retinal arterioles from rats having sustained increased blood glucose and that PKCbeta seems to play a role in mediating this effect.

Glucose-induced phosphatidylinositol 3-kinase and mitogen-activated protein kinase-dependent upregulation of the platelet-derived growth factor-B receptor potentiates vascular smooth muscle cell chemotaxis

The aim of this study was to investigate the effects of elevated D-glucose concentrations on vascular smooth muscle cell (VSMC) expression of the platelet-derived growth factor (PDGF) beta receptor and VSMC migratory behavior. Immunoprecipitation, immunofluorescent staining, and RT-PCR of human VSMCs showed that elevated D-glucose induced an increase in the PDGF beta receptor that was inhibited by phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathway inhibitors. Exposure to 25 mmol/l D-glucose (HG) induced increased phosphorylation of protein kinase B (PKB) and extracellular-regulated kinase (ERK). All HG chemotaxis assays (with either 10 days' preincubation in HG or no preincubation) in a FCS or PDGF-BB gradient showed positive chemotaxis, whereas those in 5 mmol/l D-glucose did not. Assays were also run with concentrations ranging from 5 to 25 mmol/l D-glucose. Chemotaxis was induced at concentrations >9 mmol/l D-glucose. An anti-PDGF beta receptor antibody inhibited glucose-potentiated VSMC chemotaxis, as did the inhibitors for the PI3K and MAPK pathways. This study has shown that small increases in D-glucose concentration, for a short period, increase VSMC expression of the PDGF beta receptor and VSMC sensitivity to chemotactic factors in serum, leading to altered migratory behavior in vitro. It is probable that similar processes occur in vivo with glucose-enhanced chemotaxis of VSMCs, operating through PDGF beta receptor-operated pathways, contributing to the accelerated formation of atheroma in diabetes.

Damage to plasmid DNA induced by low energy carbon ions

The damage induced in supercoiled plasmid DNA molecules by 1-6 keV carbon ions has been investigated as a function of ion exposure, energy and charge state. The production of short linear fragments through multiple double strand breaks has been demonstrated and exponential exposure responses for each of the topoisomers have been found. The cross section for the loss of supercoiling was calculated to be (2.2 +/- 0.5) x 10(-14) cm(2) for 2 keVC(+) ions. For singly charged carbon ions, increased damage was observed with increasing ion energy. In the case of 2 keV doubly charged ions, the damage was greater than for singly charged ions of the same energy. These observations demonstrate that ion induced damage is a function of both the kinetic and potential energies of the ion.

cAMP/PKA-dependent increases in Ca Sparks, oscillations and SR Ca stores in retinal arteriolar myocytes after exposure to vasopressin.

PURPOSE: To investigate the effects of arginine vasopressin (AVP) on Ca(2+) sparks and oscillations and on sarcoplasmic reticulum (SR) Ca(2+) content in retinal arteriolar myocytes. METHODS: Fluo-4-loaded smooth muscle in intact segments of freshly isolated porcine retinal arteriole was imaged by confocal laser microscopy. SR Ca(2+) store content was assessed by recording caffeine-induced Ca(2+) transients with microfluorimetry and fura-2. RESULTS: The frequencies of Ca(2+) sparks and oscillations were increased both during exposure to, and 10 minutes after washout of AVP (10 nM). Caffeine transients were increased in amplitude 10 and 90 minutes after a 3-minute application of AVP. Both AVP-induced Ca(2+) transients and the enhancement of caffeine responses after AVP washout were inhibited by SR 49059, a V(1a) receptor blocker. Forskolin, an activator of adenylyl cyclase, also persistently enhanced caffeine transients. Rp-8-HA-cAMPS, a membrane-permeant PKA inhibitor, prevented enhancement of caffeine transients by both AVP and forskolin. Forskolin, but not AVP, produced a reversible, Rp-8-HA-cAMPS insensitive reduction in basal [Ca(2+)](i). CONCLUSIONS: AVP activates a cAMP/PKA-dependent pathway via V(1a) receptors in retinal arteriolar smooth muscle. This effect persistently increases SR Ca(2+) loading, upregulating Ca(2+) sparks and oscillations, and may favor prolonged agonist activity despite receptor desensitization.

NADPH oxidase-4 mediates protection against chronic load-induced stress in mouse hearts by enhancing angiogenesis

Cardiac failure occurs when the heart fails to adapt to chronic stresses. Reactive oxygen species (ROS)-dependent signaling is implicated in cardiac stress responses but the role of different ROS sources remains unclear. Here, we report that NADPH oxidase-4 (Nox4) facilitates cardiac adaptation to chronic stress. Unlike other Nox proteins, Nox4 activity is regulated mainly by its expression level which increased in cardiomyocytes during stresses such as pressure overload or hypoxia. To investigate the functional role of Nox4 during the cardiac response to stress, we generated mice with a genetic deletion of Nox4 or a cardiomyocyte-targeted overexpression of Nox4. Basal cardiac function was normal in both models but Nox4-null animals developed exaggerated contractile dysfunction, hypertrophy and cardiac dilatation during exposure to chronic overload whereas Nox4-transgenic mice were protected. Investigation of mechanisms underlying this protective effect revealed a significant Nox4-dependent preservation of myocardial capillary density after pressure overload. Nox4 enhanced stress-induced activation of cardiomyocyte Hif1 and the release of VEGF, resulting in an increased paracrine angiogenic activity. These data indicate that cardiomyocyte Nox4 is a novel inducible regulator of myocardial angiogenesis, a key determinant of cardiac adaptation to overload stress. Our results also have wider relevance to the use of non-specific antioxidant approaches in cardiac disease and may provide an explanation for the failure of such strategies in many settings.

Chemotherapy-Induced Activation of ADAM-17: A Novel Mechanism of Drug Resistance in Colorectal Cancer

Purpose: We have shown previously that exposure to anticancer drugs can trigger the activation of human epidermal receptor survival pathways in colorectal cancer (CRC). In this study, we examined the role of ADAMs (a disintegrin and metalloproteinases) and soluble growth factors in this acute drug resistance mechanism.

Experimental Design: In vitro and in vivo models of CRC were assessed. ADAM-17 activity was measured using a fluorometric assay. Ligand shedding was assessed by ELISA or Western blotting. Apoptosis was assessed by flow cytometry and Western blotting.

Results: Chemotherapy (5-fluorouracil) treatment resulted in acute increases in transforming growth factor-a, amphiregulin, and heregulin ligand shedding in vitro and in vivo that correlated with significantly increased ADAM-17 activity. Small interfering RNA–mediated silencing and pharmacologic inhibition confirmed that ADAM-17 was the principal ADAM involved in this prosurvival response. Furthermore, overexpression of ADAM-17 significantly decreased the effect of chemotherapy on tumor growth and apoptosis. Mechanistically, we found that ADAM-17 not only regulated phosphorylation of human epidermal receptors but also increased the activity of a number of other growth factor receptors, such as insulin-like growth factor-I receptor and vascular endothelial growth factor receptor.

Conclusions: Chemotherapy acutely activates ADAM-17, which results in growth factor shedding, growth factor receptor activation, and drug resistance in CRC tumors. Thus, pharmacologic inhibition of ADAM-17 in conjunction with chemotherapy may have therapeutic potential for the treatment of CRC.

Regulation of FOS by different compartmental stresses induced by low levels of ionizing radiation

We irradiated different cellular compartments and measured changes in expression of the FOS gene at the mRNA and protein levels. [H-3]Thymidine and tritiated water were used to irradiate the nucleus and the whole cell, respectively. I-125-Concanavalin A binding was used to irradiate the cell membrane differentially. Changes in FOS mRNA and protein levels were measured using semi-quantitative RT-PCR and SDS-PAGE Western blotting, respectively, Irradiation of the nucleus or the whole cell at a dose rate of 0.075 Gy/h caused no change in the level of FOS mRNA expression, but modestly (1.5-fold) induced FOS protein after 0.5 h, Irradiation of the nucleus at a dose rate of 0.43 Gy/h induced FOS mRNA by 1.5-fold after 0.5 h, but there was no significant effect after whole-cell irradiation. FOS protein was transiently induced 2.5-fold above control levels 0.5 h after a 0.43-Gy/h exposure of the nucleus or the whole cell. Irradiation of the cell membrane at a dose rate of 1.8 Gy/h for up to 2 h caused no change in the levels of expression of FOS mRNA or protein, but a dose rate of 6.8 Gy/h transiently increased the level of FOS mRNA S-fold after 0.5 h, These data demonstrate the complexity of the cellular response to radiation-induced damage at low doses. The lack of quantitative agreement between the transcript and protein levels for FOS suggests a role for posttranscriptional regulation. (C) 2000 by Radiation Research Society.

Quantitative analysis of proton imaging measurements of laser-induced plasmas

A method for obtaining quantitative information about electric field and charge distributions from proton imaging measurements of laser-induced plasmas is presented. A parameterised charge distribution is used as target plasma. The deflection of a proton beam by the electric field of such a plasma is simulated numerically as well as the resulting proton density, which will be obtained on a screen behind the plasma according to the proton imaging technique. The parameters of the specific charge distributions are delivered by a combination of linear regression and nonlinear fitting of the calculated proton density distribution to the measured optical density of a radiochromic film screen changed by proton exposure. It is shown that superpositions of spherical Gaussian charge distributions as target plasma are sufficient to simulate various structures in proton imaging measurements, which makes this method very flexible.

Hyperglycaemia-induced pro-inflammatory responses by retinal Müller glia are regulated by the receptor for advanced glycation end-products (RAGE)

Aims/hypothesis: Up-regulation of the receptor for AGEs (RAGE) and its ligands in diabetes has been observed in various tissues. Here, we sought to determine levels of RAGE and one of its most important ligands, S100B, in diabetic retina, and to investigate the regulatory role of S100B and RAGE in Müller glia.

Methods: Streptozotocin-diabetes was induced in Sprague-Dawley rats. RAGE, S100B and glial fibrillary acidic protein (GFAP) were detected in retinal cryosections. In parallel, the human retinal Müller cell line, MIO-M1, was maintained in normal glucose (5.5 mmol/l) or high glucose (25 mmol/l). RAGE knockdown was achieved using small interfering RNA (siRNA), while soluble RAGE was used as a competitive inhibitor of RAGE ligand binding. RAGE, S100B and cytokines were detected using quantitative RT-PCR, western blotting, cytokine protein arrays or ELISA. Activation of mitogen-activated protein kinase (MAPK) by RAGE was determined by western blotting.

Results: Compared with non-diabetic controls, RAGE and S100B were significantly elevated in the diabetic retina with apparent localisation in the Müller glia, occurring concomitantly with upregulation of GFAP. Exposure of MIO-M1 cells to high glucose induced increased production of RAGE and S100B. RAGE signalling via MAPK pathway was linked to cytokine production. Blockade of RAGE prevented cytokine responses induced by high glucose and S100B in Müller glia.

Conclusions/interpretation: Hyperglycaemia in vivo and in vitro exposure to high glucose induce upregulation of RAGE and its ligands, leading to RAGE signalling, which links to pro-inflammatory responses by retinal Müller glia. These data shed light on the potential clinical application of RAGE blockade to inhibit the progression of diabetic retinopathy.

Platinum resistant cancer cells conserve sensitivity to BH3 domains and obatoclax induced mitochondrial apoptosis

Resistance to cisplatin chemotherapy remains a major hurdle preventing effective treatment of many solid cancers. BAX and BAK are pivotal regulators of the mitochondrial apoptosis pathway, however little is known regarding their regulation in cisplatin resistant cells. Cisplatin induces DNA damage in both sensitive and resistant cells, however the latter exhibits a failure to initiate N-terminal exposure of mitochondrial BAK or mitochondrial SMAC release. Both phenotypes are highly sensitive to mitochondrial permeabilisation induced by exogenous BH3 domain peptides derived from BID, BIM, NOXA (which targets MCL-1 and A1), and there is no significant change in their prosurvival BCL2 protein expression profiles. Obatoclax, a small molecule inhibitor of pro-survival BCL-2 family proteins including MCL-1, decreases cell viability irrespective of platinum resistance status across a panel of cell lines selected for oxaliplatin resistance. In summary, selection for platinum resistance is associated with a block of mitochondrial death signalling upstream of BAX/BAK activation. Conservation of sensitivity to BH3 domain induced apoptosis can be exploited by agents such as obatoclax, which directly target the mitochondria and BCL-2 family.

Experimental setup and first measurement of DNA damage induced along and around an antiproton beam

Radiotherapy employs ionizing radiation to induce lethal DNA lesions in cancer cells while minimizing damage to healthy tissues. Due to their pattern of energy deposition, better therapeutic outcomes can, in theory, be achieved with ions compared to photons. Antiprotons have been proposed to offer a further enhancement due to their annihilation at the end of the path. The work presented here aimed to establish and validate an experimental procedure for the quantification of plasmid and genomic DNA damage resulting from antiproton exposure. Immunocytochemistry was used to assess DNA damage in directly and indirectly exposed human fibroblasts irradiated in both plateau and Bragg peak regions of a 126 MeV antiproton beam at CERN. Cells were stained post irradiation with an anti-gamma-H2AX antibody. Quantification of the gamma-H2AX foci-dose relationship is consistent with a linear increase in the Bragg peak region. A qualitative analysis of the foci detected in the Bragg peak and plateau region indicates significant differences highlighting the different severity of DNA lesions produced along the particle path. Irradiation of desalted plasmid DNA with 5 Gy antiprotons at the Bragg peak resulted in a significant portion of linear plasmid in the resultant solution.

Cytosol protein regulation in H295R steroidogenesis model induced by the zearalenone metabolites, α- and β-zearalenol.

a- and b-zearalenol (a-ZOL and b-ZOL, respectively) are metabolites of the mycotoxin zearalenone (ZEN). All three individual mycotoxins have shown to be biological active i.e. being estrogenic and able to stimulate cellular proliferation albeit at different strengths. In this work, cytosol protein expression was determined by using stable-isotope labelling by amino acids in cell culture (SILAC) upon exposure of a-ZOL and b-ZOL to the steroidogenesis cell model H295R. A total of 14 and 5 individual proteins were found to be significantly regulated by a-ZOL and b-ZOL, respectively. Interestingly, there were no common protein regulations by the metabolites or the parent mycotoxin ZEN. Furthermore, the regulated proteins were assigned to networks and groups of actions that also differed from one another suggesting that the three individual mycotoxins may have unique biological activities.