Signals through the B-cell antigen receptor (BCR) are important for the survival of chronic lymphocytic leukemia (CLL) cells. Therefore, factors that influence these signals have important ...pathophysiological roles in this disease. One key mediator of BCR signaling is protein kinase C beta (PKCbeta), which regulates the activation of I-kappaB kinases and the deactivation of Bruton tyrosine kinase within the signaling pathways initiated by BCR engagement. The present study demonstrates that overexpression of the PKCbetaII isoform is a feature of CLL cells and that activity of this enzyme strongly correlates with CLL cell response to BCR engagement. Thus, intracellular Ca2+ release and increases in cell survival after BCR cross-linking were significantly greater in CLL patients with low levels than in CLL patients with high levels of active PKCbetaII. Furthermore, BCR-induced Ca2+ fluxes could be restored in CLL patients with high levels of active PKCbetaII by pretreating the cells with the PKCbeta-specific inhibitor LY379196. Conversely, BCR-mediated intracellular Ca2+ release could be inhibited in CLL cells with low levels of active PKCbetaII by pretreatment with the PKC agonist bryostatin. Taken together, these results demonstrate that overexpressed active PKCbetaII plays a role in the regulation and outcome of BCR signals that can be important for the progression of CLL.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Eating a high calorie meal is known to induce endothelial dysfunction and it is reported that consuming drinks rich in antioxidants may be protective against this. In this study we assessed the ...effects of three antioxidant drinks with considerable disparity in their antioxidant content on endothelial function. Seven apparently healthy overweight and older adults (BMI 25-35; mean age 57 ± 3 years; one male, six females) completed four trials in a randomized counterbalanced design. Water (control), orange juice, green tea, or red wine were consumed with a high calorie meal (>900 kcal). Endothelial function was measured by flow-mediated dilatation immediately before (fasted, baseline) and 2 h after the meal. Blood samples were also obtained for lipid and glucose analysis, plasma nitrite ( NO 2 - ) and oxidized low-density lipoprotein (ox-LDL). Participants returned after a minimum 3 days washout to complete the remaining arms of the study. The results found that the high calorie meal induced a substantial increase in triglycerides, but not cholesterol or glucose, at 2 h after meal ingestion. FMD was significantly reduced by ∼35% at this timepoint, but the effect was not attenuated by co-ingestion of any of the antioxidant drinks. Reduced FMD was mirrored by a reduction in NO 2 - , but ox-LDL was not increased at 2 h after the meal. None of the undertaken measures were influenced by the antioxidant drinks. We conclude that co-ingestion of none of our test antioxidant drinks protected against the substantial post-prandial endothelial dysfunction induced by an unhealthy meal challenge in our sample population at a 2 h timepoint.
Abstract
This project has been funded by the EU’s INTERREG VA programme, managed by the special EU Programmes Body (SEUPB)
Background and Aims
N-acetyl-L-cysteine (NAC) has been proposed as a ...prophylactic for the prevention of contrast induced nephropathy (CIN), a serious, adverse complication following administration of contrast media in patients with diabetes and chronic kidney disease (CKD). However, clinical trial results have failed to demonstrate consistently the beneficial qualities of NAC in this setting. The suggested mechanism by which NAC has been proposed to be effective is via antioxidant activity, but NAC itself appears not to be a powerful antioxidant; intracellular conversion of NAC to glutathione (GSH) is essential for antioxidant effects. We tested the hypothesis that oral or intravenous NAC fails to increase antioxidant activity in the plasma of healthy volunteers or CKD stage III patients undergoing elective coronary angiography.
Method
16 healthy volunteers (8 receiving contrast media) and 8 patients with chronic renal failure were recruited to 3 separate groups as a part of a crossover design study. 66 patients with CKD stage III undergoing elective coronary angiography were recruited to a parallel group randomized controlled trial. Each volunteer or patient was randomised to receive NAC (i.v.; 50 mg/kg/hr infusion for 2 h, followed by 20 mg/kg/hr infusion for a further 5 h), or NAC (oral; 1200 mg twice daily, commencing the day before the study visit) or placebo. Participants attended the Clinical Research Facility on three occasions for the crossover groups, but only once in the parallel group trial. Patients were infused with para-aminohippurate (PAH) and inulin to enable measurement of renal function parameters. Plasma and paired buffy coat samples were collected at several time points for 2 hours before contrast infusion and for 6 hours after. Samples were used to measure various clinical parameters, along with intracellular and plasma thiol concentrations and the plasma antioxidant capacity (oxygen radical antioxidant capacity; ORAC).
Results
Plasma NAC concentrations peaked at 222 µM (healthy volunteers), 354 µM (CKD patients), 271 µM (healthy volunteers receiving contrast) and 348 µM (CKD patients undergoing elective coronary angiography) following infusion of NAC. The corresponding peak concentrations were ∼2 µM following oral NAC across all groups. No comparable increase in the plasma compartment ORAC antioxidant capacity was measured in any of the treatment groups. Equally, contrast infusion itself did not significantly reduce antioxidant capacity in plasma, even in the placebo group. Buffy coat measures showed that there was a significant increase of intracellular GSH in some patients. Also, no clear additional benefit was witnessed by increasing plasma NAC ∼100-fold through infusion of NAC over oral administration.
Conclusion
NAC fails to influence ORAC antioxidant capacity in the plasma compartment of CKD stage III patients receiving contrast media. Our results question the importance of oxidative stress in contrast-induced nephropathy. Furthermore, the findings fail to demonstrate a direct antioxidant impact of NAC in the plasma compartment and question the antioxidant hypothesis related to contrast-induced nephropathy and NAC.
Figure:
The results in Chapter 2 show that cultured CLL cells secrete VEGF, and that secretion is increased as HIF-1α becomes stabilised under conditions of hypoxia. However, expression of HIF-1α is not ...related to the amount of VEGF secreted by these cells, or the presence of a p53 dysfunction mutation. In addition, CD154 stimulation of CLL cells also increased VEGF secretion. The data presented in Chapter 3, demonstrates that the VEGF secreted by CLL cells is angiogenically active. Thus, CLL-cell conditioned medium (CM) stimulated endothelial-cell proliferation in vitro and angiogenesis in vivo, effects that were abrogated by pre-incubation of CM with VEGF-neutralising antibodies. The presence of VEGF in lymph nodes and spleen from CLL patients and the elevated microvessel density observed in the lymph nodes, implicate VEGF in the increased angiogenesis that is found in this disease. Both VEGF-receptor 1 and VEGF-receptor 2 are expressed by CLL cells, and this work is shown in Chapter 4. Moreover, ligation of these receptors by VEGF or the receptor-specific ligands PLGF and VEGF-E, is accompanied by increased expression of Hsp90, thus indicating a possible autocrine role for VEGF in the activation of signalling pathways in CLL cells. Work presented in Chapter 5 explores the role of VEGF in CLL-cell survival. In culture, exogenous VEGF did not prolong CLL-cell survival nor prevent induction of cell death by fludarabine, ionising radiation of geldanamycin. Neutralising autocrine VEGF and blockade of VEGF receptors did not affect cell survival, but inhibiting VEGF receptor signalling with SU5416 significantly reduced cell viability. Furthermore, CD154-enhanced survival of CLL cells, which involves activation of NF-κB, was reduced by both anti-VEGF neutralising antibody and by SU5416.
Background
Diesel exhaust inhalation causes cardiovascular dysfunction including impaired vascular reactivity, increased blood pressure, and arterial stiffness. We investigated the role of nitric ...oxide (NO) bioavailability in mediating these effects.
Methods and Results
In 2 randomized double‐blind crossover studies, healthy nonsmokers were exposed to diesel exhaust or filtered air. Study 1: Bilateral forearm blood flow was measured during intrabrachial infusions of acetylcholine (ACh; 5 to 20 μg/min) and sodium nitroprusside (SNP; 2 to 8 μg/min) in the presence of the NO clamp (NO synthase inhibitor NG‐monomethyl‐l‐arginine (l‐NMMA) 8 μg/min coinfused with the NO donor SNP at 90 to 540 ng/min to restore basal blood flow). Study 2: Blood pressure, arterial stiffness, and cardiac output were measured during systemic NO synthase inhibition with intravenous l‐NMMA (3 mg/kg). Following diesel exhaust inhalation, plasma nitrite concentrations were increased (68±48 versus 41±32 nmol/L; P=0.006) despite similar l‐NMMA–induced reductions in basal blood flow (−20.6±14.7% versus −21.1±14.6%; P=0.559) compared to air. In the presence of the NO clamp, ACh and SNP caused dose‐dependent vasodilatation that was not affected by diesel exhaust inhalation (P>0.05 for both). Following exposure to diesel exhaust, l‐NMMA caused a greater increase in blood pressure (P=0.048) and central arterial stiffness (P=0.007), but reductions in cardiac output and increases in systemic vascular resistance (P>0.05 for both) were similar to those seen with filtered air.
Conclusions
Diesel exhaust inhalation disturbs normal vascular homeostasis with enhanced NO generation unable to compensate for excess consumption. We suggest the adverse cardiovascular effects of air pollution are, in part, mediated through reduced NO bioavailability.
Clinical Trial Registration
URL: http://www.ClinicalTrials.gov. Unique identifiers: NCT00845767 and NCT01060930.
Following bone marrow transplantation (BMT), the stroma remains host-derived, and has therefore been exposed to the high doses of chemoradiotherapy used in BMT conditioning. We have used long term ...bone marrow culture (LTBMC) to study the effect of this conditioning therapy on the stroma. Twenty-five BMT recipients were studied, comprising 13 allografts and 12 autografts. Marrow was aspirated prior to transplant (6 cases) and at 3, 6 or 12 months post-BMT. Fifteen haematologically normal subjects were studied in parallel. The stromal layer of LTBMC was visually assessed at weekly intervals and supernatant cells counted and assayed for colony forming unit-granulocyte/macrophage (CFU-GM).
Five of the six cases studied both before and after BMT formed less confluent stroma following the procedure. A successive improvement in the proportion of patients forming good stroma was observed with increasing time from BMT. Supernatant cell and CFU-GM counts were not significantly different from normal following BMT. No clear relationship was observed between stromal confluence and any of the following: supernatant cell and CFU-GM counts, transplant type, underlying disease, conditioning regime or time to engraftment. These data support the view that BMT conditioning regimes cause stromal damage, and that this damage gradually improves with time.
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