Cytokine release assays: current practices and future directions.

As a result of the CD28 superagonist biotherapeutic monoclonal antibody (TGN 1412) "cytokine storm" incident, cytokine release assays (CRA) have become hazard identification and prospective risk assessment tools for screening novel biotherapeutics directed against targets having a potential risk for eliciting adverse pro-inflammatory clinical infusion reactions. Different laboratories may have different strategies, assay formats, and approaches to the reporting, interpretation, and use of data for either decision making or risk assessment. Additionally, many independent contract research organizations (CROs), academic and government laboratories are involved in some aspect of CRA work. As a result, while some pharmaceutical companies are providing CRA data as part of the regulatory submissions when necessary, technical and regulatory practices are still evolving to provide data predictive of cytokine release in humans and that are relevant to safety. This manuscript provides an overview of different approaches employed by the pharmaceutical industry and CROs, for the use and application of CRA based upon a survey and post survey follow up conducted by ILSI-Health and Environmental Sciences Institute (HESI) Immunotoxicology Committee CRA Working Group. Also discussed is ongoing research in the academic sector, the regulatory environment, current limitations of the assays, and future directions and recommendations for cytokine release assays.

Alpha particles initiate biological production of superoxide anions and hydrogen peroxide in human cells.

The mechanism(s) by which high-linear energy transfer a particles, like those emitted by inhaled radon and radon daughters, cause lung cancer has not been elucidated. Conceivably, DNA damage that is induced by a particles may be mediated by the metabolic generation of reactive oxygen species (ROS), in addition to direct a particle-DNA interactions and hydroxyl radical-DNA interactions. Using normal human lung fibroblasts, we investigated the hypothesis that densely ionizing alpha particles may induce the intracellular generation of superoxide (O2.-) and hydrogen peroxide (H2O2). Ethidium bromide and 2',7'-dichlorofluorescein, fluorescent products of the membrane-permeable dyes hydroethidine and 2',7'-dichlorofluorescin diacetate, respectively, were used to monitor the intracellular production of O2.- and H2O2, respectively, by flow cytometry. Compared to sham-irradiated cells, fibroblasts that were exposed to alpha particles (0.4-19 cGy) had significant increases in intracellular O2.- production, along with concomitant increases in H2O2 production. Further analyses suggest that the plasma membrane-bound NADPH-oxidase is primarily responsible for this increased intracellular generation of ROS and that the ROS response does not require direct nuclear or cellular "hits" by the a particles. In this latter regard, we additionally report that unirradiated cells also show the ROS response when they are incubated with serum-containing culture medium that has been exposed to a particles or when they are incubated with supernatants from a-irradiated cells. Our overall results support the possibility that a particles, at least in part, may mediate their DNA-damaging effects indirectly via a ROS-related mechanism.

Selective estrogen receptor modulator idoxifene inhibits smooth muscle cell proliferation, enhances reendothelialization, and inhibits neointimal formation in vivo after vascular injury.

BACKGROUND: Idoxifene (ID) is a tissue-selective estrogen receptor modulator (SERM). The pharmacological profile of ID in animal studies suggests that it behaves like an estrogen receptor (ER) agonist in bone and lipid metabolism while having negligible ER activity on the reproductive system. It is unknown whether ID retains the vascular protective effects of estrogen. METHODS AND RESULTS: In cultured vascular smooth muscle cells (VSMCs), ID inhibited platelet-derived growth factor-induced DNA synthesis and mitogenesis with IC(50) values of 20.4 and 27.5 nmol/L, respectively. Treatment with ID resulted in S-phase cell cycle arrest in serum-stimulated VSMCs. ID 1 to 100 nmol/L significantly protected endothelial cells from tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis in vitro. Virgin Sprague-Dawley rats ovariectomized 1 week before the study were treated with ID (1 mg x kg(-1) x d(-1)) or vehicle by gavage for 3 days before balloon denudation in carotid artery. The SMC proliferation in injured vessels was determined by immunostaining for proliferating cell nuclear antigen (PCNA). The number of PCNA-positive SMCs was reduced by 69%, 82%, and 86% in the media at days 1, 3 and 7, respectively, and by 78% in the neointima at day 7 after injury in ID- versus vehicle-treated group (P:<0.01). ID significantly enhanced reendothelialization in the injured carotid arteries as determined by Evans blue stain and immunohistochemical analysis for von Willebrand factor. In the former assay, the reendothelialized area in injured vessels was 43% in ID-treated group versus 24% in the vehicle group (P:<0.05); in the latter assay, the numbers of von Willebrand factor-positive cells per cross section increased from 24. 8 (vehicle) to 60.5 (ID) (P:<0.01) at day 14 after injury. In addition, the production of nitric oxide from excised carotid arteries was significantly higher in ID-treated than the vehicle group (8.5 versus 2.7 nmol/g, P:<0.01). Finally, ID treatment reduced neointimal area and the ratio of intima to media by 45% and 40%, respectively (P:<0.01), at day 14 after balloon angioplasty. CONCLUSIONS: The results indicate that ID beneficially modulates the balloon denudation-induced vascular injury response. Inhibition of VSMC proliferation and acceleration of endothelial recovery likely mediate this protective effect of ID.

In vivo myocardial protection from ischemia/reperfusion injury by the peroxisome proliferator-activated receptor-gamma agonist rosiglitazone.

BACKGROUND: Diabetes is associated with increased risk of mortality as a consequence of acute myocardial infarction. This study determined whether rosiglitazone (ROSI) could reduce myocardial infarction after ischemia/reperfusion injury. METHODS AND RESULTS: Male Lewis rats were anesthetized, and the left anterior descending coronary artery was ligated for 30 minutes. After reperfusion for 24 hours, the ischemic and infarct sizes were determined. ROSI at 1 and 3 mg/kg IV reduced infarct size by 30% and 37%, respectively (P<0.01 versus vehicle). Pretreatment with ROSI (3 mg. kg(-1). d(-1) PO) for 7 days also reduced infarct size by 24% (P<0.01). ROSI also improved ischemia/reperfusion-induced myocardial contractile dysfunction. Left ventricular systolic pressure and positive and negative maximal values of the first derivative of left ventricular pressure (dP/dt) were significantly improved in ROSI-treated rats. ROSI reduced the accumulation of neutrophils and macrophages in the ischemic heart by 40% and 43%, respectively (P<0.01). Ischemia/reperfusion induced upregulation of CD11b/CD18 and downregulation of L-selectin on neutrophils and monocytes; these effects were significantly attenuated in ROSI-treated animals. Likewise, intercellular adhesion molecule-1 expression in ischemic hearts was markedly diminished by ROSI, as was the ischemia/reperfusion-stimulated upregulation of monocyte chemoattractant protein-1. CONCLUSIONS: ROSI reduced myocardial infarction and improved contractile dysfunction caused by ischemia/reperfusion injury. The cardioprotective effect of ROSI was most likely due to inhibition of the inflammatory response.

Intracellular hydrogen peroxide and superoxide anion detection in endothelial cells.

One of the objectives of studying endothelial cells in vitro is to evaluate neutrophil-endothelial cell interactions including potential consequences of oxidant-mediated damage to the endothelial cell. Current understanding of endothelial cell oxidative function is derived primarily from the measurement of extracellular products. We utilized 2 dyes, 2',7'-dichlorofluorescein diacetate (DCFH-DA) and hydroethidine (HE), which measure hydrogen peroxide (H2O2) and superoxide anion (O2-) respectively, for their suitability to monitor oxidative mechanisms in endothelial cells and to provide a reliable measure of intracellular oxidants. Endothelial cells stained with DCFH-DA and stimulated with H2O2 exhibited an increase in the fluorescent product 2',7'-dichlorofluorescein (DCF) (measure of intracellular H2O2) which peaked at 10 min. Endothelial cells stained with HE and stimulated with H2O2 exhibited an increase in the fluorescent product ethidium bromide (EB) (measure of intracellular O2-) which lasted for approximately 60 min. Superoxide dismutase increased DCF fluorescence in endothelial cells stimulated with H2O2 by 158%. Allopurinol (xanthine oxidase inhibitor) reduced DCF and EB fluorescence by 48% and 37% respectively in endothelial cells stimulated with H2O2. Catalase completely inhibited an increase in DCF or EB fluorescence in endothelial cells stimulated with H2O2. There was a direct correlation between mean DCF and EB fluorescence intensity and the concentration of H2O2 or the number of phorbol 12-myristate 13-acetate-activated neutrophils added to endothelial cells. We conclude from these studies that DCFH-DA and HE can be used to measure intracellular H2O2 and O2- in endothelial cells and that the xanthine oxidase pathway for intracellular O2- production accounts for approximately 40% of the total intracellular O2- generated in endothelial cells after stimulation with H2O2. The combination of image cytometry and flow cytometry will be important for future evaluations of endothelial cell function.

Defects in intracellular oxidative metabolism of neutrophils undergoing apoptosis.

Apoptosis permits neutrophil recognition by macrophages, thereby not only limiting potential cytotoxicity but also promoting resolution of inflammation. A direct relationship between apoptosis and intracellular hydrogen peroxide (H2O2) production was observed in phorbol 12-myristate 13-acetate (PMA) -stimulated neutrophils aged in culture. A significant decrease in intracellular H2O2 production was observed in aging neutrophils at 12, 24, and 48 h. However, intracellular superoxide anion production in response to PMA stimulation was preserved up to 24 h, implying retention of intracellular signaling pathways leading to NADPH oxidase stimulation. A significant decrease in the cytoplasmic content and activity of Cu,Zn superoxide dismutase was responsible for the observed decline in intracellular H2O2 production in apoptotic neutrophils. Intracellular glutathione content also decreased concomitantly with H2O2 production. These observations indicate that onset of apoptosis in neutrophils is in part mediated by oxidative stress resulting from the down-regulation of key antioxidant defense systems of the cell, namely superoxide dismutase and glutathione.

Impairment of human neutrophil oxidative burst by polychlorinated biphenyls: inhibition of superoxide dismutase activity.

We report evidence of a novel mechanism by which polychlorinated biphenyls might act as potent inducers of inflammation. Aroclor 1242 (A1242), a polychlorinated biphenyl mixture, and 2,2',4,4'-tetrachlorobiphenyl (PCB47), a constituent of A1242 that produces the same patterns of effects, impaired the oxidative burst of human neutrophils by inhibiting the antioxidant enzyme superoxide dismutase, which converts O2- to H2O2. Pre-incubation of neutrophils with A1242 or PCB47 before stimulation with phorbol 12-myristate 13-acetate heightened the respiratory burst, producing a significant increase in intracellular O2- production along with a significant decrease in H2O2 production compared with unexposed agonist-stimulated neutrophils. This was also evident in a physiologically relevant situation in which neutrophils pre-incubated with A1242 were subsequently stimulated with a combination of N-formyl-L-methionyl-L-leucyl-L-phenylalanine and tumor necrosis factor-alpha. Incubation of bovine copper-zinc superoxide dismutase (EC 1.15.1.1) with A1242 or PCB47 in a cell-free system reversed the enzyme-mediated inhibition of 6-hydroxydopamine autoxidation, indicating that polychlorinated biphenyls inhibited superoxide dismutase activity. Low superoxide dismutase activity in neutrophils leads to imbalances between production of free radicals and antioxidant defense mechanisms, which can in turn induce tissue damage and hasten the onset of neutrophil apoptosis.

Alpha particles induce the production of interleukin-8 by human cells.

The pulmonary microenvironment is a primary target for alpha particles like those emitted by inhaled radon and its progeny. While exposure to alpha particles has recently been associated with the generation of extracellular and intracellular reactive oxygen species (ROS; Cancer Res. 57, 3963-3971, 1997), little is known about how exposure to alpha particles may affect the generation of oxidative stress-related mediators in the respiratory tract. Interleukin-8 (IL8) is a cytokine recognized for its potent role as a chemoattractant and activator of polymorphonuclear leukocytes. Oxidative stress can up-regulate expression of the gene that encodes IL8 (IL8) in a variety of cell types. In this study, we set out to investigate a potential linkage between the generation of ROS and production of IL8 in alpha-particle-irradiated normal human lung fibroblasts. ELISA revealed that exposure of the fibroblasts to low doses of alpha particles (3.6-19 cGy) caused significant increases in generation of the IL8 protein as early as 30 min after irradiation. Northern blot analyses revealed that such increases were associated with increased IL8 mRNA levels. Cells exposed to alpha particles in the presence of antioxidants, i.e. superoxide dismutase and dimethyl sulfoxide, resulted in significant decreases in extracellular IL8 protein levels. Similar results were obtained with cells treated with dexamethasone, an inhibitor of transcription. Our results indicate that alpha-particle-induced increases in production of IL8 occur temporally in parallel with elevated production of ROS. Conceivably, such production of IL8 induced by alpha particles may contribute to an inflammatory response in the lower respiratory tract. Additionally, the promitogenic effects of IL8 may be a factor in hyperplastic responses in the airway epithelial cells to inhaled radon and radon progeny and perhaps other stresses associated with ROS.

Modulation in cell cycle and cyclin B1 expression in irradiated HeLa cells and normal human skin fibroblasts treated with staurosporine and caffeine.

The comparative effects of staurosporine or caffeine on G2-phase arrest and cyclin B1 expression in human skin fibroblasts (HSF) and transformed HeLa cells following gamma-irradiation were examined by flow cytometry. Contrary to some earlier reports with HeLa cells, the arrest in G2 after irradiation was accompanied by an increase in cyclin B1 levels in both asynchronous and synchronized HeLa cells irradiated in early S phase. Caffeine and staurosporine were equally effective in attenuating both the radiation-induced increase in cyclin B1 expression and the prolongation of G2 in synchronous and asynchronous HeLa cell populations. Staurosporine treatment was less effective in down-regulating cyclin B1 expression in asynchronous HeLa cells at earlier time points following irradiation when compared to caffeine-treated cells. In synchronized HeLa cells, down-regulation of an irradiation-induced increase in cyclin B1 expression was similar to either staurosporine or caffeine treatments, with caffeine being more effective at later time points. An increase in cyclin B1 expression was also observed in irradiated HSF cells (synchronous and asynchronous), which decreased when the cells were treated with staurosporine or caffeine. However, staurosporine was ineffective in attenuating the radiation-induced prolongation of G2 in synchronous and asynchronous HSF cells, whereas treatment of irradiated synchronous or asynchronous HSF cells with caffeine significantly reduced the prolongation of G2. These results suggest that both staurosporine and caffeine treatments act on different pathways of cell cycle control in normal and transformed cells, in terms of attenuation of G2 block and diminution of elevated levels of cyclin B1 expression, in response to radiation.

Flow cytometry in the preclinical development of biopharmaceuticals.

Novel biomarkers are often required in the preclinical development of biopharmaceuticals in order to characterize pharmacologic and toxicologic effects and to establish pharmacodynamic and pharmacokinetic relationships. Flow cytometry is uniquely suited for measurement of these biomarkers. Large numbers of single cells in a heterogeneous population can be rapidly identified and characterized with high accuracy and reproducibility. Cells are not damaged by the detection system and can be subsequently sorted for further morphologic or functional analysis. The availability of clinical instruments and a wide range of fluorescent probes have made this technology applicable for use in toxicologic clinical pathology. Flow cytometry has played an integral role in the development of a monoclonal antibody to human CD4 (keliximab, IDEC-CE9.1, SB 210396). Lymphocyte subset analysis and assays for expression, coating, and modulation of human CD4 were used for sequential assessment of the pharmacologic activity of keliximab in transgenic mice expressing human CD4.