Practical aspects of including functional endpoints in developmental toxicity studies. Case study: immune function in HuCD4 transgenic mice exposed to anti-CD4 MAb in utero.

Keliximab is a human-cynomolgus monkey chimeric (Primatized) monoclonal antibody with specificity for human and chimpanzee CD4. As the preclinical safety assessment of biopharmaceuticals requires evaluation in pharmacologically responsive species, comprehensive toxicology studies, including reproductive toxicity, of this antibody were conducted in a human CD4 transgenic mouse model. The reproductive toxicology studies included a pre- and postnatal development study that incorporated immunotoxicological evaluation of offspring (F1) mice. The potential effects of exposure to treating maternal mice (F0) with keliximab during pregnancy and lactation on offspring viability, physical growth, neurobehavioral development, reproductive function, lymphoid tissue morphological structure, lymphocyte subsets and host resistance to Candida albicans infection were assessed. The results showed no impairment of these functions. The use of F1 transgenic mice in study with keliximab provides an example of a novel practical approach to assess developmental immunotoxicity within a study of pre- and postnatal development designed in accordance with ICH Guidelines.

Preclinical efficacy and safety of pascolizumab (SB 240683): a humanized anti-interleukin-4 antibody with therapeutic potential in asthma.

The type 2 helper T cell (T(H)2) cytokine interleukin (IL)-4 is thought to play a central role in the early stages of asthma. In an effort to develop an antibody treatment for asthma that neutralizes the effects of IL-4, a murine monoclonal antibody, 3B9, was generated with specificity for human IL-4. In vitro studies demonstrated that 3B9 inhibited IL-4-dependent events including IL-5 synthesis, (T(H)2) cell activation and up-regulation of immunoglobulin E expression. 3B9 was then humanized (pascolizumab, SB 240683) to reduce immunogenicity in humans. SB 240683 demonstrated species specificity for both monkey and human IL-4 with no reactivity to mouse, rat, cow, goat or horse IL-4. Pascolizumab inhibited the response of human and monkey T cells to monkey IL-4 and effectively neutralized IL-4 bioactivity when tested against several IL-4-responsive human cell lines. Affinity studies demonstrated rapid IL-4 binding by pascolizumab with a slow dissociation rate. In vivo pharmacokinetic and chronic safety testing in cynomolgus monkeys demonstrated that pascolizumab was well tolerated, and no adverse clinical responses occurred after up to 9 months of treatment. Three monkeys developed an anti-idiotypic response that resulted in rapid pascolizumab clearance. However, in the chronic dosing study the antibody response was transient and not associated with clinical events. In conclusion, pascolizumab is a humanized anti-IL-4 monoclonal antibody that can inhibit upstream and downstream events associated with asthma, including (T(H)2) cell activation and immunoglobulin E production. Clinical trials are under way to test the clinical efficacy of pascolizumab for asthma.

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.

Pharmacokinetics and tissue distribution of SB-251353, a novel human CXC chemokine, after intravenous administration to mice.

The pharmacokinetics and tissue distribution of SB-251353, a novel truncated form of the human CXC chemokine growth-related gene product beta, were studied after intravenous administration to the mouse (0.1--250 mg/kg). At the lowest dose, the clearance exceeded blood flow to the kidney. As the dose increased, clearance approached the glomerular filtration rate in the mouse. Clearance of this chemokine may be mediated by its pharmacologic receptor, CXCR2, via endocytosis with subsequent lysosomal degradation, as has been observed for several growth and hematopoietic factors. Apparent distribution volumes were high (> or =1 l/kg). Moderate binding to the Duffy antigen/receptor for chemokines on erythrocytes was observed. Consistent with the pharmacokinetic analysis, microscopic autoradiography showed uptake into renal proximal tubule epithelial cells. Limited excretion of SB-251353 in the urine (<2%) was consistent with catabolism of the chemokine in the tubules. Binding to hepatic sinusoids and connective tissue in the dermis was observed. This possibly reflected interaction of SB-251353 with heparin sulfate proteoglycan and may explain the large distribution volumes. This first study of the disposition of a chemokine provides insight into mechanism of action and physiological factors that may influence chemokine pharmacodynamics.

Preclinical efficacy and safety of mepolizumab (SB-240563), a humanized monoclonal antibody to IL-5, in cynomolgus monkeys.

BACKGROUND: Allergic respiratory diseases are characterized by large numbers of eosinophils and their reactive products in airways and blood; these are believed to be involved in progressive airway damage and remodeling. IL-5 is the principal cytokine for eosinophil maturation, differentiation, and survival. Mepolizumab (SB-240563), a humanized monoclonal antibody (mAb) specific for human IL-5, is currently in clinical trials for treatment of asthma. OBJECTIVE: The purpose of this study was to characterize the pharmacologic activity and long-term safety profile of an anti--human IL-5 mAb to support clinical trials in asthmatic patients. METHODS: Naive and Ascaris suum -sensitive cynomolgus monkeys received various dose levels of mepolizumab and were monitored for acute and chronic pharmacologic and toxic responses. RESULTS: To support preclinical safety assessment, cynomolgus monkey IL-5 was cloned, expressed, and characterized. Although monkey IL-5 differs from human IL-5 by 2 amino acids (Ala27Gly and Asn40His), mepolizumab has comparable inhibitory activity against both monkey IL-5 and human IL-5. In A suum--sensitive monkeys, single doses of mepolizumab significantly reduced blood eosinophilia, eosinophil migration into lung airways, and levels of RANTES and IL-6 in lungs for 6 weeks. However, mepolizumab did not affect acute bronchoconstrictive responses to inhaled A suum. In an IL-2--induced eosinophilia model (up to 50% blood eosinophilia), 0.5 mg/kg mepolizumab blocked eosinophilia by >80%. Single-dose and chronic (6 monthly doses) intravenous and subcutaneous toxicity studies in naive monkeys found no target organ toxicity or immunotoxicity up to 300 mg/kg. Monkeys did not generate anti-human IgG antibodies. Monthly mepolizumab doses greater than 5 mg/kg caused an 80% to 100% decrease in blood and bronchoalveolar lavage eosinophils lasting 2 months after dosing, and there was no effect on eosinophil precursors in bone marrow after 6 months of treatment. Eosinophil decreases correlated with mepolizumab plasma concentrations (half-life = 13 days). CONCLUSION: These studies demonstrate that chronic antagonism of IL-5 by mepolizumab in monkeys is safe and has the potential, through long-term reductions in circulating and tissue-resident eosinophils, to be beneficial therapy for chronic inflammatory respiratory diseases.

Immunomodulatory effects of anti-CD4 antibody in host resistance against infections and tumors in human CD4 transgenic mice.

Anti-CD4 antibodies, which cause CD4(+) T-cell depletion, have been shown to increase susceptibility to infections in mice. Thus, development of anti-CD4 antibodies for clinical use raises potential concerns about suppression of host defense mechanisms against pathogens and tumors. The anti-human CD4 antibody keliximab, which binds only human and chimpanzee CD4, has been evaluated in host defense models using murine CD4 knockout-human CD4 transgenic (HuCD4/Tg) mice. In these mice, depletion of CD4(+) T cells by keliximab was associated with inhibition of anti-Pneumocystis carinii and anti-Candida albicans antibody responses and rendered HuCD4/Tg mice susceptible to P. carinii, a CD4-dependent pathogen, but did not compromise host defense against C. albicans infection. Treatment of HuCD4/Tg mice with corticosteroids impaired host immune responses and decreased survival for both infections. Resistance to experimental B16 melanoma metastases was not affected by treatment with keliximab, in contrast to an increase in tumor colonization caused by anti-T cell Thy1.2 and anti-asialo GM-1 antibodies. These data suggest an immunomodulatory rather than an overt immunosuppressive activity of keliximab. This was further demonstrated by the differential effect of keliximab on type 1 and type 2 cytokine expression in splenocytes stimulated ex vivo. Keliximab caused an initial up-regulation of interleukin-2 (IL-2) and gamma interferon, followed by transient down-regulation of IL-4 and IL-10. Taken together, the effects of keliximab in HuCD4/Tg mice suggest that in addition to depleting circulating CD4(+) T lymphocytes, keliximab has the capability of modulating the function of the remaining cells without causing general immunosuppression. Therefore, keliximab therapy may be beneficial in controlling certain autoimmune diseases.

Preclinical development of keliximab, a Primatized anti-CD4 monoclonal antibody, in human CD4 transgenic mice: characterization of the model and safety studies.

The preclinical safety assessment of biopharmaceuticals necessitates that studies be conducted in species in which the products are pharmacologically active. Monoclonal antibodies are a promising class of biopharmaceuticals for many disease indications; however, by design, these agents tend to have limited species cross-reactivity and tend to only be active in primates. Keliximab is a human-cynomolgus monkey chimeric (Primatized) monoclonal antibody with specificity for human and chimpanzee CD4. In order to conduct a comprehensive preclinical safety assessment of this antibody to support chronic treatment of rheumatoid arthritis in patients, a human CD4 transgenic mouse was used for chronic and reproductive toxicity studies and for genotoxic studies. In addition, immunotoxicity studies were conducted in these mice with Candida albicans, Pneumocystis carinii and B16 melanoma cells to assess the effects of keliximab on host resistance to infection and immunosurveillance to neoplasia. The results of these studies found keliximab to be well tolerated with the only effects observed being related to its pharmacologic activity on CD4+ T lymphocytes. The use of transgenic mice expressing human proteins provides a useful alternative to studies in chimpanzees with biopharmaceutical agents having limited species cross-reactivity.

Comparative pharmacodynamics of keliximab and clenoliximab in transgenic mice bearing human CD4.

Keliximab and clenoliximab are monkey/human chimeric CD4 monoclonal antibodies (mAbs) of the IgG1 and IgG4 isotypes, respectively. The pharmacokinetics (PK) and pharmacodynamics (PD) of these mAbs were evaluated in transgenic mice bearing human CD4 molecules on their T cells after a single i.v. administration at three dose levels (5-125 mg/kg). The PK of keliximab and clenoliximab were similar, dose-dependent, and adequately described by a two-compartment model with saturable elimination from both compartments. The enumeration of circulating CD4(+) T cells and density of CD4 on their surface were determined as the PD effects. An indirect response model was proposed to characterize the PD effects. With the increase in mAb dose, the maximum intensity (R(max)) of PD effects was increased, and the time to reach R(max) shifted to later times. At all three dose levels, keliximab caused a relatively rapid decline in the number of circulating CD4(+) T cells, which then recovered gradually. In contrast, clenoliximab at the lowest dose (5 mg/kg) did not produce a significant effect on CD4(+) T cell counts compared with the placebo group. At high doses, clenoliximab caused a significant decrease in the number of CD4(+) T cells. Keliximab appeared to be more potent and efficient in depleting CD4(+) T cells. Both mAbs produced similar down-modulation of CD4 at corresponding dose levels. The findings of this study are consistent with the results of a recent clinical trial that emphasize the importance of this transgenic mouse model for evaluating PK/PD to support clinical development of anti-human CD4 mAbs.

Pharmacokinetics and pharmacodynamics of SB-240563, a humanized monoclonal antibody directed to human interleukin-5, in monkeys.

The pharmacokinetics (PK) of SB-240563 have been investigated after i.v. and s.c. administration to cynomolgus monkeys. Approximately linear PK was observed following i.v. administration over a 6000-fold dose range (0.05-300 mg/kg). After i.v. dosing, SB-240563 concentration declined in a biexponential manner with a mean terminal half-life of 13 +/- 2 days. The plasma clearance and volume of distribution at steady state were approximately 0.2 ml/h/kg and 70 ml/kg, respectively. Following s.c. administration, SB-240563 was completely absorbed into the systemic circulation. Because interleukin-5 is known to stimulate production, activation, and maturation of eosinophils, eosinophil counts were measured to assess pharmacologic activity of SB-240563. The maximal response (81-96% decrease in eosinophil count relative to baseline) following a single s.c. administration occurred at 3 weeks postdosing. Suppression of eosinophil count also was observed following multiple monthly administrations of SB-240563 to monkeys. The pharmacokinetic/pharmacodynamic relationship was generally well described with an indirect pharmacologic response model with an estimated IC(50) value of 1.43 microg/ml. The combination of a low IC(50) value for reduction of circulating eosinophils and a long terminal half-life suggests the possibility of an infrequent dosing regimen for SB-240563 for treatment of diseases associated with increased eosinophil function such as asthma.

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.

Effect of SB 217242 on hypoxia-induced cardiopulmonary changes in the high altitude-sensitive rat.

The effects of SB 217242, a non-peptide endothelin (ET) receptor antagonist, were investigated against hypoxia-induced cardiopulmonary changes in high altitude-sensitive rats. In isolated pulmonary artery rings, SB 217242 (30 n m) antagonized ET-1-induced contractions with a p KB of 8.0. There was no difference in the sensitivity to ET-1 or the potency of SB 217242 in pulmonary artery from normoxic rats vs. rats exposed to hypoxia (9% O2) for 14 days. However, there was a marked reduction in the maximum response to ET-1, but not to KCl or phenylephrine, in pulmonary artery from hypoxic rats; this phenomenon was inhibited by treatment of animals with SB 217242 (10.8 mg/day, ip by osmotic pump) for the 14-day hypoxic period. Furthermore, there was a significant reduction in carbachol-induced, endothelium-dependent relaxation of precontracted pulmonary artery from hypoxic animals; SB 217242 treatment during the hypoxic period did not influence this difference. Vehicle-treated rats exposed to 14-day hypoxia had 173% higher pulmonary artery pressures and 75% higher right/left+septum ventricular mass ratios compared to normoxic animals. SB 217242 (3.6 or 10.8 mg/day, ip) markedly reduced (80 and 95%, respectively) hypoxia-induced increases in pulmonary artery pressure. Right ventricular hypertrophy was inhibited by 40% at the 10.8 mg/day dose. Marked medial thickening and luminal stenosis of small and medium-sized pulmonary arteries was observed in hypoxic rats. The SB 217242-treated, hypoxia-exposed rats had comparable small and medium-sized arteries to normoxic rats. Rats treated with SB 217242 (10.8 mg/day) for the last 14 days of a 28-day hypoxic exposure had significantly lower pulmonary artery pressures than those of vehicle-treated rats. In addition, the effects of the selective ETA receptor antagonist, SB 247083, and the selective ETB receptor antagonist, A-192621 (3.6 or 10.8 mg/day, ip), were compared against hypoxia-induced increases in pulmonary artery pressure and plasma ET concentrations. SB 247083, but not A-192621, inhibited hypoxia-induced pulmonary hypertension, whereas A-192621, but not SB 247083, significantly exacerbated hypoxia-induced increases in ET concentrations, suggesting that hypoxia-induced pulmonary pressor responses are mediated via ETA receptor activation, while ETB receptor blockade may alter clearance of hypoxia-induced elevated plasma ET. The inhibitory effects of SB 217242 on the functional and remodeling changes induced by hypoxia provide further evidence that ET may play a central role in pulmonary hypertension and that ET receptor antagonists may have a utility in the treatment of this disease.

TL1, a novel tumor necrosis factor-like cytokine, induces apoptosis in endothelial cells. Involvement of activation of stress protein kinases (stress-activated protein kinase and p38 mitogen-activated protein kinase) and caspase-3-like protease.

TL1 is a recently discovered novel member of the tumor necrosis factor (TNF) cytokine family. TL1 is abundantly expressed in endothelial cells, but its function is not known. The present study was undertaken to explore whether TL1 induces apoptosis in endothelial cells and, if so, to explore its mechanism of action. Cultured bovine pulmonary artery endothelial cells (BPAEC) exposed to TL1 showed morphological (including ultrastructural) and biochemical features characteristic of apoptosis. TL1-induced apoptosis in BPAEC was a time- and concentration-dependent process (EC50 = 72 ng/ml). The effect of TL1 was not inhibited by soluble TNF receptors 1 or 2. TL1 up-regulated Fas expression in BPAEC at 8 and 24 h after treatment, and significantly activated stress-activated protein kinase (SAPK) and p38 mitogen-activated protein kinase (p38 MAPK). The peak activities of SAPK and p38 MAPK in TL1-treated BPAEC were increased by 9- and 4-fold, respectively. TL1-induced apoptosis in the BPAEC was reduced by expression of a dominant-interfering mutant of c-Jun (62.8%, p < 0.05) or by a specific p38 inhibitor, SB203580 (1-10 microM) dose-dependently. TL1 also activated caspases in BPAEC, and TL1-induced apoptosis in BPAEC was significantly attenuated by the caspase inhibitor, ZVAD-fluromethyl-ketone. The major component activated by TL1 in BPAEC was caspase-3, which was based on substrate specificity and immunocytochemical analysis. These findings suggest that TL1 may act as an autocrine factor to induce apoptosis in endothelial cells via activation of multiple signaling pathways, including stress protein kinases as well as certain caspases.

Staurosporine-induced apoptosis in cardiomyocytes: A potential role of caspase-3.

Cardiomyocyte apoptosis has been demonstrated in animal models of cardiac injury as well as in patients with congestive heart failure or acute myocardial infarction. Therefore, apoptosis has been proposed as an important process in cardiac remodeling and progression of myocardial dysfunction. However, the mechanisms underlying cardiac apoptosis are poorly understood. The present study was designed to determine whether the family of caspase proteases and stress-activated protein kinase (SAPK/JNK) are involved in cardiac apoptosis. Cultured rat neonatal cardiac myocytes were treated with staurosporine to induce apoptosis as evidenced by the morphological (including ultrastructural) characteristics of cell shrinkage, cytoplasmic and nuclear condensation, and fragmentation. Nucleosomal DNA fragmentation in myocytes was further identified by agarose gel electrophoresis (DNA ladder) as well as in situ nick end-labeling (TUNEL). Staurosporine-induced apoptosis in myocytes was a time- and concentration-(0.25-1 micro M)-dependent process. Staurosporine-induced apoptosis in myocytes was reduced by a cell-permeable, irreversible tripeptide inhibitor of caspases, ZVAD-fmk, but not by the ICE-specific inhibitor, Ac-YVAD-CHO. At 10, 50 and 100 muM of ZVAD-fmk, staurosporine-induced myocyte apoptosis was reduced by 5.8, 39.1 (P<0.01) and 53.8% (P<0.01), respectively. Staurosporine, at 0.25-1 micro M, increased caspase activity in cardiomyocytes by five- to eight-fold, peaking at 4-8 h after stimulation. Based on substrate specificity analysis, the major component of caspases activated in myocytes was consistent with caspase-3 (CPP32). Moreover, the appearance of the 17-kD subunit of active caspase-3 in staurosporine-treated myocytes was demonstrated by immunocytochemical analysis. In contrast, staurosporine induced a rapid and transient inhibition of SAPK/JNK in myocytes. The SAPK activity in myocytes was reduced by 68.3 and 58.3% (P<0.01 v basal) at 10 and 30 min after treatment with 1 micro M of staurosporine, respectively. Our results suggest that staurosporine-induced cardiac myocyte apoptosis involves activation of caspases, mainly caspase-3, but not activation of the SAPK signaling pathway.

Chronic hypoxia-induced cardiopulmonary changes in three rat strains: inhibition by the endothelin receptor antagonist SB 217242.

The cardiopulmonary profile of three different rat strains was compared after exposure to hypoxia (9% O2) for 0, 7, or 14 days. In Sprague-Dawley (SD), Wistar (W), and high altitude-sensitive (HAS) rats, pulmonary arterial pressure (PAP) rose 30, 58, and 85% respectively, after 7 days of hypoxia, and by 108, 116, and 167%, respectively, at 14 days compared to strain- and age-matched normoxic controls. Right ventricular hypertrophy (RVH), expressed as the ratio of right free wall/left wall + septum weight, in SD, W, and HAS was increased by 24, 53, and 48%, respectively, at 7 days, and by 51, 93, and 55% at 14 days compared to normoxic littermates. Histologically, marked medial thickening and luminal stenosis of small and medium-sized arteries were observed in all hypoxic rats, being most pronounced in the HAS rats at 14 days. Treatment of HAS rats with the ET receptor antagonist SB 217242 (3.6 or 10.8 mg/day i.p. by osmotic pump) significantly inhibited the hypoxia-induced increases in PAP (70-75% decrease). RVH was inhibited by 40% at the dose of 10.8 mg/day. Histologically, the SB 217242-treated rats had almost "normal" small and medium-sized arteries, comparable to those of the normoxic HAS controls. This study demonstrates an exaggerated PAP response to chronic hypoxia in HAS compared to SD and W rats. The inhibitory influence of SB 217242 on the functional and morphologic changes induced by hypoxia provides further evidence for a role for ET and the potential utility of ET receptor antagonists in the treatment of pulmonary hypertension.

2-Methoxyestradiol, an endogenous estrogen metabolite, induces apoptosis in endothelial cells and inhibits angiogenesis: possible role for stress-activated protein kinase signaling pathway and Fas expression.

2-Methoxyestradiol (2-ME) is an endogenous metabolite of estradiol-17beta and the oral contraceptive agent 17-ethylestradiol. 2-ME was recently reported to inhibit endothelial cell proliferation. The current study was undertaken to explore the mechanism of 2-ME effects on endothelial cells, especially whether 2-ME induces apoptosis, a prime mechanism in tissue remodeling and angiogenesis. Cultured bovine pulmonary artery endothelial cells (BPAEC) exposed to 2-ME showed morphological (including ultrastructural) features characteristic of apoptosis: cell shrinkage, cytoplasmic and nuclear condensation, and cell blebbing. 2-ME-induced apoptosis in BPAEC was a time- and concentration-dependent process (EC50 = 0.45 +/- 0.09 microM, n = 8). Nucleosomal DNA fragmentation in BPAEC treated with 2-ME was identified by agarose gel electrophoresis (DNA ladder) as well as in situ nick end labeling. Under the same experimental conditions, estradiol-17beta and two of its other metabolites, estriol and 2-methoxyestriol (< or =10 microM), did not have an apoptotic effect on BPAEC. 2-ME activated stress-activated protein kinase (SAPK)/c-Jun amino-terminal protein kinase in BPAEC in a concentration-dependent manner. The activity of SAPK was increased by 170 +/- 27% and 314 +/- 22% over the basal level in the presence of 0.4 and 2 microM 2-ME (n = 3-6), respectively. The activation of SAPK was detected at 10 min, peaked at 20 min, and returned to basal levels at 60 min after exposure to 2-ME. Inhibition of SAPK/c-Jun amino-terminal protein kinase activation by basic fibroblast growth factor, insulin-like growth factor, or forskolin reduced 2-ME-induced apoptosis. Immunohistochemical analysis of BPAEC indicated that 2-ME up-regulated expression of both Fas and Bcl-2. In addition, 2-ME inhibited BPAEC migration (IC50 = 0.71 +/- 0.11 microM, n = 4) and basic fibroblast growth factor-induced angiogenesis in the chick chorioallantoic membrane model. Taken together, these results suggest that promotion of endothelial cell apoptosis, thereby inhibiting endothelial cell proliferation and migration, may be a major mechanism by which 2-ME inhibits angiogenesis.

A novel technique for mapping the lipid composition of atherosclerotic fatty streaks by en face fluorescence microscopy.

We introduce here a new fluorescence microscopy technique for en face analysis of the atherosclerotic fatty streaks (FS). This technique is semiquantitative and has the sensitivity and resolution to map lipids to individual cells in FS less than 100 microns in diameter. New Zealand White rabbits were fed an atherogenic diet for up to 26 weeks. Aortas were fixed in formalin and stained en bloc with the fluorescent dyes Nile red and filipin. Fluorescent staining was validated by correlating microfluorimetric and biochemical measurements of the lipid content in FS. To determine the cell types associated with the different staining patterns, FS were also evaluated by transmission electron microscopy (TEM) and immunohistochemistry (IH). Correlation of microfluorimetry, TEM, IH, and biochemical data indicated that regions rich in non-esterified cholesterol stained with filipin and fluoresced blue owing to accumulations of lipid vesicles and/or cholesterol crystals. Regions rich in neutral and polar lipids stained with Nile red and fluoresced yellow or orange, respectively, owing to accumulations of lipids in both macrophages and smooth muscle cells (SMC). Digital overlays of the filipin and Nile red images revealed that larger lesions (> 0.5 mm diameter) had a "nested" distribution of lipids, with a blue (filipin) fringe surrounding an orange (Nile red) fringe surrounding a yellow (Nile red) center.

Spontaneous and induced alterations in the cardiac membranous ventricular septum of fetal, weanling, and adult rats.

Alterations of the cardiac membranous ventricular septum were studied using macrodissection, scanning electron and light microscopy of fetal, weanling, and adult Sprague-Dawley rats. Membranous ventricular septal defects (VSDs) were observed in 2.0% of fetuses on day 21 postcoitus (pc) but not in weanling or adult rats. The most common observation was a nonpatent depression in the membranous septum with an incidence of 38.1, 10.5, 4.3% for fetuses on days 17, 19, or 21 pc, respectively, 11.8% for weanlings, and 9.1% for adults. VSDs were characterized by a split in the endocardial cushion cells in the interventricular component of the membranous septum. Nonpatent depressions were characterized by a split in the endocardial cushion cells in the atrioventricular component of the septum, and they persisted postnatally as a blind-ended diverticulum directed above the tricuspid valve. The cardiovascular teratogens, trimethadione and trypan blue, produced in fetuses nonpatent depressions and VSDs morphologically similar to untreated fetuses. Maternal diet restriction (25% of controls) lowered fetal (day 21 pc) body weight by 47% but did not affect the incidence of ventricular septal alterations, suggesting that intrauterine growth retardation is not necessarily associated with alterations in the development of the ventricular septum. We conclude that neither VSDs nor nonpatent depressions in Sprague-Dawley rats affect postnatal survival and that VSDs close spontaneously during neonatal life.

Purification and characterization of fully functional human osteoclast precursors.

The identification and purification of human osteoclast precursors is essential to further our understanding of the mechanisms that control human osteoclast differentiation. Osteoclastoma tissue potentially provides a rich source of human osteoclast precursors, and in previous studies we have demonstrated the existence of a population of mononuclear cells within this tissue that is reactive with osteoclast-selective vitronectin receptor monoclonal antibodies. In this study, mononuclear cells expressing the vitronectin receptor, as defined by their ability to react with a murine monoclonal antibody to the beta 3 chain of the vitronectin receptor (87MEM1), were isolated from collagenase digests of osteoclastoma tissue using a fluorescence activated cell sorter. Based on their fluorescence signal and size, approximately 2-3% of the viable cells (typically 2 x 10(5)) were obtained and prepared for further phenotyping. The isolated cells demonstrated a number of phenotypic characteristics of osteoclasts: positive tartrate-resistant acid phosphatase (TRAP) activity, reactivity with human osteoclast-selective antibodies, expression of calcitonin receptors, cathepsin K (a novel osteoclast-selective cysteine proteinase) mRNA, and osteopontin mRNA and protein. These phenotypic characteristics were also detected in mononuclear cells within cryostat sections of the native osteoclastoma tissue as well as in resorption lacunae of sections of human bone. In contrast, isolated peripheral blood monocytes were negative for TRAP activity and osteopontin expression and, unlike the osteoclastoma-derived cells, demonstrated strong nonspecific esterase activity. Significantly, when the osteoclastoma-derived 87MEM1 positive cells were cocultured on whale dentine for 1-3 weeks with stromal cells, extensive resorption of the dentine surface was observed. This is the first demonstration of the purification of human osteoclast precursors. These cells provide an homogeneous cell population for studying cellular events that occur during human osteoclast differentiation.

Characterization of CD4-gp120 activation intermediates during human immunodeficiency virus type 1 syncytium formation.

The mechanism by which cells expressing HIV envelope glycoproteins progress from binding CD4+ cells to syncytia formation is not entirely understood. The purpose of these investigations was to use physical and biochemical tools (temperature shifts, soluble CD4, protease inhibitors, and a battery of anti-CD4 monoclonal antibodies) to isolate discrete steps during syncytia formation. Previously (Fu et al., J Virol 1993;67:3818), we found that preincubation of cells stably expressing HIV-1 gp 160 (TF228.1.16) with CD4+ SupT1 cells at 16 degrees C, a temperature that is nonpermissive for syncytia formation, resulted in an increased rate of syncytia formation when the cocultures were shifted to the syncytia-permissive temperature of 37 degrees C. We have since found that syncytia formation is further enhanced by shifting the cocultures from 16 to 4 degrees C prior to incubation at 37 degrees C. Together, these data suggest that two discrete states, which we term the first and second activation intermediates (FAI and SAI), are involved in syncytia formation. We have found that acquisition of the FAI (by preincubation at 16 degree C) is sensitive to some serine protease inhibitors (PI), soluble CD4 (sCD4), shedding of gp120, and anti-CD4 monoclonal antibodies (MAb) directed toward the CDR-1/2 and CDR-3 regions of domain 1 on CD4. Expression of the FAI (formation of syncytia by shifting from 16 to 37 degrees C) remains sensitive to sCD4, shedding of gp120, and MAb directed toward CDR-1/2 but is less sensitive to MAb that bind CDR-3 and is insensitive to PI. Similarly, acquisition of the SAI (shifting cocultures from 16 to 4 degrees C), is sensitive to sCD4, shedding of gp120, and MAb directed toward CDR-1/2. In contrast, expression of the SAI (shifting cocultures from 16 to 4 to 37 degrees C) is sensitive only to MAb directed toward CDR-1/2 and cannot be blocked by sCD4, shedding of gp120, or PI. These data allow us to propose that syncytia formation, mediated by HIV-1 envelope glycoproteins, proceeds by a multistep cascade.