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Abstract-Endothelium-dependent relaxations mediated by NO are impaired in a mouse model of human atherosclerosis.Our objective was to characterize the mechanisms underlying endothelial dysfunction in aortas of apolipoprotein E (apoE)-deficient mice, treated for 26 to 29 weeks with a lipid-rich Western-type diet. Aortic rings from apoE-deficient mice showed impaired endothelium-dependent relaxations to acetylcholine (10 Ϫ9 to 10 Ϫ5 mol/L) and Ca 2ϩ ionophore (10 Ϫ9 to 10 Ϫ6 mol/L) and endothelium-independent relaxations to diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA-NONOate, 10 Ϫ10 to 10 Ϫ5 mol/L) compared with aortic rings from C57BL/6J mice (PϽ0.05). By use of confocal microscopy of an oxidative fluorescent probe (dihydroethidium), increased superoxide anion (O 2 Ϫ ) production was demonstrated throughout the aortic wall but mainly in smooth muscle cells of apoE-deficient mice. CuZn-superoxide dismutase (SOD) and Mn-SOD protein expressions were unaltered in the aorta exposed to hypercholesterolemia. A cell-permeable SOD mimetic, Mn(III) tetra(4-benzoic acid) porphyrin chloride (10 Ϫ5 mol/L), reduced O 2 Ϫ production and partially normalized relaxations to acetylcholine and DEA-NONOate in apoE-deficient mice (PϽ0.05). [ 14 C]L-Citrulline assay showed a decrease of Ca 2ϩ -dependent NOS activity in aortas from apoE-deficient mice compared with C57BL/6J mice (PϽ0.05), whereas NO synthase protein expression was unchanged. In addition, cGMP levels were significantly reduced in the aortas of apoE-deficient mice (PϽ0.05). Our results demonstrate that in apoE-deficient mice on a Western-type fat diet, impairment of endothelial function is caused by increased production of O 2 Ϫ and reduced endothelial NO synthase enzyme activity. Key Words: endothelium Ⅲ nitric oxide Ⅲ superoxide anion Ⅲ apolipoprotein E Ⅲ atherosclerosis A therosclerosis is a chronic process, which can be triggered by cardiovascular risk factors such as hypercholesterolemia, aging, hypertension, and diabetes mellitus. 1 Endothelium-derived vasoactive factors play an important regulatory role in vascular homeostasis and pathogenesis of atherosclerosis because of the strategic position of the endothelium between the vascular smooth muscle cells (VSMCs) and the circulating blood. 2,3 NO is a potent vasodilator that is formed in endothelial cells from L-arginine by endothelial NO synthase (eNOS), which is constitutively expressed. 4 -6 NO production is activated by the stimulation of cell surface receptors or by mechanical forces such as shear stress. 7,8 Accumulating evidence suggests that alterations in the NO pathway play a central role in endothelial dysfunction induced by hypercholesterolemia. This may be of major importance inasmuch as NO can substantially inhibit several components of the atherogenic process, such as VSMC contraction and proliferation, platelet aggregation, and monocyte adhesion. 9,10 Previous studies identified 3 mechanisms responsible for reduced bioavailability of NO in arteries exposed to hypercholester...

The number of human embryonic stem cell (hESC) lines available to federally funded U.S. researchers is currently limited. Thus, determining their basic characteristics and disseminating these lines is important. In this report, we recovered and expanded the earliest available cryopreserved stocks of the BG01, BG02, and BG03 hESC lines. These cultures exhibited multiple definitive characteristics of undifferentiated cells, including long-term self-renewal, expression of markers of pluripotency, maintenance of a normal karyotype, and differentiation to mesoderm, endoderm, and ectoderm. Each cell line exhibited a unique genotype and human leukocyte antigen (HLA) isotype, confirming that they were isolated independently. BG01, BG02, and BG03 maintained in feederfree conditions demonstrated self-renewal, maintenance of normal karyotype, and gene expression indicative of undifferentiated pluripotent stem cells. A survey of gene expression in BG02 cells using massively parallel signature sequencing generated a digital read-out of transcript abundance and showed that this line was similar to other hESC lines. BG01, BG02, and BG03 hESCs are therefore independent, undifferentiated, and pluripotent lines that can be maintained without accumulation of karyotypic abnormalities.

SummaryHuman peripheral blood eosinophils released eosinophil survival-enhancing activity when stimulated with the calcium ionophore, ionomycin . The release of activity was detected as early as 3 h after stimulation and was inhibited by an immunomodulating agent, cyclosporin A . The survivalenhancing activity was completely abolished by treatment with anti-interleukin 3 (IL3) and anti-granulocyte/macrophage colony-stimulating factor (GM-CSF) monoclonal antibodies . Moreover, IL-3 and GM-CSF were measurable in ionomycin-stimulated eosinophil supernatants by immunoassay. Eosinophils produced approximately one-half as much IL3 and one-fifth as much GM-CSF as ionomycin-stimulated mononuclear cells. Neutrophils also produced 11,3 and GM-CSF, but the amounts were less than those produced by eosinophils. These observations suggest a novel role for eosinophils in pathophysiology of allergic inflammation and host defense mechanisms. E osinophils are blood leukocytes associated with helminth infections and allergic diseases, especially bronchial asthma, and may mediate immunity and tissue damage (1) . Several rytokines, including IL3, 116, and granulocyte/macrophage colony-stimulating factor (GM-CSF), induce eosinophilopoiesis and activate mature peripheral blood eosinophils in vitro (reviewed in reference 2). Recently, mRNA for these rytokines was detected in allergen-induced late-phase cutaneous reactions in atopic patients, suggesting that these rytokines play an important role in allergic inflammation (3) .Because human peripheral blood eosinophils, despite their high degree of specialization, retain the ability to synthesize rytokines such as TGF-ot and IL1 (4, 5), we investigated whether they can produce IL-3, IL-5, and GM-CSF . We found that ionomycin-stimulated eosinophils produce GM-CSF and IL-3 as shown by enhanced eosinophil survival and by immunochemical measurement .Cells and Culture Conditions. Eosinophils, neutrophils, and mononuclear cells were isolated from peripheral blood of healthy volunteers or patients with mild hay fever by discontinuous Percoll density gradient as previously described (6) . Purities of eosinophils and neutrophils were 391% and 399%, respectively. Contaminating cells in the eosinophil preparations were only neutrophils and those in the neutrophil preparations were only eosinophils . Cell viabilities were >98% . Cells were incubated (2 x 105 cells per 0 .2 ml per well) in Hybri-Care medium (American Type Culture Collection, Rockville, MD) supplemented with 50 lAg/ml gentamicin and 10% defined calf serum in 96-well flat-bottomed microtiter plates (Falcon Labware, Lincoln Park, NJ) with ionomycin (Calbiochem Corp., San Diego, CA) in the presence or absence of PMA (500 pg/ml; Calbiochem Corp.) . After 24 h at 37°C, cellfree supernatants were harvested and frozen until assayed .Eosinophil Survival Assay. To measure eosinophil survival enhancing activity (7) in supernatants, eosinophils were freshly isolated from different donors and were cultured (2 .5 x 10" cells per 0 .2 ml per well) ...

Eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP) are generally regarded as eosinophil-specific proteins. We tested whether EDN and ECP are present in mature neutrophils. By indirect immunofluorescence, both eosinophils and neutrophils stained with antibodies to EDN and ECP. Lysates of purified (F0.1% eosinophil contamination) neutrophils contained EDN, 112 ؎ 4 ng/10 6 cells, and ECP, 163 ؎ 2 ng/10 6 cells, whereas eosinophil major basic protein (MBP) was not detectable. Electron microscopic examination of immunogold-labeled buffy coat cells stained with EDN antibody showed that EDN is localized to neutrophil granules. Finally, EDN mRNA was detected in lysates of highly purified neutrophils (0.001% eosinophil contamination) by the reverse transcription-polymerase chain reaction. We conclude that proteins that are either identical to or immunologically crossreactive with EDN and ECP are present in neutrophils and that EDN is synthesized and localized to neutrophil granules. Thus, caution must be exercised in interpreting the presence of EDN and ECP as specific markers of eosinophil-associated inflammation in human disease. J. Leukoc. Biol. 63: 715-722; 1998.

Eosinophils are important effector cells in defense against helminth infection and in allergic diseases. To identify novel eosinophil proteins, large scale sequencing of a cDNA library prepared from interleukin-5-stimulated umbilical cord precursor cells was performed, and the major genes expressed by maturing eosinophils were determined. This resulted in the identification of a cDNA with 64% identity to human prepro-major basic protein (hprepro-MBP). This cDNA was designated hprepro-MBP homolog (hprepro-MBPH). Interestingly, the calculated pI values for hMBPH and hMBP differed by >100-fold, with pI values of 8.7 and 11.4, respectively. Given this pronounced basicity difference, the homolog transcript's abundance (1.1%), and MBP's critical role in eosinophil biological activity, we further characterized the homolog. Reverse transcription-polymerase chain reaction detected transcription of hprepro-MBPH in bone marrow only, and this result was confirmed by analysis of a large cDNA data base (electronic Northern). hMBPH was isolated from human eosinophil granule lysates, and its identity was verified by amino acid sequencing and by mass spectrometry. Analyses of the biological activities showed that hMBPH had effects similar to hMBP in cell killing and neutrophil (superoxide anion production and interleukin-8 release) and basophil (histamine and leukotriene C 4 release) stimulation assays, but usually with reduced potency. Overall, this novel homolog's unique physical properties indicated that the high net positive charge of hMBP is important but not essential for biological activity.

Segmental bronchoprovocation (SBP) with allergen was used in an attempt to study eosinophils recruited to the airway 24 h after challenge. Unexpectedly, in the first four patients, neutrophils (rather than eosinophils) were recruited in the bronchoalveolar lavage (BAL) fluids, and we hypothesized that the allergen extracts were contaminated with endotoxin. The extracts used for challenge in the first four patients tested positive for bacterial endotoxin in a limulus amebocyte lysate assay. Rechallenge of one patient from the first group with a comparable dose of an endotoxin-free extract and SBP with endotoxin-free extract in five additional patients resulted in preferential recruitment of eosinophils rather than neutrophils. The number of neutrophils recovered from the challenged segments in the patients challenged with endotoxin-free extract was significantly less than that observed in the first four patients. Taken together, these observations suggest that neutrophil recruitment in the 24-h BAL fluids from the first four patients was probably due to endotoxin contamination of the allergen extract. We caution investigators that endotoxin contamination of allergen extract may alter the cellular inflammation during the late airway response following allergen challenge.

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