Comparative expression profiling in primary and immortalized endothelial cells: changes in gene expression in response to hydroxy methylglutaryl-coenzyme A reductase inhibition.

Immortalized cell lines offer significant logistical advantages over primary cells when used for in-vitro studies. Immortalized cells may, however, exhibit important differences relative to their primary cell counterparts. In this study, microarrays were used to make a genome-wide comparison between primary human umbilical vein endothelial cells (HUVECs) and EA.hy926, an immortalized HUVEC cell line, in their baseline properties and in their response to inhibition of the mevalonate pathway with an inhibitor of hydroxy methylglutaryl-coenzyme A reductase (statin). HUVECs and EA.hy926 were incubated with control medium, atorvastatin, mevalonate, or a combination of atorvastatin and mevalonate for 24 h. Gene expression profiles were obtained in duplicates using Affymetrix Human Genome U133A 2.0 arrays (Santa Clara, California, USA). Probe-sets were selected according to the following criteria: a twofold or greater increase/decrease in atorvastatin-treated cells compared with untreated cells; a twofold or greater reversal of the effect of atorvastatin by combined treatment with atorvastatin and mevalonate; no significant change in gene expression in cells treated with mevalonate alone compared with untreated cells. Most genes that were expressed by untreated HUVECs, were also expressed by untreated EA.hy926 cells. EA.hy926 cells, however, constitutively expressed a large number of additional genes, many of which were related to cell cycle control and apoptosis. Atorvastatin induced differential expression (> or = twofold) of 103 genes in HUVECs (10 up, 93 down) and 466 genes in EA.hy926 cells (198 up, 268 down). Applying the above selection criteria, thrombomodulin and tissue plasminogen activator were up-regulated in both cell types, whereas, connective tissue growth factor, thrombospondin-1, and cysteine-rich angiogenic inducer 61 were down-regulated. In conclusion, EA.hy926 cells retain most of the characteristics of endothelial cells under baseline conditions as well as after treatment with atorvastatin. It is necessary, however, to carefully select and validate changes in genes that are the focus of studies when using EA.hy926 cells. While this cell line is highly useful in studies on some genes, including genes encoding molecules involved in regulating thrombohemorrhagic homeostasis, they appear to be less suited for studies focused on other genes, particularly those involved in the regulation of cell proliferation and apoptosis.     

Cyclic GMP release and vasodilatation induced by EDRF and atrial natriuretic factor in the isolated perfused kidney of the rat.

1. Guanosine 3':5'-cyclic monophosphate (cyclic GMP) release and vascular tone was measured in the isolated kidney of the rat perfused at constant flow with Krebs-Henseleit solution. The effects of 3 vasodilators, acetylcholine (ACh), atrial natriuretic factor (ANF) and sodium nitroprusside (SNP) on the renal release of cyclic GMP and vascular tone were examined. The ability of the endothelial-derived relaxing factor (EDRF) inhibitors, haemoglobin and gossypol, to modify vasodilatation and vasodilator-induced changes in cyclic GMP releases from the kidney was also investigated. 2. Renal cyclic GMP release was elevated 8 fold by ANF (0.01 microM), 5 fold by SNP (1 microM) and 3 fold by ACh (0.3 microM). 3. For ACh, both the increase in renal cyclic GMP release and the vasodilatation were reduced by the EDRF inhibitors, haemoglobin (1 microM) and gossypol (15 microM). For SNP, neither the increase in renal cyclic GMP release nor vasodilatation were inhibited by gossypol (15 microM). 4. For ANF, neither the increase in cyclic GMP release from the kidney nor its vasodilator activity were affected by haemoglobin (1 microM). 5. EDRF inhibitors reduced the basal release of cyclic GMP from 0.32 +/- 0.06 pmol min-1 to 0.18 +/- 0.03 pmol min-1, gossypol being more effective than haemoglobin. 6. The results are consistent with the ability of ACh to induce EDRF-mediated vasodilatation in the isolated perfused kidney of the rat. Basal EDRF release appears to contribute approximately 50% to the basal release of cyclic GMP from this preparation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dose response and toxicity of doxorubicin microspheres in a rat tumor model.

The therapeutic response and toxic effects of chemotherapy using several doses of doxorubicin in conventional solution form or bound to an ion-exchange resin were compared in a rat tumor model, to assess the relationship of drug dose to therapeutic efficacy and associated toxicity. Single bolus injections of 3.0, 4.5, 6.0, 7.5 and 9.0 mg/kg were administered via the abdominal aorta to rats bearing hindlimb tumors. Tumor size was measured serially and the growth rates of treated groups were compared with a control growth curve. In addition, the effect of empty microspheres on tumor growth rate was assessed. The levels of circulating white blood cells were measured and compared to control levels to provide an indication of the severity of bone marrow toxicity experienced by each form of treatment. Finally, any difference in the distribution of doxorubicin to tumor, hindlimb and cardiac tissue following administration of doxorubicin as free drug or on microspheres was ascertained. Empty ion-exchange resin exerted a small although significant detrimental effect on tumor growth which may be explained by the embolization of microspheres in the precapillary blood vessels of the tumor resulting in a transient delay in tumor growth rate. The lowest dose of doxorubicin produced a significantly better therapeutic response when administered in the free drug form, but higher doses elicited an equivalent delay in tumor growth for both drug microsphere and free drug groups in a dose-dependent manner, with the maximum anti-tumor response occurring at the highest dose. Treatment with free doxorubicin at high doses resulted in significant reductions of circulating white blood cells suggesting the occurrence of bone marrow toxicity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Recombinant human nerve growth factor is biologically active and labels novel high-affinity binding sites in rat brain.

Iodinated recombinant human nerve growth factor (125I-rhNGF) stimulated neurite formation in PC12 cell cultures with a half-maximal potency of 35-49 pg/ml, compared with 39-52 pg/ml for rhNGF. In quantitative ligand autoradiography, the in vitro equilibrium binding of 125I-rhNGF to brain sections showed a 10-fold regional variation in density and was saturable, reversible, and specifically displaced by up to 74% with rhNGF or murine NGF (muNGF). At equilibrium, 125I-rhNGF bound to these sites with high affinity (Kd 52-85 pM) and low capacity (Bmax less than or equal to 13.2 fmol/mg of protein). Calculation of 125I-rhNGF binding affinity by kinetic methods gave average Kd values of 24 and 31 pM. Computer-generated maps revealed binding in brain regions not identified previously with 125I-muNGF, including hippocampus; dentate gyrus; amygdala; paraventricular thalamus; frontal, parietal, occipital, and cingulate cortices; nucleus accumbens; olfactory tubercle; subiculum; pineal gland; and medial geniculate nucleus. NGF binding sites were distributed in a 2-fold increasing medial-lateral gradient in the caudate-putamen and a 2-fold lateral-medial gradient in the nucleus accumbens. 125I-rhNGF binding sites were also found in most areas labeled by 125I-muNGF, including the interpedunucular nucleus, cerebellum, forebrain cholinergic nuclei, caudoventral caudate-putamen, and trigeminal nerve nucleus. 125I-rhNGF binding sites were absent from areas replete with low-affinity NGF binding sites, including circumventricular organs, myelinated fiber bundles, and choroid plexus. The present analysis provides an anatomical differentiation of high-affinity 125I-rhNGF binding sites and greatly expands the number of brain structures that may respond to endogenous NGF or exogenously administered rhNGF.     

The nature of the autoimmune antibody repertoire in human immunodeficiency virus type 1 infection.

Human immunodeficiency virus type 1 (HIV-1) seropositive donors typically have high serum antibody titers to a range of autoantigens, and the corresponding autoantibodies have been suggested to be of importance in the pathogenesis of HIV-1 infection. We have prepared 38 IgG human monoclonal autoantibodies from asymptomatic HIV-1 seropositive donors with elevated serum titers to autoantigens by construction of Fab combinatorial libraries on the surface of phage and affinity selection using a range of autoantigens, including double-stranded DNA, major histocompatibility complex class II, CD14, epidermal growth factor receptor, and ganglioside GD2. The autoantibodies are shown to be of moderate affinity and exhibit marked cross-reactivity with a range of antigens. This contrasts with the specific high-affinity antibodies selected (i) against infectious agents using the same libraries and (ii) against one of the autoantigens using a library from a donor with established autoimmune disease. The results lend no support to the presence of specific autoantibodies in HIV-1 infection and instead suggest attention should be focused on the pathological significance of high serum levels of antibodies capable of interacting with multiple molecular species.     

Mechanical stimulation of dorsal root ganglia induces increased production of substance P: a mechanism for pain following nerve root compromise?

The undecapeptide, substance P, is known to be synthesized in cell bodies of dorsal root ganglia. This neuropeptide is also known to modulate sensory, nociceptive transmission postsynaptically on dorsal horn interneurons. In the animal model used in the current study, experimental mechanical stimulation of dorsal root ganglia and nerve roots increased the amounts of substance P, as well as substance P immunoreactivity in cell bodies of the dorsal root ganglia and in the substantia gelatinosa of the spinal dorsal horn that the cell bodies innervated. These results were determined by using both immunohistochemistry and radioimmunoassay. This study suggests that substance P may modulate nociception when lumbar nerve roots are stimulated mechanically.     

Immunogenic and Antigenic Epitopes of Immunoglobulins Binding of Human Monoclonal Anti-D Antibodies to FcRI on the Monocyte-Like U937 Cell Line

Seventeen human monoclonal IgG1- or IgG3 anti-D-secreting clones have been examined for their ability to sensitise O+ red cells for Fc-receptor-mediated rosette formation with U937 cells. IgG3 but not IgG1 anti-D antibodies were able to mediate stable rosette formation with unstimulated U937 cells via interaction with the FcRI receptor. Decreasing FcRI density by incubating U937 cells with di-butyryl cAMP almost completely abolished rosette formation, whilst increasing FcRI density by incubating U937 cells with interferon-gamma increased the percentage of cells forming rosettes with IgG3- and IgG1-sensitised red cells. These data suggest that rosette formation between IgG anti-D-sensitised red cells and FcRI-expressing cells is dependent upon the density of IgG3 on the red cell surface, the density of FcRI on the effector cell, multiple FcRI/IgG interactions are required for stable rosette formation and that more FcRI/IgG1 than FcRI/IgG3 interactions are required.     

Wheeze in preschool children and its relation with doctor diagnosed asthma.

OBJECTIVE--To describe the characteristics of wheeze and its relation with doctor diagnosed asthma in children aged 5 years and under. DESIGN--Questionnaire survey of population based random sample of children registered on Leicestershire Health Authority's child health index for immunisation; questionnaire completed by parents. SUBJECTS--1650 white children born in 1985 to 1989 who were surveyed in 1990. MAIN OUTCOME MEASURES--Age distribution, severity, precipitants, seasonal characteristics, and diurnal variation of wheeze, family history of asthma/atopy, and their association(s) with doctor diagnosed asthma. RESULTS--There were 1422 replies (86.2%). Two hundred and twenty two (15.6%) were reported to have wheezed and of these 121 (8.6%) had formally been diagnosed as having asthma. More than 80% of the former had recurrences of wheeze and 40% (72) had three or more episodes in the preceding 12 months. Age, number of episodes per year, the severity of shortness of breath with attacks, and precipitants other than colds were the major factors determining the probability that a wheezy child will be diagnosed as having asthma. The data also suggest that despite the strong association of symptom based criteria with the label asthma, asthma was not diagnosed by these same severity criteria in one quarter of cases. CONCLUSIONS--Clinical and physiological follow up studies of children identified as asthmatic by the above criteria during the preschool years should validate or refute the predictive value of these measures of wheeze severity.