Issue published October 15, 1997

T he predominant genetic defect causing p47-phox-deficient chronic granulomatous disease (A47 degrees CGD) is a GT deletion (DeltaGT) at the beginning of exon 2. No explanation exists to account for the high incidence of this single mutation causing a rare disease in an unrelated, racially diverse population. In each of 34 consecutive unrelated normal individuals, both the normal and mutant DeltaGT sequences were present in genomic DNA, suggesting that a p47-phox related sequence carrying DeltaGT exists in the normal population. Screening of genomic bacteriophage and YAC libraries identified 13 p47-phox bacteriophage and 19 YAC clones. The GT deletion was found in 11 bacteriophage and 15 YAC clones. Only 5 exonic and 33 intronic differences distinguished all DeltaGT clones from all wild-type clones. The most striking differences were a 30-bp deletion in intron 1 and a 20-bp duplication in intron 2. These results provide good evidence for the existence of at least one highly homologous p47-phox pseudogene containing the DeltaGT mutation. The p47-phox gene and pseudogene(s) colocalize to chromosome 7q11.23. This close linkage, together with the presence within each gene of multiple recombination hot spots, suggests that the predominance of the DeltaGT mutation in A47 degrees CGD is caused by recombination events between the wild-type gene and the pseudogene(s).

I nsulin resistance is common in patients with angina pectoris, a positive exercise electrocardiogram, and normal coronary angiograms (syndrome X). It is still not known whether insulin resistance affects the cardiac muscle itself and, if so, whether insulin resistance involves myocardial hemodynamics and energy metabolism. We investigated hemodynamics as well as metabolite exchanges across the heart and the forearm in eight patients with syndrome X and eight control subjects during a baseline period after an overnight fast and during a hyperinsulinemic-euglycemic clamp. Myocardial hemodynamics and metabolism were studied at rest, during pace stress, and in the recovery period after pacing. Neither coronary sinus blood flow nor forearm blood flow differed between the groups before and during the clamp. Whole body insulin-stimulated glucose uptake was decreased in the patients (15.6+/-2.1 vs. 23.1+/-2.0 micromol x kg-1 x min-1). Insulin-stimulated glucose uptake in the forearm and the cardiac muscle was equally reduced in the patients (46+/-5 and 48+/-5%). Myocardial glucose uptake correlated with total arterial delivery in the control subjects (r = 0.63, P < 0.01), but not in patients (r = 0.22, P = 0.13). Carbohydrate and lipid oxidation was similar in the two groups at rest, and changes during the clamp were not different in control subjects and patients either at rest, during pacing, or in the recovery period. Patients with syndrome X exhibit myocardial insulin resistance, but cardiac energy metabolism remains unaffected. In patients with syndrome X, insulin-stimulated glucose uptake is independent from myocardial blood flow.

P rostaglandins (PG) are cytoprotective for gastrointestinal epithelium, possibly because they enhance mucosal repair. The objective of the present studies was to assess the role of prostaglandins in intestinal repair. Intestinal mucosa from porcine ileum subjected to 1 h of ischemia was mounted in Ussing chambers. Recovery of normal transepithelial electrical resistance occurred within 2 h, and continued to increase for a further 2 h to a value twice that of control. The latter response was blocked by inhibition of prostaglandin synthesis, and restored by addition of both carbacyclin (an analog of PGI2) and PGE2, whereas the addition of each alone had little effect. Histologically, prostaglandins had no effect on epithelial restitution or villous contraction, indicating that elevations in transepithelial resistance were associated with increases in paracellular resistance. Furthermore, prostaglandin-stimulated elevations in resistance were inhibited with cytochalasin D, an agent known to stimulate cytoskeletal contraction. Synergistic elevations in transepithelial resistance, similar to those of carbacyclin and PGE2, were also noted after treatment with cAMP and A23187 (a calcium ionophore). We conclude that PGE2 and PGI2 have a synergistic role in restoration of intestinal barrier function by increasing intracellular cAMP and Ca2+, respectively, which in turn signal cytoskeletal-mediated tight junction closure.

U nlike the uniform disease progression in inbred animals, polycystic kidney disease (PKD) progression within human families can be highly variable. This may be due to environmental or genetic factors or both. To determine if PKD severity can be influenced by modifier genes, we carried out an intercross between DBA/2-pcy/pcy and Mus m. castaneous involving 3,105 6-wk-old F2 mice. Large differences in PKD severity were observed in this cross. In addition, 23/ 800 phenotypically normal mice were pcy/pcy genotypically. These results demonstrated that PKD progression in pcy/ pcy mice is a quantitative trait that is strongly modulated by modifier genes. Whole genome quantitative trait loci mapping of 114 selected pcy/pcy mice (68 with the mild PKD and 46 with severe PKD) identified two loci, MOP1 and MOP2 that strongly modulate PKD progression. MOP1 (max LOD score = 10.3 at D4Mit111) and MOP2 (max LOD score = 13.8 at D16Mit1) accounted for 36.7 and 46.8% of the phenotypic variance, respectively. Two-factor ANOVA of the phenotypes and genotypes of all 673 pcy/pcy mice from our cross indicated that MOP1 and MOP2 alleles regulate PKD progression in a complex additive manner. Characterization of these novel modifying loci may provide additional insights into the pathogenesis of polycystic kidney diseases.

I n chronic hypercalcemia, inhibition of thick ascending limb sodium chloride reabsorption is mediated by elevated intrarenal PGE2. The mechanisms and source of elevated PGE2 in hypercalcemia are not known. We determined the effect of hypercalcemia on intrarenal expression of cytosolic phospholipase A2 (cPLA2), prostaglandin H synthase-1 (PGHS-1), and prostaglandin H synthase-2 (PGHS-2), enzymes important in prostaglandin production. In rats fed dihydrotachysterol to induce hypercalcemia, Western blot analysis revealed significant upregulation of both cPLA2 and PGHS-2 in the kidney cortex and the inner and outer medulla. Immunofluorescence localized intrarenal cPLA2 and PGHS-2 to interstitial cells of the inner and outer medulla, and to macula densa and cortical thick ascending limbs in both control and hypercalcemic rats. Hypercalcemia had no effect on intrarenal expression of PGHS-1. To determine if AT1 angiotensin II receptor activation was involved in the stimulation of cPLA2 and PGHS-2 in hypercalcemia, we treated rats with the AT1 receptor antagonist, losartan. Losartan abolished the polydipsia associated with hypercalcemia, prevented the increase in cPLA2 protein in all regions of the kidney, and diminished PGHS-2 expression in the inner medulla. In addition, losartan completely prevented the increase in urinary PGE2 excretion in hypercalcemic rats. Intrarenal levels of angiotensin II were unchanged in hypercalcemia. These data indicate that hypercalcemia stimulates intrarenal cPLA2 and PGHS-2 protein expression. Our results further support a role for angiotensin II, acting on AT1 receptors, in mediating this stimulation.

B oth rheumatoid arthritis and animal models of autoimmune arthritis are characterized by hyperactivation of synovial cells and hyperplasia of the synovial membrane. The activated synovial cells produce inflammatory cytokines and degradative enzymes that lead to destruction of cartilage and bones. Effective treatment of arthritis may require elimination of most or all activated synovial cells. The death factor Fas/Apo-1 and its ligand (FasL) play pivotal roles in maintaining self-tolerance and immune privilege. Fas is expressed constitutively in most tissues, and is dramatically upregulated at the site of inflammation. In both rheumatoid arthritis and animal models of autoimmune arthritis, high levels of Fas are expressed on activated synovial cells and infiltrating leukocytes in the inflamed joints. Unlike Fas, however, the levels of FasL expressed in the arthritic joints are extremely low, and most activated synovial cells survive despite high levels of Fas expression. To upregulate FasL expression in the arthritic joints, we have generated a recombinant replication-defective adenovirus carrying FasL gene; injection of the FasL virus into inflamed joints conferred high levels of FasL expression, induced apoptosis of synovial cells, and ameliorated collagen-induced arthritis in DBA/1 mice. The Fas-ligand virus also inhibited production of interferon-gamma by collagen-specific T cells. Coadministration of Fas-immunoglobulin fusion protein with the Fas-ligand virus prevented these effects, demonstrating the specificity of the Fas-ligand virus. Thus, FasL gene transfer at the site of inflammation effectively ameliorates autoimmune disease.

R etinoids play a critical role in cardiac morphogenesis. To examine the effects of excessive retinoid signaling on myocardial development, transgenic mice that overexpress a constitutively active retinoic acid receptor (RAR) controlled by either the alpha- or beta-myosin heavy chain (MyHC) promoter were generated. Animals carrying the alpha-MyHC-RAR transgene expressed RARs in embryonic atria and in adult atria and ventricles, but developed no signs of either malformations or disease. In contrast, beta-MyHC-RAR animals, where expression was activated in fetal ventricles, developed a dilated cardiomyopathy that varied in severity with transgene copy number. Characteristic postmortem lesions included biventricular chamber dilation and left atrial thrombosis; the incidence and severity of these lesions increased with increasing copy number. Transcript analyses showed that molecular markers of hypertrophy, alpha-skeletal actin, atrial natriuretic factor and beta-MyHC, were upregulated. Cardiac performance of transgenic hearts was evaluated using the isolated perfused working heart model as well as in vivo, by transthoracic M-mode echocardiography. Both analyses showed moderate to severe impairment of left ventricular function and reduced cardiac contractility. Thus, expression of a constitutively active RAR in developing atria and/ or in postnatal ventricles is relatively benign, while ventricular expression during gestation can lead to significant cardiac dysfunction.

A potentially fatal hemophagocytic syndrome has been noted in patients with malignant lymphomas, particularly in EBV-infected T cell lymphoma. Cytokines, such as interferon-gamma (IFN-gamma), TNF-alpha, and IL-1alpha, are elevated in patients' sera. To verify whether infection of T cells by EBV will upregulate specific cytokine genes and subsequently activate macrophages leading to hemophagocytic syndrome, we studied the transcripts of TNF-alpha, IFN-gamma, and IL-1alpha in EBV-infected and EBV-negative lymphoma tissues. By reverse transcription PCR analysis, transcripts of TNF-alpha were detected in 8 (57%) of 14 EBV-infected T cell lymphomas, higher than that detected in EBV-negative T cell lymphoma (one of six, 17%), EBV-positive B cell lymphoma (two of five, 40%) and EBV-negative B cell lymphomas (one of seven, 14%). Transcripts of IFN-gamma were consistently detected in T cell lymphoma and occasionally in B cell lymphoma, but were independent of EBV status. IL-1alpha expression was not detectable in any category. Consistent with these in vivo observations, in vitro EBV infection of T cell lymphoma lines caused upregulation of TNF-alpha gene, and increased secretion of TNF-alpha, but not IFN-gamma or IL-1alpha. Expression of TNF-alpha, IFN-gamma, and IL-1alpha was not changed by EBV infection of B cell lymphoma lines. To identify the specific cytokine(s) responsible for macrophage activation, culture supernatants from EBV-infected T cells were cocultured with a monocytic cell line U937 for 24 h. Enhanced phagocytosis and secretion of TNF-alpha, IFN-gamma, and IL-1alpha by U937 cells were observed, and could be inhibited to a large extent by anti-TNF-alpha (70%), less effectively by anti-IFN-gamma (31%), but almost completely by the combination of anti-TNF-alpha and anti-IFN-gamma (85%). Taken together, the in vivo and in vitro observations suggest that infection of T cells by EBV selectively upregulates the TNF-alpha expression which, in combination with IFN-gamma and probably other cytokines, can activate macrophages. This study not only highlights a probable pathogenesis for virus-associated hemophagocytic syndrome, but also suggests that anti-TNF-alpha will have therapeutic potential in the context of their fatal syndrome.

B oth estrogen and androgen exert their antiosteoporotic effects, at least in part, by inhibiting IL-6 production, thereby suppressing osteoclastogenesis. Several observations, however, suggest that besides increased IL-6 production, sensitivity of the osteoclastogenic process to this cytokine is altered after ovariectomy. Based on this and evidence that the ligand-binding subunit of the IL-6 receptor (gp80) is a limiting factor for the actions of IL-6 on bone, we hypothesized that sex steroids regulate expression of the IL-6 receptor as well. We report that 17beta-estradiol or dihydrotestosterone in vitro decreased the abundance of the gp80 mRNA as well as the mRNA of the signal-transducing subunit of the IL-6 receptor (gp130) in cells of the bone marrow stromal/osteoblastic lineage, and also decreased gp130 protein levels. These effects did not require new protein synthesis. In contrast to sex steroids, parathyroid hormone stimulated gp130 expression; this effect was opposed by sex steroids. Consistent with these findings, ovariectomy in mice caused an increase in expression of gp80, gp130, and IL-6 mRNAs in ex vivo bone marrow cell cultures as determined by quantitative reverse transcription (RT)-PCR, and confirmed on an individual cell basis using in situ RT-PCR. The demonstration of increased expression of the IL-6 receptor after loss of sex steroids provides an explanation for why IL-6 is important for skeletal homeostasis in the sex steroid-deficient, but not replete, state.

T o determine whether IGF-1 opposes the stimulation of myocyte death in the surviving myocardium after infarction, transgenic mice overexpressing human IGF-1B in myocytes (FVB.Igf+/-) and wild-type littermates at 1.5 and 2.5 mo of age were subjected to coronary ligation and killed 7 d later. Myocardial infarction involved an average 50% of the left ventricle, and produced cardiac failure. In the region proximate to infarction, myocyte apoptosis increased 4. 2-fold and 2.1-fold in nontransgenics at 1.5 and 2.5 mo, respectively. Corresponding increases in myocyte necrosis were 1. 8-fold and 1.6-fold. In contrast, apoptotic and necrotic myocyte death did not increase in FVB.Igf+/- mice at either age after infarction. In 2.5-mo-old infarcted nontransgenics, functional impairment was associated with a 29% decrease in wall thickness, 43% increase in chamber diameter, and a 131% expansion in chamber volume. Conversely, the changes in wall thickness, chamber diameter, and cavitary volume were 41, 58, and 48% smaller in infarcted FVB.Igf+/- than in nontransgenics. The differential response to infarction of FVB.Igf+/- mice resulted in an attenuated increase in diastolic wall stress, cardiac weight, and left and right ventricular weight-to-body wt ratios. In conclusion, constitutive overexpression of IGF-1 prevented activation of cell death in the viable myocardium after infarction, limiting ventricular dilation, myocardial loading, and cardiac hypertrophy.

I n a backcross population (n = 281) derived from a cross of the Lyon hypertensive rat with Lyon normotensive rat, we investigated whether genetic factors influence the acute cardiovascular responses to pharmacological modulation of the renin-angiotensin system, the sympathetic nervous system, and the voltage-sensitive L-type calcium channels. Using microsatellite markers, a quantitative trait locus was identified and mapped on rat chromosome 2 that specifically influences the systolic (peak LOD score 4.4) and diastolic (peak LOD score 4.1) blood pressure responses to administration of a dihydropyridine calcium antagonist, PY108-068. The locus accounted for 10.3 and 10.4% of the total variances in the systolic and diastolic responses to PY108-068, respectively. In marked contrast, the locus had no effect on either basal blood pressure or on the responses to acute administration of a ganglionic blocking agent, trimetaphan, or of an angiotensin II subtype 1 receptor antagonist, losartan. These findings provide strong direct support for the paradigm that genetic factors may influence the response to antihypertensive drugs and suggest that the heterogeneity seen in the responses to different antihypertensive agents in human essential hypertension may have a significant genetic determination.

W e explored in 51 normal subjects, distributed in various series of experiments, whether endothelium nitric oxide may play a role in insulin modulation of alpha2- and beta-adrenergic- evoked vascular responses. In particular, we examined the forearm blood flow response (FBF, ml.min-1.dl-1) to intrabrachial infusion of BHT-933 (0.5, 1, and 2 microg.min-1.dl-1) or isoproterenol (1, 3, and 6 ng. min-1.dl-1) in control conditions, during intrabrachial infusion of insulin alone (0.05 mU.kg-1.min-1) and associated with l-N-monomethylarginine (L-NMMA) (0.05 microg.min-1.dl-1), a nitric oxide synthase inhibitor. In control conditions both BHT-933 and isoproterenol induced a dose-dependent vascular response. Local hyperinsulinemia (deep venous plasma insulin 68.5+/-4 microU/ml) did not change basal FBF whereas attenuated BHT-933 vasoconstriction and enhanced isoproterenol vasodilation. L-NMMA reduced basal FBF and abolished the insulin effect on BHT-933 and isoproterenol response. To clarify whether a nitric oxide component is included in alpha2- and beta-adrenergic response and may be responsible for insulin vascular effect, we further examined BHT-933 and isoproterenol responses during nitric oxide inhibition. Interestingly, L-NMMA potentiated the BHT-933 vasoconstriction and attenuated the isoproterenol vasodilation and, in these conditions, insulin was no more able to exhibit its vascular effects. Finally, to rule out the possibility that the conteracting effect of L-NMMA may not be specifically related to insulin action, dose-response curves to phenylephrine (0.5, 1, and 2 microg.min-1.dl-1) or sodium nitroprusside (1, 2, and 4 microg.min-1.dl-1) were also performed. Both insulin and L-NMMA were unable to alter the phenylephrine-induced vasoconstriction and the sodium nitroprusside vasodilation. In conclusion, our data demonstrate an endothelial nitric oxide component in the alpha2- and beta-adrenergic vascular responses which is the target of the insulin vascular action.

T he Long-Evans Tokushima Lean (LETL) rat, characterized by rapid onset of insulin-dependent (type I) diabetes mellitus (IDDM), no sex difference in the incidence of IDDM, autoimmune destruction of pancreatic beta cells, and no significant T cell lymphopenia, is a desirable animal model for human IDDM. We have established a diabetes-prone substrain of the LETL rat, named Komeda Diabetes-Prone (KDP) rat, showing a 100% development of moderate to severe insulitis within 220 d of age. The cumulative frequency of IDDM was 70% at 120 d of age, and reached 82% within 220 d of age. Here, we performed the first genome-wide scan for non-MHC IDDM susceptibility genes in this strain. The analysis of three crosses has led to the revelation of a major IDDM susceptibility gene, termed Iddm/kdp1, on rat chromosome (Chr) 11. Homozygosity for the KDP allele at this locus is shown to be essential for the development of moderate to severe insulitis and the onset of IDDM. Comparative mapping suggests that the homologues of Iddm/ kdp1 are located on human Chr 3 and mouse Chr 16 and would therefore be different from previously reported IDDM susceptibility genes.

b eta-Chemokines and their receptors mediate the trafficking and activation of a variety of leukocytes including the lymphocyte and macrophage. An array of no less than eight beta-chemokine receptors has been identified, four of which are capable of recognizing the chemokines MIP1alpha and RANTES. Genetic deletion of one of the MIP1alpha and RANTES receptors, CCR5, is associated with protection from infection with HIV-1 in humans, while deletion of the ligand MIP1alpha protects against Coxsackie virus-associated myocarditis. In this report we show that the deletion of another receptor for MIP1alpha and RANTES, the CCR1 receptor, is associated with protection from pulmonary inflammation secondary to acute pancreatitis in the mouse. The protection from lung injury is associated with decreased levels of TNF-alpha in a temporal sequence indicating that the activation of the CCR1 receptor is an early event in the systemic inflammatory response syndrome.

F 2-Isoprostanes are prostaglandin (PG) isomers formed in situ in cell membranes by peroxidation of arachidonic acid. 8-epi PGF2alpha and IPF2alpha-I are F2-isoprostanes produced in humans which circulate in plasma and are excreted in urine. Measurement of F2-isoprostanes may offer a sensitive, specific, and noninvasive method for measuring oxidant stress in clinical settings where reactive oxygen species are putatively involved. We determined whether isoprostanes were present in human atherosclerotic lesions, where lipid peroxidation is thought to occur in vivo. 8-epi PGF2alpha ranged from 1.310-3.450 pmol/micromol phospholipid in atherectomy specimens compared with 0.045-0.115 pmol/micromol phospholipid (P < 0.001) in vascular tissue devoid of atherosclerosis. Corresponding values of IPF2alpha-I were 5.6-13.8 vs. 0.16-0.44 pmol/micromol phospholipid (P < 0.001). Levels of the two isoprostanes in vascular tissue were highly correlated (r = 0.80, P < 0.0001). Immunohistochemical studies confirmed that foam cells adjacent to the lipid necrotic core of the plaque were markedly positive for 8-epi PGF2alpha. These cells were also reactive with anti-CD68, an epitope specific for human monocyte/macrophages. 8-epi PGF2alpha immunoreactivity was also detected in cells positive for anti-alpha-smooth muscle actin antibody, which specifically recognizes vascular smooth muscle cells. Our results indicate that 8-epi PGF2alpha and IPF2alpha-I, two distinct F2-isoprostanes and markers of oxidative stress in vivo, are present in human atherosclerotic plaque. Quantitation of these chemically stable products of lipid peroxidation in target tissues, as well as in biological fluids, may aid in the rational development of antioxidant drugs in humans.

I n cardiac transplantation, chronic rejection takes the form of an occlusive vasculopathy. The mechanism underlying this disorder remains unclear. The purpose of this study was to investigate the role nitric oxide (NO) may play in the development of allograft arteriosclerosis. Rat aortic allografts from ACI donors to Wistar Furth recipients with a strong genetic disparity in both major and minor histocompatibility antigens were used for transplantation. Allografts collected at 28 d were found to have significant increases in both inducible NO synthase (iNOS) mRNA and protein as well as in intimal thickness when compared with isografts. Inhibiting NO production with an iNOS inhibitor increased the intimal thickening by 57.2%, indicating that NO suppresses the development of allograft arteriosclerosis. Next, we evaluated the effect of cyclosporine (CsA) on iNOS expression and allograft arteriosclerosis. CsA (10 mg/kg/d) suppressed the expression of iNOS in response to balloon-induced aortic injury. Similarly, CsA inhibited iNOS expression in the aortic allografts, associated with a 65% increase in intimal thickening. Finally, we investigated the effect of adenoviral-mediated iNOS gene transfer on allograft arteriosclerosis. Transduction with iNOS using an adenoviral vector suppressed completely the development of allograft arteriosclerosis in both untreated recipients and recipients treated with CsA. These results suggest that the early immune-mediated upregulation in iNOS expression partially protects aortic allografts from the development of allograft arteriosclerosis, and that iNOS gene transfer strategies may prove useful in preventing the development of this otherwise untreatable disease process.

T here is substantial evidence that dendritic cells (DC) residing within epithelial surfaces (e.g., Langerhans cells) are the initial cells infected with HIV after mucosal exposure to virus. To study DC-HIV interactions in detail, we propagated Langerhans cell-like DC from cord blood CD34(+) cells and from adult blood plastic-adherent PBMC in the presence of cytokines (GM-CSF, IL-4, and/or TNF-alpha). DC pulsed overnight with HIVBaL or HIVIIIB were infected productively with both viral subtypes (as assessed by PCR, supernatant p24 protein levels, electron microscopy, and antibody staining). Productive infection could be blocked by anti-CD4 mAbs, RANTES (regulated upon activation, normal T cell expressed and secreted) (for HIVBaL), stromal cell-derived factor-1 (for HIVIIIB), or azidothymidine added during the HIV pulse, as well as by blocking DC proliferation. However, pulsing DC with HIV under these blocking conditions had no effect on the ability of DC to capture virus and transmit infection to cocultured antigen-stimulated CD4(+) T cells. Thus, we show by several criteria that (a) productive infection of DC and (b) the ability of DC to capture virus are mediated through separate pathways. We suggest that strategies designed to block mucosal transmission of HIV should consider interfering with both virus infection and virus capture by DC.

W e demonstrated recently that the arachidonic acid (AA) cascade is involved in cytomegalovirus (CMV)-induced generation of reactive oxygen species (ROS) and the activation of nuclear factor (NF)-kappaB in human smooth muscle cells (SMCs). Since AA release from neutrophils is mediated by pertussis toxin (PTx)-sensitive guanine nucleotide-binding (G) proteins, we hypothesized by analogy that CMV stimulates ROS generation in SMCs and ultimately activates NF-kappaB via a PTx-sensitive G protein-coupled pathway. Our first test of this hypothesis demonstrated that PTx blocked AA release induced by CMV infection of SMCs, as well as blocked the terminal products of this reaction, ROS generation and NF-kappaB activation. More proximal components of the pathway were then examined. CMV infection increased phosphorylation and activity of cytosolic phospholipase A2 (cPLA2), an enzyme causing AA release; these effects were inhibited by PTx. CMV infection activated mitogen-activated protein (MAP) kinase, a key enzyme for cPLA2 phosphorylation, an effect also inhibited by PTx. Finally, inhibition of MAP kinase kinase (MAPKK), which phosphorylates and thereby activates MAP kinase, inhibited CMV-induced ROS generation. These data demonstrate that a PTx-sensitive G protein-dependent signaling pathway mediates cellular effects of CMV infection of SMCs. The downstream events include phosphorylation and activation of MAP kinase by MAPKK and subsequent phosphorylation and activation of cPLA2 (with its translocation to cell membranes), followed by stimulation of the AA cascade, which generates intracellular ROS and thereby activates NF-kappaB.

P revious studies support a role for intestinal epithelial cells (IEC) as antigen-presenting cells in mucosal immune responses. T cells activated by IEC are CD8+, suppressor in function, and dependent upon CD8-associated p56lck activation. A 180-kD glycoprotein (gp180) recognized by mAbs B9 and L12 has been identified and shown to be important in CD8+ T cell activation by IEC. Since IEC derived from patients with inflammatory bowel disease (IBD) are incapable of activating CD8+ T cells, we asked whether this correlated with gp180 expression. While frozen sections of normal bowel revealed bright gp180 staining on all IEC, both inflamed and uninflamed ulcerative colitis (UC) specimens showed patchy staining. In Crohn's disease (CD), staining was faint to absent. Flow cytometry confirmed immunohistochemical data. The staining patterns correlated with the ability of IEC to activate CD8-associated p56lck. Normal IEC induced phosphorylation of p56lck in CD8alpha but not CD4+ transfectants. In contrast, both UC and CD IEC activated CD4 and, to a much lesser extent, CD8-associated p56lck. Thus, gp180 expression by IBD IEC appears to be altered, and correlates with a functional alteration of lck activation. This defect may reflect a more proximal event in the pathogenesis of IBD.

T ie2 is a novel receptor tyrosine kinase that is expressed almost exclusively by vascular endothelium. Disruption of Tie2 function in transgenic mice resulted in embryonic lethality secondary to characteristic vascular defects; similar defects occurred after disruption of the Tie2 ligand. These findings indicate that the Tie2/Tie2 ligand pathway plays important roles during development of the embryonic vasculature. To determine whether the Tie2 pathway was involved in pathologic angiogenesis in adult tissues, a soluble form of the extracellular domain of murine Tie2 (ExTek.6His) was developed and used as a Tie2 inhibitor. After a single application of the ExTek.6His protein into a rat cutaneous window chamber, growth of a mammary tumor inside the chamber was reduced by > 75% (P < 0.005), and tumor vascular length density was reduced by 40% when compared with control-treated tumors (P < 0.01). In the rat cornea, ExTek.6His blocked angiogenesis stimulated by tumor cell conditioned media. ExTek.6His protein did not affect the viability of cultured tumor cells, indicating that the antitumor effect of ExTek.6His was due to the inhibition of tumor angiogenesis. These data demonstrate a role for the Tie2 pathway in pathologic angiogenesis, suggesting that targeting this pathway may yield effective antiangiogenic agents for treatment of cancer and other angiogenic diseases.

A synthetic peptide-1, an 18 amino acid residue peptide derived from a hydrophilic domain of a cloned platelet type I collagen receptor, was used to study the role of the receptor on types I and III collagen-induced platelet aggregation and the release of ATP. The peptide inhibits the type I, but not the type III, collagen-induced platelet aggregation and the release of ATP in a dose-dependent manner. The [125I]peptide-1 specifically binds to type I collagen-coated microtiter wells in a dose-dependent manner (with Kd = 10 nM). The binding of [125I]peptide-1 can be inhibited by an excess of unlabeled peptide-1 suggesting that the binding is specific. The labeled peptide-1 does not bind to type III collagen-coated microtiter wells. Results from an enzyme-linked immunosorbent assay show that the peptide reacts with the poly- and monoclonal antibodies raised against the purified platelet type I collagen receptor (Mr 65 kD). The peptide also inhibits the adhesion of platelets on type I collagen matrix and rabbit aortic segments in a dose-dependent manner. These results suggest that the reactive site of the platelet receptor for type I collagen resides in this portion of the molecule.

W e have studied the pathways that lead to arrest and firm adhesion of rolling PMN on activated, surface-adherent platelets. Stable arrest and adhesion strengthening of PMN on thrombin-stimulated, surface-adherent platelets in flow required distinct Ca2+- and Mg2+-dependent regions of Mac-1 (alphaMbeta2), and involved interactions of Mac-1 with fibrinogen, which was bound to platelets via alphaIIbbeta3. Mac-1 also bound to other unidentified ligands on platelets, which were not intracellular adhesion molecule-2 (ICAM-2), heparin, or heparan-sulfate proteoglycans. This was shown by inhibition with mAbs or peptides, by treatment of platelets with heparitinase, and by using platelets with defective alphaIIbbeta3 from a patient with Glanzmann thrombasthenia. Tethering of PMN on platelet ICAM-2 via LFA-1 (alphaLbeta2) was observed, which may facilitate the transition between rolling on selectins and Mac-1-dependent arrest. Arrest and adhesion strengthening was pertussis toxin sensitive and in flow was mainly induced by platelet-activating factor but not through activation of the chemokine receptor CXCR2. In stasis, spreading occurred and the CXCR2 contributed to firm adhesion.

A bnormalities contributing to the pathogenesis of non-insulin-dependent diabetes mellitus include impaired beta cell function, peripheral insulin resistance, and increased hepatic glucose production. Glucocorticoids are diabetogenic hormones because they decrease glucose uptake and increase hepatic glucose production. In addition, they may directly inhibit insulin release. To evaluate that possible role of glucocorticoids in beta cell function independent of their other effects, transgenic mice with an increased glucocorticoid sensitivity restricted to their beta cells were generated by overexpressing the glucocorticoid receptor (GR) under the control of the insulin promoter. Intravenous glucose tolerance tests showed that the GR transgenic mice had normal fasting and postabsorptive blood glucose levels but exhibited a reduced glucose tolerance compared with their control littermates. Measurement of plasma insulin levels 5 min after intravenous glucose load demonstrated a dramatic decrease in acute insulin response in the GR transgenic mice. These results show that glucocorticoids directly inhibit insulin release in vivo and identify the pancreatic beta cell as an important target for the diabetogenic action of glucocorticoids.

E ndothelial selectins mediate rolling of leukocytes on endothelium, a crucial step for leukocyte firm adhesion and emigration into sites of tissue injury and infection. To characterize the role of the endothelial selectins during bacterial sepsis in vivo, Streptococcus pneumoniae (1-10 x 10(6) colony-forming units) was inoculated intraperitoneally into wild-type mice and mice with E-, P-, or E-/P-selectin deficiencies. Mice were followed 10 d for morbidity, survival, clearance of bacteremia, and leukocyte migration to the peritoneal cavity and organs 48 h after infection. All selectin-deficient mice showed a more pronounced morbidity, a significantly higher mortality associated with persistent bacteremia, and a higher bacterial load when compared with wild-type mice. These differences were most remarkable in the E-selectin-deficient mice, which showed the highest rate of mortality and bacteremia (P </= 0.0001). No significant differences were observed among the groups in the inflammatory response present in the peritoneal cavity, brain, liver, spleen, or kidney at 48 h after inoculation. Extensive hepatic and splenic necrosis and thrombosis were noted in E- and P-selectin-deficient mice. Although the absence of endothelial selectins did not substantially impair leukocyte emigration to sites of infection 48 h after pneumococcal sepsis, it resulted in increased mortality and a higher bacterial load in the bloodstream of selectin-deficient mice. These results demonstrate a definitive phenotypic abnormality in E-selectin-deficient mice, and suggest that E- and P-selectin are important in the host defense against S. pneumoniae infection.

T o assess cellular mechanisms mediating afferent (AA) and efferent arteriolar (EA) constriction by angiotensin II (AngII), experiments were performed using isolated perfused hydronephrotic kidneys. In the first series of studies, AngII (0.3 nM) constricted AAs and EAs by 29+/-3 (n = 8, P < 0.01) and 27+/-3% (n = 8, P < 0.01), respectively. Subsequent addition of nifedipine restored AA but not EA diameter. Manganese (8 mM) reversed EA constriction by 65+/-9% (P < 0.01). In the second group, the addition of N-ethylmaleimide (10 microM), a Gi/Go protein antagonist, abolished AngII- induced EA (n = 6) but not AA constriction (n = 6). In the third series of experiments, treatment with 2-nitro-4-carboxyphenyl-N, N-diphenyl-carbamate (200 microM), a phospholipase C inhibitor, blocked both AA and EA constriction by AngII (n = 6 for each). In the fourth group, thapsigargin (1 microM) prevented AngII-induced AA constriction (n = 8) and attenuated EA constriction (8+/-2% decrease in EA diameter at 0.3 nM AngII, n = 8, P < 0.05). Subsequent addition of manganese (8 mM) reversed EA constriction. Our data provide evidence that in AAs, AngII stimulates phospholipase C with subsequent calcium mobilization that is required to activate voltage-dependent calcium channels. Our results suggest that AngII constricts EAs by activating phospholipase C via the Gi protein family, thereby eliciting both calcium mobilization and calcium entry.

T he synthesis of cholesterol and its uptake from plasma LDL are regulated by two membrane-bound transcription factors, designated sterol regulatory element binding protein-1 and -2 (SREBP-1 and SREBP-2). Here, we used the technique of homologous recombination to generate mice with disruptions in the gene encoding the two isoforms of SREBP-1, termed SREBP-1a and SREBP-1c. Heterozygous gene-disrupted mice were phenotypically normal, but 50- 85% of the homozygous (-/-) mice died in utero at embryonic day 11. The surviving -/- mice appeared normal at birth and throughout life. Their livers expressed no functional SREBP-1. There was a 1.5-fold upregulation of SREBP-2 at the level of mRNA and a two- to threefold increase in the amount of mature SREBP-2 in liver nuclei. Previous studies showed that SREBP-2 is much more potent than SREBP-1c, the predominant hepatic isoform of SREBP-1, in activating transcription of genes encoding enzymes of cholesterol synthesis. Consistent with this observation, the SREBP-1 -/- animals manifested elevated levels of mRNAs for 3-hydroxy-3-methylglutaryl coenzyme A synthase and reductase, farnesyl diphosphate synthase, and squalene synthase. Cholesterol synthesis, as measured by the incorporation of [3H]water, was elevated threefold in livers of the -/- mice, and hepatic cholesterol content was increased by 50%. Fatty acid synthesis was decreased in livers of the -/- mice. The amount of white adipose tissue was not significantly decreased, and the levels of mRNAs for lipogenic enzymes, adipocyte lipid binding protein, lipoprotein lipase, and leptin were normal in the -/- mice. We conclude from these studies that SREBP-2 can replace SREBP-1 in regulating cholesterol synthesis in livers of mice and that the higher potency of SREBP-2 relative to SREBP-1c leads to excessive hepatic cholesterol synthesis in these animals.

T his study was undertaken to determine if human recombinant growth hormone (hrGH, 6 mg/d for 2 wk) would stimulate muscle protein synthesis in AIDS wasting. Healthy controls were compared with patients who were HIV+, had AIDS without weight loss, and had AIDS with > 10% weight loss. Before hrGH, rates of skeletal muscle protein synthesis, measured with l-[2H5]phenylalanine, were the same in controls and in all stages of disease. Rates of myofibrillar protein degradation, however, assessed from urinary excretion of 3-methyl histidine, were higher in AIDS and AIDS wasting than in HIV+ or healthy individuals. The group with weight loss had significantly higher TNFalpha levels but not higher HIV viral loads. Muscle function, as determined by isokinetic knee extension and shoulder flexion, was significantly higher in controls than all infected individuals. After GH, rates of protein synthesis were stimulated 27% in controls, with a smaller increase (11%) in HIV+, and a significant depression (42%) in AIDS with weight loss, despite fourfold elevation in insulin-like growth factor-I in all groups. There was a significant correlation of hrGH-induced changes in muscle protein synthesis with severity of disease (P = 0.002). The results indicate increased basal muscle protein degradation and decreased responsiveness of muscle protein synthesis to GH in the later stages of disease.

I ndividuals with one aerodigestive tract malignancy have a high incidence of second primary aerodigestive tumors. The mechanism for this field effect has not been determined. We studied an individual with widespread dysplastic changes in the respiratory epithelium but no overt carcinoma. The entire tracheobronchial tree obtained at autopsy was embedded in paraffin, and bronchial epithelial cells were isolated by microdissection. DNA extracted from the microdissected cells was analyzed for point mutations in the p53 tumor suppressor gene. A single, identical point mutation consisting of a G:C to T:A transversion in codon 245 was identified in bronchial epithelium from 7 of 10 sites in both lungs. Epithelium at sites containing the p53 mutation was morphologically abnormal, exhibiting squamous metaplasia and mild to moderate atypia. No invasive tumor was found in the tracheobronchial tree or any other location. Cells from peripheral blood, kidney, liver, and lymph node exhibited no abnormality in the p53 gene. The widespread presence of a single somatic p53 point mutation in the bronchi of a smoker suggests that a single progenitor bronchial epithelial clone may expand to populate broad areas of the bronchial mucosa-a novel mechanism for field carcinogenesis in the respiratory epithelium that may be of importance in assessing individuals for risk of a second primary tumor as well as in devising effective strategies for chemoprevention of lung cancer.