Issue published November 1, 1997

T o investigate potential interactions between angiotensin II (AII) and the insulin signaling system in the vasculature, insulin and AII regulation of insulin receptor substrate-1 (IRS-1) phosphorylation and phosphatidylinositol (PI) 3-kinase activation were examined in rat aortic smooth muscle cells. Pretreatment of cells with AII inhibited insulin-stimulated PI 3-kinase activity associated with IRS-1 by 60%. While AII did not impair insulin-stimulated tyrosine phosphorylation of the insulin receptor (IR) beta-subunit, it decreased insulin-stimulated tyrosine phosphorylation of IRS-1 by 50%. AII inhibited the insulin-stimulated association between IRS-1 and the p85 subunit of PI 3-kinase by 30-50% in a dose-dependent manner. This inhibitory effect of AII on IRS-1/PI 3-kinase association was blocked by the AII receptor antagonist saralasin, but not by AT1 antagonist losartan or AT2 antagonist PD123319. AII increased the serine phosphorylation of both the IR beta-subunit and IRS-1. In vitro binding experiments showed that autophosphorylation increased IR binding to IRS-1 from control cells by 2.5-fold versus 1.2-fold for IRS-1 from AII-stimulated cells, suggesting that AII stimulation reduces IRS-1's ability to associate with activated IR. In addition, AII increased p85 serine phosphorylation, inhibited the total pool of p85 associated PI 3-kinase activity, and decreased levels of the p50/p55 regulatory subunit of PI 3-kinase. These results suggest that activation of the renin-angiotensin system may lead to insulin resistance in the vasculature.

L p(a) is a major inherited risk factor associated with premature heart disease and stroke. The mechanism of Lp(a) atherogenicity has not been elucidated, but likely involves both its ability to influence plasminogen activation as well as its atherogenic potential as a lipoprotein particle after receptor-mediated uptake. We demonstrate that fibroblasts expressing the human VLDL receptor can mediate endocytosis of Lp(a), leading to its degradation within lysosomes. In contrast, fibroblasts deficient in this receptor are not effective in catabolizing Lp(a). Lp(a) degradation was prevented by antibodies against the VLDL receptor, and by RAP, an antagonist of ligand binding to the VLDL receptor. Catabolism of Lp(a) was inhibited by apolipoprotein(a), but not by LDL or by monoclonal antibodies against apoB100 that block LDL binding to the LDL receptor, indicating that apolipoprotein(a) mediates Lp(a) binding to this receptor. Removal of Lp(a) antigen from the mouse circulation was delayed in mice deficient in the VLDL receptor when compared with control mice, indicating that the VLDL receptor may play an important role in Lp(a) catabolism in vivo. We also demonstrate the expression of the VLDL receptor in macrophages present in human atherosclerotic lesions. The ability of the VLDL receptor to mediate endocytosis of Lp(a) could lead to cellular accumulation of lipid within macrophages, and may represent a molecular basis for the atherogenic effects of Lp(a).

C ells proliferating from human atherosclerotic lesions are resistant to the antiproliferative effect of TGF-beta1, a key factor in wound repair. DNA from human atherosclerotic and restenotic lesions was used to test the hypothesis that microsatellite instability leads to specific loss of the Type II receptor for TGF-beta1 (TbetaR-II), causing acquired resistance to TGF-beta1. High fidelity PCR and restriction analysis was adapted to analyze deletions in an A10 microsatellite within TbetaR-II. DNA from lesions, and cells grown from lesions, showed acquired 1 and 2 bp deletions in TbetaR-II, while microsatellites in the hMSH3 and hMSH6 genes, and hypermutable regions of p53 were unaffected. Sequencing confirmed that these deletions occurred principally in the replication error-prone A10 microsatellite region, though nonmicrosatellite mutations were observed. The mutations could be identified within specific patches of the lesion, while the surrounding tissue, or unaffected arteries, exhibited the wild-type genotype. This microsatellite deletion causes frameshift loss of receptor function, and thus, resistance to the antiproliferative and apoptotic effects of TGF-beta1. We propose that microsatellite instability in TbetaR-II disables growth inhibitory pathways, allowing monoclonal selection of a disease-prone cell type within some vascular lesions.

P rotein kinase C (PKC) activation in the heart has been linked to a hypertrophic phenotype and to processes that influence contractile function. To establish whether PKC activation is sufficient to induce an abnormal phenotype, PKCbeta was conditionally expressed in cardiomyocytes of transgenic mice. Transgene expression in adults caused mild and progressive ventricular hypertrophy associated with impaired diastolic relaxation, whereas expression in newborns caused sudden death associated with marked abnormalities in the regulation of intracellular calcium. Thus, the PKC signaling pathway in cardiocytes has different effects depending on the timing of expression and, in the adult, is sufficient to induce pathologic hypertrophy.

T o explore the possibility that vitamin D status regulates sulfate homeostasis, plasma sulfate levels, renal sulfate excretion, and the expression of the renal Na-SO4 cotransporter were evaluated in vitamin D-deficient (D-D-) rats and in D-D- rats rendered normocalcemic by either vitamin D or calcium/lactose supplementation. D-D- rats had significantly lower plasma sulfate levels than control animals (0.93+/-0.01 and 1.15+/-0.05 mM, respectively, P < 0.05), and fractional sulfate renal excretion was approximately threefold higher comparing D-D- and control rats. A decrease in renal cortical brush border membrane Na-SO4 cotransport activity, associated with a parallel decrease in both renal Na-SO4 cotransport protein and mRNA content (78+/-3 and 73+/-3% decreases, respectively, compared with control values), was also observed in D-D- rats. Vitamin D supplementation resulted in a return to normal of plasma sulfate, fractional sulfate excretion, and both renal Na-SO4 cotransport mRNA and protein. In contrast, renal sulfate excretion and renal Na-SO4 cotransport activity, protein abundance, and mRNA remained decreased in vitamin D-depleted rats fed a diet supplemented with lactose and calcium, despite that these rats were normocalcemic, and had significantly lower levels of parathyroid hormone and 25(OH)- and 1,25(OH)2-vitamin D levels than the vitamin D-supplemented groups. These results demonstrate that vitamin D modulates renal Na-SO4 sulfate cotransport and sulfate homeostasis. The ability of vitamin D status to regulate Na-SO4 cotransport appears to be a direct effect, and is not mediated by the effects of vitamin D on plasma calcium or parathyroid hormone levels. Because sulfate is required for synthesis of essential matrix components, abnormal sulfate metabolism in vitamin D-deficient animals may contribute to producing some of the abnormalities observed in rickets and osteomalacia.

T he mechanism of exon skipping induced by nonsense mutations has not been well elucidated. We now report results of in vitro splicing studies which disclosed that a particular example of exon skipping is due to disruption of a splicing enhancer sequence located within the exon. A nonsense mutation (E1211X) due to a G to T transversion at the 28th nucleotide of exon 27 (G3839T) was identified in the dystrophin gene of a Japanese Becker muscular dystrophy case. Partial skipping of the exon resulted in the production of truncated dystrophin mRNA, although the consensus sequences for splicing at both ends of exon 27 were unaltered. To determine how E1211X induced exon 27 skipping, the splicing enhancer activity of purine-rich region within exon 27 was examined in an in vitro splicing system using chimeric doublesex gene pre-mRNA. The mutant sequence containing G3839T abolished splicing enhancer activity of the wild-type purine-rich sequence for the upstream intron in this chimeric pre-mRNA. An artificial polypurine oligonucleotide mimicking the purine-rich sequence of exon 27 also showed enhancer activity that was suppressed by the introduction of a T nucleotide. Furthermore, the splicing enhancer activity was more markedly inhibited when a nonsense codon was created by the inserted T residue. This is the first evidence that partial skipping of an exon harboring a nonsense mutation is due to disruption of a splicing enhancer sequence.

P ulmonary alveolar proteinosis (PAP) is a heterogeneous disorder of genetic or acquired etiologies. In some cases congenital PAP is associated with hereditary surfactant protein (SP)-B deficiency. To date, the molecular defect in the majority of patients with PAP has not been identified. In mice, PAP has been generated by targeted deletion of the genes for either the GM-CSF/IL-3/IL-5 receptor common beta chain (beta c) or GM-CSF. Here, we describe an expression defect of beta c in three of seven pediatric patients with PAP and in one patient with severe lung disease suspected to be PAP. The patients failed to express normal levels of beta c as shown by flow cytometry. Strikingly reduced or absent function of beta c was demonstrated by ligand binding studies and progenitor clonogenic assays. Analysis of beta c DNA revealed a point mutation from proline to threonine at codon 602 in one patient. Our findings provide evidence that a defect in the expression of a hematopoietic cytokine receptor is associated with human PAP.

A nimal studies indicate that the use of replication-deficient adenovirus for human gene therapy is limited by host antivector immune responses that result in transient recombinant protein expression and blocking of gene transfer when rechallenged. Therefore, we have examined immune responses to an adenoviral vector and to the beta-galactosidase protein in four patients with lung cancer given a single intratumor injection of 10(9) plaque-forming units of recombinant adenovirus. The beta-galactosidase protein was expressed in day-8 tumor biopsies from all patients at variable levels. Recombinant virus DNA was detected by PCR in day-30 and day-60 tumor biopsies from all patients except patient 1. A high level of neutralizing antiadenovirus antibodies was detected in patient 1 before Ad-beta-gal injection whereas it was low (patient 3) or undetectable in the other two patients. All patients developed potent CD4 type 1 helper T cell (Th1) responses to adenoviral particles which increased gradually over time after injection. Antiadenovirus cytotoxic T lymphocyte responses were consistently boosted in the two patients examined (patients 3 and 4). Sustained production of anti-beta-galactosidase IgG was observed in all patients except patient 1. Consistent with anti-beta-gal antibody production, all patients except patient 1 developed intense, dose-dependent Th1 responses to soluble beta-galactosidase which increased over time. Strong beta-galactosidase-specific cytotoxic T lymphocyte responses were detected in patients 2, 3, and 4. Our results clearly show that despite the intensity of antiadenovirus responses, transgene protein expression was sufficient to induce strong and prolonged immunity in three patients. Recombinant adenovirus injected directly into the tumor is a highly efficient vector for immunizing patients against the transgene protein.

C ertain HLA-DR alleles have been associated with predisposition to human rheumatoid arthritis (RA). There is also evidence that certain HLA-DQ alleles may also be important in determining susceptibility to RA. We have previously demonstrated that mice transgenic for HLA-DQ8, a DQ allele associated with susceptibility to RA, develop severe arthritis after type II collagen immunization. To investigate the influence of polymorphic difference at the DQ loci on susceptibility to arthritis, we generated mice transgenic for HLA-DQ6, an allele associated with a nonsusceptible haplotype. The DQ6 mice were found to be resistant to collagen-induced arthritis. We also assessed the combined effect of an RA-susceptible and an RA nonassociated DQ allele by producing double-transgenic mice expressing DQ6 and DQ8 molecules, representing the more prevalent condition found in humans where heterozygosity at the DQ allele is common. The double-transgenic mice developed moderate CIA when immunized with CII when compared with the severe arthritis observed in DQ8 transgenic mice, much like RA patients bearing both susceptible and nonsusceptible HLA haplotypes. These studies support a role for HLA-DQ polymorphism in human RA.

T hese studies tested the hypothesis that delivery and/or cellular uptake of L-arginine limits macula densa nitric oxide generation and actions on tubuloglomerular feedback (TGF) during salt restriction. Maximal TGF responses were assessed from reductions in proximal stop flow pressure during loop of Henle (LH) perfusion at 40 nl/min with artificial tubular fluid containing vehicles or drugs. Orthograde LH perfusion of L-arginine (10[-3] M) reduced maximal TGF significantly in rats adapted to low salt (LS: 7.9+/-0.4-6.3+/-0.4 mmHg; P < 0.05), but not high salt (HS: 5.8+/-0.3-5.9+/-0.3; NS). The effects were stereospecific and prevented by coperfusion with NG-methyl-L-arginine. Microperfusion of L-arginine (10[-3] M) into the peritubular capillaries reduced the maximum TGF response more in nephrons of LS than HS rats (deltaTGF: LS, 32+/-6 vs. HS, 13+/-4%; P < 0.05) and restored a TGF response to luminal perfusion of NG-methyl-L-arginine in LS rats. Coperfusion of nephrons with excess L-lysine or L-homoarginine, which compete with L-arginine for system y+ transport, blocked the fall in proximal stopflow pressure produced by orthograde LH perfusion of L-arginine in LS rats. Reabsorption of [3H]arginine by the perfused loop segment was similar in LS (93+/-2%) and HS (94+/-1%) rats. Coperfusion with excess L-arginine, L-lysine, or L-homoarginine, however, reduced [3H]arginine reabsorption significantly (P < 0.05) more in HS rats than in LS rats. In conclusion, blunting of maximal TGF responses in salt-restricted rats by nephron-derived NO is limited by L-arginine availability and cellular uptake via system y+.

O ptimal T cell responsiveness requires signaling through the T cell receptor (TCR) and CD28 costimulatory receptors. Previously, we showed that T cells from autoimmune nonobese diabetic (NOD) mice display proliferative hyporesponsiveness to TCR stimulation, which may be causal to the development of insulin-dependent diabetes mellitus (IDDM). Here, we demonstrate that anti-CD28 mAb stimulation restores complete NOD T cell proliferative responsiveness by augmentation of IL-4 production. Whereas neonatal treatment of NOD mice with anti-CD28 beginning at 2 wk of age inhibits destructive insulitis and protects against IDDM by enhancement of IL-4 production by islet-infiltrating T cells, administration of anti-CD28 beginning at 5-6 wk of age does not prevent IDDM. Simultaneous anti-IL-4 treatment abrogates the preventative effect of anti-CD28 treatment. Thus, neonatal CD28 costimulation during 2-4 wk of age is required to prevent IDDM, and is mediated by the generation of a Th2 cell-enriched nondestructive environment in the pancreatic islets of treated NOD mice. Our data support the hypothesis that a CD28 signal is requisite for activation of IL-4-producing cells and protection from IDDM.

I n antigen-challenged guinea pigs there is recruitment of eosinophils into the lungs and to airway nerves, decreased function of inhibitory M2 muscarinic autoreceptors on parasympathetic nerves in the lungs, and airway hyperresponsiveness. A rabbit antibody to guinea pig eosinophil major basic protein was used to determine whether M2 muscarinic receptor dysfunction, and the subsequent hyperresponsiveness, are due to antagonism of the M2 receptor by eosinophil major basic protein. Guinea pigs were sensitized, challenged with ovalbumin and hyperresponsiveness, and M2 receptor function tested 24 h later with the muscarinic agonist pilocarpine. Antigen-challenged guinea pigs were hyperresponsive to electrical stimulation of the vagus nerves compared with controls. Likewise, loss of M2 receptor function was demonstrated since the agonist pilocarpine inhibited vagally-induced bronchoconstriction in control but not challenged animals. Pretreatment with rabbit antibody to guinea pig eosinophil major basic protein prevented hyperresponsiveness, and protected M2 receptor function in the antigen-challenged animals without inhibiting eosinophil accumulation in the lungs or around the nerves. Thus, hyperresponsiveness is a result of inhibition of neuronal M2 muscarinic receptor function by eosinophil major basic protein in antigen-challenged guinea pigs.

W e developed a new mouse model of human anti-glomerular basement membrane (GBM) disease to better characterize the genetic determinants of cell-mediated injury. While all major histocompatibility complex (MHC) haplotypes (H-2a, k, s, b, and d) immunized with alpha3 NC1 domains of type IV collagen produce anti-alpha3(IV) NC1 antibodies that cross-react with human Goodpasture [anti-GBM/anti-alpha3(IV) NC1] autoantibodies, only a few strains developed nephritis and lung hemorrhage associated with Goodpasture syndrome. Crescentic glomerulonephritis and lung hemorrhage were MHC-restricted in haplotypes H-2s, b, and d (A beta/A alpha region in H-2s) and associated with the emergence of an IL-12/Th1-like T cell phenotype. Lymphocytes or anti-alpha3(IV) NC1 antibodies from nephritogenic strains transfer disease to syngeneic recipients. However, passive transfer of isogenic alpha3(IV) NC1 antibodies into -/- T cell receptor-deficient mice failed to produce nephritis. Finally, nephritis and its associated IL-12/Th1-like T cell response attenuate in disease-susceptible mice tolerized orally to alpha3(IV) collagen before immunization. Our findings suggest collectively, as a hypothesis, that anti-GBM antibodies in mice only facilitate disease in MHC haplotypes capable of generating nephritogenic lymphocytes with special T cell repertoires.

T issue factor (TF) is a transmembrane glycoprotein that initiates the coagulation cascade. Because of the potential role of TF in mediating arterial thrombosis, we have examined its expression in human aortic and coronary artery smooth muscle cells (SMC). TF mRNA and protein were induced in SMC by a variety of growth agonists. Exposure to PDGF AA or BB for 30 min provided all of the necessary signals for induction of TF mRNA and protein. This result was consistent with nuclear runoff analyses, demonstrating that PDGF-induced TF transcription occurred within 30 min. A newly developed assay involving binding of digoxigenin-labeled FVIIa (DigVIIa) and digoxigenin-labeled Factor X (DigX) was used to localize cellular TF. By light and confocal microscopy, prominent TF staining was seen in the perinuclear cytoplasm beginning 2 h after agonist treatment and persisting for 10-12 h. Surface TF activity, measured on SMC monolayers under flow conditions, increased transiently, peaking 4-6 h after agonist stimulation and returning to baseline within 16 h. Peak surface TF activity was only approximately 20% of total TF activity measured in cell lysates. Surface TF-blocking experiments demonstrated that the remaining TF was found as encrypted surface TF, and also in an intracellular pool. The relatively short-lived surface expression of TF may be critical for limiting the thrombotic potential of intact SMC exposed to growth factor stimulation. In contrast, the encrypted surface and intracellular pools may provide a rich source of TF under conditions associated with SMC damage, such as during atherosclerotic plaque rupture or balloon arterial injury.

A mphiregulin (AR) is a heparin-binding, heparin-inhibited member of the epidermal growth factor (EGF) family and an autocrine growth factor for human keratinocytes. Previous studies have shown that AR expression is increased in psoriatic epidermis. To test the hypothesis that aberrant AR expression is central to the development of psoriatic lesions, we constructed a transgene (K14-ARGE) encoding a human keratin 14 promoter-driven AR gene. Our results indicate that transgene integration and subsequent expression of AR in basal keratinocytes correlated with a psoriasis-like skin phenotype. Afflicted mice demonstrated shortened life spans, prominent scaling and erythematous skin with alopecia, and occasional papillomatous epidermal growths. Histologic examination revealed extensive areas of marked hyperkeratosis with focal parakeratosis, acanthosis, dermal and epidermal lymphocytic and neutrophilic infiltration, and dilated blood vessels within the papillary dermis. Our results reveal that AR exerts activity in the skin that is distinct from that of transgenic transforming growth factor-alpha or other cytokines, and induces skin pathology with striking similarities to psoriasis. Our observations also link the keratinocyte EGF receptor-ligand system to psoriatic inflammation, and suggest that aberrant expression of AR in the epidermis may represent a critical step in the development or propagation of psoriatic lesions.

S omatostatin-28 (S-28), secreted into the circulation from enterocytes after food, and S-14, released mainly from gastric and pancreatic D cells and enteric neurons, inhibit peripheral cellular functions. We hypothesized that S-28 is a humoral regulator of pancreatic B cell function during nutrient absorption. Consistent with this postulate, we observed in baboons a two to threefold increase in portal and peripheral levels of S-28 after meals, with minimal changes in S-14. We attempted to demonstrate a hormonal effect of these peptides by measuring their concentrations before and after infusing a somatostatin-specific monoclonal antibody (mAb) into baboons and comparing glucose, insulin, and glucagon-like peptide-1 levels before and for 4 h after intragastric nutrients during a control study and on 2 d after mAb administration (days 1 and 2). Basal growth hormone (GH) and glucagon levels and parameters of insulin and glucose kinetics were also measured. During immunoneutralization, we found that (a) postprandial insulin levels were elevated on days 1 and 2; (b) GH levels rose immediately and were sustained for 28 h, while glucagon fell; (c) basal insulin levels were unchanged on day 1 but were increased two to threefold on day 2, coincident with decreased insulin sensitivity; and (d) plasma glucose concentrations were similar to control values. We attribute the eventual rise in fasting levels of insulin to its enhanced secretion in compensation for the heightened insulin resistance from increased GH action. Based on the elevated postmeal insulin levels after mAb administration, we conclude that S-28 participates in the enteroinsular axis as a decretin to regulate postprandial insulin secretion.

M egalin/gp330 is an endocytic receptor that internalizes multiple ligands including apolipoproteins E (apo E) and B100 (apo B). Megalin is the main antigenic target in passive Heymann nephritis (pHN), where it binds circulating autoantibodies leading to the formation of subepithelial immune deposits (ID)-the hallmark of pHN. Apo E and apo B were found recently to accumulate within these IDs, and evidence was provided that their lipids may undergo peroxidation, causing glomerular basement membrane damage and proteinuria. Here we investigated if ID-forming antimegalin IgG can inhibit the binding and internalization of apo E-betaVLDL (very low density lipoprotein) by megalin, and lead to their accumulation within IDs. By immunoelectron microscopy, apo E and apo B were detected in clathrin-coated pits and multivesicular bodies of podocytes in control rats, suggesting that the uptake of lipoproteins is a constitutive function of the glomerular epithelium. When pHN was induced by intravenous injection of antimegalin IgG, apo E and apo B were found within IDs by immunofluorescence and immunoelectron microscopy. Bound antibodies eluted from glomeruli of rats with pHN were found to inhibit the binding and internalization of apo E-enriched betaVLDL by megalin. These results indicate that pHN-inducing antimegalin IgG is capable of interfering with the uptake of lipoproteins by megalin in vivo during the formation of IDs.

A ngiotensin II exerts a mitogenic effect in several in vitro models, but a direct effect on erythroid progenitors has not been documented. Angiotensin-converting enzyme inhibitors and losartan, an angiotensin II type 1 receptor (AT1) antagonist, ameliorate posttransplant erythrocytosis, without altering serum erythropoietin levels. We studied erythroid differentiation and the effect of angiotensin II on proliferation of erythroid progenitors by culturing CD34+ hematopoietic progenitor cells in liquid serum-free medium favoring growth of erythroid precursors. Aliquots of cells were collected every third day, and were used for RNA preparation. AT1 mRNA was detected after 6 d. In these same samples, erythroid-specific mRNA (erythropoietin receptor) was also detected. AT1 protein was detected in 7-d-old burst-forming units-erythroid colonies by Western blotting. The CD34+ cell liquid cultures were used to incubate erythroid precursors with angiotensin II from days 6-9. After incubation, cells were transferred to semisolid medium and cultured with erythropoietin. Angiotensin II increased proliferation of early erythroid progenitors, defined as increased numbers of burst-forming units-erythroid colonies. Losartan completely abolished this stimulatory effect of angiotensin II. Moreover, we observed increased numbers of erythroid progenitors in the peripheral blood of posttransplant erythrocytosis patients. Thus, activation of AT1 with angiotensin II enhances erythropoietin-stimulated erythroid proliferation in vitro. A putative defect in the angiotensin II/AT1 pathway may contribute to the pathogenesis of posttransplant erythrocytosis.

U sing quantitative RT-PCR in RNA from right ventricular (RV) endomyocardial biopsies from intact nonfailing hearts, and subjects with moderate RV failure from primary pulmonary hypertension (PPH) or idiopathic dilated cardiomyopathy (IDC), we measured expression of genes involved in regulation of contractility or hypertrophy. Gene expression was also assessed in LV (left ventricular) and RV free wall and RV endomyocardium of hearts from end-stage IDC subjects undergoing heart transplantation or from nonfailing donors. In intact failing hearts, downregulation of beta1-receptor mRNA and protein, upregulation of atrial natriuretic peptide mRNA expression, and increased myocyte diameter indicated similar degrees of failure and hypertrophy in the IDC and PPH phenotypes. The only molecular phenotypic difference between PPH and IDC RVs was upregulation of beta2-receptor gene expression in PPH but not IDC. The major new findings were that (a) both nonfailing intact and explanted human ventricular myocardium expressed substantial amounts of alpha-myosin heavy chain mRNA (alpha-MHC, 23-34% of total), and (b) in heart failure alpha-MHC was downregulated (by 67-84%) and beta-MHC gene expression was upregulated. We conclude that at the mRNA level nonfailing human heart expresses substantial alpha-MHC. In myocardial failure this alteration in gene expression of MHC isoforms, if translated into protein expression, would decrease myosin ATPase enzyme velocity and slow speed of contraction.

B y studying changes in the clonal composition of HIV-1 populations during the first weeks of zidovudine (ZDV) treatment before the development of ZDV resistance-conferring mutations, we demonstrated previously a selective inhibition of nonsyncytium-inducing (NSI) HIV-1, even when present as coexisting population in individuals also harboring syncytium-inducing (SI) HIV-1. In this study, we observed the opposite in individuals receiving didanosine (ddI) treatment. In these individuals (n = 7) a median -0.98 log change (range -1.55-0.08) in infectious cellular SI load was observed, whereas the coexisting NSI load was only minimally affected (median -0.15 log, range -1.27-0.50; P = 0.03). The virus phenotype-dependent treatment responses were independent of the clonal composition of HIV-1 populations at baseline. Individuals treated with a combination of ZDV and ddI revealed an equal decline of both NSI and SI infectious cellular load (n = 4; NSI: median -1.55 log, range -2.19 to -1.45; SI: median -1.47 log, range -1.81 to -0.86; P = 0.56). To test the hypothesis that the previously reported optimal activation of ZDV and ddI in activated and resting T cells, respectively, in combination with the differential T cell tropism of NSI and SI HIV-1 is the basis for the observed virus phenotype specific efficacy of nucleoside analogs, we studied the effect of treatment with a protease inhibitor that does not require intracellular activation. Individuals receiving ritonavir (n = 4) indeed showed equal declines in NSI and SI infectious cellular load (NSI: median -2.37 log, range -2.59 to -2.16; SI: median -2.82 log, range -3.14 to -2.50; P = 0.25). Our data suggest HIV-1 phenotype as an additional parameter in the design of optimal treatment regimens.

A lzheimer's disease is a neurodegenerative disorder characterized by the extracellular deposition in the brain of aggregated beta-amyloid peptide, presumed to play a pathogenic role, and by preferential loss of neurons that express the 75-kD neurotrophin receptor (p75NTR). Using rat cortical neurons and NIH-3T3 cell line engineered to stably express p75NTR, we find that the beta-amyloid peptide specifically binds the p75NTR. Furthermore, 3T3 cells expressing p75NTR, but not wild-type control cells lacking the receptor, undergo apoptosis in the presence of aggregated beta-amyloid. Normal neural crest-derived melanocytes that express physiologic levels of p75NTR undergo apoptosis in the presence of aggregated beta-amyloid, but not in the presence of control peptide synthesized in reverse. These data imply that neuronal death in Alzheimer's disease is mediated, at least in part, by the interaction of beta-amyloid with p75NTR, and suggest new targets for therapeutic intervention.

C ryptorchidism results in impaired fertility. Reduced numbers of testicular germ cells can be shown histologically during the first years of life. The process causing germ cell loss in cryptorchid prepubertal boys is unknown, but it could be the result of a form of programmed cell death known as apoptosis. 25 adult men with a history of surgically treated cryptorchidism were studied, 15 of whom had received an unsuccessful human chorionic gonadotropin (hCG) therapy before orchidopexy. Apoptotic DNA fragmentation was assayed in testis biopsies taken during orchidopexy by end-labeling, both in extracted DNA and histochemically in situ. Only a few scattered apoptotic spermatogonias were seen by end-labeling of biopsies from patients not treated with hCG, whereas more extensive labeling of spermatogonia was seen after hCG treatment. As estimated by gel electrophoresis, the amount of low molecular weight DNA was 4.3-fold higher in the hCG-treated group when compared with the level in scrotal testis of non-hCG-treated patients (P < 0.001). About 20 yr after the biopsy, the low molecular weight DNA fragmentation correlated negatively with the testis volume (r = -0.84; P < 0.001) and positively with serum FSH levels (r = 0.73; P < 0.001). Findings in the semen analysis were similar between the groups. Apoptotic loss of spermatogonia after hCG treatment of cryptorchidism warrants reevaluation of the safety of this treatment.

A ctivity of voltage-gated K+ channels (KV) in pulmonary arterial smooth muscle cells (PASMC) is pivotal in controlling membrane potential, cytoplasmic free Ca2+ concentration ([Ca2+]cyt, and pulmonary vasomotor tone. Acute hypoxia selectively inhibits KV channels, depolarizes PASMC, raises [Ca2+]cyt, and causes pulmonary vasoconstriction and vascular remodeling. Prolonged hypoxia (24-60 h) decreased significantly the mRNA levels of KV channel alpha subunits, KV1.2 and KV1.5. Consistently, the protein levels of KV1.2 and KV1.5 were also decreased significantly by hypoxia (48-72 h). Nevertheless, hypoxia affected negligibly the mRNA levels of KV channel beta subunits (KVbeta1, KVbeta2, and KVbeta3). The native K+ channels are composed of pore-forming alpha and auxiliary beta subunits. Assembly of KV beta subunits with alpha subunits confers rapid inactivation on the slowly or non-inactivating delayed rectifier KV channels. KV beta subunits also function as an open-channel blocker of KV channels. Thus, the diminished transcription and expression of KV alpha subunits may reduce the number of KV channels and decrease KC currents. Unchanged transcription of KV beta subunits may increase the fraction of the KV channel alpha subunits that are associated with beta subunits and further reduce the total KV currents. These data demonstrate a novel mechanism by which chronic hypoxia may cause pulmonary vasoconstriction and hypertension.

T he role of splanchnic glucose uptake (SGU) after oral glucose administration as a potential factor contributing to postprandial hyperglycemia in non-insulin-dependent diabetes mellitus (NIDDM) has not been established conclusively. Therefore, we investigated SGU in six patients with NIDDM and six weight-matched control subjects by means of the hepatic vein catheterization (HVC) technique. In a second part, we examined the applicability of the recently developed OG-CLAMP technique in NIDDM by comparing SGU and first-pass SGU during HVC with SGU during the OG-CLAMP experiment. The OG-CLAMP method combines a euglycemic, hyperinsulinemic clamp and an oral glucose tolerance test (75 g) during steady state glucose infusion (GINF). During HVC, SGU equals the splanchnic fractional extraction times the total (oral and arterial) glucose load presented to the liver. For OG-CLAMP, SGU was calculated as first-pass SGU by subtracting the integrated decrease in GINF over 180 min from 75 g. Cumulative splanchnic glucose output after oral glucose correlated significantly between both methods and was increased significantly in NIDDM patients (73.1+/-5.1 g for HVC, 76.5+/-5.5 for OG-CLAMP) compared with nondiabetic patients (46.7+/-4.4 g for HVC, 57.5+/-1.9 for OG-CLAMP). Thus, in NIDDM patients, SGU (7.4+/-2.1 vs. 37.8+/-5.9% in nondiabetic patients, P < 0.001) and first-pass SGU (4.7+/-1.7 vs. 26.5+/-5.1% in nondiabetic patients, P < 0.01) were decreased significantly during HVC, as was SGU during OG-CLAMP (3.9+/-1.7 vs. 23.4+/-2.5% in nondiabetic patients, P < 0.0001). SGU measured during OG-CLAMP correlated significantly with SGU (r = 0.87, P < 0.05 for NIDDM patients; r = 0.94, P < 0.01 for nondiabetic patients) and first-pass SGU (r = 0.87, P < 0.05 for NIDDM patients; r = 0.84, P < 0.05 for nondiabetic patients) during HVC. In conclusion, (a) SGU after oral glucose administration is decreased in NIDDM as measured by both methods, and (b) SGU during the OG-CLAMP is well-correlated to SGU and first-pass SGU during HVC in NIDDM. The decrease in SGU in NIDDM might contribute to postprandial hyperglycemia in diabetic subjects.

T wo isoforms of myosin heavy chain (MyHC), alpha and beta, exist in the mammalian ventricular myocardium, and their relative expression is correlated with the contractile velocity of cardiac muscle. Several pathologic stimuli can cause a shift in the MyHC composition of the rodent ventricle from alpha- to beta-MyHC. Given the potential physiological consequences of cardiac MyHC isoform shifts, we determined MyHC gene expression in human heart failure where cardiac contractility is impaired significantly. In this study, we quantitated the relative amounts of alpha- and beta-MyHC mRNA in the left ventricular free walls (LVs) of 14 heart donor candidates with no history of cardiovascular disease or structural cardiovascular abnormalities. This group consisted of seven patients with nonfailing (NF) hearts and seven patients with hearts that exhibited donor heart dysfunction (DHD). These were compared with 19 patients undergoing cardiac transplantation for chronic end-stage heart failure (F). The relative amounts of alpha-MyHC mRNA to total (i.e., alpha + beta) MyHC mRNA in the NF- and DHD-LVs were surprisingly high compared with previous reports (33.3+/-18.9 and 35.4+/-16.5%, respectively), and were significantly higher than those in the F-LVs, regardless of the cause of heart failure (2.2+/-3.5%, P < 0.0001). There was no significant difference in the ratios in NF- and DHD-LVs. Our results demonstrate that a considerable amount of alpha-MyHC mRNA is expressed in the normal heart, and is decreased significantly in chronic end-stage heart failure. If protein and enzymatic activity correlate with mRNA expression, this molecular alteration may be sufficient to explain systolic dysfunction in F-LVs, and therapeutics oriented towards increasing alpha-MyHC gene expression may be feasible.

N eonatal animals were not considered as suitable vaccine recipients either because of immune immaturity or because passively delivered antibody interferes with immune induction. In this report, we evaluated the response of neonatal mice to immunization with naked DNA encoding a herpes simplex virus (HSV) protein, and determined if maternally derived HSV antibody interfered with immunogenicity. Our results show that neonatal mice develop effective humoral and T cell responses after immunization with either DNA or inactivated vaccines. The nature of the responses to HSV immunization, however, was more Th2-like in neonates than in adults. Whereas neonatal mice from HSV-naive mothers responded well to both DNA and inactivated vaccines, only DNA immunization induced effective immunity in neonates born to immune mothers. Our results indicate that DNA vaccines might provide a useful means of immunizing young animals that still possess high levels of potentially interfering maternal antibody.

T his study was performed to test the hypothesis that cytotoxic T lymphocyte (CTL) selection of hepatitis C virus (HCV) escape variants plays a role in HCV persistence. The peripheral blood CTL responsiveness of patients with well-established chronic hepatitis C to a panel of 10 prototype HCV peptides (genotype 1a) was compared with the corresponding sequences encoded by the infecting viruses in each patient. Variant viral peptide sequences were threefold more frequent in the presence of a CTL response than in its absence, and CTL responses were detected nearly twice as often in association with variant rather than with prototype viral peptide sequences. Furthermore, over half of the patients were infected with potential CTL escape variants that contained nonimmunogenic and noncross-reactive variant peptides many of which displayed reduced HLA-binding affinity. Surprisingly, follow up analysis over a period of up to 46 mo revealed that, in contrast to the relatively high frequency of escape variants initially observed, the subsequent emergence rate of CTL escape variants was very low. Interestingly, the one escape variant that was detected proved to be a CTL antagonist. Collectively, these observations suggest that CTL selection of epitope variants may have occurred in these patients before their entrance into the study and that it may have played a role in HCV persistence. The low apparent rate of ongoing CTL selection in chronically infected patients, however, suggests that if CTL escape occurs during HCV infection it is probably an early event.

P reviously, we have shown somatostatin receptor (SSTR) subtype-specific regulation of growth hormone (GH), thyroid-stimulating hormone, and prolactin (PRL) secretion in human fetal pituitary cultures, where GH and thyroid-stimulating hormone are mediated by both SSTR2 and SSTR5, whereas SSTR2 preferentially mediates PRL secretion. We now tested SSTR subtype-selective analogues in primary human GH- and PRL-secreting pituitary adenoma cultures. Analogue affinities determined by membrane radioligand binding in cells stably expressing human SSTR forms were either SSTR2 or SSTR5-selective. Analogues preferential either for SSTR2, including octreotide, lanreotide, and novel compounds with improved affinity for SSTR2, or new SSTR5-selective compounds suppressed GH in tumor cell cultures (up to 44% of control; P < 0.0005). However, novel analogues from both groups were 30-40% more potent than octreotide and lanreotide in suppressing GH (P < 0.05). Heterologous analogue combinations containing both SSTR2- and SSTR5-selective compounds were more potent in decreasing GH than analogues used alone (P < 0.05), or than combinations of compounds specific for the same receptor subtype (P < 0.005). In contrast, SSTR2-selective analogues did not suppress PRL release from six cultured prolactinomas studied. However, new SSTR5-selective analogues suppressed in vitro PRL secretion (30-40%; P < 0.05) in four of six prolactinomas. These results suggest that both SSTR2 and SSTR5 are involved in GH regulation in somatotroph adenoma cells, whereas SSTR5 exclusively regulates PRL secretion from prolactinoma cells. Thus, somatostatin analogues with improved selective binding affinity for these receptor subtypes may be effective in the treatment of either GH- or PRL-secreting adenomas.

G lanzmann thrombasthenia is an inherited bleeding disorder characterized by absence or dysfunction of the platelet integrin alpha(IIb)beta3. Patient RM is a thrombasthenic variant whose platelets fail to aggregate in response to physiological agonists, despite the fact that they express abundant levels of alpha(IIb)beta3 on their surface. Binding of soluble fibrinogen or fibrinogen mimetic antibodies to RM platelets did not occur, except in the presence of ligand-induced binding site (LIBS) antibodies that transformed the RM integrin complex into an active conformation from outside the cell. Sequence analysis of PCR-amplified genomic DNA and platelet mRNA revealed a C2268T nucleotide substitution in the gene encoding the integrin beta3 subunit that resulted in an Arg724Ter mutation, producing a truncated protein containing only the first eight of the 47 amino acids normally present in the cytoplasmic domain. Functional analysis of both RM platelets and CHO cells stably expressing this truncated integrin revealed that the alpha(IIb)beta3Arg724Ter complex is able to mediate binding to immobilized fibrinogen, though downstream events, including cytoskeletally-mediated cell spreading and tyrosine phosphorylation of focal adhesion kinase, pp125FAK, fail to occur. These studies establish the importance of the membrane-distal portion of the integrin beta3 cytoplasmic domain in bidirectional transmembrane signaling in human platelets, and the role of integrin signaling in maintaining normal hemostasis in vivo.

C D40 ligand (CD40-L), a member of the tumor necrosis family of transmembrane glycoproteins, is rapidly and transiently expressed on the surface of recently activated CD4+ T cells. Interactions between CD40-L and CD40 induce B cell immunoglobulin production as well as monocyte activation and dendritic cell differentiation. Since these features characterize rheumatoid arthritis (RA), the expression and function of CD40-L in RA was examined. Freshly isolated RA peripheral blood (PB) and synovial fluid (SF) T cells expressed CD40-L mRNA as well as low level cell surface CD40-L. An additional subset of CD4+ RA SF T cells upregulated cell surface CD40-L expression within 15 min of in vitro activation even in the presence of cycloheximide, but soluble CD40-L was not found in SF. CD40-L expressed by RA T cells was functional, since RA PB and SF T cells but not normal PB T cells stimulated CD40-L-dependent B cell immunoglobulin production and dendritic cell IL-12 expression in the absence of prolonged in vitro T cell activation. In view of the diverse proinflammatory effects of CD40-L, this molecule is likely to play a central role in the perpetuation of rheumatoid synovitis. Of importance, blockade of CD40-L may prove highly effective as a disease modifying therapy for RA.