Issue published August 1, 1997

T he chemokines are a homologous serum protein family characterized by their ability to induce activation of integrin adhesion molecules and leukocyte migration. Chemokines interact with their receptors, which are composed of a single-chain, seven-helix, membrane-spanning protein coupled to G proteins. Two CC chemokine receptors, CCR3 and CCR5, as well as the CXCR4 chemokine receptor, have been shown necessary for infection by several HIV-1 virus isolates. We studied the effect of the chemokine monocyte chemoattractant protein 1 (MCP-1) and of a panel of MCP-1 receptor (CCR2)-specific monoclonal antibodies (mAb) on the suppression of HIV-1 replication in peripheral blood mononuclear cells. We have compelling evidence that MCP-1 has potent HIV-1 suppressive activity when HIV-1-infected peripheral blood lymphocytes are used as target cells. Furthermore, mAb specific for the MCP-1R CCR2 which recognize the third extracellular CCR2 domain inhibit all MCP-1 activity and also block MCP-1 suppressive activity. Finally, a set of mAb specific for the CCR2 amino-terminal domain, one of which mimics MCP-1 activity, has a potent suppressive effect on HIV-1 replication in M- and T-tropic HIV-1 viral isolates. We conjecture a role for CCR2 as a coreceptor for HIV-1 infection and map the HIV-1 binding site to the amino-terminal part of this receptor. This concurs with results showing that the CCR5 amino terminus is relevant in HIV-1 infection, although chimeric fusion of various extracellular domains shows that other domains are also implicated. We discuss the importance of CCR2 structure relative to its coreceptor role and the role of anti-CCR2 receptor antibodies in the prevention of HIV-1 infection.

T he present study was designed to determine if highly conserved hepatitis B virus (HBV)-derived peptides that bind multiple HLA class I alleles with high affinity are recognized as cytotoxic T lymphocyte (CTL) epitopes in acutely infected patients. Peripheral blood mononuclear cells from 67 patients with acute hepatitis B, and 12 patients convalescent from acute hepatitis B, were stimulated with three panels of peptides, each of which bind with high affinity to several class I alleles from the HLA-A2-, HLA-A3-, or HLA-B7-supertypes. In these patients, 8 of the 19 peptides tested were found to represent CTL epitopes recognized by two or more alleles in each supertype. Two sets of nested peptides were recognized in the context of alleles with completely unrelated peptide binding specificities. Finally, promiscuous recognition by the same CTL of a given peptide presented by target cells expressing different A2 subtypes was also commonly observed. In conclusion, several HBV-specific CTL epitopes, recognized by acutely infected or convalescent patients in the context of a wide range of HLA alleles have been identified. These results demonstrate the functional relevance of the supertype grouping of HLA class I molecules in a human viral disease setting. Furthermore, they represent a significant advance in the development of a totally synthetic vaccine to terminate chronic HBV infection and support the feasibility of a systematic approach to development of similar vaccines for prevention and treatment of other chronic viral infections.

A cDNA (1.6 kb) encoding a platelet protein receptor that binds type I collagen has been isolated from a human bone marrow cDNA library by using a degenerate oligonucleotide probe derived from the amino acid sequence of a CNBr fragment of the purified receptor. Computer search revealed that this cDNA represents the coding sequence of a unique protein. Using the prokaryotic expression system pKK 223-3-65 cDNA, a 54-kD recombinant protein was obtained and purified to apparent homogeneity. In an eukaryotic expression vector (pcDNA3-65 cDNA), a 65-kD protein was identified that was recognized by monoclonal anti-65 kD antibody (anti-65m). The recombinant protein binds to type I, but not to type III collagen by affinity column chromatography. The binding of the recombinant protein to type I collagen-coated Petri dishes is inhibited by anti-65m in a dose-dependent manner. The pcDNA3-65 cDNA-transfected nonadherent T cells express the protein, allowing them to attach to a type I collagen matrix, and are inhibited by anti-65m in a dose-dependent manner. Like the receptor protein purified from platelet membranes, the recombinant protein inhibits type I collagen-induced platelet aggregation and the adhesion of [14C]serotonin-labeled platelets to type I collagen in a dose-dependent manner. The recombinant protein neither binds to type III collagen-coated Petri dishes nor inhibits type III collagen and ADP-induced platelet aggregation, indicating specificity for type I collagen.

C -Reactive protein (CRP), the classic acute-phase reactant in humans, diminishes accumulation of neutrophils at inflammatory sites. To evaluate the underlying mechanisms, we have studied whether CRP and peptides derived from CRP could affect the first step of neutrophil extravasation, the L-selectin-mediated interaction of neutrophils with endothelial cells. CRP markedly attenuated attachment of human neutrophils to cultured LPS-activated human coronary artery and pulmonary microvascular endothelial cells with apparent IC50 values of 20 and 22 microg/ml, respectively. At similar concentrations, CRP rapidly downregulated the expression of L-selectin on the neutrophil surface, whereas it failed to affect expression of CD11b and CD45 or to induce granule enzyme release. The loss of L-selectin was due to cleavage and shedding of the molecule from the cell surface, as quantitated by the soluble form of L-selectin in cell-free supernatants. The effects of CRP could be prevented by an anti-CRP antiserum and by KD-IX-73-4, which inhibits shedding of L-selectin. Inhibition of adhesion with CRP was additive with function-blocking anti-E-selectin and anti-CD18 antibodies, but was not additive with anti-L-selectin antibody. Neutrophil attachment and L-selectin expression were also diminished by CRP peptides 174-185 and 201-206, but not peptide 77-82, albeit these peptides showed a weaker inhibitory effect than the parent protein. These studies indicate a specific activation-independent action of CRP and CRP peptides 174-185 and 201-206 on expression of L-selectin, and suggest that by attenuating neutrophil adhesion to the endothelium and consequently neutrophil traffic into tissues, native CRP and peptides 174-185 and 201-206 may be major mechanisms to attenuate or limit the inflammatory response.

I mpaired glucose tolerance (IGT) is associated with defects in both insulin secretion and action and carries a high risk for conversion to non-insulin-dependent diabetes mellitus (NIDDM). Troglitazone, an insulin sensitizing agent, reduces glucose concentrations in subjects with NIDDM and IGT but is not known to affect insulin secretion. We sought to determine the role of beta cell function in mediating improved glucose tolerance. Obese subjects with IGT received 12 wk of either 400 mg daily of troglitazone (n = 14) or placebo (n = 7) in a randomized, double-blind design. Study measures at baseline and after treatment were glucose and insulin responses to a 75-g oral glucose tolerance test, insulin sensitivity index (SI) assessed by a frequently sampled intravenous glucose tolerance test, insulin secretion rates during a graded glucose infusion, and beta cell glucose-sensing ability during an oscillatory glucose infusion. Troglitazone reduced integrated glucose and insulin responses to oral glucose by 10% (P = 0.03) and 39% (P = 0.003), respectively. SI increased from 1.3+/-0.3 to 2.6+/-0.4 x 10(-)5min-1pM-1 (P = 0.005). Average insulin secretion rates adjusted for SI over the glucose interval 5-11 mmol/liter were increased by 52% (P = 0.02), and the ability of the beta cell to entrain to an exogenous oscillatory glucose infusion, as evaluated by analysis of spectral power, was improved by 49% (P = 0.04). No significant changes in these parameters were demonstrated in the placebo group. In addition to increasing insulin sensitivity, we demonstrate that troglitazone improves the reduced beta cell response to glucose characteristic of subjects with IGT. This appears to be an important factor in the observed improvement in glucose tolerance.

O steoclasts are the primary cells responsible for bone resorption. They are exposed to high ambient concentrations of inorganic phosphate (Pi) during the process of bone resorption and they possess specific Pi-transport system(s) capable of taking up Pi released by bone resorption. By immunochemical studies and PCR, we confirmed previous studies suggesting the presence of an Na-dependent Pi transporter related to the renal tubular "NaPi" proteins in the osteoclast. Using polyclonal antibodies to NaPi-2 (the rat variant), an approximately 95-kD protein was detected, localized in discrete vesicles in unpolarized osteoclasts cultured on glass coverslips. However, in polarized osteoclasts cultured on bone, immunofluorescence studies demonstrated the protein to be localized exclusively on the basolateral membrane, where it colocalizes with an Na-H exchanger but opposite to localization of the vacuolar H-ATPase. An inhibitor of phosphatidylinositol 3-kinase, wortmannin, and an inhibitor of actin cytoskeletal organization, cytochalasin D, blocked the bone-stimulated increase in Pi uptake. Phosphonoformic acid (PFA), an inhibitor of the renal NaPi-cotransporter, reduced NaPi uptake in the osteoclast. PFA also elicited a dose-dependent inhibition of bone resorption. PFA limited ATP production in osteoclasts attached to bone particles. Our results suggest that Pi transport in the osteoclast is a process critical to the resorption of bone through provision of necessary energy substrates.

W e have hypothesized that T cell cytokines participate in the pathogenesis of graft arterial disease (GAD). This study tested the consequences of IFN-gamma deficiency on arterial and parenchymal pathology in murine cardiac allografts. Hearts from C-H-2(bm12)KhEg (bm12, H-2(bm12)) were transplanted into C57/B6 (B6, H-2(b)), wild-type, or B6 IFN-gamma-deficient (GKO) recipients after immunosuppression by treatment with anti-CD4 and anti-CD8 mAbs. In wild-type recipients, myocardial rejection peaked at 4 wk, (grade 2. 1+/-0.3 out of 4, mean+/-SEM, n = 9), and by 8-12 wk evolved coronary arteriopathy. At 12 wk, the GAD score was 1.4+/-0.3, and the parenchymal rejection grade was 1.2+/-0.3 (n = 8). In GKO recipients of bm12 allografts, myocardial rejection persisted at 12 wk (grade 2.5+/-0.3, n = 6), but no GAD developed (score: 0.0+/-0.0, n = 6, P < 0.01 vs. wild-type). Mice treated with anti-IFN-gamma mAbs showed similar results. Isografts generally showed no arterial changes. In wild-type recipients, arterial and parenchymal cells showed increased MHC class II molecules, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 compared to normal or isografted hearts. The allografts in GKO recipients showed attenuated expression of these molecules (n = 6). Thus, development of GAD, but not parenchymal rejection, requires IFN-gamma. Reduced expression of MHC antigens and leukocyte adhesion molecules may contribute to the lack of coronary arteriopathy in hearts allografted into GKO mice.

L ipoprotein(a) contributes to the development of atherosclerosis through the binding of its plasminogen-like apolipoprotein(a) component to fibrin and other plasminogen substrates. Apolipoprotein(a) contains a major lysine binding site in one of its kringle domains. Destruction of this site by mutagenesis greatly reduces the binding of apolipoprotein(a) to lysine and fibrin. Transgenic mice expressing this mutant form of apolipoprotein(a) as well as mice expressing wild-type apolipoprotein(a) have been created in an inbred mouse strain. The wild-type apolipoprotein(a) transgenic mice have a fivefold increase in the development of lipid lesions, as well as a large increase in the focal deposition of apolipoprotein(a) in the aorta, compared with the lysine binding site mutant strain and to nontransgenic littermates. The results demonstrate the key role of this lysine binding site in the pathogenic activity of apolipoprotein(a) in a murine model system.

A lpha toxin from Clostridium perfringens type A, a phospholipase C, has been implicated in many of the localized and systemic features of gas gangrene. We demonstrated that human endothelial cells synthesize two vasoactive lipids, platelet-activating factor (PAF) and prostacyclin, in response to alpha toxin treatment. The stimulated synthesis of PAF required the enzymatic activity of the toxin and subsequent protein kinase C activation. Alpha toxin-treated endothelial cells accumulated the products of the phospholipase C reaction, diacylglycerol and ceramide, and exhibited a decrease in the enzymatic precursors phosphatidylcholine and sphingomyelin. Furthermore, the temporal accumulation of PAF depended on the concentration of the toxin in the overlying medium and was blocked in the presence of a neutralizing antibody. The cultured endothelial cells also exhibited enhanced neutrophil adhesion in response to alpha toxin which was mediated through the PAF receptor and P-selectin. P-selectin expression by endothelial cells and extravascular neutrophil accumulation were also observed in tissue sections from alpha toxin-injected Sprague-Dawley rats. These endothelial cell-mediated processes are important in maintaining vascular homeostasis and, when activated in a dysregulated manner by C. perfringens alpha toxin, may contribute to localized and systemic manifestations of gas gangrene including enhanced vascular permeability, localized neutrophil accumulation, and myocardial dysfunction.

P revious studies of TGFbeta1 null (-/-) mice indicated that the epidermis was devoid of Langerhans cells (LC) and that the LC deficiency was not secondary to the inflammation that is the dominant feature of the -/- phenotype (Borkowski, T.A., J.J. Letterio, A.G. Farr, and M.C. Udey. 1996. J. Exp. Med. 184:2417-2422). Herein, we demonstrate that dendritic cells could be expanded from the bone marrow of -/- mice and littermate controls. Bone marrow from -/- mice also gave rise to LC after transfer into lethally irradiated recipients. Thus, the LC defect in TGFbeta1 null mice does not result from an absolute deficiency in bone marrow precursors, and paracrine TGFbeta1 production is sufficient for LC development. Several approaches were used to assess the suitability of -/- skin for LC localization. A survey revealed that although a number of cytokine mRNAs were expressed de novo, mRNAs encoding proinflammatory cytokines known to mobilize LC from epidermis (IL-1 and TNFalpha) were not strikingly overrepresented in -/- skin. In addition, bone marrow-derived LC populated full-thickness TGFbeta1 null skin after engraftment onto BALB/c nu/nu recipients. Finally, the skin of transgenic mice expressing a truncated loricrin promoter-driven dominant-negative TGFbeta type II receptor contained normal numbers of LC. Because TGFbeta1 signaling in these mice is disrupted only in keratinocytes and the keratinocyte hyperproliferative component of the TGFbeta1 -/- phenotype is reproduced, these results strongly suggest that the LC defect in TGFbeta1 null mice is not due to an epidermal abnormality but reflects a requirement of murine LC (or their precursors) for TGFbeta1.

M ultiple extracellular mitogens are involved in the pathogenesis of proliferative forms of glomerulonephritis (GN). In vitro studies demonstrate the pivotal role of extracellular signal-regulated kinase (ERK) in the regulation of cellular proliferation in response to extracellular mitogens. In this study, we examined whether this kinase, as a convergence point of mitogenic stimuli, is activated in proliferative GN in vivo. Two different proliferative forms of anti-glomerular basal membrane (GBM) GN in rats were induced and whole cortical tissue as well as isolated glomeruli examined using kinase activity assays and Western blot analysis. Administration of rabbit anti-rat GBM serum to rats, preimmunized with rabbit IgG, induced an accelerated crescentic anti-GBM GN. A significant increase in cortical, and more dramatically glomerular ERK activity was detected at 1, 3, and 7 d after induction of GN. Immunization of Wistar-Kyoto rats with bovine GBM also induced a crescentic anti-GBM GN with an increase of renal cortical ERK activity after 4, 6, and 8 wk. ERK is phosphorylated and activated by the MAP kinase/ERK kinase (MEK). We detected a significant increase in the expression of glomerular MEK in the accelerated form of anti-GBM GN, providing a possible mechanism of long-term activation of ERK in this disease model. In contrast to ERK, activation of stress-activated protein kinase was only detectable at early stages of proliferative GN, indicating these related kinases to serve distinct roles in the pathogenesis of GN. Our observations point to ERK as a putative mediator of the proliferative response to immune injury in GN and suggest that upregulation of MEK is involved in the long-term regulation of ERK in vivo.

B oth nitric oxide (NO) and carbon monoxide (CO) are vessel wall-derived messenger molecules that cause platelet inhibition and vasodilation by activating guanylyl cyclase in target cells. Since vascular smooth muscle cells (SMCs) are exposed to shear and tensile stresses, this study examined the effects of these hemodynamic forces on the enzymes that generate NO and CO in SMCs. Monolayers of cultured rat aortic SMCs were subjected to shear stress using a modified cone and plate viscometer, or cyclic elongational stretch using a compliant silastic culture membrane. Shear stress stimulated time-dependent increases in mRNA and protein for inducible heme oxygenase-1 (HO-1), the enzyme which forms CO as a byproduct of heme degradation. The threshold level of shear necessary to induce HO-1 expression was between 5 and 10 dynes/cm2. In contrast, shear stress did not stimulate inducible NO synthase (iNOS) expression. Cyclic stretch also induced the expression of HO-1 but not of iNOS mRNA. Exposure of vascular SMCs to shear stress stimulated the production and release of CO as demonstrated by the CO-dependent increase in intracellular cGMP levels in coincubated platelets. In addition, ADP-stimulated aggregation was inhibited in platelets exposed to sheared SMCs but not in platelets exposed to untreated control SMCs. Treatment of sheared SMCs with the HO-1 inhibitor, tin protoporphyrin-IX, blocked the antiaggregatory effect of the cells, whereas the iNOS inhibitor, methyl--arginine, had no effect. These results indicate that hemodynamic forces induce HO-1 gene expression and CO production in vascular SMCs, and that SMC-derived CO inhibits platelet aggregation. Thus, CO is a novel endogenous vessel wall-derived messenger molecule that may be selectively induced by hemodynamic forces to inhibit platelet reactivity and preserve blood fluidity at sites of vascular injury.

C holecystokinin (CCK) plays an important role in pancreatic carcinogenesis. While human CCK-A and -B receptors have been fully characterized, their relative roles in human pancreatic adenocarcinoma remain unclear. Thus, expression of CCK-A and -B receptors in normal human pancreas, pancreatic adenocarcinomas, and other human extrapancreatic tissues and malignancies was examined, using reverse transcription followed by the polymerase chain reaction (RT-PCR). mRNA isolated from 15 normal pancreas specimens, 22 pancreatic adenocarcinomas, and 58 extrapancreatic tissues and tumors was subjected to RT-PCR using primers specific for human CCK-A and -B receptors. Expression of CCK-B receptors was detected in all tissues arising from pancreas and in most extrapancreatic tissues and tumors. In contrast, CCK-A receptors exhibited a more selective pattern of expression in gall bladder, intestine, brain, ovary, spleen, and thymus. Of significance, CCK-A receptors were expressed selectively in all pancreatic adenocarcinomas, but not in any normal pancreas specimens. In situ hybridization, using receptor-specific riboprobes, localized CCK-A receptor expression to ductal cells, the presumed origin of most human pancreatic adenocarcinomas. Southern blot analysis revealed no evidence of CCK-A receptor gene amplification or rearrangement in pancreatic adenocarcinomas. Because of its selective expression, the CCK-A receptor may serve as selective biomarker for pancreatic adenocarcinoma.

T NF-alpha is a proinflammatory cytokine involved in many inflammatory conditions such as Crohn's disease, rheumatoid arthritis, cachexia, AIDS, and multiple sclerosis (MS). TNF-alpha is produced mainly by cells of the macrophage lineage, which includes microglia in the central nervous system. Here, we describe a mechanism through which TNF-alpha is generated by microglia. We show that activated human T lymphocytes induce the microglial production of TNF-alpha, and that is attenuated by a functional blocking antibody to CD49d, the alpha chain of the VLA-4 integrin on T cells. We also report that interferonbeta-1b (IFNbeta-1b), a drug that alleviates symptoms in MS, downregulates the expression of CD49d and reduces TNF-alpha production, mechanisms which can help account for its efficacy in MS.

A ntiphospholipid antibodies (aPL) have been associated with various neurological manifestations, but the underlying mechanism has not been elucidated. We assessed mice with induced experimental antiphospholipid syndrome (APS) for neurological and behavioral changes. After immunization with monoclonal human anticardiolipin antibody (H-3), female BALB/c mice developed elevated levels of circulating anti-negatively charged phospholipids (aPL), anti-beta2-glycoprotein I (abeta2GPI), and anti-endothelial cell antibodies (AECA), along with clinical manifestations of APS like thrombocytopenia and fetus resorption. APS mice were impaired neurologically and performed several reflexes less accurately compared to the controls, including placing reflex (P < 0.05), postural reflex (P < 0.05), and grip test (P = 0.05). The APS mice also exhibited hyperactive behavior in an open field, which tests spatial behavior (P < 0.03), and displayed impaired motor coordination on a rotating bar. aPL in combination with abeta2GPI and AECA is probably involved in the neurological and behavioral defects shown in mice with experimental APS.

D uchenne muscular dystrophy (DMD) is an X-linked, lethal disease caused by mutations of the dystrophin gene. No effective therapy is available, but dystrophin gene transfer to skeletal muscle has been proposed as a treatment for DMD. We have developed a strategy for efficient in vivo gene transfer of dystrophin cDNA into regenerating skeletal muscle. Retroviral producer cells, which release a vector carrying the therapeutically active dystrophin minigene, were mitotically inactivated and transplanted in adult nude/mdx mice. Transplantation of 3 x 10(6) producer cells in a single site of the tibialis anterior muscle resulted in the transduction of between 5.5 and 18% total muscle fibers. The same procedure proved also feasible in immunocompetent mdx mice under short-term pharmacological immunosuppression. Minidystrophin expression was stable for up to 6 mo and led to alpha-sarcoglycan reexpression. Muscle stem cells could be transduced in vivo using this procedure. Transduced dystrophic skeletal muscle showed evidence of active remodeling reminiscent of the genetic normalization process which takes place in female DMD carriers. Overall, these results demonstrate that retroviral-mediated dystrophin gene transfer via transplantation of producer cells is a valid approach towards the long-term goal of gene therapy of DMD.

T o investigate the cellular immune events contributing to airway hyperreactivity (AHR), we studied an in vivo mouse model induced by the hapten picryl (trinitrophenyl) chloride (PCl). Mice were immunized by cutaneous contact sensitization with PCl and airway challenged subsequently with picryl sulfonic acid (PSA) antigen (Ag). Increased airway resistance was produced late (24 h) after Ag challenge, disappeared by 48 h, and was associated with no decrease in diffusion capacity. AHR could be produced in PCl immune/ PSA challenged mice on day 7 or even, with challenge, as early as 1 d after contact sensitization, after adoptive transfer of immune cells lacking CD3(+) contact sensitivity effector T cells, or after transfer of Ag-specific lymphoid cells depleted of conventional T lymphocytes with surface determinants for CD3, CD4, CD8, TCR-beta, or TCR-delta molecules. Further experiments showed that development of AHR depended upon transfer of immune cells expressing surface membrane Thy-1 and B220 (CD45RA) determinants. We concluded that a novel population of Ag-specific lymphoid cells with a defined surface phenotype (Thy-1(+), CD3(-), CD4(-), CD8(-), TCR-alphabeta-, TCR-gammadelta-, and CD45RA+) is required in a mouse model for the development of AHR.

V asoactive GTP-binding protein-coupled receptor agonists (e.g., angiotensin II [AII] and alpha-thrombin) stimulate the production of mitogenic factors from vascular smooth muscle cells. In experiments to identify mitogens secreted from AII- or alpha-thrombin-stimulated rat aortic smooth muscle (RASM) cells, neutralizing antibodies directed against several growth factors (e.g., PDGF and basic fibroblast growth factor [basic FGF]) failed to inhibit the mitogenic activity of conditioned media samples derived from the cells. In this report, we found that polyclonal neutralizing antibodies directed against purified human placental basic FGF reduced the mitogenic activity of AII-stimulated RASM cell-conditioned media and in immunoblot experiments identified a 26-kD protein (14 kD under reducing conditions) that was distinct from basic FGF. After purification from RASM cell-conditioned medium, amino acid sequence analysis identified the protein as activin A, a member of the TGF-beta superfamily. Increased activin A expression was observed after treatment of the RASM cells with AII, alpha-thrombin, and the protein kinase C agonist PMA. In contrast, PDGF-BB or serum caused only a minor induction of this protein. Although activin A alone only weakly stimulated RASM cell DNA synthesis, it demonstrated a potent comitogenic effect in combination with either EGF or heparin-binding EGF-like growth factor in the RASM cells, increasing DNA synthesis by up to fourfold. Furthermore, in a rat carotid injury model, activin A mRNA was upregulated within 6 h after injury followed by increases in immunoreactive protein detected in the expanding neointima 7 and 14 d later. Taken together, these results indicate that activin A is a vascular smooth muscle cell-derived factor induced by vasoactive agonists that may, either alone or in combination with other vascular derived growth factors, have a role in neointimal formation after arterial injury.

D uring periods of intense activity such as phagocytosis, macrophages are thought to derive most of their energy from glucose metabolism under both aerobic and anaerobic conditions. To determine whether fatty acids released from lipoproteins by macrophage lipoprotein lipase (LPL) could substitute for glucose as a source of energy for phagocytosis, we cultured peritoneal macrophages from normal and LPL knockout (LPL-KO) mice that had been rescued from neonatal demise by expression of human LPL via the muscle creatine kinase promoter. Normal and LPL-KO macrophages were cultured in medium containing normal (5 mM) or low (1 mM) glucose, and were tested for their capacity to phagocytose IgG-opsonized sheep erythrocytes. LPL-KO macrophages maintained in 1 and 5 mM glucose phagocytosed 67 and 79% fewer IgG-opsonized erythrocytes, respectively, than macrophages from normal mice. Addition of VLDL to LPL-expressing macrophages maintained in 1 mM glucose enhanced the macrophages' phagocytosis of IgG-opsonized erythrocytes, but did not stimulate phagocytosis by LPL-KO macrophages. Inhibition of secreted LPL with a monoclonal anti-LPL antibody or with tetrahydrolipstatin blocked the ability of VLDL to enhance phagocytosis by LPL-expressing macrophages maintained in 1 mM glucose. Addition of oleic acid significantly enhanced phagocytosis by both LPL-expressing and LPL-KO macrophages maintained in 1 mM glucose. Moreover, oleic acid stimulated phagocytosis in cells cultured in non-glucose-containing medium, and increased the intracellular stores of creatine phosphate. Inhibition of oxidative phosphorylation, but not of glycolysis, blocked the capacity of oleic acid to stimulate phagocytosis. Receptor-mediated endocytosis of acetyl LDL by macrophages from LPL-expressing and LPL-KO mice was similar whether the cells were maintained in 5 or 1 mM glucose, and was not augmented by VLDL. We postulate that fatty acids derived from macrophage LPL-catalyzed hydrolysis of triglycerides and phospholipids provide energy for macrophages in areas that have limited amounts of ambient glucose, and during periods of intense metabolic activity.

R heumatoid arthritis (RA) is an autoimmune disease associated with HLA-DRbeta1 alleles which contain the QKRAA amino acid sequence in their third hypervariable region(s). The QKRAA sequence is also expressed by several human pathogens. We have shown previously that an Escherichia coli peptide encompassing QKRAA is a target of immune responses in RA patients. Here we address two questions: first, whether QKRAA may function as an "immunological cassette" with similar, RA-associated, immunogenic properties when expressed by other common human pathogens; and second, what is the influence of genetic background in the generation of these responses. We find that early RA patients have enhanced humoral and cellular immune responses to Epstein-Barr virus and Brucella ovis and Lactobacillus lactis antigens which contain the QKRAA sequence. These results suggest that the QKRAA sequence is an antigenic epitope on several different microbial proteins, and that RA patients recognize the immunological cassette on different backgrounds. ANOVA of immune responses to "shared epitope" antigens in monozygotic twin couples shows that, despite significantly elevated responses in affected individuals, a similarity between pairs is retained, thus suggesting a role played either by hereditary or shared environmental factors in the genesis or maintenance of these responses.

T umor necrosis factor-alpha (TNF) causes vasodilatation and a hyperdynamic state by activating nitric oxide (NO) synthesis. Tyrphostins, specific inhibitors of protein tyrosine kinase (PTK), block the signaling events induced by TNF and NO production. A hyperdynamic circulatory syndrome (HCS) is often observed in portal hypertension (PHT). TNF and NO seem to mediate these hemodynamic changes. The aim of this work was to study the effect of PTK inhibition on the systemic and portal hemodynamics, TNF and NO production, in cirrhotic rats with portal hypertension. Rats with liver cirrhosis induced by chronic inhalation of carbon tetrachloride were used. Animals were treated daily with tyrphostin AG 126 (alpha-cyano-(3-hydroxy-4-nitro) cinnamonitrile) or placebo for 5 d. Mean arterial pressure (MAP), heart rate (HR), and portal pressure (PP) were measured by indwelling catheters. Cardiac output (CI) and stroke volume (SV) were estimated by thermodilution, systemic vascular resistance (SVR) was calculated (MAP/CI), and portal systemic shunting (PSS) was quantitated using radioactive microspheres. Serum and mesenteric lymph node (MLN) TNF levels were measured using an immunoassay kit, and serum NOx was determined photometrically by its oxidation products. The AG 126-treated group showed a statistically significant increase in MAP and SVR, and decreases in CI, SV, MLN TNF, and serum NO oxidation products nitrite and nitrate (NOx) in comparison with the placebo-treated rats. No significant differences were noticed in HR, PP, PSS, or serum TNF. Significant correlations were observed between MAP and NOx, MAP and MLN TNF, PSS and NOx, and serum TNF and serum NOx. The HCS observed in PHT seems to be mediated, at least in part, by TNF and NO by the activation of PTKs and their signaling pathways. PTK activity inhibition ameliorates the hyperdynamic abnormalities that characterize animals with cirrhosis and PHT.

W e have taken the approach of introducing the muscle-specific myosin light chain (MLC)-GLUT4 transgene into the GLUT4-null background to assess the relative role of muscle and adipose tissue GLUT4 in the etiology of the GLUT4-null phenotype. The resulting MLC-GLUT4-null mice express GLUT4 predominantly in the fast-twitch extensor digitorum longus (EDL) muscle. GLUT4 is nearly absent in female white adipose tissue (WAT) and slow-twitch soleus muscle of both sexes of MLC-GLUT4-null mice. GLUT4 content in male MLC-GLUT4-null WAT is 20% of that in control mice. In transgenically complemented EDL muscle, 2-deoxyglucose (2-DOG) uptake was restored to normal (male) or above normal (female) levels. In contrast, 2-DOG uptake in slow-twitch soleus muscle of MLC-GLUT4-null mice was not normalized. With the normalization of glucose uptake in fast-twitch skeletal muscle, whole body insulin action was restored in MLC-GLUT4-null mice, as shown by the results of the insulin tolerance test. These results demonstrate that skeletal muscle GLUT4 is a major regulator of skeletal muscle and whole body glucose metabolism. Despite normal skeletal muscle glucose uptake and insulin action, the MLC-GLUT4-null mice exhibited decreased adipose tissue deposits, adipocyte size, and fed plasma FFA levels that are characteristic of GLUT4-null mice. Together these results indicate that the defects in skeletal muscle and whole body glucose metabolism and adipose tissue in GLUT4-null mice arise independently.

W e postulated that nitric oxide (NO)-mediated endothelial function would be improved by acute and short-term treatment with an angiotensin converting enzyme (ACE) inhibitor in patients with type I diabetes mellitus, in whom endothelial function is depressed. Nine type I diabetic patients and eight healthy subjects underwent forearm blood flow measurement using strain gauge plethysmography during intraarterial infusion of incremental doses of endothelium-dependent (acetylcholine [ACh]) and endothelium-independent (sodium nitroprusside [SNP]) vasodilators. Pretreatment ACh responses were depressed in diabetic patients relative to the normal subjects (P < 0.05). No difference between the groups was evident in response to SNP. Acute ACE inhibition (with intrabrachial enalaprilat) enhanced ACh responses in the diabetic patients (P < 0.005), with a further improvement evident after 1 mo of oral therapy with enalapril (P < 0.001) when ACh responses were normalized. ACE inhibition did not affect SNP responses. We conclude that acute administration of the ACE inhibitor, enalaprilat, enhances NO-mediated endothelial function in type I diabetic patients, with further improvement evident after 4 wk of enalapril therapy.

W e determined the effects of hyperosmolarity on lung microvascular barrier properties by means of the split-drop technique in single venular capillaries of the isolated, blood-perfused rat lung. Using isosmolar and hyperosmolar test solutions (colloid osmotic pressure = 21 cm H2O), we quantified transcapillary flux at a fixed absorptive capillary pressure, and the capillary hydraulic conductivity (Lp). Loss of barrier function was indicated in flux reversal from isosmolar absorption to hyperosmolar filtration (P < 0. 01), and by hyperosmolarity-induced Lp increase (P < 0.01). Barrier recovery after a 1-min hyperosmolar exposure was delayed > 25 min. The flux reversal was blocked by the tyrosine kinase inhibitors genistein and MDC (P < 0.01). Genistein also inhibited the Lp increase (P < 0.01). Immunoblots of hyperosmolarity-exposed, cultured rat lung microvascular endothelial cells (RLMEC) and of endothelial cells freshly harvested from lungs given hyperosmolar infusions indicated a genistein-inhibitable enhancement of protein tyrosine phosphorylation. Immunoprecipitation studies indicated tyrosine phosphorylation of the mitogen activated protein kinases (MAPK) ERK1 and ERK2 and the adaptor protein Shc in lysates of RLMEC exposed to hyperosmolar conditions. We conclude that in lung venular capillaries hyperosmolarity deteriorates barrier properties, possibly by inducing tyrosine phosphorylation of endothelial proteins.

T he migration of vascular smooth muscle cells (VSMCs) is thought to play a key role in the pathogenesis of many vascular diseases and is regulated by soluble growth factors/ chemoattractants as well as interactions with the extracellular matrix. We have studied the effects of antibodies to rat beta3 and human alphavbeta3 integrins on the migration of VSMCs. Both integrin antibodies as well as cyclic RGD peptides that bind to the vitronectin receptors alphavbeta3 and alphavbeta5 significantly inhibited PDGF-directed migration. This resulted in a reduction in the accumulation of inositol (1,4,5) trisphosphate and the activation of calcium/calmodulin-dependent protein kinase II (CamKII), an important regulatory event in VSMC migration identified previously. PDGF-directed VSMC migration in the presence of the anti-integrin antibodies and cyclic RGD peptides was restored when intracellular CamKII activity was elevated by either raising intracellular calcium levels with the ionophore, ionomycin, or infecting with a replication-defective recombinant adenovirus expressing a constitutively activated CamKII cDNA (AdCMV.CKIID3). Rescue of rat VSMCs was also observed in stably transfected cell lines expressing constitutively activated but not wild-type CamKII. These observations identify a key intermediate in the regulation of VSMC migration by outside-in signaling from the integrin alphavbeta3.

M embers of the superfamily of nuclear hormone receptors which are obligate heterodimeric partners of the retinoid X receptor may be important in epidermal development. Here, we examined the effects of activators of the receptors for vitamin D3 and retinoids, and of the peroxisome proliferator activated receptors (PPARs) and the farnesoid X-activated receptor (FXR), on the development of the fetal epidermal barrier in vitro. Skin explants from gestational day 17 rats (term is 22 d) are unstratified and lack a stratum corneum (SC). After incubation in hormone-free media for 3-4 d, a multilayered SC replete with mature lamellar membranes in the interstices and a functionally competent barrier appear. 9-cis or all-trans retinoic acid, 1,25 dihydroxyvitamin D3, or the PPARgamma ligands prostaglandin J2 or troglitazone did not affect the development of barrier function or epidermal morphology. In contrast, activators of the PPARalpha, oleic acid, linoleic acid, and clofibrate, accelerated epidermal development, resulting in mature lamellar membranes, a multilayered SC, and a competent barrier after 2 d of incubation. The FXR activators, all-trans farnesol and juvenile hormone III, also accelerated epidermal barrier development. Activities of beta-glucocerebrosidase and steroid sulfatase, enzymes previously linked to barrier maturation, also increased after treatment with PPARalpha and FXR activators. In contrast, isoprenoids, such as nerolidol, cis-farnesol, or geranylgeraniol, or metabolites in the cholesterol pathway, such as mevalonate, squalene, or 25-hydroxycholesterol, did not alter barrier development. Finally, additive effects were observed in explants incubated with clofibrate and farnesol together in suboptimal concentrations which alone did not affect barrier development. These data indicate a putative physiologic role for PPARalpha and FXR in epidermal barrier development.

P revious studies have shown that cytosolic-free Ca2+ (Caf) increases to at least low micromolar concentrations during ATP depletion of isolated kidney proximal tubules. However, peak levels could not be determined precisely with the Ca2+-sensitive fluorophore, fura-2, because of its high affinity for Ca2+. Now, we have used two low affinity Ca2+ fluorophores, mag-fura-2 (furaptra) and fura-2FF, to quantitate the full magnitude of Caf increase. Between 30 and 60 min after treatment with antimycin to deplete ATP in the presence of glycine to prevent lytic plasma membrane damage, Caf measured with mag-fura-2 exceeded 10 microM in 91% of tubules studied and 68% had increases to greater than 100 microM. Caf increases of similar magnitude that were dependent on influx of medium Ca2+ were also seen using the new low Ca2+ affinity, Mg2+-insensitive, fluorophore fura-2FF in tubules depleted of ATP by hypoxia, and these increases were reversed by reoxygenation. Total cell Ca2+ levels in antimycin-treated or hypoxic tubules did not change, suggesting that mitochondria were not buffering the increased Caf during ATP depletion. Considered in the context of the high degree of structural preservation of glycine-treated tubule cells during ATP depletion and the commonly assumed Ca2+ requirements for phospholipid hydrolysis, actin disassembly, and Ca2+-mediated structural damage, the remarkable elevations of Caf demonstrated here suggest an unexpected resistance to the deleterious effects of increased Caf during energy deprivation in the presence of glycine.

D NA single strand breakage and activation of the nuclear enzyme poly (ADP-ribose) synthetase (PARS) contribute to peroxynitrite-induced cellular injury. We investigated the role of PARS activation in the pathogenesis of endothelial dysfunction. In human umbilical vein endothelial cells (HUVEC), DNA strand breakage (alkaline unwinding assay), PARS activation (incorporation or radiolabeled NAD+ into proteins), mitochondrial respiration [conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to formazan] and apoptotic index (cytoplasmatic release of histones) were measured. Endotoxin shock was induced in rats by bacterial lipopolysaccharide. Vascular reactivity of thoracic aortic rings were measured in organ chambers. In HUVEC, peroxynitrite caused a dose-dependent suppression of mitochondrial respiration, induced DNA strand breakage and caused an activation of PARS. Pharmacological inhibition of PARS reduced the acute and delayed suppression of mitochondrial respiration when cells were exposed to intermediate, but not high doses of peroxynitrite. Similarly, protection against the intermediate, but not high doses of peroxynitrite was seen in fibroblasts from the PARS-/- mice, when compared to wild-type controls. These data suggest that PARS plays a role in peroxynitrite-induced cytotoxicity, but at very high levels of oxidant exposure, PARS-independent cytotoxic mechanisms become predominant. Peroxynitrite-induced apoptosis was not affected by PARS inhibition. Vascular rings exposed to peroxynitrite and rings taken from rats subjected to endotoxic shock exhibited reduced endothelium-dependent relaxant responses in response to acetylcholine. The development of this endothelial dysfunction was ameliorated by the PARS inhibitor 3-aminobenzamide. Activation of PARS by peroxynitrite, therefore, may be involved in the development of endothelial dysfunction in endotoxemia.

M aple syrup urine disease (MSUD) or branched-chain alpha-ketoaciduria is an autosomally inherited disorder in the catabolism of branched-chain amino acids leucine, isoleucine, and valine. The disease is characterized by severe ketoacidosis, mental retardation, and neurological impairments. MSUD can be classified into genetic subtypes according to the genes of the branched-chain alpha-ketoacid dehydrogenase (BCKD) complex which are affected in patients. We describe here four intronic deletions and an intronic nucleotide substitution in the E2 transacylase gene of type II MSUD, in which the E2 subunit of the BCKD complex is deficient. These new E2 mutations comprise an internal 3.2-kb deletion in intron 4 (causing a 17-bp insertion in mRNA), an internal 12-bp (ttaccttgttac) deletion in intron 4 (creating a 10-bp insertion), a 10-bp (catttctaG) deletion in intron 10/ exon 11 junction (leading to a 21-bp deletion), a 2-bp deletion in the exon 5/intron 5 junction (ATgt--> A-t) (resulting in the skipping of exon 5), and a G to A transition at nucleotide -7 of intron 9 (causing a 6-bp insertion). These intronic mutations were initially detected by secondary alterations in the mutant E2 mRNA, as a result of aberrant splicing. The 3.2-kb deletion in intron 4 was determined by the amplification of the entire intron from both a normal subject (11.2 kb) and a homozygous patient (8 kb) by long PCR, followed by subcloning and sequencing of regions flanking the deletion. Similar methods were used to identify and characterize the other intronic alterations. Our results depict heretofore undescribed splicing errors caused by the deletion of internal intronic segments, and provide an approach for detecting this class of novel and rare human mutation. The association of the thiamine-responsive phenotype with a subset of the type II MSUD patients studied is also discussed.

T he aim of this study was to investigate, in normal young men, whether gamma-hydroxybutyrate (GHB), a reliable stimulant of slow-wave (SW) sleep in normal subjects, would simultaneously enhance sleep related growth hormone (GH) secretion. Eight healthy young men participated each in four experiments involving bedtime oral administration of placebo, 2.5, 3.0, and 3.5 g of GHB. Polygraphic sleep recordings were performed every night, and blood samples were obtained at 15-min intervals from 2000 to 0800. GHB effects were mainly observed during the first 2 h after sleep onset. There was a doubling of GH secretion, resulting from an increase of the amplitude and the duration of the first GH pulse after sleep onset. This stimulation of GH secretion was significantly correlated to a simultaneous increase in the amount of sleep stage IV. Abrupt but transient elevations of prolactin and cortisol were also observed, but did not appear to be associated with the concomitant stimulation of SW sleep. Thyrotropin and melatonin profiles were not altered by GHB administration. These data suggest that pharmacological agents that reliably stimulate SW sleep, such as GHB, may represent a novel class of powerful GH secretagogues.