Issue published March 15, 1996

T he available techniques for directed gene manipulation in the mouse are unprecedented in any multicellular organism and make the mouse an invaluable tool for unraveling all aspects of mammalian biology. To realize fully the potential of these genetic tools requires that phenotypic analysis be efficient, rapid, and complete. Genetic chimeras and mosaics, in which mutant cells are mixed with wild-type cells, can be used to augment standard analysis of intact mutant animals and alleviate the time required and the expense involved in generating and maintaining multiple strains of mutant mice.

L ipoproteins can bind lipopolysaccharide (LPS) and decrease the LPS-stimulated production of pro-inflammatory cytokines. We investigated the effect of increased plasma concentrations of low-density-lipoproteins (LDL) on survival and cytokine production after a lethal challenge with either LPS or live Gram-negative bacteria in LDL receptor deficient mice (LDLR-/-). The LDLR-/- mice challenged with LPS had an eightfold increased LD50 when compared with the wild type controls (C57Bl/6J), while tumor necrosis factor alpha (TNFalpha) and interleukin-1 alpha (IL-1 alpha) plasma concentrations were decreased twofold. LDLR-/- mice had significantly lower and delayed mortality than control mice after infection with Klebsiella pneumoniae. No differences in the outgrowth of bacteria in the organs were present between the two groups, while circulating cytokine concentrations were decreased twofold in LDLR-/- mice. In contrast, the LPS-stimulated in vitro production of cytokines by peritoneal macrophages of LDLR-/- mice was significantly increased compared with controls. This increase was associated with enhanced specific binding of LPS to the macrophages of LDLR-/- mice. In conclusion, endogenous LDL can protect against the lethal effects of endotoxin and Gram-negative infection. At least part of this protection is achieved through decreased in vivo production of pro-inflammatory cytokines, in spite of increased cytokine production capacity.

T he marked tropism of human herpesvirus-6 (HHV-6) for natural killer (NK) cells and T lymphocytes has led us to investigate the effect of HHV-6 on cellular cytotoxicity. We describe here how HHV-6 infection of peripheral blood mononuclear cells (PBMC) leads to upregulation of their NK cell cytotoxicity. The induction of NK cell activity by HHV-6 was abrogated by monoclonal antibodies (mAbs) to IL-15 but not by mAbs to other cytokines (IFN-alpha, IFN-gamma, TNF-alpha, TNF-beta, IL-2, IL-12) suggesting that IL-15 secreted in response to viral infection was responsible for the observed effect. Furthermore, NK activation by HHV-6 was blocked with mAb to CD122, as well as by human anti-HHV-6 neutralizing antibodies. Using RT-PCR, we were able to detect IL-15 mRNA upregulation in purified monocyte and NK cell preparations. IL-15 protein synthesis was increased in response to HHV-6. Finally, addition of IL-15 to PBMC cultures was found to severely curtail HHV-6 expression. Taken together, our data suggest that enhanced NK activity in response to viral infection represent a natural anti-viral defense mechanism aimed at rapidly eliminating virus-infected cells.

H emophilia A patients producing antibodies towards FVIII are usually treated with infusions of high doses of FVIII in an attempt to "desensitize" them. To examine the mechanisms by which such desensitization operates, sequential plasma samples of two unrelated inhibitor patients were analyzed for anti-FVIII and antiidiotypic antibodies before and during infusions of high doses of FVIII. Anti-FVIII antibodies were separated from antiidiotypic antibodies by immunoaffinity chromatography before analysis. We show in the present study that the concentration of anti-FVIII antibodies did not change during a successful desensitization and that antibodies maintained their capacity to inhibit the procoagulant function of FVIII, even though the number of Bethesda units in plasma was reduced to undetectable levels. Using a competition assay with mAbs, we further show that the specificity of human antibodies did not vary significantly during therapy. Finally, we show that the treatment elicited antiidiotypic antibodies, which neutralized the inhibitory capacity of anti-FVIII antibodies. Inhibitor antibodies can therefore not be accurately evaluated in plasma, as their function appears to be neutralized by antiidiotypic antibodies. These findings could have implications for the design of new therapies for hemophilia A patients with inhibitors.

C hronic insulin therapy improves but does not restore impaired insulin-mediated muscle glucose uptake in human diabetes or muscle glucose uptake, transport, and transporter translocation in streptozocin diabetic rats. To determine whether this inability is due to inadequate insulin replacement, we studied fasted streptozocin-induced diabetic Lewis rats either untreated or after islet transplantation under the kidney capsule. Plasma glucose was increased in untreated diabetics and normalized by the islet transplantation (110 +/- 5, 452 +/- 9, and 102 +/- 3 mg/dl in controls, untreated diabetics, and transplanted diabetics, respectively). Plasma membrane and intracellular microsomal membrane vesicles were prepared from hindlimb skeletal muscle of basal and maximally insulin-stimulated rats. Islet transplantation normalized plasma membrane carrier-mediated glucose transport Vmax, plasma membrane glucose transporter content, and insulin-induced transporter translocation. There were no differences in transporter intrinsic activity (Vmax/Ro) among the three groups. Microsomal membrane GLUT4 content was reduced by 30% in untreated diabetic rats and normal in transplanted diabetics, whereas the insulin-induced changes in microsomal membrane GLUT4 content were quantitatively similar in the three groups. There were no differences in plasma membrane GLUT1 among the groups and between basal and insulin stimulated states. Microsomal membrane GLUT1 content was increased 60% in untreated diabetics and normalized by the transplantation. In conclusion, an adequate insulin delivery in the peripheral circulation, obtained by islet transplantation, fully restores the muscle glucose transport system to normal in streptozocin diabetic rats.

I n a proportion of atopic asthmatics, exposure to a relevant antigen is followed by chronic inflammation in the airways leading to altered airway responsiveness (AR). However, the mechanisms underlying the development of airway hyperresponsiveness still remain unclear. To elucidate the relationship between IgE-mediated reactions and airway hyperresponsiveness, a murine model of passive sensitization and airway challenge with ovalbumin (OVA) was developed using anti-OVA IgE and IgG antibodies from murine B cell hybridomas. Passive sensitization by intravenous injection of anti-OVA IgE resulted in immediate cutaneous hypersensitivity and, after airway challenge with OVA on two consecutive days, increased AR in BALB/c and SJL mice. Increased numbers of eosinophils were observed in bronchoalveolar lavage fluid, in cells extracted from the lungs, and in the peribronchial areas of BALB/c mice passively sensitized with IgE and challenged through the airways compared with nonsensitized mice. Eosinophil peroxidase activity was also elevated in lung tissue from these mice. Passive sensitization with anti-OVA IgG1 but not IgG2a or IgG3 was similarly associated with development of skin test reactivity and increased AR after airway challenge, accompanied by an increase in eosinophils in bronchoalveolar lavage fluid. These data suggest that IgE/IgG1-mediated reactions together with local challenge with antigen can result in allergic inflammation resulting in altered airway function.

W e tested the hypothesis that glycogen is preferentially oxidized in isolated working rat heart. This was accomplished by measuring the proportion of glycolytic flux (oxidation plus lactate production) specifically from glycogen which is metabolized to lactate, and comparing it to the same proportion determined concurrently from exogenous glucose during stimulation with epinephrine. After prelabeling of glycogen with either 14C or 3H, a dual isotope technique was used to simultaneously trace the disposition of glycogen and exogenous glucose between oxidative and non-oxidative pathways. Immediately after the addition of epinephrine (1 microM), 40-50% of flux from glucose was directed towards lactate. Glycogen, however, did not contribute to lactate, being almost entirely oxidized. Further, glycogen utilization responded promptly to the abrupt increase in contractile performance with epinephrine, during the lag in stimulation of utilization of exogenous glucose, suggesting that glycogen serves as substrate reservoir to buffer rapid increases in demand. Preferential oxidation of glycogen may serve to ensure efficient generation of ATP from a limited supply of endogenous substrate, or as a mechanism to limit lactate accumulation during rapid glycogenolysis.

D NA-dependent protein kinase (DNA-PK) is an important nuclear enzyme which consists of a catalytic subunit known as DNA-PKcs and a regulatory component identified as the Ku autoantigen. In the present study, we surveyed 312 patients in a search for this specificity. 10 sera immunoprecipitated a large polypeptide which exactly comigrated with DNA-PKcs in SDS-PAGE. Immunoblot analysis demonstrated that this polypeptide was recognizable by a rabbit antiserum specific for DNA-PKcs. Although the patient sera did not bind to biochemically purified DNA-PKcs in immunoblots or ELISA, they were able to deplete DNA-PK catalytic activity from extracts of HeLa cells in a dose-dependent manner. We conclude that these antibodies should be useful probes for studies which aim to define the role of DNA-PK in cells. Since six sera simultaneously contained antibodies to the Ku protein, these studies suggest that relatively intact forms of DNA-PK complex act as autoantigenic particles in selected patients.

H yperglycemia rapidly induces an increase in intracellular advanced glycation end products (AGEs) in bovine endothelial cells, causing an alteration in bFGF activity (Giardino, I., D. Edelstein, and M. Brownlee. 1994. J. Clin. Invest. 94:110-117). Because sugar or sugar-adduct autoxidation is critical for AGE formation in vitro, we evaluated the role of reactive oxygen species (ROS) in intracellular AGE formation, using bovine aortic endothelial cells. 30 mM glucose increased intracellular ROS formation by 250% and lipid peroxidation by 330%, while not affecting ROS in the media. In cells depleted of glutathione, intracellular AGE accumulation increased linearly with ROS generation as measured by immunoblotting and the fluorescent probe DCFH (AGE 0.258-3.531 AU* mm/5x10(4) cells, DCF 57-149 mean AU, r = .998, P < .002). Deferoxamine, alpha-tocopherol, and dimethylsulfoxide each inhibited hyperglycemia-induced formation of both ROS and AGE. To differentiate an effect of ROS generation on AGE formation from an effect of more distal oxidative processes, GM7373 endothelial cell lines were generated that stably expressed the peroxidation-suppressing proto-oncogene bcl-2. bcl-2 had no effect on hyperglycemia-induced intracellular ROS formation. In contrast, bcl-2 expression decreased both lipid peroxidation (100% at 3 h and 29% at 168 h) and AGE formation (55% at 168 h). These data show that a ROS-dependent process plays a central role in the generation of intracellular AGEs, and that inhibition of oxidant pathways prevents intracellular AGE formation.

T he autocrine/paracrine growth mechanism has been implicated in the regulation of bronchial epithelial cell proliferation. By inhibiting the expression of the transforming growth factor-alpha (TGF-alpha) gene product, vitamin A is able to suppress the proliferation of tracheobronchial epithelial cells in culture. Similar repressions in TGF-alpha mRNA levels by retinol were observed in airway explant cultures and in a cell line immortalized from normal human bronchial epithelial cells. Both the nuclear run-on transcriptional assay and the transfection study with the chimeric construct of the TGF-alpha promoter and chloramphenicol acetyltransferase reporter gene partly suggest a transcriptional downregulation mechanism of TGF-alpha gene expression by the retinol treatment; however, this inhibition at the transcriptional level cannot account for the total inhibition at the mRNA level. These results suggest that a downregulation of the expression of the TGF-alpha gene at the transcriptional and post-transcriptional levels by vitamin A may precede the essential event associated with the homeostasis of normal conducting airway epithelium.

M icrovascular endothelial cells (RFCs) cultured in two-dimensional (2D) cultures proliferate rapidly and exhibit an undifferentiated phenotype. Addition of transforming growth factor beta1 (TGFbeta1) increases fibronectin expression and inhibits proliferation. RFCs cultured in three-dimensional (3D) type I collagen gels proliferate slowly and are refractory to the anti-proliferative effects of TGF beta1. TGF beta1 promotes tube formation in 3D cultures. TGF beta1 increases fibronectin expression and urokinase plasminogen activator (uPA) activity and plasminogen activator inhibitor-1 (PAI-1) levels in 3D cultures. Since the TGF beta type I and II receptors have been reported to regulate different activities induced by TGF beta1, we compared the TGF beta receptor profiles on cells in 2D and 3D cultures. RFCs in 3D cultures exhibited a significant loss of cell surface type II receptor compared with cells in 2D cultures. The inhibitory effect of TGF beta1 on proliferation is suppressed in transfected 2D cultures expressing a truncated form of the type II receptor, while its stimulatory effect on fibronectin production is reduced in both 2D and 3D transfected cultures expressing a truncated form of the type I receptor. These data suggest that the type II receptor mediates the antiproliferative effect of TGF beta1 while the type I receptor mediates the matrix response of RFCs to TGF beta1 and demonstrate that changes in the matrix environment can modulate the surface expression of TGF beta receptors, altering the responsiveness of RFCs to TGF beta1.

C hronic renal failure (CRF) is associated with negative nitrogen balance and loss of lean body mass. To identify specific proteolytic pathways activated by CRF, protein degradation was measured in incubated epitrochlearis muscles from CRF and sham-operated, pair-fed rats. CRF stimulated muscle proteolysis, and inhibition of lysosomal and calcium-activated proteases did not eliminate this increase. When ATP production was blocked, proteolysis in CRF muscles fell to the same level as that in control muscles. Increased proteolysis was also prevented by feeding CRF rats sodium bicarbonate, suggesting that activation depends on acidification. Evidence that the ATP-dependent ubiquitin-proteasome pathway is stimulated by the acidemia of CRF includes the following findings: (a) An inhibitor of the proteasome eliminated the increase in muscle proteolysis; and (b) there was an increase in mRNAs encoding ubiquitin (324%) and proteasome subunits C3 (137%) and C9 (251%) in muscle. This response involved gene activation since transcription of mRNAs for ubiquitin and the C3 subunit were selectively increased in muscle of CRF rats. We conclude that CRF stimulates muscle proteolysis by activating the ATP-ubiquitin-proteasome-dependent pathway. The mechanism depends on acidification and increased expression of genes encoding components of the system. These responses could contribute to the loss of muscle mass associated with CRF.

T o examine the mechanisms by which endothelin (ET) regulates the Na/H antiporter isoform, NHE-3, OKP cells were stably transfected with ET(A) and ET(B) receptor cDNA. In cells overexpressing ET(B), but not ET(A) receptors, ET-1 increased Na/H antiporter activity (JNa/H). This effect was inhibited by a nonselective endothelin receptor blocker and by a selective ET(B) receptor blocker but was not inhibited by an ET(A) selective receptor blocker. In ET(B)-overexpressing cells, 10(-8) M ET-1 inhibited adenylyl cyclase, but protein kinase A inhibition and pertussis toxin pretreatment did not affect Na/H antiporter activation by ET-1. ET-1 caused a transient increase in cell [Ca2+], followed by a sustained increase. Increases in cell [Ca2+] were partially inhibited by pertussis toxin. ET-1-induced increases in J(Na/H) were 50% inhibited by clamping cell [Ca2+] low with BAPTA, and by KN62, a Ca-calmodulin kinase inhibitor. Inhibitors of protein kinase C, cyclooxygenase, lipoxygenase, and cytochrome P450 and cyclic GMP were without effect. In ET(A)-overexpressing cells, ET-1 increased cell [Ca2+] but did not increase JNa/H. In summary, binding of ET-1 to ET(B) receptors increases Na/H antiporter activity in OKP cells, an effect mediated in part by increases in cell [Ca2+] and Ca-calmodulin kinase. Increases in cell [Ca2+] are not sufficient for Na/H antiporter activation.

I n this study we tested the hypothesis that peptone in the intestine stimulates the secretion of the CCK-releasing peptide (CCK-RP) which mediates CCK secretion, and examined the enteric neural circuitry responsible for CCK-RP secretion. We used a "donor-recipient" rat intestinal perfusion model to quantify the CCK-RP secreted in response to nutrient stimulation. Infusion of concentrated intestinal perfusate collected from donor rat perfused with 5% peptone caused a 62 +/- 10% increase in protein secretion and an elevation of plasma CCK levels to 6.9 +/- 1.8 pM in the recipient rat. The stimulatory effect of the intestinal washings was abolished when the donor rats were pretreated with atropine or hexamethonium but not with guanethidine or vagotomy. Mucosal application of lidocaine but not serosal application of benzalkonium chloride which ablates the myenteric neurons in the donor rats also abolished the stimulatory action of the intestinal washings. Furthermore, treatment of the donor rats with a 5HT3 antagonist and a substance P antagonist also prevented the secretion of CCK-RP. These observations suggest that peptone in the duodenum stimulates serotonin release which activates the sensory substance P neurons in the submucous plexus. Signals are then transmitted to cholinergic interneurons and to epithelial CCK-RP containing cells via cholinergic secretomotor neurons. This enteric neural circuitry which is responsible for the secretion of CCK-RP may in turn play an important role in the postprandial release of CCK.

I nhibition of insulin receptor signaling by high glucose levels and by TNF-alpha was recently observed in different cell systems. The aim of the present study was to characterize the mechanism of TNF-alpha-induced insulin receptor inhibition and to compare the consequences of TNF-alpha- and hyperglycemia-induced insulin receptor inhibition for signal transduction downstream from the IR. TNF-alpha (0.5-10 nM) and high glucose (25 mM) showed similar rapid kinetics of inhibition (5-10 min, > 50%) of insulin receptor autophosphorylation in NIH3T3 cells overexpressing the human insulin receptor. TNF-alpha effects were completely prevented by the phosphotyrosine phosphatase (PTPase) inhibitors orthovanadate (40 microM) and phenylarsenoxide (35 microM), but they were unaffected by the protein kinase C (PKC) inhibitor H7 (0.1 mM), the phosphatidylinositol-3 kinase inhibitor wortmannin (5 microM), and the thiazolidindione troglitazone (CS045) (2 microgram/ml). In contrast, glucose effects were prevented by PKC inhibitors and CS045 but unaffected by PTPase inhibitors and wortmannin. To assess effects on downstream signaling, tyrosine phosphorylation of the following substrate proteins of the insulin receptor was determined: insulin receptor substrate-1, the coupling protein Shc, focal adhesion kinase (FAK125), and unidentified proteins of 130 kD, 60 kD. Hyperglycemia (25 mM glucose) and TNF-alpha showed analogous (> 50% inhibition) effects on tyrosine phosphorylation of insulin receptor substrate-1, Shc, p60, and p44, whereas opposite effects were observed for tyrosine phosphorylation of FAK125, which is dephosphorylated after insulin stimulation. Whereas TNF-alpha did not prevent insulin-induced dephosphorylation of FAK125, 25 mM glucose blocked this insulin effect completely. In summary, the data suggest that TNF-alpha and high glucose modulate insulin receptor-signaling through different mechanisms: (a) TNF-alpha modulates insulin receptor signals by PTPase activation, whereas glucose acts through activation of PKC. (b) Differences in modulation of the insulin receptor signaling cascade are found with TNF-alpha and high glucose: Hyperglycemia-induced insulin receptor inhibition blocks both insulin receptor-dependent tyrosine phosphorylation and dephosphorylation of insulin receptor substrate proteins. In contrast, TNF-alpha blocks only substrate phosphorylation, and it does not block insulin-induced substrate dephosphorylation. The different effects on FAK125 regulation allow the speculation that long-term cell effects related to FAK125 activity might develop in a different way in hyperglycemia- and TNF-alpha-dependent insulin resistance.

T ype I carbohydrate-deficient glycoprotein syndrome (CDGS) patients fail to add entire N-linked oligosaccharide chains to some serum glycoproteins. Here we show that four CDGS fibroblast cell lines have two related glycosylation abnormalities. First, they incorporate 3-10-fold less [3H] mannose into proteins, and, second, the size of the lipid-linked oligosaccharide precursor (LLO) is much smaller than in controls. Addition of exogenous mannose, but not glucose, to these CDGS cells corrects both the lowered [3H] mannose incorporation and the size of LLO. These corrections are not permanent, and the defects immediately reappear when mannose is removed. To explore further the basis of mannose correction, we analyzed the amount of 3H-labeled LLO intermediates. Except for dolichol-P-mannose, other precursors, including mannose, mannose-6-phosphate, mannose-1-phosphate, and GDP-mannose, all showed a 3-10-fold decrease in CDGS cells. Thus, there are no obvious lesions in the intracellular conversion of mannose into LLO, and, once inside the cell, [3H]mannose appeared to be metabolized normally. Initial velocities of [3H]mannose uptake were two- to threefold less in CDGS cells compared with controls, and this slower transport may partially explain the reduced [3H]mannose incorporation in CDGS cells. Since we previously showed that the enzymes converting glucose to mannose-6-phosphate appear to be normal, our results suggest that cells may acquire or generate mannose in other ways. Although we have not identified the primary defect in CDGS, these studies show that intracellular mannose is limited and that some patients might benefit from including mannose in their regular diets.

R ecently our laboratory has cloned both the rat canalicular and sinusoidal GSH transporters (RcGshT and RsGshT, respectively; Yi, J., S. Lu, J. Fernandez-Checa, and N. Kaplowitz. 1994. J. Clin. Invest. 93:1841-1845; and 1995. Proc. Natl. Acad. Sci. USA. 92:1495-1499). The current work characterized GSH transport and the expression of these two GSH transporters in various mammalian cell lines. The average cell GSH levels (nmol/10(6) cells) were 25, 22, 32, 13, and 13 in HepG2, HeLa, CaCo-2, MDCK, and Cos-1 cells, respectively. GSH efflux was temperature dependent and averaged 0.018, 0.018, 0.012, 0.007, and 0.019 nmol/10(6) cells/min from HepG2, HeLa, CaCo-2, MDCK, and Cos-1 cells, respectively. Dithiothreitol (DTT), which stimulates rat sinusoidal GSH efflux, stimulated GSH efflux only in HepG2 and HeLa cells which was partially reversed by subsequent cystine treatment. GSH uptake (1 mM plus 35S-GSH) was temperature dependent, linear up to 45 min, and Na+-independent with average rates of 1.12, 0.91, 0.45, and 0.45 nmol/10(6) cells/30 min for HepG2, HeLa, CaCo-2, MDCK, and Cos-1 cells, respectively. BSP-GSH (2mM), which cis-inhibits sinusoidal GSH uptake in rat liver and HepG2 cells, inhibited GSH uptake only in HeLa cells. mRNA and polypeptide of RcGshT are expressed in all cells whereas those of RsGshT are expressed only in HepG2 and HeLa cells. In conclusion, bidirectional GSH transport, mediated by the "canalicular" GSH transporter, is ubiquitous in mammalian cells. Sinusoidal GSH transporter expression is more restricted, being present in HepG2 and HeLa cells. DTT and BSP-GSH affect GSH transport only in cells expressing the sinusoidal transporter confirming their selective action on this transporter.

I n vitro, insulin transport across endothelial cells has been reported to be saturable, suggesting that the transport process is receptor mediated. In the present study, the transport of insulin across capillary endothelial cells was investigated in vivo. Euglycemic glucose clamps were performed in anesthetized dogs (n = 16) in which insulin was infused to achieve concentrations in the physiological range (1.0 mU/kg per min + 5 mU/kg priming bolus; n = 8) or pharmacologic range (18 mU/kg per min + 325 mU/kg priming bolus; n = 8). Insulin concentrations were measured in plasma and hindlimb lymph derived from interstitial fluid (ISF) surrounding muscle. Basal plasma insulin concentrations were twice the basal ISF insulin concentrations and were not different between the physiologic and pharmacologic infusion groups (plasma/ISF ratio 2.05 +/- 0.22 vs 2.05 +/- 0.23; p = 0.0003). The plasma/ISF gradient was, however, significantly reduced at steady-state pharmacologic insulin concentrations (1.37 +/- 0.25 vs 1.98 +/- 0.21; P = 0.0003). The reduced gradient is opposite to that expected if transendothelial insulin transport were saturable. Insulin transport into muscle ISF tended to increase with pharmacologic compared with physiologic changes in insulin concentration (41% increase; 1.37 +/- 0.18 10(-2) to 1.93 +/- 0.24 10(-2) min-1; P = 0.088), while at the same time insulin clearance out of the muscle ISF compartment was unaltered (2.53 +/- 0.26 10(-2) vs 2.34 +/- 0.28 10(-2) min-1; P = 0.62). Thus, the reduced plasma/ISF gradient at pharmacologic insulin was due to enhanced transendothelial insulin transport rather than changes in ISF insulin clearance. We conclude that insulin transport is not saturable in vivo and thus not receptor mediated. The increase in transport efficiency with saturating insulin is likely due to an increase in diffusionary capacity resulting from capillary dilation or recruitment.

C ystic fibrosis (CF) is a common autosomal recessive disease caused by mutations in the CF transmembrane conductance regulator gene. Recombinant adenoviruses have shown promise as vectors for transfer of CF transmembrane conductance regulator cDNA to airway epithelia and correction of the Cl- transport defect. However, because adenovirus-mediated gene transfer is transient, use of adenovirus as a vector for treatment of CF would require repeated administration. Therefore, we evaluated repeat administration of an adenovirus vector to the nasal epithelium of patients with CF with five escalating doses of up to 10(10) infectious units. There were no detectable adverse affects. All subjects were initially seropositive but developed additional humoral immune responses. The vector partially corrected the defect in airway epithelial Cl- transport in some subjects, although there was variability between subjects and there was less correction with subsequent administration, perhaps because the immune response limited gene transfer. Future work must focus on vectors with increased efficiency and with the ability to evade host defenses.

A ctivated macrophage/microglia may mediate tissue injury in a variety of CNS disorders. To examine this, transgenic mice were developed in which the expression of a macrophage/microglia activation cytokine, interleukin-3 (IL-3), was targeted to astrocytes using a murine glial fibrillary acidic protein fusion gene. Transgenic mice with low levels of IL-3 expression developed from 5 mo of age, a progressive motor disorder characterized at onset by impaired rota-rod performance. In symptomatic transgenic mice, multi-focal, plaque-like white matter lesions were present in cerebellum and brain stem. Lesions showed extensive primary demyelination and remyelination in association with the accumulation of large numbers of proliferating and activated foamy macrophage/microglial cells. Many of these cells also contained intracisternal crystalline pole-like inclusions similar to those seen in human patients with multiple sclerosis. Mast cells were also identified while lymphocytes were rarely, if at all present. Thus, chronic CNS production of low levels of IL-3 promotes the recruitment, proliferation and activation of macrophage/microglial cells in white matter regions with consequent primary demyelination and motor disease. This transgenic model exhibits many of the features of human inflammatory demyelinating diseases including multiple sclerosis and HIV leukoencephalopathy.

H uman blood monocytes adhere rapidly and for prolonged periods to activated platelets that display P-selectin, an adhesion protein that recognizes a specific ligand on leukocytes, P-selectin glycoprotein-1. We previously demonstrated that P-selectin regulates expression and secretion of cytokines by stimulated monocytes when it is presented in a purified, immobilized form or by transfected cells. Here we show that thrombin-activated platelets induce the expression and secretion of monocyte chemotactic protein-1 and IL-8 by monocytes. Enhanced monokine synthesis requires engagement of P-selectin glycoprotein-1 on the leukocyte by P-selectin on the platelet. Secretion of the chemokines is not, however, directly signaled by P-selectin; instead, tethering of the monocytes by P-selectin is required for their activation by RANTES (regulated upon activation normal T cell expressed presumed secreted), a platelet chemokine not previously known to induce immediate-early gene products in monocytes. Adhesion of monocytes to activated platelets results in nuclear translocation of p65 (RelA), a component of the NF-kappaB family of transcription factors that binds kappaB sequences in the regulatory regions of monocyte chemotactic protein-1, IL-8, and other immediate-early genes. However, expression of tissue factor, a coagulation protein that also has a kappaB sequence in the 5' regulatory region of its gene, is not induced in monocytes adherent to activated platelets. Thus, contact of monocytes with activated platelets differentially affects the expression of monocyte products. These experiments suggest that activated platelets regulate chemokine secretion by monocytes in inflammatory lesions in vivo and provide a model for the study of gene regulation in cell-cell interactions.

O xidation of LDL may contribute to atherogenesis, though the nature of the in vivo oxidant(s) remains obscure. Myeloperoxidase, the enzyme responsible for hypochlorous acid/hypochlorite (HOCl) production in vivo, is present in active form in human atherosclerotic lesions, and HOCl aggregates and transforms LDL into a high-uptake form for macrophages in vitro. Here we demonstrate HOCl-modified proteins in human lesions using an mAb raised against HOCl-modified LDL that recognizes HOCl-oxidized proteins but does not cross-react with Cu2+-, malondialdehyde-, or 4-hydroxynonenal-modified LDL. This antibody detected significantly more material in advanced atherosclerotic lesions than normal arteries, even though azide and methionine were included during sample work-up to inhibit myeloperoxidase and to scavenge HOCl. The epitope(s) recognized was predominantly cell associated and present in monocyte/macrophages, smooth muscle, and endothelial cells. The intima and cholesterol clefts stained more heavily than the center of the thickened vessels; adventitial staining was apparent in some cases. Immunostaining was also detected in a very early lesion from an accident victim, beside healthy areas that were unreactive. LDL oxidized by HOCl in vitro, but not native LDL, effectively competed with the epitopes in lesions for antibody binding. Density centrifugation of plaque homogenates and Western blot analysis showed that, in the apo B-containing lipoprotein fraction, the mAb recognized protein(s) of molecular mass greater than apo B, similar to those produced during oxidation of LDL with HOCl in vitro. Three major proteins were recognized by the anti-HOCl-modified protein antibody but not by an anti-apo B antibody in the apo B-free fraction. Together, these results demonstrate HOCl-oxidized proteins in human atherosclerotic lesions, implicating this oxidant in LDL modification in vivo.

T cells from HIV-1+ individuals have a defect in mounting an antigen specific response. HIV-1 Tat has been implicated as the causative agent of this immunosuppression. We have previously shown that HIV-1 Tat inhibits antigen specific proliferation of normal T cells in vitro by binding to the accessory molecule CD26, a dipeptidase expressed on the surface of activated T cells. We now demonstrate that the defective in vitro recall antigen response in HIV-1 infected individuals can be restored by the addition of soluble CD26, probably by serving as a decoy receptor for HIV-1 Tat. The restored response is comparable to that of an HIV-1- individual, suggesting that early in HIV infection there is a block in the memory cell response, rather than deletion of these cells.

D ecreased antigen (Ag)-specific T cell (TC) proliferation and IL-2 production are detected in all stages of HIV disease. To determine whether dendritic cell dysfunction and/or abnormal cytokine production contribute to HIV-induced immune dysregulation, we studies TC responses to recall Ags (influenza virus and tetanus toxoid) presented by Langerhans cells (LC) in six pairs of HIV-discordant identical twins, and the modulation of these responses by anti-IL-10 (alphaIL-10) mAbs and IL-12. LC from HIV+ twins induced IL-2 comparable to normal LC in cultures containing TC from uninfected twins. In contrast, IL-2 production was markedly decreased in cultures containing TC from HIV+ twins. IL-12 enhanced Ag-specific IL-2 production by TC from two patients with CD4+ counts > 600. In contrast, alphaIL-10 mAbs enhanced IL-2 production in influenza virus-stimulated cultures containing TC from two patients with CD4+ counts < 20. Thus, these findings suggest that immunologic dysfunction of dendritic cells does not contribute to impaired secondary immune responses in HIV+ individuals. Although few patients were studied, partial immune reconstitution in vitro, as demonstrated here, may help to predict those individuals who might benefit from cytokines or antibodies against cytokines as immunotherapy for HIV disease.