Issue published December 1, 1997

A utosomal dominant hypophosphatemic rickets (ADHR) is an inherited disorder of isolated renal phosphate wasting, the pathogenesis of which is unknown. We performed a genome-wide linkage study in a large kindred to determine the chromosome location of the ADHR gene. Two-point LOD scores indicate that the gene is linked to the markers D12S314 [Z(theta) = 3.15 at theta = 0.0], vWf [Z(theta) = 5.32 at theta = 0.0], and CD4 [Z(theta) = 3.53 at theta = 0.0]. Moreover, multilocus analysis indicates that the ADHR gene locus is located on chromosome 12p13 in the 18-cM interval between the flanking markers D12S100 and D12S397. These data are the first to establish a chromosomal location for the ADHR locus and to provide a framework map to further localize the gene. Such studies will permit ultimate identification of the ADHR gene and provide further insight into phosphate homeostasis.

C omplete interferon-gamma receptor 1 (IFNgammaR1) deficiency has been identified previously as a cause of fatal bacillus Calmette-Guérin (BCG) infection with lepromatoid granulomas, and of disseminated nontuberculous mycobacterial (NTM) infection in children who had not been inoculated with BCG. We report here a kindred with partial IFNgammaR1 deficiency: one child afflicted by disseminated BCG infection with tuberculoid granulomas, and a sibling, who had not been inoculated previously with BCG, with clinical tuberculosis. Both responded to antimicrobials and are currently well without prophylactic therapy. Impaired response to IFN-gamma was documented in B cells by signal transducer and activator of transcription 1 nuclear translocation, in fibroblasts by cell surface HLA class II induction, and in monocytes by cell surface CD64 induction and TNF-alpha secretion. Whereas cells from healthy children responded to even low IFN-gamma concentrations (10 IU/ml), and cells from a child with complete IFNgammaR1 deficiency did not respond to even high IFN-gamma concentrations (10,000 IU/ml), cells from the two siblings did not respond to low or intermediate concentrations, yet responded to high IFN-gamma concentrations. A homozygous missense IFNgR1 mutation was identified, and its pathogenic role was ascertained by molecular complementation. Thus, whereas complete IFNgammaR1 deficiency in previously identified kindreds caused fatal lepromatoid BCG infection and disseminated NTM infection, partial IFNgammaR1 deficiency in this kindred caused curable tuberculoid BCG infection and clinical tuberculosis.

U ncoupling protein-2 and -3 (UCP2 and UCP3) are mitochondrial proteins that show high sequence homology with the brown adipocyte-specific UCP1. UCP1 induces heat production by uncoupling respiration from ATP synthesis. UCP2 is widely expressed in human tissues, whereas UCP3 expression seems restricted to skeletal muscle, an important site of thermogenesis in humans. We have investigated the regulation of UCP2 and UCP3 gene expression in skeletal muscle and adipose tissue from lean and obese humans. UCP2 and -3 mRNA levels were not correlated with body mass index (BMI) in skeletal muscle, but a positive correlation (r = 0.55, P < 0.01, n = 22) was found between UCP2 mRNA level in adipose tissue and BMI. The effect of fasting was investigated in eight lean and six obese subjects maintained on a hypocaloric diet (1,045 kJ/d) for 5 d. Calorie restriction induced a similar 2-2.5-fold increase in UCP2 and -3 mRNA levels in lean and obese subjects. To study the effect of insulin on UCP gene expression, six lean and five obese subjects underwent a 3-h euglycemic hyperinsulinemic clamp. Insulin infusion did not modify UCP2 and -3 mRNA levels. In conclusion, the similar induction of gene expression observed during fasting in lean and obese subjects shows that there is no major alteration of UCP2 and -3 gene regulation in adipose tissue and skeletal muscle of obese subjects. The increase in UCP2 and -3 mRNA levels suggests a role for these proteins in the metabolic adaptation to fasting.

T his study explores the role of mevalonate inhibitors in the activation of NF-kbeta and the induction of inducible nitric oxide synthase (iNOS) and cytokines (TNF-alpha, IL-1beta, and IL-6) in rat primary astrocytes, microglia, and macrophages. Lovastatin and sodium phenylacetate (NaPA) were found to inhibit LPS- and cytokine-mediated production of NO and expression of iNOS in rat primary astrocytes; this inhibition was not due to depletion of end products of mevalonate pathway (e.g., cholesterol and ubiquinone). Reversal of the inhibitory effect of lovastatin on LPS-induced iNOS expression by mevalonate and farnesyl pyrophosphate and reversal of the inhibitory effect of NaPA on LPS-induced iNOS expression by farnesyl pyrophosphate, however, suggests a role of farnesylation in the LPS-mediated induction of iNOS. The inhibition of LPS-mediated induction of iNOS by FPT inhibitor II, an inhibitor of Ras farnesyl protein transferase, suggests that farnesylation of p21(ras) or other proteins regulates the induction of iNOS. Inhibition of LPS-mediated activation of NF-kbeta by lovastatin, NaPA, and FPT inhibitor II in astrocytes indicates that the observed inhibition of iNOS expression is mediated via inhibition of NF-kbeta activation. In addition to iNOS, lovastatin and NaPA also inhibited LPS-induced expression of TNF-alpha, IL-1beta, and IL-6 in rat primary astrocytes, microglia, and macrophages. This study delineates a novel role of the mevalonate pathway in controlling the expression of iNOS and different cytokines in rat astrocytes, microglia, and macrophages that may be important in developing therapeutics against cytokine- and NO-mediated neurodegenerative diseases.

T o determine whether oxidized LDL enhances atherogenesis by promoting monocyte recruitment into the vascular intima, we investigated whether LDL accumulation and oxidation precede intimal accumulation of monocytes in human fetal aortas (from spontaneous abortions and premature newborns who died within 12 h; fetal age 6.2+/-1.3 mo). For this purpose, a systematic assessment of fatty streak formation was carried out in fetal aortas from normocholesterolemic mothers (n = 22), hypercholesterolemic mothers (n = 33), and mothers who were hypercholesterolemic only during pregnancy (n = 27). Fetal plasma cholesterol levels showed a strong inverse correlation with fetal age (R = -0.88, P < 0.0001). In fetuses younger than 6 mo, fetal plasma cholesterol levels correlated with maternal ones (R = 0.86, P = 0.001), whereas in older fetuses no such correlation existed. Fetal aortas from hypercholesterolemic mothers and mothers with temporary hypercholesterolemia contained significantly more and larger lesions (758,651+/-87,449 and 451,255+/-37,448 micron2 per section, respectively; mean+/-SD) than aortas from normocholesterolemic mothers (61,862+/-9,555 micron2; P < 0.00005). Serial sections of the arch, thoracic, and abdominal aortas were immunostained for recognized markers of atherosclerosis: macrophages, apo B, and two different oxidation-specific epitopes (malondialdehyde- and 4-hydroxynonenal-lysine). Of the atherogenic sites that showed positive immunostaining for at least one of these markers, 58.6% were established lesions containing both macrophage/foam cells and oxidized LDL (OxLDL). 17.3% of all sites contained only native LDL, and 13.3% contained only OxLDL without monocyte/ macrophages. In contrast, only 4.3% of sites contained isolated monocytes in the absence of native or oxidized LDL. In addition, 6.3% of sites contained LDL and macrophages but few oxidation-specific epitopes. These results demonstrate that LDL oxidation and formation of fatty streaks occurs already during fetal development, and that both phenomena are greatly enhanced by maternal hypercholesterolemia. The fact that in very early lesions LDL and OxLDL are frequently found in the absence of monocyte/macrophages, whereas the opposite is rare, suggests that intimal LDL accumulation and oxidation contributes to monocyte recruitment in vivo.

B asal cell carcinoma (BCC) is the most common skin cancer in humans, and although metastasis rarely occurs, the tumor cells are nevertheless able to invade and destroy the surrounding tissue. Intralesional injection of IFN-alpha has been found to be highly effective in inducing BCC regression by an unknown mechanism. We show that in untreated patients, BCC cells express CD95 ligand, but not the receptor, which may allow tumor expansion by averting the attack of activated CD95 receptor-positive lymphoid effector cells. The CD95 ligand of BCC cells is functional as CD95-positive cells incubated on BCC cryosections become apoptotic and are lysed. In IFN-alpha-treated patients BCC cells express not only CD95 ligand but also CD95 receptor, whereas the peritumoral infiltrate that mainly consists of CD4+ T cells predominantly contains CD95 receptor and only few CD95 ligand-positive cells. Thus, in treated patients BCC most likely regresses by committing suicide through apoptosis induction via CD95 receptor-CD95 ligand interaction.

T ransgenic mice overexpressing a constitutively active human TGF-beta1 under control of the rat phosphoenolpyruvate carboxykinase regulatory sequences developed fibrosis of the liver, kidney, and adipose tissue, and exhibited a severe reduction in body fat. Expression of the transgene in hepatocytes resulted in increased collagen deposition, altered lobular organization, increased hepatocyte turnover, and in extreme cases, hemorrhage and thrombosis. Renal expression of the transgene was localized to the proximal tubule epithelium, and was associated with tubulointerstitial fibrosis, characterized by excessive collagen deposition and increased fibronectin and plasminogen activator inhibitor-1 immunoreactivity. Pronounced glomerulosclerosis was evident, and hydronephrosis developed with low penetrance. Expression of TGF-beta1 in white and brown adipose tissue resulted in a lipodystrophy-like syndrome. All white fat depots and brown fat pads were severely reduced in size, and exhibited prominent fibroplasia. This reduction in WAT was due to impaired adipose accretion. Introduction of the transgene into the ob/ob background suppressed the obesity characteristic of this mutation; however, transgenic mutant mice developed severe hepato- and splenomegaly. These studies strengthen the link between TGF-beta1 expression and fibrotic disease, and demonstrate the potency of TGF-beta1 in modulating mesenchymal cell differentiation in vivo.

A lthough bile acid transport by bile duct epithelial cells, or cholangiocytes, has been postulated, the details of this process remain unclear. Thus, we performed transport studies with [3H]taurocholate in confluent polarized monolayers of normal rat cholangiocytes (NRC). We observed unidirectional (i.e., apical to basolateral) Na+-dependent transcellular transport of [3H]taurocholate. Kinetic studies in purified vesicles derived from the apical domain of NRC disclosed saturable Na+-dependent uptake of [3H]taurocholate, with apparent Km and Vmax values of 209+/-45 microM and 1.23+/-0.14 nmol/mg/10 s, respectively. Reverse transcriptase PCR (RT-PCR) using degenerate primers for both the rat liver Na+-dependent taurocholate-cotransporting polypeptide and rat ileal apical Na+-dependent bile acid transporter, designated Ntcp and ASBT, respectively, revealed a 206-bp product in NRC whose sequence was identical to the ASBT. Northern blot analysis demonstrated that the size of the ASBT transcript was identical in NRC, freshly isolated cholangiocytes, and terminal ileum. In situ RT-PCR on normal rat liver showed that the message for ASBT was present only in cholangiocytes. Immunoblots using a well-characterized antibody for the ASBT demonstrated a 48-kD protein present only in apical membranes. Indirect immunohistochemistry revealed apical localization of ASBT in cholangiocytes in normal rat liver. The data provide direct evidence that conjugated bile acids are taken up at the apical domain of cholangiocytes via the ASBT, and are consistent with the notion that cholangiocyte physiology may be directly influenced by bile acids.

I rreversible exit from the cell cycle precludes the ability of cardiac muscle cells to increase cell number after infarction. Using adenoviral E1A, we previously demonstrated dual pocket protein- and p300-dependent pathways in neonatal rat cardiac myocytes, and have proven that E2F-1, which occupies the Rb pocket, suffices for these actions of E1A. By contrast, the susceptibility of adult ventricular cells to viral delivery of exogenous cell cycle regulators has not been tested, in vitro or in vivo. In cultured adult ventricular myocytes, adenoviral gene transfer of E2F-1 induced expression of proliferating cell nuclear antigen, cyclin-dependent protein kinase 4, cell division cycle 2 kinase, DNA synthesis, and apoptosis. In vivo, adenoviral delivery of E2F-1 by direct injection into myocardium induced DNA synthesis, shown by 5'-bromodeoxyuridine incorporation, and accumulation in G2/M, by image analysis of Feulgen-stained nuclei. In p53(-)/- mice, the prevalence of G1 exit was more than twofold greater; however, E2F-1 evoked apoptosis and rapid mortality comparably in both backgrounds. Thus, the differential effects of E2F-1 on G1 exit in wild-type versus p53-deficient mice illustrate the combinatorial power of viral gene delivery to genetically defined recipients: E2F-1 can override the G1/S checkpoint in postmitotic ventricular myocytes in vitro and in vivo, but leads to apoptosis even in p53(-)/- mice.

O besity is associated with diabetes, and leptin is known to be elevated in obesity. To investigate whether leptin has a direct effect on insulin secretion, isolated rat and human islets and cultured insulinoma cells were studied. In all cases, mouse leptin inhibited insulin secretion at concentrations within the plasma range reported in humans. Insulin mRNA expression was also suppressed in the cultured cells and rat islets. The long form of the leptin receptor (OB-Rb) mRNA was present in the islets and insulinoma cell lines. To determine the significance of these findings in vivo, normal fed mice were injected with two doses of leptin. A significant decrease in plasma insulin and associated rise in glucose concentration were observed. Fasted normal and leptin receptor-deficient db/db mice showed no response to leptin. A dose of leptin, which mimicked that found in normal mice, was administered to leptin-deficient, hyperinsulinemic ob/ob mice. This caused a marked lowering of plasma insulin concentration and a doubling of plasma glucose. Thus, leptin has a powerful acute inhibitory effect on insulin secretion. These results suggest that the action of leptin may be one mechanism by which excess adipose tissue could acutely impair carbohydrate metabolism.

H IV infection is characterized by the reduction of the CD4+, CD45RA+, CD26+, and CD28+ lymphocyte subsets and of the in vitro production of IL-2, IL-4, and interferon-gamma; on the contrary, chemokine production is usually increased. These abnormalities are only partially restored by antiretroviral chemotherapy. Therapy with interleukin-2 has been proposed to restore the functions of the immune system, but the mechanisms by which IL-2 exerts its activities are unknown. The aim of this study was to define the effects of rIL-2 administration on CD4+, CD45RA+, CD45R0+, and CD26+ lymphocytes and on the in vitro production of IL-2, IL-4, IL-10, IFN-gamma, RANTES, and sCD30 in HIV+ patients. 10 HIV+ patients with CD4 cell counts between 200 and 500 cells/mm3 were treated with six cycles of subcutaneous recombinant IL-2 administration, in combination with zidovudine and didanosine. This therapeutic regimen resulted in a remarkable increase in the number of CD4+ cells and in the prolonged reduction of the levels of viremia. CD45R01 cells were expanded during the first cycle of therapy, while CD45RA+/CD26+ cells predominated after the third cycle. At this time, the in vitro production of IL-2, IL-4, IFN-gamma, and sCD30 were significantly upregulated. These results demonstrate that rIL-2 in HIV+ patients induces the reconstitution of the CD4/CD45RA lymphocytes subtype. This expanded cell population recovered the ability to produce in vitro IL-2, IL-4, and IFN-gamma. These effects may be beneficial to HIV+ patients by improving their immune response to microorganisms or vaccines.

T ransgenic mice overexpressing the human growth hormone gene develop mammary carcinomas. Since human growth hormone gene can activate both the growth hormone receptor (GHR) and the prolactin (PRL) receptor (PRLR), it is not clear which receptor system is responsible for the malignant transformation. To clarify the receptor specificity, we created transgenic mice with two different genes: (a) transgenic mice overexpressing the bovine growth hormone (bGH) gene having high levels of bGH only activating the GHR and also high serum levels of IGF-I; and (b) transgenic mice overexpressing the rat PRL (rPRL) gene that have elevated levels of PRL (one line 150 ng/ml and one line 13 ng/ml) only binding to the PRLR and with normal IGF-I levels. When analyzed histologically, all of the PRL transgenic female mice developed mammary carcinomas at 11-15 mo of age. Only normal mammary tissue was observed among the bGH transgenic animals and the controls. Cell lines established from a tumor produced rPRL and expressed PRLR. In organ culture experiments, an auto/paracrine effect of rPRL was demonstrated. In conclusion, activation of the PRLR is sufficient for induction of mammary carcinomas in mice, while activation of the GHR is not sufficient for mammary tumor formation.

E ndothelial cells initiate the inflammatory response by recruiting and activating leukocytes. IL-6 is not an agonist for this, but we found soluble IL-6 receptor alpha-subunit (IL-6Ralpha), with their constitutive IL-6 synthesis, stimulated endothelial cells to synthesize E-selectin, intracellular adhesion molecule-1, vascular cellular adhesion molecule-1, IL-6, and IL-8, and to bind neutrophils. Neutrophils express significant amounts of IL-6Ralpha and upon stimulation shed it: this material activates endothelial cells through a newly constituted IL-6 receptor. Retrograde signaling from PMN activated in the extravascular compartment to surrounding endothelial cells will recruit more and a wider variety of leukocytes. The limiting signal is a soluble receptor, not a cytokine.

M ultiple clinical trials have shown the efficacy of adoptively transferred allogeneic antigen-specific T cells for the treatment of viral infections and relapsed hematologic malignancies. In contrast, the therapeutic potential of autologous antigen-specific T cells has yet to be established since it has been technically difficult to generate sufficient numbers of these T cells, ex vivo. A major obstacle to the success of this objective derives from our inability to simply and rapidly isolate and/or expand large numbers of highly efficient antigen presenting cells (APCs) for repetitive stimulations of antigen-specific T cells in vitro. We show that autologous CD40-activated B cells represent a readily available source of highly efficient APC that appear to have several important advantages over other APCs for ex vivo T cell expansion including: (a) methodological simplicity necessary to generate continuously large numbers of APCs from just 50 cm3 of peripheral blood without loss of APC function; (b) capacity to induce high peak T cell proliferation and interferon-gamma production without IL-10 production; (c) ease in cryopreservation; and (d) markedly reduced cost. We, therefore, contend that CD40-activated B cells are an alternative source of highly efficient APCs with which to generate antigen-specific T cells ex vivo for autologous adoptive immunotherapy.

I nflammatory bowel disease (IBD) is characterized by altered immunoregulation and augmented intestinal synthesis of nitric oxide. The purpose of this study was to determine the effects of exogenous IL-4, introduced by a recombinant human type 5 adenovirus (Ad5) vector, on the tissue injury associated with an experimental model of colonic immune activation and inflammation. Colitis was induced in rats by the intrarectal administration of trinitrobenzene sulfonic acid (TNB) dissolved in 50% ethanol, and control rats received saline via the same route. 1 h later, all rats were randomized into two groups. The first group was injected intraperitoneally (ip) with 3.0 x 10(6) plaque forming units (PFUs) of Ad5 transfected with murine interleukin-4 (Ad5IL-4) and the second group was injected ip with the same amount of Ad5 expressing the Escherichia coli Lac Z gene (Ad5LacZ). One-half of the colitic and control rats were injected again with 3.0 x 10(6) PFUs of Ad5IL-4 or Ad5LacZ on day 3 of the 6-d study. When introduced once or twice via the peritoneal route into control rats, Ad5LacZ was localized to the serosal lining of the peritoneal cavity, the diaphragm and the liver on day 6. One or two injections of Ad5IL-4 into rats also produced measurable levels of circulating IL-4. TNB-colitis in both Ad5LacZ-treated groups was associated with pronounced elevations in serum IFN-gamma, and mucosal ulceration of the distal colon. Myeloperoxidase and inducible nitric oxide synthase II (NOS II) synthetic activity were also increased by 30- and fivefold, respectively, above control levels in the distal colon. However, two injections of Ad5IL-4 into colitic rats caused the overexpression of IL-4, and significantly inhibited tissue damage, serum and colon IFN-gamma levels and myeloperoxidase activity in the distal colon. In addition, NOS II gene expression and NOS II nitric oxide synthesis was significantly inhibited. No therapeutic effect was observed in rats injected once with Ad5IL-4. Thus, IL-4, introduced by Ad5, is therapeutic during acute inflammation in the rat colon. The therapeutic effect of IL-4 was associated with an inhibition of inducible nitric oxide expression and a reduction in nitric oxide synthesis.

C ytokines, in particular tumor necrosis factor-alpha (TNF-alpha), have significant effects on energy metabolism and appetite although their mechanisms of action are largely unknown. Here, we examined whether TNF-alpha modulates the production of leptin, the recently identified fat-specific energy balance hormone, in cultured adipocytes and in mice. TNF-alpha treatment of 3T3-L1 adipocytes resulted in rapid stimulation of leptin accumulation in the media, with a maximum effect at 6 h. This stimulation was insensitive to cycloheximide, a protein synthesis inhibitor, but was completely inhibited by the secretion inhibitor brefeldin A, indicating a posttranslational effect. Treatment of mice with TNF-alpha also caused a similar increase in plasma leptin levels. Finally, in obese TNF-alpha-deficient mice, circulating leptin levels were significantly lower, whereas adipose tissue leptin was higher compared with obese wild-type animals. These data provide evidence that TNF-alpha can act directly on adipocytes to regulate the release of a preformed pool of leptin. Furthermore, they suggest that the elevated adipose tissue expression of TNF-alpha that occurs in obesity may contribute to obesity-related hyperleptinemia.

M ucin 1 (MUC1) is a large complex glycoprotein that is highly expressed in breast cancer, and as such could be a target for immunotherapy. In mice, human MUC1 is highly immunogenic, particularly when conjugated to mannan, where a high frequency of CD8(+) MHC-restricted cytotoxic T lymphocytes is induced, accompanied by tumor protection. On this basis, a clinical trial was performed in which 25 patients with advanced metastatic carcinoma of breast, colon, stomach, or rectum received mannan-MUC1 in increasing doses. After 4 to 8 injections, large amounts of IgG1 anti-MUC1 antibodies were produced in 13 out of 25 patients (with antibody titers by ELISA of 1/320-1/20,480). Most of the antibodies reacted to the epitopes STAPPAHG and PAPGSTAP. In addition, T cell proliferation was found in 4 out of 15 patients, and CTL responses were seen in 2 out of 10 patients. Mannan-MUC1 can immunize patients, particularly for antibody formation, and to a lesser extent, cellular responses. It remains to be seen whether such responses have antitumor activity.

T he endothelium plays an important role in maintaining the vascular homeostasis by releasing vasodilator substances, including prostacyclin (PGI2), nitric oxide (NO), and endothelium-derived hyperpolarizing factor (EDHF). Although the former two substances have been investigated extensively, the importance of EDHF still remains unclear, especially in human arteries. Thus we tested our hypothesis that EDHF plays an important role in human arteries, particularly with reference to the effect of vessel size, its vasodilating mechanism, and the influences of risk factors for atherosclerosis. Isometric tension and membrane potentials were recorded in isolated human gastroepiploic arteries and distal microvessels (100-150 microm in diameter). The contribution of PGI2, NO, and EDHF to endothelium-dependent relaxations was analyzed by inhibitory effects of indomethacin, NG-nitro- L-arginine, and KCl, respectively. The nature of and hyperpolarizing mechanism by EDHF were examined by the inhibitory effects of inhibitors of cytochrome P450 pathway and of various K channels. The effects of atherosclerosis risk factors on EDHF-mediated relaxations were also analyzed. The results showed that (a) the contribution of EDHF to endothelium-dependent relaxations is significantly larger in microvessels than in large arteries; (b) the nature of EDHF may not be a product of cytochrome P450 pathway, while EDHF-induced hyperpolarization is partially mediated by calcium-activated K channels; and (c) aging and hypercholesterolemia significantly impair EDHF-mediated relaxations. These results demonstrate that EDHF also plays an important role in human arteries.

P latelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) regulate mesangial cell proliferation and matrix production in vitro and in vivo and crucially participate in the pathogenesis of glomerulonephritis. We investigated whether PDGF-BB and bFGF influence nitric oxide (NO) production, another important effector molecule in inflammatory glomerular injury. Inducible NO synthase (iNOS) induction in rat glomerular mesangial cells has been described in response to two principal classes of activating signals comprising inflammatory cytokines such as interleukin 1beta (IL-1beta) or elevation of cyclic AMP (cAMP). Treatment of mesangial cells with IL-1beta induces iNOS activity measured as nitrite levels in cell culture supernatants. Coincubation of mesangial cells with PDGF-BB inhibits production of nitrite by approximately 95%. This effect can be reversed by the simultaneous incubation of PDGF-BB in the presence of calphostin C, a potent and selective inhibitor of protein kinase C. In contrast, incubation of cells in the presence of bFGF potentiates IL-1beta-induced production of NO and is functionally associated with an increased rate of apoptosis of mesangial cells. Western blot analyses reveal that PDGF-BB causes a decrease in the formation of iNOS protein which is preceded by decreases in iNOS mRNA steady state levels. bFGF drastically increases iNOS protein levels as well as the corresponding iNOS mRNA steady state levels. Nuclear run-on experiments reveal that PDGF-BB decreases the IL-1beta-induced transcription rate of the iNOS gene, whereas bFGF potentiates the transcriptional activity of the iNOS gene. Northern blot analyses demonstrate that bFGF strongly potentiates the formation of IL-1beta-induced IL-1 type I receptor mRNA levels, whereas PDGF-BB has no effect. Treatment of mesangial cells with the membrane-permeable cAMP analogue N6, O-2'-dibutyryladenosine 3',5'-phosphate (Bt2cAMP) markedly increases the production of nitrite. Whereas PDGF-BB does not affect cAMP-induced nitrite levels, bFGF strongly potentiates them. PDGF-BB alters neither cAMP-induced iNOS protein levels nor the corresponding iNOS mRNA steady state levels. By contrast, bFGF superinduces cAMP-stimulated iNOS protein and iNOS mRNA levels. These changes by bFGF are due to an increase in cAMP-induced transcriptional activity of the iNOS gene which is not affected by PDGF-BB. In summary, the results show that PDGF and bFGF differentially regulate iNOS expression in mesangial cells in a stimulus-specific way. The timely sequence of expression of PDGF and bFGF and of cytokines like IL-1 will crucially determine the amounts of NO produced and the functional consequences thereof in the course of progressive glomerular diseases.

I n cystic fibrosis (CF), defective function of the cystic fibrosis transmembrane conductance regulator (CFTR) in airway epithelial cells and submucosal glands results in chronic pulmonary infection with Pseudomonas aeruginosa. The pulmonary infection incites an intense host inflammatory response, causing progressive suppurative pulmonary disease. Mouse models of CF, however, fail to develop pulmonary disease spontaneously. We examined the effects of bronchopulmonary infection on mice homozygous for the S489X mutation of the CFTR gene using an animal model of chronic Pseudomonas endobronchial infection. Slurries of sterile agarose beads or beads containing a clinical isolate of mucoid P. aeruginosa were instilled in the right lung of normal or CF mice. The mortality of CF mice inoculated with Pseudomonas-laden beads was significantly higher than that of normal animals: 82% of infected CF mice, but only 23% of normal mice, died within 10 d of infection (P = 0.023). The concentration of inflammatory mediators, including TNF-alpha, murine macrophage inflammatory protein-2, and KC/N51, in bronchoalveolar lavage fluid in CF mice 3 d after infection and before any mortality, was markedly elevated compared with normal mice. This inflammatory response also correlated with weight loss observed in both CF and normal littermates after inoculation. Thus, this model may permit examination of the relationship of bacterial infections, inflammation, and the cellular and genetic defects in CF.

A lthough angiotensin II type 2 (AT2) receptor has recently been cloned, its functional role is not well understood. We tested the hypothesis that selective activation of AT2 receptor causes vasodilation in the preglomerular afferent arteriole (Af-Art), a vascular segment that accounts for most of the preglomerular resistance. We microperfused rabbit Af-Arts at 60 mmHg in vitro, and examined the effect of angiotensin II (Ang II; 10(-11)-10(-8) M) on the luminal diameter in the presence or absence of the Ang II type 1 receptor antagonist CV11974 (CV; 10(-8) M). Ang II was added to both the bath and lumen of preconstricted Af-Arts. Ang II further constricted Af-Arts without CV (by 74+/-7% over the preconstricted level at 10(-8) M; P < 0.01, n = 7). In contrast, in the presence of CV, Ang II caused dose-dependent dilation; Ang II at 10(-8) M increased the diameter by 29+/-2% (n = 7, P < 0.01). This dilation was completely abolished by pretreatment with an AT2 receptor antagonist PD123319 (10(-7) M, n = 6), suggesting that activation of AT2 receptor causes vasodilation in Af-Arts. The dilation was unaffected by inhibiting either nitric oxide synthase (n = 7) or cyclooxygenase (n = 7), however, it was abolished by either disrupting the endothelium (n = 10) or inhibiting the cytochrome P-450 pathway, particularly the synthesis of epoxyeicosatrienoic acids (EETs, n = 7). These results suggest that in the Af-Art activation of the AT2 receptor may cause endothelium-dependent vasodilation via a cytochrome P-450 pathway, possibly by EETs.

V ascular proliferative disorders are characterized by the proliferation of vascular smooth muscle cells (SMCs) and excessive extracellular matrix synthesis. We found that bone morphogenetic protein-2 (BMP-2) inhibited serum-stimulated increases in DNA synthesis and cell number of cultured rat arterial SMCs in a fashion quite different from that in the case of transforming growth factor-beta1 (TGF-beta1). In addition, TGF-beta1 stimulated collagen synthesis in SMCs, whereas BMP-2 did not. In an in vivo rat carotid artery balloon injury model, the adenovirus-mediated transfer of the BMP-2 gene inhibited injury-induced intimal hyperplasia. These results indicate that BMP-2 has the ability to inhibit SMC proliferation without stimulating extracellular matrix synthesis, and suggest the possibility of therapeutic application of BMP-2 for the prevention of vascular proliferative disorders.

N on-insulin-dependent diabetes mellitus (NIDDM) is caused by peripheral insulin resistance and impaired beta cell function. Phosphofructo-1-kinase (PFK1) is a rate-limiting enzyme in glycolysis, and its muscle subtype (PFK1-M) deficiency leads to the autosomal recessively inherited glycogenosis type VII Tarui's disease. It was evaluated whether PFK1-M deficiency leads to alterations in insulin action or secretion in humans. A core family of four members was evaluated for PFK1-M deficiency by DNA and enzyme-activity analyses. All members underwent oral and intravenous glucose tolerance tests (oGTT and ivGTT) and an insulin-sensitivity test (IST) using octreotide. Enzyme activity determinations in red blood cells showed that the father (46 yr, body mass index [BMI] 22. 4 kg/m2) and older son (19 yr, BMI 17.8 kg/m2) had a homozygous, while the mother (47 yr, BMI 28.4 kg/m2) and younger son (13 yr, BMI 16.5 kg/m2) had a heterozygous PFK1-M deficiency. DNA analyses revealed an exon 5 missense mutation causing missplicing of one allele in all four family members, and an exon 22 frameshift mutation of the other allele of the two homozygously affected individuals. The father showed impaired glucose tolerance, and the mother showed NIDDM. By ivGTT, both parents and the older son had decreased first-phase insulin secretion and a diminished glucose disappearance rate. The IST showed marked insulin resistance in both parents and the older, homozygous son, and moderate resistance in the younger son. PFK1-M deficiency causes impaired insulin secretion in response to glucose, demonstrating its participation in islet glucose metabolism, and peripheral insulin resistance. These combined metabolic sequelae of PFK-1 deficiency identify it as a candidate gene predisposing to NIDDM.

V itamin C concentrations in the brain exceed those in blood by 10-fold. In both tissues, the vitamin is present primarily in the reduced form, ascorbic acid. We identified the chemical form of vitamin C that readily crosses the blood-brain barrier, and the mechanism of this process. Ascorbic acid was not able to cross the blood-brain barrier in our studies. In contrast, the oxidized form of vitamin C, dehydroascorbic acid (oxidized ascorbic acid), readily entered the brain and was retained in the brain tissue in the form of ascorbic acid. Transport of dehydroascorbic acid into the brain was inhibited by d-glucose, but not by l-glucose. The facilitative glucose transporter, GLUT1, is expressed on endothelial cells at the blood-brain barrier, and is responsible for glucose entry into the brain. This study provides evidence showing that GLUT1 also transports dehydroascorbic acid into the brain. The findings define the transport of dehydroascorbic acid by GLUT1 as a mechanism by which the brain acquires vitamin C, and point to the oxidation of ascorbic acid as a potentially important regulatory step in accumulation of the vitamin by the brain. These results have implications for increasing antioxidant potential in the central nervous system.

W e investigated the mechanism by which inosine, a metabolite of adenosine that accumulates to > 1 mM levels in ischemic tissues, triggers mast cell degranulation. Inosine was found to do the following: (a) compete for [125I]N6-aminobenzyladenosine binding to recombinant rat A3 adenosine receptors (A3AR) with an IC50 of 25+/-6 microM; (b) not bind to A1 or A2A ARs; (c) bind to newly identified A3ARs in guinea pig lung (IC50 = 15+/-4 microM); (d) lower cyclic AMP in HEK-293 cells expressing rat A3ARs (ED50 = 12+/-5 microM); (e) stimulate RBL-2H3 rat mast-like cell degranulation (ED50 = 2.3+/-0.9 microM); and (f) cause mast cell-dependent constriction of hamster cheek pouch arterioles that is attenuated by A3AR blockade. Inosine differs from adenosine in not activating A2AARs that dilate vascular smooth muscle and inhibit mast cell degranulation. The A3 selectivity of inosine may explain why it elicits a monophasic arteriolar constrictor response distinct from the multiphasic dilator/constrictor response to adenosine. Nucleoside accumulation and an increase in the ratio of inosine to adenosine may provide a physiologic stimulus for mast cell degranulation in ischemic or inflamed tissues.

L eptin is thought to exert its actions on energy homeostasis through the long form of the leptin receptor (OB-Rb), which is present in the hypothalamus and in certain peripheral organs, including adipose tissue. In this study, we examined whether leptin has direct effects on the function of brown and white adipose tissue (BAT and WAT, respectively) at the metabolic and molecular levels. The chronic peripheral intravenous administration of leptin in vivo for 4 d resulted in a 1.6-fold increase in the in vivo glucose utilization index of BAT, whereas no significant change was found after intracerebroventricular administration compared with pair-fed control rats, compatible with a direct effect of leptin on BAT. The effect of leptin on WAT fat pads from lean Zucker Fa/ fa rats was assessed ex vivo, where a 9- and 16-fold increase in the rate of lipolysis was observed after 2 h of exposure to 0.1 and 10 nM leptin, respectively. In contrast, no increase in lipolysis was observed in the fat pads from obese fa/fa rats, which harbor an inactivating mutation in the OB-Rb. At the level of gene expression, leptin treatment for 24 h increased malic enzyme and lipoprotein lipase RNA 1.8+/-0.17 and 1.9+/-0.14-fold, respectively, while aP2 mRNA levels were unaltered in primary cultures of brown adipocytes from lean Fa/fa rats. Importantly, however, no significant effect of leptin was observed on these genes in brown adipocytes from obese fa/fa animals. The presence of OB-Rb receptors in adipose tissue was substantiated by the detection of its transcripts by RT-PCR, and leptin treatment in vivo and in vitro activated the specific STATs implicated in the signaling pathway of the OB-Rb. Taken together, our data strongly suggest that leptin has direct effects on BAT and WAT, resulting in the activation of the Jak/STAT pathway and the increased expression of certain target genes, which may partially account for the observed increase in glucose utilization and lipolysis in leptin-treated adipose tissue.

S ystemic delivery of specific therapeutic proteins by a parenteral route of administration is a recognized practice in the management of several gene defects and acquired diseases. As an alternative to repetitive parenteral administration, gene therapy may provide a novel means for systemic delivery of therapeutic proteins while improving patient compliance and therapeutic efficacy. However, for gene therapy to be an efficacious and safe approach to the clinical management of such diseases, gene expression must be tightly regulated. These investigations demonstrate precise in vivo control of protein expression from cells that are engineered to secrete human growth hormone (hGH) in response to stimulation by rapamycin. The cells were implanted intramuscularly into nu/nu mice and stimulated by intravenous or oral administration of rapamycin. In vivo experiments demonstrate that the activity and pharmacokinetics of rapamycin determine the level of serum hGH that result from the engineered cells. In addition, responsiveness of the cells to rapamycin, number of cells implanted, hGH expression kinetics, and the pharmacokinetics of hGH itself, also influence the circulating levels of hGH after rapamycin stimulation. Controlled manipulation of several of these parameters, either independently or in combination, allows for precise regulation of circulating hGH concentration in vivo.

F anconi anemia (FA) consists of a group of at least five autosomal recessive disorders that share both clinical (e.g., birth defects and hematopoietic failure) and cellular (e.g., sensitivity to cross-linking agents and predisposition to apoptosis) features with each other. However, a common pathogenetic link among these groups has not been established. To identify genetic pathways that are altered in FA and characterize shared molecular defects, we used mRNA differential display to isolate genes that have altered expression patterns in FA cells. Here, we report that the expression of an interferon-inducible gene, MxA, is highly upregulated in cells of FA complementation groups A, B, C, and D, but it is suppressed in FA group C cells complemented with wild-type FAC cDNA as well as in non-FA cells. A posttranscriptional mechanism rather than transcriptional induction appears to account for MxA overexpression. Forced expression of MxA in Hep3B cells enhances their sensitivity to mitomycin C and induces apoptosis, similar to the FA phenotype. Thus, MxA is a downstream target of FAC and is the first genetic marker to be identified among multiple FA complementation groups. These data suggest that FA subtypes converge onto a final common pathway, which is intimately related to the interferon signaling mechanism. Constitutive activity of this pathway may explain a number of the phenotypic features of FA, particularly the pathogenesis of bone marrow failure.

R e-epithelialization of skin wounds depends upon the migration of keratinocytes from the cut margins of the wound and is enhanced when human keratinocytes are covered with occlusive dressings that induce hypoxia. In this study, two independent migration assays were used to compare cellular motility on connective tissue components under normoxic or hypoxic conditions. Human keratinocytes apposed to collagens or fibronectin exhibited increased motility when subjected to hypoxic (0.2 or 2% oxygen) conditions compared with normoxic (9 or 20% oxygen) conditions. When compared with normoxic cells, hypoxic keratinocytes exhibited increased expression and redistribution of the lamellipodia-associated proteins (ezrin, radixin, and moesin). Furthermore, hypoxic keratinocytes demonstrated decreased secretion of laminin-5, a laminin isoform known to inhibit keratinocyte motility. Hypoxia did not alter the number of integrin receptors on the cell surface, but did induce enhanced secretion of the 92-kD type IV collagenase. These data demonstrate that hypoxia promotes human keratinocyte motility on connective tissue. Hypoxia-driven motility is associated with increased expression of lamellipodia proteins, increased expression of collagenase and decreased expression of laminin-5, the locomotion brake for keratinocytes.

M yocardial glucose use is regulated by competing substrates and hormonal influences. However, the interactions of these effectors on the metabolism of exogenous glucose and glucose derived from endogenous glycogen are not completely understood. In order to determine changes in exogenous glucose uptake, glucose oxidation, and glycogen enrichment, hearts were perfused with glucose (5 mM) either alone, or glucose plus insulin (40 microU/ml), glucose plus acetoacetate (5 mM), or glucose plus insulin and acetoacetate, using a three tracer (3H, 14C, and 13C) technique. Insulin-stimulated glucose uptake and lactate production in the absence of acetoacetate, while acetoacetate inhibited the uptake of glucose and the oxidation of both exogenous glucose and endogenous carbohydrate. Depending on the metabolic conditions, the contribution of glycogen to carbohydrate metabolism varied from 20-60%. The addition of acetoacetate or insulin increased the incorporation of exogenous glucose into glycogen twofold, and the combination of the two had additive effects on the incorporation of glucose into glycogen. In contrast, the glycogen content was similar for the three groups. The increased incorporation of glucose in glycogen without a significant change in the glycogen content in hearts perfused with glucose, acetoacetate, and insulin suggests increased glycogen turnover. We conclude that insulin and acetoacetate regulate the incorporation of glucose into glycogen as well as the relative contributions of exogenous glucose and endogenous carbohydrate to myocardial energy metabolism by different mechanisms.

W e have investigated the antidiabetic action of troglitazone in aP2/DTA mice, whose white and brown fat was virtually eliminated by fat-specific expression of diphtheria toxin A chain. aP2/DTA mice had markedly suppressed serum leptin levels and were hyperphagic, but did not gain excess weight. aP2/DTA mice fed a control diet were hyperlipidemic, hyperglycemic, and had hyperinsulinemia indicative of insulin-resistant diabetes. Treatment with troglitazone alleviated the hyperglycemia, normalized the tolerance to intraperitoneally injected glucose, and significantly decreased elevated insulin levels. Troglitazone also markedly decreased the serum levels of cholesterol, triglycerides, and free fatty acids both in wild-type and aP2/DTA mice. The decrease in serum triglycerides in aP2/DTA mice was due to a marked reduction in VLDL- and LDL-associated triglyceride. In skeletal muscle, triglyceride levels were decreased in aP2/DTA mice compared with controls, but glycogen levels were increased. Troglitazone treatment decreased skeletal muscle, but not hepatic triglyceride and increased hepatic and muscle glycogen content in wild-type mice. Troglitazone decreased muscle glycogen content in aP2/DTA mice without affecting muscle triglyceride levels. The levels of peroxisomal proliferator-activated receptor gamma mRNA in liver increased slightly in aP2/DTA mice and were not changed by troglitazone treatment. The results demonstrate that insulin resistance and diabetes can occur in animals without significant adipose deposits. Furthermore, troglitazone can alter glucose and lipid metabolism independent of its effects on adipose tissue.

T he intermediate filament vimentin might play a key role in vascular resistance to mechanical stress. We investigated the responses to pressure (tensile stress) and flow (shear stress) of mesenteric resistance arteries perfused in vitro from vimentin knockout mice. Arteries were isolated from homozygous (Vim-/-, n = 14) or heterozygous vimentin-null mice (Vim+/-, n = 5) and from wild-type littermates (Vim+/+, n = 9). Passive arterial diameter (175+/-15 micron in Vim+/+ at 100 mmHg) and myogenic tone were not affected by the absence of vimentin. Flow-induced (0-150 microl/min) dilation (e. g., 19+/-3 micron dilation at 150 mmHg in Vim+/+) was significantly attenuated in Vim-/- mice (13+/-2 micron dilation, P < 0.01). Acute blockade of nitric oxide synthesis (NG-nitro- L-arginine, 10 microM) significantly decreased flow-induced dilation in both groups, whereas acute blockade of prostaglandin synthesis (indomethacin, 10 microM) had no significant effect. Mean blood pressure, in vivo mesenteric blood flow and diameter, and mesenteric artery media thickness or media to lumen ratio were not affected by the absence of vimentin. Thus, the absence of vimentin decreased selectively the response of resistance arteries to flow, suggesting a role for vimentin in the mechanotransduction of shear stress.

T o explore mechanisms underlying triglyceride (TG) accumulation in livers of chow-fed apo E-deficient mice (Kuipers, F., J.M. van Ree, M.H. Hofker, H. Wolters, G. In't Veld, R.J. Vonk, H.M.G. Princen, and L.M. Havekes. 1996. Hepatology. 24:241-247), we investigated the effects of apo E deficiency on secretion of VLDL-associated TG (a) in vivo in mice, (b) in isolated perfused mouse livers, and (c) in cultured mouse hepatocytes. (a) Hepatic VLDL-TG production rate in vivo, determined after Triton WR1339 injection, was reduced by 46% in apo E-deficient mice compared with controls. To eliminate the possibility that impaired VLDL secretion is caused by aspecific changes in hepatic function due to hypercholesterolemia, VLDL-TG production rates were also measured in apo E-deficient mice after transplantation of wild-type mouse bone marrow. Bone marrow- transplanted apo E-deficient mice, which do not express apo E in hepatocytes, showed normalized plasma cholesterol levels, but VLDL-TG production was reduced by 59%. (b) VLDL-TG production by isolated perfused livers from apo E-deficient mice was 50% lower than production by livers from control mice. Lipid composition of nascent VLDL particles isolated from the perfusate was similar for both groups. (c) Mass VLDL-TG secretion by cultured apo E-deficient hepatocytes was reduced by 23% compared with control values in serum-free medium, and by 61% in the presence of oleate in medium (0. 75 mM) to stimulate lipogenesis. Electron microscopic evaluation revealed a smaller average size for VLDL particles produced by apo E-deficient cells compared with control cells in the presence of oleate (38 and 49 nm, respectively). In short-term labeling studies, apo E-deficient and control cells showed a similar time-dependent accumulation of [3H]TG formed from [3H]glycerol, yet secretion of newly synthesized VLDL-associated [3H]TG by apo E-deficient cells was reduced by 60 and 73% in the absence and presence of oleate, respectively. We conclude that apo E, in addition to its role in lipoprotein clearance, has a physiological function in the VLDL assembly-secretion cascade.

T he mechanisms that regulate vascular resistance in the liver are an area of active investigation. Previously, we have shown that nitric oxide (NO) modulates hepatic vascular tone in the normal rat liver. In this study, the production of NO is examined in further detail by isolating sinusoidal endothelial cells (SEC) from the rat liver. Endothelial NO synthase (eNOS) was present in SEC based on Western blotting and confocal immunofluorescence microscopy. Exposure of SEC to flow increased the release of NO. To investigate the relevance of these in vitro findings to the intact liver, we modified an in situ perfusion system to allow for direct measurement of NO release from the hepatic vasculature. NO was released from the hepatic vasculature in a time-dependent manner, and administration of N-monomethyl-L-arginine reduced NO release and increased portal pressure. Immunostaining of intact liver demonstrated eNOS localization to endothelial cells lining the hepatic sinusoids. These findings demonstrate that SEC in vitro and in vivo express eNOS and produce NO basally, and increase their production in response to flow. Additionally, an increase in portal pressure concomitant with the blockade of NO release directly demonstrates that endogenous endothelial-derived NO modulates portal pressure.