Issue published June 15, 1998

T ransforming growth factor beta is a potent immunomodulator with both pro- and antiinflammatory activities. Based on its immunosuppressive actions, exogenous TGF-beta has been shown to inhibit autoimmune and chronic inflammatory diseases. To further explore the potential therapeutic role of TGF-beta, we administered a plasmid DNA encoding human TGF-beta1 intramuscularly to rats with streptococcal cell wall-induced arthritis. A single dose of 300 microg plasmid DNA encoding TGF-beta1, but not vector DNA, administered at the peak of the acute phase profoundly suppressed the subsequent evolution of chronic erosive disease typified by disabling joint swelling and deformity (articular index = 8.17+/-0. 17 vs. 1.25+/-0.76, n = 6, day 26, P < 0.01). Moreover, delivery of the TGF-beta1 DNA even as the chronic phase commenced virtually eliminated subsequent inflammation and arthritis. Both radiologic and histopathologic as well as molecular evidence supported the marked inhibitory effect of TGF-beta1 DNA on synovial pathology, with decreases in the inflammatory cell infiltration, pannus formation, cartilage and bone destruction, and the expression of proinflammatory cytokines that characterize this model. Increases in TGF-beta1 protein were detected in the circulation of TGF-beta1 DNA-treated animals, consistent with the observed therapeutic effects being TGF-beta1 dependent. These observations provide the first evidence that gene transfer of plasmid DNA encoding TGF-beta1 provides a mechanism to deliver this potent cytokine that effectively suppresses ongoing inflammatory pathology in arthritis.

T he concept that androgens are atretogenic, derived from murine ovary studies, is difficult to reconcile with the fact that hyperandrogenic women have more developing follicles than normal-cycling women. To evaluate androgen's effects on primate follicular growth and survival, normal-cycling rhesus monkeys were treated with placebo-, testosterone-(T), or dihydrotestosterone-sustained release implants, and ovaries were taken for histological analysis after 3-10 d of treatment. Growing preantral and small antral follicles up to 1 mm in diameter were significantly and progressively increased in number and thecal layer thickness in T-treated monkeys from 3-10 d. Granulosa and thecal cell proliferation, as determined by immunodetection of the Ki67 antigen, were significantly increased in these follicles. Preovulatory follicles (> 1 mm), however, were not increased in number in androgen-treated animals. Follicular atresia was not increased and there were actually significantly fewer apoptotic granulosa cells in the T-treated groups. Dihydrotestosterone treatment had identical effects, indicating that these growth-promoting actions are mediated by the androgen receptor. These findings show that, over the short term at least, androgens are not atretogenic and actually enhance follicular growth and survival in the primate. These new data provide a plausible explanation for the pathogenesis of "polycystic" ovaries in hyperandrogenism.

T he different functions of the ventricular- and atrial-specific essential myosin light chains are unknown. Using transgenesis, cardiac-specific overexpression of proteins can be accomplished. The transgenic paradigm is more useful than originally expected, in that the mammalian heart rigorously controls sarcomeric protein stoichiometries. In a clinical subpopulation suffering from heart disease caused by congenital malformations of the outflow tract, an ELC1v-->ELC1a isoform shift correlated with increases in cross-bridge cycling kinetics as measured in skinned fibers derived from the diseased muscle. We have used transgenesis to replace the ventricular isoform of the essential myosin light chain with the atrial isoform. The ELC1v--> ELC1a shift in the ventricle resulted in similar functional alterations. Unloaded velocities as measured by the ability of the myosin to translocate actin filaments in the in vitro motility assay were significantly increased as a result of the isoform substitution. Unloaded shortening velocity was also increased in skinned muscle fibers, and at the whole organ level, both contractility and relaxation were significantly increased. This increase in cardiac function occurred in the absence of a hypertrophic response. Thus, ELC1a expression in the ventricle appears to be advantageous to the heart, resulting in increased cardiac function.

S taphylococcus aureus is a major cause of nosocomial and community-acquired infections. Morbidity and mortality due to infections such as sepsis, osteomyelitis, septic arthritis, and invasive endocarditis remain high despite the use of antibiotics. The emergence of antibiotic resistant super bugs mandates that alternative strategies for the prevention and treatment of S. aureus infections are developed. We investigated the ability of vaccination with a recombinant fragment of the S. aureus collagen adhesin to protect mice against sepsis-induced death. Actively immunized NMRI mice were intravenously inoculated with the S. aureus clinical isolate strain Phillips. 14 d after inoculation, mortality in the collagen adhesin-vaccinated group was only 13%, compared with 87% in the control antigen immunized group (P < 0.001). To determine if the protective effect was antibody mediated, we passively immunized naive mice with collagen adhesin-specific antibodies. Similar to the active immunization strategy, passive transfer of collagen adhesin-specific antibodies protected mice against sepsis-induced death. In vitro experiments indicated that S. aureus opsonized with sera from collagen adhesin immunized mice promoted phagocytic uptake and enhanced intracellular killing compared with bacteria opsonized with sera from control animals. These results indicate that the collagen adhesin is a viable target in the development of immunotherapeutics against S. aureus.

A n increase in myofibroblast number may be necessary for wound healing but may also lead to postinflammatory scarring. We have, therefore, studied the role of mediators important in inflammatory bowel disease in regulating proliferation of human colonic myofibroblasts. Using primary cultures of these cells, we have shown increases in [3H]thymidine incorporation in response to platelet-derived growth factor (EC50 = 14 ng/ml), basic fibroblast growth factor (EC50 = 2.2 ng/ml), and epidermal growth factor (EC50 = 1.1 ng/ml). Coulter counting of cell suspensions demonstrated increases in cell number with these growth factors along with insulin-like growth factor-I and -II. In addition the proinflammatory cytokines IL-1beta and TNF-alpha produced increases in [3H]thymidine incorporation. IL-1beta and platelet-derived growth factor together produced an increase in [3H]thymidine greater than either agonist alone; this effect was not, however, seen when we examined changes in cell numbers. Finally, we demonstrate a mechanism whereby these responses may be downregulated: vasoactive intestinal peptide (1 microM) elevates cyclic AwMP in these cells 4. 2-fold over control and produces a dose-related inhibition of platelet-derived growth factor-driven proliferation with a maximum inhibition of 33% at 1 microM.

T he subendothelial retention of LDLs through their interaction with proteoglycans has been proposed to be a key process in the pathogenesis of atherosclerosis. In vitro studies have identified eight clusters of basic amino acids in delipidated apo-B100, the protein moiety of LDL, that bind the negatively charged proteoglycans. To determine which of these sites is functional on the surface of LDL particles, we analyzed the proteoglycan-binding activity of recombinant human LDL isolated from transgenic mice. Substitution of neutral amino acids for the basic amino acids residues in site B (residues 3359-3369) abolished both the receptor-binding and the proteoglycan-binding activities of the recombinant LDL. Chemical modification of the remaining basic residues caused only a marginal further reduction in proteoglycan binding, indicating that site B is the primary proteoglycan-binding site of LDL. Although site B was essential for normal receptor-binding and proteoglycan-binding activities, these activities could be separated in recombinant LDL containing single-point mutation. Recombinant LDL with a K3363E mutation, in which a glutamic acid had been inserted into the basic cluster RKR in site B, had normal receptor binding but interacted defectively with proteoglycans; in contrast, another mutant LDL, R3500Q, displayed defective receptor binding but interacted normally with proteoglycans. LDL with normal receptor-binding activity but with severely impaired proteoglycan binding will be a unique resource for analyzing the importance of LDL- proteoglycan interaction in atherogenesis. If the subendothelial retention of LDL by proteoglycans is the initial event in early atherosclerosis, then LDL with defective proteoglycan binding may have little or no atherogenic potential.

C holestasis is a cardinal complication of liver disease, but most treatments are merely supportive. Here we report that the sulfonylurea glybenclamide potently stimulates bile flow and bicarbonate excretion in the isolated perfused rat liver. Video-microscopic studies of isolated hepatocyte couplets and isolated bile duct segments show that this stimulatory effect occurs at the level of the bile duct epithelium, rather than through hepatocytes. Measurements of cAMP, cytosolic pH, and Ca2+ in isolated bile duct cells suggest that glybenclamide directly activates Na+-K+-2Cl- cotransport, rather than other transporters or conventional second-messenger systems that link to secretory pathways in these cells. Finally, studies in livers from rats with endotoxin- or estrogen-induced cholestasis show that glybenclamide retains its stimulatory effects on bile flow and bicarbonate excretion even under these conditions. These findings suggest that bile duct epithelia may represent an important new therapeutic target for treatment of cholestatic disorders.

A n ethanol oral self administration paradigm showed the existence of gender differences in alcohol preference in rats: whereas males and females initiated alcohol drinking at similar rates, females maintained their preference for ethanol over a longer duration. Neonatal estrogenization of females, which effectively confers a male phenotype on a genetically female brain, resulted in patterns of drinking that were similar to those displayed by intact male rats, indicating that gender differences in alcohol drinking patterns may be, at least partially, accounted for by sexual differentiation of the brain. To test whether gonadal steroids also exert activational effects on ethanol-seeking behavior, we also examined the effects of gonadectomy alone, or in combination with gonadal steroid replacement therapy. Castration did not significantly alter ethanol consumption in males, although treatment of castrated rats with dihydrotestosterone resulted in a significant inhibition of this parameter. As compared with the situation in intact female rats, ethanol ingestion was significantly reduced in ovariectomized female rats receiving estradiol (E2) and in ovariectomized female rats receiving combined E2 and progesterone replacement therapy. However, neither ovariectomy nor progesterone replacement in ovariectomized rats resulted in ethanol drinking patterns that were different compared to those observed in intact female controls. Thus, dihydrotestosterone and E2, respectively, appear to exert modulatory influences on the male and female rats' preference for ethanol, but further investigations are necessary to determine to what extent these effects result from activational actions on the brain.

I mmunocompromised patients with disseminated human cytomegalovirus (HCMV) infection have circulating PMN carrying HCMV pp65 (antigenemia), infectious virus (viremia), and viral DNA (leukoDNAemia). Because HCMV does not fully replicate in PMN, it is generally hypothesized that virions and viral materials are taken up by phagocytosis from fully permissive HCMV-infected endothelial cells. However, no experimental evidence has ever been provided for these PMN-endothelium interactions. PMN from 11 donors were cocultured with endothelial cells infected with an endothelium-adapted HCMV strain and with human fibroblasts infected with low-passaged clinical and laboratory-adapted HCMV strains. pp65-positive PMN were detected after coculture with both HCMV-infected endothelial and fibroblast cells, provided that wild and not laboratory-adapted strains were used. In addition, cocultured PMN carried infectious virus as demonstrated by virus isolation and presence of complete virus particles by electron microscopy. Moreover, high levels of viral DNA were consistently detected by quantitative PCR in cocultured PMN. Thus, we have generated in vitro the three most important viral parameters detected in patients with disseminated HCMV infection (antigenemia, viremia, and leukoDNAemia). The failure of laboratory-adapted HCMV strain to induce this phenomenon demonstrates that important modifications have occurred in attenuated viral strains affecting basic biological functions.

T he mutant Z form of alpha1-antitrypsin (alpha1AT) is responsible for > 95% of all individuals with alpha1AT deficiency, an important inherited cause of emphysema and liver disease. Since secreted Z alpha1AT is a functional antiprotease, we hypothesized that interrupting catabolism of retained Z alpha1AT might increase its transport out of cells, causing an increase in extracellular protease protection. Both the protein translation inhibitor cycloheximide and the specific inhibitor of proteasome function, lactacystin, prevented intracellular degradation of Z alpha1AT. Moreover, this inhibition of degradation was associated with partial restoration of Z alpha1AT vesicular transport. This effect was observed in a model system of transfected CHO cells as well as in human alveolar macrophages synthesizing Z alpha1AT. This study supports the hypothesis that altering the intracellular fate of a mutant protein may be an option in the treatment of diseases associated with misfolded but potentially functional proteins.

P revious studies of osteopetrotic (op) mice lacking macrophage colony-stimulating factor (M-CSF) have revealed an inhibition of atherosclerosis development in the apolipoprotein E (apo E)-deficient model and in a diet-induced model. Using LDL receptor-deficient mice, we now show that atheroma development depends on M-CSF concentration, as not only did homozygous osteopetrotic (op/op) mice have dramatically reduced lesions (approximately 0.3% of control lesion size) but heterozygous (op/+) mice had lesions < 1% of controls. Mice heterozygous for the op mutation (op/+) had plasma levels of M-CSF about half those in controls (+/+). The finding that an approximately 2-fold reduction in M-CSF expression reduced lesion size approximately 100-fold suggests the requirement for a threshold level of M-CSF. The effect of M-CSF on atherosclerosis did not appear to be mediated either by changes in plasma lipoprotein levels or alterations in the number of circulating monocytes, since both op/op and op/+ mice exhibited higher levels of atherogenic lipoprotein particles and (op/+) mice showed a near normal number of circulating monocytes. LDL receptor-null littermates of genotypes from op/op, op/+, to +/+ showed monocyte differentials of approximately 4.5, 8, and 10%, respectively. Taken together, these results suggest that the effects of M-CSF on atherogenesis may not be mediated by expression of M-CSF systemically or by modulation of the number of circulating monocytes. These studies support the conclusion that M-CSF participates critically in fatty streak formation and progression to a complex fibrous lesion.

E ndothelial dysfunction associated with atherosclerosis has been attributed to alterations in the L-arginine-nitric oxide (NO)-cGMP pathway or to an excess of endothelin-1 (ET-1). The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) have been shown to ameliorate endothelial function. However, the physiological basis of this observation is largely unknown. We investigated the effects of Atorvastatin and Simvastatin on the pre-proET-1 mRNA expression and ET-1 synthesis and on the endothelial NO synthase (eNOS) transcript and protein levels in bovine aortic endothelial cells. These agents inhibited pre-proET-1 mRNA expression in a concentration- and time-dependent fashion (60-70% maximum inhibition) and reduced immunoreactive ET-1 levels (25-50%). This inhibitory effect was maintained in the presence of oxidized LDL (1-50 microg/ml). No significant modification of pre-proET-1 mRNA half-life was observed. In addition, mevalonate, but not cholesterol, reversed the statin-mediated decrease of pre-proET-1 mRNA levels. eNOS mRNA expression was reduced by oxidized LDL in a dose-dependent fashion (up to 57% inhibition), whereas native LDL had no effect. Statins were able to prevent the inhibitory action exerted by oxidized LDL on eNOS mRNA and protein levels. Hence, these drugs might influence vascular tone by modulating the expression of endothelial vasoactive factors.

T he potential negative impact of HLA class I antigen abnormalities on the outcome of T cell-based immunotherapy of melanoma has prompted us to investigate the mechanisms underlying lack of HLA class I antigen expression by melanoma cell lines Me18105, Me9923, and Me1386. Distinct mutations in the beta2-microglobulin (beta2m) gene were identified in each cell line which result in loss of functional beta2m. In Me18105 cells, an aberrant splicing mechanism caused by an A--> G point mutation in the splice acceptor site of intron 1 of the beta2m gene, deletes 11 bp from the beta2m mRNA creating a shift in the reading frame. In Me9923 cells a 14-bp deletion in exon 2 and in Me1386 cells a CT deletion in exon 1 of the beta2m gene produce a frameshift mutation. The beta2m gene mutations identified in Me18105, Me9923, and Me1386 cells were also detected in the surgically removed melanoma lesions from which the cell lines originated. Transfection of each melanoma cell line with a wild-type beta2m gene restored HLA class I antigen expression and, in Me18105 cells, recognition by Melan-A/MART-1-specific, HLA-A2-restricted cytotoxic T lymphocytes. Interestingly, the beta2m mutation present in Me9923 cells that were derived from a metastatic lesion was also found in the Me9923P cell line that originated from the autologous primary lesion. These data suggest that beta2m mutations in melanoma cells may be an early event in progression to the malignant phenotype.

S ickle red blood cells (RBC) are subject to a number of important cellular changes and selection pressures. In this study, we validated a biotin RBC label by comparison to the standard 51Cr label, and used it to study changes that occur in sickle cells as they age. Sickle RBC had a much shorter lifespan than normal RBC, but the two labels gave equivalent results for each cell type. A variable number of sickle, but not normal, RBC disappeared from the circulation during the first few hours after reinfusion. The number of biotinylated sickle reticulocytes was decreased by 50% after 24 h and 75% after 48 h, with a gradual decrease in the amount of reticulum per cell. The labeled sickle cells exhibited major density increases during the first 4-6 d after reinfusion, with smaller changes thereafter. A small population of very light, labeled sickle RBC was essentially constant in number after the first few days. Fetal hemoglobin (HbF) content was determined in isolated biotinylated sickle RBC after reinfusion, allowing an estimate of lifespan for RBC containing HbF (F cells) and non-F cells. The lifespan of sickle biotinylated RBC lacking HbF was estimated to be approximately 2 wk, whereas F cells survived 6-8 wk.

L iddle syndrome is an autosomal dominant form of hypertension resulting from deletion or missense mutations of a PPPxY motif in the cytoplasmic COOH terminus of either the beta or gamma subunit of the epithelial Na channel (ENaC). These mutations lead to increased channel activity. In this study we show that wild-type ENaC is downregulated by intracellular Na+, and that Liddle mutants decrease the channel sensitivity to inhibition by intracellular Na+. This event results at high intracellular Na+ activity in 1.2-2.4-fold higher cell surface expression, and 2.8-3.5-fold higher average current per channel in Liddle mutants compared with the wild type. In addition, we show that a rapid increase in the intracellular Na+ activity induced downregulation of the activity of wild-type ENaC, but not Liddle mutants, on a time scale of minutes, which was directly correlated to the magnitude of the Na+ influx into the oocytes. Feedback inhibition of ENaC by intracellular Na+ likely represents an important cellular mechanism for controlling Na+ reabsorption in the distal nephron that has important implications for the pathogenesis of hypertension.

P DGF stimulates tyrosine phosphorylation of Janus kinase 1 (JAK1) and the signal transducer and activator of transcription 1 (STAT1alpha). However, it is not known whether JAKs are required for STAT1alpha phosphorylation or if the PDGF receptor itself can directly tyrosine phosphorylate and activate STAT1alpha. In vitro immunecomplex kinase assay of PDGF beta receptor (PDGFR) or STAT1alpha immunoprecipitates from lysates of mesangial cells treated with PDGF showed phosphorylation of a 91- and an 185-kD protein. Incubation of lysates prepared from quiescent mesangial cells with purified PDGFR resulted in STAT1alpha activation. Immunodepletion of Janus kinases from the cell lysate before incubation with the purified PDGFR showed no effect on STAT1alpha activation. Moreover, lysates from mesangial cells treated with JAK2 inhibitor, retained significant STAT1alpha activity. To confirm that STAT1alpha is a substrate for PDGFR, STAT1alpha protein was prepared by in vitro transcription and translation. The addition of purified PDGFR to the translated STAT1alpha resulted in its phosphorylation. This in vitro phosphorylated and activated protein also forms a specific protein-DNA complex. Dimerization of the translated STAT1alpha protein was also required for its DNA binding. Incubation of pure STAT1alpha with autophosphorylated PDGFR resulted in physical association of the two proteins. These data indicate that activated PDGFR may be sufficient to tyrosine phosphorylate and thus directly activate STAT1alpha.

D elta-aminolevulinic acid (ALA) is the precursor of porphyrin synthesis and has been recently used in vitro and in clinical studies as an endogenous photosensitizer for photodynamic therapy in the treatment of various tumors. For this purpose, ALA is given topically, systemically, or orally. When administered by the oral route, it shows excellent intestinal absorption. ALA is also efficiently reabsorbed in the renal proximal tubule after glomerular filtration. However, the pathways and mechanisms for its transmembrane transport into epithelial cells of intestine and kidney are unknown. Here we demonstrate that ALA uses the intestinal and renal apical peptide transporters for entering into epithelial cells. Kinetics and characteristics of ALA transport were determined in Xenopus laevis ooyctes and Pichia pastoris yeast cells expressing either the cloned intestinal peptide transporter PEPT1 or the renal form PEPT2. By using radiolabeled ALA and electrophysiological techniques in these heterologous expression systems, we established that: (a) PEPT1 and PEPT2 translocate 3H-ALA by saturable and pH-dependent transport mechanisms, (b) that ALA and di-/tripeptides, but not GABA or related amino acids, compete at the same substrate-binding site of the carriers, and (c) that ALA transport is electrogenic in nature as a consequence of H+/ALA cotransport. Reverse transcriptase-PCR analysis performed with specific primers for PEPT1 and PEPT2 in rabbit tissues demonstrates that, in particular, the PEPT2 mRNA is expressed in a variety of other tissues including lung, brain, and mammary gland, which have been shown to accumulate ALA. This suggests that these tissues could take up the porphyrin precusor via expressed peptide transporters, providing the endogenous photosensitizers for efficient photodynamic therapy.

E xcessive neutrophil activation causes posttraumatic complications, which may be reduced with hypertonic saline (HS) resuscitation. We tested if this is because of modulated neutrophil function by HS. Clinically relevant hypertonicity (10-25 mM) suppressed degranulation and superoxide formation in response to fMLP and blocked the activation of the mitogen activated protein kinases (MAPK) ERK1/2 and p38, but did not affect Ca2+ mobilization. HS did not suppress oxidative burst in response to phorbol myristate acetate (PMA). This indicates that HS suppresses neutrophil function by intercepting signal pathways upstream of or apart from PKC. HS activated p38 by itself and enhanced degranulation in response to PKC activation. This enhancement was reduced by inhibition of p38 with SB203580, suggesting that p38 up-regulation participates in HS-induced enhancements of degranulation. HS had similar effects on the degranulation of cells that were previously stimulated with fMLP, but had no effect on its own, suggesting that HS enhancement of degranulation requires another signal. We conclude that depending on other stimuli, HS can suppress neutrophil activation by intercepting multiple receptor signals or augment degranulation by enhancing p38 signaling. In patients HS resuscitation may reduce posttraumatic complications by preventing neutrophil activation via chemotactic factors released during reperfusion.

H ypertension is often associated with the development of nephroangio- and glomerulo-sclerosis. This pathophysiological process is due to increased extracellular matrix protein, particularly type I collagen, accumulation. This study investigated whether nitric oxide (NO) synthesis is involved in the mechanism(s) regulating activation of the collagen I gene in afferent arterioles and glomeruli. Experiments were performed on transgenic mice harboring the luciferase gene under the control of the collagen I-alpha2 chain promoter [procolalpha2(I)]. Measurements of luciferase activity provide highly sensitive estimates of collagen I gene activation. NO synthesis was inhibited by NG-nitro-L-arginine methyl ester (L-NAME) (20 mg/kg per day) for a period of up to 14 wk. Systolic blood pressure was increased after 6 wk of treatment (117+/-2 versus 129+/-2 mmHg, P < 0.01) and reached a plateau after 10 wk (around 160 mmHg). Luciferase activity was increased in freshly isolated afferent arterioles and glomeruli as early as week 4 of L-NAME treatment (150 and 200% of baseline, P < 0.01, respectively). The activation of procolalpha2(I) became more pronounced with time, and at 14 wk increased four- and tenfold compared with controls in afferent arterioles and glomeruli, respectively (P < 0.001). In contrast, luciferase activity remained unchanged in aorta and heart up to 8 wk and was increased thereafter. Increased histochemical staining for extracellular matrix deposition, and particularly of collagen I, was detected in afferent arterioles and glomeruli after 10 wk of L-NAME treatment. This fibrogenic process was accompanied by an increased urinary excretion rate of endothelin. In separate experiments, the stimulatory effect of L-NAME on collagen I gene activation was abolished when animals were treated with bosentan, an endothelin receptor antagonist. Similarly, bosentan reduced the increased extracellular matrix deposition in afferent arterioles and glomeruli during NO inhibition. Interestingly, bosentan had no effect on the L-NAME- induced increase of systolic pressure. These data indicate that NO inhibition induces an early activation of the collagen I gene in afferent arterioles and glomeruli. This activation in the kidney precedes the increase in blood pressure and the procolalpha2(I) activation in heart and aorta, suggesting a specific renal effect of NO blockade on collagen I gene expression that is independent of increased blood pressure and, at least partly, mediated through stimulation of the endothelin receptor. Use of procolalpha2(I) transgenic mice provides a novel and efficient model to study the pathophysiological mechanism(s) regulating renal fibrosis.

T he hydrophilic bile salt ursodeoxycholic acid (UDCA) protects against the membrane-damaging effects associated with hydrophobic bile acids. This study was undertaken to (a) determine if UDCA inhibits apoptosis from deoxycholic acid (DCA), as well as from ethanol, TGF-beta1, Fas ligand, and okadaic acid; and to (b) determine whether mitochondrial membrane perturbation is modulated by UDCA. DCA induced significant hepatocyte apoptosis in vivo and in isolated hepatocytes determined by terminal transferase-mediated dUTP-digoxigenin nick end-labeling assay and nuclear staining, respectively (P < 0.001). Apoptosis in isolated rat hepatocytes increased 12-fold after incubation with 0.5% ethanol (P < 0.001). HuH-7 cells exhibited increased apoptosis with 1 nM TGF-beta1 (P < 0. 001) or DCA at >/= 100 microM (P < 0.001), as did Hep G2 cells after incubation with anti-Fas antibody (P < 0.001). Finally, incubation with okadaic acid induced significant apoptosis in HuH-7, Saos-2, Cos-7, and HeLa cells. Coadministration of UDCA with each of the apoptosis-inducing agents was associated with a 50-100% inhibition of apoptotic changes (P < 0.001) in all the cell types. Also, UDCA reduced the mitochondrial membrane permeability transition (MPT) in isolated mitochondria associated with both DCA and phenylarsine oxide by > 40 and 50%, respectively (P < 0.001). FACS(R) analysis revealed that the apoptosis-inducing agents decreased the mitochondrial transmembrane potential and increased reactive oxygen species production (P < 0.05). Coadministration of UDCA was associated with significant prevention of mitochondrial membrane alterations in all cell types. The results suggest that UDCA plays a central role in modulating the apoptotic threshold in both hepatocytes and nonliver cells, and inhibition of MPT is at least one pathway by which UDCA protects against apoptosis.

M utations in multiple cardiac sarcomeric proteins including myosin heavy chain (MyHC) and cardiac troponin T (cTnT) cause a dominant genetic heart disease, familial hypertrophic cardiomyopathy (FHC). Patients with mutations in these two genes have quite distinct clinical characteristics. Those with MyHC mutations demonstrate more significant and uniform cardiac hypertrophy and a variable frequency of sudden death. Patients with cTnT mutations generally exhibit mild or no hypertrophy, but a high frequency of sudden death at an early age. To understand the basis for these distinctions and to study the pathogenesis of the disease, we have created transgenic mice expressing a truncated mouse cTnT allele analogous to one found in FHC patients. Mice expressing truncated cTnT at low (< 5%) levels develop cardiomyopathy and their hearts are significantly smaller (18-27%) than wild type. These animals also exhibit significant diastolic dysfunction and milder systolic dysfunction. Animals that express higher levels of transgene protein die within 24 h of birth. Transgenic mouse hearts demonstrate myocellular disarray and have a reduced number of cardiac myocytes that are smaller in size. These studies suggest that multiple cellular mechanisms result in the human disease, which is generally characterized by mild hypertrophy, but, also, frequent sudden death.

T he calcium-sensing receptor (CaSR) regulates PTH secretion to control the extracellular calcium concentration in adults, but its role in fetal life is unknown. We used CaSR gene knockout mice to investigate the role of the CaSR in regulating fetal calcium metabolism. The normal calcium concentration in fetal blood is raised above the maternal level, an increase that depends upon PTH-related peptide (PTHrP). Heterozygous (+/-) and homozygous (-/-) disruption of the CaSR caused a further increase in the fetal calcium level. This increase was modestly blunted by concomitant disruption of the PTHrP gene and completely reversed by disruption of the PTH/ PTHrP receptor gene. Serum levels of PTH and 1, 25-dihydroxyvitamin D were substantially increased above the normal low fetal levels by disruption of the CaSR. The free deoxypyridinoline level was increased in the amniotic fluid (urine) of CaSR-/- fetuses; this result suggests that fetal bone resorption is increased. Placental calcium transfer was reduced, and renal calcium excretion was increased, by disruption of the CaSR. These studies indicate that the CaSR normally suppresses PTH secretion in the presence of the normal raised (and PTHrP-dependent) fetal calcium level. Disruption of the CaSR causes fetal hyperparathyroidism and hypercalcemia, with additional effects on placental calcium transfer.

S tudies on murine B lymphocytes showed that Bruton's tyrosine kinase mediates signal transduction induced via CD38, a nonlineage-restricted 45-kD ectoenzyme. This signaling is defective in B cells from X-linked immunodeficient mice affected with the analogue of human X-linked agammaglobulinemia (XLA). We performed a structural and functional analysis of CD38 in XLA and other immunodeficiencies, using EBV-immortalized B cells derived from such patients. Membrane CD38 was not significantly different from controls in structure, epitope density, enzymatic activity, and internalization upon binding of agonistic mAbs. Meanwhile, an increased release of soluble CD38 from XLA cells was observed: immunoprecipitation from XLA culture media yielded a protein of approximately 78 kD (p78), reacting also in Western blot and displaying both enzymatic activities and a peptide map similar to membrane CD38. Soluble forms and homotypic aggregations of CD38 were documented in different cell models and by crystallographic analysis of the Aplysia ADP-ribosyl cyclase, the ancestor of human CD38. p78 might represent the product of an altered turn-over of membrane CD38, a starting point for studying its association with Bruton's tyrosine kinase and its role in XLA and other B cell immunodeficiencies.

P rotein C deficiency results in a thrombotic disorder that might be treated by expressing a normal human protein C (hPC) gene in patients. An amphotropic retroviral vector with a liver-specific promoter and the hPC cDNA was delivered to rat hepatocytes in vivo during liver regeneration. Expression of hPC varied from 55 to 203 ng/ml (1.3-5.0% of normal) for 2 wk after transduction. Expression increased to an average of 900 ng/ml (22% of normal) in some rats and was maintained at stable levels for 1 yr. All of these rats developed anti-hPC antibodies and exhibited a prolonged hPC half-life in vivo. The hPC was functional as determined by a chromogenic substrate assay after immunoprecipitation. We conclude that most rats achieved hPC levels that would prevent purpura fulminans, and that hepatic gene therapy might become a viable treatment for patients with severe homozygous hPC deficiency. Anti-hPC antibodies increased the hPC half-life and plasma levels in some rats, but did not interfere with its functional activity. Thus, the development of antibodies against a plasma protein does not necessarily abrogate its biological effect in gene therapy experiments.

P latinum compounds induce apoptosis in malignant cells and are used extensively in the treatment of cancer. Total dose is limited by development of a sensory neuropathy. We now demonstrate that when rats are administered cisplatin (2 mg/kg i.p. for 5 d), primary sensory neurons in the dorsal root ganglion die by apoptosis. This was reproduced by exposure of dorsal root ganglion neurons and PC12 cells to cisplatin (3 microg/ml) in vitro. Apoptosis was confirmed by electron microscopy, DNA laddering, and inhibition by the caspase inhibitor z-VAD.fmk (100 microM). Cell death in vitro was preceded by upregulation of cyclin D1, cdk4, and increased phosphorylation of retinoblastoma protein; all are indicators of cell cycle advancement. The level of p16(INK4a), an endogenous inhibitor of the cyclin D1/cdk4 complex decreased. Exposure of PC12 cells and dorsal root ganglion neurons to increased levels of nerve growth factor (100 ng/ ml) prevented both apoptosis and upregulation of the cell cycle markers. Cancer cells without nerve growth factor receptors (gp140TrkA) were not protected by the neurotrophin. This indicated that cisplatin may kill cancer cells and neurons by a similar mechanism. In postmitotic neurons, this involves an attempt to re-enter the cell cycle resulting in apoptosis which is specifically prevented by nerve growth factor.

T he classical estrogen receptor ERalpha mediates many of the known cardiovascular effects of estrogen and is expressed in male and female vascular cells. Estrogen-independent activation of ERalpha is known to occur in cells from reproductive tissues, but has not been investigated previously in vascular cells. In this study, transient transfection assays in human saphenous vein smooth muscle cells (HSVSMC) and pulmonary vein endothelial cells (PVEC) demonstrated ERalpha-dependent activation of estrogen response element-based, and vascular endothelial growth factor-based reporter plasmids by both estrogen-deficient FBS (ED-FBS) and EGF. In nonvascular cells, ERalpha-mediated gene expression can be activated via mitogen-activated protein (MAP) kinase- induced phosphorylation of serine 118 of ERalpha. However, in vascular cells, we found that pharmacologic inhibition of MAP kinase did not alter EGF-mediated ERalpha activation. In addition, a mutant ER containing an alanine-for-serine substitution at position 118 was activated to the same degree as the wild-type receptor by ED-FBS and EGF in both HSVSMC and PVEC. Furthermore, constitutively active MAP kinase kinase (MAPKK) activated ERalpha in Cos1 cells as expected, but MAPKK inhibited ER activation in PVEC. We conclude that growth factors also stimulate ERalpha-mediated gene expression in vascular cells, but find that this occurs via a MAP kinase-independent pathway distinct from that reported previously in nonvascular cells.

I diopathic ventricular tachycardia is a generic term that describes the various forms of ventricular arrhythmias that occur in patients without structural heart disease and in the absence of the long QT syndrome. Many of these tachycardias are focal in origin, localize to the right ventricular outflow tract (RVOT), terminate in response to beta blockers, verapamil, vagal maneuvers, and adenosine, and are thought to result from cAMP-mediated triggered activity. DNA was prepared from biopsy samples obtained from myocardial tissue from a patient with adenosine-insensitive idiopathic ventricular tachycardia arising from the RVOT. Genomic sequences of the inhibitory G protein Galphai2 were determined after amplification by PCR and subcloning. A point mutation (F200L) in the GTP binding domain of the inhibitory G protein Galphai2 was identified in a biopsy sample from the arrhythmogenic focus. This mutation was shown to increase intracellular cAMP concentration and inhibit suppression of cAMP by adenosine. No mutations were detected in Galphai2 sequences from myocardial tissue sampled from regions remote from the origin of tachycardia, or from peripheral lymphocytes. These findings suggest that somatic cell mutations in the cAMP-dependent signal transduction pathway occurring during myocardial development may be responsible for some forms of idiopathic ventricular tachycardia.

M acrophage migration inhibitory factor (MIF) is a potent proinflammatory mediator that has been shown to potentiate lethal endotoxemia and to play a potentially important regulatory role in human acute respiratory distress syndrome (ARDS). We have investigated whether eosinophils are an important source of MIF and whether MIF may be involved in the pathophysiology of asthma. Unstimulated human circulating eosinophils were found to contain preformed MIF. Stimulation of human eosinophils with phorbol myristate acetate in vitro yielded significant release of MIF protein. For example, eosinophils stimulated with phorbol myristate acetate (100 nM, 8 h, 37 degreesC) released 1,539+/-435 pg/10(6) cells of MIF, whereas unstimulated cells released barely detectable levels (< 142 pg/10(6) cells, mean+/-SEM, n = 8). This stimulated release was shown to be (a) concentration- and time-dependent, (b) partially blocked by the protein synthesis inhibitor cycloheximide, and (c) significantly inhibited by the protein kinase C inhibitor Ro-31,8220. In addition, we show that the physiological stimuli C5a and IL-5 also cause significant MIF release. Furthermore, bronchoalveolar lavage fluid obtained from asthmatic patients contains significantly elevated levels of MIF as compared to nonatopic normal volunteers (asthmatic, 797.5+/-92 pg/ml; controls, 274+/-91 pg/ml). These results highlight the potential importance of MIF in asthma and other eosinophil-dependent inflammatory disorders.

I n phenylketonuria (PKU), the enzyme phenylalanine hydroxylase is deficient, resulting in a decreased conversion of phenylalanine (Phe) into tyrosine (Tyr). The severity of the disease is expressed as the tolerance for Phe at 5 yr of age. In PKU patients it is assumed that the decreased conversion of Phe into Tyr is directly correlated with the tolerance for Phe. We investigated this correlation by an in vivo stable isotope study. The in vivo residual hydroxylation was quantitated using a primed continuous infusion of L-[ring- 2H5]Phe and L-[1-13C]Tyr and the determination of the isotopic enrichments of L-[ring-2H5]Phe, L-[ring-2H4]Tyr, and L-[1-13C]Tyr in plasma. Previous reports by Thompson and coworkers (Thompson, G.N., and D. Halliday. 1990. J. Clin. Invest. 86:317-322; Thompson, G.N., J.H. Walter, J.V. Leonard, and D. Halliday. 1990. Metabolism. 39:799-807; Treacy, E., J.J. Pitt, K. Seller, G.N. Thompson, S. Ramus, and R.G.H. Cotton. 1996. J. Inherited Metab. Dis. 19:595- 602), applying the same technique, showed normal in vivo hydroxylation rates of Phe in almost all PKU patients. Therefore, our study was divided up in two parts. First, the method was re-evaluated. Second, the correlation between the in vivo hydroxylation of Phe and the tolerance for Phe was tested in seven classical PKU patients. Very low (0.13- 0.95 micromol/kg per hour) and normal (4.11 and 6.33 micromol/kg per hour) conversion rates were found in patients and controls, respectively. Performing the infusion study twice in the same patient and wash-out studies of the labels at the end of the experiment in a patient and control showed that the method is applicable in PKU patients and gives consistent data. No significant correlation was observed between the in vivo hydroxylation rates and the tolerances. The results of this study, therefore, showed that within the group of patients with classical PKU, the tolerance does not depend on the in vivo hydroxylation.

I on and fluid transport across the biliary epithelium contributes to bile secretion. Since endothelin (ET)-1 affects ion transport activities and is released by human gallbladder- derived biliary epithelial cells in primary culture, we examined the expression of ET peptides and ET receptors and the influence of ET-1 on ion transport in this epithelium ex vivo. In freshly isolated gallbladder epithelial cells, preproET-1, -2, and -3 mRNAs were detected by reverse transcription PCR and ET-1 isopeptide was identified by chromatography. The cells also displayed ET receptor mRNAs and high-affinity binding sites for ET-1, mostly of the ETB type. Electrogenic anion secretion across intact gallbladder mucosa was stimulated by forskolin, secretin, and exogenous ATP, as assessed by short-circuit current (Isc) increases in Ussing-type chambers. ET-1 inhibited forskolin- and secretin-induced changes in Isc, without affecting baseline Isc or ATP-induced changes. Accordingly, ET-1 significantly reduced the accumulation of intracellular cAMP elicited by forskolin and secretin in the epithelial cells, and this effect was abolished by pertussis toxin. This is the first evidence that ET-1 is synthesized and inhibits, via a Gi protein-coupled receptor, cAMP-dependent anion secretion in human gallbladder epithelium, indicating a role in the control of bile secretion by an autocrine/paracrine mechanism.

B ased on successful induction of donor-specific unresponsiveness by alloantigenic stimulation in several animal models of acute rejection, we hypothesized that similar immune manipulations would also inhibit the evolution of chronic rejection and transplant vasculopathy. To induce immune tolerance, DA rats received a PVG heart allograft and were immunosuppressed with cyclosporine for 30 d. At day 100 the animals were challenged with a PVG aortic allograft after either 1 or 18 h of cold ischemia. 8 wk after the aortic transplantation, the grafts were investigated for morphological changes, infiltrating cells, apoptosis, and Fas-Fas ligand expression. Control allografts showed advanced transplant arteriosclerosis, whereas tolerance-induced aortic allografts displayed reduced neointimal formation, less medial atrophy, fewer apoptotic cells, and fewer Fas- and FasL-expressing cells. Prolonged ischemic storage time did not profoundly alter the morphological changes of the allografts. Fas expression was found in T cells, macrophages, vascular smooth muscle cells, and endothelial cells, whereas FasL was expressed mainly by T cells and macrophages. FasL mRNA expression was evident throughout the entire allograft wall. In conclusion, induction of allospecific tolerance can effectively prevent transplant arteriosclerosis. Cold ischemia damage does not abrogate the beneficial effect of tolerance, but creates a separate identity of mainly endothelial lesions. Furthermore, Fas-mediated apoptosis appears to be involved in the pathological lesions seen in chronic rejection.

M yeloperoxidase (MPO) deficiency is a common inherited disorder linked to increased susceptibility to infection and malignancy. We identified a novel missense mutation in the MPO gene at codon 173 whereby tyrosine is replaced with cysteine (Y173C) that is associated with MPO deficiency and assessed its impact on MPO processing and targeting in transfectants expressing normal or mutant proteins. Although the precursor synthesized by cells expressing the Y173C mutation (MPOY173C) was glycosylated, associated with the molecular chaperones calreticulin and calnexin, and acquired heme, it was neither proteolytically processed to mature MPO subunits nor secreted. After prolonged association with calreticulin and calnexin in the endoplasmic reticulum, MPOY173C was degraded. Furthermore, the 20S proteasome inhibitor N-acetyl-L-leucinyl-L-leucinyl-L-norleucinyl inhibited its degradation, suggesting that the proteasome mediates proteolysis of MPOY173C and, thus, participates in quality control in this novel form of hereditary MPO deficiency.

A djuvant-induced arthritis (AIA) is one of many animal models of rheumatoid arthritis, a disease characterized by a T-lymphocyte and macrophage cellular infiltrate. We have characterized the development of this disease model with respect to chemokine expression. Increased levels of two chemokines, RANTES, a T-lymphocyte and monocyte chemo-attractant, and KC a chemoattractant for neutrophils, were found in whole blood and in the joint. Surprisingly, levels of MIP-1alpha, another T-lymphocyte and monocyte chemoattractant, were unchanged throughout the course of the disease in whole blood and only slightly elevated in the joint. RANTES expression plays an important role in the disease since a polyclonal antibody to RANTES greatly ameliorated symptoms in animals induced for AIA and was found to be as efficacious as treatment with indomethacin, a non-steroidal anti inflammatory. Polyclonal antibodies to either MIP-1alpha or KC were ineffective. This is the first report to show the importance of RANTES in the development of AIA.