Issue published July 1, 1997

K allistatin is a serine proteinase inhibitor which binds to tissue kallikrein and inhibits its activity. The aim of this study is to evaluate if kallistatin has a direct effect on the vasculature and on blood pressure homeostasis. We found that an intravenous bolus injection of human kallistatin caused a rapid, potent, and transient reduction of mean arterial blood pressure in anesthetized rats. Infusion of purified kallistatin (0.07-1.42 nmol/kg) into cannulated rat jugular vein produced a 20-85 mmHg reduction of blood pressure in a dose-dependent manner. Hoe 140, a bradykinin B2-receptor antagonist, had no effect on the hypotensive effect of kallistatin yet it abolished the blood pressure-lowering effect of kinin and kallikrein. Relaxation of isolated aortic rings by kallistatin was observed in the presence (ED50 of 3.4 x 10(-9) M) and in the absence of endothelium (ED50 of 10(-9) M). Rat kallikrein-binding protein, but not kinin or kallikrein, induced vascular relaxation of aortic rings. Neither Hoe 140 nor Nomega-nitro--arginine methyl ester, a nitric oxide synthase inhibitor, affected vasorelaxation induced by kallistatin. Kallistatin also caused dose-dependent vasodilation of the renal vasculature in the isolated, perfused rat kidney. Specific kallistatin-binding sites were identified in rat aorta by Scatchard plot analysis with a Kd of 0.25+/-0.07 nM and maximal binding capacity of 47.9+/-10.4 fmol/mg protein (mean+/-SEM, n = 3). These results indicate that kallistatin is a potent vasodilator which may function directly through a vascular smooth muscle mechanism independent of an endothelial bradykinin receptor. This study introduces the potential significance of kallistatin in directly regulating blood pressure to reduce hypertension.

V ascular endothelial growth factor (VEGF) is an endothelial cell mitogen involved in vascular development and angiogenesis. Recently we have observed increased VEGF expression in the normal myocardium after myocardial infarction in a rat heart. This study was designed to explore the mechanism responsible for this increase in VEGF expression. Induction of myocardial stretch in an isolated perfused Langendorff preparation by inflation of an intraventricular balloon to an end-diastolic load of 35 mmHg for 30 min resulted in a nearly sixfold increase in VEGF message level not only in the chamber subjected to stretch (left ventricle) but also in the unstretched right ventricle, thus raising the possibility of a soluble factor mediating stretch- induced induction of VEGF expression. This was further confirmed by demonstrating that coronary venous effluent collected from the stretched heart and used to perfuse isolated hearts in which no balloon was present was able to induce VEGF expression in these normal hearts. Inhibition of TGF-beta activity using a neutralizing antibody, but not antagonists/inhibitors of endothelin and angiotensin II, eliminated stretch-induced increase in VEGF expression. Staurosporine, a protein kinase C inhibitor, also blocked stretch-induced increase of VEGF expression. Measurement of TGF-beta concentration in the perfusate demonstrated increased amounts of the cytokine after myocardial stretch, and addition of TGF-beta protein to the perfusion buffer resulted in increased VEGF expression in control hearts. These results suggest that stretch-induced increase of VEGF expression in the heart is mediated at least in part by TGF-beta.

A unique subset of anti-DNA antibodies enters living cells, interacts with DNase 1, and inhibits endonuclease activity, before their nuclear localization and subsequent attenuation of apoptosis. We now report that endocytosis of these immunoglobulins is mediated by cell surface binding to brush border myosin (myosin 1). Cellular entry and internalization via this unique receptor provides initial contact for entry and sorting these immunoglobulins to translocate to the nuclear pore and enter the nucleus, interact with DNase 1 within the cytoplasm, or recycle back to the cell surface. This internalization pathway provides clues to the translocation of large proteins across cell membranes and the functional effects of intracellular antibodies on cytopathology. This is the first demonstration that brush border myosin functions as a specific cell surface receptor for internalization of large proteins.

T ransgenic mice carrying an HIV provirus, with selective deletion of all three structural genes, developed extensive lymphoid depletion which was detected not only in the spleen and lymph nodes but also in the thymus. Mice with a high level of HIV gene expression developed acute disease which resulted in premature death, and mice with a low level of viral transcripts developed chronic disease with long-term survival. Neither HIV replication nor the envelope glycoprotein (gp120) was required for T cell depletion. Despite abundant viral gene expression early in life, cell death did not become evident until about the time of full lymphoid maturation, suggesting that thymopoiesis was not affected. The more mature T cells in the peripheral lymphoid organs and in the thymic medulla were less sensitive to the apoptotic process than the immature T cells in the thymic cortex. Gradual depletion of the T cell compartment in the peripheral lymphoid organs was intimately accompanied by the reciprocal expansion of the B cell compartment, resulting in the almost complete replacement of T lymphocytes with B immunoblasts in lymph nodes. Unlike T cells, which showed abundant HIV gene expression, B cells did not. The transgenic approach may help identify the HIV nonstructural gene(s) responsible for immune deficiency and help facilitate dissection of its role in inducing apoptosis.

O steogenesis imperfecta (OI) is a disease attributable to any of a large number of possible mutations of type I collagen. The disease is clinically characterized in part by highly brittle bone, the cause of this feature being unknown. Recently a mouse model of OI, designated as osteogenesis imperfecta murine (oim), and having a well defined genetic mutation, has been studied and found to contain mineral crystals different in their alignment with respect to collagen and in their size. These observations are consistent with those reported in human OI and the unusual crystal alignment and size undoubtedly contribute to the reduced mechanical properties of OI bone. While the mineral has been investigated, no information is available on the tensile properties of oim collagen. In this study, the mechanical properties of tendon collagen under tension have been examined for homozygous (oim/oim), heterozygous (+/oim), and control (+/+) mice under native wet conditions. The ultimate stress and strain found for oim/oim collagen were only about half the values for control mice. Assuming that prestrained collagen molecules carry most of the tensile load in normal bone while the mineral confers rigidity and compression stability, the reported results suggest that the brittleness of OI bone in the mouse model may be related to a dramatic reduction of the ultimate tensile strain of the collagen.

T he BCR/ABL oncogene causes human chronic myelogenous leukemia (CML), a myeloproliferative disease characterized by massive expansion of hematopoietic progenitor cells and cells of the granulocyte lineage. When transfected into murine hematopoietic cell lines, BCR/ABL causes cytokine-independence and enhances viability. There is also growing evidence that p210(BCR/ABL) affects cytoskeletal structure. p210(BCR/ABL) binds to actin, and several cytoskeletal proteins are tyrosine phosphorylated by this oncoprotein. Also, at least one aspect of cytoskeletal function is abnormal, in that the affinity of beta1 integrins for fibronectin is altered in CML cells. However, isolated changes in beta1 integrin function would be unlikely to explain the clinical phenotype of CML. We used time-lapse video microscopy to study cell motility and cell morphology on extracellular cell matrix protein-coated surfaces of a series of cell lines before and after transformation by BCR/ABL. BCR/ABL was associated with a striking increase in spontaneous motility, membrane ruffling, formation of long actin extensions (filopodia) and accelerated the rate of protrusion and retraction of pseudopodia on fibronectin-coated surfaces. Also, while untransformed cells were sessile for long periods, BCR/ABL-transformed cells exhibited persistent motility, except for brief periods during cell division. Using cell lines transformed by a temperature-sensitive mutant of BCR/ABL, these kinetic abnormalities of cytoskeletal function were shown to require BCR/ABL tyrosine kinase activity. Similar abnormalities of cytoskeletal function on fibronectin-coated surfaces were observed when hematopoietic progenitor cells purified by CD34 selection from patients with CML were compared with CD34 positive cells from normal individuals. Interestingly, alpha-interferon treatment was found to slowly revert the abnormal motility phenotype of BCR/ABL-transformed cells towards normal. The increase in spontaneous motility and other defects of cytoskeletal function described here will be useful biological markers of the functional effects of BCR/ABL in hematopoietic cells.

T he urokinase receptor (uPAR) coordinates plasmin-mediated cell-surface proteolysis and promotes cellular adhesion via a binding site for vitronectin on uPAR. Because vitronectin also binds plasminogen activator inhibitor type 1 (PAI-1), and plasmin cleavage of vitronectin reduces PAI-1 binding, we explored the effects of plasmin and PAI-1 on the interaction between uPAR and vitronectin. PAI-1 blocked cellular binding of and adhesion to vitronectin by over 80% (IC50 approximately 5 nM), promoted detachment of uPAR-bearing cells from vitronectin, and increased cellular migration on vitronectin. Limited cleavage of vitronectin by plasmin also abolished cellular binding and adhesion and induced cellular detachment. A series of peptides surrounding a plasmin cleavage site (arginine 361) near the carboxy-terminal end of vitronectin were synthesized. Two peptides spanning res 364-380 blocked binding of uPAR to vitronectin (IC50 approximately 8-25 microM) identifying this region as an important site of uPAR-vitronectin interaction. These data illuminate a complex regulatory scheme for uPAR-dependent cellular adhesion to vitronectin: Active urokinase promotes adhesion and also subsequent detachment through activation of plasmin or complex formation with PAI-1. Excess PAI-1 may also promote migration by blocking cellular adhesion and/or promoting detachment, possibly accounting in part for the strong correlation between PAI-1 expression and tumor cell metastasis.

S ince unmethylated CpG motifs are more frequent in DNA from bacteria than vertebrates, and the unmethylated CpG motif has recently been reported to have stimulatory effects on lymphocytes, we speculated that bacterial DNA may induce inflammation in the lower respiratory tract through its content of unmethylated CpG motifs. To determine the role of bacterial DNA in lower airway inflammation, we intratracheally instilled prokaryotic and eukaryotic DNA in C3H/HeBFEJ mice and performed whole lung lavage 4 h after the exposure. Heat denatured, single stranded Escherichia coli genomic DNA (0.06 ng endotoxin/microg DNA) was compared to heat denatured, single stranded calf thymus DNA (0.007 endotoxin/microg DNA). 10 microg of bacterial DNA, in comparison to 10 microg of calf thymus DNA, resulted in a fourfold increase in the concentration of cells (P = 0.0002), a fivefold increase in the concentration of neutrophils (P = 0.0002), a 50-fold increase in the concentration of TNF-alpha (P = 0.001), and a fourfold increase in the concentration of both IL-6 (P = 0.0003) and macrophage inflammatory protein-2 (P = 0.0001) in the lavage fluid. Importantly, instillation of 0.60 ng of E. coli LPS resulted in a negligible inflammatory response. To test whether the stimulatory effects of bacterial DNA are due to its unmethylated CpG dinucleotides, we methylated the bacterial DNA and also prepared 20 base pair oligonucleotides with and without CpG motifs. In comparison to instillation of untreated bacterial DNA, methylation of the bacterial DNA resulted in a significant reduction in the concentration of cells and cytokines in the lower respiratory tract. Moreover, oligonucleotides containing embedded unmethylated CpG motifs resulted in inflammation in the lower respiratory tract that was indistinguishable from that observed with untreated bacterial DNA. In contrast, oligonucleotides without the embedded CpG motifs or with embedded but methylated CpG motifs resulted in significantly less inflammation in the lower respiratory tract. The possible relevance of these data to human disease was shown by extracting and analyzing DNA in sputum from patients with cystic fibrosis (CF). Approximately 0.1 to 1% of this sputum DNA was bacterial. Intratracheal instillation of highly purified CF sputum DNA caused acute inflammation similar to that induced by bacterial DNA. These findings suggest that bacterial DNA, and unmethylated CpG motifs in particular, may play an important pathogenic role in inflammatory lung disease.

R elationships among plasma lipoprotein cholesterol, cholesterol secretion by the isolated, perfused liver, and coronary artery atherosclerosis were examined in African green monkeys fed diets containing cholesterol and 35% of calories as fat enriched in polyunsaturated, monounsaturated, or saturated fatty acids. The livers of animals fed monounsaturated fat had significantly higher cholesteryl ester concentrations (8.5 mg/g wet wt) than the livers of the other diet groups (3.65 and 3.37 mg/g wet wt for saturated and polyunsaturated fat groups, respectively) and this concentration was highly correlated with plasma cholesterol and apoB concentrations in each diet group. Cholesteryl oleate was 58 and 74. 5% of the liver cholesteryl ester in the saturated and monounsaturated fat groups. In each diet group, perfusate cholesteryl ester accumulation rate was highly correlated to liver and plasma cholesterol concentrations, and to plasma LDL cholesteryl ester content. Cholesteryl oleate was 48 and 67% of the cholesteryl esters that accumulated in perfusate in the saturated and monounsaturated fat animals, and this percentage was very highly correlated (r = -0.9) with plasma apoB concentration. Finally, in these two diet groups, liver perfusate cholesteryl ester accumulation rate was well correlated (r >/= 0.8) to coronary artery cholesteryl ester concentration, a measure of the extent of coronary artery atherosclerosis that occurred over the five years of diet induction in these animals. These data define an important role for the liver in the cholesteryl oleate enrichment of the plasma lipoproteins in the saturated and monounsaturated fat groups, and demonstrate strong relationships among hepatic cholesteryl ester concentration, cholesteryl ester secretion, and LDL particle cholesteryl ester content. The high correlation between liver cholesteryl ester secretion and coronary artery atherosclerosis provides the first direct demonstration of the high degree of importance of hepatic cholesteryl ester secretion in the development of this disease process. The remarkable degree of enrichment of cholesteryl oleate in plasma cholesteryl esters of the monounsaturated fat group may account for the relatively high amount of coronary artery atherosclerosis in this group.

H IV-associated nephropathy (HIVAN) is a progressive glomerular and tubular disease that is increasingly common in AIDS patients and one of the leading causes of end stage renal disease in African Americans. A major unresolved issue in the pathogenesis of HIVAN is whether the kidney disease is due to renal cell infection or a "bystander" phenomenon mediated by systemically dysregulated cytokines. To address this issue, we have used two different experimental approaches and an HIV-1 transgenic mouse line that develops a progressive renal disease histologically similar to HIVAN in humans. In the murine model, kidney tissue expresses the transgene and in heterozygous adults, renal disease develops shortly thereafter. We demonstrate by terminal deoxynucleotide transferase-mediated dUTP-biotin nick-end labeling assay that similar to the disease in humans, apoptosis of renal tubular epithelial cells is a component of the molecular pathogenesis. To determine whether apoptosis is due to transgene expression or environmental factors, we treated fetal kidney explants (normal and transgenic) with UV light to induce transgene expression. Apoptosis occurred in transgenic but not normal littermates after stimulation of transgene expression. To confirm a direct effect of HIV expression on the production of HIVAN, we transplanted kidneys between normal and transgenic mice. HIVAN developed in transgenic kidneys transplanted into nontransgenic littermates. Normal kidneys remained disease free when transplanted into transgenic littermates. Thus, the renal disease in the murine model is intrinsic to the kidney. Using two different experimental approaches, we demonstrate a direct effect of transgene expression on the development of HIVAN in the mouse. These studies suggest that in humans, a direct effect of HIV-1 expression is likely the essential cause of HIVAN, rather than an indirect effect of cytokine dysregulation.

T o examine the activity of matrix metalloproteinases (MMPs) and aggrecanase in control and diseased human articular cartilage, metabolic fragments of aggrecan were detected with monospecific antipeptide antibodies. The distribution and quantity of MMP-generated aggrecan G1 fragments terminating in VDIPEN341 were compared with the distribution of aggrecanase-generated G1 fragments terminating in NITEGE373. Both types of G1 fragments were isolated from osteoarthritic cartilage. The sizes were consistent with a single enzymatic cleavage in the interglobular domain region, with no further proteolytic processing of these fragments. Both neoepitopes were also detected by immunohistochemistry in articular cartilage from patients undergoing joint replacement for osteoarthritis (OA), rheumatoid arthritis (RA), and in cartilage from adults with no known joint disease. In control specimens, the staining intensity for both G1 fragments increased with age, with little staining in cartilage from 22-wk-old fetal samples. There was also an increase with age in the extracted amount of MMP-generated neoepitope in relation to both aggrecan and collagen content, confirming the immunohistochemical results. After the age of 20-30 yr this relationship remained at a steady state. The staining for the MMP-generated epitope was most marked in control cartilage exhibiting histological signs of damage, whereas intense staining for the aggrecanase-generated fragment was often noted in adult cartilage lacking overt histological damage. Intense staining for both neoepitopes appeared in the more severely fibrillated, superficial region of the tissue. Intense immunostaining for both VDIPEN- and NITEGE- neoepitopes was also detected in joint cartilage from patients with OA or RA. Cartilage in these specimens was significantly more degraded and high levels of staining for both epitopes was always seen in areas with extensive cartilage damage. The levels of extracted VDIPEN neoepitope relative to collagen or aggrecan in both OA and RA samples were similar to those seen in age-matched control specimens. Immunostaining for both types of aggrecan fragments was seen surrounding the cells but also further removed in the interterritorial matrix. In some regions of the tissue, both neoepitopes were found while in others only one was detected. Thus, generation and/or turnover of these specific catabolic aggrecan fragments is not necessarily coordinated. Our results are consistent with the presence in both normal and arthritic joint cartilage of proteolytic activity against aggrecan based on both classical MMPs and "aggrecanase."

A polipoprotein E (apoE) plays a key role in lipoprotein metabolism and may have other important biological functions. In humans, there are three common, naturally occurring isoforms of apoE that are associated with differences in lipid levels and atherosclerosis. However, the direct in vivo effects of the apoE isoforms on lipoprotein metabolism and atherosclerosis are not yet fully understood. To investigate the effect of the apoE isoforms in vivo, we constructed second-generation recombinant adenoviruses encoding each of the apoE isoforms. These recombinant adenoviruses were injected intravenously into apoE-deficient mice fed a Western diet (mean baseline cholesterol level 1401 mg/dl) in order to study their effects in the absence of endogenous mouse apoE. Hepatic expression of apoE3 and apoE4 completely normalized the lipoprotein profile; 3 d after injection, mean plasma cholesterol levels were 194 and 217 mg/ dl, respectively, and this effect was maintained for at least 6 wk. Expression of apoE2 had much less effect on lipoprotein levels (mean cholesterol level 752 mg/dl 3 d after injection), despite much higher plasma levels of apoE2 compared with apoE3 and apoE4; by 6 wk after injection the cholesterol levels had returned to baseline levels in the apoE2-expressing mice. Expression of all three isoforms significantly increased HDL cholesterol levels by approximately threefold and was independent of the cholesterol-lowering effect. ApoE transgene expression was substantially prolonged compared with that achieved using a first generation adenovirus and apoE was readily detected in plasma 3 mo after virus injection. These studies demonstrate: (a) prolonged in vivo expression of human apoE isoforms in apoE deficient mice after second-generation recombinant adenovirus-mediated somatic gene transfer; and (b) significantly impaired ability of apoE2 in vivo to mediate clearance of remnant lipoproteins in apoE-deficient mice fed a Western diet compared with apoE3 and apoE4.

I nduction of protein kinase C (PKC) pathway in the vascular tissues by hyperglycemia has been associated with many of the cellular changes observed in the complications of diabetes. Recently, we have reported that the use of a novel, orally effective specific inhibitor of PKC beta isoform (LY333531) normalized many of the early retinal and renal hemodynamics in rat models of diabetes. In the present study, we have characterized a spectrum of biochemical and molecular abnormalities associated with chronic changes induced by glucose or diabetes in the cultured mesangial cells and renal glomeruli that can be prevented by LY333531. Hyperglycemia increased diacylglycerol (DAG) level in cultured mesangial cells exposed to high concentrations of glucose and activated PKC alpha and beta1 isoforms in the renal glomeruli of diabetic rats. The addition of PKC beta selective inhibitor (LY333531) to cultured mesangial cells inhibited activated PKC activities by high glucose without lowering DAG levels and LY333531 given orally in diabetic rats specifically inhibited the activation of PKC beta1 isoform without decreasing PKC alpha isoform activation. Glucose-induced increases in arachidonic acid release, prostaglandin E2 production, and inhibition of Na+-K+ ATPase activities in the cultured mesangial cells were completely prevented by the addition of LY333531. Oral feeding of LY333531 prevented the increased mRNA expression of TGF-beta1 and extracellular matrix components such as fibronectin and alpha1(IV) collagen in the glomeruli of diabetic rats in parallel with inhibition of glomerular PKC activity. These results suggest that the activation of PKC, predominately the beta isoform by hyperglycemia in the mesangial cells and glomeruli can partly contribute to early renal dysfunctions by alteration of prostaglandin production and Na+-K+ ATPase activity as well as the chronic pathological changes by the overexpression of TGF-beta1 and extracellular matrix components genes.

T he recently described anti-A2/RA33 autoantibodies occur in 20-40% of patients with RA, SLE, and mixed connective tissue disease (MCTD). They are directed to the A2 protein of the heterogeneous nuclear ribonucleoprotein complex (hnRNP-A2), an abundant nuclear protein associated with the spliceosome. The NH2-terminal half of the antigen contains two conserved RNA binding domains whereas its COOH-terminal part is extremely glycine-rich. The aim of this study was to characterize the autoepitopes of hnRNP-A2 and to investigate the effects of anti-A2/RA33 autoantibodies on possible functions of the antigen. Using bacterially expressed fragments, two major discontinuous epitopes were identified. One containing the complete second RNA binding domain was recognized by the majority of patients with RA and SLE but not by patients with MCTD. The second epitope contained sequences of both RNA binding domains and was preferentially targeted by patients with MCTD. When the RNA binding properties of the antigen were investigated, oligoribonucleotides containing the sequence motif r(UUAG) were found to bind to a site closely adjacent or overlapping with the epitope targeted by autoantibodies from patients with RA and SLE. Moreover, anti-A2/RA33 autoantibodies from patients with RA or SLE, but not from patients with MCTD, inhibited binding of RNA. Thus, anti-A2/RA33 autoantibodies recognize conformation-dependent epitopes located in a functionally important region of the antigen. Furthermore, the specific recognition of an epitope by MCTD patients may be used as another argument in favor of considering MCTD a distinct connective tissue disease.

T cells can recognize small molecular compounds like drugs. It is thought that covalent binding to MHC bound peptides is required for such a hapten stimulation. Sulfamethoxazole, like most drugs, is not chemically reactive per se, but is thought to gain the ability to covalently bind to proteins after intracellular drug metabolism. The purpose of this study was to investigate how sulfamethoxazole is presented in an immunogenic form to sulfamethoxazole-specific T cell clones. The stimulation of four CD4(+) and two CD8(+) sulfamethoxazole-specific T cell clones by different antigen-presenting cells (APC) was measured both by proliferation and cytolytic assays. The MHC restriction was evaluated, first, by inhibition using anti-class I and anti-class II mAb, and second, by the degree of sulfamethoxazole-induced stimulation by partially matched APC. Fixation of APC was performed with glutaraldehyde 0.05%. The clones were specific for sulfamethoxazole without cross-reaction to other sulfonamides. The continuous presence of sulfamethoxazole was required during the assay period since pulsing of the APC was not sufficient to induce proliferation or cytotoxicity. Stimulation of clones required the addition of MHC compatible APC. The APC could be fixed without impairing their ability to present sulfamethoxazole. Sulfamethoxazole can be presented in an unstable, but MHC-restricted fashion, which is independent of processing. These features are best explained by a direct, noncovalent binding of sulfamethoxazole to the MHC-peptide complex.

L ocalized inflammation of a rat's hindpaw elicits an accumulation of beta-endorphin-(END) containing immune cells. We investigated the production, release, and antinociceptive effects of lymphocyte-derived END in relation to cell trafficking. In normal animals, END and proopiomelanocortin mRNA were less abundant in circulating lymphocytes than in those residing in lymph nodes (LN), suggesting that a finite cell population produces END and homes to LN. Inflammation increased proopiomelanocortin mRNA in cells from noninflamed and inflamed LN. However, END content was increased only in inflamed paw tissue and noninflamed LN-immune cells. Accordingly, corticotropin-releasing factor and IL-1beta released significantly more END from noninflamed than from inflamed LN-immune cells. This secretion was receptor specific, calcium dependent, and mimicked by potassium, consistent with vesicular release. Finally, both agents, injected into the inflamed paw, induced analgesia which was blocked by the co-administration of antiserum against END. Together, these findings suggest that END-producing lymphocytes home to inflamed tissue where they secrete END to reduce pain. Afterwards they migrate to the regional LN, depleted of the peptide. Consistent with this notion, immunofluorescence studies of cell suspensions revealed that END is contained predominantly within memory-type T cells. Thus, the immune system is important for the control of inflammatory pain. This has implications for the understanding of pain in immunosuppressed conditions like cancer or AIDS.

D ecorin belongs to a family of secreted, small, leucine-rich proteoglycans that affect matrix assembly and cellular growth. Ectopic expression of decorin proteoglycan or protein core as a mutated form lacking any glycosaminoglycan side chains induced growth suppression in neoplastic cells of various histogenetic origins, including tumor cells derived from gastrointestinal, genital, skeletal, cutaneous, or bone marrow tissues. Exogenously added recombinant decorin also suppressed overall growth of the parental cell lines. In all stably-transfected clones, growth retardation was specifically associated with induction of the potent cyclin-dependent kinase inhibitor p21, but not p27, and subsequent translocation of p21 protein into the nuclei of decorin-expressing cells. This led to a greater proportion of the cells arrested in G1 phase of the cell cycle. These changes were independent of functional p53 or retinoblastoma protein. De novo expression of decorin in HCT116 human colon carcinoma cells harboring a disrupted p21 gene failed to induce growth suppression, in contrast to the wild-type cells in which p21 and growth arrest could be induced. These findings indicate that ectopic production of decorin protein core can retard the growth of a variety of tumor cells and that endogenous p21 is a required downstream effector of this biological axis.

O ncostatin M is a member of the IL-6 family of cytokines that is primarily known for its effects on cell growth. Endothelial cells have an abundance of receptors for oncostatin M, and may be its primary target. We determined if oncostatin M induces a key endothelial cell function, initiation of the inflammatory response. We found that subcutaneous injection of oncostatin M in mice caused an acute inflammatory reaction. Oncostatin M in vitro stimulated: (a) polymorphonuclear leukocyte (PMN) transmigration through confluent monolayers of primary human endothelial cells; (b) biphasic PMN adhesion through rapid P-selectin expression, and delayed adhesion mediated by E-selectin synthesis; (c) intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 accumulation; and (d) the expression of PMN activators IL-6, epithelial neutrophil activating peptide-78, growth-related cytokine alpha and growth-related cytokine beta without concomitant IL-8 synthesis. The nature of the response to oncostatin M varied with concentration, suggesting high and low affinity oncostatin M receptors independently stimulated specific responses. Immunohistochemistry showed that macrophage-like cells infiltrating human aortic aneurysms expressed oncostatin M, so it is present during a chronic inflammatory reaction. Therefore, oncostatin M, but not other IL-6 family members, fulfills Koch's postulates as an inflammatory mediator. Since its effects on endothelial cells differ significantly from established mediators like TNFalpha, it may uniquely contribute to the inflammatory cycle.

M ouse models of human disease can be generated by homologous recombination for germline loss-of-function mutations. However, embryonic-lethal phenotypes and systemic, indirect dysfunction can confound the use of knock-outs to elucidate adult pathophysiology. Site-specific recombination using Cre recombinase can circumvent these pitfalls, in principle, enabling temporal and spatial control of gene recombination. However, direct evidence is lacking for the feasibility of Cre-mediated recombination in postmitotic cells. Here, we exploited transgenic mouse technology plus adenoviral gene transfer to achieve Cre-mediated recombination in cardiac muscle. In vitro, Cre driven by cardiac-specific alpha-myosin heavy chain (alphaMyHC) sequences elicited recombination selectively at loxP sites in purified cardiac myocytes, but not cardiac fibroblasts. In vivo, this alphaMyHC-Cre transgene elicited recombination in cardiac muscle, but not other organs, as ascertained by PCR analysis and localization of a recombination-dependent reporter protein. Adenoviral delivery of Cre in vivo provoked recombination in postmitotic, adult ventricular myocytes. Recombination between loxP sites was not detected in the absence of Cre. These studies demonstrate the feasibility of using Cre-mediated recombination to regulate gene expression in myocardium, with efficient induction of recombination even in terminally differentiated, postmitotic muscle cells. Moreover, delivery of Cre by viral infection provides a simple strategy to control the timing of recombination in myocardium.

A ll classes of lipoproteins considered to be atherogenic contain apo-B100 or apo-B48. However, there is a distinct paucity of data regarding whether lipoproteins containing apo-B48 or apo-B100 differ in their intrinsic ability to promote the development of atherosclerosis. To address this issue, we compared the extent of atherosclerosis in three groups of animals: apo-E-deficient mice (apo-B+/+apo-E-/-) and apo-E-deficient mice that synthesize exclusively either apo-B48 (apo-B48/48apo-E-/-) or apo-B100 (apo-B100/100apo-E-/-). Mice (n = 25 in each group) were fed a chow diet for 200 days, and plasma lipid levels were assessed throughout the study. Compared with the levels in apo-B+/+apo-E-/- mice, the total plasma cholesterol levels were higher in the apo-B48/48apo-E-/- mice and were lower in the apo-B100/100apo-E-/- mice. However, the ranges of cholesterol levels in the three groups overlapped. Compared with those in the apo-B+/+apo-E-/- mice, atherosclerotic lesions were more extensive in the apo-B48/48apo-E-/- mice and less extensive in the apo-B100/100apo-E-/- mice. Once again, however, there was overlap among the three groups. The extent of atherosclerosis in each group of mice correlated significantly with plasma cholesterol levels. In mice from different groups that had similar cholesterol levels, the extent of atherosclerosis was quite similar. Thus, susceptibility to atherosclerosis was dependent on total cholesterol levels. Whether mice synthesized apo-B48 or apo-B100 did not appear to have an independent effect on susceptibility to atherosclerosis.

G alanin mRNA and peptide are not detectable in normal islets. We studied the effect of galanin antagonists on insulin secretion in the rat beta cell line, RIN5AH, and in perifused rat islets. In RIN cell membranes galanin and its antagonists showed high affinity for 125I-galanin binding sites [Kd: (galanin) 0.03+/-0.01; Ki for galanin antagonists: (C7) 0.12+/- 0.02, (M35) 0.21+/-0.04, and (M40) 0.22+/-0.03 nM, mean+/- SEM, n = 4]. Galanin (1 microM) inhibited glucose-induced insulin release in islets (control 21.2+/-1.5 vs. galanin 4.5+/-0.2 fmol/islet per min, P < 0.001, n = 6) and RIN5AH cells (control 0.26+/-0.01 vs. galanin 0.15+/-0.02 pmol/10(6) cells per h, P < 0.001, n = 9). In RIN5AH cells, all antagonists blocked the inhibitory effects of galanin and stimulated insulin release in the absence of galanin. C7 and M40 (1 microM) alone significantly stimulated glucose-induced insulin secretion. Purified porcine galanin antibody (GAb) enhanced glucose-induced insulin release from islets (control 100+/- 16.3% vs. GAb 806.1+/-10.4%, P < 0.001, n = 6), and RIN5AH cells (control 100+/-9.6% vs. GAb 149+/-6.8%, P < 0. 01, n = 6). Western blotting of dexamethasone-treated islet extracts using GAb showed a specific band of similar molecular weight to porcine galanin not detected using a rat specific galanin antibody. One possible explanation for these results is the presence of an endogenous galanin-like peptide.

S everal observations have suggested that the enhanced proteolysis and atrophy of skeletal muscle in various pathological states is due primarily to activation of the ubiquitin-proteasome pathway. To test this idea, we investigated whether peptide aldehyde inhibitors of the proteasome, N-acetyl-leucyl-leucyl-norleucinal (LLN), or the more potent CBZ-leucyl-leucyl-leucinal (MG132) suppressed proteolysis in incubated rat skeletal muscles. These agents (e.g., MG132 at 10 microM) inhibited nonlysosomal protein breakdown by up to 50% (P < 0.01), and this effect was rapidly reversed upon removal of the inhibitor. The peptide aldehydes did not alter protein synthesis or amino acid pools, but improved overall protein balance in the muscle. Upon treatment with MG132, ubiquitin-conjugated proteins accumulated in the muscle. The inhibition of muscle proteolysis correlated with efficacy against the proteasome, although these agents could also inhibit calpain-dependent proteolysis induced with Ca2+. These inhibitors had much larger effects on proteolysis in atrophying muscles than in controls. In the denervated soleus undergoing atrophy, the increase in ATP-dependent proteolysis was reduced 70% by MG132 (P < 0.001). Similarly, the rise in muscle proteolysis induced by administering thyroid hormones was reduced 40-70% by the inhibitors. Finally, in rats made septic by cecal puncture, the increase in muscle proteolysis was completely blocked by MG132. Thus, the enhanced proteolysis in many catabolic states (including denervation, hyperthyroidism, and sepsis) is due to a proteasome-dependent pathway, and inhibition of proteasome function may be a useful approach to reduce muscle wasting.

I ntestinal epithelial cells express a low level of HLA class II molecules constitutively, with elevated levels seen in the setting of mucosal inflammation including inflammatory bowel disease. The ability of intestinal epithelial cells to act as antigen presenting cells for alphabeta CD4(+) T lymphocytes was examined through a molecular analysis of the HLA class II antigen processing pathway. We have shown that intestinal epithelial cells contain abundant constitutive levels of the cathepsin proteases proven to function in HLA class II mediated antigen presentation. Activation of these cells by gamma-IFN induced the expression of invariant chain and HLA-DM alphabeta, thus facilitating the formation of compact, SDS-stable HLA- DR alphabeta heterodimers. Using HLA-DR-restricted T cells and retroviral mediated gene transfer of HLA-DR alleles into the intestinal epithelial cell lines HT-29 and T84, we demonstrated efficient antigen processing and presentation to CD4(+) T lymphocytes in the presence of the proinflammatory cytokine gamma-IFN. The class II processing pathway and presentation in the presence of gamma-IFN was indistinguishable from that observed with a conventional antigen presenting cell. Antigen processing also occurred in intestinal epithelial cells in the absence of gamma-IFN, and in contrast to that seen after stimulation with gamma-IFN, required high concentration of antigen and was not inhibited by the protease inhibitor leupeptin. These data suggest the use of two distinct pathways of HLA class II antigen processing in enterocytes with differential immunomodulatory properties in the presence or absence of mucosal inflammation.

I n the intestine, several growth factors stimulate migration of epithelial cells, contributing to the maintenance of tissue integrity. The Ras-like GTPase Rho regulates a signal transduction pathway linking growth factor receptors to the formation of actin stress fibers and focal adhesions, presumed to be important for motility. Using an in vitro wound-induced migration assay, we have examined the role of Rho GTPases in the migration of IEC-6 and Caco-2 cells, and provide evidence that the Rho GTPases play an essential role in the initial phase of mucosal wound healing. Treatment of the cells with Clostridium difficile toxins A and B, inhibitors of the Rho family GTPases inhibited migration in a dose-dependent fashion. Microinjection of the inhibitory exchange factor Rho-guanine nucleotide dissociation inhibitor (GDI), or Clostridium botulinum C3 ADP-ribosyl transferase (C3) toxin, a Rho-ADP-ribosylating exoenzyme, potently inhibited migration. Microinjection of RhoT19N, a dominant negative form of RhoA, or in vitro ADP-ribosylated RhoA impaired the ability of cells to migrate. Rho-GDI and C3 exoenzyme also inhibited EGF-induced migration of IEC-6 cells. These results demonstrate that Rho is required for endogenous and EGF-induced migration of small intestinal crypt cells, and that Rho proteins are essential elements of a mechanism by which growth factors induce cell migration to restitute mucosal integrity.

V iral respiratory infections can predispose to the development of asthma by mechanisms that are presently undetermined. Using a murine model of respiratory syncytial virus (RSV) infection, acute infection is associated with airway hyperresponsiveness as well as enhanced responses to subsequent sensitization to allergen. We demonstrate that acute viral infection results in increased airway responsiveness to inhaled methacholine and pulmonary neutrophilic and eosinophilic inflammation. This response is associated with predominant production of Th-1-type cytokines in peribronchial lymph node cells in vitro. Mice sensitized to ovalbumin via the airways after RSV infection developed increased airway responsiveness to methacholine and pulmonary eosinophilic and neutrophilic inflammation, associated with the predominant production of Th-2-type cytokines. Treatment of the mice with anti-IL-5 antibody abolished airway hyperresponsiveness and eosinophilic but not neutrophilic inflammation in both acutely infected mice and mice sensitized after infection. We conclude that RSV infection results in airway hyperresponsiveness in the acute phase and leads to changes in immune function that can enhance the effects of airway sensitization to antigen after infection. In both situations, airway hyperresponsiveness is closely associated with pulmonary eosinophilic inflammation. This model provides a means for further analyzing the influence of viral respiratory infections on airway sensitization and the development of altered airway responsiveness.