Structural Behavior of SFRC Beams strengthened with GFRP Laminates: An Experimental and Analytical Investigation

ABSTRACT This paper presents the experimental results of a reinforced concrete beams (RC) strengthened with internal steel fibers (SF) and external glass fiber reinforced polymer laminates (GFRP). The research work studied the load carrying capacity, deformation, crack width and ductility of the reinforced concrete beams strengthened with different steel fiber ratios and steel fiber reinforced concrete beams strengthened with three different glass fiber reinforced polymer laminates of two different thickness. The experimental results clearly shows that incorporating steel fibers in to the reinforced concrete beams reduced the crack width and distribute the crack evenly and also increases the bonding between tension face of the beam with glass fiber reinforced polymer laminates. The results also shows that glass fiber reinforced polymer laminates strengthened steel fiber reinforced concrete beams increases the flexural strength and ductility as compared with unstrengthened counterpart. In addition to this experimental work, theoretical calculations were done to find the ultimate load carrying capacity of the beam tested, and also compared with the experimental results.

Oncogenic tyrosine kinase NPM/ALK induces activation of the rapamycin-sensitive mTOR signaling pathway.

The mechanisms of cell transformation mediated by the nucleophosmin (NPM)/anaplastic lymphoma kinase (ALK) tyrosine kinase are only partially understood. Here, we report that cell lines and native tissues derived from the NPM/ALK-expressing T-cell lymphoma display persistent activation of mammalian target of rapamycin (mTOR) as determined by phosphorylation of mTOR targets S6rp and 4E-binding protein 1 (4E-BP1). The mTOR activation is serum growth factor-independent but nutrient-dependent. It is also dependent on the expression and enzymatic activity of NPM/ALK as demonstrated by cell transfection with wild-type and functionally deficient NPM/ALK, small interfering RNA (siRNA)-mediated NPM/ALK depletion and kinase activity suppression using the inhibitor WHI-P154. The NPM/ALK-induced mTOR activation is transduced through the mitogen-induced extracellular kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway and, to a much lesser degree, through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway. Accordingly, whereas the low-dose PI3K inhibitor wortmannin and Akt inhibitor III profoundly inhibited Akt phosphorylation, they had a very modest effect on S6rp and 4E-BP1 phosphorylation. In turn, MEK inhibitors U0126 and PD98059 and siRNA-mediated depletion of either ERK1 or ERK2 inhibited S6rp phosphorylation much more effectively. Finally, the mTOR inhibitor rapamycin markedly decreased proliferation and increased the apoptotic rate of ALK+TCL cells. These findings identify mTOR as a novel key target of NPM/ALK and suggest that mTOR inhibitors may prove effective in therapy of ALK-induced malignancies.

Nek3 kinase regulates prolactin-mediated cytoskeletal reorganization and motility of breast cancer cells.

Prolactin (PRL) stimulates the cytoskeletal re-organization and motility of breast cancer cells. During PRL receptor signaling, Vav2 becomes phosphorylated and activated, an event regulated by the serine/threonine kinase Nek3. Given the regulatory role of Vav2, the function of Nek3 in PRL-mediated motility and invasion was examined. Overexpression of Nek3 in Chinese hamster ovary transfectants potentiated cytoskeletal re-organization in response to PRL. In contrast, downregulation of Nek3 expression by small-interfering RNA (siRNA) attenuated PRL-mediated cytoskeletal reorganization, activation of GTPase Rac1, cell migration and invasion of T47D cells. In addition, PRL stimulation induced an interaction between Nek3 and paxillin and significantly increased paxillin serine phosphorylation, whereas Nek3 siRNA-transfected cells showed a marked reduction in paxillin phosphorylation. Analysis of breast tissue microarrays also demonstrated a significant up-regulation of Nek3 expression in malignant versus normal specimens. These data suggest that Nek3 contributes to PRL-mediated breast cancer motility through mechanisms involving Rac1 activation and paxillin phosphorylation.

alpha-Defensin: link between inflammation and atherosclerosis.

BACKGROUND: Several markers of inflammation predict the risk of thrombotic cardiovascular events in patients with atherosclerosis. However, the mechanism by which vascular inflammation promotes atherothrombotic disease is incompletely understood. Human neutrophil peptides 1-3, also known as alpha-defensins, are found in human atherosclerotic arteries, inhibit LDL metabolism and fibrinolysis and promote Lp(a) binding. We asked, therefore, if alpha-defensins are risk factors for the presence and severity of atherosclerosis. METHODS: alpha-Defensin was measured in skin biopsies taken from 53 male patients (age 58.7+/-11.3 years, mean+/-S.D.) immediately prior to coronary artery catheterization. Other established risk factors were measured concurrently. The correlation between alpha-defensin deposition in the skin and the severity of the coronary artery disease (CAD) was examined. RESULTS: A statistically significant correlation was observed between the amount of alpha-defensin in skin and the severity of CAD (R=0.40, p=0.003). Multiple regression analysis showed that skin alpha-defensin is an independent predictor for CAD severity (F=4.68, p=0.035). Logistic regression analysis confirmed that skin alpha-defensin independently predicted the likelihood for CAD (p=0.016, odds estimate 5.97, 95% CL 1.4-24.2). CONCLUSIONS: The deposition of alpha-defensin in the skin is a strong independent predictor of CAD in men. These results suggest a link between neutrophil activation and progression of atherosclerosis and provide a novel approach to assessment of risk factors for CAD.

Oncogenic tyrosine kinase NPM/ALK induces activation of the MEK/ERK signaling pathway independently of c-Raf.

The mechanisms of cell transformation mediated by the highly oncogenic, chimeric NPM/ALK tyrosine kinase remain only partially understood. Here we report that cell lines and native tissues derived from the NPM/ALK-expressing T-cell lymphoma (ALK+ TCL) display phosphorylation of the extracellular signal-regulated protein kinase (ERK) 1/2 complex. Transfection of BaF3 cells with NPM/ALK induces phosphorylation of EKR1/2 and of its direct activator mitogen-induced extracellular kinase (MEK) 1/2. Depletion of NPM/ALK by small interfering RNA (siRNA) or its inhibition by WHI-154 abrogates the MEK1/2 and ERK1/2 phosphorylation. The NPM/ALK-induced MEK/ERK activation is independent of c-Raf as evidenced by the lack of MEK1/2 and ERK1/2 phosphorylation upon c-Raf inactivation by two different inhibitors, RI and ZM336372, and by its siRNA-mediated depletion. In contrast, ERK1/2 activation is strictly MEK1/2 dependent as shown by suppression of the ERK1/2 phosphorylation by the MEK1/2 inhibitor U0126. The U0126-mediated inhibition of ERK1/2 activation impaired proliferation and viability of the ALK+ TCL cells and expression of antiapoptotic factor Bcl-xL and cell cycle-promoting CDK4 and phospho-RB. Finally, siRNA-mediated depletion of both ERK1 and ERK2 inhibited cell proliferation, whereas depletion of ERK 1 (but not ERK2) markedly increased cell apoptosis. These findings identify MEK/ERK as a new signaling pathway activated by NPM/ALK and indicate that the pathway represents a novel therapeutic target in the ALK-induced malignancies.

Multi-gene epigenetic silencing of tumor suppressor genes in T-cell lymphoma cells; delayed expression of the p16 protein upon reversal of the silencing.

To understand better T-cell lymphomagenesis, we examined promoter CpG methylation and mRNA expression of closely related genes encoding p16, p15, and p14 tumor suppressor genes in cultured malignant T-cells that were derived from cutaneous, adult type, and anaplastic lymphoma kinase (ALK)-expressing T-cell lymphomas. p16 gene was epigenetically silenced in all but one of the 10 malignant T-cell lines examined, p15 gene silenced in roughly half of the lines, and p14 was the least frequently affected. Extensive methylation of the p16 promoter was seen in six out of 10 cutaneous T-cell lymphoma patient samples and corresponded with lack of p16 protein expression in the cases examined. Treatment of cultured T-cells with the DNA methyltransferase inhibitor, 5-aza-2-deoxy-cytidine, resulted in reversal of the p16 gene silencing. However, expression of p16 protein was delayed in relationship to p16 promoter demethylation and required up to 3 weeks to occur, seemingly reflecting late activation of the p16 gene. These findings indicate that epigenetic silencing affects in T-cell malignancies, often simultaneously, several tumor suppressor genes that impact on key cell functions. The observed differential silencing of p16 and p14, and to a lesser degree p15 gene, indicates that the silencing is governed by precise, promoter region-specific mechanisms. The study provides also further rationale for treatment of at least some types of T-cell lymphomas with DNA methyltransferase inhibitors to target the epigenetically silenced tumor suppressor genes.

Stabilization of prolactin receptor in breast cancer cells.

The role of the hormone prolactin (PRL) in the pathogenesis of breast cancer is mediated by its cognate receptor (PRLr). Ubiquitin-dependent degradation of the PRLr that negatively regulates PRL signaling is triggered by PRL-mediated phosphorylation of PRLr on Ser349 followed by the recruitment of the beta-transducin repeats-containing protein (beta-TrCP) ubiquitin-protein isopeptide ligase. We report here for the first time that interaction between PRLr and beta-TrCP is less efficient in human breast cancer cells than in non-tumorigenic human mammary epithelial cells. Furthermore, we demonstrate that both PRLr degradation and PRLr phosphorylation on Ser349 are impaired in breast tumor cells and tissues, an observation that directly correlates with enhanced expression of the PRLr in malignant breast epithelium. These findings represent a novel mechanism through which altered PRLr stability may directly influence the pathogenesis of breast cancer.

Annexin-V imaging for noninvasive detection of cardiac allograft rejection.

Heart transplant rejection is characterized pathologically by myocyte necrosis and apoptosis associated with interstitial mononuclear cell infiltration. Any one of these components can be targeted for noninvasive detection of transplant rejection. During apoptotic cell death, phosphatidylserine, a phospholipid that is normally confined to the inner leaflet of cell membrane bilayer, gets exteriorized. Technetium-99m-labeled annexin-V, an endogenous protein that has high affinity for binding to phosphatidylserine, has been administered intravenously for noninvasive identification of apoptotic cell death. In the present study of 18 cardiac allograft recipients, 13 patients had negative and five had positive myocardial uptake of annexin. These latter five demonstrated at least moderate transplant rejection and caspase-3 staining, suggesting apoptosis in their biopsy specimens. This study reveals the clinical feasibility and safety of annexin-V imaging for noninvasive detection of transplant rejection by targeting cell membrane phospholipid alterations that are commonly associated with the process of apoptosis.

Role of signal transducer and activator of transcription 5 in nucleophosmin/ anaplastic lymphoma kinase-mediated malignant transformation of lymphoid cells.

The NPM/ALK fusion gene, formed by the t(2;5) translocation in anaplastic large-cell lymphoma, encodes a M(r) 75,000 hybrid protein that containsthe amino-terminal portion of the nucleolar phosphoprotein nucleophosmin(NPM) joined to the entire cytoplasmic portion of the receptor tyrosine kinase anaplastic lymphoma kinase (ALK). NPM/ALK encodes a constitutively activated tyrosine kinase that belongs to the family of tyrosine kinases activated by chromosomal translocation. Our studies show that NPM/ALK, similar to other members of this family, activates signal transducer and activator of transcription 5 (STAT5) and that this activation is essential for lymphomagenesis. NPM/ALK-mediated activation of STAT5 was demonstrated by detection of: (a) constitutive tyrosine phosphorylation and enhanced DNA binding ability of STAT5 in NPM/ALK-transformed cells; and (b) NPM/ALK-dependent stimulation of STAT5-mediated transactivation of the beta-casein promoter. Retroviral infection of NPM/ALK+ cells with a dominant-negative STAT5B mutant (STAT5-DNM) inhibited the antiapoptotic activity of NPM/ALK in growth factor and serum-free medium. In addition, STAT5-DNM inhibited proliferation and diminished the clonogenic properties of NPM/ALK-positive cells. Finally, SCID mice injected with NPM/ALK+ cells infected with a virus carrying STAT5-DNM survived significantly longer than mice inoculated with NPM/ALK+ cells infected with the empty virus. Necropsy identified a widespread ALK+ lymphoma in lymph nodes and liver of the affected animals. Together, our data indicate that NPM/ALK-induced activation of STAT5 may play an important role in NPM/ALK-mediated lymphomagenesis.

SHP-1 expression by malignant small B-cell lymphomas reflects the maturation stage of their normal B-cell counterparts.

SHP-1 is a protein phosphotyrosine phosphatase that plays an important role in modulating intracellular signaling, which regulates cell activation, proliferation, differentiation, and migration. It is a negative regulator of signal transduction induced by a number of cell receptors. Our immunohistochemical examination of paraffin-embedded reactive lymph nodes and lymphoid tissues revealed that B lymphocytes in follicle germinal centers do not express SHP-1. A weak staining of the B cells in the germinal center light zones was detected when an ultrasensitive amplification system was used. In contrast, normal B cells in mantle and marginal zones as well as interfollicular B lymphocytes and plasma cells displayed strong immunoreactivity. This pattern of SHP-1 expression was repeated in small B-cell lymphomas. All cases of mantle cell lymphoma (12 of 12), marginal zone lymphoma (10 of 10), and chronic lymphocytic leukemia/small lymphocytic lymphoma (13 of 13) expressed SHP-1 protein. However, only 1 of 30 cases of grade 1 follicle center cell lymphoma expressed SHP-1. Our observations highlight the biologic functions of SHP-1 and demonstrate that the SHP-1 expression pattern by small B-cell lymphomas reflects the maturation stage of their normal cell counterparts. These results indicate that determination of SHP-1 expression may help in the differential diagnosis of small B-cell lymphomas.

Lack of phosphotyrosine phosphatase SHP-1 expression in malignant T-cell lymphoma cells results from methylation of the SHP-1 promoter.

SHP-1 is an important negative regulator of signaling by several receptors including receptors for interleukin-2 (IL-2R) and other cytokines. SHP-1 acts by dephosphorylating the receptors and receptor-associated kinases such as IL-2R-associated Jak3 kinase. We found that SHP-1 protein was not detectable or greatly diminished in most (six of seven) T cell lines derived from various types of T cell lymphomas and all (eight of eight) cutaneous T-cell lymphoma tissues with a transformed, large-cell morphology. All T-cell lymphoma lines tested (eight of eight) expressed diminished amounts or no detectable SHP-1 mRNA. These T cell lines did not, however, carry any mutations in the SHP-1 gene-coding, splice-junction, and promoter regions. Importantly, SHP-1 DNA promoter region in the T cell lines was resistant to digestion with three different methylation-sensitive restriction enzymes. This resistance was reversed by treatment of the cells with a demethylating agent, 5-deoxyazacytidine. The treatment resulted also in the expression of SHP-1 mRNA and, less frequently, SHP-1 protein. The expression of SHP-1 protein was associated with dephosphorylation of the Jak3 kinase. These results show that lack of SHP-1 expression is frequent in malignant T cells and results from methylation of the SHP-1 gene promoter. Furthermore, they indicate that SHP-1 loss may play a role in the pathogenesis of T cell lymphomas by permitting persistence of signals generated by IL-2R and, possibly, other receptor complexes.

Defensin promotes the binding of lipoprotein(a) to vascular matrix.

Retention of lipoproteins within the vasculature is a central event in the pathogenesis of atherosclerosis. However, the signals that mediate this process are only partially understood. Prompted by putative links between inflammation and atherosclerosis, we previously reported that alpha-defensins released by neutrophils are present in human atherosclerotic lesions and promote the binding of lipoprotein(a) [Lp(a)] to vascular cells without a concomitant increase in degradation. We have now tested the hypothesis that this accumulation results from the propensity of defensin to form stable complexes with Lp(a) that divert the lipoprotein from its normal cellular degradative pathways to the extracellular matrix (ECM). In accord with this hypothesis, defensin stimulated the binding of Lp(a) to vascular matrices approximately 40-fold and binding of the reactants to the matrix was essentially irreversible. Defensin formed stable, multivalent complexes with Lp(a) and with its components, apoprotein (a) and low-density lipoprotein (LDL), as assessed by optical biosensor analysis, gel filtration, and immunoelectron microscopy. Binding of defensin/Lp(a) complexes to matrix was inhibited (>90%) by heparin and by antibodies to fibronectin (>70%), but not by antibodies to vitronectin or thrombospondin. Defensin increased the binding of Lp(a) (10 nmol/L) to purified fibronectin more than 30-fold. Whereas defensin and Lp(a) readily traversed the endothelial cell membranes individually, defensin/Lp(a) complexes lodged on the cell surface. These studies demonstrate that alpha-defensins released from activated or senescent neutrophils stimulate the binding of an atherogenic lipoprotein to the ECM of endothelial cells, a process that may contribute to lipoprotein accumulation in atherosclerotic lesions.

Native atherosclerosis and vein graft arterialization: association with increased urokinase receptor expression in vitro and in vivo.

Interaction of proteases with cell surface receptors may modulate cell adhesion, migration, invasion, and matrix degradation. Since the plasminogen activator system has been hypothesized to play a role in intimal thickening after various types of vascular injury, we first studied the expression of urokinase receptor (u-PAR) protein and mRNA by smooth muscle cells (SMC) grown in explant cultures from normal and diseased vessels. Using equilibrium binding studies with radiolabeled 125I-labeled single chain urokinase-type plasminogen activator (scu-PA), we determined that SMC cultured from atherosclerotic arteries expressed a higher maximal number of binding sites/cell (3.6 +/- 0.4 x 10(5) sites/cell vs. 2.1 +/- 0.3 x 10(5), +/- SEM, p < 0.05) with a similar affinity (Kd = 1.5 +/- 0.1 vs. 1.2 +/- 0.2 nM, p = ns). However, SMC subcultured from diseased saphenous vein grafts expressed the highest levels of u-PAR compared to SMC from normal saphenous vein (4.8 +/- 0.6 x 10(5) sites/cell vs. 1.6 +/- 0.9 x 10(5), +/- SEM, p < 0.05). Using binding studies and Northern analysis, we demonstrated a dose and time dependent upregulation of u-PAR protein and mRNA expression respectively in human SMC in response to serum stimulation. Using a rabbit specific u-PAR cDNA probe, we demonstrated a similar upregulation of u-PAR mRNA both in rabbit aortic SMC in culture in response to serum stimulation and up to a 20 fold increase in u-PAR mRNA in rabbit jugular veins in response to implantation as arterial grafts in vivo. Finally, to confirm that u-PAR mRNA is upregulated in human vessels after injury, we performed immunohistochemistry and in situ hybridization studies on coronary arteries, normal saphenous veins and saphenous veins from 10 weeks to 13 years after implantation as grafts. u-PAR mRNA was found mainly in the periadventitial microcirculation in normal veins, but was found to be upregulated in the neointima and media of thickened veins in both macrophages and smooth muscle cells. SMC near the internal elastic laminae in diseased coronary arteries appeared to express increased u-PAR mRNA. These data suggest that this increased expression of u-PAR may contribute to early lesion development.

Differential expression of functional protease-activated receptor-2 (PAR-2) in human vascular smooth muscle cells.

The protease-activated family of G protein-coupled receptors includes PAR-1 and PAR-3, which are activated by thrombin, and PAR-2, which is activated by trypsin and tryptase. PAR-2 has recently been shown to be expressed in human endothelial cells. In the present studies, we have examined the expression of PAR-2 in other cells, particularly vascular smooth muscle, and tested whether the receptors are functional. The results show that PAR-2 is present in human aorta and coronary artery smooth muscle cells, as well as in arteries traversing the walls of the small intestine. It was also detected in human keratinocytes, sweat glands, intestinal smooth muscle, and intestinal epithelium, but not at all in myocardial smooth muscle and only inconsistently in intestinal veins and venules. Activation of aortic smooth muscle cells in culture with PAR-2 peptide agonists caused a transient increase in the cytosolic Ca2+ concentration. In contrast, PAR-2 mRNA could not be detected in saphenous vein smooth muscle cells, and the same cells placed in culture showed little, if any, response to the PAR-2 agonist peptides. These observations show that PAR-2 is widely distributed in human vascular smooth muscle, particularly in arteries. However, this is not a universal finding and at least some venous smooth muscle cells, including those in saphenous veins, apparently do not express the receptor in detectable amounts.

Tissue factor regulates plasminogen binding and activation.

Tissue factor (TF) has been implicated in several important biologic processes, including fibrin formation, atherogenesis, angiogenesis, and tumor cell migration. In that plasminogen activators have been implicated in the same processes, the potential for interactions between TF and the plasminogen activator system was examined. Plasminogen was found to bind directly to the extracellular domain of TF apoprotein (amino acids 1-219) as determined by optical biosensor interaction analysis. A fragment of plasminogen containing kringles 1 through 3 also bound to TF apoprotein, whereas isolated kringle 4 and miniplasminogen did not. Expression of TF on the surface of a stably transfected Chinese hamster ovary (CHO) cell line stimulated plasminogen binding to the cells by 70% more than to control cells. Plasminogen bound to a site on the TF apoprotein that appears to be distinct from the binding site for factors VII and VIIa as judged by a combination of biosensor and cell assays. TF enhanced two-chain urokinase (tcuPA) activation of Glu-plasminogen, but not of miniplasminogen, in a dose-dependent, saturable manner (half maximal stimulation at 59 pmol/L). TF apoprotein induced an effect similar to that of relipidated TF, but a relatively higher concentration of the apoprotein was required (half maximal stimulation at 3.8 nmol/L). The stimulatory effect of TF on plasminogen activation was confirmed when plasmin formation was examined directly on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In accord with this, TF inhibited fibrinolysis by approximately 74% at a concentration of 14 nmol/L and almost totally inhibited the binding of equimolar concentrations of plasminogen to human umbilical vein endothelial cells and human trophoblasts. Further, CHO cells expressing TF inhibited uPA-mediated fibrinolysis relative to a wild-type control. TF apoprotein and plasminogen were found to colocalize in atherosclerotic plaque. These data suggest that plasminogen localization and activation may be modulated at extravascular sites through a high-affinity interaction between kringles 1 through 3 of plasminogen and the extracellular domain of TF.

Thermal preconditioning before rat arterial balloon injury: limitation of injury and sustained reduction of intimal thickening.

Heat shock proteins (HSPs) are a family of highly conserved proteins, essential to cell survival, that are induced during times of physiological stress. These proteins, when induced, can provide tolerance to subsequent injury. Several studies have documented that HSPs play an important role in the response of vascular cells to injury or stress. Whether the vasculature itself can be effectively preconditioned before arterial injury is unknown. Vascular HSP induction by whole-body hyperthermia (WBH) was evaluated with regard to its effects on the vascular response to balloon injury. WBH treatment of Sprague-Dawley rats (colonic temperatures of 41 to 42 degrees C for 15 minutes) resulted in maximal arterial HSP expression within 8 to 12 hours. Rats (male, 300 g, n=59) were randomly assigned to undergo either WBH or no treatment 8 hours before standard carotid balloon injury. At 14 (n=26) and 90 (n=21) days after balloon injury, histomorphometric analysis revealed a significant limitation of intimal accumulation in preconditioned arteries as compared to controls (intimal/medial area ratios+/-SEM: 14 days, 0.57+/-0.07 versus 0.86+/-0.08, P=0.01; 90 days, 0.78+/-0.12 versus 1.19+/-0.14, P<0.05). The medial cell proliferation index at 4 days (n=12) was significantly reduced in the treated group as well (3.6+/-0.9% versus 7.2+/-1.3%, P<0.05). Conversely, the mean total cell number in the media of heated arteries was higher (393+/-20 versus 328+/-17, P<0.05). Vascular preconditioning with brief WBH induces a heat shock response in the arterial wall that is associated with a significant and sustained reduction in intimal accumulation. This effect appears to be due in part to preservation of medial cell integrity and limitation of the proliferative response. These results suggest that thermal preconditioning of vascular tissue may be an effective strategy to improve long-term results after revascularization procedures.

Immunohistochemical localization of defensin in human coronary vessels.

Neutrophil defensins comprise a family of cationic peptides that possess potent antimicrobial activity. Defensins are normally sequestered in cytoplasmic granules with their primary site of action in phagolysosomes, although some peptide is released into the circulation during the course of infection or inflammation. In view of the fact that neutrophils adhere to the endothelium and that defensins have been reported to bind to human endothelial cells in vitro, we used immunohistochemistry to study the distribution of these peptides in normal and in atherosclerotic human coronary arteries. Defensin was found primarily in the intima of normal and atherosclerotic vessels, most prominently in association with intimal smooth muscle cells. Both large- and small-vessel endothelium stained focally for defensin. Defensin was also found in the media near the external elastic lamina and in some periadventitial vessels. The same distribution was seen in vessels that had been perfusion fixed immediately upon procurement, excluding diffusion of defensin from PMNs ex vivo. These data indicate that neutrophil defensin is present in the walls of human coronary arteries. The deposition of defensin in vessels may contribute to the pathophysiological consequences of inflammation in addition to their role in host defense.

Enhanced biosynthesis of extracellular matrix proteins and TGF-beta 1 by polyinosinic-polycytidylic acid during cutaneous wound healing in vivo.

In our previous study, we have shown that polyinosinic-polycytidylic acid (poly I:C), a double-stranded RNA, and a potent inducer of interferon, enhanced the wound healing in rats and mice. Increased levels of laminin and collagen, and greater influx of dermal fibroblasts were observed in poly I:C-treated wounds as compared to untreated wounds (Bhartiya et al., 1992, J. Cell. Physiol., 150:312-319). In this study, we have explored the mechanism of enhanced wound healing by poly I:C in rats. Poly I:C (1 mg/kg) in phosphate buffered saline was injected intraperitoneally 18 h prior to wound healing, and the animals were sacrificed on day 3 postwounding. Immunofluorescence studies showed increased expression of adhesion molecules that includes ICAM-1 (intercellular adhesion molecule-1;CD54) and VCAM-1 (vascular cell adhesion molecule; CD 106) in poly I:C-treated wounds as compared to untreated control. Poly I:C treatment resulted in an increase in the mRNA levels of collagen type 1 (alpha), collagen III, laminin B1, and transforming growth factor-beta 1(TGF-beta 1) in wounds compared to untreated wounds as demonstrated by in situ hybridization and PCR analysis. These studies suggests that poly I:C upregulates the biosynthesis of adhesion molecules, extracellular matrix proteins (ECM), and TGF-beta 1 in the wound bed. Adhesion molecules and ECM play a major role in wound healing, and TGF-beta 1 has been known to be a potent wound healer. Therefore, the increased expression of these molecules may play a role in the enhanced healing by poly I:C observed in rats.

Inhibition of vein graft intimal and medial thickening by periadventitial application of a sulfated carbohydrate polymer.

PURPOSE: The purpose of this study was to determine whether the wall thickening observed in vein grafts after they were placed into the arterial circulation could be inhibited by periadventitial delivery of an insoluble sulfated polymer of beta-cyclodextrin (P-CDS) capable of tightly binding heparin binding growth factors. METHODS: Thirty-four New Zealand white rabbits underwent implantation of reversed autologous jugular vein interposition grafts in the common carotid artery and were randomized to receive either 20 mg P-CDS (n = 18) topically around the graft or no additional therapy (n = 16). Before being killed at 28 days, animals were given bromodeoxyuridine to assess smooth muscle cell proliferation. Histomorphometric analyses were performed after perfusion fixation. RESULTS: Compared to controls, treatment with P-CDS was associated with reduced mean intimal thickness (24 +/- 3 vs 38 +/- 4 microns; mean SEM, p < 0.01) and intimal area (0.25 +/- 0.03 vs 0.54 +/- 0.09 mm2; p < 0.01). There was also significantly less medial thickness in the P-CDS group (45 +/- 3 vs 63 +/-3, p < 0.001). There was no significant difference in intimal or medial smooth muscle cell proliferation between P-CDS-treated and control vein grafts at 28 days. The polymer persisted in the adventitia with a mild foreign body reaction. CONCLUSION: Periadventitial placement of P-CDS, a novel, insoluble, sulfated carbohydrate polymer, inhibits intimal and medial thickening of vein bypass grafts in this model of vein grafting. The persistence of P-CDS in vivo for prolonged periods, and the ease of topical application of P-CDS during vascular bypasses may have important implications for its future use in vascular surgery.

Brefeldin A inhibits the antiviral action of interferon against encephalomyocarditis virus.

Brefeldin A (BFA), a unique fungal metabolite of a 13-membered lactone ring, exhibits various biological actions, including antitumor, antifungal and antiviral activities. In the present study, mouse LB cells were treated with various concentrations of interferon (IFN) and/or BFA overnight and infected with encephalomyocarditis virus (EMCV) after removal of IFN and BFA. Doses of BFA which neither inhibit the metabolism of the cell nor the infectivity of EMCV, decreased the IFN-induced antiviral activity against EMCV as demonstrated by virus titer from supernatants. Since 2-5A synthetase and double-stranded RNA (dsRNA)-dependent protein kinase (PKR) have been suggested to be involved in the antiviral action of IFN against EMCV, their activities were investigated in LB cells after BFA treatment. Northern blot analysis and in situ hybridization showed a decrease (2-3-fold) in the mRNA of 2'-5' oligoadenylate (2-5A) synthetase after BFA treatment. BFA also inhibited the activity of 2-5A synthetase, 2-5A dependent RNase and phosphorylation of PKR in cellular extracts, indicating that BFA may be exerting its inhibitory effect both at the transcriptional and post-transcriptional levels. This study reports a new biological action of BFA, demonstrating that BFA antagonized the antiviral action of IFN by inhibiting IFN-induced enzymatic pathways. These studies also suggest that both 2-5A and PKR are important in the antiviral activity of IFN against EMCV.