Impact of donor left ventricular hypertrophy on survival after heart transplant.

Left ventricular hypertrophy (LVH) of the donor heart is believed to increase the risk of allograft failure after transplant. However this effect is not well quantified, with variable findings from single-center studies. The United Network for Organ Sharing database was used to analyze the effect of donor LVH on recipient survival. Three cohorts, selected in accordance with the American Society of Echocardiography guidelines, were examined: recipients of allografts without LVH (<1.1 cm), with mild LVH (1.1-1.3 cm) and with moderate-severe LVH (≥ 1.4 cm). The study group included 2626 patients with follow-up of up to 3.3 years. Mild LVH was present in 38% and moderate-severe LVH in 5.6% of allografts. Predictors of mortality included a number of donor and recipient characteristics, but not LVH. However, a subgroup analysis showed an increased risk of death in recipients of allografts with LVH and donor age >55 years, and in recipients of allografts with LVH and ischemic time ≥ 4 h. In the contemporary era, close to half of all transplanted allografts demonstrate LVH, and survival of these recipients is similar to those without LVH. However, the use of allografts with LVH in association with other high-risk characteristics may result in increased mortality.

Thrombin generation in vascular tissue.

BACKGROUND: Classically, it is thought that the vast majority of thrombin is generated on the surface of platelets, however, thrombotic events occur in patients despite treatment with potent antiplatelet agents. METHODS AND RESULTS: In freshly harvested left internal mammary artery (IMA) sections, addition of CaCl2 and platelet-poor plasma (PPP) were sufficient to stimulate a profound burst of thrombin and this effect was inhibited by antitissue factor antibodies. Ultracentrifugation of PPP to remove platelet microparticles had no effect on thrombin generation. Both the extrinsic and factor VIII-dependent pathways were necessary for IMA-supported thrombin generation as PPP derived from individuals deficient in factors V, VII, VIII or X did not support thrombin production. Small amounts of thrombin were generated utilizing factor IX (FIX)-deficient plasma, however, thrombin was not generated by aorta from FIX-deficient mice when FIX-deficient plasma was used. The addition of non-lipidated tissue factor (0.6 pM) and CaCl2 to actively proliferating cultured human aortic smooth muscle cells (SMC) resulted in a pronounced burst of thrombin generation occurring between 3 and 15 min after treatment. In the absence of tissue factor, thrombin was generated but at a slower rate and with a peak value 26% of that observed in the presence of tissue factor. CONCLUSION: Significant thrombin generation can occur on vascular tissue in the absence of platelets or platelet microparticles and on the surface of non-apoptotic SMC.

Suppression of tumor necrosis factor alpha production by cAMP in human monocytes: dissociation with mRNA level and independent of interleukin-10.

BACKGROUND: Elevation of cellular cAMP inhibits lipopolysaccharide(LPS)-stimulated tumor necrosis factor alpha (TNF-alpha) production and increases the expression of interleukin (IL)-10 in mononuclear cells. TNF-alpha gene expression obligates activation of the transcription factor nuclear factor kappaB (NF-kappaB). Exogenous IL-10 inhibits NF-kappaB in monocytes and thus attenuates TNF-alpha production. We examined the role of endogenous IL-10 in the regulation of NF-kappaB activation and TNF-alpha production in human monocytes by cAMP. METHODS: Human monocytes were stimulated with Escherichia coli LPS (100 ng/ml) with and without forskolin (FSK, 50 microM) or dibutyryl cyclic AMP (dbcAMP, 100 microM). Cytokine (TNF-alpha and IL-10) release was measured by immunoassay. TNF-alpha mRNA was measured by reverse transcription polymerase chain reaction, and NF-kappaB DNA binding activity was assessed by gel mobility shift assay. RESULTS: cAMP-elevating agents inhibited LPS-stimulated TNF-alpha release (0.77 +/- 0.13 ng/10(6) cells in LPS + dbcAMP and 0.68 +/- 0.19 ng/10(6) cells in LPS + FSK, both P < 0.05 vs 1.61 +/- 0.34 ng/10(6) cells in LPS alone). Conversely, cAMP enhanced LPS-stimulated IL-10 release (100 +/- 21.5 pg/10(6) cells in LPS + dbcAMP and 110 +/- 25.2 pg/10(6) cells in LPS + FSK, both P < 0.05 vs 53.3 +/- 12.8 pg/10(6) cells in LPS alone). Neither TNF-alpha mRNA expression nor NF-kappaB activation stimulated by LPS was inhibited by the cAMP-elevating agents. Neutralization of IL-10 with a specific antibody did not attenuate the effect of cAMP-elevating agents on TNF-alpha production. CONCLUSION: The results indicate that cAMP inhibits LPS-stimulated TNF-alpha production through a posttranscriptional mechanism that is independent of endogenous IL-10.

Therapeutic implications of inflammation in atherosclerotic cardiovascular disease.

Atherosclerosis represents a spectrum of pathologic lesions with diverse clinical sequelae. In this review, we build upon the paradigm that arteriosclerosis represents an inflammatory disease. By examining mechanisms involved in the response to vascular injury, we can more effectively implement targeted therapy aimed at halting or regressing arteriosclerosis.

Current approaches to therapy for vascular injury.

Vascular injury is a ubiquitous phenomenon which can be both occult (such as with hyperlipidemia) and overt (such as with angioplasty). While the exact pathophysiology differs between acute and chronic atherosclerosis, both lesions can be mechanistically explained by the vasculature's exaggerated response to injury. Pharmacological attempts to treat atherosclerotic cardiovascular disease can be categorised by their role in modifying this inflammatory response. This manuscript reviews current therapy for cardiovascular injury at two levels: the chronic smouldering atheromatous lesion and intimal hyperplasia associated with acute vascular intervention. In addition, future therapeutic strategies, based within this inflammatory paradigm, are discussed.

The case for beta-adrenergic blockade as prophylaxis against perioperative cardiovascular morbidity and mortality.

Perioperative morbidity and mortality are frequently cardiac in origin. Many studies have prospectively attempted to define risk factors for cardiac ischemic events. Although we can now identify high-risk patients, optimal cardioprotective management strategies remain unclear. Treatment with beta-adrenergic antagonists decreases myocardial oxygen consumption and is generally well tolerated. This article reviews the physiologic and clinical basis for using these agents as prophylaxis against cardiovascular events in high-risk surgical patients.

Functional reconstitution and regulation of IL-18 activity by the IL-18R beta chain.

IL-18 and IL-12 are major IFN-gamma-inducing cytokines but the unique synergism of IL-18 and IL-12 remains unclear. In the human NK cell line NKO, IL-18R alpha, and IL-18R beta are expressed constitutively but IL-18 did not induce IFN-gamma unless IL-12 was present. COS-1 fibroblasts, which produce the chemokine IL-8 when stimulated by IL-1 beta or TNF-alpha, do not respond to IL-18, despite abundant expression of the IL-18R alpha chain. COS-1 cells lack expression of the IL-18R beta chain. The IL-18R beta cDNA was cloned from a human T-B lymphoblast cDNA library and COS-1 cells were transiently transfected with the IL-18R beta chain and a luciferase reporter. In transfected COS-1 cells, IL-18 induced IL-8 and luciferase in the absence of IL-12 and independently of IL-1 and TNF. Ab against the IL-18R alpha chain, however, prevented IL-18 responsiveness in COS-1 cells transfected with the IL-18R beta chain, suggesting that both chains be functional. In NKO cells and PBMC, IL-12 increased steady-state mRNA levels of IL-18R alpha and IL-18R beta; the production of IFN-gamma corresponded to IL-12-induced IL-18R alpha and IL-18R beta chains. We conclude that functional reconstitution of the IL-18R beta chain is essential for IL-12-independent proinflammatory activity of IL-18-induced IL-8 in fibroblasts. The synergism of IL-18 plus IL-12 for IFN-gamma production is, in part, due to IL-12 up-regulation of both IL-18R alpha and IL-18R beta chains, although postreceptor events likely contribute to IFN-gamma production.

Clinical applications of cardiovascular angiogenesis.

Angiogenesis is fundamental to both normal physiologic (wound healing) and pathologic (cancer) processes. Manipulation of divergent angiogenic signals promises effective therapy of atherosclerotic cardiovascular disease. Positive proangiogenic strategies promise collateral circulation to ischemic territories, while negative antiangiogenic strategies starve the fibromuscular proliferation within the atherosclerotic lesion. Indeed, recent phase 1 trials suggest that delivering DNA or recombinant protein to the site of vascular occlusion may stimulate physiologically significant collateral circulation in chronically ischemic myocardium. While symptomatic and functional improvement has been documented, toxicity profiles and effects on long-term patient survival are still unclear. The purposes of this article are as follows: (1) to review the pathophysiologic basis for pro- and antiangiogenic strategies in the treatment of cardiovascular disease, (2) to examine the clinical trials of proangiogenic gene or recombinant protein delivery into ischemic beds, and conversely, (3) to explore antiangiogenic strategies in the prevention and treatment of intimal neovascularization and smooth muscle proliferation within the vessel wall.

Chemokines as mediators of diseases related to surgical conditions.

Chemokines are important mediators of inflammation. Animal studies suggest that inhibition of chemokine action results in a decrease in inflammation. Novel anti-inflammatory agents directed against chemokines are now available. Surgeons are uniquely positioned to treat multiple chemokine-mediated diseases. In this article, we review the biology and nomenclature of chemokines as well as their role in neutrophil migration. Further, the potential role of chemokines in various diseases related to surgical conditions, including adult respiratory distress syndrome, atherosclerosis, inflammatory bowel disease, and solid organ rejection, is reviewed. Finally, the idea that chemokines could be targets for novel therapeutic agents is discussed.

Differential inducible nitric oxide synthase expression in systemic and pulmonary vessels after endotoxin.

Inducible nitric oxide synthase (iNOS) is associated with vascular hypocontractility in systemic vessels after endotoxin lipopolysaccharide (LPS) administration. Although lung iNOS is increased after LPS, its role in the pulmonary circulation is unclear. We hypothesized that whereas iNOS upregulation is responsible for LPS-induced vascular dysfunction in systemic vessels, iNOS does not play a significant role in the pulmonary artery (PA). Using isolated aorta (AO) and PA rings, we examined the effect of nonselective NOS inhibition [N(G)-monomethyl-L-arginine (L-NMMA); 100 micromol/l] and selective iNOS inhibition (aminoguanidine, AG; 100 micromol/l) on alpha(1)-adrenergic-mediated vasoconstriction (phenylephrine; 10(-9) to 10(-3) M) after LPS (Salmonella typhimurium, 20 mg/kg ip). We also determined the presence of iNOS using Western blot and immunohistochemistry. LPS markedly impaired AO contractility (maximal control tension 1,076 +/- 33 mg vs. LPS 412 +/- 39 mg, P < 0.05), but PA contractility was unchanged (control 466 +/- 29 mg vs. LPS 455 +/- 27 mg, P > 0.05). Selective iNOS inhibition restored the AO's response to vasoconstriction (LPS + AG 1,135 +/- 54 mg, P > 0.05 vs. control and P < 0.05 vs. LPS), but had no effect on the PA (LPS + AG 422 +/- 38 mg, P > 0.05 vs. control and LPS). Western blot and immunohistochemistry revealed increased iNOS expression in the AO after LPS, but iNOS was not detected in the PA. Our results suggest that differential iNOS expression after LPS in systemic and pulmonary vessels contributes to the phenomenon of sepsis/endotoxemia-induced systemic hypotension and pulmonary hypertension.

Chlamydia pneumoniae activates nuclear factor kappaB and activator protein 1 in human vascular smooth muscle and induces cellular proliferation.

BACKGROUND: Observational data strongly suggest an association between Chlamydia pneumoniae and atherosclerotic cardiovascular disease. However, few studies have mechanistically linked C. pneumoniae to vascular remodeling. The purpose of the present study was to examine the mechanistic relationship between C. pneumoniae and human vascular smooth muscle cell (VSMC) physiology. We sought to determine the influence of human VSMC infection by C. pneumoniae on (1) VSMC proliferation and (2) activation of the proinflammatory and proliferative transcription factors nuclear factor kappaB (NF-kappaB) and activator protein 1 (AP-1). MATERIALS AND METHODS: C. pneumoniae was grown and isolated from Hep 2 cells. Human aortic VSMCs were inoculated with C. pneumoniae in the presence and absence of the azalide antibiotic azithromycin. Cell proliferation was assayed by direct cell counting 48 h following infection. Two hours following infection, nuclear extracts were isolated, and activation of both NF-kappaB and AP-1 was assessed by electrophoretic mobility shift assay. RESULTS: Compared with control, C. pneumoniae infection stimulated VSMC proliferation (P < 0.05) and induced both NF-kappaB and AP-1 DNA binding activity. These effects were eliminated by concurrent treatment with azithromycin. CONCLUSIONS: VSMC infection with C. pneumoniae activates proliferative intracellular signals and stimulates cell growth. These data implicate C. pneumoniae as a pathogenic mediator and a potential therapeutic target in the prevention of atherosclerotic disease.

Acellular components of Chlamydia pneumoniae stimulate cytokine production in human blood mononuclear cells.

Accumulating evidence suggest that infection with Chlamydia pneumoniae is associated with atherosclerosis, but the mechanisms involved remain unclear. Inflammation is important in the initial phase of atherogenesis, and cytokines are important in the initiation and progression of inflammation. The aim of this study was to assess the capacity of acellular components of C. pneumoniae to stimulate the production of pro-inflammatory cytokines and chemokines. Peripheral blood mononuclear cells were stimulated in vitro with sonicated C. pneumoniae. Significant amounts of TNF-alpha, IL-1, IL-6, IL-8, monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1alpha (MIP-1alpha) were produced. Inhibition of endotoxin using polymyxin B revealed that chlamydial endotoxin plays a minor role in the cytokine induction. Neutralization of TNF by TNF-binding protein and blockade of IL-1 receptors by IL-1 receptor antagonist revealed that TNF, IL-1 and IL-6 production was independent from each other, whereas IL-8 synthesis was strongly dependent on endogenous TNF and IL-1. In contrast, synthesis of MCP-1 and MIP-1alpha was dependent on endogenous TNF, but not IL-1. In conclusion, acellular components of C. pneumoniae are a potent stimulus for cytokine production, and this mechanism may have an important role in the inflammatory aspects of atherogenesis.

Interleukin-1beta deficiency results in reduced NF-kappaB levels in pregnant mice.

Interleukin (IL)-1beta-deficient (IL-1beta(-/-)) mice were assessed for cytokine production during pregnancy. A significant reduction in nuclear factor (NF)-kappaB p65 protein content was observed in the uteri and spleens of pregnant IL-1beta(-/-) mice, as demonstrated by immunohistochemistry and Western immunoblot analysis. In addition, electromobility gel shift assay revealed less DNA binding activity of NF-kappaB p65-containing complex in pregnant IL-1beta(-/-) mice. To investigate differences in cytokine production regulated by NF-kappaB, the levels of tumor necrosis factor-alpha, macrophage inflammatory protein-1alpha, and interferon-gamma were measured in the uterine wall, spleen homogenates, and spleen cell cultures obtained from pregnant mice. Endocervical administration of lipopolysaccharide (LPS) increased cytokine levels in both wild-type (IL-1beta(+/+)) and IL-1beta(-/-) animals, but in IL-1beta(-/-) mice this response was 50-75% lower. Splenocytes from nonpregnant mice exhibited decreased LPS-induced cytokine production when primed in vitro with progesterone. This suppression was 25% greater in IL-1beta(-/-) than in IL-1beta(+/+) mice. These data suggest that constitutive NF-kappaB p65 protein synthesis is regulated by IL-1beta, particularly during pregnancy.

Interleukin-11 attenuates pulmonary inflammation and vasomotor dysfunction in endotoxin-induced lung injury.

Interleukin (IL)-11, like other members of the gp130 receptor class, possesses anti-inflammatory properties. We hypothesized that IL-11 pretreatment would attenuate endotoxin [lipopolysaccharide (LPS)]-induced lung inflammation and diminish injury to endothelium-dependent and -independent mechanisms of pulmonary vasorelaxation that require cGMP in Sprague-Dawley rats. LPS (20 mg/kg ip) increased lung tumor necrosis factor (TNF)-alpha compared with the saline control (0.7 +/- 0.15 ng/g lung wet wt for control vs. 3.5 +/- 0.09 ng/g lung wet wt for LPS; P < 0.05). IL-11 (200 mg/kg ip) injected 10 min before LPS administration attenuated the LPS-induced lung TNF-alpha levels (1.6 +/- 0.91 ng/g lung wet wt; P < 0.05 vs. LPS). IL-11 also diminished LPS-induced lung neutrophil sequestration as assessed by myeloperoxidase units (2.1 +/- 0.25 U/g lung wet wt for saline and 15.6 +/- 2.02 U/g lung wet wt for LPS vs. 7.07 +/- 1.65 U/g lung wet wt for LPS plus IL-11; P < 0.05). Similarly, TNF-alpha binding protein (175 mg/kg) attenuated LPS-induced myeloperoxidase activity (6.04 +/- 0.14 U/g lung wet wt; P < 0.05). Both IL-11 and TNF-alpha binding protein similarly attenuated LPS-induced endothelium-dependent vasomotor dysfunction with improved relaxation responses to 10(-7) and 10(-6) M acetylcholine and A-23187 in phenylephrine-preconstricted isolated pulmonary artery rings (P < 0.05 vs. LPS). Endothelium-independent relaxation responses to sodium nitroprusside were also improved after LPS at 10(-6) M (P < 0.05 vs. LPS). Moreover, IL-11 decreased endotoxin-induced mortality in CF1 mice from 90 to 50% (P

Inhibition of PARS attenuates endotoxin-induced dysfunction of pulmonary vasorelaxation.

Endotoxin (Etx) causes excessive activation of the nuclear repair enzyme poly(ADP-ribose) synthase (PARS), which depletes cellular energy stores and leads to vascular dysfunction. We hypothesized that PARS inhibition would attenuate injury to mechanisms of pulmonary vasorelaxation in acute lung injury. The purpose of this study was to determine the effect of in vivo PARS inhibition on Etx-induced dysfunction of pulmonary vasorelaxation. Rats received intraperitoneal saline or Etx (Salmonella typhimurium; 20 mg/kg) and one of the PARS inhibitors, 3-aminobenzamide (3-AB; 10 mg/kg) or nicotinamide (Nic; 200 mg/kg), 90 min later. After 6 h, concentration-response curves were determined in isolated pulmonary arterial rings. Etx impaired endothelium-dependent (response to ACh and calcium ionophore) and -independent (sodium nitroprusside) cGMP-mediated vasorelaxation. 3-AB and Nic attenuated Etx-induced impairment of endothelium-dependent and -independent pulmonary vasorelaxation. 3-AB and Nic had no effect on Etx-induced increases in lung myeloperoxidase activity and edema. Lung ATP decreased after Etx but was maintained by 3-AB and Nic. Pulmonary arterial PARS activity increased fivefold after Etx, which 3-AB and Nic prevented. The beneficial effects were not observed with benzoic acid, a structural analog of 3-AB that does not inhibit PARS. Our results suggest that PARS inhibition with 3-AB or Nic improves pulmonary vasorelaxation and preserves lung ATP levels in acute lung injury.

Exogenous calcium preconditions myocardium from patients taking oral sulfonylurea agents.

We have previously reported that atrial trabeculae from patients taking oral sulfonylurea hypoglycemic agents cannot be preconditioned by transient ischemia, which may, in part, explain the increased cardiovascular mortality historically associated with the use of these agents (J. C. Cleveland et al., 1997, Circulation 96, 29-32). Recently, we reported that clinically accessible and acceptable exogenous Ca(2+) pretreatment protects human atrial trabeculae from subsequent ischemia (B. S. Cain et al., 1998, Ann. Thoracic Surg. 65, 1065-1070). It remains unknown whether this preconditioning strategy could confer protection to trabeculae from patients taking oral sulfonylurea drugs. We therefore hypothesized that exogenous Ca(2+) confers ischemic protection to trabeculae from patients taking oral sulfonylureas. Human atrial trabeculae were suspended in organ baths and field stimulated at 1 Hz, and force development was recorded. Following 90 min equilibration, trabeculae from patients taking oral sulfonylurea agents (n = 6 patients) were subjected to ischemia/reperfusion (I/R; 45/120 min) with or without Ca(2+) (1 mM increase x 5 min) 10 min prior to I/R. I/R decreased postischemic human myocardial contractility in trabeculae from patients on oral hypoglycemics to 15.3 +/- 2.0% baseline developed force (%BDF). Ca(2+) pretreatment increased postischemic human myocardial developed force to 35.3 +/- 2.9 %BDF in these patients (P < 0.05 vs I/R, ANOVA and Bonferroni/Dunn). We conclude that atrial muscle from patients taking oral hypoglycemic agents can be preconditioned with exogenous Ca(2+). This therapy may offer a clinically relevant means to precondition the myocardium of diabetics taking oral hypoglycemic agents prior to clinical interventions such as coronary angioplasty or cardiac bypass.

The NFkappaB inhibitory peptide, IkappaBalpha, prevents human vascular smooth muscle proliferation.

BACKGROUND: Vessel injury results in an inflammatory response characterized by the elaboration of cytokines and growth factors, which ultimately influence vascular smooth muscle cell (VSMC) growth and contribute to atherogenesis. Nuclear factor-kappa B (NFkappaB) is a central transcription factor important in mediating stress and inflammatory-induced signals. We hypothesized that strategies aimed at inhibiting NFkappaB would abrogate mitogen-induced human VSMC proliferation. METHODS: Human aortic VSMC were stimulated with basic fibroblast growth factor (FGF) and tumor necrosis factor-alpha (TNF), and proliferation was quantified by a colormetric assay. The influence of NFkappaB on VSMC proliferation was examined by both nonspecific NFkappaB blockade with calpain inhibitor-1 (CI-1) and dexamethasone (Dex) and specific NFkappaB blockade with liposomal delivery of the NFkappaB inhibitory peptide, IkappaBalpha. RESULTS: FGF and TNF induced concentration-dependent VSMC proliferation (p < 0.002). Neither CI-1, Dex, nor liposomal IkappaBalpha influenced proliferation of unstimulated VSMC. However, both FGF- and TNF-stimulated VSMC proliferation was inhibited to the level of control with CI-1, Dex, and liposomal IkappaBalpha (p < 0.001). CONCLUSION: The mitogenic effect of FGF and TNF on human arterial VSMC may be prevented by inhibiting NFkappaB. Furthermore, liposomal delivery of endogenous inhibitory proteins such as IkappaBalpha may represent a novel, therapeutically accessible method for selective transcriptional suppression in the response to vascular injury.

Liposomal delivery of purified inhibitory-kappaBalpha inhibits tumor necrosis factor-alpha-induced human vascular smooth muscle proliferation.

Vessel injury results in the elaboration of various cytokines, including tumor necrosis factor-alpha (TNF-alpha), which may influence vascular smooth muscle cell (VSMC) function and contribute to atherogenesis. We tested the hypothesis that TNF-alpha-induced VSMC proliferation requires activation of the transcription factor nuclear factor-kappaB (NF-kappaB), which could be prevented by delivery of the NF-kappaB inhibitory peptide, IkappaBalpha. TNF-alpha induced concentration-dependent human VSMC proliferation, and neutralizing antibody to interleukin-6 reduced TNF-alpha-induced VSMC proliferation by 65%. In TNF-alpha-stimulated VSMCs, there was a 3-fold increase in NF-kappaB-dependent luciferase reporter activity that was associated with degradation of IkappaBalpha. To determine an essential role for NF-kappaB in TNF-alpha-induced VSMC proliferation, recombinant IkappaBalpha was introduced into VSMCs via liposomal delivery. Under these conditions, TNF-alpha-induced NF-kappaB nuclear translocation and DNA binding were inhibited, NF-kappaB-dependent luciferase activity was reduced by 50%, there was no degradation of native IkappaBalpha detected, interleukin-6 production was reduced by 54%, and VSMC proliferation was decreased by 60%. In conclusion, the mitogenic effect of TNF-alpha on human arterial VSMCs is dependent on NF-kappaB activation and may be prevented by exogenously delivered IkappaBalpha. Furthermore, liposomal delivery of endogenous inhibitory proteins may represent a novel, therapeutically accessible method for selective transcriptional suppression in the response to vascular injury.

LPS-Induced NF-kappaB activation and TNF-alpha release in human monocytes are protein tyrosine kinase dependent and protein kinase C independent.

BACKGROUND: Tumor necrosis factor alpha (TNF-alpha) is an important mediator of septic shock. Endotoxin (LPS) signal transduction in human monocytes leads to activation of nuclear factor-kappa B (NF-kappaB) and TNF-alpha release. Previous studies have implicated activation of both protein kinase C (PKC) and protein tyrosine kinases (PTK) in LPS-induced NF-kappaB activation and TNF-alpha production. We hypothesized that inhibition of either PKC or PTK would decrease LPS-induced NF-kappaB DNA binding and TNF-alpha release in human monocytes. MATERIALS AND METHODS: Human monocytes were stimulated with PMA (50 ng/ml) alone or LPS (100 ng/ml) with and without a nonspecific serine/threonine protein kinase inhibitor staurosporine (Stauro), a specific pan-PKC inhibitor bisindolylmaleimide (Bis), or an inhibitor of PTK genistein (Gen). TNF-alpha release in culture supernatants was measured by an ELISA. NF-kappaB DNA binding was evaluated by electrophoretic mobility shift assay. RESULTS: LPS increased NF-kappaB DNA binding and TNF-alpha release in human monocytes. Nonspecific protein kinase inhibition inhibited NF-kappaB activation and TNF-alpha release, while specific PKC inhibition with Bis had no effect on LPS-induced NF-kappaB DNA binding or TNF-alpha release. PTK inhibition with Gen attenuated both LPS-induced NF-kappaB DNA binding and TNF-alpha production in human monocytes. Direct activation of PKC with PMA induced both NF-kappaB activation and TNF-alpha production by human monocytes. CONCLUSIONS: These results suggest that LPS-induced NF-kappaB activation and TNF-alpha release in human monocytes are independent of PKC activity. Furthermore, our results provide evidence that PTK plays a role in LPS-induced NF-kappaB activation and TNF-alpha release in human monocytes and thus could be a potential therapeutic target in inflammatory states.

Utilization of endoscopic inoculation in a mouse model of intrauterine infection-induced preterm birth: role of interleukin 1beta.

A novel murine model of intrauterine infection/inflammation-induced preterm birth based on direct endoscopic intracervical inoculation is described. Using this model, we investigated infection-induced premature pregnancy loss in normal and interleukin (IL) 1beta-deficient mice. Seventy-four CD-1, HS, C57BL/6J wild type (IL-1beta+/+), and C57BL/6J IL-1beta-deficient (IL-1beta-/-) mice were inoculated intracervically using a micro-endoscope, at a time corresponding to 70% of average gestation. Intracervical injection of lipopolysaccharide (LPS) or Escherichia coli reliably induced premature birth: 100% of mice intracervically injected with LPS and 92% of mice with a positive endometrial E. coli culture delivered prematurely within 36 h after inoculation. No losses were observed in mice inoculated with saline. Pregnancy loss was associated with increased uterine tissue cyclooxygenase-2 gene expression and uterine content of IL-1beta, tumor necrosis factor alpha, macrophage inflammatory protein-1alpha, and IL-6, as well as elevation of nuclear factor-kappaB activity in uterine tissues. Although IL-1beta-/- mice exhibited decreased uterine cytokine production in response to bacteria and LPS, IL-1beta deficiency did not affect the rate of pregnancy loss. This model using direct intracervical bacterial or LPS inoculation is useful for studying preterm pregnancy loss in genetically altered mice in order to develop novel interventions for infection-associated preterm labor.