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L Kobzik

Publications and source records attributed to L Kobzik.

At least 55 records · Page 3Linked to original sources
PubMed1999

Hypoxia induces severe right ventricular dilatation and infarction in heme oxygenase-1 null mice.

Heme oxygenase (HO) catalyzes the oxidation of heme to generate carbon monoxide (CO) and bilirubin. CO increases cellular levels of cGMP, which regulates vascular tone and smooth muscle development. Bilirubin is a potent antioxidant. Hypoxia increases expression of the inducible HO isoform (HO-1) but not the constitutive isoform (HO-2). To determine whether HO-1 affects cellular adaptation to chronic hypoxia in vivo, we generated HO-1 null (HO-1(-/-)) mice and subjected them to hypoxia (10% oxygen) for five to seven weeks. Hypoxia caused similar increases in right ventricular systolic pressure in wild-type and HO-1(-/-) mice. Although ventricular weight increased in wild-type mice, the increase was greater in HO-1(-/-) mice. Similarly, the right ventricles were more dilated in HO-1(-/-) mice. After seven weeks of hypoxia, only HO-1(-/-) mice developed right ventricular infarcts with organized mural thrombi. No left ventricular infarcts were observed. Lipid peroxidation and oxidative damage occurred in right ventricular cardiomyocytes in HO-1(-/-), but not wild-type, mice. We also detected apoptotic cardiomyocytes surrounding areas of infarcted myocardium by terminal deoxynucleotide transferase-mediated dUTP nick end-labeling (TUNEL) assays. Our data suggest that in the absence of HO-1, cardiomyocytes have a maladaptive response to hypoxia and subsequent pulmonary hypertension. J.Clin. Invest. 103:R23-R29 (1999).

Animals↗
PubMed1999

Toll4 (TLR4) expression in cardiac myocytes in normal and failing myocardium.

Expression of innate immune response proteins, including IL-1beta, TNF, and the cytokine-inducible isoform of nitric oxide synthase (iNOS), have been documented in the hearts of humans and experimental animals with heart failure regardless of etiology, although the proximal events leading to their expression are unknown. Noting that expression of a human homologue of Drosophila Toll, a proximal innate immunity transmembrane signaling protein in the fly, now termed human Toll-like receptor 4 (hTLR4), appeared to be relatively high in the heart, we examined TLR4 mRNA and protein abundance in isolated cellular constituents of cardiac muscle and in normal and abnormal murine, rat, and human myocardium. TLR4 expression levels in cardiac myocytes and in coronary microvascular endothelial cells could be enhanced by either LPS or IL-1beta, an effect inhibited by the oxygen radical scavenger PDTC. Transfection of a constitutively active TLR4 construct, CD4/hTLR4, resulted in activation of a nuclear factor-kappaB reporter construct, but not of an AP-1 or an iNOS reporter construct, in cardiac myocytes. In normal murine, rat, and human myocardium, TLR4 expression was diffuse, and presumably cytoplasmic, in cardiac myocytes. However, in remodeling murine myocardium remote from sites of ischemic injury and in heart tissue from patients with idiopathic dilated cardiomyopathy, focal areas of intense TLR4 staining were observed in juxtaposed regions of 2 or more adjacent myocytes; this staining was not observed in control myocardium. Increased expression and signaling by TLR4, and perhaps other Toll homologues, may contribute to the activation of innate immunity in injured myocardium.

Amino Acid Sequence↗
PubMed1999

Intestinal ischemia-reperfusion injury is mediated by the membrane attack complex.

BACKGROUND: The dependence of intestinal ischemia-reperfusion injury on the classical complement pathway has been shown with the complement antagonist (sCR1) and complement-specific knockout mice. Using C5 deficient mice, we show that the membrane attack complex mediates local injury. METHODS: Mice underwent intestinal ischemia-reperfusion. Albumin leak and histologic evidence were compared in wildtype mice, wildtypes treated with sCR1, neutrophil-depleted wildtypes, C5-deficient mice, and C5-deficient mice reconstituted with wildtype serum. Neutrophil tissue levels in injured C5-deficient and wildtype intestines were compared. RESULTS: C5-deficient mice had a reduction in injury similar to mice treated with sCR1. Injury was restored by reconstitution with wildtype serum. Wildtype injury was unaffected by neutrophil depletion. Injured intestines of C5-deficient and wildtype mice had similar neutrophil levels. Immunohistochemistry of wildtype and reconstituted C5-deficient mice demonstrated injured intestinal epithelium although C5-deficient mice and sCr1-treated mice were similar to sham mice. CONCLUSIONS: C5-deficient animals are protected from local injury. Injury is unaffected by neutrophil depletion, and the presence of neutrophils in injured tissue is independent of C5. Local injury is C5 dependent, but the action of C5a on granulocytes is not required. Therefore the membrane attack complex mediates local injury.

Animals↗
PubMed1998

The negative inotropic effect of beta3-adrenoceptor stimulation is mediated by activation of a nitric oxide synthase pathway in human ventricle.

Beta1- and beta2-adrenoceptors in heart muscle cells mediate the catecholamine-induced increase in the force and frequency of cardiac contraction. Recently, in addition, we demonstrated the functional expression of beta3-adrenoceptors in the human heart. Their stimulation, in marked contrast with that of beta1- and beta2-adrenoceptors, induces a decrease in contractility through presently unknown mechanisms. In the present study, we examined the role of a nitric oxide (NO) synthase pathway in mediating the beta3-adrenoceptor effect on the contractility of human endomyocardial biopsies. The negative inotropic effects of a beta3-adrenoceptor agonist, BRL 37344, and also of norepinephrine in the presence of alpha- and beta1-2-blockade were inhibited both by a nonspecific blocker of NO, methylene blue, and two NO synthase (NOS) inhibitors, L-N-monomethyl-arginine and L-nitroarginine-methyl ester. The effect of the NOS inhibitors was reversed by an excess of L-arginine, the natural substrate of NOS, but not by D-arginine. Moreover, the effects of the beta3-adrenoceptor agonist on contractility were associated with parallel increases in the production of NO and intracellular cGMP, which were also inhibited by NOS inhibitors. Immunohistochemical staining of human ventricular biopsies showed the expression of the endothelial constitutive (eNOS), but not the inducible (iNOS) isoform of NOS in both ventricular myocytes and endothelial cells. These results demonstrate that beta3-adrenoceptor stimulation decreases cardiac contractility through activation of an NOS pathway. Changes in the expression of this pathway may alter the balance between positive and negative inotropic effects of catecholamines on the heart potentially leading to myocardial dysfunction.

Adrenergic beta-Agonists↗
PubMed1998

Host reactive donor T cells are associated with lung injury after experimental allogeneic bone marrow transplantation.

Noninfectious lung injury is common after allogeneic bone marrow transplantation (BMT), but its association with acute graft-versus-host disease (GVHD) is unclear. Using a murine BMT system where donor and host differ by multiple minor histocompatibility (H) antigens, we investigated the nature of lung injury and its relationship both to systemic GVHD and host-reactive donor T cells. Lethally irradiated CBA hosts received syngeneic BMT or allogeneic (B10.BR) T-cell-depleted (TCD) bone marrow (BM) with and without the addition of T cells. Six weeks after BMT, significant pulmonary histopathology was observed in animals receiving allogeneic BMT compared with syngeneic controls. Lung damage was greater in mice that received allogeneic T cells and developed GVHD, but it was also detectable after TCD BMT when signs of clinical and histologic acute GVHD were absent. In each setting, lung injury was associated with significant alterations in pulmonary function. Mature, donor (Vbeta6(+) and Vbeta3(+)) T cells were significantly increased in the broncho-alveolar lavage (BAL) fluid of all allogeneic BMT recipients compared with syngeneic controls, and these cells proliferated and produced interferon-gamma (IFN-gamma) to host antigens in vitro. These in vitro responses correlated with increased IFN-gamma and tumor necrosis factor-alpha (TNF-alpha) in the BAL fluid. We conclude that alloreactive donor lymphocytes are associated with lung injury in this allogeneic BMT model. The expansion of these cells in the BAL fluid and their ability to respond to host antigens even when systemic tolerance has been established (ie, the absence of clinical GVHD) suggest that the lung may serve as a sanctuary site for these host reactive donor T cells. These findings may have important implications with regard to the evaluation and treatment of pulmonary dysfunction after allogeneic BMT even when clinical GVHD is absent.

Animals↗
PubMed1998

A role for transforming growth factor-beta1 in the increased pneumonitis in murine allogeneic bone marrow transplant recipients with graft-versus-host disease after pulmonary herpes simplex virus type 1 infection.

To gain further insights in the pathogenesis of herpesvirus pneumonia in allogeneic bone marrow transplant recipients, transplanted mice (B10.BR --> CBA) with graft-versus-host disease (GVHD) and control mice (transplanted mice without GVHD and normal CBA mice) were infected intranasally with herpes simplex virus type 1 (HSV-1). When compared with infected control mice, infected allogeneic transplant recipients with GVHD showed increased periluminal mononuclear cell infiltrates. However, infected allogeneic transplant recipients with GVHD showed lower virus content in the lung tissue than infected control mice. High concentrations of transforming growth factor-beta 1 (TGF-beta1) were detected in the bronchoalveolar lavage (BAL) fluid of mock-infected allogeneic transplant recipients with GVHD, which increased slightly after infection. Anti-TGF-beta treatment of allogeneic transplant recipients with GVHD significantly decreased the histological evidence of pneumonitis at day 4 after HSV-1 infection. We conclude that allogeneic transplant recipients with GVHD have (1) increased pneumonia, (2) highly elevated levels of TGF-beta1 in the BAL fluid, and (3) reduced pulmonary virus content after HSV-1 infection. Our data suggest that the newly recognized dysregulation of cytokine (TGF-beta1) production may be more important than the viral load for the increased severity of HSV-1 pneumonia in allogeneic transplant recipients with GVHD.

Animals↗
PubMed1998

Environmental particulate-mediated cytokine production in lung epithelial cells (A549): role of preexisting inflammation and oxidant stress.

Epidemiologic data show that air pollution particulates cause adverse pulmonary health effects, especially in individuals with preexisting lung disease. We sought to model in vitro preexisting lung inflammation in order to investigate the hypothesis that "primed" lung epithelial cells will exhibit enhanced phlogistic responses [e.g., interleukin-8 (IL-8) production] to particulate air pollution. Exposure of tumor necrosis factor alpha (TNF-alpha) primed or control A549 cells to the air pollution particulates, residual oil fly ash (ROFA), and the known pathogenic dust alpha-quartz, but not inert TiO2, caused increased IL-8 production in primed cells compared to normal cells in a concentration-dependent manner (particle concentration range 0-200 microg/ml). We hypothesized that oxidant mechanisms may be involved in the cellular response to particulates. Addition of the antioxidant N-acetylcysteine (NAC, 1.0 mM) decreased ROFA and alpha-quartz-mediated IL-8 production by approximately 50% in normal and TNF-alpha-primed A549 cells. In addition, exposure of A549 cells to ROFA caused a substantial (and NAC inhibitable) increase in oxidant levels as measured by fluorometry (DCFH oxidation). These data suggest that (1) lung epithelial cells primed by inflammatory mediators can show enhanced cytokine production after exposure to air pollution particulates, and (2) oxidant stress is a key mechanism for this response.

Acetylcysteine↗
PubMed1998

Analysis of air pollution particulate-mediated oxidant stress in alveolar macrophages.

Adverse health effects of urban air pollution particulates may be attributable to particle-mediated oxidant stress and inflammation. Intracellular oxidant production in normal hamster alveolar macrophages (AMs) was measured upon exposure to concentrated ambient particulates (CAPs), residual oil fly ash (ROFA), and their water-soluble and particulate fractions. ROFA and CAPs caused increases in dichlorofluorescin (DCFH) oxidation, a fluorescent measure of intracellular reactive oxygen species (ROS) production, comparable to the positive control, phorbol myristate acetate (PMA). The water-soluble component of both CAPs and ROFA (CAPs, S and ROFA, S) significantly increased AM oxidant production over negative control. CAPs samples and components showed substantial day-to-day variability in their oxidant effects. Metal chelation by desferrioxamine (DF, 1 mM) caused significant inhibition of particulate-induced AM oxidant production. ROFA exposure resulted in increased macrophage inflammatory protein-2 (MIP-2) message in AMs and in increased tumor necrosis factor alpha (TNF-alpha) production by the monocyte-macrophage cell line, RAW 264.7. TNF-alpha production was inhibitable by the antioxidant N-acetylcysteine (NAC). The data suggest that metal components adsorbed to urban air pollution particulates can significantly contribute to particulate ability to cause oxidant stress and cytokine production in AMs.

Acetylcysteine↗
PubMed1998

Characteristics of nitric oxide-mediated cholinergic modulation of calcium current in rabbit sino-atrial node.

1. We have previously shown that nitric oxide (NO) production is essential for cholinergic inhibition of the beta-adrenergic stimulated L-type calcium current (ICa-L) in rabbit pacemaker (sino-atrial node (SAN)) cells. The present experiments demonstrate the presence of constitutive nitric oxide synthase (cNOS) in SAN cells, and characterize the NO-mediated cholinergic response. 2. Immunohistochemical staining, using an antibody prepared against endothelial cNOS, demonstrated that this enzyme was present in single myocytes obtained from the SAN. 3. The activation of cNOS is known to be Ca2+ and calmodulin dependent. Strongly buffering intracellular Ca2+ with the membrane-permeable chelator BAPTA-AM (10 microM) significantly reduced (and in some cases abolished) the attenuation of ICa-L by the muscarinic agonist carbamylcholine (CCh). In contrast, the CCh-induced activation of an outward K+ current, IK,ACh, was unaffected by buffering of [Ca2+]i. The calmodulin inhibitor 48/80 (20 microM) also abolished the attenuation of ICa-L by CCh, with no change in the activation of IK,ACh. 4. Neither thapsigargin nor ryanodine (5-10 microM), agents which deplete intracellular Ca2+ stores, significantly changed the attenuation of ICa-L by CCh. 5. Pertussis toxin (PTX) completely abolished both the inhibitory action of CCh on ICa-L and the activation of IK,ACh. This establishes that a PTX-sensitive GTP-binding protein links the muscarinic receptor to NO synthase activation in SAN cells. 6. Our hypothesis is that NO leads to activation of a cyclic GMP (cGMP)-activated phosphodiesterase (PDE II) as a mechanism for enhanced cyclic AMP breakdown and ICa-L attenuation. This was supported by showing that a specific inhibitor of PDE II, erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), blocks the effect of CCh on ICa-L, but not on IK,ACh. Using reverse transcriptase-polymerase chain reaction techniques, we have established that PDE II is the dominant cyclic nucleotide phosphodiesterase isoform in SAN cells.

1-Methyl-3-isobutylxanthine↗
PubMed1998

Stromelysin-3 is induced in tumor/stroma cocultures and inactivated via a tumor-specific and basic fibroblast growth factor-dependent mechanism.

Stromelysin-3 (STR-3) is a recently characterized matrix metalloproteinase (MMP) with a unique pattern of expression and substrate specificity. Unlike other MMPs, STR-3 is consistently and dramatically overexpressed by multiple epithelial malignancies, including carcinomas of the breast, lung, colon, head and neck, and skin. Recent studies suggest that STR-3 promotes the local establishment of epithelial malignancies, contributing to tumor cell survival and implantation in host tissues; however, STR-3's mechanism of action remains undefined. STR-3 is a stromal cell product, prompting speculation that infiltrating stromal cells secrete STR-3 in response to tumor-derived factors. To explore this possibility, we developed a tumor/"stroma" coculture assay in which non-small cell lung cancer (NSCLC) cell lines were grown on confluent monolayers of normal pulmonary fibroblasts. In these tumor/stroma cocultures, NSCLCs stimulate normal pulmonary fibroblasts to secrete STR-3 and release extracellular basic fibroblast growth factor. Thereafter, STR-3 is processed at a unique internal sequence via a basic fibroblast growth factor- and MMP-dependent mechanism to a previously unidentified 35-kDa protein that lacks enzymatic activity. 35-kDa STR-3 is the most abundant STR-3 protein in tumor/stroma cocultures and is only detected when normal pulmonary fibroblasts are cultured with malignant bronchial epithelial cells. Therefore, the tumor-specific processing of STR-3 to the 35-kDa protein is likely to be an important regulatory mechanism.

Amino Acid Sequence↗
PubMed1998

Nitric oxide modulates excitation-contraction coupling in the diaphragm.

We investigated the enzymatic source, cellular production, and functional importance of nitric oxide (NO) in rat diaphragm. Neuronal and endothelial isoforms of constituitive nitric oxide synthase (nc-NOS, ec-NOS) were identified by immunostaining. NOS activity measured in diaphragm homogenates averaged 5.1 pmol/min/mg. Passive diaphragm fiber bundles produced NO derivatives (NOx) at the rate of 0.9 pmol/min/mg as measured by the cytochrome c reduction assay; NO production was confirmed by photolysis/ chemiluminescence measurements. Endogenous NO depressed diaphragm contractile function. The force of submaximal contraction was increased by NOS inhibitors, an effect that was stable for up to 60 min and was reversed by NO donors. We conclude that diaphragm muscle fibers express nc-NOS, ec-NOS, or both; passive myocytes produce NOx; and NO or NO-derivatives inhibit force production by modulating excitation-contraction coupling.

Animals↗
PubMed1998

Cyclic GMP is a second messenger by which nitric oxide inhibits diaphragm contraction.

We have shown that endogenous nitrogen oxides (NOx) modulate excitation-contraction coupling in diaphragm. Because cyclic GMP (cGMP) is a second messenger for nitric oxide (NO) inhibition of smooth muscle contraction, we rested the hypothesis that NO acts via cGMP in diaphragm. Fiber bundles from rat diaphragm were studied in vitro. Immunohistochemical analysis using a cGMP-specific monoclonal antibody confirmed the presence of cGMP in the subsarcolemmal region, near nitric oxide synthase (NOS). cGMP measured by ELISA in control muscle (0.27 pmol/mg +/- 0.01 SE) was significantly increased by the NO donor S-nitroso-N-acetylcysteine 1 mM (0.55+/-0.05; N = 6; P < 0.001). Contractile studies showed that the nitric oxide synthase inhibitor N-nitro-L-arginine (L-NNA) 10 mM increased submaximal (40 Hz) tetanic force (P < 0.0001). L-NNA effects were exaggerated by the guanylate cyclase inhibitor LY83583 5-10 microM; force at 40 Hz was increased (P < 0.001). L-NNA effects were partially reversed by 8-bromo-cGMP 1 mM (8-Br-GMP; a cell-permeable cGMP analogue; P < 0.0001) or dipyridamole 10 microM (DPM; a phosphodiesterase inhibitor; P < 0.0001). 8-Br-GMP and DPM produced more-complete L-NNA reversal in combination (P < 0.0001). We conclude that cGMP functions as a second messenger by which NO inhibits diaphragm contraction.

Aminoquinolines↗
PubMed1998

Minimum protective serum concentrations of pneumococcal anti-capsular antibodies in infant rats.

Infant rats were passively immunized to determine the protective capacity of pneumococcal anticapsular antibodies. Animal-passaged strains of Streptococcus pneumoniae serotypes 1, 4, 5, 6b, 7f, 9v, 14, 18c, 19f, and 23f were used as challenge inocula (1-1500 cfu) in a model of pulmonary infection that resulted in bacteremia, meningitis, and death. From untreated control animals, histologic sections of lung demonstrated infiltrative pneumonia and lung homogenate cultures grew S. pneumoniae at concentrations of 10(3)-10(8) cfu per gram of lung tissue. A type-specific anti-capsular antibody serum concentration of 0.1-1.15 microg/mL resulted in a statistically significant reduction in mortality compared with the reduction in untreated controls, except for serotype 14, which required 2.32 microg/mL for a significant reduction in mortality. The serum antibody level that provided 50% reduction in mortality ranged from 0.1-3.5 microg/mL for all serotypes.

Animals↗
PubMed1998

Effect of TNF-alpha antisense oligomers on cytokine production by primary murine alveolar macrophages.

Antisense oligomers can inhibit expression of a single gene in a sequence-specific manner. As a result, these sequences are being developed both as powerful experimental tools in the laboratory and as a novel class of therapeutic agents. In this study, we evaluated a panel of morpholino antisense (M-AS) oligomers for their ability to inhibit tumor necrosis factor-alpha (TNF-alpha) production by primary murine alveolar macrophages (AMs) and compared them with the more commonly used phosphorothioate oligonucleotides (S-AS). We found that 25 microM of morpholino oligomers whose sequence spanned the AUG (M-AS 2, M-AS 2me, and M-AS 5) start codon of TNF-alpha significantly inhibited TNF production on stimulation by both lipopolysaccharides (LPS) (36.6 +/- 3.2%, 27.3 +/- 3.0%, and 37.7 +/- 2.0% inhibition, respectively), whereas S-AS targeted toward the same region were ineffective. M-AS 2 and M-AS 2me also significantly inhibited TNF production in AMs stimulated by adherence to a solid substrate (28.7 +/- 2.2% and 29.4 +/- 8.3% inhibition, respectively). Increasing the concentration of M-AS 2 and M-AS 2me to 50 microM improved their efficacy in both LPS-stimulated (42.7 +/- 1.5% and 45.9 +/- 2.1% inhibition, respectively) and adherence-stimulated (52.6 +/- 0.7% and 41.7 +/- 2.9% inhibition, respectively) AMs. In contrast, we showed that neither an antisense sequence targeted to a region upstream of the AUG site (M-AS 4) nor the nonsense control sequences M-NS 1 and M-NS 2 significantly inhibited TNF-alpha production by AMs on exposure to either stimulus. The data indicate that morpholino oligomers inhibit TNF-alpha production by murine AMs in a sequence-dependent and dose-dependent manner.

Animals↗
PubMed1998

Both CD80 and CD86 co-stimulatory molecules regulate allergic pulmonary inflammation.

We examined the roles of CD80 (B7-1) and CD86 (B7-2) in a model of allergic pulmonary inflammation and airway hyper-responsiveness (AHR) by selectively inhibiting either CD80 or CD86. Inhibition of co-stimulation by either CD80 or CD86 affected multiple parameters of the allergic response. Specifically, blockade of either CD80 or CD86 in ovalbumin-sensitized and challenged mice resulted in reduced expression of IL-2Ralpha (CD25) on CD4+ T lymphocytes, decreased airway eosinophilia, lower serum IgE production and diminished AHR. Importantly, blockade of CD80 and CD86 inhibited production of IL-4 and IL-2, and enhanced IFN-gamma production. Our observations support a role for both CD80- and CD86-mediated co-stimulation in development of allergic pulmonary inflammation.

Animals↗
PubMed1998

Peritonitis causes diaphragm weakness in rats.

Respiratory failure is a common and often lethal complication of severe peritonitis. Because this inflammatory process develops in the abdomen, adjacent to the diaphragm, we hypothesized that peritonitis might directly compromise diaphragm function. We tested this hypothesis using male Sprague-Dawley rats. We injected oyster glycogen into the rats' peritoneum, and 16 h later the peritoneum was lavaged for leukocyte analysis and muscle samples were excised. Contractile properties of diaphragm fiber bundles were measured in vitro. We found that neutrophils and macrophages were concentrated in peritoneal lavage fluid of experimental animals (p < 0.01) and were adherent to the abdominal surface of the diaphragm. Immunohistochemistry showed increases in inducible nitric oxide synthase in microvessels of the diaphragm and limb skeletal muscles but not in heart or spleen. Peritonitis decreased maximal force production by the diaphragm (23.6+/-0.6 versus 21.2+/-0.6 N/cm2; p < 0.05) and decreased the absolute forces developed at physiologic stimulus frequencies (> 30 Hz; p < 0.01), depressing the overall force-frequency relationship (p < 0.001). Peritonitis had little effect on acute muscular fatigue. These data demonstrate that peritonitis weakens the diaphragm in rats and suggest that humans with peritonitis may be predisposed to respiratory muscle dysfunction.

Animals↗