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N Lukacs

Publications and source records attributed to N Lukacs.

At least 19 recordsLinked to original sources
PubMed2001

Therapeutic effect of IL-13 immunoneutralization during chronic experimental fungal asthma.

IL-13 and IL-4 are key contributors to the asthmatic phenotype. The temporal role of these cytokines in airway function, inflammation, and remodeling were assessed in a chronic murine model of Asperigillus fumigatus-induced allergic asthma. IL-13 and IL-4 protein levels were significantly elevated by 30 days after conidia challenge in A. fumigatus-sensitized mice. Furthermore, IL-13Ralpha1 mRNA expression was significantly elevated 7 days after conidia challenge and remained elevated until day 21. In contrast, IL-13Ralpha2 mRNA expression, although constitutively expressed in naive lung, was absent in the lungs of A. fumigatus-sensitized mice both before and after conidia challenge. Membrane-bound IL-4R mRNA expression was significantly elevated 7 days after conidia challenge; however, soluble IL-4R mRNA expression was increased 30 days after conidia challenge. Immunoneutralization of IL-13 between days 14 and 30 or days 30 and 38 after fungal sensitization and challenge significantly attenuated airway hyperresponsiveness, collagen deposition, and goblet cell hyperplasia at day 38 after conidia challenge; however, the effects of IL-4 immunoneutralization during the same time periods were not as marked. IFN-gamma and IL-12 release after Aspergillus Ag restimulation was elevated from spleen cells isolated from mice treated with IL-4 anti-serum compared with IL-13 anti-serum or normal rabbit serum-treated mice. This study demonstrates a pronounced therapeutic effect of IL-13-immunoneutralization at extended time points following the induction of chronic asthma. Most importantly, these therapeutic effects were not reversed following cessation of treatment, and IL-13 anti-serum treatment did not alter the systemic immune response to Ag restimulation, unlike IL-4 immunoneutralization. Therefore, IL-13 provides an attractive therapeutic target in allergic asthma.

Animals↗
PubMed2000

Determination of preferential binding sites for anti-dsRNA antibodies on double-stranded RNA by scanning force microscopy.

The monoclonal anti-dsRNA antibody J2 binds double-stranded RNAs (dsRNA) in an apparently sequence-nonspecific way. The mAb only recognizes antigens with double-stranded regions of at least 40 bp and its affinity to poly(A) poly(U) and to dsRNAs with mixed base pair composition is about tenfold higher than to poly(I) poly(C). Because no specific binding site could be determined, the number, the exact dimensions, and other distinct features of the binding sites on a given antigen are difficult to evaluate by biochemical methods. We therefore employed scanning force microscopy (SFM) as a method to analyze antibody-dsRNA interaction and protein-RNA binding in general. Several in vitro-synthesized dsRNA substrates, generated from the Dictyostelium PSV-A gene, were used. In addition to the expected sequence-nonspecific binding, imaging of the complexes indicated preferential binding of antibodies to the ends of dsRNA molecules as well as to certain internal sites. Analysis of 2,000 bound antibodies suggested that the consensus sequence of a preferential internal binding site is A2N9A3N9A2, thus presenting A residues on one face of the helix. The site was verified by site-directed mutagenesis, which abolished preferential binding to this region. The data demonstrate that SFM can be efficiently used to identify and characterize binding sites for proteins with no or incomplete sequence specificity. This is especially the case for many proteins involved in RNA metabolism.

Animals↗
PubMed1999

Local production of chemokines during experimental vaginal candidiasis.

Recurrent vulvovaginal candidiasis, caused by Candida albicans, is a significant problem in women of childbearing age. Although cell-mediated immunity (CMI) due to T cells and cytokines is the predominant host defense mechanism against C. albicans at mucosal tissue sites, host defense mechanisms against C. albicans at the vaginal mucosa are poorly understood. Based on an estrogen-dependent murine model of vaginal candidiasis, our data suggest that systemic CMI is ineffective against C. albicans vaginal infections. Thus, we have postulated that local immune mechanisms are critical for protection against infection. In the present study, the kinetic production of chemokines normally associated with the chemotaxis of T cells, macrophages (RANTES, MIP-1alpha, MCP-1), and polymorphonuclear neutrophils (MIP-2) was examined following intravaginal inoculation of C. albicans in estrogen-treated or untreated mice. Results showed significant increases in MCP-1 protein and mRNA in vaginal tissue of infected mice as early as 2 and 4 days postinoculation, respectively, that continued through a 21-day observation period, irrespective of estrogen status. No significant changes were observed with RANTES, MIP-1alpha, or MIP-2, although relatively high constitutive levels of RANTES mRNA and MIP-2 protein were observed. Furthermore, intravaginal immunoneutralization of MCP-1 with anti-MCP-1 antibodies resulted in a significant increase in vaginal fungal burden early during infection, suggesting that MCP-1 plays some role in reducing the fungal burden during vaginal infection. However, the lack of changes in leukocyte profiles in vaginal lavage fluids collected from infected versus uninfected mice suggests that MCP-1 functions to control vaginal C. albicans titers in a manner independent of cellular chemotactic activity.

Animals↗
PubMed1998

Cytokine and chemokine production in HSV-1 latently infected trigeminal ganglion cell cultures: effects of hyperthermic stress.

The establishment of a primary trigeminal ganglion (TG) cell culture latently infected with herpes simplex virus type 1 (HSV-1) has been useful in studying stress-induced reactivation of the latent virus. However, the immune profile of this culture system prior to and after stress has never been established. In the present manuscript, cytokine and chemokine production were measured in primary cultures of TG cells obtained from uninfected and HSV-1 latently infected mice. Supernates from TG cell cultures contained detectable interleukin (IL)-6 but not IL-1beta, IL-2, IL-10, interferon (IFN)-gamma or tumor necrosis factor (TNF)-alpha as determined by ELISA. The basal level of IL-6 in uninfected TG cell cultures was 20.5 +/- 2.3 ng/ml, whereas latently infected TG cells produced significantly less IL-6 (12.1 +/- 1.9 ng/ml). Supernates from TG cell cultures also contained detectable levels of C-10, MCP-1 and eotaxin but little to no MIP-1alpha, MIP-1beta, or MIP-2. While there were no differences in the basal level of MCP-1 and eotaxin in TG cell cultures from HSV-1-infected and uninfected mice, C10 levels were significantly higher in TG cultures originating from infected mice compared to uninfected ones (5.86 +/- 0.61 ng/ml compared to 1.18 +/- 0.16 ng/ml). Hyperthermic stress (43 degrees C, 180 min), which induces reactivation of latent HSV-1 from TG cell cultures, significantly reduced IL-6 and C-10 levels from both uninfected and latently infected TG cell cultures. However, there was no correlation between cytokine/chemokine levels and HSV-1 reactivation. Immunofluorescent studies showed TG cell cultures contained 10% MAC-3+ staining cells (macrophage specific) but no dendritic cells. By comparison, cells from freshly isolated TG contained 6% positive dendritic cells but < 1% MAC-3 + cells. Both in vivo and in vitro TG consisted of a low percentage of CD3+ and CD8+ cells. Hyperthermic stress (43 degrees C for 3 h) eliminated the lymphocyte population as determined by RT-PCR. Whereas no spontaneous reactivation has been reported in mice, spontaneous reactivation occurred in 4.5% (10/220) of TG cell cultures surveyed over a 20 day period. Collectively, the dichotomy between HSV-1 replication and reactivation comparing the in vitro and in vivo HSV-1 latency models may reside, in part, to the differences in the levels of cytokines, chemokines and immune cell populations within the microenvironment of the in vitro and in vivo TG.

Animals↗
PubMed1998

Interferon-induced protein 10 and interleukin 8. C-X-C chemokines present in proliferative diabetic retinopathy.

OBJECTIVE: To determine vitreous levels of interleukin 8 (IL-8) and interferon-induced protein 10 (IP-10), which are members of the C-X-C chemokine family that promote and inhibit neovascularization, respectively. METHODS: We measured the levels of IL-8 and IP-10 by specific enzyme-linked immunosorbent assays in the vitreous from 30 patients with proliferative diabetic retinopathy (PDR) and 10 control patients undergoing vitrectomy for idiopathic macular holes or idiopathic macular puckers. RESULTS: Detectable levels of IL-8 were found in 23 of 24 patients with active PDR, 4 of 6 patients with inactive PDR, and 6 of 10 controls. Levels of IL-8 were significantly increased in vitreous samples from the patients with active PDR (P = .02) when compared with vitreous samples from the controls. The IL-8 levels detected in vitreous samples from patients with inactive PDR were not significantly elevated over those found in the control samples. Interferon-induced protein 10 was detected in the vitreous samples from 23 of 24 patients with active PDR, all patients with inactive PDR, and 9 of 10 controls. Significant elevations of IP-10 were measured in samples from patients with active PDR (P = .004) and in those with inactive PDR (P = .00) over those from controls. In addition, levels of IP-10 were significantly elevated in vitreous samples from patients with inactive PDR compared with vitreous samples from patients with active PDR (P = .02). CONCLUSION: Both IL-8 and IP-10 participate in the pathogenesis of PDR.

Adult↗
PubMed1998

The role of IL-5 in bleomycin-induced pulmonary fibrosis.

Eosinophils are known to express cytokines capable of promoting fibrosis. Interleukin-5 (IL-5) is important in regulating eosinophilopoiesis, eosinophil recruitment and activation. Lung IL-5 expression is elevated in pulmonary fibrosis, wherein the eosinophil is a primary source of fibrogenic cytokines. To determine the role of IL-5 in pulmonary fibrosis, the effects of anti-IL-5 antibody were investigated in a model of bleomycin-induced pulmonary fibrosis. Fibrosis was induced in mice by endotracheal bleomycin treatment. Animals were also treated with either anti-IL-5 antibody or control IgG. Lungs were then analyzed for fibrosis, eosinophil influx, chemotactic activity, and cytokine expression. The results show that a primary chemotactic activity at the height of eosinophil recruitment is IL-5. Furthermore, anti-IL-5 antibody caused significant reduction in lung eosinophilia, cytokine expression, and fibrosis. These findings taken together suggest an important role for IL-5 in pulmonary fibrosis via its ability to regulate eosinophilic inflammation, and thus eosinophil-dependent fibrogenic cytokine production.

Animals↗
PubMed1998

Tumor necrosis factor up-regulates intercellular adhesion molecule 1, which is important in the neutrophil-dependent lung and liver injury associated with hepatic ischemia and reperfusion in the rat.

Tumor necrosis factor (TNF) is released during hepatic ischemia/reperfusion (I/R) and plays an important role in the ensuing neutrophil-mediated lung and liver injury. Since TNF is not a direct neutrophil chemotaxin, we hypothesized that TNF may up-regulate neutrophil adhesion molecules, specifically intercellular adhesion molecule-1 (ICAM-1), following hepatic I/R, and that this molecule then plays an important role in tissue neutrophil influx. Rats underwent 90 min of lobar hepatic ischemia with reperfusion. Pulmonary and hepatic ICAM-1 expression were assessed by reverse transcription-polymerase chain reaction, Western blot analysis, and immunohistochemical staining. Increases in hepatic ICAM-1 were demonstrated within 1 h of reperfusion, while increases in pulmonary ICAM-1 were not seen until 6 h of reperfusion. Next, rats were treated with anti-TNF antibody or control antibody without TNF neutralizing properties prior to hepatic I/R. Pretreatment with anti-TNF antibody significantly decreased pulmonary and hepatic ICAM-1 expression after hepatic I/R. We next investigated the effects of pretreatment with anti-ICAM-1 antibodies on the lung and liver injury that follows hepatic I/R. Lung injury was assessed by changes in pulmonary capillary permeability as estimated by extravasation of Evans Blue dye and pulmonary neutrophil influx as measured by lung myeloperoxidase levels. Liver injury was assessed by hepatic neutrophil morphometrics and plasma liver enzymes (alanine aminotransferase). Pretreatment with anti-ICAM-1 antibodies significantly decreased pulmonary capillary permeability, pulmonary myeloperoxidase, hepatic neutrophil influx, and plasma alanine aminotransferase, as compared to animals pretreated with control antibody. These data suggest that TNF is a proximal trigger for pulmonary and hepatic ICAM-1 up-regulation following hepatic ischemia with reperfusion, and that ICAM-1 is important for pulmonary and hepatic neutrophil influx, with the resultant tissue injury, following hepatic I/R.

Alanine Transaminase↗
PubMed1998

Therapeutic effects of nitric oxide inhibition during experimental fecal peritonitis: role of interleukin-10 and monocyte chemoattractant protein 1.

This study demonstrates that the therapeutic effect of a nitric oxide inhibitor in a murine model of fecal peritonitis is mediated in part by increased levels of interleukin-10 (IL-10) and monocyte chemoattractant protein 1 (MCP-1). Female CD1 mice were subjected to cecal ligation and puncture (CLP) with a 21-gauge needle and, immediately following surgery, were injected intraperitoneally with saline, N(G)-nitro-L-arginine methyl ester (L-NAME; 8 mg/kg), or N(G)-nitro-D-arginine methyl ester (D-NAME; 8 mg/kg). At 96 h after surgery and drug treatment, 20% of mice that received D-NAME had survived whereas 60% of mice that received L-NAME were alive. To elucidate the effect of L-NAME treatment on chemokine and cytokine production during fecal peritonitis, the levels of macrophage inflammatory protein 2 (MIP-2), IL-10, and MCP-1 were measured in peritoneal washings from additional groups of mice 24 h after the CLP surgery. Peritoneal fluids from L-NAME-treated mice contained significantly higher levels of IL-10 and MCP-1 than did those from D-NAME-treated mice. To elucidate the effect of nitric oxide inhibition on potential cellular sources of IL-10 and MCP-1 in the CLP model, cultured alveolar and peritoneal macrophages were activated with bacterial lipopolysaccharide in the presence of L-NAME; these macrophages produced significantly more MCP-1 than did similarly activated macrophages in the presence of D-NAME. In the CLP surgery model, immunoneutralization of IL-10 alone or IL-10 and MCP-1 together with polyclonal antibodies prior to surgery significantly reduced the survival rates in L-NAME-treated groups compared with L-NAME-treated groups that received preimmune serum. Taken together, these data demonstrate that the inhibition of nitric oxide following experimental CLP fecal peritonitis is therapeutic, in part through the modulatory effect of this treatment on the synthesis of IL-10 and MCP-1.

Animals↗
PubMed1998

TNF and IL-6 mediate MIP-1alpha expression in bleomycin-induced lung injury.

Previously, macrophage inflammatory protein-1alpha (MIP-1alpha), a member of the C-C chemokine family, has been implicated in bleomycin-induced pulmonary fibrosis, a model of the human disease idiopathic pulmonary fibrosis. Neutralization of MIP-1alpha protein with anti-MIP-1alpha antibodies significantly attenuated both mononuclear phagocyte recruitment and pulmonary fibrosis in bleomycin-challenged CBA/J mice. However, the specific stimuli for MIP-1alpha expression in the bleomycin-induced lesion have not been characterized. In this report, two mediators of the inflammatory response to bleomycin, tumor necrosis factor (TNF) and interleukin-6 (IL-6), were evaluated as putative stimuli for MIP-1alpha expression after bleomycin challenge in CBA/J mice. Elevated levels of bioactive TNF and IL-6 were detected in bronchoalveolar lavage (BAL) fluid and lung homogenates from bleomycin-treated CBA/J mice at time points post-bleomycin challenge, which precede MIP-1alpha protein expression. Treatment of bleomycin-challenged mice with soluble TNF receptor (sTNFr) or anti-IL-6 antibodies significantly decreased MIP-1alpha protein expression in the lungs. Furthermore, normal alveolar macrophages secreted elevated levels of MIP-1alpha protein in response to treatment with TNF plus IL-6 or bleomycin plus IL-6, but not TNF, bleomycin, or IL-6 alone. Finally, leukocytes recovered from the BAL fluid of bleomycin-challenged mice secreted higher levels of MIP-1alpha protein, compared to controls, when treated with TNF alone. Based on the data presented here, we propose that TNF and IL-6 are part of a cytokine network that modulates MIP-1alpha protein expression in the profibrotic inflammatory lesion during the response to intratracheal bleomycin challenge.

Animals↗
PubMed1997

Mycobacterial and schistosomal antigen-elicited granuloma formation in IFN-gamma and IL-4 knockout mice: analysis of local and regional cytokine and chemokine networks.

Type 1 (IFN-gamma/TNF-dominant) and 2 (IL-4/IL-5-dominant) granulomatous inflammation were analyzed in mice with knockout of IFN-gamma or IL-4 genes. Lung granulomas were elicited by beads coated with purified protein derivative (PPD) of Mycobacteria bovis or soluble Schistosoma mansoni egg Ags. Parameters included granuloma size, composition, and macrophage function; white blood cell differentials; lymph node cytokine profiles; and cytokine/chemokine mRNA expression by lungs. Type 1 (PPD) and 2 (soluble Schistosoma mansoni egg Ags) responses showed characteristic cytokine and chemokine profiles in control mice. IFN-gamma knockout converted the PPD response to a type 2-like pattern with eosinophil infiltration and decreased TNF and RANTES, but increased IL-4, IL-5, IL-10, IL-13, monocyte chemoattractant protein-3 (MCP-3), and eotaxin expression. IL-4 knockout exacerbated type 1 inflammation with increased IL-2/IFN-gamma production by lymph nodes and IL-1 production by granuloma macrophages, but unexpectedly, IFN-gamma transcripts were reduced in lungs. Regarding the type 2 response, IL-4 was needed for maximal blood eosinophilia, but surprisingly, its absence had a minimal effect on type 2 granuloma size and composition despite regional reductions of IL-5 and IL-10 as well as local reductions of TNF-alpha, MCP-1, MCP-3, and eotaxin. Thus, the type 2 granuloma was not converted to a type 1 composition with IL-4 knockout, but showed persistent expression of IL-13 and some degree of IL-5 and MCP-3, suggesting that these cytokines could potentially support a compensatory type 2 response. IFN-gamma knockout did not augment type 2 granuloma size or Th2 cytokines in lymph nodes and unexpectedly reduced IL-4 transcripts in lungs. This study offers important implications regarding inflammation and its relationship to local and regional cytokine expression.

Animals↗
PubMed1996

The role of chemokines in inflammatory joint disease.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by the elicitation and activation of a number of leukocyte populations within both the synovial space and joint tissue. The recruited leukocytes subsequently play an instrumental role in synovial cell proliferation, pannus formation, and bone erosion. Although it is know that leukocytes are important participants in the evolving joint pathology, the mechanism responsible for the successful elicitation of cells to the joint is not clear. A number of studies have identified an association of specific cytokines, including chemokines, with active arthritis, but longitudinal analyses of cytokine expression and the causal role of these mediators have not been defined. Animal models and cell culture systems have proved useful in identifying the expression of various cytokines during the maintenance of chronic joint inflammation. In addition, animal models have provided important information regarding the kinetic production and contribution of specific mediators to the development of experimental arthritis. These studies provide insights into the potential mechanisms for leukocyte involvement in inflammatory joint disease.

Animals↗
PubMed1995

CD8+ and CD45RA+ human peripheral blood lymphocytes are potent sources of macrophage inflammatory protein 1 alpha, interleukin-8 and RANTES.

The chemokines macrophage inflammatory protein 1 alpha (MIP 1 alpha), interleukin-8 (IL-8) and RANTES are potent regulators of leukocyte trafficking. Examination of chemokine secretion by human peripheral blood lymphocytes after stimulation with anti-CD3 or phorbol 12, 13 myristate acetate and ionomycin showed CD8+ cells were the dominant source of MIP 1 alpha and RANTES. Although production of MIP 1 alpha and IL-8 were similar in pharmacologically stimulated CD4+ CD45RA+, CD4+ CD45RO+, and CD8+ CD45RA+ cells, the largest amounts of MIP 1 alpha and RANTES were secreted by CD8+ CD45RO+ lymphocytes. A parallel pattern of prolonged chemokine mRNA expression for at least 18 h after activation was observed in the T cells subsets. These results confirm that human T lymphocytes have a unique capacity for secretion of these three chemokines. In addition, CD8+ cells have an unrecognized role in recruiting cells to sites of inflammation, and adult human CD45RA+ cells have a physiologically significant secretory capacity.

Blotting, Northern↗
PubMed1995

Chemokines and their role in human disease.

The recruitment of leukocyte populations to an area of inflammation is one of the most fundamental processes of immune reactivity, yet a number of the mechanisms which are important to this process are not clearly understood. Investigations directed at understanding the mechanisms of leukocyte elicitation have centered around classical chemotactic factors such as C5a and fMLP, however, these known agents have demonstrated little specificity for recruiting particular leukocyte populations. Recent advances in this field have been made with the discovery of a novel supergene family of chemotactic cytokines or chemokines. These cytokines are important as they possess a high degree of specificity for the recruitment of specific subpopulations of leukocytes.

Adult↗
PubMed1995

Expression and biology of neutrophil and endothelial cell-derived chemokines.

The elicitation of polymorphonuclear leukocytes or neutrophils to an area of tissue injury is one of the most fundamental of all cell responses during the initiation of host defense. Historically, the neutrophil has been identified as an important phagocytic cell with a limited capacity to synthesize de-novo proteins. However, recent investigations have now shown that stimulated neutrophils can express a variety of cytokines, such as tumor necrosis factor, interleukin-1, interferon-alpha and interleukin-8. The ability of neutrophils and endothelial cells to produce interleukin-8 is extremely important, as this chemokine can contribute to the continued maintenance of cell infiltrates into the developing inflammatory lesion.

Base Sequence↗
PubMed1991

Monoclonal antibodies to double-stranded RNA as probes of RNA structure in crude nucleic acid extracts.

We describe four monoclonal antibodies (MAB) which specifically recognize double-stranded RNA (dsRNA) together with their use in new methods for detecting and characterizing dsRNA in unfractionated nucleic acid extracts. The specificity of the antibodies was analyzed using a panel of 27 different synthetic and naturally occurring nucleic acids. All four antibodies reacted in a highly specific manner with long dsRNA helices, irrespective of their sequence; no binding to single-stranded RNA homopolymers or to DNA or RNA-DNA hybrids was observed. The apparent affinity of the antibodies to short (less than or equal to 11 bp) RNA helices was very low in all test systems used: only background levels of binding were obtained on single-stranded RNA species which contain double-helical secondary structures (e.g. rRNA, tRNA, viroid RNA). A sandwich ELISA and a dsRNA-immunoblotting procedure have been established which allow detection and characterization of dsRNA by MAB even in the presence of a large excess of other nucleic acids. In combination with temperature-gradient gelelectrophoresis (TGGE) not only the molecular weights but also the highly characteristic Tm-values of conformational transitions of individual dsRNA species could be determined by immunoblotting. An example of the general use of these methods for the detection of plant virus infections is demonstrated with groundnut rosette virus (GRV) dsRNAs. We were able to estimate the dsRNA content of infected leaves, identify the dsRNA species present in crude extracts and to determine the Tm- values of GRV dsRNA-3.

Animals↗
PubMed1989

Degree of biotinylation in nucleic acids estimated by a gel retardation assay.

A simple and nonradioactive gel retardation assay was established in order to separate weakly biotinylated nucleic acids according to the number of biotin molecules with which they are labeled. The method uses streptavidin to retard biotinylated RNA (Bio-RNA) in nondenaturing polyacrylamide gels; the streptavidin-Bio-RNA complexes are detected by silver staining. From the quantitative analysis of the band distribution, the average number of biotinylated nucleotides incorporated in a particular preparation may be calculated. The degree of biotinylation was analyzed with this method by studying the incorporation of Bio-4-UTP or Bio-11-UTP into RNA by in vitro transcription with T7 RNA polymerase. We found that both Bio-UTPs were incorporated into RNA by T7 polymerase three to four times less efficiently than unmodified UTP and that the average degree of biotinylation depended in a simple and calculable way on the Bio-UTP to total UTP ratio during transcription. Therefore, RNA transcripts with a defined average degree of biotinylation can be synthesized by T7 RNA polymerase simply by setting the Bio-UTP to total UTP ratio to the appropriate value. The gel retardation assay was also used to determine the average number and distribution of biotinyl residues in photobiotinylated RNA.

Bacterial Proteins↗