Exogenous Tumor Necrosis Factor-alpha Research Articles

MP84-08 TUMOR NECROSIS FACTOR ALPHA IS INCREASED AFTER CAVERNOUS NERVE INJURY AND IMPAIRS REGENERATION OF NITRERGIC NEURONS AND MODULATES PENILE SMOOTH MUSCLE TONE

You have accessJournal of UrologySexual Function/Dysfunction: Basic Research & Pathophysiology (MP84)1 Apr 2020MP84-08 TUMOR NECROSIS FACTOR ALPHA IS INCREASED AFTER CAVERNOUS NERVE INJURY AND IMPAIRS REGENERATION OF NITRERGIC NEURONS AND MODULATES PENILE SMOOTH MUSCLE TONE Hotaka Matsui*, Nikolai A. Sopko, Jefferey D. Campbell, Xiaopu Liu, Emmanuel Weyne, Maarten Albersen, Fabio Castiglione, Johanna L. Hannan, and Bivalacqua Trinity J Hotaka Matsui*Hotaka Matsui* More articles by this author , Nikolai A. SopkoNikolai A. Sopko More articles by this author , Jefferey D. CampbellJefferey D. Campbell More articles by this author , Xiaopu LiuXiaopu Liu More articles by this author , Emmanuel WeyneEmmanuel Weyne More articles by this author , Maarten AlbersenMaarten Albersen More articles by this author , Fabio CastiglioneFabio Castiglione More articles by this author , Johanna L. HannanJohanna L. Hannan More articles by this author , and Bivalacqua Trinity JBivalacqua Trinity J More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000976.08AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Erectile dysfunction is induced after cavernous nerve (CN) injury by inflammation in pelvic ganglia. We examined levels of tumor necrosis factor alpha (TNFA), after bilateral CN injury (BCNI) in vivo, and effect of exogenous TNFA (eTNFA) on neurite outgrowth from major pelvic ganglion (MPG) ex vivo. Additionally, we evaluated effect of TNFA signal inhibition to penile smooth muscle tone with TNFA receptor knockout mice (TNFRKO). METHODS: 70 male rats underwent sham or BCNI. MPGs of sham rats were collected after 48 hours. BCNI groups had MPG harvested at 6 h, 12 h, 24 h, 48 h, 7 days, or 14 days after surgery. We performed qPCR to examine gene expression of TNFA and Western blot to examine protein amount of TNFA in MPGs. Immunofluorescence(IF) for TNFA was performed in MPG. For ex vivo study, MPGs were harvested from control rats and cultured in Matrigel with eTNFA (0, 10, 20, 30 ng/mL). Neurite lengths from MPGs were measured 72 h after culture. MPGs were processed for qPCR of Tyrosine hydroxylase (TH) and neuronal nitric oxidase (nNOS). IF was assessed for TH and nNOS in MPG. To examine effect of TNFA inhibition on MPG and penis after BCNI, wild type mice (WT) and TNFRKO were randomized to undergo sham or BCNI. MPGs were harvested 48 hours after surgery to examine gene expression of nNOS and TH by qPCR. Penises were used to examine smooth muscle tone to electrical field stimulation (EFS). RESULTS: TNFA’s gene/protein expression was increased in MPGs after BCNI, with peaks at 2 and 7 days, respectively. IF showed TNFA localized to perivascular areas at 48 h and neuronal cell body at 14 days after BCNI. eTNFA inhibited neurite outgrowth in a dose-dependent fashion, and impaired gene expression of nNOS in MPG (p<0.05). MPGs cultured with eTNFA showed significantly shorter neurite lengths of nNOS+ neurons than TH+ neurons (p<0.01), while this difference was not seen in controls. TNFRKO showed enhanced gene expression of nNOS compared to WT (p<0.01). Penises of WT showed enhanced smooth-muscle contraction to EFS, compared to TNFKO (p<0.01). TNFRKO had enhanced relaxation response to EFS after maximal contraction, compared to WT (p<0.01). CONCLUSIONS: TNFA was increased following BCNI and inhibited neurite outgrowth from MPGs in a dose-dependent fashion. eTNFA lowered gene and protein expression of nNOS. TNFRKO mice showed enhanced gene expression of nNOS and enhanced penile smooth muscle relaxation. These results suggest that TNFA may selectively inhibit nNOS+ neurons, and impairs penile smooth muscle tone. Source of Funding: NIH © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e1267-e1267 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Hotaka Matsui* More articles by this author Nikolai A. Sopko More articles by this author Jefferey D. Campbell More articles by this author Xiaopu Liu More articles by this author Emmanuel Weyne More articles by this author Maarten Albersen More articles by this author Fabio Castiglione More articles by this author Johanna L. Hannan More articles by this author Bivalacqua Trinity J More articles by this author Expand All Advertisement PDF downloadLoading ...

97. Effect of placental growth factor on trophoblast integration into endothelial cell networks in the presence of inflammation

Introduction Invasion of maternal spiral arterioles by trophoblast cells is fundamental to normal placental development. Abnormal spiral arteriole remodelling is often present in pregnancies affected by preeclampsia. Placental growth factor (PlGF) is produced by the placenta and this molecule is decreased during early pregnancy of women with preeclamptic pregnancy as compared to unaffected women. The influence of PlGF on the processes of early placentation is not known. Objectives To observe the effect of supplemental PlGF upon capillary-like uterine endothelial cell networks and a first trimester human trophoblast cell line in the presence or absence of inflammation induced by exogenous tumour necrosis factor-alpha (TNF- α ) . Methods 24-well tissue culture plates were coated with 300 uL of undiluted Matrigel and allowed to gelatinise at 37°C for 30 min. Fluorescent labelled uterine microvascular endothelial cells and HTR8/SVNeo cells were co-cultured (1 X 10 5 per well) for 20 h treated with PlGF (10 ng/mL) and/or TNF- α (0.5 ng/mL). Images were captured by fluorescence microscopy and analysed using ImageJ. Experiments were repeated 8 times, data was analysed using SPSS v24. Results TNF alpha reduced trophoblast cell integration into endothelial networks (Control vs TNF- α p = 0.006) but this was not ameliorated by addition of PlGF (TNF- α vs TNF- α + PlGF p > 0.9). Furthermore, PlGF supplementation itself did not improve cell integration (Control vs PlGF p = 0.2). Discussion PlGF not improve trophoblast cell integration into endothelial cell networks nor reverse the inhibitory effect of TNF- α .

Exogenous tumor necrosis factor-alpha could induce egress ofToxoplasma gondiifrom human foreskin fibroblast cells

Toxoplasma gondii is an intra-cellular protozoan parasite that can infect almost all nucleated cells, eliciting host immune responses against infection. Host tissue damage is mainly caused by cellular lysis when T. gondii egresses from infected cells. However, the effects of cytokines released by host immune cells on egression of T. gondii remain elusive. This study aimed to investigate the role of tumor necrosis factor-alpha (TNF-α) on the egress of T. gondii from infected human foreskin fibroblast (HFF) cells and to elucidate the underlying mechanisms that regulate TNF-α-induced egress. Using flow cytometry to count tachyzoites of T. gondii released into cell culture medium, we found that egress of T. gondii from infected HFF cells could be induced by 10 ng/mL TNF-α in a time-dependent manner. Pre-treatment of infected HFF cells with BAPTA-AM to chelate intra-parasitic calcium could greatly inhibit TNF-α-induced egress. Similar results were obtained when using cytochalasin D to block parasite motility before the TNF-α-induced egress assay. In addition, blocking host apoptosis by Z-VAD-FMK could decrease TNF-α induced egress, while blocking necroptosis by necrostatin-1 has little impact on TNF-α-induced egress. The egressed tachyzoites displayed a normal growth rate and lost no virulence. Our results suggest that host cytokines could influence the cellular lytic processes of T. gondii, providing new insights into the relationship between host TNF-α and T. gondii pathogenesis.

Tumor Necrosis Factor-alpha-mutant Mice Exhibit High Frequency Hearing Loss

Exogenous tumor necrosis factor-alpha (TNF-α) plays a role in auditory hair cell death by altering the expression of apoptosis-related genes in response to noxious stimuli. Little is known, however, about the function of TNF-α in normal hair cell physiology. We, therefore, investigated the cochlear morphology and auditory function of TNF-α-deficient mice. Auditory evoked brainstem response showed significantly higher thresholds, especially at higher frequencies, in 1-month-old TNF-α(-/-) mice as compared to TNF-α(+/-) and wild type (WT); hearing loss did not progress further from 1 to 4 months of age. There was no difference in the gross morphology of the organ of Corti, lateral wall, and spiral ganglion cells in TNF-α(-/-) mice compared to WT mice at 4 months of age, nor were there differences in the anatomy of the auditory ossicles. Outer hair cells were completely intact in surface preparations of the organ of Corti of TNF-α(-/-) mice, and synaptic ribbon counts of TNF-α(-/-) and WT mice at 4 months of age were similar. Reduced amplitudes of distortion product otoacoustic emissions, however, indicated dysfunction of outer hair cells in TNF-α(-/-) mice. Scanning electron microscopy revealed that stereocilia were sporadically absent in the basal turn and distorted in the middle turn. In summary, our results demonstrate that TNF-α-mutant mice exhibit early hearing loss, especially at higher frequencies, and that loss or malformation of the stereocilia of outer hair cells appears to be a contributing factor.

In Vitro and In Vivo Effect of 5-FC Combined Gene Therapy with TNF-α and CD Suicide Gene on Human Laryngeal Carcinoma Cell Line Hep-2

This study was aimed to investigate the effect of combined cancer gene therapy with exogenous tumor necrosis factor-alpha (TNF-α) and cytosine deaminase (CD) suicide gene on laryngeal carcinoma cell line Hep-2 in vitro and in vivo. Transfection of the recombinant eukaryotic vectors of pcDNA3.1 (+) containing TNF-α and/or CD into Hep-2 cells resulted in expression of TNF-α and/or CD gene in vitro. The significant increase in apoptotic Hep-2 cells and decrease of Hep-2 cell proliferation were observed using 5-FC treatment combined with TNF-a expression by CD/5-FC suicide system. Moreover, bystander effect was also observed in the TNF-α and CD gene co-expression group. Laryngeal squamous cell carcinoma (LSCC) mice model was established by using BALB/c mice which different transfected Hep-2 cells with pcDNA3.1 (+) containing TNF-α and/or CD were applied subcutaneously. So these mice are divided into four groups, namely, Hep-2/TIC group; Hep-2/CD group; Hep-2/TNF-α group; Hep-2/0 group. At day 29 after cell inoculation, volume of grafted tumor had significant difference between each two of them (P<0.05). These results showed that the products of combined CD and TNF-α genes inhibited the growth of transplanted LSCC in mice model. So by our observed parameters and many others results, we hypothesized that 5-FC combined gene therapy with TNF-αand CD suicide gene should be an effective treatment on Laryngeal carcinoma.

Th1 cytokines promote T-cell binding to antigen-presenting cells via enhanced hyaluronan production and accumulation at the immune synapse.

Hyaluronan (HA) production by dendritic cells (DCs) is known to promote antigen presentation and to augment T-cell activation and proliferation. We hypothesized that pericellular HA can function as intercellular 'glue' directly mediating T cell-DC binding. Using primary human cells, we observed HA-dependent binding between T cells and DCs, which was abrogated upon pre-treatment of the DCs with 4-methylumbelliferone (4-MU), an agent which blocks HA synthesis. Furthermore, T cells regulate HA production by DCs via T cell-derived cytokines in a T helper (Th) subset-specific manner, as demonstrated by the observation that cell-culture supernatants from Th1 but not Th2 clones promote HA production. Similar effects were seen upon the addition of exogenous Th1 cytokines, IL-2, interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha). The critical factors which determined the extent of DC-T cell binding in this system were the nature of the pre-treatment the DCs received and their capacity to synthesize HA, as T-cell clones which were pre-treated with monensin, added to block cytokine secretion, bound equivalently irrespective of their Th subset. These data support the existence of a feedforward loop wherein T-cell cytokines influence DC production of HA, which in turn affects the extent of DC-T cell binding. We also document the presence of focal deposits of HA at the immune synapse between T-cells and APC and on dendritic processes thought to be important in antigen presentation. These data point to a pivotal role for HA in DC-T cell interactions at the IS.

Effects of Exogenous Tumour Necrosis Factor‐α on the Secretory Function of the Bovine Reproductive Tract Depend on Tumour Necrosis Factor‐α Concentrations

The aim of study was to correlate tumour necrosis factor-alpha (TNF) infused doses used with the TNF concentrations achieved and with the secretory function of both the ovary and the uterus in cows. We evaluated the concentrations of progesterone (P4), prostaglandin (PG)F(2alpha), PGE(2) nitric oxide (NO) and TNF in the jugular vein and vena cava caudalis as parameters of exogenous TNF action on the female reproductive tract. Aortae abdominalis of cows (n = 18) were infused with saline or two doses of TNF (luteolytic--1 microg or luteotrophic--10 microg). In the peripheral blood, 1 microg TNF concentrations achieved within the range of 30-45 pg/ml, and 10 microg TNF provoked a sharp increase in achieved concentrations at a range of 250-450 pg/mL). The TNF concentrations achieved in vena cava caudalis were five to six times higher than that in peripheral blood (p < 0.001). One microgram TNF increased PGF(2alpha) and NO (p < 0.001) and decreased P4 (p < 0.05). The higher TNF dose stimulated P4 and PGE(2) (p < 0.01). TNF infusion at luteolytic dose achieved its concentrations at the physiological range previously observed in cows. Luteotrophic TNF dose achieved the concentrations in vena cava caudalis that are much higher than physiological level and were previously noted in pathological circumstances (i.e. mastitis, metritis).

Genetic variation in neutrophil accumulation in mice is not mediated through immigrant regulatory cells.

Genetic variation of induced peritoneal neutrophilia in mice was accompanied by parallel variation in macrophage responses. The timing of the macrophage responses in high responder (C57B1/10) mice indicated a potential role for these cells in mediating the enhanced neutrophil response. However, adoptive transfer of inflammatory macrophages did not induce neutrophilia. Analysis of peritoneal cytokine levels in high and low responder mice further indicated that IL-1, IL-3, GM-CSF, G-CSF and interferon-gamma (IFN-gamma) were not involved in mediating the genetic variation observed. Exogenous tumour necrosis factor-alpha (TNF-alpha) was effective in inducing the high responder phenotype, despite the absence of detectable TNF-alpha in either peritoneal fluid or serum. A role for genetically determined differential expression of endothelial adhesion molecules in high and low responders is suggested.

Suppression of lymphokine-activated killer (LAK) cell induction mediated by interleukin-4 and transforming growth factor-β1: effect of addition of exogenous tumour necrosis factor-alpha and interferon-gamma, and measurement of their endogenous production

Recombinant human interleukin-4 (rhIL-4) and transforming growth factor-beta 1 (TGF-beta 1) suppressed the induction of lymphokine-activated killer (LAK) activity induced by recombinant human interleukin-2 (rhIL-2) in peripheral blood lymphocytes. DNA synthesis and the expression of the p55 alpha chain of the IL-2 receptor (Tac antigen) were also inhibited. The inhibitory effect was greatest when these factors were added during the first 48 h of a 4-day culture, with reduced cytolytic activity against both natural killer (NK) resistant and NK-sensitive tumour cell line targets. The suppressive action of both cytokines was accompanied by a reduction in tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) levels in lymphocyte culture supernatants. Recombinant human IFN-gamma (rhIFN-gamma), but not recombinant human TNF-alpha (rhTNF-alpha) was able to overcome the inhibitory effect of recombinant human interleukin-4 (rhIL-4) on LAK induction and DNA synthesis but not Tac antigen expression. However, cytotoxicity induced by rhIFN-gamma alone was also suppressed by rhIL-4 and TGF-beta 1, inferring that rhIFN-gamma-mediated abrogation of rhIL4 suppression was not simply a direct IL-2-independent effect on cytotoxicity. In addition, rhIL-4 did not increase TGF-beta production from rhIL-2-activated peripheral blood mononuclear cells, suggesting that rhIL-4 did not mediate reduction of rhIL-2 responses through the induction of TGF-beta release.

Tumor necrosis factor alpha inhibits the suppressive effect of regulatory T cells on the hepatitis B virus–specific immune response

Chronicity of hepatitis B virus (HBV) infection is characterized by a weak immune response to the virus. CD4+CD25+ regulatory T cells (Treg) are present in increased numbers in the peripheral blood of chronic HBV patients, and these Treg are capable of suppressing the HBV-specific immune response. The aim of this study was to abrogate Treg-mediated suppression of the HBV-specific immune response. Therefore, Treg and a Treg-depleted cell fraction were isolated from peripheral blood of chronic HBV patients. Subsequently, the suppressive effect of Treg on the response to HBV core antigen (HBcAg) and tetanus toxin was compared, and the effect of exogenous tumor necrosis factor alpha (TNF-alpha), interleukin-1-beta (IL-1beta), or neutralizing antibodies against interleukin-10 (IL-10) or transforming growth factor beta (TGF-beta) on Treg-mediated suppression was determined. The results show that Treg of chronic HBV patients had a more potent suppressive effect on the response to HBcAg compared with the response to tetanus toxin. Neutralization of IL-10 and TGF-beta or exogenous IL-1beta had no effect on Treg-mediated suppression of the anti-HBcAg response, whereas exogenous TNF-alpha partially abrogated Treg-mediated suppression. Preincubation of Treg with TNF-alpha demonstrated that TNF-alpha had a direct effect on the Treg. No difference was observed in the type II TNF receptor expression by Treg from chronic HBV patients and healthy controls. Treg-mediated suppression of the anti-HBV response can be reduced by exogenous TNF-alpha. Because chronic HBV patients are known to produce less TNF-alpha, these data implicate an important role for TNF-alpha in the impaired antiviral response in chronic HBV.

Involvement of transient receptor potential vanilloid 1 in the vascular and hyperalgesic components of joint inflammation

To investigate the endogenous involvement of transient receptor potential vanilloid 1 (TRPV1) in a model of knee joint inflammation in the mouse. Following characterization of wild-type (WT) and TRPV1-knockout mice, inflammation was induced via intraarticular (IA) injection of Freund's complete adjuvant (CFA). Knee swelling was assessed as diameter, and inflammatory heat hyperalgesia was determined using the Hargreaves technique, for up to 3 weeks. At 18 hours, acute hyperpermeability was measured with 125I-albumin, and cytokines and myeloperoxidase activity, a marker of neutrophils, were assayed in synovial fluid extracts. The possibility that exogenous tumor necrosis factor alpha (TNFalpha) was involved in influencing TRPV1 activation was investigated in separate experiments. Increased levels of knee swelling, hyperpermeability, leukocyte accumulation, and TNFalpha were found in WT mice 18 hours after IA CFA treatment compared with saline treatment. Significantly less knee swelling and hyperpermeability were found in TRPV1-/- mice, but leukocyte accumulation and TNFalpha levels were similar in WT and TRPV1-/- mice. Knee swelling in response to CFA remained significantly higher for a longer period in WT mice compared with TRPV1-/- mice, with thermal hyperalgesic sensitivity observed at 24 hours and at 1 week in WT, but not TRPV1-/-, mice. Knee swelling was attenuated (P < 0.05) in TRPV1-/- compared with WT mice 4 hours after IA administration of TNFalpha. Our findings indicate that TRPV1 has a role in acute and chronic inflammation in the mouse knee joint. Thus, selective antagonism of TRPV1 should be considered as a potential target for treatment of acute and chronic joint inflammation.

Mitochondrial Dysfunction in Hepatitis C

Chronic hepatitis C induces a state of hepatic oxidative stress that is more pronounced than that present in many other inflammatory liver diseases. This review summarizes recent information that the hepatitis C virus (HCV) core protein plays an important role in this phenomenon. Core protein localizes to mitochondria, particularly at the points of contact between mitochondrial outer membrane and endoplasmic reticulum. Its expression causes inhibition of electron transport at complex I, increased complex I reactive oxygen species (ROS) production, decreased mitochondrial glutathione, and increased mitochondrial permeability transition in response to exogenous oxidants and tumor necrosis factor-alpha. Possible mechanisms of the core protein effects include direct interaction with electron carriers and indirect effects mediated by changes in mitochondrial calcium. These results suggest that antioxidant approaches may prove beneficial for patients with chronic hepatitis C.

Protective effect of exogenous recombinant mouse interferon-gamma and tumour necrosis factor-alpha on ectromelia virus infection in susceptible BALB/c mice.

The resistance to mousepox is correlated with the production of type I cytokines: interleukin (IL)-2, IL-12, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha. We intend to describe the modulation of generalized ectromelia virus (EV) infection with exogenous administration of mrIFN-gamma and mrTNF-alpha separately and in combination using susceptible BALB/c mice. The treatment schemes presented resulted in the localization of the generalized EV infection and its development into non-fatal sloughing of the infected limb. This was accompanied by low virus titres in the treated mice due to control of systemic virus replication and virus clearance. The balance of type I versus type II cytokines was dominated by a type I response in the treated groups. The group treated with the combination of IFN-gamma and TNF-alpha exhibited the best survival with Th1-dominant (IFN-gamma and IL-12) cytokine profiles, whereas the TNF-alpha-treated group of mice was less successful in clearance of virus and demonstrated the lowest survival rate. The successful cytokine treatment schemes in this orthopoxvirus model system may have important implications in the treatment of viral diseases in humans and, in particular, of variola virus infection.

Intramuscular injection of tumor necrosis factor-alpha induces muscle hyperalgesia in rats

The role of proinflammatory cytokines in neuropathic and inflammatory pain is well established. Recent studies suggest that cytokines such as tumor necrosis factor-alpha (TNF) may also be involved in the development of muscle pain. To investigate the pathophysiology of intramuscular TNF, exogenous TNF (0.1–10 μg), formalin (9%) or vehicle was injected into the gastrocnemius or biceps brachii muscles of rats. To quantify muscle hyperalgesia, changes in forelimb grip force or withdrawal thresholds to increasing pressure applied to the gastrocnemius muscle were measured. TNF evoked a time- and dose-dependent muscle hyperalgesia within several hours after injection that was totally reversed by systemic treatment with the non-opioid analgesic metamizol. Paw withdrawal thresholds or latencies to mechanical and thermal stimuli, respectively, were unchanged after intramuscular injection of TNF or formalin. In contrast to formalin, which induced significant muscle tissue damage, macrophage infiltration, swelling and partial motor impairment demonstrated in rotarod tests, TNF induced neither histopathological tissue damage nor motor dysfunction. To investigate the effect of TNF and formalin on other potentially algesic mediators, muscles were analyzed for calcitonin-gene related peptide (CGRP), prostaglandin E 2 (PGE 2) and nerve growth factor (NGF) 1 day after injection. TNF and formalin evoked intramuscular upregulation of CGRP and NGF, whereas PGE 2 was increased exclusively after TNF injection. These findings allow us to speculate that endogenous TNF may play a role in the development of muscle hyperalgesia. Targeting proinflammatory cytokines might be beneficial for the treatment of musculoskeletal pain syndromes.

Airway Wall Remodeling Induced by Occupational Mineral Dusts and Air Pollutant Particles

COPD has been reported in workers exposed to particulates, and there is increasing evidence that high levels of ambient particulate pollutants may also be associated with COPD. The studies here investigate the hypothesis that particulates, including air pollution particles, can induce airway wall fibrosis, a process that can lead to COPD. Rat tracheal explants were exposed to various occupationally encountered dusts, air pollution particles, and model air pollution particles. In some experiments, iron was loaded onto the particle surface. Gene expression and nuclear factor (NF)-kappaB activation were measured after 7 days of air culture. Adhesion to and uptake of dusts by the tracheal epithelium were also evaluated. Known fibrogenic dusts such as amosite asbestos produced increased gene expression of procollagen, transforming growth factor-beta, and platelet-derived growth factor, and increased hydroxyproline in the explants, and the addition of iron increased these effects. The addition of iron also converted nonfibrogenic TiO2 into a fibrogenic dust. Dusts with surface complexed iron activated NF-kappaB via an oxidant mechanism. However, an ultrafine TiO2 with very low iron was also fibrogenic. In separate experiments, exogenous tumor necrosis factor-alpha increased dust adhesion to, and exogenous ozone increased dust uptake by, tracheal epithelial cells. Mineral dusts can directly induce fibrosis in the airway wall. Exogenous inflammatory cells and exogenous agents are not required, but they probably exaggerate the fibrogenic effects. An iron-mediated oxidant mechanism underlies the fibrogenic effects of some, but not all, of these dusts. Particle-induced airway wall fibrosis may lead to COPD.

Inhibition of IL-12 synthesis of peripheral blood mononuclear cells (PBMC) stimulated with a bacterial superantigen by pooled human immunoglobulin: implications for its effect on Kawasaki disease (KD).

The aim of this study was to further assess the role of pooled human immunoglobulin (PHIG) on cytokine production from PBMC stimulated with a bacterial superantigen. Human PBMC were cultured with Streptococcus pyrogenic exotoxin A (SPE-A) with or without PHIG and several proinflammatory cytokine levels of culture supernatants were measured. Serum cytokine levels of KD patients before and after PHIG therapy were also examined. PHIG greatly reduced the production of IL-12, interferon-gamma (IFN-gamma) and other cytokines from SPE-A-stimulated PBMC, while exogenous IL-12, but neither IL-1 nor tumour necrosis factor-alpha (TNF-alpha), restored IFN-gamma production inhibited by PHIG. Although PHIG partially adsorbed SPE-A, its inhibitory effect on cytokine production was not played by anti-SPE-A antibody. Although purified CD4+ T cells cultured with human HLA-DR-transfected mouse L cells and SPE-A could not effectively produce IFN-gamma, they produced large amounts of IFN-gamma if exogenous IL-12 was introduced. KD patients in the acute phase had higher levels of serum IFN-gamma than did controls and patients with bacterial infection. Although IL-12 levels of children with or without KD were not significantly different, IL-12 levels of children were much higher than those of adults. However, serum levels of IL-12 of KD patients were transiently but significantly decreased by PHIG therapy and IFN-gamma amounts subsequently reverted to basal levels thereafter. These findings indicate that PHIG inhibits IL-12 production of SPE-A-activated monocytes and thereby decreases IFN-gamma synthesis by T cells and suggest that inhibition of IL-12 and IFN-gamma production is an important part of the mechanisms underlying PHIG therapy on KD.

2000 Volvo Award winner in basic science studies: Exogenous tumor necrosis factor-alpha mimics nucleus pulposus-induced neuropathology. Molecular, histologic, and behavioral comparisons in rats.

This study tested the hypothesis that the 17-kDa form of tumor necrosis factor-alpha is the pathophysiologic agent expressed by herniated nucleus pulposus in vivo that is primarily responsible for the histologic and behavioral manifestations of experimental sciatica associated with herniated lumbar discs. The authors determined the molecular weight and concentration of active tumor necrosis factor-alpha in rat herniated disc and used exogenous tumor necrosis factor-alpha at the same molecular weight to study its neuropathologic effect on rat nerve root and dorsal root ganglion preparations in vivo. Expressed by herniated nucleus pulposus in culture, tumor necrosis factor-alpha causes neuropathologic injury in nerve roots and neuropathic pain states in which mechanical allodynia is seen in response to peripheral stimuli. Western blotting was used to identify the molecular weight of the operative tumor necrosis factor-alpha protein form, and measures of optical density were used for semiquantitative determination of concentration. Plastic-embedded nerve roots and dorsal root ganglion were used for neuropathologic evaluation, and von Frey stimulation was used to quantify mechanical allodynia. The 17-kDa form of tumor necrosis factor-alpha is expressed by herniated nucleus pulposus at a concentration of approximately 0.48 ng per herniated rat lumbar disc. Exogenous tumor necrosis factor-alpha applied in vivo to rat nerve roots produced neuropathologic changes and behavior deficits that mimicked experimental studies with herniated nucleus pulposus applied to nerve roots. The data reinforce other evidence that tumor necrosis factor-alpha is involved in mechanisms of neuropathic pain.

Tumor necrosis factor receptors types 1 and 2 differentially regulate osteoclastogenesis.

The potent osteoclastogenic agent, tumor necrosis factor-alpha (TNF), exerts its biological effects via two receptors, namely TNF receptors 1 (p55r) and 2 (p75r), each present on osteoclast precursors. Thus, we asked if p55r and p75r differentially impact the osteoclastogenic process. Marrow derived from mice expressing only p55r generates substantially more osteoclasts, in the basal state, than does wild type, while marrow expressing only p75r, produces substantially fewer. Reflecting its preferential activation of p55r, exogenous TNF stimulates osteoclast formation by p55r(+/+)p75r(-/-), but not p55r(-/-)p75r(+/+), marrow. Consistent with the fact that NF-kappaB is essential for osteoclastogenesis, this transcription complex is activated, relative to wild type, in p55r(+/+)p75r(-/-) osteoclast precursors and suppressed in those expressing only p75r. Because p55r enhances, and p75r suppresses, osteoclastogenesis, we asked if their principal ligands, namely soluble and membrane-residing TNF, respectively, differentially impact basal osteoclast recruitment. We find, in contrast to the significant level of osteoclast formation in wild type marrow, osteoclastogenesis by that derived from mice expressing membrane, but not soluble, TNF, is negligible. Thus, optimal therapeutic inhibition of bone resorption may entail selective TNF receptor modulation and/or arrested cleavage of membrane TNF to its soluble form.

EARLY RENAL ISCHEMIA, WITH OR WITHOUT REPERFUSION, ACTIVATES NFκB AND INCREASES TNF-α BIOACTIVITY IN THE KIDNEY

Acute tubular necrosis (ATN) and the ensuing renal failure induced by ischemia and reperfusion injury (I/R) remain a major cause of morbidity and mortality among patients in the intensive care unit. Although it is well established that exogenous tumor necrosis factor-alpha (TNF) induces renal injury, it remains unknown whether ischemia and/or reperfusion activates the signaling mechanisms required for renal TNF production. We hypothesized that ischemia and/or reperfusion would activate the oxidant sensitive TNF transcription factor, nuclear factor kappa B (NFkappaB), and thereby lead to renal TNF production. Male Sprague-Dawley rats were anesthetized with sodium pentobarbital, after which various periods of renal ischemia, with or without reperfusion, were induced in rats. At different time intervals, kidneys were harvested and NFkappaB activation (electrophoretic mobility shift assay), TNF mRNA content (RT-PCR), and TNF bioactivity (WEHI-164 cell clone cytotoxicity assay) were determined. Results indicate that 15 minutes of ischemia alone activates NFkappaB, whereas peak activation occurred at 30 minutes of ischemia alone. NFkappaB remained activated through 60 minutes reperfusion. Thirty minutes of ischemia is required to induce renal TNF mRNA production; however, renal TNF protein expression and bioactivity peaked following 1 hour of ischemia and 2 hours reperfusion. These results are the initial demonstration that renal ischemia, with or without reperfusion, activates the TNF transcription factor NFkappaB and increases TNF bioactivity in the kidney.

Expression of plasminogen activator inhibitor-1 in human adipose tissue: a role for TNF- α?

Elevated plasminogen activator inhibitor-1 (PAI-1) plasma levels, responsible for reduced fibrinolysis, are associated with animal and human obesity and with increased cardiovascular disease. The expression of PAI-1 has been found recently in animal and human adipose tissue. Factors and mechanisms regulating such an expression remain to be elucidated. In omental and/or subcutaneous biopsies from obese non-diabetic patients, incubated in Medium 199, we have confirmed that human adipose tissue expresses PAI-1 protein and mRNA; furthermore we have demonstrated that such an expression is clearly evident also in collagenase isolated human adipocytes and that it is stimulated by incubation itself and enhanced by exogenous human tumor necrosis factor-alpha (h-TNF- α). Since human adipose tissue produces TNF- α, to further characterize the relationship of PAI-1 to TNF- α, human fat biopsies were also incubated with Pentoxifylline (PTX) or Genistein, both known to inhibit endogenous TNF- α through different mechanisms. PTX caused a dose-dependent decrease of basal PAI-1 protein release, reaching 80% maximal inhibitory effect at 10 −3 M, the same inhibitory effect caused by Genistein at 100 μg/ml. This was associated to a marked inhibition of PAI-1 mRNA and of endogenous TNF- α production. Furthermore, when human fat biopsies were incubated in the presence of polyclonal rabbit neutralizing anti-human TNF- α antibody (at a concentration able to inhibit 100 UI/ml human TNF- α activity), a modest but significant decrease of the incubation induced expression of PAI-1 mRNA was observed (19.8±19.0% decrease, P=0.04, n=7). In conclusion, the results of this study demonstrate that PAI-1 expression is present in human isolated adipocytes and that it is enhanced in human adipose tissue in vitro by exogenous TNF- α. Furthermore our data support the possibility of a main role of endogenous TNF- α on human adipose tissue PAI-1 expression. This cytokine, produced by human adipose tissue and causing insulin resistance, may be a link in the clinical relationship between insulin-resistance syndrome and increased PAI-1 plasma levels.