Involvement of the α7-nicotinic acetylcholine receptors in the anti-inflammatory action of the thymulin-related peptide (PAT).

BACKGROUND AND PURPOSE: Peptide analog of thymulin (PAT) has been shown to have anti-hyperalgesic and anti-inflammatory properties in animal models of inflammation. Recent reports suggest that the peripheral cholinergic system has an anti-inflammatory role mediated by α7-nicotinic acetylcholine receptor (α7-nAChR). Our aim is to investigate whether the action of PAT is mediated, via the cholinergic pathway. EXPERIMENTAL APPROACH: The anti-hyperalgesic and anti-inflammatory action of PAT was assessed in rat models of inflammatory nociceptive hyperactivity (carrageenan and endotoxin) and in a mice air-pouch model for localized inflammation, respectively; the possible attenuation of PAT's effects by pretreatment with the α7-nAchR specific antagonist methyllycaconitine citrate (MLA) was also investigated. In another series of experiments, using two electrode recordings, the effect of PAT on the α7-nAChRs, expressed in Xenopus Oocytes, was also determined. KEY RESULTS: Administration of PAT reversed inflammatory nociceptive hyperactivity and cold and tactile hyperactivity in rats. This effect was partially or totally prevented by MLA, as assessed by different behavioral pain tests. Treatment with PAT also reduced the alteration of cytokines and NGF levels by carrageenan injection in the mouse air pouch model; this effect was partially antagonized by MLA. Electrophysiological recording demonstrated that PAT significantly potentiated the α7-nAchR expressed in Xenopus Oocytes. These effects were not observed when a control peptide, with a reverse sequence (rPAT), was utilized. CONCLUSIONS AND IMPLICATIONS: The behavioral and electrophysiological observations described in this report demonstrate that PAT mediates, at least partially, its anti-inflammatory action by potentiating the α7-nAChR. These results indicate that PAT has a potential for new therapeutic applications as anti-inflammatory and analgesic agent.

Transient attenuation of neuropathic manifestations in rats following lesion or reversible block of the lateral thalamic somatosensory nuclei.

BACKGROUND AND AIMS: Nociceptive behavior in animal models for mononeuropathy has been shown to be altered by spinal tract lesions which suggest a possible supraspinal modulation. The thalamus constitutes a chief center for the processing of nociception. We have, therefore, investigated the effects of transient or permanent blocks of the lateral somatosensory thalamic nuclei (the ventrobasal complex) on the neuropathic manifestations in rats. METHODS: Different groups of rats (n = 5-6) were subjected to mononeuropathy, following the spared nerve injury model, known to produce sustained heat hyperalgesia and tactile and cold allodynia which peaked about 2 weeks after nerve injury. This was followed by stereotaxic placement of either electrolytic or chemical lesions or implantation of mini osmotic pump for slow release of lidocaine in the ventrobasal complex. RESULTS: Chronic electrolytic and chemical lesions or reversible block of the lateral somatosensory thalamus produced transient (1-2 weeks) attenuation of neuropathic manifestations along with a persistent decrease of the hot plate latency. The most pronounced effect was observed on heat hyperalgesia, and the least significant and short-lived effect was observed on cold allodynia. CONCLUSION: We conclude that the lateral somatosensory thalamic complex is involved in the processing of neuropathic manifestations but cannot be considered as an obligatory or exclusive relay center for the neuropathic syndromes.

Involvement of substance P, CGRP and histamine in the hyperalgesia and cytokine upregulation induced by intraplantar injection of capsaicin in rats.

Intraplantar (i.pl.) injection of small doses of capsaicin has been shown to produce hyperalgesia and upregulation of the levels of proinflammatory cytokines. The present work aimed at investigating the possible mediation of these effects by sensory neuropeptides and mast cells. Various groups of rats received i.pl. injection of capsaicin alone or preceded by the injection of antagonists to substance P (SP), calcitonin gene-related protein (CGRP) and histamine (H1, H2) or the mast cell blocker ketotifen. All pretreatments prevented, in a dose-related manner, the capsaicin-induced hyperalgesia. The SP, H2 antagonists and ketotifen prevented the upregulation of all cytokines and nerve growth factor (NGF) levels, while the CGRP and H1 antagonists showed only attenuation of the NGF level.

The effect of endotoxin on functional parameters of mammary CID-9 cells.

The effect of endotoxin on mammary CID-9 cells, which differentiate in culture and express beta-casein, was investigated. Cells in culture supplemented with lactogenic hormones and dripped with EMS-Matrix (EMS-drip), were treated daily with endotoxin (0.5-500 microg/ml). Endotoxin at concentrations of less or equal to 10 microg/ml did not affect cell growth and viability up to 5 days post endotoxin treatment. Endotoxin (0.01-10 microg/ml) was added to the culture medium, upon confluence, and functional parameters were examined within 48 h post endotoxin treatment. Nuclear factor-kappaB (NF-kappaB) (p52) increased in nuclear extracts from endotoxin-stimulated cells within 1 h of treatment, while beta-casein mRNA and protein expression decreased in a concentration-dependent manner at 24 and 48 h post treatment. Zymography showed that the 72 and 92 kDa gelatinase activity increased in cells at 24 and 48 h post endotoxin treatment at 10 and 50 microg/ml. At the latter concentration, the active form of 72 kDa gelatinase was induced at 48 h. Interleukin-6 and tumor necrosis factor-alpha levels increased at 1-3 h post endotoxin treatment and peaked at 6 h in cells on plastic and EHS-drip. Nerve growth factor (NGF) levels increased in control and endotoxin-treated cells in a time-dependent manner, and endotoxin increased NGF levels in culture at 6 and 9 h post endotoxin treatment. This study shows that endotoxin activated NF-kappaB, suppressed beta-casein expression and upregulated gelatinases, cytokines and NGF. This model could be used to investigate the role of mammary cells in initiating and propagating inflammation and to test candidate molecules for potential anti-inflammatory properties.

Thymulin reverses inflammatory hyperalgesia and modulates the increased concentration of proinflammatory cytokines induced by i.c.v. endotoxin injection.

The immunomodulatory thymic hormone thymulin has been shown previously to possess anti-inflammatory actions in the periphery. In this study, we have examined the effect of i.c.v. injections of either endotoxin (ET) or thymulin, in separate groups of conscious rats, on pain-related behavior and cytokine levels in different areas of the brain. Furthermore, we investigated the effect of pretreatment with either i.c.v. or i.p. injections of thymulin on endotoxin-induced hyperalgesia and the effect of pretreatment with i.c.v. thymulin on endotoxin-induced up-regulation of cytokine levels. Our results demonstrate that i.c.v. injection of endotoxin (1 microg in 5 microl saline) resulted in a significant decrease in the nociceptive thresholds as assessed by different pain tests, with peak hyperalgesia at 3 h. However, thymulin at different doses, when injected (i.c.v.), had no significant effect on pain related behavior. Pretreatment (i.c.v.) with thymulin (0.1, 0.5 and 1 microg in 5 microl saline) 20 min before endotoxin (i.c.v.) injection (1 microg in 5 microl saline) reduced, in a dose dependent manner, the endotoxin-induced hyperalgesia and exerted differential effects on the up-regulated levels of cytokines in different areas of the brain. The results provide behavioral and immunochemical characterization of a rat model for intracerebral inflammation and indicates a neuroprotective role for thymulin in the CNS.

Upregulation of proinflammatory cytokines and nerve growth factor by intraplantar injection of capsaicin in rats.

Capsaicin-sensitive primary afferents (CSPA) are known to be involved in nociception and neurogenic inflammation. Extensive research has been devoted to the sensory role of these fibres but less attention has been paid to their local effector function. This study aimed at gaining more insight into the molecular mechanisms underlying the neurogenic inflammation induced by this special group of afferent fibres. Different groups of rats (n = 5 in each group), either naive or subjected to selective ablation of their CSPA, received individual intraplantar injections of saline, capsaicin, its vehicle or capsaicin preceded by its antagonist, capsazepine. Acute tests for nociception were used to assess the variations of the nociceptive thresholds. Variations of the levels of proinflammatory cytokines and nerve growth factor (NGF) were measured by enzyme-linked immunosorbent assay (ELISA). Intraplantar injection of capsaicin (10 microg in 50 microl) produced a sustained thermal and mechanical hyperalgesia that peaked at 3-6 h and disappeared 24 h following the injection. Similar capsaicin injection in further groups of rats produced an early upregulation of the proinflammatory cytokines and NGF, which peaked at 30-60 min and returned to control levels within 2-5 h. Similar effects were observed following the application of either capsaicin or intense electrical stimulation on the cut end of the distal portion of the sciatic nerve. The effects of capsaicin were abolished in rats subjected to selective ablation of their CSPA. These results demonstrate that CSPA can simultaneously challenge the immune system through the release of proinflammatory mediators and the central nervous system through nociceptive signalling and can therefore serve as a common afferent pathway to both immune and nervous systems.

The role of the sympathetic efferents in endotoxin-induced localized inflammatory hyperalgesia and cytokine upregulation.

The sympathetic system (SNS) is considered to be a major component of the neurogenic contribution to inflammation and hyperalgesia. We have investigated the role of the SNS in the local inflammatory pain induced by intraplantar (i.pl) injections of bacterial endotoxin (ET). Treatment of rats with an alpha-adrenoceptor antagonist (phentolamine, 0.25-1 mg/kg, i.p.), a beta-adrenoceptor antagonist (propranolol, 1-10 mg/kg, p.o.) or a sympathetic neuron-blocking agent (guanethedine, 30 mg/kg, s.c.) resulted in a dose-dependent reduction of the thermal hyperalgesia induced by ET. Mechanical hyperalgesia, however, was less sensitive to inhibition by propranolol and guanethedine but significantly inhibited by phentolamine. ET injection produced significant upregulation of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), IL-6, and nerve growth factor (NGF). Treatment with any one of the three sympatholytics abolished the upregulation of NGF and IL-6, while phentolamine and guanethedine also reversed the upregulation of TNF-alpha. IL-1 beta was resistant to all of the sympatholytic treatments. We conclude that the SNS can contribute to the local inflammation and hyperalgesia following injection of ET. The resistance to sympatholytics shown by IL-1 beta, known to play a key role in the inflammatory cascade, suggests that ET can initiate inflammation and hyperalgesia independently of peripheral and central sympathetic mechanisms.

Immunopotentiation of a developed Salmonella enterica serotype enteritidis vaccine by thymulin and zinc in meat chicken breeders.

The humoral immunity, spleen and thymus weight indices, lymphocyte count in the thymus cortex, and granuloma diameter at vaccination sites were assessed in four differently immunopotentiated groups of meat chicken breeders. Breeders in the first two groups were given a killed Salmonella enterica serotype Enteritidis (SE) vaccine subcutaneously at 15 and 19 weeks of age. Breeders in the third and fourth groups were left unvaccinated. Breeders in the first group were further immunopotentiated with zinc and thymulin. Each bird in the first group was given the immunopotentiators intraperitoneally in a volume of 0.1 ml at intervals of 3 days for a period of 3 weeks, starting at 15 weeks of age. At each time, each bird in the first group received thymulin (10 ng) and ZnCl2 (1 micromol/L), using a carboxymethyl cellulose carrier, totalling 90 ng thymulin and 9 micromol of ZnCl2 per bird. Each bird in the first three groups was challenged orally with 6.7 x 10(6) cfu/ml of highly virulent SE organisms, at an age of 22 weeks. The first group, which had received zinc and thymulin, had the earliest and highest humoral immune response to SE (p<0.05). This was observed at 2 and 4 weeks after the first vaccination. In addition, the first group had the highest mean thymus weight index, and the highest mean lymphocyte count in the thymus cortex. No significant difference was observed between the first two vaccinated groups in the mean granuloma diameter developed at the two vaccination sites 48 h after administration of the vaccine (p>0.05).

The biphasic immunoregulation of pyrimidylpiperazine (Y-40138) is IL-10 sensitive and requires NF-kappa B targeting in the alveolar epithelium.

1. Pyrimidylpiperazine (Y-40138), a synthetic derivative of N-[1-(4-([4-(pyrimidin-2-yl)piperazin-1-yl]methyl)phenyl)cyclopropyl] acetamide, is a novel dual regulator of pro- and anti-inflammatory cytokines in vivo. The aim of the present study was to determine the signal transduction mechanisms implicated in vitro. 2. In alveolar epithelial cells, pre-treatment (30 min) with Y-40138 reduced LPS-induced biosynthesis of IL-1 beta, IL-6 and TNF-alpha, an effect paralleled by up-regulating an anti-inflammatory counter-loop mediated through IL-10. 3. This differential regulation of pro- and anti-inflammatory signals was accompanied by an inhibition of the nuclear localization of selective NF-kappa B subunits, particularly NF-kappa B(1) (p50), RelA (p65), the major transactivating member of the Rel family, RelB (p68) and c-Rel (p75). In addition, Y-40138 blockaded, in a dose-dependent manner, the LPS-induced nuclear activation of NF-kappa B. 4. Analysis of the upstream pathway involved in Y-40138-dependent retardation of LPS-induced NF-kappa B translocation/activation revealed the involvement of an I kappa B-alpha sensitive pathway. Pre-treatment with Y-40138 ameliorated LPS-induced degradation of I kappa B-alpha in the cytosolic compartment and retarded its phosphorylation, suggesting the involvement of an upstream kinase. 5. Recombinant IL-10 (0 -- 10 ng ml(-1)) blockaded, in a dose-dependent manner, LPS-induced biosynthesis of IL-1 beta, IL-6 and TNF-alpha. Furthermore, rhIL-10 reduced the DNA binding activity of NF-kappa B. Immunoneutralization of endogenous IL-10 by a polyclonal alpha IL-10 (5 microg ml(-1)) reversed the inhibitory effect of Y-40138 on pro-inflammatory cytokines and partially restored the DNA binding activity of NF-kappa B. 6. These results indicate that Y-40138 mediated dual immunoregulation of pro- and anti-inflammatory cytokines is IL-10 sensitive and mediated through the I kappa B-alpha/NF-kappa B signal transduction pathway.

Alpha-melanocyte-related tripeptide, Lys-d-Pro-Val, ameliorates endotoxin-induced nuclear factor kappaB translocation and activation: evidence for involvement of an interleukin-1beta193-195 receptor antagonism in the alveolar epithelium.

The potential anti-inflammatory role of alpha-melanocyte-stimulating hormone (alpha-MSH)-related tripeptide, lysine(11)-D-proline-valine(13) (KDPV), an analogue of interleukin (IL)-1beta(193-195) and an antagonist of IL-1beta/prostaglandin E(2), is not well characterized in the alveolar epithelium. In a model of foetal alveolar type II epithelial cells in vitro, we showed that lipopolysaccharide endotoxin (LPS) differentially, but selectively, induced the nuclear subunit composition of nuclear factor kappaB(1) (NF-kappaB(1)) (p50), RelA (p65) and c-Rel (p75), in parallel to up-regulating the DNA-binding activity (supershift indicating the presence of the p50-p65 complex). LPS accelerated the degradation of inhibitory kappaB-alpha (IkappaB-alpha), accompanied by enhancing its phosphorylation in the cytosolic compartment but not in the nucleus. KDPV suppressed, in a dose-dependent manner, the nuclear localization of p50, p65 and p75, an effect that led to the subsequent inhibition of NF-kappaB activation. Interleukin-1 receptor antagonist (IL-1ra) decreased the nuclear abundance of p50, p65 and p75, and subsequently depressed the DNA-binding activity induced by LPS. Analysis of the mechanism involved in the KDPV- and IL-1ra-mediated inhibition of NF-kappaB nuclear localization revealed a reversal in IkappaB-alpha phosphorylation and degradation, followed by cytosolic accumulation. LPS induced endogenous IL-1beta biosynthesis in a time-dependent manner; the administration of exogenous recombinant human interleukin 1 (rhIL-1) resulted in a dose-dependent activation of NF-kappaB. KDPV and IL-1ra abrogated the effect of rhIL-1. Pretreatment with the non-steroidal anti-inflammatory drug (NSAID) indomethacin, an inhibitor of cyclo-oxygenase, blocked the LPS-induced activation of NF-kappaB. These results indicate the involvement of prostanoid-dependent (NSAID-sensitive) and IL-1-dependent (IL-1ra-sensitive) mechanisms mediating LPS-induced NF-kappaB translocation and activation, a pathway that is regulated, in part, by a negative feedback mechanism transduced through IkappaB-alpha, the major cytosolic inhibitor of NF-kappaB.

Chemioxyexcitation (delta pO2/ROS)-dependent release of IL-1 beta, IL-6 and TNF-alpha: evidence of cytokines as oxygen-sensitive mediators in the alveolar epithelium.

The signalling mechanisms in oxidative stress mediated by cytokines in the perinatal alveolar epithelium are not well known. In an in vitro model of fetal alveolar type II epithelial cells, we investigated the profile of cytokines in response to ascending Deltap O(2)regimen (oxyexcitation). The peak of TNF-alpha (4 h) preceded IL-1beta and IL-6 (6-9 h), indicating a positive feedback autocrine loop confirmed by exogenous rmTNF-alpha. Reactive oxygen species (ROS) induced a dose-dependent release of cytokines, an effect specifically obliterated by selective antioxidants of the hydroxyl radical (*OH) and superoxide anion (O(2)-). Actinomycin and cycloheximide blocked the induced production of cytokines, implicating transcriptional and translational control. Whilst the dismutating enzymes superoxide dismutase (SOD) and catalase were ineffective in reducing ROS-induced cytokines, MnP, a cell-permeating SOD mimetic, abrogated xanthine/xanthine oxidase-dependent cytokine release. Desferrioxamine mesylate, which inhibits the iron-catalysed generation of *OH via the Fenton reaction, exhibited a mild effect on the release of cytokines. Dynamic variation in alveolar p O(2)constitutes a potential signalling mechanism within the perinatal lung allowing upregulation of cytokines in an ROS-dependent manner.

Thiol regulation of pro-inflammatory cytokines reveals a novel immunopharmacological potential of glutathione in the alveolar epithelium.

The therapeutic immunopharmacological potential of glutathione in the alveolar epithelium is not well characterized. We developed an in vitro model of fetal alveolar type II epithelial cells to investigate the effect of redox disequilibrium on chemioxyexcitation (DeltapO(2)/ROS) induced up-regulation of pro-inflammatory cytokines. Buthionine sulfoximine, an irreversible inhibitor of gamma-glutamylcysteine synthetase, the rate-limiting enzyme in glutathione (GSH) biosynthesis, induced intracellular reactive oxygen species (ROS) and the release of interleukin-1beta (IL-1beta), IL-6, and tumor necrosis factor-alpha. Chloroethyl nitrosourea, which blocks the NADPH-dependent recycling of oxidized glutathione (GSSG), reduced ROS-induced cytokine production, similar to pyrrolidine dithiocarbamate, an antioxidant/pro-oxidant thiuram, which elevates GSSG. The antioxidant and GSH precursor, acetylcysteine, abrogated cytokine release concomitant with suppression of ROS, an effect mimicked by gamma-glutamylcysteinyl-ethyl ester, a cell permeant GSH. Cysteine, the rate-limiting amino acid in the de novo synthesis of GSH, administered as oxothiazolidine carboxylate and adenosylmethionine, mitigated the chemioxyexcitation-dependent cytokine release. Ebselen, an anti-inflammatory antioxidant, which mimics the effect of glutathione peroxidase, neutralized ROS by the GSH-peroxidase-coupled reaction, thereby blocking the pathway to cytokine enhancement. Our results indicate that modulating redox equilibrium by pharmacological thiols exhibits differential regulation on pro-inflammatory cytokines, thus bearing clinical consequences for the therapeutic treatment of pediatric distresses in pathophysiology.

A perfusion technique for the determination of pro-inflammatory mediators induced by intradental application of irritants.

INTRODUCTION: Several morphological and functional features contribute to the consideration of the tooth as a separate compartment having special type of innervation and special immune mechanisms. This study describes a new method allowing the intradental perfusion of rat incisors for the in vivo assessment of pulpal reaction to inflammatory agents. METHODS: Under deep anesthesia, the distal 2-3 mm of each of the rat lower incisors was cut and wrapped in a polyethylene tubing connected to a perfusion chamber made of tigone tubing (ID 1/8 in., volume 100-150 microl). Several groups of rats (n=5 each) were used for intradental application of either saline, capsaicin (100 microg in 100 microl), or endotoxin (ET, 20 microg in 100 microl) for a period of 40 min followed by filling the tooth chamber with saline and collecting the perfusate every 40 min for a period of 8 h. The collected perfusates were stored at -70 degrees C for subsequent determination of the concentration of prostaglandin E(2) (PGE(2)) and nerve growth factor (NGF) by enzyme-linked immunosorbent assay (ELISA). RESULTS: Dentinal injury produced a moderate increase in the levels of NGF and PGE(2) in incisors perfused with saline. Application of ET or capsaicin, however, produced a highly significant increase in the levels of both mediators. These effects peaked at 1.5-3 h for PGE(2) and at 5 h for NGF. Capsaicin showed the most significant effects. DISCUSSION: The reported results cannot be attributed to any factor other than the inflammation of the incisor's pulp, because the described chamber does not allow any spread or leak of the applied irritants. Further studies using other reagents can allow the determination of the variation of the levels of the various pro-inflammatory mediators and their modulation by treatment with anti-inflammatory drugs.

Modulation of ultraviolet-induced hyperalgesia and cytokine upregulation by interleukins 10 and 13.

1. Exposure to midrange ultraviolet radiation (UVB) is known to produce skin inflammation similar to sunburn. The aim of this study was to characterize the hyperalgesia and cytokine upregulation induced by UVB and their modulation by antiinflammatory cytokines. 2. Acute exposure of the dorsal skin of mice to UVB (200, 250 and 300 mJ cm(2)) resulted in a dose-dependent decrease in the latencies of the hot plate and tail flick tests, without evident signs of skin lesions. 3. The observed hyperalgesia displayed a biphasic temporal evolution with an acute phase (3 - 6 h) and a late (48 - 96 h) phase. 4. Exposure to UVB (300 mJ cm(2)) elicited significant upregulation of interleukin (IL)-1 beta, tumour necrosis factor (TNF)-alpha and nerve growth factor (NGF), determined by ELISA in the exposed skin. This upregulation was more important during the acute phase of hyperalgesia. 5. Daily treatment of mice, with i.p. injections of either IL-10 or IL-13 (1.5, 7.5 and 15 ng in 100 microl saline) produced a dose-dependent attenuation of the UVB-induced hyperalgesia. 6. Treatment with the highest doses of either IL-10 or IL-13, produced significant attenuation of the levels of the cytokines and NGF by UVB, with relatively more pronounced effects by IL-13. 7. Acute exposure to moderate amounts of UVB results in a systemic hyperalgesia related to the upregulation of cytokine and NGF levels, since both were prevented by treatment with antiinflammatory cytokines.

Immunomodulatory potential of thymulin-Zn(2+) in the alveolar epithelium: amelioration of endotoxin-induced cytokine release and partial amplification of a cytoprotective IL-10-sensitive pathway.

The immunomodulatory potential of thymulin in the perinatal epithelium is not well characterized. In an in vitro model of fetal alveolar type II epithelial cells, we investigated the exhibition of an anti-inflammatory activity of this peptide hormone. Thymulin selectively ameliorated, in a dose-dependent manner, the endotoxin-induced release of IL-1 beta (IC(50) = 657 ng. ml(-1)), but showed no inhibitory effect on IL-6 and TNF-alpha. Zinc, an anti-inflammatory antioxidant, which is required for the biological activity of thymulin, reduced the secretion of IL-1 beta (IC(50) = 62 microM), TNF-alpha (IC(50) = 1000 microM), and, to a lesser extent, IL-6. This cation (100 microM) amplified the effect of thymulin on IL-1 beta and TNF-alpha (IC(50) < 0.1 ng. ml(-1)), but not on IL-6. Analysis of whether thymulin is up-regulating a counterpart anti-inflammatory signaling loop revealed the involvement of an IL-10-sensitive pathway. These results indicate that thymulin acts as a novel dual immunoregulator by enhancing an anti-inflammatory cytoprotective response and depressing an inflammatory signal, an effect synergistically amplified, in part, by cationic zinc.

The role of cytokines and prostaglandin-E(2) in thymulin induced hyperalgesia.

We have recently reported that intraperitoneal (i.p.) injection of thymulin at low doses (50 ng) resulted in thermal and mechanical hyperalgesia and upregulation of the level of interleukin-1beta in the liver. In this study, we demonstrate that such injections of thymulin result in a significant elevation in the levels of TNF-alpha (P<0.01), NGF (P<0.01) and PGE(2) (P<0.01) in the liver of the treated rats, in addition to the increase in the levels of IL-1beta. Pretreatment with specific antagonists to each of these factors (polyclonal anti-TNF-alpha, anti-NGF antiserum and IL-1 receptor antagonist) did not result in the abolition of the hyperalgesia as assessed by the paw pressure, hot plate, paw immersion and tail flick tests. However, pretreatment with a combination of the above antagonist and antisera almost completely prevented thymulin-induced hyperalgesia. The cyclooxygenase inhibitor, meloxicam, reversed in a dose dependent manner (0.2, 0.4 and 2 mg/kg) thymulin effects as assessed by the different pain tests. It also abolished the thymulin-induced increase in the level of cytokines and NGF in the liver. Our results indicate that PGE(2) could be the key mediator of the hyperalgesic action of thymulin and the observed upregulation of proinflammatory cytokines and NGF.

Functional interplay between gelatinases and hyperalgesia in endotoxin-induced localized inflammatory pain.

The role of ECM-degrading proteinases in normal developmental processes and in pathological conditions is extensively studied. However, few reports describe the role ECM-degrading proteinases play in modulating hyperalgesia. The goal of this study is to describe the regulation of gelatinases during endotoxin mediated local inflammation, induced by intra plantar endotoxin (ET; 1.25 microg/50 microl) injection in Balb/c mice, and to correlate that with hyperalgesia. ET injections induced hyperalgesia, as determined by hot plate and paw pressure tests, which peaked by 24 h and recovered by 48 h post-injection. Contralateral paw of ET injected mice and saline injected paws in control mice elicited no hyperalgesia. Zymography showed that ET and saline injected paws elicited increased gelatinase activity by 9 h after injection. However, only the former maintained high levels of expression of a 90 kD gelatinase up to at least 96 h post ET injection, while in the latter gelatinase expression was down regulated by 24 h. Interestingly, the 90-kD gelatinase was upregulated in the contralateral paw of the ET-injected mice beyond 48 h post injection. Saline injection in that paw, during a time when gelatinases are upregulated, induced hyperalgesia. Intraperitoneal injection of either ZnCl(2) (100 microM), thymulin (5 microg/100 microl), or morphine (2 mg/kg/100 microl) reversed the ET-induced hyperalgesia and suppressed gelatinase activity. Furthermore, intraperitoneal injection of MPI, an ECM-degrading proteinase inhibitor, reversed ET induced hyperalgesia. Taken together, the above suggests that a functional interplay exists between gelatinase upregulation triggered by ET injections and hyperalgesia. The exact mechanism underlying such correlation remains to be determined.

Macrophage recruitment and activation: a model for comparing resistance to Salmonella enteritidis in different broiler breeds.

A model for comparing resistance to Salmonella Enteritidis was evaluated in different broiler breeds. The recruitment and phagocytic activity of peritoneal macrophages were assessed in three different broiler breeds (A, B and C) which are farmed world-wide. Assessment was performed after three days of intraperitoneal (i.p.) administration of 3% Sephadex G-200 (10 ml), initiated at twenty-one days of age, followed by contact with i.p. live S. Enteritidis (10 ml, 1.2 x 10(8) colony forming units/ml) for 45 min. Assessment included determination of the number of i.p. macrophages recruited, the number of i.p. phagocytized S. Enteritidis cells per macrophage, the levels of degranulated i.p. beta-glucuronidase and beta-galactosidase, and the count of surviving S. Enteritidis cells. Confirmation of the significance of the model was obtained by comparing resistance to field infection by S. Enteritidis in the three broiler breeds. The recruitment of i.p. macrophages in response to challenge with Sephadex and S. Enteritidis was significantly higher (P < 0.05) in birds of breed A (mean cumulative i.p. macrophage count, in 10 fields of microscopic slide smear magnified at x1,000, was equal to 81.7), compared to recruitment in birds of breed B (33.3) or breed C (41.2). The mean number of phagocytized S. Enteritidis cells per i.p. macrophage in birds of breed A (2.68) was significantly higher (P < 0.05) than in breed B (0.83) and insignificantly higher (P > 0.05) than in breed C (2.35). In addition, the highest level of recruitment and phagocytic activity of macrophages, in birds of breed A, was associated with a higher significant mean i.p. beta-glucuronidase activity (10,425.5 units/ml) than in breed B (3,438.2 units/ml) or breed C (3,356.94 units/ml) (P < 0.05). Moreover, birds of breed A demonstrated a higher mean i.p. beta-galactosidase activity (2.225 units/ml) than birds of breed B (0.852 units/ml) or breed C (1.852 units/ml) (P > 0.05). The higher level of recruitment and activity of i.p. macrophages and the higher rate of degranulation of i.p. enzymes in breed A were associated with a greater number of surviving i.p. S. Enteritidis cells. In response to outbreaks of S. Enteritidis in the field, the average mortality was significantly higher in flocks of breed A (3.2%) than in flocks of breed B (1.2%) or breed C (0.96%) (P < 0.05). These data provide an indication of the significance of the model in reflecting the differences in resistance of S. Enteritidis of broiler breeds reared in a farm environment.

Altered plasma levels of nerve growth factor and transforming growth factor-beta2 in type-1 diabetes mellitus.

Nerve growth factor (NGF) and transforming growth factor-beta2 (TGF-beta2) are cytokines which have known immunological effects. An elevated level of NGF has been reported in certain autoimmune diseases, whereas TGF-beta2 is an immunosuppressor which is known to play a role in regulating cell proliferation. A role of this cytokine has been proposed in the pathogenesis of type-1 diabetes mellitus (IDDM), but no clinical studies have yet measured its serum level in this disease. In this study we measured the levels of NGF and TGF-beta2 in the sera of patients with IDDM (n = 26) and values were compared to those of age-matched normal subjects (n = 27) and also to patients with type-2 diabetes mellitus (NIDDM) (n = 26) with similar HbA1c levels and an equal duration of diabetes. Serum NGF levels were significantly elevated in IDDM patients compared to those of age-matched controls (p <.001) and NIDDM controls (p <.01). TGF-beta2 levels were lower in IDDM patients when compared with the healthy control (p <.001) and the NIDDM control (p <.05). There was no correlation between the levels of NGF and TGF-beta2. The duration of diabetes and the level of HbA1c did not affect the NGF and TGF-beta2 levels in the IDDM patients. We conclude that an increase in NGF and a suppression in TGF-beta2 levels are present in patients with type-1 diabetes mellitus and that both cytokines may play independent roles in the pathogenesis of this disease.

Thymulin induces c-fos expression in the spinal cord of rats which is reversed by meloxicam and morphine.

Intraplantar (i.pl.) injections of thymulin have been shown to produce hyperalgesia in rats through a prostaglandin E2-dependent mechanism. This study aimed at investigating if such injections can produce sustained activation of spinal neurons by mapping the fos-like-immunoreactivity (FLI) as a marker for this activation. Our results showed that thymulin produces significant and sustained FLI in neurons located in spinal laminae known to be involved in nociception. Pretreatment with either morphine or meloxicam (a cyclooxygenase inhibitor) revealed differential effects on FLI and the hyperalgesia induced by thymulin. These findings support the hypothesis that thymulin can affect central neurons either directly or through the peripheral nerve terminals.