The peripheral pro-nociceptive state induced by repetitive inflammatory stimuli involves continuous activation of protein kinase A and protein kinase C epsilon and its Na(v)1.8 sodium channel functional regulation in the primary sensory neuron

In the present study, the participation of the Na(v)1.8 sodium channel was investigated in the development of the peripheral pro-nociceptive state induced by daily intraplantar injections of PGE(2) in rats and its regulation in vivo by protein kinase A (PKA) and protein kinase C epsilon (PKC epsilon) as well. In the prostaglandin E(2) (PGE(2))-induced persistent hypernociception, the Na(v)1.8 mRNA in the dorsal root ganglia (DRG) was up-regulated. The local treatment with dipyrone abolished this persistent hypernociception but did not alter the Na(v)1.8 mRNA level in the DRG. Daily intrathecal administrations of antisense Na(v)1.8 decreased the Na(v)1.8 mRNA in the DRG and reduced ongoing persistent hypernociception. once the persistent hypernociception had been abolished by dipyrone, but not by Na(v)1.8 antisense treatment, a small dose of PGE(2) restored the hypernociceptive plateau. These data show that, after a period of recurring inflammatory stimuli, an intense and prolonged nociceptive response is elicited by a minimum inflammatory stimulus and that this pro-nociceptive state depends on Na(v)1.8 mRNA up-regulation in the DRG. in addition, during the persistent hypernociceptive state, the PKA and PKC epsilon expression and activity in the DRG are up-regulated and the administration of the PKA and PKC epsilon inhibitors reduce the hypernociception as well as the Na(v)1.8 mRNA level. In the present study, we demonstrated that the functional regulation of the Na(v)1.8 mRNA by PKA and PKC epsilon in the primary sensory neuron is important for the development of the peripheral pro-nociceptive state induced by repetitive inflammatory stimuli and for the maintenance of the behavioral persistent hypernociception. (C) 2008 Elsevier Inc. All rights reserved.

Different inflammatory stimuli in the footpad of mice influence the kinetics of resident peritoneal cells

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Response of splenic melanomacrophage centers of Oreochromis niloticus (Linnaeus, 1758) to inflammatory stimuli by BCG and foreign bodies

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cytokine inhibition and time-related influence of inflammatory stimuli on the hyperalgesia induced by the nucleus pulposus

The symptoms of lumbar disc herniation, such as low back pain and sciatica, have been associated with local release of cytokines following the inflammatory process induced by the contact of the nucleus pulposus (NP) with the spinal nerve. Using an animal experimental model of intervertebral disc herniation and behavioral tests to evaluate mechanical (electronic von Frey test) and thermal (Hargreaves Plantar test) hyperalgesia in the hind paw of rats submitted to the surgical model, this study aimed to detect in normal intervertebral disc the cytokines known to be involved in the mechanisms of inflammatory hyperalgesia, to observe if previous exposure of the intervertebral disc tissue to specific antibodies could affect the pain behavior (mechanical and thermal hyperalgesia) induced by the NP, and to observe the influence of the time of contact of the NP with the fifth lumbar dorsal root ganglion (L5-DRG) in the mechanical and thermal hyperalgesia. The cytokines present at highest concentrations in the rat NP were TNF-alpha, IL-1 beta and CINC-1. Rats submitted to the disc herniation experimental model, in which a NP from the sacrococcygeal region is deposited over the right L5-DRG, showed increased mechanical and thermal hyperalgesia that lasted at least 7 weeks. When the autologous NP was treated with antibodies against the three cytokines found at highest concentrations in the NP (TNF-alpha, IL-1 beta and CINC-1), there was decrease in both mechanical and thermal hyperalgesia in different time points, suggesting that each cytokine may be important for the hyperalgesia in different steps of the inflammatory process. The surgical remotion of the NP from herniated rats 1 week after the implantation reduced the hyperalgesia to the level similar to the control group. This reduction in the hyperalgesia was also observed in the group that had the NP removed 3 weeks after the implantation, although the intensity of the hyperalgesia did not decreased totally. The removal of the NP after 5 weeks did not changed the hyperalgesia observed in the hind paw, which suggests that the longer the contact of the NP with the DRG, the greater is the possibility of development of chronic pain. Together our results indicate that specific cytokines released during the inflammatory process induced by the herniated intervertebral disc play fundamental role in the development of the two modalities of hyperalgesia (mechanical and thermal) and that the maintenance of this inflammation may be the most important point for the chronification of the pain.

Role of complement C5a in mechanical inflammatory hypernociceptio: potential use of C5a receptor antagonists to control inflammatory pain

particularly neutrophil chemoattraction. Herein, the role of C5a in the genesis of inflammatory hypernociception was investigated in rats and mice using the specific C5a receptor antagonist PMX53 (AcF-[OP(D-Cha)WR]). Experimental approach: Mechanical hypernociception was evaluated with a modification of the Randall-Selitto test in rats and electronic pressure meter paw test in mice. Cytokines were measured by ELISA and neutrophil migration was determined by myeloperoxidase activity. Key results: Local pretreatment of rats with PMX53 (60-180 mg per paw) inhibited zymosan-, carrageenan-, lipopolysaccharide (LPS)- and antigen-induced hypernociception. These effects were associated with C5a receptor blockade since PMX53 also inhibited the hypernociception induced by zymosan- activated serum and C5a but not by the direct-acting hypernociceptive mediators, prostaglandin E-2 and dopamine. Underlying the C5a hypernociceptive mechanisms, PMX53 did not alter the cytokine release induced by inflammatory stimuli. However, PMX53 inhibited cytokine-induced hypernociception. PMX53 also inhibited the recruitment of neutrophils induced by zymosan but not by carrageenan or LPS, indicating an involvement of neutrophils in the hypernociceptive effect of C5a. Furthermore, the C5a-induced hypernociception was reduced in neutrophil-depleted rats. Extending these findings in rats, blocking C5a receptors also reduced zymosan- induced joint hypernociception in mice. Conclusions and implications: These results suggest that C5a is an important inflammatory hypernociceptive mediator, acting by a mechanism independent of hypernociceptive cytokine release, but dependent on the presence of neutrophils. Therefore, we suggest that inhibiting the action of C5a has therapeutic potential in the control of inflammatory pain.

Commensal microbiota is fundamental for the development of inflammatory pain

The ability of an individual to sense pain is fundamental for its capacity to adapt to its environment and to avoid damage. The sensation of pain can be enhanced by acute or chronic inflammation. In the present study, we have investigated whether inflammatory pain, as measured by hypernociceptive responses, was modified in the absence of the microbiota. To this end, we evaluated mechanical nociceptive responses induced by a range of inflammatory stimuli in germ-free and conventional mice. Our experiments show that inflammatory hypernociception induced by carrageenan, lipopolysaccharide, TNF-alpha, IL-1 beta, and the chemokine CXCL1 was reduced in germfree mice. In contrast, hypernociception induced by prostaglandins and dopamine was similar in germ-free or conventional mice. Reduction of hypernociception induced by carrageenan was associated with reduced tissue inflammation and could be reversed by reposition of the microbiota or systemic administration of lipopolysaccharide. Significantly, decreased hypernociception in germ-free mice was accompanied by enhanced IL-10 expression upon stimulation and could be reversed by treatment with an anti-IL-10 antibody. Therefore, these results show that contact with commensal microbiota is necessary for mice to develop inflammatory hypernociception. These findings implicate an important role of the interaction between the commensal microbiota and the host in favoring adaptation to environmental stresses, including those that cause pain.

Retinal thickening in HLA-B27 associated acute anterior uveitis: evolution with time and association with severity of inflammatory activity.

Purpose: To describe the evolution of retinal thickness in eyes affected with acute anterior uveitis (AAU) in the course of follow-up and to assess its correlation with severity of inflammatory activity in the anterior chamber. Methods: Design: Prospective, cohort study Setting: Institutional study Patient population: 72 eyes (affected and fellow eyes) of 36 patients Observation procedure: Patients were followed daily until beginning of resolution of inflammatory activity and weekly thereafter. Optical coherence tomography and laser flare photometry were performed at each visit. Treatment consisted of topical corticosteroids Main outcome measures: Retinal thickness of affected eyes, difference in retinal thickness between affected and fellow eyes and their evolution in time, association between maximal retinal thickness and initial laser flare photometry. Results: Difference in retinal thickness between affected and fellow eyes became significant on average seven days from baseline and remained so through-out follow-up (p<0.001). There was a steep increase in retinal thickness of affected eyes followed by a progressive decrease after reaching a peak value. Maximal difference in retinal thickness between affected and fellow eyes was observed between 17 and 25 days from baseline and exhibited a strong, positive correlation with initial laser flare photometry values (p=0.015). Conclusions: Retinal thickness in eyes affected with AAU presents a steep increase over 3 to 4 weeks and then gradually decreases. Severity of inflammation at baseline predicts the amount of retinal thickening in affected eyes. A characteristic pattern of temporal response of retinal anatomy to inflammatory stimuli seems to arise.

Identification of human IKK-2 inhibitors of natural origin (Part II): in Silico prediction of IKK-2 inhibitors in natural extracts with known anti-inflammatory activity.

Human inhibitor NF-κB kinase 2 (hIKK-2) is the primary component responsible for activating NF-κB in response to various inflammatory stimuli. Thus, synthetic ATP-competitive inhibitors for hIKK-2 have been developed as anti-inflammatory compounds. We recently reported a virtual screening protocol (doi:10.1371/journal.pone.0016903) that is able to identify hIKK-2 inhibitors that are not structurally related to any known molecule that inhibits hIKK-2 and that have never been reported to have anti-inflammatory activity. In this study, a stricter version of this protocol was applied to an in-house database of 29,779 natural products annotated with their natural source. The search identified 274 molecules (isolated from 453 different natural extracts) predicted to inhibit hIKK-2. An exhaustive bibliographic search revealed that anti-inflammatory activity has been previously described for: (a) 36 out of these 453 extracts; and (b) 17 out of 30 virtual screening hits present in these 36 extracts. Only one of the remaining 13 hit molecules in these extracts shows chemical similarity with known synthetic hIKK-2 inhibitors. Therefore, it is plausible that a significant portion of the remaining 12 hit molecules are lead-hopping candidates for the development of new hIKK-2 inhibitors.

Treatment of hemorrhagic shock with hypertonic saline solution modulates the inflammatory response to live bacteria in lungs

Shock and resuscitation render patients more susceptible to acute lung injury due to an exacerbated immune response to subsequent inflammatory stimuli. To study the role of innate immunity in this situation, we investigated acute lung injury in an experimental model of ischemia-reperfusion (I-R) followed by an early challenge with live bacteria. Conscious rats (N = 8 in each group) were submitted to controlled hemorrhage and resuscitated with isotonic saline (SS, 0.9% NaCl) or hypertonic saline (HS, 7.5% NaCl) solution, followed by intratracheal or intraperitoneal inoculation of Escherichia coli. After infection, toll-like receptor (TLR) 2 and 4 mRNA expression was monitored by RT-PCR in infected tissues. Plasma levels of tumor necrosis factor α and interleukins 6 and 10 were determined by ELISA. All animals showed similar hemodynamic variables, with mean arterial pressure decreasing to nearly 40 mmHg after bleeding. HS or SS used as resuscitation fluid yielded equal hemodynamic results. Intratracheal E. coli inoculation per se induced a marked neutrophil infiltration in septa and inside the alveoli, while intraperitoneal inoculation-associated neutrophils and edema were restricted to the interseptal space. Previous I-R enhanced lung neutrophil infiltration upon bacterial challenge when SS was used as reperfusion fluid, whereas neutrophil influx was unchanged in HS-treated animals. No difference in TLR expression or cytokine secretion was detected between groups receiving HS or SS. We conclude that HS is effective in reducing the early inflammatory response to infection after I-R, and that this phenomenon is achieved by modulation of factors other than expression of innate immunity components.

Role of M2 muscarinic receptor in the airway response to methacholine of mice selected for minimal or maximal acute inflammatory response

Airway smooth muscle constriction induced by cholinergic agonists such as methacholine (MCh), which is typically increased in asthmatic patients, is regulated mainly by muscle muscarinic M3 receptors and negatively by vagal muscarinic M2 receptors. Here we evaluated basal (intrinsic) and allergen-induced (extrinsic) airway responses to MCh. We used two mouse lines selected to respond maximally (AIRmax) or minimally (AIRmin) to innate inflammatory stimuli. We found that in basal condition AIRmin mice responded more vigorously to MCh than AIRmax. Treatment with a specific M2 antagonist increased airway response of AIRmax but not of AIRmin mice. The expression of M2 receptors in the lung was significantly lower in AIRmin compared to AIRmax animals. AIRmax mice developed a more intense allergic inflammation than AIRmin, and both allergic mouse lines increased airway responses to MCh. However, gallamine treatment of allergic groups did not affect the responses to MCh. Our results confirm that low or dysfunctional M2 receptor activity is associated with increased airway responsiveness to MCh and that this trait was inherited during the selective breeding of AIRmin mice and was acquired by AIRmax mice during allergic lung inflammation

Most lymphoid organ dendritic cell types are phenotypically and functionally immature

Dendritic cells (DCs) have been thought to follow a life history, typified by Langerhans cells (LCs), with 2 major developmental stages: an immature stage that captures antigens in the periphery and a mature stage that presents those antigens in the lymphoid organs. However, a systematic assessment of the maturity of lymphoid organ DCs has been lacking. We have analyzed the maturity of the DC types found in the steady state in the spleen, lymph nodes (LNs), and thymus. The DCs that migrate into the iliac, mesenteric, mediastinal, or subcutaneous LNs from peripheral tissues were mature and therefore could not process and present newly encountered antigens. However, all the other DC types were phenotypically and functionally immature: they expressed low levels of surface major histocompatibility complex class 11 (MHC 11) and CD86, accumulated MHC 11 in their endosomes, and could present newly encountered antigens. These immature DCs could 1346 induced to mature by culture in vitro or by Inoculation of inflammatory stimuli in vivo. Therefore, the lymphoid organs contain a large cohort of immature DCs, most likely for the maintenance of peripheral tolerance, which can respond to infections reaching those organs and mature in situ. (C) 2003 by The American Society of Hematology.

The protease inhibitor cystatin C is differentially expressed among dendritic cell populations, but does not control antigen presentation

Dendritic cells (DC) undergo complex developmental changes during maturation. The MHC class H (MHC H) molecules of immature DC accumulate in intracellular compartments, but are expressed at high levels on the plasma membrane upon DC maturation. It has been proposed that the cysteine protease inhibitor cystatin C (CyC) plays a pivotal role in the control of this process by regulating the activity of cathepsin S, a protease involved in removal of the MHC H chaperone E, and hence in the formation of MHC H-peptide complexes. We show that CyC is differentially expressed by mouse DC populations. CD8(+) DC, but not CD4(+) or CD4(-)CD8(-) DC, synthesize CyC, which accumulates in MHC II(+)Lamp(+) compartments. However, II processing and MHC H peptide loading proceeded similarly in all three DC populations. We then analyzed MHC H localization and Ag presentation in CD8(+) DC, bone marrow-derived DC, and spleen-derived DC lines, from CyC-deficient mice. The absence of CyC did not affect the expression, the subcellular distribution, or the formation of peptide-loaded MHC II complexes in any of these DC types, nor the efficiency of presentation of exogenous Ags. Therefore, CyC is neither necessary nor sufficient to control MHC II expression and Ag presentation in DC. Our results also show that CyC expression can differ markedly between closely related cell types, suggesting the existence of hitherto unrecognized mechanisms of control of CyC expression.

MET is required for the recruitment of anti-tumoural neutrophils

Mutations or amplification of the MET proto-oncogene are involved in the pathogenesis of several tumours, which rely on the constitutive engagement of this pathway for their growth and survival. However, MET is expressed not only by cancer cells but also by tumour-associated stromal cells, although its precise role in this compartment is not well characterized. Here we show that MET is required for neutrophil chemoattraction and cytotoxicity in response to its ligand hepatocyte growth factor (HGF). Met deletion in mouse neutrophils enhances tumour growth and metastasis. This phenotype correlates with reduced neutrophil infiltration to both the primary tumour and metastatic sites. Similarly, Met is necessary for neutrophil transudation during colitis, skin rash or peritonitis. Mechanistically, Met is induced by tumour-derived tumour necrosis factor (TNF)-a or other inflammatory stimuli in both mouse and human neutrophils. This induction is instrumental for neutrophil transmigration across an activated endothelium and for inducible nitric oxide synthase production upon HGF stimulation. Consequently, HGF/MET-dependent nitric oxide release by neutrophils promotes cancer cell killing, which abates tumour growth and metastasis. After systemic administration of a MET kinase inhibitor, we prove that the therapeutic benefit of MET targeting in cancer cells is partly countered by the pro-tumoural effect arising from MET blockade in neutrophils. Our work identifies an unprecedented role of MET in neutrophils, suggests a potential 'Achilles' heel' of MET-targeted therapies in cancer, and supports the rationale for evaluating anti-MET drugs in certain inflammatory diseases.

Macrophage migration inhibitory factor: a counter-regulator of glucocorticoid action and critical mediator of septic shock.

Recent studies have led to the discovery of a mediator that acts as an endogenous counter-regulator of glucocorticoid action within the immune system. Isolated as a product of anterior pituitary cells, this protein was found to have the sequence of macrophage migration inhibitory factor (MIF), one of the first cytokine activities to be described. Macrophages and T cells release MIF in response both to various inflammatory stimuli and upon incubation with low concentrations of glucocorticoids. The glucocorticoid-induced secretion of MIF is tightly regulated and decreases at high, anti-inflammatory steroid concentrations. Once secreted, MIF "overrides" the anti-inflammatory and immunosuppressive effects of steroids on macrophage and T-cell cytokine production. The physiological role of MIF thus appears to be to counter-balance steroid inhibition of the inflammatory response. Anti-MIF antibodies fully protect animals from experimentally induced gram-negative or gram-positive septic shock, an effect that may be the result of the increased anti-inflammatory effects of glucocorticoids after neutralization of endogenous MIF. Anti-MIF therapeutic strategies are presently under development and may prove to be a means to modulate cytokine production in septic shock as well as in other inflammatory disease states.

Local anaesthetic medication for the treatment of asthma

It is presumed that drugs able to prevent bronchial spasm and/or inflammation may have therapeutic potential to control asthma symptoms. The local anaesthetic lidocaine has recently received increased attention as an alternative form of treatment for asthmatic patients. This paper reviews the major findings on the topic and summarizes the putative mechanisms underlying the airway effects of local anaesthetic agents. We think that lidocaine extends the spectrum of options in asthma therapy, probably by counteracting both spasmogenic and inflammatory stimuli in the bronchial airways. The possibility of development of new anti-asthma compounds based on the synthesis of lidocaine derivatives is also on the horizon.