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Osteoclasts play a key role in the regulation of bone mass and are highly active metabolically. Here we show that a metabolic reprogramming toward the hexosamine biosynthetic pathway (HBP) is required not only for osteoclast differentiation but also to determine the bone resorption mode during physiological and pathological bone remodeling. We found that pharmacological inhibition of O-GlcNAc transferase (OGT) significantly reduced protein O-GlcNAcylation and osteoclast differentiation. Accordingly, genetic deletion of OGT also inhibited osteoclast formation and downregulated critical markers related to osteoclasts differentiation and function (NFATc1, αvintegrin, cathepsin K). Indeed, cells treated with OSMI-1, an OGT inhibitor, also reduced nuclear translocation of NFATc1. Furthermore, the addition of exogenous N-acetylglucosamine (GlcNAc) strongly increased osteoclast formation and demineralization ability. Strikingly, our data show for the first time that O-GlcNAcylation facilitates an aggressive trench resorption mode in human cells. The incubation of osteoclasts with exogenous GlcNAc increases the percentage of erosion by trench while having no effect on pit resorption mode. Through time-lapse recording, we documented that osteoclasts making trenches moving across the bone surface are sensitive to GlcNAcylation. Finally, osteoclast-specific Ogt -deficient mice show increased bone density and reduced inflammation-induced bone loss during apical periodontitis model. We show that osteoclast-specific Og t-deficient mice are less susceptible to develop bacterial-induced periapical lesion. Consistent with this, Ogt -deleted mice showed a decreased number of tartrate-resistant acid phosphatase-positive cells lining the apical periodontitis site. In summary, here we describe a hitherto undiscovered role of the HBP/O-GlcNAcylation axis tuning resorption mode and dictating bone resorption outcome. [ABSTRACT FROM AUTHOR]

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 germ-free 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-lL-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. cytokines | germ-free mice | hyperalgesia | nociception

The reduction of circulating neutrophil migration to infection sites is associated with a poor outcome of severe sepsis. [alpha]-1-Acid glycoprotein (AGP) was isolated from the sera of severely septic patients by HPLC and acrylamide gel electrophoresis and identified by mass spectrometry. Both the isolated protein and commercial AGP inhibited carrageenin-induced neutrophil migration into the rat peritoneal cavity when administered i.v. at a dose of 4.0 [micro]g per rat (95 pmol per rat). Analysis by intravital microscopy demonstrated that both proteins inhibited the rolling and adhesion of leukocytes in the mesenteric microcirculation. The inhibitory activity was blocked by 50 mg/kg aminoguanidine, s.c., and was not demonstrable in inducible nitric oxide synthase (iNOS) knockout mice. Incubation of AGP with neutrophils from healthy subjects induced the production of NO and inhibited the neutrophil chemotaxis by an iNOS/NO/cyclic guanosine 3,5-monophosphate-dependent pathway. In addition, AGP induced the L-selectin shedding by neutrophils. The administration of AGP to rats with mild cecal ligation puncture sepsis inhibited neutrophil migration and reduced 7-day survival from [approximately equal to] 80% to 20%. These data demonstrate that AGP, an acute-phase protein, inhibits neutrophil migration by an NO-dependent process and suggest that AGP also participates in human sepsis.

The hypernociceptive effects of cytokines [TNF-[alpha], keratinocyte-derived chemokine (KC), and IL-1[beta]] and their participation in carrageenan (Cg)-induced inflammatory hypernociception in mice were investigated. Nociceptor sensitization (hypernociception) was quantified with an electronic version of the von Frey filament test in WT and TNF receptor type 1 knockout mice (TNF-R[1.sup.-/-]). TNF-[alpha]-induced hypernociception was abolished in TNF-R[1.sup.-/-] mice, partially inhibited by pretreatment with IL-1 receptor antagonist (IL-1ra) or indomethacin and unaffected by Ab against KC (AbKC) or guanethidine. IL-1ra and indomethacin pretreatment strongly inhibited the hypernociception induced by IL-1[beta], which was not altered by AbKC or guanethidine or by knocking out TNF-R1. KC-induced hypernociception was abolished by AbKC, inhibited by pretreatment with indomethacin plus guanethidine, and partially inhibited by IL-1ra, indomethacin, or guanethidine. In contrast, KC-induced hypernociception was not altered by knocking out TNF-R1. Cg-induced hypernociception was abolished by administration of indomethacin plus guanethidine, diminished in TNF-R[1.sup.-/-] mice, and partially inhibited in WT mice pretreated with AbKC, IL-1ra, indomethacin, or guanethidine. TNF-[alpha], KC, and IL-1[beta] concentrations were elevated in the skin of Cg-injected paws. The TNF-[alpha] and KC concentrations rose concomitantly and peaked before that of IL-1[beta]. In mice, the cytokine cascade begins with the release of TNF-[alpha] (acting on TNF-R1 receptor) and KC, which stimulate the release of IL-1[beta]. As in rats, the final mediators of this cascade were prostaglandins released by IL-1[beta] and sympathetic amines released by KC. These results extend to mice the concept that the release of primary mediators responsible for hypernociception is preceded by a cascade of cytokines. inflammation | prostaglandins | TNF | hyperalgesia | pain

The role of tumour necrosis factor alpha (TNF-alpha) and inducible nitric oxide synthase (iNOS) generated nitric oxide (NO) in gastric damage and granulocyte infiltration is examined using tumour necrosis factor receptor 1 (TNF-R1-/-) or iNOS (iNOS-/-) deficient mice. It is concluded that TNF-alpha, acting via TNF-R1, is involved in indomethacin induced gastric damage and granulocyte infiltration.

Glucocorticoids induce interleukin-8 (IL-8) inhibition to enhance the drug's antipyretic effect in fever unaffected by cyclooxygenase inhibitors. Phagocytes and several other cells, when subjected to inflammatory stimuli and exposed to such endogenous mediators as IL-1 and tumor necrosis factor-alpha1, release cytokine IL-8. IL-8 triggers neutrophil chemotaxis, neutrophil activation and histamine release.

Preclinical and clinical evidence suggests pro-inflammatory cytokines might play an important role in the neurobiology of schizophrenia and stress-related psychiatric disorders. Interleukin-18 (IL-18) is a member of the IL-1 family of cytokines and it is widely expressed in brain regions involved in emotional regulation. Since IL-18 involvement in the neurobiology of mental illnesses, including schizophrenia, remains unknown, this work aimed at investigating the behavior of IL-18 null mice (KO) in different preclinical models: 1. the prepulse inhibition test (PPI), which provides an operational measure of sensorimotor gating and schizophrenic-like phenotypes; 2. amphetamine-induced hyperlocomotion, a model predictive of antipsychotic activity; 3. resident-intruder test, a model predictive of aggressive behavior. Furthermore, the animals were submitted to models used to assess depressive- and anxiety-like behavior. IL-18KO mice showed impaired baseline PPI response, which was attenuated by d -amphetamine at a dose that did not modify PPI response in wild-type (WT) mice, suggesting a hypodopaminergic prefrontal cortex function in those mice. d -Amphetamine, however, induced hyperlocomotion in IL-18KO mice compared to their WT counterparts, suggesting hyperdopaminergic activity in the midbrain. Moreover, IL-18KO mice presented increased basal levels of IL-1β levels in the hippocampus and TNF-α in the prefrontal cortex, suggesting an overcompensation of IL-18 absence by increased levels of other proinflammatory cytokines. Although no alteration was observed in the forced swimming or in the elevated plus maze tests in naïve IL-18KO mice, these mice presented anxiogenic-like behavior after exposure to repeated forced swimming stress. In conclusion, deletion of the IL-18 gene resembled features similar to symptoms observed in schizophrenia (positive and cognitive symptoms, aggressive behavior), in addition to increased susceptibility to stress. The IL-18KO model, therefore, could provide new insights into how changes in brain immunological homeostasis induce behavioral changes related to psychiatric disorders, such as schizophrenia. [ABSTRACT FROM AUTHOR]

The NOD-like receptors are cytoplasmic proteins that sense microbial by-products released by invasive bacteria. Although NOD1 and NOD2 are functionally expressed in cells from oral tissues and play a role triggering immune responses, the role of NOD2 receptor in the bone resorption and in the modulation of osteoclastogenesis is still unclear. We show that in an experimental model of periodontitis with Porphyromonas gingivalis W83, NOD2-/- mice showed lower bone resorption when compared to wild type. Quantitative polymerase chain reaction analysis revealed that wild-type infected mice showed an elevated RANKL/OPG ratio when compared to NOD2-/- infected mice. Moreover, the expression of 2 osteoclast activity markers—cathepsin K and matrix metalloproteinase 9—was significantly lower in gingival tissue from NOD2-/- infected mice compared to WT infected ones. The in vitro study reported an increase in the expression of the NOD2 receptor 24 hr after stimulation of hematopoietic bone marrow cells with M-CSF and RANKL. We also evaluated the effect of direct activation of NOD2 receptor on osteoclastogenesis, by the activation of this receptor in preosteoclasts culture, with different concentrations of muramyl dipeptide. The results show no difference in the number of TRAP-positive cells. Although it did not alter the osteoclasts differentiation, the activation of NOD2 receptor led to a significant increase of cathepsin K expression. We confirm that this enzyme was active, since the osteoclasts resorption capacity was enhanced by muramyl dipeptide stimulation, evaluated in osteoassay plate. These results show that the lack of NOD2 receptor impairs the bone resorption, suggesting that NOD2 receptor could contribute to the progression of bone resorption in experimental model of periodontitis. The stimulation of NOD2 by its agonist, muramyl dipeptide, did not affect osteoclastogenesis, but it does favor the bone resorption capacity identified by increased osteoclast activity. [ABSTRACT FROM PUBLISHER]

Background Neutrophil recruitment mediated by the CXCL1/KC chemokine and its receptors CXCR1/CXCR2 plays a critical role in inflammatory diseases. Recently, neutrophil migration and activation triggered by CXCL1-CXCR1/2 signalling was implicated in inflammatory nociception; however, their role in post-surgical pain has not been elucidated. In this study, we addressed the function of neutrophils in the genesis of post-incisional pain in an experimental model of post-surgical pain. Methods Mechanical hyperalgesia was determined with an electronic von Frey test in a mouse hindpaw incisional model. Neutrophil accumulation and the level of CXCL1/KC in the plantar tissue were determined by myeloperoxidase activity assay and enzyme-linked immunosorbent assay, respectively. Results An incision in the mouse hindpaw produces long-lasting mechanical hyperalgesia that persists for at least 72 h after surgery. Following surgery, there was an increase in both neutrophil accumulation and the CXCL1/KC level in the incised paws. The depletion of the mouse neutrophils by vinblastine sulphate or anti-neutrophil antibody treatments reduced the mechanical hyperalgesia after paw incision. Furthermore, the treatment of mice with ladarixin, an orally acting CXCR1/2 antagonist, also reduced both the mechanical hyperalgesia and the infiltration of neutrophils in the incised paws. Conclusion In conclusion, it appears that after surgical processes, neutrophils are recruited by CXCL1-CXCR1/2 signalling and participate in the cascade of events, leading to mechanical hyperalgesia. These results suggest that blocking neutrophil migration through the inhibition of CXCL1-CXCR1/2 signalling might be a target to control post-surgical pain. [ABSTRACT FROM AUTHOR]

The activation of inflammatory cells and consequent release of mediators play an important role in the resorption of mineralized tissues. In the present study, we examined the ability of dentin extracts to induce inflammatory cell recruitment and activation. We showed here that dentin extracts triggered an intense cell migration and progressive cell maturation, in a time- and dose-dependent manner. Expression of interleukin-1β (IL-1b), tumor necrosis factor-α (TNF-α), nitric oxide (NO), and hydrogen peroxide (H2O2) was also up-regulated by dentin extracts. These results show that inflammatory events can be elicited in response to dentin, which may suggest a possible involvement of dentin molecules in the inflammatory events, coupled with their release at the root resorption sites. [ABSTRACT FROM AUTHOR]

1 Sepsis induced by S. aureus was used to investigate whether neutrophil migration failure to infectious focus correlates with lethality in Gram-positive bacteria-induced sepsis in mice. 2 By contrast with the sub-lethal (SL-group), the lethal (L-group) intraperitoneal inoculum of S. aureua caused failure of neutrophil migration (92% reduction), high CFU in the exudate, bacteremia and impairment of in vitro neutrophil chemotactic activity. 3 Pre-treatments of L-group with adequate doses of aminoguanidine prevented the neutrophil migration failure and improved the survival of the animals (pre-treated: 43%; untreated: 0% survival). Thus, the impairment of neutrophil migration in the L-group appears to be mediated by nitric oxide (NO). 4 The injection of 5. aureus SL-inoculum in iNOS deficient (- / -) or aminoguanidine-treated wild-type mice (pre- and post-treatment), which did not present neutrophil migration failure, paradoxically caused severe peritonitis and high mortality. This fact is explainable by the lack of NO dependent microbicidal activity in migrated neutrophils. 5 In conclusion, although the NO microbicidal mechanism is active in neutrophils. the failure of their migration to the infectious focus may be responsible for the severity and outcome of sepsis. [ABSTRACT FROM AUTHOR]

Examines the effects of saccharomyces boulardii (S. boulardii) monoassociation on the immune responses of gnotobiotic mice. Evaluation on the effect of S. boulardii on the immune system; Colonization of the gut-free mice; Survival of S.boulardii on the gastrointestinal conditions of the mice.

In the present study, we investigated the role of resident cells in the neutrophil migration induced by ovalbumin (OVA) in immunized rats, OVA administration induced dose-dependent neutrophil migration, which was inhibited by pretreating the animals with dexamethasone, but not with indomethacin or BW 70C. Lymphocytes, but not macrophages or mast cells, obtained from sensitized animals and stimulated in vitro with OVA released a factor that induced neutrophil migration in vivo and in vitro. Both the release of this factor in vitro and the neutrophil migration induced in vivo were inhibited by dexamethasone, thus explaining the inhibitory effect of glucocorticoids on the neutrophil migration induced by OVA in immunized animals. Neither indomethacin nor BW 70C had any such effect. The fact that actinomycin D also inhibited the release of the factor from OVA-stimulated lymphocytes suggests that this substance is of a proteinaceous nature. The importance of lymphocytes for neutrophil recruitment in OVA immunized rats was supported by the fact that homologous lymphocyte transfer into air pouches rendered these cavities responsive to OVA. Lymphocytes obtained from naive rats and stimulated with the lectins concanavalin A (Con A) or phytohaemagglutinin (PHA) were also able to release a factor that induced neutrophil migration in vivo. In vitro incubation of the supernatant from OVA stimulated lymphocytes with antisera to interleukin-1β (IL-1β), IL-8 and tumour necrosis factor-α (TNF-α) did not inhibit the neutrophil chemotactic activity. These data suggest that IL-1β, IL-8 and TNF-α are not involved in the neutrophil chemotactic activity of the supernatant. Overall, these results indicate the importance of lymphocyte participation in neutrophil recruitment during inflammatory immune reactions, through the release of a neutrophil chemotactic factor different from IL-1β, IL-8 and TNF-α. [ABSTRACT FROM AUTHOR]

BALB/c mice injected intraperitoneally with bacterial lipopolysaccharide (LPS) developed lethal septic shock. This was accompanied by significantly elevated concentrations of nitrite and nitrate in the plasma and expression of high levels of nitric oxide (NO) synthase activity in the lungs, heart, spleen and peritoneal macrophages. Mice pretreated with anti-turnout necrosis factor-α (TNF-α) monoclonal antibody or anti-interleukin-1β (IL-1β) polyclonal antibody were protected, in a dosedependent manner, from endotoxin-induced mortality. This effect was accompanied by a significant reduction in plasma levels of nitrite and nitrate. Antibody treatment also reduced the level of NO synthase activity in peritoneal macrophages, spleen and heart but had no effect on enzyme expression in the lung. These results demonstrate that TNF-α and IL-1β play an important role in the induction of NO following administration of LPS and in the development of endotoxin-induced shock. In addition, NO synthase activity is differentially expressed in various organs and this may not always require TNF-α and IL-1β. [ABSTRACT FROM AUTHOR]

Abstract: Ethnopharmacological relevance: Peperomia serpens (Piperaceae), popularly known as “carrapatinho”, is an epiphyte herbaceous liana grown wild on different host trees in the Amazon rainforest. Its leaves are largely used in Brazilian folk medicine to treat inflammation, pain and asthma. Aim of the study: This study investigated the effects of essential oil of Peperomia serpens (EOPs) in standard rodent models of pain and inflammation. Materials and methods: The antinociceptive activity was evaluated using chemical (acetic acid and formalin) and thermal (hot plate) models of nociception in mice whereas the anti-inflammatory activity was evaluated by carrageenan- and dextran-induced paw edema tests in rats croton oil-induced ear edema, as well as cell migration, rolling and adhesion induced by carrageenan in mice. Additionally, phytochemical analysis of the EOPs has been also performed. Results: Chemical composition of the EOPs was analyzed by gas chromatography and mass spectrometry (GC/MS). Twenty-four compounds, representing 89.6% of total oil, were identified. (E)-Nerolidol (38.0%), ledol (27.1%), α-humulene (11.5%), (E)-caryophyllene (4.0%) and α-eudesmol (2.7%) were found to be the major constituents of the oil. Oral pretreatment with EOPs (62.5–500mg/kg) significantly reduced the writhing number evoked by acetic acid injection, with an ED50 value of 188.8mg/kg that was used thereafter in all tests. EOPs had no significant effect on hot plate test but reduced the licking time in both phases of the formalin test, an effect that was not significantly altered by naloxone (0.4mg/kg, s.c.). EOPs inhibited the edema formation induced by carrageenan and dextran in rats. In mice, EOPs inhibited the edema formation by croton oil as well as the leukocyte and neutrophil migration, the rolling and the adhesion of leukocytes. Conclusions: These data show for the first time that EOPs has a significant and peripheral antinociceptive effect that seems unrelated to interaction with the opioid system. EOPs also displays a significant anti-inflammatory effect in acute inflammation models. This effect seems to be related to components which inhibit the production of several inflammatory mediators. These results support the widespread use of Peperomia serpens in popular medicine to treat inflammation and pain. [Copyright &y& Elsevier]

Abstract: This study assessed the effect of the agonist 15d-PGJ2 administered into the rat temporomandibular joint (TMJ) on nociceptive behavioral and the anti-inflammatory potential of this prostaglandin on TMJ. It was observed that 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) significantly reduced formalin-induced nociceptive behavior in a dose dependent manner, however injection of 15d-PGJ2 into the contralateral TMJ failed to reduce such effects. This antinociceptive effect is dependent on peroxisome proliferator-activated receptors-γ (PPAR-γ) since pre-treatment with GW9662 (PPAR-γ receptor antagonist) blocked the antinociceptive effect of 15d-PGJ2 in the TMJ. In addition, the antinociceptive effect of 15d-PGJ2 was also blocked by naloxone suggesting the involvement of peripheral opioids in the process. Confirming this hypothesis pre-treatment with κ, δ, but not μ receptor antagonists significantly reduced the antinociceptive effect of 15d-PGJ2 in the TMJ. Similarly to opioid agonists, the 15d-PGJ2 antinociceptive action depends on the nitric oxide (NO)/guanilate cyclase (cGMP)/ATP-sensitive potassium channel blocker(K+ ATP) channel pathway since it was prevented by the pre-treatment with the inhibitors of nitric oxide synthase (NOS; aminoguanidine), cGMP (ODQ), or the K+ ATP (glibenclamide). In addition, 15d-PGJ2 (100 ng/TMJ) inhibits 5-HT-induced TMJ hypernociception. Besides, TMJ treated with 15d-PGJ2 showed lower vascular permeability, assessed by Evan''s Blue extravasation, and also lower neutrophil migration induced by carrageenan administration. Taken together, these results demonstrate that 15d-PGJ2 has a potential peripheral antinociceptive and anti-inflammatory effect in the TMJ via PPAR-γ activation. The results also suggest that 15d-PGJ2 induced-peripheral antinociceptive response in the TMJ is mediated by κ/δ opioid receptors by the activation of the intracellular l-arginine/NO/cGMP/K+ ATP channel pathway. The pharmacological properties of the peripheral administration of 15d-PGJ2 highlight the potential use of this PPAR-γ agonist on TMJ inflammatory pain conditions. [Copyright &y& Elsevier]

Abstract: Aim of the study: The latex of Calotropis procera has been used in the traditional medicinal system for the treatment of leprosy, ulcers, tumors, piles and diseases of liver, spleen, abdomen and toothache. It comprises of a non-dialyzable protein fraction (LP) that exhibits anti-inflammatory properties and a dialyzable fraction (DF) exhibiting pro-inflammatory properties. The present study was carried out to evaluate the effect of LP sub-fractions on neutrophil functions and nociception in rodent models and to elucidate the mediatory role of nitric oxide (NO). Material and methods: The LP was subjected to ion exchange chromatography and the effect of its three sub-fractions (LPPI, LPPII and LPPIII) thus obtained was evaluated on leukocyte functions in the rat peritonitis model and on nociception in the mouse model. Results: LP sub-fractions exhibit distinct protein profile and produce a significant decrease in the carrageenan and DF induced neutrophil influx and exhibit anti-nociceptive property. The LP and its sub-fractions produced a marked reduction in the number of rolling and adherent leukocytes in the mesenteric microvasculature as revealed by intravital microscopy. The anti-inflammatory effect of LPPI, the most potent anti-inflammatory fraction of LP, was accompanied by an increase in the serum levels of NO. Further, our study shows that NO is also involved in the inhibitory effect of LPPI on neutrophil influx. Conclusions: Our study shows that LP fraction of Calotropis procera comprises of three distinct sets of proteins exhibiting anti-inflammatory and anti-nociceptive properties of which LPPI was most potent in inhibiting neutrophil functions and its effects are mediated through NO production. [Copyright &y& Elsevier]