Diabetes and obesity are associated with inflammation and activation of the immune system with infiltration of adipose tissue by macrophages. This is mainly studied in adipose tissue, with limited information to clarify immune-skeletal muscle interactions in these conditions. We show that exposure of L6 rat skeletal muscle cells to saturated fatty acid palmitate results in insulin resistance, activation of inflammatory pathways, upregulation of pro-inflammatory cytokine and chemokine gene expression and secretion. We identified monocyte chemoattractant protein-1 [MCP-1] as the main factor responsible for macrophage attraction, as blocking it reduced macrophage migration to muscle cells. When macrophages are exposed to palmitate, a similar response ensues with production of macrophage chemoattractants and activation of inflammatory pathways and gene expression profiles, and secretion of multiple cytokines. Our work identifies MCP-1 chemokine produced in response to palmitate treatment by both muscle cells and macrophages and provides a potential link in immune-metabolic crosstalk in diabetogenic environment.

Inflammation is an ubiquitous response of the body to cellular damage and injury. It not only leads to increased production of inflammatory molecules such as prostaglandins, bradykinins, histamine, and nerve growth factor (NGF) that sensitize the neurons, but is also associated with acidosis due to a local physiological decrease in extracellular pH, to as low as 5.3. The addition of protons to the sensitized area results in increased membrane conductance and depolarization in sensory neurons involving the acid sensing ion channels (ASICs), the capsaicin sensitive transient receptor potential vanilloid type 1 receptor (TRPV1), and blockade of background potassium conductance. Collectively, this combination heightens the pain state. As expression and activation of the NGF receptor tropomyosin-related kinase A (TrkA) are critically linked to inflammation-associated nociceptor sensitization, this led us to hypothesize that decreased pH may contribute to this process by depolarizing the neuron and mobilizing more TrkA to the cell membrane. This hypothesis is premised by previous experiments in other neuronal populations demonstrating that increased neuronal activity mobilizes more of the neurotrophin receptor TrkB to the membrane. We explored this question utilizing an in vitro model of acidosis akin to that observed with inflammation. Primary sensory neurons were grown on coverslips at a physiological pH of 7.4. The pH of the media was decreased to an acidic pH of 6.5 in half of the cultures for 30 minutes, followed by fixation under permeablizing or non-permeablizing conditions. Immunocytochemical analysis revealed a significant increase in the mobilization of TrkA to the plasma membrane in response to acidosis (confirmed using a biotinylation assay) and an enhanced level of TrkA activation in response to brief NGF challenge. This rapid mobilization of TrkA was attenuated with the addition of proton-sensitive channel blockers capsazepine and amiloride, for the TRPV and ASIC channels respectively. Unexpectedly, the amount of activated TrkA was also increased at pH 6.5 in the absence of NGF challenge. Taken together, the data suggests that sensory neurons can be rapidly sensitized to NGF in response to a decrease in pH and as such likely plays a role in the sensitization and hyperalgesia associated with an inflammatory state.

The tumour suppressor p27kip1 (p27) is a potent inhibitor of cell growth and proliferation. We identified NF-κB activator, IKKα, as a novel interacting partner of p27 in a protein microarray screen. Both the IKKα and IKKβ components of the IKK complex were mapped to the C-terminal domain of p27. To investigate the physiological function of the p27-IKK interaction, we employed a well-established model of LPS-induced sepsis which is known to activate the IKK/NF-κB pathway. Lentivirally-mediated overexpression of p27 blocked LPS activation of NF-κB. Furthermore, in LPS-injected animals transduced with TAT-p27, a significant improvement in the left ventricular function of the heart was observed. TAT-p27 treatment was also shown to attenuate the endotoxin effect and significantly improve survival compared to both saline and TAT-LacZ controls. Our results indicate that p27 attenuates inflammation, possibly through inhibiting the IKK-dependent activation of NF-κB, thus supporting a novel link between both cell cycle regulation and inflammation.

Inflammation can be up-regulated by microglia and macrophages through the release of pro-inflammatory cytokines such as tumour necrosis factor-alpha (TNF-α). Excess production of TNF-α can lead to a variety of diseases and even tissue necrosis. Recently, the expression of alpha seven acetylcholine receptors (α7AChR) by microglia have been shown to decrease the amount of TNF-α released. This anti-inflammatory pathway has been studied extensively where researchers are able to reduce TNF-α concentration through α7AChR expression and increases in the concentration of its ligand. I have shown that Neuregulin is able to increase the expression of α7AChR in microglia and macrophages. Using three immortalized cell lines, BV-2, EOC-20 and RAW 264.7, and primary microglial cells harvest from mice I investigated the role that neuregulin plays in the anti-inflammatory process. Neuregulin signals through the ErbB receptors, a family of tyrosine kinase receptors, to facilitate the effects on ACh expression. My results show that ErbB4 is expressed in BV-2, EOC-20 and RAW 264.7 cell lines while ErbB2-4 receptors are expressed in primary microglia. As well, I was able to show that ErbB4 became phosphorylated upon binding to NRG in immortalized cell lines. Using an Enzyme Linked Immunsorbent Assay to analyze TNF- α concentration in microglia and macrophages, I was able to demonstrate that increased levels of α7AChRs did not result in a reduction in TNF-α concentration. These results showed that NRG is able to increase α7AChRs in microglia and macrophages after the phosphorylation of the ErbB4 receptors. As well, this increase in α7AChR does not relate to a reduction in TNF-α, thus under these experimental conditions does not have an effect on the anti-inflammatory pathway.

Inflammation is an ubiquitous response of the body to cellular damage and injury. It not only leads to increased production of inflammatory molecules such as prostaglandins, bradykinins, histamine, and nerve growth factor (NGF) that sensitize the neurons, but is also associated with acidosis due to a local physiological decrease in extracellular pH, to as low as 5.3. The addition of protons to the sensitized area results in increased membrane conductance and depolarization in sensory neurons involving the acid sensing ion channels (ASICs), the capsaicin sensitive transient receptor potential vanilloid type 1 receptor (TRPV1), and blockade of background potassium conductance. Collectively, this combination heightens the pain state. As expression and activation of the NGF receptor tropomyosin-related kinase A (TrkA) are critically linked to inflammation-associated nociceptor sensitization, this led us to hypothesize that decreased pH may contribute to this process by depolarizing the neuron and mobilizing more TrkA to the cell membrane. This hypothesis is premised by previous experiments in other neuronal populations demonstrating that increased neuronal activity mobilizes more of the neurotrophin receptor TrkB to the membrane. We explored this question utilizing an in vitro model of acidosis akin to that observed with inflammation. Primary sensory neurons were grown on coverslips at a physiological pH of 7.4. The pH of the media was decreased to an acidic pH of 6.5 in half of the cultures for 30 minutes, followed by fixation under permeablizing or non-permeablizing conditions. Immunocytochemical analysis revealed a significant increase in the mobilization of TrkA to the plasma membrane in response to acidosis (confirmed using a biotinylation assay) and an enhanced level of TrkA activation in response to brief NGF challenge. This rapid mobilization of TrkA was attenuated with the addition of proton-sensitive channel blockers capsazepine and amiloride, for the TRPV and ASIC channels respectively. Unexpectedly, the amount of activated TrkA was also increased at pH 6.5 in the absence of NGF challenge. Taken together, the data suggests that sensory neurons can be rapidly sensitized to NGF in response to a decrease in pH and as such likely plays a role in the sensitization and hyperalgesia associated with an inflammatory state.

Inflammation can be up-regulated by microglia and macrophages through the release of pro-inflammatory cytokines such as tumour necrosis factor-alpha (TNF-α). Excess production of TNF-α can lead to a variety of diseases and even tissue necrosis. Recently, the expression of alpha seven acetylcholine receptors (α7AChR) by microglia have been shown to decrease the amount of TNF-α released. This anti-inflammatory pathway has been studied extensively where researchers are able to reduce TNF-α concentration through α7AChR expression and increases in the concentration of its ligand. I have shown that Neuregulin is able to increase the expression of α7AChR in microglia and macrophages. Using three immortalized cell lines, BV-2, EOC-20 and RAW 264.7, and primary microglial cells harvest from mice I investigated the role that neuregulin plays in the anti-inflammatory process. Neuregulin signals through the ErbB receptors, a family of tyrosine kinase receptors, to facilitate the effects on ACh expression. My results show that ErbB4 is expressed in BV-2, EOC-20 and RAW 264.7 cell lines while ErbB2-4 receptors are expressed in primary microglia. As well, I was able to show that ErbB4 became phosphorylated upon binding to NRG in immortalized cell lines. Using an Enzyme Linked Immunsorbent Assay to analyze TNF- α concentration in microglia and macrophages, I was able to demonstrate that increased levels of α7AChRs did not result in a reduction in TNF-α concentration. These results showed that NRG is able to increase α7AChRs in microglia and macrophages after the phosphorylation of the ErbB4 receptors. As well, this increase in α7AChR does not relate to a reduction in TNF-α, thus under these experimental conditions does not have an effect on the anti-inflammatory pathway.

Mastitis is the most prevalent and major cause of economic losses in dairy farms. Bovine mastitis caused by strains of <i>S. aureus</i> is a major economically important disease affecting the dairy industry worldwide. <i>S. aureus</i> is one of the most common udder pathogens that cause either clinical or sub-clinical mammary gland infections. Different treatment regimes have failed to cure <i>S. aureus</i> intramammary infections. Most mastitis vaccination strategies have focused on the enhancement of systemic humoral immunity rather than strengthening local intramammary immunity. Vaccines aimed at enhancing intramammary immunity of dairy cows against <i>S. aureus</i> mastitis have had limited success. Commercially available vaccines show various degrees of success and work in research laboratories with experimental vaccines suggest that in part, the failure of these vaccines lies in the limited antigenic repertoire contained in the vaccine formulations. Moreover, not only does variation in the antigenic composition but also presence of capsular polysaccharide in most pathogenic strains and decreased activity of immune effectors in milk affect the success of vaccines. In addition to these, the ability of <i>S. aureus</i> to attach and internalize into mammary epithelial cells, enables bacteria to escape from the effect of immunity and antibiotics by being hidden in the intracellular niche and thereby causing chronic recurrent intramammary infection. <i>S. aureus</i> also has the ability to become electron-transport-defective and to form slow-growing small colonies that are non haemolytic and less virulent. These small colony variants might hide from the immune surveillance in the intracellular area and revert to the parental strain causing chronic recurrent infections. If immunization targets antigenic molecules that are conserved throughout all pathogenic strains, even the small colony variants can be controlled since the immune system will clear the parental strain which causes lethal infection. Thus, immunization trials should focus on conserved immunogenic antigen molecules among pathogenic strains formulated with an adjuvant and delivered by a route of immunization to induce maximum stimulation of the immune system. Moreover, immunization should focus on inducing Th1 responses, which is protective against <i>S. aureus</i> mastitis. It has been reported that proteins with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity might be used as such antigens to induce protection against parasitic and microbial infections. Previous study in our laboratory on mastitis-causing streptococci indicates that GapC proteins of <i>S. uberis</i> and <i>S. dysgalactiae</i> have potential as vaccine antigens to protect dairy cows against mastitis caused by environmental streptococci. Two conserved cell wall associated proteins with iii glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, GapB and GapC have been identified from <i>S. aureus</i> isolates from bovine intramammary infections. The overall goal of this study was to improve our understanding on intramammary immunity using the GapC and GapB proteins of <i>S. aureus</i> as model antigens for mastitis and to determine the regulation of expression of <i>gapB</i> and <i>gapC</i> genes and their roles in the pathogenesis of bovine <i>S. aureus</i> mastitis. We hypothesized that strengthening local intramammary immunity using GapB and GapC proteins of <i>S. aureus</i> as antigens will protect against bovine <i>S. aureus</i> mastitis. To test this hypothesis we took the approach of using the <i>gapB</i> and <i>gapC</i> genes and constructed plasmids encoding GapB, GapC and GapB::GapC (GapC/B) chimeric proteins. We set six objectives to test our hypothesis using these proteins to enhance the intramammary immunity. In aim 1 we constructed plasmids encoding the GapB, GapC proteins and also constructed a chimeric gene encoding the GapC and GapB proteins as a single entity (GapC/B chimera) as the basis for a multivalent vaccine. In this objective the humoral and cellular immune responses to GapC/B were compared to the responses to the individual proteins alone or in combination in C57 BL/6 mice. Our results showed that the GapC/B protein elicited strong humoral and cellular immune responses as judged by the levels of total IgG, IgG1, IgG2a, IL-4 and IFN-ã secretion and lymphocyte proliferation. These results strongly suggest the potential of this chimeric protein as a target for vaccine production to control mastitis caused by <i>S. aureus</i>. In aim 2 we continued our studies on GapC/B by testing the effects of DNA vaccination with plasmids encoding the individual gapB and gapC genes as well as the gapC/B protein gene with or without a boost with the recombinant proteins. The results showed that DNA vaccination alone was unable to elicit a significant humoral response and barely able to elicit a detectable cell-mediated response to the recombinant antigens but subsequent immunization with the proteins elicited an excellent response. In addition, we found that DNA vaccination using a plasmid encoding the GapC/B chimera followed by a boost with the same protein, although successful, is less effective than priming with plasmids encoding GapB or GapC followed by a boost with the individual antigens. In aim 3 we optimized immune responses in cows by comparing route of vaccination (subcutaneous versus intradermal), site of vaccination (locally at the area drained by the supramammary lymph node versus distantly at area drained by parotid lymph node. Our results showed that both subcutaneous and intradermal immunizations with the GapC/B protein at the area drained by the supramammary and parotid lymph nodes resulted in significantly increased serum and milk titers of total IgG, IgG1, IgG2, iv and IgA in all vaccinated groups as compared to placebo. The anti-GapC/B IgG1 serum and milk titers were significantly higher in all vaccinated group as compared to the placebo group. These results indicated that vaccination at the area drained by the supramammary lymph node resulted in better immune responses. In aim 4 we tested different formulations of the GapC/B antigen with adjuvants such as PCPP, CpG, PCPP + CpG and VSA-3. We found that the VSA-3 formulation induced the best immune responses in cows. In this objective we also monitored immune responses longitudinally over one lactation cycle to determine the duration of immune responses by measuring IgG, IgG1, IgG2, and IgA on monthly blood and milk samples. We found that the duration of immune responses was about four months. In aim 5 we tested the role of GapC in the virulence of <i>S. aureus</i> mastitis using the <i>S. aureus</i> wild type strain RN6390 and its isogenic GapC mutant strain H330. Our results from both in vitro adhesion and invasion assays on MAC- T cells and in vivo infection of ovine mammary glands showed that GapC is an important virulence factor in <i>S. aureus</i> mastitis. In aim 6 we examined the role of sar and agr loci on the expression of <i>gapC</i> and <i>gapB</i> genes by qRT- PCR using <i>S. aureus</i> RN6390 and its isogenic mutants defective in agrA, sarA and sar/agr (double mutant) at exponential and stationary phases of growth. Our results showed that both <i>gapB</i> and <i>gapC</i> expression were down regulated in the mutant strains, indicating that the expression of the <i>gapB</i> and <i>gapC</i> genes is controlled by the universal virulence gene regulators, agr and sar. We also checked the role of environmental factors such as pH, growth media, and oxygen tension on the expression of <i>gapB</i> and <i>gapC</i> using q-RT-PCR. Our results showed that the expression of <i>gapB</i> and <i>gapC</i> genes in different strains of <i>S. aureus</i> was not consistent under the above-mentioned environmental conditions.

Glu-Leu-Arg (ELR)-CXC chemokine-mediated neutrophil migration and activation plays a key role in many inflammatory diseases. Dysregulated neutrophil activation often leads to inflammatory responses such as acute lung injury (ALI) or acute respiratory distress syndrome (ARDS).<p> Previously, we generated a bovine drug (i.e., bovine CXCL8(3-74)K11R/G31P, bG31P) by mutating the first two amino acids at the beginning of the N-terminus of bovine CXCL8/IL-8 and later substituting Arg for Lys11 and Pro for Gly31. Bovine G31P was shown to be a highly effective ELR-CXC chemokine and neutrophil antagonist in cattle & guinea pigs, but a human equivalent thereof would be of significantly more use in human medicine. Published studies on the structure and function of human CXCL8 suggest that human CXCL8(3-72)K11R/G31P (i.e., hG31P) would not be a particularly effective chemokine antagonist. Thus, development of a humanized form of bG31P became a primary goal. I first examined the effect of wholesale ligation of the carboxy half of hCXCL8 onto the amino half of bG31P and generated a human-bovine chimeric G31P (hbG31P; i.e., bCXCL8(3-44)K11R/G31P-hCXCL8(45-72)). I also made substitutions at each remaining human-discrepant amino acid (i.e., T3K, H13Y, T15K, E35A, and S37T) within the 5 half of the hbG31P cDNA. The results showed that hbG31P and its analogues blocked CXCL8-induced human neutrophil chemotactic responses, reactive oxygen intermediate (ROI) release, and intracellular calcium flux. Humanized bovine G31P was also shown to significantly block pulmonary neutrophilic pathology in a guinea pig model of airway endotoxemia.<p> As bG31P, hbG31P and its further humanized forms showed essentially equivalent ELR-CXC chemokine antagonist activity, Dr. Fang Li, Ms Jennifer Town and I then generated a fully human form of bG31P, hG31P. <i>In vitro</i>, hG31P was shown to effectively inhibit CXCL-1-, -5-, and -8-induced neutrophil chemotactic responses, intracellular Ca2+ flux, and ROI release. Human G31P also desensitized heterologous G protein-coupled receptors (GPCR) including bacterial peptides (e.g., N-formyl-methionine-leucine-phenylalanine, fMLP), anaphylatoxin (e.g., complement 5a, C5a), lipid mediators (e.g., leukotriene B4, LTB4; platelet-activating factor, PAF) receptors. Moreover, hG31P, in a dose-dependent manner suppressed CXCL1 and CXCL8 expression by LPS-challenged airway epithelial cells and reversed the anti-apoptotic influence of ELR-CXC chemokines on neutrophils. <i>In vivo</i>, hG31P was significantly effective in blocking the pathology associated with airway endotoxemia, aspiration pneumonia, and intestinal ischemia and reperfusion injury, including neutrophil recruitment (70-95% reduction) into, and activation within, the airways or gut, chemokine or cytokine expression, and pulmonary vascular complications. The blockade of neutrophil recruitment by hG31P in aspiration pneumonia animals did not increase airway bacterial growth. The G31P treatment was protective in both mesenteric (i.e., local) and remote organ injury. These findings suggest that hG31P is not only a potent neutrophil antagonist, but an effective blocker of other inflammatory responses. These comprehensive anti-inflammatory effects indicate that hG31P could potentially provide a viable therapeutic approach for inflammatory diseases such as ALI /ARDS.

Background The mechanism by which allergies trigger asthma occurs through the interaction of antigen, IgE and the FcεR1 receptor on mast cells resulting in the release of mediators that exert their effects on various surrounding tissues causing bronchoconstriction, plasma exudation and mucus hypersecretion. The response is usually maximal within 30 minutes and resolves spontaneously within two hours. At least half of the individuals who exhibit this so called early response also manifest a late response which is a subsequent episode of bronchoconstriction that is usually maximal around six hours following exposure and involves airway inflammation. Montelukast has proven efficacious in the management of asthma and desloratadine is effective in the treatment of allergic rhinitis and chronic idiopathic urticaria. Since the early response involves the actions of multiple mediators, including histamine and the leukotrienes, the question of whether concurrent mediator blockade would be superior to either agent alone was raised. Additionally, the recent evidence supporting anti-inflammatory activity for these agents suggested potential efficacy against the late airway response. Methods Two double-blind, randomized, placebo-controlled, 4-way crossover allergen inhalation challenge investigations were conducted in twenty (10 per investigation) mild atopic asthmatics. The early response investigation involved the administration of either 5 mg desloratadine, 10 mg montelukast, the combination , or placebo (Vitamin B1) at 26 hours and 2 hours prior to allergen inhalation. The late response investigation involved single dose administration of each agent, alone or in combination, 2 hours prior to allergen inhalation. Measurements of changes in airway responsiveness and inflammation were also conducted. Results The early response was significantly inhibited by montelukast and the combination. Desloratadine did not differ from placebo. The late response was significantly decreased by desloratadine and montelukast and completely blocked with the combination. Desloratadine decreased sputum eosinophils at 7 hours, montelukast at 24 hours, and the combination at both time points. Airway responsiveness to methacholine trended lower with montelukast and the combination. Montelukast was the only treatment to significantly decrease exhaled nitric oxide levels. Conclusion The combination of desloratadine and montelukast provides inhibition that is superior to both monotherapies on the early and the late airway responses to inhaled allergen in people with mild atopic asthma.

In mammals, the neurogenic inflammatory response can be induced by stimulation or activation on the peripheral sensory C-fibers to release neuropeptides from the peripheral terminals, at the same time their afferent functions are enhanced. There are several neuropeptides stored and released from peripheral terminals of the afferent fibers, such as substance P (SP), neurokinin A, and calcitonin gene related peptide (CGRP). SP is one of the major inflammatory mediators of neurogenic inflammation that can act on neurokinin-1 receptors on smooth muscles and endothelial cells of blood vessels, causing vasodilatation, endothelial gap formation, and local plasma leakage. There are many studies and reports indicate that animals pretreated with a short period non-lethal hyperthermia can induce heat shock response and activate the expression of a group of inducible proteins called heat shock proteins (HSPs), and this stress response reduces the injury by same or other following stresses. In this study, the hyperthermia treatment (HT) was implemented by 42¢J hot water bath and the core body temperature of anesthetized rat was elevated and maintained around 42.0¡Ó0.5¢J for 15 min, and the normothermia control treatment (NT) was implemented by 37¢J warm water bath with the same period. 24 hours after NT or HT, the neurogenic plasma leakage was induced by intravascular injection with capsaicin (90 £gg/kg), SP (3 £gg/kg), or electrical stimulation on the right thoracic vagus nerve. The blood pressures of each animal were continually recorded during the neurogenic inflammation induction or sham operation. The amount of neurogenic inflammation of airway was evaluated by the area density leaky blood vessels. The leaking vessels were labeled with India ink and quantitative analysis by morphometric method. Plasma leakage was also measured by interstitial Evans blue concentration. The results indicated that HT could reduce plasma leakage and hypotension of the neurogenic inflammation that induced by capsaicin, SP or electrical stimulation on vagus nerve. Animals pretreated with aminoguanidine (a selective inhibitor of iNOS) had no significant effect on the neurogenic inflammation by following systemic SP infusion, but that could eliminate the anti-neurogenic inflammatory effect of HT. Animal applied with diphenhydramine (an antagonist of histamine H1 receptor) could attenuate the neurogenic inflammation by following systemic SP infusion, and HT could attenuate the neurogenic inflammation that with or without H1 receptor antagonist. This result indicates that NO synthesis and the activity of iNOS have few effects on neurogenic inflammation of airway, but it plays a critical factor on the initiation of heat shock response. The neurogenic inflammation induced by SP not only direct act on blood vessels but have other indirect effect by the histamine H1 receptor to enhance inflammation. Neonatal rats received high dose capsaicin treatment would induce irreversible sensory C-fiber denervation. The adult rats that were neonatally treated with capsaicin showed a more serious inflammatory response to systemic SP infusion as compared with animals neonatally treated with vehicle. HT still had the anti-inflammatory effects on the neurogenic inflammation that induced by SP. The results indicated that animals with sensory C-fiber denervation might conserve their neurogenic inflammatory responses and were hypersensitive to SP. In conclusion, the HT could attenuate the neurogenic inflammation that induced by different drugs or methods, and the anti-inflammatory effects were correlated with the increase in HSP72 expression. In the neurogenic inflammation induced by SP, the activation of histamine H1 receptors may enhance inflammation, but the activity of endogenous iNOS was less effective.