Total Number Of Inflammatory Cells Research Articles

Policy Forum: Putting People First: Services and Supports for People with Developmental Disabilities

### Objectives The implantable biomaterial devices are used for long term performance for the replacement of damaged or diseased tissues, especially for vascular grafts and artificial skin. Due to the rising number of patients with cardiovascular diseases (CVD) and the shortage of suitable autologous vessels, the demand for clinically effective synthetic small diameter vascular grafts is increasing. Improving biocompatibility of biomaterial is the goal of tissue engineering scientists attempt to minimise inflammatory responses into tolerance. Silk and Polyurethane (PU) are currently being trailed in the animal experiment as potential biomaterial used in different implantable medical device. Interleukin 1 beta (IL-1β) luciferase mouses are generated by incorporating the firefly luciferase gene driven by a 4.5-kb fragment of human IL-1β gene promoter (cHS4l-hIL-1β). In this study, IL-1β transgenic mice have been used to determine the IL-1β production after biomaterial implantation. The aim of this study is determine host responses to Silk or PU implants ± coating recombinant human tropoelastin (rhTE) in transgenic IL-1β mouse model. ### Methods The back of each IL-1β mouse (n = 18) was subcutaneously implanted with received 4 different treated implants, control Silk, rhTE-coated Silk, control PU and rhTE-coated PU implants. The in vivo luciferase signal of IL-1β expression in each mouse was imaged daily for 5 days post-surgery, and then 9mice were euthanised at 10 days (10D) and 3 weeks (3W) post-surgery. The implants were harvested and stained with H&E and Milligan's trichrome to assess inflammatory cell infiltration and collagen depositions. Performing immunohistochemistry stains, Von Willebrand factor (vWF) for neovascularisation, F480 for macrophage, Ly-6G and Ly-6C for neutrophil and proliferation maker Ki67 for proliferating cells. ### Results Significantly reduced the levels of IL-1β production was observed in rhTE-coated Silk implants compared to control Silk implants at 2 and 3days post-surgery. Comparing to control implants, less number of total inflammatory cells were found in rhTE-coated Silk (∼47% 3W) and rhTE-coated PU (∼54.64% 10D). Also a reduction of neovascularisation (∼43% 3w) and less proliferating inflammatory cells (∼58% 3W) were observed in rhTE-coated Silk. ### Conclusions Our results suggested that rhTE coating improves biocompatibility of Silk and PU, reducing inflammatory responses.

Celastrol suppresses allergen-induced airway inflammation in a mouse allergic asthma model

Celastrol has anti-inflammatory and immunomodulatory activities, but its anti-allergic effects remain poorly understood. Therefore, we aimed to investigate the ability of celastrol to inhibit asthmatic reactions in a mouse allergic asthma model. BALB/c mice were sensitized and challenged with ovalbumin to induce asthma. We measured the recruitment of inflammatory cells into the bronchoalveolar lavage fluid or lung tissues by Diff-Quik and hematoxylin and eosin staining, respectively, goblet cell hyperplasia by periodic acid-Schiff (PAS) staining, airway hyperresponsiveness by Flexvent system, mRNA and protein expression of cytokines, matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) by reverse transcriptase polymerase chain reaction and ELISA, respectively, and the activities of mitogen-activated protein (MAP) kinases and nuclear factor-kappa B (NF-κB) in the bronchoalveolar lavage cells and lung tissues by Western blot and electrophoretic mobility shift assay (EMSA), respectively. Celastrol reduced the total number of inflammatory cells in the bronchoalveolar lavage fluid and in peribronchial areas, and decreased the airway hyperresponsiveness, mRNA and protein expression levels for inflammatory cytokines such as interleukin (IL)-4, IL-13, TNF-α and IFN-γ, and for MMPs and TIMPs, MAP kinases and NF-κB activities in the bronchoalveolar lavage cells and in the lung tissues increased in ovalbumin-induced allergic asthma in mice. Our data suggest that oral administration of celastrol suppresses ovalbumin-induced airway inflammation, hyperresponsiveness, and tissue remodeling by regulating the imbalance of MMP-2/-9 and TIMP-1/-2 by inflammatory cytokines via MAP kinases/NF-κB in inflammatory cells. Based on our findings, we suggest that celastrol may be used as a therapeutic agent for allergy-induced asthma.

An increased proportion of inflammatory cells express tumor necrosis factor alpha in idiopathic achalasia of the esophagus

Achalasia is a motility disorder characterized by the absence of coordinated peristalsis and incomplete relaxation of the lower esophageal sphincter. The etiology remains unclear although dense inflammatory infiltrates within the myenteric plexus have been described. The nature of these infiltrating cells is unknown. The aim of this study was to evaluate the expression of proinflammatory cytokines - namely, tumor necrosis factor alpha and interleukin-2 - in the distal esophageal muscle in patients with achalasia. Lower esophageal sphincter muscle from eight patients undergoing myotomy or esophagectomy for achalasia of the esophagus were obtained at the time of surgery. Control specimens consisted of similar muscle taken from eight patients undergoing operation for cancer or Barrett's esophagus. The expression of tumor necrosis factor alpha and interleukin-2 were assessed by immunohistochemistry. The total number of inflammatory cells within the myenteric plexus were counted in five high power fields. The percentage of infiltrating cells expressing tumor necrosis factor alpha or interleukin-2 was calculated. Clinical data including demographics, preoperative lower esophageal sphincter pressure, duration of symptoms, and dysphagia score (1 = no dysphagia to 5 = dysphagia to saliva) were obtained through electronic medical records. Statistical comparisons between the groups were made using the unpaired t-test, Fisher's exact test, or Mann-Whitney U test, with a two-tailed P-value less than 0.05 being considered significant. The total number of inflammatory cells was found to be similar between the groups. A significantly higher proportion of inflammatory cells expressed tumor necrosis factor alpha in achalasia as compared with controls (22 vs. 11%; P= 0.02). A similar percentage of infiltrating cells expressed interleukin-2 (40 vs. 41%; P= 0.87). Age, gender, preoperative lower esophageal sphincter pressure, or dysphagia score were not correlated to expression of these cytokines. There was, however, a significant inverse correlation between duration of symptoms and the proportion of inflammatory cells expressing tumor necrosis factor alpha in achalasia (P= 0.007). In conclusion, a higher proportion of infiltrating inflammatory cells expressed tumor necrosis factor alpha in achalasia. Furthermore, this proportion appears to be highest early in the disease process. Further studies are required to more clearly delineate the role of tumor necrosis factor alpha in the pathogenesis of this idiopathic disease.

SH2‐Bβ expression in alveolar macrophages in BAL fluid of asthmatic guinea pigs and its role in NGF–TrkA‐mediated asthma

Nerve growth factor (NGF)/tyrosine kinase receptor A (TrkA) signalling may play an important role in the pathogenesis of asthma, and SH2-B beta, a TrkA-binding protein, modulates the NGF signalling pathway. In this study, SH2-B beta expression in alveolar macrophages (AM) in guinea pig BAL fluid and its role in asthma pathogenesis through the NGF-TrkA signalling pathway were investigated. Guinea pigs were randomized into five groups: control, a model of asthma, anti-SH2-B beta antibody treatment, anti-NGF antibody treatment and anti-TrkA antibody treatment. The asthmatic model was established in guinea pigs by inhalation of ovalbumin. Specific anti-SH2-B beta, anti-NGF and anti-TrkA antibodies were administered and AM were isolated from BAL fluid to assess SH2-B beta expression using an immunofluorescence assay. SH2-B beta and TrkA protein expression were determined by western blotting, IL-1 beta and IL-4 levels in the BAL fluid supernatants were determined by ELISA, and pathological changes in the bronchi and lung tissues were examined by HE staining. Lymphocyte, eosinophil and total inflammatory cell numbers in BAL fluid were significantly higher in the asthma model group than in the other groups (P < 0.01). NGF expression in the asthma model group was significantly higher than that in the PBS control group (P < 0.01). SH2-B beta was expressed in AM of control animals and expression was significantly higher in the asthma model than in the other groups (P < 0.01). TrkA protein expression was significantly higher in the asthma model group than in the PBS group (P < 0.01), and treatment with anti-NGF antibody resulted in significant reduction of TrkA expression (P < 0.01). SH2-B beta is expressed in AM of normal guinea pigs, and SH2-B beta may participate in asthma pathogenesis through the NGF-TrkA signalling pathway.

The inhaled Rho kinase inhibitor Y-27632 protects against allergen-induced acute bronchoconstriction, airway hyperresponsiveness, and inflammation

Recently, we have shown that allergen-induced airway hyperresponsiveness (AHR) after the early (EAR) and late (LAR) asthmatic reaction in guinea pigs could be reversed acutely by inhalation of the Rho kinase inhibitor Y-27632. The present study addresses the effects of pretreatment with inhaled Y-27632 on the severity of the allergen-induced EAR and LAR, the development of AHR after these reactions, and airway inflammation. Using permanently instrumented and unrestrained ovalbumin (OA)-sensitized guinea pigs, single OA challenge-induced EAR and LAR, expressed as area under the lung function (pleural pressure, P(pl)) time-response curve, were measured, and histamine PC(100) (provocation concentration causing a 100% increase of P(pl)) values were assessed 24 h before, and at 6 and 24 h after, the OA challenge (after the EAR and LAR, respectively). Thirty minutes before and 8 h after OA challenge, saline or Y-27632 (5 mM) was nebulized. After the last PC(100) value, bronchoalveolar lavage (BAL) was performed, and the inflammatory cell profile was determined. It was demonstrated that inhalation of Y-27632 before allergen challenge markedly reduced the immediate allergen-induced peak rise in P(pl), without significantly reducing the overall EAR and LAR. Also, pretreatment with Y-27632 considerably protected against the development of AHR after the EAR and fully prevented AHR after the LAR. These effects could not be explained by a direct effect of Y-27632 on the histamine responsiveness, because of the short duration of the acute bronchoprotection of Y-27632 (<90 min). In addition, Y-27632 reduced the number of total inflammatory cells, eosinophils, macrophages, and neutrophils recovered from the BAL. Altogether, inhaled Y-27632 protects against acute allergen-induced bronchoconstriction, development of AHR after the EAR and LAR, and airway inflammation in an established guinea pig model of allergic asthma.

Effect of Topical Cyclosporin A on Herpetic Stromal Keratitis in a Mouse Model

To study the effect of topical cyclosporin A on herpetic stromal keratitis (HSK) in a mouse model. The corneas of BALB/c mice were infected with herpes simplex type 1 virus. The mice were divided into 4 groups according to topical treatment methods: vehicle solution, 0.01% cyclosporin A, 0.1% cyclosporin A, and 1% cyclosporin A. The severity of stromal keratitis was graded clinically at 3, 7, 10, and 14 days after treatment. Histologic and immunohistochemical examinations were performed to analyze infiltrating inflammatory cells and T lymphocytes at 14 days after treatment. Flow cytometry was performed to count CD4 T cells at 14 days after treatment. On day 3 after treatment, there were no significant differences in mean severity scores of HSK between the vehicle and cyclosporin A-treated groups. On days 7, 10, and 14, the severity scores in the groups treated with 0.1% or 1% cyclosporin A decreased significantly compared with the vehicle group (P < 0.05). However, the scores in the group treated with 0.01% cyclosporin A did not change up to 14 days after treatment. By histologic and immunohistochemical analysis, a significant decrease in the total number of inflammatory cells and T lymphocytes was detected in the groups treated with 0.1% or 1% cyclosporin A. Flow cytometry also showed similar findings. Topical cyclosporin A effectively reduces stromal haze and inflammation in experimental HSK, and cyclosporin A eyedrops with a >0.1% concentration can be used for the treatment of HSK.

Physalis angulata extract exerts anti-inflammatory effects in rats by inhibiting different pathways

Physalis angulata is a popular medicine used in Brazil due to its anti-inflammatory effects, but the pharmacological mechanisms underlying these actions remain to be better understood. In the present work, lyophilized aqueous extract from the roots of Physalis angulata Linneu (AEPa) was used to control the inflammatory response induced by the injection of 1% carrageenan into subcutaneous rat's air pouches. Adenosine deaminase (ADA) activity, nitrite level, and prostaglandin E 2 (PGE 2) level were used to evaluate the action of inflammatory mediators. Tumor growth factor-β (TGF-β) level was used as a bioindicator of immunomodulatory response. Rats were injected with vehicle, indomethacin, or AEPa (0.5 mg/kg, 1 mg/kg, and 5 mg/kg i.p.), 1 h before carrageenan administration. AEPa at 0.5 mg/kg had no effect. However, 1 mg/kg of AEPa showed significant anti-inflammatory effects, decreasing exudate volume, total number of inflammatory cells, ADA activity, nitrite level, and PGE 2 level in 50%, 41%, 20%, 60%, and 41%, respectively. The anti-inflammatory effects of 5 mg/kg AEPa appeared to be more effective than those of 1 mg/kg AEPa (84%, 80%, 43%, 70%, and 75%, respectively). In addition, TGF-β level was upregulated to 9700 pg/ml after 5 mg/kg AEPa, in comparison with 160 pg/ml in the vehicle-treated group, and 137 pg/ml in the indomethacin-treated group. The results indicate that AEPa exerts powerful anti-inflammatory and immunomodulatory activities, interfering with the cyclooxygenase pathway, lymphocyte proliferation, NO, and TGF-β production.

Changes in Upper and Lower Airway Inflammation Following Administration of Mometasone Furoate in Allergen-Challenged Brown Norway Rats

RationaleTo assess inhibitory effects of mometasone furoate (MF) on allergen-provoked upper and lower airway inflammation, we evaluated inflammatory cell infiltration and reductions in forced vital capacity (FVC) and peak expiratory flow (PEF) in allergen-challenged Brown Norway rats.MethodsFour experiments were performed on ovalbumin (OVA)-sensitized rats: Group 1) intranasal (i.n.) MF or vehicle given for 3 consecutive days, last treatment dose given 2 hr before i.n. OVA (1%) challenge on last day; Group 2) and Group 3) intratracheal (i.t.) MF or vehicle given 5 hr before aerosolized OVA (1%) challenge; Group 4) nose-only inhalation of dry powder MF, given 5 hr before aerosolized OVA (1%) challenge. In groups 1 and 2, nasal lavage (NL) and bronchoalveolar lavage (BAL) samples were collected 24 hr after OVA challenge. In groups 3 and 4, FVC and PEF were assessed 24 hr after OVA challenge.ResultsIntranasal MF (0.01-10 ng/ml) reduced number of total inflammatory cells in the NL induced by OVA challenge with significant effects seen at all doses. Intratracheal MF (0.01-0.3 mg/kg, i.t.) significantly attenuated number of total BAL inflammatory cells. MF (0.1-1 mg/kg, i.t.) also inhibited reductions in FVC and PEF induced by the OVA challenge. MF attenuated reductions in FVC (32%, 45%, and 92% inhibition) and PEF (50%, 59%, and 100% inhibition) when given by nose-only inhalation (estimated pulmonary deposition of MF, 1.4, 4.1, and 13.3 μg/kg, respectively).ConclusionsIn these animal models of upper and lower airway inflammation, MF significantly attenuated cellular lung inflammation and normalized lung function following allergen provocation. RationaleTo assess inhibitory effects of mometasone furoate (MF) on allergen-provoked upper and lower airway inflammation, we evaluated inflammatory cell infiltration and reductions in forced vital capacity (FVC) and peak expiratory flow (PEF) in allergen-challenged Brown Norway rats. To assess inhibitory effects of mometasone furoate (MF) on allergen-provoked upper and lower airway inflammation, we evaluated inflammatory cell infiltration and reductions in forced vital capacity (FVC) and peak expiratory flow (PEF) in allergen-challenged Brown Norway rats. MethodsFour experiments were performed on ovalbumin (OVA)-sensitized rats: Group 1) intranasal (i.n.) MF or vehicle given for 3 consecutive days, last treatment dose given 2 hr before i.n. OVA (1%) challenge on last day; Group 2) and Group 3) intratracheal (i.t.) MF or vehicle given 5 hr before aerosolized OVA (1%) challenge; Group 4) nose-only inhalation of dry powder MF, given 5 hr before aerosolized OVA (1%) challenge. In groups 1 and 2, nasal lavage (NL) and bronchoalveolar lavage (BAL) samples were collected 24 hr after OVA challenge. In groups 3 and 4, FVC and PEF were assessed 24 hr after OVA challenge. Four experiments were performed on ovalbumin (OVA)-sensitized rats: Group 1) intranasal (i.n.) MF or vehicle given for 3 consecutive days, last treatment dose given 2 hr before i.n. OVA (1%) challenge on last day; Group 2) and Group 3) intratracheal (i.t.) MF or vehicle given 5 hr before aerosolized OVA (1%) challenge; Group 4) nose-only inhalation of dry powder MF, given 5 hr before aerosolized OVA (1%) challenge. In groups 1 and 2, nasal lavage (NL) and bronchoalveolar lavage (BAL) samples were collected 24 hr after OVA challenge. In groups 3 and 4, FVC and PEF were assessed 24 hr after OVA challenge. ResultsIntranasal MF (0.01-10 ng/ml) reduced number of total inflammatory cells in the NL induced by OVA challenge with significant effects seen at all doses. Intratracheal MF (0.01-0.3 mg/kg, i.t.) significantly attenuated number of total BAL inflammatory cells. MF (0.1-1 mg/kg, i.t.) also inhibited reductions in FVC and PEF induced by the OVA challenge. MF attenuated reductions in FVC (32%, 45%, and 92% inhibition) and PEF (50%, 59%, and 100% inhibition) when given by nose-only inhalation (estimated pulmonary deposition of MF, 1.4, 4.1, and 13.3 μg/kg, respectively). Intranasal MF (0.01-10 ng/ml) reduced number of total inflammatory cells in the NL induced by OVA challenge with significant effects seen at all doses. Intratracheal MF (0.01-0.3 mg/kg, i.t.) significantly attenuated number of total BAL inflammatory cells. MF (0.1-1 mg/kg, i.t.) also inhibited reductions in FVC and PEF induced by the OVA challenge. MF attenuated reductions in FVC (32%, 45%, and 92% inhibition) and PEF (50%, 59%, and 100% inhibition) when given by nose-only inhalation (estimated pulmonary deposition of MF, 1.4, 4.1, and 13.3 μg/kg, respectively). ConclusionsIn these animal models of upper and lower airway inflammation, MF significantly attenuated cellular lung inflammation and normalized lung function following allergen provocation. In these animal models of upper and lower airway inflammation, MF significantly attenuated cellular lung inflammation and normalized lung function following allergen provocation.

Correlations Between Clinical and Histological Aspects in Nasal Polyposis

The histological study of nasal polyps does not reveal any specific lesions but eosinophilic infiltration nasal mucosae seems to be characteristic of nasal polyposis. The aim of this work is to study possible links between certain histological and clinical aspects in nasal polyposis. Furthermore, we attempt to compare the quantification of tissue eosinophilia according to the number of eosinophils per field with the percentage figure obtained with respect to the total of inflammatory cells. We have studied 40 patients with idiopathic bilateral nasal polyposis, assessing the correlations between various clinical aspects such as their endoscopic and radiological status, association with asthma and intolerance to NSAIDs, against histological aspects of nasal polyps such as the frequency of metaplasia, fibrosis and the degree of eosinophilic infiltration. A group of 12 healthy subjects allowed comparison of our results with healthy nasal mucosa. Tissue eosinophilia correlates statistically with clinical staging and tends to be higher in patients with ASA triad. The quantitative measurement of tissue eosinophilia (number of eosinophils per field) correlates with the percentage figure obtained (with respect to the total number of inflammatory cells in the infiltrate). Eosinophil infiltration of the nasal mucosa is, together with oedema, the most constant histological characteristic of nasal polyposis and seems to be an important factor in the clinical behaviour of sinonasal polyposis. Quantitative measurement of tissue eosinophilia is easier and quicker to perform and equivalent to percentage evaluation.

Comparison of tracheal aspirates before and after high‐speed treadmill exercise in racehorses

To determine whether percentages of neutrophils in tracheal aspirate (TA) samples collected from racehorses are increased after exercise and whether interpretation of results from TA samples taken before and after exercise agree. Case series of 40 young Thoroughbred and Standardbred racehorses in race training presented for evaluation of poor performance. TA samples were collected endoscopically from racehorses presented for poor performance 24 h before and 1 to 2 h after high speed treadmill exercise testing. Aliquots of the retrieved fluid were cytocentrifuged and smears were stained with Diff-Quik. Mean neutrophil counts were expressed as percentages of the total number of inflammatory cells counted and subsequently were categorised as either above or below an accepted cut-off of 20%. Comparisons between percentages of neutrophils before and after exercise were made. Percentage of neutrophils from TA samples obtained from racehorses after exercise was significantly higher than neutrophil percentages from TA samples collected from the same horse before exercise. In horses with TA specimens that were categorised as having < or = 20% neutrophils before treadmill exercise, the percentage of neutrophils in their TA specimens after exercise was, on average, significantly higher and was greater than the cut-off value of 20%. Recent strenuous exercise may change the proportion of neutrophils in lower airways of racehorses and practitioners should be aware of this when collecting and interpreting the results from TA samples. The most practical time for collection of a TA sample to obtain the most diagnostically useful information might be after a suitable washout period of at least 1 to 2 h post-exercise.

Effect of molecular mobility of polymeric implants on soft tissue reactions: An in vivo study in rats

Although numerous different polymers are used as implants or otherwise studied for many other biotechnical applications, there is a lack of basic models that correlate polymer characteristics with foreign body reactions. This study aims at developing one such model by systematically studying surface molecular mobility of polymeric implants in soft tissues in vivo. Changing the length of the alkyl side chain of poly(alkyl methacrylates) (PAMAs), provides an interesting opportunity to study the surface molecular mobility with minimal changes of the hydrophobicity of the surface. Thus, in this study three different PAMAs, with increasingly surface mobility; poly (isobutyl methacrylate) (PIBMA), poly(butyl methacrylate) (PBMA), and poly(lauryl methacralate) (PLMA) along with pure titanium (Ti) substrates were implanted in the dorsum of Sprague-Dawley rats. Inflammatory cell recruitment, cell adhesion, and cytokine release were studied after 1, 3, and 28 days of implantation. Total number of inflammatory cells in the exudate was measured but no correlation between surface mobility and cell recruitment where found. However, the number of surface associated cells where significantly lower on the surfaces with high molecular mobility (PLMA and PBMA). The histological evaluation performed after 28 days revealed thicker fibrous capsule and a higher number of blood vessels on the low molecular mobility surface (PIBMA). After 28 days the cell activity was higher on the high molecular mobility surfaces (PLMA and PBMA) compared with PIBMA, based on the cytokine release. None of the surfaces induced any significant cell-death. On the basis of the results of this study we conclude that there is a significant difference in biological response to surfaces with different in molecular mobility. This might affect the wound healing process and the biocompatibility of biomaterials.

Vitamin E deficiency enhances pulmonary inflammatory response and oxidative stress induced by single-walled carbon nanotubes in C57BL/6 mice

Exposure of mice to single-walled carbon nanotubes (SWCNTs) induces an unusually robust pulmonary inflammatory response with an early onset of fibrosis, which is accompanied by oxidative stress and antioxidant depletion. The role of specific components of the antioxidant protective system, specifically vitamin E, the major lipid-soluble antioxidant, in the SWCNT-induced reactions has not been characterized. We used C57BL/6 mice, maintained on vitamin E-sufficient or vitamin E-deficient diets, to explore and compare the pulmonary inflammatory reactions to aspired SWCNTs. The vitamin E-deficient diet caused a 90-fold depletion of α-tocopherol in the lung tissue and resulted in a significant decline of other antioxidants (GSH, ascorbate) as well as accumulation of lipid peroxidation products. A greater decrease of pulmonary antioxidants was detected in SWCNT-treated vitamin E-deficient mice as compared to controls. Lowered levels of antioxidants in vitamin E-deficient mice were associated with a higher sensitivity to SWCNT-induced acute inflammation (total number of inflammatory cells, number of polymorphonuclear leukocytes, released LDH, total protein content and levels of pro-inflammatory cytokines, TNF-α and IL-6) and enhanced profibrotic responses (elevation of TGF-β and collagen deposition). Exposure to SWCNTs markedly shifted the ratio of cleaved to full-length extracellular superoxide dismutase (EC-SOD). Given that pulmonary levels of vitamin E can be manipulated through diet, its effects on SWCNT-induced inflammation may be of practical importance in optimizing protective strategies.

Anti-inflammatory mechanism of simvastatin in mouse allergic asthma model

Statins have anti-inflammatory property and immunomodulatory activity. In this study we aimed to investigate the inhibitory mechanism of simvastatin in allergic asthmatic symptoms in mice. BALB/c mice were sensitized and challenged by ovalbumin to induce asthma. Ovalbumin-specific serum IgE levels were measured by enzyme-linked immunosorbent assay (ELISA), and the recruitment of inflammatory cells into bronchoalveolar lavage fluid or lung tissues was measured by Diff-Quik staining and hematoxylin and eosin (H&E) staining, respectively, the expressions of CD40, CD40 ligand (CD40L), and vascular cell adhesion molecule-1 (VCAM-1) by immunohistochemistry, the mRNA and protein expressions of cytokines in lung tissues by reverse transcriptase-polymerase chain reaction (RT-PCR) or ELISA, epithelial hyperplasia by periodic acid-Schiff (PAS) staining, activities of matrix metalloproteinases (MMPs) by zymography, the activities of small G proteins, mitogen-activated protein (MAP) kinases and nuclear factor-kappa B (NF-κB) in bronchoalveolar lavage cells and lung tissues by western blot and EMSA, respectively. Simvastatin reduced ovalbumin-specific IgE level, the number of total inflammatory cells, macrophages, neutrophils, and eosinophils into bronchoalveolar lavage fluid, the expressions of CD40, CD40L or VCAM-1, the mRNA and protein levels of interleukin (IL)-4, IL-13 and tumor necrosis factor (TNF)-α, the numbers of goblet cells, activities of MMPs, and further small G proteins, MAP kinases and NF-κB activities in bronchoalveolar lavage cells and lung tissues increased in ovalbumin-induced allergic asthma in mice. Our data suggest that simvastatin may be used as a therapeutic agent in asthma, based on reductions of various allergic responses via regulating small G proteins/MAP kinases/NF-κB in mouse allergic asthma.

Protease-Activated Receptor-2 Activation: A Major Role in the Pathogenesis of Porphyromonas gingivalis Infection

Protease-Activated Receptor-2 Activation: A Major Role in the Pathogenesis of Porphyromonas gingivalis Infection

Human Periprosthetic Tissues Implanted in Severe Combined Immunodeficient Mice Respond to Gene Transfer of a Cytokine Inhibitor

Periprosthetic tissue formation and local inflammation that are associated with wear debris contribute to the pathogenesis of aseptic loosening of a prosthesis. This study evaluated a retrovirus-mediated gene therapy with use of a novel xenograft-based animal model. Human periprosthetic tissues obtained from patients during revision arthroplasty performed because of aseptic loosening of a prosthetic joint were transplanted into the left quadriceps and paravertebral muscles of severe combined immunodeficient (SCID) mice. The engrafted tissues were recovered seven, fifteen, or thirty days after implantation for histological and molecular analyses. The periprosthetic tissues were incubated with retroviruses encoding for human interleukin-1 receptor antagonist (hIL-1Ra) or bacteria beta-galactosidase (LacZ) at 37 degrees C for three hours prior to implantation to evaluate their responses to gene modification. The human periprosthetic tissues were well accepted in SCID mice for up to thirty days, with angiogenesis occurring in the majority of the implanted tissue sections. The histological appearance was consistent between the recovered graft tissue and the original donor tissue. Strong expression of interleukin-1, tumor necrosis factor, and interleukin-6 was detected in the xenografts with use of immunohistochemical stains. Histological analysis revealed that interleukin-1 receptor antagonist gene modification significantly decreased the total number of inflammatory cells (p < 0.01) in engrafted human tissue containing implant wear debris. Real-time reverse transcription-polymerase chain reaction and immunohistochemical staining showed declining expression levels of interleukin-1 and tumor necrosis factor following interleukin-1 receptor antagonist gene transfer in comparison with LacZ-transduced or virus-free controls. Human periprosthetic tissue can survive in the SCID mouse host for up to thirty days and responds to the interleukin-1 receptor antagonist gene transfer with the amelioration of inflammation.

Melatonin promoted chemotaxins expression in lung epithelial cell stimulated with TNF-α

BackgroundPatients with asthma demonstrate circadian variations in the airway inflammation and lung function. Pinealectomy reduces the total inflammatory cell number in the asthmatic rat lung. We hypothesize that melatonin, a circadian rhythm regulator, may modulate the circadian inflammatory variations in asthma by stimulating the chemotaxins expression in the lung epithelial cell.MethodsLung epithelial cells (A549) were stimulated with melatonin in the presence or absence of TNF-α(100 ng/ml). RANTES (Regulated on Activation Normal T-cells Expressed and Secreted) and eotaxin expression were measured using ELISA and real-time RT-PCR, eosinophil chemotactic activity (ECA) released by A549 was measured by eosinophil chemotaxis assay.ResultsTNF-α increased the expression of RANTES (307.84 ± 33.56 versus 207.64 ± 31.27 pg/ml of control, p = 0.025) and eotaxin (108.97 ± 10.87 versus 54.00 ± 5.29 pg/ml of control, p = 0.041). Melatonin(10-10 to 10-6M) alone didn't change the expression of RNATES (204.97 ± 32.56 pg/ml) and eotaxin (55.28 ± 6.71 pg/ml). However, In the presence of TNF-α (100 ng/ml), melatonin promoted RANTES (410.88 ± 52.03, 483.60 ± 55.37, 559.92 ± 75.70, 688.42 ± 95.32, 766.39 ± 101.53 pg/ml, treated with 10-10, 10-9, 10-8, 10-7,10-6M melatonin, respectively) and eotaxin (151.95 ± 13.88, 238.79 ± 16.81, 361.62 ± 36.91, 393.66 ± 44.89, 494.34 ± 100.95 pg/ml, treated with 10-10, 10-9, 10-8, 10-7, 10-6M melatonin, respectively) expression in a dose dependent manner in A549 cells (compared with TNF-α alone, P < 0.05). The increased release of RANTES and eotaxin in A549 cells by above treatment were further confirmed by both real-time RT-PCR and the ECA assay.ConclusionTaken together, our results suggested that melatonin might synergize with pro-inflammatory cytokines to modulate the asthma airway inflammation through promoting the expression of chemotaxins in lung epithelial cell.

Influence of dimethylfumarate on experimental HSV-1 necrotizing keratitis.

This study was performed to investigate the influence of fumaric acid esters on the course of herpes stromal keratitis (HSK). The corneas of BALB/c mice were inoculated with 105 plaque-forming units of herpes simplex virus 1 (HSV-1, KOS strain). Groups of mice were treated intraperitoneally with phosphate buffered saline (PBS) (control mice), or with dimethylfumarate (DMF) at 15 mg/kg of body weight dissolved in PBS daily for 28 days pre-infection and for 14 days post-infection. The course of HSV-1 keratitis was studied clinically. Corneal sections were examined for inflammatory cell infiltration. The numbers of CD3, GR-1, CD11b and F4/80-expressing cells infiltrating the corneas were analyzed by immunohistochemistry. On day 14 after HSV infection, 72% of the mice in the control group had severe HSK. The development of HSK was reduced by DMF treatment in the DMF group (22%) (P=0.004). The total number of inflammatory cells and infiltration of polymorphonuclear-neutrophils (PMNs) were reduced in the corneas of DMF-treated mice. Compared to the PBS-treated mice, numbers of CD3, CD11b, GR-1 and F4/80-positive cells were reduced in the DMF group of mice. The course of experimental herpes stromal keratitis can be improved with systemic fumaric acid ester treatment. The improvement of keratitis correlates with a reduced corneal infiltration of T cells and mononuclear cells.

Effects of Amphetamine on Immune-Mediated Lung Inflammatory Response in Rats

Objective: The present study analyzed the effects of acute amphetamine (AMPH) treatment on immune-mediated lung inflammatory response in rats. Methods: There were four experiments. In the first and second experiments, rats were treated with AMPH (1 mg/kg) or 0.9% NaCl, and locomotor activity (experiment 1) and serum AMPH concentrations (experiment 2) were measured 1 or 12 h after treatment. In the third experiment, rats which were immunized with ovalbumin (OVA) were treated 14 days later with 0.9% NaCl or AMPH (1 mg/kg). Twelve hours after these treatments, all animals were submitted to challenge by 1% OVA inhalation being analyzed afterwards for bronchoalveolar lavage fluid (BAL), peripheral blood and bone marrow cellularity. In the fourth and final experiment, rats were treated and studied as for experiment 3, except that half of the animals within each group were previously treated with metyrapone prior to the OVA challenge. Results: In the non- immunized rats, AMPH treatment induced an increase in locomotor activity synchronized to high serum AMPH concentrations 1 h after, but not 12 h after treatment. In OVA-challenged rats, AMPH treatment decreased the total number of inflammatory cells, recovered in both BAL and peripheral blood and increased the total number of bone marrow cells. These effects, observed 1 day after OVA challenge, were abrogated by previous metyrapone treatment. Conclusion: AMPH treatment changed HPA-axis responsiveness to the stress condition imposed by the OVA challenge decreasing lung and blood leukocytes cellularity most probably via corticosterone actions on bone marrow activity.

Experimental murine mycobacteriosis: evaluation of the functional activity of alveolar macrophages in thalidomide- treated mice

Thalidomide is a selective inhibitor of tumor necrosis factor-alpha (TNF-alpha), a cytokine involved in mycobacterial death mechanisms. We investigated the role of this drug in the functional activity of alveolar macrophages in the presence of infection induced by intranasal inoculation of Mycobacterium avium in thalidomide-treated and untreated adult Swiss mice. Sixty animals were inoculated with 5 x 10(6) M. avium by the respiratory route. Thirty animals received daily thalidomide (30 mg/kg mouse) and 30 received water by gavage up to sacrifice. Ten non-inoculated mice were used as a control group. Lots of animals from each group were evaluated until 6 weeks after inoculation. Infection resulted in an increased total number of inflammatory cells as well as increased activity of pulmonary macrophages. Histologically, intranasal inoculation of bacilli resulted in small mononuclear infiltrates located at the periphery of the organ. Culture of lung fragments revealed the presence of bacilli only at the beginning and at the end of the experimental period. Thalidomide administration did not affect the microbiological or histological features of the infection. Thalidomide-treated and untreated animals showed the same amount of M. avium colonies 3 weeks after infection. Although it did not affect bacillary clearance, thalidomide administration resulted in a decreased percent of spread cells and release of hydrogen peroxide, suggesting that factors other than TNF-alpha play a role in the killing of mycobacteria by alveolar macrophages. Thalidomide administration also reduced the number of spread cells among resident macrophages, suggesting a direct effect of the drug on this phenomenon.

The influence of dexamethasone on the proliferation and apoptosis of pulmonary inflammatory cells in bleomycin-induced pulmonary fibrosis in rats.

The aim of this study was to investigate the inhibitory effect of dexamethasone on the state of proliferation/apoptosis of the pulmonary inflammatory cells in a rat pulmonary fibrosis model induced by bleomycin. Seventy-five pathogen-free Sprague-Dawley (SD) rats were randomly divided into three groups: control, bleomycin (BLM) and dexamethasone (DXM) groups with 25 rats in each group. Each group was then divided into five subgroups based on time of study (1-, 3-, 7-, 14- and 28-days). BAL fluid was obtained, the cells were counted and a differential was performed. A lower DNA content in apoptotic cells was detected and quantitated by flow cytometry. Haematoxylin and eosin staining was performed to observe the extent of alveolitis and fibrosis of lung tissue; the morphological changes in apoptotic cells were discerned by transmission electron-microscopy and a semi-quantitative assessment of apoptotic cells in lung tissue was performed using in situ TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick endlabelling). The total number of inflammatory cells and the percentage of neutrophils in BAL fluid in almost every subgroup of the DXM group were significantly lower than those in corresponding subgroups of the BLM group (P < 0.01). The percentages of apoptotic cells in BAL fluid in the 14-day and 28-day subgroups of the DXM group were higher than those in corresponding subgroups of the BLM group (P < 0.05). The peak of alveolitis in the DXM group shifted backward and the extent of fibrosis was less than that in the BLM group. The apoptosis index (AI) of inflammatory cells in each of the DXM subgroups was higher than that in corresponding BLM subgroups except for day 14. Dexamethasone can induce apoptosis of pulmonary inflammatory cells and reduce the extent of alveolitis and fibrosis in bleomycin-induced pulmonary fibrosis of rats.