Protective Effect of RC-3095, an Antagonist of the Gastrin-Releasing Peptide Receptor, in Experimental Arthritis

Objective. To evaluate the antiinflammatory effects of RC-3095 in 2 experimental models of arthritis, collagen-induced arthritis (CIA) and antigen-induced arthritis (AIA), and to determine the mechanisms of action involved. Methods. RC-3095 was administered daily to mice with CIA and mice with AIA, after induction of disease with methylated bovine serum albumin. Disease incidence and severity were assessed using a clinical index and evaluation of histologic features, respectively. In mice with CIA, gastrin-releasing peptide receptor (GRPR) was detected by immunohistochemical analysis, while in mice with AIA, migration of neutrophils, presence of glycosaminoglycans, and lymphocyte proliferation, determined using the MTT assay, were assessed. Expression of cytokines interleukin-17 (IL-17), IL-1 beta, and tumor necrosis factor alpha (TNF alpha) was evaluated in all mouse knees using enzyme-linked immunosorbent assay. Treg cell production was assessed by flow cytometry in the joints of mice with AIA. Results. In mice with AIA, administration of RC-3095 reduced neutrophil migration, mechanical hypernociception, and proteoglycan loss. These findings were associated with inhibition of the levels of all 3 proinflammatory cytokines, decreased lymphocyte proliferation, and increased Treg cell numbers. In the CIA model, treatment with RC-3095 led to a significant reduction in arthritis clinical scores and the severity of disease determined histologically. Synovial inflammation, synovial hyperplasia, pannus formation, and extensive erosive changes were all dramatically reduced in the arthritic mice treated with RC-3095. Furthermore, arthritic mice treated with RC-3095 showed a significant reduction in the concentrations of IL-17, IL-1 beta, and TNF alpha, and showed a diminished expression of GRPR. Conclusion. These findings suggest that the GRP pathway has a significant role in chronic arthritis, and its inhibition can be explored as a possible therapeutic strategy in rheumatoid arthritis.

Cannabidiol decreases bone resorption by inhibiting RANK/RANKL expression and pro-inflammatory cytokines during experimental periodontitis in rats

Cannabidiol (CBD) is a cannabinoid component from Cannabis sativa that does not induce psychotomimetic effects and possess anti-inflammatory properties. In the present study we tested the effects of CBD in a periodontitis experimental model in rats. We also investigated possible mechanisms underlying these effects. Periodontal disease was induced by a ligature placed around the mandible first molars of each animal. Male Wistar rats were divided into 3 groups: control animals; ligature-induced animals treated with vehicle and ligature-induced animals treated with CBD (5 mg/kg, daily). Thirty days after the induction of periodontal disease the animals were sacrificed and mandibles and gingival tissues removed for further analysis. Morphometrical analysis of alveolar bone loss demonstrated that CBD-treated animals presented a decreased alveolar bone loss and a lower expression of the activator of nuclear factor-kappa B ligand RANKL/RANK. Moreover, gingival tissues from the CBD-treated group showed decreased neutrophil migration (MPO assay) associated with lower interleukin (IL)-1 beta and tumor necrosis factor (TNF)-alpha production. These results indicate that CBD may be useful to control bone resorption during progression of experimental periodontitis in rats. Crown Copyright (C) 2008 Published by Elsevier B.V. All rights reserved.

Morphologic evaluation and expression of matrix metalloproteinases-2 and 9 and nitric oxide during experimental periodontal disease in rat

The immunopathologic and inflammatory mechanisms involved in periodontal disease (PD) include the participation of host resident, inflammatory cells and chemical mediators. Metalloproteinases (MMPs) and nitric oxide (NO) play essential role in extracellular matrix turnover of periodontal tissue destruction. In this study, by means of RT-PCR through semi-quantitative densitometric scanning methods, the expression of MMPs -2 and -9 and inducible NO synthase (iNOS) was temporally and spatially investigated during the destructive mechanisms of experimentally induced PD in rats. Samples from different periods were microscopically analyzed and compared with the contralateral side (control). Our results showed significant expression of MMP-9 and iNOS in tissues affected by PD, as compared with controls, three days after PD induction, simultaneously with the beginning of alveolar bone loss. At 7 days post induction, only the MMP-9 mRNA presented a significantly higher expression, as compared with the respective controls. Thus, in the rat ligature-induced PD, MMP-9 and iNOS might importantly participate in the early stages of the disease, including inflammatory cell migration, tissue destruction and alveolar bone resorption. Also, we may suggest that the exuberant presence of PMNs may be related to the important expression of iNOS and MMP-9 found at 3 days post induction.

Increased activities of cardiac matrix metalloproteinases matrix metalloproteinase (MMP)-2 and MMP-9 are associated with mortality during the acute phase of experimental Trypanosoma cruzi infection

The strong inflammatory reaction that occurs in the heart during the acute phase of Trypanosoma cruzi infection is modulated by cytokines and chemokines produced by leukocytes and cardiomyocytes. Matrix metalloproteinases (MMPs) have recently emerged as modulators of cardiovascular inflammation. In the present study we investigated the role of MMP-2 and MMP-9 in T. cruzi-induced myocarditis, by use of immunohistochemical analysis, gelatin zymography, enzyme-linked immunosorbent assay, and real-time polymerase chain reaction to analyze the cardiac tissues of T. cruzi-infected C57BL/6 mice. Increased transcripts levels, immunoreactivity, and enzymatic activity for MMP-2 and MMP-9 were observed by day 14 after infection. Mice treated with an MMP inhibitor showed significantly decreased heart inflammation, delayed peak in parasitemia, and improved survival rates, compared with the control group. Reduced levels of cardiac tumor necrosis factor-alpha, interferon-gamma, serum nitrite, and serum nitrate were also observed in the treated group. These results suggest an important role for MMPs in the induction of T. cruzi-induced acute myocarditis.

Hematopoietic SCT modulates gut inflammation in experimental inflammatory bowel disease

Hematopoietic SCT (HSCT) and high-dose chemotherapy are being explored as therapy for various human refractory immune-mediated conditions, including inflammatory bowel diseases (IBD). Nevertheless, the exact immunological mechanisms by which the BM cells (BMCs) or immunosuppression provide remission from these diseases is not yet clear. In this work, we investigated the role of these therapies in the modulation of gut mucosal inflammation in an experimental model of IBD. Colitis was induced in mice by 2,4,6-trinitrobenzenesulfonic acid and after CY was administered (200 mg/kg) alone (CY group) or followed by BMCs infusion (HSCT group). Animals were followed for 60 days. Both HSCT and CY reduced the histopathological features of colitis significantly. Infused cells were localized in the gut, and a marked decrease of CD4(+) leukocytes in the inflammatory infiltrate on days +7 and +14 and of CD8(+) cells on day +7 was found in both treatments allied to impressive reduction of proinflammatory Th1 and Th17 cytokines. Although chemotherapy alone was the best treatment regarding the induction of immunosuppressive molecules, only HSCT resulted in increased survival rates compared with the control group. Our findings indicate that high-dose CY followed by HSCT is effective in the modulation of mucosal immunity and in accelerating immune reconstitution after BMT, thus providing valuable tools to support the development and understanding of novel therapeutic strategies for IBD. Bone Marrow Transplantation (2010) 45, 1562-1571; doi:10.1038/bmt.2010.6; published online 15 March 2010

Experimental autoimmune encephalomyelitis evolution was not modified by multiple infections with Strongyloides venezuelensis

P>According to the hygiene hypothesis, the increased incidence of allergic and autoimmune diseases in developed countries is mainly explained by the decreased contact between the human population and certain environmental agents as lactobacillus, mycobacteria and helminths. In this study, we evaluated the effect of multiple infections with Strongyloides venezuelensis on the development of experimental autoimmune encephalomyelitis (EAE) in Lewis rats. Multiple infections before EAE induction were not able to change the evolution of the disease. No alterations were observed in weight loss, clinical score and inflammation intensity at the central nervous system. The presence of significant levels of parasite-specific IgG1 but not IgG2b suggested a Th2 polarization. However, the percentage and absolute number of CD4+CD25+Foxp3+ T cells were not changed, being their levels in the spleen and lymph nodes of infected rats comparable to the ones found in normal animals. These results suggest that a Th2-polarized response without concomitant expansion of Foxp3+ regulatory T cells was not able to modify EAE progression. Even though these results do not threaten the hygiene hypothesis, they suggest that this paradigm might be an oversimplification. They also emphasize the need of a study to compare the immunoregulatory ability associated with different helminth spp.

Transient disruption of liver gap junctional intercellular communication and induction of apoptosis after administration of 1,4-bis[2-(3,5 dichloropyridyloxy)]benzene in mice

Gap junctional intercellular communication (GJIC) and connexin expression (Cx26 and Cx32) in mouse liver were studied after administration of 4-bis[2-(3,5 dichloropyridyloxy)]benzene (TCPOBOP), a phenobarbital-like enzyme inducer. Female C57BI/6 mice were administered TCPOBOP (5.8 mg/kg BW) and euthanized 0, 24, 48 and 72 hours later. Liver samples were snap frozen, or fixed in formalin, or submitted to GJIC analysis. The proliferating cell nuclear antigen (PCNA) immunohistochemistry and the Western blotting for Cx26 and Cx32 were performed. After 48 and 72 h of drug administration the liver-to-body weight ratio was increased 70% and 117% (p < 0.0001), respectively. There were temporal-dependent alterations in liver histopathology and a significant increase in cell proliferation was noted after 48h and sustained after 72h, though to a lesser extent (p < 0.0001). In addition. TCPOBOP administration induced apoptosis, which appeared to be time-dependent showing statistical significance only after 72h (p < 0.0001). Interestingly, a transient disruption by nearly 50% of GJIC capacity was detected after 48 h of drug ingestion, which recovered after 72 h (p = 0.003). These GJIC changes were due to altered levels of Cx26 and Cx32 in the livers of TCPOBOP-treated mice. We concluded that a single administration of TCPOBOP transiently disrupted the levels of GJIC due to decreased expression of connexins and increased apoptotic cell death in mouse liver. (C) 2009 Elsevier GmbH. All rights reserved.

Influence of Antiplatelet Drugs in the Pathogenesis of Experimental Periodontitis and Periodontal Repair in Rats

Background: Platelets contain an array of biologic mediators that can modulate inflammation and repair processes including proinflammatory mediators and growth factors. Previous studies have shown that periodontitis and periodontal repair are associated with platelet activation. We hypothesized that drug-induced platelet inactivation may interfere in the processes of inflammation and repair in experimental periodontitis in rats by suppressing the release of biologic mediators from platelets to the site of injury. Methods: To measure the effects on periodontitis, ligatures were placed around first molars, and aspirin (Asp, 30 mg/kg) or clopidogrel (Clo, 75 mg/kg) was given intragastrically once daily for 15 days. Interleukin-6 (IL-6), tumor necrosis factor-a (TNF-alpha), and thromboxane A(2) levels were measured by enzyme-linked immunosorbent assay. To evaluate the effects of antiplatelet drugs on periodontal repair, ligatures were removed after 15 days of periodontitis induction, and Asp or Clo were administered beginning the following day for 15 days. Periodontal repair was assessed by microcomputed tomography. Results: On periodontitis phase, Asp and Clo significantly reduced levels of TNF-alpha and II-6 (P < 0.05), but only Asp decreased thromboxane A(2) (P < 0.05). Asp and Clo decreased inflammatory infiltration; however, this reduction was more pronounced with Clo treatment (P < 0.05). Histometric analysis showed that Asp and Clo impaired alveolar bone resorption. During the repair phase and after removal of the ligatures, microcomputed tomography analysis demonstrated that treatment with Asp and Clo did not impair alveolar bone repair. Conclusion: Systemic administration of Asp and Clo attenuates the inflammation associated with periodontitis without affecting the repair process when stimulus is removed. J Periodontol 2011;82:767-777.

Growth regime map for liquid-bound granules: further development and experimental validation

An attempt was made to quantify the boundaries and validate the granule growth regime map for liquid-bound granules recently proposed by Iveson and Litster (AlChE J. 44 (1998) 1510). This regime map postulates that the type of granule growth behaviour is a function of only two dimensionless groups: the amount of granule deformation during collision (characterised by a Stokes deformation number, St(def)) and the maximum granule pore saturation, s(max). The results of experiments performed with a range of materials (glass ballotini, iron ore fines, copper chalcopyrite powder and a sodium sulphate and cellulose mixture) using both drum and high shear mixer granulators were examined. The drum granulation results gave good agreement with the proposed regime map. The boundary between crumb and steady growth occurs at St(def) of order 0.1 and the boundary between steady and induction growth occurs at St(def) of order 0.001. The nucleation only boundary occurs at pore saturations that increase from 70% to 80% with decreasing St(def). However, the high shear mixer results all had St(def) numbers which were too large. This is most likely to be because the chopper tip-speed is an over-estimate of the average impact velocity granules experience and possibly also due to the dynamic yield strength of the materials being significantly greater than the yield strengths measured at low strain rates. Hence, the map is only a useful tool for comparing the granulation behaviour of different materials in the same device. Until we have a better understanding of the flow patterns and impact velocities in granulators, it cannot be used to compare different types of equipment. Theoretical considerations also revealed that several of the regime boundaries are also functions of additional parameters not explicitly contained on the map, such as binder viscosity. (C) 2001 Elsevier Science B.V. All rights reserved.

Induction of suppressor cells following mucosal presentation of Actinomyces viscosus in mice

Mucosal presentation of Actinomyces viscosus results in antigen-specific systemic immune suppression, known as oral tolerance. The aim of the present study was to determine the mechanism by which this oral tolerance is induced. DBA/2 mice were gastrically immunized with A. viscosus. Serum, Peyer's patch (PP) and spleen cells were transferred to syngeneic recipients which were then systemically challenged with the sameiA. viscosus strain. To determine antigen-specificity of cells from gastrically immunized mice, recipients which received immune spleen cells were also challenged with Porphyromonas gingivalis. One week after the last systemic challenge, the delayed type hypersensitivity (DTH) response was determined by footpad swelling and the level of serum IgG, IgA and IgM antibodies to A. viscosus or P. gingivalis measured by an ELISA. No suppression of DTH response or of specific serum antibodies was found in recipients which received serum from gastrically immunized mice. Systemic immune suppression to A. viscosus was observed in recipients which had been transferred with PP cells obtained 2 days but not 4 and 6 days after gastric immunization with A. viscosus. Conversely, suppressed immune response could be seen in recipients transferred with spleen cells obtained 6 days after gastric immunization. The immune response to P. gingivalis remained unaltered in mice transferred with A. viscosus-gastrically immunized cells. The results of the present study suggest that oral tolerance induced by A. viscosus may be mediated by antigen-specific suppressor cells which originate in the PP and then migrate to the spleen.

Exogenous induction of ovarian activity and ovulation and transfer of fresh embryos of domestic cat (Felis catus)

The objective of the present study was the exogenous stimulation of ovarian activity and definition of embryo collection, and transfer protocols, in the domestic cat for potential application in non-domestic endangered species. Sixteen adult queens and two adult male reproducers kept in the experimental cat house at the Morphology sector at the Veterinary Department (DVT), UFV, were used in this study. All the queens received a single application of 150 IU Equine Chorionic Gonadotropin (eCG) in the post estrus to induce ovarian activity and 80 to 84 hours later, received a single application of 100 UI Human Chorionic Gonadotropin (hCG) to induce ovulation. After hCG application, only the donor queens were naturally mated. The receptor queens received extra stimulus for induction of ovulation through manipulation of an intravaginal swab. Five to six days after hCG application, the donor queens were subjected to a laparotomy for embryo collection that was performed by trans-horn uterine washing. On average, six embryos were surgically inovulated. They were classified as type I and III compact morula and blastocysts in four receptor queens. Three animals presented pregnancy confirmed by ultrasound at day 36 and two of these animals gave birth to litters of two and four offsprings, respectively, at 66 and 63 days after induction of ovulation. Except for one still birth, all the offspring developed normally.

A new control strategy with saturation effect compensation for an autonomous induction generator drivenby wide speed range turbines

This paper presents a variable speed autonomous squirrel cage generator excited by a current-controlled voltage source inverter to be used in stand-alone micro-hydro power plants. The paper proposes a system control strategy aiming to properly excite the machine as well as to achieve the load voltage control. A feed-forward control sets the appropriate generator flux by taking into account the actual speed and the desired load voltage. A load voltage control loop is used to adjust the generated active power in order to sustain the load voltage at a reference value. The control system is based on a rotor flux oriented vector control technique which takes into account the machine saturation effect. The proposed control strategy and the adopted system models were validated both by numerical simulation and by experimental results obtained from a laboratory prototype. Results covering the prototype start-up, as well as its steady-state and dynamical behavior are presented. (C) 2011 Elsevier Ltd. All rights reserved.

Follow-up study of morphology and cardiac function in rats undergoing induction of supravalvular aortic stenosis

OBJECTIVE: To characterize the follow-up of an experimental model of left ventricular hypertrophy (LVH) induced by supravalvular ascending aortic stenosis in young rats. METHODS: Wistar rats were submitted to thoracotomy and aortic stenosis was created by placing a clip on the ascending aorta (AoS group, n=12). Age-matched control animals underwent a sham operation (C group, n=12). Cardiac function was analysed by echocardiograms performed 6, 12, and 21 weeks after aortic banding. Myocardial morphological features and myocardial hydroxyproline concentration (HOP) were evaluated 2, 6, 12, and 21 weeks after surgery in additional animals. RESULTS: Aortic banding promoted early concentric LVH and a progressive increase in HOP. Under light microscopy, we observed myocyte hypertrophy and wall thickening of the intramural branches of the coronary arteries due to medial hypertrophy. Cardiac function was supranormal after 6 weeks (percentage of fractional shortening - EAo6: 70.3±10.8; C6: 61.3±5.4; p<0.05), and depressed in the last period. Diastolic dysfunction was detected after 12 weeks (ratio of early-to-late filling velocity - EAo12: 4.20±3.25; C12: 1.61±0.16; p<0.05). CONCLUSION: Ascending aortic stenosis promotes concentric LVH with myocardial fibrosis and minimal histological changes. According to the period of evaluation, cardiac function may be improved, normal, or depressed. The model is suitable and useful for studies on pathophysiology and treatment of the different phases of cardiac hypertrophy.

Fractal Dimension in Quantifying Experimental-Pulmonary-Hypertension-Induced Cardiac Dysfunction in Rats

Abstract Background: Right-sided heart failure has high morbidity and mortality, and may be caused by pulmonary arterial hypertension. Fractal dimension is a differentiated and innovative method used in histological evaluations that allows the characterization of irregular and complex structures and the quantification of structural tissue changes. Objective: To assess the use of fractal dimension in cardiomyocytes of rats with monocrotaline-induced pulmonary arterial hypertension, in addition to providing histological and functional analysis. Methods: Male Wistar rats were divided into 2 groups: control (C; n = 8) and monocrotaline-induced pulmonary arterial hypertension (M; n = 8). Five weeks after pulmonary arterial hypertension induction with monocrotaline, echocardiography was performed and the animals were euthanized. The heart was dissected, the ventricles weighed to assess anatomical parameters, and histological slides were prepared and stained with hematoxylin/eosin for fractal dimension analysis, performed using box-counting method. Data normality was tested (Shapiro-Wilk test), and the groups were compared with non-paired Student t test or Mann Whitney test (p < 0.05). Results: Higher fractal dimension values were observed in group M as compared to group C (1.39 ± 0.05 vs. 1.37 ± 0.04; p < 0.05). Echocardiography showed lower pulmonary artery flow velocity, pulmonary acceleration time and ejection time values in group M, suggesting function worsening in those animals. Conclusion: The changes observed confirm pulmonary-arterial-hypertension-induced cardiac dysfunction, and point to fractal dimension as an effective method to evaluate cardiac morphological changes induced by ventricular dysfunction.

Nitric oxide synthase 2 is required for conversion of pro-fibrogenic inflammatory CD133+ progenitors into F4/80+ macrophages in experimental autoimmune myocarditis.

AIMS: Experimental autoimmune myocarditis (EAM) model mirrors important mechanisms of inflammatory dilated cardiomyopathy (iDCM). In EAM, inflammatory CD133(+) progenitors are a major cellular source of cardiac myofibroblasts in the post-inflammatory myocardium. We hypothesized that exogenous delivery of macrophage-colony-stimulating factor (M-CSF) can stimulate macrophage lineage differentiation of inflammatory progenitors and, therefore, prevent their naturally occurring myofibroblast fate in EAM. METHODS AND RESULTS: EAM was induced in wild-type (BALB/c) and nitric oxide synthase 2-deficient (Nos2(-/-)) mice and CD133(+) progenitors were isolated from inflamed hearts. In vitro, M-CSF converted inflammatory CD133(+) progenitors into nitric oxide-producing F4/80(+) macrophages and prevented transforming growth factor-β-mediated myofibroblast differentiation. Importantly, only a subset of heart-infiltrating CD133(+) progenitors expresses macrophage-specific antigen F4/80 in EAM. These CD133(+)/F4/80(hi) cells show impaired myofibrogenic potential compared with CD133(+)/F4/80(-) cells. M-CSF treatment of wild-type mice with EAM at the peak of disease markedly increased CD133(+)/F4/80(hi) cells in the myocardium, and CD133(+) progenitors isolated from M-CSF-treated mice failed to differentiate into myofibroblasts. In contrast, M-CSF was not effective in converting CD133(+) progenitors from inflamed hearts of Nos2(-/-) mice into macrophages, and M-CSF treatment did not result in increased CD133(+)/F4/80(hi) cell population in hearts of Nos2(-/-) mice. Accordingly, M-CSF prevented post-inflammatory fibrosis and left ventricular dysfunction in wild-type but not in Nos2(-/-) mice. CONCLUSION: Active and NOS2-dependent induction of macrophage lineage differentiation abrogates the myofibrogenic potential of heart-infiltrating CD133(+) progenitors. Modulating the in vivo differentiation fate of specific progenitors might become a novel approach for the treatment of inflammatory heart diseases.