HET-CAM bioassay as in vitro alternative to the croton oil test for investigating steroidal and non-steroidal compounds.
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In this study the irritation phenomena at the chorioallantoic membrane of incubated hen's eggs as an in vitro model (HET-CAM assay) were investigated in comparison to the in vivo croton oil test by including hydrocortisone, indomethacin, phenylbutazone, acetylsalicylic acid, rutin, quercetin, apigenin, and p-coumaric acid as steroidal and non-steroidal test substances. For the first time the two methods were compared in a valid way with the perspective of a realistic reduction of animal experiments. It should be investigated whether an in vitro-in vivo correlation exists and, if there is any possibility, to replace the in vivo model by an in vitro test system. Both bioassays were able to demonstrate the anti-inflammatory potency of the constituents tested. The determination of the anti-inflammatory activity of all compounds in the two test systems showed individual trends of inhibitory effects. However, the in vitro HET-CAM test was much more sensitive in comparison to the in vivo croton oil test. The croton oil test gave dose-effect correlations in the anti-inflammatory substances investigated. The modified HET-CAM assay did not provide clear dose-effect ratios. The HET-CAM assay is an inexpensive test being easy to manage after a short practical training. Because of its sensitivity the HET-CAM assay could be considered a suitable tool for qualitative testing of the anti-inflammatory activity of substances if no appropriate dose-effect curves are required. From these results it can be concluded that the different courses of the dose-effect curves may be primarily due to different mechanisms of action. (+info)
C-reactive-protein-associated increase in myocardial infarct size after ischemia/reperfusion.
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C-Reactive protein (CRP), a marker for acute inflammation, is associated with increased risk of cardiovascular events. The mechanism underlying this association is uncertain. An acute inflammatory response was induced in rabbits by subcutaneous injection of croton oil (CO) 1 to 3 days before 30 min of regional myocardial ischemia/180 min of reperfusion. CO treatment increased plasma CRP from below the limit of detection to 2.5 +/- 0.5 mg/dl and was associated with an increase in infarct size expressed as percentage of risk region [32 +/- 6% vehicle controls (n = 7) to 47 +/- 9% CO-treated rabbits (n = 7; P < 0.05]. After 10 min of ischemia and 180 min reperfusion, no infarct was found in controls; however, an infarct of 7 +/- 1% was found in CO-treated rabbits (P < 0.05; CRP, 2.3 +/- 0.4 mg/dl). The CRP-related increase in infarct size was not observed in croton oil-treated, C6-deficient rabbits (n = 5/group), indicating the involvement of complement. In these rabbits, infarct size was 22 +/- 2% (P < 0.05) despite having plasma CRP of 4.3 +/- 0.4 mg/dl. The CRP-associated increase in infarct size was ameliorated by pretreatment with heparin (n = 7; infarct size 33 +/- 3%; CRP, 2.3 +/- 0.3 mg/dl; P < 0.05) or N-acetylheparin (n = 7; infarct size 23 +/- 4%; CRP, 3.1 +/- 0.5 mg/dl; P < 0.05). These observations may explain why increased serum CRP is associated with an augmented risk for cardiovascular events. (+info)
An aspirin-triggered lipoxin A4 stable analog displays a unique topical anti-inflammatory profile.
(19/87)
Lipoxins and 15-epi-lipoxins are counter-regulatory lipid mediators that modulate leukocyte trafficking and promote the resolution of inflammation. To assess the potential of lipoxins as novel anti-inflammatory agents, a stable 15-epi-lipoxin A(4) analog, 15-epi-16-p-fluorophenoxy-lipoxin A(4) methyl ester (ATLa), was synthesized by total organic synthesis and examined for efficacy relative to a potent leukotriene B(4) (LTB(4)) receptor antagonist (LTB(4)R-Ant) and the clinically used topical glucocorticoid methylprednisolone aceponate. In vitro, ATLa was 100-fold more potent than LTB(4)R-Ant for inhibiting neutrophil chemotaxis and trans-epithelial cell migration induced by fMLP, but was approximately 10-fold less potent than the LTB(4)R-Ant in blocking responses to LTB(4). A broad panel of cutaneous inflammation models that display pathological aspects of psoriasis, atopic dermatitis, and allergic contact dermatitis was used to directly compare the topical efficacy of ATLa with that of LTB(4)R-Ant and methylprednisolone aceponate. ATLa was efficacious in all models tested: LTB(4)/Iloprost-, calcium ionophore-, croton oil-, and mezerein-induced inflammation and trimellitic anhydride-induced allergic delayed-type hypersensitivity. ATLa was efficacious in mouse and guinea pig skin inflammation models, exhibiting dose-dependent effects on edema, neutrophil or eosinophil infiltration, and epidermal hyperproliferation. We conclude that the LXA(4) and aspirin-triggered LXA(4) pathways play key anti-inflammatory roles in vivo. Moreover, these results suggest that ATLa and related LXA(4) analogs may have broad therapeutic potential in inflammatory disorders and could provide an alternative to corticosteroids in certain clinical settings. (+info)
Modulation of cutaneous inflammation by angiotensin-converting enzyme.
(20/87)
Cutaneous neurogenic inflammation is a complex biological response of the host immune system to noxious stimuli. Present evidence suggests that zinc metalloproteases may play an important role in the regulation of neurogenic inflammation by controlling the local availability of neuropeptides, such as substance P (SP), that are capable of initiating or amplifying cutaneous inflammation after release from sensory nerves. To address the hypothesis that the dipeptidyl carboxypeptidase angiotensin-converting enzyme (ACE) is capable of modulating skin inflammation, we have analyzed murine allergic contact dermatitis (ACD) and irritant contact dermatitis (ICD) using wild-type C57BL/6J (ACE(+/+)) or genetically engineered mice with a heterozygous deletion of somatic ACE (ACE(+/-)). In 2,4-dinitro-1-fluorobenzene-sensitized ACE(+/-) mice, ACD was significantly augmented in comparison to ACE(+/+) controls as determined by the degree of ear swelling after exposure to hapten. Likewise, systemic treatment of ACE(+/+) mice with the ACE inhibitor captopril before sensitization or elicitation of ACD significantly augmented the ACD response. In contrast, local damage and neuropeptide depletion of sensory nerves following capsaicin, injection of a bradykinin B(2), or a SP receptor antagonist before sensitization significantly inhibited the augmented effector phase of ACD in mice with functionally absent ACE. However, in contrast to ACD, the response to the irritant croton oil was not significantly altered in ACE(+/-) compared with ACE(+/+) mice. Thus, ACE by degrading bradykinin and SP significantly controls cutaneous inflammatory responses to allergens but not to irritants, which may explain the frequently observed exacerbation of inflammatory skin disease in patients under medication with ACE inhibitors. (+info)
Exacerbated and prolonged allergic and non-allergic inflammatory cutaneous reaction in mice with targeted interleukin-18 expression in the skin.
(21/87)
Interleukin 18 induces both T helper 1 and T helper 2 cytokines, proinflammatory cytokines, chemokines, and IgE and IgG1 production. A role of interleukin 18 in inflammatory cutaneous reactions is still unclear, however. Here we generated keratin 5/interleukin 18 transgenic mice overexpressing mature murine interleukin 18 in the skin using a human keratin 5 promoter. In the contact hypersensitivity model, trinitrochlorobenzene elicited a stronger ear swelling in keratin 5/interleukin 18 transgenic mice compared with control littermate wild-type or immunoglobulin/interleukin 18 transgenic mice in which mature interleukin 18 was expressed by B and T cells under the control of the immunoglobulin promoter. Application of an irritant, croton oil, induced stronger and more sustained ear swelling in keratin 5/interleukin 18 transgenic mice than in immunoglobulin/interleukin 18 transgenic or wild-type mice. Repetitive topical application (weekly for six consecutive weeks) of trinitrochlorobenzene to their ears also elicited a stronger cutaneous inflammation in keratin 5/interleukin 18 transgenic mice than seen in immunoglobulin/interleukin 18 transgenic or wild-type mice. After these six trinitrochlorobenzene applications, the expression of interferon-gamma, interleukin-4, and CCL20 mRNA in the ear tissue was increased and dermal changes, such as acanthosis and eosinophilic, neutrophilic, and mast cell infiltration, were greater in keratin 5/interleukin 18 transgenic mice than in wild-type mice. Furthermore, the repetitive application elicited a significant increase in serum IgE levels and the number of B cells in the draining lymph node in keratin 5/interleukin 18 transgenic mice. These results suggest that overexpression of interleukin 18 in the skin aggravates allergic and nonallergic cutaneous inflammation, which is accompanied by high expression of T helper 1 and T helper 2 cytokines and chemokines in the skin. (+info)
The fine structure of the basement membrane in epidermal tumors.
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Epidermal tumors were induced in Swiss female mice by a topical application of 9,10-dimethyl-1,2-benzanthracene solution followed by repeated applications of croton oil solutions. Fourteen benign and malignant tumors were sampled 25 weeks after the treatment had begun, fixed in osmium tetroxide or potassium permanganate, and embedded in Epon. Sections stained with lead hydroxide were examined. In three tumors defects of the epidermal basement membrane were seen. These defects accompanied local invasion of the tumors. The possible mechanisms of the development of this unusual anatomical situation are discussed. (+info)
Inhibition of DMBA-croton oil two-stage mouse skin carcinogenesis by diphenylmethyl selenocyanate through modulation of cutaneous oxidative stress and inhibition of nitric oxide production.
(23/87)
Selenium, an essential micronutrient, plays important roles against different diseases, including several types of cancer. In the present study, antioxidative and chemopreventive properties of a synthetic organoselenium compound, diphenylmethyl selenocyanate, were evaluated with a 7,12-dimethylbenz (a) anthracene - croton oil induced two-stage mouse skin carcinogenesis model. The compound was administered orally to carcinogen-treated mice at two different non-toxic doses, 2mg/kg. b.w. and 3mg/kg. b.w. Significant inhibition in the incidence of papilloma formation (53-80%) as well as in the cumulative numbers of papillomas per papilloma bearing mouse were observed in the treated groups as compared to the carcinogen control group. The compound was also found to upregulate significantly different phase II detoxifying enzymes such as glutathione-S-transferase (p<0.01) and superoxide dismutase (p<0.01) in skin cytosol when measured after 15 days and also after 12 weeks of the first 7,12-dimethylbenz (a) anthracene treatment. Lipid peroxidation measured with reference to thiobarbituric acid reactive substances in skin microsomes was significantly inhibited (p<0.05) in a dose dependent manner by diphenylmethyl selenocyanate. Considerable inhibition of the level of nitric oxide production in peritoneal macrophages was observed after 12 weeks (p<0.05). Thus the compound appears to exert chemopreventive activity in terms of papilloma formation, which may be through modulation of cutaneous lipid peroxidation, the phase II detoxifying enzyme system and nitric oxide production. (+info)
Platelet endothelial cell adhesion molecule deficiency or blockade significantly reduces leukocyte emigration in a majority of mouse strains.
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PECAM is a molecule used specifically during the diapedesis step when neutrophils and monocytes leave the blood compartment. Anti-PECAM reagents, such as Abs and soluble fusion proteins, block diapedesis both in vivo and in vitro. However, the PECAM knockout mouse in C57BL/6 strain has no serious defects in most models of inflammation. We show in this study that the same PECAM knockout backcrossed into the FVB/n strain clearly has reduced leukocyte emigration in two models of inflammation. Furthermore, we show that anti-PECAM reagents can block leukocyte emigration in several other wild-type strains of mice like FVB/n, SJL, and the outbred strain Swiss Webster. This clearly shows that the C57BL/6 strain is uniquely able to compensate for the loss of PECAM function. Murine models of inflammatory disease that have been studied using C57BL/6 mice should be re-evaluated using FVB/n or other mouse strains to determine whether PECAM plays a role in those models. (+info)