Chemically-induced Inflammation Research Articles

839 Early biomarker identification for immune sensitization and prevention with oral vitamin D3

Reaction to chemical irritants is a common cause of dermatology consultation and a challenge for topical medication utility. Cholecalciferol (D3) mitigates inflammation from experimental sunburn in humans and chemical injury in mice. To investigate the clinical utility of D3 for chemical injury in humans, we conducted a double-blinded, placebo-controlled trial. 28 healthy subjects had 3x8mm2 of arm skin exposed to 0.0016% topical nitrogen mustard (NM) under occlusion. 2 weeks later, subjects were randomized to receive 200,000 IU oral D3 or placebo after repeat NM exposure on the other arm. By proteomic analysis of skin biopsies, placebo subjects have 22% more differentially expressed inflammatory proteins (DEP) than D3 subjects after 3 days and 650% after 6 weeks. PCA of RNAseq separates samples by exposure yet shows 2 intervention-independent subclusters in NM exposure 2. One shows a normal skin profile (NS) and the other shows immune memory cell enrichment, suggesting immune sensitization (S). S subjects show more inflammation than NS by skin erythema (p<0.01), edema (p=0.05), and histopathologic score (p<1e-4). S and NS D3 subjects express fewer DEPs than placebo at 3 days (S:41%;NS:82%) and 6 weeks (S:86%;NS:100%). Subgroup analysis of exposure 1 identifies early S-associated biomarkers. After exposure 2, NS subjects express no S markers if given D3 vs 75% if given placebo. Results identify biomarkers of sensitization that may have predictive clinical utility and demonstrate that oral D3 mitigates chemically-induced inflammation for up to 6-weeks. Thus, D3 may have preventative properties against chemical sensitization and serve as a low-risk treatment and prevention in those undergoing repetitive chemical exposure.

Chemically-Induced Inflammation Changes the Number of Nitrergic Nervous Structures in the Muscular Layer of the Porcine Descending Colon.

Simple SummaryThe enteric nervous system (ENS) is the part of the nervous system that is located in the wall of the gastrointestinal tract and regulates the majority of the functions of the stomach and intestine. The ENS is characterized by a complex structure and a high degree of independence from the brain. It is known that the ENS changes under the impact of physiological and pathological stimuli. One of the active substances synthetized by enteric neurons is nitric oxide (NO), which is involved in the regulation of intestinal motility, blood flow, secretory activity, and immunological processes in the gastrointestinal tract. In the present study, the influence of chemically-induced inflammatory process on a number of nitrergic neuronal structures located in the muscular layer of the descending colon is investigated. An increase in the number of structures that nitric oxide takes part in is correlated with the inflammatory processes.The enteric nervous system (ENS) is the part of the nervous system that is located in the wall of the gastrointestinal tract and regulates the majority of the functions of the stomach and intestine. Enteric neurons may contain various active substances that act as neuromediators and/or neuromodulators. One of them is a gaseous substance, namely nitric oxide (NO). It is known that NO in the gastrointestinal (GI) tract may possess inhibitory functions; however, many of the aspects connected with the roles of this substance, especially during pathological states, remain not fully understood. An experiment is performed here with 15 pigs divided into 3 groups: C group (without any treatment), C1 group (“sham” operated), and C2 group, in which experimental inflammation was induced. The aim of this study is to investigate the influence of inflammation on nitrergic nervous structures in the muscular layer of the porcine descending colon using an immunofluorescence method. The obtained results show that inflammation causes an increase in the percentage of nitric oxide synthase (nNOS)-positive neurons in the myenteric plexus of the ENS, as well as the number of nitrergic nerve fibers in the muscular layer of the descending colon. The obtained results suggest that NO is involved in the pathological condition of the large bowel and probably takes part in neuroprotective and/or adaptive processes.

Spirometric Pulmonary Restriction in Herbicide-Exposed U.S. Vietnam War Veterans.

Spirometric restriction in herbicide-exposed U.S. Army Chemical Corps Vietnam War veterans was examined because no published research on this topic in Vietnam War veterans exists. Spirometry was conducted on 468 veterans who served in chemical operations in a 2013 study assessing the association between chronic obstructive pulmonary disease (COPD) and herbicide exposure. Exposure was verified based on blood serum values of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Further, the association between herbicide exposure and spirometry restriction (forced expiratory volume in one second (FEV1)/forced vital capacity (FVC) ≥ lower limit of normal (LLN) and FVC < LLN) was tested after adjustment for military characteristics, selected anthropometrics, and other predictors using multivariable regression. Spirometric restriction in herbicide sprayers (15.7%, 95% CI: 10.6, 20.9) was almost twice that of nonsprayers (9.91%, 95% CI: 5.9, 13.9) (p = 0.081). While spirometric restriction was not significantly associated with herbicide exposure (adjusted odds ratio (aOR) = 1.64, 95% CI: 0.82, 3.29) despite the greater prevalence of restriction in sprayers versus nonsprayers, spirometric restriction was significantly associated with race/ethnicity (aOR = 3.04, 95% CI: 1.36, 6.79) and waist circumference (aOR = 2.46, 95% CI: 1.25, 4.85). Because restrictive pulmonary disease may result from chemically-induced inflammation or sensitivity, research on chemical exposures and restriction in veterans should continue. Future study should include full pulmonary function testing, targeted research designs, and a wider set of explanatory variables in analysis, such as other determinants of health.

Psychotria leiocarpa Extract and Vincosamide Reduce Chemically-Induced Inflammation in Mice and Inhibit the Acetylcholinesterase Activity.

Species from Psychotria are used in folk medicine against inflammatory diseases, respiratory disturbances, and anti-hallucinogenic. In the present study, the compound vincosamide (PL-1) was identified for the first time in methanolic extract of the Psychotria leiocarpa (ME-PL) leaves, as well as the anti-inflammatory and anticholinesteric effects in rodents and molecular docking simulations. The fractionation of the chloroform fraction (CF-PL) through chromatographic methods afforded the known compound PL-1. The anti-inflammatory activity of the ME-PL (30, 100, and 300mg/kg) and PL-1 (3, 30, and 100mg/kg) was analyzed using experimental models: paw edema, pleurisy, and mechanical and thermal hyperalgesia induced by carrageenan. The anticholinesterase activity of the ME-PL (30 and 100mg/kg) and PL-1 (30mg/kg) was showed by acetylcholinesterase (AChE) inhibitory in brain structures. The molecular docking simulations were performed using Molegro Virtual Docker v6.0. Overall, the results indicated that ME-PL and PL-1 demonstrated an anti-edematogenic effect in Cg-induced paw edema, leukocyte migration in the pleurisy model, and significantly reduced mechanical hyperalgesia, cold response to acetone in mice. The samples exhibited maximal inhibition of enzyme acetylcholinesterase (AChE) in the frontal cortex. The molecular coupling of PL-1 with the AChE showed significant interactions with the catalytic and peripheral site, corroborating the activity presented in the inhibition assay. The acute administration of ME-PL did not cause signs of toxicity in the treated animals. The results showed that P. leiocarpa inhibited AChE and anti-inflammatory activity, and alkaloid vincosamide could be responsible, at least in part, for the observed effects, supporting the popular use of this genus.

Antioxidant extracts of coffee leaves and its active ingredient 5-caffeoylquinic acid reduce chemically-induced inflammation in mice

Leaves from Coffea arabica L. have been considered an agricultural residue of little value; however, these leaves are a potentially sustainable source of phenolic compounds. Investigation of the properties of these leaves is a crucial strategy for identifying agents that may be beneficial to the health. This study evaluates the antioxidant and anti-inflammatory activity of extracts of C. arabica leaves and 5-caffeoylquinic acid (5-CQA) in a mouse model. Hexane (HFCAL), dichloromethane (DFCAL), ethyl acetate (EFCAL), and butanol (BFCAL) fractions were obtained by partition of the methanol extract (MECAL). High performance liquid chromatography with UV-diode array detection (HPLC-UV-DAD) and spectrophotometric methods were used to identify and quantify the chemical constituents. The antioxidant activity of the extracts were determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP), and β-carotene/linoleic acid assays. Ear edema was induced in Swiss mice by the topical application of Croton oil, phenol, or histamine. Tissues were analyzed histopathologically, and myeloperoxidase and N-acetyl-β-d-glucosaminidase activities were assessed. 5-CQA and mangiferin were identified in MECAL, EFCAL, and BFCAL. The total phenolic and flavonoid contents ranged from 1.65 to 20.60 g gallic acid equivalents/100 g of extract and 0.26 to 14.96 g of rutin equivalents/100 g of extract, respectively. The content of 5-CQA ranged from 2.29 to 5.50 g/100 g of extract, while that of mangiferin was 1.46–4.00 g/100 g of extract. The IC50 values ranged from 7.47 ± 0.12 to 122.76 ± 1.38 μg/mL as determined by DPPH assay and from 4.67 ± 0.02 to 71.90 ± 0.22 μg/mL as determined by FRAP. The greatest inhibitor of lipid peroxidation was HFCAL (49.19 ± 1.10%). The MECAL and 5-CQA (0.1, 0.5, and 1.0 mg/ear) reduced the edema thickness and weight induced by croton oil and phenol. After histamine application, the edema thickness and weight were inhibited by MECAL but not by 5-CQA. Treatment with either MECAL or 5-CQA decreased inflammatory parameters and the activity of myeloperoxidase and N-acetyl-β-d-glucosaminidase. The results suggest that 5-CQA and extracts from coffee leaves (C. arabica) possess antioxidant and anti-inflammatory properties, indicating new possibilities for the treatment of disorders involving oxidative and cutaneous damage.

The influence of experimental inflammation and axotomy on leucine enkephalin (leuENK) distribution in intramural nervous structures of the porcine descending colon

BackgroundThe enteric nervous system (ENS), located in the intestinal wall and characterized by considerable independence from the central nervous system, consists of millions of cells. Enteric neurons control the majority of functions of the gastrointestinal tract using a wide range of substances, which are neuromediators and/or neuromodulators. One of them is leucine–enkephalin (leuENK), which belongs to the endogenous opioid family. It is known that opioids in the gastrointestinal tract have various functions, including visceral pain conduction, intestinal motility and secretion and immune processes, but many aspects of distribution and function of leuENK in the ENS, especially during pathological states, remain unknown.ResultsDuring this experiment, the distribution of leuENK – like immunoreactive (leuENK-LI) nervous structures using the immunofluorescence technique were studied in the porcine colon in physiological conditions, during chemically-induced inflammation and after axotomy. The study included the circular muscle layer, myenteric (MP), outer submucous (OSP) and inner submucous plexus (ISP) and the mucosal layer. In control animals, the number of leuENK-LI neurons amounted to 4.86 ± 0.17%, 2.86 ± 0.28% and 1.07 ± 0.08% in the MP, OSP and ISP, respectively. Generally, both pathological stimuli caused an increase in the number of detected leuENK-LI cells, but the intensity of the observed changes depended on the factor studied and part of the ENS. The percentage of leuENK-LI perikarya amounted to 11.48 ± 0.96%, 8.71 ± 0.13% and 9.40 ± 0.76% during colitis, and 6.90 ± 0.52% 8.46 ± 12% and 4.48 ± 0.44% after axotomy in MP, OSP and ISP, respectively. Both processes also resulted in an increase in the number of leuENK-LI nerves in the circular muscle layer, whereas changes were less visible in the mucosa during inflammation and axotomy did not change the number of leuENK-LI mucosal fibers.ConclusionsLeuENK in the ENS takes part in intestinal regulatory processes not only in physiological conditions, but also under pathological factors. The observed changes are probably connected with the participation of leuENK in sensory and motor innervation and the neuroprotective effects of this substance. Differences in the number of leuENK-LI neurons during inflammation and after axotomy may suggest that the exact functions of leuENK probably depend on the type of pathological factor acting on the intestine.

Zinc Transporter 3 (ZnT3) in the Enteric Nervous System of the Porcine Ileum in Physiological Conditions and during Experimental Inflammation.

Zinc transporter 3 (ZnT3) is a member of the solute-linked carrier 30 (SLC 30) zinc transporter family. It is closely linked to the nervous system, where it takes part in the transport of zinc ions from the cytoplasm to the synaptic vesicles. ZnT3 has also been observed in the enteric nervous system (ENS), but its reactions in response to pathological factors remain unknown. This study, based on the triple immunofluorescence technique, describes changes in ZnT3-like immunoreactive (ZnT3-LI) enteric neurons in the porcine ileum, caused by chemically-induced inflammation. The inflammatory process led to a clear increase in the percentage of neurons immunoreactive to ZnT3 in all “kinds” of intramural enteric plexuses, i.e., myenteric (MP), outer submucous (OSP) and inner submucous (ISP) plexuses. Moreover, a wide range of other active substances was noted in ZnT3-LI neurons under physiological and pathological conditions, and changes in neurochemical characterisation of ZnT3+ cells in response to inflammation depended on the “kind” of enteric plexus. The obtained results show that ZnT3 is present in the ENS in a relatively numerous and diversified neuronal population, not only in physiological conditions, but also during inflammation. The reasons for the observed changes are not clear; they may be connected with the functions of zinc ions and their homeostasis disturbances in pathological processes. On the other hand, they may be due to adaptive and/or neuroprotective processes within the pathologically altered gastrointestinal tract.

RAGE: A novel target for breast cancer growth and metastasis.

Metastasis is a major cause of mortality in Breast Cancer (BC) patients in part due to lack of clinically established targeted therapies. Among the different types of BC, triple negative BC (TNBC) (ER-, PR-, HER2-) has been associated the most with poor prognosis and survival due to early metastasis to other organs and a lack of clinically established targeted therapies. Hence, elucidating novel mechanisms that regulate metastasis would lead to the development of targeted therapies and new treatments for TNBC and metastatic breast cancers. It is now well accepted that solid tumors, including those in the breast, have an inflammatory microenvironment. Receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily of cell surface molecules which has been associated with chronic inflammation, which in turn enhances the progression of various cancers [1]. We have shown that RAGE is expressed in a panel of aggressive BC cell lines and TNBC tissues [3]. High RAGE expression was also observed in lymph node and distant metastases patient samples. In addition, we observed that high RAGE expression was associated with poor prognosis in breast cancer [3]. RAGE has also been shown to play important role in various cancers including pancreatic cancer. Under hypoxic environment, RAGE was shown to interact directly to mutant KRAS and upregulate HIF1α that leads to the development of pancreatic cancer [2]. We have shown that RAGE deficiency inhibits the growth of murine breast cancer tumor cells [3]. RAGEdeficient mice have shown less chemically-induced inflammation [1]. Furthermore, we have shown that RAGE neutralizing antibody treatment significantly inhibited breast cancer metastasis in an intracardial mouse model [3]. RAGE is a multi-ligand receptor and binds to several inflammatory ligands such as advanced glycation end products (AGE), high mobility group box 1 peptide (HMGB-1), amyloid-β peptide and the S100 family of proteins. Our mechanistic investigation has revealed that RAGE mediates its functional effects in breast cancer by binding to S100A7 [3]. S100A7 is a small molecular weight calcium-binding protein [4]. Although a number of putative functions have been proposed for S100A7, its biological role, particularly in BC, remains to be defined [4]. Phylogenetic analyses have shown the mouse ancestor mS100a7a15 to be most related to human S100A7 [5]. It has been shown that mS100a7a15 is up-regulated during carcinogen-induced mammary tumorigenesis. However, the direct functional role of mS100a7a15 in disease progression is not well-characterized. We have shown that mS100a7a15 overexpression induced hyperplasia in mammary glands of these transgenic mice [5]. Upon binding to ligands, RAGE activates its downstream signaling mechanisms that augment and maintain chronic inflammatory conditions [1]. We have also shown that S100A7 enhances NF-kB activation and its nuclear translocation in TNBC cells. These features of RAGE make it an ideal candidate for therapeutic strategies against inflammation induced cancers. The link between inflammation and cancer has been postulated for a long time because of the presence of inflammatory cells in the tumor microenvironment, including myeloid cells and tumor associated macrophages (TAMs) [6]. These cells have been shown to significantly enhance tumor growth and the metastasis of various tumors and are key inhibitors of anti-tumor immunity [6]. In addition, collaborative interactions of tumor cells with TAMs have been associated with poor prognosis in BC [6]. TAMs have also been shown to play an important role in enhancing metastasis and drug resistance [6]. Therefore, inhibition of TAM accumulation and recruitment is a promising strategy for enhancing the effectiveness of immune-based therapies against tumors [6]. We and others have shown that RAGE is also expressed on immune cells [3, 5, 7]. RAGE expression was detected in different cell types present within the tumor microenvironment including macrophages. In addition, RAGE has been shown to be present in the microglia in the gliomas. Chen et al (2014) have shown that RAGE signaling in glioma TAMs regulates angiogenesis most likely by MMP9 [8]. We also showed in breast cancer mouse model that blocking of RAGE signaling significantly inhibited tumor growth, metastasis and angiogenesis through inhibition of MMP9+ TAMs [3]. In addition, RAGE also binds to S100A8/A9 to recruit myeloid derived suppressor cells (MDSCs) and thereby enhance cancer growth and metastasis [7]. These studies suggest that RAGE could enhance tumor growth and metastasis through regulating tumor microenvironment. Overall, our results indicate that RAGE could be used as a novel therapeutic target and biomarker for TNBCs. Furthermore RAGE/S100A7 signaling through paracrine and endocrine mechanisms in epithelial as well as TAMs plays an important role in linking inflammation to breast cancer development and metastasis (Fig. ​(Fig.1).1). In addition, RAGE also binds to S100A8/A9 to recruit myeloid derived suppressor cells (MDSCs) and thereby enhance breast cancer growth and metastasis. Figure 1 Role of S100A7/RAGE-signaling axis in breast cancer growth and metastasis

Afadin requirement for cytokine expressions in keratinocytes during chemically induced inflammation in mice.

Afadin is a filamentous actin-binding protein and a mediator of nectin signaling. Nectins are Ig-like cell adhesion molecules, and the nectin family is composed of four members, nectin-1 to nectin-4. Nectins show homophilic and heterophilic interactions with other nectins or proteins on adjacent cells. Nectin signaling induces formation of cell–cell junctions and is required for the development of epithelial tissues, including skin. This study investigated the role of afadin in epithelial tissue development and established epithelium-specific afadin-deficient (CKO) mice. Although showing no obvious abnormality in the skin development and homeostasis, the mice showed the reduced neutrophil infiltration into the epidermis during chemical-induced inflammation with 12-O-tetradecanoylphorbol 13-acetate (TPA). Immunohistochemical and quantitative real-time PCR analyses showed that the expression levels of cytokines including Cxcl2, Il-1β and Tnf-α were reduced in CKO keratinocytes compared with control keratinocytes during TPA-induced inflammation. Primary-cultured skin keratinocytes from CKO mice also showed reduced expression of these cytokines and weak activation of Rap1 compared with those from control mice after the TPA treatment. These results suggested a remarkable function of afadin, which was able to enhance cytokine expression through Rap1 activation in keratinocytes during inflammation.

Proteome dynamics in neutrophils of adult zebrafish upon chemically-induced inflammation

Neutrophils are the most abundant polymorphonuclear leukocytes, presenting the first line of defence against infection or tissue damage. To characterize the molecular changes on the protein level in neutrophils during sterile inflammation we established the chemically-induced inflammation (ChIn) assay in adult zebrafish and investigated the proteome dynamics within neutrophils of adult zebrafish upon inflammation. Through label-free proteomics we identified 48 proteins that were differentially regulated during inflammation. Gene ontology analysis revealed that these proteins were associated with cell cycle, nitric oxide signalling, regulation of cytoskeleton rearrangement and intermediate filaments as well as immune-related processes such as antigen presentation, leucocyte chemotaxis and IL-6 signalling. Comparison of protein expression dynamics with transcript expression dynamics suggests the existence of regulatory mechanisms confined to the protein level for some genes. This is the first proteome analysis of adult zebrafish neutrophils upon chemically-induced inflammation providing a valuable reference for future studies using zebrafish inflammation models.

Substance P as a neuronal factor in the enteric nervous system of the porcine descending colon in physiological conditions and during selected pathogenic processes

The present investigation pertains to changes in substance P-like immunoreactive (SP-LI) nerve structures of the enteric nervous system (ENS) in the porcine descending colon, caused by chemically-induced inflammation and nerve injury (axotomy). The distribution pattern of SP-LI structures was studied using the double immunofluorescence technique in the myenteric (MP), outer submucous (OSP) and inner submucous (ISP) plexuses, as well as in the circular muscle and mucosal layers. Under physiological conditions, SP-LI neurons have been shown to constitute 4.13 ± 0.24%, 3.36 ± 0.26%, and 7.92 ± 0.16% in the MP, OSP, and ISP, respectively. Changes in SP-immunoreactivity depended on the pathological factor studied. The numbers of the SP-LI perikarya amounted to 7.89 ± 0.34, 5.56 ± 0.30, and 19.96 ± 0.57 in chemically-induced colitis, and 4.28 ± 0.13%, 7.18 ± 20%, and 11.62 ± 0.48% after axotomy in MP, OSP, and ISP, respectively. The both studied processes generally resulted in an increase in the number of SP-LI nerve fibers in the circular muscle and mucosal layers. The obtained results suggest that SP-LI nerve structures of the ENS may participate in various pathological processes in the porcine descending colon and exact functions of SP probably depend on the type of the pathological factor.

Highly heated food rich in Maillard reaction products limit an experimental colitis in mice

Maillard reaction products (MRPs) are a mixture of compounds generated after the heat treatment of food. High circulating levels of MRPs have been associated with degenerative pathologies such as diabetes, but little is known about their effect on the gut, the main organ in contact with food-derived MRPs. This study was aimed at determining whether repeated low-level exposure to MRPs, generated via two different heat treatments, can contribute to the modulation of experimental colitis in mice. In the first series of experiments, we tested whether pellets rich in MRPs would increase plasmatic and faecal concentration of MRPs. In the second series, we assessed whether two levels of pellet-derived MRPs would be able to modulate chemically-induced inflammation and affect tissue healing. The ingestion of MRPs correlates with the increase of its plasmatic and faecal concentration. Highly treated pellets were proved to significantly protect against inflammation whereas standard or moderately heated pellets had no effect on the inflammatory course. The chemical analysis of the different pellets indicated that high heating generates more melanoidins. There is a correlation between the exposure to highly heated foods and the reduction of murine inflammation, of which the mechanisms remain to be elucidated.

Protective Effects of Black Rice Bran against Chemically-Induced Inflammation of Mouse Skin

We investigated the inhibitory effects of black rice (cv. LK1-3-6-12-1-1) bran against 12-O-tetradecanolylphorbol-13-acetate (TPA)-induced skin edema and 2,4-dinitrofluorobenzene (DNFB)-induced allergic contact dermatitis (ACD) in inflammatory mouse models. We also determined the effects of the bran extract on the following biomarkers: pro-inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), eicosanoids leukotriene B4 (LTB4), and prostaglandin E2 (PGE2). Topical application of TPA to ears of CD-1 mice induced inflammation accompanied with substantial increase in TNF-α, IL-1β, IL-6, LTB4, and PGE2 levels and an elevation in intercellular adhesion molecule-1 (ICAM-1) gene expressions in ear skin tissues. Intraperitoneal injection of black rice bran extract prior to TPA application in mice significantly suppressed TPA-induced inflammation (edema) and induced a marked decrease in the production of TNF-α, IL-1β, IL-6, and LTB4. Feeding mice a standard diet with added 10% black rice bran also significantly suppressed DNFB-induced allergic contact dermatitis on the skin of the mice. By contrast, a nonpigmented brown rice bran extract did not inhibit the TPA-induced edema and failed to significantly suppress production of pro-inflammatory biomarkers (mediators). These in vivo findings further demonstrate the potential value of black rice bran as an anti-inflammatory and antiallergic food ingredient and possibly also as a therapeutic agent for the treatment and prevention of diseases associated with chronic inflammation.

Effect of manoalide on human 5-lipoxygenase activity.

The marine natural product manoalide (MLD) has been described to inactivate phospholipase A2 (PLA2) from several sources as well as to inhibit synthesis of eicosanoids in human polymorphonuclear leukocytes (HPMNL). MLD also reduces chemically-induced inflammation in vivo. In this investigation we have examined the effect of MLD on A23187-induced generation of leukotriene B4 (LTB4) and thromboxane B2 (TXB2) in HPMNL as well as 5-lipoxygenase (5-LO) activity from HPMNL sonicated preparations. In the intact cell system, MLD inhibited with similar potency biosynthesis of LTB4 and TXB2 (IC50 1.7 and 1.4 microM, respectively). In order to discern if inhibition of 5-LO is involved in the effect of MLD, we examined the action of this compound on 5-LO activity from 10,000 x g and 100,000 x g supernatants of sonicated HPMNL homogenates. The enzymatic activity was not affected at concentrations of MLD up to 50 microM. These data indicate that MLD is not a direct inhibitor of 5-LO activity from HPMNL and support the hypothesis that its anti-inflammatory action could be related with a reduction of eicosanoid biosynthesis via inhibition of PLA2.

The valdivones, anti-inflammatory diterpene esters from the South African soft coral alcyonium valdivae

The South African soft coral Alcyonium valdivae contains five diterpene esters, valdivones A ( 1) and B ( 2), the corresponding methoxy ketals ( 3) and ( 4), and dihydrovaldivone A ( 5). Although the producing organisms are unrelated taxonomically, the valdivones are most closely related to the sarcodictins (e.g. 6) from the stoloniferan coral Sarcodictyon roseum. Valdivones A ( 1) and B ( 2) inhibit chemically-induced inflammation in the mouse ear assay.

Inflammatory mediators applied to in vitro toxicology: Studies on mediator release and two-cell systems

Release of mediators is a manifestation of tissue stimulation or damage applicable to in vitro toxicological investigations. Intercell mediators, or cytokines, are released on tissue stimulation or as a result of mild damage. Intracell mediators, or enzymes of respiration and metabolism, released from damaged cells, participate in inflammatory change and are the usual mediator determinants in in vitro tests. Cells examined for in vitro mediator release are mast cells, which are applicable to anaphylactic allergy and acute inflammation, macrophages, which are particularly applicable to toxicity of inhalable dusts, and fibroblasts/fibrocytes, which are widely used as a general model for cytotoxicity. In tests on organs, lungs may be perfused to identify cytokines released by test substances, which may be used to distinguish between effects due to allergy and those resulting from chemically-induced inflammation. Skin, both human and animal, as full depth or keratome slices of epidermis, has an application in the detection of irritant and corrosive substances. Weak irritants are identified by release of enzymes, for example acid phosphatase, neutral protease and lactate dehydrogenase. Strong irritants and corrosive substances inhibit release or destroy the enzymes. There are inconsistencies in the identification of moderate irritants, both chemicals and bacterial toxins, in which the rank order of severity of effect does not correspond to other in vitro features or in vivo potency. Detection of cytokines (prostaglandins, leukotrienes and interleukin-1) is possible with appropriate conditions of culture, but has been little applied to toxicology. Studies on two-organ/cell systems are briefly reported in which biological mediator release is detectable without specific identification. Cultured skin preparations were treated in vitro with irritants. The culture media were transferred to cultures of normal skin (examined for stimulus to DNA synthesis), to fibroblasts (examined for DNA and polysaccharide/heparan sulphate synthesis) and to suspensions of neutrophils in Boyden chambers (for chemotaxis). The media were also examined for histamine (none found), acid protease cathepsins, acid phosphatase and lactate dehydrogenase. As before, weak chemical irritants were detectable by stimulation of normal skin and fibroblasts to DNA synthesis, fibroblasts to increased heparan sulphate-like synthesis, and analytically by release of enzymes. Moderate irritants and Clostridium perfringens type C toxins depressed these features. Reference is made to the possibility to identify substances likely to induce progressive pulmonary fibrosis, by transfer to fibrocyte cultures of media from macrophages ingesting the dusts or co-culturing macrophages and fibrocytes, to detect a sustained stimulus to fibrogenesis.