Concomitant occurence of multiple autoantibodies against human cytochromes P450.

Cytochromes P450 (CYPs) are a large superfamily of heme-containing enzymes that are essential for the metabolism of a variety of endogenous and xenobiotic compounds. The role and the possible diagnostic or prognostic value of the occurrence of anti-CYP autoantibodies (aAbs) in cancer patients are essentially unclear. Recently we reported the monitoring of aAbs against CYP4Z1 and CYP19A1 in breast cancer patients and healthy controls. In the present study, we extended this investigation by screening the sera of 47 lung cancer patients (17 female and 30 male; age range 49-84) and 119 healthy controls (60 female and 59 male; age range 21-72) for the presence of aAbs directed against CYP2D6, CYP4Z1, or CYP17A1, respectively. Determination of anti-CYP aAb levels was done using our previously established ELISA method. Most sera gave low signals while a small fraction showed stronger responses; however, there were no statistically significant differences between the different test groups. Also, there was no significant difference in aAb signals between the various subtypes of lung cancer. Unexpectedly, sera from two female lung cancer patients (age 67 (adenocarcinoma) and 70 (small cell carcinoma)) and from four healthy controls (one female and three male; age range 34-48) showed significantly elevated signals for more than one of the three CYPs tested. These findings corroborate earlier reports that anti-CYP aAbs occur with low frequency in the general population and, moreover, suggest that the simultaneous presence of multiple aAbs targeting different CYPs should be taken into consideration when evaluating anti-CYP aAbs as biomarkers.

Dual effect of selenium loaded chitosan nanoparticles on growth, antioxidant, immune related genes expression, transcriptomics modulation of caspase 1, cytochrome P450 and heat shock protein and Aeromonas hydrophila resistance of Nile Tilapia (Oreochromis niloticus).

Nowadays there is a great attention for nanotechnology in aquaculture production. It has an efficient role in nutrients and drugs delivery, ponds sterilization, water treatment and aquatic diseases reduction. Till now, there is no available data on impact of selenite-loaded chitosan nanoparticles (SeChNPs) on Nile tilapia. Hence, the current study investigated the effects of selenite-loaded chitosan nanoparticles supplementation on the growth, immune, antioxidant and apoptotic related genes as well as resistance to Aeromonas hydrophila of Nile tilapia, Oreochromis niloticus. A total of 400 fish were randomly divided into four groups, and each group retained five replicates. The control group was fed a basal diet (with inorganic se), other groups fed diets supplemented with SeChNPs 0.5, 1 and 2 g/kg diet. The loading concentration of Se to ChNPs was 0.3, 0.6 and 1.2 mg/0.5, 1 and 2 gm respectively. Fish groups fed SeChNPs (0.5 and 1 g/kg) exhibited the highest final body gain, better feed utilization. Additionally, the expression of myostatin gene was down-regulated by 0.2 and 0.3 fold in group fed 0.5 and 1 g/kg SeChNPs when compared with control group. Dietary inclusion of SeChNPs increased serum lysozyme, alternative complement and myeloperoxidase activities and immunoglobulin type M level. Supplementation of SeChNPs at the level of 2 g/kg up-regulated glutathione peroxidase, superoxide dismutase and catalase expression by 1.12, 4.9 and 2.31 folds respectively, in comparison with control group. In contrast, the levels of C- reactive protein and malondialdehyde were reduced. The expression of IL-10, IL-8, TNF-α and IL-1ß genes was up-regulated after dietary inclusion of different levels of SeChNPs in a dose dependent manner. Post-challenge, the highest survival rate was detected in group fed 2 g/kg SeChNPs (93%) in contrast, the control group was displayed the lowest survival rate (45%). After challenge with A. hydrophila, the expression of caspase 1 was up-regulated in groups fed 1 and 2 g/kg of SeChNPs. Moreover, the maximum down-regulation of cytochromes P450 and heat shock protein were found in 2 g/kg SeChNPs supplemented group (reduced by 0.4 and 0.6-fold, respectively, when compared with control group). In conclusion, the ameliorative effects of SeChNPs on Nile tilapia growth resulted from immune stimulatory and free radicals scavenging effects of selenium loaded chitosan nano composite.

Prognostic value of prostaglandin I2 synthase and its correlation with tumor-infiltrating immune cells in lung cancer, ovarian cancer, and gastric cancer.

BACKGROUND: Prostaglandin I2 synthase (PTGIS) is a crucial gene for the synthesis of prostaglandin I2, which has multiple roles in inflammation and immune modulation. However, studies on the prognostic value of PTGIS and its correlation with tumor-infiltrating immune cells in multiple cancers are still rare. RESULTS: Multiple datasets of the Oncomine database showed that PTGIS was expressed at low levels in lung cancer and ovarian cancer compared to the levels in normal tissues. Kaplan-Meier plotter showed that high PTGIS was associated with poor overall survival and progression-free survival in lung, ovarian, and gastric cancers. Moreover, PTGIS expression was significantly positively correlated with infiltrating levels of macrophages and was strongly associated with a variety of immune markers, especially tumor-associated macrophages (TAMs) and T-regulatory cells (Tregs). CONCLUSIONS: High expression of PTGIS could promote the infiltration of TAMs and Tregs in the tumor microenvironment and deteriorate outcomes of patients with lung, ovarian, and gastric cancers. These findings suggest that PTGIS could be taken as a potential biomarker of prognosis and tumor-infiltrating immune cells. METHODS: PTGIS expression was investigated in different datasets of the Oncomine database, and its expression levels in various tumors and corresponding normal tissues were analyzed by the Tumor Immune Estimation Resource (TIMER). Then, the clinical prognostic value of PTGIS was assessed with online public databases. In addition, we initially explored the correlation between PTGIS and tumor-infiltrating immune cells by TIMER and Gene Expression Profiling Interactive Analysis (GEPIA).

Xenobiotica-metabolizing enzyme induction potential of chemicals in animal studies: NanoString nCounter gene expression and peptide group-specific immunoaffinity as accelerated and economical substitutions for enzyme activity determinations?

Xenobiotica-metabolizing enzyme (XME) induction is a relevant biological/biochemical process vital to understanding the toxicological profile of xenobiotics. Early recognition of XME induction potential of compounds under development is therefore important, yet its determination by traditional XME activity measurements is time consuming and cost intensive. A proof-of-principle study was therefore designed due to the advent of faster and less cost-intensive methods for determination of enzyme protein and transcript levels to determine whether two such methods may substitute for traditional measurement of XME activity determinations. The results of the study show that determination of enzyme protein levels by peptide group-specific immunoaffinity enrichment/MS and/or determination of gene expression by NanoString nCounter may serve as substitutes for traditional evaluation methodology and/or as an early predictor of potential changes in liver enzymes. In this study, changes of XME activity by the known standard XME inducers phenobarbital, beta-naphthoflavone and Aroclor 1254 were demonstrated by these two methods. To investigate the applicability of these methods to demonstrate XME-inducing activity of an unknown, TS was also examined and found to be an XME inducer. More specifically, TS was found to be a phenobarbital-type inducer (likely mediated by CAR rather than PXR as nuclear receptor), but not due to Ah receptor-mediated or antioxidant response element-mediated beta-naphthoflavone-type induction. The results for TS were confirmed via enzymatic activity measurements. The results of the present study demonstrate the potential applicability of NanoString nCounter mRNA quantitation and peptide group-specific immunoaffinity enrichment/MS protein quantitation for predicting compounds under development to be inducers of liver XME activity.

Microwaving and Fluorophore-Tyramide for Multiplex Immunostaining on Mouse Adrenals - Using Unconjugated Primary Antibodies from the Same Host Species.

Immunostaining is widely used in biomedical research to show the cellular expression pattern of a given protein. Multiplex immunostaining allows labeling using multiple primary antibodies. To minimize antibody cross-reactivity, multiplex immunostaining using indirect staining requires unlabeled primary antibodies from different host species. However, the appropriate combination of different species antibodies is not always available. Here, we describe a method of using unlabeled primary antibodies from the same host species (e.g., in this case both antibodies are from rabbit) for multiplex immunofluorescence on formalin-fixed paraffin-embedded (FFPE) mouse adrenal sections. This method uses the same procedure and reagents used in the antigen retrieval step to strip the activity of the previously stained primary antibody complex. Slides were stained with the first primary antibody using a general immunostaining protocol followed by a binding step with a biotinylated secondary antibody. Then, an avidin-biotin-peroxidase signal development method was used with fluorophore-tyramide as the substrate. The immunoactivity of the first primary antibody complex was stripped through immersion in a microwaved boiling sodium citrate solution for 8 min. The insoluble fluorophore-tyramide deposition remained on the sample, which allowed the slide to be stained with other primary antibodies. Although this method eliminates most false positive signals, some background from antibody cross-reactivity may remain. If the samples are enriched with endogenous biotin, a peroxidase-conjugated secondary antibody may be used to replace the biotinylated secondary antibody to avoid the false positive from recovered endogenous biotin.

DEHP induces immunosuppression through disturbing inflammatory factors and CYPs system homeostasis in common carp neutrophils.

Di-(2-ethylhexyl) phthalate (DEHP), a common pollutant in the water environment, has been reported to be associated with immune functions, especially aquatic organisms. However, whether DEHP exposure causes neutrophils toxicity in common carp is still unclear. To investigate the toxic effect of DEHP on immune functions, common carp neutrophils were exposed to DEHP (40 µmol/L and 200 µmol/L) for 2 h. The common carp neutrophils exposed to DEHP showed a decrease in neutrophil phagocytosis rate compared with control group. DEHP exposure induced a significant decrease in mRNA expression levels of inflammatory cytokines-related genes (Interleukin-6, Interleukin-8, transforming growth factor, tumor necrosis factor (TNF)-α, TNF-R1, TNF-T1, Interferon (IFN)-2a, IFN-g2b, IFN-g1) in common carp neutrophils, while the expression levels of IL-1ß and IL-10 were increased compared with control group (P < 0.05). Furthermore, the detection of cytochrome P450 enzyme related genes showed that the mRNA expression levels of CYP (cytochrome P450 proteins)-1A, CYP-1B1, CYP-C1, CYP-2K were significantly decreased, and the mRNA expression level of CYP-3A was significantly reduced (P < 0.05). The results indicated that DEHP could affect the phagocytic ability of neutrophils by regulating the expression of inflammatory cytokines and disrupting cytochrome P450 homeostasis, which caused the immunosuppression in common carp.

Hepatic Cytochrome P450 Activity and Nitric Oxide Production During Multiple Ovalbumin Challenges.

BACKGROUND AND OBJECTIVES: Mast cell-mediated allergic diseases are a significant global health problem. Nitric oxide (NO) produced by acute type 1 allergies greatly suppresses hepatic cytochrome P450 (CYP) metabolism. A recent in vitro study demonstrated that repeated FcεRI-mediated activation intrinsically modulates mast cell function. We investigated the effect of ovalbumin (OVA) challenges on CYP activity and NO production under real immune responses. METHODS: After repeated sensitization with OVA once a week, serum nitrate plus nitrite (NOx) and total plasma immunoglobulin E concentrations were measured using commercially available kits. Hepatic microsomal CYP-specific activities and protein expression were determined using typical substrates and by western blot, respectively. In the liver, the levels of inducible NO synthase (iNOS), F4/80, and c-kit mRNA were determined by real-time polymerase chain reaction. Hepatic total NOS activity was measured using a colorimetric assay kit. RESULTS: When mice received multiple OVA challenges, the 11th sensitization elevated NOx concentrations in serum and suppressed the activities of five major CYPs without altering protein expression levels. After the 7th, 11th, and 15th sensitizations, F4/80-positive Kupffer cell and hepatic c-kit-dependent mast cell mRNA levels were similar to those of the control. The 7th and 11th sensitizations increased hepatic iNOS mRNA expression to 15-fold and threefold above control levels, respectively, but did not enhance the total NOS activity in the liver. CONCLUSIONS: Multiple OVA challenges, unlike acute sensitization, greatly reduced serum NOx levels. The challenge-suppressed hepatic CYP metabolism was likely related to the increased serum NOx. Serum NOx may be an endogenous marker for CYP metabolism inhibition in type 1 allergic diseases.

[Effect of B-vitamin deficiency on biochemical, immunologic markers and trace element status of rats and mice of various lines].

Biochemical, vitamin, trace element and immunological changes were searched for the combined nutritional deficiency of vitamins B1, B2, B6 on in vivo models in rats and mice. Female rats of Wistar (W) strain and hybrids of the 1st generation of Dark Aguti and Wistar (DA x W) strains, female mice of BALB/c strain and DBCB tetrahybrids were used in experiment. Animals received for 35 days a balanced diet (control) according to AIN-93 or a similar diet with the exception of vitamins B1, B2, B6 (experimental groups). The content of vitamins B1, B2 in liver, riboflavin blood plasma level and urinary excretion of thiamine, riboflavin and 4-pyridoxic acid were determined, as well as in rats: blood and liver content of α-tocopherol and retinol, blood biochemical indices of lipid and nitrogen metabolism, activity of cytochrome P isoforms-450 (CYP) in liver; in mice: the circulating levels of pro- and anti-inflammatory cytokines of blood plasma, in animals of both species - the content of essential and toxic elements in the kidneys. DAxW rats compared to W and DBCB mice compared to BALB/c were more sensitive to the development of B-vitamin deficiency judging by the B-vitamin status indicators. In the rats of the experimental groups, there were signs of a deterioration in blood and liver levels of vitamin E, multidirectional shifts in vitamin A sufficiency, increased activity of the CYP3A isoform (6ß-TG), a decrease in triglycerides, total protein and albumin fraction levels with an increase in urea level. Manifestation degree of these effects depended on the choice of the animal's line. In mice, the B-vitamin deficiency was characterized by an increase in the levels of proinflammatory cytokines TNF-α, IL-10, IL-Ιß, IL-6 and a decrease in IFN-γ and IL-17A. The content of magnesium, copper, zinc, chromium and silver was lowered, of cesium - was increased in the kidneys of the rats of the experimental groups. In mice, B-vitamin deficiency resulted in diminishment of magnesium, copper, zinc, chromium, selenium, cadmium and lead content, excess accumulation of cobalt and cesium. Some of these biomarkers are supposed to be used in pre-clinical evaluation of the effectiveness of new vitamin complexes, specialized foods and dietary supplements, as well as studies of interactions of various vitamins.

Hepatic cytochrome P450 metabolism suppressed by mast cells in type 1 allergic mice.

Mast cells and Kupffer cells secrete interleukin (IL)-1ß, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α, which stimulate excess nitric oxide (NO) producing-inducible NO synthase (iNOS). Unlike Kupffer cells, immunoglobulin E-sensitized mast cells elicit sustained NO production. We investigated the participation of mast cell-released NO and cytokine-derived iNOS activation in type 1 allergy-suppressed hepatic cytochrome P450 (CYP) metabolism. Aminoguanidine, a selective iNOS inhibitor, completely suppressed serum nitrate plus nitrite (NOx) concentrations after primary and secondary sensitization of ICR mice and markedly attenuated allergy-suppressed hepatic CYP1A2, CYP2C, CYP2E1, and CYP3A activities. In the liver, primary and secondary sensitization enhanced iNOS-stimulating IFN-γ (5-15-fold) and TNF-α (3-5-fold) mRNA levels more than IL-1ß (2-fold) and F4/80-positive Kupffer cell (2-fold) mRNA levels. When mast cell-deficient (-/-) mice were sensitized, hepatic CYP activities were not suppressed. Serum NOx levels in the sensitized -/- mice were similar with those in saline-treated ICR and -/- mice. In the liver of -/- mice, secondary sensitization markedly enhanced mRNA expression of iNOS (20-fold), IFN-γ (15-fold), and TNF-α (3-fold). However, hepatic total NOS activities in -/- mice were not significantly different between saline treatment and sensitization. Similarly, primary and secondary ICR mice did not significantly enhance total NOS activities in the liver and hepatocytes. The total NOS activities observed did not relate to the high levels of iNOS, IFN-γ, and TNF-α mRNA in the liver. Hepatic c-kit-positive mast cells in sensitized ICR mice were maintained at control levels. Therefore, our data suggest that mast cell-released NO participates in type 1 allergy-suppressed CYP1A2, CYP2C, CYP2E1, and CYP3A metabolism.

A longitudinal study of autoantibodies against cytochrome P450 side-chain cleavage enzyme in dogs (Canis lupus familiaris) affected with hypoadrenocorticism (Addison's disease).

Autoantibodies directed against the P450 side chain cleavage enzyme (P450scc) have been recently described in dogs affected with hypoadrenocorticism, consistent with an immune-mediated pathogenesis of this endocrinopathy. In human autoimmune Addison's disease, autoantibodies may have a predictive value, being detectable before clinical signs developing, and have been shown to persist for a period of time after diagnosis. Furthermore, an autoantibody positive status post-diagnosis has been associated with successful remission of Addison's disease following B-cell depletion, suggesting active immunopathology in these cases. The current study was designed to investigate changes in serum P450scc autoantibody status over time in dogs diagnosed with spontaneous hypoadrenocorticism. P450scc autoantibodies were measured using a species-specific radioimmunoprecipitation assay in an initial cohort of 213 dogs, indicating a prevalence of 24%. Thirty two of these dogs had repeat samples (n = 80 in total) available for analysis. Five dogs were consistently P450scc autoantibody positive in all samples, for up to 425 days following first sampling. Three dogs were initially autoantibody positive, then became seronegative at later time points. One dog, a 1 year old female entire standard poodle, was initially negative for P450scc autoantibodies, but seroconverted 18 months after diagnosis. The remaining 23 dogs with multiple samples available were consistently P450scc autoantibody negative. Persistence was not associated with sex (p = .673). This study demonstrates persistence of P450scc autoantibodies in a subset of dogs affected with hypoadrenocorticism and seroconversion over one year post-diagnosis. P450scc autoantibody reactivity in human autoimmune Addison's disease has been associated with sex, with females having a higher prevalence, possibly due to P450scc expression in the ovary acting as an additional source of antigenic stimulation. However, there was no sex difference in autoantibody persistence in the dogs affected with hypoadrenocorticism. Autontibody persistence in dogs with hypoadrenocorticism might represent persistent pathology, due to residual antigenic stimulation and autoimmune inflammation in the adrenal gland.

Evaluation of cellular immunogenicity of recombinant cytochrome p450 cyp141 protein of Mycobacterium tuberculosis in human and mouse model.

Mycobacterium tuberculosis (Mtb) is still considered one of the unsolved problems for the World Health Organization Identifying and selecting an immunogenic antigen capable of generating specific immune responses is generally the goal of all studies being carried out in to designing new vaccines. Accordingly, the present study was conducted to evaluate the immunogenicity of a M. tuberculosis recombinant protein which exist in the regions of the bacterium genome and may be an immunogenic protein. Immunogenicity of purified proteins was measured by PBMC and mouse spleen lymphocytes culturing methods using ELISA after an appropriate amount of time of incubation with Recombinant cytochrome P450 CYP141 protein. Cellular immune responses were determined and compared by measuring IFN-γ and IL4 in human, and mouse groups. The results revealed a high level of IFN-γ in PPD + individuals and the mice immunized with protein and adjuvant. Recombinant cytochrome P450 CYP141 protein proved capable of generating an immune response in mice and people with a history of previous encounters with Mycobacterium tuberculosis bacteria. It, could be considered a tuberculosis vaccine candidate in order to induce a specific effective immune response in both mice and humans.

Activity of Zearalenone in the Porcine Intestinal Tract.

This study demonstrates that low doses (somewhat above the No Observed Adverse Effect Level, NOAEL) of the mycoestrogen zearalenone (ZEN) and its metabolites display multispecificity towards various biological targets in gilts. The observed responses in gilts were surprising. The presence of ZEN and zearalenols (ZELs) did not evoke a response in the porcine gastrointestinal tract, which was attributed to dietary tolerance. Lymphocyte proliferation was intensified in jejunal mesenteric lymph nodes, and lymphocyte counts increased in the jejunal epithelium with time of exposure. In the distal digestive tract, fecal bacterial counts decreased, the activity of fecal bacterial enzymes and lactic acid bacteria increased, and cecal water was characterized by higher genotoxicity. The accompanying hyperestrogenism led to changes in mRNA activity of selected enzymes (cytochrome P450, hydroxysteroid dehydrogenases, nitric oxide synthases) and receptors (estrogen and progesterone receptors), and it stimulated post-translational modifications which play an important role in non-genomic mechanisms of signal transmission. Hyperestrogenism influences the regulation of the host's steroid hormones (estron, estradiol and progesteron), it affects the virulence of bacterial genes encoding bacterial hydroxysteroid dehydrogenases (HSDs), and it participates in detoxification processes by slowing down intestinal activity, provoking energy deficits and promoting antiporter activity at the level of enterocytes. In most cases, hyperestrogenism fulfils all of the above roles. The results of this study indicate that low doses of ZEN alleviate inflammatory processes in the digestive system, in particular in the proximal and distal intestinal tract, and increase body weight gains in gilts.

Application of substrate depletion assay to evaluation of CYP isoforms responsible for stereoselective metabolism of carvedilol.

To evaluate the relative contribution of cytochrome P450 (CYP) isoforms responsible for carvedilol (CAR) oxidation, enantioselective metabolism of CAR was investigated in human liver microsomes (HLMs) and recombinant human CYPs by using the substrate depletion assay. CYP2D6 exhibited the highest contribution to the metabolism of R-CAR, followed by CYP3A4, CYP1A2, and CYP2C9, whereas the metabolism of the S-enantiomer was mainly mediated by CYP1A2, followed by CYP2D6 and CYP3A4. In HLMs, metabolism of R- and S-CAR was markedly inhibited by quinidine; R-CAR metabolism (57-61% decrease) was more inhibited than S-CAR metabolism (37-43% decrease), and furafylline and ketoconazole almost equally inhibited metabolism of both enantiomers by 25-32% and 30-50%, respectively. The absence of CYP2D6 in a mixture of five major recombinant CYP isoforms at the approximate ratio as in HLMs resulted in a 42% and 25% decrease in the metabolic activities for R- and S-CAR, respectively. Moreover, the absence of CYP1A2 in the mixture resulted in a 16% and 39% decrease in the metabolic activities for R- and S-CAR, respectively. Our results suggest the stereoselective metabolism of CAR is determined by not only the activity of CYP2D6 but also of CYP1A2 and CYP3A4.

Cytochrome P450-Mediated Estrogen Metabolites and Autoimmunity: Relationship and Link to Free Radicals.

Cytochrome P450 enzymes are responsible for the hydroxylation of various endogenous estrogens of the Phase I metabolic pathway. Cytochrome P450s produce hormonally active estrogen metabolites that are typically reactive and mutagenic. Although these metabolites are known to have important roles in autoimmunity, the underlying mechanism of this remains unknown. Here we report that cytochrome P450-mediated estrogen metabolites produce high ROS concentrations that can result in DNA damage. Such DNA damage can alter its immunogenicity, resulting in the induction and elevation of autoantibody concentrations, thus generating various autoimmune conditions. Here we focus on the mechanisms through which cytochrome P450-catalyzed estrogen metabolites induce immune responses and subsequently produce the autoimmune phenomenon.

CYP4F18-Deficient Neutrophils Exhibit Increased Chemotaxis to Complement Component C5a.

CYP4Fs were first identified as enzymes that catalyze hydroxylation of leukotriene B4 (LTB4). CYP4F18 has an unusual expression in neutrophils and was predicted to play a role in regulating LTB4-dependent inflammation. We compared chemotaxis of wild-type and Cyp4f18 knockout neutrophils using an in vitro assay. There was no significant difference in the chemotactic response to LTB4, but the response to complement component C5a increased 1.9-2.25-fold in knockout cells compared to wild-type (P < 0.01). This increase was still observed when neutrophils were treated with inhibitors of eicosanoid synthesis. There were no changes in expression of other CYP4 enzymes in knockout neutrophils that might compensate for loss of CYP4F18 or lead to differences in activity. A mouse model of dextran sodium sulfate colitis was used to investigate the consequences of increased C5a-dependent chemotaxis in vivo, but there was no significant difference in weight loss, disease activity, or colonic tissue myeloperoxidase between wild-type and Cyp4f18 knockout mice. This study demonstrates the limitations of inferring CYP4F function based on an ability to use LTB4 as a substrate, points to expanding roles for CYP4F enzymes in immune regulation, and underscores the in vivo challenges of CYP knockout studies.

Xenobiotic and Endobiotic Mediated Interactions Between the Cytochrome P450 System and the Inflammatory Response in the Liver.

The liver is a unique organ in the body as it has significant roles in both metabolism and innate immune clearance. Hepatocytes in the liver carry a nearly complete complement of drug metabolizing enzymes, including numerous cytochrome P450s. While a majority of these enzymes effectively detoxify xenobiotics, or metabolize endobiotics, a subportion of these reactions result in accumulation of metabolites that can cause either direct liver injury or indirect liver injury through activation of inflammation. The liver also contains multiple populations of innate immune cells including the resident macrophages (Kupffer cells), a relatively large number of natural killer cells, and blood-derived neutrophils. While these cells are primarily responsible for clearance of pathogens, activation of these immune cells can result in significant tissue injury during periods of inflammation. When activated chronically, these inflammatory bouts can lead to fibrosis, cirrhosis, cancer, or death. This chapter will focus on interactions between how the liver processes xenobiotic and endobiotic compounds through the cytochrome P450 system, and how these processes can result in a response from the innate immune cells of the liver. A number of different clinically relevant diseases, as well as experimental models, are currently available to study mechanisms related to the interplay of innate immunity and cytochrome P450-mediated metabolism. A major focus of the chapter will be to evaluate currently understood mechanisms in the context of these diseases, as a way of outlining mechanisms that dictate the interactions between the P450 system and innate immunity.

Eicosanoid storm in infection and inflammation.

Controlled immune responses to infection and injury involve complex molecular signalling networks with coordinated and often opposing actions. Eicosanoids and related bioactive lipid mediators derived from polyunsaturated fatty acids constitute a major bioactive lipid network that is among the most complex and challenging pathways to map in a physiological context. Eicosanoid signalling, similar to cytokine signalling and inflammasome formation, has primarily been viewed as a pro-inflammatory component of the innate immune response; however, recent advances in lipidomics have helped to elucidate unique eicosanoids and related docosanoids with anti-inflammatory and pro-resolution functions. This has advanced our overall understanding of the inflammatory response and its therapeutic implications. The induction of a pro-inflammatory and anti-inflammatory eicosanoid storm through the activation of inflammatory receptors by infectious agents is reviewed here.

Development of a lateral flow test to detect metabolic resistance in Bemisia tabaci mediated by CYP6CM1, a cytochrome P450 with broad spectrum catalytic efficiency.

Cotton whitefly, Bemisia tabaci (Genn.) (Homoptera: Aleyrodidae) is a major sucking pest in many agricultural and horticultural cropping systems globally. The frequent use of insecticides of different mode of action classes resulted in populations resisting treatments used to keep numbers under economic damage thresholds. Recently it was shown that resistance to neonicotinoids such as imidacloprid is linked to the over-expression of CYP6CM1, a cytochrome P450 monooxygenase detoxifying imidacloprid and other neonicotinoid insecticides when recombinantly expressed in insect cells. However over-expression of CYP6CM1 is also known to confer cross-resistance to pymetrozine, an insecticide not belonging to the chemical class of neonicotinoids. In addition we were able to demonstrate by LC-MS/MS analysis the metabolisation of pyriproxyfen by recombinantly expressed CYP6CM1. Based on our results CYP6CM1 is one of the most versatile detoxification enzymes yet identified in a pest of agricultural importance, as it detoxifies a diverse range of chemical classes used to control whiteflies. Therefore we developed a field-diagnostic antibody-based lateral flow assay which detects CYP6CM1 protein at levels providing resistance to neonicotinoids and other insecticides. The ELISA based test kit can be used as a diagnostic tool to support resistance management strategies based on the alternation of different modes of action of insecticides.

Preparation and application of anti-peptide antibodies for detection of orphan cytochromes P450.

OBJECTIVES: Cytochromes P450 (CYP) are monooxygenases, which metabolize mostly hydrophobic endogenous and exogenous compounds. CYPs without any clear connection to metabolism are called "orphans". Interestingly, these "orphan" CYPs are over-expressed in tumor tissues. Thus, the main aim of the paper is the development of antibodies for immunodetection of these CYPs as potential malignancy markers. METHODS: Unique sequences of CYP2S1 and 2W1 were selected and peptides synthesized. Chickens were immunized with peptides bound to hemocyanin (KLH). The antibodies were isolated from egg yolks and their reactivity was tested by ELISA. Antibodies were further affinity purified on immobilized peptides. Western blots containing CYP2S1 and 2W1 standards were developed with purified antibodies. RESULTS: Using unique peptide immunogens of CYP2S1 and 2W1 the antibodies were developed. As judged from ELISA all chickens produced specific antibodies against the respective peptides. Both affinity purified antibodies against CYP2S1 peptide recognized the CYP2S1 standard on Western blots, but only one of four anti-peptide antibodies against CYP2W1 reacted with CYP2W1 standard. The antibodies were used for the detection of CYPs in cancer cell lines and human tissues samples. Although both CYPs were frequently co-expressed in cancer cells, CYP2S1 was solely induced in the cell line BxPC3, while CYP2W1 was predominantly present in cell lines MCF7 and HeLa. Our data show that anti-peptide antibodies are an indispensable tool for detection of homologous CYPs. CONCLUSIONS: The anti-peptide antibodies successfully recognized CYP2S1 and 2W1 in the cancer cell lines and tissue samples.