Calprotectin, a sensitive marker of inflammation, is robustly assessed in plasma from patients with early or established rheumatoid arthritis by use of different laboratory methods.

Calprotectin (S100A8/S100A9, MRP8/MRP14) is a major leukocyte protein found to be more sensitive than C-Reactive Protein (CRP) and Erythrocyte Sedimentation Rate (ESR) as a marker of inflammation in patients with rheumatoid arthritis (RA). The present objective was to explore the robustness of calprotectin assessments by comparing two different laboratory methods assessing calprotectin in plasma samples from patients with early or established RA. A total of 212 patients with early RA (mean (SD) age 52(13.3) years, disease duration 0.6(0.5) years) and 177 patients with established RA (mean (SD) age 52.9(13.0) years, disease duration 10.0(8.8) years) were assessed by clinical, laboratory, and ultrasound examinations. Frozen plasma samples (-80 °C) were analysed for calprotectin levels at baseline, 1, 2, 3, 6 and 12 months by use of either enzyme-linked immunosorbent assay (ELISA) or fluoroenzyme immunoassay (FEIA). The ELISA technique used kits from Calpro AS and the FEIA technology was assessed on an automated Thermo Fisher Scientific instrument. The results showed high correlations between the two methods at baseline and during follow-up, with Spearman correlation at baseline 0.93 (p < 0.001) in the early and 0.96 (p < 0.001) in the established RA cohorts. The correlations between each of the two calprotectin assessments and clinical examinations had similar range. Calprotectin correlated well with clinical examinations, with at least as high correlations as CRP and ESR. The present study showed similar results for the two analytical methods, supporting the robustness of calprotectin analyses, and suggest calprotectin in plasma to be included in the assessments offered by clinical routine laboratories.

From cohorts to molecules: Adverse impacts of endocrine disrupting mixtures.

Convergent evidence associates exposure to endocrine disrupting chemicals (EDCs) with major human diseases, even at regulation-compliant concentrations. This might be because humans are exposed to EDC mixtures, whereas chemical regulation is based on a risk assessment of individual compounds. Here, we developed a mixture-centered risk assessment strategy that integrates epidemiological and experimental evidence. We identified that exposure to an EDC mixture in early pregnancy is associated with language delay in offspring. At human-relevant concentrations, this mixture disrupted hormone-regulated and disease-relevant regulatory networks in human brain organoids and in the model organisms Xenopus leavis and Danio rerio, as well as behavioral responses. Reinterrogating epidemiological data, we found that up to 54% of the children had prenatal exposures above experimentally derived levels of concern, reaching, for the upper decile compared with the lowest decile of exposure, a 3.3 times higher risk of language delay.

Outcomes of a uniformly active approach to infants born at 22-24 weeks of gestation.

OBJECTIVE: To determine survival and outcomes in infants born at 22-24 weeks of gestation in a centre with a uniformly active approach to management of extremely preterm infants. STUDY DESIGN: Single-centre retrospective cohort study including infants born 2006-2015. Short-term morbidities assessed included retinopathy of prematurity, necrotising enterocolitis, patent ductus arteriosus, intraventricular haemorrhage, periventricular malacia and bronchopulmonary dysplasia. Neurodevelopmental outcomes assessed included cerebral palsy, visual impairment, hearing impairment and developmental delay. RESULTS: Total survival was 64% (143/222), ranging from 52% at 22 weeks to 70% at 24 weeks. Of 133 (93%) children available for follow-up at 2.5 years corrected age, 34% had neurodevelopmental impairment with 11% classified as moderately to severely impaired. Treatment-requiring retinopathy of prematurity, severe bronchopulmonary dysplasia, visual impairment and developmental delay correlated with lower gestational age. CONCLUSIONS: A uniformly active approach to all extremely preterm infants results in survival rates that are not distinctly different across the gestational ages of 22-24 weeks and more than 50% survival even in infants at 22 weeks. The majority were unimpaired at 2.5 years, suggesting that such an approach does not result in higher rates of long-term adverse neurological outcome.

Foreign-born women's lifestyle and health before and during early pregnancy in Sweden.

Objectives: The aims of the study were to investigate foreign-born women's lifestyle and health before and during early pregnancy and compare them with those of Nordic-born women.Methods: Women recruited at antenatal clinics in Sweden answered a questionnaire in Swedish, English or Arabic or by telephone interview with an interpreter. Questions covered pregnancy planning and periconceptional lifestyle and health. The responses of women born in or outside Europe were compared with those of Nordic-born women. The impact of religiousness and integration on periconceptional lifestyle and health was also investigated.Results: Twelve percent of participants (N = 3389) were foreign-born (n = 414). Compared with Nordic women, European and non-European women consumed less alcohol before conception (respectively, adjusted odds ratio [aOR] 0.38; 95% confidence interval [CI] 0.24, 0.58 and aOR 0.14; 95% CI 0.10, 0.19) and during early pregnancy (respectively, aOR 0.61; 95% CI 0.40, 0.91 and aOR 0.20; 95% CI 0.14, 0.29). Non-European women used less tobacco and were less physically active, but body mass index (BMI) did not differ between groups. Self-perceived health, stress and anxiety during early pregnancy did not differ, but non-European women more often had depressive symptoms (aOR 1.67; 95% CI 1.12, 2.51). Non-European women's healthy lifestyle was associated with religiousness but not with the level of integration.Conclusions: Non-European women were overall less likely to engage in harmful lifestyle habits before and during early pregnancy but were more likely to suffer from depressive symptoms in comparison with Nordic women.

Placental location and pregnancy outcomes in nulliparous women: A population-based cohort study.

INTRODUCTION: The impact of placenta previa on pregnancy, delivery and infant outcomes has been extensively studied. However, less is known about the possible association of placental location other than previa with pregnancy outcomes. The aim of this study was to investigate if placental location other than previa is associated with adverse pregnancy, delivery and infant outcomes. MATERIAL AND METHODS: This is a population-based cohort study, with data from the regional population-based Stockholm-Gotland Obstetric Cohort, Sweden, from 2008 to 2014. The study population included 74 087 nulliparous women with singleton pregnancies resulting in live-born infants, with information about placental location from the second-trimester ultrasound screening. The association between placental location (fundal, lateral, anterior or posterior) and pregnancy outcomes was estimated using logistic regression analysis. Odds ratios (OR) with 95% confidence intervals (95% CI) were calculated, and adjustments were made for maternal age, height, country of birth, smoking in early pregnancy, sex of the infant and in vitro fertilization. Main outcome measures were pregnancy, delivery and infant outcomes. RESULTS: Compared with posterior placental location, fundal and lateral placental locations were associated with a number of adverse pregnancy outcomes, the most important being: very preterm birth (<32 weeks of gestation) (adjusted OR [aOR] 1.78, 95% CI 1.18-2.63 and aOR 2.12, 95% CI 1.39-2.25, respectively), moderate preterm birth (32-36 weeks of gestation) (aOR 1.23, 95% CI 1.001-1.51 and aOR 1.62, 95% CI 1.32-2.00, respectively), small-for-gestational-age birth (aOR 1.67, 95% CI 1.34-2.07 and aOR 1.77, 95% CI 1.39-2.25, respectively) and manual removal of the placenta in vaginal births (aOR 3.27, 95% CI 2.68-3.99 and aOR 3.27, 95% CI 2.60-4.10, respectively). Additionally, lateral placental location was associated with preeclampsia (aOR 1.30, 95% CI 1.03-1.65) and severe postpartum hemorrhage (aOR 1.42, 95% CI 1.27-1.82). CONCLUSIONS: Compared with posterior placental location, fundal and lateral placental locations are associated with a number of adverse pregnancy, delivery and infant outcomes.

Risk of fistula formation and long-term health effects after a benign hysterectomy complicated by organ injury: A population-based register study.

INTRODUCTION: There is a paucity of data on the impact of organ injury on long-term outcomes after a hysterectomy for benign indications. The aim of this study was to investigate fistula formation and patient-reported long-term health outcomes after organ injury at the time of a hysterectomy. MATERIAL AND METHODS: This was a population-based study of 22 538 women undergoing a hysterectomy between 2000 and 2014 in Sweden. Their medical history, characteristics of their surgery, and patient-reported outcomes were retrieved from Swedish national health and quality registers. Predictors for fistula formation were investigated with logistic regression and are presented as odds ratios with a 95% CI. RESULTS: Fistulas were reported in 7% of women with organ injuries, compared with 0.4% of those without organ injuries (adjusted odds ratio 15.29 [9.81-23.85]). Laparotomy and postoperative infection were associated with postoperative fistulas. Most of the women reported having better health 1 year after the hysterectomy, but 7% of those with organ injuries and 24% of those with fistulas reported deteriorated health, compared with 2% of women without injuries. CONCLUSIONS: Organ injury at the time of hysterectomy is associated with the development of fistulas involving the female genital tract and increases the proportion of women reporting deteriorated health 1 year after surgery.

The role of anti-citrullinated protein antibody reactivities in an inception cohort of patients with rheumatoid arthritis receiving treat-to-target therapy.

BACKGROUND: Anti-citrullinated protein antibody (ACPA) reactivities precede clinical onset of rheumatoid arthritis (RA), and it has been suggested that ACPA reactivities towards distinct target proteins may be associated with differences in RA phenotypes. We aimed to assess the prevalence of baseline ACPA reactivities in an inception cohort of patients with early RA, and to investigate their associations with disease activity, treatment response, ultrasound findings and radiographic damage. METHODS: Disease-modifying antirheumatic drug (DMARD)-naïve patients with early RA, classified according to the 2010 American College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) criteria, were included in the ARCTIC trial and assessed in the present analysis. During follow up, patients were monitored frequently and treatment was adjusted according to a predetermined protocol, starting with methotrexate monotherapy with prednisolone bridging. Analysis of 16 different ACPA reactivities targeting citrullinated peptides from fibrinogen, alpha-1 enolase, vimentin, filaggrin and histone was performed using a multiplex chip-based assay. Samples from 0, 3, 12 and 24 months were analysed. Controls were blood donors with similar characteristics to the patients (age, gender, smoking status). RESULTS: A total of 217 patients and 94 controls were included. Median [25, 75 percentile] number of ACPA reactivities in all patients was 9 [4, 12], and were most prevalent in anti-cyclic citrullinated peptide /rheumatoid factor-positive patients 10 [7, 12]. Disease activity measures and ultrasound scores at baseline were lower in ACPA reactivity-positive compared to ACPA reactivity-negative patients. ACPA reactivity levels decreased after 3 months of DMARD treatment, most pronounced for fibrinogenß 60-74 to 62% of baseline antibody level, with least change in filaggrin 307-324 to 81% of baseline antibody level, both p < 0.001. However, outcomes in disease activity measures, ultrasound and radiographic scores after 12 and 24 months were not associated with baseline levels or changes in ACPA reactivity levels and/or seroreversion after 3 months. CONCLUSIONS: The clinical relevance of analysing ACPA reactivities in intensively treated and closely monitored early RA was limited, with no apparent associations with disease activity, prediction of treatment response or radiographic progression. Further studies in larger patient materials are needed to understand the role of ACPA reactivities in patients with RA classified according to the 2010 ACR/EULAR criteria and treated according to modern treatment strategies. TRIAL REGISTRATION: www.ClinicalTrials.gov, NCT01205854 . Registered on 21 September 2010.

Effect of remote cesarean delivery on complications during hysterectomy: a cohort study.

BACKGROUND: Cesarean delivery is performed frequently worldwide, and follow-up studies that report complications at subsequent surgery are warranted. OBJECTIVES: The aim of the study was to investigate the association between a previous abdominal delivery and complications during a subsequent hysterectomy and to estimate the fraction of complications that are driven by the presence of adhesions. STUDY DESIGN: This was a longitudinal population-based register study of 25354 women who underwent a benign hysterectomy at 46 hospital units in Sweden 2000-2014. RESULTS: Adhesions were found in 45% of the women with a history of cesarean delivery. Organ injury affected 2.2% of the women. The risk of organ injury (adjusted odds ratio, 1.74; 95% confidence interval, 1.41-2.15) and postoperative infection (adjusted odds ratio, 1.26; 95% confidence interval, 1.15-1.39) was increased with previous cesarean delivery, irrespective of whether adhesions were present or not. The direct effect on organ injury by a personal history of cesarean delivery was estimated to 73%, and only 27% was mediated by the presence of adhesions. Previous cesarean delivery was a predictor of bladder injury (adjusted odds ratio, 1.86; 95% confidence interval, 1.40-2.47) and bowel injury (adjusted odds ratio, 1.83; 95% confidence interval, 1.10-3.03), but not ureter injury. A personal history of other abdominal surgeries was associated with bowel injury (adjusted odds ratio, 2.27; 95% confidence interval, 1.37-3.78), and the presence of endometriosis increased the risk of ureter injury (adjusted odds ratio, 2.15; 95% confidence interval, 1.34-3.44). CONCLUSION: Previous cesarean delivery is associated with an increased risk of complications during a subsequent hysterectomy, but the risk is only partly attributable to the presence of adhesions. Previous cesarean delivery and presence of endometriosis were major predisposing factors of organ injury at the time of the hysterectomy, whereas background and perioperative characteristics were of minor importance.

Ahr2-dependence of PCB126 effects on the swim bladder in relation to expression of CYP1 and cox-2 genes in developing zebrafish.

The teleost swim bladder is assumed a homolog of the tetrapod lung. Both swim bladder and lung are developmental targets of persistent aryl hydrocarbon receptor (AHR(2)) agonists; in zebrafish (Danio rerio) the swim bladder fails to inflate with exposure to 3,3',4,4',5-pentachlorobiphenyl (PCB126). The mechanism for this effect is unknown, but studies have suggested roles of cytochrome P450 1 (CYP1) and cyclooxygenase 2 (Cox-2) in some Ahr-mediated developmental effects in zebrafish. We determined relationships between swim bladder inflation and CYP1 and Cox-2 mRNA expression in PCB126-exposed zebrafish embryos. We also examined effects on ß-catenin dependent transcription, histological effects, and Ahr2 dependence of the effect of PCB126 on swim bladder using morpholinos targeting ahr2. One-day-old embryos were exposed to waterborne PCB126 or carrier (DMSO) for 24h and then held in clean water until day 4, a normal time for swim bladder inflation. The effects of PCB126 were concentration-dependent with EC(50) values of 1.4 to 2.0 nM for induction of the CYP1s, 3.7 and 5.1 nM (or higher) for cox-2a and cox-2b induction, and 2.5 nM for inhibition of swim bladder inflation. Histological defects included a compaction of the developing bladder. Ahr2-morpholino treatment rescued the effect of PCB126 (5 nM) on swim bladder inflation and blocked induction of CYP1A, cox-2a, and cox-2b. With 2nM PCB126 approximately 30% of eleutheroembryos(3) failed to inflate the swim bladder, but there was no difference in CYP1 or cox-2 mRNA expression between those embryos and embryos showing inflated swim bladder. Our results indicate that PCB126 blocks swim bladder inflation via an Ahr2-mediated mechanism. This mechanism seems independent of CYP1 or cox-2 mRNA induction but may involve abnormal development of swim bladder cells.

Epithelial expression of vasoactive intestinal peptide in ulcerative colitis: down-regulation in markedly inflamed colon.

BACKGROUND: Vasoactive intestinal peptide (VIP) has a number of important effects in intestinal physiology and pathology, including in ulcerative colitis (UC). The expression patterns of the predominant VIP receptor in the mucosa (the VPAC1 receptor) are unknown for the mucosa in UC. It is assumed that the sources of VIP in the intestine are the innervation and the inflammatory cells. AIMS: The VIP and VPAC1 receptor expression patterns in the epithelial layer of UC and non-UC patients were examined in the present study. The influence of marked inflammation of the mucosa was evaluated. METHODS: Specimens of the human colon, including the colon of UC patients, were examined concerning expressions of VIP and VPAC1 receptor, focusing on the epithelial layer. Immunohistochemistry and in situ hybridization were utilized. RESULTS: There were VIP mRNA reactions and also marked VPAC1 receptor immunoreactions in the normal and slightly/moderately affected epithelium. VIP mRNA reactions were not detected and VPAC1 immunoreactions were minimal in response to marked mucosal derangement. CONCLUSIONS: The findings suggest that there is a local production of VIP in the epithelial cells in normal and slightly/moderately inflamed mucosa but not in severely inflamed mucosa. Furthermore, a marked downregulation in VPAC1 receptor expressions occurs in the epithelium in severe UC. Based on the knowledge that VIP can have trophic, healing and anti-inflammatory effects, it is likely that the decrease in VIP mRNA and VPAC1 receptor reactions seen in severely affected mucosa in UC may be associated with adverse effects on intestinal function.

Cytochrome p450 1 genes in birds: evolutionary relationships and transcription profiles in chicken and Japanese quail embryos.

BACKGROUND: Cytochrome P450 1 (CYP1) genes are biomarkers for aryl hydrocarbon receptor (AHR) agonists and may be involved in some of their toxic effects. CYP1s other than the CYP1As are poorly studied in birds. Here we characterize avian CYP1B and CYP1C genes and the expression of the identified CYP1 genes and AHR1, comparing basal and induced levels in chicken and quail embryos. METHODOLOGY/PRINCIPAL FINDINGS: We cloned cDNAs of chicken CYP1C1 and quail CYP1B1 and AHR1. CYP1Cs occur in several bird genomes, but we found no CYP1C gene in quail. The CYP1C genomic region is highly conserved among vertebrates. This region also shares some synteny with the CYP1B region, consistent with CYP1B and CYP1C genes deriving from duplication of a common ancestor gene. Real-time RT-PCR analyses revealed similar tissue distribution patterns for CYP1A4, CYP1A5, CYP1B1, and AHR1 mRNA in chicken and quail embryos, with the highest basal expression of the CYP1As in liver, and of CYP1B1 in eye, brain, and heart. Chicken CYP1C1 mRNA levels were appreciable in eye and heart but relatively low in other organs. Basal transcript levels of the CYP1As were higher in quail than in chicken, while CYP1B1 levels were similar in the two species. 3,3',4,5,5'-Pentachlorobiphenyl induced all CYP1s in chicken; in quail a 1000-fold higher dose induced the CYP1As, but not CYP1B1. CONCLUSIONS/SIGNIFICANCE: The apparent absence of CYP1C1 in quail, and weak expression and induction of CYP1C1 in chicken suggest that CYP1Cs have diminishing roles in tetrapods; similar tissue expression suggests that such roles may be met by CYP1B1. Tissue distribution of CYP1B and CYP1C transcripts in birds resembles that previously found in zebrafish, suggesting that these genes serve similar functions in diverse vertebrates. Determining CYP1 catalytic functions in different species should indicate the evolving roles of these duplicated genes in physiological and toxicological processes.

Cytochrome P450 1A, 1B, and 1C mRNA induction patterns in three-spined stickleback exposed to a transient and a persistent inducer.

Cytochrome P450 1 (CYP1) mRNA induction patterns in three-spined stickleback (Gasterosteus aculeatus) were explored for use in environmental monitoring of aryl hydrocarbon receptor (AHR) agonists. The cDNAs of stickleback CYP1A, CYP1B1, CYP1C1, and CYP1C2 were cloned and their basal and induced expression patterns were determined in the brain, gill, liver and kidney. Also, their induction time courses were compared after waterborne exposure to a transient (indigo) or a persistent (3,3',4,4',5-pentacholorbiphenyl PCB 126) AHR agonist. The cloned stickleback CYP1s exhibited a high amino acid sequence identity compared with their zebrafish orthologs and their constitutive tissue distribution patterns largely agreed with those reported in other species. PCB 126 (100 nM) induced different CYP1 expression patterns in the four tissues, suggesting tissue-specific regulation. Both indigo (1 nM) and PCB 126 (10 nM) induced a strong CYP1 expression in gills. However, while PCB 126 gave rise to a high and persistent induction in gills and liver, induction by indigo was transient in both organs. The number of putative dioxin response elements found in each CYP1 gene promoter roughly reflected the induction levels of the genes. The high responsiveness of CYP1A, CYP1B1, and CYP1C1 observed in several organs suggests that three-spined stickleback is suitable for monitoring of pollution with AHR agonists.

Eosinophils express muscarinic receptors and corticotropin-releasing factor to disrupt the mucosal barrier in ulcerative colitis.

BACKGROUND & AIMS: Altered intestinal barrier function has been implicated in the pathophysiology of ulcerative colitis (UC) in genetic, functional, and epidemiological studies. Mast cells and corticotropin-releasing factor (CRF) regulate the mucosal barrier in human colon. Because eosinophils are often increased in colon tissues of patients with UC, we assessed interactions among mast cells, CRF, and eosinophils in the mucosal barrier of these patients. METHODS: Transmucosal fluxes of protein antigens (horseradish peroxidase) and paracellular markers ((51)Cr-EDTA, fluorescein isothiocyanate-dextran 4000) were studied in noninflamed, colonic mucosal biopsy samples collected from 26 patients with UC and 53 healthy volunteers (controls); samples were mounted in Ussing chambers. We also performed fluorescence and electron microscopy of human tissue samples, assessed isolated eosinophils, and performed mechanistic studies using in vitro cocultured eosinophils (15HL-60), mast cells (HMC-1), and a colonic epithelial cell line (T84). RESULTS: Colon tissues from patients with UC had significant increases in permeability to protein antigens compared with controls. Permeability was blocked by atropine (a muscarinic receptor antagonist), α-helical CRF(9-41) (a CRF receptor antagonist), and lodoxamide (a mast-cell stabilizer). Eosinophils were increased in number in UC tissues (compared with controls), expressed the most M2 and M3 muscarinic receptors of any mucosal cell type, and had immunoreactivity to CRF. In coculture studies, carbachol activation of eosinophils caused production of CRF and activation of mast cells, which increased permeability of T84 epithelial cells to macromolecules. CONCLUSIONS: We identified a neuroimmune intercellular circuit (from cholinergic nerves, via eosinophils to mast cells) that mediates colonic mucosal barrier dysfunction in patients with UC. This circuit might exacerbate mucosal inflammation.

Characterization of three lactic acid bacteria and their isogenic ldh deletion mutants shows optimization for YATP (cell mass produced per mole of ATP) at their physiological pHs.

Several lactic acid bacteria use homolactic acid fermentation for generation of ATP. Here we studied the role of the lactate dehydrogenase enzyme on the general physiology of the three homolactic acid bacteria Lactococcus lactis, Enterococcus faecalis, and Streptococcus pyogenes. Of note, deletion of the ldh genes hardly affected the growth rate in chemically defined medium under microaerophilic conditions. However, the growth rate was affected in rich medium. Furthermore, deletion of ldh affected the ability for utilization of various substrates as a carbon source. A switch to mixed acid fermentation was observed during glucose-limited continuous growth and was dependent on the growth rate for S. pyogenes and on the pH for E. faecalis. In S. pyogenes and L. lactis, a change in pH resulted in a clear change in Y(ATP) (cell mass produced per mole of ATP). The pH that showed the highest Y(ATP) corresponded to the pH of the natural habitat of the organisms.

Induction patterns of new CYP1 genes in environmentally exposed rainbow trout.

The cytochrome P4501 (CYP1) gene family comprises four subfamilies in fish: CYP1A, CYP1B, CYP1C, and CYP1D. Only two CYP1 genes, CYP1A1 and CYP1A3, are so far known in rainbow trout (Oncorhynchus mykiss). The present study aimed to identify other CYP1 subfamily genes in rainbow trout, to establish methods for quantitative mRNA expression analysis of these genes, and to determine their basal and induced mRNA expression in gills and liver. Another goal was to examine their mRNA expression in environmentally exposed fish. We cloned four new transcripts, denoted rbCYP1B1, rbCYP1C1, rbCYP1C2, and rbCYP1C3. Levels of these and the previously known rbCYP1A transcripts were determined by real-time PCR in unexposed fish, fish exposed to the potent aryl hydrocarbon receptor (AhR) agonist 3,3',4,4',5-pentachlorobiphenyl (PCB126), and fish caged in various waters in the Uppsala region (Sweden). The mRNA expression patterns observed in unexposed rainbow trout (basal levels) were markedly similar to those reported for orthologous genes in other species. All six transcripts were induced by PCB126 in gills and liver, suggesting all genes to be AhR regulated. The caged fish showed clear rbCYP1 induction in gills at all monitoring sites (up to 70-fold the basal level), whereas the liver responses were weak; induction (up to 5-fold) was recorded only at the Uppsala municipal sewage treatment plant outlet. Gill filament EROD activity was induced at all caging sites. Most interestingly, the rbCYP1 gene response patterns in gills differed among caging sites and among subfamilies. The EROD induction seemed to only reflect induction of rbCYP1A transcription. Response patterns of multiple CYP1 genes in gills and liver could provide an improved monitoring strategy. Such patterns could be used to characterize complex mixtures of AhR agonists and antagonists in aquatic environments.

Induction of cytochrome P450 1 genes and stress response genes in developing zebrafish exposed to ultraviolet radiation.

Ultraviolet (UV) radiation damages cell molecules, and has been suggested to up-regulate mammalian cytochrome P4501 (CYP1) genes through an aryl hydrocarbon receptor (AHR) mediated mechanism. In this study, embryos and larvae of zebrafish (Danio rerio) were exposed to UV to determine the effects on expression of CYP1 and stress response genes in vivo in these fish. Zebrafish embryos were exposed for varying times to UV on two consecutive days, with exposure beginning at 24 and 48h post-fertilization (hpf). Embryos exposed for 2, 4 or 6h twice over 2 days to UVB (0.62 W/m(2); 8.9-26.7 kJ/m(2)) plus UVA (2.05 W/m(2); 29.5-144.6 kJ/m(2)) had moderately (2.4+/-0.8-fold) but significantly up-regulated levels of CYP1A. UVA alone had no effect on CYP1A expression. Proliferating cellular nuclear antigen (PCNA) and Cu-Zn superoxide dismutase (SOD1) transcript levels were induced (2.1+/-0.2 and 2.3+/-0.5-fold, respectively) in embryos exposed to two 6-h pulses of 0.62 W/m(2) UVB (26.8 kJ/m(2)). CYP1A was induced also in embryos exposed to higher intensity UVB (0.93 W/m(2)) for two 3-h or two 4-h pulses (20.1 or 26.8 kJ/m(2)). CYP1B1, SOD1 and PCNA expression was induced by the two 3-h pulses of the higher intensity UVB, but not after two 4-h pulses of the higher intensity UVB, possibly due to impaired condition of surviving embryos, reflected in a mortality of 34% at that UVB dose. A single 8-h long exposure of zebrafish larvae (8dpf) to UVB at 0.93 W/m(2) (26.8 kJ/m(2)) significantly induced CYP1A and CYP1B1 expression, but other CYP1 genes (CYP1C1, CYP1C2 and CYP1D1) showed no significant increase. The results show that UVB can induce expression of CYP1 genes as well stress response genes in developing zebrafish, and that UVB intensity and duration influence the responses.

Cannabinoid receptor-independent cytotoxic effects of cannabinoids in human colorectal carcinoma cells: synergism with 5-fluorouracil.

Cannabinoids (CBs) have been found to exert antiproliferative effects upon a variety of cancer cells, including colorectal carcinoma cells. However, little is known about the signalling mechanisms behind the antitumoural effect in these cells, whether the effects are shared by endogenous lipids related to endocannabinoids, or whether such effects are synergistic with treatment paradigms currently used in the clinic. The aim of this preclinical study was to investigate the effect of synthetic and endogenous CBs and their related fatty acids on the viability of human colorectal carcinoma Caco-2 cells, and to determine whether CB effects are synergistic with those seen with the pyrimidine antagonist 5-fluorouracil (5-FU). The synthetic CB HU 210, the endogenous CB anandamide, the endogenous structural analogue of anandamide, N-arachidonoyl glycine (NAGly), as well as the related polyunsaturated fatty acids arachidonic acid and eicosapentaenoic acid showed antiproliferative and cytotoxic effects in the Caco-2 cells, as measured by using [(3)H]-thymidine incorporation assay, the CyQUANT proliferation assay and calcein-AM fluorescence. HU 210 was the most potent compound examined, followed by anandamide, whereas NAGly showed equal potency and efficacy as the polyunsaturated fatty acids. Furthermore, HU 210 and 5-FU produced synergistic effects in the Caco-2 cells, but not in the human colorectal carcinoma cell lines HCT116 or HT29. The compounds examined produced cytotoxic, rather than antiproliferative effects, by a mechanism not involving CB receptors, since the CB receptor antagonists AM251 and AM630 did not attenuate the effects, nor did pertussis toxin. However, alpha-tocopherol and the nitric oxide synthase inhibitor L-NAME attenuated the CB toxicity, suggesting involvement of oxidative stress. It is concluded that the CB system may provide new targets for the development of drugs to treat colorectal cancer.

New aspects concerning ulcerative colitis and colonic carcinoma: analysis of levels of neuropeptides, neurotrophins, and TNFalpha/TNF receptor in plasma and mucosa in parallel with histological evaluation of the intestine.

BACKGROUND: The levels of neuropeptides, neurotrophins, and TNFalpha (TNFalpha)/TNF receptor in plasma and mucosa for patients with ulcerative colitis (UC) and colonic carcinoma, and concerning plasma also for healthy controls, were examined. Moreover, the relationships between the different substances and the influence of mucosal derangement on the levels were analyzed. METHODS: The levels of VIP, SP, CGRP, BDNF, NGF, and TNFalpha/TNF receptor 1 were measured using ELISA/EIA. RESULTS: Patients with UC demonstrated the highest levels of all analyzed substances in plasma, with the exception of BDNF. However, there were differences within the UC group, patients treated with corticosteroids, and/or nonsteroid antiinflammatory/immunosuppressive treatment having higher plasma levels than those not given these treatments. Patients with colonic carcinoma showed higher SP and TNF receptor 1 levels in plasma compared to healthy controls. Concerning mucosa, the levels of almost all analyzed substances were elevated for patients with UC compared to noncancerous mucosa of colonic carcinoma patients. There were correlations between many of the substances in both plasma and mucosa, especially concerning the 3 neuropeptides examined. There were also marked associations with mucosa derangement. CONCLUSIONS: Via analysis of correlations for the respective patients and via comparisons between the different patient groups, new and original information was obtained. Interestingly, the degree of mucosal affection was markedly correlated with tissue levels of the substances and the treatments were found to be of importance concerning plasma but not tissue levels of these. Combined plasma analysis of neuropeptides, neurotrophins, and TNF receptor 1 may help to distinguish UC and colonic carcinoma patients.

Decrease in binding for the neuropeptide VIP in response to marked inflammation of the mucosa in ulcerative colitis.

The neuropeptide vasoactive intestinal peptide (VIP) is involved in the neuroimmunomodulation of the intestine. In the present study, specimens from the sigmoid colon of ulcerative colitis (UC) and non-UC patients were examined for immunohistochemistry and in vitro receptor autoradiography. Marked occurrence of VIP binding was observed in the mucosa. However, there were very low levels of binding in areas showing pronounced inflammation/derangement. The study shows that marked derangement of the mucosa leads to a distinct decrease in VIP binding. Thus, it is possible that a decrease in trophic and anti-inflammatory VIP effects occurs in areas exhibiting a very marked inflammation.

Role of AHR2 in the expression of novel cytochrome P450 1 family genes, cell cycle genes, and morphological defects in developing zebra fish exposed to 3,3',4,4',5-pentachlorobiphenyl or 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Halogenated agonists for the aryl hydrocarbon receptor (AHR), such as 3,3',4,4',5-pentachlorobiphenyl (PCB126) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), cause developmental toxicity in fish. AHR dependence of these effects is known for TCDD but only presumed for PCB126, and the AHR-regulated genes involved are known only in part. We defined the role of AHR in regulation of four cytochrome P450 1 (CYP1) genes and the effect of PCB126 on cell cycle genes (i.e., PCNA and cyclin E) in zebra fish (Danio rerio) embryos. Basal and PCB126-induced expression of CYP1A, CYP1B1, CYP1C1, and CYP1C2 was examined over time as well as in relation to cell cycle gene expression and morphological effects of PCB126 in developing zebra fish. The four CYP1 genes differed in the time for maximal basal and induced expression, i.e., CYP1B1 peaked within 2 days postfertilization (dpf), the CYP1Cs around hatching (3 dpf), and CYP1A after hatching (14-21 dpf). These results indicate developmental periods when the CYP1s may play physiological roles. PCB126 (0.3-100nM) caused concentration-dependent CYP1 gene induction (EC50: 1.4-2.7nM, Lowest observed effect concentration [LOEC]: 0.3-1nM) and pericardial edema (EC50: 4.4nM, LOEC: 3nM) in 3-dpf embryos. Blockage of AHR2 translation significantly inhibited these effects of PCB126 and TCDD. PCNA gene expression was reduced by PCB126 in a concentration-dependent manner, suggesting that PCB126 could suppress cell proliferation. Our results indicate that the four CYP1 genes examined are regulated by AHR2 and that the effect of PCB126 on morphology in zebra fish embryos is AHR2 dependent. Moreover, the developmental patterns of expression and induction suggest that CYP1 enzymes could function in normal development and in developmental toxicity of PCB126 in fish embryos.