Induction of a putative monooxygenase of crabs (Carcinus spp.) by polycyclic aromatic hydrocarbons

As part of a programme to develop biomarker assays for polycyclic aromatic hydrocarbons (PAHs) in marine invertebrates, two species of crabs, Carcinus maenas and Carcinus aestuarii were exposed to benzo(a)pyrene (B(a)P) or crude oil. Microsomes were prepared from the midgut gland (hepatopancreas), examined by gel electrophoresis and Western blotting and assayed for B(a)P monooxygenase activity. In early experiments there was evidence of protein degradation and results were inconsistent and inconclusive. However, when steps were taken to minimize this in subsequent experiments, including the inclusion of four protease inhibitors in the homogenization buffer, there was consistent evidence for an increase of proteins of estimated molecular weight 45-60 kDa, and particularly of a distinct band at c. 48 kDa, following exposure to PAH at levels down to 0.1 ppm in ambient water. In C. aestuarii the increase in this band was found to coincide with an 8-12-fold increaseof B(a)P monooxygenase activity in midgut gland microsomes. These results suggest that one or more forms of cytochrome P450 may be induced by PAHs in these species. However, Western blotting using antibodies raised to vertebrate P450s, and representing four different gene families, failed to recognize any proteins in either the PAH-treated samples or in the controls. The isolation and characterization of induced protein, and the production of antibodies may provide the basis for a biomarker assay to measure a response to environmental PAHs in crabs.     

Genetic Determinants of Height Growth Assessed Longitudinally from Infancy to Adulthood in the Northern Finland Birth Cohort 1966

Recent genome-wide association (GWA) studies have identified dozens of common variants associated with adult height. However, it is unknown how these variants influence height growth during childhood. We derived peak height velocity in infancy (PHV1) and puberty (PHV2) and timing of pubertal height growth spurt from parametric growth curves fitted to longitudinal height growth data to test their association with known height variants. The study consisted of N=3,538 singletons from the prospective Northern Finland Birth Cohort 1966 with genotype data and frequent height measurements (on average 20 measurements per person) from 0–20 years. Twenty-six of the 48 variants tested associated with adult height (p<0.05, adjusted for sex and principal components) in this sample, all in the same direction as in previous GWA scans. Seven SNPs in or near the genes HHIP, DLEU7, UQCC, SF3B4/SV2A, LCORL, and HIST1H1D associated with PHV1 and five SNPs in or near SOCS2, SF3B4/SV2A, C17orf67, CABLES1, and DOT1L with PHV2 (p<0.05). We formally tested variants for interaction with age (infancy versus puberty) and found biologically meaningful evidence for an age-dependent effect for the SNP in SOCS2 (p=0.0030) and for the SNP in HHIP (p=0.045). We did not have similar prior evidence for the association between height variants and timing of pubertal height growth spurt as we had for PHVs, and none of the associations were statistically significant after correction for multiple testing. The fact that in this sample, less than half of the variants associated with adult height had a measurable effect on PHV1 or PHV2 is likely to reflect limited power to detect these associations in this dataset. Our study is the first genetic association analysis on longitudinal height growth in a prospective cohort from birth to adulthood and gives grounding for future research on the genetic regulation of human height during different periods of growth.     

Corticotropin-releasing hormone receptor 2-deficient mice have reduced intestinal inflammatory responses

Corticotropin-releasing hormone (CRH) and urocortins (Ucn) bind with various affinities to two G-protein-coupled receptors, CRHR1 and CRHR2, which are expressed in brain and in peripheral tissues, including immune cells. CRHR2-deficient mice display anxiety-like behavior, hypersensitivity to stress, altered feeding behavior and metabolism, and cardiovascular abnormalities. However, the phenotype of these mice in inflammatory responses has not been determined. In the present study we found that compared with wild-type CRHR2-null mice developed substantially reduced intestinal inflammation and had lower intestinal mRNA expression of the potent chemoattractants keratinocyte chemokine and monocyte chemoattractant protein 1 following intraluminal exposure to Clostridium difficile toxin A, a potent enterotoxin that mediates antibiotic-associated diarrhea and colitis in humans. This effect was recapitulated by administration of astressin 2B, a selective CRHR2 antagonist, before toxin A exposure. Moreover, Ab array analysis revealed reduced expression of several inflammatory chemokines, including keratinocyte chemokine and monocyte chemoattractant protein 1 in toxin A-exposed mice pretreated with astressin 2B. Real-time RT-PCR of wild-type mouse intestine showed that only UcnII, but not other Ucn, was significantly up-regulated by ileal administration of toxin A at 4 h compared with buffer exposure. We also found that human colonic epithelial HT-29 cells express CRHR2alpha mRNA, whereas expression of beta and gamma spliced variants was minimal. Moreover, treatment of HT-29 cells with UcnII, which binds exclusively to CRHR2, stimulated expression of IL-8 and monocyte chemoattractant protein 1. Taken together, these results provide direct evidence that CRHR2 mediates intestinal inflammatory responses via release of proinflammatory mediators at the colonocyte level.     

ST-segment elevation associated with allergic reaction to echocardiographic contrast agent administration

We report a case of an allergic reaction after the administration of an echocardiographic contrast agent which resulted in ST-segment elevation. Hypersensitivity and allergic reactions are known causes of acute cardiovascular events. However, only limited reports are available which suggest the exact mechanism of the occurrence of angina or myocardial infarction during severe allergic reactions. In our case, through invasive imaging (coronary angiography and IVUS) we have shown for the first time a transient coronary spasm in the absence of intra-coronary thrombus and only minimal neointimal hyperplasia.     

Graft-versus-host disease is independent of innate signaling pathways triggered by pathogens in host hematopoietic cells

Graft-versus-host disease (GVHD) is initiated by APCs that prime alloreactive donor T cells. In antipathogen responses, Ag-bearing APCs receive signals through pattern-recognition receptors, including TLRs, which induce the expression of costimulatory molecules and production of inflammatory cytokines, which in turn mold the adaptive T cell response. However, in allogeneic hematopoietic stem cell transplantation (alloSCT), there is no specific pathogen, alloantigen is ubiquitous, and signals that induce APC maturation are undefined. To investigate APC activation in GVHD, we used recipient mice with hematopoietic cells genetically deficient in pathways critical for APC maturation in models in which host APCs are absolutely required. Strikingly, CD8-mediated and CD4-mediated GVHD were similar whether host APCs were wild-type or deficient in MyD88, TRIF, or MyD88 and TRIF, which excludes essential roles for TLRs and IL-1β, the key product of inflammasome activation. Th1 differentiation was if anything augmented when APCs were MyD88/TRIF(-/-), and T cell production of IFN-γ did not require host IL-12. GVHD was also intact when APCs lacked the type I IFNR, which amplifies APC activation pathways that induce type I IFNs. Thus in GVHD, alloreactive T cells can be activated when pathways critical for antipathogen T cell responses are impaired.     

Model-Based Tumor Growth Dynamics and Therapy Response in a Mouse Model of De Novo Carcinogenesis

Tumorigenesis is a complex, multistep process that depends on numerous alterations within the cell and contribution from the surrounding stroma. The ability to model macroscopic tumor evolution with high fidelity may contribute to better predictive tools for designing tumor therapy in the clinic. However, attempts to model tumor growth have mainly been developed and validated using data from xenograft mouse models, which fail to capture important aspects of tumorigenesis including tumor-initiating events and interactions with the immune system. In the present study, we investigate tumor growth and therapy dynamics in a mouse model of de novo carcinogenesis that closely recapitulates tumor initiation, progression and maintenance in vivo. We show that the rate of tumor growth and the effects of therapy are highly variable and mouse specific using a Gompertz model to describe tumor growth and a two-compartment pharmacokinetic/ pharmacodynamic model to describe the effects of therapy in mice treated with 5-FU. We show that inter-mouse growth variability is considerably larger than intra-mouse variability and that there is a correlation between tumor growth and drug kill rates. Our results show that in vivo tumor growth and regression in a double transgenic mouse model are highly variable both within and between subjects and that mathematical models can be used to capture the overall characteristics of this variability. In order for these models to become useful tools in the design of optimal therapy strategies and ultimately in clinical practice, a subject-specific modelling strategy is necessary, rather than approaches that are based on the average behavior of a given subject population which could provide erroneous results.     

Isolation and primary cultures of human intrahepatic bile ductular epithelium

Summary A technique for the isolation of human intrahepatic bile ductular epithelium, and the establishment of primary cultures using a serum- and growth-factor-supplemented medium combined with a connective tissue substrata is described. Initial cell isolates and monolayer cultures display phenotypic characteristics of biliary epithelial cells (low molecular weight prekeratin positive; albumin, alphafetoprotein, and Factor VIII-related antigen negative). Ultrastructural features of the cultured cells show cell polarization with surface microvilli, numerous interepithelial junctional complexes and cytoplasmic intermediate prekeratin filaments.     

The effect of Mycoplasma arthritidis infection on the kinetics of colloidal carbon clearance in mice

Abstract The activity of phagocytes from A/J mice was estimated by the carbon clearance test following injection of Mycoplasma arthritidis . Phagocytic activity was significantly depressed 12 h post-infection ( P =0.001) and returned to normal values at 24 h. For animals examined 2 and 7 days post-infection, the overall phagocytic activity increased significantly ( P <10⁻⁴). Phagocytic activity gradually decreased and returned to that of the control group by the end of the fourth week. The relative weights of liver and spleen were significantly increased from the 2nd day post infection ( P =0.0028 and P =0.0014 respectively) and remained increased until the end of the experiment. The early depressive effect on phagocytic activity may be related to superantigen activity with the production of mediators such as macrophage deactivating factor. The later expansion of the macrophage population might bring about the stimulation of autoreactive clones of T and B cells and be responsible for the chronic arthritis that developed in the mycoplasma treated mice.     

Corticotropin-releasing hormone contributes to the peripheral inflammatory response in experimental autoimmune encephalomyelitis

Peripheral corticotropin-releasing hormone (CRH) is thought to have proinflammatory effects. We used the model of experimental autoimmune encephalomyelitis (EAE) to study the role of CRH in an immune-mediated disease. We showed that CRH-deficient mice are resistant to EAE, with a decrease in clinical score as well as decreased cellular infiltration in the CNS. Furthermore, Ag-specific responses of primed T cells as well as anti-CD3/anti-CD28 TCR costimulation were decreased in crh(-/-) mice with decreased production of Th1 cytokines and increased production of Th2 cytokines. Wild-type mice treated in vivo with a CRH antagonist showed a decrease in IFN-gamma production by primed T cells in vitro. This effect of CRH is independent of its ability to increase corticosterone production, because adrenalectomized wild-type mice had similar disease course and severity as control mice. We found that IkappaBalpha phosphorylation induced by TCR cross-linking was decreased in crh(-/-) T cells. We conclude that peripheral CRH exerts a proinflammatory effect in EAE with a selective increase in Th1-type responses. These findings have implications for the treatment of Th1-mediated diseases such as multiple sclerosis.