Surface Marker-Defined Head Kidney Granulocytes and B Lymphocytes of Rainbow Trout Express Benzo[a]pyrene-Inducible Cytochrome P4501A Protein

Polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (BaP) are immunotoxic to fish. Metabolism of PAHs in immune cells has been implicated in PAH immunotoxicity in mammals, but for fish the presence of metabolic enzymes in immune cells is less clear. The objective of this study was to examine localization and induction of the BaP-metabolizing biotransformation enzyme, cytochrome P4501A (CYP1A), in head kidney immune cells of rainbow trout (Oncorhynchus mykiss). In the first step, we measured induction of CYP1A-dependent 7-ethoxyresorufin-O-deethylase (EROD) activity and CYP1A protein in head kidney of rainbow trout treated with a single intraperitoneal (ip) injection of 25 mg BaP/kg body weight. From days 3 to 10 postinjection, the BaP treatment led to a significant elevation of EROD and CYP1A protein in head kidney and liver, with CYP1A expression levels in the head kidney being much lower than in the liver. Next, we examined the cellular localization of CYP1A protein in the head kidney cell types: vascular endothelial, endocrine and lymphoid cells. CYP1A immunoreactivity was detectable only in BaP-treated trout, where it was localized in endothelial and lymphoid cells. Finally, we aimed to clarify which of the hematopoietic cell types possess CYP1A protein. Using double immunostaining for CYP1A and surface markers of rainbow trout immune cells, we identified B lymphocytes and granulocytes expressing inducible CYP1A protein and being the likely sites of BaP metabolism in the head kidne

A systematic immunohistochemical survey of the distribution patterns of GH, prolactin, somatolactin, β-TSH, β-FSH, β-LH, ACTH, and α-MSH in the adenohypophysis of Oreochromis niloticus , the Nile tilapia

Fish pituitary plays a central role in the control of growth, development, reproduction and adaptation to the environment. Several types of hormone-secreting adenohypophyseal cells have been characterised and localised in diverse teleost species. The results suggest a similar distribution pattern among the species investigated. However, most studies deal with a single hormone or hormone family. Thus, we studied adjacent sections of the pituitary of Oreochromis niloticus, the tilapia, by conventional staining and immunohistochemistry with specific antisera directed against growth hormone (GH), prolactin (PRL), somatolactin (SL), thyrotropin (β-TSH), follicle-stimulating hormone (β-FSH), luteinising hormone (β-LH), adrenocorticotropic hormone (ACTH) and melanocyte-stimulating hormone (α-MSH). The pituitary was characterised by a close interdigitating neighbourhood of neurohypophysis (PN) and adenohypophysis. PRL-immunoreactive and ACTH-immunoreactive cells were detected in the rostral pars distalis. GH-immunoreactive cells were present in the proximal pars distalis (PPD). A small region of the PPD contained β-TSH-immunoreactive cells, and β-LH-immunoreactive cells covered approximately the remaining parts. Centrally, β-FSH-immunoreactive cells were detected in the vicinity of the GH-containing cells. Some of these cells also displayed β-LH immunoreactivity. The pars intermedia was characterised by branches of the PN surrounded by SL-containing and α-MSH-immunoreactive cells.The ACTH and α-MSH antisera were observed to cross-react with the respective antigens. This cross-reactivity was abolished by pre-absorption. We present a complete map of the distinct localisation sites for the classical pituitary hormones, thereby providing a solid basis for future research on teleost pituitar

Surface Marker-Defined Head Kidney Granulocytes and B Lymphocytes of Rainbow Trout Express Benzo[a]pyrene-Inducible Cytochrome P4501A Protein

Polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (BaP) are immunotoxic to fish. Metabolism of PAHs in immune cells has been implicated in PAH immunotoxicity in mammals, but for fish the presence of metabolic enzymes in immune cells is less clear. The objective of this study was to examine localization and induction of the BaP-metabolizing biotransformation enzyme, cytochrome P4501A (CYP1A), in head kidney immune cells of rainbow trout (Oncorhynchus mykiss). In the first step, we measured induction of CYP1A-dependent 7-ethoxyresorufin-O-deethylase (EROD) activity and CYP1A protein in head kidney of rainbow trout treated with a single intraperitoneal (ip) injection of 25 mg BaP/kg body weight. From days 3 to 10 postinjection, the BaP treatment led to a significant elevation of EROD and CYP1A protein in head kidney and liver, with CYP1A expression levels in the head kidney being much lower than in the liver. Next, we examined the cellular localization of CYP1A protein in the head kidney cell types: vascular endothelial, endocrine and lymphoid cells. CYP1A immunoreactivity was detectable only in BaP-treated trout, where it was localized in endothelial and lymphoid cells. Finally, we aimed to clarify which of the hematopoietic cell types possess CYP1A protein. Using double immunostaining for CYP1A and surface markers of rainbow trout immune cells, we identified B lymphocytes and granulocytes expressing inducible CYP1A protein and being the likely sites of BaP metabolism in the head kidney

Micro-CT imaging of Thiel-embalmed and iodine-stained human temporal bone for 3D modeling

Introduction This pilot study explores whether a human Thiel-embalmed temporal bone is suitable for generating an accurate and complete data set with micro-computed tomography (micro-CT) and whether solid iodine-staining improves visualization and facilitates segmentation of middle ear structures. Methods A temporal bone was used to verify the accuracy of the imaging by first digitally measuring the stapes on the tomography images and then physically under the microscope after removal from the temporal bone. All measurements were compared with literature values. The contralateral temporal bone was used to evaluate segmentation and three-dimensional (3D) modeling after iodine staining and micro-CT scanning. Results The digital and physical stapes measurements differed by 0.01–0.17 mm or 1–19%, respectively, but correlated well with the literature values. Soft tissue structures were visible in the unstained scan. However, iodine staining increased the contrast-to-noise ratio by a factor of 3.7 on average. The 3D model depicts all ossicles and soft tissue structures in detail, including the chorda tympani, which was not visible in the unstained scan. Conclusions Micro-CT imaging of a Thiel-embalmed temporal bone accurately represented the entire anatomy. Iodine staining considerably increased the contrast of soft tissues, simplified segmentation and enabled detailed 3D modeling of the middle ear

Human dendritic cells process and present Listeria antigens for in vitro priming of autologous CD4+ T lymphocytes

The role of human dendritic cells (DC) in the immune response toward intracellularly growing Listeria was analyzed under in vitro conditions using several morphological and functional methods. DC incubated with Listeria innocua and L. monocytogenes, respectively, readily phagocytosed the bacteria. Listeria did not impair viability and immunogenic potential of human DC. Listerial antigens were found to be processed within the lysosomal compartment of DC and colocalized with major histocompatibility complex (MHC) class II molecules, as shown by fluorescence and transmission electron microscopy. DC challenged with apathogenic L. innocua were highly effective in priming autologous naïve T cells (mainly CD4+) in vitro. The T cells strongly proliferated in the presence of DC incubated with L. innocua, which could be significantly inhibited by anti-MHC II mAb. L. innocua-primed T cells were also successfully stimulated by DC harboring the pathogenic L. monocytogenes, either the wild-type strain EGD or the p60 reduced mutant strain RIII. From our results, we conclude that human DC infected with nonpathogenic intracellular bacteria are able to efficiently prime naïve T cells, which are then suitable for recognition of antigens derived from related virulent bacterial species. This in vitro human model provides an interesting tool for basic research in infectious immunology and possibly for a new immunotherap

Organ-specific expression of IGF-I during early development of bony fish as revealed in the tilapia, Oreochromis niloticus , by in situ hybridization and immunohistochemistry: indication for the particular importance of local IGF-I

The cellular sites of insulin-like growth factor I (IGF-I) synthesis in the early developing tilapia (0-140 days post fertilization, DPF) were investigated. IGF-I mRNA and peptide appeared in liver as early as 4 DPF and in gastro-intestinal epithelial cells between 5-9 DPF. In exocrine pancreas, the expression of IGF-I started at 4 DPF and continued until 90DPF. IGF-I production was detected in islets at 6 DPF in non-insulin cells and occurred throughout life. In renal tubules and ducts, IGF-I production started at 8 DPF. IGF-I production in chondrocytes had its onset at 4 DPF, was more pronounced in growing regions and was also found in adults. IGF-I mRNA and peptide appeared in the cytoplasm of skeletal muscle cells at 4 DPF. In gill chloride cells, IGF-I production started at 6 DPF. At 13 DPF, IGF-I was detected in cardiac myocytes. IGF-I-producing epidermal cells appeared at 5 DPF. In brain and ganglia, IGF-I was expressed in virtually all neurones from 6 to 29 DPF, their number decreasing with age. Neurosecretory IGF-I-immunoreactive axons were first seen in the neurohypophysis around 17 DPF. Endocrine cells of the adenohypophysis exhibited IGF-I mRNA at 28 DPF and IGF-I immunoreactivity at 40 DPF. Thus, IGF-I appeared early (4-5 DPF), first in liver, the main source of endocrine IGF-I, and then in organs involved in growth or metabolism. The expression of IGF-I was more pronounced during development than in juvenile and adult life. Local IGF-I therefore seems to have a high functional impact in early growth, metabolism and organogenesi

Distinct organ-specific up- and down-regulation of IGF-I and IGF-II mRNA in various organs of a GH-overexpressing transgenic Nile tilapia

Several lines of GH-overexpressing fish have been produced and characterized concerning organ integrity, growth, fertility and health but few and contradictory data are available on IGF-I that mediates most effects of GH. Furthermore, nothing is known on IGF-II. Thus, the expression of both IGFs in liver and various extrahepatic sites of adult transgenic (GH-overexpressing) tilapia and age-matched wild-type fish was determined by real-time PCR. Both IGF-I and IGF-II mRNA were found in all organs investigated and were increased in gills, kidney, intestine, heart, testes, skeletal muscle and brain of the transgenics (IGF-I: 1.4-4-fold; IGF-II: 1.7-4.2-fold). Except for liver, brain and testis the increase in IGF-I mRNA was higher than that in IGF-II mRNA. In pituitary, no significant change in IGF-I or IGF-II mRNA was detected. In spleen, however, IGF-I and IGF-II mRNA were both decreased in the transgenics, IGF-I mRNA even by the 19-fold. In agreement, in situ hybridisation revealed a largely reduced number of IGF-I mRNA-containing leukocytes and macrophages when compared to wild-type. These observations may contribute to better understanding the reported impaired health of GH-transgenic fish. Growth enhancement of the transgenics may be due to the increased expression of both IGF-I and IGF-II in extrahepatic sites. It is also reasonable that the markedly enhanced expression of liver IGF-II mRNA that may mimick an early developmental stage is a further reason for increased growt

The effect of hypophysectomy on pancreatic islet hormone and insulin-like growth factor I content and mRNA expression in rat

The growth arrest after hypophysectomy in rats is mainly due to growth hormone (GH) deficiency because replacement of GH or insulin-like growth factor (IGF) I, the mediator of GH action, leads to resumption of growth despite the lack of other pituitary hormones. Hypophysectomized (hypox) rats have, therefore, often been used to study metabolic consequences of GH deficiency and its effects on tissues concerned with growth. The present study was undertaken to assess the effects of hypophysectomy on the serum and pancreatic levels of the three major islet hormones insulin, glucagon, and somatostatin, as well as on IGF-I. Immunohistochemistry (IHC), in situ hybridization (ISH), radioimmunoassays (RIA), and Northern blot analysis were used to localize and quantify the hormones in the pancreas at the peptide and mRNA levels. IHC showed slightly decreased insulin levels in the β cells of hypox compared with normal, age-matched rats whereas glucagon in α cells and somatostatin in δ cells showed increase. IGF-I, which localized to α cells, showed decrease. ISH detected a slightly higher expression of insulin mRNA and markedly stronger signals for glucagon and somatostatin mRNA in the islets of hypox rats. Serum glucose concentrations did not differ between the two groups although serum insulin and C-peptide were lower and serum glucagon was higher in the hypox animals. These changes were accompanied by a more than tenfold drop in serum IGF-I. The pancreatic insulin content per gram of tissue was not significantly different in hypox and normal rats. Pancreatic glucagon and somatostatin per gram of tissue were higher in the hypox animals. The pancreatic IGF-I content of hypox rats was significantly reduced. Northern blot analysis gave a 2.6-, 4.5-, and 2.2-fold increase in pancreatic insulin, glucagon, and somatostatin mRNA levels, respectively, in hypox rats, and a 2.3-fold decrease in IGF-I mRNA levels. Our results show that the fall of serum IGF-I after hypophysectomy is accompanied by a decrease in pancreatic IGF-I peptide and mRNA but by partly discordant changes in the serum concentrations of insulin and glucagon and the islet peptide and/or mRNA content of the three major islet hormones. It appears that GH deficiency resulting in a "low IGF-I state” affects translational efficiency of these hormones as well as their secretory responses. The maintenance of normoglycemia in the presence of reduced insulin and elevated glucagon serum levels, both of which would be expected to raise blood glucose, may result mainly from the enhanced insulin sensitivity, possibly due to GH deficiency and the subsequent decrease in IGF-I productio

Insulin-like growth factor I (IGF-I) in a growth-enhanced transgenic (GH-overexpressing) bony fish, the tilapia ( Oreochromis niloticus ): indication for a higher impact of autocrine/paracrine than of endocrine IGF-I

Several lines of growth hormone (GH)-overexpressing fish have been produced and analysed for growth and fertility parameters. However, only few data are available on the growth-promoting hormone insulin-like growth factor I (IGF-I) that mediates most effects of GH, and these are contradictory. Using quantitative real-time RT-PCR, radioimmunoassay, in situ hybridization, immunohistochemistry, and radiochromatography we investigated IGF-I and IGF binding proteins (IGFBPs) in an adult (17 months old) transgenic (GH-overexpressing) tilapia (Oreochromis niloticus). The transgenics showed an around 1.5-fold increase in length and an approximately 2.3-fold higher weight than the non-transgenics. Using radioimmunoassay, the serum IGF-I levels were lower (6.22±0.75ng/ml) in transgenic than in wild-type (15.01±1.49ng/ml) individuals (P=0.0012). Radioimmunoassayable IGF-I in transgenic liver was 4.2-times higher than in wild-type (16.0±2.21 vs. 3.83±0.71ng/g, P=0.0017). No hepatocytes in wild-type but numerous hepatocytes in transgenic liver contained IGF-I-immunoreactivity. RT-PCR revealed a 1.4-times higher IGF-I mRNA expression in the liver of the transgenics (10.51±0.82 vs. 7.3±0.49pg/μg total RNA, P=0.0032). In correspondence, in situ hybridization showed more IGF-I mRNA containing hepatocytes in the transgenics. A twofold elevated IGF-I mRNA expression was determined in the skeletal muscle of transgenics (0.33±0.02 vs. 0.16±0.01pg/μg total RNA, P<0.0001). Both liver and serum of transgenics showed increased IGF-I binding. The increased IGFBP content in the liver may lead to retention of IGF-I, and/or the release of IGF-I into the circulation may be slower resulting in accumulation of IGF-I in the hepatocytes. Our results indicate that the enhanced growth of the transgenics likely is due to enhanced autocrine/paracrine action of IGF-I in extrahepatic sites, as shown here for skeletal muscl

Microglia at center stage: a comprehensive review about the versatile and unique residential macrophages of the central nervous system

Microglia cells are the unique residential macrophages of the central nervous system (CNS). They have a special origin, as they derive from the embryonic yolk sac and enter the developing CNS at a very early stage. They play an important role during CNS development and adult homeostasis. They have a major contribution to adult neurogenesis and neuroinflammation. Thus, they participate in the pathogenesis of neurodegenerative diseases and contribute to aging. They play an important role in sustaining and breaking the blood-brain barrier. As innate immune cells, they contribute substantially to the immune response against infectious agents affecting the CNS. They play also a major role in the growth of tumours of the CNS. Microglia are consequently the key cell population linking the nervous and the immune system. This review covers all different aspects of microglia biology and pathology in a comprehensive way