Biological basis of the behavior of sick animals

Summary: The discovery of novel small-molecule antibacterial drugs has been stalled for many years. The purpose of this review is to underscore and illustrate those scientific problems unique to the discovery and optimization of novel antibacterial agents that have adversely affected the output of the effort. The major challenges fall into two areas: (i) proper target selection, particularly the necessity of pursuing molecular targets that are not prone to rapid resistance development, and (ii) improvement of chemical libraries to overcome limitations of diversity, especially that which is necessary to overcome barriers to bacterial entry and proclivity to be effluxed, especially in Gram-negative organisms. Failure to address these problems has led to a great deal of misdirected effort.

Challenges of Antibacterial Discovery

This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell-derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.

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Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME.

The acute phase proteins (APP) are a group of blood proteins that contribute to restoring homeostasis and limiting microbial growth in an antibody-independent manner in animals subjected to infection, inflammation, surgical trauma or stress. In the last two decades, many advances have been made in monitoring APP in both farm and companion animals for clinical and experimental purposes. Also, the mechanism of the APP response is receiving attention in veterinary science in connection with the innate immune systems of animals. This review describes the results of recent research on animal APP, with special reference to their induction and regulatory mechanisms, their biological functions, and their current and future applications to veterinary diagnosis and animal production.

Current research on acute phase proteins in veterinary diagnosis: an overview.

Physiology of mastitis and factors affecting somatic cell counts.

Summary Metabolic disturbances following intravenous and intramammary administration of endotoxins in ruminants are described. In contrast to the similarity in response of blood biochemical parameters after intravenous and intramammary administrations of endotoxins, responses in plasma concentrations of enzyme activities, the thyroid hormones, cortisol, and somatotropin differ markedly. Biochemical changes in blood after endotoxin administration are predominantly dose‐dependent; thus some of the biochemical parameters ‐ especially plasma concentrations of Fe and Zn ‐ serve also to evaluate the effects of certain drugs in endotoxin models. Changes in milk composition have been documented only after intramammary infusion of endotoxins and can partly be explained by the increased permeability of the blood/milk barrier. Appearance and production of milk returns to normal within a week after intramammary endotoxin treatment, indicating that the mammary gland is only temporarily damaged by endotoxin‐induced mas...

Pathophysiological effects of endotoxins in ruminants. 1. Changes in body temperature and reticulo-rumen motility, and the effect of repeated administration.

Suppression of cytochrome P450- and UDP glucuronosyl transferase-dependent enzyme activities by proinflammatory cytokines and possible role of nitric oxide in primary cultures of pig hepatocytes.

For several years it is known that β-adrenergic receptor agonists have anti-inflammatory effects. However, little is known about the role of β- adrenergic receptors on macrophages in the modulation of cytokine production by β-agonists during inflammation. In this study, the presence of β- receptors on PMA-differentiated U937 human macrophages, and the participation of these receptors in the modulation of LPS-mediated cytokine production by β-agonists was investigated. Total β-receptor expression on undifferentiated (monocyte) and PMA-differentiated U937 cells was established using receptor binding studies on membrane fractions with a radio ligand. The expression of β-receptors proved to be significantly lower on monocytes than on macrophages, additionally a predominant expression of β2-receptors was found. Production of the cytokines TNF-α, IL-6, and IL-10 by LPS-stimulated differentiated U937 cells was measured in time. Peak concentrations for TNF- α, IL-76 and IL-10 occurred at 3, 12 and 9 hrs, respectively. When differentiated U937 cells were incubated with both LPS and the β-agonist clenbuterol the production of TNF-α and IL-6 was significantly reduced. However the production of IL-10 was increased. To study the mechanism of modulation of cytokine production in more detail, U937 macrophages were incubated with LPS/clenbuterol in combination with selective β1- and β2- antagonists. These results indicated that the β2- and not the β1- receptor is involved in the anti-inflammatory activity of clenbuterol.

Participation of β-Adrenergic Receptors on Macrophages in Modulation of LPS-Induced Cytokine Release

Objective and Design: To investigate the suppressive effects of the β-agonist clenbuterol on the release of TNF-α and IL-6 in a lipopolysaccharide (LPS)-model of inflammation, both in vitro and in vivo. Material and Subjects: Human U-937 cell line (monocyte-derived macrophages), and male Wistar rats (200-250 g). Treatment: U-937 macrophages were incubated with LPS at 1 μg/ml, with or without 1.0 mM-0.1 nM test drugs (clenbuterol and other cAMP elevating agents) for 1-24 h. Rats were administered either 1 or 10 μg/kg clenbuterol (or saline) orally, 1 h before intraperitoneal administration of 2 mg/kg LPS. Methods and Results: TNF-α and IL-6 time-concentration profiles were determined both in culture media and plasma, using ELISA's and bioassays. LPS-mediated release of both cytokines was significantly suppressed by clenbuterol. Conclusions: The β-agonist clenbuterol very potently suppresses the LPS-induced release of the pro-inflammatory cytokines TNF-α and IL-6 both in vitro and in vivo.

The β-adrenoceptor agonist clenbuterol is a potent inhibitor of the LPS-induced production of TNF-α and IL-6 in vitro and in vivo

Summary

After a review of the literature the reactions of the different animal species to the parenteral administration of a single batch of E. coli 0111 B 4 lipopolysaccharide are described. Special attention has been given to the effects on body temperature, circulating white blood cells and the gastrointestinal system (vomiting, diarrhoea, hypomotility of the rumen). It is concluded that for these effects and this pyrogen the following list of diminishing sensitivity can be given: rabbit, horse, goat, dog, cow, sheep, cat, swine and chicken. The mouse, rat and guinea pig take a separate place by the absence of a fever reaction or a leucocyte response.



The development of tolerance to repeated injections was studied in the goat.



Acknowledgements



We acknowledge the technical assistance of Miss. C. Th. M. van Duin, P. I. Lagendijk, J. M. Eyndhoven and the help of the veterinary students J. Atsma, H. H. Stigter and W. de Haan.



Animals and experimental facilities were provided by the Institute of Veterinary Physiology and the Clinics for Veterinary Obstetrics, Surgery and Internal Medicine of the Veterinary Faculty of the University of Utrecht, the Institute for Animal Physiology of the Agricultural University Wageningen, and the Hoorn Institute of Feeding Research.



Zusammenfassung

Die Reaktion verschiedener Tierarten auf bakterielle Pyrogene



Nach der Besprechung der einschlagigen Literatur werden die Reaktionen verschiedener Tierarten auf die Applikation von E. coli 0111 B 4-Lipopolysaccharid beschrieben.



Besondere Beachtung fanden hierbei: die Korpertemperatur, die Leukozyten im zirkulierenden Blut und das Verhalten des Magendarmtraktes (Erbrechen, Durchfall, herabgesetzte Pansenmotorik).



Nach ihrer Empfindlichkeit — Reihenfolge entsprechend abnehmender Sensitivitat — kann folgende Liste aufgestellt werden: Kaninchen, Pferd, Ziege, Hund, Rind, Schaf, Katze, Schwein und Huhn. Maus, Ratte und Meerschweinchen nehmen eine Sonderstellung ein, indem sie weder Fieber noch leukozytare Reaktionen aufweisen.



Resume

La reaction de differentes especes animales aux pyrogenes bacteriens



Apres un compte-rendu de la litterature concernant ce sujet, on decrit les reactions observees chez differentes especes animales apres application de lipopolysaccharide de E. coli 0111 B 4.



On examine tout particulierement la temperature du corps, les leucocytes dans la circulation sanguine et le comportement du tractus digestif (vomissements, diarrhee, motricite diminuee de le rumen).



Par ordre decroissant de sensibilite, nous nommons lapin, cheval, chevre, chien, boeuf, mouton, chat, porc et poule. Souris, rat et cobaye ont une position speciale, car ils ne produisent ni fievre, ni reactions leucocytaires.



Resumen

La reaccion de diversas especies animales frente a diversos pirogenos bacterianos



Tras discutir la bibliografia correspondiente, describense las reacciones de varias especies animales frente a la aplicacion del lipopolisacarido de E. coli 0111 B 4.



Consideracion especial merecieron a este respecto: la temperatura corporal, los leucocitos en la sangre circulante y el comportamiento del tracto gastrointestinal (vomito, diarrea, movimientos rebajados de la panza).



Con arreglo a su sensibilidad — sucesion correspondiendo a la sensitividad — se puede establecer la lista siguiente: conejo, caballo, cabra, perro, vaca, oveja, gato, cerdo y gallina. El raton, rata y cobaya ocupan una posicion un tanto especial, ya que no manifiestan fiebre ni reacciones leucocitarias.

A. S. J. P. A. M. van Miert

Papers

Pathophysiological effects of endotoxins in ruminants. 1. Changes in body temperature and reticulo-rumen motility, and the effect of repeated administration.

Suppression of cytochrome P450- and UDP glucuronosyl transferase-dependent enzyme activities by proinflammatory cytokines and possible role of nitric oxide in primary cultures of pig hepatocytes.

Participation of β-Adrenergic Receptors on Macrophages in Modulation of LPS-Induced Cytokine Release

The β-adrenoceptor agonist clenbuterol is a potent inhibitor of the LPS-induced production of TNF-α and IL-6 in vitro and in vivo

The reaction of different animal species to bacterial pyrogens.