Loss of JUNB/AP-1 promotes invasive prostate cancer.

Prostate cancer is a frequent cause of male death in the Western world. Relatively few genetic alterations have been identified, likely owing to disease heterogeneity. Here, we show that the transcription factor JUNB/AP-1 limits prostate cancer progression. JUNB expression is increased in low-grade prostate cancer compared with normal human prostate, but downregulated in high-grade samples and further decreased in all metastatic samples. To model the hypothesis that this downregulation is functionally significant, we genetically inactivated Junb in the prostate epithelium of mice. When combined with Pten (phosphatase and tensin homologue) loss, double-mutant mice were prone to invasive cancer development. Importantly, invasive tumours also developed when Junb and Pten were inactivated in a small cell population of the adult anterior prostate by topical Cre recombinase delivery. The resulting tumours displayed strong histological similarity with human prostate cancer. Loss of JunB expression led to increased proliferation and decreased senescence, likely owing to decreased p16(Ink4a) and p21(CIP1) in epithelial cells. Furthermore, the tumour stroma was altered with increased osteopontin and S100 calcium-binding protein A8/9 expression, which correlated with poor prognoses in patients. These data demonstrate that JUNB/AP-1 cooperates with PTEN signalling as barriers to invasive prostate cancer, whose concomitant genetic or epigenetic suppression induce malignant progression.

Associations between functional polymorphisms in the NFκB signaling pathway and response to anti-TNF treatment in Danish patients with inflammatory bowel disease.

Antitumor necrosis factor-α (TNF-α) is used for treatment of severe cases of inflammatory bowel diseases (IBD), including Crohn's disease (CD) and ulcerative colitis (UC). However, one-third of the patients do not respond to the treatment. Genetic markers may predict individual response to anti-TNF therapy. Using a candidate gene approach, 39 mainly functional single nucleotide polymorphisms (SNPs) in 26 genes regulating inflammation were assessed in 738 prior anti-TNF-naive Danish patients with IBD. The results were analyzed using logistic regression (crude and adjusted for age, gender and smoking status). Nineteen functional polymorphisms that alter the NFκB-mediated inflammatory response (TLR2 (rs3804099, rs11938228, rs1816702, rs4696480), TLR4 (rs5030728, rs1554973), TLR9 (rs187084, rs352139), LY96 (MD-2) (rs11465996), CD14 (rs2569190), MAP3K14 (NIK) (rs7222094)), TNF-α signaling (TNFA (TNF-α) (rs361525), TNFRSF1A (TNFR1) (rs4149570), TNFAIP3(A20) (rs6927172)) and other cytokines regulated by NFκB (IL1B (rs4848306), IL1RN (rs4251961), IL6 (rs10499563), IL17A (rs2275913), IFNG (rs2430561)) were associated with response to anti-TNF therapy among patients with CD, UC or both CD and UC (P ⩽ 0.05). In conclusion, the results suggest that polymorphisms in genes involved in activating NFκB through the Toll-like receptor (TLR) pathways, genes regulating TNF-α signaling and cytokines regulated by NFκB are important predictors for the response to anti-TNF therapy among patients with IBD. Genetically strong TNF-mediated inflammatory response was associated with beneficial response. In addition, the cytokines IL-1ß, IL-6 and IFN-γ may be potential targets for treating patients with IBD who do not respond to anti-TNF therapy. These findings should be examined in independent cohorts before these results are applied in a clinical setting.

Formation of radicals during heating lysine and glucose in solution with an intermediate water activity.

Heating glucose with lysine under alkaline conditions (pH 7.0-10.0) was found to take place with consumption of oxygen together with formation of brown-colored compounds. Highly reactive intermediary radicals were detected when lysine and glucose were heated at intermediate water activity at pH 7.0 and 8.0. The detection was based on initial trapping of highly reactive radicals by ethanol followed by spin trapping of 1-hydroxyethylradicals with α-(4-pyridyl N-oxide)-N-tert-butylnitrone (POBN) and Electron Spin Resonance (ESR) spectroscopy. The generation of reactive intermediary radicals from the Maillard reactions was favored by enhancing alkaline conditions (pH 8.0) and stimulated by presence of the transition metal ion Fe²âº. The stability of the nitrone spin traps, N-tert-butyl-α-phenylnitrone and POBN was examined in buffered aqueous solutions within the pH range 1-12, and found to be less temperature dependent at acidic pH compared to alkaline conditions. A low rate (kobs) of hydrolysis of POBN was found at the used experimental conditions of 70°C and pH 7.0 and 8.0, which made this spin trap method suitable for the detection of radicals in the Maillard reaction system.

Lipid oxidation in fish oil enriched mayonnaise: calcium disodium ethylenediaminetetraacetate, but not gallic acid, strongly inhibited oxidative deterioration.

The antioxidative effects of gallic acid, EDTA, and extra emulsifier Panodan DATEM TR in mayonnaise enriched with 16% fish oil were investigated. EDTA reduced the formation of free radicals, lipid hydroperoxides, volatiles, and fishy and rancid off-flavors. The antioxidative effect of EDTA was attributed to its ability to chelate free metal ions and iron from egg yolk located at the oil-water interface. Gallic acid reduced the levels of both free radicals and lipid hydroperoxides but promoted slightly the oxidative flavor deterioration in mayonnaise and influenced the profile of volatiles. Gallic acid may therefore promote the decomposition of lipid hydroperoxides to volatile oxidation products. Addition of extra emulsifier reduced the lipid hydroperoxide levels but did not influence the level of free radicals or the oxidative flavor deterioration in mayonnaisse; however, it appeared to alter the profile of volatiles. The effect of the emulsifier on the physical structure and rheological properties depended on the presence of antioxidants.

[Azathioprine treatment of Crohn disease]. / Azathioprinbehandling af Crohns sygdom.

Azathioprine/6MP (AZA/6MP) is effective in long-term treatment (> 3 months) of Crohn's disease and superior to other established medical treatments. The optimal dose remains to be defined. So far, effect has been demonstrated with 2-2.5 mg azathioprine/kg/day, but not with 1 mg/kg/day. A disease controlling effect has been demonstrated during up to four years of continuous treatment, after which data remains to be established. As part of remission-inducing combination therapy the effect of AZA/6MP can not be detected until two-three months after treatment start. High dose intravenous AZA/6MP administration does not shorten this interval. Reversible dose dependent side effects may require dose reduction or termination of treatment. Reversible dose independent side effects exclude further or repeated treatment. Some 10-15% stop treatment due to side effects. There is no increased death rate due to cancer in AZA/6MP treated Crohn patients. When giving the above full dose of AZA/6MP, monthly blood tests are recommended for the entire treatment period, more often during the first three months.

Selectivity of prandial glucose regulators: nateglinide, but not repaglinide, accelerates exocytosis in rat pancreatic A-cells.

The effects of the two prandial glucose regulators, repaglinide and nateglinide, on ATP-sensitive K(+) (K(ATP)) channel activity, membrane potential and exocytosis in single rat pancreatic A-cells were investigated using the patch-clamp technique. K(ATP) channel activity was reversibly blocked by repaglinide (K(d)=22 nM) and nateglinide (K(d)=410 nM) and this was associated with membrane depolarisation and initiation of electrical activity. The effect of repaglinide and nateglinide on stimulation of glucagon secretion by direct interference with the exocytotic machinery was investigated by the use of capacitance measurements. Nateglinide, but not repaglinide, at concentrations similar to those required to block K(ATP) channels potentiated Ca(2+)-evoked exocytosis 3-fold. In alphaTC1-9 glucagonoma cells addition of nateglinide, but not repaglinide, was associated with stimulation of glucagon secretion. These results indicate that the fast-acting insulin secretagogue nateglinide is glucagonotropic primarily by stimulating Ca(2+)-dependent exocytosis.

Regeneration of phenolic antioxidants from phenoxyl radicals: an ESR and electrochemical study of antioxidant hierarchy.

Radicals from the flavonoids quercetin, (+)-catechin, (+/-)-taxifolin and luteolin, and from all-rac-alpha-tocopherol have been generated electrochemically by one-electron oxidation in deaerated dimethylformamide (DMF), and characterised by electron spin resonance spectroscopy (ESR) after spin-trapping by 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Simulations of the ESR spectrum based on estimated coupling constants of the spin-trapped quercetin radical, confirmed that this antioxidant radical is oxygen-centered. The complex mixture of radicals, quinoid intermediates and stable two-electron oxidation products, were for each antioxidant allowed to react with each of the four other antioxidants, and the progression of reaction followed by ESR after addition of DMPO, and the product solution further analysed by HPLC. All-rac-alpha-tocopherol was found to be most efficient in regenerating each of the other antioxidants from their oxidation products with a regeneration index (defined as moles regenerated of the oxidised phenolic antioxidant divided with moles of all-rac-alpha-tocopherol consumed) of 0.90+/-0.16 for quercetin, 0.48+/-0.11 for (+)-catechin, 0.48+/-0.06 for (+/-)-taxifolin and 0.50+/-0.10 for luteolin in equimolar 1.00 mM solution. Quercetin was found to have the highest regeneration index among the flavonoids: 0.88+/-0.13 for (+/-)-catechin, 0.41+/-0.03 for (+/-)-taxifolin and 0.41+/-0.02 for luteolin. The antioxidant hierarchy based on the reduction potentials determined by cyclic voltammetry under similar conditions (0.93 V for all-rac-alpha-tocopherol, 1.07 V for quercetin, 1.15 V for luteolin, 1.16V for (+)-catechin and 1.20 V for (+/-)-taxifolin) is compared with the observed over-all regeneration (34% for quercetin, 34% for (+)-catechin, 52% for (+/-)-taxifolin and 43% for luteolin by all-rac-alpha-tocopherol).

[Mechanisms of action of repaglinide at a cellular level]. / Mécanismes d'action du répaglinide au niveau cellulaire.

In type 2 diabetic patients, the important post-prandial hyperglycemic peak combined with the defective insulin secretion emphasizes the need for restoring a physiological insulin profile during meals, characterized by insulinemia peaking within 1 hour and returning to basal levels within 4 hours. During type 1 diabetes mellitus, short acting insulin analogs aim at counteracting on post-prandial hyperglycemic peak. During type 2 diabetes mellitus, repaglinide is the first fast-acting oral antidiabetic drug able to stimulate endogenous insulin secretion during meal by mimicking physiological insulin secretion pattern.

Effects of heparin and aminoguanidine on glomerular basement membrane thickening in diabetic rats.

The effects of heparin and aminoguanidine on glomerular basement membrane thickening were studied in streptozotocin diabetic Sprague-Dawley rats. A placebo-treated group and a non-diabetic group served as controls. All diabetic rats remained severely hyperglycaemic (23 mmol/l) throughout the 8-month study period. At the end of this time relative kidney weight was significantly increased in diabetic control rats (4.9 +/- 0.5 g/kg b.w.) compared with non-diabetic rats (3.3 +/- 0.3 g/kg). This increase was not affected by the intervention treatments. Glomerular basement membrane thickness increased 32% in diabetic control rats (240 +/- 24 nm) compared with non-diabetic rats (182 +/- 20 nm). This increase was prevented by s.c. treatment with both unfractionated and low molecular weight heparins, while basement membrane thickness was the same in animals treated with oral heparins and aminoguanidine and untreated diabetic rats. Macroscopic malignant kidney tumours were seen in three aminoguanidine-treated rats. In conclusion, subcutaneously administered heparin prevents diabetes-induced glomerular basement membrane thickening.

The delayed-type hypersensitivity reaction is dependent on IL-8. Inhibition of a tuberculin skin reaction by an anti-IL-8 monoclonal antibody.

Cell-mediated immune reactions are essential to our immune response toward foreign organisms such as microorganisms, or in the response toward foreign tissue Ags, as seen in the rejection of allogeneic transplanted organs. Similar reactions form the basis for the development and the progression of delayed-type hypersensitivity (DTH) reactions. We found that the alpha-chemokine IL-8 plays an important pathophysiologic role for the development of a DTH reaction because infusion of a neutralizing anti-IL-8 mAb (WS-4) was able to suppress the development of a tuberculin skin reaction in rabbits, as judged by histologic, biochemical, and clinical examinations. Thus, the number of neutrophil granulocytes and lymphocytes at the site of tuberculin injection was decreased considerably, and the clinical signs of inflammation were suppressed almost completely at 24 h after intracutaneous injection of tuberculin, as judged by the size of the infiltrates. In contrast, we did not see any effect on the visible erythema of the skin. We found that the tissue content of myeloperoxidase (MPO), reflecting the number of infiltrating neutrophils, was lowered significantly. Furthermore, immunohistochemical analysis confirmed that IL-8 immunoreactivity is actually enhanced in the skin of positive tuberculin reactions. The results indicate that IL-8 plays an important role for the early accumulation of leukocytes in the skin and for the clinical signs of a DTH reaction.

Pharmacological characterization of a biosynthetic trisulfide-containing hydrophobic derivative of human growth hormone: comparison with standard 22 K growth hormone.

Growth hormone is the classical anabolic hormone which promotes organ growth after binding to somatogenic target cell receptors, present in various target tissues. The present study elucidated the pharmacological characteristics in vitro and in vivo of human growth hormone and a recently identified by-product of a recombinant human growth hormone preparation; i.e. a trisulfide-containing (cys 182-cys 189) hydrophobic, folding derivative of growth hormone, hydrophobic derivative-growth hormone. Standard growth hormone and hydrophobic derivative-growth hormone possessed similar characteristics in vitro, both as regards binding to the somatogenic receptor on the human IM-9 cell line, and the prolactin receptor-mediated proliferation of rat Nb2 cells. This indicates that no change occurs in the binding characteristics in spite of a change in conformation of the molecule. Using an ELISA assay that detected standard and hydrophobic derivative-growth hormone equally well, the plasma pharmacokinetical profiles of the preparations following a single intravenous or subcutaneous dose were indistinguishable. Thus, following initial disposition of hydrophobic derivative-growth hormone and standard growth hormone into a volume, V1, of one to two times the plasma volume, almost 90% of either compound disappeared from plasma during the alpha-phase of the plasma decay curve. Similar half-lives of 4-5 min. were found for hydrophobic derivative-growth hormone and standard growth hormone during this phase, indicating rapid removal of drug from the circulation. Also, the AUC and Cmax values for standard and hydrophobic derivative-growth hormone did not differ following intravenous or subcutaneous administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacokinetics of recombinant factor VIIa in the rat--a comparison of bio-, immuno- and isotope assays.

Recombinant human factor VIIa (rFVIIa) is an activated coagulation factor for intravenous use as a haemostatic agent in haemophiliacs who generate antibodies against factor VIII or IX. Plasma kinetic studies are important for the understanding of the action of rFVIIa which is exerted in the vascular compartment of the body, more specifically on the vessel walls at the site of injury. In the present study, rats were dosed 100 or 500 micrograms/kg 125I-rFVIIa i.v., without any side effects being observed, and the plasma profile of rFVIIa was studied by 3 different assays that were shown to correlate well at early times post-dose: trichloroacetic acid (TCA)-precipitable drug-related radioactivity, rFVIIa antigen determination by ELISA technique, and the assay of clot activity which is the only clinically applicable assay. The plasma concentration curve could be resolved into 1-3 exponentials, depending on the FVIIa detection principle that was employed. Initially, there was a short (ca. 10 min) phase of increasing concentrations before the attainment of Cmax. This was followed by a plasma recovery (Cmax x plasma volume/dose) in the vicinity of one half of the administered dose. The initial volume of distribution (V1) corresponded to the vascular compartment whereas the volume of distribution at steady state (Vss) was somewhat larger. Whole body clearance (CL-B) of rFVIIa was approx. 1 ml/min per kg, and mean residence time (MRT) and the half-life assumed to be associated with the loss of biological activity was approx. 1 h and 20-45 min, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of the recombinant coagulation factor 125I-rFVIIa in rats.

Recombinant human factor VIIa (rFVIIa; NovoSeven) is a two-chain activated clotting factor that is used in the treatment of haemophilia. The distribution of radioactivity in male and pregnant and non-pregnant female rats has been examined by whole-body autoradiography (WBA) after single intravenous doses of 125I-radiolabelled rFVIIa at a dosage level of ca. 0.1 mg/kg. Concentrations of radioactivity were highest in the blood and the highly perfused major thoracic and visceral organs and gonads. This distribution of radioactivity was generally similar in pregnant and non-pregnant females, and although radioactivity was concentrated in the foetal thyroid, it was present in other foetal tissues only at trace levels. Radioactivity in thyroid, urinary bladder and gastrointestinal tract of all rats was apparently associated with detached 125I-iodide. At early sacrifice times (up to 2 h), radioactivity was present in the bone marrow, but at later times (6-24 h) it was apparently associated with the mineralised bone structures. The quantitative distribution of total and trichloroacetic acid precipitable radioactivity in the tissues of rats also was studied after single intravenous doses of 125I-rFVIIa and 125I-rFVII, the non-activated single chain precursor of FVIIa, which is normally present in the circulation. These studies confirmed the WBA findings and showed that the tissue distribution of 125I-rFVII and 125I-rFVIIa was similar, indicating that the distribution of rFVIIa during therapy would be similar to that produced from endogenous FVII as a physiological response to vascular injury.

Accumulation of the recombinant factor VIIa in rat bone: importance of the Gla-domain and relevance to factor IX, another vitamin K-dependent clotting factor.

The vitamin K-dependent clotting factors II, VII, IX, and X are proteins which undergo gamma-carboxylation of specific glutamic acid residues prior to secretion from the liver. These unique Ca2+ binding amino acids allow the interaction of the proteins with cell surface phospholipids, a function that is crucial for expression of full procoagulant activity of the proteins. The N-terminal region of the molecule contains the gamma-carboxylation sites and is termed the Gla-domain. A preliminary observation in rats suggested that mineralized bone accumulated activated recombinant FVII (rFVIIa: NovoSeven) as well as the non-activated, single chain rFVII. The present study investigated the role of the Gla-domain in the accumulation of rFVII in bone, as well as the influence of the activation state of FVII on this phenomenon. Rats were treated with 125I-labelled rFVII, rFVIIa, Gla-domainless rFVIIa, factor IX, iodide, or recombinant human growth hormone (rhGH). Following sacrifice, radioactivity was measured in mineralized bone, among other tissues. Following administration of 125I-radiolabelled rFVII, rFVIIa and factor IX, but not Gla-domainless rFVIIa, iodide or rhGH, extensive sequestration occurred in endochondrally as well as intramenbranously ossified bones. The results indicate that the proteins containing a Gla-domain, and only these, are sequestered in bone. Additionally, the normally occurring form of FVII in the circulation, the single-chain FVII, exhibited similar kinetics in rat bone and plasma, as the two-chain rFVIIa. The half-life of rFVII/rFVIIa in mineralized bone was between 3 and 4 days, implying that significant bone accumulation of the factor will take place at steady state.(ABSTRACT TRUNCATED AT 250 WORDS)

Polymorphonuclear neutrophil granulocyte chemotactic hyperresponsiveness in a case of canine acromegaly.

Growth hormone (GH) has recently been shown to affect polymorphonuclear neutrophil granulocyte (PMN) function and to be secreted by mononuclear cells, indicating that the hormone may be active in an immunophysiologic network, acting as an endo- or paracrine priming agent. The purpose of the present study was to evaluate the chemotactic responsiveness of canine peripheral PMN in a dog with acromegaly, caused by spontaneous, progesterone-induced hypersecretion of GH and, secondary to this, a seven-fold increase in insulin-like growth factor I (IGF-I). The chemotactic responsiveness towards zymosan-activated serum (ZAS) and leukotriene B4 (LTB4) was evaluated at a time when the dog suffered from acromegaly and again 57 days after corrective surgery (ovariohysterectomy). The experiments showed that PMN from the patient exhibited enhanced chemotactic migration that appeared to be associated with the hypersomatotropic condition as judged from the reversibility of the phenomenon. The glucose intolerance and elevated serum alkaline phosphatase that were observed in the acromegalic dog were also shown to be reversible following surgery.

ETH615, a synthetic inhibitor of leukotriene biosynthesis and function, also inhibits the production of and biological responses towards interleukin-8.

ETH615 (4-(2-quinolylmethoxy)-N-(3-fluorobenzyl-phenyl-amino-methyl -4- benzoic-acid), a synthetic inhibitor of leukotriene B4 production and activities, was tested for its effect on the production of and biological responses towards human interleukin-8. We found that ETH615 inhibits lipopolysaccharide-induced (LPS-induced) expression of interleukin-8 messenger-RNA (mRNA) and interleukin-8 production in human peripheral blood mononuclear cells. We also observed that ETH615 completely inhibited interleukin-8 as well as leukotriene B4 directed chemotaxis of human neutrophils in a dose-dependent manner. A moderate effect on fMLP-directed neutrophil chemotaxis was observed. Further, no significant effect on either interleukin-8, leukotriene B4 or fMLP-directed T-cell migration was observed. These results further support the concept of a cytokine-leukotriene regulatory circuit and encourage the establishment of clinical trials testing the effect of ETH615 on inflammatory skin diseases, which are characterized by high levels of interleukin-8 and leukotriene B4 in lesional skin.

Purification and characterization of hepatic microsomal cytochrome P-450 in phenobarbital- and beta-naphthoflavone-treated pigs.

Different cytochrome P-450 isoenzymes from hepatic microsomes of phenobarbital (PB) and beta-naphthoflavone (beta-NF) treated pigs and rats were isolated, purified, and characterized. The physico-chemical properties of the porcine isoenzymes were similar to properties of forms isolated from other species. The molecular sizes ranged from 52.5 to 59.5 kD and, in the ferrous-carbonyl state, the isoenzymes had absorbance maxima between 447 and 451 nm. Antigenic similarities were found between the isoenzymes present in PB-induced pigs, and between the isoenzymes present in beta-NF-induced pigs. Cross-reactivity was not observed between PB- and beta-NF-inducible isoenzymes, but beta-NF-inducible isoenzymes in pigs and rats possessed antigenic similarities.

Substance P: a neurogenic mediator of acute cellular inflammation in the dog?

Substance P (SP) is a neuropeptide that has recently been implicated in the pathogenesis of neurogenic inflammation. SP has been shown to activate polymorphonuclear leukocytes (PMN) as well as other inflammatory cells. The present study investigated the direct stimulatory and priming effects of SP on canine PMN aggregation and migration. Direct stimulation of cell migration by SP was present at an unphysiologically high concentration of the mediator. However, when micromolar concentrations of SP were added to PMN prior to stimulation with sub-optimal concentrations of leukotriene B4 (LTB4), the cells exhibited enhanced aggregation and migration, i.e. priming, when stimulated with the latter. Since SP has been reported to act via the formyl-Met-Leu-Phe (fMLP) chemotaxin receptor, this mediator was also studied and found not to possess any effects similar to SP. Thus, the results indicate that SP acts as a primer of canine PMN functions in vitro via a receptor different from that for fMLP. Before ascribing SP a mediator role in canine neurogenic inflammation, in vivo studies determining the concentrations of, and responses to SP in inflamed tissue should be performed.

Reassessment of two Boyden chamber methods for measuring canine neutrophil migration: the leading front and the lower surface count assays.

Laboratory investigation of neutrophil locomotion has attracted a great deal of attention owing to its potential usefulness to veterinary clinical medicine. Two of the most important principles for measurement of chemotaxis are the leading front and lower surface count techniques. The latter assay has become increasingly popular following the introduction of multi-well chambers utilizing polycarbonate filters. In the present study, this method was compared quantitatively and qualitatively to the leading front assay. Further, the potential usefulness of a simple shape-change assay as a rapid measure of chemotactic activation of neutrophils was assessed and compared with the migration assays. It was concluded that the two migration assays are superior to the shape-change assay, even though both suffer from certain methodological drawbacks. This may be relevant to the elucidation of clinical cases of neutrophil dysfunction.