Effects of methotrexate upon inflammatory parameters induced by carrageenan in the mouse model of pleurisy

BACKGROUND: The model of pleurisy induced by carrageenan exhibits a biphasic response (4 and 48 h) and permits the quantification of exudate, cell migration and certain enzymes such as myeloperoxidase (MPO) and adenosine-deaminase (ADA) that are markers of activated leukocytes. AIMS: The present study evaluates whether there exists, in the pleurisy model, a significant inhibition of ADA and MPO enzymes, leukocyte kinetics and other markers of inflammation [nitric oxide (NO) levels, exudation] caused by methotrexate treatment by the intraperitoneal (i.p.) route. METHODS: The pleurisy was induced by carrageenan (1%) in mice, and the parameters were analyzed 4 and 48 h after. RESULTS: After the induction of inflammation (4 h), methotrexate (20 mg/kg, i.p., 24 h before pleurisy induction) inhibited the leukocyte infiltration (p < 0.05), NO levels and MPO activity (p < 0.01), but not ADA activity and fluid leakage (p > 0.05). Regarding the second phase of pleurisy (48 h), methotrexate (40 mg/kg, i.p., 0.5 h before pleurisy induction) inhibited the leukocyte infiltration (p < 0.05), fluid leakage, NO levels (p < 0.01), and ADA and MPO activity (p < 0.05). CONCLUSIONS: These findings support the evidence that the acute administration of methotrexate has an important systemic anti-inflammatory activity in the studied inflammatory model. This effect was due to a significant inhibition on both neutrophil and mononuclear cells, being less marked in relation to exudation 48 h after. In relation to the enzymes studied and to NO levels, the findings support the evidence that methotrexate inhibits both enzymes (MPO and ADA) from leukocytes at the site of injury, thus reflecting the activation of both neutrophils and lymphocytes, respectively. Furthermore, the inhibiting effect on NO in both phases of pleurisy induced by carrageenan (4 and 48 h) indicates that methotrexate acts on constitutive and/or inducible NO synthases by means of different cells of the pleural cavity.     

The proteinase inhibitor camostat mesilate suppresses pancreatic pain in rodents

Camostat mesilate, an orally available proteinase inhibitor, is clinically used for treatment of pancreatitis. Given recent evidence that pancreatic proteinases including trypsin and/or proteinase-activated receptor-2 (PAR2) might be involved in pancreatic pain, we examined if camostat mesilate could suppress spinal Fos expression, a marker for neuronal activation, following specific application of trypsin to the pancreas, and pancreatitis-related referred allodynia. Trypsin, administered into the pancreatic duct, caused delayed expression of Fos proteins in the superficial layer of the bilateral T8 and T9 spinal dorsal horns in rats. The trypsin-induced spinal Fos expression was completely abolished by oral pre-administration of camostat mesilate at 300 mg/kg. After hourly repeated (6 times in total) administration of caerulein, mice showed typical symptoms of pancreatitis, accompanied by mechanical allodynia in the upper abdomen (i.e., referred hyperalgesia/allodynia), as assessed by use of von Frey filaments. Camostat mesilate at 100-300 mg/kg, given orally twice before the 1st and 4th doses of caerulein, abolished the pancreatitis-related abdominal allodynia, while it partially prevented the inflammatory signs. The same doses of camostat mesilate, when administered once after the final dose of caerulein, also revealed significant anti-allodynic effect. These data suggest that camostat mesilate prevents and/or depresses pancreatitis-induced pain and/or referred hyperalgesia/allodynia, in which proteinases including trypsin would play a critical role.     

Distribution and function of monoacylglycerol lipase in the gastrointestinal tract

The endogenous cannabinoid system plays an important role in the regulation of gastrointestinal function in health and disease. Endocannabinoid levels are regulated by catabolic enzymes. Here, we describe the presence and localization of monoacylglycerol lipase (MGL), the major enzyme responsible for the degradation of 2-arachidonoylglycerol. We used molecular, biochemical, immunohistochemical, and functional assays to characterize the distribution and activity of MGL. MGL mRNA was present in rat ileum throughout the wall of the gut. MGL protein was distributed in the muscle and mucosal layers of the ileum and in the duodenum, proximal colon, and distal colon. We observed MGL expression in nerve cell bodies and nerve fibers of the enteric nervous system. There was extensive colocalization of MGL with PGP 9.5 and calretinin-immunoreactive neurons, but not with nitric oxide synthase. MGL was also present in the epithelium and was highly expressed in the small intestine. Enzyme activity levels were highest in the duodenum and decreased along the gut with lowest levels in the distal colon. We observed both soluble and membrane-associated enzyme activities. The MGL inhibitor URB602 significantly inhibited whole gut transit in mice, an action that was abolished in cannabinoid 1 receptor-deficient mice. In conclusion, MGL is localized in the enteric nervous system where endocannabinoids regulate intestinal motility. MGL is highly expressed in the epithelium, where this enzyme may have digestive or other functions yet to be determined.     

The modulatory role played by TNF-alpha and IL-1 beta in the inflammatory responses induced by carrageenan in the mouse model of pleurisy

We describe here the modulation caused by intrapleural (i.pl.) injection of the cytokines TNF-alpha and IL-1beta and their specific antibodies in the early (4 h) and late (48 h) inflammatory responses caused by injection of carrageenan (Cg) into the mouse pleural cavity. The antibodies against TNF-alpha and IL-1beta, when injected 30 min prior to Cg, reduced, in a graded and significant manner, both exudation and cell migration in the early (4 h) phase, while they potentiated or had no effect in the late (48 h) phase of Cg response. The natural IL-1 receptor antagonist IL-1RA, given 30 min prior to Cg, reduced the exudation by about 50% and abolished the total and differential cell migration in the early (4 h) and late (48 h) phases of the Cg responses. The i.pl. injection of TNF-alpha or IL-1beta, 5 min prior to Cg, caused graded increase in the exudation of the early (4 h) and late (48 h) phases of the Cg-induced inflammatory responses. In contrast, these treatments markedly reduced the total and differential cell migration at 4 h, while having little or no effect on the late (48 h) phase of the Cg pleurisy. These findings extend previous results and demonstrate that the pro-inflammatory cytokines TNF-alpha and IL-1beta have a critical role in controlling both cell migration and exudation caused by injection of Cg in the mouse pleural cavity. Together, these findings may be relevant to the understanding of the mechanisms involved in airway inflammatory responses.     

Spontaneous inflammatory pain model from a mouse line with N-ethyl-N-nitrosourea mutagenesis

Background

Most filarial nematodes contain Wolbachia symbionts. The purpose of this study was to examine the effects of doxycycline on gene expression in Wolbachia and adult female Brugia malayi.

Methods

Brugia malayi infected gerbils were treated with doxycycline for 6-weeks. This treatment largely cleared Wolbachia and arrested worm reproduction. RNA recovered from treated and control female worms was labeled by random priming and hybridized to the Version 2- filarial microarray to obtain expression profiles.

Results and discussion

Results showed significant changes in expression for 200 Wolbachia (29% of Wolbachia genes with expression signals in untreated worms) and 546 B. malayi array elements after treatment. These elements correspond to known genes and also to novel genes with unknown biological functions. Most differentially expressed Wolbachia genes were down-regulated after treatment (98.5%). In contrast, doxycycline had a mixed effect on B. malayi gene expression with many more genes being significantly up-regulated after treatment (85% of differentially expressed genes). Genes and processes involved in reproduction (gender-regulated genes, collagen, amino acid metabolism, ribosomal processes, and cytoskeleton) were down-regulated after doxycycline while up-regulated genes and pathways suggest adaptations for survival in response to stress (energy metabolism, electron transport, anti-oxidants, nutrient transport, bacterial signaling pathways, and immune evasion).

Conclusions

Doxycycline reduced Wolbachia and significantly decreased bacterial gene expression. Wolbachia ribosomes are believed to be the primary biological target for doxycycline in filarial worms. B. malayi genes essential for reproduction, growth and development were also down-regulated; these changes are consistent with doxycycline effects on embryo development and reproduction. On the other hand, many B. malayi genes involved in energy production, electron-transport, metabolism, anti-oxidants, and others with unknown functions had increased expression signals after doxycycline treatment. These results suggest that female worms are able to compensate in part for the loss of Wolbachia so that they can survive, albeit without reproductive capacity. This study of doxycycline induced changes in gene expression has provided new clues regarding the symbiotic relationship between Wolbachia and B. malayi.     

The relationship between the carboxylesterase and monoacylglycerol lipase activities of chicken liver microsomes

The carboxylesterase (carboxylic-ester hydrolase, EC 3.1.1.1) and monoacylglycerol lipase (glycerol-monoester acylhydrolase, EC 3.1.1.23) activities, measured against ethyl butyrate and emulsified monooleoylglycerol respectively, were determined for chicken liver microsomes and highly purified chicken liver carboxylesterase. The activity ratio (ethyl butyrate activity/monooleoylglycerol activity) was approx. 5 for microsomes and approx. 400 for carboxylesterase. Homogenization of microsomes in 0.1 M Tris-HCl buffer (pH 7.92) released all of the ethyl butyrate activity and about half of the monooleoylglycerol activity into a soluble form. Both activities eluted from a Sephadex G-200 column with the same elution volume as that of pure carboxylesterase. This fraction (fraction B) had an activity ratio of approx. 15, an average pI of 5.01 (cf. 4.75 for carboxylesterase), and ran on polyacrylamide gel electrophoresis at pH 8.6 as a number of closely spaced esterase bands with mobilities considerably less than those of the esterase bands present in the carboxylesterase. Fraction B activities against both substrates were completely inhibited by diethyl p-nitrophenyl phosphate and completely precipitated by antibody to carboxylesterase. The remaining half of the monoacylglycerol lipase activity of microsomes was solubilized by treatment with 1.5% (w/v) Triton X-100. This solubilized monoacylglycerol lipase was completely inhibited by diethyl p-nitrophenyl phosphate, showing it to be a serine-dependent enzyme like the carboxylesterases. However, it had no detectable activity against ethyl butyrate, indicating that it is not closely related to the carboxylesterases.     

The effects of IL-6 and IL-10 and their specific antibodies in the acute inflammatory responses induced by carrageenan in the mouse model of pleurisy

This study evaluates the effects of intrapleural (i.pl.) injection of interleukin (IL-6) and IL-10 and their specific antibodies on the early (4 h) and late (48 h) inflammatory responses caused by carrageenan (Cg) injected into the mouse pleural cavity. The i.pl. injection of IL-6, 5 min prior to Cg, reduced in a dose-dependent and significant manner, the exudation and total and differential leukocyte migration according to assessment in both the early (4 h) and the late (48 h) phases of Cg inflammatory response (P<0.01). Intrapleural injection of IL-10, 5 min prior to i.pl. injection of Cg, resulted in a significant inhibition of the early phase (4 h) (P<0.01), but had no significant effect in relation to the late (48 h) phase of Cg response. The antibodies anti-IL-6 (given i.pl. 30 min prior to Cg) caused a significant decrease in both total and differential leukocyte influx, but significantly increased exudation according to assessment 4 h after pleurisy induction by Cg (P<0.01). In contrast, anti-IL-10 antibody caused graded and marked increase of both total and differential leukocyte influx and also increased fluid leakage as assessed 4 h after Cg injection (P<0.01). In the late phase (48 h) these antibodies increased the inflammatory parameters (anti-IL-6) studied or had no effect (anti-IL-10). Taken together, the current results confirm and extend previous data from the literature by showing that IL-6 and IL-10 regulate several signs of inflammatory response, here characterized by marked inhibition of polymorphonuclear cell influx and blockage of fluid leakage to the site of Cg-induced pleurisy in the mouse.     

Lipoprotein lipase in rat heart--I. Characterization of tri-, di- and monoacylglycerol lipase activities in post-heparin effluents

1. The lipolytic activities that sequentially hydrolyze tri-, di- and monoacylglycerol in rat post-heparin heart effluents were examined. 2. Properties of triacylglycerol lipase (TAGL) activity were typical of lipoprotein lipase. Diacylglycerol lipase (DAGL) behaved similarly to TAGL, suggesting that both activities refer to the same catalytic entity. 3. Differences, particularly in thermal stability, between TAGL and DAGL activities on one hand, and monoacylglycerol lipase (MAGL) activity on the other, may reflect different intrinsic molecular properties. 4. TAGL, DAGL and MAGL activities could not be separated by physical means and appeared to belong to a single unit at the same site on the capillary wall.     

Brivanib,a multitargeted small-molecule tyrosine kinase inhibitor,suppresses laser-induced CNV in a mouse model of neovascular AMD

In age-related macular degeneration (AMD), choroidal neovascularization (CNV), a major pathologic feature of neovascular AMD (nAMD), affects 10% of patients, potentially causing serious complications, including vision loss. Vascular endothelial growth factor receptor 2 (VEGFR2) and fibroblast growth factor receptor 1 (FGFR1) contribute to the pathogenesis of CNV. Brivanib is an oral selective dual receptor tyrosine kinase (RTK) inhibitor of FGFRs and VEGFRs, especially VEGFR2 and FGFR1. In this study, brivanib inhibited zebrafish embryonic angiogenesis without impairing neurodevelopment. In a mouse CNV model, brivanib intravitreal injection blocked phosphorylation of FGFR1 and VEGFR2 and reduced CNV leakage, area, and formation without causing intraocular toxicity. Moreover, brivanib oral gavage reduced CNV leakage and area. Accordingly, brivanib remained at high concentrations (above 14,000 ng/ml) in retinal/choroidal/scleral tissues following intravitreal injection. Similarly, brivanib remained at high concentrations (over 10,000 ng/ml) in retinal/choroidal/scleral tissues following oral gavage. Finally, in vitro cell experiments demonstrated that brivanib inhibited the proliferation, migration and tube formation of microvascular endothelial cells. In conclusion, our study suggested that brivanib treatment could be a novel therapeutic strategy for nAMD.     

Myeloperoxidase and adenosine-deaminase levels in the pleural fluid leakage induced by carrageenan in the mouse model of pleurisy

BACKGROUND: Although myeloperoxidase (MPO) and adenosine-deaminase (ADA) levels are markers of activated leukocytes, both enzymes have not been currently addressed in inflammation models. AIMS: This study evaluates whether the concentrations of these enzymes are significantly correlated with the content of leukocytes in a pleurisy model. METHODS: The pleurisy was induced by carrageenan (1%) in mice, and the parameters analyzed 4 and 48 h after. RESULTS: After the induction of inflammation (4h), MPO and ADA levels peaked in parallel to neutrophils (p<0.01). Regarding the second phase of pleurisy (48 h), the highest concentrations of ADA were detected in parallel to the highest levels of mononuclears (p<0.01). At this time, MPO levels and neutrophils remained elevated, although at lower levels than those found at 4 h. A significant positive correlation was found among neutrophiLs and MPO, and mononuclears and ADA (p<0.01). CONCLUSIONS: These findings support the evidence that both enzymes are markers of the inflammatory process, and provide new tools for a better understanding of the immunoregulatory pathways that occur in inflammation.     

Inflammatory response in white adipose tissue in the non-obese hormone-sensitive lipase null mouse model

In the present study, a local inflammatory response in white adipose tissue from the nonobese HSL-null mouse model is demonstrated. The protein levels of several well-known markers of inflammation, like TNFalpha and ferritin HC, were highly increased and accompanied by an activation of NFkappaB. A number of macrophage proteins, i.e., gal-3, Capg, and MCP-4, were expressed at increased levels and immunohistochemical analyses revealed an increased infiltration of F4/80+ cells.     

The lipase inhibitor tetrahydrolipstatin binds covalently to the putative active site serine of pancreatic lipase

Tetrahydrolipstatin (THL) is a selective inhibitor of fat absorption. In animal models, it has anti-obesity and anti-hypercholesterolemic activity and is presently in clinical trials for these indications. THL binds covalently to pancreatic lipase. Complete inhibition of lipolytic activity is obtained concomitant with the incorporation of 1 mol of THL/mol of enzyme. Pancreatic lipase is the best studied lipase, but published results concerning its catalytic mechanism are still controversial. In order to learn more about the inhibitory mechanism of THL, a selective lipase inhibitor interacting at or near the catalytic site, and therefore, to obtain more information on the catalytic mechanism of lipase, we have determined the amino acid residue to which THL is bound. After proteolytic degradation of porcine pancreatic lipase inhibited with radioactively labeled THL, the labeled peptides were isolated and analyzed by quantitative amino acid analysis, N-terminal sequencing, and by mass spectrometry with fast atom bombardment ionization. The data clearly show that THL is bound as an ester to the serine 152 of the lipase.     

The lipase C-terminal domain. A novel unusual inhibitor of pancreatic lipase activity

In vertebrates, dietary fat digestion mainly results from the combined effect of pancreatic lipase, colipase, and bile. It has been proposed that in vivo lipase adsorption on oil-water emulsion is mediated by a preformed lipase-colipase-mixed micelle complex. The main lipase-colipase binding site is located on the C-terminal domain of the enzyme. We report here that in vitro the isolated C-terminal domain behaves as a potent noncovalent inhibitor of lipase and that the inhibitory effect is triggered by the presence of micelles. Lipase inhibition results from the formation of a nonproductive C-terminal domain-colipase-micelle ternary complex, which competes for colipase with the active lipase-colipase-micelle ternary complex, thus diverting colipase from its lipase-anchoring function. The formation of such a complex has been evidenced by molecular sieving experiments. This nonproductive complex lowers the amount of active lipase thus reducing lipolysis. Preliminary experiments performed in rats show that the C-terminal domain also behaves as an inhibitor in vivo and thus could be considered a potential new tool for specifically reducing intestinal lipolysis.     

Autophagy impairment in a mouse model of neuropathic pain

Background

High frequency electrical stimulation (HFS) of primary nociceptive afferents in humans induce a heightened sensitivity in the surrounding non-stimulated skin area. Several studies suggest that this heterotopic effect is the result of central (spinal) plasticity. The aim of this study is to investigate HFS-induced central plasticity of sensory processing at the level of the brain using the electroencephalogram (EEG). To this end we measured evoked potentials in response to noxious electrical pinprick-like stimuli applied in the heterotopic skin area before, directly after and 30 minutes after HFS.

Results

We observed potential cortical electrophysiological correlates of heterotopic facilitation. Two different cortical correlates were found; the first one was a lateralized effect, i.e. a larger N100 amplitude on the conditioned arm than the control arm 30 minutes after end of HFS. This was comparable with the observed lateralized effect of visual analogue scale (VAS) scores as response to the mechanical punctate stimuli. The second correlate seems to be a more general (non-lateralized) effect, because the result affects both arms. On average for both arms the P200 amplitude increased significantly 30 minutes after end of HFS with respect to baseline.

Conclusions

We suggest that for studying heterotopic nociceptive facilitation the evoked brain response is suitable and relevant for investigating plasticity at the level of the brain and is perhaps a more sensitive and reliable marker than the perceived pain intensity (e.g. VAS).     

X-linked inhibitor of apoptosis deficiency in the TRAMP mouse prostate cancer model

Deregulation of apoptotic pathways plays a central role in cancer pathogenesis. X-linked inhibitor of apoptosis protein (XIAP), is an antiapoptotic molecule, whose elevated expression has been observed in tumor specimens from patients with prostate carcinoma. Studies in human cancer cell culture models and xenograft tumor models have demonstrated that loss of XIAP sensitizes cancer cells to apoptotic stimuli and abrogates tumor growth. In view of these findings, XIAP represents an attractive antiapoptotic therapeutic target for prostate cancer. To examine the role of XIAP in an immunocompetent mouse cancer model, we have generated transgenic adenocarcinoma of the mouse prostate (TRAMP) mice that lack XIAP. We did not observe a protective effect of Xiap deficiency in TRAMP mice as measured by tumor onset and overall survival. In fact, there was an unexpected trend toward more aggressive disease in the Xiap-deficient mice. These findings suggest that alternative mechanisms of apoptosis resistance are playing a significant oncogenic role in the setting of Xiap deficiency. Our study has implications for XIAP-targeting therapies currently in development. Greater understanding of these mechanisms will aid in combating resistance to XIAP-targeting treatment, in addition to optimizing selection of patients who are most likely to respond to such treatment.     

Genital transmission of HPV in a mouse model is potentiated by nonoxynol-9 and inhibited by carrageenan

Genital human papillomavirus (HPV) infection is the most common sexually transmitted infection, and virtually all cases of cervical cancer are attributable to infection by a subset of HPVs (reviewed in ref. 1). Despite the high incidence of HPV infection and the recent development of a prophylactic vaccine that confers protection against some HPV types, many features of HPV infection are poorly understood. It remains worthwhile to consider other interventions against genital HPVs, particularly those that target infections not prevented by the current vaccine. However, productive papillomavirus infection is species- and tissue-restricted, and traditional models use animal papillomaviruses that infect the skin or oral mucosa. Here we report the development of a mouse model of cervicovaginal infection with HPV16 that recapitulates the establishment phase of papillomavirus infection. Transduction of a reporter gene by an HPV16 pseudovirus was characterized by histology and quantified by whole-organ, multispectral imaging. Disruption of the integrity of the stratified or columnar genital epithelium was required for infection, which occurred after deposition of the virus on the basement membrane underlying basal keratinocytes. A widely used vaginal spermicide, nonoxynol-9 (N-9), greatly increased susceptibility to infection. In contrast, carrageenan, a polysaccharide present in some vaginal lubricants, prevented infection even in the presence of N-9, suggesting that carrageenan might serve as an effective topical HPV microbicide.