Correction to: AM404, paracetamol metabolite, prevents prostaglandin synthesis in activated microglia by inhibiting COX activity.

After publication of the article [1], it has been brought to our attention that the caption for Figure 2 has been mistakenly replaced with a reproduction of the Figure 4 caption.

AM404, paracetamol metabolite, prevents prostaglandin synthesis in activated microglia by inhibiting COX activity.

BACKGROUND: N-arachidonoylphenolamine (AM404), a paracetamol metabolite, is a potent agonist of the transient receptor potential vanilloid type 1 (TRPV1) and low-affinity ligand of the cannabinoid receptor type 1 (CB1). There is evidence that AM404 exerts its pharmacological effects in immune cells. However, the effect of AM404 on the production of inflammatory mediators of the arachidonic acid pathway in activated microglia is still not fully elucidated. METHOD: In the present study, we investigated the effects of AM404 on the eicosanoid production induced by lipopolysaccharide (LPS) in organotypic hippocampal slices culture (OHSC) and primary microglia cultures using Western blot, immunohistochemistry, and ELISA. RESULTS: Our results show that AM404 inhibited LPS-mediated prostaglandin E2 (PGE2) production in OHSC, and LPS-stimulated PGE2 release was totally abolished in OHSC if microglial cells were removed. In primary microglia cultures, AM404 led to a significant dose-dependent decrease in the release of PGE2, independent of TRPV1 or CB1 receptors. Moreover, AM404 also inhibited the production of PGD2 and the formation of reactive oxygen species (8-iso-PGF2 alpha) with a reversible reduction of COX-1- and COX-2 activity. Also, it slightly decreased the levels of LPS-induced COX-2 protein, although no effect was observed on LPS-induced mPGES-1 protein synthesis. CONCLUSIONS: This study provides new significant insights about the potential anti-inflammatory role of AM404 and new mechanisms of action of paracetamol on the modulation of prostaglandin production by activated microglia.

Blockade of Multidrug Resistance-Associated Proteins Aggravates Acute Pancreatitis and Blunts Atrial Natriuretic Factor's Beneficial Effect in Rats: Role of MRP4 (ABCC4).

We previously reported that atrial natriuretic factor (ANF) stimulates secretin-evoked cAMP efflux through multidrug resistance-associated protein 4 (MRP4) in the exocrine pancreas. Here we sought to establish in vivo whether this mechanism was involved in acute pancreatitis onset in the rat. Rats pretreated with or without probenecid (MRPs general inhibitor) were infused with secretin alone or with ANF. A set of these animals were given repetitive cerulein injections to induce acute pancreatitis. Plasma amylase and intrapancreatic trypsin activities were measured and histological examination of the pancreas performed. Secretin alone activated trypsinogen but induced no pancreatic histological changes. Blockade by probenecid in secretin-treated rats increased trypsin and also induced vacuolization, a hallmark of acute pancreatitis. ANF prevented the secretin response but in the absence of probenecid. In rats with acute pancreatitis, pretreatment with secretin aggravated the disease, but ANF prevented secretin-induced changes. Blockade of MRPs in rats with acute pancreatitis induced trypsinogen activation and larger cytoplasmic vacuoles as well as larger areas of necrosis and edema that were aggravated by secretin but not prevented by ANF. The temporal resolution of intracellular cAMP levels seems critical in the onset of acute pancreatitis, since secretin-evoked cAMP in a context of MRP inhibition makes the pancreas prone to injury in normal rats and aggravates the onset of acute pancreatitis. Present findings support a protective role for ANF mediated by cAMP extrusion through MRP4 and further suggest that the regulation of MRP4 by ANF would be relevant to maintain pancreatic acinar cell homeostasis.

Detection of MYD88 L265P mutations in formalin-fixed and decalcified BM biopsies from patients with lymphoplasmacytic lymphoma.

The diagnosis of bone marrow (BM) infiltration by Waldenström macroglobulinemia (WM)/lymphoplasmacytic lymphoma (LPL) poses a diagnostic challenge in hematopathology. No definitive morphology or immunophenotype is able to distinguish between infiltration of paraffin-embedded BM sections by WM/LPL and other indolent lymphomas, in particular those of the splenic marginal zone (SMZL) which may also show plasmacytic maturation. An oncogenic gain-of-function mutation (L265P) in the human MYD88 gene has been found to be present in most cases of WM/LPL, yet is absent in most other cases of B-cell chronic lymphoproliferative disorders (LPD), including SMZL. Here, we compare two newly developed diagnostic protocols for detection of this mutation in paraffin-embedded archival tissues which are particularly applicable to decalcified BM biopsies. Sanger sequencing can easily detect levels of BM infiltration above 15% by WM lymphoplasmacytic cells, while the allele-specific PCR can detect the L265P mutation in BM infiltrations below 1% of lymphoma cells. We show that these methods are easily applicable to archival BM specimens and markedly improve diagnostic accuracy of BM infiltrations by indolent B-cell lymphomas.

Oxidative stress and hippocampus in a low-grade hepatic encephalopathy model: protective effects of curcumin.

AIM: The present study was performed on prehepatic portal hypertensive rats, a model of low-grade hepatic encephalopathy, designed to evaluate whether oxidative stress was a possible pathway implicated in hippocampal damage and if so, the effect of an anti-oxidant to prevent it. METHODS: Prehepatic portal hypertension was induced by a regulated portal vein stricture. Oxidative stress was investigated by assessing related biochemical parameters in rat hippocampus. The effect of the anti-oxidant curcumin, administered in a single i.p. dose of 100 mg/kg on the seventh, ninth and eleventh days after surgery, was evaluated. RESULTS: Oxidative stress in the rat hippocampal area was documented. Curcumin significantly decreased tissue malondialdehyde levels and significantly increased glutathione peroxidase, catalase and superoxide dismutase activities in the hippocampal tissue of portal hypertensive rats. CONCLUSION: Oxidative stress was found to be implicated in the hippocampal damage and curcumin protected against this oxidative stress in low-grade hepatic encephalopathic rats. These protective effects may be attributed to its anti-oxidant properties.

Involvement of serum apolipoprotein AI and B100 and lecithin cholesterol acyl transferase in alcoholic cirrhotics.

Lipoproteins are synthesized by the liver and secreted to plasma. Chronic alcoholic intoxication produces frequently cirrhosis and concomitantly alterations in liver metabolism. Thirty patients with alcoholic cirrhosis and 83 healthy controls were selected for this study. Apolipoprotein A1, B100, lecithin cholesterol acyltransferase, responsible for cholesterol esterification and seudocholinesterase enzyme activity not related to lipid metabolism, as a referent of proteins synthesized by the liver were analyzed. In 7 patients serum tiobarbituric acids, catalase, glutathione peroxidase were measured, as exponent of the presence of oxidative stress. Our results showed a significant decrease in lipoproteins, lecithin cholesterol acyltransferase and seudocholinesterase activities. An increase in serum tiobarbituric acids and a decrease in both antioxidant enzymes were found as well. In conclusion, alcohol cirrhotic liver decreases the production of liver proteins including those related to lipid metabolism, allowing the formation of steatosis and/or necrosis. Moreover oxidative stress participate possible as a major mechanism in liver damage.

Hepatic encephalopathy: Ever closer to its big bang.

Hepatic encephalopathy (HE) is a neuropsychiatric disorder that commonly complicates the course of patients with liver disease. Despite the fact that the syndrome was probably first recognized hundreds of years ago, the exact pathogenesis still remains unclear. Minimal hepatic encephalopathy (MHE) is the earliest form of HE and is estimated to affect more that 75% of patients with liver cirrhosis. It is characterized by cognitive impairment predominantly attention, reactiveness and integrative function with very subtle clinical manifestations. The development of MHE is associated with worsen in driving skills, daily activities and the increase of overall mortality. Skeletal muscle has the ability to shift from ammonia producer to ammonia detoxifying organ. Due to its large size, becomes the main ammonia detoxifying organ in case of chronic liver failure and muscular glutamine-synthase becomes important due to the failing liver and brain metabolic activity. Gut is the major glutamine consumer and ammonia producer organ in the body. Hepatocellular dysfunction due to liver disease, results in an impaired clearance of ammonium and in its inter-organ trafficking. Intestinal bacteria, can also represent an extra source of ammonia production and in cirrhosis, small intestinal bacterial overgrowth and symbiosis can be observed. In the study of HE, to get close to MHE is to get closer to its big bang; and from here, to travel less transited roads such as skeletal muscle and intestine, is to go even closer. The aim of this editorial is to expose this road for further and deeper work.

Hippocampal mitochondrial dysfunction with decreased mtNOS activity in prehepatic portal hypertensive rats.

Portal hypertension is a major complication of human cirrhosis that frequently leads to central nervous system dysfunction. In our study, rats with prehepatic portal hypertension developed hippocampal mitochondrial dysfunction as indicated by decreased respiratory rates, respiratory control and mitochondrial nitric oxide synthase (mtNOS) activity in mitochondria isolated from the whole hippocampus. Succinate-dependent respiratory rates decreased by 29% in controlled state 4 and by 42% in active state 3, and respiratory control diminished by 20%. Portal hypertensive rats showed a decreased mtNOS activity of 46%. Hippocampal mitochondrial dysfunction was associated with ultrastructural damage in the mitochondria of hippocampal astrocytes and endothelial cells. Swollen mitochondria, loss of cristae and rupture of outer and inner membrane was observed in astrocytes and endothelial cells of the blood-brain barrier in parallel with the ammonia gradient. It is concluded that the moderate increase in plasma ammonia that followed portal hypertension was the potential primary cause of the observed alterations.

Pharmacodynamic study of the 7,8-dihydroxy-4-methylcoumarin-induced selective cytotoxicity toward U-937 leukemic cells versus mature monocytes: cytoplasmic p21(Cip1/WAF1) as resistance factor.

The development of tumor-selective drugs with low systemic toxicity has always been a major challenge in cancer treatment. Our group previously identified the 7,8-dihydroxy-4-methylcoumarin (DHMC) as a potential chemotherapeutic agent due to its potent, selective anti-proliferative and apoptosis-inducing effects on several cancer cell lines over peripheral blood mononuclear cells. However, there are still no published reports that can explain such selectivity of action. Herein, we addressed this question by using the U-937 promonocytic leukemia cell line, which can be forced to differentiate into a monocyte-like phenotype in vitro. U-937 cells differentiation is dependent on the nuclear expression of p21(Cip1/WAF1), a protein that is absent in immature U-937 cells but present in both the nucleus and the cytoplasm of normal DHMC-resistant monocytes. Considering that induction of differentiation rendered U-937 cells resistant to DHMC, we evaluated the possible causal role of cytoplasmic p21(Cip1/WAF1) in the onset of such resistance by employing U-937 cells stably transfected with a ZnCl2-inducible p21(Cip1/WAF1) variant lacking the nuclear localization signal (U-937/CB6-ΔNLS-p21 cells). Expression of cytoplasmic p21(Cip1/WAF1) did not induce differentiation of the cells but turned them resistant to DHMC through inhibition of JNK, a crucial mediator of DHMC-induced apoptosis in U-937 cells. Sub-acute toxicity evaluation of DHMC in Balb/c mice indicated that DHMC administered intraperitoneally at doses up to 100mg/kg induced no systemic damage. Collectively, our results explain for the first time the selective cytotoxicity of DHMC for tumor cells over normal monocytes, and encourage further in vivo studies on this compound as potential anti-leukemic agent.

Hepatic encephalopathy: An approach to its multiple pathophysiological features.

Hepatic encephalopathy (HE) is a neuropsychiatric complex syndrome, ranging from subtle behavioral abnormalities to deep coma and death. Hepatic encephalopathy emerges as the major complication of acute or chronic liver failure. Multiplicity of factors are involved in its pathophysiology, such as central and neuromuscular neurotransmission disorder, alterations in sleep patterns and cognition, changes in energy metabolism leading to cell injury, an oxidative/nitrosative state and a neuroinflammatory condition. Moreover, in acute HE, a condition of imminent threat of death is present due to a deleterious astrocyte swelling. In chronic HE, changes in calcium signaling, mitochondrial membrane potential and long term potential expression, N-methyl-D-aspartate-cGMP and peripheral benzodiazepine receptors alterations, and changes in the mRNA and protein expression and redistribution in the cerebral blood flow can be observed. The main molecule indicated as responsible for all these changes in HE is ammonia. There is no doubt that ammonia, a neurotoxic molecule, triggers or at least facilitates most of these changes. Ammonia plasma levels are increased two- to three-fold in patients with mild to moderate cirrhotic HE and up to ten-fold in patients with acute liver failure. Hepatic and inter-organ trafficking of ammonia and its metabolite, glutamine (GLN), lead to hyperammonemic conditions. Removal of hepatic ammonia is a differentiated work that includes the hepatocyte, through the urea cycle, converting ammonia into GLN via glutamine synthetase. Under pathological conditions, such as liver damage or liver blood by-pass, the ammonia plasma level starts to rise and the risk of HE developing is high. Knowledge of the pathophysiology of HE is rapidly expanding and identification of focally localized triggers has led the development of new possibilities for HE to be considered. This editorial will focus on issues where, to the best of our knowledge, more research is needed in order to clarify, at least partially, controversial topics.

Cambios funcionales en el sistema nervioso central de ratas hipertensas portales prehepáticas / Functional alterations in central nervous system of prehepatic portal hypertensive rats

La hipertensión portal prehepática experimental produce alteraciones morfológicas en la barrera hematoencefálica (BHE), astrogliosis y angiogénesis en áreas CA4 y CA1 del hipocampo. El objetivo fue estudiar los posibles cambios funcionales de la BHE en ratas HP. Métodos: Se usaron ratas Wistar Kyoto macho de 240g las que se dividieron en: GI (n=8) hipertensas portales por estrechez reglada de la vena porta; GII (n=6) con operación simulada (sham). Se determinaron la integridad funcional de la BHE por Trypan Blue (TB, Reynolds), concentración proteica (CP) en líquido cefaloraquídeo (LCR, Bradford), actividad eléctrica cortical (EEG), contenido de agua en cerebro (gravidimetría) y test conductuales: Animex, rigthing reflex, dolor y Rotarod. El TB fue positivo en áreas perivasculares e hipocampo solo en el GI; la CP en LCR (ug/ml) fue (X±ESM); GI: 40.6±6.8 y GII: 16.5±4.2 (p<0.005) y en plasma (mg/ml): GI: 108.8±7.6 y GII: 87.4±2 (NS). El EEG se mostró alterado con predominancia de onda delta para GI: 0.551±0.033 y GII: 0.342±0.031 (p<0.008), el porcentaje de agua por g/tejido cerebral fue GI: 79.21±0.2, GII: 78.95±0.18 (NS). Estos resultados demuestran la existencia de cambios funcionales en la BHE con una presentación subclínica de encefalopatía hepática en ratas con HP.