Detection of PrPres in genetically susceptible fetuses from sheep with natural scrapie

Scrapie is a transmissible spongiform encephalopathy with a wide PrPres dissemination in many non-neural tissues and with high levels of transmissibility within susceptible populations. Mechanisms of transmission are incompletely understood. It is generally assumed that it is horizontally transmitted by direct contact between animals or indirectly through the environment, where scrapie can remain infectious for years. In contrast, in utero vertical transmission has never been demonstrated and has rarely been studied. Recently, the use of the protein misfolding cyclic amplification technique (PMCA) has allowed prion detection in various tissues and excretions in which PrPres levels have been undetectable by traditional assays. The main goal of this study was to detect PrPres in fetal tissues and the amniotic fluid from natural scrapie infected ewes using the PMCA technique. Six fetuses from three infected pregnant ewes in an advanced clinical stage of the disease were included in the study. From each fetus, amniotic fluid, brain, spleen, ileo-cecal valve and retropharyngeal lymph node samples were collected and analyzed using Western blotting and PMCA. Although all samples were negative using Western blotting, PrPres was detected after in vitro amplification. Our results represent the first time the biochemical detection of prions in fetal tissues, suggesting that the in utero transmission of scrapie in natural infected sheep might be possible.

Anoxic-ischemic encephalopathy: association of predictors with the vital and functional prognosis of patients

Improve the prediction of the vital and functional prognosis of comatose patients suffering from anoxic-ischemic encephalopathy after successful resuscitation from a cardiac arrest, addmitted to the Intensive Care and Coronary Units of the Dr. Josep Trueta Hospital, based on clinical, neurophysiological and biochemical results.The results of these different tests, revised and combined all together, will improve the prediction of the patients' prognosis, leading to an accurate vital and functional outcome, as they only have been studied separately so far. Anoxia is the third most frequent cause of coma, and the most common cause of post-anoxic coma in adults is the cardiac arrest. The incidence of hypoxic-ischemic brain injury is not well known, but it is certain that cardiac arrest, the most common cause of post-anoxic coma, affects approximately 24000 to 50000 Spanish people every year, most of them occuring out of the hospital. A cardiac arrest is the abrupt cessation of normal circulation of the blood due to failure of the heart to contract effectively during systole. It is different from, but may be caused by, a heart attack or myocardial infarction, where blood flow to the still-beating heart is interrupted. Arrested blood circulation prevents delivery of oxygen to all parts of the body. Cerebral hypoxia, or lack of oxygen supply to the brain, causes victims to lose consciousness and to stop normal breathing, although agonal breathing may still occur. Brain injury is likely if cardiac arrest is untreated for more than five minutes

Funcions de les molècules associades a la mielina i la proteïna priònica cel•lular (PrPc) en neurodegeneració (Malaltia d’Alzheimer) i neuroinflamació (Esclerosi Múltiple)

Les proteïnes associades a la mielina (MAIS), Nogo-A, MAG i OMgp, són molècules que presenten una capacitat inhibitòria molt important per el recreixement axonal i la neuroreparació després de lesió. No obstant des de fa anys les seves funcions han estat ampliades i s’han involucrat en diferents processos degeneratius del sistema nerviós o en processos neuroinflamatoris del sistema nerviós central i el perifèric com ara l'Escleresi Múltiple (MS). La base neurobiològica d’indicadors moleculars que són responsables del dany axonal en MS segueixen sense estar plenament descrits. Recentment s’ha publicat que el mecanisme de senyalització Nogo-A pot regir els primers canvis de la desmielinització immunomediada del sistema nerviós central en el model animal de MS, l’encefalomielitis autoimmune experimental (EAE). De la mateixa forma la proteïna priònica cel•lular és una proteïna que s’ha associat majoritàriament a malalties espongiformes, però que recentment s’ha vinculat (no sense controvèrsia) amb la seva possible relació amb la Malaltia d'Alzheimer (AD), ja que seria capaç de reclutar els oligòmers d’Aβ (ADDLs), els quals correlacionen millor amb el grau de demència, i amb els que interacciona directament, actuant així com un possible mediador de la fosforilació de tau en la malaltia. No obstant, les funcions de les MAIS i de la PrPc en aquests models de la malaltia no estan clarament definits i, per altra banda, es desconeixen els mecanismes de senyalització implicats, no descartant de forma clara el component neural i l’immune.

Neuroprotective role of PrPC against kainate-induced epileptic seizures and cell death depends on the modulation of JNK3 activation by GluR6/7-PSD-95 binding

Cellular prion protein (PrPC) is a glycosyl-phosphatidylinositol¿anchored glycoprotein. When mutated or misfolded, the pathogenic form (PrPSC) induces transmissible spongiform encephalopathies. In contrast, PrPC has a number of physiological functions in several neural processes. Several lines of evidence implicate PrPC in synaptic transmission and neuroprotection since its absence results in an increase in neuronal excitability and enhanced excitotoxicity in vitro and in vivo. Furthermore, PrPC has been implicated in the inhibition of N-methyl-D-aspartic acid (NMDA)¿mediated neurotransmission, and prion protein gene (Prnp) knockout mice show enhanced neuronal death in response to NMDA and kainate (KA). In this study, we demonstrate that neurotoxicity induced by KA in Prnp knockout mice depends on the c-Jun N-terminal kinase 3 (JNK3) pathway since Prnpo/oJnk3o/o mice were not affected by KA. Pharmacological blockage of JNK3 activity impaired PrPC-dependent neurotoxicity. Furthermore, our results indicate that JNK3 activation depends on the interaction of PrPC with postsynaptic density 95 protein (PSD-95) and glutamate receptor 6/7 (GluR6/7). Indeed, GluR6¿PSD-95 interaction after KA injections was favored by the absence of PrPC. Finally, neurotoxicity in Prnp knockout mice was reversed by an AMPA/KA inhibitor (6,7-dinitroquinoxaline-2,3-dione) and the GluR6 antagonist NS-102. We conclude that the protection afforded by PrPC against KA is due to its ability to modulate GluR6/7-mediated neurotransmission and hence JNK3 activation.

Regulation of GABA(A) and glutamate receptor expression, synaptic facilitation and long-term potentiation in the hippocampus of prion mutant mice

Background: Prionopathies are characterized by spongiform brain degeneration, myoclonia, dementia, and periodic electroencephalographic (EEG) disturbances. The hallmark of prioniopathies is the presence of an abnormal conformational isoform (PrP(sc)) of the natural cellular prion protein (PrP(c)) encoded by the Prnp gene. Although several roles have been attributed to PrP(c), its putative functions in neuronal excitability are unknown. Although early studies of the behavior of Prnp knockout mice described minor changes, later studies report altered behavior. To date, most functional PrP(c) studies on synaptic plasticity have been performed in vitro. To our knowledge, only one electrophysiological study has been performed in vivo in anesthetized mice, by Curtis and coworkers. They reported no significant differences in paired-pulse facilitation or LTP in the CA1 region after Schaffer collateral/commissural pathway stimulation. Principal Findings: Here we explore the role of PrP(c) expression in neurotransmission and neural excitability using wild-type, Prnp -/- and PrP(c)-overexpressing mice (Tg20 strain). By correlating histopathology with electrophysiology in living behaving mice, we demonstrate that both Prnp -/- mice but, more relevantly Tg20 mice show increased susceptibility to KA, leading to significant cell death in the hippocampus. This finding correlates with enhanced synaptic facilitation in paired-pulse experiments and hippocampal LTP in living behaving mutant mice. Gene expression profiling using Illumina microarrays and Ingenuity pathways analysis showed that 129 genes involved in canonical pathways such as Ubiquitination or Neurotransmission were co-regulated in Prnp -/- and Tg20 mice. Lastly, RT-qPCR of neurotransmission-related genes indicated that subunits of GABA(A) and AMPA-kainate receptors are co-regulated in both Prnp -/- and Tg20 mice. Conclusions/Significance: Present results demonstrate that PrP(c) is necessary for the proper homeostatic functioning of hippocampal circuits, because of its relationships with GABA(A) and AMPA-Kainate neurotransmission. New PrP(c) functions have recently been described, which point to PrP(c) as a target for putative therapies in Alzheimer's disease. However, our results indicate that a "gain of function" strategy in Alzheimer's disease, or a "loss of function" in prionopathies, may impair PrP(c) function, with devastating effects. In conclusion, we believe that present data should be taken into account in the development of future therapies.

Neuroprotective role of PrPC against kainate-induced epileptic seizures and cell death depends on the modulation of JNK3 activation by GluR6/7-PSD-95 binding

Cellular prion protein (PrPC) is a glycosyl-phosphatidylinositol¿anchored glycoprotein. When mutated or misfolded, the pathogenic form (PrPSC) induces transmissible spongiform encephalopathies. In contrast, PrPC has a number of physiological functions in several neural processes. Several lines of evidence implicate PrPC in synaptic transmission and neuroprotection since its absence results in an increase in neuronal excitability and enhanced excitotoxicity in vitro and in vivo. Furthermore, PrPC has been implicated in the inhibition of N-methyl-D-aspartic acid (NMDA)¿mediated neurotransmission, and prion protein gene (Prnp) knockout mice show enhanced neuronal death in response to NMDA and kainate (KA). In this study, we demonstrate that neurotoxicity induced by KA in Prnp knockout mice depends on the c-Jun N-terminal kinase 3 (JNK3) pathway since Prnpo/oJnk3o/o mice were not affected by KA. Pharmacological blockage of JNK3 activity impaired PrPC-dependent neurotoxicity. Furthermore, our results indicate that JNK3 activation depends on the interaction of PrPC with postsynaptic density 95 protein (PSD-95) and glutamate receptor 6/7 (GluR6/7). Indeed, GluR6¿PSD-95 interaction after KA injections was favored by the absence of PrPC. Finally, neurotoxicity in Prnp knockout mice was reversed by an AMPA/KA inhibitor (6,7-dinitroquinoxaline-2,3-dione) and the GluR6 antagonist NS-102. We conclude that the protection afforded by PrPC against KA is due to its ability to modulate GluR6/7-mediated neurotransmission and hence JNK3 activation.

Prognostic Importance of the Cuse of Renal Failure in Patients With Cirrhosis

BACKGROUND & AIMS: The prognostic value of the different causes of renal failure in cirrhosis is not well established. This study investigated the predictive value of the cause of renal failure in cirrhosis. METHODS: Five hundred sixty-two consecutive patients with cirrhosis and renal failure (as defined by serum creatinine 1.5 mg/dL on 2 successive determinations within 48 hours) hospitalized over a 6-year period in a single institution were included in a prospective study. The cause of renal failure was classified into 4 groups: renal failure associated with bacterial infections, renal failure associated with volume depletion, hepatorenal syndrome (HRS), and parenchymal nephropathy. The primary end point was survival at 3 months. RESULTS: Four hundred sixty-three patients (82.4%) had renal failure that could be classified in 1 of 4 groups. The most frequent was renal failure associated with infections (213 cases; 46%), followed by hypovolemia-associated renal failure (149; 32%), HRS (60; 13%), and parenchymal nephropathy (41; 9%). The remaining patients had a combination of causes or miscellaneous conditions. Prognosis was markedly different according to cause of renal failure, 3-month probability of survival being 73% for parenchymal nephropathy, 46% for hypovolemia-associated renal failure, 31% for renal failure associated with infections, and 15% for HRS (P .0005). In a multivariate analysis adjusted for potentially confounding variables, cause of renal failure was independently associated with prognosis, together with MELD score, serum sodium, and hepatic encephalopathy at time of diagnosis of renal failure. CONCLUSIONS: A simple classification of patients with cirrhosis according to cause of renal failure is useful in assessment of prognosis and may help in decision making in liver transplantation.

New insights into cellular prion protein (PrPc) functions: the 'ying and yang' of a relevant protein.

The conversion of cellular prion protein (PrPc), a GPI-anchored protein, into a protease-K-resistant and infective form (generally termed PrPsc) is mainly responsible for Transmissible Spongiform Encephalopathies (TSEs), characterized by neuronal degeneration and progressive loss of basic brain functions. Although PrPc is expressed by a wide range of tissues throughout the body, the complete repertoire of its functions has not been fully determined. Recent studies have confirmed its participation in basic physiological processes such as cell proliferation and the regulation of cellular homeostasis. Other studies indicate that PrPc interacts with several molecules to activate signaling cascades with a high number of cellular effects. To determine PrPc functions, transgenic mouse models have been generated in the last decade. In particular, mice lacking specific domains of the PrPc protein have revealed the contribution of these domains to neurodegenerative processes. A dual role of PrPc has been shown, since most authors report protective roles for this protein while others describe pro-apoptotic functions. In this review, we summarize new findings on PrPc functions, especially those related to neural degeneration and cell signaling.

Coexistence of protease sensitive and resistant prion protein in 129VV homozygous sporadic Creutzfeldt-Jakob disease: a case report

Introduction: The coexistence of different molecular types of classical protease-resistant prion protein in the same individual have been described, however, the simultaneous finding of these with the recently described protease-sensitive variant or variably protease-sensitive prionopathy has, to the best of our knowledge, not yet been reported. Case presentation: A 74-year-old Caucasian woman showed a sporadic Creutzfeldt-Jakob disease clinical phenotype with reactive depression, followed by cognitive impairment, akinetic-rigid Parkinsonism with pseudobulbar syndrome and gait impairment with motor apraxia, visuospatial disorientation, and evident frontal dysfunction features such as grasping, palmomental reflex and brisk perioral reflexes. She died at age 77. Neuropathological findings showed: spongiform change in the patient"s cerebral cortex, striatum, thalamus and molecular layer of the cerebellum with proteinase K-sensitive synaptic-like, dot-like or target-like prion protein deposition in the cortex, thalamus and striatum; proteinase K-resistant prion protein in the same regions; and elongated plaque-like proteinase K-resistant prion protein in the molecular layer of the cerebellum. Molecular analysis of prion protein after proteinase K digestion revealed decreased signal intensity in immunoblot, a ladder-like protein pattern, and a 71% reduction of PrPSc signal relative to non-digested material. Her cerebellum showed a 2A prion protein type largely resistant to proteinase K. Genotype of polymorphism at codon 129 was valine homozygous. Conclusion: Molecular typing of prion protein along with clinical and neuropathological data revealed, to the best of our knowledge, the first case of the coexistence of different protease-sensitive prion proteins in the same patient in a rare case that did not fulfill the current clinical diagnostic criteria for either probable or possible sporadic Creutzfeldt-Jakob disease. This highlights the importance of molecular analyses of several brain regions in order to correctly diagnose rare and atypical prionopathies

Management of varices and variceal hemorrhage in cirrhosis.

Variceal hemorrhage is a lethal complication of cirrhosis, particularly in patients in whom clinical decompensation (i.e., ascites, encephalopathy, a previous episode of hemorrhage, or jaundice) has already developed. Practice guidelines for the management of varices and variceal hemorrhage1 in cirrhosis are mostly based on evidence in the literature that has been summarized and prioritized at consensus conferences...

New insights into cellular prion protein (PrPc) functions: the 'ying and yang' of a relevant protein.

The conversion of cellular prion protein (PrPc), a GPI-anchored protein, into a protease-K-resistant and infective form (generally termed PrPsc) is mainly responsible for Transmissible Spongiform Encephalopathies (TSEs), characterized by neuronal degeneration and progressive loss of basic brain functions. Although PrPc is expressed by a wide range of tissues throughout the body, the complete repertoire of its functions has not been fully determined. Recent studies have confirmed its participation in basic physiological processes such as cell proliferation and the regulation of cellular homeostasis. Other studies indicate that PrPc interacts with several molecules to activate signaling cascades with a high number of cellular effects. To determine PrPc functions, transgenic mouse models have been generated in the last decade. In particular, mice lacking specific domains of the PrPc protein have revealed the contribution of these domains to neurodegenerative processes. A dual role of PrPc has been shown, since most authors report protective roles for this protein while others describe pro-apoptotic functions. In this review, we summarize new findings on PrPc functions, especially those related to neural degeneration and cell signaling.

Extracorporeal liver support in severe alcoholic hepatitis

The severity of alcoholic hepatitis (AH) which may coexist with cirrhosis varies greatly, from asymptomatic forms which are detected in alcoholic patients without any sign of liver disease, except laboratory abnormalities, to severe forms characterised by deep jaundice, ascites, hepatic encephalopathy and low prothrombin index. In hospitalized patients the mortality could be as high as 75%. The elevated number of therapeutic proposals reported for more than forty years reveals the lack of efficacy of a particular modality. Even in the most favorable trials, the survival is already very poor and in some cases related to the development of renal failure or hepatorenal syndrome. There are some motivating reports concerning albumin dialysis as a support treatment in patients with severe AH, either alone or in combination with other pharmacological therapies. The favorable effects of albumin dialysis in patients with severe AH suggest that the procedure used alone or in combination with other therapies may have a role in this clinical condition. This will be particularly relevant to offer an alternative therapy in these patients, thus being a potential bridge to recovery or to be listed for liver transplantation.

Prognostic Importance of the Cuse of Renal Failure in Patients With Cirrhosis

BACKGROUND & AIMS: The prognostic value of the different causes of renal failure in cirrhosis is not well established. This study investigated the predictive value of the cause of renal failure in cirrhosis. METHODS: Five hundred sixty-two consecutive patients with cirrhosis and renal failure (as defined by serum creatinine 1.5 mg/dL on 2 successive determinations within 48 hours) hospitalized over a 6-year period in a single institution were included in a prospective study. The cause of renal failure was classified into 4 groups: renal failure associated with bacterial infections, renal failure associated with volume depletion, hepatorenal syndrome (HRS), and parenchymal nephropathy. The primary end point was survival at 3 months. RESULTS: Four hundred sixty-three patients (82.4%) had renal failure that could be classified in 1 of 4 groups. The most frequent was renal failure associated with infections (213 cases; 46%), followed by hypovolemia-associated renal failure (149; 32%), HRS (60; 13%), and parenchymal nephropathy (41; 9%). The remaining patients had a combination of causes or miscellaneous conditions. Prognosis was markedly different according to cause of renal failure, 3-month probability of survival being 73% for parenchymal nephropathy, 46% for hypovolemia-associated renal failure, 31% for renal failure associated with infections, and 15% for HRS (P .0005). In a multivariate analysis adjusted for potentially confounding variables, cause of renal failure was independently associated with prognosis, together with MELD score, serum sodium, and hepatic encephalopathy at time of diagnosis of renal failure. CONCLUSIONS: A simple classification of patients with cirrhosis according to cause of renal failure is useful in assessment of prognosis and may help in decision making in liver transplantation.

Thioflavin-S staining of bacterial inclusion bodies for the fast, simple, and inexpensive screening of amyloid aggregation inhibitors

Amyloid aggregation is linked to a large number of human disorders, from neurodegenerative diseases as Alzheimer"s disease (AD) or spongiform encephalopathies to non-neuropathic localized diseases as type II diabetes and cataracts. Because the formation of insoluble inclusion bodies (IBs) during recombinant protein production in bacteria has been recently shown to share mechanistic features with amyloid self-assembly, bacteria have emerged as a tool to study amyloid aggregation. Herein we present a fast, simple, inexpensive and quantitative method for the screening of potential anti-aggregating drugs. This method is based on monitoring the changes in the binding of thioflavin-S to intracellular IBs in intact Eschericchia coli cells in the presence of small chemical compounds. This in vivo technique fairly recapitulates previous in vitro data. Here we mainly use the Alzheimer"s related beta-amyloid peptide as a model system, but the technique can be easily implemented for screening inhibitors relevant for other conformational diseases simply by changing the recombinant amyloid protein target. Indeed, we show that this methodology can be also applied to the evaluation of inhibitors of the aggregation of tau protein, another amyloidogenic protein with a key role in AD.

Thioflavin-S staining of bacterial inclusion bodies for the fast, simple, and inexpensive screening of amyloid aggregation inhibitors

Amyloid aggregation is linked to a large number of human disorders, from neurodegenerative diseases as Alzheimer"s disease (AD) or spongiform encephalopathies to non-neuropathic localized diseases as type II diabetes and cataracts. Because the formation of insoluble inclusion bodies (IBs) during recombinant protein production in bacteria has been recently shown to share mechanistic features with amyloid self-assembly, bacteria have emerged as a tool to study amyloid aggregation. Herein we present a fast, simple, inexpensive and quantitative method for the screening of potential anti-aggregating drugs. This method is based on monitoring the changes in the binding of thioflavin-S to intracellular IBs in intact Eschericchia coli cells in the presence of small chemical compounds. This in vivo technique fairly recapitulates previous in vitro data. Here we mainly use the Alzheimer"s related beta-amyloid peptide as a model system, but the technique can be easily implemented for screening inhibitors relevant for other conformational diseases simply by changing the recombinant amyloid protein target. Indeed, we show that this methodology can be also applied to the evaluation of inhibitors of the aggregation of tau protein, another amyloidogenic protein with a key role in AD.