Review: PrP 106-126 - 25 years after.

A quarter of a century ago, we proposed an innovative approach to study the pathogenesis of prion disease, one of the most intriguing biomedical problems that remains unresolved. The synthesis of a peptide homologous to residues 106-126 of the human prion protein (PrP106-126), a sequence present in the PrP amyloid protein of Gerstmann-Sträussler-Scheinker syndrome patients, provided a tractable tool for investigating the mechanisms of neurotoxicity. Together with several other discoveries at the beginning of the 1990s, PrP106-126 contributed to underpin the role of amyloid in the pathogenesis of protein-misfolding neurodegenerative disorders. Later, the role of oligomers on one hand and of prion-like spreading of pathology on the other further clarified mechanisms shared by different neurodegenerative conditions. Our original report on PrP106-126 neurotoxicity also highlighted a role for programmed cell death in CNS diseases. In this review, we analyse the prion research context in which PrP106-126 first appeared and the advances in our understanding of prion disease pathogenesis and therapeutic perspectives 25 years later.

Mono and dually decorated nanoliposomes for brain targeting, in vitro and in vivo studies.

PURPOSE: Mono- and dual-decorated (DUAL) liposomes (LIP) were prepared, by immobilization of MAb against transferrin (TfR[OX26 or RI7217]) and/or a peptide analogue of ApoΕ3 (APOe) -to target low-density lipoprotein receptor(LPR)-, characterized physicochemically and investigated for BBB-targeting, in-vitro and in-vivo. METHODS: Human microvascular endothelial cells (hCMEC/D3) were used as BBB model, and brain targeting was studied by in-vivo imaging of DiR-labelled formulations (at two doses and surface ligand densities), followed by ex-vivo organ imaging. RESULTS: LIP diameter was between 100 nm and 150 nm, their stability was good and they were non-cytotoxic. LIP uptake and transport across the hCMEC/D3 cell monolayer was significantly affected by decoration with APOe or MAb, the DUAL exerting an additive effect. Intact vesicle-transcytosis was confirmed by equal transport of hydrophilic and lipophilic labels. In-vivo and ex-vivo results confirmed MAb and DUAL-LIP increased brain targeting compared to non-targeted PEG-LIPs, but not for APOe (also targeting ability of DUAL-LIP was not higher than MAb-LIP). The contradiction between in-vitro and in-vivo results was overruled when in-vitro studies (uptake and monolayer transport) were carried out in presence of serum proteins, revealing their important role in targeted-nanoformulation performance. CONCLUSIONS: A peptide analogue of ApoΕ3 was found to target BBB and increase the targeting potential of TfR-MAb decorated LIP, in-vitro, but not in-vivo, indicating that different types of ligands (small peptides and antibodies) are affected differently by in-vivo applying conditions. In-vitro tests, carried out in presence of serum proteins, may be a helpful predictive "targetability" tool.

A biodistribution study of PEGylated PCL-based nanoparticles in C57BL/6 mice bearing B16/F10 melanoma.

One of the major drawbacks that limits the clinical application of nanoparticles is the lack of preliminary investigations related to their biocompatibility, biodegradability and biodistribution. In this work, biodegradable PEGylated polymer nanoparticles (NPs) have been synthesized by using macromonomers based on poly(ε-caprolaconte) oligomers. More in detail, NPs have been produced by adopting a surfactant-free semibatch emulsion polymerization process using PEG chains as a stabilizing agent. The NPs were also labeled with rhodamine B covalently bound to the NPs to quantitatively study their biodistribution in vivo. NPs were investigated in both in vitro and in vivo preclinical systems to study their biodistribution in mice bearing B16/F10 melanoma, as well as their biocompatibility and biodegradability. The NP concentration was evaluated in different tissues at several times after intravenous injection. The disappearance of the NPs from the plasma was biphasic, with distribution and elimination half-lives of 30 min and 15 h, respectively. NPs were retained in tumors and in filter organs for a long time, were still detectable after 7 d and maintained a steady concentration in the tumor for 120 h. 48 h after injection, 70 ± 15% of the inoculated NPs were excreted in the feces. The favorable tumor uptake, fast excretion and absence of cytotoxicity foster the further development of produced NPs as drug delivery carriers.

Cytomegalovirus infection in intensive care unit patients with hematological malignancies: Characteristics and clinical outcomes.

BACKGROUND: Cytomegalovirus (CMV) infection is associated with poor outcome in ICU patients. However, data on immunocompromised patients are scarce. This study aims to describe characteristics and outcomes of critically ill hematological patients and CMV infection. CMV disease characteristics and relationship between CMV viral load, CMV disease, coinfections by other pathogens and outcomes are described. METHODS: Retrospective single center study (Jan 2010-Dec 2017). Adult patients, admitted to the ICU, having underlying hematological malignancy and CMV infection were included. Results are reported as median (interquartile) or n (%). Factors associated with hospital mortality or CMV disease were analysed using logistic regression. RESULTS: 178 patients were included (median age 55y [42-64], 69.1% male). Hospital mortality was 53% (n = 95). Median viral load was 2.7 Log [2.3-3.5]. CMV disease occurred in 44 (24.7%) patients. Coinfections concerned 159 patients (89.3%). After adjustment for confounders, need for vasopressors (OR 2.53; 95%CI 1.11-5.97) and viral load (OR 1.88 per Log; 95%CI 1.29-2.85) were associated with hospital mortality. However, neither CMV disease nor treatment were associated with outcomes. Allogeneic stem cell transplantation (OR 2.55; 95%CI 1.05-6.16), mechanical ventilation (OR 4.11; OR 1.77-10.54) and viral load (OR 1.77 per Log; 95%CI 1.23-2.61) were independently associated with CMV disease. Coinfections were not associated with CMV disease or hospital mortality. CONCLUSION: In critically-ill hematological patients, CMV viral load is independently associated with hospital mortality. Conversely, neither CMV disease nor treatment was associated with outcome suggesting viral load to be a surrogate for immune status rather than a cause of poor outcome.

Acetylcholinesterase as an amyloid enhancing factor in PrP82-146 aggregation process.

Acetylcholinesterase (AChE) triggers beta amyloid plaques formation and is associated with amyloid plaques in the brain. Recent studies have demonstrated that AChE promotes the aggregation of PrP106-126, a peptide deduced from the prion protein sequence. In the present study we show that AChE triggers also the fibrillization of the main component of the amyloid plaques -the peptide spanning residues 82-146 (PrP82-146)- found in patients with Gerstmann-Sträussler-Scheinker disease (GSS). The kinetics of PrP82-146 aggregate formation was directly correlated with AChE concentration and mature fibrils showed the tinctorial and optical properties of amyloid. Atomic force microscopy analysis showed that oligomer and amyloid fibril formation were significantly accelerated by AChE. This effect was mediated by the peripheral site of the enzyme since propidium iodide inhibited the fibrillization process. Present results strongly support the role of AChE in triggering amyloidogenesis and the potential therapeutic relevance of peripheral site blocker compounds.

Phylogenetic investigation of HCV-4d epidemic in Paris MSM HIV population reveals a still active outbreak and a strong link to the Netherlands.

OBJECTIVES: The hepatitis C virus (HCV) epidemic is evolving quickly despite new treatments, and due to behaviour changes increasing at-risk situations. We investigated potential origins and evolution of the HCV-4d French emergence among human immunodeficiency virus (HIV)-infected men who have sex with men (MSM), in Paris in 2003. METHODS: We analysed all HCV sequences from the initial Paris outbreak with all newly available sequences publicly available, including sampling date and geographical location, resulting in 184, 68, 156, 107, 13 and 2 sequences from France, The Netherlands, other European countries, Africa, the Middle East or Turkey, Americas and Asia, respectively. Phylogenetic reconstruction was performed using maximum likelihood and Bayesian approaches. RESULTS: HCV-4d sequences from Europe were strongly separated from non-European sequences. Sequences from the initial Paris outbreak were all included into two well-separated and supported clusters with branch support at 100%, mean genetic distance <2.8 substitutions/100 nucleotides and >3.4 substitutions/100 nucleotides between their common ancestor and the previous node. The largest cluster interleaved French (n = 98) and Dutch (n = 28) sequences, suggesting several translocations between these countries. This cluster included 41 French sequences from Lyon sampled after 2014, highlighting its continuous spread within France since the initial outbreak. The smallest cluster included one Paris sequence with UK sequences (n = 9). DISCUSSION: A few previous works have shown HCV-4d transmissions occurring between a few countries. In our work, we suggest a new and large connection between France and The Netherlands MSM communities and highlight a well-separated pan-European transmission network. Large collaborative networks are needed to investigate ongoing transmissions across countries and help specific prevention measures.

c-Jun N-terminal kinase binding domain-dependent phosphorylation of mitogen-activated protein kinase kinase 4 and mitogen-activated protein kinase kinase 7 and balancing cross-talk between c-Jun N-terminal kinase and extracellular signal-regulated kinase pathways in cortical neurons.

The c-Jun N-terminal kinase (JNK) is a mitogen-activated protein kinase (MAPK) activated by stress-signals and involved in many different diseases. Previous results proved the powerful effect of the cell permeable peptide inhibitor d-JNKI1 (d-retro-inverso form of c-Jun N-terminal kinase-inhibitor) against neuronal death in CNS diseases, but the precise features of this neuroprotection remain unclear. We here performed cell-free and in vitro experiments for a deeper characterization of d-JNKI1 features in physiological conditions. This peptide works by preventing JNK interaction with its c-Jun N-terminal kinase-binding domain (JBD) dependent targets. We here focused on the two JNK upstream MAPKKs, mitogen-activated protein kinase kinase 4 (MKK4) and mitogen-activated protein kinase kinase 7 (MKK7), because they contain a JBD homology domain. We proved that d-JNKI1 prevents MKK4 and MKK7 activity in cell-free and in vitro experiments: these MAPKK could be considered not only activators but also substrates of JNK. This means that d-JNKI1 can interrupt downstream but also upstream events along the JNK cascade, highlighting a new remarkable feature of this peptide. We also showed the lack of any direct effect of the peptide on p38, MEK1, and extracellular signal-regulated kinase (ERK) in cell free, while in rat primary cortical neurons JNK inhibition activates the MEK1-ERK-Ets1/c-Fos cascade. JNK inhibition induces a compensatory effect and leads to ERK activation via MEK1, resulting in an activation of the survival pathway-(MEK1/ERK) as a consequence of the death pathway-(JNK) inhibition. This study should hold as an important step to clarify the strong neuroprotective effect of d-JNKI1.

Regulation of the macrophage content of neoplasms by chemoattractants.

Factor chemotactic for mononuclear phagocytes was found in supernatant fluids of cultured human and mouse tumor cells. In 11 mouse tumors there was a correlation observed between chemotactic activity and macrophage content of neoplastic tissues. Tumor-derived chemoattractants appear to participate in the regulation of tumor-associated macrophages.

Effectiveness of anthracycline against experimental prion disease in Syrian hamsters.

Prion diseases are transmissible neurodegenerative conditions characterized by the accumulation of protease-resistant forms of the prion protein (PrP), termed PrPres, in the brain. Insoluble PrPres tends to aggregate into amyloid fibrils. The anthracycline 4'-iodo-4'-deoxy-doxorubicin (IDX) binds to amyloid fibrils and induces amyloid resorption in patients with systemic amyloidosis. To test IDX in an experimental model of prion disease, Syrian hamsters were inoculated intracerebrally either with scrapie-infected brain homogenate or with infected homogenate coincubated with IDX. In IDX-treated hamsters, clinical signs of disease were delayed and survival time was prolonged. Neuropathological examination showed a parallel delay in the appearance of brain changes and in the accumulation of PrPres and PrP amyloid.

In Situ Tissue Labeling of Cerebral Amyloid Using HIV-Related Tat Peptide.

Delivering peptide-based drugs to the brain is a major challenge because of the existence of the blood-brain barrier (BBB). To overcome this problem, cell-penetrating peptides derived from proteins that are able to cross biological membranes have been used as cell-permeable and brain-penetrant compounds. An example is the transactivator of transcription protein transduction domain (Tat) of the human immunodeficiency virus. The basic domain of Tat is formed of arginine and lysine amino acid residues. Tat has been used as brain-penetrant carrier also in therapies for Alzheimer disease (AD), the most common form of dementia characterized by extracellular cerebral deposits of amyloid made up of Aß peptide. The aim of our study was to assess whether Tat bind to amyloid deposits of AD and other amyloidoses. An in situ labeling using biotinylated Tat 48-57 peptide was employed in the brain tissue with amyloid deposits made up of Aß (patients with AD and transgenic AD mice), of prion protein (patients with Gerstmann-Straussler-Scheinker disease), and other amyloidosis, processed by different fixations and pretreatments of histological sections. Our results showed that Tat peptide binds amyloid deposits made up of Aß, PrP, and immunoglobulin lambda chains in the brain and other tissues processed by alcoholic fixatives but not in formalin-fixed tissue. The fact that biotinylated Tat peptide stains amyloid of different biochemical composition and the specific charge characteristics of the molecules suggests that Tat may bind to heparan sulfate glicosaminoglicans, that are present in amyloid deposits. Inhibition of the binding by Tat pre-incubation with protamine reinforces this hypothesis. Binding of Tat to amyloid deposits should be kept in mind in interpreting the results of studies employing this molecule as brain-penetrating compound for the treatment of cerebral amyloidoses. Our results also suggest that Tat may be helpful for the analysis of the mechanisms of amyloidogenesis, and in particular, the interactions between specific amyloid peptides and glicosaminoglicans.

HHV-6 reactivation as a cause of fever in autologous hematopoietic stem cell transplant recipients.

OBJECTIVES: We report the biological and clinical impacts possibly associated with HHV-6 reactivation in autologous hematopoietic stem cell transplant (AHSCT) recipients after intensive chemotherapy regimen for lymphoma. METHODS: We retrospectively reviewed clinical, biological, radiological, treatment and outcomes of patients with positive HHV-6 DNA in whole blood following autologous hematopoietic stem cell transplantation. RESULTS: Blood HHV-6 reactivation was reported in 27 (8.5%) patients among 316 AHSCT recipients after high dose therapy for lymphoma. Thirteen (4.1%) patients were symptomatic with fever (100%), diarrhea (61.5%), skin rash (46.1%), and pneumonia (23.1%). Antiviral treatment was administered in 9 (69%) patients and outcome was favorable in all cases. CONCLUSION: Our study suggests a possible pathogenic role of HHV-6 in AHSCT recipients and suggests an impact of antiviral treatments on viral replication and clinical signs resolution.

Effect of acetylcholinesterase inhibitors on AChE-induced PrP106-126 aggregation.

Transmissible spongiform encephalopaties are caused by an extracellular surface protein, the scrapie prion protein (PrPsc), which is an aberrant form of normal and functional cellular PrP (PrPc). The pathological hallmarks of these diseases are the accumulation and deposition of PrPsc in the form of amyloid fibrils in the central nervous system (Tateishi et al., 1988), similar to amyloid-beta (Abeta) protein in Alzheimer's disease (AD). In some patients, Abeta and prion pathology can coexist (Hainfellner et al., 1998), and a common spatial pattern of protein deposition has been described (Armstrong et al., 2001). In addition, it is well-known that acetylcholinesterase (AChE) colocalizes with Abeta deposits of brains in AD patients and accelerates assembly of Abeta peptides through the peripheral site of the enzyme (Inestrosa et al., 1996). The aim of the present study was to analyze time course and concentration dependence of the AChE proaggregating effect on synthetic peptide-spanning residues 106-126 of human PrP (PrP106-126) and the reversion of this effect by different AChE inhibitors (AChEIs).

Amyloid in Alzheimer's disease and prion-related encephalopathies: studies with synthetic peptides.

Deposition of amyloid-beta protein (beta A) in brain parenchyma and vessel walls is a major pathological feature of Alzheimer's disease (AD). In prion-related encephalopathies (PRE), too, an altered form of prion protein (PrPsc) forms amyloid fibrils and accumulates in the brain. In both conditions the amyloid deposition is accompanied by nerve cell loss, whose pathogenesis and molecular basis are not understood. Neuropathological, genetic and biochemical studies indicate a central role of beta A in the AD pathogenesis. Synthetic peptides homologous to beta A and its fragments contribute to investigate the mechanisms of beta A deposit formation and the role played by beta A in AD pathogenesis. The physicochemical studies on the beta-sheet conformation and self-aggregation properties of beta A peptides indicate the conditions and the factors influencing the formation of beta A deposits. The neurotoxic activity of beta A and its fragments support the causal relationship between beta A deposits and the neuropathological events in AD. Numerous studies were performed to clarify the mechanism of neuronal death induced by exposure to beta A peptides. A similar approach has been used to investigate the role of PrPsc in PRE; in these diseases, the association between accumulation of PrPsc and neuropathology is evident and numerous data indicate that PrPsc itself might be the infectious agent responsible for disease transmission. Thus, PrP peptides were used to investigate the pathogenic role of PrPsc in PRE and the conformational change responsible for the conversion PrPc to PrPsc that makes the molecule apparently infectious. In particular, we synthesized a peptide homologous to residues 106-126, an integral part of all abnormal PrP isoforms that accumulate in the brain of subjects' PRE. This peptide is fibrillogenic, has secondary structure largely composed of beta-sheet and proteinase-resistant properties, is neurotoxic and induces astrogliosis. In this review, we summarize and compare the data obtained with beta A and PrP peptides and analyze the significance in terms of amyloidogenic proteins and neurodegeneration.

p21WAF1-derived peptides linked to an internalization peptide inhibit human cancer cell growth.

We tested the ability of synthetic peptides derived from p21(WAF1), fused to the internalization peptide sequence derived from Antennapedia, to inhibit the growth of cancer cells in two human ovarian cancer cell lines expressing wild-type p53 or not. Two fused peptides corresponding to p21(WAF1) regions 17-33 and 63-77 inhibited cell growth in both cell lines while the same peptides without the internalization sequence were inactive. The fused peptides prevented growth at concentrations which inhibited cyclin-dependent kinase 2 and cdc2 activity, thus demonstrating that the peptides act by mimicking the action of p21(WAF1) on kinases. This study illustrates the potential pharmacological use of small peptides fused with the Antennapedia internalization sequence in proliferative disorders. The approach may be extended to other diseases in which cell penetration of a peptide may be of therapeutic benefit. More stable drug-like molecules with better pharmacological properties could be designed based on the results obtained with peptides.

Role of reactive oxygen intermediates in the interferon-mediated depression of hepatic drug metabolism and protective effect of N-acetylcysteine in mice.

Interferon (IFN) and IFN inducers are known to depress hepatic microsomal cytochrome P-450 levels, and the liver toxicity of IFN was reported to be lethal in newborn mice. We have observed that administration to mice of IFN and IFN inducers caused a marked increase in liver xanthine oxidase activity. Because this enzyme is well known to produce reactive oxygen intermediates and cytochrome P-450 was reported to be sensitive to the oxidative damage, we have tested the hypothesis that a free radical mechanism could mediate the depression of cytochrome P-450 levels by IFN. Administration to mice of the IFN inducer polyinosinic-polycytidylic acid (2 mg/kg i.p.) caused a 29 to 52% decrease in liver cytochrome P-450. Concomitant p.o. administration of the free radical scavenger, N-acetylcysteine (as a 2.5% solution in drinking water), or the xanthine oxidase inhibitor, allopurinol (100 mg/kg), protected against the IFN-mediated depression of P-450 kg), protected against the IFN-mediated depression of P-450 levels. The results suggest that an increased endogenous generation of free radicals, possibly due to the induction of xanthine oxidase, is implicated in the IFN-mediated depression of liver drug metabolism. The relevance of these data also extends to cases in which this side effect is observed in pathological situations (e.g., viral diseases and administration of vaccines) associated with an induction of IFN.

Prostaglandin endoperoxide synthetase and the activation of benzo(a)pyrene to reactive metabolites in vivo in guinea pigs.

The role of prostaglandin endoperoxide synthetase in the in vivo activation of benzo(a)pyrene to reactive metabolites capable of interacting irreversibly with cellular macromolecules was studied in guinea pig liver, lung, kidney, spleen, small intestine, colon, and brain. DNA and protein covalent binding experiments were made after systemic administration of acetylsalicylic acid (200 mg/kg) followed by radiolabeled benzo(a)pyrene (4 microgram/kg). Results are compared with a control situation in which the prostaglandin endoperoxide synthetase inhibitor (acetylsalicylic acid) was not administered. No decrease in the level of DNA or protein benzo(a)pyrene-derived covalent binding was observed in any of the tissues studied.

Inhibition of cancer cell growth and c-Myc transcriptional activity by a c-Myc helix 1-type peptide fused to an internalization sequence.

c-Myc is a nuclear protein with important roles in cell transformation, cell proliferation, and gene transcription. It has been previously shown that a 14-amino acid (aa) modified peptide (H1-S6A,F8A) derived from the helix 1 (H1) carboxylic region of c-Myc can interfere in vitro with specific c-Myc DNA binding. Here, we have linked the above Myc-derived 14-aa peptide to a 16-aa sequence from the third helix of Antennapedia (Int). It has been repeatedly reported that this 16-aa Antennapedia peptide is able to cross mammalian cell membranes and to work as a vector for short peptides. Using fluorescent (dansylated or rhodaminated) peptides, we have shown that the fusion peptide with the Antennapedia fragment (Int-H1-S6A,F8A) but not the c-Myc derived fragment alone (H1-S6A,F8A) was capable of internalization inside MCF-7 human breast cancer cells. Int-H1-S6A,F8A and H1-S6A,F8A were the only two peptides capable of inhibiting coimmunoprecipitation of the c-Myc/Max heterodimer in vitro. We have treated (continuously for 10-11 days) MCF-7 cells with four different peptides: Int, H1-S6A,F8A, Int-H1-S6A,F8A, and Int-H1wt [a peptide differing from Int-H1-S6A,F8A by 2 aa (S6 and F8) in the H1 region]. In intact MCF-7 cells, Int-H1-S6A,F8A was the only active peptide capable of inducing the following biological effects: (a) inhibition of cloning efficiency on plates; (b) inhibition of cell growth and induction of apoptosis in subconfluent/confluent cells; and (c) inhibition of transcription of two c-Myc-regulated genes (ODC and p53). Int-H1-S6A,F8A was active in the 1-10 microM range. Int-H1-S6A,F8A may represent a lead molecule for peptidomimetic compounds that have a similar three-dimensional structure but are more resistant to peptidases and, therefore, suitable for in vivo treatment of experimentally induced tumors.

Role of Nrf2, HO-1 and GSH in Neuroblastoma Cell Resistance to Bortezomib.

The activation of Nrf2 has been demonstrated to play a crucial role in cancer cell resistance to different anticancer therapies. The inhibition of proteasome activity has been proposed as a chemosensitizing therapy but the activation of Nrf2 could reduce its efficacy. Using the highly chemoresistant neuroblastoma cells HTLA-230, here we show that the strong reduction in proteasome activity, obtained by using low concentration of bortezomib (BTZ, 2.5 nM), fails in reducing cell viability. BTZ treatment favours the binding of Nrf2 to the ARE sequences in the promoter regions of target genes such as heme oxygenase 1 (HO-1), the modulatory subunit of γ-glutamylcysteine ligase (GCLM) and the transporter for cysteine (x-CT), enabling their transcription. GSH level is also increased after BTZ treatment. The up-regulation of Nrf2 target genes is responsible for cell resistance since HO-1 silencing and GSH depletion synergistically decrease BTZ-treated cell viability. Moreover, cell exposure to all-trans-Retinoic acid (ATRA, 3 µM) reduces the binding of Nrf2 to the ARE sequences, decreases HO-1 induction and lowers GSH level increasing the efficacy of bortezomib. These data suggest the role of Nrf2, HO-1 and GSH as molecular targets to improve the efficacy of low doses of bortezomib in the treatment of malignant neuroblastoma.