Caenorhabditis elegans RAC1/ced-10 mutants as a new animal model to study very early stages of Parkinson's disease.

Patients with Parkinson's disease (PD) display non-motor symptoms arising prior to the appearance of motor signs and before a clear diagnosis. Motor and non-motor symptoms correlate with progressive deposition of the protein alpha-synuclein (Asyn) both within and outside of the central nervous system, and its accumulation parallels neurodegeneration. The genome of Caenorhabditis elegans does not encode a homolog of Asyn, thus rendering this nematode an invaluable system with which to investigate PD-related mechanisms in the absence of interference from endogenous Asyn aggregation. CED-10 is the nematode homolog of human RAC1, a small GTPase needed to maintain the function and survival of dopaminergic neurons against human Asyn-induced toxicity in C. elegans. Here, we introduce C. elegans RAC1/ced-10 mutants as a predictive tool to investigate early PD symptoms before neurodegeneration occurs. Deep phenotyping of these animals reveals that, early in development, they displayed altered defecation cycles, GABAergic abnormalities and an increased oxidation index. Moreover, they exhibited altered lipid metabolism evidenced by the accumulation of lipid droplets. Lipidomic fingerprinting indicates that phosphatidylcholine and sphingomyelin, but not phosphatidylethanolamine or phosphatidylserine, were elevated in RAC1/ced-10 mutant nematodes. These collective characteristics reflect the non-motor dysfunction, GABAergic neurotransmission defects, upregulation of stress response mechanisms, and metabolic changes associated with early-onset PD. Thus, we put forward an easy-to-manipulate preclinical animal model to deepen our understanding of early-stage PD and accelerate the translational path for therapeutic target discovery.

TNFα induces survival through the FLIP-L-dependent activation of the MAPK/ERK pathway.

Activation of tumor necrosis factor receptor-1 can trigger survival or apoptosis pathways. In many cellular models, including the neuronal cell model PC12, it has been demonstrated that inhibition of protein synthesis is sufficient to render cells sensitive to apoptosis induced by TNFα. The survival effect is linked to the translocation of the transcription factor nuclear factor-kappa B (NF-κB) to the nucleus and activation of survival-related genes such as FLICE-like inhibitory protein long form (FLIP-L) or IAPs. Nonetheless, we previously reported an NF-κB-independent contribution of Bcl-xL to cell survival after TNFα treatment. Here, we demonstrate that NF-κB-induced increase in FLIP-L expression levels is essential for mitogen-activated protein kinases/extracellular signal-regulated kinases (MAPK/ERK) activation. We demonstrate that FLIP-L behaves as a Raf-1 activator through both protein-protein interaction and Raf-1 kinase activation, without the requirement of the classical Ras activation. Importantly, prevention of FLIP-L increase by NF-κB inhibition or knockdown of endogenous FLIP-L blocks MAPK/ERK activation after TNFα treatment. From a functional point of view, we show that inhibition of the MAPK/ERK pathway and the NF-κB pathway are equally relevant to render PC12 cells sensitive to cell death induced by TNFα. Apoptosis induced by TNFα under these conditions is dependent on jun nuclear kinase1/2 JNK1/2-dependent Bim upregulation. Therefore, we report a previously undescribed and essential role for MAPK/ERK activation by FLIP-L in the decision between cell survival and apoptosis upon TNFα stimulation.

Safety and pharmacokinetics of subcutaneously administered recombinant activated factor VII (rFVIIa).

BACKGROUND: Recombinant activated factor VIIa (rFVIIa) is used to treat bleeds in hemophilia patients with inhibitors. A subcutaneous formulation could potentially improve its half-life and make it suitable for prophylactic treatment. OBJECTIVES: A study was conducted to determine the safety of subcutaneously administered rFVIIa in patients with hemophilia and the pharmacokinetic profile (including bioavailability). PATIENTS/METHODS: This was a multicenter, open-label, cross-over comparison of single doses of intravenous rFVIIa 90µgkg(-1) and a new formulation of rFVIIa for subcutaneous injection at dose levels of 45, 90, 180, 270 and 360µgkg(-1) . Sixty subjects (12 per dose cohort) with hemophilia A or B were enrolled. RESULTS: Subcutaneously administered rFVIIa showed lower mean peak plasma concentrations and prolonged FVII activity (C(max) , 0.44-5.16IU mL(-1) [across doses]; t(1/2) , 12.4h; t(max) , 5.6h) compared with intravenously administered rFVIIa (C(max) , 51.7IUmL(-1) ; t(1/2) , 2.7h; t(max) , <10min). The absolute bioavailability of subcutaneous rFVIIa ranged from 21.1 to 30.1% across dose levels. Dose proportionality was observed within a 2-fold dose increase but not across the full dose range. No thromboembolic events, drug-related serious adverse events, severe injection-site reactions or neutralizing antibodies were reported (primary endpoint). Mild and moderate injection-site reactions were more frequent with subcutaneous than with intravenous injections. CONCLUSION: This phase I clinical trial did not identify safety concerns of prolonged exposure to rFVIIa administered subcutaneously in single doses to hemophilia patients.

Risk stratification for inhibitor development at first treatment for severe hemophilia A: a tool for clinical practice.

BACKGROUND: Replacement therapy in severe hemophilia A patients is complicated by formation of inhibitory antibodies against factor VIII (inhibitors) in around 25% of children. Management of bleeds and eradicating inhibitors is complicated, costly and not always successful. OBJECTIVE: To develop a simple score that stratifies untreated patients with severe hemophilia according to their risk of developing inhibitory antibodies. METHODS: The study population consisted of 332 children, with severe hemophilia A, selected from a retrospective multicentre cohort (the CANAL study). The score was based on risk factors available at the first treatment episode. The score was validated in an external population. RESULTS: A total of 87 patients (25%) developed inhibitory antibodies. The selected risk score comprised positive family history (two points), high risk factor VIII gene mutations (two points), and intensive treatment at initial treatment (three points). Inhibitor incidence was 6% (six of 95) in patients without risk factor, 23% (38 of 170) in those with two points, and 57% (38 of 67) in patients with three points or more. The discriminative ability of the score was good (area under the receiver operating curve 0.74). The score performed equally well in the external validation population. CONCLUSION: These findings suggest that the development of inhibitory antibodies in untreated patients with severe hemophilia A can validly be predicted with the presented risk stratification score.

Fanhdi, efficacy and safety in von Willebrand's disease: prospective international study results.

UNLABELLED: Recently, three multicentre prospective international studies have been carried out to evaluate the clinical efficacy and safety of Fanhdi [high-purity, double-inactivated plasma-derived factor VIII/von Willebrand factor (VWF) concentrate] in patients with von Willebrand's disease (VWD). Pharmacokinetic parameters, clinical efficacy and safety of Fanhdi in acute bleedings episodes or invasive procedures were determined in this population. RESULTS: Pharmacokinetic parameters observed were similar to previous reported for other highly purified plasma-derived FVIII/VWF concentrate. The mean in vivo recovery (IU dL(-1) per IU kg(-1)) was 1.9 +/- 0.6 for VWF:RCof; 2.1 +/- 0.6 for VWF:Ag and 2.6 +/- 0.6 for FVIII:C. The mean half-life (h) was 14.4 +/- 10.5 for VWF:RCof; 27.5 +/- 11.0 for VWF:Ag and 33.4 +/- 16.4 for FVIII:C. Therapeutic benefit of Fanhdi in VWD patients treated during bleeding episodes was clearly demonstrated. The achievement of haemostasis was excellent or good in 100% of the cases (major or minor bleeding episodes). Also, the clinical efficacy of Fanhdi in preventing excessive bleeding during surgery showed a very good profile. Efficacy was rated as excellent in six cases (three major/three minor surgical procedures) and good in three cases (two major/one minor surgical procedures). In addition, the product was well tolerated and no adverse events potentially related to the study drug were reported. CONCLUSIONS: Fanhdi is an effective and safe plasma-derived FVIII/VWF concentrate that can be used as an alternative to the current replacement therapy in patients with VWD to provide an adequate haemostasis during surgical procedures and treatment of bleeding episodes.

Regulation of apoptosis/necrosis execution in cadmium-treated human promonocytic cells under different forms of oxidative stress.

Pulse-treatment of U-937 human promonocytic cells with cadmium chloride followed by recovery caused caspase-9/caspase-3-dependent, caspase-8-independent apoptosis. However, pre-incubation with the glutathione (GSH)-suppressing agent DL-buthionine-(S,R)-sulfoximine (cadmium/BSO), or co-treatment with H2O2 (cadmium/H2O2), switched the mode of death to caspase-independent necrosis. The switch from apoptosis to necrosis did not involve gross alterations in Apaf-1 and pro-caspase-9 expression, nor inhibition of cytochrome c release from mitochondria. However, cadmium/H2O2-induced necrosis involved ATP depletion and was prevented by 3-aminobenzamide, while cadmium/BSO-induced necrosis was ATP independent. Pre-incubation with BSO increased the intracellular cadmium accumulation, while co-treatment with H2O2 did not. Both treatments caused intracellular peroxide over-accumulation and disruption of mitochondrial transmembrane potential (delta psi m). However, while post-treatment with N-acetyl-L-cysteine or butylated hydroxyanisole reduced the cadmium/BSO-mediated necrosis and delta psi m disruption, it did not reduce the effects of cadmium/H2O2. Bcl-2 over-expression, which reduced peroxide accumulation without affecting the intracellular GSH content, attenuated necrosis generation by cadmium/H2O2 but not by cadmium/BSO. By contrast, AIF suppression, which reduced peroxide accumulation and increased the GSH content, attenuated the toxicity of both treatments. These results unravel the existence of two different oxidation-mediated necrotic pathways in cadmium-treated cells, one of them resulting from ATP-dependent apoptosis blockade, and the other involving the concurrence of multiple regulatory factors.

Isolation of AmphiCASP-3/7, an ancestral caspase from amphioxus (Branchiostoma floridae). Evolutionary considerations for vertebrate caspases.

Caspases are a large family of cysteine proteases that play an essential role as effectors of apoptosis in metazoans. Thirteen different caspases have been identified in vertebrates so far, and their function in apoptotic or inflammatory responses is well documented. We have taken advantage of the broadly accepted condition of amphioxus (Cephalochordata, Branchiostoma floridae) as the closest living relative to vertebrates to study the molecular evolution of caspases. Here we report for the first time the pattern of programmed cell death during development of cephalochordates. We also describe the isolation and functional characterisation of the first caspase related gene in amphioxus, which we named AmphiCASP-3/7. The amphioxus caspase is expressed throughout development, from the gastrula to larva stage. AmphiCASP-3/7 induced cell death when ectopically expressed in human HEK 293T cells, and the recombinant protein was inhibited by DEVD peptides. AmphiCASP-3/7 reflects the primitive condition of the executor vertebrates caspases -3 and -7, prior to vertebrate specific duplication. Interestingly, AmphiCASP-3/7 is functionally closer to vertebrate caspase-7, as shown by substrate specificity both in vitro and in MCF7 cells. Our phylogenetic and functional data help in drawing the evolutionary history of caspases, and illustrates an example of acquisition in vertebrates of novel functional properties after gene duplication.

Neuronal survival induced by neurotrophins requires calmodulin.

It has been reported that phosphoinositide 3-kinase (PI 3-kinase) and its downstream target, protein kinase B (PKB), play a central role in the signaling of cell survival triggered by neurotrophins (NTs). In this report, we have analyzed the involvement of Ca2+ and calmodulin (CaM) in the activation of the PKB induced by NTs. We have found that reduction of intracellular Ca2+ concentration or functional blockade of CaM abolished NGF-induced activation of PKB in PC12 cells. Similar results were obtained in cultures of chicken spinal cord motoneurons treated with brain-derived neurotrophic factor (BDNF). Moreover, CaM inhibition prevented the cell survival triggered by NGF or BDNF. This effect was counteracted by the transient expression of constitutive active forms of the PKB, indicating that CaM regulates NT-induced cell survival through the activation of the PKB. We have investigated the mechanisms whereby CaM regulates the activation of the PKB, and we have found that CaM was necessary for the proper generation and/or accumulation of the products of the PI 3-kinase in intact cells.

The absence of oligonucleosomal DNA fragmentation during apoptosis of IMR-5 neuroblastoma cells: disappearance of the caspase-activated DNase.

Caspase-activated DNase is responsible for the oligonucleosomal DNA degradation during apoptosis. DNA degradation is thought to be important for multicellular organisms to prevent oncogenic transformation or as a mechanism of viral defense. It has been reported that certain cells, including some neuroblastoma cell lines such as IMR-5, enter apoptosis without digesting DNA in such a way. We have analyzed the causes for the absence of DNA laddering in staurosporine-treated IMR-5 cells, and we have found that most of the molecular mechanisms controlling apoptosis are well preserved in this cell line. These include degradation of substrates for caspases, blockade of cell death by antiapoptotic genes such as Bcl-2 or Bcl-X(L), or normal levels and adequate activation of caspase-3. Moreover, these cells display normal levels of caspase-activated DNase and its inhibitory protein, inhibitor of caspase-activated DNase, and their cDNA sequences are identical to those reported previously. Nevertheless, IMR-5 cells lose caspase-activated DNase during apoptosis and recover their ability to degrade DNA when human recombinant caspase-activated DNase is overexpressed. Our results lead to the conclusion that caspase-activated DNase is processed during apoptosis of IMR-5 cells, making these cells a good model to study the relevance of this endonuclease in physiological or pathological conditions.

Binding patterns of lectins with GalNAc specificity in the mouse dorsal root ganglia and spinal cord.

To localize membrane glycoconjugates in neurons of the mouse spinal cord and dorsal root ganglia (DRG), cryostat sections of newborn (P0), 7 day-old (P7), P14, P21 and P31 animals were stained with ten FITC-conjugated plant lectins, the majority of them recognizing N-acetyl-D-galactosamine (GalNAc) terminal sugar residues. In the dorsal root ganglia of P0 animals, the different lectins showed distinct patterns of labeling in either cells of the nervous system, including neurons, or other structures such as nerves or blood vessels. Moreover, some of these lectins showed important changes in their pattern of labeling during postnatal development. This was especially relevant for lectins that label a subpopulation of small-sized cells that have been previously identified as the nociceptive cells of the DRG. Enzymatic digestion of sections with neuraminidase removes sialic acid from the carbohydrate chains of glycoconjugates thus exposing novel sugar residues. When this treatment was applied to DRG sections from postnatal animals the pattern of lectin staining was either changed or eliminated and heterogeneous subsets of glycoconjugates normally masked by this sugar were exposed. In the spinal cord of PO animals, none of the lectins labeled cells in the central gray matter. However, after the enzymatic digestion of sections with neuraminidase, spinal cord motoneurons and some other cells were labeled by two of the lectins suggesting that GalNAc residues present in these cells are normally masked by terminal sialic acid. Altogether, these results show important changes in the temporal and spatial expression of glycoconjugates that may be relevant for the postnatal development of the CNS and PNS of mice.

A prospective randomized trial of granulocyte colony-stimulating factor therapy after autologous blood stem cell transplantation in adults.

In order to assess the potential clinical benefit of filgrastim (G-CSF) after peripheral blood stem cell (PBSC) autotransplantation a randomized study was begun in our center in July 1997: 62 patients were involved (30 received filgrastim after PBSC infusion and 32, the control group, received no cytokines). All were adults (median 40 years, range 18-65). Patients with one of three different pathologies were recruited: 28 had advanced breast carcinoma, 23 had lymphomas (12 Hodgkin's disease and 11 non-Hodgkin's lymphoma) and 11 had de novo AML. All of them were transplanted using myeloablative chemotherapy conditioning regimens. G-CSF was administered subcutaneously from day +5 in the treated group at a dose of 5 microg/kg body weight/day. The numbers of CD34+ and mononuclear (MNC) cells infused were similar in each group. Only minor differences regarding the use of G-CSF could be inferred from the analysis of the data. Faster granulocyte engraftment was evident in the treated group (mean of 10 vs 12 days to achieve >0.5 x 109/l granulocytes, P = 0.0008), without differences in incidence and severity of infections, days of fever or duration of antibiotic treatment between groups. There was slightly slower platelet engraftment (mean of 15 days in the group with G-CSF vs 12 days in the other group to achieve >20 x 109/l platelets, P = NS) in this series, but there were no differences in incidence and severity of haemorrhage or platelet transfusion support. Considering the economical costs, the median expenditure per inpatient stay was Eur5961 (range Eur4386-Eur17186) in the G-CSF group compared with Eur5751 (range Eur3676-Eur15640) in the control group (P = 0.47). From our data it could be concluded that for adult patients transplanted with PBSC there is no clear beneficial impact of post-infusion G-CSF administration.

Development of survival responsiveness to brain-derived neurotrophic factor, neurotrophin 3 and neurotrophin 4/5, but not to nerve growth factor, in cultured motoneurons from chick embryo spinal cord.

During embryonic development, most neuronal populations undergo a process usually referred to as naturally occurring neuronal death. For motoneurons (MTNs) of the lumbar spinal cord of chick embryos, this process takes place in a well defined period of time, between embryonic days 6 and 10 (E6-E10). Neurotrophins (NTs) are the best characterized family of neurotrophic factors and exert their effects through activation of their specific Trk receptors. In vitro and in vivo studies have demonstrated that rodent motoneurons survive in response to BDNF, NT3, and NT4/5. In contrast, the trophic dependencies of chicken motoneurons have been difficult to elucidate, and various apparently conflicting reports have been published. In the present study, we describe how freshly isolated motoneurons from E5.5 chick embryos did not respond to any neurotrophin in vitro. Yet, because motoneurons were maintained alive in culture in the presence of muscle extract, they developed a delayed specific survival response to BDNF, NT3, and NT4/5 that is clearly dose-dependent, reaching saturation at doses of 100 pg/ml. This trophic response correlated with increasing expression of the corresponding functional receptors TrkB and TrkC. Moreover, TrkB receptor is able to become autophosphorylated and to activate classical intracellular signaling pathways such as the extracellular signal-regulated protein kinase when it is stimulated with its cognate ligand BDNF. Therefore, our results reconcile the reported differences between in vivo and in vitro studies on the ability of chicken MTNs to respond to some members of the neurotrophin family of trophic factors.

Characterization of the cell death process induced by staurosporine in human neuroblastoma cell lines.

Staurosporine is a potent and non-specific inhibitor of protein kinases. There is also evidence of staurosporine being a potent inducer of apoptosis. In several human neuroblastoma cell lines (SH-SY5Y, NB69, IMR-5 and IMR-32) we have found 100 nM staurosporine to induce cell death in half the population (EC50). Electron microscopy of these cells, fluorescence microscopy after Hoechst-33258 staining of chromatin and agarose-electrophoresis of DNA, show two different types of cell death. SH-SY5Y and NB69 die by apoptosis and display all the characteristic features of it. IMR-5 and IMR-32 lack some of these features and a ladder pattern of DNA degradation is not found. Different morphological types of apoptosis have been described during the development of vertebrates; the possibility of finding a similar diversity in cell culture is suggested. On the other hand, staurosporine is a potent promoter of neurite outgrowth. In all the neuroblastoma cell lines we have tested, neurite-promoting and cell death-inducing staurosporine concentrations mostly overlap. This fact has not been reported before, probably because of an early versus late timing of these two different phenomena. The neuritogenic effect has prompted the suggestion that staurosporine could be a prototype of drugs for neurodegenerative diseases; the present study raises several concerns about such a proposal.

Visceral leishmaniasis in haemophiliac patients with HIV: four case reports.

Visceral leishmaniasis (VL), an endemic parasitosis in Spain, is increasing as an important opportunistic infection in AIDS patients. We describe four cases of severe haemophilic patients with end-stage HIV disease who present with visceral leishmaniasis. We report the clinical course, methods of diagnosis and response to therapy. From the assessed clinical data it does not appear to be any difference with regard to VL either in other HIV risk groups or in immunocompetent groups. In contrast with previous reports, we have found that despite their immunodepressed state, positive serology for Leishmania was found in three of the four cases. A similar observation has been made in patients with VL who are immunodepressed because of other reasons. We also confirm previous reports of poor response and intolerance of antimony treatment, the deteriorating course of the disease in AIDS patients and that there is only a slight relationship between the disease and the cause of patient death. We agree with the proposition that this pathology ought to be included in the definition critera for AIDS.