Biotherapies in stroke.

Stroke is the second leading cause of death worldwide and the most common cause of severe disability. Neuroprotection and repair mechanisms supporting endogenous brain plasticity are often insufficient to allow complete recovery. While numerous neuroprotective drugs trials have failed to demonstrate benefits for patients, they have provided interesting translational research lessons related to neurorestorative therapy mechanisms in stroke. Stroke damage is not limited to neurons but involve all brain cell type including the extracellular matrix in a "glio-neurovascular niche". Targeting a range of host brain cells, biotherapies such as growth factors and therapeutic cells, currently hold great promise as a regenerative medical strategy for stroke. These techniques can promote both neuroprotection and delayed neural repair through neuro-synaptogenesis, angiogenesis, oligodendrogliogenesis, axonal sprouting and immunomodulatory effects. Their complex mechanisms of action are interdependent and vary according to the particular growth factor or grafted cell type. For example, while "peripheral" stem or stromal cells can provide paracrine trophic support, neural stem/progenitor cells (NSC) or mature neurons can act as more direct neural replacements. With a wide therapeutic time window after stroke, biotherapies could be used to treat many patients. However, guidelines for selecting the optimal time window, and the best delivery routes and doses are still debated and the answers may depend on the chosen product and its expected mechanism including early neuroprotection, delayed neural repair, trophic systemic transient effects or graft survival and integration. Currently, the great variety of growth factors, cell sources and cell therapy products form a therapeutic arsenal that is available for stroke treatment. Their effective clinical use will require prior careful considerations regarding safety (e.g. tumorgenicity, immunogenicity), potential efficacy, cell characterization, delivery route and in vivo biodistribution. Bone marrow-derived cell populations such as mesenchymal stromal/stem cells (MSC) or mononuclear cells (MNC), umbilical cord stem cells and NSC are most investigated notably in clinical trials. Finally, we discuss perspectives concerning potential novel biotherapies such as combinatorial approaches (growth factor combined with cell therapy, in vitro optimization of cell products, or co-transplantation) and the development of biomaterials, which could be used as injectable hydrogel scaffold matrices that could protect a cell graft or selectively deliver drugs and growth factors into the post-stroke cavity at chronic stages. Considering the remaining questions about the best procedure and the safety cautions, we can hope that future translational research about biotherapies will bring more efficient treatments that will decrease post-stroke disability for many patients.

How mutations in the nAChRs can cause ADNFLE epilepsy.

PURPOSE: The linkage between autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) and neuronal nicotinic acetylcholine receptor has been strongly reinforced by the report of five distinct mutations in the two genes coding for the major brain alpha4beta2 nicotinic acetylcholine (ACh) receptors. As a first step toward understanding the basic mechanisms underlying this genetically transmissible neurologic disorder, we examined the similarities and differences of the functional properties displayed by naturally occurring mutant forms of this ligand-gated channel. METHODS: Functional studies of neuronal nicotinic ACh receptors reconstituted in Xenopus oocytes were designed to analyze the common traits displayed by the different mutations associated with ADNFLE. RESULTS: Coexpression of the control and mutated alleles harboring the alpha4S248F mutation obtained from patient DNAs yielded ACh-evoked currents of amplitude comparable to the control responses but with a higher sensitivity and desensitization to the natural agonist. Alternatively, the other mutants (alpha4L776ins3, alpha4S252L, and beta2V287M) displayed an increased ACh sensitivity without pronounced desensitization. In addition, whereas a reduction of calcium permeability was observed for the mutants (alpha4S248F and alpha4L776ins3), no significant modification of ionic selectivity could be detected in the alpha4S252L mutation. Hence increase in ACh sensitivity is the only common characteristic so far observed between the four naturally occurring mutant receptors investigated. CONCLUSIONS: Analyses of functional properties of four nAChR mutants associated with ADNFLE indicate that a gain of function of these mutant receptors may be at the origin of the neuronal network dysfunction that causes the epileptic seizures. These data are discussed in the context of our latest knowledge of the pyramidal cell function.

Ion channel variation causes epilepsies.

The discovery of genetically transmissible form of epilepsy associated with a mutation in a gene that codes for a subunit of a ligand-gated channel shined a new light in this field of neurological diseases. Because this gene (CHRNA4) codes for a neuronal nicotinic acetylcholine receptor subunit, functional studies could be designed to evaluate the alterations caused by this mutation. Since this initial observation, five mutations were identified and determination of their functional properties initiated. These experiments were extended to pairwise expression of the control and mutated allele to mimic the heterozygote human genotype. The first common functional trait identified so far, in four of these mutants, is an increased sensitivity to the acetylcholine, suggesting that these mutations may cause a gain of function. An alternative possibility that cannot be excluded is that conditions in the brain are such that these higher responding receptors may be more prone to desensitization. The importance of ionic channels as cause of epilepsies was further demonstrated with the identification of the association between the benign neonatal epilepsy and mutations in genes coding for potassium channel subunits (KCNQ2, KCNQ3). Additional evidences were brought by the identification of mutations in voltage-dependent sodium channels (SCN1A, SCN1B) in a form of generalized epilepsy with febrile seizures.

Ca2+-binding activity of a COOH-terminal fragment of the Drosophila BK channel involved in Ca2+-dependent activation.

Mutational and biophysical analysis suggests that an intracellular COOH-terminal domain of the large conductance Ca(2+)-activated K(+) channel (BK channel) contains Ca(2+)-binding site(s) that are allosterically coupled to channel opening. However the structural basis of Ca(2+) binding to BK channels is unknown. To pursue this question, we overexpressed the COOH-terminal 280 residues of the Drosophila slowpoke BK channel (Dslo-C280) as a FLAG- and His(6)-tagged protein in Escherichia coli. We purified Dslo-C280 in soluble form and used a (45)Ca(2+)-overlay protein blot assay to detect Ca(2+) binding. Dslo-C280 exhibits specific binding of (45)Ca(2+) in comparison with various control proteins and known EF-hand Ca(2+)-binding proteins. A mutation (D5N5) of Dslo-C280, in which five consecutive Asp residues of the "Ca-bowl" motif are changed to Asn, reduces (45)Ca(2+)-binding activity by 56%. By electrophysiological assay, the corresponding D5N5 mutant of the Drosophila BK channel expressed in HEK293 cells exhibits lower Ca(2+) sensitivity for activation and a shift of approximately +80 mV in the midpoint voltage for activation. This effect is associated with a decrease in the Hill coefficient (N) for activation by Ca(2+) and a reduction in apparent Ca(2+) affinity, suggesting the loss of one Ca(2+)-binding site per monomer. These results demonstrate a functional correlation between Ca(2+) binding to a specific region of the BK protein and Ca(2+)-dependent activation, thus providing a biochemical approach to study this process.

CHRNB2 is the second acetylcholine receptor subunit associated with autosomal dominant nocturnal frontal lobe epilepsy.

Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is an uncommon, idiopathic partial epilepsy characterized by clusters of motor seizures occurring in sleep. We describe a mutation of the beta2 subunit of the nicotinic acetylcholine receptor, effecting a V287M substitution within the M2 domain. The mutation, in an evolutionary conserved region of CHRNB2, is associated with ADNFLE in a Scottish family. Functional receptors with the V287M mutation are highly expressed in Xenopus oocytes and characterized by an approximately 10-fold increase in acetylcholine sensitivity. CHRNB2 is a new gene for idiopathic epilepsy, the second acetylcholine receptor subunit implicated in ADNFLE.

Permeation of large tetra-alkylammonium cations through mutant and wild-type voltage-gated sodium channels as revealed by relief of block at high voltage.

Many large organic cations are potent blockers of K(+) channels and other cation-selective channels belonging to the P-region superfamily. However, the mechanism by which large hydrophobic cations enter and exit the narrow pores of these proteins is obscure. Previous work has shown that a conserved Lys residue in the DEKA locus of voltage-gated Na(+) channels is an important determinant of Na(+)/K(+) discrimination, exclusion of Ca(2+), and molecular sieving of organic cations. In this study, we sought to determine whether the Lys(III) residue of the DEKA locus interacts with internal tetra-alkylammonium cations (TAA(+)) that block Na(+) channels in a voltage-dependent fashion. We investigated block by a series of TAA(+) cations of the wild-type rat muscle Na(+) channel (DEKA) and two different mutants of the DEKA locus, DEAA and DERA, using whole-cell recording. TEA(+) and larger TAA(+) cations block both wild-type and DEAA channels. However, DEAA exhibits dramatic relief of block by large TAA(+) cations as revealed by a positive inflection in the macroscopic I-V curve at voltages greater than +140 mV. Paradoxically, relief of block at high positive voltage is observed for large (e.g., tetrapentylammonium) but not small (e.g., TEA(+)) symmetrical TAA(+) cations. The DEKA wild-type channel and the DERA mutant exhibit a similar relief-of-block phenomenon superimposed on background current rectification. The results indicate: (a) hydrophobic TAA(+) cations with a molecular diameter as large as 15 A can permeate Na(+) channels from inside to outside when driven by high positive voltage, and (b) the Lys(III) residue of the DEKA locus is an important determinant of inward rectification and internal block in Na(+) channels. From these observations, we suggest that hydrophobic interfaces between subunits, pseudosubunits, or packed helices of P-region channel proteins may function in facilitating blocker access to the pore, and may thus play an important role in the blocking and permeation behavior of large TAA(+) cations and potentially other kinds of local anesthetic molecules.

Structure-activity relationships for the interaction of bovine pancreatic trypsin inhibitor with an intracellular site on a large conductance Ca(2+)-activated K(+) channel.

Large conductance Ca(2+)-activated K(+) channels (BK(Ca)) contain an intracellular binding site for bovine pancreatic trypsin inhibitor (BPTI), a well-known inhibitor of various serine proteinase (SerP) enzymes. To investigate the structural basis of this interaction, we examined the activity of 11 BPTI mutants using single BK(Ca) channels from rat skeletal muscle incorporated into planar lipid bilayers. All of the mutants induced discrete substate events at the single-channel level. The dwell time of the substate, which is inversely related to the dissociation rate constant of BPTI, exhibited relatively small changes (<9-fold) for the various mutants. However, the apparent association rate constant varied up to 190-fold and exhibited a positive correlation with the net charge of the molecule, suggesting the presence of a negative electrostatic surface potential in the vicinity of the binding site. The substate current level was unaffected by most of the mutations except for substitutions of Lys15. Different residues at this position were found to modulate the apparent conductance of the BPTI-induced substate to 0% (K15G), 10% (K15F), 30% (K15 wild-type), and 55% (K15V) of the open state at +20 mV. Lys15 is located on a loop of BPTI that forms the primary contact region for binding to many SerPs such as trypsin, chymotrypsin, and elastase. The finding that Lys15 is a determinant of the conductance behavior of the BK(Ca) channel when BPTI is bound implies that the same inhibitory loop that contacts SerP's is located close to the protein interface in the BK(Ca) channel complex. This supports the hypothesis that the C-terminal region of the BK(Ca) channel protein contains a domain homologous to SerP's. We propose a domain interaction model for the mechanism of substate production by Kunitz inhibitors based on current ideas for allosteric activation of BK(Ca) channels by voltage and Ca(2+).

Primary motor cortex involvement in Alzheimer disease.

In Alzheimer disease (AD) the involvement of entorhinal cortex, hippocampus, and associative cortical areas is well established. Regarding the involvement of the primary motor cortex the reported data are contradictory. In order to determine whether the primary motor cortex is involved in AD, the brains of 29 autopsy cases were studied, including, 17 cases with severe cortical AD-type changes with definite diagnoses of AD, 7 age-matched cases with discrete to moderate cortical AD-type changes, and 5 control cases without any AD-type cortical changes. Morphometric analysis of the cortical surface occupied by senile plaques (SPs) on beta-amyloid-immunostained sections and quantitative analysis of neurofibrillary tangles (NFTs) on Gallyas-stained sections was performed in 5 different cortical areas including the primary motor cortex. The percentage of cortical surface occupied by SPs was similar in all cortical areas, without significant difference and corresponded to 16.7% in entorhinal cortex, 21.3% in frontal associative, 16% in parietal associative, and 15.8% in primary motor cortex. The number of NFTs in the entorhinal cortex was significantly higher (41 per 0.4 mm2), compared with those in other cortical areas (20.5 in frontal, 17.9 in parietal and 11.5 in the primary motor cortex). Our findings indicate that the primary motor cortex is significantly involved in AD and suggest the appearance of motor dysfunction in late and terminal stages of the disease.

[Dependence, abuse and misuse of drugs: knowledge and attitudes of private physicians in the Ile-de-France]. / Dépendance, cumul, abus et détournement de médicaments: connaissances et attitudes des médecins libéraux d'Ile-de-France.

BACKGROUND: The rise in drug consumption and poorly adapted prescription practices are currently a growing problem. In the face of this trend, "L'Union Professionnelle des Médecins Libéraux d'Ile-de-France" investigated the awareness and the attitude of physicians towards drug dependency, abuse and misuse. METHODS: One hundred physicians and 50 specialists, randomly selected, were contacted by phone. They were asked to give their own description of dependency, abuse and misuse based on patients' attitudes. They were to describe cases of their patients classifying them according to each category. In the second part of the phone interview, the physicians were to give their view about existing and potential tools to solve these problems. RESULTS: Dependency, abuse and misuse problems were well known and frequent: 94% of physicians have such experience and among those, 60% saw such a patient more than once a week. The population concerned by these problems were generally middle-aged women with chronic disease. Misuse behavior was less easily identified (53% of the physicians described such patients). They generally concerned young men, most often drug addicts. From a global point of view, psychotropes were the most frequently misuse drugs, followed by the pain drugs and nsaids. These types of drugs are used routinely, some of them being sold without prescription. Diseases concerned were essentially psychiatric disorders, sleeping troubles and long lasting pains well classified. To face these problem, physicians recommend a dialogue between medical members and patients as well as a changing in professional (physicians and pharmacists) behavior such as firmness, education and listening. CONCLUSION: Situations evoked during the survey were frequent but non systematically discerned as damageable. As a matter of fact, these observations are only a piece of an "intuitive" estimation of the risk versus benefit of the prescription of the drugs mentioned above. Actual regulation and control tools seem to be somewhat irrelevant to solve the problem.

[Competition for the promotion of essential generic drugs in Africa]. / Concours pour la promotion des médicaments essentiels génériques en Afrique.

STATEMENT OF THE PROBLEM: in most sub-Saharan countries, an extensive economic crisis coupled with a low level of existing resources has put most pharmaceuticals beyond the reach of the general public. This situation was worsened by the devaluation of the Franc CFA in 1994. The supply of lowprice drugs and the improvement of rational drug use is now a priority. Unfortunately, essential generic drugs are little known and used in Africa. OBJECTIVE: 1. To improve the knowledge of and confidence in essential and generic drugs among providers, prescribers and consumers, through the participation of the general public in an incentive-based, creative competition; 2. To collect locally-adapted promotional material, to be used in the future promotion of essential and generic drugs in Africa. Design, setting and method: a competition was announced in June 1995, via various networks, in French-speaking Africa: the population was invited to create one or several items promoting essential generic drugs. These items included slogans, posters, songs and short plays. Over 550 entries were received before the deadline (15 October 1995), from 22 countries (mostly Sub-Saharan and French-speaking). The entries included 387 slogans, 94 posters, 22 plays, and various (photos, comic strips, songs, poems). RESULTS: 1. Greater awareness of essential generic drugs in Western Africa, through the publicity given to the competition; 2. Selection of a pool of approximately 200 promotional items, produced by the target populations, to be published in a catalog and distributed in African countries; 3. Insights into popular practices and representations of Western medications, and local re-interpretation of the concept. CONCLUSION: this public competition was effective at achieving three important goals: 1. Dynamic promotion (the competition was the opportunity for school projects, radio talkshows, etc. on generic drugs); 2. Re appropriation of a policy, thanks to a participative approach; 3. Analysis of the population's general comprehension of pharmaceuticals, through analysis of competition entries.

Simultaneous binding of basic peptides at intracellular sites on a large conductance Ca2+-activated K+ channel. Equilibrium and kinetic basis of negatively coupled ligand interactions.

The homologous Kunitz inhibitor proteins, bovine pancreatic trypsin inhibitor (BPTI) and dendrotoxin I (DTX-I), interact with large conductance Ca2+-activated K+ channels (maxi-KCa) by binding to an intracellular site outside of the pore to produce discrete substate events. In contrast, certain homologues of the Shaker ball peptide produce discrete blocking events by binding within the ion conduction pathway. In this study, we investigated ligand interactions of these positively charged peptide molecules by analysis of single maxi-KCa channels in planar bilayers recorded in the presence of DTX-I and BPTI, or DTX-I and a high-affinity homologue of ball peptide. Both DTX-I (Kd, 16.5 nM) and BPTI (Kd, 1, 490 nM) exhibit one-site binding kinetics when studied alone; however, records in the presence of DTX-I plus BPTI demonstrate simultaneous binding of these two molecules. The affinity of BPTI (net charge, +6) decreases by 11.7-fold (Kd, 17,500 nM) when DTX-I (net charge, +10) is bound and, conversely, the affinity of DTX-I decreases by 10.8-fold (Kd, 178 nM) when BPTI is bound. The ball peptide homologue (BP; net charge, +6) exhibits high blocking affinity (Kd, 7.2 nM) at a single site when studied alone, but has 8.0-fold lower affinity (Kd, 57 nM) for blocking the DTX-occupied channel. The affinity of DTX-I likewise decreases by 8.4-fold (Kd, 139 nM) when BP is bound. These results identify two types of negatively coupled ligand-ligand interactions at distinct sites on the intracellular surface of maxi-KCa channels. Such antagonistic ligand interactions explain how the binding of BPTI or DTX-I to four potentially available sites on a tetrameric channel protein can exhibit apparent one-site kinetics. We hypothesize that negatively coupled binding equilibria and asymmetric changes in transition state energies for the interaction between DTX-I and BP originate from repulsive electrostatic interactions between positively charged peptide ligands on the channel surface. In contrast, there is no detectable binding interaction between DTX-I on the inside and tetraethylammonium or charybdotoxin on the outside of the maxi-KCa channel.

On the structural basis for size-selective permeation of organic cations through the voltage-gated sodium channel. Effect of alanine mutations at the DEKA locus on selectivity, inhibition by Ca2+ and H+, and molecular sieving.

Recent evidence indicates that ionic selectivity in voltage-gated Na+ channels is mediated by a small number of residues in P-region segments that link transmembrane elements S5 and S6 in each of four homologous domains denoted I, II, III, and IV. Important determinants for this function appear to be a set of conserved charged residues in the first three homologous domains, Asp(I), Glu(II), and Lys(III), located in a region of the pore called the DEKA locus. In this study, we examined several Ala-substitution mutations of these residues for alterations in ionic selectivity, inhibition of macroscopic current by external Ca2+ and H+, and molecular sieving behavior using a series of organic cations ranging in size from ammonium to tetraethylammonium. Whole-cell recording of wild-type and mutant channels of the rat muscle micro1 Na+ channel stably expressed in HEK293 cells was used to compare macroscopic current-voltage behavior in the presence of various external cations and an intracellular reference solution containing Cs+ and very low Ca2+. In particular, we tested the hypothesis that the Lys residue in domain III of the DEKA locus is responsible for restricting the permeation of large organic cations. Mutation of Lys(III) to Ala largely eliminated selectivity among the group IA monovalent alkali cations (Li+, Na+, K+, Rb+, Cs+) and permitted inward current of group IIA divalent cations (Mg2+, Ca2+, Sr2+, Ba2+). This same mutation also resulted in the acquisition of permeability to many large organic cations such as methylammonium, tetramethylammonium, and tetraethylammonium, all of which are impermeant in the native channel. The results lead to the conclusion that charged residues of the DEKA locus play an important role in molecular sieving behavior of the Na+ channel pore, a function that has been previously attributed to a hypothetical region of the channel called the "selectivity filter." A detailed examination of individual contributions of the Asp(I), Glu(II), and Lys(III) residues and the dependence on molecular size suggests that relative permeability of organic cations is a complex function of the size, charge, and polarity of these residues and cation substrates. As judged by effects on macroscopic conductance, charged residues of the DEKA locus also appear to play a role in the mechanisms of block by external Ca2+ and H+, but are not essential for the positive shift in activation voltage that is produced by these ions.

On the structural basis for ionic selectivity among Na+, K+, and Ca2+ in the voltage-gated sodium channel.

Voltage-sensitive sodium channels and calcium channels are homologous proteins with distinctly different selectivity for permeation of inorganic cations. This difference in function is specified by amino acid residues located within P-region segments that link presumed transmembrane elements S5 and S6 in each of four repetitive Domains I, II, III, and IV. By analyzing the selective permeability of Na+, K+, and Ca2+ in various mutants of the mu 1 rat muscle sodium channel, the results in this paper support the concept that a conserved motif of four residues contributed by each of the Domains I-IV, termed the DEKA locus in sodium channels and the EEEE locus in calcium channels, determines the ionic selectivity of these channels. Furthermore, the results indicate that the Lys residue in Domain III of the sodium channel is the critical determinant that specifies both the impermeability of Ca2+ and the selective permeability of Na+ over K+. We propose that the alkylammonium ion of the Lys(III) residue acts as an endogenous cation within the ion binding site/selectivity filter of the sodium channel to tune the kinetics and affinity of inorganic cation binding within the pore in a manner analogous to ion-ion interactions that occur in the process of multi-ion channel conduction.

Specificity for block by saxitoxin and divalent cations at a residue which determines sensitivity of sodium channel subtypes to guanidinium toxins.

bTyrosine 401 of the skeletal muscle isoform (mu 1) of the rat muscle Na channel is an important determinant of high affinity block by tetrodotoxin (TTX) and saxitoxin (STX) in Na-channel isoforms. In mammalian heart Na channels, this residue is substituted by cysteine, which results in low affinity for TTX/STX and enhanced sensitivity to block by Zn2+ and Cd2+. In this study, we investigated the molecular basis for high affinity block of Na channels by STX and divalent cations by measuring inhibition of macroscopic Na+ current for a series of point mutations at residue Tyr401 of the rat mu 1 Na channel expressed in Xenopus oocytes. Substitution of Tyr401 by Gly, Ala, Ser, Cys, Asp, His, Trp, and Phe produced functional Na+ currents without major perturbation of gating or ionic selectivity. High affinity block by STX and neosaxitoxin (NEO) with Ki values in the range of 2.6-18 nM required Tyr, Phe, or Trp, suggestive of an interaction between an aromatic ring and a guanidinium group of the toxin. The Cys mutation resulted in a 7- and 23-fold enhancement of the dissociation rate of STX and NEO, respectively, corresponding to rapid toxin dissociation rates of cardiac Na channels. High affinity block by Zn2+ (Ki = 8-23 microM) required Cys, His, or Asp, three residues commonly found to coordinate directly with Zn2+ in metalloproteins. For the Cys mutant of mu 1 and also for the cardiac isoform Na channel (rh1) expressed in the L6 rat muscle cell line, inhibition of macroscopic Na+ conductance by Zn2+ reached a plateau at 85-90% inhibition, suggesting the presence of a substate current. The Asp mutant also displayed enhanced affinity for inhibition of conductance by Ca2+ (Ki = 0.3 mM vs approximately 40 mM in wild type), but block by Ca2+ was incomplete, saturating at approximately 69% inhibition. In contrast, Cd2+ completely blocked macroscopic current in the Cys mutant and the L6 cell line. These results imply that the magnitude of substate current depends on the particular residue at position 401 and the species of divalent cation. The His mutant also exhibited enhanced sensitivity to block by H+ with a pKa of approximately 7.5 for the His imidazole group. Our findings provide further evidence that residue 401 of mu 1 is located within the outer vestibule of the Na channel but external to the single-filing region for permeant ions.

[Congenital cystic dilatation of the choledochus: apropos of a case diagnosed in an adult Senegalese women]. / La dilatation kystique congénitale du cholédoque: à propos d'un cas diagnostiqué chez une adulte sénégalaise.

The authors report a case of congenital cystic dilatation of the choledochus diagnosed on a 37 years old senegalese woman. It is an uncommon affection in Africa. The clinical presentation with various signs is reviewed. Ultrasound or cholangiography confirms the diagnosis. Surgical excision of the cystic dilatation is the best treatment because of the high risk of cancerisation.