Evaluation of occupational exposure: comparison of biological and environmental variabilities using physiologically based toxicokinetic modeling

PURPOSE: Few studies compare the variabilities that characterize environmental (EM) and biological monitoring (BM) data. Indeed, comparing their respective variabilities can help to identify the best strategy for evaluating occupational exposure. The objective of this study is to quantify the biological variability associated with 18 bio-indicators currently used in work environments. METHOD: Intra-individual (BV(intra)), inter-individual (BV(inter)), and total biological variability (BV(total)) were quantified using validated physiologically based toxicokinetic (PBTK) models coupled with Monte Carlo simulations. Two environmental exposure profiles with different levels of variability were considered (GSD of 1.5 and 2.0). RESULTS: PBTK models coupled with Monte Carlo simulations were successfully used to predict the biological variability of biological exposure indicators. The predicted values follow a lognormal distribution, characterized by GSD ranging from 1.1 to 2.3. Our results show that there is a link between biological variability and the half-life of bio-indicators, since BV(intra) and BV(total) both decrease as the biological indicator half-lives increase. BV(intra) is always lower than the variability in the air concentrations. On an individual basis, this means that the variability associated with the measurement of biological indicators is always lower than the variability characterizing airborne levels of contaminants. For a group of workers, BM is less variable than EM for bio-indicators with half-lives longer than 10-15��h. CONCLUSION: The variability data obtained in the present study can be useful in the development of BM strategies for exposure assessment and can be used to calculate the number of samples required for guiding industrial hygienists or medical doctors in decision-making.

Pollution stress on aquatic microbial communities

Thousands of chemical compounds enter the natural environment but many have unknown effects and consequences, in particular at low concentrations. This thesis work contributes to our understanding of pollution effects by using bacteria as test organisms. Bacteria are important for this question because some of them degrade and transform pollutants into less harmful compounds, but secondly because they themselves can be inhibited in their reproduction by exposure to toxic compounds. When inhibitory effects occur this may change the composition of the microbial com��munity in the long run, leading to altered or diminished ecosystem services by those communities. As a result chemicals of anthropogenic origin may accumulate and per��sist in the environment, and finally, affect higher organisms as well. In addition to acquiring basic understanding of pollutant effects at low concentrations on bacterial communities an applied goal of this thesis work was to develop bacteria-based tests to screen new organic chemicals for toxicity and biod��gradation. In the first part of this work we developed a flow cytometry-based assay on SYT09 plus ethidium-bromide or propidium-iodide stained cells of Pseudomonas ��uorescens exposed or not to a variety of pollutants under oligotrophic growth conditions. Flow cytometry (FC) allows fast and accurate counting of bacterial cells under simul��taneous assessment of their physiological state, in particular in combination with different fluorescent dyes. Here we employed FC and fluorescent dyes to monitor the effect that pollutants may exert on Pseudomonas ��uorescens SV3. First we designed an oligotrophic growth test, which enabled us to follow population growth at low densities (104 - 10 7 cells per ml) using 0.1 mM sodium acetate as carbon source. Cells in the oligotrophic milieu were then exposed or not to a variety of common pollutants, such as 2-chlorobiphenyl (2CBP), naphthalene (NAH), 4-chlorophenol (4CP), tetradecane (TD), mercury chloride (HgCl2) or benzene, in different dosages. Exposed culture samples were stained with SYT09 (green fluorescent dye binding nucleic acids, generally staining all cells) in combination with propidium iodide (PI) or ethidium bromide (EB), both dyes being membrane integrity indicators. We ob- served that most of the tested compounds decreased population growth in a dosage- dependent manner. SYT09/PI or SYT09/EB staining then revealed that chemical exposure led to arisal of subpopulations of live and injured or dead cells. By modeling population growth on the total cell numbers in population or only the subpopulation of live cells we inferred that even in stressed populations live cells multiply at rates no different to unexposed controls. The net decrease in population growth would thus be a consequence of more and more cells being not able to multiply at all, rather than all cells multiplying at slower rates. In addition, the proportion of injured cells correlated to the compound dosage. We concluded that the oligotrophic test may be useful to asses toxicity of unknown chemicals on a variety of model bacteria. Mul��tiple tests can be run in parallel and effects are rapidly measured within a period of 8 hours. Interestingly, in the same exposure tests with P. fluorescens SV3 we observed that some chemicals which did not lead to a reduction of net population growth rates did cause measurable effects on live cells. This was mainly observed in cells within the live subpopulation as an increase of the EB fluorescence signal. We showed that SYT09/EB is a more useful combination of dyes than SYT09/PI because PI fluorescence tend to increase only when cells are effectively dead, but not so much in live cells (less then twofold). In contrast, EB geometric mean fluorescence in live cells increased up to eightfold after exposure to toxic compounds. All compounds even at the lowest concentration caused a measurable increase in EB geometric mean fluorescence especially after 2 h incubation time. This effect was found to be transient for cells exposed to 2CBP and 4CP, but chronic for cells incubated with TD and NAH (ultimately leading to cell death). In order to understand the mechanism underlying the observed effects we used known membrane or energy uncouplers. The pattern of EB signal increase in chemical-exposed populations resembled mostly that of EDTA, although EB fluorescence in EDTA-treated or pasteurized cells was even higher than after exposure to the four test chemicals. We conclude that the ability of cells to efflux EB under equilibrium conditions is an appropriate measure for the potential of a chemical to exert toxicity. Since most bacterial species possess efflux systems for EB that all require cellular energy, our test should be more widely relevant to infer toxicity effects of chemical exposure on the physiological status of the bacterial cell. To better understand the effect of toxicant exposure on efflux defense systems, we studied 2-hydroxybiphenyl toxicity to Pseudomonas azeiaica HBP1. We showed that 2-HBP exerts toxicity even to P. azelaica HBP1, but only at concentrations higher than 0.5 mM. Above this concentration transient loss of membrane polarization and integrity occurred, which we conclude from staining of growing cells with fluorescent dyes. Cells finally recover and resume growth on 2HBP. The high resistance of P. azelaica HBP1 to 2-HBP was found to be the result of an efficient MexABOprM- type efflux pump system counteracting passive influx of this compound into the membrane and cellular interior. Mutants with disrupted mexA, mexB and oprM genes did no longer grow on 2-HBP at concentrations above 100 μΜ, whereas below this concentration we found 2-HBP-concentration dependent decrease of growth rate. The MexAB-OprM system in P. azeiaica HBP1 is indeed an efflux pump for ethidium bromide as well. By introducing gfp reporter fusions responsive to intracellular 2- HBP concentrations into HBP1 wild-type or the mutants we demonstrated that 2HBP enters into the cells in a similar way. In contrast, the reporter system in the wild-type cells does not react to 2-HBP at an outside concentration of 2.4 μΜ, whereas in mutant cells it does. This suggests that wild-type cells pump 2-HBP to the outside very effectively preventing accumulation of 2-HBP. 2HBP metabolism, therefore, is not efficient enough to lower the intracellular concentration and prevent toxicity. We conclude that P. azelaica HBP1 resistance to 2-HBP is mainly due to an efficient efflux system and that 2HBP in high concentrations exerts narcotic effects on the bacterial membrane. In the part of this thesis, we investigated the possibilities of bacteria to degrade pollutants at low concentrations (1 mg per L and below). As test components we used 2-hydroxybiphenyl, antibiotics and a variety of fragrances, many of which are known to be difficult to biodegrade. By using accurate counting of low numbers of bacterial cells we could demonstrate that specific growth on these compounds is possible. We demonstrated the accuracy of FC counting at low cell numbers (down to 103 bacterial cells per ml). Then we tested whether bacterial population growth could be specifically monitored at the expense of low substrate concentrations, us��ing P. azelaica HBP1. A perfect relationship was found between growth rate, yield and 2-HBP concentrations in the range of 0.1 up to 5 mg per L. Mixing P. azelaica within sludge, however, suggested that growth yields in a mixed community can be much lower than in pure culture, perhaps because of loss of metabolic intermediates. We then isolated new strains from activated sludge using 2-HBP or antibiotics (Nal, AMP, SMX) at low concentrations (0.1-1 mg per L) as sole carbon and energy sub��strate and PAO microdishes. The purified strains were then examined for growth on their respective substrate, which interestingly, showed that all strains can not with��stand higher than 1 or 10 mg per L concentrations of target substrate. Thus, bacteria must exist that contribute to compound degradation at low pollutant concentrations but are inhibited at higher concentrations. Finally we tested whether specific biomass growth (in number of cells) at the expense of pollutants can also be detected with communities as starting material. Hereto, we focused on a number of fragrance chemicals and measured community biomass increase by flow cytometry cell counting on two distinct starter communities: (i) diluted Lake Geneva water, and dilute activated sludge from a wastewater treatment plant. We observed that most of the test compounds indeed resulted in significant biomass increase in the starter community compared to a no-carbon added control, but activated sludge and lake Geneva water strongly differed (almost mutually ex��clusive) in their capacity to degrade the test chemicals. In two cases for activated sludge the same type of microbial community developed upon compound exposure, as concluded from transcription fragment length polymorphism analysis on community purified and PCR amplified 16S rRNA gene fragments. To properly test compound biodegradability it is thus important to use starter communities of different origin. We conclude that FC counting can be a valuable tool to screen chemicals for their biodegradability and toxicity. - Des milliers de produits chimiques sont lib��r��s dans l'environnement mais beaucoup ont des effets inconnus, en particulier �� basses concentrations. Ce travail de th��se contribue �� notre comprehension des effets de la pollution en utilisant des bacteries comme des organismes-tests. Les bacteries sont importantes pour etudier cette ques��tion car certaines d'entre elles peuvent degrader ou transformer les polluants, mais ��galement parce qu'elles-mmes peuvent tre inhibees dans leur reproduction apr��s avoit ete exposees �� ces composes toxiques. Quand des effets inhibiteurs ont lieu, la composition de la communaut�� microbienne peut tre changee �� long terme, ce qui m��ne �� une reduction du service d'ecosyst��me offert par ces communaut��s. En consequence, apr��s leur liberation dans l'environnement, les produits chimiques d'origine anthropogenique peuvent soit s'y accumuler et per��sister, exerant ainsi des effets encore inconnus sur les organismes vivants. En plus d'acqu��rir des connaissances de base sur les effets des polluants �� basses concentra��tions sur les communaut��s microbiennes, un but applique de cette th��se ��tait de d��velopper des tests bases sur les bacteries afin d'identifier de nouveau composes pour leur toxicit�� ou leur biod��gradation. Dans la premi��re partie de ce travail, nous avons developpe un test base sur la cytometrie de flux (FC) sur des cellules de Pseudomonas fluorescens colorees par du bromure d'ethidium ou de l'iodure de propidium et exposees ou non �� une palette de polluants sous des conditions de croissance oligotrophique. La cytometrie de flux est une technique qui conna��t de nombreuses applications dans la microbiologie environ��nementale. Cela est principalement du au fait qu'elle permet un comptage rapide et precis ainsi que l'��valuation de l'��tat physiologique, en particulier lorsqu'elle est combin��e h des colorations fluorescentes. Ici, nous avons utilise la technique FC et des colorants fluorescents afin de mesurer l'effet que peuvent exercer certains pollu��ants sur Pseudomonas ��uorescens SV3 . D'abord nous avons conu des tests oligo- trophiques qui nous permettent de suivre la croissance compl��te de cellules en culture h des densites faibles (104 -10 7 cellules par ml), sur de l'acetate de sodium �� 0.1 mM, en presence ou absence de produits chimiques (2-chlorobiphenyl (2CBP), naphthal��ne (NAH), 4-chlorophenol (4CP), tetradecane (TD), chlorure de mercure(II) (HgCl2)) �� diff��rentes concentrations. Afin de montrer le devenir des bacteries tant au niveau de la cellule individuelle que celui de la population globale, apr��s exposition �� des series de composes chimiques, nous avons compte les cellules colorees avec du SYT09 (col��orant fluorescent vert des acides nucl��iques pour la discrimination des cellules par rapport au bruit de fond) en combinaison avec l'iodure de propidium (PI) ou le bromure d'ethidium (EB), indicateurs de l'int��grit�� de la membrane cellulaire avec FC. Nous avons observe que de nombreux composes testes avaient un effet sur la croissance bacterienne, resultant en une baisse du taux de reproduction de la pop��ulation. En outre, la double coloration que nous avons utilisee dans cette etude SYT09/PI ou SYT09/EB a montre que les produits chimiques testes induisaient une reponse heterog��ne des cellules dans la population, divisant celle-ci en sous- populations "saine", "endommagee" ou "morte". Les nombres de cellules �� partir du comptage et de la proportion de celles "saines" et "endommagees/mortes" ont ensuite ete utilises pour modeliser la croissance de P. ��uorescens SV3 exposee aux produits chimiques. La reduction nette dans la croissance de population est une consequence du fait que de plus en plus de cellules sont incapables de se reproduire, plutt que du fait d'une croissance plus lente de l'ensemble de la population. De plus, la proportion de cellules endommagees est correllee au dosage du compose chimique. Les r��sultats obtenus nous ont permis de conclure que le test oligotrophique que nous avons developpe peut tre utilise pour l'��valuation de la toxicit�� de produits chimiques sur diff��rents mod��les bacteriens. Des tests multiples peuvent tre lances en parall��le et les effets sont mesures en l'espace de huit heures. Par ailleurs, nous en d��duisons que les produits chimiques exerc��nt un effet sur la croissance des cellules de P. ��uorescens SV3, qui est heterog��ne parmi les cellules dans la population et depend du produit chimique. Il est int��ressant de noter que dans les mmes tests d'exposition avec P. ��uorescens SV3, nous avons observe que certains composes qui n'ont pas conduit �� une reduction du taux de la croissance nette de la population, ont cause des effets mesurables sur les cellule saines. Ceci a ete essentiellement observe dans la portion "saine" des cellules en tant qu'augmentation du signal de la fluorescence de 1ΈΒ. D'abord nous avons montre que SYT09/EB ��tait une com��binaison de colorants plus utile que celle de SYT09/PI parce que la fluorescence du PI a tendance �� augmenter uniquement lorsque les cellules sont effectivement mortes, et non pas dans les cellules saines (moins de deux fois plus). Par opposi��tion, la fluorescence moyenne de l'EB dans les cellules saines augmente jusqu'�� huit fois plus apr��s exposition aux composes toxiques. Tous les composes, mme aux plus basses concentrations, induisent une augmentation mesurable de la fluorescence moy��enne de 1ΈΒ, plus particuli��rement apr��s deux heures d'incubation. Cet effet s'est revele tre transitoire pour les cellules exposees aux 2CNP et 4CP, mais est chro��nique pour les cellules incubees avec le TD et le NAH (entranant la mort cellulaire). Afin de comprendre les m��canismes qui sous-tendent les effets observes, nous avons utilise des decoupleurs d'energie ou de membrane. L'augmentation du signal EB dans les populations causee par des produits chimiques ressemblait �� celle exerce par le chelateur des ions divalents EDTA. Cependant, les intensit��s du signal EB des cellules exposees aux produits chimiques testees n'ont jamais atteint les valeurs des cellules traitees avec l'EDTA ou pasteurises. Nous en concluons que le test oli- gotrophique utilisant la coloration (SYT09/)EB des cellules exposees ou non �� un produit chimique est utile afin d'evaluer l'effet toxique exerce par les polluants sur la physiologie bacterienne. Afin de mieux comprendre la reaction d'un syst��me de defense par pompe �� efflux apr��s exposition �� une toxine, nous avons ��tudi�� la toxicit�� du 2-hydroxybiphenyl (2-HBP) sur Pseudomonas azeiaica HBP1. Nous avons montre que le 2-HBP exerce une toxicit�� mme sur HBP1, mais uniquement �� des concentrations sup��rieures �� 0.5 mM. Au-dessus de cette concentration, des pertes transitoires d'int��grit�� et de polarization membranaire ont lieu, comme cela nous a ete montre par coloration des cellules en croissance. Les cellules sont finalement capables de se r��tablir et de reprendre leur croissance sur 2-HBP. La forte resistance de P. azeiaica HBP1 h 2-HBP physiologie bacterienne s'est revele tre le r��sultat d'un syst��me de pompe h efflux de type MexABOprM qui contre-balance l'influx passif de ce compose h travers la membrane. Nous avons montre, en construisant des mutants avec des insertions dans les g��nes mexA, mexB and oprM et des fusions avec le g��ne rapporteur gfp, que l'alt��ration de n'importe quelle partie du syst��me d'efflux conduisait �� accro��tre l'accumulation de 2-HBP dans la cellule, en comparaison avec la souche sauvage HBP1, provoquant une diminution de la resistance au 2-HBP ainsi qu'une baisse du taux de reproduction des cellules. Des syst��mes d'efflux similaires sont r��pandus chez de nombreuses esp��ces bact��riennes. Ils seraient responsables de la resistance aux produits chimiques tels que les colorants fluorescents (bromure d'ethidium) et des antibiotiques. Nous concluons que la resistance de P. azelaica HBP1 �� 2-HBP est principalement due �� un syst��me d'efflux efficace et que 2-HBP, �� des concentrations elevees, exerce un effet delet��re sur la membrane bacterienne. En se basant sur le comptage des cellules avec la FC, nous avons developpe ensuite une methode pour evaluer la biodegradabilite de polluants tels que le 2-HBP ainsi que les antibiotiques (acide nalidixique (Nal), ampicilline (AMP) ou sulfamethoxazole (SMX)) �� de faibles concentrations lmg par L et moins), par le suivi de la croissance sp��cifique sur le compose de cultures microbiennes pures et mixtes. En utilisant un comptage precis de faibles quantit��s de cellules nous avons pu demontrer que la croissance sp��cifique sur ces composes est possible. Nous avons pu illustrer la precision du comptage par cytometrie de flux �� faible quantit�� de cellules (jusqu'�� 10 3 cellules par ml). Ensuite, nous avons teste s'il ��tait possible de suivre dynamiquement la croissance de la population de cellules sur faibles concentrations de substrats, en utilisant P. azelaica HBP1. Une relation parfaite a ete trouvee entre le taux de croissance, le rendement et les concentrations de 2-HBP (entre 0.1 et 5 mg par L). En m��langeant HBP1 �� de la boue active, nous avons pu montrer que le rendement en communaut�� mixtes pouvait tre bien inf��rieur qu'en culture pure. Ceci ��tant peut tre le r��sultat d'une perte d'interm��diaires m��taboliques. Nous avons ensuite isole de nouvelles souches �� partir de la boue active en utilisant le 2-HBP ou des antibiotiques (Nal, AMP, SMX) h basses concentrations (0.1-1 mg par L) comme seules sources de carbone et d'energie. En combinaison avec ceci, nous avons ��galement utilise des microplaques PAO. Les souches purifiees ont ensuite ete examinees pour leurs croissances sur leurs substrats respectifs. De faon int��ressante, toutes ces souches ont montre qu'elles ne pouvaient pas survivre �� des concentrations de substrats sup��rieures �� 1 ou 10 mg par L. Ainsi, il existe des bacteries qui contribuent �� la degradation de composes �� basses concentrations de polluant mais sont inhibes lorsque ces concentrations deviennent plus hautes. Finalement, nous avons cherche �� savoir s'il est possible de detecter une croissance sp��cifique �� une biomasse au depend d'un polluant, en partant d'une communaut�� microbienne. Ainsi, nous nous sommes concentre sur certains composes et avons mesure l'augmentation de la biomasse d'une communaut�� grce �� la cytometrie de flux. Nous avons compte deux communaut��s de depart distinctes: (i) une dilution d'eau du Lac L��man, et une dilution de boue active d'une station d'��puration. Nous avons observe que la plupart des composes testes ont entrane une augmentation de la biomasse de depart par rapport au control sans addition de source de carbone. N��anmoins, les ��chantillons du lac L��man et de la station d'��puration diff��raient largement (s'excluant mutuellement l'un l'autre) dans leur capacit�� �� degrader les composes chimiques. Dans deux cas provenant de la station d'��puration, le mme type de communaut�� microbienne s'est developpe apr��s exposition aux composes, comme l'a d��montr�� l'analyse TRFLP sur les fragments d'ARN 16S purifie de la communaut�� et amplifie par PCR. Afin de tester correctement la biodegradabilite d'un compose, il est donc important d'utiliser des communaut��s de depart de diff��rentes origines Nous en concluons que le comptage par cytometrie de flux peut tre un outil de grande utilit�� pour mettre en valeur la biodegradabillite et la toxicit�� des composes chimiques.

Molecular modeling of peptide-MHC class I complexes : applications to peptide based immunotherapy

Summary The specific CD8+ T cell immune response against tumors relies on the recognition by the T cell receptor (TCR) on cytotoxic T lymphocytes (CTL) of antigenic peptides bound to the class I major histocompatibility complex (MHC) molecule. Such tumor associated antigenic peptides are the focus of tumor immunotherapy with peptide vaccines. The strategy for obtaining an improved immune response often involves the design of modified tumor associated antigenic peptides. Such modifications aim at creating higher affinity and/or degradation resistant peptides and require precise structures of the peptide-MHC class I complex. In addition, the modified peptide must be cross-recognized by CTLs specific for the parental peptide, i.e. preserve the structure of the epitope. Detailed structural information on the modified peptide in complex with MHC is necessary for such predictions. In this thesis, the main focus is the development of theoretical in silico methods for prediction of both structure and cross-reactivity of peptide-MHC class I complexes. Applications of these methods in the context of immunotherapy are also presented. First, a theoretical method for structure prediction of peptide-MHC class I complexes is developed and validated. The approach is based on a molecular dynamics protocol to sample the conformational space of the peptide in its MHC environment. The sampled conformers are evaluated using conformational free energy calculations. The method, which is evaluated for its ability to reproduce 41 X-ray crystallographic structures of different peptide-MHC class I complexes, shows an overall prediction success of 83%. Importantly, in the clinically highly relevant subset of peptide-HLAA*0201 complexes, the prediction success is 100%. Based on these structure predictions, a theoretical approach for prediction of cross-reactivity is developed and validated. This method involves the generation of quantitative structure-activity relationships using three-dimensional molecular descriptors and a genetic neural network. The generated relationships are highly predictive as proved by high cross-validated correlation coefficients (0.78-0.79). Together, the here developed theoretical methods open the door for efficient rational design of improved peptides to be used in immunotherapy. R��sum�� La r��ponse immunitaire sp��cifique contre des tumeurs d��pend de la reconnaissance par les r��cepteurs des cellules T CD8+ de peptides antig��niques pr��sent��s par les complexes majeurs d'histocompatibilit�� (CMH) de classe I. Ces peptides sont utilis��s comme cible dans l'immunoth��rapie par vaccins peptidiques. Afin d'augmenter la r��ponse immunitaire, les peptides sont modifi��s de fa��on �� am��liorer l'affinit�� et/ou la r��sistance �� la d��gradation. Ceci n��cessite de conna��tre la structure tridimensionnelle des complexes peptide-CMH. De plus, les peptides modifi��s doivent ��tre reconnus par des cellules T sp��cifiques du peptide natif. La structure de l'��pitope doit donc ��tre pr��serv��e et des structures d��taill��es des complexes peptide-CMH sont n��cessaires. Dans cette th��se, le th��me central est le d��veloppement des m��thodes computationnelles de pr��diction des structures des complexes peptide-CMH classe I et de la reconnaissance crois��e. Des applications de ces m��thodes de pr��diction �� l'immunoth��rapie sont ��galement pr��sent��es. Premi��rement, une m��thode th��orique de pr��diction des structures des complexes peptide-CMH classe I est d��velopp��e et valid��e. Cette m��thode est bas��e sur un ��chantillonnage de l'espace conformationnel du peptide dans le contexte du r��cepteur CMH classe I par dynamique mol��culaire. Les conformations sont ��valu��es par leurs ��nergies libres conformationnelles. La m��thode est valid��e par sa capacit�� �� reproduire 41 structures des complexes peptide-CMH classe I obtenues par cristallographie aux rayons X. Le succ��s pr��dictif g��n��ral est de 83%. Pour le sous-groupe HLA-A*0201 de complexes de grande importance pour l'immunoth��rapie, ce succ��s est de 100%. Deuxi��mement, �� partir de ces structures pr��dites in silico, une m��thode th��orique de pr��diction de la reconnaissance crois��e est d��velopp��e et valid��e. Celle-ci consiste �� g��n��rer des relations structure-activit�� quantitatives en utilisant des descripteurs mol��culaires tridimensionnels et un r��seau de neurones coupl�� �� un algorithme g��n��tique. Les relations g��n��r��es montrent une capacit�� de pr��diction remarquable avec des valeurs de coefficients de corr��lation de validation crois��e ��lev��es (0.78-0.79). Les m��thodes th��oriques d��velopp��es dans le cadre de cette th��se ouvrent la voie du design de vaccins peptidiques am��lior��s.

Glibenclamide enhances neurogenesis and improves long-term functional recovery after transient focal cerebral ischemia

Glibenclamide is neuroprotective against cerebral ischemia in rats. We studied whether glibenclamide enhances long-term brain repair and improves behavioral recovery after stroke. Adult male Wistar rats were subjected to transient middle cerebral artery occlusion (MCAO) for 90 minutes. A low dose of glibenclamide (total 0.6mg) was administered intravenously 6, 12, and 24 hours after reperfusion. We assessed behavioral outcome during a 30-day follow-up and animals were perfused for histological evaluation. In vitro speci���c binding of glibenclamide to microglia increased after pro-in���ammatory stimuli. In vivo glibenclamide was associated with increased migration of doublecortin-positive cells in the striatum toward the ischemic lesion 72 hours after MCAO, and reactive microglia expressed sulfonylurea receptor 1 (SUR1) and Kir6.2 in the medial striatum. One month after MCAO, glibenclamide was also associated with increased number of NeuN-positive and 5-bromo-2-deoxyuridine-positive neurons in the cortex and hippocampus, and enhanced angiogenesis in the hippocampus. Consequently, glibenclamide-treated MCAO rats showed improved performance in the limb-placing test on postoperative days 22 to 29, and in the cylinder and water-maze test on postoperative day 29. Therefore, acute blockade of SUR1 by glibenclamide enhanced long-term brain repair in MCAO rats, which was associated with improved behavioral outcome.

Intermittent hemodialysis treatment in cefepime-induced neurotoxicity: Case report, pharmacokinetic modeling, and review of the literature.

Cefepime is a broad-spectrum cephalosporin indicated for in-hospital treatment of severe infections. Acute neurotoxicity, an increasingly recognized adverse effect of this drug in an overdose, predominantly affects patients with reduced renal function. Although dialytic approaches have been advocated to treat this condition, their role in this indication remains unclear. We report the case of an 88-year-old female patient with impaired renal function who developed life-threatening neurologic symptoms during cefepime therapy. She was treated with two intermittent 3-hour high-flux, high-efficiency hemodialysis sessions. Serial pre-, post-, and peridialytic (pre- and postfilter) serum cefepime concentrations were measured. Pharmacokinetic modeling showed that this dialytic strategy allowed for serum cefepime concentrations to return to the estimated nontoxic range 15 hours earlier than would have been the case without an intervention. The patient made a full clinical recovery over the next 48 hours. We conclude that at least 1 session of intermittent hemodialysis may shorten the time to return to the nontoxic range in severe clinically patent intoxication. It should be considered early in its clinical course pending chemical confirmation, even in frail elderly patients. Careful dosage adjustment and a high index of suspicion are essential in this population.

Improved multiexponential transient spectroscopy by iterative deconvolution

The analysis of multiexponential decays is challenging because of their complex nature. When analyzing these signals, not only the parameters, but also the orders of the models, have to be estimated. We present an improved spectroscopic technique specially suited for this purpose. The proposed algorithm combines an iterative linear filter with an iterative deconvolution method. A thorough analysis of the noise effect is presented. The performance is tested with synthetic and experimental data.

Dynamic compact thermal models with multiple power sources: application to an ultrathin chip stacking technology

Whereas numerical modeling using finite-element methods (FEM) can provide transient temperature distribution in the component with enough accuracy, it is of the most importance the development of compact dynamic thermal models that can be used for electrothermal simulation. While in most cases single power sources are considered, here we focus on the simultaneous presence of multiple sources. The thermal model will be in the form of a thermal impedance matrix containing the thermal impedance transfer functions between two arbitrary ports. Eachindividual transfer function element ( ) is obtained from the analysis of the thermal temperature transient at node �� �� after a power step at node �� .�� Different options for multiexponential transient analysis are detailed and compared. Among the options explored, small thermal models can be obtained by constrained nonlinear least squares (NLSQ) methods if the order is selected properly using validation signals. The methods are applied to the extraction of dynamic compact thermal models for a new ultrathin chip stack technology (UTCS).

Measurements of seismic attenuation and transient fluid pressure in partially saturated Berea sandstone: Evidence of fluid flow on the mesoscopic scale

A novel laboratory technique is proposed to investigate wave-induced fluid flow on the mesoscopic scale as a mechanism for seismic attenuation in partially saturated rocks. This technique combines measurements of seismic attenuation in the frequency range from 1 to 100?Hz with measurements of transient fluid pressure as a response of a step stress applied on top of the sample. We used a Berea sandstone sample partially saturated with water. The laboratory results suggest that wave-induced fluid flow on the mesoscopic scale is dominant in partially saturated samples. A 3-D numerical model representing the sample was used to verify the experimental results. Biot's equations of consolidation were solved with the finite-element method. Wave-induced fluid flow on the mesoscopic scale was the only attenuation mechanism accounted for in the numerical solution. The numerically calculated transient fluid pressure reproduced the laboratory data. Moreover, the numerically calculated attenuation, superposed to the frequency-independent matrix anelasticity, reproduced the attenuation measured in the laboratory in the partially saturated sample. This experimental?numerical fit demonstrates that wave-induced fluid flow on the mesoscopic scale and matrix anelasticity are the dominant mechanisms for seismic attenuation in partially saturated Berea sandstone.

Local transient myocardial liposomal gene transfer of inducible nitric oxide synthase does not aggravate myocardial function and fibrosis and leads to moderate neovascularization in chronic myocardial ischemia in pigs.

Microcirculation (2010) 17, 69-78. doi: 10.1111/j.1549-8719.2010.00002.x Abstract Background: This study was designed to explore the effect of transient inducible nitric oxide synthase (iNOS) overexpression via cationic liposome-mediated gene transfer on cardiac function, fibrosis, and microvascular perfusion in a porcine model of chronic ischemia. Methods and Results: Chronic myocardial ischemia was induced using a minimally invasive model in 23 landrace pigs. Upon demonstration of heart failure, 10 animals were treated with liposome-mediated iNOS-gene-transfer by local intramyocardial injection and 13 animals received a sham procedure to serve as control. The efficacy of this iNOS-gene-transfer was demonstrated for up to 7 days by reverse transcriptase-polymerase chain reaction in preliminary studies. Four weeks after iNOS transfer, magnetic resonance imaging showed no effect of iNOS overexpression on cardiac contractility at rest and during dobutamine stress (resting ejection fraction: control 27%, iNOS 26%; P = ns). Late enhancement, infarct size, and the amount of fibrosis were similar between groups. Although perfusion and perfusion reserve in response to adenosine and dobutamine were not significantly modified by iNOS-transfer, both vessel number and diameter were significantly increased in the ischemic area in the iNOS-treated group versus control (point score: control 15.3, iNOS 34.7; P < 0.05). Conclusions: Our findings demonstrate that transient iNOS overexpression does not aggravate cardiac dysfunction or postischemic fibrosis, while potentially contributing to neovascularization in the chronically ischemic heart.

Modeling water movement in horizontal columns using fractal theory

Fractal mathematics has been used to characterize water and solute transport in porous media and also to characterize and simulate porous media properties. The objective of this study was to evaluate the correlation between the soil infiltration parameters sorptivity (S) and time exponent (n) and the parameters dimension (D) and the Hurst exponent (H). For this purpose, ten horizontal columns with pure (either clay or loam) and heterogeneous porous media (clay and loam distributed in layers in the column) were simulated following the distribution of a deterministic Cantor Bar with fractal dimension H" 0.63. Horizontal water infiltration experiments were then simulated using Hydrus 2D software. The sorptivity (S) and time exponent (n) parameters of the Philip equation were estimated for each simulation, using the nonlinear regression procedure of the statistical software package SAS��. Sorptivity increased in the columns with the loam content, which was attributed to the relation of S with the capillary radius. The time exponent estimated by nonlinear regression was found to be less than the traditional value of 0.5. The fractal dimension estimated from the Hurst exponent was 17.5 % lower than the fractal dimension of the Cantor Bar used to generate the columns.

Multi-level modeling of aspects associated with poor mental health in a sample of prehospital emergency professionals.

BACKGROUND: The goal of this paper is to investigate the respective influence of work characteristics, the effort-reward ratio, and overcommitment on the poor mental health of out-of-hospital care providers. METHODS: 333 out-of-hospital care providers answered a questionnaire that included queries on mental health (GHQ-12), demographics, health-related information and work characteristics, questions from the Effort-Reward Imbalance Questionnaire, and items about overcommitment. A two-level multiple regression was performed between mental health (the dependent variable) and the effort-reward ratio, the overcommitment score, weekly number of interventions, percentage of non-prehospital transport of patients out of total missions, gender, and age. Participants were first-level units, and ambulance services were second-level units. We also shadowed ambulance personnel for a total of 416 hr. RESULTS: With cutoff points of 2/3 and 3/4 positive answers on the GHQ-12, the percentages of potential cases with poor mental health were 20% and 15%, respectively. The effort-reward ratio was associated with poor mental health (P < 0.001), irrespective of age or gender. Overcommitment was associated with poor mental health; this association was stronger in women (&#946; = 0.054) than in men (&#946; = 0.020). The percentage of prehospital missions out of total missions was only associated with poor mental health at the individual level. CONCLUSIONS: Emergency medical services should pay attention to the way employees perceive their efforts and the rewarding aspects of their work: an imbalance of those aspects is associated with poor mental health. Low perceived esteem appeared particularly associated with poor mental health. This suggests that supervisors of emergency medical services should enhance the value of their employees' work. Employees with overcommitment should also receive appropriate consideration. Preventive measures should target individual perceptions of effort and reward in order to improve mental health in prehospital care providers.

Groundwater Availability Modeling of the Lower Dakota Aquifer in Northwest Iowa, October 2008

The 2007 Iowa General Assembly, recognizing the increased demand for water to support the growth of industries and municipalities, approved funding for the first year of a multi-year evaluation and modeling of Iowa���s major aquifers by the Iowa Department of Natural Resources. The task of conducting this evaluation and modeling was assigned to the Iowa Geological and Water Survey (IGWS). The first aquifer to be studied was the Lower Dakota aquifer in a sixteen county area of northwest Iowa.