Construction and analysis of a new conditional ferritin H knockout mouse strain/

Résumé Le fer joue un rôle important dans la plupart des fonctions biologiques mais sa présence excessive provoque la production de molécules réactives d'oxygène (ROS) qui peuvent contribuer à diverses maladies. La protéine de stockage du fer, la ferritine H, capte l'excès en fer et le stocke sous forme non-toxique, ce qui empêche des dommages potentiels. La délétion de la ferritine H dans des souris knock-out a été essayée antérieurement, mais ces souris mouraient au stade précoce du développement embryonnaire. Pour étudier l'importance du fer, et en particulier son stockage dans la ferritine, et pour pouvoir mieux comprendre les fonctions de la ferritine H, nous avons créé un modèle de souris knock-out conditionnelles de la ferritine H, selon le système classique de Cre-LoxP. Le premier exon et la région du promoteur du gène de la ferritine H ont été entourés de sites loxP. La mortalité embryonnaire provoquée par la délétion constitutive du gène de la ferritine H a été confirmée en croisant nos souris avec des souris exprimant nestin-Cre1. En croisant nos souris avec des souris transgéniques Mx-Cre, nous avons observé que l'induction de Cre par injection de polyI-polyC provoque la délétion presque complète de la ferritine H dans le foie (> 99%) et la rate (> 88%). Ces tissus ont également perdu une grande partie de leur réserve de fer. Cette observation apporte pour la première fois la preuve in vivo que la ferritine H est indispensable pour le stockage du fer, que les fonctions de la ferritine H et de la ferritine L ne sont pas équivalentes, et que la ferritine L ne peut pas assumer seule la fonction de stockage du fer. Dans le foie des souris knock-out, l'expression de l'ARN messager de l'hepcidine a été induite après 10 jours. En même temps, l'expression de l'ARN messager des gènes codant pour des protéines de l'absorption de fer (DMT1, ferroportin, Dcytb1 et hephaestin) a été réprimée mais dans le duodénum seulement. L'expression d'hepcidine est inversément corrélée avec celle des gènes liés à l'absorption de fer. Cette observation corrobore des études antérieures. Mais, en plus, elle montre également que cette répression se produit seulement dans l'intestin. Nous pouvons ainsi tirer la conclusion suivante : ou bien l'hepcidine a un récepteur spécifique dans le duodénum ou bien les gènes liés à l'absorption de fer dans le duodénum ont un facteur spécifique de transcription sensible à l'hepcidine. Aucune répression de DMT1 et de ferroportin n'a été observée dans les macrophages de la rate après l'induction d'hepcidine. La délétion de ferritine H a entraîné une augmentation du taux de mortalité des cellules hépatiques, ainsi que des altérations dans l'architecture normale du tissu de la rate. Vu par l'immunohistologie, le nombre de lymphocytes B et T était réduit dans la rate, tendant à démontrer que la ferritine H et l'homéostase du fer jouent un rôle dans l'immunité. En conclusion, le modèle de souris knock-out conditionnelles de la ferritine H nous fournit un outil précieux pour l'étude in vivo du rôle joué par la ferritine dans l'homéostase du fer, dans les dommages créés par les ROS, ainsi que dans l'apoptose et l'immunité. Summary Iron plays an important role in most biological functions. However, excess of iron results in production of reactive oxygen species (ROS) which could substantially contribute to pathology of various diseases. Ferritin H scavenges excess of iron and stores it in non-toxic form and potentially prevents the damage. Fenitin H targeting in mice has been attempted before, however, straight knockout was lethal in early embryonic stage. To study the role of iron and its storage protein ferritin and to further elucidate ferritin H functions, we aimed at creating a conditional ferritin H knockout mouse model by classical Cre-LoxP system. First exon along with promoter region of the ferritin H gene was foxed. Embryonic lethality of the constitutive ferritin H deletion was confirmed by crossing the foxed mice with mice expressing nestin Cre-1 as transgene. Almost complete deletion was observed in liver (> 99%) and spleen (>88%) upon induction of Cre by injecting polyI-polyC in Fth Lox/Lox; MxCre mice. These tissues also lost substantial fraction of their iron stores. This provides first in vivo evidence that ferritin H is required for iron storage, ferritin H and L functions are not redundant and that ferritin L cannot perform iron storage function alone. Hepcidin mRNA expression was induced after 10 days in the livers of deleted mice and, simultaneously, mRNA expression of iron absorption related genes (DMT 1, ferroportin, Dcytb1 and hephaestin) was repressed in duodenum only. Hepcidin expression is inversely correlated with that of duodenal iron absorption related genes. This is in agreement with previous studies. However, we also show that this repression happens only in intestine. This leads to the conclusion that either hepcidin has a specific receptor in duodenum or the iron absorption related genes have duodenum specific transcription factor that is responsive to hepcidin. No repression of DMT1 and ferroportin was observed in spleen macrophages upon hepcidin induction. Ferritin H deletion showed increased cell death in liver and disruption of normal architecture of spleen. B lymphocytes were reduced in spleen on immunohistology which point towards a role of ferritin H and iron homeostasis in immunity. In conclusion, ferritin H conditional knockout mouse model provides us with an invaluable tool to study the in vivo role of ferritin H in iron homeostasis, ROS mediated damage, apoptosis and immunity.

Thrombosis in paroxysmal nocturnal hemoglobinuria at a glance: a clinical review.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired stem cell disorder, with its primary clinical manifestations being hemolytic anemia, marrow failure and thrombophilia. Chronic hemolysis, failures of the fibrinolytic system, increased leukocyte-derived tissue factor levels in plasma, procoagulant microparticles generated through complement-mediated damage of platelets and venous endothelium are related to the acquired hypercoagulable state. Visceral thrombosis (including hepatic veins and mesenteric veins), cerebrovascular events and pulmonary embolism predict a poor outcome. Thrombosis is also associated with significant morbidity during pregnancy. Depending on the sites of thrombosis, a score-based probability to predict outcome can be assigned. Abdominal vein thromboses account for the majority of morbidity and mortality related to thrombosis, and time-dependent trends suggest that mortality rates tend to decline, with the advent of evolution of therapeutic and diagnostic strategies. In contrast, mortality rates from cerebrovascular events display no significant decline. Prompt diagnosis requires both clinical suspicion and sophisticated imaging techniques, along with multidisciplinary therapeutic intervention. In the eculizumab era, a significant reduction of thrombotic events was observed during therapy, and long-term follow up is needed to establish any benefit in rates and pattern of this complication. However, up to now, only bone marrow transplantation permanently abolishes the coagulation defect.

Fas and Fas ligand in embryos and adult mice: ligand expression in several immune-privileged tissues and coexpression in adult tissues characterized by apoptotic cell turnover.

The cell surface receptor Fas (FasR, Apo-1, CD95) and its ligand (FasL) are mediators of apoptosis that have been shown to be implicated in the peripheral deletion of autoimmune cells, activation-induced T cell death, and one of the two major cytolytic pathways mediated by CD8+ cytolytic T cells. To gain further understanding of the Fas system., we have analyzed Fas and FasL expression during mouse development and in adult tissues. In developing mouse embryos, from 16.5 d onwards, Fas mRNA is detectable in distinct cell types of the developing sinus, thymus, lung, and liver, whereas FasL expression is restricted to submaxillary gland epithelial cells and the developing nervous system. Significant Fas and FasL expression were observed in several nonlymphoid cell types during embryogenesis, and generally Fas and FasL expression were not localized to characteristic sites of programmed cell death. In the adult mouse, RNase protection analysis revealed very wide expression of both Fas and FasL. Several tissues, including the thymus, lung, spleen, small intestine, large intestine, seminal vesicle, prostate, and uterus, clearly coexpress the two genes. Most tissues constitutively coexpressing Fas and FasL in the adult mouse are characterized by apoptotic cell turnover, and many of those expressing FasL are known to be immune privileged. It may be, therefore, that the Fas system is implicated in both the regulation of physiological cell turnover and the protection of particular tissues against potential lymphocyte-mediated damage.

TRAIL receptor-mediated JNK activation and Bim phosphorylation critically regulate Fas-mediated liver damage and lethality.

TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family with potent apoptosis-inducing properties in tumor cells. In particular, TRAIL strongly synergizes with conventional chemotherapeutic drugs to induce tumor cell death. Thus, TRAIL has been proposed as a promising future cancer therapy. Little, however, is known regarding what the role of TRAIL is in normal untransformed cells and whether therapeutic administration of TRAIL, alone or in combination with other apoptotic triggers, may cause tissue damage. In this study, we investigated the role of TRAIL in Fas-induced (CD95/Apo-1-induced) hepatocyte apoptosis and liver damage. While TRAIL alone failed to induce apoptosis in isolated murine hepatocytes, it strongly amplified Fas-induced cell death. Importantly, endogenous TRAIL was found to critically regulate anti-Fas antibody-induced hepatocyte apoptosis, liver damage, and associated lethality in vivo. TRAIL enhanced anti-Fas-induced hepatocyte apoptosis through the activation of JNK and its downstream substrate, the proapoptotic Bcl-2 homolog Bim. Consistently, TRAIL- and Bim-deficient mice and wild-type mice treated with a JNK inhibitor were protected against anti-Fas-induced liver damage. We conclude that TRAIL and Bim are important response modifiers of hepatocyte apoptosis and identify liver damage and lethality as a possible risk of TRAIL-based tumor therapy.

Role of pparß in irradiation mediated intestinal damage

Les récepteurs activés proliférateurs de peroxisomes (PPARs) appartiennent à la grande famille des récepteurs nucléaires et ont été impliqué dans plusieurs processus physiologiques. Parmi les trois isotypes PPAR, PPARβ est bien connu pour son rôle dans les décisions déterminant le destin des cellules, notamment dans les processus de prolifération, de différentiation et d'apoptose. Ce rôle a particulièrement été souligné comme protecteur dans les contextes de survie cellulaire et de cicatrisation. Il est fortement exprimé dans l'intestin grêle. Notre groupe a déjà rapporté sa présence importante dans les cryptes duodénales, où se trouvent les cellules souches intestinales. Précédemment, nous avons aussi fait remarquer le rôle de PPARβ dans la differentiation des cellules de Paneth, par la régulation négative de la signalisation Ihh de l'épithélium intestinal. Malgré sa capacité de figurer parmi les tissus du corps qui se régénèrent le plus rapidement, l'épithélium intestinal est particulièrement sensible aux attaques cytotoxiques, surtout celles dues à la radiothérapie des cancers abdomino-pelviens. Cela peut donner lieu à des lésions gastro-intestinal en tant qu'effet indésirable d'une exposition aiguë et chronique à l'irradiation. En raison du rôle protecteur de PPARβ le but de cette étude était de comprendre les voies de signalisation moléculaires régulées par PPARβ qui sont impliquées dans les réponses des cellules intestinales aux dommages causés par l'irradiation.Afin de déchiffrer les mécanismes moléculaires sous-jacents, un modèle in-vitro d'une lignée cellulaire - HT-29 a été utilisée. Il n'y a cependant pas de preuve d'un effet protecteur de PPARβ dans divers contextes d'endommagement cellulaire testés in-vitro. Ceci contraste avec les observations in-vivo qui indiquent que l'irradiation provoque une létalité supérieure dans les souris PPARβ-/- par rapport aux souris PPARβ+/+, entre autre correlée avec une apoptose augmentée des cellules souches intestinales à 4h après irradiation. En plus, le décès plus important de cellules mésenchymateuses a été observé dans les souris PPARβ-/-, 8 jours après irradiation. Moins nombreuses, ces cellules se sont également détachées de la matrice extracellulaire reliant l'épithélium et le mésenchyme. Nous stipulons qu'in-vivo, PPARβ participe au dialogue entre le mésenchyme et l'épithélium, ce qui est concordant avec le délai observé lors de la réparation tissulaire. Ce dialogue entre l'épithélium et le mésenchyme, n'existe pas de la même manière in-vitro. Il en résulte donc un défaut de réponse mésenchymale médiée par PPARβ, d'où le paradoxe entre les conditions in-vivo et in-vitro.Ces observations indiquent l'implication possible de PPARβ dans les lesions actiniques, en tant que conséquence naturelle de la radiothérapie de patients avec un cancer. Les mécanismes précis de l'action de PPARβ nécessitent une exploration approfondie de son rôle physiologique dans ce contexte.

A peptide inhibitor of C-jun N-terminal kinase modulates hepatic damage and the inflammatory response after hemorrhagic shock and resuscitation

Hemorrhage and resuscitation (H/R) leads to phosphorylation of mitogen-activated stress kinases, an event that is associated with organ damage. Recently, a specific, cell-penetrating, protease-resistant inhibitory peptide of the mitogen-activated protein kinase c-JUN N-terminal kinase (JNK) was developed (D-JNKI-1). Here, using this peptide, we tested if inhibition of JNK protects against organ damage after H/R. Male Sprague-Dawley rats were treated with D-JNKI-1 (11 mg/kg, i.p.) or vehicle. Thirty minutes later, rats were hemorrhaged for 1 h to a MAP of 30 to 35 mmHg and then resuscitated with 60% of the shed blood and twice the shed blood volume as Ringer lactate. Tissues were harvested 2 h later. ANOVA with Tukey post hoc analysis or Kruskal-Wallis ANOVA on ranks, P < 0.05, was considered significant. c-JUN N-terminal kinase inhibition decreased serum alanine aminotransferase activity as a marker of liver injury by 70%, serum creatine kinase activity by 67%, and serum lactate dehydrogenase activity by 60% as compared with vehicle treatment. The histological tissue damage observed was blunted after D-JNKI-1 pretreatment both for necrotic and apoptotic cell death. Hepatic leukocyte infiltration and serum IL-6 levels were largely diminished after D-JNKI-1 pretreatment. The extent of oxidative stress as evaluated by immunohistochemical detection of 4-hydroxynonenal was largely abrogated after JNK inhibition. After JNK inhibition, activation of cJUN after H/R was also reduced. Hemorrhage and resuscitation induces a systemic inflammatory response and leads to end-organ damage. These changes are mediated, at least in part, by JNK. Therefore, JNK inhibition deserves further evaluation as a potential treatment option in patients after resuscitated blood loss.

Drug refractory epilepsy in brain damage: effect of dextromethorphan on EEG in four patients.

High doses of dextromethorphan (20-42 mg/kg/day) were given to four critically ill children with seizures and frequent epileptiform abnormalities in the EEG that were refractory to antiepileptic drugs. Their acute diseases (hypoxia, head trauma and hypoxia, neurodegenerative disease, hypoglycaemia) were thought to be due in part to N-methyl-D-aspartate (NMDA) receptor mediated processes. Treatment with dextromethorphan, an NMDA receptor antagonist, was started between 48 hours and 14 days after the critical incident. In three patients the EEG improved considerably within 48 hours and seizures ceased within 72 hours. In the patient with neurodegenerative disease the effect on the EEG was impressive, but the seizures were not controlled. Despite the improvement of the EEG the clinical outcome was poor in all children: three died in the critical period or due to the progressing disease; the patient with hypoglycaemia survived with severe neurological sequelae. Plasma concentrations of dextromethorphan varied between 74-1730 ng/ml and its metabolite dextrorphan varied between 349-3790 ng/ml. In one patient corresponding concentrations in CSF were lower than those in plasma. The suppression of epileptic discharges by the doses of dextromethorphan given suggests that such doses are sufficient to block NMDA receptors.

Visualization of recombination intermediates produced by RAD52-mediated single-strand annealing.

Double-strand breaks (DSBs) occur frequently during DNA replication. They are also caused by ionizing radiation, chemical damage or as part of the series of programmed events that occur during meiosis. In yeast, DSB repair requires RAD52, a protein that plays a critical role in homologous recombination. Here we describe the actions of human RAD52 protein in a model system for single-strand annealing (SSA) using tailed (i.e. exonuclease resected) duplex DNA molecules. Purified human RAD52 protein binds resected DSBs and promotes associations between complementary DNA termini. Heteroduplex intermediates of these recombination reactions have been visualized by electron microscopy, revealing the specific binding of multiple rings of RAD52 to the resected termini and the formation of large protein complexes at heteroduplex joints formed by RAD52-mediated annealing.

NLRP3 Suppresses NK Cell-Mediated Responses to Carcinogen-Induced Tumors and Metastases.

The NLRP3 inflammasome acts as a danger signal sensor that triggers and coordinates the inflammatory response upon infectious insults or tissue injury and damage. However, the role of the NLRP3 inflammasome in natural killer (NK) cell-mediated control of tumor immunity is poorly understood. Here, we show in a model of chemical-induced carcinogenesis and a series of experimental and spontaneous metastases models that mice lacking NLRP3 display significantly reduced tumor burden than control wild-type (WT) mice. The suppression of spontaneous and experimental tumor metastases and methylcholanthrene (MCA)-induced sarcomas in mice deficient for NLRP3 was NK cell and IFN-γ-dependent. Focusing on the amenable B16F10 experimental lung metastases model, we determined that expression of NLRP3 in bone marrow-derived cells was necessary for optimal tumor metastasis. Tumor-driven expansion of CD11b(+)Gr-1(intermediate) (Gr-1(int)) myeloid cells within the lung tumor microenvironment of NLRP3(-/-) mice was coincident with increased lung infiltrating activated NK cells and an enhanced antimetastatic response. The CD11b(+)Gr-1(int) myeloid cells displayed a unique cell surface phenotype and were characterized by their elevated production of CCL5 and CXCL9 chemokines. Adoptive transfer of this population into WT mice enhanced NK cell numbers in, and suppression of, B16F10 lung metastases. Together, these data suggested that NLRP3 is an important suppressor of NK cell-mediated control of carcinogenesis and metastases and identify CD11b(+)Gr-1(int) myeloid cells that promote NK cell antimetastatic function. Cancer Res; 72(22); 5721-32. ©2012 AACR.

Hexyl-aminolevulinate mediated photodynamic therapy: how to spare normal urothelium. An in vitro approach

Résumé Objectifs : La thérapie photodynamique a pour but la destruction sélective du tissu néoplasique par interaction de lumière, d'oxygène et d'une substance photosensibilisatrice (la Protoporphyrine IX dans notre étude). Malgré une accumulation sélective du photosensibilisateur dans le tissu tumoral, la thérapie photodynamique du carcinome urothélial de la vessie peut endommager les cellules normales de l'épithélium urinaire. La prévention de ces lésions est importante pour la régénération de la muqueuse. Notre étude sur un modèle in vitro d'urothélium porcin étudie l'influence de la concentration du photosensibilisateur, des paramètres d'irradiation et de la production d'intermédiaires réactifs de l'oxygène (ROS) sur les effets photodynamique. Le but était de déterminer les conditions seuil pour épargner l'urothélium sain. Méthode: Dans une chambre de culture transparente à deux compartiments, des muqueuses vésicales de porc maintenues en vie ont été incubées avec une solution d'hexyl-aminolévulinate (HAL), le précurseur de la Protoporphyrine IX. Ces muqueuses ont ensuite été irradiées avec des doses lumineuses croissantes en lumière bleue et en lumière blanche, et les altérations cellulaires ont été évaluées par microscopie électronique à balayage et par un colorant fluorescent, le Sytox green. Nous avons également évalué la production d'intermédiaires réactifs de l'oxygène parla mesure de la fluorescence intracellulaire de Rhodamine 123 (R123), produit de l'oxydation de la Dihydrorhodamine 123 (DHR123) non fluorescente. Ces valeurs ont été corrélées avec celles du photo blanchiment de la PAIX. Résultats : Le taux de mortalité cellulaire était dépendant de la concentration de PAIX. Après 3 heures d'incubation, la valeur seuil de dose lumineuse pour la lumière bleu était de 0.15 et 0.75 J/cm2 (irradiance 30 et 75 mW/cm2, respectivement) et pour la lumière blanche de 0.55 J/cm2 (irradiante 30 mW/cm2). Le taux de photo blanchiment était inversement proportionnel à l'irradiante. Le système de détection des intermédiaires réactifs de l'oxygène DHR123/R123 a démontré une bonne corrélation avec les valeurs seuil pour toutes les conditions d'irradiation utilisées. Conclusions : Nous avons déterminé les doses lumineuses permettant d'épargner 50% des cellules urothéliales saines. L'utilisation d'une faible irradiante associée à des systèmes permettant de mesurer la production d'intermédiaires réactifs de l'oxygène dans les tissus irradiés pourrait améliorer la dosimétrie in vivo et l'efficacité de la thérapie photodynamique. Abstract Background and Objectives: Photodynamic therapy of superficial bladder cancer may cause damages to the normal surrounding bladder wall. Prevention of these is important for bladder healing. We studied the influence of photosensitizes concentration, irradiation parameters and production of reactive oxygen species (ROS) on the photodynamically induced damage in the porcine urothelium in vitro. The aim was to determine the threshold conditions for the cell survival. Methods: Living porcine bladder mucosae were incubated with solution of hexylester of 5-aminolevulinic acid (HAL). The mucosae were irradiated with increasing doses and cell alterations were evaluated by scanning electron microscopy and by Sytox green fluorescence. The urothelial survival score was correlated with Protoporphyrin IX (PpIX) photobleaching and intracellular fluorescence of Rhodamine 123 reflecting the ROS production. Results: The mortality ratio was dependent on PpIX concentration. After 3 hours of incubation, the threshold radiant exposures for blue light were 0.15 and 0.75 J/cm2 (irradiance 30 and 75 mW/cm2, respectively) and for white light 0.55 J/cm2 (irradiance 30 mW/cm2). Photobleaching rate increased with decreasing irradiance. Interestingly, the DHR123/R123 reporter system correlated well with the threshold exposures under all conditions used. Conclusions: we have determined radiant exposures sparing half of normal urothelial cells. We propose that the use of low irradiance combined with systems reporting the ROS production in the irradiated tissue could improve the in vivo dosimetry and optimize the PDT.

Adenosine A2B receptor-mediated leukemia inhibitory factor release from astrocytes protects cortical neurons against excitotoxicity.

ABSTRACT: BACKGROUND: Neuroprotective and neurotrophic properties of leukemia inhibitory factor (LIF) have been widely reported. In the central nervous system (CNS), astrocytes are the major source for LIF, expression of which is enhanced following disturbances leading to neuronal damage. How astrocytic LIF expression is regulated, however, has remained an unanswered question. Since neuronal stress is associated with production of extracellular adenosine, we investigated whether LIF expression in astrocytes was mediated through adenosine receptor signaling. METHODS: Mouse cortical neuronal and astrocyte cultures from wild-type and adenosine A2B receptor knock-out animals, as well as adenosine receptor agonists/antagonists and various enzymatic inhibitors, were used to study LIF expression and release in astrocytes. When needed, a one-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test was used for statistical analysis. RESULTS: We show here that glutamate-stressed cortical neurons induce LIF expression through activation of adenosine A2B receptor subtype in cultured astrocytes and require signaling of protein kinase C (PKC), mitogen-activated protein kinases (MAPKs: p38 and ERK1/2), and the nuclear transcription factor (NF)-κB. Moreover, LIF concentration in the supernatant in response to 5'-N-ethylcarboxamide (NECA) stimulation was directly correlated to de novo protein synthesis, suggesting that LIF release did not occur through a regulated release pathway. Immunocytochemistry experiments show that LIF-containing vesicles co-localize with clathrin and Rab11, but not with pHogrin, Chromogranin (Cg)A and CgB, suggesting that LIF might be secreted through recycling endosomes. We further show that pre-treatment with supernatants from NECA-treated astrocytes increased survival of cultured cortical neurons against glutamate, which was absent when the supernatants were pre-treated with an anti-LIF neutralizing antibody. CONCLUSIONS: Adenosine from glutamate-stressed neurons induces rapid LIF release in astrocytes. This rapid release of LIF promotes the survival of cortical neurons against excitotoxicity.

RecA-mediated strand exchange traverses substitutional heterologies more easily than deletions or insertions.

RecA protein in bacteria and its eukaryotic homolog Rad51 protein are responsible for initiation of strand exchange between homologous DNA molecules. This process is crucial for homologous recombination, the repair of certain types of DNA damage and for the reinitiation of DNA replication on collapsed replication forks. We show here, using two different types of in vitro assays, that in the absence of ATP hydrolysis RecA-mediated strand exchange traverses small substitutional heterologies between the interacting DNAs, whereas small deletions or insertions block the ongoing strand exchange. We discuss evolutionary implications of RecA selectivity against insertions and deletions and propose a molecular mechanism by which RecA can exert this selectivity.

Antibody-Mediated Trapping of Helminth Larvae Requires CD11b and Fcγ Receptor I.

Infections with intestinal helminths severely impact on human and veterinary health, particularly through the damage that these large parasites inflict when migrating through host tissues. Host immunity often targets the motility of tissue-migrating helminth larvae, which ideally should be mimicked by anti-helminth vaccines. However, the mechanisms of larval trapping are still poorly defined. We have recently reported an important role for Abs in the rapid trapping of tissue-migrating larvae of the murine parasite Heligmosomoides polygyrus bakeri. Trapping was mediated by macrophages (MΦ) and involved complement, activating FcRs, and Arginase-1 (Arg1) activity. However, the receptors and Ab isotypes responsible for MΦ adherence and Arg1 induction remained unclear. Using an in vitro coculture assay of H. polygyrus bakeri larvae and bone marrow-derived MΦ, we now identify CD11b as the major complement receptor mediating MΦ adherence to the larval surface. However, larval immobilization was largely independent of CD11b and instead required the activating IgG receptor FcγRI (CD64) both in vitro and during challenge H. polygyrus bakeri infection in vivo. FcγRI signaling also contributed to the upregulation of MΦ Arg1 expression in vitro and in vivo. Finally, IgG2a/c was the major IgG subtype from early immune serum bound by FcγRI on the MΦ surface, and purified IgG2c could trigger larval immobilization and Arg1 expression in MΦ in vitro. Our findings reveal a novel role for IgG2a/c-FcγRI-driven MΦ activation in the efficient trapping of tissue-migrating helminth larvae and thus provide important mechanistic insights vital for anti-helminth vaccine development.

False recognition following frontal lobe damage: The role of encoding factors

This paper reports a series of experiments on patient JB, a man with memory difficulties following damage to the left frontal lobe. The primary characteristic of JB's recognition memory impairment is a high level of false recognition together with a normal hit rate. The hypothesis that JB's false recognition reflects an over-reliance on familiarity is considered, but discounted on the basis that the false alarm rate is not affected by increasing the similarity between distracters and targets, and remains high when nonword stimuli are used. It is suggested, instead, that JB relies on a poorly focused memory description, which lacks item-specific detail but contains more general, low-level properties of the target items-these properties being held by many distracter items as well. This deficit is considered to arise because of damage to frontally mediated control processes involved in the selection of elements for memory encoding. An encoding deficit is supported by the fact that JB's false recognition is significantly reduced by orienting instructions, and is eliminated when his remote memory is subjected to recognition testing. In contrast, it is shown that manipulations at the level of retrieval (e.g. restricting the number of "old" responses) have little effect on his false recognition.

Hyperactivation of retina by light in mice leads to photoreceptor cell death mediated by VEGF and retinal pigment epithelium permeability.

Light toxicity is suspected to enhance certain retinal degenerative processes such as age-related macular degeneration. Death of photoreceptors can be induced by their exposure to the visible light, and although cellular processes within photoreceptors have been characterized extensively, the role of the retinal pigment epithelium (RPE) in this model is less well understood. We demonstrate that exposition to intense light causes the immediate breakdown of the outer blood-retinal barrier (BRB). In a molecular level, we observed the slackening of adherens junctions tying up the RPE and massive leakage of albumin into the neural retina. Retinal pigment epithelial cells normally secrete vascular endothelial growth factor (VEGF) at their basolateral side; light damage in contrast leads to VEGF increase on the apical side - that is, in the neuroretina. Blocking VEGF, by means of lentiviral gene transfer to express an anti-VEGF antibody in RPE cells, inhibits outer BRB breakdown and retinal degeneration, as illustrated by functional, behavioral and morphometric analysis. Our data show that exposure to high levels of visible light induces hyperpermeability of the RPE, likely involving VEGF signaling. The resulting retinal edema contributes to irreversible damage to photoreceptors. These data suggest that anti-VEGF compounds are of therapeutic interest when the outer BRB is altered by retinal stresses.