Immunization of BALB/c mice with Helicobacter urease B induces a T helper 2 response absent in Helicobacter infection.

BACKGROUND & AIMS: Infection with Helicobacter induces a T helper type 1 response in mice and humans. Mice can be cured or protected from infection with Helicobacter by mucosal immunization with recombinant H. pylori urease B subunit (rUreB). This study characterizes the immune response of infected mice immunized with rUreB. METHODS: BALB/c mice were infected with H. felis. Two weeks later, they were orally immunized four times with rUreB and cholera toxin (CT) at weekly intervals. Controls were only infected or sham-immunized with CT. Animals were killed at various times after immunization. Splenic CD4(+) cells were obtained and cultured in vitro with rUreB to evaluate antigen-specific proliferation and induction of interferon gamma and interleukin 4 secretion. RESULTS: All rUreB-immunized mice (n = 8) were cured from infection 3 weeks after the fourth immunization. Immunization induced a proliferative response of splenic CD4(+) cells, a progressive decrease in interferon gamma secretion, and a concomitant increase in interleukin 4 secretion after each immunization. A simultaneous increase in rUreB specific serum immunoglobulin G1 levels was observed in infected/immunized mice. CONCLUSIONS: In BALB/c mice, therapeutic mucosal immunization with rUreB induces progressively a Th2 CD4(+) T cell response resulting in the elimination of the pathogen.

Excitotoxicity-induced endocytosis confers drug targeting in cerebral ischemia.

OBJECTIVE: Targeting neuroprotectants specifically to the cells that need them is a major goal in biomedical research. Many peptidic protectants contain an active sequence linked to a carrier such as the transactivator of transcription (TAT) transduction sequence, and here we test the hypothesis that TAT-linked peptides are selectively endocytosed into neurons stressed by excitotoxicity and focal cerebral ischemia. METHODS: In vivo experiments involved intracerebroventricular injection of TAT peptides or conventional tracers (peroxidase, fluorescein isothiocyanate-dextran) in young rats exposed to occlusion of the middle cerebral artery at postnatal day 12. Cellular mechanisms of uptake were analyzed in dissociated cortical neuronal cultures. RESULTS: In both models, all tracers were taken up selectively into stressed neurons by endocytosis. In the in vivo model, this was neuron specific and limited to the ischemic area, where the neurons displayed enhanced immunolabeling for early endosomal antigen-1 and clathrin. The highly efficient uptake of TAT peptides occurred by the same selective mechanism as for conventional tracers. All tracers were targeted to the nucleus and cytoplasm of neurons that appeared viable, although ultimately destined to die. In dissociated cortical neuronal cultures, an excitotoxic dose of N-methyl-D-aspartate induced a similar endocytosis. It was 100 times more efficient with TAT peptides than with dextran, because the former bound to heparan sulfate proteoglycans at the cell surface, but it depended on dynamin and clathrin in both cases. INTERPRETATION: Excitotoxicity-induced endocytosis is the main entry route for protective TAT peptides and targets selectively the neurons that need to be protected.

Topical and intravitreous administration of cationic nanoemulsions to deliver antisense oligonucleotides directed towards VEGF KDR receptors to the eye.

Antisense oligonucleotides (ODNs) specific for VEGFR-2-(17 MER) and inhibiting HUVEC proliferation in-vitro were screened. One efficient sequence was selected and incorporated in different types of nanoemulsions the potential toxicity of which was evaluated on HUVEC and ARPE19 cells. Our results showed that below 10 microl/ml, a 2.5% mid-chain triglycerides cationic DOTAP nanoemulsion was non-toxic on HUVEC and retinal cells. This formulation was therefore chosen for further experiments. In-vitro transfection of FITC ODNs in ARPE cells using DOTAP nanoemulsions showed that nanodroplets do penetrate into the cells. Furthermore, ODNs are released from the nanoemulsion after 48 h and accumulate into the cell nuclei. In both ex-vivo and in-vivo ODN stability experiments in rabbit vitreous, it was noted that the nanoemulsion protected at least partially the ODN from degradation over 72 h. The kinetic results of fluorescent ODN (Hex) distribution in DOTAP nanoemulsion following intravitreal injection in the rat showed that the nanoemulsion penetrates all retinal cells. Pharmacokinetic and ocular tissue distribution of radioactive ODN following intravitreal injection in rabbits showed that the DOTAP nanoemulsion apparently enhanced the intraretinal penetration of the ODNs up to the inner nuclear layer (INL) and might yield potential therapeutic levels of ODN in the retina over 72 h post injection.

Epigenetic regulation of the bacterial cell cycle.

N(6)-methyl-adenines can serve as epigenetic signals for interactions between regulatory DNA sequences and regulatory proteins that control cellular functions, such as the initiation of chromosome replication or the expression of specific genes. Several of these genes encode master regulators of the bacterial cell cycle. DNA adenine methylation is mediated by Dam in gamma-proteobacteria and by CcrM in alpha-proteobacteria. A major difference between them is that CcrM is cell cycle regulated, while Dam is active throughout the cell cycle. In alpha-proteobacteria, GANTC sites can remain hemi-methylated for a significant period of the cell cycle, depending on their location on the chromosome. In gamma-proteobacteria, most GATC sites are only transiently hemi-methylated, except regulatory GATC sites that are protected from Dam methylation by specific DNA-binding proteins.

Synapse formation in adult barrel cortex following naturalistic environmental enrichment.

Environmental enrichment paradigms in adult laboratory animals, consisting of physical, perceptual, and social stimulation, have been shown to affect synapse and cell morphology in sensory cortex and enhance learning ability, whereas enrichment, which is in harmony with the animal's natural habitat may have even greater implications for plasticity. Previous studies in our laboratory have shown that whisker stimulation induced the formation of synapses and spines in the corresponding barrel. In the present study adult C57/Bl6J female laboratory mice at 6 weeks of age were placed during 2 months in a protected enrichment enclosure in a forest clearing at the Chisti Les Biological Station, Tvier, Russia. We analyzed neuropil ultrastructure in the C2 barrel using serial-section electron microscopy on a total of eight mice (n=4 enriched, n=4 standard cagemate controls). Quantitative analyses of volumes of neuropil showed a significant increase in excitatory and inhibitory synapses on spines and excitatory synapses on dendritic shafts in the C2 barrel in the enriched group compared with standard cagemate controls. These results demonstrate that naturalistic experience alters the synaptic circuitry in layer IV of the somatosensory cortex, the first cortical relay of sensory information, leaving a lasting trace that may guide subsequent behavior.

Analyse des modifications de la chromatine impliquées dans l'effet barrière de CTF1 aux télomères humains

Eukaryotic genomes are compartmentalized in different structural domains that can affect positively or negatively gene expression. These regions of euchromatin and heterochromatin are characterized by distinct histones marks which can facilitate or repress gene transcription. The chromatin environment represents thus one of the main problems to control gene expression in biotechnological applications or gene therapy, since its expression is affected by the chromatin neighboring its locus of insertion. Some chromatin regions like telomeres are composed of constitutive heterochromatin which leads to the telomeric position effect (TPE) that silences genes adjacent to the telomere. TPE is known to spread by the selfrecruitment of the SIR histone deacetylase complex from the telomere in S.cerevisiae, but the histone marks that are associated to telomeric chromatin in mammalian cells remain mostly unknown. The transcription factor CTF1 has shown antisilencing properties in mammalian cells and also a boundary activity against TPE in yeast cells when fused to the yeast Gal4 DNA binding domain. In the work presented here, we describe a dual-reporter system to assess the boundary activity of proteins such as CTF1 at human telomeres. When located between the two reporter genes, CTF1 shields the telomere distal gene from TPE, while the telomereproximal gene remains silenced by telomeric heterochromatin. The boundary activity of CTF1 is shown to act regardless its function of transcriptional activator, by opposition to the transcriptional activator VP16 which activates indifferently both transgenes. Moreover, this study shows that CTF1 boundary activity is linked to its H3 binding function, as expected from a chromatin remodeler. ChIP experiments showed that histone deacetylation is the main histone modification involved in gene silencing at mammalian cell telomeres. Distinctly to yeast cells, the histone deacetylation signal in human cells extented over a short range along the chromosome. CTF1 may help to block this propagation and therefore to restore histones acetylation level on telomere protected locus. Surprisingly, other histone marks such as trimethyl-H3K9 or trimethyl-H4K20 were found on telomere protected locus, while in another clone, unsilencing of telomere distal transgene was associated with recruitment of the histone variant H2A.Z. Thus, I conclude that CTF1 displays a chromatin boundary function which is independent of its transcriptional activity and therefore exhibit features required for use as chromatin insulator in biotechnological applications. RESUME Les génomes eucaryotes sont compartementalisés en domaines structurels qui peuvent affecter positivement ou négativement l'expression des gènes avoisinants. Ces régions dites d'euchromatine ou d'hétérochromatine sont caractérisées par des modifications posttraductionnelles des histones qui peuvent faciliter ou au contraire inhiber la transcription des gènes qui s'y trouvent. Ainsi, isoler un gène de son environnement chromatinien est problème fréquent lorsqu'il s'agit de contrôler son expression dans le cadre d'applications en biotechnologie ou encore en thérapie génique. Certaines régions de chromatine telles que les télomères sont composées d'hétérochromatine constitutive qui mène au silençage des gènes avoisinants. Cet effet de position télomérique (TPE) est connu dans la levure S.cerevisiae comme se propageant par auto-recrutement du complexe de déacétylation d'histone SIR, alors que peu de modifications de chromatine ont pu être associées à ce phénomène dans les cellules de mammifères. Le facteur de transcription CTF1 a montré des propriétés d'anti-silençage dans les cellules de mammifères, ainsi qu'une activité barrière contre le silençage télomérique dans les cellules de levures lorsqu'il est fusionné au domaine de liaison à l'ADN de la protéine de levure Gal4. Dans le travail présenté ci-après est décrit un système à deux gènes rapporteurs permettant de mesurer l'activité barrière de protéines telles que CTF1 aux télomères humains, et les modifications de chromatine qui y sont associées. Lorsque CTF1 est placé entre les deux gènes rapporteurs, le gène distant du télomère est protégé du silençage qui lui est associé, alors que le gène proche du télomère reste soumis à ce silençage induit par l'hétérochromatine télomérique. L'activité barrière de CTF1 est montrée ici comme agissant indépendamment de son activité transcriptionnelle, par opposition à l'activateur transcriptionnel VP16 qui active indifféremment les deux transgènes. En outre, cette étude appuie l'hypothèse stipulant que CTF1 agisse comme remodeleur chromatinien puisqu'elle démontre que son activité barrière est directement dépendante de son activité de liaison avec l'histone H3. De plus, des expériences d'immuno-précipitation de la chromatine démontrent que la déacétylation des histones est le majeur phénomène intervenant dans le silençage télomérique. Par opposition à la levure, ce signal de déacétylation ne se propage dans les cellules humaines que sur une courte distance le long du chromosome. CTF1 agit ainsi en bloquant cette propagation et en restaurant le niveau d'acétylation des histones sur le locus protégé du télomère. De manière surprenante et inattendue, d'autres modifications d'histones telles que 4 les H3K9 et H4K20 triméthylées sont aussi observées à ce locus, tandis le recrutement du variant H2A.Z peut aussi être suffisant à restaurer l'expression du gène distant du télomère. En terme de cette analyse, CTF1 exhibe ainsi une fonction de barrière chromatinienne qui exclue une activité transcriptionnelle non désirée - propriété qui est requise dans l'établissement des isolateurs visant à permettre le contrôle d'un transgène dans le cadre d'applications en biotechnologies.

The role of endogenous and exogenous RasGAP-derived fragment N in protecting cardiomyocytes from peroxynitrite-induced apoptosis.

Peroxynitrite (PN) is a potent nitrating and oxidizing agent generated during various pathological situations affecting the heart. The negative effects of PN result, at least in part, from its ability to activate caspases and apoptosis. RasGAP is a ubiquitously expressed protein that is cleaved sequentially by caspase-3. At low caspase-3 activity, RasGAP is cleaved into an N-terminal fragment, called fragment N, that protects cells by activating the Ras/PI3K/Akt pathway. At high caspase-3 activity, fragment N is further cleaved and this abrogates its capacity to stimulate the antiapoptotic Akt kinase. Fragment N formation is crucial for the survival of cells exposed to a variety of stresses. Here we investigate the pattern of RasGAP cleavage upon PN stimulation and the capacity of fragment N to protect cardiomyocytes. PN did not lead to sequential cleavage of RasGAP. Indeed, PN did not allow accumulation of fragment N because it induced its rapid cleavage into smaller fragments. No situations were found in cells treated with PN in which the presence of fragment N was associated with survival. However, expression of a caspase-resistant form of fragment N in cardiomyocytes protected them from PN-induced apoptosis. Our results indicate that the antiapoptotic pathway activated by fragment N is effective at inhibiting PN-induced apoptosis (as seen when cardiomyocytes express a capase-3-resistant form of fragment N) but because fragment N is too transiently generated in response to PN, no survival response is effectively produced. This may explain the marked deleterious consequences of PN generation in various organs, including the heart.

Coping with continuous human disturbance in the wild: insights from penguin heart rate response to various stressors.

ABSTRACT: BACKGROUND: A central question for ecologists is the extent to which anthropogenic disturbances (e.g. tourism) might impact wildlife and affect the systems under study. From a research perspective, identifying the effects of human disturbance caused by research-related activities is crucial in order to understand and account for potential biases and derive appropriate conclusions from the data. RESULTS: Here, we document a case of biological adjustment to chronic human disturbance in a colonial seabird, the king penguin (Aptenodytes patagonicus), breeding on remote and protected islands of the Southern ocean. Using heart rate (HR) as a measure of the stress response, we show that, in a colony with areas exposed to the continuous presence of humans (including scientists) for over 50 years, penguins have adjusted to human disturbance and habituated to certain, but not all, types of stressors. When compared to birds breeding in relatively undisturbed areas, birds in areas of high chronic human disturbance were found to exhibit attenuated HR responses to acute anthropogenic stressors of low-intensity (i.e. sounds or human approaches) to which they had been subjected intensely over the years. However, such attenuation was not apparent for high-intensity stressors (i.e. captures for scientific research) which only a few individuals experience each year. CONCLUSIONS: Habituation to anthropogenic sounds/approaches could be an adaptation to deal with chronic innocuous stressors, and beneficial from a research perspective. Alternately, whether penguins have actually habituated to anthropogenic disturbances over time or whether human presence has driven the directional selection of human-tolerant phenotypes, remains an open question with profound ecological and conservation implications, and emphasizes the need for more knowledge on the effects of human disturbance on long-term studied populations.

Nasal immunization of mice with virus-like particles protects offspring against rotavirus diarrhea.

Rotavirus is the major cause of diarrhea among young infants in both humans and animals. Immune protection of newborns by vaccination is difficult to achieve since there is not enough time to mount an immune response before exposure to the virus. We have designed a vaccination strategy mediating transfer of neutralizing antibodies from the mother to the offspring during pregnancy and/or lactation. Adult female mice were nasally immunized with virus-like particles (VLPs) made of viral proteins VP2 and 6 (VLP2/6) or VP 2, 6, and 7 (VLP2/6/7) derived from the RF rotavirus strain in the presence or absence of cholera toxin. Both vaccines elicited serum and milk antibodies against the respective VPs. Four days after parturition, suckling pups were challenged orally with RF rotavirus. Pups from mothers immunized with VLP2/6/7 but not VLP2/6 were protected against rotavirus diarrhea, indicating that VP7 plays a key role in protection. Protection was mediated by milk rather than serum antibodies, and mucosal adjuvants were not required. In conclusion, VLPs containing VP7 administered nasally to mothers represent a promising vaccine candidate for the protection of suckling newborns against rotavirus-induced diarrhea, even in the absence of a mucosal adjuvant.

Protection against Plasmodium falciparum challenge induced in Aotus monkeys by liver-stage antigen-3-derived long synthetic peptides.

The vaccine potential of Plasmodium falciparum liver stage antigen-3 (LSA3) was investigated in Aotus monkeys using two long synthetic peptides corresponding respectively to an N-terminal non-repeat peptide (NRP) and repeat 2 (R2) region of the LSA3, adjuvanted by ASO2. Both 100-222 (NRP) and 501-596 repeat peptides induced effector B- and T-cell responses in terms of antigen-driven antibodies and/or specific IFN-gamma secretion. Animals challenged with P. falciparum sporozoites were protected following immunization with either the NRP region alone or the NRP combined with the R2 repeat region, as compared with controls receiving the adjuvant alone. These results indicate that the NRP may be sufficient to induce full, sterile protection and confirm the vaccine potential of LSA3 previously demonstrated in chimpanzees and in Aotus.

GLUT2 in pancreatic and extra-pancreatic gluco-detection (review).

Detection of variations in blood glucose concentrations by pancreatic beta-cells and a subsequent appropriate secretion of insulin are key events in the control of glucose homeostasis. Because a decreased capability to sense glycemic changes is a hallmark of type 2 diabetes, the glucose signalling pathway leading to insulin secretion in pancreatic beta-cells has been extensively studied. This signalling mechanism depends on glucose metabolism and requires the presence of specific molecules such as GLUT2, glucokinase and the K(ATP) channel subunits Kir6.2 and SUR1. Other cells are also able to sense variations in glycemia or in local glucose concentrations and to modulate different physiological functions participating in the general control of glucose and energy homeostasis. These include cells forming the hepatoportal vein glucose sensor, which controls glucose storage in the liver, counterregulation, food intake and glucose utilization by peripheral tissues and neurons in the hypothalamus and brainstem whose firing rates are modulated by local variations in glucose concentrations or, when not protected by a blood-brain barrier, directly by changes in blood glucose levels. These glucose-sensing neurons are involved in the control of insulin and glucagon secretion, food intake and energy expenditure. Here, recent physiological studies performed with GLUT2-/- mice will be described, which indicate that this transporter is essential for glucose sensing by pancreatic beta-cells, by the hepatoportal sensor and by sensors, probably located centrally, which control activity of the autonomic nervous system and stimulate glucagon secretion. These studies may pave the way to a fine dissection of the molecular and cellular components of extra-pancreatic glucose sensors involved in the control of glucose and energy homeostasis.

Inhibition of inducible nitric oxide synthase protects against liver injury induced by mycobacterial infection and endotoxins.

BACKGROUND/AIMS: Bacillus Calmette Guerin (BCG) infection causes hepatic injury following granuloma formation and secretion of cytokines which render mice highly sensitive to endotoxin-mediated hepatotoxicity. This work investigates the role of inducible nitric oxide synthase (iNOS) in liver damage induced by BCG and endotoxins in BCG-infected mice. METHODS: Liver injury and cytokine activation induced by BCG and by LPS upon BCG infection (BCG/LPS) were compared in wild-type and iNOS-/- mice. RESULTS: iNOS-/- mice infected with living BCG are protected from hepatic injury when compared to wild-type mice which express iNOS protein in macrophages forming hepatic granulomas. In addition, iNOS-/- mice show a decrease in BCG-induced IFN-gamma serum levels. LPS challenge in BCG-infected mice strongly activates iNOS in the liver and spleen of wild-type mice which show important liver damage associated with a dramatic increase in TNF and IL-6 and also Th1 type cytokines. In contrast, iNOS-/- mice are protected from liver injury after BCG/LPS challenge and their TNF, IL-6 and Th1 type cytokine serum levels raise moderately. CONCLUSIONS: These results demonstrate that nitric oxide (NO) from iNOS is involved in hepatotoxicity induced by both mycobacterial infection and endotoxin effects upon BCG infection and that inhibition of NO from iNOS protects from liver injuries.

New concepts in the pathophysiology of infective endocarditis.

Endocarditis pathogens colonize valves with pre-existing sterile vegetations or valves with minimal endothelial lesions. Inflamed endothelia produce cytokines, integrins, and tissue factor, which in turn attract fibronectin, monocytes, and platelets. Bacteria attaching to such structures further activate the cascade, becoming embedded and protected from host defenses. Staphylococcus aureus also actively invade the endothelium, causing apoptosis and endothelial damage. Knowledge of this interplay identifies host factors as potential therapeutic targets. Blocking infection by modulating host factors might be opportune because host factors are conserved. In contrast, interfering with bacterial virulence factors might be more complicated because they vary among different bacteria.

Haemophilia registry of the medical committee of the Swiss Haemophilia Society. Update and annual survey 2009.

The Swiss Haemophilia Registry of the Medical Committee of the Swiss Haemophilia Society started in 1996 but was set as an internet-based, double password-protected facility in the year 2000. With the inclusion of patients' data from two new centres in 2009, we assume a coverage rate of about 90% of all patients with inherited bleeding disorders in our country. Data concerning the phenotype and genotype of the disorder, its severity, its therapy, the prevalence of inhibitors are readily available to the registered users, allowing quality control of haemophilia therapy at a national level, but also rapid care of the patient visiting the emergency room of another treatment centre. Basing on the available data, about two thirds of the WFH global survey can be answered; the mortality statistics shows that bleeding remains a cause of death in haemophiliacs, also in the 21th century. The Registry allows for comparisons with international datasets, especially with respect to treatment (prophylaxis vs. on-demand therapy), factor consumption and costs.

Global methylation state at base-pair resolution of the Caulobacter genome throughout the cell cycle.

The Caulobacter DNA methyltransferase CcrM is one of five master cell-cycle regulators. CcrM is transiently present near the end of DNA replication when it rapidly methylates the adenine in hemimethylated GANTC sequences. The timing of transcription of two master regulator genes and two cell division genes is controlled by the methylation state of GANTC sites in their promoters. To explore the global extent of this regulatory mechanism, we determined the methylation state of the entire chromosome at every base pair at five time points in the cell cycle using single-molecule, real-time sequencing. The methylation state of 4,515 GANTC sites, preferentially positioned in intergenic regions, changed progressively from full to hemimethylation as the replication forks advanced. However, 27 GANTC sites remained unmethylated throughout the cell cycle, suggesting that these protected sites could participate in epigenetic regulatory functions. An analysis of the time of activation of every cell-cycle regulatory transcription start site, coupled to both the position of a GANTC site in their promoter regions and the time in the cell cycle when the GANTC site transitions from full to hemimethylation, allowed the identification of 59 genes as candidates for epigenetic regulation. In addition, we identified two previously unidentified N(6)-methyladenine motifs and showed that they maintained a constant methylation state throughout the cell cycle. The cognate methyltransferase was identified for one of these motifs as well as for one of two 5-methylcytosine motifs.