Ana Cardona

Secretory component: a new role in secretory IgA-mediated immune exclusion in vivo.

Secretory immunoglobulin (Ig) A (SIgA) is essential in protecting mucosal surfaces. It is composed of at least two monomeric IgA molecules, covalently linked through the J chain, and secretory component (SC). We show here that a dimeric/polymeric IgA (IgA(d/p)) is more efficient when bound to SC in protecting mice against bacterial infection of the respiratory tract. We demonstrate that SC ensures, through its carbohydrate residues, the appropriate tissue localization of SIgA by anchoring the antibody to mucus lining the epithelial surface. This in turn impacts the localization and the subsequent clearance of bacteria. Thus, SC is directly involved in the SIgA function in vivo. Therefore, binding of IgA(d/p) to SC during the course of SIgA-mediated mucosal response constitutes a crucial step in achieving efficient protection of the epithelial barrier by immune exclusion.

Absence of the gut microbiota enhances anxiety-like behavior and neuroendocrine response to acute stress in rats

BACKGROUND AND AIMS Establishment of the gut microbiota is one of the most important events in early life and emerging evidence indicates that the gut microbiota influences several aspects of brain functioning, including reactivity to stress. To better understand how the gut microbiota contributes to a vulnerability to the stress-related psychiatric disorders, we investigated the relationship between the gut microbiota, anxiety-like behavior and HPA axis activity in stress-sensitive rodents. We also analyzed the monoamine neurotransmitters in the brain upper structures involved in the regulation of stress and anxiety. METHODS Germfree (GF) and specific pathogen free (SPF) F344 male rats were first subjected to neurological tests to rule out sensorimotor impairments as confounding factors. Then, we examined the behavior responses of rats to social interaction and open-field tests. Serum corticosterone concentrations, CRF mRNA expression levels in the hypothalamus, glucocorticoid receptor (GR) mRNA expression levels in the hippocampus, and monoamine concentrations in the frontal cortex, hippocampus and striatum were compared in rats that were either exposed to the open-field stress or not. RESULTS GF rats spent less time sniffing an unknown partner than SPF rats in the social interaction test, and displayed a lower number of visits to the aversive central area, and an increase in latency time, time spent in the corners and number of defecations in the open-field test. In response to the open-field stress, serum corticosterone concentrations were 2.8-fold higher in GF than in SPF rats. Compared to that of SPF rats, GF rats showed elevated CRF mRNA expression in the hypothalamus and reduced GR mRNA expression in the hippocampus. GF rats also had a lower dopaminergic turnover rate in the frontal cortex, hippocampus and striatum than SPF rats. CONCLUSIONS In stress-sensitive F344 rats, absence of the gut microbiota exacerbates the neuroendocrine and behavioral responses to acute stress and the results coexist with alterations of the dopaminergic turnover rate in brain upper structures that are known to regulate reactivity to stress and anxiety-like behavior.

Localization of [3H]nicotine, [3H]cytisine, [3H]epibatidine, and [125I]α‐bungarotoxin binding sites in the brain of Macaca mulatta

We determined the localization of [3H]nicotine, [3H]cytisine, [3H]epibatidine, and [125I]α‐bungarotoxin binding sites in the brain of rhesus monkey by means of receptor autoradiography. The labelings by [3H]nicotine, [3H]cytisine, and [3H]epibatidine were highly concordant, except for epibatidine. Layer IV of some cortical areas, most thalamic nuclei, and presubiculum displayed high levels of labeling for the three ligands. Moderate levels of binding were detected in the subiculum, the septum, and the mesencephalon. Low levels were present in layers I–II and VI of the cortex, the cornu Ammonis, the dentate gyrus, and the amygdala. In addition, the level of epibatidine labeling was very high in the epithalamic nuclei and the interpeduncular nucleus, whereas labeling by nicotine and cytisine was very weak in the same regions. The distribution of [125I]α‐bungarotoxin binding differed from the binding of the three agonists. The labeling was dense in layer I of most cortical areas, dentate gyrus, stratum lacunosum‐moleculare of CA1 field, several thalamic nuclei, and medial habenula. A moderate labeling was found in layers V and VI of the prefrontal and frontal cortices, layer IV of primary visual cortex, amygdala, septum, hypothalamus, and some mesencenphalic nuclei. A weak signal was also detected in subiculum, claustrum, stratum oriens, and stratum lucidum of cornu Ammonis and also in some mesencephalic nuclei. The distribution of nicotine, cytisine, and epibatidine bindings corresponds broadly to the patterns observed in rodents, with the marked exception of the epithalamus. However, in monkey, those distributions match the distribution of α2 messenger RNA, rather than that of α4 transcripts as it exists in rodent brains. The distribution of the binding sites for α‐bungarotoxin is larger in the brain of rhesus monkeys than in rodent brain, suggesting a more important role of α7 receptors in primates. J. Comp. Neurol. 461:49–60, 2003.

DYRK1A interacts with the REST/NRSF-SWI/SNF chromatin remodelling complex to deregulate gene clusters involved in the neuronal phenotypic traits of Down syndrome

The molecular mechanisms that lead to the cognitive defects characteristic of Down syndrome (DS), the most frequent cause of mental retardation, have remained elusive. Here we use a transgenic DS mouse model (152F7 line) to show that DYRK1A gene dosage imbalance deregulates chromosomal clusters of genes located near neuron-restrictive silencer factor (REST/NRSF) binding sites. We found that Dyrk1a binds the SWI/SNF complex known to interact with REST/NRSF. The mutation of a REST/NRSF binding site in the promoter of the REST/NRSF target gene L1cam modifies the transcriptional effect of Dyrk1a-dosage imbalance on L1cam. Dyrk1a dosage imbalance perturbs Rest/Nrsf levels with decreased Rest/Nrsf expression in embryonic neurons and increased expression in adult neurons. Using transcriptome analysis of embryonic brain subregions of transgenic 152F7 mouse line, we identified a coordinated deregulation of multiple genes that are responsible for dendritic growth impairment present in DS. Similarly, Dyrk1a overexpression in primary mouse cortical neurons induced severe reduction of the dendritic growth and dendritic complexity. We propose that DYRK1A overexpression-related neuronal gene deregulation via disturbance of REST/NRSF levels, and the REST/NRSF-SWI/SNF chromatin remodelling complex, significantly contributes to the neural phenotypic changes that characterize DS.

Helicobacter pylori Exploits Cholesterol-Rich Microdomains for Induction of NF-κB-Dependent Responses and Peptidoglycan Delivery in Epithelial Cells

ABSTRACT Infection with Helicobacter pylori cag pathogenicity island (cagPAI)-positive strains is associated with more destructive tissue damage and an increased risk of severe disease. The cagPAI encodes a type IV secretion system (TFSS) that delivers the bacterial effector molecules CagA and peptidoglycan into the host cell cytoplasm, thereby inducing responses in host cells. It was previously shown that interactions between CagL, present on the TFSS pilus, and host α5β1 integrin molecules were critical for CagA translocation and the induction of cytoskeletal rearrangements in epithelial cells. As the α5β1 integrin is found in cholesterol-rich microdomains (known as lipid rafts), we hypothesized that these domains may also be involved in the induction of proinflammatory responses mediated by NOD1 recognition of H. pylori peptidoglycan. Indeed, not only did methyl-β-cyclodextrin depletion of cholesterol from cultured epithelial cells have a significant effect on the levels of NF-κB and interleukin-8 (IL-8) responses induced by H. pylori bacteria with an intact TFSS (P < 0.05), but it also interfered with TFSS-mediated peptidoglycan delivery to cells. Both of these effects could be restored by cholesterol replenishment of the cells. Furthermore, we demonstrated for the first time the involvement of α5β1 integrin in the induction of proinflammatory responses by H. pylori. Taking the results together, we propose that α5β1 integrin, which is associated with cholesterol-rich microdomains at the host cell surface, is required for NOD1 recognition of peptidoglycan and subsequent induction of NF-κB-dependent responses to H. pylori. These data implicate cholesterol-rich microdomains as a novel platform for TFSS-dependent delivery of bacterial products to cytosolic pathogen recognition molecules.

Fate of Entamoeba histolytica during Establishment of Amoebic Liver Abscess Analyzed by Quantitative Radioimaging and Histology

ABSTRACT The protozoan parasite Entamoeba histolytica is the causative agent of amoebiasis, a human disease characterized by dysentery and liver abscess. The physiopathology of hepatic lesions can be satisfactorily reproduced in the hamster animal model by the administration of trophozoites through the portal vein route. Hamsters were infected with radioactively labeled amoebas for analysis of liver abscess establishment and progression. The radioimaging of material from parasite origin and quantification of the number inflammation foci, with or without amoebas, described here provides the first detailed assessment of trophozoite survival and death during liver infection by E. histolytica. The massive death of trophozoites observed in the first hours postinfection correlates with the presence of a majority of inflammatory foci without parasites. A critical point for success of infection is reached after 12 h when the lowest number of trophozoites is observed. The process then enters a commitment phase during which parasites multiply and the size of the infection foci increases fast. The liver shows extensive areas of dead hepatocytes that are surrounded by a peripheral layer of parasites facing inflammatory cells leading to acute inflammation. Our results show that the host response promotes massive parasite death but also suggest also that this is a major contributor to the establishment of inflammation during development of liver abscess.

In situ imaging of specific binding of [3H]isatin in rat brain

Isatin is an endogenous indole that influences a range of processes both in vivo and in vitro. It has a distinct and discontinuous distribution in the brain, as well as in other mammalian tissues and body fluids. However, the distribution of isatin binding sites in the brain is not known. Using a real‐time β‐imager we have investigated the distribution of [3H]isatin‐specific binding in rat brain sections. The highest labeling was found in hypothalamic nuclei and in the cortex, hippocampus, and cerebellum. Administration of the mechanism based monoamine oxidase inhibitor, pargyline, reduced but did not abolish the specific binding of [3H]isatin in the rat brain. The distribution became cortex, cerebellum, hypothalamus > hippocampus > brain stem > thalamus ≈ striatum.

Isatin binding proteins in rat brain: In situ imaging, quantitative characterization of specific [3H]isatin binding, and proteomic profiling

Isatin (indole‐2,3‐dione) is an endogenous indole that has a distinct and discontinuous distribution in the brain and in other mammalian tissues and body fluids. Its output is increased under conditions of stress and anxiety. Its biological targets remain poorly characterized, although [3H]isatin binding sites have been demonstrated in various brain structures. In this study, by using a real‐time beta‐imager, [3H]isatin radioligand binding analysis, and proteomic identification of proteins specifically bound to the affinity sorbent 5‐aminocaproyl‐isatin‐Sepharose, we have investigated the distribution of [3H]isatin specific binding sites in the rat brain, characterized their Kd and Bmax, and identified some individual brain isatin binding proteins. The binding of [3H]isatin to rat brain sections was saturable and characterized by Kd values (of 0.2–0.3 μM) consistent with physiological concentrations. The highest Bmax was found in the hypothalamus, consistent with a role in stress. In most brain regions, the homologous inhibition of [3H]isatin binding by increasing concentrations of cold isatin demonstrated complex behavior suggesting involvement of various binding proteins characterized by different affinity to isatin. Affinity chromatography of Triton X‐100 lysates of whole‐brain homogenates on 5‐aminocaproyl‐isatin‐Sepharose followed by subsequent proteomic analysis resulted in identification of 25 individual proteins, including glyceraldehyde‐3‐phosphate dehydrogenase, one of few previously reported isatin binding proteins, and a group of cytoskeleton‐related proteins. These binding sites may be related to the known antiproliferative and proapoptotic activities of isatin.

Regional differential effects of chronic nicotine on brain α4-containing and α6-containing receptors

Chronic nicotine upregulates central nicotinic acetylcholine receptors (nAChRs), a plasticity process thought to contribute to its addictive properties. To analyze this process in vivo, we chronically exposed mice to nicotine using minipump delivering nicotine at concentrations close to those found in tobacco smokers. Binding studies show upregulation of high-affinity nAChRs after 21 days of treatment in cortical areas, caudate putamen, nucleus accumbens, hippocampus, ventral tegmental area, and superior colliculi. No upregulation was observed in thalamus and discrete cortical areas. Using wild type and &agr;6−/− mice, we observed a downregulation of &agr;6*-nAChRs in superior colliculi and no effects in other structures. The complex pattern of upregulation/downregulation observed in this study depends on both nAChR composition and regional distribution.

Substantial N diversity is generated in T cell receptor α genes at birth despite low levels of terminal deoxynucleotidyl transferase expression in mouse thymus

N region diversity in antigen receptors is a developmentally regulated process in B and T lymphocytes, which correlates with the differential expression of terminal deoxynucleotidyl transferase (TdT). To precisely determine the onset of TdT gene activation during T cell differentiation and thymic ontogeny, TdT expression was directly detected at the cellular level by in situ hybridization and TdT function was assessed by analyzing the distribution of N additions in α and β TCR genes at early stages of development. Even though TdT transcripts were undetectable at birth, substantial N additions were observed in VαJα junctions and 3 days later in VβDβJβ junctions, indicating that TdT expression could be induced in immature thymocytes much earlier than expected. Indeed low TdT expression level was found in TN3/4 and DP from fetal day 17, suggesting that the onset of TdT expression occurs simultaneously in both populations and may depend on microenvironmental cues. Moreover significant increase in the proportion of thymocytes expressing high levels of TdT mRNA during the first week after birth without a similar increase in the level of N diversity suggests that TdT expression and TdT function in the generation of N diversity are not strictly correlated.