Intragastric administration of a superoxide dismutase-producing recombinant Lactobacillus casei BL23 strain attenuates DSS colitis in mice.

Human immune cells release large amounts of reactive oxygen species (ROS) such as superoxide radical and hydrogen peroxide via respiratory burst. In inflammatory bowel diseases, a sustained and abnormal activation of the immune response results in oxidative stress of the digestive tract and in a loss of intestinal homeostasis. We previously reported that heterologous production of the Lactobacillus plantarum manganese catalase (MnKat) enhances the survival of Lb. casei BL23 when exposed to oxidative stress. Anti-inflammatory effects were observed after Lb. casei BL23 oral administrations in moderate murine dextran sodium sulfate (DSS)-induced colitis, without added effects of the MnKat production. Here, we evaluated the protective effects obtained by an improved antioxidative strategy. The Lactococcus lactis sodA gene was expressed in Lb. casei BL23 which acquired an efficient manganese superoxide dismutase (MnSOD) activity. The effects of Lb. casei MnSOD alone or in combination with Lb. casei MnKat were compared first in eukaryotic cell PMA-induced oxidative stress model and then in severe murine DSS-induced colitis. Based on ROS production assays as well as colonic histological scores, a significant reduction of both oxidative stress and inflammation was observed with Lb. casei MnSOD either alone or in combination with Lb. casei MnKat. No added effect of the presence of Lb. casei MnKat was observed. These results suggest that Lb. casei BL23 MnSOD could have anti-inflammatory effects on gut inflammation.

Efficient infection of buffalo rat liver-resistant cells by encephalomyocarditis virus requires binding to cell surface sialic acids.

In contrast to the production of virus and cell lysis seen in baby hamster kidney cells (BHK-21) infected with the strain 1086C of encephalomyocarditis virus (EMCV), in buffalo rat liver cells (BRL) neither virus replication nor cytopathic effects were observed. After 29 passages in BRL cells, each alternating with boosts of the recovered virus in BHK-21 cells, the virus acquired the ability to replicate effectively in BRL cells, attaining virus titres comparable to those in BHK-21 cells and producing complete cell destruction. The binding of virus on BRL cells was increased after adaptation and was similar to that observed on BHK-21 cells. Treatment of BRL cells with sialidase resulted in an 87 % reduction in virus binding and inhibition of infection. Sequence analyses revealed three mutations in the VP1 amino acid sequence of the adapted virus at positions 49 (Lys-->Glu), 142 (Leu-->Phe) and 180 (Ile-->Ala). The residue 49 is exposed at the surface of the capsid and is known to be part of a neutralization epitope. These results suggest that the adaptation of EMCV to BRL cells may have occurred through a mutation in a neutralizing site that confers to the virus a capacity to interact with cell surface sialic acid residues. Taken together, these data suggest a link between virus neutralization site, receptor binding and cell permissivity to infection.

Molecular analysis of the sex chromosomes of the platyfish Xiphophorus maculatus: Towards the identification of a new type of master sexual regulator in vertebrates.

In contrast to mammals and birds, fish display an amazing diversity of genetic sex determination systems, with frequent changes during evolution possibly associated with the emergence of new sex chromosomes and sex-determining genes. To better understand the molecular and evolutionary mechanisms driving this diversity, several fish models are studied in parallel. Besides the medaka (Oryzias latipes Temminck and Schlegel, 1846) for which the master sex-determination gene has been identified, one of the most advanced models for studying sex determination is the Southern platyfish (Xiphophorus maculatus, Günther 1966). Xiphophorus maculatus belongs to the Poeciliids, a family of live-bearing freshwater fish, including platyfish, swordtails and guppies that perfectly illustrates the diversity of genetic sex-determination mechanisms observed in teleosts. For X. maculatus, bacterial artificial chromosome contigs covering the sex-determination region of the X and Y sex chromosomes have been constructed. Initial molecular analysis demonstrated that the sex-determination region is very unstable and frequently undergoes duplications, deletions, inversions and other rearrangements. Eleven gene candidates linked to the master sex-determining gene have been identified, some of them corresponding to pseudogenes. All putative genes are present on both the X and the Y chromosomes, suggesting a poor degree of differentiation and a young evolutionary age for platyfish sex chromosomes. When compared with other fish and tetrapod genomes, syntenies were detected only with autosomes. This observation supports an independent origin of sex chromosomes, not only in different vertebrate lineages but also between different fish species.

Plasmacytoid dendritic cells migrate in afferent skin lymph.

Conventional dendritic cells enter lymph nodes by migrating from peripheral tissues via the lymphatic route, whereas plasmacytoid dendritic cells (pDC), also called IFN-producing cells (IPC), are described to gain nodes from blood via the high endothelial venules. We demonstrate here that IPC/pDC migrate in the afferent lymph of two large mammals. In sheep, injection of type A CpG oligodinucleotide (ODN) induced lymph cells to produce type I IFN. Furthermore, low-density lymph cells collected at steady state produced type I IFN after stimulation with type A CpG ODN and enveloped viruses. Sheep lymph IPC were found within a minor B(neg)CD11c(neg) subset expressing CD45RB. They presented a plasmacytoid morphology, expressed high levels of TLR-7, TLR-9, and IFN regulatory factor 7 mRNA, induced IFN-gamma production in allogeneic CD4(pos) T cells, and differentiated into dendritic cell-like cells under viral stimulation, thus fulfilling criteria of bona fide pDC. In mini-pig, a CD4(pos)SIRP(pos) subset in afferent lymph cells, corresponding to pDC homologs, produced type I IFN after type A CpG-ODN triggering. Thus, pDC can link innate and acquired immunity by migrating from tissue to draining node via lymph, similarly to conventional dendritic cells.

Transcytosis of Streptococcus iniae through skin epithelial barriers: an in vitro study.

By constructing a biological model based on in vitro culture of polarized rainbow trout primary skin epithelial cell monolayers, the series of early events that precede Streptococcus iniae infection, particularly colonization and translocation through external barriers, were analyzed. Streptococcus iniae promptly invades skin epithelial cells, but the rapid decline of viable intracellular bacteria points out the limited capability of intracellular survival for this bacterium. Translocation assays, supported by electron microscopy microphotographs, demonstrate that following successful in vitro invasion of skin epithelial cell, the bacterium exists free in the cytoplasm after release from the endosome, and translocates through the skin barrier. Bacterial invasion and transcytosis is not accompanied by apparent cell-line damages or disruption of host cells' tight junctions. It is hypothesized that the phenomenon of epithelial invasion coupled to the rapid translocation through the barrier plays a crucial role in Streptococcus iniae infection.

The nine C-terminal amino acids of the respiratory syncytial virus protein P are necessary and sufficient for binding to ribonucleoprotein complexes in which six ribonucleotides are contacted per N protein protomer.

The respiratory syncytial virus (RSV) phosphoprotein (P) is a major polymerase co-factor that interacts with both the large polymerase fragment (L) and the nucleoprotein (N). The N-binding domain of RSV P has been investigated by co-expression of RSV P and N proteins in Escherichia coli. Pull-down assays performed with a series of truncated forms of P fused to glutathione S-transferase (GST) revealed that the region comprising the last nine C-terminal amino acid residues of P (233-DNDLSLEDF-241) is sufficient for efficient binding to N. Site-directed mutagenesis shows that the last four residues of this peptide are crucial for binding and must be present at the end of a flexible C-terminal tail. The presence of the P oligomerization domain (residues 100-160) was an important stabilizing factor for the interaction. The tetrameric full-length P fused to GST was able to pull down both helical and ring structures, whereas a monomeric C-terminal fragment of P (residues 161-241) fused to GST pulled down exclusively RNA-N rings. Electron-microscopy analysis of the purified rings showed the presence of two types of complex: undecamers (11N) and decamers (10N). Mass-spectrometry analysis of the RNA extracted from rings after RNase A treatment showed two peaks of 22,900 and 24,820 Da, corresponding to a mean RNA length of 67 and 73 bases, respectively. These results suggest strongly that each N subunit contacts 6 nt, with an extra three or four bases further protected from nuclease digestion by the ring structure at both the 5' and 3' ends.

Phenotypic and functional similarity of gut intraepithelial and systemic T cells in a teleost fish.

Gut-associated lymphocytes were described in fish, but their involvement in immune responses is still unknown. In rainbow trout, intraepithelial lymphocytes (IELs) are scattered between gut epithelial cells, but neither Peyer's patches nor mesenteric lymph nodes were identified. Rainbow trout IELs contain mainly T cells, because they expressed transcripts of T cell marker homologs of CD8, CD4, CD28, CD3epsilon, TCRzeta, TCRgamma, and TCRbeta and lacked IgM. However, trout IELs did not show specific homing to the gut mucosa, which in mammals defines IELs as a distinctive mucosal population. A detailed analysis of the TCRbeta repertoire of rainbow trout IELs was performed in both naive and virus-infected animals. TCRbeta transcripts of rainbow trout IELs were highly diverse and polyclonal in adult naive individuals, in sharp contrast with the restricted diversity of IEL oligoclonal repertoires described in birds and mammals. Significant modifications of the trout IEL TCRbeta repertoire were observed after a systemic infection with a fish rhabdovirus and were especially marked for Vbeta4-bearing receptors as previously reported for spleen cells. Thus, we could not find any specific properties of the trout IEL TCRbeta repertoire compared with the spleen and pronephros TCRbeta repertoire, which questions the reality of a distinct IEL compartment in teleosts. Our findings suggest that a highly diversified alphabeta TauCR repertoire is maintained in fish IELs in the absence of Peyer's patches and mesenteric lymph nodes, whereas the restricted diversity of mouse alphabeta IELs is attributed to multiple cycles of activation and recirculation, allowing a progressive narrowing of the repertoire.

Molecular analysis of the sex-determining region of the platyfish Xiphophorus maculatus.

Due to the presence of genetically well-defined sex chromosomes, with a relatively restricted sex-determination region containing markers identified at the molecular level, the platyfish Xiphophorus maculatus is one of the best models for the positional cloning of a master sex-determining gene in fish. Both male and female heterogametes and three different types of sex chromosomes have been described in the platyfish, with several loci involved in pigmentation, melanoma formation, and sexual maturity closely linked to the master sex-determining locus. Using the melanoma-inducing oncogene Xmrk, its protooncogenic counterpart egfrb, as well as other X- and Y-linked molecular markers, bacterial artificial chromosome (BAC) contigs have been assembled for the sex-determining region of X. maculatus, which was mapped by fluorescent in situ hybridization to the subtelomeric region of the sex chromosomes. Initial sequence analysis of these contigs revealed several gene candidates and uncovered syntenies with different mammalian and chicken autosomes, supporting an independent origin of sex chromosomes in platyfish and tetrapods. Strikingly, the sex determination region of the platyfish is very instable and frequently undergoes duplications, deletions, and transpositions. This instability might be linked to the high genetic variability affecting sex determination and other sex-linked traits in Xiphophorus.

Clonality and diversity of the fish pathogen Lactococcus garvieae in Mediterranean countries.

Infection with Lactococcus garvieae is considered the most important risk factor for the European trout industry, and the losses are approximately 50% of the total production. To improve our understanding of the genetic links among strains originating from different countries, we examined the population structure of L. garvieae by comparing 81 strains isolated from different sources and ecosystems (41 farms in six countries) in which the bacterium is commonly found. Genetic similarities (as assessed with molecular tools, including restriction fragment length polymorphism ribotyping with two endonucleases) were compared with serological data. The combined results reveal that in endemic sites the bacterial population displays a clonal structure, whereas bacterial diversity characterizes sites where the infection is sporadic.

Trojan horse effect: phagocyte-mediated Streptococcus iniae infection of fish.

The salmonid macrophage-like cell line RTS-11 and purified trout pronephros phagocytes were used to analyze in vitro entry and survival of two Streptococcus iniae serotypes. Efficient invasion by S. iniae occurred in both cells, but only the type II strain persisted in pronephros phagocytes for at least 48 h. Ex vivo models of opsonin-dependent phagocytosis by pronephros phagocytes demonstrated increased phagocytosis efficacy. Analysis of phagocytes collected from diseased fish demonstrated that approximately 70% of the bacteria contained in the blood during the septic phase of the disease were located within phagocytes, suggesting an in vivo intracellular lifestyle. In addition to the augmented levels of bacteremia and enhanced survival within phagocytes, S. iniae type II induces considerable apoptosis of phagocytes. These variabilities in intramacrophage lifestyle might explain differences in the outcomes of infections caused by different serotypes. The generalized septic disease associated with serotype II strains is linked not only to the ability to enter and multiply within macrophages but also to the ability to cause considerable death of macrophages via apoptotic processes, leading to a highly virulent infection. We assume that the phenomenon of survival within phagocytes coupled to their apoptosis plays a crucial role in S. iniae infection. In addition, it may provide the pathogen an efficient mechanism of translocation into the central nervous system.