Glycan-modifying bacteria-derived soluble factors from Bacteroides thetaiotaomicron and Lactobacillus casei inhibit rotavirus infection in human intestinal cells

Rotaviruses attach to intestinal cells in a process that requires glycan recognition. Some bacteria from the gut microflora have been shown to modify cell-surface glycans. In this study, human intestinal cultured cells were incubated with bacteria-derived soluble factors and infected with rotavirus. Results show that only bacterial soluble factors that increase cell-surface galactose namely, those of Bacteroides thetaiotaomicron and Lactobacillus casei were able to efficiently block rotavirus infections. Increasing cell-surface galactose using galactosyltransferase resulted in a similar blockage of rotavirus infections. These results indicate that manipulation of cell-surface intestinal glycans by bacterial soluble factors can prevent rotavirus infection in a species-specific manner, and should now be considered a potential therapeutic approach against rotavirus infection.     

Cutting edge: a naturally occurring mutation in CD1e impairs lipid antigen presentation

The human CD1a-d proteins are plasma membrane molecules involved in the presentation of lipid Ags to T cells. In contrast, CD1e is an intracellular protein present in a soluble form in late endosomes or lysosomes and is essential for the processing of complex glycolipid Ags such as hexamannosylated phosphatidyl-myo-inositol, PIM(6). CD1e is formed by the association of beta(2)-microglobulin with an alpha-chain encoded by a polymorphic gene. We report here that one variant of CD1e with a proline at position 194, encoded by allele 4, does not assist PIM(6) presentation to CD1b-restricted specific T cells. The immunological incompetence of this CD1e variant is mainly due to inefficient assembly and poor transport of this molecule to late endosomal compartments. Although the allele 4 of CD1E is not frequent in the population, our findings suggest that homozygous individuals might display an altered immune response to complex glycolipid Ags.     

Expression of nonstructural rotavirus protein NSP4 mimics Ca2+ homeostasis changes induced by rotavirus infection in cultured cells

Rotavirus infection modifies Ca²⁺ homeostasis, provoking an increase in Ca²⁺ permeation, the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_cyto), and total Ca²⁺ pools and a decrease in Ca²⁺ response to agonists. A glycosylated viral protein(s), NSP4 and/or VP7, may be responsible for these effects. HT29 or Cos-7 cells were infected by the SA11 clone 28 strain, in which VP7 is not glycosylated, or transiently transfected with plasmids coding for NSP4-enhanced green fluorescent protein (EGFP) or NSP4. The permeability of the plasma membrane to Ca²⁺ and the amount of Ca²⁺ sequestered in the endoplasmic reticulum released by carbachol or ATP were measured in fura-2-loaded cells at the single-cell level under a fluorescence microscope or in cell suspensions in a fluorimeter. Total cell Ca²⁺ pools were evaluated as ⁴⁵Ca²⁺ uptake. Infection with SA11 clone 28 induced an increase in Ca²⁺ permeability and ⁴⁵Ca²⁺ uptake similar to that found with the normally glycosylated SA11 strain. These effects were inhibited by tunicamycin, indicating that inhibition of glycosylation of a viral protein other than VP7 affects the changes of Ca²⁺ homeostasis induced by infection. Expression of NSP4-EGFP or NSP4 in transfected cells induced the same changes observed with rotavirus infection, whereas the expression of EGFP or EGFP-VP4 showed the behavior of uninfected and untransfected cells. Increased ⁴⁵Ca²⁺ uptake was also observed in cells expressing NSP4-EGFP or NSP4, as evidenced in rotavirus infection. These results indicate that glycosylated NSP4 is primarily responsible for altering the Ca²⁺ homeostasis of infected cells through an initial increase of cell membrane permeability to Ca²⁺.     

Modulation of Macrophage Activation State Protects Tissue from Necrosis during Critical Limb Ischemia in Thrombospondin-1-Deficient Mice

Background

Macrophages, key regulators of healing/regeneration processes, strongly infiltrate ischemic tissues from patients suffering from critical limb ischemia (CLI). However pro-inflammatory markers correlate with disease progression and risk of amputation, suggesting that modulating macrophage activation state might be beneficial. We previously reported that thrombospondin-1 (TSP-1) is highly expressed in ischemic tissues during CLI in humans. TSP-1 is a matricellular protein that displays well-known angiostatic properties in cancer, and regulates inflammation in vivo and macrophages properties in vitro. We therefore sought to investigate its function in a mouse model of CLI.

Methods and Findings

Using a genetic model of tsp-1 ^−/− mice subjected to femoral artery excision, we report that tsp-1 ^−/− mice were clinically and histologically protected from necrosis compared to controls. Tissue protection was associated with increased postischemic angiogenesis and muscle regeneration. We next showed that macrophages present in ischemic tissues exhibited distinct phenotypes in tsp-1 ^−/− and wt mice. A strong reduction of necrotic myofibers phagocytosis was observed in tsp-1 ^−/− mice. We next demonstrated that phagocytosis of muscle cell debris is a potent pro-inflammatory signal for macrophages in vitro. Consistently with these findings, macrophages that infiltrated ischemic tissues exhibited a reduced postischemic pro-inflammatory activation state in tsp-1 ^−/− mice, characterized by a reduced Ly-6C expression and a less pro-inflammatory cytokine expression profile. Finally, we showed that monocyte depletion reversed clinical and histological protection from necrosis observed in tsp-1 ^−/− mice, thereby demonstrating that macrophages mediated tissue protection in these mice.

Conclusion

This study defines targeting postischemic macrophage activation state as a new potential therapeutic approach to protect tissues from necrosis and promote tissue repair during CLI. Furthermore, our data suggest that phagocytosis plays a crucial role in promoting a deleterious intra-tissular pro-inflammatory macrophage activation state during critical injuries. Finally, our results describe TSP-1 as a new relevant physiological target during critical leg ischemia.     

MCL-1 inhibits BAX in the absence of MCL-1/BAX Interaction

The BCL-2 family of proteins plays a major role in the control of apoptosis as the primary regulator of mitochondrial permeability. The pro-apoptotic BCL-2 homologues BAX and BAK are activated following the induction of apoptosis and induce cytochrome c release from mitochondria. A second class of BCL-2 homologues, the BH3-only proteins, is required for the activation of BAX and BAK. The activity of both BAX/BAK and BH3-only proteins is opposed by anti-apoptotic BCL-2 homologues such as BCL-2 and MCL-1. Here we show that anti-apoptotic MCL-1 inhibits the function of BAX downstream of its initial activation and translocation to mitochondria. Although MCL-1 interacted with BAK and inhibited its activation, the activity of MCL-1 against BAX was independent of an interaction between the two proteins. However, the anti-apoptotic function of MCL-1 required the presence of BAX. These results suggest that the pro-survival activity of MCL-1 proceeds via inhibition of BAX function at mitochondria, downstream of its activation and translocation to this organelle.     

Collagen fibril network and elastic system remodeling in a reconstructed skin transplanted on nude mice.

Wound healing of deep and extensive burns can induce hypertrophic scar formation, which is a detrimental outcome for skin functionality. These scars are characterized by an impaired collagen fibril organization with fibril bundles oriented parallel to each other, in contrast with a basket weave pattern arrangement in normal skin. We prepared a reconstructed skin made of a collagen sponge seeded with human fibroblasts and keratinocytes and grown in vitro for 20 days. We transplanted it on the back of nude mice to assess whether this reconstructed skin could prevent scar formation. After transplantation, murine blood vessels had revascularized one-third of the sponge thickness on the fifth day and were observed underneath the epidermis at day 15. The reconstructed skin extracellular matrix was mostly made of human collagen I, organized in loosely packed fibrils 5 days after transplantation, with a mean diameter of 45 nm. After 40-90 days, fibril bundles were arranged in a basket weave pattern while their mean diameter increased to 56 nm, therefore exactly matching mouse skin papillary dermis organization. Interestingly, we showed that an elastic system remodeling was started off in this model. Indeed, human elastin deposits were organized in thin fibrils oriented perpendicular to epidermis at day 90 whereas elastic system usually took years to be re-established in human scars. Our reconstructed skin model promoted in only 90 days the remodeling of an extracellular matrix nearly similar to normal dermis (i.e. collagen fibril diameter and arrangement, and the partial reconstruction of the elastic system).     

Role of I-region gene products in T cell activation. I. Stimulation of T lymphocyte proliferative responses by subcellular membrane preparations containing Ia alloantigens

A model system has been developed for exploring the requirements for activation of T cells by subcellular forms of Ia alloantigen. Lymph node cells from mice recently primed subcutaneously with viable allogeneic cells show strong proliferative responses in vitro to membrane preparations derived from cells bearing the appropriate I-region-encoded glycoproteins. This stimulation shows kinetics characteristic of a secondary response, with a peak at 24 to 48 hr. Primary responses to alloantigen-bearing membranes are weak or absent under these conditions. The predominant cell type involved in the secondary response is the Lyt-1+ T lymphocyte, and the major antigenic stimulus is the I-A subregion-encoded Ia glycoprotein. Syngeneic Ia+ accessory cells do not appear necessary for activation to occur. Detergent solubilized reconstituted membrane vesicles also will stimulate primed T lymphocytes to respond by proliferation. The applications of this approach to the study of T cell recognition of antigen and the role of nonspecific lymphokines in T cell triggering are discussed.     

Influence des inflorescences de la plante hôte (Vigna unguiculata) sur la levée de la diapause reproductrice de Bruchidius atrolineatus

Bruchidius atrolineatus (Pic) présente en zone sahélienne une diapause reproductrice durant la saison sèche et une partie de la saison des pluies. Les femelles diapausantes ne produisent pas de vitellogénine et le germarium des ovarioles est seul développé. Chez les mâles la spermatogenèse est très ralentie et les glandes annexes sont inactives. Lorsque les insectes diapausants sont placés en présence d'inflorescences de Vigna unguiculata Walp, leurs organes reproducteurs deviennent fonctionnels après un temps de latence de 15 à 20 jours. Il n'y a par contre aucune levée de la diapause chez des bruches placées en présence de gousses sèches de V. unguiculata dans une atmosphère saturée. Des informations sensorielles issues des pièces florales ou (et) des facteurs nutritionnels sans doute liés a la consommation de nectar semblent être à l'origine de cette levée de la diapause. Le pollen fort peu consommé n'a par contre aucun rôle. Cette régulation du cycle reproducteur de B. atrolineatus par les inflorescences de V. unguiculata permet l'émission des oeufs dès que les gousses commencent à se former à la fin de la saison des pluies.

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Summary Bruchidius atrolineatus (Pic) is a widely distributed bruchid in the Sahelian zone which shows a reproductive diapause during the dry season and part of the rainy season. Diapausing females do not produce vitellogenin and their ovaries are reduced to the germarium. Spermatogenesis is very much reduced and male accessory glands are inactive. When these insects were placed in the presence of inflorescences of Vigna unguiculata which were renewed daily, the reproductive diapause of both males and females was interrupted after 15–20 days. Vitellogenesis occurred in the females and spermatogenesis increased in the males whilst their accessory glands became functional. When diapausing bruchids, found in stores of on V. unguiculata seeds during the dry season, were placed near the host plant's inflorescences, diapause was also terminated. In all cases, diapause was not interrupted when the insects were offered dry pods of V. unguiculata in a water-saturated atmosphere. The pollen, which is hardly eaten by this bruchid, did not seem to stimulate termination of diapause. Sensory stimulations derived from the flowers or/and nutritional factors may be the cause of the development of the reproductive organs. After termination of the diapause the males showed normal sexual activity whereas female fecundity was rather low, at least in our experimental conditions. This type of reproductive regulation allows this sahelian bruchid to resume its sexual activity when the host plant's flowers appear in the field at the end of the rainy season. Then the beetles lay their eggs on the pods as soon as the pods are developed.