The gastrointestinal tract functions as a barrier against antigens from microorganisms and food. The generation of immunophysiologic regulation in the gut depends on the establishment of indigenous microflora. This has led to the introduction of novel therapeutic interventions based on the consumption of cultures of beneficial live microorganisms that act as probiotics. Among the possible mechanisms of probiotic therapy is promotion of a nonimmunologic gut defense barrier, which includes the normalization of increased intestinal permeability and altered gut microecology. Another possible mechanism of probiotic therapy is improvement of the intestine's immunologic barrier, particularly through intestinal immunoglobulin A responses and alleviation of intestinal inflammatory responses, which produce a gut-stabilizing effect. Many probiotic effects are mediated through immune regulation, particularly through balance control of proinflammatory and anti-inflammatory cytokines. These data show that probiotics can be used as innovative tools to alleviate intestinal inflammation, normalize gut mucosal dysfunction, and down-regulate hypersensitivity reactions. More recent data show that differences exist in the immunomodulatory effects of candidate probiotic bacteria. Moreover, distinct regulatory effects have been detected in healthy subjects and in patients with inflammatory diseases. These results suggest that specific immunomodulatory properties of probiotic bacteria should be characterized when developing clinical applications for extended target populations.
Integrin ␣21 mediates the binding of several epithelial and mesenchymal cell types to collagen. The composition of the surrounding plasma membrane, especially caveolin-1-and cholesterol-containing membrane structures called caveolae, may be important to integrin signaling. On cell surface ␣21 integrin was located in the raft like membrane domain, rich in GPI-anchored proteins, rather than in caveolae. However, when antibodies were used to generate clusters of ␣21 integrin, they started to move laterally on cell surface along actin filaments. During the lateral movement small clusters fused together. Finally ␣21 integrin was found inside caveolae and subsequently internalized into caveosome-like perinuclear structures. The internalization process, unlike cluster formation or lateral redistribution, was dependent on protein kinase C␣ activity. Caveolae are known to be highly immobile structures and ␣21 integrin clusters represent a previously unknown mechanism to activate endocytic trafficking via caveolae. The process was specific to ␣21 integrin, because the antibody-mediated formation of ␣V integrin clusters activated their internalization in coated vesicles and early endosomes. In addition to natural ligands human echovirus-1 (EV1) gains entry into the cell by binding to ␣21 and taking advantage of ␣21 internalization via caveolae. INTRODUCTIONCaveolae are cave-like invaginations of the cell surface (for reviews see Kurzchalia and Parton, 1999;Parton 2003). They have been described as highly immobile and not involved in constitutive endocytic trafficking (Thomsen et al., 2002). Their internalization can be activated, for example, by the phosphatase inhibitor okadaic acid (Parton et al., 1994). In endothelial cells the interaction of albumin docking protein gp60 and caveolin-1 initiates the endocytosis of caveolae (Minshall et al., 2000). Caveolins are membrane proteins that are molecular markers of caveolae and shown to be crucial for the formation of these structures (Fra et al., 1995). Caveolins can be associated with cell surface growth factor receptors (Couet et al., 1997) and cell adhesion receptors (Wary et al., 1996(Wary et al., , 1998Wei et al., 1999) and caveolae might therefore play a role in cellular signaling. In addition, caveolae have been suggested to participate in the uptake of folate by pinocytosis, but this is still under discussion (Parton, 2003). Caveolin-deficient mice have given novel insight into the function of caveolae. Caveolin-1 gene knockout leads to pulmonary defects, vasoconstriction, or dilatation abnormalities and resistance to diet-induced obesity (Drab et al., 2001;Razani et al., 2001;Schubert et al., 2001;Sotgia et al., 2002;Razani et al., 2002). Simian virus 40 (SV40) has been shown to be internalized through caveolae and detailed studies focused on the virus entry mechanism have produced a lot of new information about the biology of caveolae (Pelkmans et al., 2001. We have recently shown that human echovirus 1 (EV1) is another virus using caveolae in its entry (Mar...
We have previously shown that a human picornavirus echovirus 1 (EV1) is transported to caveosomes during 2 h together with its receptor ␣21 integrin. Here, we show that the majority of early uptake does not occur through caveolae. ␣21 integrin, clustered by antibodies or by EV1 binding, is initially internalized from lipid rafts into tubulovesicular structures. These vesicles accumulate fluid-phase markers but do not initially colocalize with caveolin-1 or internalized simian virus 40 (SV40). Furthermore, the internalized endosomes do not contain glycosylphosphatidylinositol (GPI)-anchored proteins or flotillin 1, suggesting that clustered ␣21 integrin does not enter the GPI-anchored protein enriched endosomal compartment or flotillin pathways, respectively. Endosomes mature further into larger multivesicular bodies between 15 min to 2 h and concomitantly recruit caveolin-1 or SV40 inside. Cell entry is regulated by p21-activated kinase (Pak)1, Rac1, phosphatidylinositol 3-kinase, phospholipase C, and actin but not by dynamin 2 in SAOS-␣21 cells. An amiloride analog, 5-(N-ethyl-N-isopropanyl) amiloride, blocks infection, causes integrin accumulation in early tubulovesicular structures, and prevents their structural maturation into multivesicular structures. Our results together suggest that ␣21 integrin clustering defines its own entry pathway that is Pak1 dependent but clathrin and caveolin independent and that is able to sort cargo to caveosomes.
Lactobacillus rhamnosus GG and Lactobacillus rhamnosus LC-705, previously shown to effectively bind to aflatoxin B1, were subjected to various chemical and physical treatments to examine the effects of these treatments on the binding affinity of these strains toward aflatoxin B1. Treatment of bacterial pellets of both strains with hydrochloric acid significantly (P < 0.05) enhanced the binding ability to bind aflatoxin B1 was also observed when the bacterial pellets were subjected to heat treatment by either autoclaving or boiling at 100 degrees C in a water bath, put the impact of these two treatments was not as effective as the acid treatment. Ethanol, UV radiation, sonication, alkaline, or pH treatments either had not effect or reduced the binding ability of the bacteria.
Baculoviruses are enveloped insect viruses that can carry large quantities of foreign DNA in their genome. Baculoviruses have proved to be very promising gene therapy vectors but little is known about their transduction mechanisms in mammalian cells. We show in this study that Autographa californica multiple nuclear polyhedrosis virus capsid is compatible with the incorporation of desired proteins in large quantities. Fusions can be made to the N-terminus or C-terminus of the major capsid protein vp39 without compromising the viral titer or functionality. As an example of the baculovirus capsid display we show a tracking of the baculovirus transduction in mammalian cells by an enhanced green fluorescent protein (EGFP)-displaying virus. Our confocal and electron microscopy results suggest that the transduction block in mammalian cells is not in the endosomal escape, as previously proposed, but rather in the cytoplasmic transport or nuclear entry of the virus capsid. Our results also suggest that the EGFP-tagged virus can be used for visualization of the virus biodistribution in vivo. Furthermore, capsid-modified baculoviruses hold great promise for the nuclear and subcellular targeting of transgenes and as a novel peptide display system for a variety of eukaryotic applications.
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