A strain of Lactobacillus casei inhibits the effector phase of immune inflammation.

Some nonpathogenic bacteria were found to have protective effects in mouse models of allergic and autoimmune diseases. These "probiotics" are thought to interact with dendritic cells during Ag presentation, at the initiation of adaptive immune responses. Many other myeloid cells are the effector cells of immune responses. They are responsible for inflammation that accounts for symptoms in allergic and autoimmune diseases. We investigated in this study whether probiotics might affect allergic and autoimmune inflammation by acting at the effector phase of adaptive immune responses. The effects of one strain of Lactobacillus casei were investigated in vivo on IgE-induced passive systemic anaphylaxis and IgG-induced passive arthritis, two murine models of acute allergic and autoimmune inflammation, respectively, which bypass the induction phase of immune responses, in vitro on IgE- and IgG-induced mouse mast cell activation and ex vivo on IgE-dependent human basophil activation. L. casei protected from anaphylaxis and arthritis, and inhibited mouse mast cell and human basophil activation. Inhibition required contact between mast cells and bacteria, was reversible, and selectively affected the Lyn/Syk/linker for activation of T cells pathway induced on engagement of IgE receptors, leading to decreased MAPK activation, Ca(2+) mobilization, degranulation, and cytokine secretion. Also, adoptive anaphylaxis induced on Ag challenge in mice injected with IgE-sensitized mast cells was abrogated in mice injected with IgE-sensitized mast cells exposed to bacteria. These results demonstrate that probiotics can influence the effector phase of adaptive immunity in allergic and autoimmune diseases. They might, therefore, prevent inflammation in patients who have already synthesized specific IgE or autoantibodies.

Cytosolic phospholipase A2alpha mediates Pseudomonas aeruginosa LPS-induced airway constriction of CFTR -/- mice.

BACKGROUND: Lungs of cystic fibrosis (CF) patients are chronically infected with Pseudomonas aeruginosa. Increased airway constriction has been reported in CF patients but underplaying mechanisms have not been elucidated. AIM: To examine the effect of P. aeruginosa LPS on airway constriction in CF mice and the implication in this process of cytosolic phospholipase A2alpha (cPLA2alpha), an enzyme involved in arachidonic acid (AA) release. METHODS: Mice were instilled intra-nasally with LPS. Airway constriction was assessed using barometric plethysmograph. MIP-2, prostaglandin E2 (PGE2), leukotrienes and AA concentrations were measured in BALF using standard kits and gas chromatography. RESULTS: LPS induced enhanced airway constriction and AA release in BALF of CF compared to littermate mice. This was accompanied by increased levels of PGE2, but not those of leukotrienes. However, airway neutrophil influx and MIP-2 production remained similar in both mouse strains. The cPLA2alpha inhibitor arachidonyl trifluoro-methyl-ketone (ATK), but not aspirin which inhibit PGE2 synthesis, reduced LPS-induced airway constriction. LPS induced lower airway constriction and PGE2 production in cPLA2alpha -/- mice compared to corresponding littermates. Neither aspirin nor ATK interfered with LPS-induced airway neutrophil influx or MIP-2 production. CONCLUSIONS: CF mice develop enhanced airway constriction through a cPLA2alpha-dependent mechanism. Airway inflammation is dissociated from airway constriction in this model. cPLA2alpha may represent a suitable target for therapeutic intervention in CF. Attenuation of airway constriction by cPLA2alpha inhibitors may help to ameliorate the clinical status of CF patients.

Targeted expression of the dominant-negative prolactin receptor in the mammary gland of transgenic mice results in impaired lactation.

The F3-short form of the rat PRL receptor (F3-SPRLR) form acts as a dominant negative inhibitor in vitro. We have developed a transgenic mouse model in which the rat F3-SPRLR was expressed in mammary epithelium under the control of the mouse mammary tumor virus promoter. Two lines of mice were characterized and shown to express the transgene in the mammary gland. No developmental abnormalities or differences from wild-type littermates were observed on the basis of size, activity, or fertility. Mice with a low level of transgene expression had a mammary phenotype similar to the wild type. However, mice overexpressing the transgene (levels much higher than those of the endogenous long PRLR transcript) had impaired mammary gland differentiation and lactation. In these mice, whole-mount and histological analyses demonstrated normal ductal development, but severely reduced lobuloalveolar outgrowth. signal transducer and activator of transcription-5 phosphorylation and expression of beta-casein and whey acidic protein gene were decreased. In vivo bromodeoxyuridine incorporation at midpregnancy showed that the reduction in mammary development was not due to an inhibition of ductal growth and side-branching. This model demonstrates for the first time in vivo a function of the SPRLR and a local and targeted effect of PRL on the mammary gland that are essential for its function, but not for its development.

Severe osteopenia in CFTR-null mice.

Osteoporosis is a common complication in cystic fibrosis (CF) patients. In this study, we performed a histomorphometric analysis of the bones of a mouse genetic model of human CF in which both copies of the cystic fibrosis transmembrane conductance regulator (CFTR) gene are inactivated. We find that, even in the absence of obvious nutritional and therapeutic differences, the CFTR mutation is associated with severe osteopenia. Bone mineral density (BMD) of total body and of individual bones is significantly diminished. CFTR mutants display a striking significant (50%) reduction of cortical bone width and thinner trabeculae. Analysis of dynamic parameters indicates a significant reduction of bone formation and a concomitant strong increase in bone resorption. Active osteoclasts where found mostly associated with cortical bone. Our data support the concept that CF-associated osteoporosis is part of the syndromic symptoms associated with the CFTR mutation.

Oral probiotic control skin inflammation by acting on both effector and regulatory T cells.

Probiotics are believed to alleviate allergic and inflammatory skin disorders, but their impact on pathogenic effector T cells remains poorly documented. Here we show that oral treatment with the probiotic bacteria L. casei (DN-114 001) alone alleviates antigen-specific skin inflammation mediated by either protein-specific CD4(+) T cells or hapten-specific CD8(+) T cells. In the model of CD8(+) T cell-mediated skin inflammation, which reproduces allergic contact dermatitis in human, inhibition of skin inflammation by L. casei is not due to impaired priming of hapten-specific IFNgamma-producing cytolytic CD8(+) effector T cells. Alternatively, L. casei treatment reduces the recruitment of CD8(+) effector T cells into the skin during the elicitation (i.e. symptomatic) phase of CHS. Inhibition of skin inflammation by L. casei requires MHC class II-restricted CD4(+) T cells but not CD1d-restricted NK-T cells. L casei treatment enhanced the frequency of FoxP3(+) Treg in the skin and increased the production of IL-10 by CD4(+)CD25(+) regulatory T cells in skin draining lymph nodes of hapten-sensitized mice. These data demonstrate that orally administered L. casei (DN-114 001) efficiently alleviate T cell-mediated skin inflammation without causing immune suppression, via mechanisms that include control of CD8(+) effector T cells and involve regulatory CD4(+) T cells. L. casei (DN-114 001) may thus represent a probiotic of potential interest for immunomodulation of T cell-mediated allergic skin diseases in human.

Transfection of multiple pulmonary cell types following intravenous injection of PEI-DNA in normal and CFTR mutant mice.

BACKGROUND: The polycationic vector polyethylenimine (PEI) has been shown to be a powerful agent for transfecting the mouse lung after injection of plasmid-based polyplexes through the tail vein. These findings raise therapeutic prospects for a number of lung conditions. For such potentials to be realised, the precise identity of the transfected cells remains to be determined; however, so far, no ultrastructural analysis has been performed on PEI-transfected lungs. The definition of which pulmonary cells are transfected is particularly critical for certain pulmonary diseases which might require transfection of defined cell types such as epithelial cells for cystic fibrosis (CF). METHODS: Here, we use a combination of light and electron microscopy to determine which cells are transfected in the lung after PEI-mediated gene delivery through the intravenous route. Furthermore, we extend the same experimental setting to a mouse model of CF to provide proof of principle that this approach can be used in genetic models of the disease. RESULTS: We show that within 18-20 h after injection through the tail vein, DNA/PEI complexes have already crossed the capillary barrier resulting in high levels of expression of reporter genes in the lungs. Transgene expression is observed in endothelial cells, in type I and type II pneumocytes, and in septal cells. Coexpression of the transgene and of the endogenous CF transmembrane conductance regulator (CFTR) gene is observed in some of the targeted epithelial cells. Levels and sites of expression are similar in normal and in CFTR-mutant mice. CONCLUSIONS: The results demonstrate that PEI-mediated gene delivery leads to transfection of epithelial cells beyond the endothelial barrier and show that this method can be used for lung gene delivery in CF fragile mutant mice.