SummaryIn epithelial cells, the intracellular pathogen Brucella abortus escapes from the endocytic pathway, exploits the autophagic machinery of the host cell and establishes a unique replication niche in the endoplasmic reticulum. The molecular mechanisms underlying these processes are still poorly understood. Recently, a B. abortus type IV-related secretion system encoded by the virB operon has been described as being involved in the intracellular trafficking of the bacteria. In this study, we have analysed the intracellular pathway of B. abortus virB10 mutant strains by confocal microscopy. We demonstrate that a functional virB operon is essential for the biogenesis of the Brucella-containing vacuole. Polar mutation preventing the transcription of virB10 and downstream sequences did not allow Brucella to bypass the endocytic pathway. Consequently, polar mutant-containing vacuoles fused with lysosomes in which bacteria underwent a degradation process. In contrast, virB10 non-polar mutants were capable of avoiding interactions with the endocytic pathway but, diverging to wild-type Brucella, were unable to reach the endoplasmic reticulum to establish their intracellular replication niche and seemed to be recycled to the cell surface. Based on the two particular phenotypes described in this work, a model of maturation of the Brucella-containing vacuole is proposed.

SummaryAfter uptake by host cells, the pathogen Brucella transits through early endosomes, evades phago± lysosome fusion and replicates in a compartment associated with the endoplasmic reticulum (ER). The molecular mechanisms underlying these processes are still poorly understood. To identify new bacterial factors involved in these processes, a library of 1800 Brucella melitensis 16M mini-Tn5catkm mutants was screened for intracellular survival and multiplication in HeLa cells and J774A.1 macrophages. Thirteen mutants were identified as defective for their intracellular survival in both cell types. In 12 of them, the transposon had inserted in the virB operon, which encodes a type IV-related secretion system. The preponderance of virB mutants demonstrates the importance of this secretion apparatus in the intracellular multiplication of B. melitensis. We also examined the intracellular fate of three virB mutants (virB2, virB4 and virB9) in HeLa cells by immunofluorescence. The three VirB proteins are not necessary for penetration and the inhibition of phago±lysosomal fusion within non-professional phagocytes. Rather, the virB mutants are unable to reach the replicative niche and reside in a membranebound vacuole expressing the late endosomal marker, LAMP1, and the sec61b protein from the ER membrane, proteins that are present in autophagic vesicles originating from the ER.

Preformed Fas ligand (FasL) and APO2 ligand (APO2L)/TNF-related apoptosis-inducing ligand (TRAIL) are stored in the cytoplasm of the human Jurkat T cell line and of normal human T cell blasts. The rapid release of these molecules in their bioactive form is involved in activation-induced cell death. In this study, we show by confocal microscopy that FasL and APO2L/TRAIL are mainly localized in lysosomal-like compartments in these cells. We show also by immunoelectron microscopy that FasL and APO2L/TRAIL are stored inside cytoplasmic compartments ∼500 nm in diameter, with characteristics of multivesicular bodies. Most of these compartments share FasL and APO2L/TRAIL, although exclusive APO2L/TRAIL labeling can be also observed in separate compartments. Upon PHA activation, the mobilization of these compartments toward the plasma membrane is evident, resulting in the secretion of the internal microvesicles loaded with FasL and APO2L/TRAIL. In the case of activation with anti-CD59 mAb, the secretion of microvesicles labeled preferentially with APO2L/TRAIL predominates. These data provide the basis of a new and efficient mechanism for the rapid induction of autocrine or paracrine cell death during immune regulation and could modify the interpretation of the role of FasL and APO2L/TRAIL as effector mechanisms in physiological and pathological situations.

Maturation and maintenance of the intracellular vacuole in which Salmonella replicates is controlled by virulence proteins including the type III secretion system encoded by Salmonella pathogenicity island 2 (SPI‐2). Here, we show that, several hours after bacterial uptake into different host cell types, Salmonella induces the formation of an F‐actin meshwork around bacterial vacuoles. This structure is assembled de novo from the cellular G‐actin pool in close proximity to the Salmonella vacuolar membrane. We demonstrate that the phenomenon does not require the Inv/Spa type III secretion system or cognate effector proteins, which induce actin polymerization during bacterial invasion, but does require a functional SPI‐2 type III secretion system, which plays an important role in intracellular replication and systemic infection in mice. Treatment with actin‐depolymerizing agents significantly inhibited intramacrophage replication of wild‐type Salmonella typhimurium. Furthermore, after this treatment, wild‐type bacteria were released into the host cell cytoplasm, whereas SPI‐2 mutant bacteria remained within vacuoles. We conclude that actin assembly plays an important role in the establishment of an intracellular niche that sustains bacterial growth.

The interaction of Fas with Fas ligand (FasL) mediates activation-induced cell death (AICD) of T hybridomas and of mature T lymphocytes. The TNF/TNF receptor system also plays a significant role in AICD of mature T cells and in the maintenance of peripheral tolerance. We previously demonstrated that in human Jurkat leukemia cells, AICD is triggered mainly by the rapid release of preformed FasL upon TCR stimulation. In the present work, we show that the cytotoxic cytokine APO2 ligand (APO2L; also known as TRAIL) is constitutively expressed as an intracytoplasmic protein in Jurkat T cells and derived sublines. APO2L is also detected in fresh human peripheral blood mononuclear cells (PBMC) from a significant number of donors, and the amount of both FasL and APO2L substantially increases upon blast generation. A neutralizing anti-APO2L monoclonal antibody (mAb) partially suppresses the cytotoxicity induced by supernatants of phytohemagglutinin (PHA)-prestimulated Jurkat or human PBMC on non-activated Jurkat cells, indicating that APO2L is released by these cells and contributes to AICD. A combination of neutralizing anti-APO2L and anti-Fas mAb blocks around 60 % of the toxicity associated with supernatants from PHA-activated human PBMC. These results show that FasL and APO2L account for the majority of cytotoxic activity released during AICD, and suggest that additional uncharacterized factors may also contribute to this process.

SUMMARYInteraction of Fas/APO-1 (CD95) and its ligand (FasL) plays an important role in the activation-induced cell death (AICD) of T lymphocytes. In the present work, the contribution of soluble FasL to AICD of the human T-cell line Jurkat has been studied. Jurkat cells prestimulated with phytohaemagglutinin (PHA) induced the death of non-activated Jurkat cells, and also of L1210Fas, but not that of Fasnegative L1210 cells. Culture supernatants from prestimulated Jurkat cells were highly toxic to their non-activated counterparts. Time-course analysis revealed that PHA-stimulated Jurkat cells quickly release (less than 15 min) to the medium a toxic molecule following a biphasic pattern, with maximal cytotoxic activities at 1 hr and 7 hr after stimulation. The cytotoxic effect of those supernatants was prevented by the addition of a blocking anti-Fas monoclonal antibody, suggesting that PHA-stimulated Jurkat cells exert Fas-based cytotoxicity mainly through the release of soluble FasL. The constitutive intracellular expression of FasL in non-activated Jurkat cells and its release as a consequence of PHA activation were detected by immunostaining and immunoblotting using an anti-FasL antibody. These data indicate that, at least in Jurkat cells, AICD is mainly mediated by the rapid release of preformed FasL in soluble form upon stimulation.

Objective. We previously observed that T lymphocytes present in synovial fluid (SF) from patients with rheumatoid arthritis (RA) were sensitive to APO2L/ TRAIL. In addition, there was a drastic decrease in the amount of bioactive APO2L/TRAIL associated with exosomes in SF from RA patients. This study was undertaken to evaluate the effectiveness of bioactive APO2L/TRAIL conjugated with artificial lipid vesicles resembling natural exosomes as a treatment in a rabbit model of antigen-induced arthritis (AIA).Methods. We used a novel Ni 2؉-(N-5-amino-1-carboxypentyl)-iminodiacetic acid)-containing liposomal system. APO2L/TRAIL bound to liposomes was intraarticularly injected into the knees of animals with AIA. One week after treatment, rabbits were killed, and arthritic synovial tissue was analyzed.Results. Tethering APO2L/TRAIL to the liposome membrane increased its bioactivity and resulted in more effective treatment of AIA compared with soluble, unconjugated APO2L/TRAIL, with substantially reduced synovial hyperplasia and inflammation in rabbit knee joints. The results of biophysical studies suggested that the increased bioactivity of APO2L/TRAIL associated with liposomes was due to the increase in the local concentration of the recombinant protein, augmenting its receptor crosslinking potential, and not to conformational changes in the protein. In spite of this increase in bioactivity, the treatment lacked systemic toxicity and was not hepatotoxic.Conclusion. Our findings indicate that binding APO2L/TRAIL to the liposome membrane increases its bioactivity and results in effective treatment of AIA.

Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiating into several mesoderm lineages. They have been isolated from different tissues, such as bone marrow, adult peripheral blood, umbilical cord blood, and adipose tissue. The aim of this study was to analyze the differences in proliferation and phenotype of adipose tissue-derived MSCs from three different species, and to evaluate their capacity to differentiate into chondrocytes in vitro. A comparative study of cultured human, rabbit, and sheep mesenchymal cells from adipose tissue was carried out, and the main morphological parameters, proliferative activity, and expression of surface markers were characterized. Proliferation and flow cytometry data showed species-related differences between animal and human MSCs. Histological staining suggested that rabbit and sheep mesenchymal cells were able to differentiate into chondrocytic lineages. Human mesenchymal cells, though they could also differentiate, accomplished it with more difficulty than animal MSCs. These results could help to explain the differences in the chondrogenic capacity of sheep and rabbit MSCs when they are used as animal models compared to human mesenchymal cells in a clinical assay. ß