Invasion and destruction of a murine fibrosarcoma by Salmonella-induced effector CD8 T cells as a therapeutic intervention against cancer.

We have developed a new vaccination strategy by using the Salmonella type III secretion system (T3SS) to translocate heterologous antigens into the cytosol of host cells. This leads to an efficient antigen-specific CD8 T cell induction. Recently, we have demonstrated the use of Salmonella's T3SS for the immunoprophylaxis of a solid tumor. The murine fibrosarcoma WEHI 164 was transfected with the DNA sequence encoding the MHC class I-peptide p60(217-225) from Listeria monocytogenes. In the present study, we used this tumor model to investigate the potential of vaccination with recombinant Salmonella in a therapeutic setting. BALB/c mice were subcutaneously challenged with WEHI-p60 cells. Simultaneously or 4 days later, these mice received either an orogastric or intravenous immunization with Salmonella translocating p60. Interestingly, 71-80% of the intravenously and 50-52% of the orogastrically immunized mice showed a complete tumor regression after 14 days. In addition, the distribution of tetramer-positive p60(217-225)-specific CD8 T cell subpopulations in blood and tumor tissue was analyzed. Co-staining with CD62L and CD127 revealed that the frequencies of p60(217-225)-specific effector and effector memory CD8 T cells in blood and in fibrosarcoma tissue were related to the kinetics of tumor regression. In summary, our study demonstrates that therapeutic vaccination with Salmonella leads to efficient induction of tumor-invading effector CD8 T cells that may result in significant tumor regression

Zinc sequestration by the neutrophil protein calprotectin enhances Salmonella growth in the inflamed gut

Neutrophils are innate immune cells that counter pathogens by many mechanisms, including release of antimicrobial proteins such as calprotectin to inhibit bacterial growth. Calprotectin sequesters essential micronutrient metals such as zinc, thereby limiting their availability to microbes, a process termed nutritional immunity. We find that while calprotectin is induced by neutrophils during infection with the gut pathogen Salmonella Typhimurium, calprotectin-mediated metal sequestration does not inhibit S. Typhimurium proliferation. Remarkably, S. Typhimurium overcomes calprotectin-mediated zinc chelation by expressing a high affinity zinc transporter (ZnuABC). A S. Typhimurium znuA mutant impaired for growth in the inflamed gut was rescued in the absence of calprotectin. ZnuABC was also required to promote the growth of S. Typhimurium over that of competing commensal bacteria. Thus, our findings indicate that Salmonella thrives in the inflamed gut by overcoming the zinc sequestration of calprotectin and highlight the importance of zinc acquisition in bacterial intestinal colonization

Monomeric myosin V uses two binding regions for the assembly of stable translocation complexes

Myosin-motors are conserved from yeast to human and transport a great variety of cargoes. Most plus-end directed myosins, which constitute the vast majority of all myosin motors, form stable dimers and interact constitutively with their cargo complexes. To date, little is known about regulatory mechanisms for cargo-complex assembly. In this study, we show that the type V myosin Myo4p binds to its cargo via two distinct binding regions, the C-terminal tail and a coiled-coil domain-containing fragment. Furthermore, we find that Myo4p is strictly monomeric at physiologic concentrations. Because type V myosins are thought to require dimerization for processive movement, a mechanism must be in place to ensure that oligomeric Myo4p is incorporated into cargo-translocation complexes. Indeed, we find that artificial dimerization of the Myo4p C-terminal tail promotes stabilization of myosin-cargo complexes, suggesting that full-length Myo4p dimerizes in the cocomplex as well. We also combined the Myo4p C-terminal tail with the coiled-coil region, lever arm, and motor domain from a different myosin to form constitutively dimeric motor proteins. This heterologous motor successfully translocates its cargo in vivo, suggesting that wild-type Myo4p may also function as a dimer during cargo-complex transport

Uremic Plasma Impairs Barrier Function and Depletes the Tight Junction Protein Constituents of Intestinal Epithelium

BACK GROUND: CKD causes intestinal barrier dysfunction which by allowing influx of endotoxin and other noxious products contributes to the CKD-associated systemic inflammation and uremic toxicity. We have recently shown that intestinal barrier dysfunction in CKD animals is due to degradation of trans-cellular [claudin-1 and occludin] and intra-cellular [ZO1] constituents of epithelial tight junction (TJ). This study determined whether CKD-associated disruption of TJ is mediated by retained uremic toxins/metabolites and if so whether they are removed by hemodialysis. METHODS: The TJ-forming human enterocytes (T84 cells) were seeded on the Transwell plates and utilized when trans-epithelial electrical resistance (TER) exceeded 1000 mΩ/cm2 to ensure full polarization and TJ formation. The cells were then incubated for 24 hr in media containing 10% pre- or post-hemodialysis plasma from ESRD patients or healthy individuals. TER was then measured and cells were processed for Western blot and immunohistological analyses. RESULTS: Compared with the control plasma, incubation in media containing pre-dialysis plasma from ESRD patients resulted in a marked drop in TER pointing to increased epithelial permeability. This was accompanied by significant reductions in cluadin-1 (85%), occludin (15%), and ZO1 (70%) abundance. The severity of TJ damage and dysfunction was significantly less in cells exposed to the post-dialysis than per-dialysis plasma. These findings point to the presence of as-yet unidentified product(s) in the uremic plasma capable of depleting epithelial TJ. CONCLUSIONS: Exposure to uremic milieu damages the intestinal epithelial TJ and impairs its barrier function, events which are mediated by agents which are partially removed by hemodialysis