Critical cancer pathways often cannot be targeted because of limited efficiency crossing cell membranes. Here we report the development of a Salmonella-based intracellular delivery system to address this challenge. We engineer genetic circuits that (1) activate the regulator flhDC to drive invasion and (2) induce lysis to release proteins into tumor cells. Released protein drugs diffuse from Salmonella containing vacuoles into the cellular cytoplasm where they interact with their therapeutic targets. Control of invasion with flhDC increases delivery over 500 times. The autonomous triggering of lysis after invasion makes the platform self-limiting and prevents drug release in healthy organs. Bacterial delivery of constitutively active caspase-3 blocks the growth of hepatocellular carcinoma and lung metastases, and increases survival in mice. This success in targeted killing of cancer cells provides critical evidence that this approach will be applicable to a wide range of protein drugs for the treatment of solid tumors.

Interleukin-1β (IL-1β) production is impaired in cord blood monocytes. However, the mechanism underlying this developmental attenuation remains unclear. Here, we analyzed the extent of variability within the Toll-like receptor (TLR)/NLRP3 inflammasome pathways in human neonates. We show that immature low CD14 expressing/CD16 pos monocytes predominate before 33 weeks of gestation, and that these cells lack production of the pro-IL-1β precursor protein upon LPS stimulation. In contrast, high levels of pro-IL-1β are produced within high CD14 expressing monocytes, although these cells are unable to secrete mature IL-1β. The lack of secreted IL-1β in these monocytes parallels a reduction of NLRP3 induction following TLR stimulation resulting in a lack of caspase-1 activity before 29 weeks of gestation, whereas expression of the apoptosisassociated speck-like protein containing a CARD and function of the P2×7 receptor are preserved. Our analyses also reveal a strong inhibitory effect of placental infection on LPS/ATP-induced caspase-1 activity in cord blood monocytes. Lastly, secretion of IL-1β in preterm neonates is restored to adult levels during the neonatal period, indicating rapid maturation of these responses after birth. Collectively, our data highlight important developmental mechanisms regulating IL-1β responses early in gestation, in part due to a downregulation of TLR-mediated NLRP3 expression. Such mechanisms may serve to limit potentially damaging inflammatory responses in a developing fetus.Keywords: Human r Inflammasome r Interleukin-1 beta (IL-1β) r Neonate r Toll-like receptor Additional supporting information may be found in the online version of this article at the publisher's web-site Correspondence: Dr. Pascal M. Lavoie e-mail: plavoie@cw.bc.ca * These authors contributed equally to this work.C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu Eur. J. Immunol. 2015. 45: 238-249 Innate immunity 239 IntroductionNewborns are at high risk of infections, in part due to attenuated innate immune defenses [1]. The cytokine interleukin-1β (IL-1β) is an important inflammatory mediator in response to infections [2]. Mice lacking IL-1β display impaired acute phase and pyrogenic responses [3], and increased susceptibility to pathogens commonly encountered in the neonatal period [4,5]. In contrast, high levels of IL-1β in a fetus can result in autoimmune organ damage [2], as well as lethal metabolic disturbances including severe weight loss and hypoglycemia [6]. Together, these data illustrate the evolutionary importance of a tight regulation of IL-1β in order to avoid inflammation-mediated organ damage, neurological injury [7], or even premature birth in a developing human [2,8].Monocytes are primarily responsible for the production of IL-1β in circulating blood [9,10]. Three subsets predominate in humans: "Classical" monocytes express CD14, but lack expression of the immunoglobulin receptor CD16. These CD14 high /CD16 neg monocytes make up the majority of monocytes in peripheral adult bloo...

Dysfunction of the intestinal mucus barrier causes disorders such as ulcerative colitis and Crohn’s disease. The function of this essential barrier may be affected by the periodically changing luminal environment. We hypothesized that the pH and ion concentration in mucus control its porosity, molecular permeability, and the penetration of microbes. To test this hypothesis, we developed a scalable method to extract porcine small intestinal mucus (PSIM). The aggregation and porosity of PSIM were determined using rheometry, spectrophotometry, and microscopy. Aggregation of PSIM at low pH increased both the elastic (G′) and viscous (G″) moduli, and it slowed the transmigration of pathogenic Salmonella. Molecular transport was dependent on ion concentration. At moderate concentrations, many microscopic aggregates (2–5 μm in diameter) impeded diffusion. At higher concentrations, PSIM formed aggregate islands, increasing both porosity and diffusion. This in vitro model could lead to a better understanding of mucus barrier functions and improve the treatment of intestinal diseases.

The mucosal barrier in combination with innate immune system are the first line of defense against luminal bacteria at the intestinal mucosa. Dysfunction of the mucus layer and bacterial infiltration are linked to tissue inflammation and disease. To study host–bacterial interactions at the mucosal interface, we created an experimental model that contains luminal space, a mucus layer, an epithelial layer, and suspended immune cells. Reconstituted porcine small intestinal mucus formed an 880 ± 230 µm thick gel layer and had a porous structure. In the presence of mucus, sevenfold less probiotic and nonmotile VSL#3 bacteria transmigrated across the epithelial barrier compared to no mucus. The higher bacterial transmigration caused immune cell differentiation and increased the concentration of interleukin‐8 (IL‐8) and tumor necrosis factor‐alpha (TNF‐α; p < .01). Surprisingly, the mucus layer increased transmigration of pathogenic Salmonella and increased secretion of TNF‐α and IL‐8 (p < .05). Nonmotile, flagella knockout Salmonella had lower transmigration and caused lower IL‐8 and TNF‐α secretion (p < .05). These results demonstrate that motility enables pathogenic bacteria to cross the mucus and epithelial layers, which could lead to infection. Using an in vitro coculture platform to understand the interactions of bacteria with the intestinal mucosa has the potential to improve the treatment of intestinal diseases.