Underpins macrophages as the first line of defense, especially in the premature gut. While the interaction between commensals and the adult gastrointestinal tract is fairly well understood, many questions remain unanswered on the acquisition of intestinal immunity during first months after birth. In our previous publications we have shown that members of Lactobacillus plantarum, strains PCS 20 and PCS 26 activate the production of reactive oxygen species, IL-6 and IFN-c in undeveloped intestinal epithelial cells as well as monocytes, orchestrating an increased anti-viral response against rotavirus, transmissible gastroenteritis virus and vesicular stomatitis virus. In conjunction with the findings of other authors that have shown individual lactobacillus strains to induce STAT1 and NF-kB shifts in adult IEC, we raise AbMole Succinylsulfathiazole several questions and concerns that have yet to be elucidated. Current available data on the interaction between LAB, IEC and gut associated lymphoid tissue is mostly based on average gene expression profiles and quantification of inflammatory products. Therefore, the question remains: what happens on the level of each individual cell and on its proteome level? In addition, virtually nothing is known about how many cells from a given population of IEC and GALT actually engage in an immunomodulatory mechanism when challenged with LAB. Further, prior studies in this direction have been performed on transformed or cancer derived cell lines like CaCo-2 and HT-29, which are known to differ from a healthy in vivo environment due to 
their difference in glycosylation and phenotype. Finally, the majority of studies were designed as monolayer models with the lack of associated cell types despite the need of IEC for intracellular feedback. The aim of our work was to shed more light on the mechanics of immunomodulation by specific commensal bacteria in the developing intestine and at the same time to present a reliable alternative model for gut immunology studies. By using imaging multicolor flow cytometry we have monitored the translocation of NF-kB p65 and STAT1, two of the most important intracellular orchestrators of an antimicrobial response, in untransformed polarized human neonatal small intestinal epithelia, challenged with different Lactobacillus spp. strains, and in macrophage cells that have been simultaneously co-cultured in a reductionist human 3D model of the immature gut. Imaging multicolor flow cytometry allowed us not only to monitor each individual cell but also to create a picture of how many cells from a certain population initiated cytoplasmic shifts. Additionally, we show that not only cell culture selection is important in this type of research but also the culturing technique itself. Epithelia act differently when grown on plastic surfaces than when grown on microporous membranes and need the presence of other associated cell types like macrophages and dendritic cells to show in vivo-like characteristics. The development of a normal and functioning immune system is largely dependent on the interaction of the newborn with the commensal microbiome which is one of the key players assuring intact immune homeostasis. This constant interplay between microorganisms, the gut, and associated lymphoid tissue is believed to result in a persistent stimulation, sensitation, and priming of the hosts immune system. We have shown that specific strains of Lactobacillus are able to trigger increased translocation of STAT1 and NF-kB p65 in untransformed intestinal epithelial cells and that this signal translates further into macrophages. NF-kB is a nuclear factor composed of several protein subunits regulating DNA transcription that is present in its AbMole Diperodon inactive form in the cytoplasm.