Natural killer (NK) cells are classically viewed as lymphocytes that provide innate surveillance against virally-infected cells and tumor cells through release of cytolytic mediators and IFN-γ. In humans, blood CD56dim NK cells specialize in lysis of cell targets1. In lymph nodes, CD56bright NK cells secrete IFN-γ cooperating with dendritic cells (DC) and T cells in the generation of adaptive responses1, 2. Here we report the characterization of a human NK cell subset located in mucosa-associated lymphoid tissues (MALT), such as tonsils and Payer’s patches, which is hard-wired to secrete interleukin (IL)-22, IL-26, and leukaemia inhibitory factor (LIF). These NK cells, which we refer to as NK-22 cells, are triggered by acute exposure to IL-23. In vitro, NK-22-secreted cytokines stimulate epithelial cells to secrete IL-10, proliferate and express a variety of mitogenic and anti-apoptotic molecules. NK-22 cells are also found in mouse MALT and appear in the small intestine lamina propria during bacterial infection suggesting that NK-22 cells provide an innate source of IL-22 that may help constrain inflammation and protect mucosal sites.

We have identified cellular and molecular features of the stem cell niche required for marked amplification of mouse colonic epithelial progenitors (ColEPs) that occurs in response to wounding of the epithelium with dextran sodium sulfate. This regenerative response in areas adjacent to breaches in the epithelial barrier depends on the gut microbiota because ColEP proliferation is markedly diminished in germ-free animals. Analysis of conventionally raised C57BL͞6 (B6) knockout mice lacking the Toll-like receptor signal transduction pathway component Myd88 and wild-type animals transplanted with Myd88 ؊/؊ bone marrow, revealed that Myd88-mediated signaling through mesenchymal cells is also required for the ColEP response. Studies of B6 Csf1 op/op (lacking macrophages) mice, Rag1 ؊/؊ mice, and wild-type mice treated with neutrophil-specific Gr1 mAbs, disclosed that macrophages but not lymphocytes or neutrophils are necessary. GeneChip analysis of laser-capture-microdissected mesenchymal cells coupled with immunohistochemical and electron microscopic studies showed that, during the regenerative response, macrophages in the pericryptal stem cell niche express genes associated with their activation and extend processes to directly contact ColEPs near the crypt base. GeneChip analysis also identified a number of potential molecular mediators of regeneration expressed in the pericryptal progenitor niche, including secreted factors that stimulate epithelial proliferation and proteins involved in extracellular matrix and basement membrane function, stability, and growth factor binding. Together, these studies indicate that the colonic epithelial progenitor niche is a dynamic structure in which macrophages function as mobile ''cellular transceivers'' that coordinate inputs from luminal microbes and injured epithelium and transmit regenerative signals to neighboring ColEPs.laser capture microdissection ͉ epithelial-mesenchymal cross-talk ͉ macrophage activation ͉ stem cell ͉ stem cell niche

Continuous regeneration of digestive enzyme (zymogen)-secreting chief cells is a normal aspect of stomach function that is disrupted in precancerous lesions (e.g. metaplasias, chronic atrophy). The cellular and genetic pathways that underlie zymogenic cell (ZC) differentiation are poorly understood. Here,we describe a gene expression analysis of laser capture microdissection purified gastric cell populations that identified the bHLH transcription factor Mist1 as a potential ZC regulatory factor. Our molecular and ultrastructural analysis of proliferation, migration and differentiation of the gastric unit in Mist1-/- and control mice supports a model whereby wild-type ZC progenitors arise as neck cells in the proliferative (isthmal) zone of the gastric unit and become transitional cells(TCs) with molecular and ultrastructural characteristics of both enzyme-secreting ZCs and mucus-secreting neck cells as they migrate to the neck-base zone interface. Thereafter, they rapidly differentiate into mature ZCs as they enter the base. By contrast, Mist1-/- neck cells differentiate normally, but ZCs in the mature, basal portion of the gastric unit uniformly exhibit multiple apical cytoplasmic structural abnormalities. This defect in terminal ZC differentiation is also associated with markedly increased abundance of TCs, especially in late-stage TCs that predominantly have features of immature ZCs. Thus, we present an in vivo system for analysis of ZC differentiation, present molecular evidence that ZCs differentiate from neck cell progenitors and identify Mist1 as the first gene with a role in this clinically important process.

We identified cellular and molecular mechanisms within the stem cell niche that control the activity of colonic epithelial progenitors (ColEPs) during injury. Here, we show that while WT mice maintained ColEP proliferation in the rectum following injury with dextran sodium sulfate, similarly treated Myd88 -/-(TLR signaling-deficient) and prostaglandin-endoperoxide synthase 2 -/-(Ptgs2 -/-) mice exhibited a profound inhibition of epithelial proliferation and cellular organization within rectal crypts. Exogenous addition of 16,16-dimethyl PGE 2 (dmPGE 2 ) rescued the effects of this injury in both knockout mouse strains, indicating that Myd88 signaling is upstream of Ptgs2 and PGE 2 . In WT and Myd88 -/-mice, Ptgs2 was expressed in scattered mesenchymal cells. Surprisingly, Ptgs2 expression was not regulated by injury. Rather, in WT mice, the combination of injury and Myd88 signaling led to the repositioning of a subset of the Ptgs2-expressing stromal cells from the mesenchyme surrounding the middle and upper crypts to an area surrounding the crypt base adjacent to ColEPs. These findings demonstrate that Myd88 and prostaglandin signaling pathways interact to preserve epithelial proliferation during injury using what we believe to be a previously undescribed mechanism requiring proper cellular mobilization within the crypt niche.

Garbow JR, Doherty JM, Schugar RC, Travers S, Weber ML, Wentz AE, Ezenwajiaku N, Cotter DG, Brunt EM, Crawford PA. Hepatic steatosis, inflammation, and ER stress in mice maintained long term on a very low-carbohydrate ketogenic diet. Am J Physiol Gastrointest Liver Physiol 300: G956 -G967, 2011. First published March 31, 2011 doi:10.1152/ajpgi.00539.2010.-Low-carbohydrate diets are used to manage obesity, seizure disorders, and malignancies of the central nervous system. These diets create a distinctive, but incompletely defined, cellular, molecular, and integrated metabolic state. Here, we determine the systemic and hepatic effects of longterm administration of a very low-carbohydrate, low-protein, and high-fat ketogenic diet, serially comparing these effects to a highsimple-carbohydrate, high-fat Western diet and a low-fat, polysaccharide-rich control chow diet in C57BL/6J mice. Longitudinal measurement of body composition, serum metabolites, and intrahepatic fat content, using in vivo magnetic resonance spectroscopy, reveals that mice fed the ketogenic diet over 12 wk remain lean, euglycemic, and hypoinsulinemic but accumulate hepatic lipid in a temporal pattern very distinct from animals fed the Western diet. Ketogenic diet-fed mice ultimately develop systemic glucose intolerance, hepatic endoplasmic reticulum stress, steatosis, cellular injury, and macrophage accumulation, but surprisingly insulin-induced hepatic Akt phosphorylation and whole-body insulin responsiveness are not impaired. Moreover, whereas hepatic Pparg mRNA abundance is augmented by both high-fat diets, each diet confers splice variant specificity. The distinctive nutrient milieu created by long-term administration of this low-carbohydrate, low-protein ketogenic diet in mice evokes unique signatures of nonalcoholic fatty liver disease and whole-body glucose homeostasis. nutrient state; magnetic resonance spectroscopy; peroxisome proliferator activated receptor-␥; insulin resistance; unfolded protein response; x-box-binding protein-1 splicing; high-fat diets; fatty liver disease THERAPEUTIC USE OF REDUCED-CARBOHYDRATE diets has been extensively studied for the amelioration of a multitude of clinical states, including obesity and its metabolic complications, seizure disorders, and malignancies of the central nervous system (21,23,26,36,41,54,55,59,64). Nonetheless, the full scope of metabolic effects incurred by low-, and very low (ketogenic)-carbohydrate diets (KD) has only been preliminarily characterized. In wild-type mice, KDs result in weight loss and increased hepatic and myocardial fatty acid oxidation, compared with mice maintained on standard chow diets rich in polysaccharides (5,35,62). Whereas leptin-deficient obese (ob/ob) animals maintained on KD for 7 wk exhibit persistent weight gain, glucose tolerance and insulin resistance improve compared with ob/ob mice maintained on standard chow diet (3, 57). Collectively, encouraging preclinical and clinical studies of low-carbohydrate KDs have favored their continued study for anticonv...

*Molecular mechanisms that regulate the generation of hematopoietic and endothelial cells from mesoderm are poorly understood. To define the underlying mechanisms, we compared gene expression profiles between embryonic stem (ES) cell-derived hemangioblasts (Blast-Colony-Forming Cells, BL-CFCs) and their differentiated progeny, Blast cells. Bioinformatic analysis indicated that BL-CFCs resembled other stem cell populations. A role for Gata2, one of the BL-CFC-enriched transcripts, was further characterized by utilizing the in vitro model of ES cell differentiation. Our studies revealed that Gata2 was a direct target of BMP4 and that enforced GATA2 expression upregulated Bmp4, Flk1 and Scl. Conditional GATA2 induction resulted in a temporal-sensitive increase in hemangioblast generation, precocious commitment to erythroid fate, and increased endothelial cell generation. GATA2 additionally conferred a proliferative signal to primitive erythroid progenitors. Collectively, we provide compelling evidence that GATA2 plays specific, contextual roles in the generation of Flk-1 + mesoderm, the Flk-1 + Scl + hemangioblast, primitive erythroid and endothelial cells.

Alterations in the tissue microenvironment collaborate with cell autonomous genetic changes to contribute to neoplastic progression. The importance of the microenvironment in neoplastic progression is underscored by studies showing that fibroblasts isolated from a tumor stimulate the growth of preneoplastic and neoplastic cells in xenograft models. Similarly, senescent fibroblasts promote preneoplastic cell growth in vitro and in vivo. Because senescent cells accumulate with age, their presence is hypothesized to facilitate preneoplastic cell growth and tumor formation in older individuals. To identify senescent stromal factors directly responsible for stimulating preneoplastic cell growth, we carried out whole-genome transcriptional profiling and compared senescent fibroblasts with their younger counterparts. We identified osteopontin (OPN) as one of the most highly elevated transcripts in senescent fibroblasts. Importantly, reduction of OPN protein levels by RNA interference did not affect senescence induction in fibroblasts; however, it dramatically reduced the growth-promoting activities of senescent fibroblasts in vitro and in vivo, showing that OPN is necessary for paracrine stimulation of preneoplastic cell growth. In addition, we found that recombinant OPN was sufficient to stimulate preneoplastic cell growth. Finally, we show that OPN is expressed in senescent stroma within preneoplastic lesions that arise following 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate treatment of mice, suggesting that stromal-derived OPN-mediated signaling events affect neoplastic progression. [Cancer Res 2009;69(3):1230-9]

The lack of reliable molecular markers for normal differentiated epithelial cells limits understanding of human gastric carcinogenesis. Recognized precursor lesions for gastric adenocarcinoma are intestinal metaplasia and spasmolytic polypeptide expressing metaplasia (SPEM), defined here by ectopic CDX2 and TFF2 expression, respectively. In mice, expression of the bHLH transcription factor MIST1, normally restricted to mature chief cells, is down-regulated as chief cells undergo experimentally induced metaplasia. Here, we show MIST1 expression is also a specific marker of human chief cells. The mainstays of therapy in gastric carcinoma are early recognition, resection, and neoadjuvant or adjuvant therapy. However, gastric cancer remains the second largest cause of cancer-related mortality worldwide, 1 which drastically illustrates our lack of understanding of the sequence and progression of preneoplastic conditions. The traditional linear progression model of cellular changes, such as (Helicobacter-mediated) inflammation, atrophy, intestinal metaplasia (IM), dysplasia, and carcinoma, 2-4 does not apply to all cases and does not allow incorporation of more recently recognized entities. For example, there are distinct types of IMs, not all carrying definitive preneoplastic potential, and some authors have argued that IM in general is a paraneoplastic condition because the earliest gastric carcinomas arise from gasSupported by