The tumor microenvironment of colorectal carcinoma is a complex community of genomically altered cancer cells, nonneoplastic cells, and a diverse collection of microorganisms. Each of these components may contribute to carcinogenesis; however, the role of the microbiota is the least well understood. We have characterized the composition of the microbiota in colorectal carcinoma using whole genome sequences from nine tumor/normal pairs. Fusobacterium sequences were enriched in carcinomas, confirmed by quantitative PCR and 16S rDNA sequence analysis of 95 carcinoma/normal DNA pairs, while the Bacteroidetes and Firmicutes phyla were depleted in tumors. Fusobacteria were also visualized within colorectal tumors using FISH. These findings reveal alterations in the colorectal cancer microbiota; however, the precise role of Fusobacteria in colorectal carcinoma pathogenesis requires further investigation.

Epithelial regeneration is critical for barrier maintenance and organ function after intestinal injury. The intestinal stem cell (ISC) niche provides Wnt, Notch, and epidermal growth factor (EGF) signals supporting Lgr5+ crypt base columnar ISCs for normal epithelial maintenance1,2. However, little is known about the regulation of the ISC compartment after tissue damage. Utilizing ex vivo organoid cultures, we provide evidence that innate lymphoid cells (ILCs), potent producers of Interleukin-22 (IL-22) after intestinal injury3,4, increased the growth of murine small intestine (SI) organoids in an IL-22-dependent fashion. Recombinant IL-22 directly targeted ISCs, augmenting the growth of both murine and human intestinal organoids, increasing proliferation, and promoting ISC expansion. IL-22 induced Stat3 phosphorylation in Lgr5+ ISCs, and Stat3 was critical for both organoid formation and IL-22-mediated regeneration. Treatment with IL-22 in vivo after murine allogeneic bone marrow transplantation (BMT) enhanced recovery of ISCs, increased epithelial regeneration, and reduced intestinal pathology and mortality from graft vs. host disease (GVHD). Atoh1-deficient organoid culture demonstrated that IL-22 induced epithelial regeneration independent of the Paneth cell niche. Our findings reveal a fundamental mechanism by which the immune system is able to support intestinal epithelium, activating ISCs to promote regeneration.

Background Intravascular red cell hemolysis impairs NO-redox homeostasis, producing endothelial dysfunction, platelet activation and vasculopathy. Red blood cell storage under standard conditions results in reduced integrity of the erythrocyte membrane, with formation of exocytic microvesicles or “microparticles” and hemolysis, which we hypothesized could impair vascular function and contribute to the putative “storage lesion” of banked blood. Methods and Results We now find that storage of human red blood cells under standard blood banking conditions results in the accumulation of cell free and microparticle-encapsulated hemoglobin which, despite 39 days of storage, remains in the reduced ferrous oxyhemoglobin redox state and stoichiometrically reacts with and scavenges the vasodilator nitric oxide (NO). Using stopped-flow spectroscopy and laser triggered NO release from a caged NO compound we found that both free hemoglobin and microparticles react with NO about 1000 times faster than with intact erythrocytes. In complementary in vivo studies we show that hemoglobin, even at concentrations below 10 μM (in heme), produces potent vasoconstriction when infused into the rat circulation, while controlled infusions of methemoglobin and cyanomethemoglobin, which do not consume NO, have substantially reduced vasoconstrictor effects. Infusion of the plasma from stored human red cell units into the rat circulation produces significant vasoconstriction related to the magnitude of storage related hemolysis. Conclusions The results of these studies suggest new mechanisms for endothelial injury and impaired vascular function associated with the most fundamental of storage lesions, hemolysis.

Apoptosis resistance is to a large extent a major obstacle leading to chemotherapy failure during cancer treatment. Bypassing the apoptotic pathway to induce cancer cell death is considered to be a promising approach to overcoming this problem. Necroptosis is a regulated necrotic cell death modality in a caspase-independent fashion and is mainly mediated by Receptor-Interacting Protein 1 (RIP1), RIP3, and Mixed Lineage Kinase Domain-Like (MLKL). Necroptosis serves as an alternative mode of programmed cell death overcoming apoptosis resistance and may trigger and amplify antitumor immunity in cancer therapy. The role of necroptosis in cancer is complicated. The expression of key regulators of the necroptotic pathway is generally downregulated in cancer cells, suggesting that cancer cells may also evade necroptosis to survive; however, in certain types of cancer, the expression level of key mediators is elevated. Necroptosis can elicit strong adaptive immune responses that may defend against tumor progression; however, the recruited inflammatory response may also promote tumorigenesis and cancer metastasis, and necroptosis may generate an immunosuppressive tumor microenvironment. Necroptosis also reportedly promotes oncogenesis and cancer metastasis despite evidence demonstrating its antimetastatic role in cancer. In addition, necroptotic microenvironments can direct lineage commitment to determine cancer subtype development in liver cancer. A plethora of compounds and drugs targeting necroptosis exhibit potential antitumor efficacy, but their clinical feasibility must be validated. Better knowledge of the necroptotic pathway mechanism and its physiological and pathological functions is urgently required to solve the remaining mysteries surrounding the role of necroptosis in cancer. In this review, we briefly introduce the molecular mechanism and characteristics of necroptosis, the interplay between necroptosis and other cell death mechanisms, crosstalk of necroptosis and metabolic signaling and detection methods. We also summarize the intricate role of necroptosis in tumor progression, cancer metastasis, prognosis of cancer patients, cancer immunity regulation, cancer subtype determination and cancer therapeutics.

The pineal hormone melatonin elicits two effects on the suprachiasmatic nuclei (SCN): acute neuronal inhibition and phase-shifting. Melatonin evokes its biological effects through G protein-coupled receptors. Since the Mel1a melatonin receptor may transduce the major neurobiological actions of melatonin in mammals, we examined whether it mediates both melatonin effects on SCN function by using mice with targeted disruption of the Mel1a receptor. The Mel1a receptor accounts for all detectable, high affinity melatonin binding in mouse brain. Functionally, this receptor is necessary for the acute inhibitory action of melatonin on the SCN. Melatonin-induced phase shifts, however, are only modestly altered in the receptor-deficient mice; pertussis toxin still blocks melatonin-induced phase shifts in Mel1a receptor-deficient mice. The other melatonin receptor subtype, the Mel1b receptor, is expressed in mouse SCN, implicating it in the phase-shifting response. The results provide a molecular basis for two distinct, mechanistically separable effects of melatonin on SCN physiology.

The CD4 ؉ CD25 ؉ regulatory T lymphocytes have been implicated in suppressing T cell immune responses. Our aim was to characterize the frequency, phenotype, function, and specificity of CD4 ؉ CD25 ؉ T cells in hepatitis C virus (HCV) infection. Peripheral CD4 ؉ CD25 ؉ cells from recovered (n ‫؍‬ 15), chronic infected (n ‫؍‬ 30), and normal control (n ‫؍‬ 15) subjects were analyzed ex vivo for quantitation, phenotype, and effect on HCVspecific interferon gamma production and proliferation. CD4 ؉ CD25 ؉ specificity was determined by intracellular cytokine staining for interleukin 10 (IL-10). A higher proportion of CD4 ؉ CD25 ؉ were found in chronic infection (mean, 3.02%) when compared with recovered (1.64%, P ‫؍‬ .001) and normal controls (2.27%, P ‫؍‬ .02). CD4 ؉ CD25 ؉ cells display CD45RO high , CD45RA low , CD28 high , CD62L high , and CD95 high phenotype. HCVspecific interferon gamma activity was enhanced in peripheral blood mononuclear cells depleted of CD4 ؉ CD25 ؉ and suppressed in peripheral blood mononuclear cells enriched with CD4 ؉ CD25 ؉ . Depletion of CD4 ؉ CD25 ؉ cells also enhanced HCV-specific CD4 ؉ and CD8 ؉ T cell proliferation. Cytokine analysis suggested CD4 ؉ CD25 ؉ cells secrete transforming growth factor beta (TGF-␤ 1 ) and IL-10. The inhibitory role for TGF-␤ 1 was confirmed by anti-TGF-␤ 1 . Transwell studies showed CD4 ؉ CD25 ؉ mediated suppression to be dose dependent and requiring cell contact. CD4 ؉ CD25 ؉ cells showed HCV-specificity through IL-10 production, with a frequency ranging from 1.9% to 5.3%. A positive correlation was detected between CD4 ؉ CD25 ؉ T cell frequency and HCV RNA titer, whereas an inverse relation was found with liver inflammatory activity. In conclusion, CD4 ؉ CD25 ؉ T lymphocytes constitute a highly differentiated population and appear to play a role in viral persistence by suppressing HCV-specific T cell responses in a cell-cell contact manner.

Summary Little is known about the maintenance of intestinal stem cells (ISCs) and progenitors during immune-mediated tissue damage or about the susceptibility of transplant recipients to tissue damage mediated by the donor immune system during graft vs. host disease (GVHD). We demonstrate here that deficiency of recipient-derived IL-22 increased acute GVHD tissue damage and mortality, that ISCs were eliminated during GVHD, and that ISCs as well as their downstream progenitors expressed the IL-22 receptor. Intestinal IL-22 was produced after bone marrow transplant by IL-23-responsive innate lymphoid cells (ILCs) from the transplant recipients, and intestinal IL-22 increased in response to pre-transplant conditioning. However, ILC frequency and IL-22 amounts were decreased by GVHD. Recipient IL-22 deficiency led to increased crypt apoptosis, depletion of ISCs, and loss of epithelial integrity. Our findings reveal IL-22 as a critical regulator of tissue sensitivity to GVHD and a protective factor for ISC during inflammatory intestinal damage.

GVHD is associated with significant shifts in the composition of the intestinal microbiota in human and mouse models; manipulating the microbiota can alter the severity of GVHD in mice.