Altered differentiation of tumor-associated monocytes and macrophages following genetic ablation of disabled homolog 2 gene

Tumor progression is usually associated with abnormal myelopoiesis and recruitment of several myeloid cell subsets into tissues. These cells heavily infiltrate the primary tumor and sustain its growth by providing local immune suppression and promoting angiogenesis. They also assist metastatic spreading by favoring the tumor invasion of adjacent tissues, and by supporting cancer cell seeding into distant sites. Recent advances have partially highlighted the mechanisms through which myeloid cells are recruited into the tumor mass and suppress the immune response against tumor cells, thus laying the basis for new antitumor immunotherapeutic approaches. However, very little is known about the molecular pathways which regulate myeloid cell differentiation and functions within the tumor microenvironment, especially in the context of the metastatic process. To obtain starting cues about new relevant molecular pathways acting in tumor-infiltrating myeloid cells, we performed gene expression analysis in purified CD11b+ intratumoral myeloid cells isolated from different transplantable murine tumor models. Among the most upregulated genes, we got particularly interested in the disabled homolog 2, mitogen-responsive phosphoprotein (Dab2) gene. The DAB2 protein is a molecular adaptor which participates to endocytosis and signal transduction pathways. Its main function is to link membrane receptors with clathrin assemblies, allowing selective clathrin-mediated endocytosis of transmembrane proteins. The vesicular trafficking has the important function to distribute and organize the protein content of the plasma membrane, allowing the cell to spatially react to external stimuli. We thought that this process is of key-importance within the tumor microenvironment, where complex cell-to-cell interactions occur and oriented stimuli are released. In this work we show that, once within the tumor, both monocytes and macrophages upregulated the DAB2 protein. Both the cytokines GM-CSF and M-CSF, which regulate the development of mononuclear phagocytes, were able to induce DAB2 expression by myeloid cells in vitro, through a mechanism requiring the transcription factor C/EBPβ. Conditional knockout of the Dab2 gene in the hematopoietic system resulted in a strong reduction of tumor-infiltrating monocytes and macrophages. In vivo tracking experiments showed that Dab2-knockout (Dab2-/-) monocytes were less efficient to generate tumor-associated macrophages than wild type ones, and this was accompanied by reduced recovery of Dab2-/- monocytes within the tumor mass. In vitro differentiation of bone marrow-derived macrophages indicated that Dab2-/- monocytes cannot efficiently carry out the autophagy process, suggesting a molecular mechanism that could explain their macrophage differentiation defects. Moreover, Dab2-/- tumor-associated macrrophages had increased expression of genes and membrane markers associated with the M2 macrophage polarization. Finally, we found that spontaneous generation of metastases was impaired in Dab2-/- mice. These data strongly indicate that DAB2 is required for correct differentiation of tumor-associated macrophages, and suggest that this protein may be an optimal molecular target to obstruct myeloid cell-assisted dissemination of metastases in tumor-bearing host

Modulation of Morphology and Glycan Composition of Mucins in Farmed Guinea Fowl (Numida meleagris) Intestine by the Multi-Strain Probiotic Slab51®

Probiotics have become highly recognized as supplements for poultry.Since gut health can be considered synonymous withanimal health, the effects of probiotic Slab51® on the morphology and the glycan composition of guineafowlintestine were examined. The probiotics were added in drinking water (2 x 1011 UFC/L) throughout the grow-out cycle.Birds were individually weighed andslaughtered after four months. Samples from the duodenum, ileum and caecum were collected and processed for morphological, morphometric, conventional and lectin glycohistochemical studies. The results were analyzed for statistical significance by Student’s t test. Compared with control samples, probiotic group revealed (1) significant increase in villus height (p < 0.001 in duodenum and ileum; p < 0.05 in caecum), crypt depth (p < 0.001 in duodenum and caecum; p < 0.05 in ileum) and goblet cells (GCs) per villus (p < 0.001) in all investigated tracts; (2) increase in galactosel,3Nacetylgalacyosamine( Gall,3GalNAc)terminating O-glycans and l,2-fucosylated glycans secretory GCs in the duodenum; (3) increase in 2,6-sialoglycans and high-mannose N-linked glycans secretory GCs but reduction in GCs-secreting sulfoglycans in the ileum; (4) increase in Gall,3GalNAc and high-mannose N-linked glycans secretory GCs and decrease in GCs-producing sulfomucins in the caecum; (5) increase in the numbers of crypt cells containing sulfate and non-sulfated acidic glycans. Overall, dietary Slab51® induces morphological and region-specific changes in glycoprotein composition of guinea fowl intestine, promoting gut health

Positive Influence of a Probiotic Mixture on the Intestinal Morphology and Microbiota of Farmed Guinea Fowls (Numida meleagris)

To understand the effectiveness of a probiotic mixture on intestinal morphology, mucus layer composition, and cecal microbiota diversity, 40 10-day-old Guinea fowls (Numida meleagris) were assigned to two groups: the control group (C), receiving drinking water, and the treated group (P), receiving water plus a commercial multi-strain probiotic (Slab51®, 2 × 1011 CFU/L). Birds were slaughtered after 4 months, and the intestines were collected. Samples from the duodenum, ileum, and cecum were processed for morphological and morphometric studies, and conventional glycohistochemistry. Cecal samples were also used to assess the microbiota by 16S metataxonomic approach. Group P showed significant increase in the villus height (p < 0.001 in the duodenum and p < 0.05 in the ileum and cecum), villus width (p < 0.05 in all investigated tracts), depth of crypts (p < 0.001 in the duodenum and cecum; p < 0.05 in the ileum), and goblet cells per villus (p < 0.001 in all investigated tracts) compared with group C. Cecal microbiota of the birds varied considerably and comparing the relative abundance of the main observational taxonomic units (OTUs), a positive enrichment of several beneficial taxa, such as Oscillospira, Eubacterium, Prevotella, and members of the Ruminococcaceae, was observed. The enrichment of those taxa can improve microbiota stability and resilience facing environmental stresses, enhancing its resistance against invading pathogens. Ruminococcaceae, which represent the most important taxon in both groups, and Prevotella have a key role in the gut physiology due to the production of short-chain fatty acids (SCFAs), which are a vital energy source for enterocytes, improve glucose metabolism, and exert an overall anti-inflammatory effect. Probiotic administration enriches the presence of Coprococcus, Oscillospira, and Eubacterium taxa that produce butyrate, which exerts a beneficial effect on growth performance, structure of villi, and pathogen control and has anti-inflammatory properties too. This study indicates that Slab51® supplementation positively affects the morphology and microbiota diversity of the guinea fowl intestine

T Cell Cancer Therapy Requires CD40-CD40L Activation of Tumor Necrosis Factor and Inducible Nitric-Oxide-Synthase-Producing Dendritic Cells

Effective cancer immunotherapy requires overcoming immunosuppressive tumor microenvironments. We\ua0found that local nitric oxide (NO) production by tumor-infiltrating myeloid cells is important for adoptively transferred CD8(+) cytotoxic T\ua0cells to destroy tumors. These myeloid cells are phenotypically similar to inducible nitric oxide synthase (NOS2)- and tumor necrosis factor (TNF)-producing dendritic cells (DC), or Tip-DCs. Depletion of immunosuppressive, colony stimulating factor 1 receptor (CSF-1R)-dependent arginase 1(+) myeloid cells enhanced NO-dependent tumor killing. Tumor elimination via NOS2 required the CD40-CD40L pathway. We also uncovered a strong correlation between survival of colorectal cancer patients and NOS2, CD40, and TNF expression in their tumors. Our results identify a network of pro-tumor factors that can be targeted to boost cancer immunotherapies

Expanded circulating hematopoietic stem/progenitor cells as novel cell source for the treatment of TCIRG1 osteopetrosis

Allogeneic hematopoietic stem cell transplantation is the treatment of choice for autosomal recessive osteopetrosis caused by defects in the TCIRG1 gene. Despite recent progress in conditioning, a relevant number of patients are not eligible for allogeneic stem cell transplantation because of the severity of the disease and significant transplant-related morbidity. We exploited peripheral CD34+ cells, known to circulate at high frequency in the peripheral blood of TCIRG1-deficient patients, as a novel cell source for autologous transplantation of gene corrected cells. Detailed phenotypical analysis showed that circulating CD34+ cells have a cellular composition that resembles bone marrow, supporting their use in gene therapy protocols. Transcriptomic profile revealed enrichment in genes expressed by hematopoietic stem and progenitor cells (HSPCs). To overcome the limit of bone marrow harvest/ HSPC mobilization and serial blood drawings in TCIRG1 patients, we applied UM171-based ex-vivo expansion of HSPCs coupled with lentiviral gene transfer. Circulating CD34+ cells from TCIRG1-defective patients were transduced with a clinically-optimized lentiviral vector (LV) expressing TCIRG1 under the control of phosphoglycerate promoter and expanded ex vivo. Expanded cells maintained long-term engraftment capacity and multi-lineage repopulating potential when transplanted in vivo both in primary and secondary NSG recipients. Moreover, when CD34+ cells were differentiated in vitro, genetically corrected osteoclasts resorbed the bone efficiently. Overall, we provide evidence that expansion of circulating HSPCs coupled to gene therapy can overcome the limit of stem cell harvest in osteopetrotic patients, thus opening the way to future gene-based treatment of skeletal diseases caused by bone marrow fibrosis

Evacetrapib and Cardiovascular Outcomes in High-Risk Vascular Disease

BACKGROUND: The cholesteryl ester transfer protein inhibitor evacetrapib substantially raises the high-density lipoprotein (HDL) cholesterol level, reduces the low-density lipoprotein (LDL) cholesterol level, and enhances cellular cholesterol efflux capacity. We sought to determine the effect of evacetrapib on major adverse cardiovascular outcomes in patients with high-risk vascular disease. METHODS: In a multicenter, randomized, double-blind, placebo-controlled phase 3 trial, we enrolled 12,092 patients who had at least one of the following conditions: an acute coronary syndrome within the previous 30 to 365 days, cerebrovascular atherosclerotic disease, peripheral vascular arterial disease, or diabetes mellitus with coronary artery disease. Patients were randomly assigned to receive either evacetrapib at a dose of 130 mg or matching placebo, administered daily, in addition to standard medical therapy. The primary efficacy end point was the first occurrence of any component of the composite of death from cardiovascular causes, myocardial infarction, stroke, coronary revascularization, or hospitalization for unstable angina. RESULTS: At 3 months, a 31.1% decrease in the mean LDL cholesterol level was observed with evacetrapib versus a 6.0% increase with placebo, and a 133.2% increase in the mean HDL cholesterol level was seen with evacetrapib versus a 1.6% increase with placebo. After 1363 of the planned 1670 primary end-point events had occurred, the data and safety monitoring board recommended that the trial be terminated early because of a lack of efficacy. After a median of 26 months of evacetrapib or placebo, a primary end-point event occurred in 12.9% of the patients in the evacetrapib group and in 12.8% of those in the placebo group (hazard ratio, 1.01; 95% confidence interval, 0.91 to 1.11; P=0.91). CONCLUSIONS: Although the cholesteryl ester transfer protein inhibitor evacetrapib had favorable effects on established lipid biomarkers, treatment with evacetrapib did not result in a lower rate of cardiovascular events than placebo among patients with high-risk vascular disease. (Funded by Eli Lilly; ACCELERATE ClinicalTrials.gov number, NCT01687998 .)

Effects of a probiotic on the morphology and mucin composition of pig intestine

Although the use of probiotics in human and animal medicine is growing, their mode of action remains poorly understood. This study examined the effects of a multi-strain probiotic (SLAB51™) on the morphology and carbohydrate composition of mucins secreted by goblet cells of intestinal crypts in growingfinishing pigs. Sections of duodenum, caecum and colon from pigs fed for 12 weeks with an orally administered control basal diet (No-Pro) or one with a probiotic blend (Pro) were processed for microscopic analysis and stained with (1) haematoxylin-eosin for structural and morphometrical investigation; (2) conventional histochemistry (periodic acid-Schiff, Alcian Blue pH 2.5, high iron diamine staining) for neutral, acidic nonsulphated, and sulphated mucin analysis; and (3) FITClabelled MAA-II and SNA lectins for α2,3- and α2,6- sialomucin identification. Compared with No-Pro samples, Pro samples displayed (1) increased goblet cell numbers in all investigated tract crypts; (2) an increase in acidic non-sulphomucins but a decrease in neutral, sulphated and α2,6-sialomucin-secreting goblet cells in the duodenum; (3) decreased crypt depth, an increase in α2,6-sialomucin secretory goblet cells, and a loss of goblet cell-secreting α2,3-sialomucins, which appeared on the apical surface of crypt fundus epithelial cells in the caecum; and (4) an increase in α2,6-sialomucinproducing goblet cells in the colon. Results suggest that treatment with SLAB51™ induces region-specific changes in the morphology and carbohydrate composition of mucins secreted along intestinal tracts of growing-finishing pigs. These changes could ameliorate the health status of the animals, which displayed higher growth performance and meat quality than controls (Tufarelli et al., 2017)

Probiotic supplementation affects the glycan composition of mucins secreted by Brunner's glands of the pig duodenum.

The effect of a dietary probiotic blend on the carbohydrate composition of mucins secreted by the Brunner's glands in the duodenum of growing-finishing pigs was investigated by means of conventional (periodic acid-Schiff, Alcian Blue pH 2.5, high iron diamine staining) and lectin (15 lectins) histochemistry. Pigs were assigned to two dietary treatments: a control basal diet without the probiotic blend (No-Pro) and a test diet that included the probiotic blend (Pro). Duodenal tissue fragments were fixed in 4% phosphate-buffered-saline-buffered paraformaldehyde, dehydrated through a graded alcohol series, and embedded in paraffin wax. The secretory cells of the Brunner's glands from No-Pro pigs primarily produced neutral glycoproteins and a small amount of acidic non-sulphated mucins. This glycan pattern was opposite that of the Brunner's glands from Pro animals. A comparison of lectin-binding profiles of the secretory cells of Brunner's glands in these two groups showed that in Pro pigs, there was (i) a decrease in N-linked glycans containing α1,2-linked fucose (Con A, UEA I); (ii) a loss of complex types of N-glycans (PHA-L, PHA-E) terminating with lactosamine (RCA120), α1,6- and α1,3-linked fucose (LTA), and α-galactose (GSA I-B4), as well as of O-glycans with terminal Galβ1,3GalNAc (PNA); and (iii) an increase in O-glycans containing GalNAc HPA. No-Pro and Pro samples showed no change in the expression of α2,6 sialoglycans and terminal GlcNAc residues and no affinity for MAL II, DBA, and SBA. These results indicate that probiotic supplementation affects the glycan composition of mucins produced in the Brunner's glands of growing-finishing pigs. These changes could effectively act on the gastrointestinal function and health status of these animals because the probiotic blend induced higher growth performance and meat quality in the test probiotic group than it did in the control basal diet group (Tufarelli et al., 2017)

PD-L1 is a novel direct target of HIF-1α, and its blockade under hypoxia enhanced MDSC-mediated T cell activation

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