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Cardiac tissue macrophages (cTMs) are a previously uncharacterised cell type that we have identified and characterise here as an abundant GFP(+) population within the adult Cx(3)cr1(GFP/+) knock-in mouse heart. They comprise the predominant myeloid cell population in the myocardium, and are found throughout myocardial interstitial spaces interacting directly with capillary endothelial cells and cardiomyocytes. Flow cytometry-based immunophenotyping shows that cTMs exhibit canonical macrophage markers. Gene expression analysis shows that cTMs (CD45(+)CD11b(+)GFP(+)) are distinct from mononuclear CD45(+)CD11b(+)GFP(+) cells sorted from the spleen and brain of adult Cx(3)cr1(GFP/+) mice. Gene expression profiling reveals that cTMs closely resemble alternatively-activated anti-inflammatory M2 macrophages, expressing a number of M2 markers, including Mrc1, CD163, and Lyve-1. While cTMs perform normal tissue macrophage homeostatic functions, they also exhibit a distinct phenotype, involving secretion of salutary factors (including IGF-1) and immune modulation. In summary, the characterisation of cTMs at the cellular and molecular level defines a potentially important role for these cells in cardiac homeostasis.

Monocytes and macrophages are major cellular components of the innate immunity that play essential roles in tissue homeostasis. The contribution of different subsets of monocytes/macrophages to periodontal health and disease has not been fully elucidated. Type 2 diabetes mellitus (T2DM) is a risk factor for periodontitis. We hypothesized that the monocyte/macrophage signaling is perturbed in periodontitis-affected sites versus periodontally healthy sites and that this perturbation plays a critical role in the pathogenesis of periodontitis. Pairs of gingival tissue samples (each from a periodontally healthy and a periodontitis-affected site of the same patient) were harvested from 27 periodontitis patients, with and without T2DM. Each sample was processed to form a single-cell suspension, and a flow-cytometry panel was designed and validated to study monocyte and macrophage phenotypes. In separate experiments, the transcriptional changes associated with a pro-inflammatory phenotype were also examined in monocyte/macrophage subsets obtained from peripheral blood of patients with T2DM versus diabetes-free controls. A significantly higher proportion of intermediate (CD14 CD16 ) monocytes was observed in periodontitis-affected tissues compared to healthy tissues. These monocytes overexpressed HLA-DR and PDL1 molecules, suggesting their activated inflammatory status. PDL1 increase was specific to intermediate monocytes. The ratio of M1/M2 macrophages was also significantly higher in periodontally affected sites, signifying an imbalance between inflammatory and repair mechanisms. We found a significantly higher expression of PDL1 in overall monocytes and M1 macrophages in periodontitis-affected sites compared to controls. Importantly, we identified a subpopulation of M1 macrophages present in periodontally affected tissues which expressed high levels of CD47, a glycoprotein of the immunoglobulin family that plays a critical role in self-recognition and impairment of phagocytosis. Analysis of the transcriptional landscape of monocytes/macrophages in gingival tissue of T2DM patients with periodontitis revealed a significant disruption in homeostasis toward a proinflammatory phenotype, elevation of pro-inflammatory transcription factors STAT1 and IRF1, and repression of anti-inflammatory JMJD3 in circulating monocytes. Taken together, our results demonstrate disruption of myeloid-derived cell homeostasis in periodontitis, with or without T2DM, and highlight a potentially significant role of these cell types in its pathogenesis. The impact of macrophage and monocyte signaling pathways on the pathobiology of periodontitis should be further evaluated.

V-domain Ig suppressor of T cell activation (VISTA), a novel immune checkpoint regulatory molecule, suppresses T cell mediated immune responses. The aim of the present study was to profile the immunological expression, clinical significance and correlation of VISTA in human oral squamous cell carcinoma (OSCC). Human tissue microarrays, containing 165 primary OSCCs, 48 oral epithelial dysplasias and 43 normal oral mucosae, were applied to investigate the expression levels of VISTA, CD8, cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), programmed death ligand 1 (PD-L1), PI3Kα p110, IL13Rα2, phospho-STAT3 at tyrosine 705 (p-STAT3) and myeloid-derived suppressor cell (MDSC) markers (CD11b and CD33) by immunohistochemistry and digital pathology analysis. The results demonstrated that the protein level of VISTA was significantly higher in human OSCC specimens, and that VISTA expression in primary OSCCs was correlated with lymph node status. VISTA expression did not serve as an independent predictor for poor prognosis, while patient subgroup with VISTA high and CD8 low expression (22/165) had significantly poorer overall survival compared with other subgroups based on the multivariate and Cox hazard analyses among the primary OSCC patients in the present cohort. Additionally, the expression of VISTA was significantly correlated with PD-L1, CTLA-4, IL13Rα2, PI3K, p-STAT3, CD11b and CD33 according to Pearson’s correlation coefficient test. Taken together, the results indicated that the VISTA high and CD8 low group, as an immunosuppressive subgroup, might be associated with a poor prognosis in primary OSCC. These findings indicated that VISTA might be a potential immunotherapeutic target in OSCC treatment.

Several techniques have been devised for the dissociation of tissues for primary culture. These techniques can affect the quantity and quality of the isolated cells. The aim of our study was to develop the most appropriate method for the isolation of human umbilical cord-derived mesenchymal (hUCM) cells. In the present study, we compared four methods for the isolation of hUCM cells: three enzymatic methods; collagenase/hyaluronidase/trypsin (CHT), collagenase/trypsin (CT) and trypsin (Trp), and an explant culture (Exp) method. The trypan blue dye exclusion test, the water-soluble tetrazolium salt-1 (WST-1) assay, flow cytometry, alkaline phosphatase activity and histochemical staining were used to evaluate the results of the different methods. The hUCM cells were successfully isolated by all methods but the isolation method used profoundly altered the cell number and proliferation capacity of the isolated cells. The cells were successfully differentiated into adipogenic and osteogenic lineages and alkaline phosphatase activity was detected in the hUCM cell colonies of all groups. Flow cytometry analysis revealed that CD44, CD73, CD90 and CD105 were expressed in all groups, while CD34 and CD45 were not expressed. The expression of C-kit in the enzymatic groups was higher than in the explant group, while the expression of Oct-4 was higher in the CT group compared to the other groups. We concluded that the collagenase/trypsin method of cell isolation yields a higher cell density than the others. These cells expressed a higher rate of pluripotent cell markers such as C-kit and Oct-4, while the explant method of cell isolation resulted in a higher cell proliferation rate and activity compared to the other methods.

We find that CD11c+cells with many markers of dendritic cells (DCs) are a major cell type in the skin lesions of psoriasis. These CD11c+cells, which are evident in both epidermis and dermis, are the sites for the expression of two mediators of inflammation, inducible nitric oxide synthase (iNOS) and$TNF-\\alpha$in diseased skin. These cells express HLA-DR, CD40, and CD86, lack the Langerin and CD14 markers of Langerhans cells and monocytes, respectively, and to a significant extent express the DC maturation markers DC-LAMP and CD83. Treatment of psoriasis with efalizumab (anti-CD11a, Raptiva) strongly reduces infiltration by these DCs in patients responding to this agent. Disease activity after therapy was more related to DC infiltrates and iNOS mRNA levels than T cell infiltrates, and CD11c+cells responded more quickly to therapy than epidermal keratinocytes. Our results suggest that a type of DC, which resembles murine \"Tip-DCs\" that can accumulate during infection, has proinflammatory effects in psoriasis through nitric oxide and$TNF-\\alpha$production, and can be an important target for suppressive therapies.

In an interim analysis of this phase 3 trial, the addition of pembrolizumab to chemotherapy resulted in longer progression-free survival than chemotherapy alone among patients with advanced triple-negative breast cancer whose tumors expressed programmed death ligand 1 (PD-L1) with a combined positive score (CPS; the number of PD-L1-staining tumor cells, lymphocytes, and macrophages, divided by the total number of viable tumor cells, multiplied by 100) of 10 or more. The results of the final analysis of overall survival have not been reported. We randomly assigned patients with previously untreated locally recurrent inoperable or metastatic triple-negative breast cancer in a 2:1 ratio to receive pembrolizumab (200 mg) every 3 weeks plus the investigator's choice of chemotherapy (nanoparticle albumin-bound paclitaxel, paclitaxel, or gemcitabine-carboplatin) or placebo plus chemotherapy. The primary end points were progression-free survival (reported previously) and overall survival among patients whose tumors expressed PD-L1 with a CPS of 10 or more (the CPS-10 subgroup), among patients whose tumors expressed PD-L1 with a CPS of 1 or more (the CPS-1 subgroup), and in the intention-to-treat population. Safety was also assessed. A total of 847 patients underwent randomization: 566 were assigned to the pembrolizumab-chemotherapy group, and 281 to the placebo-chemotherapy group. The median follow-up was 44.1 months. In the CPS-10 subgroup, the median overall survival was 23.0 months in the pembrolizumab-chemotherapy group and 16.1 months in the placebo-chemotherapy group (hazard ratio for death, 0.73; 95% confidence interval [CI], 0.55 to 0.95; two-sided P = 0.0185 [criterion for significance met]); in the CPS-1 subgroup, the median overall survival was 17.6 and 16.0 months in the two groups, respectively (hazard ratio, 0.86; 95% CI, 0.72 to 1.04; two-sided P = 0.1125 [not significant]); and in the intention-to-treat population, the median overall survival was 17.2 and 15.5 months, respectively (hazard ratio, 0.89; 95% CI, 0.76 to 1.05 [significance not tested]). Adverse events of grade 3, 4, or 5 that were related to the trial regimen occurred in 68.1% of the patients in the pembrolizumab-chemotherapy group and in 66.9% in the placebo-chemotherapy group, including death in 0.4% of the patients in the pembrolizumab-chemotherapy group and in no patients in the placebo-chemotherapy group. Among patients with advanced triple-negative breast cancer whose tumors expressed PD-L1 with a CPS of 10 or more, the addition of pembrolizumab to chemotherapy resulted in significantly longer overall survival than chemotherapy alone. (Funded by Merck Sharp and Dohme; KEYNOTE-355 ClinicalTrials.gov number, NCT02819518.).

The broad use of transgenic and gene-targeted mice has established bone marrow-derived macrophages (BMDM) as important mammalian host cells for investigation of the macrophages biology. Over the last decade, extensive research has been done to determine how to freeze and store viable hematopoietic human cells; however, there is no information regarding generation of BMDM from frozen murine bone marrow (BM) cells. Here, we establish a highly efficient protocol to freeze murine BM cells and further generate BMDM. Cryopreserved murine BM cells maintain their potential for BMDM differentiation for more than 6 years. We compared BMDM obtained from fresh and frozen BM cells and found that both are similarly able to trigger the expression of CD80 and CD86 in response to LPS or infection with the intracellular bacteria Legionella pneumophila. Additionally, BMDM obtained from fresh or frozen BM cells equally restrict or support the intracellular multiplication of pathogens such as L. pneumophila and the protozoan parasite Leishmania (L.) amazonensis. Although further investigation are required to support the use of the method for generation of dendritic cells, preliminary experiments indicate that bone marrow-derived dendritic cells can also be generated from cryopreserved BM cells. Overall, the method described and validated herein represents a technical advance as it allows ready and easy generation of BMDM from a stock of frozen BM cells.

The epithelium of the adult prostate contains 3 distinct cell types: basal, luminal, and neuroendocrine. Tissue-regenerative activity has been identified predominantly from the basal cells, isolated by expression of CD49f and stem cell antigen-1 (Sca-1). An important question for the field is whether all basal cells have stem cell characteristics. Prostate-specific microarray databases were interrogated to find candidate surface antigens that could subfractionate the basal cell population. Tumor-associated calcium signal transducer 2 (TACSTD2/Trop2/M1S1/GA733-1) was identified because it was enriched after castration, in prostate sphere cells and in the basal fraction. In the murine prostate, Trop2 shows progenitor characteristics such as localization to the region of the gland proximal to the urethra and enrichment for sphere-forming and colony-forming cells. Trop2 subfractionates the basal cells into 2 populations, both of which express characteristic basal cell markers by quantitative PCR. However, only the basal cells expressing high levels of Trop2 were able to efficiently form spheres in vitro. In the human prostate, where Sca-1 is not expressed, sphere-forming progenitor cells were also isolated based on high expression of Trop2 and CD49f. Trop2-expressing murine basal cells could regenerate prostatic tubules in vivo, whereas the remaining basal cells had minimal activity. Evidence was found for basal, luminal, and neuroendocrine cells in prostatic tubules regenerated from ${\\rm Trop}2^{{\\rm hi}}$ basal cells. In summary, functionally distinct populations of cells exist within the prostate basal compartment and an epithelial progenitor can give rise to neuroendocrine cells in vivo.

Inadequate immunomodulatory potency of mesenchymal stem cells (MSC) may limit their therapeutic efficacy. We report glucocorticoid steroids augment MSC expression and activity of indoleamine-2,3-dioxygenase (IDO), a primary mediator of MSC immunomodulatory function. This effect depends on signaling through the glucocorticoid receptor and is mediated through up-regulation of FOXO3. Treatment of MSCs with glucocorticoids, budesonide or dexamethasone, enhanced IDO expression following IFN-γ stimulation in multiple donors and was able to restore IDO expression in over-passaged MSCs. As IDO enhancement was most notable when cells were continuously exposed to budesonide, we engineered MSC with budesonide loaded PLGA microparticles. MSC efficiently internalized budesonide microparticles and exhibited 4-fold enhanced IDO activity compared to budesonide preconditioned and naïve MSC, resulting in a 2-fold improvement in suppression of stimulated peripheral blood mononuclear cells in an IDO-dependent manner. Thus, the augmentation of MSC immune modulation may abrogate challenges associated with inadequate potency and enhance their therapeutic efficacy.

Background Tumor-associated macrophages (TAMs) play an important role in growth, progression and metastasis of tumors. In non-small cell lung cancer (NSCLC), TAMs' anti-tumor or pro-tumor role is not determined. Macrophages are polarized into M1 (with anti-tumor function) and M2 (with pro-tumor function) forms. This study was conducted to determine whether the M1 and M2 macrophage densities in NSCLC are associated with patient's survival time. Methods Fifty patients with an average of 1-year survival (short survival group) and 50 patients with an average of 5-year survival (long survival group) were included in this retrospective study. Paraffin-embedded NSCLC specimens and their clinicopathological data including up to 8-year follow-up information were used. Immunohistochemical double-staining of CD68/HLA-DR (markers for M1 macrophages) and CD68/CD163 (markers for M2 macrophages) was performed and evaluated in a blinded fashion. The M1 and M2 macrophage densities in the tumor islets, stroma, or islets and stroma were determined using computer-aided microscopy. Correlation of the macrophage densities and patient's survival time was analyzed using the Statistical Package for the Social Sciences. Results Approximately 70% of TAMs were M2 macrophages and the remaining 30% were M1 macrophages in NSCLC. The M2 macrophage densities (approximately 78 to 113 per mm 2 ) in the tumor islets, stroma, or islets and stroma were not significantly different between the long survival and short survival groups. The M1 macrophage densities in the tumor islets (approximately 70/mm 2 ) and stroma (approximately 34/mm 2 ) of the long survival group were significantly higher than the M1 macrophage densities in the tumor islets (approximately 7/mm 2 ) and stroma (13/mm 2 ) of the short survival group (P < 0.001 and P < 0.05, respectively). The M2 macrophage densities were not associated with patient's survival time. The M1 macrophage densities in the tumor islets, stroma, or islets and stroma were positively associated with patient's survival time in a univariate analysis (P < 0.01 or 0.001). In a multivariate Cox proportional hazards analysis, the M1 macrophage density in the tumor islets was an independent predictor of patient's survival time. Conclusions The M1 macrophage density in the tumor islets is an independent predictor of survival time in NSCLC patients.