TREM1 promotes cancer associated malignant phenotype through activated MAPK signaling pathway and predicts poor prognosis in gastric cancer.

Background: CD molecules plays a vital role in gastric cancer (GC). We used bioinformatics analysis methods to develop prognosis related CD molecules risk signature; On the other hand, we used the experiments to further explore the function and mechanism of differentially expressed prognostic CD molecules (TREM1) in GC. Methods: Kaplan-Meier survival and univariate Cox regression analysis were used to evaluate the overall survival of CD molecule genes in gastric cancer. ROC curve and Kaplan-Meier curves were used to analyze the predictive value of CD molecule related genes risk signature by "survival and timeROC" R packages. GSEA, and Cibersortx software were used to analyze the functional enrichment. Finally, we verified the function and mechanism of TREM1 in GC by gene silencing and MAPK inhibitor (SB203580) in vitro and vivo. Results: A total of 41 prognosis related risk factors in gastric cancer were identified based on CD molecules, including TREM1 and ect. The high-risk patients had higher risk score and shorter survival time. ROC curves revealed that this risk signature accurately predicted survival times of gastric cancer patients at the 1-, 2-, 3-, 4- and 5-year. The frequency of T cells follicular helper and NK cells activated were added in low-risk group. Next, differentially expressed prognostic CD molecules analysis revealed that TREM1 was identified as key genes in GC progression based on TCGA and GES158662 and GSE15459 datasets of GC. In vitro experiments, TREM1 silencing significantly inhibited GC cell proliferation and migration, induced cell apoptosis. GSEA revealed that TREM1 activated cancer related signaling pathway, including MAPK signaling pathway and ect. High expression of TREM1 was related Macrophages M2 and Mast cells resting in GC tissues. Moreover, knockdown of TREM1 inhibited tumor growth through downregulated MAPK signaling pathway in vivo. Conclusion: These results identified that CD molecule related genes as a novel prognostic and diagnostic biomarker in gastric cancer. TREM1 acts as an oncogene role in GC by activated MAPK signaling pathway.

Significance of Complement Regulatory Protein Tetraspanins in the Male Reproductive System and Fertilization.

Fertilization is a very sophisticated and unique process involving several key steps resulting in a zygote's formation. Recent research has indicated that some immune system-related cell surface molecules (CD molecules from the tetraspanin superfamily) may have a role in fertilization. Extracellular vesicles are undeniably involved in a variety of cellular functions, including reproduction. Tetraspanin proteins identified in extracellular vesicles are now used mostly as markers; mounting evidence indicates that they also participate in cell targeting, cargo selection, and extracellular vesicle formation. Their significance and potential in mammalian reproduction are currently being studied extensively. Despite the fact that the current data did not establish any theory, the crucial function of tetraspanins in the fertilization process was not ruled out, and the specific role of tetraspanins is still unknown. In this review, we bring insight into the existing knowledge regarding the expression of tetraspanins in spermatozoa and seminal fluid and their role in gamete binding and fusion.

Pregnancy stage-dependent modulation of neutrophil function may impact embryo survivability and pregnancy outcome in crossbred cows.

Pregnancy is a complicated physiological process that involves synchronized coordination between immune and endocrine systems. Neutrophils have been suggested as a critical immune cell for embryo implantation and pregnancy maintenance. The present study was conducted to evaluate the dynamic changes in the mRNA expressions of the cluster of designation (CD11b, CD31, CD44 and CD62L) molecules and interferon-stimulated genes (ISG15, MX1 and OAS1) in blood neutrophils throughout pregnancy in dairy cows and correlate them with the outcome of pregnancy. Blood samples were taken from negative control (NC) group, and non-pregnant (NP) group at the time of artificial insemination (AI, day zero) and on days 10, 14, 16, 18, and 21 post-AI. In pregnant (P) cows, samples were taken as described above and after every 30 days until the time of parturition. In aborted cows, samples were collected until the time of the abortion. Comparison between pregnant, non-pregnant and aborted cows revealed that the expression of CD molecules increased (p < 0.05) on days 14, 16, 18 and 21 post-AI only in NP cows as compared to other groups. Although the expression of CD molecules remained constant throughout the study period in pregnant and aborted cows, the expression of CD11b, CD31 and CD62L increased (p < 0.05) on the day of abortion and parturition. Unlike CD molecules, the expression of CD44 decreased significantly (p < 0.05) at the time of abortion. There was a significant (p < 0.05) increase in the expression of interferon-stimulated genes including MX1, OAS1 and ISG15 during the peri-implantation period in pregnant cows, and at the time of abortion in aborted cows. However, the expression of ISGs was lower (p < 0.05) in non-pregnant cows as compared to the other groups. The results revealed the critical role played by neutrophils during pregnancy and form the basis to unravel the underlying mechanism for neutrophil associated immunological infertility in bovines.

Identification of cluster of differentiation molecule-associated microRNAs as potential therapeutic targets for gastrointestinal cancer immunotherapy.

BACKGROUND: Cluster of differentiation molecules are markers of immune cells that have been identified as a potential immunotherapeutic target for cancer treatment. MicroRNAs are small non-coding RNAs that act as tumor suppressors or oncogenes whose importance in diagnosis, prognosis, and treatment of gastric and colorectal cancers has been widely reported. However, their association with cluster of differentiation molecules in gastrointestinal cancers has not been well studied. Therefore, our study aimed to analyze the relationship between microRNAs and cluster of differentiation molecules in gastrointestinal cancers, and to identify cluster of differentiation molecule-associated microRNAs as prognostic biomarkers for gastrointestinal cancer patients. METHODS: Targetscan, Starbase, DIANA microT, and miRDB were used to investigate microRNA profiles that might be correlated with cluster of differentiation molecules in gastrointestinal cancers. Moreover, The Cancer Genome Atlas data analysis was used to investigate the association between cluster of differentiation molecules and microRNA expression in patients with gastric, colon, rectal, pancreatic, and esophageal cancers. The Kaplan-Meier plotter was used to identify the association between overall survival and cluster of differentiation molecule-associated microRNA expression in gastrointestinal cancer patients. RESULTS: miR-200a, miR-559, and miR-1236 were negatively associated with CD86, CD81, and CD160, respectively, in almost all types of gastrointestinal cancers, which were further verified in the in vitro studies by transfecting microRNA mimics in gastric cancer, colon cancer, pancreatic, and esophageal cell lines. CONCLUSION: Our study showed that miR-200a, miR-1236, and miR-559 are identified as cluster of differentiation-associated microRNAs in gastrointestinal cancers, providing a novel perspective to identify new therapeutic targets for cancer immunotherapy in gastrointestinal cancer patients.

A comparative study on various immunological parameters influencing embryo survivability in crossbred dairy cows.

Since long embryonic mortality has remained an area of concern affecting the reproduction, production, and profitability of dairy cows. We investigated the possible interaction between interleukins, hormones, and neutrophil associated CD markers during the implantation window in Karan Fries (KF) cows naturally coming to heat. Blood collection was done on days 0 i.e. day of Artificial Insemination (AI), 10, 18, 21, 30 and on day 40 post-AI. Total leucocyte count (TLC) and neutrophil to lymphocyte (N:L) ratio were recorded. Blood neutrophils were isolated and their number, phagocytic activity (PA), myeloperoxidase (MPO) concentration and relative mRNA expression of cell adhesion molecules (CD-11b, CD-31, CD-44, CD-62L) as well as progesterone-inducing-blocking-factor (PIBF) and glucocorticoid receptor alpha (GRα) were examined. Plasma progesterone, cortisol, IL-2, IL-8, IL-6, and IL-10 were also measured. Pregnancy was confirmed by non-return to heat, ultrasonography and per rectal examination along with progesterone assay. Cows were further divided into pregnant (P), early embryonic mortality (EEM) and late embryonic mortality (LEM) groups. Embryonic losses cows showed lower plasma concentration of IL-10 (<100 pg/ml) and a higher concentration of IL-2 (>500 pg/ml). Also, a 4 fold increase in the relative mRNA expression of CD-11b and 2.5 fold changes in CD-44 expression were observed in embryonic mortality. We observed a 1.5 fold increase in the relative mRNA expression of PIBF and a 0.5 fold increase in GRα expression in pregnant cows compared to EEM (on day 21) and LEM (on days 30 and 40) cows. Our results depicted that the hyperimmune status of the dam which could be due to multifactorial events that led to the pregnancy failure. The above basic values may be used for checking the immune status and thus timely management strategies can be taken to prevent embryonic losses.

Selected CD molecules and the phagocytosis of microvesicles released from erythrocytes ex vivo.

BACKGROUND AND OBJECTIVES: The accumulation of microvesicles in erythrocyte concentrates during storage or irradiation may be responsible for clinical symptoms such as inflammation, coagulation and immunization. Our aim was to determine whether any of the cluster of differentiation (CD) molecules responsible for important functions are present on microvesicles, and if their expression level is dependent on the storage period of erythrocyte concentrates. MATERIAL AND METHODS: Erythrocyte microvesicles were isolated from 'fresh' (2nd day) and 'old' (42nd day) stored erythrocyte concentrates. Qualitative cytometric analysis of 0·5 µm, erythrocyte-derived, PS-exposing vesicles was performed using the annexin V-FITC, anti-CD235a-PE antibody and calibrated beads. The microvesicles were also visualized under a confocal microscope. The expression of the molecules CD235a, CD44, CD47, CD55, CD59 and of phosphatidylserine (PS) was compared using flow cytometry. Measurements of microvesicle phagocytosis by human monocytes were carried out using a flow cytometer and a confocal microscope. RESULTS: The analysis of the microvesicles with calibration beads allowed us to identify these structures with a diameter of about 0·5 µm in the 'fresh' and 'old' samples. At day 2, the microvesicles had elevated expression levels of CD47, reduced expression levels of PS, CD55 and CD59. The phagocytosis index was higher for the microvesicles isolated from the 42-day-old erythrocyte concentrates. CONCLUSION: This research may bring us closer to understanding the factors responsible for erythrocyte ageing and to evaluate the quality of stored red blood concentrates intended for transfusion.

The cell surface phenotype of human dendritic cells.

Dendritic cells (DC) are bone marrow derived leucocytes that are part of the mononuclear phagocytic system. These are surveillance cells found in all tissues and, as specialised antigen presenting cells, direct immune responses. Membrane molecules on the DC surface form a landscape that defines them as leucocytes and part of the mononuclear phagocytic system, interacts with their environment and directs interactions with other cells. This review describes the DC surface landscape, reflects on the different molecules confirmed to be on their surface and how they provide the basis for manipulation and translation of the potent functions of these cells into new diagnostics and immune therapies for the clinic.

Defining Genome-Wide Expression and Phenotypic Contextual Cues in Macrophages Generated by Granulocyte/Macrophage Colony-Stimulating Factor, Macrophage Colony-Stimulating Factor, and Heat-Killed Mycobacteria.

Heat-killed (HK) Mycobacterium obuense (NCTC13365) is currently being evaluated in the clinic as an immunotherapeutic agent for cancer treatment. Yet, the molecular underpinnings underlying immunomodulatory properties of HK M. obuense are still largely undefined. To fill this void, we sought to perform immunophenotyping, chemokine/cytokine release analysis and genome-wide characterization of monocyte-derived macrophages (MDM) in which monocytes were originally isolated from healthy donors and differentiated by HK M. obuense (Mob-MDM) relative to macrophage colony-stimulating factor (M-MDM) and granulocyte/macrophage colony-stimulating factor (GM-MDM). Immunophenotyping and cytokine release analysis revealed downregulated surface expression of CD36, decreased spontaneous release of CCL2 and increased spontaneous secretion of CCL5, CXCL8/IL-8, IL-6, and TNF-α in Mob-MDM relative to M-MDM and GM-MDM. Analysis of cytostatic activity showed that Mob-MDM exhibited similar growth inhibitory effects on immortalized and malignant epithelial cells compared with GM-MDM but at an elevated rate relative to M-MDM. To understand global cues in Mob-MDM, we performed comparative RNA-sequencing (RNA-Seq) analysis of Mob-MDM relative to GM-MDM and M-MDM (n = 4 donors). Clustering analysis underscored expression profiles (n = 256) that were significantly modulated in Mob-MDM versus both M-MDM and GM-MDM including, among others, chemokines/cytokines and their receptors, enzymes and transcriptions factors. Topological functional analysis of these profiles identified pathways and gene sets linked to Mob-MDM phenotype including nitric oxide production, acute phase response signaling and microbe recognition pathways as well as signaling cues mediated by the proinflammatory cytokine, interferon-gamma, and the intracellular pattern recognition receptor, nucleotide-binding oligomerization domain-containing protein 2. Taken together, our study highlights molecular immune phenotypes and global signaling cues in Mob-MDM that may underlie immunomodulatory properties of HK M. obuense. Such properties could be of valuable use in immunotherapy approaches such as adoptive cell therapy against cancer.

Immunomodulatory effects of heat-killed Mycobacterium obuense on human blood dendritic cells.

Heat-killed (HK) Mycobacterium obuense is a novel immunomodulator, currently undergoing clinical evaluation as an immunotherapeutic agent in the treatment of cancer. Here, we examined the effect of in vitro exposure to HK M. obuense on the expression of different categories of surface receptors on human blood myeloid (m) and plasmacytoid (p) DCs. Moreover, we have characterized the cytokine and chemokine secretion patterns of purified total blood DCs stimulated with HK M. obuense. HK M. obuense significantly up-regulated the expression of CD11c, CD80, CD83, CD86, CD274 and MHC class II in whole-blood mDCs and CD80, CD123 and MHC class II in whole-blood pDCs. Down-regulation of CD195 expression in both DC subpopulations was also noted. Further analysis showed that HK M. obuense up-regulated the expression of CD80, CD83 and MHC class II on purified blood DC subpopulations. TLR2 and TLR1 were also identified to be engaged in mediating the HK M. obuense-induced up-regulation of surface receptor expression on whole blood mDCs. In addition, our data demonstrated that HK M. obuense augmented the secretion of CCL4, CCL5, CCL22, CXCL8, IL-6, IL-12p40 and TNF-α by purified total blood DCs. Taken together, our data suggest that HK M. obuense exerts potent differential immunomodulatory effects on human DC subpopulations.

Differential regulation of surface receptor expression, proliferation, and apoptosis in HaCaT cells stimulated with interferon-γ, interleukin-4, tumor necrosis factor-α, or muramyl dipeptide.

Keratinocytes are routinely subjected to both internal and external stimulation. This study investigates the effects of interferon gamma, interleukin-4, tumor necrosis factor alpha, and the synthetic immunomodulator muramyl dipeptide on the human keratinocyte cell line, HaCaT. Following HaCaT stimulation with cytokines or muramyl dipeptide for different time periods, changes in the expression of different cell surface receptors, cell proliferation, and cell apoptosis were evaluated by flow cytometry, tritiated thymidine uptake, and annexin-V staining, respectively. A significant decrease in the expression of CD49d was found upon treatment with interleukin-4. Interferon gamma and tumor necrosis factor alpha increased the expression of intercellular adhesion molecule 1 and major histocompatibility complex class I, whereas major histocompatibility complex class II and CD1b were only upregulated by interferon gamma. Interferon gamma and tumor necrosis factor alpha had opposite effects regarding CD119 expression, with the former downregulating, while the latter upregulating its expression. Of the stimuli tested, only interferon gamma and tumor necrosis factor alpha significantly inhibited proliferation of HaCaT cells, yet only interferon gamma played a significant role in inducing HaCaT cell apoptosis. Our data demonstrate differential effects of the three tested cytokines on keratinocytes and reveal that the absence of HaCaT cell responses to muramyl dipeptide is associated with undetectable levels of its cytoplasmic receptor, nucleotide-binding oligomerization domain-containing protein 2.

Immunophenotype of mouse cerebral hemispheres-derived neural precursor cells.

Postnatally isolated neural precursor cells (piNPCs) from mouse cerebral tissue have been studied in cell-based therapeutic approaches for Experimental Autoimmune Encephalomyelitis (EAE). Transplantation experiments in EAE rodents revealed that piNPCs manage to integrate into the host tissue and ameliorate clinical symptoms. When cultured in vitro, mouse cerebral piNPCs form neurospheres consisting of immature cells positive for polysialylated neural adhesion molecule (PSA-NCAM) that differentiate mainly towards glial cells, but also neurons. Herein, we have characterized piNPCs immunophenotype, with flow cytometry. NPCs were positive for CD24, CD44, and CD133 though negative for CD15, CD184 and CD49d. This immunophenotype, determined for the first time, among cells isolated from neonates might be useful for the identification of NPC population aiming at the development of transplantation protocols.

Evaluation of toxicity and adjuvant effects of peptidoglycan microspheres orally administered to mice.

In this study, peptidoglycan microspheres were evaluated for their toxicity and adjuvant effects after oral administration to mice. The liver and spleen indexes, CD cell content in peripheral blood and spleen, and immunoglobulin content in peripheral blood were measured by flow cytometry and indirect ELISA, respectively. Peptidoglycan microspheres with a loading capacity of 46.41 ± 0.83 g/100 g were prepared. In vivo tests showed that peptidoglycan microspheres revealed an immuno-enhancing profile as indicated by the slow increase of IgG content in peripheral blood compared with that of the untreated peptidoglycan group. In conclusion, peptidoglycan microspheres may be used as a new oral adjuvant in the host.

Characterizing the human hematopoietic CDome.

In this study, we performed extensive semi-automated data collection from the primary and secondary literature in an effort to characterize the expression of all membrane proteins within the CD scheme on hematopoietic cells. Utilizing over 6000 data points across 305 CD molecules on 206 cell types, we seek to give a preliminary characterization of the "human hematopoietic CDome." We encountered severe gaps in the knowledge of CD protein expression, mostly resulting from incomplete and unstructured data generation, which we argue inhibit both basic research as well as therapies seeking to target membrane proteins. We detail these shortcomings and propose strategies to overcome these issues. Analyzing the available data, we explore the functional characteristics of the CD molecules both individually and across the groups of hematopoietic cells on which they are expressed. We compare protein and mRNA data for a subset of CD molecules, and explore cell functions in the context of CD protein expression. We find that the presence and function of CD molecules serve as good indicators for the overall function of the cells that express them, suggesting that increasing our knowledge about the cellular CDome may serve to stratify cells on a more functional level.