Expression Levels Of CD86 Research Articles

Inecalcitol Induces CD38 Expression in Multiple Myeloma Cell Lines More Efficiently Than All-Trans Retinoic Acid (ATRA)

Targeting CD38 has proven to be an effective strategy for the treatment of multiple myeloma (MM), with the therapeutic success of the first anti-CD38 monoclonal antibody: daratumumab. Interestingly, pretreatment levels of CD38 expression on MM cells were significantly higher in patients who achieved at least partial response (PR) as compared to patients who achieved less than PR (Nijhof et al., 2016, Blood). Therefore, increasing the levels of CD38 expression is a relevant pharmacological objective, which ATRA has already been shown to achieve in MM cell lines (Nijhof et al., 2015, Leukemia).Inecalcitol (INE: 14epi-,19nor-,23yne-,1,25dihydroxy-cholecalciferol), a vitamin D receptor agonist characterized by a high anti-proliferative effect and a low calcemic potential (Okamoto R et al ., 2012, Int J Cancer; Ma Y et al ., 2013, Cell Cycle, Medioni et al . 2014, Clin Cancer Res), has also been shown to stimulate CD38 expression levels in several MM cell lines (ASH 2016, Abstract#3521). The aim of the present study was to further screen a large collection of 38 human MM cell lines. After culture for 72 hrs with or without 10 nM INE, cell surface CD38 labeling was measured with a fluorescent anti-human CD38 mouse FITC antiboby (eBiosciences, 11-0389). On 3 independent experimental days, cells from 3 different wells of either control (ethanol 0.1% v/v) or treated conditions were analyzed by flow cytometry to measure mean fluorescence intensity (MFI). Paired Student's t test was performed to check the unilateral hypothesis that INE had stimulated CD38 expression. Basal control CD38 labeling among the 38 MM cell lines was heterogeneous and ranged from minimal (MFI < 150) to very high (MFI > 10,000). The stimulation of CD38 expression by INE reached statistical significance in 37 out of the 38 (97%) tested MM cell lines, with a mean ± SEM of [4,75 ± 1.05]-fold increase in MFI. Only the U266 cell line remained insensitive to INE and was subsequently shown to entirely lack vitamin D receptors.INE was then compared head-to-head with ATRA at 5 concentrations (0.3, 1, 3, 10 and 30 nM) for 72 hrs on 8 MM cell lines. At all tested concentrations on any cell line (40 experimental conditions), INE induced a higher increase in CD38 than ATRA, the difference reaching statistical significance in 36 out of the 40 bilateral paired Student's t test comparisons. At the threshold concentration of 0.3 nM, CD38 expression was significantly increased by INE on 7 out of 8 cell lines with a [3.7 ± 1.1]-fold stimulation, and on 6 cell lines by ATRA with a [1.4 ± 0.09]-fold increase. Maximal stimulation was achieved by INE at 3 nM, 10 nM and 30 nM on 2, 2 and 4 cell lines, respectively, with a [7.9 ± 3.3]-fold increase, ranging from 3.0 to 31. Maximal stimulation was observed only at 30 nM of ATRA in all cell lines with a [2.9 ± 0.5]-fold increase, ranging from 1.3 to 5.9. INE was on average 2.6 - 2.7 times more efficient than ATRA to induce CD38 in MM cell lines, both in terms of potency and efficacy.These results suggest that inecalcitol could be a therapeutic agent of choice to potentiate the clinical response of a large majority of MM patients to anti-CD38 antibodies, such as daratumumab. DisclosuresMouly:Hybrigenics: Employment. Menoret:Hybrigenics: Research Funding; Myelomax: Employment, Equity Ownership. Kervoelen:Hybrigenics: Research Funding; Myelomax: Employment. Planquette:hybrigenics: Employment, Equity Ownership, Patents & Royalties: Inventorship without royalties. Rousseau:Hybrigenics: Employment. Delansorne:Hybrigenics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Inventorship.

Microrna-31 Promotes T-Cell Pathogenicity in Chronic Graft-Versus-Host Disease

MicroRNAs (miRs) are short, non-coding RNAs that repress gene expression at post-transcriptional level. Certain miRs play critical roles on T-cell responses in inflammation and autoimmunity. To understand the biology of miRs in T-cell allo-responses, we measured expression profiles of miRs in donor T cells in allogeneic vs. syngeneic recipients following bone marrow transplantation (BMT). We found miR-31 was dramatically up-regulated in T cells under alloantigen-driven vs. homeostatic proliferation in vivo(Fig. A). MiR-31 was shown to regulate autoimmunity and cancer. However, its roles in regulating T-cell response remain largely undetermined. Recent study shows miR-31 represses peripheral Treg differentiation in EAE development. In contrast, miR-31 inhibits CD8 T-cell function in anti-virus response. We hypothesize that miR-31 is required for CD4 T-cell pathogenicity in chronic graft-versus-host disease (cGVHD), but is dispensable for CD8 T-cell cytolytic function in graft-versus-leukemia (GVL) activity.To study how miR-31 regulates T-cell allo-response, we used mice with miR-31 conditional knock out (KO) in T cells (miR-31fl/fl CD4Cre). No differences were observed in the baseline composition of naïve WT (miR-31fl/fl) and miR-31 KO splenocytes. After being transferred into allogeneic recipients, KO T cells had impaired ability to proliferate, reflected by fewer divided (% CFSElow) cells and reduced donor T-cell expansion in recipient spleens (Fig. B). To further evaluate how miR-31 contributes to T-cell pathogenicity in cGVHD, we used a murine model of BMT (B6 → BALB/c), in which low dose spleonocyes can induce scleroderma manifestation. Significant reduction of clinical scores (Fig. C), including improved skin scores and body weight loss, were found in the recipients of miR-31 KO grafts compared to those with WT grafts. This finding was validated in another haploidentical BMT model of scleroderma (B6 → B6D2F1), indicating miR-31 is required for T-cell to induce cGVHD. Consistently, the pathological damage was dramatically reduced in skin, gut and lung in the recipients of miR-31 KO grafts (Fig. D).Mechanistically, miR-31 deficiency in donor T cells resulted in significantly reduced numbers of CD4 T cells that produce IFNγ, IL-4/5 or IL-17 in recipient spleen and mesenteric lymph nodes. In absence of miR-31, fewer effector and central memory cells were generated from donor CD4 T cells. Further, T cells that are capable of localizing to B-cell follicles (CXCR5+PD-1hi) were significantlyreduced in spleen (Fig. E) and peripheral lymph nodes in recipients of miR-31 KO grafts. Interestingly, among these cells, more foxp3+ follicular T-regulatory (Tfr) but fewer foxp3- follicular T-helper (Tfh) cells were observed in the recipients of KO grafts (Fig. F), indicating miR-31 contributes to the unbalance of Tfr and Tfh cells during cGVHD development. Consistently with the role of miR-31 in regulating Tfr and Tfh cells, we found Blimp1 is the predicted targets of miR-31, which is critically involved in the homeostasis of Tfr and Tfh cells. On donor B-cell compartment, lower levels of CD86 and MHC-II expression were observed in the recipients of miR-31 KO grafts. In addition, germinal center B cells and plasma cells were also reduced in those recipients, indicating miR-31 indirectly regulates B-cell activation and differentiation through controlling T-cell pathogenicity.For translational purpose, we evaluated how miR-31 impacts T cell-mediated GVL effect. Using B6 → BDF1 BMT model, we consistently found attenuated skin manifestation in recipients of KO T cells. Importantly, in the ongoing experiment, miR-31 KO T cells were capable of controlling P815(mastocytoma) growth as efficiently as WT T cells (Fig. G), suggesting miR-31 may be dispensable for T cells to mediating GVL activity after allogeneic BMT.Taken together, the current work reveals that miR-31 plays pleotropic roles in enhancing CD4 T-cell pathogenicity in cGVHD. MiR-31 represents a promising therapeutic target for the control of cGVHD while sparing GVL effects after allogeneic hematopoietic stem cell transplantation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Adaptability of Antibody-Coupled T Cell Receptor (ACTR) Engineered Autologous T Cells in Combination with Daratumumab over CAR-Based Approaches

Chimeric antigen receptor (CAR)-T cells have demonstrated profound clinical responses against CD19+ B cell malignancies, with ongoing trials in multiple myeloma (MM). Although BCMA CAR-T cells show promise in MM, development of CAR-T against other MM targets has been problematic due to target expression on T cells themselves. Specifically, efforts to develop CAR-T cell therapies targeting CD38 have been hampered by the upregulation of CD38 on activated T cells and consequent induction of CAR-T mediated autolysis that occurs during manufacturing. To overcome this challenge during CAR-T production, investigators have taken several approaches including multiple rounds of T cell expansion, addition of CD38 blocking antibody, and CD38 gene knockout in T cells prior to CAR transduction (1,2,3).The Antibody-Coupled T Cell Receptor (ACTR) technology is a universal, engineered T cell therapy that combines the Fc receptor, CD16, with endogenous T cell signaling and costimulatory domains (4). ACTR T cells function by engaging the Fc domain of therapeutic antibodies opsonized on target cells, resulting in T cell activation and tumor cell killing. In contrast to CAR-T cell constructs that express the antigen targeting single chain variable fragment (scFv), ACTR T cell activity is regulated through antibody administration and is therefore not susceptible to target-mediated autolysis during T cell production.The recent approval of the CD38-targeting antibody, daratumumab, has improved the treatment of multiple myeloma through its tumor targeting and immunomodulating capabilities. Here, we leveraged the ability of the ACTR T cell platform to decouple the targeting antibody from the engineered T cell signaling moieties, and evaluated the anti-myeloma activity of ACTR T cells in combination with daratumumab. We demonstrated that in contrast to CD38-targeting CAR-T cells, ACTR T cells exhibited enhanced viability and expansion of the chimeric construct throughout the manufacturing process. We then evaluated the anti-myeloma activity of ACTR T cells in combination with daratumumab. ACTR T cells mediated robust cytotoxicity, cytokine production, and proliferation in response to a range of daratumumab-opsonized target cell lines. Notably, the target specific killing and cytokine production mediated by ACTR T cells in combination with daratumumab was greater than the activity mediated by daratumumab alone. Recognizing the augmented activity of ACTR T cells in combination with daratumumab, we systematically investigated the potential for on-target off-tumor toxicity. In a peripheral blood mononuclear cell coculture assay, ACTR T cells in combination with daratumumab selectively targeted CD38-expressing multiple myeloma cells over CD38-expressing normal B cells, T cells, and natural killer cells. Furthermore, despite low levels of CD38 expression reported on erythrocytes, ACTR T cells in combination with daratumumab did not mediate hemolysis. Taken together, our findings demonstrate the adaptability of the ACTR platform to function in combination with daratumumab, and bypass the challenges of scFv-based CAR-T cell production to safely and effectively target CD38-positive multiple myeloma.

Aqueous extracts from cultivated Cistanche deserticola Y.C. Ma as polysaccharide adjuvant promote immune responses via facilitating dendritic cell activation

Ethnopharmacological relevanceHerbal polysaccharides have exhibited great immune-enhancing potential. Adjuvants are a key tool for developing efficacious vaccines. In our previous study, a water-soluble polysaccharide extracted from wild Cistanche deserticola Y.C. Ma showed potent immunostimulatory activity. Aim of studyIn this study, the immune profiles and efficacy of aqueous extracts of cultivated Cistanche deserticola Y.C. Ma (AECCD) on ICR mice against ovalbumin (OVA) were investigated. In vitro experiments, the possible DC activation mechanism by AECCD was evaluated. Materials and methodsAECCD were extracted using hot water after which the crude polysaccharides were precipitated by ethanol. Mice were firstly immunized subcutaneously with OVA (10 μg per mouse) alone or OVA (10 μg per mouse) respectively containing different dose of AECCD (200, 400 and 800 μg per mouse) on Days 1 and 14 and the magnitude and kinetics of antibodies and cell-mediated responses were then assessed. ResultsAECCD elicited vigorous and long-term IgG responses with mixed Th1/Th2 responses and up-regulated levels of Th-associated cytokines (CD4+IL-4, CD4+IFN-γ and CD8+IFN-γ). Moreover, AECCD induced the strong cellular immune response characterized by increased splenocyte proliferation as well as the activated T cell response. Notably, AECCD significantly enhanced the maturation of dendritic cells (DCs) and inhibited Tregs. In vitro experiments, Preliminary tests indicated that AECCD induced DC activation by promoting phenotypic maturation, cytokine section and allostimulatory activity. Toll-like receptor 4 (TLR4) was an essential receptor for DCs to directly bind AECCD. The inhibitors of NF-κB decreased the expression levels of CD40, CD80, CD86 and MHC-II and the production of IFN-γ, TNF-α and IL-6 through DCs. ConclusionsFinally, these findings suggested that AECCD could elicit potent and durable antigen specific immune responses through DC activation, which was involved in the regulation of maturation markers and cytokine expression via TLR4-related NF-κB pathway. The study indicates that AECCD is a potential immunomodulator.

Systems biology unravels the relationship of lncRNA OIP5-AS1 with CD25

The regulatory T cells function in the immune homeostasis, these cells express high level of CD25. It is known that CD25 expression levels are various among different cancers. Thus, we intended to dissect systems biology of a lncRNA pertained to CD25 and CD25 protein interactors-targeting miRNAs. Using RNA-seq data, co-expression analyses of the lncRNA pertained to some cancers were performed. Our analysis was done for protein interactors of CD25 by STRING 11.0, ShinyGO v0.60 and KEGG web servers were used for enrichment and network analysis of CD25. TargetScan 7.2, miRTargetLink Human and mirDIP were applied for determining the CD25 and CD25 interactors-targeting miRNAs. To find the lncRNA-miRNA and lncRNA-protein interactions, starBase v3.0, LncBase Predicted v.2 and SFPEL-LPI were utilized, respectively. Also, using Co-LncRNA, the co-expressed lncRNA analysis and the relative signaling pathways in some cancers including bladder, breast, head and neck, kidney, liver, lung, prostate and thyroid cancers using RNA-seq data were achieved. OIP5-AS1 was shown to have the interaction with CD25 and CD25 protein interactors-targeting miRNAs. In addition, the co-expression of OIP5-AS1 in cancers and their signaling pathways was identified. In conclusion, OIP5-AS1 can effect on CD25 expression in all relative signaling pathways of these cancers.

Both in vitro T cell proliferation and telomere length are decreased, but CD25 expression and IL-2 production are not affected in aged men.

IntroductionAging is a natural process involving dysfunction of multiple organs and is characterized by increased susceptibility to infections, cancer and autoimmune diseases. The functionality of the immune system depends on the capacity of lymphocytes to proliferate in response to antigenic challenges, and telomere length has an important role regulating the number of cell divisions. The aim of this study was to determine the possible relationship between telomere length, interleukin 2 (IL-2) production, CD25 expression and proliferation of peripheral blood mononuclear cells (PBMCs) in aged men.Material and methodsTelomere length was measured by RT-PCR in PBMCs from young and aged men. IL-2 production and CD25 expression were determined by ELISA and flow cytometry, respectively. Cell proliferation was measured by CFSE dilution assays upon in vitro stimulation with concanavalin A (Con A).ResultsPBMCs from aged men showed a shorter telomere length and a reduced capacity to proliferate in vitro, compared to young men. In contrast, no significant differences in the level of CD25 expression on T lymphocytes, and in vitro production of IL-2 were detected in both groups. In addition, no significant correlation was detected between levels of CD25 expression, IL-2 production, cell proliferation, and telomere length in aged men.ConclusionsIn aged men the telomere length shortening and the reduced T cell proliferation are not related to the capacity of IL-2 production and CD25 expression on T lymphocytes.

Human skin mucosal associated invariant T (MAIT) cells show increased pro-inflammatory cytokine expression following burn trauma

Abstract The role of T cells in burn trauma is an understudied research area explored primarily in mice, with sparse data in humans. Patients surviving an initial burn injury are at a high risk of infection, sepsis, and death. Unconventional T-cells such as mucosal-associated invariant T (MAIT) cells are innate-like lymphocytes that respond rapidly to pathogens. Circulating MAIT cells are primarily interferon (IFN)-gamma and tumor necrosis factor (TNF) producing when presented with a microbial-derived ligand on MHC class-1 related protein (MR1) or activated by cytokines interleukin (IL)-12 &amp; IL-18. In this study, we obtained discarded burn (n=6) and normal (from discarded autograft, n=4) skin tissue from burn patients and compared unconventional T cell functionality by flow cytometry and single-cell RNA-sequencing (scRNA-seq). We did not find any significant differences between the frequency of total MAIT cells between burn injury or normal skin, 1.51 ± 1.42 and 2.98 ± 4.40, respectively. There is a significant higher level of CD38 expression in MAIT cells from burn injury tissue and a significant higher level of CD69 expression in MAIT cells from normal skin tissue. Skin MAIT cells in a burn injury show significantly higher levels of IFN-g and TNF before and after PMA-ionomycin stimulation compared to normal skin MAIT cells, which mainly produce IL-6 &amp; IL-17A, suggesting that skin MAIT cells of burn patients show a MAIT cell phenotype that is more responsive to infection. We are characterizing these MAIT cell subtypes in further detail with scRNA-seq and anticipate presentation of that data at time of the conference. Our findings provide the means towards an understanding of the role that skin MAIT cells play in skin of patients with burn trauma.

CD1d deficiency limits tolerogenic properties of peritoneal macrophages

Invariant natural killer T (iNKT) cells are involved in various autoimmune diseases. Although iNKT cells are arthritogenic, transforming growth factor beta (TGFβ)-treated tolerogenic peritoneal macrophages (Tol-pMφ) from wild-type (WT) mice are more tolerogenic than those from CD1d knock-out iNKT cell-deficient mice in a collagen-induced arthritis (CIA) model. The underlying mechanism by which pMφ can act as tolerogenic antigen presenting cells (APCs) is currently unclear. To determine cellular mechanisms underlying CD1d-dependent tolerogenicity of pMφ, in vitro and in vivo characteristics of pMφ were investigated. Unlike dendritic cells or splenic Mφ, pMφ from CD1d+/− mice showed lower expression levels of costimulatory molecule CD86 and produced lower amounts of inflammatory cytokines upon lipopolysaccharide (LPS) stimulation compared to pMφ from CD1d-deficient mice. In a CIA model of CD1d-deficient mice, adoptively transferred pMφ from WT mice reduced the severity of arthritis. However, pMφ from CD1d-deficient mice were unable to reduce the severity of arthritis. Hence, the tolerogenicity of pMφ is a cell-intrinsic property that is probably confer-red by iNKT cells during pMφ development rather than by interactions of pMφ with iNKT cells during antigen presentation to cognate T cells.

Clinical Relevance of CD4 Cytotoxic T Cells in High-Risk Neuroblastoma.

Neuroblastoma is the most common extracranial childhood solid tumor. The majority of high-risk neuroblastoma is resistant/refractory to the current high intensity therapy, and the survival of these patients remains poor for the last three decades. To effectively treat these extremely unfavorable neuroblastomas, innovative immunotherapy approaches would be the most promising. In this article, we discuss the identity of tumor-infiltrating effector cells and immunosuppressive cells in high-risk neuroblastoma. Neuroblastoma is unique in that it expresses little or no classical HLA Class I and II. In contrast, high-risk neuroblastomas express the stress-responsive non-classical Class I, HLA-E molecule. HLA-E is the ligand of activating receptors NKG2C/E that are expressed on memory NK cells, CD8+T cells and CD4 CTLs. By examining a comprehensive RNA-seq gene expression dataset, we detected relatively high levels of CD4 expression in high-risk neuroblastoma tissues. The majority of CD4+ cells were CD3+, and thus they were likely tumor-associated CD4+T cells. In addition, high-level of both CD4 and NKG2C/E expression was associated with prolonged survival of the high-risk neuroblastoma patients, but CD8 levels were not, further suggesting that the CD4+ NKG2C/E+ T cells or CD4 CTL conferred cytotoxicity against the neuroblastoma cells. However, this T cell mediated- “protective effect” declined over time, in part due to the progressive formation of immunosuppressive tumor microenvironment. These observations suggest that to improve survival of high-risk neuroblastoma patients, it is essential to gain insights into how to enhance CD4 CTL cytotoxicity and control the immunosuppressive tumor microenvironment during the course of the disease.

The Human Cytomegalovirus US31 Gene Predicts Favorable Survival and Regulates the Tumor Microenvironment in Gastric Cancer.

Human cytomegalovirus (HCMV) is an oncogenic virus associated with tumorigenesis. Our previous study revealed that the HCMV US31 gene interacted with NF-κB2 and mediated inflammation through macrophages. However, there are few reports on the role of US31 in gastric cancer (GC). The aim of this study was to investigate the expression of the US31 gene in GC tissue and assess its role in the occurrence and development of GC. US31 expression in 573 cancer tissues was analyzed using immunohistochemistry. Results showed that US31 was significantly associated with tumor size (P = 0.005) and distant metastasis (P < 0.001). Higher US31 expression indicated better overall survival in GC patients. Overexpression of US31 significantly inhibited the proliferation, migration, and invasion of GC cells in vitro (P < 0.05). Furthermore, expression levels of CD4, CD66b, and CD166 were positively correlated with US31, suggesting that it was involved in regulating the tumor immune microenvironment of GC. RNA sequencing, along with quantitative real-time polymerase chain reaction, confirmed that the expression of US31 promoted immune activation and secretion of inflammatory cytokines. Overall, US31 inhibited the malignant phenotype and regulated tumor immune cell infiltration in GC; these results suggest that US31 could be a potential prognostic factor for GC and may open the door for a new immunotherapy strategy.

An IgD-Fc-Ig fusion protein restrains the activation of T and B cells by inhibiting IgD-IgDR-Lck signaling in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by synovitis and the destruction of small joints. Emerging evidence shows that immunoglobulin D (IgD) stimulation induces T-cell activation, which may contribute to diseases pathogenesis in RA. In this study, we investigated the downstream signaling pathways by which IgD activated T cells as well as the possible role of IgD in the T-B interaction. Peripheral blood mononuclear cells were isolated from peripheral blood of healthy controls and RA patients. We demonstrated that IgD activated T cells through IgD receptor (IgDR)-lymphocyte-specific protein tyrosine kinase (Lck)-zeta-associated protein 70 (ZAP70)/phosphatidylinositol 3-kinase (PI3K)/nuclear factor kappa-B (NF-κB) signaling pathways; IgD-induced CD4+ T cells promoted the proliferation of CD19+ B cells in RA patients. A novel fusion protein IgD-Fc-Ig (composed of human IgD-Fc domain and IgG1 Fc domain, which specifically blocked the IgD-IgDR binding) inhibited the coexpression of IgDR and phosphorylated Lck (p-Lck) and the expression levels of p-Lck, p-ZAP70, p-PI3K on CD4+ T cells, and decreased NF-κB nuclear translocation in Jurkat cells. Meanwhile, IgD-Fc-Ig downregulated the expression levels of CD40L on CD4+ T cells as well as CD40, CD86 on CD19+ B cells in RA patients and healthy controls. It also decreased the expression levels of CD40L on CD4+ T cells and CD40 on CD19+ B cells from spleens of collagen-induced arthritis (CIA) mice and reduced IL-17A level in mouse serum. Moreover, administration of IgD-Fc-Ig (1.625-13 mg/kg, iv, twice a week for 4 weeks) in CIA mice dose-dependently decreased the protein expression levels of CD40, CD40L, and IgD in spleens. IgD-Fc-Ig restrains T-cell activation through inhibiting IgD-IgDR-Lck-ZAP70-PI3K-NF-κB signaling, thus inhibiting B-cell activation. Our data provide experimental evidences for application of IgD-Fc-Ig as a highly selective T cell-targeting treatment for RA.

Evaluation of TNF Family Gene Expression under the Influence of Single-Walled and Multi-Walled Carboxylated Carbon Nanotubes in Jurkat Cell Line and Rat.

Background: Nanomaterials, e.g.carbon nanotubes (CNTs), have broad usage in medicine for diagnosis, treatment, and drug delivery. Prior to the widespread use of CNTs, any potential toxicity issues must be considered. Apoptosis is an important issue in toxicological studies, and tumor necrosis factor (TNF) family members execute crucial roles in apoptosis and inflammation. We examined the survival of Jurkat cells under the influence of single-walled CNTs (SWCNTs) and multi-walled CNTs (MWCNTs) as well as their impacts on the mRNA levels of TNF family transcripts in Jurkat cells and rats.Objective: To evaluate the toxicity or safety of a specific concentration and form of CNT on the expression of one of the gene families of the apoptotic pathway.Materials and Methods: Jurkat cells were exposed to SWCNTs and MWCNTs in carboxylated form (SWCNTS-COOH and MWCNTs-COOH). MTT assay assessed the cell survival, and using qRT-PCR, the expression levels of TNF, CD40LG, TNFSF10, TNFSF8, CD40, TNFRSF10A, TNFRSF10B, TNFRSF11B, TNFRSF1A, TNFRSF21, TNFRSF25, and TNFRSF9 were examined. The housekeeping genes β-actin and glyceraldehyde 3-phosphate dehydrogenase was utilized for normalization. We also evaluated the expression levels of TNF and TNFRSF10A in rats in vivo 30 and 60 days after being injected with CNTs.Results: After 72 h of carboxylated CNTs at 100 µg. mL-1, no significant change was observed in the survival rate of treated Jurkat cells. The expression of two genes (TNF and TNFRSF10A) changed significantly. Examining the expression profiles of these two genes in rats demonstrated an insignificant change in the expression of any of these genes after 30 and 60 days. The qRT-PCR analysis exhibited the elevated levels of TNF and TNFRSF10A mRNA in the CNT-treated cells, while expression of other TNF family members did not significantly differ from control (untreated) Jurkat cells. There was also no significant change in the gene expression levels of TNF and TNFRSF10A in CNT-treated rats after 30 and 60 days.Conclusions: Administration of SWCNTs-COOH and MWCNTs-COOH could result in the up-regulation of TNF and TNFRSF10A but did not initiate apoptosis in Jurkat cells. Carboxylated SWCNTs showed more potent activity than MWCNTs in activating TNF gene expression and probably trigger cell death through external apoptotic pathways.

Monocyte TREM-1 Levels Associate With Anti-TNF Responsiveness in IBD Through Autophagy and Fcγ-Receptor Signaling Pathways

The expression of Triggering Receptor Expressed on Myeloid cells (TREM)-1 has been described as a predictive marker for anti-Tumor Necrosis Factor (TNF)-α monoclonal antibody (mAb) therapy responsiveness in patients with inflammatory bowel disease (IBD). Here we investigated expression of TREM-1 specifically in CD14+ monocytes in relation to anti-TNF response. The pretreatment TREM-1 expression levels of CD14+ monocytes of Crohn’s disease (CD) patients were predictive of outcome to anti-TNF mAb therapy, with low TREM-1 expression associated with response to anti-TNF. FACSorting of CD14+ monocytes with different TREM-1 levels showed that differentiation towards regulatory CD206+ M2 type macrophages by anti-TNF was suppressed in CD14+ monocytes with high TREM-1 expression. Activity of the Fcγ-Receptor and autophagy pathway, both necessary for M2 type differentiation and the response to anti-TNF, were decreased in CD14+ monocytes with high expression of TREM-1. We confirmed that the activity of the Fcγ-Receptor pathway was decreased in the CD patients that did not respond to anti-TNF therapy and that it was negatively correlated with TREM-1 expression levels in the CD patient cohort. In conclusion, our results indicate that TREM-1 expression levels in CD14+ monocytes associate with decreased autophagy and FcγR activity resulting in decreased differentiation to M2 type regulatory macrophages upon anti-TNF mAb treatment, which may explain anti-TNF non-response in IBD patients with high expression levels of TREM-1.

Immunological impact of chemotherapy on the tumor microenvironment in gastric cancer.

This study aimed to investigate alterations in pre- and post-neoadjuvant chemotherapy (NACT) tumor-infiltrating immune cells and subsequent evaluation of the predictive and prognostic value of these changes in gastric cancer (GC). Fifty patients with GC underwent three cycles of S-1 and oxaliplatin (SOX regimen)-NACT. Paired samples from tumor lesions before and after NACT were available for all patients participating in the study. Immunohistochemistry was performed for T cell subsets (CD3+ and CD8+ ) and macrophages (CD68+ and CD163+ ). After NACT, the average expression levels of CD3, CD8, CD68, and CD163 were significantly increased (p < .001). However, neither expression levels pre- nor post-chemotherapy correlated with treatment response. Multivariate Cox regression analysis demonstrated that upregulation of CD8/CD3 levels (hazard ratio [HR] = 0.117; 95% confidence interval [CI] = 0.031-0.446; p = 0.002) and CD163 levels after chemotherapy (HR = 2.258; 95% CI = 1.047-4.867; p = 0.038) were independent prognostic factors of overall survival. Chemotherapy in GC is useful to induce CD3+ and CD8+ T lymphocytes as well as CD68+ and CD163+ macrophages in the tumor microenvironment in combination with its direct cytotoxic effects. These results indicate that chemotherapy may play a role in tumor immune microenvironment remodeling.

G protein-coupled estrogen receptor increases vascular endothelial inflammatory response after ovariectomized mice

To investigate the effects of calcium supplementation on the inflammatory response of vascular endothelial cells and the possible role and mechanism of G protein-coupled estrogen receptor(GPER)under the physiological conditions of estrogen deficiency(ovary removal). Forty healthy 8-week-old female C57 BL/6 mice were divided into 4 groups by random number table according to body weight, which were respectively the control group, ovarian removal group, ovarian removal high calcium group, and G1 ovarian removal high calcium group, each group ten. One week after the basal diet, the control group underwent sham surgery, and the ovaries-removed group, ovarian-removed high-calcium group, and G1 ovarian-removed high-calcium group were treated for ovarian removal. The control group and the ovary-removed group were fed basic feed(AIN-93 G, calcium content 0. 5%), the ovarian-removed high-calcium group and the G1 implanted ovarian-removed high-calcium group were fed high-calcium feed(adjusted AIN-93 G, calcium content 1. 5%). Three months later, mice in the control, ovarian-removed, and ovarian-reduced high-calcium groups were implanted with a placebo subcutaneously, and G1 ovarian-removed high-calcium groups were implanted with a GPER specific agonist G1, intervention for 28 days, under the anesthesia state, collect blood from 40 mice, isolate serum to measure vascular endothelial adhesion factor-1(VCAM-1), tumor necrosis factor-α(TNF-α), estradiol and blood calcium concentration; take aortic arch for immunization histochemical detection of transient receptor potential channel 1(TRPC1), phosphorylated-extracellular regulated kinase(p-ERK)1/2, VCAM-1 and CD68 expression levels. SPSS software performs statistical analysis and draws conclusion. The differences in weight gain between the three groups and the control group were statistically significant(P&lt;0. 05), while the differences in weight between the three groups were not statistically significant(P&gt;0. 05). There were statistically significant differences in serum estradiol levels between the three groups, the ovariectomized high calcium group, and the G1 ovariectomized group implanted with the control group(P&lt;0. 05). The difference was not statistically significant(P&gt;0. 05). The differences in serum VCAM-1 concentrations between the control group and the other three groups were statistically significant(P&lt;0. 05). The difference in serum VCAM-1 concentration was statistically significant between the ovariectomized group and the implanted G1 ovariectomized high calcium group and the ovariectomized high calcium group(P&lt;0. 05). There was no significant difference in serum VCAM-1 concentration in the group(P&gt;0. 05). There was no significant difference in TNF-α concentration between the four groups(P&gt;0. 05). By immunohistochemistry, the percentages of TRPC1, p-ERK1/2, VCAM-1, and CD68-positive cell areas in the ovarian high-calcium group were significantly higher than those in the ovarian-removed group and the G1-ovarian-removed high-calcium group. The difference was statistically significant(P&lt;0. 05), and the difference in the percentage of positive cell area between the ovarian-removed group and the G1-removed high-calcium group was not statistically significant(P&gt;0. 05). In the state of estrogen deficiency, calcium supplementation causes an increase in inflammatory response, which may be related to the change in GPER activity, and then affect the TRPC1/ERK1/2 pathway.

Bioinformatics analysis and verification of gene targets for renal clear cell carcinoma

BackgroundIt is estimated that there are 338,000 new renal-cell carcinoma releases every year in the world. Renal cell carcinoma (RCC) is a heterogeneous tumor, of which more than 70% is clear cell renal cell carcinoma (ccRCC). It is estimated that about 30% of new renal-cell carcinoma patients have metastases at the time of diagnosis. However, the pathogenesis of renal clear cell carcinoma has not been elucidated. Therefore, it is necessary to further study the pathogenesis of ccRCC. MethodsTwo expression profiling datasets (GSE68417, GSE71963) were downloaded from the GEO database. Differentially expressed genes (DEGs) between ccRCC and normal tissue samples were identified by GEO2R. Functional enrichment analysis was made by the DAVID tool. Protein-protein interaction (PPI) network was constructed. The hub genes were excavated. The clustering analysis of expression level of hub genes was performed by UCSC (University of California Santa Cruz) Xena database. The hub gene on overall survival rate (OS) in patients with ccRCC was performed by Kaplan-Meier Plotter. Finally, we used the ccRCC renal tissue samples to verify the hub genes. Results1182 common DEGs between the two datasets were identified. The results of GO and KEGG analysis revealed that variations in were predominantly enriched in intracellular signaling cascade, oxidation reduction, intrinsic to membrane, integral to membrane, nucleoside binding, purine nucleoside binding, pathways in cancer, focal adhesion, cell adhesion molecules. 10 hub genes ITGAX, CD86, LY86, TLR2, TYROBP, FCGR2A, FCGR2B, PTPRC, ITGB2, ITGAM were identified. FCGR2B and TYROBP were negatively correlated with the overall survival rate in patients with ccRCC (P < 0.05). RT-qPCR analysis showed that the relative expression levels of CD86, FCGR2A, FCGR2B, TYROBP, LY86, and TLR2 were significantly higher in ccRCC samples, compared with the adjacent renal tissue groups. ConclusionsIn summary, bioinformatics technology could be a useful tool to predict the progression of ccRCC. In addition, there are DEGs between ccRCC tumor tissue and normal renal tissue, and these DEGs might be considered as biomarkers for ccRCC.

Synthesis of Carboxamide-Containing Tranylcypromine Analogues as LSD1 (KDM1A) Inhibitors Targeting Acute Myeloid Leukemia.

Lysine-specific demethylase 1 (LSD1/KDM1A) oxidatively removes methyl groups from histone proteins, and its aberrant activity has been correlated with cancers including acute myeloid leukemia (AML). We report a novel series of tranylcypromine analogues with a carboxamide at the 4-position of the aryl ring. These compounds, such as 5 a and 5 b with benzyl and phenethylamide substituents, respectively, had potent sub-micromolar IC50 values for the inhibition of LSD1 as well as cell proliferation in a panel of AML cell lines. The dose-dependent increase in cellular expression levels of H3K4me2, CD86, CD11b and CD14 supported a mechanism involving LSD1 inhibition. The tert-butyl and ethyl carbamate derivatives of these tranylcypromines, although inactive in LSD1 inhibition, were of similar potency in cell-based assays with a more rapid onset of action. This suggests that carbamates can act as metabolically labile tranylcypromine prodrugs with superior pharmacokinetics.

Interleukin-37 improves T-cell-mediated immunity and chimeric antigen receptor T-cell therapy in aged backgrounds.

Aging‐associated declines in innate and adaptive immune responses are well documented and pose a risk for the growing aging population, which is predicted to comprise greater than 40 percent of the world's population by 2050. Efforts have been made to improve immunity in aged populations; however, safe and effective protocols to accomplish this goal have not been universally established. Aging‐associated chronic inflammation is postulated to compromise immunity in aged mice and humans. Interleukin‐37 (IL‐37) is a potent anti‐inflammatory cytokine, and we present data demonstrating that IL‐37 gene expression levels in human monocytes significantly decline with age. Furthermore, we demonstrate that transgenic expression of interleukin‐37 (IL‐37) in aged mice reduces or prevents aging‐associated chronic inflammation, splenomegaly, and accumulation of myeloid cells (macrophages and dendritic cells) in the bone marrow and spleen. Additionally, we show that IL‐37 expression decreases the surface expression of programmed cell death protein 1 (PD‐1) and augments cytokine production from aged T‐cells. Improved T‐cell function coincided with a youthful restoration of Pdcd1, Lat, and Stat4 gene expression levels in CD4+ T‐cells and Lat in CD8+ T‐cells when aged mice were treated with recombinant IL‐37 (rIL‐37) but not control immunoglobin (Control Ig). Importantly, IL‐37‐mediated rejuvenation of aged endogenous T‐cells was also observed in aged chimeric antigen receptor (CAR) T‐cells, where improved function significantly extended the survival of mice transplanted with leukemia cells. Collectively, these data demonstrate the potency of IL‐37 in boosting the function of aged T‐cells and highlight its therapeutic potential to overcome aging‐associated immunosenescence.

Macrophage differentiation in the tissues of myomatous nodes, depending on MRI pattern

Macrophage differentiation is known to be regulated by specific microenvironment invaded by these cells. However, despite numerous studies on pathogenesis of uterine leiomyoma, a common benign tumor of reproductive system, the features of macrophage polarization within myoma nodes are still scarcely studied. The aim of our work was to reveal some differentiation features of macrophages which invade the tissues of myomatous nodes in the patients with different types of uterine leiomyoma (UL) dependent on MRI patterns observed. We have performed a study of 42 patients in their reproductive age with intramural UL. All the patients were subjected to MRI of pelvic area. Twelve samples of endometrium have been taken from healthy women without any signs of UL, being used as controls. UL biopsies and endometrium in its projection served as study material. Phenotype of endometrial and UL-invading macrophages was evaluated by means of multi-color flow cytometry. Expression of Activin А and RARα mRNAs was estimated in endometrial and myoma node macrophages. Likewise, collagen type 1 mRNA expression was evaluated by means of reverse-transcription real time PCR. The collagen type 1 concentration in myomatous nodes was assessed by ELISA technique. We have revealed that peripheral blood monocytes, macrophages in endometrium, and UL nodes each consist of three different subpopulations, dependent on expression levels of expression levels of CD14 and CD16 membrane receptors. For endometrium projected onto myomatous node, the ratios of “intermediate” macrophages (CD14++CD16+), and alternatively activated macrophages (CD36+) was increased, thus exerting potentially negative effects upon reproductive functions in women with uterine leiomyoma. Immediately in myomatous tissue, we have found a shift of macrophage differentiation from ‘classic’ forms towards “intermediate” and “non-classical” macrophages associated with alternative activation. However, the percentage of scavenger receptor-expressing macrophages (CD36+, CD206+) was decreased in the myoma nodes. Enhanced expression of RARα mRNA was observed in macrophages invading the myomatous nodes, whereas Activin А synthesis was higher in the macrophages invading leiomyomas with MRI pattern of simple and degenerative nodes. Imbalance between “intermediate” and “non-classical” UL-invading macrophages was associated with fibrosis, or degenerative changes of myomatous tissues, thus, probably, representing an important pathogenetic link in development of different clinical variants of uterine leiomyoma.

Enhanced Bruton\u2019s tyrosine kinase activity in the kidney of patients with IgA nephropathy

PurposeBruton’s tyrosine kinase (BTK) is a vital biological molecule that contributes to immune regulation. Previous studies have showed that BTK can be detected in patients with lupus nephritis and rheumatoid arthritis. However, the role of BTK in IgA nephropathy (IgAN) has not yet been elucidated. The purpose of this research was to investigate the role of BTK activation in macrophages in IgAN.MethodsPeripheral blood and renal tissue samples were collected from 63 patients with IgAN, and peritumoral normal tissues were collected from 20 patients after surgical resection of renal tumor for use as control. Additionally, 20 healthy volunteers were recruited as control. The levels of BTK, CD68, phosphorylated BTK (pBTK), phosphorylated NF-κB (p-NF-κB p65), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and monocyte chemotactic protein (MCP)-1 were measured by immunohistochemistry (IHC), real-time polymerase chain reaction (RT-PCR), western blotting, and enzyme-linked immunosorbent assay (ELISA).ResultsCompared to peritumoral normal tissues, the expression levels of CD68 and BTK were significantly increased in IgAN group (p < 0.001) and the differences between M0 and M1, E0 and E1, S0 and S1, T0 and T1-2, C0 and C1-2 were statistically significant in the updated Oxford Classification (p < 0.05). Also, CD68 and BTK were positively correlated with Katafuchi semi-quantitative glomerular and tubulointerstitial scores (r = 0.580, 0.637 and 0.442, 0.489, respectively, p < 0.05). The expression of BTK was significantly higher in C3b- and C4d-positive renal tissues of patients with IgAN (p < 0.05). In addition, BTK was positively correlated with 24-h urine protein, serum creatinine levels (r = 0.456 and 0.453, respectively, p < 0.001), and negatively correlated with serum albumin (r = 0.357, p < 0.05). The intensity of expression of pBTK and p-NF-κB p65 was observably increased in renal tissues and monocytes of patients with IgAN compared to the control group. The results of IHC, RT-PCR, and ELISA indicated that the levels of TNF-ɑ, IL-1β, and MCP-1 were markedly increased in the IgAN group (p < 0.05).ConclusionThe results of this study indicate that activation of BTK in macrophages may play an important role in promoting the progression of renal inflammation in IgAN.