Cell CD4 Research Articles

Interaction of human gut microbiota and local immune system in progression of colorectal adenoma (MIMICA-1): a protocol for a prospective, observational cohort study.

The current understanding of colorectal carcinogenesis is based on the adenoma-carcinoma sequence, where genetics, intestinal microbiota changes and local immunity shifts seem to play the key roles. Despite the emerging evidence of dysbiotic intestinal state and immune-cell infiltration changes in patients with colorectal adenocarcinoma, early and advanced adenoma as precursors of colorectal cancer, and carcinoma in situ as the following progression, are rather less studied. The newly colon-site adapted AI-based analysis of immune infiltrates is able to predict long-term outcomes of colon carcinoma. Though it could also facilitate the pathologic evaluation of precancerous lesion's potential to progress. Therefore, the purpose of this prospective cohort study (MIMICA-1) is, firstly, to identify the intestinal microbiota and immune infiltration patterns around the normal bowel tissue, early and advanced adenoma, carcinoma in situ, and adenocarcinoma, and secondly, to analyze the immune - microbiome interplay along the steps of conventional colorectal tumorigenesis. This study aims to prospectively recruit 40 patients (10 per group) with confirmed colorectal dysplasia undergoing endoscopic polypectomy, endoscopic mucosal resection for colorectal small (≤1cm), and large (>1cm) adenoma or carcinoma in situ, or biopsy and subsequent colon resection for invasive colorectal cancer, and 10 healthy patients undergoing screening colonoscopy. Stool samples will be collected prior to bowel preparation for the analysis of fecal (luminal) microbiota composition. Biopsy specimens will be taken from the terminal ileum, right colon, left colon, and a pathological lesion in the colon (if present) to assess mucosa-associated microbiota composition and intestinal immunity response. DNA will be extracted from all samples and sequenced using the Illumina MiSeq platform. Unifrac and Bray-Curtis methods will be used to assess microbial diversity. The intestinal immune system response will be examined using digital image analysis where primarily immunohistochemistry procedures for CD3, CD8, CD20 and CD68 immune cell markers will be performed. Thereafter, the count, density and distribution of immunocompetent cells in epithelial and stromal tissue compartments will be evaluated using AI-based platform. The interaction between the microbial shifts and intestinal immune system response in adenoma-carcinoma sequence and the healthy patients will be examined. In addition, fecal samples will be explored for gut microbiota's composition, comparing fecal- and tissue-derived bacterial patterns in healthy gut and along the adenoma-carcinoma sequence. We hypothesize that changes within the human gut microbiota led to detectable alterations of the local immune response and correlate with the progression from normal mucosa to colorectal adenoma and invasive carcinoma. It is expectable to find more severe gut immune infiltration at dysplasia site, though analyzing invasive colorectal cancer we expect to detect broader mucosa-associated and luminal microbiota changes with subsequent local immune response at near-lesion site and possibly throughout the entire colon. We believe that specific compositional differences detected around premalignant colorectal lesions are critically important for its primary role in initiation and acceleration of colorectal carcinogenesis. Thus, these microbial patterns could potentially supplement fecal immunohistochemical tests for the early non-invasive detection of colorectal adenoma. Moreover, AI-based analysis of immune infiltrates could become additional diagnostic and prognostic tool in precancerous lesions prior to the development of colorectal cancer. The study is registered at the Australian New Zealand Clinical Trials Registry (ACTRN12624000976583) https://www.anzctr.org.au/.

Chaperonin containing TCP1 subunit 6A may activate Notch and Wnt pathways to facilitate the malignant behaviors and cancer stemness in oral squamous cell carcinoma

ABSTRACT Chaperonin containing TCP1 subunit 6A (CCT6A) was recently discovered to be involved in cancer pathogenesis and stemness; however, its role in oral squamous cell carcinoma (OSCC) has not been reported. The current study aimed to investigate the impact of CCT6A on OSCC cell malignant behaviors and stemness and to explore its potentially interreacted pathways. SCC-15 and HSC-3 cells were transfected with the plasmid loading control overexpression, CCT6A overexpression, control knockout, or CCT6A knockout. Wnt4 overexpression or Notch1 overexpression plasmids were transfected into CCT6A-knockout SCC-15 cells. Cell proliferation, apoptosis, invasion, stemness, Notch, and Wnt pathways were detected in both cell lines, whereas RNA sequencing was only performed in SCC-15 cells. CCT6A was upregulated in five OSCC cell lines, including SCC-15, HSC-3, SAT, SCC-9, and KON, compared to that in the control cell line. In SCC-15 and HSC-3 cells, CCT6A overexpression increased cell proliferation, invasion, sphere formation, CD133, and Sox2 expression, but decreased cell apoptosis; on the contrary, CCT6A knockout exhibited an opposite effect on the above indexes. RNA-sequencing data revealed that the Wnt and Notch pathways were involved in the CCT6A’effect on SCC-15 cell functions. CCT6A positively regulates the Wnt and Notch pathways in SCC-15 and HSC-3 cells. Importantly, it was shown that activation of the Wnt or Notch pathways attenuated the effect of CCT6A knockout on SCC-15 cell survival, invasion, and stemness. CCT6A may promote OSCC malignant behavior and stemness by activating the Wnt and Notch pathways.

Influence of graphene oxide nanoparticles on functional activity of Jurkat cell line

Graphene oxide offers properties that make it a promising material for numerous biomedical applications, including various cancer treatment methods. Nanomaterials may overcome certain limitations of conventional chemotherapy. In cancer research, cell lines like Jurkat (T cell acute lymphoblastic leukemia cells) serve as models for investigating tumor treatment strategies. Previous studies have explored the effect of pegylated graphene oxide nanoparticles on some parameters of Jurkat cells, such as metabolism and apoptosis. This study aims to investigate how pegylated graphene oxide nanoparticles, varying in size, surface functionalization, and concentration, influence the functional activity of Jurkat cells, including cell viability, IL-2 production, and CD69 expression, both spontaneously, and following external activation. Jurkat cells were cultured with different types of graphene oxide nanoparticles (100-200 nm and 1-5 ìm; functionalized with linear and branched polyethylene glycol (PEG)) at concentrations of 5 ìg/ mL and 25 ìg/ mL for 24 hours. For assessment of cell parameters under stimulation with different types of particles, phytohemagglutinin (PHA, 50 ìg/mL) was used. Subsequently, the cells were stained with Zombie Aqua and CD69-APC antibody, followed by flow cytometry analysis (CytoFlex S) to determine the percentage of viable cells and CD69-expressing cells. The presence of IL-2 in cell culture supernatants was quantified using ELISA tests. It was observed that nanoparticles at low concentrations did not induce cytotoxic effects; cell viability improved after PHA stimulation. Small particles (100-200 nm) coated with linear PEG induced IL-2 production and CD69 expression. However, 1-5 ìm graphene oxide modified with branched PEG at a concentration of 25 ìg/mL led to a decrease in CD69 expression following PHA-stimulation. It was shown for the first time that pegylated graphene oxide nanoparticles affect the functional activity of Jurkat cells. The influence of particles is dependent on the size, concentration, surface functionalization of graphene oxide, and activation by PHA.

Phenotypic characterization of NK cells in 5-year-old children exposed to maternal HIV and antiretroviral therapy in early-life

BackgroundHIV-exposed uninfected (HEU) children are at increased risk of morbidity during the first years of life. Although the immune responses of HEU infants in early-life are relatively well described, studies of natural killer (NK) cells in older HEU children are lacking. NK cell subsets were analysed in HEU children and compared to those in HIV unexposed uninfected (HUU) children aged ~ five years.MethodsMulti-parametric flow cytometry was used to characterize peripheral blood-derived NK cell CD56, CD16, CD57, NKG2A and KIR3DL1/KIR2DL2/L3 expression, including intracellular perforin and granzyme B. NK cell subsets were compared between HEU children exposed to prenatal antiretroviral therapy (ART) from conception [long-term (HEULT)]; those exposed to ART during pregnancy [medium-term (HEUMT)] with continued exposure throughout the breastfeeding period and HUU peers. Furthermore, clinical data of the children, including sick clinic visits and hospitalizations documented in morbidity diaries from birth to 5 years were compared between HEU and HUU groups. Frequencies of CD56bright and CD56dim NK cell were correlated with these clinical parameters.Results139 children were enrolled however, 133 comprising 43 HEULT, 38 HEUMT and 52 HUU were included in the main analyses. Total NK cell, CD56bright nor CD56dim NK cell proportions differed between HEU and HUU children. However, HEULT children had lower frequencies of CD56dim NK cells compared to HEUMT children, (p = 0.002) which maintained significance after controlling for preterm birth, p = 0.012. No differences were observed between HEULT and HUU. The expressions of NKG2A, KIR3DL1/KIR2DL2/L3 and CD57 on CD56bright and CD56dim NK cells were similar between the three groups. Furthermore, the frequencies of granzyme B and perforin double positive NK cells were similar between the HUU with HEULT and HEUMT children. CD56dim NK cell counts had a significant moderate negative correlation with recurrent respiratory infections (rho=-0.38; p = 0.010) in HUU children and negatively correlated with total sick clinic visits in HEUMT (rho=-0.40, p = 0.064).ConclusionThe proportions of total NK cell, CD56bright and CD56dim NK cells, NK cells inhibitory and differentiation surface marker expression and cytolytic granule-positive cells were similar between HEU and HUU children. These data suggest that early-life HIV/ART exposure may not result in major changes in NK cell subsets at 5 years of age.

Rituximab-IgG2 is a phagocytic enhancer in antibody-based immunotherapy of B-cell lymphoma by altering CD47 expression.

Antibody-dependent cellular phagocytosis (ADCP) by monocytes and macrophages contributes significantly to the efficacy of many therapeutic monoclonal antibodies (mAbs), including anti-CD20 rituximab (RTX) targeting CD20+ B-cell non-Hodgkin lymphomas (NHL). However, ADCP is constrained by various immune checkpoints, notably the anti-phagocytic CD47 molecule, necessitating strategies to overcome this resistance. We have previously shown that the IgG2 isotype of RTX induces CD20-mediated apoptosis in B-cell lymphoma cells and, when combined with RTX-IgG1 or RTX-IgG3 mAbs, can significantly enhance Fc receptor-mediated phagocytosis. Here, we report that the apoptotic effect of RTX-IgG2 on lymphoma cells contributes to changes in the tumor cell's CD47 profile by reducing its overall expression and altering its surface distribution. Furthermore, when RTX-IgG2 is combined with other lymphoma-targeting mAbs, such as anti-CD59 or anti-PD-L1, it significantly enhances the ADCP of lymphoma cells compared to single mAb treatment. In summary, RTX-IgG2 acts as a potent phagocytic enhancer by promoting Fc-receptor mediated phagocytosis through apoptosis and reduction of CD47 in CD20+ malignant B-cells. RTX-IgG2 represents a valuable therapeutic component in enhancing the effectiveness of different mAbs targeting B-cell NHL.

ICU patient-on-a-chip emulating orchestration of mast cells and cerebral organoids in neuroinflammation

Propofol and midazolam are the current standard of care for prolonged sedation in Intensive Care Units (ICUs). However, the effects and mechanism of these sedatives in brain tissue are unclear. Herein, the development of an ICU patient-on-a-chip platform to elucidate those effects is reported. The humanized neural tissue compartment combines mast cells differentiated from human induced pluripotent stem cells (hiPSCs) with cerebral organoids in a three-dimensional (3D) matrix, which is covered with a membrane populated with human cerebral microvascular endothelial cells (hCMEC/D3) that separates the tissue chamber from the vascular lumen, where sedatives were infused for four days to evaluate neurotoxicity and cell-mediated immune responses. Subsequent to propofol administration, gene expressions of CD40 and TNF-α in mast cells, AIF1 in microglia and GFAP/S100B/OLIG2/MBP in macroglia were elevated, as well as NOS2, CD80, CD40, CD68, IL6 and TNF-α mediated proinflammation is noted in cerebral organoids, which resulted in higher expressions of GJB1, GABA-A and NMDAR1 in the tissue construct of the platform. Besides, midazolam administration stimulated expression of CD40 and CD203c+ reactivated mast cell proliferation and compromised BBB permeability and decreased TEER values with higher barrier disruption, whereas increased populations of CD11b+ microglia, higher expressions of GFAP/DLG4/GJB1 and GABA-A-/NMDAR1- identities, as well as glutamate related neurotoxicity and IL1B, IFNG, IFNA1, IL6 genes mediated proinflammation, resulting in increased apoptotic zones are observed in cerebral organoids. These results suggest that different sedatives cause variations in cell type activation that modulate different pathways related to neuroinflammation and neurotoxicity in the ICU patient-on-chip platform.

Inhibiting intracellular CD28 in cancer cells enhances antitumor immunity and overcomes anti-PD-1 resistance via targeting PD-L1.

Deciphering mechanisms for cancer immune escape may provide targets for improving immunotherapy efficacy. By invivo genome-wide CRISPR loss-of-function screening in a mouse model of triple negative breast cancer (TNBC), we uncovered a non-classical function of Cd28 in cancer cells to promote immune escape. Knocking out Cd28 in cancer cells increased infiltration of type I conventional DC (cDC1) and activated tumor-specific CD8+ Tcells, and pharmaceutical inducible knockdown of Cd28 inhibited pre-established tumor growth and overcame anti-PD-1 resistance invivo. Furthermore, high expression of cancer cell CD28 in human TNBC tissues correlated with elevated PD-L1 expression, less CD8+ Tcell infiltration, and poor prognosis. Mechanistically, intracellular CD28 directly bound to Cd274 mRNA and recruited spliceosomal factor SNRPB2 to stabilize Cd274 mRNA in nucleus, promoting PD-L1 expression and immune escape. Therefore, disrupting cancer cell CD28-mediated immune escape may provide a potential approach to improve breast cancer immunotherapy.

CD2 expressing innate lymphoid and T cells are critical effectors of immunopathogenesis in hidradenitis suppurativa

Hidradenitis suppurativa (HS) is a chronic, debilitating inflammatory skin disease with a poorly understood immunopathogenesis. Here, we report that HS lesional skin is characterized by the expansion of innate lymphocytes and T cells expressing CD2, an essential activation receptor and adhesion molecule. Lymphocytes expressing elevated CD2 predominated with unique spatial distribution throughout the epidermis and hypodermis in the HS lesion. CD2+ cells were mainly innate lymphocytes expressing the NK cell marker, CD56, and CD4+ T cells. Importantly, these CD2+ cells interacted with CD58 (LFA3) expressing epidermal keratinocytes and fibroblasts in the hypodermis. Granzyme Abright NKT cells (CD2+CD3+CD56bright) clustered with α-SMA expressing fibroblasts juxtaposed to epithelialized tunnels and fibrotic regions of the hypodermis. Whereas NK cells (CD2+CD56dim) were perforin+, granzymes A+ and B+, and enriched adjacent to hyperplastic follicular epidermis and tunnels of HS showing presence of apoptotic cells. The cytokines IL-12, IL-15, and IL-18, which enhance NK cell maturation and function were significantly elevated in HS. Ex vivo HS skin explant cultures treated with CD2:CD58 interaction-blocking anti-CD2 monoclonal antibody attenuated secretion of inflammatory cytokines/chemokines and suppressed inflammatory gene signature. Additionally, CD2:CD58 blockade altered miRNAs involved in NK/NKT differentiation and/or function. In summary, we show that a cellular network of heterogenous NKT and NK cell populations drives inflammation and is critical in the pathobiology of HS, including tunnel formation and fibrosis. Finally, CD2 blockade is a viable immunotherapeutic approach for the effective management of HS.

Study on the correlation between viral load and activation and exhaustion levels of CD8+T cells in HIV/AIDS patients

Objective: To investigate and analyze the correlation between the expression levels of CD38, HLA-DR and programmed cell death-1 (PD-1) on peripheral blood CD8+T cells and HIV-1 RNA viral load, immune activation and exhaustion in patients with human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS). Methods: A total of 81 HIV/AIDS patients (64 without antiretroviral therapy and 17 with therapy) and 40 healthy donors in the same period were enrolled as the control group. Flow cytometry was used to analyze the CD4+T lymphocyte count and the expression levels of activation markers CD38 and HLA-DR and apoptosis marker PD-1 on CD8+T cells. HIV-1 RNA in the plasma of HIV-1 infected patients was quantitatively detected by real-time fluorescence quantitative polymerase chain reaction. Variance analysis was used to compare the expression levels of CD38, HLA-DR and PD-1 on CD8+T cells between HIV/AIDS patients and healthy controls. Spearman correlation analysis was used to analyze the correlation between different T lymphocyte counts and HIV RNA viral load, and the correlation between HIV RNA viral load and peripheral blood CD8+T cell CD38, HLA-DR and PD-1. Results: Among the 81 HIV/AIDS patients, 69 (85.19%) were males and 12 (14.81%) were females, with an age M (Q1, Q3) of 58 (36.5, 65.0) years. There were 60 HIV/AIDS patients over 55 years old (74.07%) and 21 HIV/AIDS patients between 18 and 55 years old (25.93%). The results of variance analysis showed that compared with the healthy control group, the expression levels of CD38, HLA-DR and PD-1 on CD8+T cells in HIV/AIDS patients increased, and the differences were statistically significant (all P<0.05). In addition, the expression of CD38, HLA-DR and PD-1 increased significantly in patients with CD4+T cell count less than 350 cells/μl, and the differences were statistically significant (all P<0.05). Spearman correlation analysis showed that CD4+and CD4+/CD8+were negatively correlated with viral load in HIV/AIDS patients (r=-0.407 and -0.378, respectively, both P<0.05), and CD8+was positively correlated with viral load (r=0.356, P<0.05). When the HIV RNA level was≤105 CPs/ml, there was no correlation between the HIV RNA level and the expression levels of CD38, HLA-DR and PD-1 on CD8+T cells (all P>0.05). However, when the level of HIV RNA was>105 CPs/ml, the level of HIV RNA was positively correlated with the expression levels of CD38, HLA-DR and PD-1 on CD8+T cells (r=0.412, 0.387, 0.395, respectively, all P<0.05). Conclusions: The activation levels of CD38 and HLA-DR and the expression of PD-1 on CD8+T cells in the peripheral blood of HIV/AIDS patients are increased. When the viral load is high, the HIV RNA viral load is positively correlated with the activation and exhaustion levels of CD8+T cells.

Bispecific killer engager for targeted depletion of PD-1 positive lymphocytes: A new avenue for autoimmune disease treatment

Bispecific killer cell engagers (BiKEs) are a powerful tool to incite the killing power of natural killer (NK) cells. Here, we posited that the BiKE technology could be utilized to deplete activated immune cells expressing programmed death-1 (PD-1+ cells), and hence treat autoimmune diseases since these cells drive the disorders. We designed and generated PD-1 BiKE that targets an activating NK cell receptor, CD16, and PD-1. PD-1 BiKE showed specific binding to PD-1+ cells and engaged CD16 simultaneously. PD-1 BiKE enhanced NK cell-mediated apoptosis and depletion of PD-1+ Raji cells, but not PD-1- Raji cells. Further, PD-1 BiKE induced apoptosis of primary PD-1+ T lymphocytes that are highly relevant to autoimmune disease progression. The BiKE depleted 42% of primary T cells that were stimulated in vitro. Importantly, those ablated primary T cells were activated cells. Meanwhile, naive T cells were spared by the BiKE treatment, supporting the crucial selectivity of PD-1 BiKE-directed cell depletion. Lastly, PD-1 BiKE is more effective than a conventional depleting antibody in the depletion of PD-1+ cells. The current work supports PD-1 BiKE is a selective, potent, and safe tool to deplete PD-1+ cells.

Genomes and epigenomes of matched normal and tumor breast tissue reveal diverse evolutionary trajectories and tumor-host interactions

Normal tissues adjacent to the tumor (NATs) may harbor early breast carcinogenesis events driven by field cancerization. Although previous studies have characterized copy-number (CN) and transcriptomic alterations, the evolutionary history of NATs in breast cancer (BC) remains poorly characterized. Utilizing whole-genome sequencing (WGS), methylation profiling, and RNA sequencing (RNA-seq), we analyzed paired germline, NATs, and tumor samples from 43 individuals with BC in Hong Kong (HK). We found that single-nucleotide variants (SNVs) were common in NATs, with one-third of NAT samples exhibiting SNVs in driver genes, many of which were present in paired tumor samples. The most frequently mutated genes in both tumor and NAT samples were PIK3CA, TP53, GATA3, and AKT1. In contrast, large-scale aberrations such as somatic CN alterations (SCNAs) and structural variants (SVs) were rarely detected in NAT samples. We generated phylogenetic trees to investigate the evolutionary history of paired NAT and tumor samples. They could be categorized into tumor only, shared, and multiple-tree groups, the last of which is concordant with non-genetic field cancerization. These groups exhibited distinct genomic and epigenomic characteristics in both NAT and tumor samples.Specifically, NAT samples in the shared-tree group showed higher number of mutations, while NAT samples belonging to the multiple-tree group showed a less inflammatory tumor microenvironment (TME), characterized by a higher proportion ofregulatory Tcells (Tregs) and lower presence of CD14 cell populations. In summary, our findings highlight the diverse evolutionary history in BC NAT/tumor pairs and the impact of field cancerization and TME in shaping the genomic evolutionary historyof tumors.

Immunomodulatory role of Trichinella spiralis-derived antigen on imiquimod-induced psoriasis in mice model.

The immunomodulatory activity of parasites has been extensively investigated in multiple immune-related diseases. However, dermatological diseases have been off the list for a long time despite their vast incidence and the deleterious consequences of some of them. This study explored the immunomodulatory role of autoclaved Trichinella spiralis (T. spiralis) larvae antigen (ATSLA) as a psoriasis immunotherapeutic candidate in a mice model. Psoriasis was induced in Swiss albino mice using commercial imiquimod cream (IMQ). Mice were randomly divided into the IMQ untreated control group and the IMQ treated group that was treated with ATSLA twice, on day 0 and day 3. Additional mice served as normal controls. Assessment of skin thickness, erythema, and scales was recorded. Total skin scores were calculated. Skin MDA levels, splenic indices, serum and skin IL-23, and tumor necrosis factor alpha (TNF-α) were measured. Skin sections were stained with H&E and immune stained for CD68-positive cells using immunohistochemistry. Treatment with ATSLA significantly reduced skin thickness, erythema, scales, and total skin scores in the IMQ-treated group compared to the untreated control. This was accompanied by a reduction in the splenic index, skin MDA levels, IL-23, and TNF-α in both the skin and serum of the treated group. Pathologically, skin sections of the treated group showed less epidermal thickness, acanthosis, hyperkeratosis, and CD68 cell count. The study concluded the immunotherapeutic activity of ATSLA in experimental psoriatic skin lesions. This will enrich the psoriasis immunotherapeutic list with novel candidates of parasitic origin.

Expression of EGFRvIII and its co‑expression with wild‑type EGFR, or putative cancer stem cell biomarkers CD44 or EpCAM are associated with poorer prognosis in patients with hepatocellular carcinoma.

The aberrant expression of HER family members and cancer stem cells (CSCs) have been associated with tumour progression and resistance to therapy. At present, several HER inhibitors have been approved for the treatment of patients with a range of cancers but not for the treatment of patients with hepatocellular carcinoma (HCC). The present study investigated the co‑expression and prognostic significance of HER family members, type‑III deletion mutant EGFR (EGFRvIII), and the putative CSC biomarkers CD44 and epithelial cell adhesion molecule (EpCAM) in 43 patients with HCC. The relative expression of these biomarkers was determined using immunohistochemistry. At a cut off value of >5% of tumour cells stained for these biomarkers, 35% [wild‑type (wt)EGFR], 58% (HER‑2), 0% (HER‑3), 19% (HER‑4), 26% (EGFRvIII), 40% (CD44) and 33% (EpCAM) of patients were positive. In 23, 14 and 9% of the patients, wtEGFR expression was accompanied by co‑expression with HER‑2, EGFRvIII and HER‑2/EGFRvIII, respectively. EGFRvIII expression, membranous expression of CD44 and co‑expression of wtEGFR/EGFRvIII were associated with poor overall survival (OS). By contrast, cytoplasmic CD44 expression was associated with a longer OS time. The present study also investigated the effect of several agents targeting one or more members of the HER family, other growth factor receptors and cell signalling proteins on the proliferation of HCC cell lines. Among agents targeting one or more members of the HER family, the pan‑HER family blocker afatinib was the most effective, inhibiting the proliferation of three out of seven human liver cancer cell lines (LCCLs), while the CDK inhibitor dinacicilib was the most effective agent, inhibiting the proliferation of all human LCCLs tested. Taken together, the present results suggested that EGFRvIII expression and its co‑expression with wtEGFR or CD44 was of prognostic significance. These results also support further investigations of the therapeutic potential of drugs targeting EGFRvIII and other members of the HER family in patients with HCC.

CD36 cell surface expression as a surrogate marker to identify ABL/JAK-class kinase fusions in pediatric BCP-ALL.

Genetic alterations are the cornerstone of risk stratification in B-cell precursor acute lymphoblastic leukemia (BCP-ALL), and their accurate identification is critical for optimal treatment. Most cases with ABL-class fusion are classified as high-risk yet display good responses to tyrosine kinase inhibitors (TKIs). Current clinical protocols recommend adding a TKI to chemotherapy as soon as possible, making it mandatory to rapidly identify these alterations. We investigated here whether the identification of immunophenotypic features associated with these molecular alterations could be a valuable screening tool. CD36 expression was shown to be a characteristic feature of ABL- or JAK-class kinase fusions. The main genetic subgroups clustering in the subset with Philadelphia (Ph)-like features were also found to display specific immunophenotypic characteristics. A predictive multiparameter scoring system was generated, segregating genetic subtypes with aberrant kinase activation (PAX5/CRLF2alt, BCR::ABL1, ABL/JAK-class). The most robust markers identified were the TSLPR with CD19/22/9/38/81/304 and CD49f. As TKI adjunction is currently limited to the ABL-class kinase fusions, immunophenotypes distinguishing ABL from JAK-class were also investigated. The flow cytometry method reported here, accessible to most hematology departments, is thus a new useful tool to quickly screen for Ph-like kinase fusion with a good sensitivity (95%) and specificity (96%).

Effects of dietary Lactobacillus postbiotics and bacitracin on the modulation of mucosa-associated microbiota and pattern recognition receptors affecting immunocompetence of jejunal mucosa in pigs challenged with enterotoxigenic F18+ Escherichia coli

BackgroundEnterotoxigenic Escherichia coli (E. coli) is a threat to humans and animals that causes intestinal disorders. Antimicrobial resistance has urged alternatives, including Lactobacillus postbiotics, to mitigate the effects of enterotoxigenic E. coli.MethodsForty-eight newly weaned pigs were allotted to NC: no challenge/no supplement; PC: F18+ E. coli challenge/no supplement; ATB: F18+ E. coli challenge/bacitracin; and LPB: F18+ E. coli challenge/postbiotics and fed diets for 28 d. On d 7, pigs were orally inoculated with F18+ E. coli. At d 28, the mucosa-associated microbiota, immune and oxidative stress status, intestinal morphology, the gene expression of pattern recognition receptors (PRR), and intestinal barrier function were measured. Data were analyzed using the MIXED procedure in SAS 9.4.ResultsPC increased (P < 0.05) Helicobacter mastomyrinus whereas reduced (P < 0.05) Prevotella copri and P. stercorea compared to NC. The LPB increased (P < 0.05) P. stercorea and Dialister succinatiphilus compared with PC. The ATB increased (P < 0.05) Propionibacterium acnes, Corynebacterium glutamicum, and Sphingomonas pseudosanguinis compared to PC. The PC tended to reduce (P = 0.054) PGLYRP4 and increased (P < 0.05) TLR4, CD14, MDA, and crypt cell proliferation compared with NC. The ATB reduced (P < 0.05) NOD1 compared with PC. The LPB increased (P < 0.05) PGLYRP4, and interferon-γ and reduced (P < 0.05) NOD1 compared with PC. The ATB and LPB reduced (P < 0.05) TNF-α and MDA compared with PC.ConclusionsThe F18+ E. coli challenge compromised intestinal health. Bacitracin increased beneficial bacteria showing a trend towards increasing the intestinal barrier function, possibly by reducing the expression of PRR genes. Lactobacillus postbiotics enhanced the immunocompetence of nursery pigs by increasing the expression of interferon-γ and PGLYRP4, and by reducing TLR4, NOD1, and CD14.

Investigation of local stimulation effects of embedding PGLA at Zusanli (ST36) acupoint in rats based on TRPV2 and TRPV4 ion channels.

Acupoint Catgut Embedding (ACE) is an extended and developed form of traditional acupuncture that serves as a composite stimulation therapy for various diseases. However, its local stimulation effects on acupoints remain unclear. Acupuncture can activate mechanically sensitive calcium ion channels, TRPV2 and TRPV4, located on various cell membranes, promoting Ca2+ influx in acupoint tissues to exert effects. Whether ACE can form mechanical physical stimulation to regulate these channels and the related linkage effect requires validation. This study investigates the influence of TRPV2 and TRPV4 ion channels on the local stimulation effects of ACE by embedding PGLA suture at the Zusanli (ST36) acupoint in rats and using TRPV2 and TRPV4 inhibitors. Flow cytometry, immunofluorescence, Western blot, and Real-time quantitative PCR were employed to detect intracellular Ca2+ fluorescence intensity, the expression of macrophage (Mac) CD68 and mast cell (MC) tryptase, as well as the protein and mRNA expression of TRPV2 and TRPV4 in acupoint tissues after PGLA embedding. The results indicate that ACE using PGLA suture significantly increases the mRNA and protein expression of TRPV2 and TRPV4, Ca2+ fluorescence intensity, and the expression of Mac CD68 and MC tryptase in acupoint tissues, with these effects diminishing over time. The increasing trends are reduced after using inhibitors, particularly when both inhibitors are used simultaneously. Furthermore, correlation analysis shows that embedding PGLA suture at the ST36 acupoint regulates Mac and MC functions through Ca2+ signaling involving not only TRPV2 and TRPV4 but multiple pathways. These results suggest that embedding PGLA suture at the ST36 acupoint generates mechanical physical stimulation and regulates TRPV2 and TRPV4 ion channels, which couple with Ca2+ signaling to form a linkage effect that gradually weakens over time. This provides new reference data for further studies on the stimulation effects and clinical promotion of ACE.

Investigation of TLR4, TLR6, TLR7, and CD36 expression on T lymphocytes in coronary artery disease

Background: Coronary artery disease (CAD) is among the significant causes of death globally, caused by fatty deposits in blood vessel walls. Increasing evidence indicates that toll-like receptors (TLRs) are pivotal to atherosclerosis progression. The function of CD36 as a glycoprotein in atherosclerosis was also suggested. This study aimed to investigate the levels of TLR4, TLR6, and TLR7, as well as CD36 cell surface markers in CAD. Methods: This study included 64 patients undergoing angiography to determine whether atherosclerosis was present. The patients were categorized into two groups. The three main coronary arteries are all stenosed at over 50% in patients with CAD+ (n=22) as cases and CAD− (n=42) with smooth angiography as controls. Each patient's syntax score (SS) and Gensini (vessel score) were calculated. The TLR-4, TLR-6, TLR-7, and CD36 expression were measured using Flow cytometry. Results: TLR4 and TLR6 showed a significant link with the Gensini score in the subject group. In addition, TLR7 had an association with Syntax scoring in this study. Conclusion: The findings illustrated the association between TLR4, TLR6, and TLR7 cell surface markers with Gensini and Syntax scoring in individuals with coronary artery disease.

Tannic acid elicits differential gene regulation in prostate cancer apoptosis.

Prostate cancer is a significant global health concern that requires innovative therapeutic investigations. Here, the potential anticancer properties of tannic acid were evaluated by examining its effects on apoptosis in prostate cancer cell lines. PC-3 and LnCaP prostate adeno carcinoma cells, along with PNT1A prostate control cells, were cultured and divided into untreated and tannic acid-treated groups. Cell proliferation, cytotoxicity, and effects of tannic acid on the cell death mechanism were evaluated. mRNA expression levels of 84 genes were explored in cells following tannic acid treatment. Notably, tannic acid-induced down-regulation of several pro-survival genes, including ATM, BCL2, BCL2A1, BIK, BIRC2, BIRC3, BRE, CASP3, CASP6, CASP8, CHEK2, CRADD, PPIA, RPA3, TNFSF18, TRAF1, TRAF2, TRAF4, and TRAF5 in both cell lines. Moreover, tannic acid treatment led to the up-regulation of various pro-apoptotic genes, such as BCL10, BIRC3, BNIP3, CASP1, CASP5, CD40, CIDEB, DAPK2, FASLG, GADD45A, MYD88, RPA 3, TNFRSF10D, TNFRSF17, TNFRSF8, TNFSF13B, TNFSF4, TNFSF7, TNFSF8, TNFSF9, TP53, TRAF1, and TRAF2 in both PC-3 and LnCap cells. These findings highlight tannic acid's ability to induce apoptosis in prostate cancer cells through pro-apoptotic pathways. This study concludes that tannic acid selectively inhibits prostate cancer cell growth.

Abstract P390: The Adipokine Chemerin Does Not Promote Activation or Cytokine Secretion by T Cells Stimulated With a Strong Polyclonal Activator

High fat diet has long been implicated as a risk factor for obesity and other conditions associated with metabolic syndrome, including hypertension. The mechanism by which high fat diet increases risk of hypertension is likely multifactorial, but corresponding low-grade inflammation may play an important role. Low-grade inflammation associated with high fat diet is often accompanied by altered adipokine levels, including an increase in circulating chemerin. However, our understanding of the effects of chemerin and other adipokines on immune cells is limited. Thus, the purpose of these studies was to test the hypothesis that the adipokine chemerin promotes CD4 and CD8 T cell activation and cytokine secretion. To test this hypothesis, primary mouse splenocytes were treated with chemerin or vehicle 30 min prior to activation with a T cell-specific activator (anti-CD3/anti-CD28) for 24 h. T cell activation causes a rapid upregulation of cell surface proteins that are commonly used as experimental markers of activation. We found that chemerin did not impact the induction of the T cell activation markers, CD25, CD69, CD44, CD134, CD137 and CD154, in either CD4 or CD8 T cells. Likewise, we did not see a difference in the downregulation of CD62L, which is rapidly internalized by T cells following activation. Consistent with this, chemerin caused no difference in cytokine secretion by activated T cells. Specifically, the concentrations of IL-2, IL-17A, IFNγ, and TNFα were similar between groups. Overall, this study shows that the adipokine chemerin does not promote acute changes in activation or cytokine expression in primary mouse splenic T cells fully activated with anti-CD3/anti-CD28.

CD11b/CD86 involved in the microenvironment of colorectal cancer by promoting Wnt signaling activation.

Colorectal cancer (CRC) is a malignancy that arises within the gastrointestinal tract. Despite ongoing research, the etiology and pathogenesis of CRC remain elusive; particularly, the distribution and characteristics of tumor-associated macrophages is currently an active area of investigation in understanding the pathological progression and prevention of CRC. This study utilized CRC patient surgical samples, mouse models of colitis-associated cancer, colonic organoid, and co-culture cell line to examine the changes in CD11b/CD86 at different pathological region and detect the Wnt signaling pathway activity. Our findings revealed a sensitive and increased expression of CD11b from the early to the advanced CRC tissues and correlated with poor prognosis, while CD86 expression was reduced in advanced CRC tissues. CD133 expression was also elevated in advanced CRC tissues and mainly co-localized with CD11b, suggesting a positive regulatory effect of CD11b and CD133 expression that may contribute to CRC progression. In AOM/DSS mouse models, activation of the Wnt signaling pathway was associated with increased CD133 and CD11b expression. Invitro, THP-1 cell was induced to high expression of CD11b, and the above conditional cultural medium enhanced HCT116 cell colony number and CD133 protein expression. Furthermore, colonic crypts from AOM/DSS mouse models were isolated to culture, and the colonic organoids exhibited dilation and significant increases expression of CD133 and β-Catenin/N-P-B-Catenin. CD11b might be an important factor to participate the progress of CRC. And the high CD11b of CRC microenviroment might potentially promote CD133 expression and associate with Wnt signal activation.