Abundance Of Cells Research Articles

Binding of interleukin-1 receptor antagonist to cholinergic receptor muscarinic 4 promotes immunosuppression and neuroendocrine differentiation in prostate cancer

The tumor microenvironment (TME) of prostate cancer (PCa) is characterized by high levels of immunosuppressive molecules, including cytokines and chemokines. This creates a hostile immune landscape that impedes effective immune responses. The interleukin-1 (IL-1) receptor antagonist (IL1RN), a key anti-inflammatory molecule, plays a significant role in suppressing IL-1-related immune and inflammatory responses. Our research investigates the oncogenic role of IL1RN in PCa, particularly its interactions with muscarinic acetylcholine receptor 4 (CHRM4), and its involvement in driving immunosuppressive pathways and M2-like macrophage polarization within the PCa TME. We demonstrate that following androgen deprivation therapy (ADT), the IL1RN-CHRM4 interaction in PCa activates the MAPK/AKT signaling pathway. This activation upregulates the transcription factors E2F1 and MYCN, stimulating IL1RN production and creating a positive feedback loop that increases CHRM4 abundance in both PCa cells and M2-like macrophages. This ADT-driven IL1RN/CHRM4 axis significantly enhances immune checkpoint markers associated with neuroendocrine differentiation and treatment-resistant outcomes. Higher serum IL1RN levels are associated with increased disease aggressiveness and M2-like macrophage markers in advanced PCa patients. Additionally, elevated IL1RN levels correlate with better clinical outcomes following immunotherapy. Clinical correlations between IL1RN and CHRM4 expression in advanced PCa patients and neuroendocrine PCa organoid models highlight their potential as therapeutic targets. Our data suggest that targeting the IL1RN/CHRM4 signaling could be a promising strategy for managing PCa progression and enhancing treatment responses.

FKBP3, a poor prognostic indicator, promotes the progression of LUAD via regulating ferroptosis and immune infiltration.

Ferroptosis was reported to possess the therapeutic potentials in various human cancers. In the present study, we explored the expression, clinical significance and the molecular mechanism of FK506 binding protein 3 (FKBP3) in the progression of lung adenocarcinoma (LUAD). Cox regression was performed to obtain the prognosis related to differentially expressed genes (DEGs) in LUAD datasets from TCGA. We also downloaded the ferroptosis-related gene datasets from GeneCards. Venn diagram was performed to find the intersecting genes and FKBP3 was selected as the targeted gene by analyzing the diagnostic and prognostic values of Top10 intersecting genes. Moreover, univariate and multivariate analyses were performed to evaluate the association between clinicopathological factors and survival rates. GO/KEGG and GSEA analysis was performed to explore the function of FKBP3 in LUAD progression. Protein-protein interaction (PPI) network was performed via STRING database and the top10 hub genes were selected. Finally, the relationship between FKBP3 and immune infiltration was explored by ssGSEA analysis. Firstly, 184 genes associated with the prognosis of LUAD and ferroptosis were obtained. FKBP3 was found to be significantly associated with a poor overall survival rate of LUAD patients. Immunohistochemical staining results showed that FKBP3 was highly located in cytoplasm and membrane of cells in LUAD tissues. PPI network analysis results showed that HDAC1, YY1, HDAC2, MTOR, PSMA3, PIN1, NCL, C14orf166, PIN4, and LARP6 were the top10 hub genes. Furthermore, spearman analysis results showed that the expression of FKBP3 was positively correlated with the abundance of Th2 cells and T helper cells. High level of FKBP3 was associated with poor prognostic outcomes of LUAD patients, which also inhibited immune infiltration in LUAD tissues. Additionally, FKBP3 was involved in regulating the ferroptosis process in LUAD patients. Thus, FKBP3 possessed the tumor promotion role might be involving in regulating ferroptosis and immune infiltration in LUAD progression.

Long-term follow-up of levonorgestrel intrauterine device for atypical hyperplasia and early endometrial cancer reveals relapse characterized by immune exhaustion.

Nonsurgical treatment options are increasingly needed for endometrial atypical hyperplasia (AH) and endometrioid endometrial cancer (EEC). Despite promising initial response rates, prospective long-term data and determinants for relapse are limited. Follow-up data from patients in our prospective phase II trial of LIUD for AH/G1EEC were collected from medical records. Spatial transcriptomics (Nanostring GeoMX digital spatial profiling) with in silico cell type deconvolution and pathway analyses were employed on longitudinal biopsy samples from five patients across pre-treatment, on-treatment, and relapse. Of 43 participants exhibiting initial response to LIUD, 41 had follow-up data. Sixteen (39%) experienced relapse. Clinical factors associated with shorter response duration included younger age, initial diagnosis of G1EEC, lack of response at six months, premenopausal status, and Hispanic ethnicity (p<0.05), but only six-month response status remained a significant predictor in a multivariate model (p=0.023). LIUD increased abundance of NK cells (DMCP-counter score=46.13, FDR=0.004) and cytotoxic lymphocytes (DMCP-counter score=277.67, FDR=0.004), as well as lymphocyte cytotoxicity markers PRF1 (log2FC=1.62, FDR=0.025) and GZMA (log2FC=2.47, FDR=0.008). NK cells were reduced at relapse (DMCP-counter score=-55.96, FDR=0.02). Immune-related pathways (IFNα-response and TGFβ-signaling) were enriched at relapse (FDR<0.05). IDO1 expression, reflecting immune exhaustion, was upregulated at relapse (FDR<0.05). Upfront resistance and relapse after initial response to LIUD for AH/G1EEC impacts nearly half of patients, remaining a major hurdle for non-surgical treatment of AH/G1EEC. Molecular studies evaluating longitudinal biopsies from a small cohort implicate immune mechanisms at relapse, including reversal of progestin-related immunomodulation and increased immune exhaustion.

Antibody-mediated immunological memory correlates with long-term Lyme veterinary vaccine protection in mice

Lyme disease, caused by the bacterium Borrelia burgdorferi, is the most common tick-borne illness in the United States. Despite the rise in Lyme disease incidence, there is no vaccine against B. burgdorferi approved for human use. Little is known about the immune correlates of protection needed to prevent Lyme disease. In this work, a mouse model was used to characterize the immune response and compare the protection provided by two USDA-approved vaccines for use in canines: Duramune (bacterin vaccine) and Vanguard crLyme (subunit vaccine composed of two outer surface proteins, OspA and OspC). C3H/HeNCrl mice were immunized with two doses of either Duramune or Vanguard, and immune responses and protection against B. burgdorferi were assessed in short (35 days) and long-term (120 days) studies. Flow cytometry, ELISPOT detection of antibody-producing cells, and antibody affinity studies were performed to identify correlates of vaccine-mediated protection. Both vaccines induced humoral responses, with high IgG titers against B. burgdorferi. However, the levels of anti-B. burgdorferi antibodies decayed over time in Vanguard-vaccinated mice. While both vaccines triggered the production of antibodies against both OspA and OspC, antibody levels against these proteins were also lower in Vanguard-vaccinated mice 120 days post-vaccination. Both vaccines only provided partial protection against B. burgdorferi at the dose used in this model. The protection provided by Duramune was superior to Vanguard 120 days post-vaccination, and was characterized by higher antibody titers, higher abundance of long-lived plasma cells, and higher avidity antibodies than Vanguard. Overall, these studies provide insights into the importance of the humoral memory response to veterinary vaccines against Lyme disease and will help inform the development of future human vaccines.

Uptake of dissolved inorganic nitrogen and N2 fixation by Crocosphaera watsonii under climate change scenarios

The response of N2 fixation to projected future conditions in the ocean cannot be reliably predicted to date. We conducted a minicosm experiment with pre-acclimated cultures of the globally significant diazotroph Crocosphaera watsonii strain WH8501 (“Crocosphaera”). PH and temperature were altered simultaneously to match the RCP scenarios 4.5 and 6 and investigate a more realistic future scenario compared to studies that focus on changes of a single stressor only. The cell abundance and nitrogen metabolism of Crocosphaera was monitored over 5 days. Our results imply that Crocosphaera is able to simultaneously perform N2 fixation and assimilate dissolved inorganic nitrogen (DIN, i.e., nitrate and ammonium) under all the conditions tested and implies a competition with non-diazotrophic phytoplankton for DIN, which should be further investigated. Using NanoSIMS analysis of single cells, our results point towards a preference for DIN assimilation over N2 fixation under more acidic and warmer conditions. Overall, our results show that while the combined alteration of pH and temperature had a negative effect on the diazotroph’s growth and N2 fixation, Crocosphaera is likely to cope well with conditions in the future ocean. The high intra-population variability in nitrogen assimilation pathways may give this species the flexibility to quickly react to environmental changes.

Network-Based Transcriptome Analysis Reveals FAM3C as a Novel Potential Biomarker for Glioblastoma.

Glioblastoma (GBM) is the most common form of malignant primary brain tumor with a high mortality rate. The aim of the present study was to investigate the clinical significance of Family with Sequence Similarity 3, Member C, FAM3C, in GBM using bioinformatic-integrated analysis. First, we performed the transcriptomic integration analysis to assess the expression profile of FAM3C in GBM using several data sets (RNA-sequencingand scRNA-sequencing), which were obtained from TCGA and GEO databases. By using theSTRING platform, we investigated FAM3C-coregulated genes to construct the protein-protein interaction network. Next,Metascape, Enrichr, and CIBERSORT databases were used. We found FAM3C high expression in GBM with poor survival rates. Further, we observed, via FAM3C coexpression network analysis, that FAM3C plays key roles in several hallmarks of cancer. Surprisingly, we also highlighted five FAM3C‑coregulated genes overexpressed in GBM. Specifically, we demonstrated the association between the high expression of FAM3C and the abundance of the different immune cells, which may markedly worsen GBM prognosis. For the first time, our findings suggest that FAM3C not only can be a new emerging biomarker with promising therapeutic values to GBM patientsbut also gavea new insight into a potential resource for future GBM studies.

Natural Killer Cell Presence in Antibody-Mediated Rejection.

Transcript analyses highlight an important contribution of natural killer (NK) cells to microvascular inflammation (MVI) in antibody-mediated rejection (ABMR), but only few immunohistologic studies have quantified their spatial distribution within graft tissue. This study included 86 kidney transplant recipients who underwent allograft biopsies for a positive donor-specific antibody (DSA) result. NK cells were visualized and quantified within glomeruli and peritubular capillaries (PTC), using immunohistochemistry for CD34 alongside CD16/T-bet double-staining. Staining results were analyzed in relation to histomorphology, microarray analysis utilizing the Molecular Microscope Diagnostic System, functional NK cell genetics, and clinical outcomes. The number of NK cells in glomeruli per mm2 glomerular area (NKglom) and PTC per mm2 cortical area (NKPTC) was substantially higher in biopsies with ABMR compared to those without rejection, and correlated with MVI scores (NKglom Spearman's correlation coefficient [SCC] = 0.55, p < 0.001, NKPTC 0.69, p < 0.001). In parallel, NK cell counts correlated with molecular classifiers reflecting ABMR activity (ABMRprob: NKglom 0.59, NKPTC 0.75) and showed a trend towards higher levels in association with high functional FCGR3A and KLRC2 gene variants. Only NKPTC showed a marginally significant association with allograft function and survival. Our immunohistochemical results support the abundance of NK cells in DSA-positive ABMR.

Integrated profiling identifies DXS253E as a potential prognostic marker in colorectal cancer

BackgroundIncreasing evidence suggests that DXS253E is critical for cancer development and progression, but the function and potential mechanism of DXS253E in colorectal cancer (CRC) remain largely unknown. In this study, we evaluated the clinical significance and explored the underlying mechanism of DXS253E in CRC.MethodsDXS253E expression in cancer tissues was investigated using the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. The Kaplan-Meier plot was used to assess the prognosis of DXS253E. The cBioPortal, MethSurv, and Tumor Immune Estimation Resource (TIMER) databases were employed to analyze the mutation profile, methylation, and immune infiltration associated with DXS253E. The biological functions of DXS253E in CRC cells were determined by CCK-8 assay, plate cloning assay, Transwell assay, flow cytometry, lactate assay, western blot, and qRT-PCR.ResultsDXS253E was upregulated in CRC tissues and high DXS253E expression levels were correlated with poor survival in CRC patients. Our bioinformatics analyses showed that high DXS253E gene methylation levels were associated with the favorable prognosis of CRC patients. Furthermore, DXS253E levels were linked to the expression levels of several immunomodulatory genes and an abundance of immune cells. Mechanistically, the overexpression of DXS253E enhanced proliferation, migration, invasion, and the aerobic glycolysis of CRC cells through the AKT/mTOR pathway.ConclusionsWe demonstrated that DXS253E functions as a potential role in CRC progression and may serve as an indicator of outcomes and a therapeutic target for regulating the AKT/mTOR pathway in CRC.

Characteristics of phytoplankton assemblages and their relationship with environmental factors in the Qiongzhou Strait, China

The characteristics and influencing factors of phytoplankton assemblages were analyzed in July (rainy season) and November (dry season) 2022 in the Qiongzhou Strait, China. A total of 68 species of phytoplankton belonging to 33 genera and 3 phyla were recorded. Diatom was the most dominant group. The broad-temperature widespread species were the most abundant. The dominant species in the rainy season were Skeletonema costatum, S. tropicum, Chaetoceros preseudocurvisetus and Thalassionema nitzschioides while those in the dry season were T. frauenfeldii, T. nitzschioides and Coscinodiscus granii. Phytoplankton cell abundance was significantly positively correlated with nitrite and total nitrogen concentrations, and negatively correlated with temperature and pH. Red tide algae occurred with high frequency. All sites were at eutrophic level except for 3 sites in the rainy season, which were at mesotrophic level. Therefore, there is a potential risk of red tides in the Qiongzhou Strait, which requires long-term monitoring and precautions.

C-terminally phosphorylated p27 activates self-renewal driver genes to program cancer stem cell expansion, mammary hyperplasia and cancer

In many cancers, a stem-like cell subpopulation mediates tumor initiation, dissemination and drug resistance. Here, we report that cancer stem cell (CSC) abundance is transcriptionally regulated by C-terminally phosphorylated p27 (p27pT157pT198). Mechanistically, this arises through p27 co-recruitment with STAT3/CBP to gene regulators of CSC self-renewal including MYC, the Notch ligand JAG1, and ANGPTL4. p27pTpT/STAT3 also recruits a SIN3A/HDAC1 complex to co-repress the Pyk2 inhibitor, PTPN12. Pyk2, in turn, activates STAT3, creating a feed-forward loop increasing stem-like properties in vitro and tumor-initiating stem cells in vivo. The p27-activated gene profile is over-represented in STAT3 activated human breast cancers. Furthermore, mammary transgenic expression of phosphomimetic, cyclin-CDK-binding defective p27 (p27CK-DD) increases mammary duct branching morphogenesis, yielding hyperplasia and microinvasive cancers that can metastasize to liver, further supporting a role for p27pTpT in CSC expansion. Thus, p27pTpT interacts with STAT3, driving transcriptional programs governing stem cell expansion or maintenance in normal and cancer tissues.

Identification and validation of the role of ZNF281 in 5-fluorouracil chemotherapy of gastric cancer

BackgroundThe early diagnosis of gastric cancer (GC) and overcoming chemotherapy resistance is challenging. The aberrant expression of zinc finger protein 281 (ZNF281) and the over-activation of the Wnt/β-catenin pathway are oncogenic factors and confer tumor chemoresistance. ZNF281 modulates the Wnt/β-catenin pathway to influence malignant tumor behavior. However, the role of ZNF281 in GC chemotherapy and the relationship with the Wnt/β-catenin pathway have not been elucidated by researchers.MethodsWe explored differences in ZNF281 expression in Pan-cancer and normal tissues, the effect of its expression on prognosis of patients treated with 5-fluorouracil (5-FU). Cox regression was utilized to determine whether ZNF281 is an independent prognostic factor. Enrichment analysis was performed to explore the mechanism underlying ZNF281’s role in 5-FU treatment. We assessed the relationship between ZNF281 and the tumour microenvironment (TME) and combined bulk-RNA and single-cell RNA data to analyse the relationship between ZNF281 and immune infiltration. In vitro experiments verified the effects of ZNF281 knockdown on proliferation, invasion, migration, apoptosis, DNA damage of GC cells with 5-FU treated and the Wnt/β-catenin pathway proteins.ResultsZNF281 was highly expressed in seven cancers and correlates with the prognosis. It is an independent prognostic factor in 5-FU treatment. ZNF281 correlates with TME score, CD8T cell abundance. ZNF281 is primarily associated with DNA repair and the Wnt/β-catenin pathway. ZNF281 knockdown enhanced the effect of 5-FU on phenotypes of GC cells.ConclusionWe identified and verified ZNF281 as one of the potential influencing factors of 5-FU treatment in GC and may be associated with the Wnt/β-catenin pathway. Low ZNF281 may contribute to improved 5-FU sensitivity in GC patients.

Immune cell signatures and causal association with irritable bowel syndrome: A mendelian randomization study

BACKGROUND The mucosal barrier's immune-brain interactions, pivotal for neural development and function, are increasingly recognized for their potential causal and therapeutic relevance to irritable bowel syndrome (IBS). Prior studies linking immune inflammation with IBS have been inconsistent. To further elucidate this relationship, we conducted a Mendelian randomization (MR) analysis of 731 immune cell markers to dissect the influence of various immune phenotypes on IBS. Our goal was to deepen our understanding of the disrupted brain-gut axis in IBS and to identify novel therapeutic targets. AIM To leverage publicly available data to perform MR analysis on 731 immune cell markers and explore their impact on IBS. We aimed to uncover immunophenotypic associations with IBS that could inform future drug development and therapeutic strategies. METHODS We performed a comprehensive two-sample MR analysis to evaluate the causal relationship between immune cell markers and IBS. By utilizing genetic data from public databases, we examined the causal associations between 731 immune cell markers, encompassing median fluorescence intensity, relative cell abundance, absolute cell count, and morphological parameters, with IBS susceptibility. Sensitivity analyses were conducted to validate our findings and address potential heterogeneity and pleiotropy. RESULTS Bidirectional false discovery rate correction indicated no significant influence of IBS on immunophenotypes. However, our analysis revealed a causal impact of IBS on 30 out of 731 immune phenotypes (P &lt; 0.05). Nine immune phenotypes demonstrated a protective effect against IBS [inverse variance weighting (IVW) &lt; 0.05, odd ratio (OR) &lt; 1], while 21 others were associated with an increased risk of IBS onset (IVW ≥ 0.05, OR ≥ 1). CONCLUSION Our findings underscore a substantial genetic correlation between immune cell phenotypes and IBS, providing valuable insights into the pathophysiology of the condition. These results pave the way for the development of more precise biomarkers and targeted therapies for IBS. Furthermore, this research enriches our comprehension of immune cell roles in IBS pathogenesis, offering a foundation for more effective, personalized treatment approaches. These advancements hold promise for improving IBS patient quality of life and reducing the disease burden on individuals and their families.

N6-methyladenosine methylation regulates the tumor microenvironment of Epstein-Barr virus-associated gastric cancer.

N6-methyladenosine (m6A) methylation modification exists in Epstein-Barr virus (EBV) primary infection, latency, and lytic reactivation. It also modifies EBV latent genes and lytic genes. EBV-associated gastric cancer (EBVaGC) is a distinctive molecular subtype of GC. We hypothesized EBV and m6A methylation regulators interact with each other in EBVaGC to differentiate it from other types of GC. To investigate the mechanisms of m6A methylation regulators in EBVaGC to determine the differentiating factors from other types of GC. First, The Cancer Gene Atlas and Gene Expression Omnibus databases were used to analyze the expression pattern of m6A methylation regulators between EBVaGC and EBV-negative GC (EBVnGC). Second, we identified Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment of m6A-related differentially expressed genes. We quantified the relative abundance of immune cells and inflammatory factors in the tumor microenvironment (TME). Finally, cell counting kit-8 cell proliferation test, transwell test, and flow cytometry were used to verify the effect of insulin-like growth factor binding protein 1 (IGFBP1) in EBVaGC cell lines. m6A methylation regulators were involved in the occurrence and development of EBVaGC. Compared with EBVnGC, the expression levels of m6A methylation regulators Wilms tumor 1-associated protein, RNA binding motif protein 15B, CBL proto-oncogene like 1, leucine rich pentatricopeptide repeat containing, heterogeneous nuclear ribonucleoprotein A2B1, IGFBP1, and insulin-like growth factor 2 binding protein 1 were significantly downregulated in EBVaGC (P < 0.05). The overall survival rate of EBVaGC patients with a lower expression level of IGFBP1 was significantly higher (P = 0.046). GO and KEGG functional enrichment analyses showed that the immunity pathways were significantly activated and rich in immune cell infiltration in EBVaGC. Compared with EBVnGC, the infiltration of activated CD4+ T cells, activated CD8+ T cells, monocytes, activated dendritic cells, and plasmacytoid dendritic cells were significantly upregulated in EBVaGC (P < 0.001). In EBVaGC, the expression level of proinflammatory factors interleukin (IL)-17, IL-21, and interferon-γ and immunosuppressive factor IL-10 were significantly increased (P < 0.05). In vitro experiments demonstrated that the expression level of IGFBP1 was significantly lower in an EBVaGC cell line (SNU719) than in an EBVnGC cell line (AGS) (P < 0.05). IGFBP1 overexpression significantly attenuated proliferation and migration and promoted the apoptosis levels in SNU719. Interfering IGFBP1 significantly promoted proliferation and migration and attenuated the apoptosis levels in AGS. m6A regulators could remodel the TME of EBVaGC, which is classified as an immune-inflamed phenotype and referred to as a "hot" tumor. Among these regulators, we demonstrated that IGFBP1 affected proliferation, migration, and apoptosis.

An In vitro Model of Chlamydia trachomatis Infection in Human Dendritic Cells

Chlamydia trachomatis (CT) is an obligate intracellular bacterial pathogen known to cause trachoma, sexually transmitted infections, and reactive arthritis. The more serious sequelae of these diseases, blindness from trachoma and pelvic inflammatory disease from chlamydial STD are immunopathological responses to chronic infections. With the abundance of dendritic cells in CT infection sites and their central importance in immune regulation, the susceptibility of dendritic cells to CT infection was investigated in vitro. CT (serovar F) was grown and extracted from HeLa 229 cells for infection of human dendritic cells (DCs). Human DCs were generated from peripheral blood mononuclear cells in the presence of GM-CSF and IL-4. Infected DCs were harvested at 0, 4, 24, and 48 h after infection and immunostained. Results from fluorescence microscopy showed that DC can be infected and can support the growth of CT in vitro. Intracellular elementary and reticulate bodies of CT were detected, and reticulate bodies were shown to multiply over the 48 h-cycle in DC. At 48 h, intracellular elementary bodies were detected in some DCs. These results may provide new insights into the possible participation of DC in sustaining chronic or latent infection, or even dissemination of CT infection to other sites

Detailed Profiling of the Tumor Microenvironment in Ethnic Breast Cancer, Using Tissue Microarrays and Multiplex Immunofluorescence.

Breast cancer poses a global health challenge, yet the influence of ethnicity on the tumor microenvironment (TME) remains understudied. In this investigation, we examined immune cell infiltration in 230 breast cancer samples, emphasizing diverse ethnic populations. Leveraging tissue microarrays (TMAs) and core samples, we applied multiplex immunofluorescence (mIF) to dissect immune cell subtypes across TME regions. Our analysis revealed distinct immune cell distribution patterns, particularly enriched in aggressive molecular subtypes triple-negative and HER2-positive tumors. We observed significant correlations between immune cell abundance and key clinicopathological parameters, including tumor size, lymph node involvement, and patient overall survival. Notably, immune cell location within different TME regions showed varying correlations with clinicopathologic parameters. Additionally, ethnicities exhibited diverse distributions of cells, with certain ethnicities showing higher abundance compared to others. In TMA samples, patients of Chinese and Caribbean origin displayed significantly lower numbers of B cells, TAMs, and FOXP3-positive cells. These findings highlight the intricate interplay between immune cells and breast cancer progression, with implications for personalized treatment strategies. Moving forward, integrating advanced imaging techniques, and exploring immune cell heterogeneity in diverse ethnic cohorts can uncover novel immune signatures and guide tailored immunotherapeutic interventions, ultimately improving breast cancer management.

Effects of the commensal microbiota on spleen and mesenteric lymph node immune function: investigation in a germ-free piglet model.

Commensal microbial-host interaction is crucial for host metabolism, growth, development, and immunity. However, research on microbial-host immunity in large animal models has been limited. This study was conducted to investigate the effects of the commensal microbiota on immune function in two model groups: germ-free (GF) and specific-pathogen-free (SPF) piglets. The weight and organ index of the spleen of the GF piglet were larger than those in the SPF piglet (P < 0.05). The histological structure of the red pulp area and mean area of germinal centers were larger in the SPF piglet than in the GF piglet (P < 0.05), whereas the areas of staining of B cells and T cells in the spleen and mesenteric lymph nodes (MLNs) were lower in the GF piglet (P < 0.05). We identified immune-related genes in the spleen and MLNs using RNA sequencing, and used real-time quantitative PCR to analyze the expression of core genes identified in gene set enrichment analysis. The expression levels of genes in the transforming growth factor-β/SMAD3 signaling pathway, Toll-like receptor 2/MyD88/nuclear factor-κB signaling pathway, and pro-inflammatory factor genes IL-6 and TNF-α in the spleen and MLNs were higher in the SPF piglet and in splenic lymphocytes compared with those in the GF and control group, respectively, under treatment with acetic acid, propionic acid, butyric acid, lipopolysaccharide (LPS), or concanavalin A (ConA). The abundances of plasma cells, CD8++ T cells, follicular helper T cells, and resting natural killer cells in the spleen and MLNs were significantly greater in the SPF piglet than in the GF piglet (P < 0.05). In conclusion, the commensal microbiota influenced the immune tissue structure, abundances of immune cells, and expression of immune-related pathways, indicating the importance of the commensal microbiota for spleen and MLNs development and function. In our study, GF piglet was used as the research model, eliminating the interference of microbiota in the experiment, and providing a suitable and efficient large animal research model for exploring the mechanism of "microbial-host" interactions.

The miRNA Contribution in Adipocyte Maturation.

Mesenchymal stem cells, due to their multipotent ability, are considered one of the best candidates to be used in regenerative medicine. To date, the most used source is represented by the bone marrow, despite the limited number of cells and the painful/invasive procedure for collection. Therefore, the scientific community has investigated many alternative sources for the collection of mesenchymal stem cells, with the adipose tissue representing the best option, given the abundance of mesenchymal stem cells and the easy access. Although adipose mesenchymal stem cells have recently been investigated for their multipotency, the molecular mechanisms underlying their adipogenic potential are still unclear. In this scenario, this communication is aimed at defining the role of miRNAs in adipogenic potential of adipose-derived mesenchymal stem cells via real-time PCR. Even if preliminary, our data show that cell culture conditions affect the expression of specific miRNA involved in the adipogenic potential of mesenchymal stem cells. The in vitro/in vivo validation of these results could pave the way for novel therapeutic strategies in the field of regenerative medicine. In conclusion, our research highlights how specific cell culture conditions can modulate the adipogenic potential of adipose mesenchymal stem cells through the regulation of specific miRNAs.

MRGPRX2-mediated mast cell activation by substance P from overloaded human tenocytes induces inflammatory and degenerative responses in tendons

Mast cells are immune cells minimally present in normal tendon tissue. The increased abundance of mast cells in tendinopathy biopsies and at the sites of tendon injury suggests an unexplored role of this cell population in overuse tendon injuries. Mast cells are particularly present in tendon biopsies from patients with more chronic symptom duration and a history of intensive mechanical loading. This study, therefore, examined the cross talk between mast cells and human tendon cells in either static or mechanically active conditions in order to explore the potential mechanistic roles of mast cells in overuse tendon injuries. A coculture of isolated human tenocytes and mast cells (HMC-1) combined with Flexcell Tension System for cyclic stretching of tenocytes was used. Additionally, human tenocytes were exposed to agonists and antagonists of substance P (SP) receptors. Mast cell degranulation was assessed by measuring β-hexosaminidase activity. Transwell and cell adhesion assays were used to evaluate mast cell migration and binding to tendon extracellular matrix components (collagen and fibronectin), respectively. Gene expressions were analyzed using real time qRT-PCR. Our results indicate that mechanical stimulation of human tenocytes leads to release of SP which, in turn, activates mast cells through the Mas-related G-protein-coupled receptor X2 (MRGPRX2). The degranulation and migration of mast cells in response to MRGPRX2 activation subsequently cause human tenocytes to increase their expression of inflammatory factors, matrix proteins and matrix metalloproteinase enzymes. These observations may be important in understanding the mechanisms by which tendons become tendinopathic in response to repetitive mechanical stimulation.

Development of novel lysosome-related signatures and their potential target drugs based on bulk RNA-seq and scRNA-seq for diabetic foot ulcers

BackgroundDiabetic foot ulcers (DFU) is the most serious complication of diabetes mellitus, which has become a global health problem due to its high morbidity and disability rates and the poor efficacy of conventional treatments. Thus, it is urgent to identify novel molecular targets to improve the prognosis and reduce disability rate in DFU patients.ResultsIn the present study, bulk RNA-seq and scRNA-seq associated with DFU were downloaded from the GEO database. We identified 1393 DFU-related DEGs by differential analysis and WGCNA analysis together, and GO/KEGG analysis showed that these genes were associated with lysosomal and immune/inflammatory responses. Immediately thereafter, we identified CLU, RABGEF1 and ENPEP as DLGs for DFU using three machine learning algorithms (Randomforest, SVM-RFE and LASSO) and validated their diagnostic performance in a validation cohort independent of this study. Subsequently, we constructed a novel artificial neural network model for molecular diagnosis of DFU based on DLGs, and the diagnostic performance in the training and validation cohorts was sound. In single-cell sequencing, the heterogeneous expression of DLGs also provided favorable evidence for them to be potential diagnostic targets. In addition, the results of immune infiltration analysis showed that the abundance of mainstream immune cells, including B/T cells, was down-regulated in DFUs and significantly correlated with the expression of DLGs. Finally, we found latamoxef, parthenolide, meclofenoxate, and lomustine to be promising anti-DFU drugs by targeting DLGs.ConclusionsCLU, RABGEF1 and ENPEP can be used as novel lysosomal molecular signatures of DFU, and by targeting them, latamoxef, parthenolide, meclofenoxate and lomustine were identified as promising anti-DFU drugs. The present study provides new perspectives for the diagnosis and treatment of DFU and for improving the prognosis of DFU patients.

Exploring the role of pyroptosis and immune infiltration in sepsis based on bioinformatic analysis

PurposeSepsis is a disease that is typically treated in intensive care units with high mortality and morbidity. Pyroptosis is a newly identified type of programmed cell death and is characterized by inflammatory cytokine secretion. However, the role of pyroptosis in sepsis remains unclear. MethodsGSE28750 and GSE134347 datasets were obtained from the Gene Expression Omnibus (GEO) database. Differentially expressed pyroptosis genes (DEPGs) were identified between sepsis and healthy controls. Machine learning was used to further narrow the gene range. Receiver operating curves (ROC) were generated to estimate the diagnostic efficacy. Immune infiltration levels were estimated via single-sample gene set enrichment analysis (ssGSEA). A network database was used to predict the upstream transcription factors and miRNAs of DEPGs. Finally, the expression of the genes was validated by qRT-PCR between sepsis patients and healthy controls. ResultsWe found that the pyroptosis pathway was enriched and activated in sepsis. 8 DEPGs were identified. A heatmap showed that the genes, NLRC4, NAIP, IL-18, AIM2 and ELANE, were abundant in the sepsis samples, and the genes, NLRP1, CHMP7 and TP53, were abundant in the healthy control samples. The ssGSEA results showed that the abundances of activated dendritic cells, MDSC, macrophage, plasmacytoid dendritic cells, regulatory T-cells, and Th17-cells were significantly higher, while the activated B-cell, activated CD8 T-cell, CD56 dim tural killer cell, immature B-cell, monocyte, and T follicular helper cell abundances were lower in sepsis samples compared to healthy controls. The qRT-PCR results showed that the expression levels of NAIP, IL-18, TP53, CHMP7, NLRC4, ELANE and NLRP1 were consistant with the bioinformatic analyses, while the expression level of AIM2 has no significant difference. ConclusionOur study identified seven potential pyroptosis-related genes, NAIP, IL-18, TP53, CHMP7, NLRC4, ELANE and NLRP1. This study revealed that pyroptosis may promote sepsis development by activating the immune response.