BACKGROUND

The topical application of mitomycin C has been evaluated as a complementary therapy for eosinophilic nasal polyposis (ENP). However, the mechanism underlying the additional benefits of mitomycin C for the control of eosinophilic inflammation and prevention of posttherapeutic relapse remains to be elucidated. In this work, the aim was to characterize the gene expression profile by quantitative real-time polymerase chain reaction (qPCR) of proinflammatory and regulatory biomarkers that are typically associated with ENP and to assess the impact of the topical application of mitomycin C on the nasal mucosal tissue immunologic milieu after ENP surgery.

METHODS

We have selected 20 patients with ENP that were recommended to undergo surgical intervention. Normal mucosal tissue was obtained from healthy nasal mucosa from six patients with absence of eosinophilic infiltration. To test the effect of mitomycin C, one side of the maxillary sinus mucosa was selected for topical application of this drug and the other received no further treatment and acted as the control. The genes interleukin-4 (IL-4), IL-5, IL-10, IL-13, chemokine (C-C motif) ligand 5 (CCL5), CCL24, colony-stimulating factor 2 (CSF2), transforming growth factor beta 1 (TGFB1), tumor necrosis factor alpha (TNF-alpha), and beta actin (ACTB) were selected for gene expression analysis by qPCR.

RESULTS

The data showed higher expression of proinflammatory biomarkers and lower levels of regulatory TGFB1 transcripts in ENP mucosal tissue. Surgery with topical application of mitomycin C induced a prominent transcriptional down-regulation of the immunologic biomarkers, CCL24, TNF-alpha, CSF2, and IL-5, in ENP mucosal tissue. Additionally, this treatment restored the levels of chemokines and cytokines to those observed in the nasal mucosal tissue of control subjects, except for TGFB1, which remained below the reference pattern. Moreover, CSF2 was identified as a putative biomarker with significant predictive value for complementary prophylactic purposes after surgery in ENP patients.

CONCLUSIONS

After the characterization of the expression signatures of immunologic biomarkers in ENP, we observed that the topical use of mitomycin C is important for the reestablishment of the immunologic microenvironment of a normal expression profile of biomarkers involved in ENP mucosal tissue.

Background: Farrowing induction with prostaglandin F2 analogue cloprostenol is commonly used on commercial farms to manage the timing of farrowing. When labour induction is applied, the questions arise about possible side effects of such a hormonal intervention on physiological processes connected with labour and lactation, including colostral immunity.

Results: In this study, immune cells composition, lysozyme concentration, complement bacteriolytic activity and proinflamatory (GM-CSF2, IL-1β, IL-6, a TNFα) and anti-inflammatory (IL-4, IL-10, TGFβ1 a TGFβ2) cytokines were measured in colostrum samples from sows farrowing naturally (NP) and from sows with farrowing induced using cloprostenol administration on day 113 of gestation (IP). A significantly higher proportion of lymphocytes was found in colostrum of induced sows compared to colostrum of non-induced sows. No significant differences between NP and IP were found in complement activity, in the proportions of granulocytes, macrophages and lymphocyte subpopulations. Lower lysozyme concentration and higher IL-1β, IL-6, TGFβ1 and TNFα concentrations were found in IP sow colostrum compared to colostrum from NP sows.

Conclusions: An increased proportion of colostral lymphocytes can positively influence the cellular immunity transmission from sow to her offspring. On the other hand, a lower lysozyme concentration can adversely affect newborn's intestinal immunity, as well as changes in cytokine concentrations can have an adverse effect on newborn piglet intestinal epithelium development and its defence function.

Keywords: Colostrum; Complement; Cytokines; Lymphocytes; Lysozyme; Sow.

Background: Haptoglobin (Hp) binds to and facilitates clearance of heme. Compared with HP 1-1 and 1-2 genotypes, HP 2-2 has a weaker binding affinity and has been linked with increased inflammation and vasospasm after aneurysmal subarachnoid hemorrhage (SAH).

Objective: To assess levels of inflammatory cytokines in the context of different HP genotypes.

Methods: Patients were enrolled among those presenting with spontaneous aneurysmal SAH. Blood was drawn at four time points; <24 hours (T1), 24-48 hours (T2), 3-5 days (T3), and 6-8 days (T4). Blood was analyzed for levels of 41 cytokines at each time point, as well as for HP genotypes. These data were analyzed using mixed-effect models to assess the association between HP genotypes and cytokine levels. The modified Rankin Scale (mRS) score was obtained at discharge, 3 months, and 6 months.

Results: Fifty-seven patients were enrolled. Compared with HP 1-1 and 1-2, subjects encoding HP 2-2 had elevated levels of the following cytokines at all time points: FLT3L, IFNγ, IL-17A, TGFα, and VEGF-A. Elevations were also seen at some time points for IL-8, CSF2, FGF2, IL-7, IL-12p70, and TNFα. This study was not powered to detect differences in functional outcome; however, there were no significant differences in dichotomized mRS score between patients with HP 1- 1/1-2 or HP 2-2.

Conclusion: Our findings indicate that HP 2-2 genotype leads to increased proinflammatory cytokine levels compared with HP 1-1/1-2 genotypes. These data may provide guidance for further studies seeking to identify testable markers for functional prognosis or targets for treatment.

Keywords: Haptoglobin; cytokines; genotype; inflammation; subarachnoid hemorrhage.

"Humanized" immunodeficient mice generated via the transplantation of CD34+ human hematopoietic stem cells (hHSC) are an important preclinical model system. The triple transgenic NOD.Cg-Prkdc^scidIl2rg^tm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ (NSGS) mouse line is increasingly used as recipient for CD34+ hHSC engraftment. NSGS mice combine the features of the highly immunodeficient NSG mice with transgenic expression of the human myeloid stimulatory cytokines GM-CSF, IL-3, and Kit ligand. While generating humanized NSGS (huNSGS) mice from two independent cohorts, we encountered a fatal macrophage activation syndrome (MAS)-like phenotype resulting from the transplantation of CD34+ hHSC. huNSGS mice exhibiting this phenotype declined clinically starting at approximately 10 weeks following CD34+ hHSC engraftment, with all mice requiring euthanasia by 16 weeks. Gross changes comprised small, irregular liver, splenomegaly, cardiomegaly, and generalized pallor. Hematological abnormalities included severe thrombocytopenia and anemia. Pathologically, huNSGS spontaneously developed a disseminated histiocytosis with infiltrates of activated macrophages and hemophagocytosis predominantly affecting the liver, spleen, bone marrow, and pancreas. The infiltrates were chimeric with a mixture of human and mouse macrophages. Immunohistochemistry suggested activation of the inflammasome in both human and murine macrophages. Active Epstein-Barr virus infection was not a feature. Although the affected mice exhibited robust chimerism of the spleen and bone marrow, the phenotype often developed in the face of low chimerism of the peripheral blood. Given the high penetrance and early lethality associated with the MAS-like phenotype here described, we urge caution when considering the use of huNSGS mice for the development of long-term studies.

Keywords: Chimeric mouse; HLH; Humanized model; Inflammasome; Macrophage activation syndrome; NSG-SGM3; NSGS; PDX model.

The pathological mechanisms that lead to the onset and reactivation of celiac disease (CD) remain largely unknown. While gluten free diet (GFD) improves the intestinal damage and associated clinical symptoms in majority of cases, it falls short of providing full recovery. Additionally, late or misdiagnosis is also common as CD presents with a wide range of symptoms. Clear understanding of CD pathogenesis is thus critical to address both diagnostic and treatment concerns. We aimed to study the molecular impact of short gluten exposure in GFD treated CD patients, as well as identify biological pathways that remain altered constitutively in CD regardless of treatment. Using RNAseq profiling of PBMC samples collected from treated CD patients and gluten challenged patient and healthy controls, we explored the peripheral transcriptome in CD patients following a short gluten exposure. Short gluten exposure of just three days was enough to alter the genome-wide PBMC transcriptome of patients. Pathway analysis revealed gluten-induced upregulation of mainly immune response related pathways, both innate and adaptive, in CD patients. We evaluated the perturbation of biological pathways in sample-specific manner. Compared to gluten exposed healthy controls, pathways related to tight junction, olfactory transduction, metabolism of unsaturated fatty acids (such as arachidonic acid), metabolism of amino acids (such as cysteine and glutamate), and microbial infection were constitutively altered in CD patients regardless of treatment, while GFD treatment appears to mostly normalize immune response pathways to "healthy" state. Upstream regulator prediction analysis using differentially expressed genes identified constitutively activated regulators relatively proximal to previously reported CD associated loci, particularly SMARCA4 on 19p13.2 and CSF2 on 5q31. We also found constitutively upregulated genes in CD that are in CD associated genetic loci such as MEF2BNB-MEF2B (BORCS8-MEF2B) on 19p13.11 and CSTB on 21q22.3. RNAseq revealed strong effects of short oral gluten challenge on whole PBMC fraction and constitutively altered pathways in CD PBMC suggesting important factors other than gluten in CD pathogenesis.

Keywords: celiac disease; celiac disease RNA sequencing; celiac disease gene expression analysis; celiac disease transcriptomics; pathway analysis, gluten challenge.

Purpose: To explore the consequences of innate interference on intraocular inflammatory responses during Bacillus endophthalmitis.

Methods: Bacillus endophthalmitis was induced in mice. Innate immune pathway activation was interfered by injecting S layer protein-deficient (∆slpA) B. thuringiensis or by treating wild-type (WT)-infected mice with a TLR2/4 inhibitor (WT+OxPAPC). At 10 hours postinfection, eyes were harvested and RNA was purified. A NanoString murine inflammation panel was used to compare gene expression in WT-infected, WT+OxPAPC, ∆slpA-infected, and uninfected eyes.

Results: In WT-infected eyes, 56% of genes were significantly upregulated compared to uninfected controls. Compared to WT-infected eyes, the expression of 27% and 50% of genes were significantly reduced in WT+OxPAPC and ∆slpA-infected eyes, respectively. Expression of 61 genes that were upregulated in WT-infected eyes was decreased in WT+OxPAPC and ∆slpA-infected eyes. Innate interference resulted in blunted expression of complement factors (C3, Cfb, and C6) and several innate pathway genes (TLRs 2, 4, 6, and 8, MyD88, Nod2, Nlrp3, NF-κB, STAT3, RelA, RelB, and Ptgs2). Innate interference also reduced the expression of several inflammatory cytokines (CSF2, CSF3, IL-6, IL-1β, IL-1α, TNFα, IL-23α, TGFβ1, and IL-12β) and chemokines (CCL2, CCL3, and CXCLs 1, 2, 3, 5, 9, and 10). All of the aforementioned genes were significantly upregulated in WT-infected eyes.

Conclusions: These results suggest that interfering with innate activation significantly reduced the intraocular inflammatory response in Bacillus endophthalmitis. This positive clinical outcome could be a strategy for anti-inflammatory therapy of an infection typically refractory to corticosteroid treatment.

DNA methylation has been demonstrated to play significant roles in the etiology and pathogenesis of head and neck squamous cell carcinoma (HNSCC). In this study, methylation microarray dataset (GSE87053) and gene expression microarray dataset (GSE23558) were downloaded from GEO database and analyzed through R language. A total of 255 hypermethylated-downregulated genes and 114 hypomethylated-upregulated genes were finally identified. Functional enrichment analyses were performed and a comprehensive protein-protein interaction (PPI) network was constructed. Subsequently, the top ten hub genes selected by Cytoscape software were subjected to further analyses. It was illustrated that the expression level of CSF2, CTLA4, ETS1, PIK3CD, and CFTR was intimately associated with HNSCC. Survival analysis suggested that CTLA4 and FGFR2 could serve as effective independent prognostic biomarkers for HNSCC patients. Overall, our study lay a groundwork for further investigation into the underlying molecular mechanisms in HNSCC carcinogenesis, providing potential biomarkers and therapeutic targets for HNSCC.

Objective: To investigate the inflammatory mechanism of nasal instillation of fine particulate matter (PM_2.5)on hippocampal tissue injury in mice.Methods: Thirty C57BL/6J mice were randomly divided into 3 groups(n=10):control group, low-dose group, high-dose group. The nasal instillation doses of PM_2.5 in the low-dose group and the high-dose group were 1.5 mg/kg BW and 7.5 mg/kg BW, respectively, and the control group was given saline with an equal volume. Saline was sprayed once every other time for 12 times. The serum levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) were determined by ELISA method. HE staining and electron microscopy were used to observe the pathological changes and ultrastructure of lung tissue and hippocampus. The inflammatory cytokine levels in hippocampus were detected by antibody chip technique. Results: There was no significant effect of PM_2.5 nasal instillation on serum TNF-α, IL-1β and IL-6 levels (P＞0.05), and there was no obvious pathological changes in lung tissue structure. In hippocampus, low-dose and high-dose PM_2.5 exposure could lead to disordered neuronal arrangement in the hippocampal CA3 region, and there were neurological changes around the neuron cells and ultrastructural changes such as edema around small blood vessels. Compared with the control group, the levels of inflammatory cytokines such as CX3CL1, CSF2 and TECK in the low-dose group were increased significantly (P ＜0.05), while sTNFR1 was decreased significantly (P＜0.05); the inflammatory factors CX3CL1, CSF2, and TCA-3 were significantly increased in the high-dose group (P＜0.05), while leptin, MIG, and FASLG were significantly decreased (P＜0.05). Conclusion: Nasal instillation of PM_2.5 can induce tissue damage in the hippocampus of mice, and its mechanism of action may be the olfactory brain pathway. The increasing of TNF-α and IL-6 and the decreasing of sTNFR1 and FASLG may be involved in inflammatory mechanisms.

目的: 探讨鼻腔滴注不同浓度的PM_2.5对小鼠海马组织损伤的炎性机制。方法: 30只C57BL/6J小鼠随机分为3组(n=10):对照组、低剂量组、高剂量组。采用鼻腔滴注方法进行染毒,每次染毒前测量体重,低剂量组和高剂量组PM_2.5染毒剂量分别为1.5 mg/kg BW和7.5 mg/kg BW,对照组给予等体积的生理盐水,隔天染毒一次,共12次。用ELISA法测定血清中肿瘤坏死因子-α(TNF-α)、白介素-1β(IL-1β)和白介素-6(IL-6)水平。HE染色和电镜观察肺和海马组织的病理变化及超微结构。用抗体芯片技术测定海马组织炎性细胞因子水平。结果: PM_2.5鼻腔滴注染毒对小鼠血清中TNF-α、IL-1β和IL-6水平无显著影响(P＞0.05),肺组织结构也无明显病理改变。而在海马组织中,低剂量和高剂量PM_2.5暴露均能导致海马CA3区神经元排列紊乱,并存在神经元细胞周围突触数量减少,小血管周围水肿等超微结构变化。利用抗体芯片检测海马组织炎性细胞因子表达变化,结果显示,与对照组比较,低剂量组海马组织中CX3CL1、CSF2和TECK等炎性细胞因子水平显著升高(P＜0.05),而MIG和sTNFR1显著降低(P＜0.05);高剂量组中炎性因子CX3CL1、CSF2和TCA-3等显著升高(P＜0.05),而Leptin、MIG和FASLG等显著降低(P＜0.05)。结论: PM_2.5鼻腔滴注可诱导小鼠海马组织结构损伤,其作用途径可能为嗅脑通路,引起海马损伤的炎性机制可能与TNF-α和IL-6等促炎因子的显著升高和sTNFR1 、FASLG等炎性疾病标志物的显著降低有关。.

Keywords: PM 2.5; antibody chip; hippocampus; inflammatory factor; mice; olfactory pathway.

It is well known that as the king of cancer, pancreatic ductal adenocarcinoma (PDAC) has relatively malignant biological behavior and poor prognosis. The interaction between pancreatic stellate cells and PDAC cells promotes the development of PDAC. The aim of this study was to describe gene characteristics in pancreatic stellate cell (PSCs) after cross-talked with BXPC-3 and unravel their underlying mechanisms. The expression profiling analysis of genes in PSCs was completed after co-cultured with primary BXPC-3 for 48h. The Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) enrichment analysis and gene ontology (GO) analysis were performed, and the differentially expressed genes (DEGs) were identified by Agilent GeneSpring GX program. In total, 1804 DEGs were filtered out in PSCs, including 958 up-regulated genes and 846 downregulated genes. GO analysis showed that the up-regulated DEGs were significantly enriched in biological processes (BP) such as defense response, immune system process and immune response; the down-regulated DEGs were significantly enriched in biological regulation and cytoskeleton organization. KEGG pathway analysis showed that 28 pathways were upregulated and 5 were downregulated. By constructing PPI network, we selected out 10 key genes (IL6，IL8, IL1B, BCL2, CCL2, CSF2, KIT, ICAM1, PTPRC and IGF1) and significant enriched pathways. In conclusion, the current study suggests that the filtered DEGs contribute to our understanding of the molecular mechanisms underlying the interaction between PSCs and pancreatic cancer cells, and might be used as molecular targets to further the study the role of tumor microenvironment in the progression of PDAC.

Collisions between cold cesium atoms and background gas atoms at ambient temperature reduce the cold atom signal in a fountain clock and at the same time produce a shift in the measured clock frequency. We evaluate the shift in the NPL-CsF2 cesium fountain primary frequency standard based on measurements of the fractional loss of cold atoms from the atomic cloud during the interrogation time combined with a model by Gibble that quantifies the relationship between the loss and the frequency shift.

Previously, we have reported that interleukin (IL)-4, granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-11, but not IL-33, are up-regulated in two strains of mice with immune thrombocytopenia (ITP) that are responsive to intravenous immunoglobulin (IVIg) treatment. Previously, IL-4 was ruled out in the mechanism of IVIg; however, other publications have suggested this cytokine as a major player in the mechanism of IVIg action. Thus, we sought to further investigate a role for IL-4 and, in addition, GM-CSF and IL-11 in the mechanism of action of IVIg using a murine model of ITP. A passive platelet antibody model was used to generate ITP in IL-4 receptor knock-out (IL-4R-/- ), IL-11 receptor knock-out (IL-11Rα-/- ) and GM-CSF knock-out (Csf2-/- ) mice. We also used a neutralizing antibody to IL-11 and recombinant human IL-11 (rhIL-11) in addition to depleting basophils in vivo to study the effect of IVIg to ameliorate ITP. Our results showed that basophils, IL-4 and GM-CSF were unimportant in both ITP induction and its amelioration by IVIg. The role of IL-11 in these processes was less clear. Even though IL-11Rα-/- mice with ITP responded to IVIg similarly to wild-type (WT) mice, treatment of ITP WT mice with rhIL-11 instead of IVIg showed an increase in platelet numbers and WT mice administered anti-IL-11 showed a significant reduction in the ability of IVIg to ameliorate the ITP. Our findings indicate that neither IL-4, basophils or GM-CSF have roles in IVIg amelioration of ITP; however, a role for IL-11 requires further study.

Aberrant activation of cytokine and growth factor signal transduction pathways confers enhanced survival and proliferation properties to acute myeloid leukemia (AML) cells. However, the mechanisms underlying the deregulation of signaling pathways in leukemia cells are unclear. To identify genes capable of independently supporting cytokine-independent growth, we employed a genome-wide CRISPR/Cas9-mediated loss-of-function screen in GM-CSF-dependent human AML TF-1 cells. More than 182 genes (p < 0.01) were found to suppress the cytokine-independent growth of TF-1 cells. Among the top hits, genes encoding key factors involved in sialylation biosynthesis were identified; these included CMAS, SLC35A1, NANS, and GNE. Knockout of either CMAS or SLC35A1 enabled cytokine-independent proliferation and survival of AML cells. Furthermore, NSG (NOD/SCID/IL2Rγ^-/-) mice injected with CMAS or SLC35A1-knockout TF-1 cells exhibited a shorter survival than mice injected with wild-type cells. Mechanistically, abrogation of sialylation biosynthesis in TF-1 cells induced a strong activation of ERK signaling, which sensitized cells to MEK inhibitors but conferred resistance to JAK inhibitors. Further, the surface level of α2,3-linked sialic acids was negatively correlated with the sensitivity of AML cell lines to MEK/ERK inhibitors. We also found that sialylation modulated the expression and stability of the CSF2 receptor. Together, these results demonstrate a novel role of sialylation in regulating oncogenic transformation and drug resistance development in leukemia. We propose that altered sialylation could serve as a biomarker for targeted anti-leukemic therapy.

Keywords: AML; CMAS; Loss of function screening; SLC35A1; Sialylation.

The lung is exposed to airborne fungal spores, and fungi that colonize the oral cavity such as Candida albicans, but does not develop disease to opportunistic fungal pathogens unless the immune system is compromised. The Group IVA cytosolic phospholipase A2 (cPLA2α) is activated in response to Candida albicans infection resulting in the release of arachidonic acid for eicosanoid production. Although eicosanoids such as prostaglandins and leukotrienes modulate inflammation and immune responses, the role of cPLA2α and eicosanoids in regulating C. albicans lung infection is not understood.

The responses of cPLA2α(+/+) and cPLA2α(-/-) Balb/c mice to intratracheal instillation of C. albicans were compared. After challenge, we evaluated weight loss, organ fungal burden, and the recruitment of cells and the levels of cytokines and eicosanoids in bronchoalveolar lavage fluid. The ability of macrophages and neutrophils from cPLA2α(+/+) and cPLA2α(-/-) mice to recognize and kill C. albicans was also compared.

After C. albicans instillation, cPLA2α(+/+) mice recovered a modest weight loss by 48 h and completely cleared fungi from the lung by 12 h with no dissemination to the kidneys. In cPLA2α(-/-) mice, weight loss continued for 72 h, C. albicans was not completely cleared from the lung and disseminated to the kidneys. cPLA2α(-/-) mice exhibited greater signs of inflammation including higher neutrophil influx, and elevated levels of albumin and pro-inflammatory cytokines/chemokines (IL1α, IL1β, TNFα, IL6, CSF2, CXCL1, CCL20) in bronchoalveolar lavage fluid. The amounts of cysteinyl leukotrienes, thromboxane B2 and prostaglandin E2 were significantly lower in bronchoalveolar lavage fluid from C. albicans-infected cPLA2α(-/-) mice compared to cPLA2α(+/+) mice. Alveolar macrophages and neutrophils from uninfected cPLA2α(-/-) mice exhibited less killing of C. albicans in vitro than cells from cPLA2α(+/+) mice. In addition alveolar macrophages from cPLA2α(-/-) mice isolated 6 h after instillation of GFP-C. albicans contained fewer internalized fungi than cPLA2α(+/+) macrophages.

The results demonstrate that cPLA2α contributes to immune surveillance and host defense in the lung to prevent infection by the commensal fungus C. albicans and to dampen inflammation.

Cranberry contains high levels of nutrients and bioactive molecules that have health-promoting properties. The purpose of the present studies was to determine if cranberry extracts (CEs) contain phytochemicals that exert anti-inflammatory effects. The human monocytic cell line THP-1 was treated with two CEs (CE and 90MX) and subsequently challenged with Lipopolysaccharides (LPS). Tumor necrosis factor α (TNF α) expression was decreased in the CE-treated cells, indicative of an anti-inflammatory effect. Gene expression microarrays identified several immune-related genes that were responsive to CEs including interferon-induced protein with tetratricopeptide repeats 1 and 3 (IFIT 1 and 3), macrophage scavenger receptor 1 (MSR1) and colony-stimulating factor 2 (CSF2). In addition, in the CE-treated cells, metallothionein 1F and other metal-responsive genes were induced. Taken together, this data indicates that CEs contain bioactive components that have anti-inflammatory effects and may protect cells from oxidative damage.

Kupffer cells (KCs), which are liver-resident macrophages, originate from the fetal yolk sac and represent one of the largest macrophage populations in the body. However, the current data on the origin of the cells that restore macrophages during liver injury and regeneration remain controversial. Here, we address the question of whether liver macrophage restoration results from circulating monocyte infiltration or local KC proliferation in regenerating livers after partial hepatectomy (PHx) and uncover the underlying mechanisms. By using several strains of genetically modified mice and performing immunohistochemical analyses, we demonstrated that local KC proliferation mainly contributed to the restoration of liver macrophages after PHx. Peak KC proliferation was impaired in Il6-knockout (KO) mice and restored after the administration of IL-6 protein, whereas KC proliferation was not affected in Il4-KO or Csf2-KO mice. The source of IL-6 was identified using hepatocyte- and myeloid-specific Il6-KO mice and the results revealed that both hepatocytes and myeloid cells contribute to IL-6 production after PHx. Moreover, peak KC proliferation was also impaired in myeloid-specific Il6 receptor-KO mice after PHx, suggesting that IL-6 signaling directly promotes KC proliferation. Studies using several inhibitors to block the IL-6 signaling pathway revealed that sirtuin 1 (SIRT1) contributed to IL-6-mediated KC proliferation in vitro. Genetic deletion of the Sirt1 gene in myeloid cells, including KCs, impaired KC proliferation after PHx. In conclusion, our data suggest that KC repopulation after PHx is mainly driven by local KC proliferation, which is dependent on IL-6 and SIRT1 activation in KCs.

Keywords: IL-6; Kupffer cells; Liver regeneration; Myeloid cells; Sirtuin 1.

Background: Basal-like breast cancer (BLBC) is associated with a poor clinical outcome; however, the mechanism of BLBC aggressiveness is still unclear. It has been shown that a linker histone functions as either a positive or negative regulator of gene expression in tumors. Here, we aimed to investigate the possible involvement and mechanism of HIST1H1B in BLBC progression.

Experimental design: We analyzed multiple gene expression datasets to determine the relevance of HIST1H1B expression with BLBC. We employed quantitative real-time PCR, transwell assay, colony formation assay, and mammosphere assay to dissect the molecular events associated with the expression of HIST1H1B in human breast cancer. We studied the association of HIST1H1B with CSF2 by ChIP assay. Using tumorigenesis assays, we determine the effect of HIST1H1B expression on tumorigenicity of BLBC cells.

Results: Here, we show that the linker histone HIST1H1B is dramatically elevated in BLBC due to HIST1H1B copy number amplification and promoter hypomethylation. HIST1H1B upregulates colony-stimulating factor 2 (CSF2) expression by binding the CSF2 promoter. HIST1H1B expression promotes, whereas knockdown of HIST1H1B expression suppresses tumorigenicity. In breast cancer patients, HIST1H1B expression is positively correlated with large tumor size, high grade, metastasis and poor survival.

Conclusion: HIST1H1B contributes to basal-like breast cancer progression by modulating CSF2 expression, indicating a potential prognostic marker and therapeutic target for this disease.

Keywords: CSF2; DNA methylation; HIST1H1B; basal-like breast cancer (BLBC); copy number variant (CNV).

The mammary gland is a unique apocrine gland made up of a branching network of ducts that end in alveoli. It is an ideal system to study the molecular mechanisms associated with cell proliferation, differentiation, and oncogenesis. MFG-E8, also known as Lactadherin, is a vital glycoprotein related to the milk fat globule membrane and initially identified to get secreted in bovine milk. Our previous report suggests that a high level of MFG-E8 is indicative of high milk yield in dairy animals. Here, we showed that MFG-E8 controls the cell growth and morphology of epithelial cells through a network of regulatory transcription factors. To understand the comprehensive action, we downregulated its expression in MECs by MFG-E8 specific shRNA. We generated a knockdown proteome profile of differentially expressed proteins through a quantitative iTRAQ experiment on a high-resolution mass spectrometer (Q-TOF). The downregulation of MFG-E8 resulted in reduced phagocytosis and cell migration ability, whereas it also leads to more lifespan to knockdown vis-a-vis healthy cells, which is confirmed through BrdU, MTT, and Caspase 3/7. The bioinformatics analysis revealed that MFG-E8 knockdown perturbs a large number of intracellular signaling, eventually leading to cessation in cell growth. Based on the directed network analysis, we found that MFG-E8 is activated by CX3CL1, TP63, and CSF2 and leads to the activation of SOCS3 and CCL2 for the regulation of cell proliferation. We further proved that the depletion of MFG-E8 resulted in activated cytoskeletal remodeling by MFG-E8 knockdown, which results in the activation of three independent pathways ZP4/JAK-STAT5, DOCK1/STAT3, and PIP3/AKT/mTOR. Overall, this study suggests that MFG-E8 expression in mammary epithelial cells is an indication of intracellular deterioration in cell health. To date, to the best of our knowledge, this is the first study that explores the downstream targets of MFG-E8 involved in the regulation of mammary epithelial cell health.

Keywords: KEGG; cell proliferation; cytoscape; milk fat globule epithelial growth factor-8; oncogenesis; quantitative proteomics.

Breast cancer (BRCA) is a serious public health problem, as it is the most frequent malignant tumor in women worldwide. BRCA is a molecularly heterogeneous disease, particularly at gene expression (mRNAs) level. Recent evidence shows that coding RNAs represent only 34% of the total transcriptome in a human cell. The rest of the 66% of RNAs are non-coding, so we might be missing relevant biological, clinical or regulatory information. In this report, we identified two novel tumor types from TCGA with LINC00460 deregulation. We used survival analysis to demonstrate that LINC00460 expression is a marker for poor overall (OS), relapse-free (RFS) and distant metastasis-free survival (DMFS) in basal-like BRCA patients. LINC00460 expression is a potential marker for aggressive phenotypes in distinct tumors, including HPV-negative HNSC, stage IV KIRC, locally advanced lung cancer and basal-like BRCA. We show that the LINC00460 prognostic expression effect is tissue-specific, since its upregulation can predict poor OS in some tumors, but also predicts an improved clinical course in BRCA patients. We found that the LINC00460 expression is significantly enriched in the Basal-like 2 (BL2) TNBC subtype and potentially regulates the WNT differentiation pathway. LINC00460 can also modulate a plethora of immunogenic related genes in BRCA, such as SFRP5, FOSL1, IFNK, CSF2, DUSP7 and IL1A and interacts with miR-103-a-1, in-silico, which, in turn, can no longer target WNT7A. Finally, LINC00460:WNT7A ratio constitutes a composite marker for decreased OS and DMFS in Basal-like BRCA, and can predict anthracycline therapy response in ER-BRCA patients. This evidence confirms that LINC00460 is a master regulator in BRCA molecular circuits and influences clinical outcome.

Keywords: LINC00460; WNT7A; basal-like; biomarker; breast cancer; increased prognosis; mir-103a; sponge.

Dendritic cells (DCs) are the major specialized antigen-presenting cells, thereby connecting innate and adaptive immunity. Because of their role in establishing adaptive immunity, they constitute promising targets for immunotherapy. Monocytes can differentiate into DCs in vitro in the presence of colony-stimulating factor 2 (CSF2) and interleukin 4 (IL4), activating four signalling pathways (MAPK, JAK/STAT, NFKB and PI3K). However, the downstream transcriptional programme responsible for DC differentiation from monocytes (moDCs) remains unknown. By analysing the scientific literature on moDC differentiation, we established a preliminary logical model that helped us identify missing information regarding the activation of genes responsible for this differentiation, including missing targets for key transcription factors (TFs). Using ChIP-seq and RNA-seq data from the Blueprint consortium, we defined active and inactive promoters, together with differentially expressed genes in monocytes, moDCs and macrophages, which correspond to an alternative cell fate. We then used this functional genomic information to predict novel targets for previously identified TFs. By integrating this information, we refined our model and recapitulated the main established facts regarding moDC differentiation. Prospectively, the resulting model should be useful to develop novel immunotherapies targeting moDCs.

Keywords: dendritic cells; differentiation; logical modelling; regulatory networks.

Background: Autologous T cells engineered to express a chimeric antigen receptor (CAR) specific for CD19 molecule have transformed the therapeutic landscape in patients with highly refractory leukemia and lymphoma, and the use of donor-generated allogeneic CAR T is paving the way for further breakthroughs in the treatment of cancer. However, it remains unknown how the intrinsic heterogeneities of these engineered cells mediate therapeutic efficacy and whether allogeneic products match the effectiveness of autologous therapies.

Methods: Using single-cell mRNA sequencing in conjunction with CITE-seq, we performed multiomics characterization of CAR T cells generated from healthy donor and patients with acute lymphoblastic leukemia. CAR T cells used in this study were manufactured at the University of Pennsylvania through lentiviral transduction with a CD19-4-1BB-CD3ζ construct. Besides the baseline condition, we engineered NIH-3T3 cells with human CD19 or mesothelin expression to conduct ex vivo antigen-specific or non-antigen stimulation of CAR T cells through 6-hour coculture at a 1:1 ratio.

Results: We delineated the global cellular and molecular CAR T landscape and identified that transcriptional CAR tonic signaling was regulated by a mixture of early activation, exhaustion signatures, and cytotoxic activities. On CD19 stimulation, we illuminated the disparities of CAR T cells derived from different origins and found that donor CAR T had more pronounced activation level in correlation with the upregulation of major histocompatibility complex class II genes compared with patient CAR T cells. This finding was independently validated in additional datasets from literature. Furthermore, GM-CSF(CSF2) expression was found to be associated with functional gene productions, but it induced little impact on the CAR T activation.

Conclusions: Through integrated multiomics profiling and unbiased canonical pathway analyses, our results unveil heterogeneities in the transcriptional, phenotypic, functional, and metabolic profiles of donor and patient CAR T cells, providing mechanistic basis for ameliorating clinical outcomes and developing next-generation 'off- the-shelf' allogeneic products.

Keywords: chimeric antigen; immunotherapy; receptors; translational medical research.

Objective: to establish the connection of radiation-induced changes in gene expression with the realized pathology of the broncho-pulmonary and cardiovascular systems in Chornobyl clean-up workers.

Materials and methods: We examined 314 male Chornobyl clean-up workers (main group; age (58.94 ± 6.82) years(M ± SD); min 33, max 79 years; radiation dose (411.82 ± 625.41) mSv (M ± SD); min 1.74, max 3600 mSv) with various nosological forms of cardiovascular and broncho-pulmonary pathology (BPP) and 50 subjects of the controlgroup: age (50.50 ± 5.73) years (M ± SD); min 41, max 67 years. The relative level of BCL2, CDKN2A, CLSTN2, GSTM1,IFNG, IL1B, MCF2L, SERPINB9, STAT3, TERF1, TERF2, TERT, TNF, TP53, CCND1, CSF2, VEGFA genes expression was determined inperipheral blood leukocytes by real-time PCR (7900 HT Fast Real-Time PCR System (Applied Biosystems, USA)). The«gene-disease» association was determined on statistical models stratified separately for each disease and gene.Logistic regression was used to calculate the odds ratio.

Results: Increased GSTM1 gene expression and no changes in angiogenesis-related VEGFA gene expression werefound in the main group of patients with coronary heart disease (CHD). It was established overexpression of TP53,VEGF and IFNG genes in the group of patients with arterial hypertension (AH). At combination of these diseases anincrease of expression of СSF2, TERF1, TERF2 genes was established. The detected changes demonstrate an activationof the antioxidative defense system in patients with CHD, while AH is associated with the expression of genes ofangiogenesis and immune inflammation. It was shown an increase in the expression of genes associated with apoptosis and kinase activity (BCL2, CLSTN2, CDKN2), immune inflammation (CSF2, IL1B, TNF) in Chornobyl clean-upworkers with BPP. Expression of TP53 and GSTM1 (gene, associated with the glutathione system) was significantlyupregulated in the group of individuals with chronic bronchitis, whereas in patients with chronic obstructive pulmonary disease, no increase was detected; the expression of SERPINB9 and MCF2L genes was downregulated.

Conclusions: Changes in the expression of genes, associated with the development of somatic pathology in theremote period after irradiation, in particular the genes of the immune response and inflammatory reactions CSF2,IFNG, IL1B, TNF; expression of genes that regulate cell proliferation, aging and apoptosis TP53, BCL2, MCF2L, CDKN2A,SERPINB9, TERF1, TERF2, TERT; genes that regulate cell adhesion and angiogenesis CLSTN2, VEGF.

Meta: vstanovyty zv’iazok radiatsiĭno-asotsiĭovanykh porushen' gennoï ekspresiï z realizovanoiu patologiieiubronkholegenevoï ta sertsevo-sudynnoï system v uchasnykiv likvidatsiï naslidkiv avariï (LNA) na ChAES.Materialy i metody. Obstezheno 314 uchasnykiv LNA na ChAES 1986–1987 rr. (osnovna grupa) cholovichoï stati zriznymy nozologichnymy formamy sertsevo-sudynnoï ta bronkholegenevoï patologiĭ ta 50 osib kontrol'noï grupy.Vikova kharakterystyka: osnovna grupa – (58,94 ± 6,82) rokiv (M ± SD); 33/79 rokiv (min/max), kontrol'nagrupa – (50,50 ± 5,73) rokiv (M ± SD); 41/67 rokiv (min/max). Dozy zovnishn'ogo oprominennia osib osnovnoïgrupy: (411,82 ± 625,41) mZv (M ± SD); 1,74/3600 mZv (min/max). Provedeno vyznachennia vidnosnogo rivniaekspresiï geniv: BCL2, CDKN2A, CLSTN2, GSTM1, IFNG, IL1B, MCF2L, SERPINB9, STAT3, TERF1, TERF2, TERT, TNF, TP53, CCND1,CSF2, VEGFA u leĭkotsytakh peryferychnoï krovi metodom polimeraznoï lantsiugovoï reaktsiï u real'nomu chasi (7900HT Fast Real-Time PCR System (Applied Biosystems, USA)). Vyznachennia asotsiatsiï «gen-zakhvoriuvannia» provedenona statystychnykh modeliakh, stratyfikovanykh okremo dlia kozhnogo zakhvoriuvannia ta gena. Logistychna regresiia bulavykorystana dlia rozrakhunku koefitsiientiv shansiv.Rezul'taty. V osib osnovnoï grupy, khvorykh na ishemichnu khvorobu sertsia vyiavleni pidvyshchennia ekspresiï genaGSTM1 ta vidsutnist' zmin gennoï ekspresiï VEGFA. V grupi khvorykh na gipertonichnu khvorobu vyznacheno giperekspresiiu geniv TP53, VEGFA ta IFNG. Pry poiednanni danykh zakhvoriuvan' vstanovleno pidvyshchennia ekspresiï genivCSF2, TERF1, TERF2. Vyiavleni zminy demonstruiut' vkliuchennia systemy okysnogo gomeostazu u khvorykh na ishemichnukhvorobu sertsia, todi iak gipertonichna khvoroba asotsiiuiet'sia z ekspresiieiu geniv angiogenezu ta imunnogo zapalennia.V uchasnykiv LNA na ChAES z bronkholegenevoiu patologiieiu vstanovleno zrostannia ekspresiï geniv, pov’iazanykh zapoptozom ta kinaznoiu aktyvnistiu (BCL2, CLSTN2, CDKN2A), imunnym zapalenniam (CSF2, IL1B, TNF); ekspresiia genaTP53 ta gena GSTM1, pov’iazanogo z systemoiu glutationu, bula dostovirno pidvyshchenoiu v grupi osib z khronichnymbronkhitom, todi iak u khvorykh na khronichne obstruktyvne zakhvoriuvannia legen' pidvyshchennia ne vyznachalosia;znyzhenoiu bula ekspresiia geniv SERPINB9 ta MCF2L.Vysnovky. Vyznacheno zminy ekspresiï geniv, asotsiĭovani z rozvytkom somatychnoï patologiï u viddalenomuperiodi pislia oprominennia, zokrema geniv imunnoï vidpovidi ta zapal'nykh reaktsiĭ CSF2, IFNG, IL1B, TNF; ekspresiïgeniv-reguliatoriv klitynnoï proliferatsiï, starinnia ta apoptozu TP53, BCL2, MCF2L, CDKN2A, SERPINB9, TERF1, TERF2,TERT; geniv-reguliatoriv klitynnoï adgeziï ta angiogenezu CLSTN2, VEGFA.

Keywords: Chornobyl; gene expression; radiation; somatic pathology.

Hyperleukocytic acute leukemia (HLAL) circulating exosomes are delivered to hematopoietic stem cells (HSCs) and bone marrow mesenchymal stem cells (BM-MSCs), thereby inhibiting the normal hematopoietic process. In this paper, we have evaluated and explored the effects of miR-125b, which is carried by HLAL-derived exosomes, on the hematopoietic function of HSCs and BM-MSCs. For this purpose, we have isolated exosomes from the peripheral blood of HLAL patients and healthy volunteers. Then, we measured the level of miR-125b in exosomes cocultured exosomes with HSCs and BM-MSCs. Moreover, we have used miR-125b inhibitors/mimic for intervention and then measured miR-125b expression and colony forming unit (CFU). Apart from it, HSC and BM-MSC hematopoietic-related factors α-globulin, γ-globulin, CSF2, CRTX4 and CXCL12, SCF, IGF1, and DKK1 expression were measured. Evaluation of the miR-125b and BAK1 targeting relationship, level of miR-125b, and expression of hematopoietic-related genes was performed after patients are treated with miR-125b mimic and si-BAK1. We have observed that miR-125b was upregulated in HLAL-derived exosomes. After HLAL-exosome acts on HSCs, the level of miR-125b is upregulated, reducing CFU and affecting the expression of α-globulin, γ-globulin, CSF2, and CRCX4. For BM-MSCs, after the action of HLAL-exo, the level of miR-125b is upregulated and affected the expression of CXCL12, SCF, IGF1, and DKK1. Exosomes derived from HLAL carry miR-125b to target and regulate BAK1. Further study confirmed that miR-125b and BAK1mimic reduced the expression of miR-125b and reversed the effect of miR-125b mimic on hematopoietic-related genes. These results demonstrated that HLAL-derived exosomes carrying miR-125b inhibit the hematopoietic differentiation of HSC and hematopoietic support function of BM-MSC through BAK1.

Laminin-332 pemphigoid is a rare and severe autoimmune blistering disease, caused by IgG autoantibodies targeting laminin-332 in the dermal-epidermal basement zone. Laminin-332 pemphigoid is characterized by variable inflammatory infiltrate and the predominance of non-complement-fixing antibodies. Given these findings, we hypothesized that IgG autoantibodies to laminin-332 directly resulted in keratinocyte expression of inflammatory factors. We performed RNA-seq on primary human keratinocytes treated with IgG from patients with laminin-332 pemphigoid. Genes for numerous cytokines and chemokines were upregulated, including CSF2, CSF3, CXCL1, CXCL5, CXCL3, CXCL8, CXCL10, CXCL1, IL6, IL7, IL15, IL23, IL32, IL37, TGFB2 as well as metalloproteases. Considering the pro-inflammatory and proteolytic effect of autoantibodies from patients with laminin-332 pemphigoid identified in our initial experiment, we next questioned whether the reactivity against specific laminin subunits dictates the inflammatory and proteolytic keratinocyte response. Then, we treated keratinocytes with IgG from a separate cohort of patients with reactivity against individual subunits of laminin-332. We identified upregulation of IL-1α, IL-6, IL-8, CXCL1, MMP9, TSLP, and GM-CSF at the protein level, most notably in keratinocytes treated with IgG from laminin β3-reactive patients. We for the first time demonstrated a pro-inflammatory response, similar to that described in keratinocytes treated with IgG autoantibodies from patients with bullous pemphigoid, providing novel insight into the pathogenesis of laminin-332 pemphigoid and laminin-332 biology.

Keywords: RNA-seq; autoimmune blistering diseases; immunobullous disease; keratinocyte; laminin-332 pemphigoid.

Background: ATRX inactivation occurs with IDH1 R132H and p53 mutations in over 80% of Grade II/III astrocytomas. It is believed that ATRX loss contributes to oncogenesis by dysregulating epigenetic and telomere mechanisms but effects on anti-glioma immunity have not been explored. This paper examines how ATRX loss contributes to the malignant and immunosuppressive phenotypes of IDH1 R132H/p53mut glioma cells and xenografts.

Methods: Isogenic astrocytoma cells (+/-IDH1 R132H/+/-ATRXloss) were established in p53mut astrocytoma cell lines using lentivirus encoding doxycycline inducible IDH1 R132H, ATRX shRNA or Lenti-CRISPR/Cas9 ATRX. Effects of IDH1 R132H+/- ATRXloss on cell migration, growth, DNA repair and tumorigenicity were evaluated by clonal growth, transwell and scratch assays, MTT, immunofluorence and immunoblotting assays and xenograft growth. Effects on the expression and function of modulators of the immune microenvironment were quantified by qRT-PCR, immunoblot, T-cell function, macrophage polarization and flow cytometry assays. Pharmacologic inhibitors were used to examine epigenetic drivers of the immunosuppressive transcriptome of IDH1 R132H/p53mut/ATRXloss cells.

Results: Adding ATRX loss to the IDH1 R132H/p53mut background promoted astrocytoma cell aggressiveness, induced expression of BET proteins BRD3/4 and an immune suppressive transcriptome consisting of up-regulated immune checkpoints (e.g. PD-L1, PD-L2) and altered cytokine/chemokine profiles (e.g. IL33, CXCL8, CSF2, IL6, CXCL9). ATRX loss enhanced the capacity of IDH1 R132H/p53mut cells to induce T-cell apoptosis, tumorigenic/anti-inflammatory macrophage polarization and Treg infiltration. The transcriptional and biological immune suppressive responses to ATRX loss were enhanced by temozolomide and radiation and abrogated by pharmacologic BET inhibition.

Conclusions: ATRX loss activates a BRD-dependent immune suppressive transcriptome and immune escape mechanism in IDH1 R132H/p53mut astrocytoma cells.

Keywords: ATRX loss; BRD-dependent-immune suppressive transcriptome; IDH1 mutant astrocytoma; glioma immune microenvironment.