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Objective: To screen and obtain specific anti-lymphocyte activation gene-3 (LAG3) nanobody sequences, purify and express recombinant anti-LAG3 nanobody, and verify its effect on promoting T cells to kill tumor cells., Methods: Based on the camel derived natural nanobody phage display library constructed by the research group, the biotinylated LAG3 antigen was used as the target, and the anti-LAG3 nanobody sequences were screened by biotin-streptavidin liquid phase screening, phage-ELISA and sequencing. The sequence-conjμgated human IgG1 Fc fragment was obtained, the recombinant anti-LAG3 nanobody expression vector was constructed, the expression of the recombinant anti-LAG3 nanobody was induced by IPTG and purified, and the characteristics and functions of the recombinant anti-LAG3 nanobody were verified by SDS-PAGE, Western blot, cytotoxicity assay, etc. RESULTS: One anti-LAG3 nanobody sequence was successfully screened, and the corresponding recombinant anti-LAG3 nanobody-expressing bacteria were constructed. The results of SDS-PAGE, Western blot and cytotoxicity assay showed that the recombinant anti-LAG3 nanobody was successfully expressed, which was specific, and it could promote the killing ability of T cells against tumor cells, and the optimal concentration was 200 μg/mL., Conclusion: The recombinant anti-LAG3 nanobody screened and expressed has specific and auxiliary anti-tumor cell effects, which lays a foundation for its subsequent application., Competing Interests: Declaration of competing interest We do not have any recognized conflicting economic interests or personal relationships that influence the research presented in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Upregulation of the Wilms' tumour 1 (WT1) gene is common in acute myeloid leukaemia (AML) and is associated with poor prognosis. WT1 generates 12 primary transcripts through different translation initiation sites and alternative splicing. The short WT1 transcripts express abundantly in primary leukaemia samples. We observed that overexpression of short WT1 transcripts lacking exon 5 with and without the KTS motif (sWT1+/- and sWT1-/-) led to reduced cell growth. However, only sWT1+/- overexpression resulted in decreased CD71 expression, G1 arrest, and cytarabine resistance. Primary AML patient cells with low CD71 expression exhibit resistance to cytarabine, suggesting that CD71 may serve as a potential biomarker for chemotherapy. RNAseq differential expressed gene analysis identified two transcription factors, HOXA3 and GATA2, that are specifically upregulated in sWT1+/- cells, whereas CDKN1A is upregulated in sWT1-/- cells. Overexpression of either HOXA3 or GATA2 reproduced the effects of sWT1+/-, including decreased cell growth, G1 arrest, reduced CD71 expression and cytarabine resistance. HOXA3 expression correlates with chemotherapy response and overall survival in NPM1 mutation-negative leukaemia specimens. Overexpression of HOXA3 leads to drug resistance against a broad spectrum of chemotherapeutic agents. Our results suggest that WT1 regulates cell proliferation and drug sensitivity in an isoform-specific manner., (© 2024 British Society for Haematology and John Wiley & Sons Ltd.)

Most high-grade ovarian carcinomas (HGOCs) are sensitive to carboplatin (CBP)-based chemotherapy but frequently recur within 24 months. Recurrent tumors remain CBP-sensitive and acquire resistance only after several treatment rounds. Recurrences arise from a small number of residual tumor cells not amenable to investigation in patients. We developed patient-derived xenografts (PDXs) that allow the study of these different stages of CBP-sensitive recurrence and acquisition of resistance. We generated PDX models from CBP-sensitive and intrinsically resistant HGOC. PDXs were CBP- or mock-treated and tumors were sampled, after treatment and at recurrence. We also isolated models with acquired-resistance from CBP-sensitive PDXs. Tumors were characterized at the histological and transcriptome levels. PDX models reproduced treatment response seen in the patients. CBP-sensitive residual tumors contained nonproliferating tumor cell clusters embedded in a fibrotic mesh. In nontreated PDX tumors and treated CBP-resistant tumors, fibrotic tissue was not prevalent. Residual tumors had marked differences in gene expression when compared to naïve and recurrent tumors, indicating downregulation of the cell cycle and proliferation and upregulation of interferon response and the epithelial-mesenchymal transition. This gene expression pattern resembled that described in embryonal diapause and 'drug-tolerant persister' states. Residual and acquired-resistance tumors share the overexpression of three genes: CEACAM6, CRYAB, and SOX2. Immunostaining analysis showed strong CEACAM6, CRYAB, and SOX2 protein expression in CBP-sensitive residual and acquired-resistance PDX, thus confirming the RNA profiling results. In HGOC PDX, CBP-sensitive recurrences arise from a small population of quiescent, drug-tolerant, residual cells embedded in a fibrotic mesh. These cells overexpress CEACAM6, CRYAB, and SOX2, whose overexpression is also associated with acquired resistance and poor patient prognosis. CEACAM6, CRYAB, and SOX2 may thus serve as a biomarker to predict recurrence and emergence of resistant disease in CBP-treated HGOC patients. © 2022 The Pathological Society of Great Britain and Ireland., (© 2022 The Pathological Society of Great Britain and Ireland.)

Acute respiratory distress syndrome (ARDS) results in catastrophic lung failure and has an urgent, unmet need for improved early recognition and therapeutic development. Neutrophil influx is a hallmark of ARDS and is associated with the release of tissue-destructive immune effectors, such as matrix metalloproteinases (MMPs) and membrane-anchored metalloproteinase disintegrins (ADAMs). Here, we observed using intravital microscopy that Adam8-/- mice had impaired neutrophil transmigration. In mouse pneumonia models, both genetic deletion and pharmacologic inhibition of ADAM8 attenuated neutrophil infiltration and lung injury while improving bacterial containment. Unexpectedly, the alterations of neutrophil function were not attributable to impaired proteolysis but resulted from reduced intracellular interactions of ADAM8 with the actin-based motor molecule Myosin1f that suppressed neutrophil motility. In 2 ARDS cohorts, we analyzed lung fluid proteolytic signatures and identified that ADAM8 activity was positively correlated with disease severity. We propose that in acute inflammatory lung diseases such as pneumonia and ARDS, ADAM8 inhibition might allow fine-tuning of neutrophil responses for therapeutic gain.

Hyperinsulinemia is commonly viewed as a compensatory response to insulin resistance, yet studies have demonstrated that chronically elevated insulin may also drive insulin resistance. The molecular mechanisms underpinning this potentially cyclic process remain poorly defined, especially on a transcriptome-wide level. Transcriptomic meta-analysis in >450 human samples demonstrated that fasting insulin reliably and negatively correlated with INSR mRNA in skeletal muscle. To establish causality and study the direct effects of prolonged exposure to excess insulin in muscle cells, we incubated C2C12 myotubes with elevated insulin for 16 h, followed by 6 h of serum starvation, and established that acute AKT and ERK signaling were attenuated in this model of in vitro hyperinsulinemia. Global RNA-sequencing of cells both before and after nutrient withdrawal highlighted genes in the insulin receptor (INSR) signaling, FOXO signaling, and glucose metabolism pathways indicative of 'hyperinsulinemia' and 'starvation' programs. Consistently, we observed that hyperinsulinemia led to a substantial reduction in Insr gene expression, and subsequently a reduced surface INSR and total INSR protein, both in vitro and in vivo. Bioinformatic modeling combined with RNAi identified SIN3A as a negative regulator of Insr mRNA (and JUND, MAX, and MXI as positive regulators of Irs2 mRNA). Together, our analysis identifies mechanisms which may explain the cyclic processes underlying hyperinsulinemia-induced insulin resistance in muscle, a process directly relevant to the etiology and disease progression of type 2 diabetes., (© 2021 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Tumour-associated macrophages (TAMs) support cancer cell survival and suppress anti-tumour immunity. Tumour infiltration by CD163 pos TAMs is associated with poor outcome in several human malignancies, including multiple myeloma (MM). Signal transducer and activator of transcription 3 (STAT3) is over-activated in human cancers, and specifically within TAMs activation of STAT3 may induce an immunosuppressive (M2-like) phenotype. Therefore, STAT3-inhibition in TAMs may be a future therapeutic strategy.We investigated TAM markers CD163, CD206, and activated STAT3 (pSTAT3) in patients with MGUS (n = 32) and MM (n = 45), as well as healthy controls (HCs, n = 13).Blood levels of the macrophage biomarkers sCD163 and sCD206, and circulating cytokines, as well as bone marrow mRNA expression of CD163 and CD206, were generally increased in MGUS and MM patients, compared to HCs, but to highly similar levels. By immunohistochemistry, bone marrow levels of pSTAT3 were increased specifically within CD163 pos cells in both MGUS and MM patients.In conclusion, macrophage-related inflammatory changes, including activation of STAT3, were present already at the MGUS stage, at similar levels as in MM. Specific increase in pSTAT3 levels within CD163 pos cells supports that the CD163 scavenger receptor may be a useful target for future delivery of STAT3-inhibitory drugs to TAMs in MM patients., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

HIV-1 virus release from infected cells is blocked by human BST-2, but HIV-1 Vpu efficiently antagonises BST-2 due to direct transmembrane domain interactions that occur between each protein. Targeting the interaction between these two proteins is seen as viable for HIV-1 antiviral intervention. This study describes the successful over-expression and purification of a recombinant full-length human BST-2 from inclusion bodies using affinity and anion exchange chromatography. Two milligrams of purified full-length BST-2 were produced per litre of BL21 (DE3) T7 Express® pLysY E. coli culture. Far-UV circular dichroism validated the renaturing of the recombinant protein and retention of its secondary structure. Furthermore, through ELISA, a known human BST-2 binding partner, HIV-1 Vpu, was shown to bind to the renatured and purified protein, further validating its folding. To our knowledge this is the first report of the purification of a wild-type, full-length human BST-2 from Escherichia coli., (Copyright © 2021. Published by Elsevier Inc.)

Decellularized and de-epithelialized placenta membranes have widely been used as scaffolds and grafts in tissue engineering and regenerative medicine. Exceptional pro-angiogenic and biomechanical properties and low immunogenicity have made the amniochorionic membrane a unique substrate which provides an enriched niche for cellular growth. Herein, an optimized combination of enzymatic solutions (based on streptokinase) with mechanical scrapping is used to remove the amniotic epithelium and chorion trophoblastic layer, which resulted in exposing the basement membranes of both sides without their separation and subsequent damages to the in-between spongy layer. Biomechanical and biodegradability properties, endothelial proliferation capacity, and in vivo pro-angiogenic capabilities of the substrate were also evaluated. Histological staining, immunohistochemistry (IHC) staining for collagen IV, and scanning electron microscope demonstrated that the underlying amniotic and chorionic basement membranes remained intact while the epithelial and trophoblastic layers were entirely removed without considerable damage to basement membranes. The biomechanical evaluation showed that the scaffold is suturable. Proliferation assay, real-time polymerase chain reaction for endothelial adhesion molecules, and IHC demonstrated that both side basement membranes could support the growth of endothelial cells without altering endothelial characteristics. The dorsal skinfold chamber animal model indicated that both side basement membranes could promote angiogenesis. This bi-sided substrate with two exposed surfaces for cultivating various cells would have potential applications in the skin, cardiac, vascularized composite allografts, and microvascular tissue engineering., (© 2021. The Author(s).)

To investigate whether expression-based polygenic risk scores for the insulin receptor gene network (ePRS-IRs) modifiy the association between type of depressive symptoms and health-related quality of life (HRQoL). This cross-sectional study includes 1558 individuals from the Helsinki Birth Cohort Study. Between 2001 and 2004, the Short Form-36 questionnaire was employed to assess mental and physical components of HRQoL and Beck Depression Inventory (BDI) to assess depressive symptoms. Depressive symptoms were categorized into minimal (BDI < 10), non-melancholic and melancholic types of depression. The ePRS-IRs were calculated for the hippocampal (hePRS-IR) and the mesocorticolimbic (mePRS-IR) regions of the brain. General linear regression models adjusted for age, sex, population stratification, lifestyle factors and body mass index were applied to analyze the data. Both types of depressive symptoms were associated with lower HRQoL (p < 0.0001). HePRS-IR modified the association between the types of depression and mental HRQoL (p for interaction = 0.005). Melancholic type of depressive symptoms was associated with higher mental HRQoL compared to the non-melancholic symptoms among individuals with low hePRS-IR (adjusted mean 4.1, 95% CI 0.7-7.4, p = 0.018). However, no such difference was evident in moderate or high hePRS-IR groups as higher hePRS-IR was associated with lower mental HRQoL (B = - 3.4, 95% CI - 5.6 to - 1.2) in individuals with melancholic type of depressive symptoms. No direct associations were detected between the ePRS-IRs and type of depressive symptoms or HRQoL. Variations in the glucose-insulin metabolism can lower HRQoL in individuals with melancholic depressive symptoms., (© 2021. The Author(s).)

Background and Aims: Several studies demonstrated a role of active chronic inflammatory infiltrate in carotid plaques progression suggesting a possible link between cardiovascular risk factors and inflammation-related plaque instability. The aim of this study is therefore to evaluate the possible effects of cardiovascular risk factors on in situ expression of proinflammatory markers associated with carotid plaque instability., Methods and Results: A tissue microarray containing carotid plaques from 36 symptomatic (major stroke or transient ischemic attack) and 37 asymptomatic patients was built. Serial sections were employed to evaluate the expression of some inflammatory markers by immunohistochemistry [CD3, CD4a, CD8, CD20, CD86, CD163, interleukin (IL)-2, IL-6, IL-17]. Immunohistochemical data were analyzed to study the possible associations between in situ expression of inflammatory biomarker and the main cardiovascular risk factors. Our data demonstrated that plaque instability is associated with the high in situ expression of some cytokines, such as IL-2, IL-6, IL-17. Besides the female sex, none of the risk factors analyzed showed a significant association between the in situ expression of these markers and unstable plaques. A significant increase of IL-6-positive and IL-17-positive cells was observed in unstable atheromatous plaques of female patients, as compared with unstable plaques of male patients., Conclusions: Plaque destabilization is certainly correlated with the presence of the major cardiovascular risk factors, however, our results showed that, with the exception of sex, their action in the evolutive process of plaque instability seems rather nonspecific, favoring a general release of proinflammatory cytokines., Competing Interests: The authors declare no conflict of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Human genetic studies have pointed to a prominent role for innate immunity and lipid pathways in immunological and neurodegenerative disorders. Our understanding of the composition and function of immunomodulatory lipid networks in innate immune cells, however, remains incomplete. Here, we show that phospholipase Cγ2 (PLCγ2 or PLCG2)-mutations in which are associated with autoinflammatory disorders and Alzheimer's disease-serves as a principal source of diacylglycerol (DAG) pools that are converted into a cascade of bioactive endocannabinoid and eicosanoid lipids by DAG lipase (DAGL) and monoacylglycerol lipase (MGLL) enzymes in innate immune cells. We show that this lipid network is tonically stimulated by disease-relevant human mutations in PLCγ2, as well as Fc receptor activation in primary human and mouse macrophages. Genetic disruption of PLCγ2 in mouse microglia suppressed DAGL/MGLL-mediated endocannabinoid-eicosanoid cross-talk and also caused widespread transcriptional and proteomic changes, including the reorganization of immune-relevant lipid pathways reflected in reductions in DAGLB and elevations in PLA2G4A. Despite these changes, Plcg2 -/- mice showed generally normal proinflammatory cytokine and chemokine responses to lipopolysaccharide treatment, instead displaying a more restricted deficit in microglial activation that included impairments in prostaglandin production and CD68 expression. Our findings enhance the understanding of PLCγ2 function in innate immune cells, delineating a role in cross-talk with endocannabinoid/eicosanoid pathways and modulation of subsets of cellular responses to inflammatory stimuli., Competing Interests: The authors declare no competing interest.

Cadherin switch (CS) outlined by downregulation of E-cadherin and upregulation of N-cadherin is an established epithelial-mesenchymal transition (EMT) hallmark, being a common signature in wound healing and carcinogenesis. It is intriguing to explore the EMT-associated CS pattern in precancerous phases as well as variably aggressive bladder cancer categories. In this study, we tested CS signified by a reduction in urothelial cells E-cadherin expression and/or aberrant N-cadherin expression in proliferative epithelial changes (PEC) associating inflammation, non-muscle-invasive bladder cancer (NMIBC), and muscle-invasive bladder cancer (MIBC). Immunohistochemical study of both E-cadherin and N-cadherin was performed for 60 cases: 15 PEC, 8 NMIBC, and 37 MIBC. CS patterns were analyzed: abnormal CS patterns were expressed as deviated, hybrid, co-negative, and full CS patterns. E-cadherin expression was significantly preserved in PEC (86.7%) followed by NMIBC (62.5%) and then MIBC (37.8%) (P=0.004), whereas N-cadherin showed obvious aberrant expression in MIBC (51.4%) as compared with PEC (33.3%) and NMIBC (25%). In the MIBC group, abnormal cadherin patterns were the highest (70.3%) and was associated with adverse prognostic indicators. In the context of NMIBC progression to MIBC, combined E and N-cadherin evaluation showed highest sensitivity (70.3%) and NPV (31.3%), whereas aberrant expression of N-cadherin presented highest specificity (75%) and positive predictive value (90.5%). For cancer prediction, combined E-cadherin and N-cadherin evaluation showed the highest sensitivity (64.4%); abnormal E-cadherin offered highest specificity (86.7%), positive predictive value (92.9%), and negative predictive value (40.6%). In posttherapy follow-up setting, a metastable EMT signature in the form of partial CS was noted and might reflect resistant dormant populations., Competing Interests: The authors declare no conflict of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Metastasis is the main cause of poor prognosis in the advanced prostate cancer in clinic. Accumulating evidences have proposed that cell motility greatly contributes to the multiple steps of the metastatic process. MicroRNA-145 (miR-145) has been found to be downregulated in prostate cancer and serve as a putative tumor suppressor via decrease of cell growth and augmentation of cell death; however, the effects and the underlying mechanisms of miR-145 in prostate cancer cell motility have not been completely clarified. In the current study, we first demonstrated that miR-145 exerted inhibitory effects on the aggressive phenotype of the prostate cancer cells. Based on the bioinformatics analysis of the putative target genes of miR-145, we further experimentally identified a novel mechanism of miR-145 suppressing the aggressive phenotype of prostate cancer cells via directly targeting cadherin-2 (CDH2) protein translation. Re-expression of CDH2 could rescue miR-145-triggered cell migration and invasion defects. Our results suggested that miR-145 suppressed the motility of prostate cancer cells via post-transcriptional downregulation of CDH2 expression, and miR-145-CDH2 pair might serve as a potential target for intervention of prostate cancer metastasis., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Interferon-γ (IFN-γ) is the sole representative of type II IFNs, with well recognized role in numerous inflammatory processes. Lately, its significant pleiotropic nature has been recognized in many scenarios, where IFN-γ contributes to maintenance or induction of tolerogenic responses in context of various immune cell types. In this manuscript we demonstrate, that IFN-γ-mediated induction of programmed death ligand 1 (PD-L1) on human monocyte-derived dendritic cells (DCs) represents an important tolerogenic aspect in immunological network of type II IFNs. When fully differentiated, immature DCs were treated with increasing concentrations of IFN-γ there was no sign of maturation, as revealed by CD80, CD83 and CD86 expression. In terms of co-stimulatory receptor response, we did observe a dose-dependent increase in CD40 expression. Phenotypic analysis of inhibitory molecules revealed that PD-L1 expression is particularly sensitive to IFN-γ, as its expression can be induced almost 10-fold in comparison to non-treated DCs. Functional analysis of such PD-L1 high DCs revealed significant immunosuppressive properties in a mixed lymphocyte reaction with whole or memory CD4 + T cells. When IFN-γ treated DCs were co-cultured with naive CD4 + CD45RA + T cells, they induced an increased percentage of CD4 + CD25 + CD127 - FoxP3 + Tregs. Inhibition of PD-1/PD-L1 axis using neutralizing anti-PD-L1 mAbs, reversed the immunosuppressive effect of IFN-γ-treated DCs to suppress CD4 + T cell proliferation and to induce Tregs. In summary, our findings demonstrate the importance of IFN-γ-mediated tolerogenic effects, exerted on DCs by inducing increased expression of PD-L1, which enhances their regulatory function., (Copyright © 2021 Elsevier B.V. All rights reserved.)

CD303 + plasmacytoid dendritic cells (pDCs) play an important role in the induction of immune tolerance and antitumor immunity. Here, we focused on the effect of NSCLC cells on the development of CD303 + pDC subsets expressing CD205 and/or CD103. The NSCLC cell line H1299 and primary NSCLC cells were incubated with DCs. The protein expression of costimulatory molecules on CD303 + pDCs, the production of pro-inflammatory and anti-inflammatory cytokines by CD303 + pDCs and the development of CD303 + pDC subsets were detected by using flow cytometry. Coculture with NSCLC cells modulates the protein expression of CD86 and HLA-DR on CD303 + pDCs. Moreover, NSCLC cells suppressed the production of IL-12 and IL-23 but facilitated the secretion of IL-27 and TGF-β by CD303 + pDCs. There were new CD303 + pDC subsets expressing CD205 and/or CD103 in healthy donors and NSCLC patients: CD303 + CD205 + CD103 + , CD303 + CD205 + CD103 - , CD303 + CD205 - CD103 + and CD303 + CD205 - CD103 - pDCs. NSCLC cells modulated the differentiation of CD303 + pDC subpopulations by regulating the protein expression of CD205 and/or CD103 on CD303 + pDCs. NSCLC cells may regulate the immune functions of CD303 + pDCs by modulating the expression of costimulatory molecules on DCs and the production of pro-inflammatory/anti-inflammatory cytokines by DCs. NSCLC cells also regulate the development of CD303 + pDC subsets expressing CD205 and/or CD103. These outcomes may reveal a new cellular mechanism leading to the NSCLC-induced immune-suppressive microenvironment., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Richter syndrome (RS), an aggressive lymphoma occurring in the context of chronic lymphocytic leukaemia/small lymphocytic lymphoma, is associated with poor prognosis when treated with conventional immunochemotherapy, therefore, improved treatments are required. Immune checkpoint blockade has shown efficacy in some B-cell malignancies and modest responses in early clinical trials for RS. We investigated the immune checkpoint profile of RS as a basis to inform rational therapeutic investigations in RS. Formalin-fixed, paraffin-embedded biopsies of RS (n = 19), de novo diffuse large B-cell lymphoma (DLBCL; n = 58), transformed indolent lymphomas (follicular [tFL], n = 16; marginal zone [tMZL], n = 24) and non-transformed small lymphocytic lymphoma (SLL; n = 15) underwent gene expression profiling using the NanoString Human Immunology panel. Copy number assessment was performed using next-generation sequencing. Immunohistochemistry (IHC) for LAG3 and PD-1 was performed. LAG3 gene expression was higher in RS compared to DLBCL (P = 0·0002, log2FC 1·96), tFL (P < 0·0001, log2FC 2·61), tMZL (P = 0·0004, log2FC 1·79) and SLL (P = 0·0057, log2FC 1·45). LAG3 gene expression correlated with the gene expression of human leukocyte antigen Class I and II, and related immune genes and immune checkpoints. IHC revealed LAG3 protein expression on both malignant RS cells and tumour-infiltrating lymphocytes. Our findings support the investigation of LAG3 inhibition to enhance anti-tumour responses in RS., (© 2021 British Society for Haematology and John Wiley & Sons Ltd.)

Objective: The secondary injury caused by RBC autolysis after intracerebral hemorrhage (ICH) can be reduced by increasing the efficiency of microglia (MG)/macrophages (Mø) phagocytizing red blood cells (RBCs). CD47 is an important regulator of MG/Mø phagocytosis. This study aims to clarify whether anti-CD47 antibody administrated into the cisterna magna after ICH can transfer to the hematoma site, promote MG/Mø gathering to phagocytize RBCs and ultimately reduce cell death., Methods: Forty male Wistar rats were divided into sham, ICH, low-dosage (group A, 0.3 μg), medium-dosage (group B, 0.9 μg) and high-dosage (group C, 1.8 μg) anti-CD47 antibody groups. For the rats in group A, B and C, anti-CD47 antibody solution was administrated into the cisterna magna at 10 min after ICH. Brain tissue was harvested 3 days after the operation. Western blotting was performed to detect the expression of Caspase-3 and Bcl-2. Immunofluorescence was performed to detect the CD68 expression. TUNEL was performed to detect the cell death., Results: The hematoma of the ICH rats was located in the basal ganglia, with a good homogeneity of hematoma volume. Low-dosage anti-CD47 antibody in group A had no effects on the perihematomal CD68 (P = 0.338), Caspase-3 (P = 0.769), Bcl-2 (P = 0.176) expression and cell death (P = 0.698), compared with the ICH group. CD68 and Bcl-2 expression increased and Caspase-3 expression decreased significantly in group B (P < 0.001 for all) and group C (P < 0.001 for all). The increase of CD68 expression in group C was greater than that in group B (P < 0.01) by a large margin, while there was no difference for Bcl-2 (P = 0.908) and Caspase-3 (P = 0.913) expression between the 2 groups. Compared with the ICH group, medium-dosage of anti-CD47 antibody in group B significantly reduced the number of TUNEL-positive cells (P < 0.005), but not for group C (P = 0.311)., Conclusion: The results suggested that anti-CD47 antibody administration into the cisterna magna in proper dosage (0.9 μg) can effectively reach the hematoma, induce more MG/Møs to gather around the hematoma, and reduce cell death in perihematomal brain tissue. The results of this study has provided a basic theory for improving the efficiency of MG/Mø phagocytizing RBCs and hematoma clearance after ICH by administrating anti-CD47 antibody via the cisterna magna., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The purpose of this study was to determine whether administration of the microorganism Eubacterium rectale ( E. rectale ) could regulate dendritic cell (DC) activation and systemic inflammation in herpes simplex virus type 1-induced Behçet's disease (BD). E. rectale , butyrate-producing bacteria, was administered to BD mice. Peripheral blood leukocytes (PBL) and lymph node cells were isolated and analyzed by flow cytometry. 16S rRNA metagenomic analysis was performed in the feces of mice to determine the differences in the composition of the microbial population between normal and BD mice. Serum cytokine levels were measured by enzyme-linked immunosorbent assay. The frequency of DC activation marker CD83 positive cells was significantly increased in PBL of BD mice. Frequencies of CD83+ cells were also significantly increased in patients with active BD. 16S rRNA metagenomic analysis revealed different gut microbiota composition between normal and BD mice. The administration of E. rectale to BD mice reduced the frequency of CD83+ cells and significantly increased the frequency of NK1.1+ cells with the improvement of symptoms. The co-administration of colchicine and E. rectale also significantly reduced the frequency of CD83+ cells. Differences in gut microbiota were observed between normal mice and BD mice, and the administration of E. rectale downregulated the frequency of CD83, which was associated with BD deterioration. These data indicate that E. rectale could be a new therapeutic adjuvant for BD management., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Islam, Ryu, Sayeed, Byun, Jung, Kim, Suh and Sohn.)

In breast cancer, humoral immune responses may contribute to clinical outcomes, especially in more immunogenic subtypes. Here, we investigated B lymphocyte subsets, immunoglobulin expression, and clonal features in breast tumors, focusing on aggressive triple-negative breast cancers (TNBC). In samples from patients with TNBC and healthy volunteers, circulating and tumor-infiltrating B lymphocytes (TIL-B) were evaluated. CD20 + CD27 + IgD - isotype-switched B lymphocytes were increased in tumors, compared with matched blood. TIL-B frequently formed stromal clusters with T lymphocytes and engaged in bidirectional functional cross-talk, consistent with gene signatures associated with lymphoid assembly, costimulation, cytokine-cytokine receptor interactions, cytotoxic T-cell activation, and T-cell-dependent B-cell activation. TIL-B-upregulated B-cell receptor (BCR) pathway molecules FOS and JUN, germinal center chemokine regulator RGS1, activation marker CD69, and TNFα signal transduction via NFκB, suggesting BCR-immune complex formation. Expression of genes associated with B lymphocyte recruitment and lymphoid assembly, including CXCL13, CXCR4, and DC-LAMP, was elevated in TNBC compared with other subtypes and normal breast. TIL-B-rich tumors showed expansion of IgG but not IgA isotypes, and IgG isotype switching positively associated with survival outcomes in TNBC. Clonal expansion was biased toward IgG, showing expansive clonal families with specific variable region gene combinations and narrow repertoires. Stronger positive selection pressure was present in the complementarity determining regions of IgG compared with their clonally related IgA in tumor samples. Overall, class-switched B lymphocyte lineage traits were conspicuous in TNBC, associated with improved clinical outcomes, and conferred IgG-biased, clonally expanded, and likely antigen-driven humoral responses. SIGNIFICANCE: Tumor-infiltrating B lymphocytes assemble in clusters, undergoing B-cell receptor-driven activation, proliferation, and isotype switching. Clonally expanded, IgG isotype-biased humoral immunity associates with favorable prognosis primarily in triple-negative breast cancers., (©2021 The Authors; Published by the American Association for Cancer Research.)

Background: Tissue resident memory T cells (TRMs) persist long-term in peripheral tissues without recirculation, triggering an immediate protective inflammatory state upon the re-recognition of the antigen. Despite evidence incriminating the dysregulation of TRMs in autoimmune diseases, few studies have examined their expression in cutaneous lupus erythematosus (CLE)., Objectives: We aimed to examine whether there are differences among TRM populations in CLE depending on different clinical conditions, such as the CLE subtype or association with systemic lupus erythematosus, and to determine the effect of type I interferon (IFN) on the development of TRMs in CLE., Methods: CLE disease activity was evaluated using the Cutaneous Lupus Erythematosus Disease Area and Severity Index. The expression of the TRM markers CD69 and CD103 in CLE lesions was evaluated by immunofluorescence. Flow cytometry was performed on peripheral blood mononuclear cells after IFNα treatment., Results: The number of TRMs expressing either CD69 or CD103 was significantly higher in CLE lesions than in control skin; however, it was not significantly different between discoid lupus erythematosus and subacute CLE, or dependent on the presence of concomitant systemic lupus. Lesional severity was not correlated with an increase in TRMs in CLE. IFNα treatment induced a conspicuous increase in CD69 expression in skin-homing T cells, more profoundly in CD4+ T cells than in CD8+ T cells., Conclusions: Skin TRMs, either CD69 or CD103-positive cells, showed increased levels in the lesional skin of CLE, and IFNα increased the expression of CD69 in T cells.

Pregnancy is an immunological paradox whereby maternal immunity accepts a genetically unique fetus (or fetuses), while maintaining protective innate and adaptive responses to infectious pathogens. This close contact between the genetically diverse mother and fetus requires numerous mechanisms of immune tolerance initiated by trophoblast cell signals. However, in a placental condition known as villitis of unknown etiology (VUE), there appears to be a breakdown in this tolerance allowing maternal cytotoxic T-cells to traffic into the placenta to destroy fetal villi. VUE is associated with several gestational complications and an increased risk of recurrence in a subsequent pregnancy, making it a significant obstetrical diagnosis. The cause of VUE remains unclear, but dysfunctional signaling through immune checkpoint pathways, which have a critical role in blunting immune responses, may play an important role. Therefore, using placental tissue from normal pregnancy (n=8), VUE (n=8) and cytomegalovirus (CMV) infected placentae (n=4), we aimed to identify differences in programmed cell death 1 (PD-1), programmed death ligand-1 (PD-L1), LAG3 and CTLA4 expression between these etiologies by immunohistochemistry (IHC). Results demonstrated significantly lower expression of PD-L1 on trophoblast cells from VUE placentae compared to control and CMV infection. Additionally, we observed significantly higher counts of PD-1+ (>100 cells/image) and LAG3+ (0-120 cells/image) cells infiltrating into the villi during VUE compared to infection and control. Minimal CTLA4 staining was observed in all placentae, with only a few Hofbauer cells staining positive. Together, this suggests that a loss of tolerance through immune checkpoint signaling may be an important mechanism leading to the activation and trafficking of maternal cells into fetal villi during VUE. Further mechanistic studies are warranted to understand possible allograft rejection more clearly and in developing effective strategies to prevent this condition from occurring in utero ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Shahi, Mamber Czeresnia, Cheek, Quinton, Chakraborty and Enninga.)

Purpose: Tumor-infiltrating lymphocytes (TIL) are strongly associated with survival in most cancers; however, the tumor-reactive subset that drives this prognostic effect remains poorly defined. CD39, CD103, and PD-1 have been independently proposed as markers of tumor-reactive CD8 + TIL in various cancers. We evaluated the phenotype, clonality, and prognostic significance of TIL expressing various combinations of these markers in high-grade serous ovarian cancer (HGSC), a malignancy in need of more effective immunotherapeutic approaches., Experimental Design: Expression of CD39, CD103, PD-1, and other immune markers was assessed by high-dimensional flow cytometry, single-cell sequencing, and multiplex immunofluorescence of primary and matched pre/post-chemotherapy HGSC specimens., Results: Coexpression of CD39, CD103, and PD-1 ("triple-positive" phenotype) demarcated subsets of CD8 + TIL and CD4 + regulatory T cells (Treg) with a highly activated/exhausted phenotype. Triple-positive CD8 + TIL exhibited reduced T-cell receptor (TCR) diversity and expressed genes involved in both cytolytic and humoral immunity. Triple-positive Tregs exhibited higher TCR diversity and a tumor-resident phenotype. Triple-positive TIL showed superior prognostic impact relative to TIL expressing other combinations of these markers. TIGIT was uniquely upregulated on triple-positive CD8 + effector cells relative to their CD4 + Treg counterparts., Conclusions: Coexpression of CD39, CD103, and PD-1 demarcates highly activated CD8 + and CD4 + TIL with inferred roles in cytolytic, humoral, and regulatory immune functions. Triple-positive TIL demonstrate exceptional prognostic significance and express compelling targets for combination immunotherapy, including PD-1, CD39, and TIGIT., (©2021 American Association for Cancer Research.)

Endothelial-to-mesenchymal transition (EndMT) and fibroblast-to-myofibroblast (FibroMF) differentiation are frequently reported in organ fibrosis. Stromelysin1, a matrix metalloprotease-3 (MMP3) has been indicated in vascular pathologies and organ injuries that often lead to fibrosis. In the current study, we investigated the role of stromelysin1 in EndMT and FibroMF differentiation, which is currently unknown. In our results, whereas TGFβ2 treatment of endothelial cells (ECs) induced EndMT associated with increased expression of stromelysin1 and mesenchymal markers such as α-smooth muscle actin (αSMA), N-cadherin, and activin linked kinase-5 (ALK5), inhibition of stromelysin1 blunted TGFβ2-induced EndMT. In contrast, treatment of NIH-3T3 fibroblasts with TGFβ1 promoted FibroMF differentiation accompanied by increased expression of αSMA, N-cadherin, and ALK5. Intriguingly, stromelysin1 inhibition in TGFβ1-stimulated myofibroblasts further exacerbated fibroproliferation with increased FibroMF marker expression. Gene Expression Omnibus (GEO) data analysis indicated increased stromelysin1 expression associated with EndMT and decreased stromelysin1 expression in human pulmonary fibrosis fibroblasts. In conclusion, our study has identified that EndMT and FibroMF differentiation are reciprocally regulated by stromelysin1., (© 2020 Wiley Periodicals LLC.)

Epithelial-mesenchymal transition (EMT) is an essential biological process involved in the initiation and progression of cancer by which epithelial tumor cells lose their differentiated characteristics, such as cell-cell adhesion and apical-basal polarity and acquire a more invasive and∕or metastatic mesenchymal phenotype. The present study investigated the expression of immunomarkers with a role in EMT of non-melanoma skin cancers (NMSCs), such as E-cadherin, fibronectin and Slug, for a number of 50 NMSCs, represented by 30 cases of basal cell carcinomas (BCCs) and 20 cases of squamous cell carcinomas (SCCs). For BCC, the statistical analysis of the investigated immunomarkers indicated significantly differences in relation to the depth of invasion, and for E-cadherin and fibronectin with the degree of risk. In the case of SCC, the statistical analysis indicated significant differences of E-cadherin and Slug with the degree of tumor differentiation, and for fibronectin and Slug with the depth of invasion. The analysis of the distribution for the percentage values of the investigated immunomarkers in the case of BCC indicated a significant negative linear relation between E-cadherin/fibronectin and E-cadherin/Slug, and in SCC a significant negative linear relation between E-cadherin∕fibronectin, E-cadherin∕Slug and a positive linear one in the case of fibronectin∕Slug. The study indicates through the statistically significant relation between E-cadherin∕fibronectin and E-cadherin∕Slug, the EMT intervention in carcinogenesis of NMSC.

Integrin α2β1 plays an important role in cellular migration and metastasis processes associated with prostate cancer. The aim of this study was to assess whether selective inhibition of integrin α2β1 is an effective strategy to target metastatic prostate cancer cells. In this regard, we examined the effects of the inhibitor BTT-3033, which selectively interferes with the connection between integrin a2b1 and its ligand, on migration, epithelial-mesenchymal transition (EMT), cell cycle arrest, apoptosis, and specific intracellular signaling pathways using LNcap-FGC and DU-145 prostate cancer cell lines. Western blot analysis and immunocytochemistry assays showed that inhibition of integrin a2b1 inhibits EMT, through the increased expression of E-cadherin and decreased expression of N-cadherin and vimentin. Scratch wound healing assays revealed a direct effect on integrin α2β1 in the migration capacity of cells. In addition, treatment with BTT-3033 induced a reduction in cell viability and proliferation, as assessed by MTT and BrdU assays. In addition, the results show that BTT-3033 inhibits cell proliferation by inducing G1 cell cycle arrest. Moreover, inhibition of integrin α2β1 induces apoptosis through the activation of ROS, Bax protein upregulation, caspase-3 activation, and depletion of ΔΨm.  Molecular signaling studies showed that integrin α2β1 was a positive regulator of MKK7 phosphorylation. In conclusion, our results reveal a critical role for integrin a2b1 in the proliferation of prostate cancer cells, as demonstrated by EMT inhibition, cell cycle arrest, and apoptosis induction in response to treatment with its specific inhibitor BT-3033., (© 2020 Wiley Periodicals LLC.)

Decades after identifying cannabinoids and their beneficial effects on Parkinson's disease (PD), many gaps are still missing. Although, CB 2 -dependent actions have been shown as underlying positive effects of cannabinoid treatment, in recent years, another receptor of cannabinoids, CB 1 , emerged as a valuable player in cannabinoid-induced neuroprotection. Remarkably, the effects of CB 1 are mainly related to immune cells in the CNS, microglia, and astrocytes. However, oxidative stress, α-syn accumulation, and immune disbalance are essential aspects of both neurons and glial cells. Therefore, in this study, we investigated the effects of the CB 1 on both α-syn and rotenone-treated SH-SY5Y and C8-D1A cells. ACEA and AM-251 were used as CB 1 agonists and antagonists. Cell viability, IL-1β, IL-6, TNF-α levels, and CD200 expressions were determined in culture mediums. Our results demonstrated that preformed fibril form (pFF) of α-syn did not cause any significant change in SH-SY5Y cells compared to C8-D1A cells. Rotenone significantly increased the expression of IL-1β, IL-6, and TNF-α levels in both cells. pFF α-syn and rotenone treatment caused a decrease in CD200 expression. Surprisingly both ACEA and AM-251 alleviated rotenone-induced increase in cytokine levels in both cell lines. Although ACEA prevented pFF α-syn induced increase in cytokine levels and decrease in CD200 expression in C8-D1A cells, AM-251 failed to affect CD200 expression levels. Additionally, ACEA + AM-251 abolished the protective effects of both ACEA and AM-251 against rotenone and α-syn insults in both cell lines. The current study suggests that cannabinoid receptor agonism alleviates rotenone and α-syn-dependent inflammation in neurons and astrocytes., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Objective: To observe the protective effect of AC-YVAD-CMK on sepsis-induced acute kidney injury in mice and to explore its possible mechanisms primarily., Methods: Eighteen male C57BL/6 mice were randomly divided into sham-operated group (Control), cecal ligation and puncture group (CLP), and CLP model treated with AC-YVAD-CMK group (AC-YVAD-CMK) ( n = 6 in each group). Mice were sacrificed at 24 h after operation, and blood and kidney tissue samples were collected for analyses. Histologic changes were determined microscopically following HE staining. The expression of Ly-6B and CD68 was investigated using immunohistochemistry. Serum concentrations of creatinine (sCR) and blood urea nitrogen (BUN) were measured. Serum levels of interleukin-1 β (IL-1 β ), interleukin-18 (IL-18), TNF- α , and interleukin-6 (IL-6) were determined by ELISA. The expressions of Caspas-1, NLRP-1, IL-1 β , and IL-18 in renal tissues were investigated using Western blot. Immunofluorescence staining was used to detect the expression of GSDMD protein in renal tissues., Results: AC-YVAD-CMK treatment significantly alleviates sepsis-induced acute kidney injury, with decreased histological injury in renal tissues, suppresses the accumulation of neutrophils and macrophages in renal tissues, and decreased sCR and BUN level ( P < 0.05). Attenuation of sepsis-induced acute kidney injury was due to the prohibited production of inflammatory cytokines and decrease expression of Caspas-1, NLRP-1, IL-1 β , and IL-18 in renal tissues. In addition, AC-YVAD-CMK treatment significantly reduced the expression of GSDMD in renal tissues compared to those observed in controls ( P < 0.05)., Conclusions: We demonstrated a marked renoprotective effect of caspase-1-inhibitor AC-YVAD-CMK in a rat model of sepsis by inhibition of pyroptosis., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2021 Mei Yang et al.)

Objective: Many nutrients essential to the fetus and for proper function of the placenta itself cannot freely diffuse across membrane barriers, and their transplacental transfer depends on transporters. Our previous studies provided evidence for altered expression of transporters for folic acid in trophoblasts exposed to antiseizure medications (ASMs). The goal of the current study was to explore the effects of older and newer ASMs on the expression and function of uptake transporters for choline, which interacts with folate at pathways for methyl group donation., Methods: BeWo cells were incubated for 2 or 5 days with valproate (42, 83, or 166 µg/ml), carbamazepine (6 or 12 µg/ml), levetiracetam (10 or 30 µg/ml), lamotrigine (3 or 12 µg/ml), lacosamide (5, 10, or 20 µg/ml), or their vehicles (n = 6/treatment group). Quantitative polymerase chain reaction (PCR) analysis was utilized to study the effects of ASMs on the transcript levels of the choline transporters SLC44A1 (CTL1) and SLC44A2 (CTL2). Transporter protein expression in valproate-treated cells was assessed by western blot analysis. Choline and acetylcholine were quantified in cell lysates by a choline/acetylcholine assay kit., Results: Compared with controls, valproate and levetiracetam at high therapeutic concentrations (83 and 30 µg/ml, respectively) lowered choline transporter transcript levels by up to 42% and 26%, and total choline levels by 20% and 21%, respectively (p < .05). At 83 μg/ml, valproate additionally reduced CTL1 and CTL2 protein expression, by 39 ± 21% and 61 ± 13% (mean ± SD), respectively (p < .01). Carbamazepine reduced SLC44A1 transcript levels, whereas lacosamide modestly decreased the expression of SLC44A2. Lamotrigine did not alter choline transporter expression., Significance: Antiseizure medications, particularly at high therapeutic concentrations, can interfere with the placental uptake of choline. In line with current knowledge from pregnancy registries and clinical studies, the present in vitro findings further support careful adjustment of maternal ASM doses during pregnancy., (© 2021 International League Against Epilepsy.)

There is overwhelming clinical evidence that the extracellular-regulated protein kinase 5 (ERK5) is significantly dysregulated in human breast cancer. However, there is no definite understanding of the requirement of ERK5 in tumor growth and metastasis due to very limited characterization of the pathway in disease models. In this study, we report that a high level of ERK5 is a predictive marker of metastatic breast cancer. Mechanistically, our in vitro data revealed that ERK5 was critical for maintaining the invasive capability of triple-negative breast cancer (TNBC) cells through focal adhesion protein kinase (FAK) activation. Specifically, we found that phosphorylation of FAK at Tyr397 was controlled by a kinase-independent function of ERK5. Accordingly, silencing ERK5 in mammary tumor grafts impaired FAK phosphorylation at Tyr397 and suppressed TNBC cell metastasis to the lung without preventing tumor growth. Collectively, these results establish a functional relationship between ERK5 and FAK signaling in promoting malignancy. Thus, targeting the oncogenic ERK5-FAK axis represents a promising therapeutic strategy for breast cancer exhibiting aggressive clinical behavior.

Persistent immune activation and inflammation in people living with HIV (PLWH) are associated with immunosenescence, premature aging and increased risk of non-AIDS comorbidities, with the underlying mechanisms not fully understood. In this study, we show that downregulation of the T-cell immunoglobulin receptor CD96 on CD8 + T cells from PLWH is associated with decreased expression of the co-stimulatory receptors CD27 and CD28, higher expression of the senescence marker CD57 and accumulation of a terminally differentiated T-cell memory phenotype. In addition, we show that CD96-low CD8 + T-cells display lower proliferative potential compared to their CD96-high counterparts and that loss of CD96 expression by HIV-specific CD8 + T-cells is associated with a suboptimal response to HIV antigens. In conclusion, our results suggest that CD96 marks CD8 + T-cells with competent responses to HIV and the loss of its expression might be used as a biomarker for CD8 + T-cell senescence and dysfunction in PLWH., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Bunet, Nayrac, Ramani, Sylla, Durand, Chartrand-Lefebvre, Routy, Landay, Gauchat, Chomont, Ancuta, Kaufmann, Bernard, Tremblay and El-Far.)

We characterised patients with mantle cell lymphoma (MCL) with poor prognosis based on differences in immune infiltration. Different expressions of the tumour cell markers Cyclin D1 and sex-determining region Y-box transcription factor 11 (SOX11), and the immune markers cluster of differentiation 3 (CD3), CD4, CD8, CD25, forkhead box protein P3 (FoxP3), T-box transcription factor TBX21 (T-bet), programmed cell death protein 1 (PD-1), programmed-death ligand 1 (PD-L1) and CD163 were investigated for all-cause mortality in 282 patients with MCL and time-to-progression (TTP) in 106 clinical trial patients. With increasing age, a significantly lower infiltration of CD3 + T lymphocytes was seen. T-cell infiltration was independent of cellular tumour antigen p53 (p53) expression, Ki-67, morphology and frequency of tumour cells. The all-cause mortality was higher in patients with PD-L1-expression above cut-off [hazard ratio (HR) 1·97, 95% confidence interval (CI) 1·18-3·25, adjusted for sex and MCL International Prognostic Index (MIPI)] and a higher frequency of CD163 + cells (continuously, HR 1·51, 95% CI 1·03-2·23, adjusting for age, sex, morphology, Ki-67 and p53). In patients treated within the Nordic Lymphoma Group MCL2/3 trials, TTP was shorter in patients with a higher frequency of FoxP3 + cells (HR 3·22, 95% CI 1·40-7·43) and CD163 + cells (HR 6·09, 95% CI 1·84-20·21), independent of sex and MIPI. When combined a higher frequency of CD163 + macrophages and PD-L1 + cells or high CD163 + macrophages and FoxP3 + regulatory T cells indicated worse outcome independent of established risk factors. The T-cell infiltrate was in turn independent of molecular characteristics of the malignant cells and decreased with age., (© 2021 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

Platelets (PLTs) can modulate the immune system through the release of soluble mediators or through interaction with immune cells. Monocytes are the main immune cells that bind with PLTs, and this interaction is increased in several inflammatory and autoimmune conditions, including systemic lupus erythematosus (SLE). Our aim was to characterize the phenotypic and functional consequences of PLT binding to monocytes in healthy donors (HD) and in SLE and to relate it to the pathogenesis of SLE. We analyzed the phenotypic and functional features of monocytes with non-activated and activated bound PLTs by flow cytometry. We observed that monocytes with bound PLTs and especially those with activated PLTs have an up-regulated HLA-DR, CD86, CD54, CD16 and CD64 expression. Monocytes with bound PLTs also have an increased capacity for phagocytosis, though not for efferocytosis. In addition, monocytes with bound PLTs have increased IL-10, but not TNF-α, secretion. The altered phenotypic and functional features are comparable in SLE and HD monocytes and in bound PLTs. However, the percentages of monocytes with bound PLTs are significantly higher in SLE patients and are associated with undetectable levels of anti-dsDNA antibodies and hematuria, and with normal C3 and albumin/creatinine levels. Our results suggest that PLTs have a modulatory influence on monocytes and that this effect may be highlighted by an increased binding of PLTs to monocytes in autoimmune conditions.

Background : Extracellular vesicles (EVs) are secreted by cells from their membrane within circulation and body fluids. Knowledge of the involvement of EVs in pathogenesis of lung diseases is increasing. The present study aimed to evaluate the expression of exosomal surface epitopes in a cohort of idiopathic pulmonary fibrosis (IPF) patients followed in two Italian Referral Centres for Interstitial Lung Diseases, comparing them with a group of healthy volunteers. Materials and Methods : Ninety IPF patients (median age and interquartile range (IQR) 71 (66-75) years; 69 males) were selected retrospectively. Blood samples were obtained from patients before starting antifibrotic therapy. A MACSPlex Exosome Kit, human, (Miltenyi Biotec, Bergisch-Gladbach, Germany), to detect 37 exosomal surface epitopes, was used. Results: CD19, CD69, CD8, and CD86 were significantly higher in IPF patients than in controls ( p = 0.0023, p = 0.0471, p = 0.0082, and p = 0.0143, respectively). CD42a was lower in IPF subjects than in controls ( p = 0.0153), while CD209, Cd133/1, MCSP, and ROR1 were higher in IPF patients than in controls ( p = 0.0007, p = 0.0050, p = 0.0139, and p = 0.0335, respectively). Kaplan-Meier survival analysis for IPF patients: for median values and a cut-off of 0.48 for CD25, the two subgroups showed a significant difference in survival rate ( p = 0.0243, hazard ratio: 0.52 (95%CI 0.29-0.92); the same was true for CD8 (cut-off 1.53, p = 0.0309, hazard ratio: 1.39 (95%CI 0.75-2.53). Conclusion: Our multicenter study showed for the first time the expression of surface epitopes on EVs from IPF patients, providing interesting data on the communication signatures/exosomal profile in serum from IPF patients and new insights into the pathogenesis of the disease and a promising reliability in predicting mid-term survival of IPF patients.

Mycosis fungoides (MF) is the most common primary cutaneous T-cell lymphoma. While initially restricted to the skin, malignant cells can appear in blood, bone marrow and secondary lymphoid organs in later disease stages. However, only little is known about phenotypic and functional properties of malignant T cells in relationship to tissue environments over the course of disease progression. We thus profiled the tumor micromilieu in skin, blood and lymph node in a patient with advanced MF using single-cell RNA sequencing combined with V-D-J T-cell receptor sequencing. In skin, we identified clonally expanded T-cells with characteristic features of tissue-resident memory T-cells (T RM , CD69 + CD27 - NR4A1 + RGS1 + AHR + ). In blood and lymph node, the malignant clones displayed a transcriptional program reminiscent of a more central memory-like phenotype ( KLF2 + TCF7 + S1PR1 + SELL + CCR7 + ), while retaining tissue-homing receptors (CLA , CCR10 ). The skin tumor microenvironment contained potentially tumor-permissive myeloid cells producing regulatory ( IDO1 ) and Th2-associated mediators ( CCL13, CCL17, CCL22 ). Given their expression of PVR, TNFRSF14 and CD80/CD86 , they might be under direct control by TIGIT + CTLA4 + CSF2 + TNFSF14 + tumor cells. In sum, this study highlights the adaptive phenotypic and functional plasticity of MF tumor cell clones. Thus, the T RM -like phenotype enables long-term skin residence of MF cells. Their switch to a T CM -like phenotype with persistent skin homing molecule expression in the circulation might explain the multi-focal nature of MF., Competing Interests: CJ is an employee of the Medical University of Vienna, and has received personal fees from AbbVie, Almirall, Amgen, Eli Lilly, Janssen, LEO Pharma, Mallinckrodt/Therakos, Pfizer, Novartis, Sandoz, Takeda, and UCB. CJ is an investigator for Eli Lilly and Company, Novartis and 4SC (grant paid to her institution). PB is an employee of the Medical University of Vienna, and has received personal fees from LEO Pharma, Pfizer, Sanofi Genzyme, Eli Lilly, Novartis, Celgene, UCB Pharma, Biotest, Boehringer Ingelheim, AbbVie, Amgen and Arena Pharmaceuticals. PB is an investigator for Novartis (grant paid to his institution). WB is an employee of the Medical University of Vienna and has received personal fees from Takeda, Abbvie, GSK/ViiV and Gilead. SP has received personal fees from Takeda. MM has received personal fees (speaker honoraria) from Siemens and Bristol Myers Squibb. WW is an employee of the Medical University of Vienna and has received personal fees from LEO Pharma, Pfizer, Sanofi Genzyme, Eli Lilly, Novartis, Boehringer Ingelheim, AbbVie, and Janssen. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Rindler, Bauer, Jonak, Wielscher, Shaw, Rojahn, Thaler, Porkert, Simonitsch-Klupp, Weninger, Mayerhoefer, Farlik and Brunner.)

Numerous studies have elucidated the impact of long non‑coding (lnc)RNAs in carcinogenesis; however, the role and the mechanism of the lncRNA LOC284454 in hepatocellular carcinoma (HCC) remain unknown. In the present study, reverse transcription‑quantitative PCR assay, χ 2  analysis and Kaplan‑Meier analysis were performed to assess the role of LOC284454 in HCC. Furthermore, MTT and Transwell assays were performed to measure the function of LOC284454 on HCC cell proliferation, invasion and migration. RNA immunoprecipitation, chromatin immunoprecipitation, RNA pull‑down, fluorescence in situ hybridization and luciferase reporter assays were performed to explore the mechanism of LOC284454. The results revealed that LOC284454 expression was aberrantly elevated in HCC and increased LOC284454 expression was markedly associated with aggressive clinicopathological factors and shorter survival time in patients with HCC, suggesting that LOC284454 behaved as an oncogenic factor in HCC. Mechanistically, LOC284454 could bind with the enhancer of zeste homolog 2 (EZH2) mRNA and subsequently inhibit E‑cadherin expression by binding to its promoter region. The rescue assay demonstrated that E‑cadherin was essential for the oncogenic function of LOC284454 in HCC cells. The present results suggested that the LOC284454/EZH2/E‑cadherin axis may be an alternative therapeutic target for patients with HCC.

Semaphorin 4D (Sema4D) is highly expressed in a variety of tumors and is associated with high invasion, poor prognosis and poor therapeutic response. However, the expression and role of Sema4D in leukemia remains unclear. The present study investigated the expression of Sema4D in pediatric leukemia and its effects in leukemia cells. The results demonstrated that Sema4D protein was highly expressed in peripheral blood mononuclear cells of patients with pediatric leukemia, and high levels of soluble Sema4D were also observed in the plasma of these patients. Sema4D knockdown induced cell cycle arrest in G 0 /G 1 phase, inhibited proliferation and promoted apoptosis in BALL‑1 cells, while Sema4D overexpression exhibited the opposite effect. In Jurkat cells, Sema4D knockdown inhibited proliferation and promoted apoptosis, while Sema4D overexpression decreased the abundance of the cells in the G 0 /G 1 phase of the cell cycle and promoted proliferation. Sema4D overexpression also increased the migratory capacity of Jurkat cells and the invasive capacity of BALL‑1 cells. The phosphorylation level of PI3K was decreased in both Sema4D knocked‑down Jurkat and BALL‑1 cells, and the phosphorylation level of ERK was decreased in Sema4D knocked‑down BALL‑1 cells. The phosphorylation levels of PI3K, ERK and AKT were elevated in patients with pediatric leukemia, and were correlated to the increased Sema4D expression. Sema4D overexpression was associated with a shorter overall survival in patients with acute myeloid leukemia. Overall, the results of the present study indicated that Sema4D serves an important role in leukemia development by activating PI3K/AKT and ERK signaling, and it may be used as a potential target for the diagnosis and treatment of leukemia.

Background: Pancreatic neuroendocrine tumors (pNETs) are a heterogeneous group of neoplasms with malignant behaviors that can develop from inert slow growth or low malignancy to aggressive metastasis during follow-up. Recently, vimentin and E-cadherin were shown to be prognostic markers in some malignant tumors but were not evaluated in pNETs. The aim of this study was to evaluate the expression and prognostic significance of vimentin and E-cadherin in grade 1 and 2 pNETs., Methods: A retrospective review of 227 patients with grade 1 and 2 pNETs undergoing surgical resection was conducted. Tumor specimens were immunohistochemically stained for vimentin and E-cadherin. Correlations between vimentin and E-cadherin expression and other clinicopathological features were then analyzed. Furthermore, overall survival (OS) and disease-free survival (DFS) were evaluated using Kaplan-Meier and univariate and multivariate Cox regression methods., Results: Among 227 patients, 55 (24.2%) harbored tumors with high vimentin expression, while 117 (51.5%) harbored tumors with loss of E-cadherin expression. Patients with high vimentin expression and loss of E-cadherin expression had significantly elevated risks of lymph node metastasis, distant metastasis, perineural invasion and an advanced American Joint Committee on Cancer (AJCC) stage compared with those with low vimentin expression and preserved E-cadherin expression, high vimentin expression and preserved E-cadherin expression, or low vimentin expression and loss of E-cadherin expression. Furthermore, multivariate analysis showed that high vimentin expression with loss of E-cadherin expression was an independent predictor of OS and DFS in patients with grade 1 and 2 pNETs who underwent resection (both P < 0.001)., Conclusions: The current study demonstrated that high vimentin expression with loss of E-cadherin expression was correlated with lymph node metastasis, distant metastasis, disease progression and a poor prognosis in patients with grade 1 and 2 pNETs who underwent resection.

Objective: Recent studies have indicated that CD47, interacting with SIRP-α, conveys "don't eat me" signal in evasion of tumor cells and serves as a potential target for cancer immunotherapy. The purpose of this study was to investigate the clinical correlation of CD47 and uncover prognostic implications of CD47 and CD68 in non-small cell lung cancer (NSCLC)., Methods: The specimens from 384 patients with completely resected NSCLC were collected for immunohistochemical assays of CD47 and CD68. Cox multivariate proportion hazard analyses were conducted to confirm the independent prognostic value of CD47 and CD68. TCGA database and GSE37745 were used to identify the association between CD47 and immune cells., Results: In 186 pairs of lung cancer and adjacent tissues, the RNA of CD47 was overexpressed in lung cancer tissues (P < 0.001). High expression of CD47 was associated with worse recurrence-free survival in RNA and protein level (P = 0.032 and P < 0.001, respectively). High expression of CD47 was significantly associated with large tumor size (P = 0.004), advanced pathologic TNM stage (P < 0.001), and histology (P = 0.003). Further analyses demonstrated that CD47 and CD68 predicted outcomes of patients independently. In addition, the expression of CD47 correlated with neutrophils, and did not correlated with B cells and CD4 + T cells in the TCGA database and GSE37745., Conclusion: Combined use of CD47 and CD68 exhibited excellent performance in predicting survival of patients with NSCLC. CD47 was a potential therapeutic target for immune therapy of lung cancer.

Osteoporosis is a prevalent systemic skeletal disorder entailing bone fragility and increased fracture risk, often emerging in post-menopausal life. Emerging evidence implicates the dysregulation of microRNAs (miRNAs or miRs) in the progression of osteoporosis. This study investigated the effect of miR-199a-3p on osteoporosis and its underlying mechanism. We first examplished an ovariectomized (OVX)-induced rat osteoporosis model, and then isolated mesenchymal stem cells (MSCs) from bone marrow of the model rats. The overexpression and knock down of miR-199a-3p were conducted in OVX rats and MSCs to verify the role of miR-199a-3p on MSC differentiation. Calcium nodules were measured using alizarin red S (ARS) staining. RT-qPCR and Western blot assay were performed to measure the expression of miR-199a-3p, Kdm3a and osteogenic differentiation-related markers in rat tissues and cells. The correlation between miR-199a-3p and Kdm3a was confirmed using dual-luciferase reporter assay. The enrichment of Kdm3a at the Erk2 and Klf2 promoter was assessed using chromatin immunoprecipitation (ChIP) assay. Isolated MSCs were positive for CD29, CD44, CD90, and CD45, suggesting successful isolation of MSCs. There was increased expression of miR-199a-3p and inhibited osteogenic differentiation in OVX rats. Kdm3a was negatively targeted by miR-199a-3p. Our results also demonstrated that Kdm3a elevated the expression of Erk2 and Erk2 by promoting Erk2 and Klf2 demethylation, which further contributed to osteogenic differentiation. Overall, our results revealed a regulatory network of miR-199a-3p in osteogenic differentiation, highlighting miR-199a-3p as a potential target for therapeutic interventions in osteoporosis., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Macrophages can initiate innate immune responses against microbes and cancer. The aim of this study was to elucidate the effects of Brassica rapa L. on macrophages. The production of interleukin (IL)-6, tumor necrosis factor (TNF)-α, and interferon-γ induced by the insoluble fraction of B. rapa L. was decreased in macrophage-depleted spleen cells compared with controls. The insoluble fraction of B. rapa L. induced expression of H-2Kb, I-Ab, CD40, and CD86, production of cytokines and nitric oxide, and phagocytic activity in RAW264 cells. After treatment with the insoluble fraction, IL-6 and TNF-α production was significantly decreased by anti-Toll-like receptor (TLR)2 mAb or polymyxin B compared with the control. Furthermore, insoluble fraction-mediated cytokine production was significantly lower in peritoneal macrophages from TLR2-/- and TLR4-/- mice compared with wild-type mice. These results suggest that B. rapa L. is a potentially effective immunomodulator for activating macrophages to prevent infections., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Sialic acid-binding immunoglobulin-like lectins (Siglecs) are involved in various immune cell-mediated diseases. Their role in cancer is poorly investigated, and research focusses on Siglec-expression on immune cells interacting with tumor cells. This study evaluates the role of Siglec-8 in breast cancer (BC). Siglec-8 expression was analyzed immunohistochemically on 235 primary BC cases and was correlated with clinical and pathological parameters and outcome. Cell culture experiments were performed with various BC cell lines. Siglec-8 was expressed in 215 BC cases and expression was lowest in triple-negative BC. It correlated with estrogen receptor-status, grading and the prognostic factors galectin (Gal)-7 and tumor-associated mucin-1 (TA-MUC1). However, Gal-7 and TA-MUC1 were only prognosticators for clinical outcome in the cohort expressing high (Immunoreactivity score IRS > 3) Siglec-8 levels but not in the low-expressing cohort. Siglec-8 knockdown led to a significantly reduced Gal-7 expression in MCF7 cells. All BC cell lines expressed low Siglec-8-levels, that could be elevated in MCF7 by Peroxisome proliferator-activated receptor (PPARγ)-stimulation. This study demonstrates that Siglec-8 is expressed in BC cells and correlates with known clinical and prognostic parameters. It is probably associated with Gal-7 and TA-MUC1 and might be regulated via PPARγ. Further analyses focusing on functional associations will clarify Siglec-8's eligibility as a possible therapeutic target.

Both, aberrant mast cell responses and complement activation contribute to allergic diseases. Since mast cells are highly responsive to C3a and C5a, while Interleukin-33 (IL-33) is a potent mast cell activator, we hypothesized that IL-33 critically regulates mast cell responses to complement anaphylatoxins. We sought to understand whether C3a and C5a differentially activate primary human mast cells, and probe whether IL-33 regulates C3a/C5a-induced mast cell activities. Primary human mast cells were generated from peripheral blood precursors or isolated from healthy human lung tissue, and mast cell complement receptor expression, degranulation, mediator release, phosphorylation patterns, and calcium flux were assessed. Human mast cells of distinct origin express constitutively higher levels of C3aR1 than C5aR1, and both receptors are downregulated by anaphylatoxins. While C3a is a potent mast cell degranulation inducer, C5a is a weaker secretagogue with more delayed effects. Importantly, IL-33 potently enhances the human mast cell reactivity to C3a and C5a (degranulation, cytokine and chemokine release), independent of changes in C3a or C5a receptor expression or the level of Ca 2+ influx. Instead, this reflects differential dynamics of intracellular signaling such as ERK1/2 phosphorylation. Since primary human mast cells respond differentially to anaphylatoxin stimulation, and that IL-33 is a key regulator of mast cell responses to complement anaphylatoxins, this is likely to aggravate Th2 immune responses. This newly identified cross-regulation may be important for controlling exacerbated complement- and mast cell-dependent Th2 responses and thus provides an additional rationale for targeting anti-IL33 therapeutically in allergic diseases., Competing Interests: LR is an employee at GSK. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 West, Bahri, Garcia-Rodriguez, Sweetland, Wileman, Shah, Montero, Rapley and Bulfone-Paus.)

Dim light at night (dLAN) has become a pervasive part of the modern world, and growing evidence shows its association with increased health risks. Though this link is attributed to a disturbed circadian clock, the underlying mechanisms that can explain how circadian disruption from dLAN causes negative health effects remain unclear. Here, we exposed rats to a light-dark cycle (12:12 h) with low-intensity light at night (~2 lx) for 2 and 5 weeks and explored the steady-state pattern of circulating immune cells and renal immune-related markers, which are well controlled by the circadian clock. After 5 weeks, dLAN impaired the daily variation in several types of white blood cells, especially monocytes and T cells. Two-week dLAN caused a reduction in blood monocytes and altered gene expression of macrophage marker Cd68 and monocyte-attracting chemokine Ccl2 in the kidney. Interestingly, dLAN decreased renal 3-nitrotyrosine levels and resulted in up-regulation of the main endogenous antioxidant pathways, indicating a disturbance in the renal redox balance and an activation of compensatory mechanisms. These effects paralleled the altered renal expression of the molecular clock components and increased plasma corticosterone levels. Together, our results show that chronic exposure to dLAN weakened the circadian control of daily variation of circulating immune cells and disturbed renal immune and redox homeostasis. Consequences of this dLAN-disturbed immune balance on the ability of the immune system to cope with other challenges should by clarified in further studies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Okuliarova, Mazgutova, Majzunova, Rumanova and Zeman.)

Background: The disappointing activity of single agent immune-checkpoint inhibitors in epitherlial ovarian cancer (EOC) has been attributed in part to its unique tumor microenvironment (TME). IDO, PDL1, LAG3 and TIM3 have been implicated in the immunotolerance of EOC. We investigated the expression of these co-regulators, their change with neoadjuvant chemotherapy (NACT), and their association with outcome., Method: We identified 98 patients with EOC treated with NACT and performed IDO, PDL1, LAG3 and TIM3 immunohistochemistry on samples obtained before and after NACT. The cut-off threshold to consider a positive sample was set at 5%., Results: In our cohort, TIM3 was the most prevalent co-regulator, with more than 75% of the samples being TIM3 positive. In comparison, only 22%, 28% and 17% of the samples were considered IDO, PDL1 and LAG3 positive. More than half of ovarian tumors expressed 2, 3 or even all 4 co-inhibitory molecules. However, biomarkers were not correlated with each other. NACT had a marked impact on immune co-regulator expression with over 70% of patients showing a change in biomarker status from negative to positive or vice versa. There was no significant difference in the pattern of co-regulator expression between platinum-sensitive and resistant patients. Co-expression of multiple inhibitory molecules did not appear to affect overall and progression-free survival., Conclusion: TIM3 is the most abundant co-inhibitory molecule in OC and may represent an attractive target. In addition, OC frequently co-expressed 2 or more markers supporting ICI combinatorial approaches. Finally, NACT significantly altered the expression of immunosuppressive molecules suggesting that the choice of ICI combinations should be adapted to the composition of the post-NACT immune TME., Competing Interests: Declaration of Competing Interest AL declares potential competing interests with Astrazenca (Advisory board, travel expenses), Clovis (Advisory board), Tesaro/GSK (Advisory board, travel expenses), Merck Serono (Advisory board), biocad (Advisory board), MSD (Advisory board), Roche (travel expenses). The rest authors declare no potential conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

In vertebrates, the semaphorin family of proteins is composed of 21 members that are divided into five subfamilies, i.e. classes 3 to 7. Semaphorins play crucial roles in regulating multiple biological processes, such as neural remodeling, tissue regeneration, cancer progression, and, especially, in immunological regulation. Semaphorin 4D (SEMA4D), also known as CD100, is an important member of the semaphorin family and was first characterized as a lymphocyte-specific marker. SEMA4D has diverse effects on immunologic processes, including immune cell proliferation, differentiation, activation, and migration, through binding to its specific membrane receptors CD72, PLXNB1, and PLXNB2. Furthermore, SEMA4D and its underlying signaling have been increasingly linked with several immunological diseases. This review focuses on the significant immunoregulatory role of SEMA4D and the associated underlying mechanisms, as well as the potential application of SEMA4D as a diagnostic marker and therapeutic target for the treatment of immunological diseases., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Long noncoding RNAs (lncRNAs) promote invasion and migration by glioblastoma (GBM) cells. In this study, quantitative real-time polymerase chain reaction was used to detect expression levels of the lncRNA HOTAIRM1 in GBM tissue samples and cells. The function of HOTAIRM1 was examined using wound healing assays, transwell assays, and in vivo experiments after GBM cells were transfected with either sh-ctrl or sh-HOTAIRM1. Luciferase reporter assays and RIP assays were performed to determine the interactions between HOTAIRM1 and miR-153-5p and between miR-153-5p and SNAI2. We also used luciferase reporter assays and ChIP assays to assess the transcriptional regulation of HOTAIRM1 by SNAI2 and CDH1. HOTAIRM1 was significantly overexpressed in GBM tissues and cells. HOTAIRM1 knockdown significantly weakened the migration and invasion by GBM cells. HOTAIRM1 was found to sponge miR-153-5p, and SNAI2 is a direct target of miR-153-5p. In addition, SNAI2 was shown to force HOTAIRM1 expression through directly promoting transcription and suppressing the negative regulation of CDH1 on transcription. Our results indicate a positive feedback loop between HOTAIRM1 and SNAI2, and suggest that the lncRNA HOTAIRM1 is a potential biomarker and therapeutic target in GBM.

Background: Iron is required for the proliferation of cancer cells, and its depletion suppresses tumor growth. Eribulin mesylate (eribulin), a non-taxane microtubule inhibitor, disrupts the tumor microenvironment via vascular remodeling and obstruction of the epithelial-mesenchymal transition (EMT). Herein, we investigated the effects of the iron chelator on tumor-related properties of breast cancer cells and the effects of iron chelator plus eribulin on tumor growth in vivo., Methods: Two triple-negative breast cancer (TNBC) cell lines, MDA-MB-231 and BT-549, and one hormone-receptor positive breast cancer cell line, MCF-7, were used in our study. Cell proliferation, cell migration, cell cycle position, and gene expression were analyzed via MTT assays, wound-healing assays, flow cytometry, and quantitative real-time-polymerase chain reaction, respectively. For the in vivo experiments, mice with breast cancer xenografts were treated with the inhibitors, alone or together, and tumor volume was determined., Results: Iron chelator inhibited breast cancer cell proliferation and decreased the proportion of S-phase cells. Conversely, it induced hypoxia, angiogenesis, EMT, and immune checkpoints, as determined by quantifying the expression of marker mRNAs in MDA-MB-231 and MCF-7 cells. Eribulin suppressed the expression of the hypoxia and EMT related marker mRNAs in the presence of iron chelator. Iron chelator plus eribulin inhibited tumor growth in vivo to a greater extent than did either inhibitor alone., Conclusions: Although iron chelator induces oncogenic events (hypoxia, angiogenesis, EMT, and immune checkpoints), it may be an effective treatment for breast cancer when administered in combination with eribulin.

BST2 is a single-pass type II transmembrane (TM) protein, which has a cytoplasmic domain, a transmembrane domain, and an extracellular domain, each domain is important for biologic function of BST2. BST2 is a host restriction factor that can effectively inhibit retrovirus release. Rhesus monkeys are considered as relevant natural animal models for studying AIDS in humans. In order to recognize rhesus BST2 (RhBST2) protein and detect its function accurately, we prepared a polyclonal antibody (pAb) especially for RhBST2. Meanwhile, we constructed RhBST2 proteins with the addition of an HA-tag at the N-terminus (RhBST2-NHA) or inside of the ectodomain (RhBST2-IHA) to compare the recognition ability of rabbit anti-RhBST2 pAb and anti-HA mAb. The results showed that the anti-HA mAb and rabbit anti-RhBST2 pAb had the same ability to identify RhBST2. RhBST2 demonstrated antiviral activity and the ability to activate NF-κB. Moreover, the N-glycosylation states, cell surface level and intracellular localization of RhBST2 were detected. However, HA tags relatively changed part of the biological function of RhBST2. These results show that the RhBST2 polyclonal antibody is more suitable for analyzing the properties and functions of RhBST2, and the natural domain of RhBST2 is very important for its function., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Objective: This study aimed to explore the effects of activating GABAB1 receptor by baclofen on proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) of ovarian cancer cells., Results: One hundred μmol/L, 200 μmol/L and 300 μmol/L were selected as low, medium and high baclofen concentrations respectively. Cells were divided into four groups: Control, 100 μmol/L, 200 μmol/L and 300 μmol/L. Compared with the control group, the viability, colony formation, migration and invasion of SKOV3 cells were inhibited, and the apoptosis of SKOV3 cells were enhanced significantly at 200 μmol/L and 300 μmol/L baclofen. Moreover, they changed significantly with the increase of baclofen concentration. Compared with the control group, the expression of E-cadherin and GABAB1 increased and the N-cadherin expression decreased significantly in 200 μmol/L and 300 μmol/L groups. Higher concentration of baclofen induced higher expression of E-cadherin and lower expression of N-cadherin., Conclusion: Baclofen inhibited the proliferation, cloning, migration, invasion and EMT of ovarian cancer cells by activating GABAB1 receptor. These results might contribute a lot to clarify the role and possible mechanism of GABAB1 receptor in ovarian cancer.

Aims: To explore the mechanisms of erythropoietin (EPO)'s protection on inner blood-retinal barrier (iBRB) in experimental diabetic retinopathy., Material and Methods: Male SD rats were rendered diabetic with streptozotocin, followed by intravitreal injection of EPO. The permeability of iBRB was examined with fluorescein isothiocyanate (FITC)-dextran. Human retinal microvascular endothelial cells (HRMECs) and human umbilical vein endothelial cells (HUVECs) were treated with glyoxal and studied for cell viability and barrier function. The expressions of vascular endothelial (VE)-cadherin, Src kinase, vascular endothelial growth factor (VEGF) and VEGF receptor-2 (VEGFR2) were analyzed with Western blot, ELISA, qPCR, or immunofluorescence., Key Findings: VE-cadherin in rat retinas was down-regulated with diabetes progression. EPO treatment could increase VE-cadherin expression at week 8 and week 16. The expressions of p-Src and p-VE-cadherin were increased at week 2, while decreased at week 8 of diabetes; which were prevented by EPO. The leakage of FITC-dextran in 8-week diabetic rat retinas was ameliorated by EPO. In vitro results showed the expressions of VEGF, p-Src and p-VE-cadherin were increased significantly, accompanied with the decreased barrier function, which were prevented by EPO. Ranibizumab and CGP77675 also inhibited the glyoxal-induced phosphorylation of Src and VE-cadherin. Cellular fractionation showed EPO mitigated the VE-cadherin internalization in glyoxal-treated cells., Significance: EPO maintained the expression of VE-cadherin in experimental diabetic retinopathy by inhibiting its phosphorylation and internalization through VEGF/VEGFR2/Src pathway, thus improved the integrity of iBRB., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2020. Published by Elsevier Inc.)