Membrane Expression Research Articles

4-Phenylbutyrate promoted wild-type γ-aminobutyric acid type A receptor trafficking, reduced endoplasmic reticulum stress, and mitigated seizures in Gabrg2+/Q390X mice associated with Dravet syndrome.

γ-Aminobutyric acid type A (GABAA ) receptor subunit gene mutations are major causes of various epilepsy syndromes, including severe kinds such as Dravet syndrome. Although the GABAA receptor is a major target for antiseizure medications, treating GABAA receptor mutations with receptor channel modulators is ineffective. Here, we determined the effect of a novel treatment with 4-phenylbutyrate (PBA) in Gabrg2+/Q390X knockin mice associated with Dravet syndrome. We used biochemistry in conjunction with differential tagging of the wild-type and the mutant alleles, live brain slice surface biotinylation, microsome isolation, patch-clamp whole-cell recordings, and video-monitoring synchronized electroencephalographic (EEG) recordings in Gabrg2+/Q390X mice to determine the effect of PBA invitro with recombinant GABAA receptors and invivo with knockin mice. We found that PBA reduced the mutant γ2(Q390X) subunit protein aggregates, enhanced the wild-type GABAA receptor subunits' trafficking, and increased the membrane expression of the wild-type receptors. PBA increased the current amplitude of GABA-evoked current in human embryonic kidney 293T cells and the neurons bearing the γ2(Q390X) subunit protein. PBA also proved to reduce endoplasmic reticulum (ER) stress caused by the mutant γ2(Q390X) subunit protein, as well as mitigating seizures and EEG abnormalities in the Gabrg2+/Q390X mice. This research has unveiled a promising and innovative approach for treating epilepsy linked to GABAA receptor mutations through an unconventional antiseizure mechanism. Rather than directly modulating the affected mutant channel, PBA facilitates the folding and transportation of wild-type receptor subunits to the cell membrane and synapse. Combining these findings with our previous study, which demonstrated PBA's efficacy in restoring GABA transporter 1 (encoded by SLC6A1) function, we propose that PBA holds significant potential for a wide range of genetic epilepsies. Its ability to target shared molecular pathways involving mutant protein ER retention and impaired protein membrane trafficking suggests broad application in treating such conditions.

Soluble and Membrane P-Glycoprotein Expression in Lymphocytes from Diffuse Large B Cell Non-Hodgkin's Lymphoma Patients Treated with R-CHOP

Introduction Diffuse large B cell lymphoma (DLBC) accounts for 25% of all Non-Hodgkin's Lymphoma (NHL) in the United States. Rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone (R-CHOP) has been the first-line therapy for patients with DLBC-NHL since the early 2000s. Nearly 40% relapse in the following 2-years after complete response and 15% are refractory to first-line therapy. Despite many efforts, other chemotherapy combinations have failed to improve the response rate of NHL patients. Mechanisms of resistance to cytotoxic drugs are vast and include augmented drug efflux mediated through ATP-dependent pumpsas P-glycoprotein (P-gp), which has been related to lesser efficacy. This is due to an over-expression of the glycoprotein with the consequent increase of the drug's efflux from the cytosol. Almost all components of the R-CHOP therapy are P-gp substratesand thereby are affected by P-gp activity. Objectives Analyze serum, plasmatic levels, and membrane expression of P-gp in lymphocytes from patients with DLBC-NHL. Methods A case-control study was conducted to measure serum and plasmatic levels of P-gp, and membrane P-gp expression at the end of treatment on DLBC-NHL patients at “Hospital Civil de Guadalajara Fray Antonio Alcalde,” diagnosed from April to September 2022. Inclusion criteria were age ≥ 18 years, without cancer history and without P-gp inductors or inhibitors. Exclusion criteria were pregnancy, hepatitis, autoimmune disease, Richter syndrome and infection 15 days before blood draw. Using whole blood samples, we identified T and NK cells expressing membrane P-gp by flow cytometry on Attune® NxT (Life Technologies) using the following antibodies and fluorochrome conjugates: CD3-APC/Cy7, CD4-AlexaFlour488, CD8-APC, CD56-BrilliantViolet, CD243-PE. Data was analyzed with FlowJo v10 software. Serum and plasmatic P-gp measurement were done using the Human Permeability Glycoprotein (P-gp) ELISA kit (MyBioSource). Statistical Analysis Proportion comparison between groups was made using Chi-Square or Fisher exact tests. A quantitative variable comparison was done using the U Mann-Witney test. Correlation analysis was made using the Spearman correlation coefficient. Statistical significance was calculated using the p<0.05 two-tailed test. All statistical analysis was made using the software R version 4.1.2 and ggpurb/ggplot2 package. Results Only 15 patients and 30 control individuals were included. Group characteristics and comparisons are summarized in table 1. Regarding the DLBC-NHL group, 27% patients were classified as Lugano I, II and IV stages respectively and 20% as Lugano III stage. Nearly 27% of the patients received radiotherapy as consolidation therapy. Also, 40% of patients showed lymphopenia with a median of 1.23 x10 3/μL (IQR 0.94 - 1.34). Serum and plasmatic P-gp concentrations were similar between groups (p>0.05). The CD8+, CD4+, and CD56+ lymphocyte percentage with membrane P-gp expression comparison between the DLBC-NHL group and the control group presented no significant differences (p>0.05). However, the P-gp median fluorescence index (MFI) in CD8+, CD4+, and CD56+ lymphocytes was higher in the control group than DLBC-NHL group (p<0.05), as displayed in figure 1. Discussion When analyzing only the DLBC-NHL group, we identified a negative correlation between serum P-gp concentration and its expression in NK cell membrane and a positive correlation with membrane expression in CD4+ and CD8+ lymphocytes. Kato et al. reported that P-gp can be secreted through exosomes in patients with drug resistance. Lettau et al. found that CD4+ and CD8+ T lymphocytes have higher exosome expulsion compared to NK cells. These could be related to our finding of negative correlation between serum P-gp and membrane P-gp expression in NK lymphocytes. Ghetie et al. found that Rituximab-an anti-CD20 monoclonal antibody-inhibits P-gp efflux function and therefore makes multi-drug-resistant cell cultures chemosensitive again. B-cells affected by rituximab may influence T-cell P-gp expression and this may be the reason why MFI was lower on DLBC-NHL group than control group. This study helps to expand the understanding of P-gp's role in DLBC-NHL patients as well as R-CHOP therapy effects on the expression of the P-gp in circulating T-cells.

Capturing Endothelial-to-Hematopoietic Transition Using a Highly Efficient in Vitro Platform

Introduction De novo generation of hematopoietic stem and progenitor cells (HSPCs) for therapeutic use has been a long-standing goal of regenerative medicine. During development, the first definitive hematopoietic stem cells (HSCs) derive from a small number of specialized vascular endothelial cells (EC), named hemogenic endothelial cells (HECs), located in the Aorta-Gonado-Mesonephrose (AGM). The process of HEC conversion to HSPCs is known as endothelial-to-hematopoietic transition (EHT). During EHT, endothelial features are lost while switching on hematopoietic transcriptional programs leading to drastic functional and morphological changes that generate free-floating, round hematopoietic cells from large, flat, anchorage-dependent ECs. Key steps regulating EHT are difficult to elucidate using in vivo models because the hemogenic capacity of HECs occurs within a 2-day developmental window. Only a tiny number of cells undergo EHT in vivo. New model systems that overcome current in vivo obstacles are required so the details of EHT and the mechanisms that lead to the generation of HSPCs can be better understood. We previously reported that fully-committed ECs can be converted to functional HSPCs by ectopic expression of transcription factors Spi1, FOSB, GFI1, and Runx1 (SFGR) and an appropriate vascular niche (Sandler et al. Nature, 2014; Lis et al. Nature, 2017). But conversion using this method required several weeks and was associated with variable and low efficiency. Here, we show that our new in vitro EHT platform (ivEHT), very closely recapitulates the process of EHT in vivo and can be used to uncover heretofore unrecognized steps necessary for EC conversion to HSPCs. Methods All steps in the in vitro conversion of ECs to HSPCs were systematically analyzed and optimized. The refined model utilized new media constituents and polycistronic constructs to ensure ectopic expression of transcription factors Spi1, FOSB, GFI1 and Runx1 (SFGR)in human umbilical vein-ECs (HUVECs) in appropriate ratios and sequence. Results The new ivEHT platform yields efficient EC conversion to hematopoietic like CD45+ cells (≥80%) and high temporal reproducibility of conversion that is largely complete within 96 hrs. The ivEHT sequence of conversion closely follows the timing and sequence of EHT in vivo. Initially, transduced HUVECs are large, flat cells with CDH5-marked intercellular junctions and firm adhesion to basement membrane. Within the first 24hrs of induction, SFGR-HUVECs downregulate ECs markers CD31, KDR, CDH5 and activate the endogenous RUNX1 locus detected by the expression of nuclear GFP (GFPn) under control of the human RUNX1+24 enhancer (detected using a lentiviral reporter vector). Day 2 (D2) is characterized by complete clearance of endothelial-specific Weibel-Palade Bodies (WPBs, marked by Von Willebrand Factor, VWF) and excess mitochondria. This window also initiates morphologic changes in RUNX1-GFPn marked cells with loss of intercellular junctions and attachment to basement membrane and initial expression of hematopoietic markers such as CD45. Days 3-4 are marked by loss of cell volume and cytoplasm, cellular rounding and detachment from matrix, and increased expression of CD45 and other hematopoietic markers like cKIT, MPL, CD43. These stages of conversion are only identified in RUNX1-GFPn marked cells and by the end of D5, ~80% of cells have fully converted to CD45+ cells. Conversion is not accompanied by cell proliferation, but after D5 cell numbers expand rapidly. The ivEHT platform is scalable and highly reproducible making it amenable to higher-throughput testing in 96-well format using flow cytometry readouts. Using the ivEHT platform, we have captured and validated in AGM explants previously unobserved aspects of early EHT that is complete between D2-D3. Discussion This work promises a reproducible, faithful, and scalable platform which generates hematopoietic like cells while successfully recapitulating early steps of the EHT in the AGM. Importantly, this platform is readily accessible and is easily customized for discovery of EHT molecular details currently hidden to science, allowing significant advances in the understanding of how HSPCs emergence in vivo.

Identification of LPAR1/LPAR5 As Novel GPCR Partners of GPRC5D for the Efficient CAR-T Therapy of Multiple Myeloma

Introduction: The emergence of chimeric antigen receptor T-cell (CAR T) therapy has brought hope to the treatment of refractory or relapsed multiple myeloma (RRMM). GPRC5D is an orphan G protein-coupled receptor (GPCR), which has a seven-transmembrane structure, and really high expression in multiple myeloma cells. So that CAR-T cells that target GPRC5D and this has been validated in a phase I/II clinical trials with encouraging clinical efficacy and manageable safety profiles of anti-GPRC5D CAR T-cell therapy for RRMM. It is of great clinical significance to find ways to further improve the efficacy of anti-GPRC5D CAR T-cell therapy. Methods: Our previous study based on a single-cell transcriptome analysis revealed a strong co-expression correlation of GPRC5D and lysophosphatidic acid receptor 1/5 (LPAR1/5). To further confirm the interaction with GPRC5D, LPAR1 and LPAR5 were tested by co-immunoprecipitation (co-IP) screening, bimolecular fluorescence complementation (BiFC), multiple pharmacological ELISA assays, and flow cytometry (FACS) experiments. The MM cell lines (RPMI 8226, L363 and KMS11) were treated with activator or antagonists of LPAR1 or LPAR5, and then co-cultured with anti-GPRC5D CAR T-cells. The cytotoxicity and cytokine analysis were determined by luciferase-base killing assay and FACS. Results: The results showed the obvious interactions between LPAR1/LPAR5 and GPRC5D from co-IP and BiFC assays, strongly supported the prediction from the bioinformatic analysis. The heterodimers of GPRC5D and LPAR1/LPAR5 influenced membrane expression of GPRC5D to different degrees based on ELISA assay. The HEK293T surface expression of GPRC5D was reduced in the presence of LPA receptors activator (1-Oleoyl lysophosphatidic acid, 1-Oleoyl-LPA), which indicated that both LPAR1 and LPAR5 had trafficking effect on GPRC5D. To further confirm these effects, the MM cell lines, RPMI 8226, L363 and KMS11, were cultured with 1-Oleoyl-LPA or combining antagonists of LPAR1 (AM966) or LPAR5 (TC LPA5 4). The cell-surface change of GPRC5D expression had no statistical difference when MM cells were only treated with 1-Oleoyl-LPA. However, the surface expression of GPRC5D of MM cell lines was increased with the combination of LPAR1/LPAR5 activator and antagonists by FACS detection To further investigate the above effects could promote the efficacy of anti-GPRC5D CAR T-cell therapy, MM cell lines and anti-GPRC5D CAR T-cells were co-cultured, with or without the treatment of activator and antagonists. The results showed that the CAR T-cell killing capacity to MM cell lines was reduced after the treatment of 1-Oleoyl-LPA, while its killing capacity was significantly recovered after performing a combination of 1-Oleoyl-LPA and AM966/TC LPA5 4. Notably, the levels of IL-2 and TNF-α in 1-Oleoyl-LPA groups were lower than the mock groups, and increased after the combination of AM966/TC LPA5 4. But, IFN-γ did not display significant difference. These data indicated that the regulation of LPAR1 and LPAR5 function might have enhance the efficacity in anti-GPRC5D CAR T-cell therapy. Conclusions: Our study provides hint evidence that LPAR1 and LPAR5 increase the MM cell-surface expression of GPRC5D and improve the efficacy of anti-GPRC5D CAR T-cell therapy, which might be as novel GPCR partners of GPRC5D for the efficient CAR-T therapy of multiple myeloma.

High GLUT1 membrane expression and low PSMA membrane expression in Ductal Adenocarcinoma and Intraductal Carcinoma of the prostate.

Both Ductal Adenocarcinoma (DAC) and Intraductal Carcinoma (IDC) of the prostate are generally associated with aggressive clinical behavior and poor prognosis, which were linked with discordant FDG positivity and low Prostate-Specific Membrane Antigen (PSMA) expression. A recent study only cited a DAC patient with low 68Ga-PSMA-11 PET/CT uptake but high 18F-FDG PET/CT uptake, however, there is lack of directly compared articles nor large data sets. Hence, the objective of this study was to investigate the expression of PSMA and GLUT1 in DAC and IDC-P patients. The study was conducted on 87 DAC or/and IDC-P patients without any treatment and 97 PAC patients with a Gleason score ≥8 of prostate biopsies and prostatectomy samples between August 2017 and August 2022. We performed immunohistochemical staining and scoring of various cancer component samples from the patients to reflect the protein expression levels of PSMA and GLUT1. PSMA expression in PAC was significantly higher than in DAC/IDC-P (141.2 vs 78.6, p < 0.001). There was no significant difference in PSMA expression between DAC/IDC-P and adjacent PAC (78.6 vs 93.4, p = 0.166). GLUT1 expression was higher in DAC/IDC-P than in adjacent PAC (68.6 vs 51.3, p = 0.007), but was still lower than that in pure PAC (68.6 vs 93.1, p = 0.0014). It is worth noting that GLUT1 membrane expression in DAC/IDC-P was significantly increased than in pure PAC (13.0 vs 6.6, p = 0.025), and in PAC adjacent to DAC/IDC-P (13.0 vs 2.0, p < 0.001). In DAC/IDC-P tissues, PSMA expression is low, while GLUT1 expression, especially GLUT1 membrane expression is high. These findings imply that DAC/IDC-P may have higher glucose metabolic and raise interest in targeting membrane GLUT1 as a novel anticancer strategy for DAC/IDC-P and other prostate cancer with high glucose metabolism.

POMC Neuron BBSome Regulation of Body Weight is Independent of its Ciliary Function.

The BBSome, a complex of several Bardet-Biedl syndrome (BBS) proteins including BBS1, has emerged as a critical regulator of energy homeostasis. Although the BBSome is best known for its involvement in cilia trafficking, through a process that involve BBS3, it also regulates the localization of cell membrane receptors underlying metabolic regulation. Here, we show that inducible Bbs1 gene deletion selectively in proopiomelanocortin (POMC) neurons cause a gradual increase in body weight, which was associated with higher fat mass. In contrast, inducible deletion of Bbs3 gene in POMC neurons failed to affect body weight and adiposity. Interestingly, loss of BBS1 in POMC neurons led to glucose intolerance and insulin insensitivity, whereas BBS3 deficiency in these neurons is associated with slight impairment in glucose handling, but normal insulin sensitivity. BBS1 deficiency altered the plasma membrane localization of serotonin 5-HT2C receptor (5-HT2CR) and ciliary trafficking of neuropeptide Y2 receptor (NPY2R).In contrast, BBS3 deficiency, which disrupted the ciliary localization of the BBSome, did not interfere with plasma membrane expression of 5-HT2CR, but reduced the trafficking of NPY2R to cilia. We also show that deficiency in BBS1, but not BBS3, alters mitochondria dynamics and decreased total and phosphorylated levels of dynamin-like protein 1 (DRP1) protein. Importantly, rescuing DRP1 activity restored mitochondria dynamics and localization of 5-HT2CR and NPY2R in BBS1-deficient cells. The contrasting effects on energy and glucose homeostasis evoked by POMC neuron deletion of BBS1 versus BBS3 indicate that BBSome regulation of metabolism is not related to its ciliary function in these neurons.

Deficiency of N-linked glycosylation impairs immune function of B7-H6.

B7-H6 is a novel immune checkpoint molecule that triggers NK cell cytotoxicity, but the role of N-glycosylation in B7-H6 is poorly understood. We here identified the existence of N-glycosylation of B7-H6 in different cell lines and exogenous expression cells by PNGase F digestion and tunicamycin blockage. Subsequently, we demonstrated that B7-H6 contains 6 functional N-linked glycosylation sites by single site mutation and electrophoresis. Phylogenetical and structural analysis revealed that N43 and N208 glycan are conserved in jawed vertebrates and may thus contribute more to the biological functions. We further demonstrated that N43 and N208 glycosylation are essential for B7-H6 to trigger NK cell activation. Mechanistically, we found that N43 and N208 glycan contributed to the stability and membrane expression of B7-H6 protein. Lack of N208 glycosylation led to membrane B7-H6 shedding, while N43 mutation resulted in impaired B7-H6/NKp30 binding affinity. Together, our findings highlight the significance of N-linked glycosylation in B7-H6 biological functions and suggest potential targets for modulating NK cell-mediated immunity.

UMG1/CD3ε-bispecific T-cell engager redirects T-cell cytotoxicity against diffuse large B-cell lymphoma.

UMG1 is a unique epitope of CD43, not expressed by normal cells and tissues of haematopoietic and non-haematopoietic origin, except thymocytes and a minority (<5%) of peripheral blood T lymphocytes. By immunohistochemistry analysis of tissue microarray and pathology slides, we found high UMG1 expression in 20%-24% of diffuse large B-cell lymphomas (DLBCLs), including highly aggressive BCL2high and CD20low cases. UMG1 membrane expression was also found in DLBCL bone marrow-infiltrating cells and established cell lines. Targeting UMG1 with a novel asymmetric UMG1/CD3ε-bispecific T-cell engager (BTCE) induced redirected cytotoxicity against DLBCL cells and was synergistic with lenalidomide. We conclude that UMG1/CD3ε-BTCE is a promising therapeutic for DLBCLs.

Glut4 expression in pro-atherogenic monocytes is associated with estrogen induced ROS formation

Abstract Background Glucose transporter (GLUT) 4 was shown to be expressed on monocytes with dynamic alterations. Moreover, increased formation of monocyte related reactive oxygen species (ROS) is an important marker for atherosclerosis and estrogen is known to increase ROS. However, the impact of estrogen on human monocytes have not been investigated. This is of great importance as a high number of aging women receive hormone replacement therapy (HRT) due to meno-pause symptoms. This was shown to be associated with high risk for ischemic events. In this study, we investigated the impact of estrogen on human monocytes about membrane expression of GLUT4 and ROS formation. Methods In this pilot study, we investigated human leukocytes from 88 healthy volunteers (54 female and 34 male) and 114 patients (56 female and 58 male) treated in the department of Cardiology, Pneumology and Vascular medicine, University Hospital Düsseldorf. Buffy coat was extracted out of whole blood by centrifugation and analysed by flow-cytometry. Amount of CD14+ and CD16+ monocytes was measured in % of all leukocytes in all participants. ROS and GLUT4 expression were assessed for monocyte type in dependence from increasing estrogen concentrations in a sub cohort by in-vitro incubation representing physiological peak and HRT concentrations (0, 500 and 2000 pM). Results Mean age of healthy volunteers was 31,2 years. Mean age of patients was 75,9 years. In patients, 51% were male, 58% had arterial hypertension, 33% diabetes mellitus type II, 16% were smokers and 19% were obese. Antiatherogenic monocytes (CD16+) were stable in males but decreased with age in females (female: r=-0.3130 p=0.0010; male: r=-0.1011 p=0.3946). In contrast, pro-atherogenic monocytes (CD14+) increased with age in females (female: r=0.4013 p=&amp;lt;0.0001; male: r=0.09135 p=0.4389). Increasing estrogen concentrations induced ROS formation in all monocytes in a dose dependent manner (0 – 947±124, 500 - 1312±258, 2000 - 1248±629 geometric mean, p=0.0327 [one-way ANOVA]). Separation in CD14+ and CD16+ monocytes revealed that this effect was only driven by increase in CD14+ monoctyes (0 - 1021±325, 500- 1160±239, 2000 – 513 geometric mean, p=0.0179), whereby CD16+ monocytes did not show relevant increase. GLUT4 membrane expression was inversely related to increasing estrogen levels with decrease in CD14+ (0 - 578±81, 500- 498±55, 2000- 507±41 geometric mean, p=0.0131) and numerical reduction in CD16+ monocytes by high estrogen levels (0- 592±77, 500 - 544±80, 2000- 537±72 geometric mean, p=0.0784) Conclusion In this study, we could show that high estrogen levels induce decrease of GLUT4 membrane expression in human monocytes. This might be associated with increased ROS formation in pro-atherogenic CD14+ monocytes which are already increased with age in women. This might reflect a relevant mechanism for the increased rate of ischemic events by HRT.

Nuclear overexpression of DNA damage-inducible transcript 4 (DDIT4) is associated with aggressive tumor behavior in patients with pancreatic tumors

DNA damage-inducible transcript 4 (DDIT4) is induced in various cellular stress conditions. Several studies showed that the dysregulation of DDIT4 is involved in different malignancies with paradoxical expressions and roles. Therefore, this study investigated the clinical significance, prognostic, and diagnostic value of DDIT4 in different types of pancreatic tumors (PT). The expression of DDIT4 and long non-coding RNA (TPTEP1) in mRNA level was examined in 27 fresh PT samples using Real-time quantitative PCR (RT-qPCR). Moreover, 200 formalin-fixed paraffin-embedded PT tissues, as well as 27 adjacent normal tissues, were collected to evaluate the clinical significance, prognostic, and diagnosis value of DDIT4 expression by immunohistochemistry (IHC) on tissue microarrays (TMA) slides. The results of RT-qPCR showed that the expression of DDIT4 in tumor samples was higher than in normal samples which was associated with high tumor grade (P = 0.015) and lymphovascular invasion (P = 0.048). Similar to this, IHC findings for nucleus, cytoplasm, and membrane localization showed higher expression of DDIT4 protein in PT samples rather than in nearby normal tissues. A statistically significant association was detected between a high level of nuclear expression of DDIT4 protein, and lymphovascular invasion (P = 0.025), as well as advanced TNM stage (P = 0.034) pancreatic ductal adenocarcinoma (PDAC) and in pancreatic neuroendocrine tumor (PNET), respectively. In contrast, a low level of membranous expression of DDIT4 protein showed a significant association with advanced histological grade (P = 0.011), margin involvement (P = 0.007), perineural invasion (P = 0.023), as well as lymphovascular invasion (P = 0.005) in PDAC. No significant association was found between survival outcomes and expression of DDIT4 in both types. It was found that DDIT4 has rational accuracy and high sensitivity as a diagnostic marker. Our results revealed a paradoxical role of DDIT4 expression protein based on the site of nuclear and membranous expression. The findings of this research indicated that there is a correlation between elevated nuclear expression of DDIT4 and the advancement and progression of disease in patients with PT. Conversely, high membranous expression of DDIT4 was associated with less aggressive tumor behavior in patients with PDAC. However, further studies into the prognostic value and biological function of DDIT4 are needed in future studies.

Dysregulation of neutrophil oxidant production and interleukin-1-related cytokines in granulomatosis with polyangiitis.

Neutrophils play a key role in ANCA-associated vasculitis, both as targets of autoimmunity and as facilitators of vascular damage. In granulomatosis with polyangiitis (GPA), the data regarding the production of reactive oxygen species (ROS) in neutrophils are unclear. Further, recent data suggests that ROS production could have an anti-inflammatory effect through the regulation of inflammasomes and IL-1-related cytokines. We aimed to analyse ROS production in neutrophils from patients with GPA and investigate its association with IL-1-related cytokines and the autoantigen PR3. Seventy-two GPA patients with disease flare were included in the NEUTROVASC prospective cohort study. ROS production in whole blood of patients with active GPA was evaluated and compared with that in the same patients in remission or healthy controls. Associations between ROS production, PR3 membrane expression on neutrophils, serum levels of IL-1-related cytokines as well as inflammasome-related proteins were analysed. We observed a robust defect in ROS production by neutrophils from patients with active GPA compared with healthy controls, independent of glucocorticoid treatment. Serum levels of IL-1-related cytokines were significantly increased in GPA patients, particularly in patients with kidney involvement, and levels of these cytokines returned to normal after patients achieved remission. Further, inflammasome-related proteins were significantly dysregulated in the cytosol of neutrophils as well as the serum from GPA patients. Our data suggests that ROS production and regulation of inflammasomes in neutrophils from patients with GPA are disturbed and may be a potential therapeutic target. ClinicalTrials.gov, https://www.clinicaltrials.gov, NCT01862068.

Cancer and Autism: How PTEN Mutations Degrade Function at the Membrane and Isoform Expression in the Human Brain

Mutations causing loss of PTEN lipid phosphatase activity can promote cancer, benign tumors (PHTS), and neurodevelopmental disorders (NDDs). Exactly how they preferentially trigger distinct phenotypic outcomes has been puzzling. Here, we demonstrate that PTEN mutations differentially allosterically bias P loop dynamics and its connection to the catalytic site, affecting catalytic activity. NDD-related mutations are likely to sample conformations of the functional wild-type state, while sampled conformations for the strong, cancer-related driver mutation hotspots favor catalysis-primed conformations, suggesting that NDD mutations are likely to be weaker, and our large-scale simulations show why. Prenatal PTEN isoform expression data suggest exons 5 and 7, which harbor NDD mutations, as cancer-risk carriers. Since cancer requires more than a single mutation, our conformational and genomic analysis helps discover how same protein mutations can foster different clinical manifestations, articulates a role for co-occurring background latent driver mutations, and uncovers relationships of splicing isoform expression to life expectancy.

Mycoplasma synoviae lipid-associated membrane proteins identification and expression changes when exposed to chicken cells.

Mycoplasma synoviae is a significant cause of respiratory disease and synovitis among chickens, and has an adverse economic impact on broiler breeding efforts. The present study was designed to develop a systematic understanding of the role that M. synoviae lipid-associated membrane proteins (LAMPs) may play in the virulence of this pathogen. Bioinformatics tools were used to identify 146 predicted membrane proteins and lipoproteins in the M. synoviae proteome. Then, Triton X-114 was used to extract LAMPs that were subsequently identified via LC-MS/MS. This approach enabled the detection of potential LAMPs, and the top 200 most abundant proteins detected using this strategy were subject to further analysis. M. synoviae cells (100 MOI) were exposed to chicken fibroblasts (DF-1) and macrophages (HD-11) in a 1:1 mixed culture. Analysis of LAMP transcripts identified 72 up-regulated LAMP genes which were analyzed in depth by bioinformatics. GO analysis revealed these genes to be enriched in the nucleotide binding, sulfur amino acid transmembrane transporter activity, tRNA binding, rRNA modification, and transition metal ion transport pathways. Moreover, KEGG enrichment analysis suggested that these genes were enriched in the biosynthesis of secondary metabolites, carbon metabolism, glycolysis/gluconeogenesis, and nitrogen metabolism pathways.

GHB toxicokinetics and renal monocarboxylate transporter expression are influenced by the estrus cycle in rats

BackgroundThe illicit use and abuse of gamma-hydroxybutyric acid (GHB) occurs due to its sedative/hypnotic and euphoric effects. Currently, there are no clinically available therapies to treat GHB overdose, and care focuses on symptom treatment until the drug is eliminated from the body. Proton- and sodium-dependent monocarboxylate transporters (MCTs (SLC16A) and SMCTs (SLC5A)) transport and mediate the renal clearance and distribution of GHB. Previously, it has been shown that MCT expression is regulated by sex hormones in the liver, skeletal muscle and Sertoli cells. The focus of the current study is to evaluate GHB toxicokinetics and renal monocarboxylate transporter expression over the estrus cycle in females, and in the absence of male and female sex hormones.MethodsGHB toxicokinetics and renal transporter expression of MCT1, SMCT1 and CD147 were evaluated in females over the estrus cycle, and in ovariectomized (OVX) female, male and castrated (CST) male rats. GHB was administered iv bolus (600 and 1000 mg/kg) and plasma and urine samples were collected for six hours post-dose. GHB concentrations were quantified using a validated LC/MS/MS assay. Transporter mRNA and protein expression was quantified by qPCR and Western Blot.ResultsGHB renal clearance and AUC varied between sexes and over the estrus cycle in females with higher renal clearance and a lower AUC in proestrus females as compared to males (intact and CST), and OVX females. We demonstrated that renal MCT1 membrane expression varies over the estrus cycle, with the lowest expression observed in proestrus females, which is consistent with the observed changes in GHB renal clearance.ConclusionsOur results suggest that females may be less susceptible to GHB-induced toxicity due to decreased exposure resulting from increased renal clearance, as a result of decreased renal MCT1 expression.

Altered E-cadherin/β-catenin expression in feline cutaneous squamous cell carcinomas

Cutaneous squamous cell carcinoma (CSCC) is the most common malignant skin tumour in cats and the nature of the molecular mechanisms involved is poorly defined. Included among the molecular mechanisms in human and canine CSCCs is altered expression of E-cadherin/β-catenin. This study aimed to explore the immunohistochemical expression pattern of E-cadherin and β-catenin in 43 samples of feline CSCC by using a tissue microarray to elucidate whether expression of these molecules is dysregulated. Membrane expression of E-cadherin and membrane and cytoplasmic expression of β-catenin were significantly reduced in the CSCCs. Cytoplasmic expression of E-cadherin and nuclear expression of β-catenin were also found in some CSCCs. These findings indicate that altered expression of E-cadherin and β-catenin is a frequent event in feline CSCCs, suggesting that these molecules play an important role in acquisition of the malignant phenotype in feline patients with CSCC. The results also suggest the existence of a subpopulation of feline patients with CSCC in which the Wnt pathway may contribute to epidermal carcinogenesis.

Effects of Progesterone and Selective Ligands of Membrane Progesterone Receptors in HepG2 Cells of Human Hepatocellular Carcinoma.

Progesterone exerts multiple effects in different tissues through nuclear receptors (nPRs) and through membrane receptors (mPRs) of adiponectin and progestin receptor families. The effect of progesterone on the cells through different types of receptors can vary significantly. At the same time, it affects the processes of proliferation and apoptosis in normal and tumor tissues in a dual way, stimulating proliferation and carcinogenesis in some tissues, suppressing them and stimulating cell death in others. In this study, we have shown the presence of high level of mPRβ mRNA and protein in the HepG2 cells of human hepatocellular carcinoma. Expression of other membrane and classical nuclear receptors was not detected. It could imply that mPRβ has an important function in the HepG2 cells. The main goal of the work was to study functions of this protein and mechanisms of its action in human hepatocellular carcinoma cells. Previously, we have identified selective mPRs ligands, compounds LS-01 and LS-02, which do not interact with nuclear receptors. Their employment allows differentiating the effects of progestins mediated by different types of receptors. Effects of progesterone, LS-01, and LS-02 on proliferation and death of HepG2 cells were studied in this work, as well as activating phosphorylation of two kinases, p38 MAPK and JNK, under the action of three steroids. It was shown that all three progestins after 72 h of incubation with the cells suppressed their viability and stimulated appearance of phosphatidylserine on the outer surface of the membranes, which was detected by binding of annexin V, but they did not affect DNA fragmentation of the cell nuclei. Progesterone significantly reduced expression of the proliferation marker genes and stimulated expression of the p21 protein gene, but had a suppressive effect on the expression of some proapoptotic factor genes. All three steroids activated JNK in these cells, but had no effect on the p38 MAPK activity. The effects of progesterone and selective mPRs ligands in HepG2 cells were the same in terms of suppression of proliferation and stimulation of apoptotic changes in outer membranes, therefore, they were mediated through interaction with mPRβ. JNK is a member of the signaling cascade activated in these cells by the studied steroids.

Vulvo-vaginal epithelial tumors in mares: A preliminary investigation on epithelial-mesenchymal transition and tumor-immune microenvironment.

Vulvo-vaginal epithelial tumors are uncommon in mares, and data on the epithelial-to-mesenchymal transition (EMT) and the tumor-immune microenvironment (TIME) are still lacking. This is a study investigating the equus caballus papillomavirus type 2 (EcPV2) infection state as well as the EMT process and the tumor microenvironment in vulvo-vaginal preneoplastic/ benign (8/22) or malignant (14/22) epithelial lesions in mares. To do this, histopathological, immunohistochemical, transcriptomic, in situ hybridization, and correlation analyses were carried out. Immunohistochemistry quantification showed that cytoplasmic E-cadherin and β-catenin expression as well as nuclear β-catenin expression were features of malignant lesions, while benign/preneoplastic lesions were mainly characterized by membranous E-cadherin and β-catenin expression. Despite this, there were no differences between benign and malignant equine vulvo-vaginal lesions in the expression of downstream genes involved in the canonical and noncanonical wnt/β-catenin pathways. In addition, malignant lesions were characterized by a lower number of cells with cytoplasmic cytokeratin expression as well as a slightly higher cytoplasmic vimentin immunolabeling. The TIME of malignant lesions was characterized by more numerous CD204+ M2-polarized macrophages. Altogether, our results support the hypothesis that some actors in TIME such as CD204+ M2-polarized macrophages may favor the EMT process in equine vulvo-vaginal malignant lesions providing new insights for future investigations in the field of equine EcPV2-induced genital neoplastic lesions.

P1.22-19 Determination of the Membranous Expression of cMET in Patients with Advanced NSCLC and RET Fusions

P1.22-19 Determination of the Membranous Expression of cMET in Patients with Advanced NSCLC and RET Fusions

HER-3 surface expression increases in advanced colorectal cancer representing a potential therapeutic target

HER-3 (also known as ErbB-3) is a human epidermal growth factor receptor tyrosine kinases family member, and its expression in CRC (colorectal cancer) tissues was previously associated with poor prognosis. In this study, HER-3 expression was analyzed by immunohistochemistry in two cohorts of early and advanced metastatic CRC patients. The first cohort included 180 patients diagnosed with CRC in absence of lymph nodes or distant metastases (Stage I and Stage II), while the second was obtained from 53 advanced metastatic CRC patients who developed synchronous (SM) and metachronous (MM) liver metastases. In the first early-stage CRC cohort, 86 out of 180 (47.8%) tumors showed membranous expression of HER-3, with a mean percentage of positive tumor cells of 25.7%; conversely, in advanced metastatic CRC primary tumors, HER-3 was detected in all specimens, with a mean percentage of positive tumor cells of 76.1%. Kaplan–Meier curves showed that in the advanced metastatic CRC group, patients with HER-3high tumors had a significantly lower Cancer-Specific Survival (CSS) rate compared to patients with HER-3low tumors (p = 0.021). Importantly, this worse CSS rate was observed only in the MM subgroup of patients with HER-3high tumors (p = 0.002). Multivariate analysis confirmed that high HER-3 expression represents a significant and strong risk factor for death in patients developing MM liver metastases (Hazard Ratio = 64.9; 95% Confidence Interval, 4.7–886.6; p = 0.002). In addition, using a specific anti-HER-3 antibody-drug conjugate, named EV20/MMAF, we showed that HER-3 + CRC cells can be efficiently targeted in vitro and in vivo. Overall, this study confirms that surface HER-3 is highly expressed in CRC and reveals that HER-3 expression increases in metastatic CRC patients compared to early stage. Importantly, the results suggest that HER-3 has a prognostic and therapeutic value in patients developing MM liver metastases.

Mitochondrial transplantation attenuates traumatic neuropathic pain, neuroinflammation, and apoptosis in rats with nerve root ligation.

Traumatic neuropathic pain (TNP) is caused by traumatic damage to the somatosensory system and induces the presentation of allodynia and hyperalgesia. Mitochondrial dysfunction, neuroinflammation, and apoptosis are hallmarks in the pathogenesis of TNP. Recently, mitochondria-based therapy has emerged as a potential therapeutic intervention for diseases related to mitochondrial dysfunction. However, the therapeutic effectiveness of mitochondrial transplantation (MT) on TNP has rarely been investigated. Here, we validated the efficacy of MT in treating TNP. Both in vivo and in vitro TNP models by conducting an L5 spinal nerve ligation in rats and exposing the primary dorsal root ganglion (DRG) neurons to capsaicin, respectively, were applied in this study. The MT was operated by administrating 100 µg of soleus-derived allogeneic mitochondria into the ipsilateral L5 DRG in vivo and the culture medium in vitro. Results showed that the viable transplanted mitochondria migrated into the rats' spinal cord and sciatic nerve. MT alleviated the nerve ligation-induced mechanical and thermal pain hypersensitivity. The nerve ligation-induced glial activation and the expression of pro-inflammatory cytokines and apoptotic markers in the spinal cord were also repressed by MT. Consistently, exogenous mitochondria reversed the capsaicin-induced reduction of mitochondrial membrane potential and expression of pro-inflammatory cytokines and apoptotic markers in the primary DRG neurons in vitro. Our findings suggest that MT mitigates the spinal nerve ligation-induced apoptosis and neuroinflammation, potentially playing a role in providing neuroprotection against TNP.