PSGL-1: a novel immune checkpoint driving T-cell dysfunction in obstructive sleep apnea.

Introduction: Although higher incidence of cancer represents a major burden for obstructive sleep apnea (OSA) patients, the molecular pathways driving this association are not completely understood. Recently, the adhesion receptor P-selectin glycoprotein-1 (PSGL 1) has been identified as a novel immune checkpoint, which are recognized major hallmarks in several types of cancer and have revolutionized cancer therapy. Methods: The expression of PSGL-1 and its ligands VISTA and SIGLEC-5 was assessed in the leucocytes of OSA patients and control subjects exploring the role of intermittent hypoxia (IH) using in vitro models. In addition, PSGL-1 impact on T-cells function was evaluated by ex vivo models. Results: Data showed PSGL-1 expression is upregulated in the T-lymphocytes from patients with severe OSA, indicating a relevant role of hypoxemia mediated by intermittent hypoxia. Besides, results suggest an inhibitory role of PSGL-1 on T-cell proliferation capacity. Finally, the expression of SIGLEC-5 but not VISTA was increased in monocytes from OSA patients, suggesting a regulatory role of intermittent hypoxia. Discussion: In conclusion, PSGL-1 might constitute an additional immune checkpoint leading to T-cell dysfunction in OSA patients, contributing to the disruption of immune surveillance, which might provide biological plausibility to the higher incidence and aggressiveness of several tumors in these patients.

NK cells require immune checkpoint receptor LILRB4/gp49B to control neurotropic Zika virus infections in mice.

Immune cells express an array of inhibitory checkpoint receptors that are upregulated upon activation and limit tissue damage associated with excessive response to pathogens or allergens. Mouse leukocyte immunoglobulin like receptor B4 (LILRB4), also known as glycoprotein 49B (gp49B), is an inhibitory checkpoint receptor constitutively expressed in myeloid cells and upregulated in B cells, T cells, and NK cells upon activation. Here, we report that expression of LILRB4, which binds Zika virus (ZIKV), was increased in microglia and myeloid cells infiltrating the brains of neonatal mice with ZIKV-associated meningoencephalitis. Importantly, while C57BL/6 mice developed transient neurological symptoms but survived infection, mice lacking LILRB4/gp49B (LILRB4 KO) exhibited more severe signs of neurological disease and succumbed to disease. Their brains showed increased cellular infiltration but reduced control of viral burden. The reduced viral clearance was associated with altered NK cell function in the absence of LILRB4/gp49B. In naive animals, this manifested as reduced granzyme B responses to stimulation, but in ZIKV-infected animals, NK cells showed phenotypic changes that suggested altered maturation, diminished glucose consumption, reduced IFN-γ and granzyme B production, and impaired cytotoxicity. Together, our data reveal LILRB4/gp49B as an important regulator of NK cell function during viral infections.

SELPLG Expression Was Potentially Correlated With Metastasis and Prognosis of Osteosarcoma.

Background: Osteosarcoma (OS) is the most prevalent malignant primary bone tumor in children. Selectin P ligand gene (SELPLG) has been studied in several cancers. Our research aimed to explore the role of SELPLG in OS. Methods: All OS patient data was obtained from TARGET and GEO databases. Differential expression analyses were conducted in limma package of R. Functional analyses included GO and KEGG enrichment analyses. Immune cell infiltration analysis was done in CIBERSORT software. The overall survival was calculated using survival and survminer package of R. Results: Significantly lower SELPLG expression was observed in metastatic OS samples compared with non-metastatic OS samples, both in TARGET and in GSE21257. Low SELPLG expression was an independent undesirable prognostic factor for OS patients, in both TARGET and GEO datasets. Totally 62 differentially expressed gene (DEG) overlaps were found between high SELPLG vs. low SELPLG and non-metastatic vs. metastatic OS samples, affecting metastases and thereby influencing the prognosis, which were significantly enriched in 40 GO and six KEGG terms. Five types of immune cells were significantly differentially infiltrated between high and low SELPLG expression OS patients. Conclusion: SELPLG is closely correlated with metastases and prognosis of OS patients. The OS patients with low SELPLG expression have relatively poorer prognosis and SELPLG is a potential prognostic biomarker for OS.

Human CD206+ macrophages associate with diabetes and adipose tissue lymphoid clusters.

Increased adipose tissue macrophages (ATMs) correlate with metabolic dysfunction in humans and are causal in development of insulin resistance in mice. Recent bulk and single-cell transcriptomics studies reveal a wide spectrum of gene expression signatures possible for macrophages that depends on context, but the signatures of human ATM subtypes are not well defined in obesity and diabetes. We profiled 3 prominent ATM subtypes from human adipose tissue in obesity and determined their relationship to type 2 diabetes. Visceral adipose tissue (VAT) and s.c. adipose tissue (SAT) samples were collected from diabetic and nondiabetic obese participants to evaluate cellular content and gene expression. VAT CD206+CD11c- ATMs were increased in diabetic participants, were scavenger receptor-rich with low intracellular lipids, secreted proinflammatory cytokines, and diverged significantly from 2 CD11c+ ATM subtypes, which were lipid-laden, were lipid antigen presenting, and overlapped with monocyte signatures. Furthermore, diabetic VAT was enriched for CD206+CD11c- ATM and inflammatory signatures, scavenger receptors, and MHC II antigen presentation genes. VAT immunostaining found CD206+CD11c- ATMs concentrated in vascularized lymphoid clusters adjacent to CD206-CD11c+ ATMs, while CD206+CD11c+ were distributed between adipocytes. Our results show ATM subtype-specific profiles that uniquely contribute to the phenotypic variation in obesity.

Lymphocyte HLA-DR/CD-38 co-expression correlates with Hodgkin lymphoma cell cytotoxicity in vitro independent of PD-1/PD1-L pathway.

The interactions between Hodgkin and Reed Sternberg cells and tumor microenvironment, the changes that occur with therapy and, in particular, checkpoint inhibition are not fully understood. Understanding these is key to optimizing outcomes for patients with Hodgkin lymphoma (HL). We evaluated the immunophenotypic characteristics of cytotoxic, helper T and NK lymphocytes upon in vitro stimulation, cell-mediated cytotoxicity against HL cells, HDLM-2 and KM-H2, and the association with effector cell activation state, as well as changes in cytotoxicity following PD-1 or PDL-1 blockade. Higher HLA-DR/CD38 expression on effector cells was associated with increased cytotoxicity against HL cells. All effector cell types were cytotoxic of HL cells, though achieved maximum activation and cytotoxicity at variable timepoints. HLA-DR/CD38 co-expression correlated with cytotoxicity, but PD-1 expression did not. There was no significant change in cell-mediated cytotoxicity following PD-1/PDL-1 blockade. The mechanism of action of checkpoint inhibitors may not be limited to direct PD-1/PDL-1 blockade.

Targeting hippocampal amyloidogenesis with SV2A protein modulator levetiracetam.

Cerebral amyloid ß (Aß) proteostasis is compromised under neuronal overexcitation, long-term neuroinflammation and brain aging. Using the animal model of LPS-induced neuroinflammation we demonstrated that treatment with levetiracetam, a specific modulator of synaptic vesicle glycoprotein SV2A, rescues abnormal synaptic vesicle (SV) fusion and neurotransmitter release, decreasing elevated hippocampal APP levels in vivo. Therapy with levetiracetam upregulates the SV2A in hippocampus and restores the level of apolipoprotein E, involved in brain Aß aggregation/clearance and resolution of inflammation. We demonstrated that oligomers of Aß1-42 and Aß1-40 peptides promote SV clustering, which reduces the rate and plateau level of subsequent homo- and heterotypic SNARE-mediated SV fusion. Oligomeric Aß1-42 lowered ΔpH gradient across the vesicular membrane, thus affecting their neurotransmitter storage capacity. In contrast, monomers of Aß1-42 and Aß1-40 had negligible impact on studied processes. Our data suggests that in the course of progression of neuroinflammation oligomeric forms of Aß1-42 and Aß1-40 can compromise the SV fusion machinery and that antiepileptic agent levetiracetam, acting on SV recycling and restricting overexcitation, is able to affect APP processing and Aß generation within the hippocampus in vivo.

Downregulation of TREM2 expression exacerbates neuroinflammatory responses through TLR4-mediated MAPK signaling pathway in a transgenic mouse model of Alzheimer's disease.

Activation of glial cells and neuroinflammation play an important role in the onset and development of Alzheimer's disease (AD). Triggering receptor expressed on myeloid cells 2 (TREM2) is a microglia-specific receptor in the brain that is involved in regulating neuroinflammation. However, the precise effects of TREM2 on neuroinflammatory responses and its underlying molecular mechanisms in AD have not been studied in detail. Here, we employed a lentiviral-mediated strategy to downregulation of TREM2 expression on microglia in the brain of APPswe/PS1dE9 (APP/PS1) transgenic mice and BV2 cells. Our results showed that downregulation of TREM2 significantly aggravated AD-related neuropathology including Aß accumulation, peri-plaque microgliosis and astrocytosis, as well as neuronal and synapse-associated proteins loss, which was accompanied by a decline in cognitive ability. The further mechanistic study revealed that downregulation of TREM2 expression initiated neuroinflammatory responses through toll-like receptor 4 (TLR4)-mediated mitogen-activated protein kinase (MAPK) signaling pathway and subsequent stimulating the production of pro-inflammatory cytokines in vivo and in vitro. Moreover, blockade of p38, JNK, and ERK1/2 inhibited the release of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6) induced by Aß1-42 in TREM2-knocked down BV2 cells. Taken together, these findings indicated that TREM2 might be a potential therapeutic target for AD and other neuroinflammation-related diseases.

Diagnostic significance of CyclinD1 and D2-40 expression for follicular neoplasm of the thyroid.

OBJECTIVES: To evaluate the expression and differential diagnostic significance of CyclinD1 and D2-40 in follicular neoplasm (FN) and other thyroid adenomatoid lesions. METHODS: A total of 144 cases of thyroid adenomatoid lesions were enrolled. Immunohistochemistry for CyclinD1 and D2-40 was performed. RESULTS: We found two patterns of CyclinD1 expression: nuclear (N) and cytoplasmic (C). The expression of N-CyclinD1 / C-CyclinD1 in FN (77.4%, 48/62; 50.0%, 31/62) was much higher than that in multinodular goiters with dominant nodules (MNG-DN) (16.4%, 10/61; 4.9%, 3/61) (p < 0.05). In contrast, the expression of D2-40 in MNG-DN (82.0%,50/61) was much higher than that in FN (4.8%, 3/62) (p < 0.05). In addition, unique staining patterns were observed: CyclinD1 showed no immunostaining only in all 8 cases of oncocytic cell tumors (OCT); D2-40 staining showed the characteristic wide distribution of lymphatic vessels in all 8 cases of poorly differentiated thyroid carcinoma (PDTC). Finally, the expression of CyclinD1 and D2-40 did not differ among follicular thyroid adenoma and follicular thyroid carcinoma / noninvasive follicular thyroid neoplasm with papillary-like nuclear features (p > 0.05). CONCLUSIONS: CyclinD1 and D2-40 are helpful diagnostic markers of FN, which can assist to discern FN from MNG-DN / OCT / PDTC.

GPNMB plays an active role in the M1/M2 balance.

The phenotypic function of macrophages varies with the local microenvironment. Macrophages play an important role in the development of periodontitis. As one of the sources of GPNMB protein, the phenotype of macrophages is affected by GPNMB expression. In this study, activated macrophages were evaluated by flow cytometry, RT-qPCR and WB, and M2a macrophages had higher GPNMB expression than M0 and M1 macrophages. On this basis, a macrophage model with overexpression of GPNMB was established, and it was observed that GPNMB overexpression promoted the secretion of anti-inflammatory factors by macrophages and inhibited the secretion of pro-inflammatory factors by M1 macrophages.

Impact of the molar activity and PSMA expression level on [18F]AlF-PSMA-11 uptake in prostate cancer.

This two-part preclinical study aims to evaluate prostate specific membrane antigen (PSMA) as a valuable target for expression-based imaging applications and to determine changes in target binding in function of varying apparent molar activities (MAapp) of [18F]AlF-PSMA-11. For the evaluation of PSMA expression levels, male NOD/SCID mice bearing prostate cancer (PCa) xenografts of C4-2 (PSMA+++), 22Rv1 (PSMA+) and PC-3 (PSMA-) were administered [18F]AlF-PSMA-11 with a medium MAapp (20.24 ± 3.22 MBq/nmol). SUVmean and SUVmax values were respectively 3.22 and 3.17 times higher for the high versus low PSMA expressing tumors (p < 0.0001). To evaluate the effect of varying MAapp, C4-2 and 22Rv1 xenograft bearing mice underwent additional [18F]AlF-PSMA-11 imaging with a high (211.2 ± 38.9 MBq/nmol) and/or low MAapp (1.92 ± 0.27 MBq/nmol). SUV values showed a significantly increasing trend with higher MAapp. Significant changes were found for SUVmean and SUVmax between the high versus low MAapp and medium versus low MAapp (both p < 0.05), but not between the high versus medium MAapp (p = 0.055 and 0.25, respectively). The effect of varying MAapp was more pronounced in low expressing tumors and PSMA expressing tissues (e.g. salivary glands and kidneys). Overall, administration of a high MAapp increases the detection of low expression tumors while also increasing uptake in PSMA expressing tissues, possibly leading to false positive findings. In radioligand therapy, a medium MAapp could reduce radiation exposure to dose-limiting organs with only limited effect on radionuclide accumulation in the tumor.

Placental Expression of Bile Acid Transporters in Intrahepatic Cholestasis of Pregnancy.

Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-related condition characterized by increased maternal circulating bile acids (BAs) having adverse fetal effects. We investigated whether the human placenta expresses specific regulation patterns to prevent fetal exposition to harmful amounts of BAs during ICP. Using real-time quantitative PCR, we screened placentae from healthy pregnancies (n = 12) and corresponding trophoblast cells (n = 3) for the expression of 21 solute carriers and ATP-binding cassette transporter proteins, all acknowledged as BA- and/or cholestasis-related genes. The placental gene expression pattern was compared between healthy women and ICP patients (n = 12 each). Placental SLCO3A1 (OATP3A1) gene expression was significantly altered in ICP compared with controls. The other 20 genes, including SLC10A2 (ASBT) and EPHX1 (EPOX, mEH) reported for the first time in trophoblasts, were comparably abundant in healthy and ICP placentae. ABCG5 was undetectable in all placentae. Placental SLC10A2 (ASBT), SLCO4A1 (OATP4A1), and ABCC2 mRNA levels were positively correlated with BA concentrations in ICP. Placental SLC10A2 (ASBT) mRNA was also correlated with maternal body mass index. We conclude that at the transcriptional level only a limited response of BA transport systems is found under ICP conditions. However, the extent of the transcriptional response may also depend on the severity of the ICP condition and the magnitude by which the maternal BA levels are increased.

Eubacterium rectale Attenuates HSV-1 Induced Systemic Inflammation in Mice by Inhibiting CD83.

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.

TLR7 is expressed by support cells, but not sensory neurons, in ganglia.

BACKGROUND: Toll-like receptor 7 (TLR7) is an innate immune receptor that detects viral single-stranded RNA and triggers the production of proinflammatory cytokines and type 1 interferons in immune cells. TLR7 agonists also modulate sensory nerve function by increasing neuronal excitability, although studies are conflicting whether sensory neurons specifically express TLR7. This uncertainty has confounded the development of a mechanistic understanding of TLR7 function in nervous tissues. METHODS: TLR7 expression was tested using in situ hybridization with species-specific RNA probes in vagal and dorsal root sensory ganglia in wild-type and TLR7 knockout (KO) mice and in guinea pigs. Since TLR7 KO mice were generated by inserting an Escherichia coli lacZ gene in exon 3 of the mouse TLR7 gene, wild-type and TLR7 (KO) mouse vagal ganglia were also labeled for lacZ. In situ labeling was compared to immunohistochemistry using TLR7 antibody probes. The effects of influenza A infection on TLR7 expression in sensory ganglia and in the spleen were also assessed. RESULTS: In situ probes detected TLR7 in the spleen and in small support cells adjacent to sensory neurons in the dorsal root and vagal ganglia in wild-type mice and guinea pigs, but not in TLR7 KO mice. TLR7 was co-expressed with the macrophage marker Iba1 and the satellite glial cell marker GFAP, but not with the neuronal marker PGP9.5, indicating that TLR7 is not expressed by sensory nerves in either vagal or dorsal root ganglia in mice or guinea pigs. In contrast, TLR7 antibodies labeled small- and medium-sized neurons in wild-type and TLR7 KO mice in a TLR7-independent manner. Influenza A infection caused significant weight loss and upregulation of TLR7 in the spleens, but not in vagal ganglia, in mice. CONCLUSION: TLR7 is expressed by macrophages and satellite glial cells, but not neurons in sensory ganglia suggesting TLR7's neuromodulatory effects are mediated indirectly via activation of neuronally-associated support cells, not through activation of neurons directly. Our data also suggest TLR7's primary role in neuronal tissues is not related to antiviral immunity.

Reductions in Synaptic Vesicle Glycoprotein 2 Isoforms in the Cortex and Hippocampus in a Rat Model of Traumatic Brain Injury.

Traumatic brain injury (TBI) can produce lasting cognitive, emotional, and somatic difficulties that can impact quality of life for patients living with an injury. Impaired hippocampal function and synaptic alterations have been implicated in contributing to cognitive difficulties in experimental TBI models. In the synapse, neuronal communication is facilitated by the regulated release of neurotransmitters from docking presynaptic vesicles. The synaptic vesicle glycoprotein 2 (SV2) isoforms SV2A and SV2B play central roles in the maintenance of the readily releasable pool of vesicles and the coupling of calcium to the N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex responsible for vesicle docking. Recently, we reported the findings of TBI-induced reductions in presynaptic vesicle density and SNARE complex formation; however, the effect of TBI on SV2 is unknown. To investigate this, rats were subjected to controlled cortical impact (CCI) or sham control surgery. Abundance of SV2A and SV2B were assessed at 1, 3, 7, and 14 days post-injury by immunoblot. SV2A and SV2B were reduced in the cortex at several time points and in the hippocampus at every time point assessed. Immunohistochemical staining and quantitative intensity measurements completed at 14 days post-injury revealed reduced SV2A immunoreactivity in all hippocampal subregions and reduced SV2B immunoreactivity in the molecular layer after CCI. Reductions in SV2A abundance and immunoreactivity occurred concomitantly with motor dysfunction and spatial learning and memory impairments in the 2 weeks post-injury. These findings provide novel evidence for the effect of TBI on SV2 with implications for impaired neurotransmission neurobehavioral dysfunction after TBI.

Upregulation of leukocyte immunoglobulin-like receptor B4 on interstitial macrophages in COPD; their possible protective role against emphysema formation.

BACKGROUND: Leukocyte immunoglobulin-like receptor B4 (LILRB4) is one of the inhibitory receptors in various types of immune cells including macrophages. Previous reports suggested that LILRB4 could be involved in a negative feedback system to prevent excessive inflammatory responses. However, its role has been unclear in chronic obstructive pulmonary disease (COPD), in which macrophages play a crucial role in the pathogenesis. In this study, we aimed to examine the changes of LILRB4 on macrophages both in the lung specimens of COPD patients and the lungs of a mouse emphysema model. We then tried to compare the differences in both inflammation and emphysematous changes of the model between wild-type and LILRB4-deficient mice in order to elucidate the role of LILRB4 in the pathogenesis of COPD. METHODS: We prepared single-cell suspensions of resected lung specimens of never-smokers (n = 21), non-COPD smokers (n = 16), and COPD patients (n = 14). The identification of LILRB4-expressing cells and the level of LILRB4 expression were evaluated by flow cytometry. We analyzed the relationships between the LILRB4 expression and clinical characteristics including respiratory function. In the experiments using an elastase-induced mouse model of emphysema, we also analyzed the LILRB4 expression on lung macrophages. We compared inflammatory cell accumulation and emphysematous changes induced by elastase instillation between wild-type and LILRB4-deficient mice. RESULTS: The levels of surface expression of LILRB4 are relatively high on monocyte linage cells including macrophages in the human lungs. The percentage of LILRB4+ cells in lung interstitial macrophages was increased in COPD patients compared to non-COPD smokers (p = 0.018) and correlated with the severity of emphysematous lesions detected by CT scan (rs = 0.559, p < 0.001), whereas the amount of smoking showed no correlation with LILRB4 expression. Increased LILRB4 on interstitial macrophages was also observed in elastase-treated mice (p = 0.008). LILRB4-deficient mice showed severer emphysematous lesions with increased MMP-12 expression in the model. CONCLUSIONS: LILRB4 on interstitial macrophages was upregulated both in human COPD lungs and in a mouse model of emphysema. This upregulated LILRB4 may have a protective effect against emphysema formation, possibly through decreasing MMP-12 expression in the lungs.

FLRT2 and FLRT3 Cooperate in Maintaining the Tangential Migratory Streams of Cortical Interneurons during Development.

Neuron migration is a hallmark of nervous system development that allows gathering of neurons from different origins for assembling of functional neuronal circuits. Cortical inhibitory interneurons arise in the ventral telencephalon and migrate tangentially forming three transient migratory streams in the cortex before reaching the final laminar destination. Although migration defects lead to the disruption of inhibitory circuits and are linked to aspects of psychiatric disorders such as autism and schizophrenia, the molecular mechanisms controlling cortical interneuron development and final layer positioning are incompletely understood. Here, we show that mouse embryos with a double deletion of FLRT2 and FLRT3 genes encoding cell adhesion molecules exhibit an abnormal distribution of interneurons within the streams during development, which in turn, affect the layering of somatostatin+ interneurons postnatally. Mechanistically, FLRT2 and FLRT3 proteins act in a noncell-autonomous manner, possibly through a repulsive mechanism. In support of such a conclusion, double knockouts deficient in the repulsive receptors for FLRTs, Unc5B and Unc5D, also display interneuron defects during development, similar to the FLRT2/FLRT3 mutants. Moreover, FLRT proteins are chemorepellent ligands for developing interneurons in vitro, an effect that is in part dependent on FLRT-Unc5 interaction. Together, we propose that FLRTs act through Unc5 receptors to control cortical interneuron distribution in a mechanism that involves cell repulsion.SIGNIFICANCE STATEMENT Disruption of inhibitory cortical circuits is responsible for some aspects of psychiatric disorders such as schizophrenia or autism. These defects include interneuron migration during development. A crucial step during this process is the formation of three transient migratory streams within the developing cortex that determine the timing of interneuron final positioning and the formation of functional cortical circuits in the adult. We report that FLRT proteins are required for the proper distribution of interneurons within the cortical migratory streams and for the final laminar allocation in the postnatal cortex. These results expand the multifunctional role of FLRTs during nervous system development in addition to the role of FLRTs in axon guidance and the migration of excitatory cortical neurons.

Three-Dimensional Reconstructed Bone Marrow Matrix Culture Improves the Viability of Primary Myeloma Cells In-Vitro via a STAT3-Dependent Mechanism.

Primary myeloma (PM) cells are short-lived in conventional culture, which limited their usefulness as a study model. Here, we evaluated if three-dimensional (3D) culture can significantly prolong the longevity of PM cells in-vitro. We employed a previously established 3D model for culture of bone marrow mononuclear cells isolated from 15 patients. We assessed the proportion of PM cells, viability and proliferation using CD38 staining, trypan blue exclusion assays and carboxy fluorescein succinimidyl ester (CFSE) staining, respectively. We observed significantly more CD38+ viable cells in 3D than in conventional culture (65% vs. 25%, p = 0.006) on day 3. CFSE staining showed no significant difference in cell proliferation between the two culture systems. Moreover, we found that PM cells in 3D culture are more STAT3 active by measure of pSTAT3 staining (66% vs. 10%, p = 0.008). Treatment of IL6, a STAT3 activator significantly increased CD38+ cell viability (41% to 68%, p = 0.021). In comparison, inhibition of STAT3 with Stattic significantly decreased PM cell viability in 3D culture (38% to 17% p = 0.010). Neither IL6 nor Stattic affected the PM cell viability in conventional culture. This study suggests that 3D culture can significantly improve the longevity of PM cells in-vitro, and STAT3 activation can further improve their viability.

Phenotypic and Functional Consequences of PLT Binding to Monocytes and Its Association with Clinical Features in SLE.

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.

Comprehensive Analysis of Differential m6A RNA Methylomes in the Hippocampus of Cocaine-Conditioned Mice.

N6-methyladenosine (m6A) is the most prevalent internal modification found in mRNAs and lncRNA and plays a vital role in posttranscriptional regulation in mammals. m6A is abundant in the nervous system, where it modulates neuronal development and hippocampus-dependent learning and memory. However, the roles of RNAs m6A modification and its related enzymes in cocaine reward are still not fully understood. In this study, we found that the fat mass and obesity-associated gene (FTO) demethylase, but not methyltransferase-like 3 (METTL3) and 14 (METTL14), was downregulated in the hippocampus following cocaine-induced conditioned place preference (CPP), and the level of m6A is notably higher in the hippocampus of cocaine CPP training mice. Using methylated m6A RNA immunoprecipitation sequencing (MeRIP-m6A-seq), we identified a total of 6516 m6A peaks within 4460 mRNAs, and 3083 m6A peaks within 850 lncRNAs were significantly dysregulated. Intriguingly, the altered m6A peaks within mRNAs and lncRNAs were enriched in synapse maturation and localization processes. Our study uncovers a critical role for an m6A epitranscriptomic dysregulation and downregulation of FTO expression in the hippocampus following cocaine-induced CPP.

Antiseizure medications and fetal nutrients: Effects on choline transporters in a human placental cell line.

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.