Levels Of CD86 Research Articles

CD44 Drives M1 Macrophage Polarization in Diabetic Retinopathy

Purpose Diabetic retinopathy is a typical complication of diabetes, which can facilitate the risk of blindness in severe cases. We sought to determine the function of CD44 in inflammatory responses of human retinal microvascular endothelial cells (HRMECs) and macrophage polarization during diabetic retinopathy (DR). Methods The hub genes were tested based on two datasets from the Gene Expression Omnibus database. Gene Ontology and pathway enrichment analysis was conducted on the base of differentially expressed genes (DEGs). The infiltration score and infiltration of the immune cells were assessed, and the link between key genes and macrophages was analyzed. The role of CD44 in HRMECs and macrophage polarization was determined by quantitative reverse transcription polymerase chain reaction, western blot, cell counting kit-8, Enzyme-linked immunosorbent assay, flow cytometry, and immunofluorescence. Results DEGs were enriched in several pathways linked to DR, such as cellular response to retinoic acid, retinol metabolic process, retina homeostasis, PI3K-AKT signaling pathway, and leukocyte transendothelial migration. A total of 144 DEGs were identified by up-regulation both in GSE102485 and GSE160306. Moreover, the infiltration of macrophages was greater in the DR group than that in the control group. We highlighted an obvious increase in the expression of CD44 and CD86 in patients with DR, and distinct positive associations were found between levels of macrophages and levels of CD44 and CD86. Furthermore, CD44 expression was substantially increased in HRMECs under high glucose (HG) conditions and CD44 knockdown markedly inhibited HG-induced inflammatory responses of HRMECs. HG-induced HRMECs remarkably influenced M1 polarization of macrophages, but CD44 knockdown significantly nullified this effect. Conclusions CD44 influenced the advancement of DR via meditating M1 polarization of macrophages. Our findings could enhance the understanding of the mechanism of DR, which might offer a therapeutic target for DR patients.

CD64 as a diagnostic marker for bacterial infection in acute bronchiolitis

Objectives: The aim of this study was to determine the association between neutrophils and monocytes CD 64 molecules and cases of bronchiolitis with an associated bacterial infection. Methods: Blood samples were obtained from patients younger than 2 years old diagnosed with bronchiolitis. Complete blood count (CBC), blood culture, procalcitonin, and CD64 surface expression in neutrophils and monocytes were examined using flow cytometry. We also assessed CBC, procalcitonin, and CD64 levels in neutrophils and monocytes in 31 healthy control subjects. Results: Sixty-two patients with lower respiratory tract infections were included in the first group. The patients were divided into 34 with non-bacterial infections and 28 with possible bacterial infections. We found a significant increase in the number of positive cells in the Mean Fluorescence Intensity (MFI) of both nCD64 and mCD64 in the Possible Bacterial Infection (PBI) group. Procalcitonin levels correlated with nCD64 and nCD64%, but not with mCD64. The AUC of nCD64 was 0.873 (at a cut-off point of 4489.4; the MFI had a specificity of 73% and a sensitivity of 94%). Conclusion: Neutrophil CD64 may be a marker for bacterial infection in children with bronchiolitis.

Fucoidan (A Sulfated Polysaccharide) and Cerebroprotein in Combination Alleviate the Neuroinflammation-mediated Neural Damage and Functional Deficits in the Focal Cerebral Ischemia Model of Rat

Cerebral ischemic reperfusion injury could emanate a cascade of events ensuing in neural death and severe neurobehavioural deficits. The currently available interventions have failed to target the multimodal, interlinked mechanisms that operate cerebral ischemia-induced damage and functional loss. So an integrative intervention has become a mandate to overcome the deleterious mechanisms involved in cerebral ischemic pathophysiology. In this study, adult male Sprague dawley rats were exposed to 2 hours of right middle cerebral artery occlusion (rMCAo) followed by reperfusion, and the intervention group received Fucoidan alone at a dose of 50 mg/kg, i.p (intraperitoneal), Cerebrolysin alone at a dose of 2.5 mg/kg body weight and the combination of both. The sham rats were exposed to surgical procedures, except for the rMCAo. The assessments of the groups were made 24 h after the rMCAo. The stand-alone treatment with Fucoidan, Cerebrolysin has shown a better outcome in the neurobehavioral and, histopathological assessments and the combination has made a significant reduction in the neurological deficits and the infarct volume when compared to the standalone groups. The BBB integrity was well preserved in the combination group when compared with the lesion and standalone groups. Moreover, the combined intervention reduced the level of pro-inflammatory cytokines TNFα, NFkB, IL1α, IL1-β, IL-6, CD68, COX-2, and mRNA expression of inflammatory genes IL1α, IL1-β, IL-6, IBA-1, and COX-2 effectively. In conclusion, the present study suggests that rMCAo induced neuroinflammation and neurobehavioural alterations were attenuated by intervention with a combination of Fucoidan and cerebrolysin; Further, Fucoidan and Cerebrolysin combination improved the ischemic tolerance level by promoting the proteins and genes that regulate the inflammatory cytokines and in aiding better recovery after ischemic reperfusion injury.

Effects of herbal cake separated moxibustion on Tim-4 and ubiquitination of PD-1 in rabbits with immunosuppression

To observe the effects of herbal cake separated moxibustion on macrophage effector molecule T-cell immunoglobulin and mucin-domain containing-4 (Tim-4) and ubiquitination of programmed cell death protein 1 (PD-1) in rabbits with immunosuppression, and to explore the possible mechanism on herbal cake separated moxibustion in improving immunosuppression.Thirty-two big-ear white rabbits were randomly divided into a normal group, a model group, a moxa stick moxibustion group and a herbal cake separated moxibustion group, 8 rabbits in each group. Except the normal group, the immunosuppression model was established by intraperitoneal injection of cyclophosphamide of60 mg/kg in the other 3 groups. "Shenque" (CV 8), "Shenshu" (BL 23), "Zusanli" (ST 36), etc. were selected in both the moxa stick moxibustion group and the herbal cake separated moxibustion group. Moxa stick moxibustion was applied in the moxa stick moxibustion group, one cone at each acupoint; herbal cake separated moxibustion was applied in the herbal cake separated moxibustion group, 5 cones at each acupoint. The intervention was given once every other day for 10 times in both groups. Leukocyte content in peripheral blood was detected by blood cell analyzer; the positive expression of PD-1 in CD+4 T lymphocytes, CD+8T lymphocytes and CD+68 macrophages in peripheral blood was measured by flow cytometry, the serum levels of interleukin 2 (IL-2), CD8, CD68 and Tim-4 were detected by ELISA, and the expression of Tim-4 and F-box only protein 38 (FBXO38) in the liver and spleen tissues was measured by immunohistochemistry.Compared with the normal group, in the model group, white blood cell count (WBC) and percentage of neutrophils (NEU%) were decreased while percentage of lymphocyte (LYM%) was increased (P<0.01) in peripheral blood; the positive expression rates of PD-1 in CD+4 T lymphocytes, CD+8T lymphocytes and CD+68 macrophages in peripheral blood were increased (P<0.01); the serum levels of IL-2, CD68 and Tim-4 were increased (P<0.01), the serum level of CD8 was decreased (P<0.01); the average optical density (AOD) of Tim-4 in the liver tissue and FBXO38 in the liver and spleen tissues was increased (P<0.01). Compared with the model group, in the moxa stick moxibustion group and the herbal cake separated moxibustion group, WBC and NEU% were increased (P<0.01); the positive expression rates of PD-1 in CD+4 T lymphocytes, CD+8T lymphocytes and CD+68 macrophages in peripheral blood were decreased (P<0.01); the serum levels of IL-2, CD68 and Tim-4 were decreased (P<0.01), the serum levels of CD8 were increased (P<0.01); the AOD of Tim-4 and FBXO38 in the liver tissue and FBXO38 in the spleen tissue was decreased (P<0.01, P<0.05). Compared with the moxa stick moxibustion group, in the herbal cake separated moxibustion group, the positive expression rate of PD-1 in CD+68 macrophages in peripheral blood was increased (P<0.05); serum level of Tim-4 was increased (P<0.01); AOD of Tim-4 in the liver tissue was decreased (P<0.05).Herbal cake separated moxibustion can improve immunosuppression by regulating the expression of macrophage effector molecule Tim-4 and the FBXO38 mediated ubiquitination of PD-1, Tim-4 may be one of the specific indexes of immunomodulation involving with herbal cake separated moxibustion.目的：观察隔药饼灸对免疫抑制兔巨噬细胞效应分子T细胞免疫球蛋白黏蛋白分子-4（Tim-4）及细胞程序性死亡受体1（PD-1）泛素化的影响，探讨隔药饼灸改善免疫抑制的可能机制。方法：将32只大耳白兔随机分为空白组、模型组、艾条灸组及隔药饼灸组，每组8只。除空白组外，其余3组均以60 mg/kg剂量腹腔注射环磷酰胺制备免疫抑制模型。艾条灸组予艾条灸，每穴灸1壮；隔药饼灸组予隔药饼灸，每穴灸5壮。均选取“神阙”“肾俞”“足三里”等穴，隔日1次，共干预10次。采用血细胞分析仪检测外周血白细胞含量；流式细胞术检测外周血PD-1在CD+4、CD+8T淋巴细胞及CD+68巨噬细胞中的阳性表达；ELISA法检测血清白细胞介素2 （IL-2）、CD8、CD68及Tim-4含量；免疫组化法检测肝脏、脾脏Tim-4及F-box蛋白38（FBXO38）表达。结果：与空白组比较，模型组外周血白细胞计数（WBC）及中性粒细胞百分比（NEU%）降低、淋巴细胞百分比（LYM%）升高（P<0.01）；外周血PD-1在CD+4、CD+8T淋巴细胞及CD+68巨噬细胞中的阳性表达率升高（P<0.01）；血清IL-2、CD68及Tim-4含量升高（P<0.01），CD8含量降低（P<0.01）；肝脏Tim-4及肝脏、脾脏FBXO38平均吸光度值增高（P<0.01）。与模型组比较，艾条灸组与隔药饼灸组WBC及NEU%升高（P<0.01）；外周血PD-1在CD+4、CD+8T淋巴细胞及CD+68巨噬细胞中的阳性表达率降低（P<0.01）；血清IL-2、CD68与Tim-4含量降低（P<0.01），CD8含量升高（P<0.01）；肝脏Tim-4、FBXO38及脾脏FBXO38平均吸光度值降低（P<0.01，P<0.05）。与艾条灸组比较，隔药饼灸组外周血PD-1在CD+68巨噬细胞中的阳性表达率升高（P<0.05）；血清Tim-4含量增高（P<0.01）；肝脏Tim-4平均吸光度值降低（P<0.05）。结论：隔药饼灸可通过调节巨噬细胞效应分子Tim-4表达及FBXO38介导的PD-1泛素化改善免疫抑制状态，Tim-4可能是其参与免疫调控的特异性指标之一。.

Effects of herbal cake separated moxibustion on Tim-4 and ubiquitination of PD-1 in rabbits with immunosuppression

To observe the effects of herbal cake separated moxibustion on macrophage effector molecule T-cell immunoglobulin and mucin-domain containing-4 (Tim-4) and ubiquitination of programmed cell death protein 1 (PD-1) in rabbits with immunosuppression, and to explore the possible mechanism on herbal cake separated moxibustion in improving immunosuppression. Thirty-two big-ear white rabbits were randomly divided into a normal group, a model group, a moxa stick moxibustion group and a herbal cake separated moxibustion group, 8 rabbits in each group. Except the normal group, the immunosuppression model was established by intraperitoneal injection of cyclophosphamide of60 mg/kg in the other 3 groups. "Shenque" (CV 8), "Shenshu" (BL 23), "Zusanli" (ST 36), etc. were selected in both the moxa stick moxibustion group and the herbal cake separated moxibustion group. Moxa stick moxibustion was applied in the moxa stick moxibustion group, one cone at each acupoint; herbal cake separated moxibustion was applied in the herbal cake separated moxibustion group, 5 cones at each acupoint. The intervention was given once every other day for 10 times in both groups. Leukocyte content in peripheral blood was detected by blood cell analyzer; the positive expression of PD-1 in CD+4 T lymphocytes, CD+8T lymphocytes and CD+68 macrophages in peripheral blood was measured by flow cytometry, the serum levels of interleukin 2 (IL-2), CD8, CD68 and Tim-4 were detected by ELISA, and the expression of Tim-4 and F-box only protein 38 (FBXO38) in the liver and spleen tissues was measured by immunohistochemistry. Compared with the normal group, in the model group, white blood cell count (WBC) and percentage of neutrophils (NEU%) were decreased while percentage of lymphocyte (LYM%) was increased (P<0.01) in peripheral blood; the positive expression rates of PD-1 in CD+4 T lymphocytes, CD+8T lymphocytes and CD+68 macrophages in peripheral blood were increased (P<0.01); the serum levels of IL-2, CD68 and Tim-4 were increased (P<0.01), the serum level of CD8 was decreased (P<0.01); the average optical density (AOD) of Tim-4 in the liver tissue and FBXO38 in the liver and spleen tissues was increased (P<0.01). Compared with the model group, in the moxa stick moxibustion group and the herbal cake separated moxibustion group, WBC and NEU% were increased (P<0.01); the positive expression rates of PD-1 in CD+4 T lymphocytes, CD+8T lymphocytes and CD+68 macrophages in peripheral blood were decreased (P<0.01); the serum levels of IL-2, CD68 and Tim-4 were decreased (P<0.01), the serum levels of CD8 were increased (P<0.01); the AOD of Tim-4 and FBXO38 in the liver tissue and FBXO38 in the spleen tissue was decreased (P<0.01, P<0.05). Compared with the moxa stick moxibustion group, in the herbal cake separated moxibustion group, the positive expression rate of PD-1 in CD+68 macrophages in peripheral blood was increased (P<0.05); serum level of Tim-4 was increased (P<0.01); AOD of Tim-4 in the liver tissue was decreased (P<0.05). Herbal cake separated moxibustion can improve immunosuppression by regulating the expression of macrophage effector molecule Tim-4 and the FBXO38 mediated ubiquitination of PD-1, Tim-4 may be one of the specific indexes of immunomodulation involving with herbal cake separated moxibustion.

Naringenin Impedes the Differentiation of Mouse Hematopoietic Stem Cells Derived from Bone Marrow into Mature Dendritic Cells, thereby Prolonging Allograft Survival.

The use of immature dendritic cells (imDCs) to induce donor-specific immunotolerance following in vivo stimulation is limited by their low rate of induction and their tendency to undergo maturation. We derived imDCs from bone marrow hematopoietic stem cells (HSCs-imDCs). We then tested the ability of naringenin (Nar) to impede the maturation of HSCs-imDCs for inducing transplantation immune tolerance. HSCs derived from bone marrow were collected and induced to differentiate into imDCs by treating with Nar (Nar-HSCs-imDCs). Flow cytometry was used to evaluate DC surface markers, apoptosis, and endocytic ability. The ability of DCs to influence the in vitro proliferation of T cells and of regulatory T cells (Tregs) was analyzed by mixed lymphocyte reaction assays. Enzyme-linked immunoassays were used to quantify cytokine levels in supernatants from co-cultured DCs and Tregs, as well as in the serum of experimental animals. The level of immunotolerance induced by Nar-HSCs-imDCs was evaluated by skin grafting in recipient Balb/c mice, while the Kaplan-Meier method was used to statistically evaluate graft survival. Compared with HSC-imDCs, Nar-HSCs-imDCs showed higher expression of cluster of differentiation 11c (CD11c), but lower expression levels of CD80, CD86, and major histocompatibility complex class II. Nar-HSCs-imDCs also showed stronger inhibition of T cells and higher Treg cell proliferation. Interleukin 2 (IL-2) and interferon gamma levels were downregulated in Nar-HSCs-imDCs, whereas IL-4, IL-10, and transforming growth factor beta levels were upregulated. The rate of apoptosis and endocytic capacity of Nar-HSCs-DCs increased significantly after treatment with lipopolysaccharide. HSCs-imDCs or Nar-HSCs-imDCs were injected into Balb/c mice via the tail vein 7 days before skin grafting. Significantly reduced donor-specific CD4+ T cells and induced proliferation of CD4+CD25+FoxP3+ Treg cells were observed in the spleen of mice from the Nar-HSCs-imDCs group, especially at a dose of 106 Nar-HSCs-imDCs. The latter group also showed significantly prolonged survival of skin grafts. Nar-HSCs-imDCs markedly improved the acceptance of organ allografts, offering a potentially new strategy for inducing immune tolerance in transplantation.

Soluble CD163 is a predictor of fibrosis and hepatocellular carcinoma development in nonalcoholic steatohepatitis

BackgroundNonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease. The serum level of soluble CD163 (sCD163), a macrophage activation marker, is associated with liver tissue changes; however, its prognostic value is unknown. Here, we determined the utility of sCD163 as a marker for hepatocellular carcinoma (HCC) and prognostic marker for NAFLD.MethodsThis retrospective study obtained data regarding serum sCD163 levels, liver histology, and background factors associated with NAFLD in 287 patients (men/women, 140/147; average age, 53 ± 14 years) with NAFLD who underwent liver biopsy. Repeated liver biopsies of 287 patients with NAFLD (5.0 ± 2.7 years) were compared regarding serum sCD163 levels and liver tissue changes (stage, grade, steatosis, and NAFLD activity score).ResultsSerum sCD163 levels increased with the progression of liver fibrosis and inflammation (both P < 0.05) and were particularly helpful in distinguishing cases of Grade 4 fibrosis (P < 0.001). Levels of sCD163 significantly decreased in patients with NAFLD exhibiting alleviated fibrosis and inflammation (P < 0.05). We could also predict the development of HCC and associated mortality based on serum sCD163 levels at the time of NAFLD diagnosis. Serum sCD163 levels were higher in patients with HCC than in patients without HCC (1074 ± 379 ng/ml vs. 669 ± 261 ng/ml; P < 0.0001), and the same trend was observed for mortality.ConclusionsThe serum sCD163 level reflects the progression of fibrosis and inflammation in liver tissues, showing much promise as a noninvasive biomarker for nonalcoholic steatohepatitis and NAFLD as well as a possible predictor of HCC development and patient prognosis.

Intestinal Permeability in Patients Early after Kidney Transplantation Treated with Two Different Formulations of Once-Daily Tacrolimus.

Adequate tacrolimus blood exposure is crucial in the early post-renal transplant period and a gut epithelial barrier integrity may play a role. We prospectively investigated several markers of intestinal permeability in recent kidney transplant recipients (KTRs) treated with different tacrolimus extended-release formulations. Within each of the 49 KTR pairs that received grafts from the same donor, an early randomized conversion was performed from twice-daily (Prograf) to once-daily tacrolimus formulation: Advagraf or Envarsus. Plasma zonulin, calprotectin, circulating lipopolysaccharide (LPS), LPS-binding protein (LBP), intestinal fatty acid binding protein (FABP-2), and CD-14 levels were measured. There was no difference in the recipient age, dialysis vintage, BMI, and residual diuresis between Advagraf and Envarsus groups. FABP-2 and LPS levels were significantly associated with tacrolimus trough level, 3-h level, and area under the curve (AUC) in the Envarsus but not in the Advagraf group. AUC was independently increased by LPS and decreased by age, FABP-2 concentration, and the use of Envarsus formulation as compared with Advagraf. Functional changes of gastrointestinal tract in patients treated with Envarsus may influence intestinal tacrolimus absorption to a greater extent than in Advagraf-treated KTRs and may lead to inadequate variability of tacrolimus exposure early after kidney transplantation.

Poly-L-Lactic Acid Fillers Improved Dermal Collagen Synthesis by Modulating M2 Macrophage Polarization in Aged Animal Skin.

Poly-L-lactic acid (PLLA) fillers correct cutaneous volume loss by stimulating fibroblasts to synthesize collagen and by augmenting the volume. PLLA triggers the macrophage-induced activation of fibroblasts that secrete transforming growth factor-β (TGF-β). However, whether M2 macrophage polarization is involved in PLLA-induced collagen synthesis via fibroblast activation in aged skin is not known. Therefore, we evaluated the effect of PLLA on dermal collagen synthesis via M2 polarization in an H2O2-induced cellular senescence model and aged animal skin. H2O2-treated macrophages had increased expression levels of the M1 marker CD80 and decreased expression levels of the M2 marker CD163, which were reversed by PLLA. The expression levels of interleukin (IL)-4 and IL-13, which mediate M2 polarization, were decreased in H2O2-treated macrophages and increased upon the PLLA treatment. CD163, IL-4, and IL-13 expression levels were decreased in aged skin, but increased after the PLLA treatment. The expression levels of TGF-β, pSMAD2/SMAD2, connective tissue growth factor (CTGF), alpha-smooth muscle actin (α-SMA), collagen type 1A1 (COL1A1), and COL3A1 were also decreased in aged skin, but increased after the PLLA treatment. Moreover, PLLA upregulated phosphatidylinositol 3-kinase p85α (PI3-kinase p85α)/protein kinase B (AKT) signaling, leading to fibroblast proliferation. PLLA decreased the expression of matrix metalloproteinase (MMP) 2 and MMP3, which destroy collagen and elastin fibers in aged skin. The amount of collagen and elastin fibers in aged skin increased following the PLLA treatment. In conclusion, PLLA causes M2 polarization by increasing IL-4 and IL-13 levels and upregulating TGF-β expression and collagen synthesis in aged skin.

Macrophage polarization during Streptococcus agalactiae infection is isolate specific.

Streptococcus agalactiae (Group B Streptococcus, GBS), a Gram-positive commensal in healthy adults, remains a major cause of neonatal infections, usually manifesting as sepsis, meningitis, or pneumonia. Intrapartum antibiotic prophylaxis has greatly reduced the incidence of early-onset disease. However, given the lack of effective measures to prevent the risk of late-onset disease and invasive infections in immunocompromised individuals, more studies investigating the GBS-associated pathogenesis and the interplay between bacteria and host immune system are needed. Here, we examined the impact of 12 previously genotyped GBS isolates belonging to different serotypes and sequence types on the immune response of THP-1 macrophages. Flow cytometry analysis showed isolate-specific differences in phagocytic uptake, ranging from 10% for isolates of serotype Ib, which possess the virulence factor protein β, to over 70% for isolates of serotype III. Different isolates also induced differential expression of co-stimulatory molecules and scavenger receptors with colonizing isolates inducing higher expression levels of CD80 and CD86 compared to invasive isolates. In addition, real-time measurements of metabolism revealed that macrophages enhanced both glycolysis and mitochondrial respiration after GBS infection, with isolates of serotype III being the most potent activators of glycolysis and glycolytic ATP production. Macrophages also showed differential resistance to GBS-mediated cell cytotoxicity as measured by LDH release and real-time microscopy. The differences were evident both between serotypes and between isolates obtained from different specimens (colonizing or invasive isolates) demonstrating the higher cytotoxicity of vaginal compared with blood isolates. Thus, the data suggest that GBS isolates differ in their potential to become invasive or remain colonizing. In addition, colonizing isolates appear to be more cytotoxic, whereas invasive isolates appear to exploit macrophages to their advantage, avoiding the immune recognition and antibiotics.

Characterization of extracellular vesicles in AIDS-associated non-Hodgkin lymphoma.

Abstract Even with the use of anti-retroviral therapy, HIV +subjects develop non-Hodgkin lymphoma (NHL) at a higher frequency than uninfected subjects. Extracellular vesicles (EVs) secreted by cells may contain immunomodulatory molecules; for example, tumor cells can secrete EVs containing bioactive PD-L1 that can inhibit anti-tumor responses. We have shown EVs bearing PD-L1, TNF-RII or IL-6Rα can be biomarkers of NHL risk. Here we aim to characterize EVs isolated from EBV +and EBV −lymphoma cell lines to define their involvement in lymphomagenesis and identify more EV-associated biomarkers for NHL risk (like immune checkpoint molecules CD80, CD86, CD28). EVs were isolated from AIDS-NHL and NHL cell lines using a total exosome isolation kit and analyzed by Western blot. A subset of these were also analyzed by proteomics using Metascape, Reactome, and R. EVs were also isolated from archived serum of HIV +individuals before NHL onset (n = 51) and matched HIV +controls (n = 53) from participants of the MACS/WIHS Combined Cohort study, with a total exosome isolation kit and characterized by Western blot. EV-specific markers were detected in EVs isolated from cell lines and serum. EVs isolated from EBV +cell lines largely expressed LMP1, an EBV CD40 homolog that induces EBV-driven B cell transformation. In EVs isolated from serum, levels of CD80, CD86, or CD28 were comparable between pre-NHL samples and matched controls, though control samples tended to have greater CD80 and CD86 expression. Our findings shed light on the EV marker expression profiles of AIDS-NHL and NHL cell lines and show LMP1 expression in EBV +cell lines. Additionally, this research provides initial insight into the complex composition of EVs and their role in lymphomagenesis. Supported by grants from NIH (R01CA228157, P30CA016042-S, R01CA228157S)

Pathogenesis of flares in liver inflammation in hepatitis B virus-infected women during and after pregnancy

Abstract Women with chronic hepatitis B (CHB) are at risk of increased liver inflammation during pregnancy. To investigate the pathogenesis of these hepatic flares we studied immune responses and gut microbiota in 19 women with CHB (12 without and 7 with antiviral (NUC) treatment) and 3 uninfected controls in each trimester and at two and six months postpartum. Increased serum alanine aminotransferase (ALT) activity as a liver inflammation marker was defined as a value greater than the upper limit of normal and twice the baseline value. While increased plasma levels of soluble CD163, inflammatory cytokines and chemokines generally occurred during episodes of liver inflammation, increases of IL-22 and IFN-γ levels during pregnancy preceded a postpartum ALT increase. In contrast, levels of IL-27, a cytokine produced by the trophoblast, increased also in women with normal ALT levels. The fecal microbiota of HBV-infected patients with and without NUC treatment had lower α-diversity (inverse Simpson index) than those of uninfected controls. 16srRNA amplicon analysis and PCA showed patient-specific clustering of microbiota. To identify factors predictive of a postpartum ALT increase, we compared 16srRNA DNA data from patients with a postpartum ALT increase to those with normal postpartum ALT levels (four patients per group). Combining all time points, the gut microbiota of those with postpartum ALT increase differed significantly from those with normal ALT activities (UMAP, PERMANOVA p&amp;lt;0.003). In conclusion, the composition of the fecal microbiota was patient-rather than time point-specific and differed between patients with and without postpartum ALT increase. Supported by NIH Bench-to-Bedside Award (B.R. and D.T.Y.L.)

Cryptococcosis Inhibits the Immune Response of Dendritic Cells Through the snhg1-miR-145a-3p-Bcl2 Axis.

Dendritic cells are one of the first host cells that cryptococcus encounters. However, the correlations among cryptococcus, dendritic cells, and long noncoding RNA remain unclear. This study was undertaken to investigate the effects of long noncoding RNAs on dendritic cells with cryptococcus infection. We treated dendritic cells with cryptococcus and then detected expression of CD80, CD86, and major histocompatibility complex class II in dendritic cells with a real-time fluorescent quantitative polymerase chain reaction assay. We used nextgeneration sequencing and bioinformatics analysis to determine the competitive endogenous RNA mechanisms, confirmed via real-time polymerase chain reaction, dual luciferase reporter, and RNA-binding protein immunoprecipitation assays. After treatment of dendritic cells with 1 × 108 CFU/mL cryptococcus for 12 hours, dendritic cell viability was normal, whereas mRNA expression levels of CD80, CD86, and major histocompatibility complex class II in dendritic cells were substantially increased. With next-generation sequencing, we discovered 4 small nucleolar RNA host genes (snhg1, snhg3, snhg4, and snhg16) in cryptococcus-treated dendritic cells compared with wild-type dendritic cells. Bioinformatics analysis combined with real-time polymerase chain reaction led us to speculate that cryptococcus may affect the maturation and apoptosis of dendritic cells by regulating snhg1-miR-145a-3p-Bcl2. Further polymerase chain reaction, dual luciferase reporter, and RNA-binding protein immunoprecipitation experiments revealed that snhg1 acted as a sponge for miR145a-3p to inhibit the expression of miR-145a-3p and that miR-145a-3p promoted the expression of Bcl2 by directly targeting the 3'-UTR of Bcl2. Functional recovery experiments showed that cryptococcus promoted the maturation and apoptosis and inhibited the proliferation of dendritic cells through the snhg1-Bcl2 pathway. This study lays a foundation for the further understanding of the pathogenic role of snhg1-miR-145a-3p-Bcl2 axis in cryptococcosis.

Microglia Cell Line SIM-A9 Features – New Data

SIM-A9 is a line of spontaneously immortalized mouse microglia cells obtained from newborn C57BL/6 mice’s cerebrum. The aim of this work is to characterize SIM-A9 line by the ratio of cells with the resting and activated microglia phenotype, to analyze the expression of stem/progenitor cell markers CD133 and nestin, growth factors receptors CSF-1R and EGFR, and the karyotype of this line. The light microscopy, immunocytochemistry, flow cytometry and RT/PCR were used to analyze the morphology, phenotype, and gene expression levels of pro-inflammatory cytokines, and the mFISH method was used to analyze the karyotype. It was shown for the first time that SIM-A9 cells express a high level of TSPO protein, CD68, CD11b and CD45 markers on the surface membrane of cells, which corresponds to the phenotype of activated microglia. Despite this, the cells of this line respond with additional activation to LPS stimulation, which leads to an increase in the pro-inflammatory cytokine genes IL-1β, TNFα, IL-6 expression and a high level of active oxygen and nitrogen metabolites formation. It was shown that SIM-A9 cells express stem and progenitor cells markers, CD133+ and nestin, which allows us to consider the cells of this line as early poorly differentiated progenitor cells, despite their phenotype corresponding to activated microglia. It was also found that SIM-A9 cells express receptors of two growth factors CSF-1 and EGF, CSF-1R and EGFR, which indicates the possibility of SIM-A9 cells proliferation stimulation by two alternative mechanisms under the action of the corresponding factors. SIM-A9 cells have a hypotetraploid karyotype with a large number of structural and quantitative chromosome anomalies.

Inhibition of the mevalonate pathway causes accumulation of CD14 in the endoplasmic reticulum of macrophages

Abstract Mevalonate kinase deficiency (MKD) is an autoinflammatory disease caused by mutations in the mevalonate kinase enzyme, which acts early in the mevalonate pathway to produce sterol and non-sterol isoprenoid compounds. Patients with MKD have episodes of inflammatory symptoms accompanied by increased plasma levels of pro-inflammatory cytokines. CD14 is a receptor that binds to lipopolysaccharide (LPS) and induces the release of cytokines. Isoprenoid depletion was previously shown to increase cellular and decrease extracellular levels of CD14 in macrophages after LPS stimulation. The objective of this study is to characterize the cellular localization of CD14 following isoprenoid depletion. RAW 264.7 macrophages were treated with lovastatin to deplete isoprenoids and CD14 localization was examined using immunofluorescence and western blotting after LPS stimulation. CD14 co-localized with the endoplasmic reticulum (ER) protein calnexin following lovastatin treatment. To quantitate ER CD14, cell lysates were digested with endoglycosidase (Endo) H, which removes high mannose glycans found on ER proteins, but not complex glycans added in the Golgi apparatus. Western blot results showed that the level of Endo H sensitive CD14 was higher in lovastatin as compared to carrier-treated cells. Co-incubation of lovastatin-treated cells with mevalonate or geranylgeranyl pyrophosphate (GGPP), but not cholesterol, prevented the ER accumulation of CD14 indicating that GGPP depletion may cause this effect. Understanding how mevalonate pathway products contribute to protein trafficking in macrophages will expand our understanding of inflammatory diseases.

Nasal administration of recombinant Neospora caninum secreting IL-15/IL-15Rα inhibits metastatic melanoma development in lung

BackgroundMetastases are the leading cause of mortality in many cancer types and lungs are one of the most common sites of metastasis alongside the liver, brain, and bones. In melanoma,...

Relation between Soluble CD14 Levels, Inflammation, Subclinical Atherosclerosis and Mortality in Hemodialysis Patients.

Chronic kidney disease (CKD) is characterized by persistent lowgrade inflammation. Soluble CD14 (sCD14) is involved in many pathological conditions, including inflammation and atherosclerosis. The present study aimed to assess the relationship between sCD14 levels, subclinical atherosclerosis (SCA), inflammation and mortality in Egyptian hemodialysis (HD) patients. The present longitudinal study included 62 HD patients. All patients were submitted to careful history taking, thorough clinical examination and laboratory assessment for high-sensitivity C-reactive protein (hsCRP) and sCD14. Carotid intima-media thickness (CIMT) was also assessed. Patients were followed for a maximum of 18 months. The primary outcome is patients' mortality. Data were statistically analyzed using standard descriptive, comparative, correlative and regression methods. The present study was conducted on 62 HD patients. They comprised 34 males and 28 females with an age of 54.6 ± 9.0 years. At the end of follow-up, 12 patients (19.4 %) died. It was shown that survivors had significantly lower hsCRP levels (104.2 ± 38.2 versus 134.1 ± 15.3 mg/dL, p < 0.001), lower sCD14 levels (32.7 ± 10.3 versus 47.4 ± 18.4 μg/mL, p = 0.02) and lower CIMT (1.32 ± 0.5 versus 1.5 ± 0.2 mm, p = 0.049). sCD14 levels were significantly correlated with hsCRP (r = 0.4, p = 0.001) and CIMT (r = 0.31, p = 0.013). Multivariate analysis identified HD duration [HR (95% CI): 1.02 (1.0-1.04), p = 0.021] and sCD14 levels [HR (95% CI): 1.06 (1.0-1.12), p = 0.026] as significant predictors of patients' survival. sCD14 levels in this cohort of HD patients are well-correlated with hsCRP levels and CIMT. In addition, they are significant predictors of patients' mortality.

Platelet and Monocyte Activation After Transcatheter Aortic Valve Replacement (POTENT-TAVR): A Mechanistic Randomized Trial of Ticagrelor Versus Clopidogrel

BackgroundInflammation and thrombosis are often linked mechanistically and are associated with adverse events after transcatheter aortic valve replacement (TAVR). High residual platelet reactivity (HRPR) is especially common when clopidogrel is used in this setting, but its relevance to immune activation is unknown. We sought to determine whether residual activity at the purinergic receptor P2Y12 (P2Y12) promotes prothrombotic immune activation in the setting of TAVR. MethodsThis was a randomized trial of 60 patients (enrolled July 2015 through December 2018) assigned to clopidogrel (300mg load, 75mg daily) or ticagrelor (180mg load, 90 mg twice daily) before and for 30 days following TAVR. Co-primary endpoints were P2Y12-dependent platelet activity (Platelet Reactivity Units; VerifyNow) and the proportion of inflammatory (cluster of differentiation [CD] 14+/CD16+) monocytes 1 day after TAVR. ResultsCompared to clopidogrel, those randomized to ticagrelor had greater platelet inhibition (median Platelet Reactivity Unit [interquartile range]: (234 [170.0-282.3] vs. 128.5 [86.5-156.5], p < 0.001), but similar inflammatory monocyte proportions (22.2% [18.0%-30.2%] vs. 25.1% [22.1%-31.0%], p = 0.201) 1 day after TAVR. Circulating monocyte-platelet aggregates, soluble CD14 levels, interleukin 6 and 8 levels, and D-dimers were also similar across treatment groups. HRPR was observed in 63% of the clopidogrel arm and was associated with higher inflammatory monocyte proportions. Major bleeding events, pacemaker placement, and mortality did not differ by treatment assignment. ConclusionsResidual P2Y12 activity after TAVR is common in those treated with clopidogrel but ticagrelor does not significantly alter biomarkers of prothrombotic immune activation. HRPR appears to be an indicator (not a cause) of innate immune activation in this setting.

Gut Microbiome and Associated Metabolites Following Bariatric Surgery and Comparison to Healthy Controls.

The gut microbiome plays a significant role in regulating the host's ability to store fat, which impacts the development of obesity. This observational cohort study recruited obese adult men and women scheduled to undergo sleeve gastrectomy and followed up with them 6 months post-surgery to analyse their microbial taxonomic profiles and associated metabolites in comparison to a healthy control group. There were no significant differences in the gut bacterial diversity between the bariatric patients at baseline and at follow-up or between the bariatric patients and the cohort of healthy controls. However, there were differential abundances in specific bacterial groups between the two cohorts. The bariatric patients were observed to have significant enrichment in Granulicatella at baseline and Streptococcus and Actinomyces at follow-up compared to the healthy controls. Several operational taxonomic units assigned to commensal Clostridia were significantly reduced in the stool of bariatric patients both at baseline and follow-up. When compared to a healthy cohort, the plasma levels of the short chain fatty acid acetate were significantly higher in the bariatric surgery group at baseline. This remained significant when adjusted for age and sex (p = 0.013). The levels of soluble CD14 and CD163 were significantly higher (p = 0.0432 and p = 0.0067, respectively) in the bariatric surgery patients compared to the healthy controls at baseline. The present study demonstrated that there are alterations in the abundance of certain bacterial groups in the gut microbiome of obese patients prior to bariatric surgery compared to healthy individuals, which persist post-sleeve gastrectomy.

Ginkgolide C attenuates cerebral ischemia/reperfusion-induced inflammatory impairments by suppressing CD40/NF-κB pathway

Ethnopharmacological relevanceGinkgo biloba L. (Ginkgoaceae), a traditional Chinese medicine, has been applied for thousands of years for the treatment of cardio-cerebral vascular diseases in China. It is written in Compendium of Materia Medica that Ginkgo has the property of “dispersing poison”, which is now referred to as anti-inflammatory and antioxidant. Ginkgolides are important active ingredients in Ginkgo biloba leaves and ginkgolide injection has been frequently applied in clinical practice for the treatment of ischemic stroke. However, few studies have explored the effect and mechanism of ginkgolide C (GC) with anti-inflammatory activity in cerebral ischemia/reperfusion injury (CI/RI). Aim of the studyThe present study aimed to demonstrate whether GC was capable of attenuating CI/RI. Furthermore, the anti-inflammatory effect of GC in CI/RI was explored around the CD40/NF-κB pathway. Materials and methodsIn vivo, middle cerebral artery occlusion/reperfusion (MCAO/R) model was established in rats. The neuroprotective effect of GC was assessed by neurological scores, cerebral infarct rate, microvessel ultrastructure, blood-brain barrier (BBB) integrity, brain edema, neutrophil infiltration, and levels of TNF-α, IL-1β, IL-6, ICAM-1, VCAM-1, and iNOS. In vitro, rat brain microvessel endothelial cells (rBMECs) were preincubated in GC before hypoxia/reoxygenation (H/R) culture. The cell viability, levels of CD40, ICAM-1, MMP-9, TNF-α, IL-1β, and IL-6, and activation of NF-κB pathway were examined. In addition, the anti-inflammatory effect of GC was also investigated by silencing CD40 gene in rBMECs. ResultsGC attenuated CI/RI as demonstrated by decreasing neurological scores, reducing cerebral infarct rate, improving microvessel ultrastructural features, ameliorating BBB disruption, attenuating brain edema, inhibiting MPO activity, and downregulating levels of TNF-α, IL-1β, IL-6, ICAM-1, VCAM-1, and iNOS. Coherently, in rBMECs exposed to H/R GC enhanced cell viability and downregulated levels of ICAM-1, MMP-9, TNF-α, IL-1β, and IL-6. Furthermore, GC suppressed CD40 overexpression and hindered translocation of NF-κB p65 from the cytosol to the nucleus, phosphorylation of IκB-α, and activation of IKK-β in H/R rBMECs. However, GC failed to protect rBMECs from H/R-induced inflammatory impairments and suppress activation of NF-κB pathway when CD40 gene was silenced. ConclusionsGC attenuates cerebral ischemia/reperfusion-induced inflammatory impairments by suppressing CD40/NF-κB pathway, which may provide an available therapeutic drug for CI/RI.