Ia Expression Research Articles

Fiber rich food suppressed airway inflammation, GATA3 + Th2 cells, and FcεRIα+ eosinophils in asthma.

Allergic Asthma is a disease presenting various endotypes and no current therapies act curative but alleviate disease symptoms. Dietary interventions are gaining increasing importance in regulating immune responses. Furthermore, short chain fatty acids (SFCA), as the main products of dietary fiber's fermentation by the gut bacteria, ameliorate the pathogenesis and disease burden of different illnesses including asthma. Nevertheless, the connection and crosstalk between the gut and lung is poorly understood. In this work, the role of high fiber diet on the development of allergic asthma at baseline and after exacerbation of disease induced by respiratory viruses was investigated. Hereby, SCFA in serum of asthmatic and non-asthmatic pre-school children before and after airway disease symptoms were analyzed. Moreover, the effect of high fiber diet in vivo in a murine model of house dust mite extract (HDM) induced allergic asthma and in the end in isolated lung and spleen cells infected ex vivo with Rhinovirus was analyzed. In this study, a decrease of the SCFA 3-Hydroxybutyric acid in serum of asthmatic children after symptomatic episodes at convalescent visit as compared to asthmatic and control children at baseline visit was observed. In experimental asthma, in mice fed with high fiber diet, a reduced lung GATA3 + Th2 type mediated inflammation, mucus production and collagen deposition and expression of Fc epsilon receptor Ia (FcεRIa) in eosinophils was observed. By contrast, the CD8+ memory effector T cells were induced in the lungs of asthmatic mice fed with high fiber diet. Then, total lung cells from these asthmatic mice fed with either standard food or with fiber rich food were infected with RV ex vivo. Here, RV1b mRNA was found significantly reduced in the lung cells derived from fiber rich food fed mice as compared to those derived from standard food fed asthmatic mice. Looking for the mechanism, an increase in CD8+ T cells in RV infected spleen cells derived from fiber rich fed asthmatic mice, was observed. Convalescent preschool asthmatic children after a symptomatic episode have less serum ß-Hydroxybutyric acid as compared to control and asthmatic children at baseline visit. Fiber rich diet associated with anti-inflammatory effects as well as anti-allergic effects by decreasing Type 2 and IgE mediated immune responses and inducing CD8+ memory effector T cells in a murine model of allergic asthma. Finally, ex vivo infection with Rhinovirus (RV) of total lung cells from asthmatic mice fed with fiber rich food led to a decreased RV load as compared to mice fed with standard food. Moreover, spleen cells derived from asthmatic mice fed with fiber rich food induced CD8+ T cells after ex vivo infection with RV. Dietary interventions with increased content in natural fibers like pectins would ameliorate asthma exacerbations. Moreover, respiratory infection in asthma downregulated SCFA in the gut contributing to asthma exacerbations.

A microengineered biomimetic model of the placental barrier to study environmental exposures during pregnancy

Exposure to toxic heavy metals contributes to the development of pregnancy complications that can adversely affect fetal development. Despite increasing evidence suggesting the negative health impact of heavy metals, investigating their adverse effects in the context of human pregnancy remains a major challenge due to the difficulty of human subject research and the limited capacity of animal models to properly represent the anatomy and function of the human reproductive system. Motivated by problem, here we describe a microengineered in vitro model designed to reverse engineer the maternal-fetal interface in the human placenta. This biomimetic system enables advanced capabilities to grow human trophoblast cells and fetal vascular endothelial cells in a physiological spatial arrangement and dynamic culture conditions to engineer the placental barrier in vitro that can emulate regulated material transport between maternal and fetal circulations. Using cadmium (CdCl2 ) as a representative example of heavy metals, we investigate whether and how this environmental chemical affects the human placental barrier and its physiological function as a gatekeeper. Our study also examines the role of membrane transporters in the maternal-to-fetal transfer of cadmium to provide insights into efflux transporter-medicated protection against environmental exposures during pregnancy.We used soft lithography to construct a compartmentalized elastomeric device consisting of two overlapping chambers separated by a thin semipermeable membrane. After sterilization and surface coating with extracellular matrix protein, the device was seeded with BeWo b30 human trophoblast cells and primary human villous endothelial cells on the opposite sides of the membrane. The bi-layer tissue was then cultured under perfusion conditions by using syringe pumps connected to each chamber. To mimic the physiological process of placental development, we treated the trophoblast cell layer in the maternal chamber with forskolin (50 µM) to induce syncytialization.Our microengineered model of the human placental barrier formed a tight barrier (evaluated by expression of E-cadherin and VE-cadherin), secreted the pregnancy hormone human chorionic gonadotropin beta (hCGβ), and supported physiological maternal-to-fetal transport of glucose. Following CdCl2 treatment (0.5-50 µM), we observed concentration-dependent deleterious responses of the maternal and fetal tissues as demonstrated by reduced cell viability and placental hormone secretion, compromised barrier function, and increased secretion of proinflammatory cytokines. The results also indicated upregulated expression of metallothioneins IA and IIA (MT-IA/IIA) metal binding proteins.The placenta-on-a-chip is an innovative and translational in vitro platform for the study of environmental chemical toxicity in the human placenta. With further improvements, this technology may advance our ability to model, interrogate, and predict the potential of xenobiotics to disrupt placentation and maintenance of human pregnancy.

Effects of dietary leucine and valine levels on growth performance, glycolipid metabolism and immune response in Tilapia GIFT Oreochromis niloticus

An 8-week feeding trial was performed to evaluate the effects of dietary leucine (Leu) and valine (Val) levels on growth performance, glycolipid metabolism and immune response in Oreochromis niloticus. Fish (15.23 ± 0.05 g) were randomly fed four diets containing two Leu levels (1.2% and 2.3%) and two Val levels (0.7% and 1.4%) as a 2 × 2 experimental design (LL-LV, LL-HV, HL-LV and HL-HV). Compared with LL-LV group, the growth parameters (final weight, daily growth coefficient (DGC) and growth rate per metabolic body weight (GRMBW)), feed conversion rate (FCR), the activities of intestinal amylase, lipase, creatine kinase (CK) and Na+, K+-ATPase, liver NAD+/NADH ratio, as well as the expression of SIRT1, GK, PK, FBPase, PPARα, CPT IA, ACO and IL10 all increased significantly in the HL-LV group; however, in the high Val group, final weight, DGC, GRMBW, intestinal enzyme activities, as well as the expression of PEPCK, SREBP1, FAS, IL8 and IL10 of the HL-HV group were significantly lower than those of the LL-HV group, while the opposite was true for the remaining indicators. Significant interactions between dietary Leu and Val were observed in final weight, DGC, GRMBW, plasma IL1β and IL6 levels, intestinal amylase and CK activities, liver NAD+/NADH ratio, as well as the expression of SIRT1, PK, PEPCK, FBPase, SREBP1, FAS, PPARα, CPT IA, ACO, NF-κB1, IL1β, IL6 and IL10. The highest values of growth parameters, intestinal enzyme activities and expression of SIRT1, FBPase, PPARα, CPT IA and ACO were observed in the HL-LV group, while the opposite was true for the expression of SREBP1, FAS, PPARα, NF-κB1, IL1β and IL6. Overall, our findings indicated that dietary Leu and Val can effect interactively, and fish fed with diets containing 2.3% Leu with 0.7% Val had the best growth performance and hepatic health status of O. niloticus.

Inhibitory mechanism of icariin against oxidative stress-induced calcification in aortic vascular smooth muscle cells

This study aimed to observe the inhibitory effect of icariin against oxidative stress-induced calcification in aortic vascular smooth muscle cells(VSMCs) and elucidate the molecular mechanism of icariin in inhibiting endoplasmic reticulum stress(ERS)-mediated atherosclerotic calcification, so as to provide new ideas for exploring the anti-atherosclerotic mechanism of Epimedii Folium. The VSMCs in rat thoracic aorta were subjected to adherent culture and then treated with the complete calcification DMEM containing high glucose and hydrogen peroxide(H_2O_2) for three weeks. The resulting calcified VSMCs were divided into different treatment groups. Icariin was added one week after calcification induction for protecting the VSMCs, whose viability was then detected using cell counting kit-8(CCK-8). Alizarin red-S staining was conducted to observe the calcification degree. The activity of alkaline phosphatase(ALP) in VSMCs was measured using the disodium phenyl phosphate substrate and the calcium content was measured by arsenazo Ⅲ method. The mRNA expression levels of ossification-related factors including osteocalcin(OC), osteopontin(OPN), Runt-related transcription factor 2(Runx2), and type Ⅰ collagen(Col Ⅰa) were detected by real-time PCR. Western blot was carried out to determine the protein expression levels of α-smooth muscle actin(α-SMA), Runx2, activating transcription factor 4(ATF4), and eukaryotic translation initiation factor(eIF)-2α. The results showed that H_2O_2 significantly induced the calcification of VSMCs, increased the ALP activity and calcium content in VSMCs, promoted OC, OPN, Runx2, and Col Ⅰa mRNA expression and Runx2 protein expression, and reduced α-SMA protein expression. The ATF4 protein expression and eIF2α phosphorylation were also elevated significantly. Icariin reversed the calcification of VSMCs induced by H_2O_2, inhibited ALP activity and calcium content in VSMCs, down-regulated the mRNA expression levels of OC, OPN, Runx2 and Col Ⅰa and Runx2 protein expression, and relatively up-regulated the expression of α-SMA. The expression of ATF4 and phosphorylation of eIF2α also declined significantly. All these have demonstrated that icariin inhibited VSMCs calcification by down-regulating the ossification-related factors and lowering ALP activity and calcium content in VSMCs. Besides, the down-regulation of Runx2 expression and the inhibition of ATF4 and eIF2α-mediated cellular calcification pathway in ERS might also be involved in such calcification-suppressing process.

Role of the β-Catenin/REG Iα Axis in the Proliferation of Sessile Serrated Adenoma/Polyps Associated with Fusobacterium nucleatum.

Although sessile serrated adenoma/polyps (SSA/Ps) may arise through a pathway different from the traditional adenoma–carcinoma sequence, details of SSA/P tumorigenesis still remain unclear. Fusobacterium nucleatum (Fn) is frequently detected in colorectal cancer (CRC) tissues and may play a pivotal role in colorectal carcinogenesis. Here, we investigated the relationship between Fn and the β-catenin/REG Iα axis in SSA/Ps and their involvement in the proliferation of these lesions. Fn was detected in SSA/Ps by fluorescence in situ hybridization using a Fn-targeted probe, and expression of β-catenin, REG Iα and Ki67 was examined using immunohistochemistry. Sixteen of 30 SSA/P lesions (53.3%) were positive for Fn. Eighteen SSA/P lesions (60%) showed β-catenin immunoreactivity in the tumor cell nuclei. A significant majority of Fn-positive lesions showed nuclear expression of β-catenin (87.5%) and higher REG Iα scores and Ki67 labeling indices relative to Fn-negative lesions. The SSA/P lesions expressing β-catenin in nuclei had significantly higher REG Iα scores and Ki67 labeling indices than those expressing β-catenin on cytomembranes. The REG Iα score was positively correlated with the Ki67 labeling index in SSA/P lesions. The treatment with Wnt agonist SKL2001 promoted nuclear β-catenin translocation and enhanced REG Ia expression in Caco2 cells. Fn may play a role in the proliferation of SSA/P lesions through promotion of β-catenin nuclear translocation and REG Iα expression.

Identification of a Dps contamination in Mitomycin-C–induced expression of Colicin Ia

Colicins are bacterial toxins targeting Gram-negative bacteria, including E. coli and related Enterobacteriaceae strains. Some colicins form ion-gated pores in the inner membrane of attacked bacteria that are lethal to their target. Colicin Ia was the first pore-forming E. coli toxin, for which a high-resolution structure of the monomeric full-length protein was determined. It is so far also the only colicin, for which a low-resolution structure of its membrane-inserted pore was reported by negative-stain electron microscopy. Resolving this structure at the atomic level would allow an understanding of the mechanism of toxin pore formation. Here, we report an observation that we made during an attempt to determine the Colicin Ia pore structure at atomic resolution. Colicin Ia was natively expressed by mitomycin-C induction under a native SOS promotor and purified following published protocols. The visual appearance in the electron microscope of negatively stained preparations and the lattice parameters of 2D crystals obtained from the material were highly similar to those reported earlier resulting from the same purification protocol. However, a higher-resolution structural analysis revealed that the protein is Dps (DNA-binding protein from starved cells), a dodecameric E. coli protein. This finding suggests that the previously reported low-resolution structure of a “Colicin Ia oligomeric pore” actually shows Dps.

Protective effects of astragaloside IV against hypertension-induced vascular remodeling involves the DNMT1 and TET2 signaling pathway

Proliferation, migration, and the phenotypic switch of vascular smooth muscle cells (VSMCs) play an important role in vascular remodeling induced by hypertension. Astragaloside IV (AS-IV), the active ingredient of Astragalus membranaceus, has been shown to exert a beneficial role in cardiovascular disease. The present study aimed to investigate the mechanism responsible for the protective effects of AS-IV on hypertension-induced vascular remodeling. AS-IV significantly reduced blood pressure and aortic media thickness in two-kidney, one-clip (2K1C) hypertensive rats. AS-IV administration downregulated the expression levels of DNA methyltransferase1 (DNMT1), matrix metalloproteinase (MMP2) and proliferating cell nuclear antigen (PCNA) and upregulated the expression of smooth muscle 22? protein (SM22?), ?-smooth muscle actin (ACTA2) and ten-eleven translocation 2 (TET2) in the aorta of hypertensive rats. AS-IV inhibited the proliferation and migration in VSMCs treated with angiotensin II (Ang II). AS-IV increased the expression of SM22?, ACTA2 and TET2, and decreased the expression of collagen Ia (COL-1a), collagen IIIa (COL-3a), DNMT1, MMP2 and PCNA in vitro. Reduction in 5-methylcytosine (5-mC) was observed in VSMCs treated with AS-IV. Knockdown of DNMT1 induced the expression of TET2, while the level of DNMT1 did not change after knockdown of TET2. These results suggest that AS-IV reversed hypertension-induced vascular remodeling by inhibiting DNMT1 and upregulating TET2.

Ia Antigen Expression by Human Malignant Lymphomas: Correlation With Conventional Lymphoid Markers

The Ia (p23,30) antigens are useful in determining the B- or T-cell origin of normal peripheral blood lymphocytes. However, exceptions to the preferential B-cell expression of Ia antigens exist, i.e., many plasma cells are Ia– and T cells are rarely Ia+. Therefore, we investigated Ia expression (assayed by direct immunofluorescence using heteroantisera) by cells isolated from 33 malignant lymphomas and 7 benign lymph nodes. This was compared with surface immunoglobulin (SIg) and sheep erythrocyte (SRBC) receptor (E rosette) expression. Results with normal lymph nodes were similar to those described for peripheral blood. Most lymph node B cells were Ia+SIg+. Occasional Ia+SIg– B cells and rare Ia+ T cells were present. Ia antigens were expressed in parallel with SIg in 15 of 19 B-cell lymphomas. In 4 B-cell lymphomas, substantial numbers of neoplastic Ia+SIg– cells were also present, suggesting the presence of neoplastic cells at varying stages of differentiation. Heterogeneity was also seen in those lymphomas associated with monoclonal proteins, as small numbers of Ia+ and Ia– plasma cells were present. Seven cases expressed the T-cell (la–SIg–E+) phenotype. The common acute lymphoblastic leukemia phenotype (Ia+SIg–E–) was rarely expressed and may be uncommon among lymphomas. Three lymphomas expressing the Ia+E+ phenotype were shown to be B-cell malignancies whose SIg had SRBC specificity. One Ia 4 T-cell lymphoproliferative disorder was found. Thus, Ia antigens are useful B-cell markers in most instances. Furthermore, their enumeration allows the demonstration of phenotype heterogeneity in B-cell malignancies, analogous to that described for T-cell malignancies.

Changes in expression of antimicrobial peptides and Fc receptors in the small intestines of neonatal calves during the passive immunity period

The contribution of colostrum to passive immunity transfer and intestinal protection is well known; however, the effects of colostrum intake on the expression of antimicrobial peptides (AP) and Fc receptors in the intestine of neonatal calves are unclear. Our aim was to investigate changes in the expression of AP and Fc receptor in the small intestine of calves in the first 36 h postpartum. Twenty-four Holstein bull calves were used in this study, of which 18 calves were administered 3.2 L of pooled colostrum for each calf per meal via an esophageal tube. Calves were slaughtered at 8 h (1 meal at 1-2 h), 24 h (2 meals at 1-2 h and 10-12 h), and 36 h (3 meals at 1-2 h, 10-12 h, and 22-24 h) postpartum. The remaining 6 calves without any milk administration were slaughtered at 2 h postpartum. Samples of blood and jejunum digesta were collected to determine immunoglobulin concentration using ELISA. Samples of the duodenum, jejunum, and ileum tissues after slaughter were collected to determine AP and Fc receptor expression using quantitative real-time PCR. In calves administered colostrum, IgG concentration in jejunum digesta rapidly decreased in an age-dependent manner (33.41, 9.47, and 0.34 mg/mL at 8, 24, and 36 h, respectively), whereas serum IgG concentration increased significantly, from 0.25 μg/mL at 2 h to 21.72 mg/mL at 24 h. Cathelicidin-4, β-defensin (DEFB)-7, and enteric β-defensin expression was upregulated at 8 h postpartum in the duodenum and jejunum compared with that at 2 h, but progressive recovery was detected from 24 h onward. Higher expression of cathelicidin-4, regenerating family member 3γ, lysozyme (LYZ), LYZ1, and LYZ2 and lower expression of DEFB, DEFB1, DEFB7, DEFB10, and enteric β-defensin were observed in the duodenum and jejunum compared with the ileum. Differences in AP expression between intestinal regions suggested that the innate immune defense mechanism varied significantly among the duodenum, jejunum, and ileum. No difference in the expression of Fc fragment of the IgG receptor was observed either among ages or small intestinal regions. The Fcγ receptor (FcγR)Ia and FcγRIIb expression was the highest at 8 h compared with that at 2, 24, and 36 h, and expression of FcγRIa, FcγRIIb, and FcγRIIIa was higher in the duodenum and jejunum than in the ileum. These results indicated that AP and Fcγ receptors might play important roles in intestinal defense during the passive immunity period.

Characterization of HLA-G Regulation and HLA Expression in Breast Cancer and Malignant Melanoma Cell Lines upon IFN-γ Stimulation and Inhibition of DNA Methylation.

The potential role of human leukocyte antigen (HLA)-G as a target for new cancer immunotherapy drugs has increased the interest in the analysis of mechanisms by which HLA-G expression is regulated, and how the expression can be manipulated. We characterized HLA expression in breast cancer and malignant melanoma cell lines and investigated the induction of HLA-G expression by two distinct mechanisms: stimulation with interferon (IFN)-γ or inhibition of methylation by treatment with 5-aza-2’-deoxycytidine (5-aza-dC). The effect of IFN-γ and 5-aza-dC on HLA expression was dependent on the cancer cell lines studied. However, in general, surface expression of HLA class Ia was induced on all cell lines. Surface expression of HLA-G was inconclusive but induction of HLA-G mRNA was prevalent upon treatment with 5-aza-dC and a combination of IFN-γ and 5-aza-dC. IFN-γ alone failed to induce HLA-G expression in the HLA-G-negative cell lines. The results support that HLA-G expression is regulated partly by DNA methylation. Furthermore, IFN-γ may play a role in the maintenance of HLA-G expression rather than inducing expression. The study demonstrates the feasibility of manipulating HLA expression and contributes to the exploration of mechanisms that can be potential targets for immunotherapy in breast cancer and malignant melanoma.

The Regulation of Bone Morphogenetic Protein/Smads Signaling Pathway in Spinal Cord Injury

The incidence of spinal cord injury (SCI) increases year by year. SCI is characterized as high disability rate and poor prognosis. BMP/Smads signaling participates in the formation of osteoblasts and renal failure. This article will explore the regulation of BMP/Smads signaling pathway in SCI. Wistar rats were divided into control group; SCI group; and BMP-2 treatment group that were treated by tail vein injection of BMP-2 antisense oligonucleotide BMP-2 phosphorothioate AODN at 30 min after modeling. Real-time PCR and Western blot were used to detect BMP-2, Smad1, and Smad5 expressions. Hematoxylin-eosin (HE) staining was applied to analyze the change of SCI in each group. Immunohistochemistry (IHC) was selected to test BMPR Ia expression. Basso, Beattie Bresnahan-cocomotor rating scale (BBB) scale and Reuter score were compared. Enzyme-linked immunosorbent assay (ELISA) was adopted to detect TNF-α and Interleukin-2 (IL-2) expressions. Compared with the control group, BMP-2, Smad1, and Smad5 mRNA and protein expressions increased, BBB score declined, Reuter score elevated, and TNF-α and IL-2 secretion enhanced in the SCI group (P < 0.05). HE staining showed spinal cord injury, and IHC exhibited increased expression of BMPR Ia. The TGF-β treatment group significantly reduced the expressions of BMP-2, Smad1, and Smad5 mRNA and protein, increased BBB score, reduced Reuter score, and weakened the secretions of TNF-α and IL-2 (P < 0.05). HE staining demonstrated decreased reduction of spinal cord tissue and declined expression of BMPR Ia. SCI activated BMP/Smads signaling pathway, up-regulated BMPR Ia expression, and promoted inflammation. Regulation of BMP/Smads signaling pathway can downregulate BMPR Ia expression and inhibit inflammation to effectively relieve SCI.

GZ17-6.02 initiates DNA damage causing autophagosome-dependent HDAC degradation resulting in enhanced anti-PD1 checkpoint inhibitory antibody efficacy.

Our studies examined the molecular mechanisms by which the novel cancer therapeutic GZ17-6.02 (NCT03775525) killed GI tumor cells. TZ17-6.02 activated ATM which was responsible for increased phosphorylation of nuclear γH2AX and AMPKα T172. ATM-AMPK signaling was responsible for the subsequent inactivation of mTORC1 and mTORC2, dephosphorylation of ULK1 S757, and increased phosphorylation of ULK1 S317 and of ATG13 S318, which collectively caused enhanced autophagosome formation. GZ17-6.02 interacted with 5-fluorouracil in an additive to greater than additive fashion to kill all of the tested GI tumor cell types. This was associated with greater ATM activation and a greater mammalian target of rapamycin inactivation and autophagosome induction. As a result, autophagy-dependent degradation of multiple histone deacetylase (HDAC) proteins and chaperone proteins occurred. Loss of HDAC expression was causal in reduced expression of programed death ligand 1 (PD-L1), ornithine decarboxylase, and indole amine 2,3-dioxygenase (IDO1) and in the elevated expression of major histocompatibility complex Class IA (MHCA). Treatment with GZ17-6.02 also resulted in enhanced efficacy of a subsequently administered anti-PD1 checkpoint inhibitory antibody. Thus, the primary mode of GZ17-6.02 action is to induce a DNA damage response concomitant with ATM activation, that triggers a series of interconnected molecular events that result in tumor cell death and enhanced immunogenicity.

Abstract 085: Loss of Microglia Enhances Neuronal Activity in the Sympathetic Paraventricular Nucleus

Microglia, the primary immune cells in the CNS, are critical in the maintenance of brain homeostasis such as participating neurogenesis and pruning synapses. Our previous work indicates that microglia are activated in hypertension; depletion of microglia in different activation status have opposite effects on blood pressure regulation, which could be due to tuning sympathetic outflow. To further delineate the mechanism how microglia modulate sympathetic nerve activity, we investigated the effect of microglial depletion on sympathetic neuronal activity in the hypothalamic paraventricular nucleus (PVN). In transgenic CD11b-DTR mice, intracerebroventricular (ICV) infusion of diphtheria toxin resulted in over 90% loss of microglia in the brain. Using the transgenic mouse brain slices, we examined the excitability of RVLM-PVN neurons by electrophysiological technique. Patch-clamp recordings obtained from the RVLM-projecting PVN neurons showed significant increases in firing frequency (3.59±0.72 vs. 1.56±0.51; P<0.05 by t-test) and half-width of action potential (2.53 ± 0.11 vs. 1.65 ± 0.07; P<0.001 by t-test); and a dramatic reduction in the outward K currents (I A and I k ) in the absence of microglia compared to control. Single-cell RNA sequencing analysis of RVLM- PVN neurons revealed a significant decreased expression of I A (Kcnd2 abd Kcnd3) and I k (kcna2, Kcna3 and Kcnc2) subunits in neurons without microglia vs. the controls. These results indicate that loss of microglia increases PVN sympathetic neuronal activity, which could be mediated, at least in part, by the inhibition of outward K currents. Collectively, this study suggests a critical role of microglia in the maintaining the homeostasis of sympathetic PVN neurons in normal condition.

SnapshotDx Quiz: December 2018

SnapshotDx Quiz: December 2018

Cardiac myofibroblast induces decreased expression of major histocompatibility complex class II (Ia) on rat monocyte/macrophages.

The up-regulation of HLA antigens is important during heart inflammatory events and myofibroblasts may modulate the expression of this molecule in tissues. To test this possibility, the effect of cardiac myofibroblast:macrophage contact and the production of myofibroblast inhibitor factor(s) on the macrophage HLA (Ia) expression were studied. Listeria monocytogenes-elicited Ia + peritoneal macrophages (high Ia expression) were co-cultured with cardiac myofibroblasts for 3 and 7 days (myofibroblast contact). Proteosa peptone-elicited macrophages (low Ia expression) were cultured for 3 days with interferon gamma (INF-γ) and myofibroblast conditioned medium (FCM). Ia expression was analyzed by immunofluorescence and by radioimmune assay. Myofibroblast contact induced decreased expression of Ia molecule on macrophages (p < 0.001). This was confirmed by the radioimmune analysis in macrophage: myofibroblast co-cultures (p < 0.001). Double staining for Ia and CD14 showed that only CD14 positive cells (macrophages) expressed Ia molecule. FCM was capable of diminishing Ia expression induced by INF-γ on macrophages (p < 0.001). Decreased Ia macrophage expression induced by myofibroblasts could be important in the heart inflammation's resolution, probably involving Ia redistribution on cell: cell contact and myofibroblast inhibitor factor production.

Biolistic co-transformation of rice using gold nanoparticles

ABSTRACT- In order to produce transgenic rice lines lacking selectable marker gene, biolistic co-transformation technique using gold nanoparticles was adopted. In the first step, the efficiency of different sizes of gold particles was evaluated. The results showed that the efficiency of the nanoparticles in the transformation was comparable to that of the micro particles. Subsequently, two separate plasmids including p UBC harboring cry IA (c) gene and pTRA132 carrying hph gene as selectable marker gene were mixed and coated with gold nanoparticles. Embryo genic calli originated from mature rice seeds were targeted with plasmid-coated nan projectiles. Putative transgenic cells were selected from selection media after three subcultures in MS medium were supplemented with 50 mg hygromycine B l-1 in three weeks' intervals. The selected calli were then grown into putative transgenic plantlets. Molecular analysis performed on the regenerated lines showed that at least one copy of both genes was inserted into the genome of some transgenic lines. RT-PCR analysis also confirmed successful transcription of the cry IA (c) gene. Accordingly, it could be concluded that the biolistic co-transformation method using gold nanoparicles could be a successful method for gene delivery into rice varieties. Nevertheless, expression of cry IA (c) gene and loci of both genes should still be investigated in future studies.

Bone Morphogenetic Protein Receptor IA and IB expression in the adult rat trigeminal nuclei

Bone Morphogenetic Protein Receptor IA and IB expression in the adult rat trigeminal nuclei

Abstract A014: CIITA dependent MHC class II IA expression in tumor cells triggers CD4 T cell protective and long lasting antitumor immunity

Abstract We previously demonstrated the generation of a CD4 T cell-specific long lasting anti-tumor immune response in the H-2d BALB/c mouse model by using tumor cells that have been genetically modified with CIITA to express MHC-II I-E and I-A molecules. We have now investigated the pertinence of this approach in the H-2b C57BL/6 mouse model despite the defect in their I-Eα gene and thus the lack of expression of I-E subset. To this purpose we injected in vivo the CIITA-driven I-A-only MHC-II-positive LLC (lewis Lung Carcinoma) and MC38 colon carcinoma in the C57BL/6 mice and their growth rate along with the recipient's immune response were analyzed. The CIITA-transfected, MHC-II-positive tumor cells were either completely rejected or showed a significant growth retardation compared to the MHC-II-negative parental tumor. The protected mice were re-injected with the parental tumors and a complete rejection was obtained proving that a specific long lasting immune response was triggered by the CIITA-transfected tumors. Furthermore, total splenocytes or purified CD4+, CD8+ T cells and B cells were transferred from the vaccinated recipients to naïve recipients that were co-injected with the parental tumor cells and the results showed that CD4+ TH cells were the main effectors of the immune response against the tumor cells. Interestingly, similar results were obtained in C57BL/6-DOG transgenic mice whose dendritic cells could be conditionally ablated after administration of Diphteria toxin. These results demonstrate the validity of triggering a specific, long-lasting anti-tumor immune response using CIITA-driven MHC class II positive tumor cells of different MHC haplotype as stimulators. Importantly, the results establish that expression of a single MHC-II restriction element, I-A, in tumor cells is sufficient to trigger CD4+ TH cell protective immune response, strongly suggesting that the relevant tumor-associated antigenic repertoire can be displayed without the need of the I-E restriction element. Finally, these results strongly suggest that CIITA-modified tumor cells can act as antigen presenting cells in vivo to prime naïve CD4+ TH cells against tumor antigens. Citation Format: Farah Bou Nasser Eddine, Greta Forlani, Giovanna Tosi, Roberto S. Accolla. CIITA dependent MHC class II IA expression in tumor cells triggers CD4 T cell protective and long lasting antitumor immunity [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A014.

Selective depletion of CD11c+ CD11b+ dendritic cells partially abrogates tolerogenic effects of intravenous MOG in murine EAE.

Intravenous (i.v.) injection of a soluble myelin antigen can induce tolerance, which effectively ameliorates experimental autoimmune encephalomyelitis (EAE). We have previously shown that i.v. myelin oligodendrocyte glycoprotein (MOG) induces tolerance in EAE and expands a subpopulation of tolerogenic CD11c+ CD11b+ dendritic cells (DCs) with an immature phenotype having low expression of IA and co-stimulatory molecules CD40, CD86, and CD80. Here, we further investigate the role of tolerogenic DCs in i.v. tolerance by injecting clodronate-loaded liposomes, which selectively deplete CD11c+ CD11b+ and immature DCs, but not CD11c+ CD8+ DCs and mature DCs. I.v. MOG-induced suppression of EAE was partially, yet significantly, blocked by CD11c+ CD11b+ DC depletion. While i.v. MOG inhibited IA, CD40, CD80, CD86 expression and induced TGF-β, IL-27, IL-10 production in CD11c+ CD11b+ DCs, these effects were abrogated after injection of clodronate-loaded liposomes. Depletion of CD11c+ CD11b+ DCs also precluded i.v. autoantigen-induced T-cell tolerance, such as decreased production of IL-2, IFN-γ, IL-17 and numbers of IL-2+ , IFN-γ+ , and IL-17+ CD4+ T cells, as well as an increased proportion of CD4+ CD25+ Foxp3+ regulatory T cells and CD4+ IL-10+ Foxp3- Tr1 cells. CD11c+ CD11b+ DCs, through low expression of IA and costimulatory molecules as well as high expression of TGF-β, IL-27, and IL-10, play an important role in i.v. tolerance-induced EAE suppression.

High-fat diet feeding promotes stemness and precancerous changes in murine gastric mucosa mediated by leptin receptor signaling pathway

Obesity increases the risk for gastric cancers. However, the occurrence and mechanisms of precancerous atrophic gastritis induced by high-fat diet (HFD) remain unclear. Here, we show that HFD-associated lipotoxicity induces precancerous lesions that are accompanied by the disruption of organelle homeostasis, tissue integrity, and deregulated expression of stemness genes in the gastric epithelium mediated by leptin receptor (ObR) signaling. Following HFD feeding, ectopic fat accumulated and expression of LAMP2A in lysosome and COX IV in mitochondria increased in the gastric mucosa. HFD feeding also led to enhanced expression of activated-Notch1 and stem cell markers Lgr5, CD44, and EpCAM. In addition, HFD-fed mice showed intracellular β-catenin accumulation in the gastric mucosa with increased expression of its target genes, Nanog, Oct4, and c-Myc. These observations were abrogated in the leptin-deficient ob/ob mice and ObR-mutated db/db mice, indicating that these HFD-induced changes were responsible for effects downstream of the ObR. Consistent with this, the expression of the Class IA and III PI3Ks was increased following ObR activation in the gastric mucosa of HFD-fed mice. Together, these results suggest that HFD-induced lipotoxicity and deregulated organelle biosynthesis confer cancer stem cell-like properties to the gastric mucosa via signaling pathway mediated by leptin, PI3K and β-catenin.