scholarly journals Control of Astrocyte Quiescence and Activation in a Synthetic Brain Hydrogel

2019 ◽  
Author(s):  
Sualyneth Galarza ◽  
Alfred J. Crosby ◽  
ChangHui Pak ◽  
Shelly R. Peyton
Keyword(s):  
Physiological Role ◽  
Protein Composition ◽  
Culture Conditions ◽  
Quiescent State ◽  
Dependent Manner ◽  
Synthetic Hydrogel ◽  
Health And Disease ◽  
And Control ◽  
First Time

Bioengineers designed numerous instructive brain extracellular matrix (ECM) environments that have tailored and tunable protein composition and biomechanics in vitro to study astrocyte reactivity during trauma and inflammation. However, a major limitation of both protein-based and model microenvironments is that astrocytes within fail to retain their characteristic stellate morphology and quiescent state without becoming activated under “normal” culture conditions. Here we introduce a synthetic hydrogel, that for the first time demonstrates maintenance of astrocyte quiescence, and control over activation on demand. With this synthetic brain hydrogel, we show the brain-specific integrin-binding and matrix metalloprotease (MMP)-degradable domains of proteins control astrocyte star-shaped morphologies, and we can achieve an ECM condition that maintains astrocyte quiescence with minimal activation. In addition, we can induce activation in a dose-dependent manner via both defined cytokine cocktails and low molecular weight hyaluronic acid. We envision this synthetic brain hydrogel as a new tool to study the physiological role of astrocytes in health and disease.

scholarly journals Secreted KIAA1199 promotes the progression of rheumatoid arthritis by mediating hyaluronic acid degradation in an ANXA1-dependent manner

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Wei Zhang ◽  
Guoyu Yin ◽  
Heping Zhao ◽  
Hanzhi Ling ◽  
Zhen Xie ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Hyaluronic Acid ◽  
Dependent Manner ◽  
Collagen Induced Arthritis ◽  
Intracellular Degradation ◽  
Main Pathway ◽  
First Time

AbstractIn inflamed joints, enhanced hyaluronic acid (HA) degradation is closely related to the pathogenesis of rheumatoid arthritis (RA). KIAA1199 has been identified as a hyaladherin that mediates the intracellular degradation of HA, but its extracellular function remains unclear. In this study, we found that the serum and synovial levels of secreted KIAA1199 (sKIAA1199) and low-molecular-weight HA (LMW-HA, MW < 100 kDa) in RA patients were significantly increased, and the positive correlation between them was shown for the first time. Of note, treatment with anti-KIAA1199 mAb effectively alleviated the severity of arthritis and reduced serum LMW-HA levels and cytokine secretion in collagen-induced arthritis (CIA) mice. In vitro, sKIAA1199 was shown to mediate exogenous HA degradation by attaching to the cell membrane of RA fibroblast-like synoviosytes (RA FLS). Furthermore, the HA-degrading activity of sKIAA1199 depended largely on its adhesion to the membrane, which was achieved by its G8 domain binding to ANXA1. In vivo, kiaa1199-KO mice exhibited greater resistance to collagen-induced arthritis. Interestingly, this resistance could be partially reversed by intra-articular injection of vectors encoding full-length KIAA1199 instead of G8-deleted KIAA119 mutant, which further confirmed the indispensable role of G8 domain in KIAA1199 involvement in RA pathological processes. Mechanically, the activation of NF-κB by interleukin-6 (IL-6) through PI3K/Akt signaling is suggested to be the main pathway to induce KIAA1199 expression in RA FLS. In conclusion, our study supported the contribution of sKIAA1199 to RA pathogenesis, providing a new therapeutic target for RA by blocking sKIAA1199-mediated HA degradation.

scholarly journals IMMUNE RESPONSES IN VITRO

1971 ◽  
Vol 134 (2) ◽  
pp. 395-416 ◽  
Author(s):  
Carl W. Pierce ◽  
Barbara M. Johnson ◽  
Harriet E. Gershon ◽  
Richard Asofsky
Keyword(s):  
Culture Conditions ◽  
Antibody Concentration ◽  
Spleen Cells ◽  
Immunoglobulin Class ◽  
Cell Densities ◽  
Erythrocyte Antigen ◽  
First Time ◽  
Kinetics Of

We have demonstrated for the first time that mouse spleen cells stimulated in vitro with heterologous erythrocytes developed immunoglobulin class-specific γM, γ1, γ2a+2b, and γA plaque-forming cell (PFC) responses. A modification of the hemolytic plaque technique, the addition of goat anti-mouse µ-chain antibody to the assay preparation, specifically prevented development of all γM PFC and enabled accurate and reproducible enumeration of immunoglobulin class-specific PFC after treatment with appropriate monospecific anti-globulins and complement. Culture conditions, with regard to medium, atmosphere, agitation, and spleen cell densities, were similar to those previously shown to support only γM PFC responses. Evaluation of the kinetics of appearance of PFC showed that γM PFC reached maximum numbers on days 4–5; the magnitude of this response was 3–10 times greater than γ1 γ2a+2b, or γA PFC which reached maximum numbers on days 5–6. Optimal erythrocyte antigen dose for γM PFC responses was 107/culture, whereas a dose of 106 erythrocytes/culture consistently stimulated optimal γ1 γ2a+2b, or γA PFC responses. Investigations of the effects of anti-erythrocyte antibody on γM and γG PFC responses indicated that antibody suppressed these responses by neutralizing the effective antigenic stimulus at the macrophage-dependent phase of the response. At the same antibody concentration, γG PFC responses were more effectively suppressed than γM PFC responses. Further, γG responses could be almost completely suppressed by antibody as long as 48 hr after initiation of cultures, whereas γM PFC responses could only be completely suppressed during the first 24 hr. These results were discusssed in terms of the role of antigen in the stimulation γM and γG antibody.

scholarly journals INTEGRINS MEDIATE PLACENTAL EXTRACELLULAR VESICLE TRAFFICKING TO LUNG AND LIVER IN VIVO

2020 ◽  
Author(s):  
Sean L. Nguyen ◽  
Soo Hyun Ahn ◽  
Jacob W. Greenberg ◽  
Benjamin W. Collaer ◽  
Dalen W. Agnew ◽  
...  
Keyword(s):  
Immune Cells ◽  
Extracellular Vesicle ◽  
Dependent Manner ◽  
Cellular Phenotype ◽  
Reporter Mouse ◽  
Membrane Bound ◽  
First Time

ABSTRACTMembrane-bound extracellular vesicles (EVs) mediate intercellular communication in all organisms, and those produced by placental mammals have become increasingly recognized as significant mediators of fetal-maternal communication. Here, we aimed to identify maternal cells targeted by placental EVs and elucidate the mechanisms by which they traffic to these cells. Exogenously administered pregnancy-associated EVs traffic specifically to the lung; further, placental EVs associate with lung interstitial macrophages and liver Kupffer cells in an integrin-dependent manner. Localization of EV to maternal lungs was confirmed in unmanipulated pregnancy using a transgenic reporter mouse model, which also provided in situ and in vitro evidence that fetally-derived EVs, rarely, may cause genetic alteration of maternal cells. These results provide for the first time direct in vivo evidence for targeting of placental EVs to maternal immune cells, and further, evidence that EVs can alter cellular phenotype.

scholarly journals Regulatory B cells (Bregs) inhibit osteoclastogenesis and prevent bone loss in osteoporotic mice model

2021 ◽  
Author(s):  
Leena Sapra ◽  
Asha Bhardwaj ◽  
Pradyumna K. Mishra ◽  
Bhupendra K. Verma ◽  
Rupesh K. Srivastava
Keyword(s):  
B Cells ◽  
Bone Marrow Cells ◽  
Dependent Manner ◽  
Regulatory B Cells ◽  
Mice Model ◽  
Post Menopausal Osteoporosis ◽  
Post Menopausal ◽  
First Time

AbstractIncreasing evidences in recent years have suggested that regulatory B cells (Bregs) are crucial modulator in various inflammatory disease conditions. However, the role of Bregs in case of postmenopausal osteoporosis remains unknown. Also, no study till date have ever investigated the significance of Bregs in modulating osteoclastogenesis. In the present study, we for the first time examined the anti-osteoclastogenic potential of Bregs under in vitro conditions and we observed that Bregs suppressed RANKL mediated osteoclastogenesis in bone marrow cells in a dose dependent manner. We further elucidated the mechanism behind the suppression of osteoclasts differentiation by Bregs and found that Bregs inhibit osteoclastogenesis via IL-10 production. To further confirm the bone health modulating potential of Bregs we employed post-menopausal osteoporotic mice model. Remarkably, our in vivo data clearly suggest a significant reduction (p < 0.01) in both CD19+IL-10+ and CD19+CD1dhiCD5+IL-10+ B10 Bregs in case of osteoporotic mice model. Moreover, our serum cytokine data further confirms the significant reduction of IL-10 levels in osteoporotic mice. Taken together, the present study for the first time unravels and establish the unexplored role of regulatory B cells in case of osteoporosis and provide new insight into Bregs biology in the context of post-menopausal osteoporosis.

scholarly journals Increased Bone Mass in Mice Lacking the Adipokine Apelin

Endocrinology ◽  
2013 ◽  
Vol 154 (6) ◽  
pp. 2069-2080 ◽  
Author(s):  
Lalita Wattanachanya ◽  
Wei-Dar Lu ◽  
Ramendra K. Kundu ◽  
Liping Wang ◽  
Marcia J. Abbott ◽  
...  
Keyword(s):  
Bone Mass ◽  
Bone Cells ◽  
Physiological Role ◽  
In Vitro Study ◽  
Maximum Effect ◽  
Dependent Manner ◽  
Primary Mouse ◽  
Skeletal Homeostasis

Abstract Adipose tissue plays an important role in skeletal homeostasis, and there is interest in identifying adipokines that influence bone mass. One such adipokine may be apelin, a ligand for the Gi-G protein-coupled receptor APJ, which has been reported to enhance mitogenesis and suppress apoptosis in MC3T3-E1 cells and primary human osteoblasts (OBs). However, it is unclear whether apelin plays a physiological role in regulating skeletal homeostasis in vivo. In this study, we compared the skeletal phenotypes of apelin knockout (APKO) and wild-type mice and investigated the direct effects of apelin on bone cells in vitro. The increased fractional cancellous bone volume at the distal femur was observed in APKO mice of both genders at 12 weeks of age and persisted until the age of 20. Cortical bone perimeter at the femoral midshaft was significantly increased in males and females at both time points. Dynamic histomorphometry revealed that APKO mice had increased rates of bone formation and mineral apposition, with evidences of accelerated OB proliferation and differentiation, without significant alteration in osteoclast activity. An in vitro study showed that apelin increased proliferation of primary mouse OBs as well as suppressed apoptosis in a dose-dependent manner with the maximum effect at 5nM. However, it had no effect on the formation of mineralized nodules. We did not observed significantly altered in osteoclast parameters in vitro. Taken together, the increased bone mass in mice lacking apelin suggested complex direct and paracrine/endocrine effects of apelin on bone, possibly via modulating insulin sensitivity. These results indicate that apelin functions as a physiologically significant antianabolic factor in bone in vivo.

Comparison of gene expression and activation of transcription factors in organoid-derived monolayer intestinal epithelial cells and organoids

2021 ◽  
Author(s):  
Yu Takahashi ◽  
Yu Inoue ◽  
Keitaro Kuze ◽  
Shintaro Sato ◽  
Makoto Shimizu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Gene Expression Regulation ◽  
Intestinal Epithelial Cells ◽  
Three Dimensional ◽  
Culture Conditions ◽  
Dependent Manner ◽  
Intestinal Epithelial

Abstract Intestinal organoids better represent in vivo intestinal properties than conventionally used established cell lines in vitro. However, they are maintained in three-dimensional culture conditions that may be accompanied by handling complexities. We characterized the properties of human organoid-derived two-dimensionally cultured intestinal epithelial cells (IECs) compared with those of their parental organoids. We found that the expression of several intestinal markers and functional genes were indistinguishable between monolayer IECs and organoids. We further confirmed that their specific ligands equally activate intestinal ligand-activated transcriptional regulators in a dose-dependent manner. The results suggest that culture conditions do not significantly influence the fundamental properties of monolayer IECs originating from organoids, at least from the perspective of gene expression regulation. This will enable their use as novel biological tools to investigate the physiological functions of the human intestine.

POSSIBLE ROLE FOR THE CA2+-DEPENDENT PROTEASE (CALPAIN I) AS AN IRREVERSIBLE ACTIVATOR OF CA2+/CALMODULIN-MEDIATED REACTIONS IN THE HUMAN PLATELET

1987 ◽  
Author(s):  
Robert W Wallace ◽  
E Ann Tallant ◽  
Lynn M Brumley
Keyword(s):  
Myosin Light Chain ◽  
Binding Proteins ◽  
Human Platelet ◽  
Limited Proteolysis ◽  
Physiological Role ◽  
Human Platelets ◽  
Dependent Manner ◽  
Protease Activation ◽  
Stimulation Of

Calmodulin (CaM)-binding proteins have been identified in human platelets using Western blotting techniques and 125I-CaM. Ten proteins of 245, 225. 175, 150, 90. 82(2), 60 and 41(2) kilodaltons (kDa) bind 125I-CaM in a Ca2+-dependent manner; the binding is blocked by both trifluoperazine and nonradiolabeled CaM. The 225 and 90 kDa proteins are labeled by antisera against myosin light chain kinase (MLCK); the 60 kDa and one of the 82 kDa proteins have been identified as the CaM-dependent phosphatase (calcineurin) and caldesmon. The other proteins are presumed to be other Ca2+/CaM regulated enzymes and proteins which may be important in platelet function. Most of the CaM-binding proteins are degraded upon addition of Ca2+ to a platelet homogenate; the degradation may be blocked by either EGTA, leupeptin or N-ethylmaleimide which suggests that the degradation is due to a Ca2+-dependent protease. Activation of intact platelets under conditions which promote platelet aggregation (i.e. stirring with extracellular Ca2+) also results in limited proteolysis of CaM-binding proteins including those labeled with anti sera against MLCK and the phosphatase. In vitro studies utilizing purified phosphatase and calpain I indicate that the phosphatase is irreversibly activated upon Ca2+-dependent proteolysis. The proteolytically-activated enzyme is insensitive to either Ca2+ or Ca2+/CaM; in addition, its activity in the absence of Ca2+ is even greater than the activity of the unproteolyzed enzyme in the presence of Ca2+ and CaM. Proteolytic stimulation of the phosphatase is accompanied by degradation of the 60 kDa subunit of the enzyme (subunit A) to 56, 52 and 45 kDa fragments, sequentially; proteolysis results in the loss of CaM binding to the enzyme. These results suggest that the Ca2+-dependent protease may have a physiological role in platelet activation as an irreversible activator of Ca2+/ CaM-dependent reactions. Supported by NIH grant HL29766.

scholarly journals The Influence of Bee Venom Melittin on the Functioning of the Immune System and the Contractile Activity of the Insect Heart—A Preliminary Study

Toxins ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 494 ◽  
Author(s):  
Jan Lubawy ◽  
Arkadiusz Urbański ◽  
Lucyna Mrówczyńska ◽  
Eliza Matuszewska ◽  
Agata Światły-Błaszkiewicz ◽  
...  
Keyword(s):  
Immune System ◽  
Contractile Activity ◽  
Model Organism ◽  
Dependent Manner ◽  
Physiological Studies ◽  
Almost All ◽  
First Time ◽  
Dose Dependent ◽  
Positive Effect

Melittin (MEL) is a basic polypeptide originally purified from honeybee venom. MEL exhibits a broad spectrum of biological activity. However, almost all studies on MEL activity have been carried out on vertebrate models or cell lines. Recently, due to cheap breeding and the possibility of extrapolating the results of the research to vertebrates, insects have been used for various bioassays and comparative physiological studies. For these reasons, it is valuable to examine the influence of melittin on insect physiology. Here, for the first time, we report the immunotropic and cardiotropic effects of melittin on the beetle Tenebrio molitor as a model insect. After melittin injection at 10−7 M and 10−3 M, the number of apoptotic cells in the haemolymph increased in a dose-dependent manner. The pro-apoptotic action of MEL was likely compensated by increasing the total number of haemocytes. However, the injection of MEL did not cause any changes in the percent of phagocytic haemocytes or in the phenoloxidase activity. In an in vitro bioassay with a semi-isolated Tenebrio heart, MEL induced a slight chronotropic-positive effect only at a higher concentration (10−4 M). Preliminary results indicated that melittin exerts pleiotropic effects on the functioning of the immune system and the endogenous contractile activity of the heart. Some of the induced responses in T. molitor resemble the reactions observed in vertebrate models. Therefore, the T. molitor beetle may be a convenient invertebrate model organism for comparative physiological studies and for the identification of new properties and mechanisms of action of melittin and related compounds.

scholarly journals The Transcriptional Regulator VqmA Increases Expression of the Quorum-Sensing Activator HapR in Vibrio cholerae

2006 ◽  
Vol 188 (7) ◽  
pp. 2446-2453 ◽  
Author(s):  
Zhi Liu ◽  
Ansel Hsiao ◽  
Adam Joelsson ◽  
Jun Zhu
Keyword(s):  
Quorum Sensing ◽  
Vibrio Cholerae ◽  
Diarrheal Disease ◽  
Constitutive Expression ◽  
Physiological Role ◽  
Transcriptional Regulator ◽  
Protease Production ◽  
Dependent Manner ◽  
Cell Densities

ABSTRACT Vibrio cholerae is the causative agent of the severe diarrheal disease cholera. A number of environmental stimuli regulate virulence gene expression in V. cholerae, including quorum-sensing signals. At high cell densities, quorum sensing in V. cholerae invokes a series of signal transduction pathways in order to activate the expression of the master regulator HapR, which then represses the virulence regulon and biofilm-related genes and activates protease production. In this study, we identified a transcriptional regulator, VqmA (VCA1078), that activates hapR expression at low cell densities. Under in vitro inducing conditions, constitutive expression of VqmA represses the virulence regulon in a HapR-dependent manner. VqmA increases hapR transcription as measured by the activity of the hapR-lacZ reporter, and it increases HapR production as measured by Western blotting. Using a heterogenous luxCDABE cosmid, we found that VqmA stimulates quorum-sensing regulation at lower cell densities and that this stimulation bypasses the known LuxO-small-RNA regulatory circuits. Furthermore, we showed that VqmA regulates hapR transcription directly by binding to its promoter region and that expression of vqmA is cell density dependent and autoregulated. The physiological role of VqmA is also discussed.

scholarly journals A C21-Steroidal Glycoside from Cynanchum atratum Attenuates Concanavalin A-Induced Liver Injury in Mice

Molecules ◽  
2019 ◽  
Vol 24 (6) ◽  
pp. 1087 ◽  
Author(s):  
Jian Yang ◽  
Bin Wang ◽  
Chao-feng Zhang ◽  
Xiang-hong Xu ◽  
Mian Zhang
Keyword(s):  
T Lymphocytes ◽  
Concanavalin A ◽  
Underlying Mechanism ◽  
Dependent Manner ◽  
Histopathological Changes ◽  
Con A ◽  
Concentration Dependent Manner ◽  
Steroidal Glycoside ◽  
First Time

Cynatratoside A (CyA) is a C21 Steroidal glycoside with pregnane skeleton isolated from the root of Cynanchum atratum Bunge (Asclepiadaceae). This study aimed to investigate the effects of CyA on concanavalin A (Con A)-induced autoimmune hepatitis (AIH) and the underlying mechanism. CyA was orally administered to mice at 10 and 40 mg/kg 8 h before and 1 h after Con A treatment. The effects of CyA on Con A-induced spleen and liver in mice were assessed via histopathological changes, T lymphocyte amounts and the expressions of IL-1β and ICAM-1. Con A-induced L-02 hepatocytes were used to evaluate whether CyA (0.1–10 μM) can directly protect hepatocytes from cytotoxicity and the possible mechanism. The results revealed that CyA treatment could significantly improve the histopathological changes of spleen and liver, reduce the proliferation of splenic T lymphocytes, and decrease the expressions of IL-1β and ICAM-1 in liver. The experiment in vitro showed that CyA inhibited Con A-induced hepatotoxicity in a concentration-dependent manner. CyA (10 μM) significantly increased/decreased the expression of Bcl-2/Bax and reduced the levels of cleaved caspases-9 and -3. Our study demonstrated for the first time that CyA has a significant protective effect on Con A-induced AIH by inhibiting the activation and adhesion of T lymphocytes and blocking hepatocyte apoptosis.

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