Boyden Chamber System Research Articles

OVERLOAD-INDUCED EXTRACELLULAR MITOCHONDRIA RELEASING FROM TENDON ORGANOID LEADS TO INFLAMMATION

Tendinopathy is the most frequent musculoskeletal disease that requires medical attention. Mechanical overload has been considered as a key driver of its pathology. However, the underline mechanism on how overload induces tendinopathy and inflammation is unclear. Extracellular mitochondria (EM) are newly identified as cell-to-cell communicators. The aim of this study is to elucidate the role of mitochondria in overload-induced inflammation.We performed three-dimensional uniaxial stretching to mouse tendon organoid in bioreactors. Cyclic strain of uniaxial loadings included underload, normal load, and overload, according to previous work. We then harvested microvesicles including EM, from the bioreactor by differential centrifugation and evaluated their characteristics by flow cytometry and super-resolution confocal microscopy. Raw 264.7 mouse macrophage cell line was used for chemotaxis assay in a Boyden Chamber System with Magnetic-Activated Cell Sorting Technology. EM induced cytokines secretion by macrophages was analyzed by a bead-based multiplex assay panel. N-Acetyl-L-cysteine (NAC) was used as the antioxidant to tendon organoid to regulate mitochondrial fitness.We showed mechanical load induced tendon organoid to release microvesicles including mitochondria. The size of microvesicles is mainly in the range from 220nm to 880nm. More than 75% of microvesicles could be stained by PKH26, confirming they were with lipophilic membrane. Super-resolution confocal microscopy identified two forms of mitochondria, including mitochondria encapsulated in vesicles and free mitochondria. Overload led to the degeneration of the organoid and induced microvesicles release containing most EM. Chemotaxis assay showed that EM from overloaded tendon organoid induced macrophages chemotaxis. In addition, microvesicles extracted from overloaded tendon organoid induced the production of proinflammatory cytokines including IL-6, KC (Keratinocyte-Derived Chemokine) and IL-18. NAC treatment to tendon cells could attenuate overload-induced macrophage chemotaxis.Overload induces EM releasing from tendon cells, which leads to chemotaxis of macrophages toward tendon, resulting in induction of inflammation.

Effects of the Co-Administration of Morphine and Lipopolysaccharide on Toll-Like Receptor-4/Nuclear Factor Kappa β Signaling Pathway of MDA-MB-231 Breast Cancer Cells.

The Toll-like receptor 4 (TLR4) gene promotes migration in adenocarcinoma cells. Morphine is an agonist for TLR4 that has a dual role in cancer development. The promoter or inhibitor role of morphine in cancer progression remains controversial. This study aims to evaluate the effects of morphine on the TLR4, myeloid differentiation primary response protein 88-dependent (MyD88), and nuclear factor-kappa B (NF-κB) expressions in the human MDA-MB-231 breast cancer cell line. The cells were examined after 24 hours of incubation with morphine using the Boyden chamber system. TLR4, MyD88, and NF-κB mRNA expressions were assessed using quantitative real-time polymerase chain reaction (RT-PCR). The concentration of interleukin-2 beta was also measured using the ELISA assay. According to the findings, three doses of morphine (0.25, 1.25, and 0.025 μM) increased the expression of the TLR4 and NF-κB genes, whereas no significant change was observed in the mRNA expression of MyD88. Furthermore, treatment with morphine and lipopolysaccharide (LPS) significantly decreased the expression of TLR4, MyD88, and NF-κB. However, no significant change was observed in interleukin 2 beta concentration. These findings confirmed the excitatory effects of morphine on TRL4 expression and the MYD88 signaling pathway in vitro.

Abstract 15156: Statistical Modeling of Cardiac-Derived Progenitor Cell Extracellular Vesicles to Predict Clinical Trial Outcomes

Introduction: Cardiac-derived c-kit+ progenitor cells (CPCs) are under investigation in the CHILD phase I clinical trial (NCT03406884) for the treatment of hypoplastic left heart syndrome (HLHS). Therapeutic efficacy of CPCs can be attributed to the release of extracellular vesicles (EVs), carrying beneficial RNA cargo. Despite some successes with pediatric cardiac cell therapy, large variation in cell populations and patient outcomes remains a problem. To investigate these discrepancies and understand sources of cell therapy variability we took a machine learning approach: combining bulk CPC-derived EV (CPC-EV) RNA sequencing and cardiac-relevant in vitro experiments to build a predictive model. Methods: We isolated CPCs from cardiac biopsies of congenital heart disease patients (n=30) and the lead-in HLHS patients in the CHILD trial (n=7). We cultured CPCs, collected EVs from the media, and sequenced EV miRNA and total RNA. We treated cardiac endothelial cells and fibroblasts with CPC-EVs and measured inflammatory or fibrotic gene expression with qRT-PCR. We treated mesenchymal stromal cells with CPC-EVs and assessed migration in a Boyden chamber system. We treated cardiac endothelial cells seeded on Matrigel with CPC-EVs and measured tube formation. Finally, we put together the CPC-EV sequencing and experimental data in partial least squares and regularized regression models. Results: With our models we determined that the most important CPC-EV RNA signals involved in pro-reparative outcomes had significant fit to cardiac development and signaling pathways. Furthermore, using a model trained on previously collected CPC-EVs, we were able to predict in vitro outcomes for the CHILD clinical samples. Finally, we formed rank-based predictions for the CHILD lead-in patients, based on the in vitro assays. Conclusions: The CHILD phase I trial is set to conclude next year. We will validate our predictions with the release of these clinical data. With this work, we (1) investigated the EV RNA signals involved in pediatric cardiac repair and (2) will use our in vitro results to predict CHILD clinical trial outcomes. Our results highlight the importance of quantitative studies in determining the underlying variance in pediatric cardiac cell therapy.

Reparative effect of mesenchymal stromal cells on endothelial cells after ischemic and inflammatory injury

Background & Aim Renal endothelium is damaged by ischemia reperfusion injury (IRI) during transplantation as a result of the hypoxic metabolism and the resumption of oxygen supply after reperfusion. It has been shown that mesenchymal stromal cells (MSC) are effective in enhancing kidney function after IRI. However, the cellular targets and mechanisms of action behind MSC effects are still poorly understood. Therefore, our objective was to evaluate the role of MSC in repairing injured endothelium as it is the first tissue with which they interact after infusion and to determine the specific interactions involved in this process. The role of physical communication and cytokine secretion in the reparative action of MSC was studied as well as its molecular mechanisms. Methods, Results & Conclusion Human umbilical vein endothelial cells (HUVEC) were submitted to hypoxic and inflammatory insult (TNF-a). This increased the expression of activation molecules such as CD54, CD62e and HLA-II on HUVEC's cell surface. Consequently, an increase of almost 20% in the adherence of MSC to HUVEC was observed in comparison to untreated HUVEC. Moreover, an increase of around 20% in the migratory capacity of MSC towards injured HUVEC was observed. MSC showed a 15 to 20% increase in their ability to transmigrate through injured HUVEC monolayers. Once MSC were in contact with HUVEC, the oxidative stress of HUVEC decreased as observed by a decrease in reactive oxygen species levels, their proliferative status improved and MSC restored the angiogenic potential to levels close to those from healthy HUVEC. To determine whether physical interaction or soluble factors released by MSC are fundamental for MSC regenerative capacity, a Boyden chamber system experiment was performed and we observed that both were required to achieve the full reparative effect of MSC. Moreover, we observed an increase in the expression of CD44 and CD29 adhesion molecules on MSC membrane after co-culture with injured HUVEC, as well as their ligands CD62e and CD106 on HUVEC membrane. We conclude that MSC are able to repair the injury caused by ischemic and inflammatory injury on HUVEC via a mechanism that requires both physical and paracrine interaction between MSC and HUVEC. We are currently studying the specific effector molecules involved in MSC and HUVEC cell-to-cell interaction which will allow targeted modification to improve the regenerative effects of MSC therapy.

SAT-160 Fetuin-a Secreted From Pancreatic β-cells Plays Significant Role In Macrophage Accumulation And Inflammation In The Islets During Hyperlipidemic Condition

Inflammation in the pancreatic islets by means of increased macrophage migration and elevated levels of pro-inflammatory cytokines in the circulating serum occurs during Type 2 Diabetes. Another mechanism underlying increase in inflammation is polarization of macrophages from anti-inflammatory M2 to pro-inflammatory M1 form. Whether such changes occur in the infiltrating macrophages in the islets and what were the factors leading to these changes arte yet unknown. Fetuin-A, a glycoprotein known to be secreted from liver and adipocytes has also been found to be secreted from pancreatic β-cells by our group recently. Role of fetuin-A in macrophage polarisation has been worked upon but still more work is yet to be done. Our focus of interest was whether the β-cell derived fetuin-A has any role in polarisation of macrophages migrating to the islets leading to increase in inflammation. For in vivo model we used high fat diet mice model and for in vitro studies we used MIN6 pancreatic β cell line and RAW 264.7 murine macrophage cell line. In the in vivo mice model immunohistochemistry studies revealed increase in number of M1 macrophages in the pancreatic islets and fetuin-A secretion in comparison to the control standard diet fed mice (CD11c, arginase, F4/80). Also there was decrease in islet mass, specifically in number of β-cells. Fetuin-A KD mice using vivo morpholino showed decrease in infiltrating M1 macrophages in the islets as well improved β-cell survival. For replicating hyperlipidemic condion in vitro we treated the MIN6 cells with various doses of palmitate (0, 0.25, 0.50 and 0.75 mM) and assessed the increase in number of infiltrating RAW 264.7 macrophages (4 folds) in a boyden chamber system along with increasing level of fetuin-A (3 folds) in the media as determined by ELISA. Next the effect of this β-cell derived fetuin-A was observed upon macrophage polarisation. Originally RAW264.7 is anti-inflammatory M2 type. The cells were treated with fetuin-A secreted from β-cells, palmitate alone and LPS plus TNFα (positive control), while a set of macrophages were left untreated as negative control. The cells were collected and subjected to qualitative PCR for identifying the expression of M1 and M2 markers like CD11c, arginase, F4/80 and it was found that the pro-inflammatory markers increased in fetuin-A treated RAW 264.7 cells in similar levels like that of LPS+TNFα levels. These results indicated possible role of β-cell derived fetuin-A in macrophage polarisation in the islets. Another important point was that β-cells also express MCP1 another chemoattractant. Blocking of MCP1 by si-RNA decreased macrophage migration a little but expression of fetuin-A was enough for significant number of macrophages to migrate, however blocking fetuin-A by si-RNA in MIN6 cells decreased the number of migrating macrophages significantly.

Stimulation of Peritoneal Mesothelial Cells to Secrete Matrix Metalloproteinase-9 (MMP-9) by TNF-α: A Role in the Invasion of Gastric Carcinoma Cells.

It has recently been recognized that inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), upregulate the secretion of matrix metalloproteinase-9 (MMP-9) from cancer cells and thereby promote peritoneal dissemination. In this study, we found that TNF-α also stimulated peritoneal mesothelial cells to secrete MMP-9 as assessed by zymography. MMP-9 gene expression in mesothelial cells induced by TNF-α was confirmed by quantitative RT-PCR analysis. We then utilized the reconstituted artificial mesothelium, which was composed of a monolayer of mesothelial cells cultured on a Matrigel layer in a Boyden chamber system, to examine the effects of TNF-α on carcinoma cell invasion. The transmigration of MKN1 human gastric carcinoma cells through the reconstituted mesothelium was promoted by TNF-α in a dose-dependent manner. The increased MKN1 cell migration was partially inhibited by the anti-α3 integrin antibody, indicating that the invasion process involves an integrin-dependent mechanism. Finally, we observed that the invasion of MMP-9-knockdown MKN1 cells into Matrigel membranes was potentiated by the exogenous addition of purified proMMP-9. These results suggest that TNF-α-induced MMP-9 secretion from mesothelial cells plays an important role in the metastatic dissemination of gastric cancer.

Effect of Sorbus commixta on the invasion and migration of human hepatocellularcarcinoma Hep3Bcells.

Tumor metastasis is a main cause of cancer-related morbidity and mortality. Thus, a number of medicinal herbs and phytochemicals have been investigated as possible candidates for the inhibition of cancer metastasis. Sorbus commixtaHedl.(SC) is a traditional medicinal plant used in the treatment of inflammatory diseases, as it has antioxidant, anti-inflammatory, anti-atherosclerotic and anti-hepatotoxic activities. In this study, we demonstrate that the water extract of SC exerts inhibitory effect on the invasion and migration of hepatocellular carcinoma Hep3Bcells. The activity and expression of matrix metalloproteinase(MMP)-9, which is responsible for the invasion of cancercells, was decreased by SC treatment. The invasive and migratory potentials of the Hep3Bcells were also decreased, as evidence by invitro assay using the Boyden chamber system. In addition, the expression of the chemokine receptors, C-X-C chemokine receptor type4(CXCR)4 and C-X-C chemokine receptor type6(CXCR6), were inhibited by SC in Hep3Bcells. Furthermore, actin fiber organization was markedly suppressed by SC treatment. Taken together, the findings of this study suggest for the first time, to the best of our knowledge, that SC suppresses the invasion and migration of highly metastatic Hep3Bcells.

Plasma matrix metalloprotease 9 correlates with blood lymphocytosis, leukemic cell invasiveness, and prognosis in B-cell chronic lymphocytic leukemia

The complex biology underlying chronic lymphocytic leukemia cell migration and tissue invasiveness is not yet completely understood and might provide novel predictive markers and therapeutic targets. A total of 36 patients out of treatment from at least 3 months were enrolled and followed up for a median period of 44.2 months (range: 4.4-99.2). Matrix metalloprotease 9 and tissue inhibitor of metalloproteases 1 plasma levels and production/release from lymphoid cells were measured by zymography and enzyme-linked immunosorbent assay (ELISA) analysis. Malignant and normal lymphocyte mobility and matrix-degradation capability were studied using a Boyden chamber system, with and without autologous plasma. Free matrix metalloprotease 9 plasma levels were related with blood lymphocytosis, especially in more advanced stages (p = 0.003), and higher concentrations were associated with an increased disease progression risk (hazard ratio = 9.0, 95% confidence interval = 1.5-13.8). Leukemic cells expressed and secreted very little matrix metalloprotease 9. On the contrary, normal lymphocytes derived from the same leukemic patients showed matrix metalloprotease 9 intracellular levels that were lower in subjects with higher blood lymphocytosis (p = 0.024) and more advanced stages (p = 0.03); the released quantities were inversely associated with matrix metalloprotease 9 plasma concentrations (p = 0.035). Leukemic cells had a reduced spontaneous mobility and matrix-degradation capability that were stimulated by autologous plasma (p = 0.001) and normal lymphocytes (p = 0.005), respectively. Matrix metalloprotease 9 affected cell invasiveness depending on concentration and disease stage. In conclusion, chronic lymphocytic leukemia cells have a reduced mobility, matrix-degradation capability, and matrix metalloprotease 9 production compared to their own autologous normal lymphocytes. They are exposed to matrix metalloprotease 9 of prevalently systemic origin whose higher levels are associated with both leukemic and normal lymphocyte accumulation in the peripheral blood and have a negative prognostic value.

A novel anti-adhesion peptide (β3) inhibits hepatocellular carcinoma activity in vitro and in vivo.

The present study aimed to investigate the blocking of tumor cell adhesion to the extracellular matrix, the prevention of tumor metastasis by the β peptide trimer β3, as well as the influence of β3 on the recurrence and survival time of hepatocellular carcinoma (HCC) nude mice model LCI-D20 after early resection. To this end, the DNA fragment of the β3 peptide (DLYYLMDLSYSMKGGDLYYLMDLSYSMKGGDLYYLMDLSYSMK) was cloned into the expression vector pET-His and the fusion protein His-β3 was expressed in E. coli BL21 (DE3) plysS. The anti-adhesion effect of β3 on the highly metastatic HCC cell line HCCLM6 to fibronectin (FN) was measured by MTT assay. The inhibition of HCCLM6 cell invasion by β3 was analyzed using a Transwell (modified Boyden chamber) system and Matrigel. The influence of β3 on the recurrence of HCC and mouse survival time after early resection was investigated using the HCC metastasis nude mice model LCI-D20. HCCLM6 cells incubated with 10, 20, 50 or 100 µmol/l β3 for 3 h demonstrated a marked reduction in adhesion to FN. The adhesion inhibition rates were 11.8, 21.7, 37.5 and 66.4%, respectively. In addition, cell invasion was reduced by 51.3% in HCCLM6 cells cultured with 100 µmol/l β3. Treatment with β3 also inhibited tumor recurrence at the incisal edge and prolonged the survival time of LCI-D20 mice following early resection. The present study provided evidence that β3 peptide specifically blocked the adhesion and invasion of HCCLM6 cells, inhibited HCC recurrence in vivo and prolonged the survival time of HCC nude mice LCI-D20 following hepatectomy. Therefore, β3 may be further investigated as a novel anti-tumor drug.

Ginsenoside 20(S)-Rh2 exerts anti-cancer activity through targeting IL-6-induced JAK2/STAT3 pathway in human colorectal cancer cells

Ethnopharmacological relevancePanax ginseng is one of the most well-known medicinal herbs in Korea and China, which has been used for treatment and prevention of cancer, obesity, diabetes, and cardiovascular diseases. Ginsenosides are the major components of P. ginseng, having a wide range of pharmacological activities. Among the ginsenosides, protopanaxadiol (PPD)-types reportedly have potent anti-cancer effects. Rh2 is PPD-type ginsenoside, and two stereoisomeric forms of Rh2 as 20(S)- and 20(R)-Rh2 were selectively isolated recently. Aim of the studyThe biological activities of Rh2 ginsenosides are known to depend on their differences in stereochemistry. Colorectal cancer (CRC) is one of the most lethal neoplasm, and cancer-related death is usually associated with metastasis to other organs. We aimed this study to investigate whether 20(S)- and 20(R)-Rh2 can suppress tumor invasion in human CRC cells. Materials and methods20(S)- and 20(R)-Rh2 were isolated from the roots of ginseng. Human CRC cells were incubated with 20(S)- or 20(R)-Rh2 in the presence or absence of interleukin-6. An MTT assay was used to measure cell viability. Western blot and quantitative real-time PCR analyses were performed to determine levels of expression and phosphorylation. An invasion assay was performed using a Boyden chamber system with the Matrigel-coated membrane to measure cancer cell invasion. Results20(S)- and 20(R)-Rh2 showed differential cytotoxic activity. Only 20(S)-Rh2 decreased cancer cell viability. Additionally, 20(S)-Rh2 effectively inhibited IL-6-induced signal transducer and activator of transcription 3 (STAT3) phosphorylation and the expression of matrix metalloproteinases (MMPs), including MMP-1, −2, and −9, resulting in inhibition of cancer cell invasion. Interestingly, these pharmacological activities of 20(S)-Rh2 were more potent than those of 20(R)-Rh2. Furthermore, combination treatment showed that 20(S)-Rh2 enhanced the sensitization of doxorubicin-treated anti-cancer activities in CRC cells. ConclusionOur results demonstrated that ginsenoside 20(S)-Rh2 has therapeutic potential for the treatment with CRC and may be valuable as a combination partner with more classic chemotherapeutic agents, such as doxorubicin, to treat CRC.

Silencing the Nucleocytoplasmic O-GlcNAc Transferase Reduces Proliferation, Adhesion, and Migration of Cancer and Fetal Human Colon Cell Lines.

The post-translational modification of proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) is regulated by a unique couple of enzymes. O-GlcNAc transferase (OGT) transfers the GlcNAc residue from UDP-GlcNAc, the final product of the hexosamine biosynthetic pathway (HBP), whereas O-GlcNAcase (OGA) removes it. This study and others show that OGT and O-GlcNAcylation levels are increased in cancer cell lines. In that context, we studied the effect of OGT silencing in the colon cancer cell lines HT29 and HCT116 and the primary colon cell line CCD841CoN. Herein, we report that OGT silencing diminished proliferation, in vitro cell survival and adhesion of primary and cancer cell lines. SiOGT dramatically decreased HT29 and CCD841CoN migration, CCD841CoN harboring high capabilities of migration in Boyden chamber system when compared to HT29 and HCT116. The expression levels of actin and tubulin were unaffected by OGT knockdown but siOGT seemed to disorganize microfilament, microtubule, and vinculin networks in CCD841CoN. While cancer cell lines harbor higher levels of OGT and O-GlcNAcylation to fulfill their proliferative and migratory properties, in agreement with their higher consumption of HBP main substrates glucose and glutamine, our data demonstrate that OGT expression is not only necessary for the biological properties of cancer cell lines but also for normal cells.

Megakaryocytes Support Viability Proliferation and Protection of Primary Pre-B ALL Cells from Chemotherapy

BACKGROUND: The bone marrow is known to shelter leukemia cells from chemotherapy and contributes to the survival of chemotherapy resistant residual cells, termed minimal residual disease (MRD). We have studied in situ the location of MRD+ ALL cells using a xenograft model of primary ALL cells and have found a novel co-localization of megakaryocytes (MK) with ALL cells. Mature hematopoietic cells have been implicated in modifying the local normal hematopoietic stem cell environment including MK. We hypothesize that MK are associated with the survival of MRD+ ALL cells. For this purpose, we tested the role of MK cells in maintenance and chemoprotection of ALL cells.METHODS: Patient-derived (primary) pre-B ALL cells were engrafted into non-irradiated NOD/SCID IL2Rγ-/- (NSG) mice. Leukemia-bearing mice received 4 weeks of chemotherapy treatment (Vincristine, Dexamethasone, L-Asparaginase; VDL). MRD status of mice was confirmed by detection of human CD45 + CD19+ leukemia cells in the bone marrow by flow cytometry. In situ location of the MRD+ ALL cells was determined by histological analysis and quantitation was performed by Fiji Image J. For in vitro studies, primary pre-B ALL cells were co-cultured for up to 2 days with murine calvaria-derived stromal OP9 cells or MK isolated from C57/BL6 BM primed for with murine thrombopoietin (mTPO) and sorted by flow cytometry for CD41+ MK. Annexin V/7-AAD staining was used for viability determination by flow cytometry. Boyden chamber system with either OP9 cells or MK cells seeded on the bottom and ALL cells on the top of the system was used for migration assays.RESULTS:In situ analysis of MRD+ ALL recipient mice showed that cells MRD+ ALL cells (huCD45+) are located in close proximity to MKs, with 11.57±2.94% of MRD+ ALL cells lying directly adjacent to MKs (0-5μm distance to MK). To further assess the role of MKs in ALL survival in vitro, we compared if MK cells can sustain proliferation and viability of primary leukemia cells like the OP stromal co-culture model that we have established previously. MKs were isolated from BM of C57BL/6 mice by FACS sorting for CD41+ cells from BM primed with mTPO. The sorted population showed a 90% purity of CD41+ cells. MKs were able to maintain ALL cell proliferation 1.90e6 cell count ± 0.48e6 cell count on day 2) and provide chemoprotection from VDL treatment (77.24 ± 2.03% on day 2), which was similar to the effect of OP9 cells on sustained proliferation and viability. Interestingly ALL cells cultured with MKs had a slight reduction in G2/M-phase (8.46±0.31%) 3 days after culture set up without treatment compared to cultures with OP9 stromal cells (13.59±0.14%; P-value < 0.0005). In a migration assay, MKs stimulated migration of ALL cells (5.42e5 ±0.72 migrated cells) significantly more than OP9 stromal cells (2.92e5 ±0.72 migrated cells) over a 24 hour period (P-value = 0.0132). Using the SDF1α inhibitor AMD3100 (100µM), migration of ALL cells was only partially inhibited (2.92e5 ±0.72), suggesting additional MK produced factors influence mobilization of human leukemia cells besides SDF1α. Using the recombinant forms of stromal cell-derived factor 1 (SDF1α) and von Willebrand factor (VWF), ALL cell migration was successfully stimulated over 24 hours (6.25e4 ±1.25 and 3.33e4 ±0.72 migrated cells, respectively), and this effect was inhibited using AMD3100, small molecule inhibitor of CXCR4, and anti-VWF antibody respectively.CONCLUSION: Here we show for the first time that co-culture of MK and primary pre-B ALL cells supports their proliferation, viability and protection from chemotherapy similar to murine OP9 stromal cells. Our data warrants further investigation of the underlying mechanism. DisclosuresNo relevant conflicts of interest to declare.

Discrete roles of intracellular phospholipases A2 in human neutrophil cytotoxicity.

The role of calcium-independent phospholipase A2 (iPLA2), a component of the three major PLA2 families, in acute/chronic inflammatory processes remains elusive. Previous investigations have documented iPLA2-mediated respiratory burst of neutrophils (PMNs); however, the causative isoform of iPLA2 is unidentified. We also demonstrated that the iPLA2γ-specific inhibitor attenuates trauma/hemorrhagic shock-induced lung injury. Therefore, iPLA2γ may be implicated in acute inflammation. In addition, arachidonic acid (AA), which is primarily produced by cytosolic PLA2 (cPLA2), is known to display PMN cytotoxicity, although the relationship between AA and the cytotoxic function is still being debated on. We therefore hypothesized that iPLA2γ regulates PMN cytotoxicity via AA-independent signaling pathways. The study aim was to distinguish the role of intracellular phospholipases A2, iPLA2, and cPLA2, in human PMN cytotoxicity and explore the possibility of the presence of signaling molecule(s) other than AA. Isolated human PMNs were incubated with the PLA2 inhibitor selective for iPLA2β, iPLA2γ, or cPLA2 and then activated with formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol 12-myristate 13-acetate (PMA). Superoxide production was assayed according to the superoxide dismutase-inhibitable cytochrome c reduction method, and the degree of elastase release was measured using a p-nitroanilide-conjugated elastase-specific substrate. In addition, chemotaxis toward platelet activating factor/fMLP was determined with a modified Boyden chamber system. The iPLA2γ-specific inhibitor reduced the fMLP/PMA-stimulated superoxide generation by 90% and 30%, respectively; in addition, the inhibitor completely blocked the fMLP/PMA-activated elastase release. However, the cPLA2-specific inhibitor did not abrogate these effects to any degree at all concentrations. Likewise, the inhibitor for iPLA2γ, but not iPLA2β or cPLA2, completely inhibited the platelet activating factor/fMLP-induced chemotaxis. iPLA2 is involved in extracellular reactive oxygen species production, elastase release, and chemotaxis in response to well-defined stimuli. In addition, the ineffectiveness of the cPLA2 inhibitor suggests that AA may not be relevant to these cytotoxic functions.

Tracking mesenchymal stem cell tumor-homing using fluorescent silica nanoparticles.

Stem cell tracking can reveal the underlying biological processes of stem-cell-based therapies such as the migration and biodistribution of human mesenchymal stem cells (hMSCs) in cancer therapy. Nanoparticle-based contrast agents offer unprecedented opportunities for achieving this goal due to their unique and tunable imaging capabilities. However, most nanoparticles are still in the process of development due to challenges such as retention time and safety issues, and are inaccessible to most researchers. In this article, we investigate the potential application of core-shell fluorescent silica nanoparticles (i.e. C dots), which are commercially available and approved by the FDA for clinical trials. Specifically we demonstrate that 500 nm C dots have prolonged cellular retention (up to one month), minimal contrast agent transfer (at least three weeks) between cells in a co-culture Boyden chamber system, and minimal influence on the hMSC properties including viability, proliferation, differentiation, and tropism to tumor cells.

Helicobacter pylori dwelling on the apical surface of gastrointestinal epithelium damages the mucosal barrier through direct contact.

Epithelial junctions and mucins compose a major portion of the mucosal barrier. Helicobacter pylori (H. pylori) infections induce alterations of the tight junctions and adherens junctions in epithelial cells, although the precise mechanisms underlying this process are not fully understood. The expression of adhesion molecules and MUC1 was systematically investigated in gastrointestinal epithelial cells infected with H. pylori in vitro and in vivo. Furthermore, we developed several new in vitro methods to study the relationships between the bacterium and the dysfunction of tight junctions using Boyden Chambers. The expression of a series of junctional molecules and MUC1 decreased in the cultured cells that were infected with H. pylori. According to the degree of damage at the tight junctions, direct contact of H. pylori with the apical membrane of the cells resulted in the greatest increase in permeability compared to basal membrane binding or non-binding of H. pylori to the cells. Similarly, we noted that H. pylori infection could reduce the expression and glycosylation of MUC1. Helicobacter pylori dwelling on the apical surface of the gastrointestinal epithelium could directly induce serious injury of the mucosal barrier, and the new methods outlined here, based on the Boyden Chamber system, could be very useful for studying the relationships between bacteria and their target cells.

The effect of pharmacological PI3Kγ inhibitor on eotaxin-induced human eosinophil functions

BackgroundAsthma is characterized by chronic inflammation caused by activation of immune cells including Th2 lymphocytes and eosinophils. Phosphoinositide 3-kinase (PI3K) γ deficient asthmatic mice did not develop lung eosinophilia, although the detailed mechanisms are not well known. A CC chemokine eotaxin (CCL11) plays a prominent role in developing eosinophilic inflammation through CCR3. In this study, we tested the roles of PI3Kγ in eotaxin-induced eosinophil functions using a pharmacological inhibitor. MethodHuman peripheral blood eosinophils were isolated by CD16-negative selection method. The effect of AS605240, synthetic PI3Kγ inhibitor on eotaxin-induced adhesion, chemotaxis, and degranulation were studied using intracellular adhesion molecule-1 (ICAM-1)-coated plates, Boyden chamber system, ELISA for eosinophil-derived neurotoxin (EDN) levels in the culture supernatant, respectively. CCR3 expression levels and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation were assessed by flowcytometry. Involvement of PI3Kγ in spontaneous apoptosis was studied using flowcytometry. ResultsAlthough AS605240 did not affect the eosinophil spontaneous apoptosis, eotaxin-induced chemotaxis, adhesion to ICAM-1 coated plate, and EDN release were inhibited by AS605240. AS605240 also inhibited the eotaxin-induced ERK1/2 phosphorylation without down-regulation of surface CCR3 expression. ConclusionThese results indicate that PI3Kγ inhibitor attenuates eotaxin-induced eosinophil functions by suppressing the downstream signaling of CCR3 without significant cytotoxicity. PI3Kγ plays an important role in the development of eosinophilic inflammation and blockade of PI3Kγ might be a therapeutic strategy for treatment of eosinophil-related diseases including asthma.

Estrogen modulates the influence of cardiac inflammatory cells on function of cardiac fibroblasts

BackgroundInflammatory cells play a major role in the pathology of heart failure by stimulating cardiac fibroblasts to regulate the extracellular matrix in an adverse way. In view of the fact that inflammatory cells have estrogen receptors, we hypothesized that estrogen provides cardioprotection by decreasing the ability of cardiac inflammatory cells to influence fibroblast function.MethodsMale rats were assigned to either an untreated or estrogen-treated group. In the treated group, estrogen was delivered for 2 weeks via a subcutaneous implanted pellet containing 17β-estradiol. A mixed population of cardiac inflammatory cells, including T-lymphocytes (about 70%), macrophages (about 12%), and mast cells (about 12%), was isolated from each rat and cultured in a Boyden chamber with cardiac fibroblasts from untreated adult male rats for 24 hours. To examine if tumor necrosis factor-alpha (TNF-α) produced by inflammatory cells represents a mechanism contributing to the stimulatory effects of inflammatory cells on cardiac fibroblasts, inflammatory cells from the untreated group were incubated with cardiac fibroblasts in a Boyden chamber system for 24 hours in the presence of a TNF-α-neutralizing antibody. Cardiac fibroblasts were also incubated with 5 ng/mL of TNF-α for 24 hours. Fibro-blast proliferation, collagen synthesis, matrix metalloproteinase activity, β1 integrin protein levels, and the ability of fibroblasts to contract collagen gels were determined in all groups and statistically compared via one-way analysis of variance.ResultsInflammatory cells from the untreated group resulted in: 1) an increased fibroblast proliferation, collagen production and matrix metalloproteinase activity; and 2) a loss of β1 integrin protein and a reduced ability to contract collagen gels. In contrast, inflammatory cells from the treated group resulted in: 1) an attenuated fibroblast proliferation; 2) a nonsignificant reduction in collagen production; 3) the prevention of matrix metalloproteinase activation and the loss of β1 integrin by fibroblasts and 4) a preservation of the fibroblasts’ ability to contract collagen gels. The TNF-α neutralizing antibody attenuated or prevented the untreated inflammatory cell-induced fibroblast proliferation, collagen production, matrix metalloproteinase activation and loss of β1 integrin protein as well as preserved fibroblast contractile ability. Incubation with TNF-α yielded changes in the cardiac fibroblast parameters that were directionally similar to the results obtained with untreated inflammatory cells.ConclusionThese results and those of our previous in vivo studies suggest that a major mechanism by which estrogen provides cardioprotection is its ability to modulate synthesis of TNF-α by inflammatory cells, thereby preventing inflammatory cell induction of cardiac fibroblast events that contribute to adverse extracellular matrix remodeling.

Adiponectin attenuates human eosinophil adhesion and chemotaxis: implications in allergic inflammation

Objective: Growing evidence has shown an association between obesity and asthma. Adiponectin, an adipocyte-derived cytokine, is known to have anti-inflammatory effects with reduced concentrations in obese subjects. Recent findings raised the intriguing possibility that adiponectin might play a role in allergic inflammation, although the mechanistic basis for their relationship remains unclear. The purpose of this study was to examine whether adiponectin might affect functions of eosinophils, which play an important role in the pathogenesis of asthma. Methods: Human peripheral blood eosinophils were purified to study expression of adiponectin receptors AdipoR1 and AdipoR2 using RT-PCR and flow cytometry. The effect of adiponectin on eosinophil survival was investigated using annexin V and propidium iodide staining. Eotaxin-induced cell adhesion was investigated using ICAM-1-coated plates. A Boyden chamber and real-time horizontal migration system were used for eotaxin-directed chemotaxis assay. Expression of eotaxin receptor CCR3 and intracellular calcium influx were assessed by flow cytometry. Results: AdipoR1 and AdipoR2 were expressed in human eosinophils. Adiponectin did not affect eosinophil survival or CCR3 expression; however, eotaxin-enhanced adhesion was inhibited by pretreatment with adiponectin. Adiponectin also diminished eotaxin-directed chemotactic responses by disturbing both velocity and directionality. Calcium influx in response to eotaxin was attenuated by adiponectin. Conclusions: These results indicate that adiponectin attenuates the eosinophil functions induced by eotaxin without affecting cell viability. The inhibitory effect was associated with diminished calcium signaling rather than altering of surface receptor expression. Increasing circulating adiponectin might be a novel therapeutic modality for treatment of asthma, especially in obese asthmatics.

THU0084 C5A and C5AR are Elevated in Joints of Rheumatoid and Psoriatic Arthritis Patients, and C5AR Blockade Attenuates Leukocyte Activation and Migration

BackgroundInvolvement of the complement system in the pathogenesis of rheumatoid arthritis (RA) has been proposed, as complement activation correlate with disease activity1. More specifically, C5a levels were elevated in synovial...

A naturally occurring carotenoid, lutein, reduces PDGF and H2O2 signaling and compromises migration in cultured vascular smooth muscle cells

BackgroundPlatelet-derived growth factor (PDGF) is a potent stimulator of growth and motility of vascular smooth muscle cells (VSMCs). Abnormalities of PDGF/PDGF receptor (PDGFR) are thought to contribute to vascular diseases and malignancy. We previously showed that a carotenoid, lycopene, can directly bind to PDGF and affect its related functions in VSMCs. In this study we examined the effect of the other naturally occurring carotenoid, lutein, on PDGF signaling and migration in VSMCs.MethodsWestern blotting was performed to examine PDGF and H2O2 signaling. Flowcytometry was used to determine PDGF binding to VSMCs. Fluorescence microscopy was performed to examine intracellular ROS production. Modified Boyden chamber system (Transwell apparatus) was used for migration assay.ResultsLutein reduced PDGF signaling, including phosphorylation of PDGFR-β and its downstream protein kinases/enzymes such as phospholipase C-γ, Akt, and mitogen-activated protein kinases (MAPKs). Although lutein possesses a similar structure to lycopene, it was striking that lutein inhibited PDGF signaling through a different way from lycopene in VSMCs. Unlike lycopene, lutein not only interacted with (bound to) PDGF but also interfered with cellular components. This was evidenced that preincubation of PDGF with lutein and treatment of VSMCs with lutein followed by removing of lutein compromised PDGF-induced signaling. Lutein reduced PDGF-induced intracellular reactive oxygen species (ROS) production and attenuated ROS- (H2O2-) induced ERK1/2 and p38 MAPK activation. A further analysis indicated lutein could inhibit a higher concentration of H2O2-induced PDGFR signaling, which is known to act through an oxidative inhibition of protein tyrosine phosphatase. Finally, we showed that lutein functionally inhibited PDGF-induced VSMC migration, whereas its stereo-isomer zeaxanthin did not, revealing a special action of lutein on VSMCs.ConclusionsOur study reveals a differential action mechanism of lutein from other reported caroteinoids and suggests a possible beneficial effect of lutein but not zeaxanthin on prevention of vascular diseases.