scholarly journals Harnessing the Cross-talk between Tumor Cells and Tumor-associated Macrophages with a Nano-drug for modulation of Glioblastoma Immune Microenvironment

2017 ◽  
Author(s):  
Tong-Fei Li ◽  
Ke Li ◽  
Chao Wang ◽  
Xin Liu ◽  
Yu Wen ◽  
...  
Keyword(s):  
Cross Talk ◽  
Culture Medium ◽  
Enzyme Linked Immunosorbent Assay ◽  
Immune Microenvironment ◽  
Potent Inducer ◽  
The Cross ◽  
Molecular Patterns ◽  
Damage Associated Molecular Patterns

AbstractGlioblastoma (GBM) is the most frequent and malignant brain tumor with a high mortality rate. The presence of a large population of macrophages (Mφ) in the tumor microenvironment is a prominent feature of GBM and these so-called tumor-associated Mφ (TAM) closely interact with the GBM cells to promote the survival, progression and therapy resistance of the GBM. Various therapeutic strategies have been devised either targeting the GBM cells or the TAM but few have addressed the cross-talks between the two cell populations. The present study was carried out to explore the possibility of exploiting the cross-talks between the GBM cells (GC) and TAM for modulation of the GBM microenvironment through using Nano-DOX, a drug composite based on nanodiamonds bearing doxorubicin. In the in vitro work on human cell models, Nano-DOX-loaded TAM were first shown to be viable and able to infiltrate three-dimensional GC spheroids and release cargo drug therein. GC were then demonstrated to encourage Nano-DOX-loaded TAM to unload Nano-DOX back into GC which consequently emitted damage-associated molecular patterns (DAMPs) that are powerful immunostimulatory agents as well as indicators of cell damage. Nano-DOX was next proven to be a more potent inducer of GC DAMPs emission than doxorubicin. As a result, Nano-DOX-damaged GC exhibited an enhanced ability to attract both TAM and Nano-DOX-loaded TAM. Most remarkably, Nano-DOX-damaged GC reprogrammed the TAM from a pro-GBM phenotype to an anti-GBM phenotype that suppressed GC growth. Finally, the in vivo relevance of the in vitro findings was tested in animal study. Mice bearing orthotopic human GBM xenografts were intravenously injected with Nano-DOX-loaded mouse TAM which were found releasing drug in the GBM xenografts 24 h after injection. GC damage was evidenced by the induction of DAMPs emission within the xenografts and a shift of TAM phenotype was detected as well. Taken together, our results demonstrate a novel way with therapeutic potential to harness the cross-talk between GBM cells and TAM for modulation of the tumor immune microenvironment.AbbreviationsATP, adenosine triphosphate; BBB, blood-brain barrier; BCA, bicinchoninic acid; BMDM, bone marrow derived macrophages; CD, cluster of differentiation; CFSE, 5(6)-carboxyfluorescein diacetate, succinimidyl ester; CM, conditioned culture medium; CNS, central nervous system; CRT, calreticulin; DAMPs, damage-associated molecular patterns; DAB, diaminobenzidine; DOX, doxorubicin; ECL, enhanced chemiluminescence; ELISA, enzyme-linked immunosorbent assay; HMGB1, high mobility group protein B1; HSP90, heat shock protein 90; FACS, flow cytometry; GBM, glioblastoma; Guanylate Binding Protein 5 (GBP5); GC, glioblastoma cells; IHC, immunohistochemical; IL, interleukin; Mφ, macrophages; mBMDM, mouse BMDM; mBMDM2, Type-2 mBMDM; M1, Type-1 Mø; M2, Type-2 Mø; Nano-DOX, ND-PG-RGD-DOX; ND, nanodiamonds; Nano-DOX-mBMDM, Nano-DOX-loaded mouse BMDM; NGCM, Nano-DOX-treated-GC-conditioned medium; PBS, phosphate buffered saline; PG, polyglycerol; PMA, phorbol 12-myristate 13-acetate; PVDF, polyvinylidene fluoride; RGD, tripeptide of L-arginine, glycine and L-aspartic acid; RM, regular culture medium; SD, standard deviation; TAM, tumor-associated Mφ; TBST, Tris Buffered Saline with Tween® 20.Graphic abstract

scholarly journals Avidity, Potency, and Cross-Reactivity of Monoclonal Antibodies to Pneumococcal Capsular Polysaccharide Serotype 6B

2001 ◽  
Vol 69 (1) ◽  
pp. 336-344 ◽  
Author(s):  
Yan Sun ◽  
Young-il Hwang ◽  
Moon H. Nahm
Keyword(s):  
Monoclonal Antibodies ◽  
Capsular Polysaccharide ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antigen Binding ◽  
Strongly Correlated ◽  
Pneumococcal Infections ◽  
The Cross ◽  
Binding Titer

ABSTRACT Many pneumococcal capsular polysaccharides (PSs) are similar in structure, and a pneumococcal antibody often binds to all of the PSs with a similar structure. Yet, these cross-reactive antibodies may bind to the structurally related pneumococcal capsular PSs with an avidity too low to be effective. If memory B cells producing such weakly cross-reactive antibodies are elicited with pneumococcal conjugate vaccines, the memory cells for low-avidity antibodies could compromise the subsequent immune responses to the cross-reactive PS (original antigenic sin). To investigate these issues, we produced 14 hybridomas secreting monoclonal antibodies (MAbs) to the capsular PS ofStreptococcus pneumoniae serotype 6B by immunizing BALB/c mice with antigens containing 6B PS and studied their epitope, avidity, in vitro opsonizing capacity, in vivo protective capacity, and “antigen binding titer” by enzyme-linked immunosorbent assay (ELISA) of 6A and 6B capsular PSs. Six MAbs bound to the non-cross-reactive 6B-specific epitope, and seven MAbs bound to the cross-reactive epitope present in both 6A and 6B PSs One MAb (Hyp6BM6) revealed a novel epitope. This epitope was found on 6A PS in solution, but not on 6A PS adsorbed onto the plastic surface of the ELISA plates. The avidity of the MAb for 6A or 6B PS ranged from 7.8 × 106 M−1 to 4.1 × 1011M−1. No MAbs were weakly cross-reactive, since none of the cross-reactive MAbs showed any tendency toward having less avidity to 6A PS (the cross-reactive PS) than to 6B PS. Avidity influenced the results of several antibody assays. When all of the hybridomas were examined, avidity strongly correlated with the titer of a unit amount of MAb to bind antigen-coated ELISA plates (r = 0.91) or to opsonize pneumococci in vitro (r = −0.85). Because both assay results are avidity dependent, the ELISA and the opsonization assay results were strongly correlated (r= 0.91), regardless of avidity. Avidity also correlated with the potency of a MAb to passively protect mice against pneumococcal infections. When only the immunoglobulin G hybridomas were examined, little increase in opsonizing capacity and in vivo protective potency was observed above 109 M−1. Taken together, an ELISA measuring antigen binding titer may be an adequate measure of the protective immunity induced with pneumococcal vaccines, and the absence of a partially cross-reactive MAb suggests that antigenic sin may not be significant in responses to vaccines against the S. pneumoniae 6B serotype.

scholarly journals KIOM-79, an Inhibitor of AGEs–Protein Cross-linking, Prevents Progression of Nephropathy in Zucker Diabetic Fatty Rats

2011 ◽  
Vol 2011 ◽  
pp. 1-10 ◽  
Author(s):  
Young Sook Kim ◽  
Junghyun Kim ◽  
Chan-Sik Kim ◽  
Eun Jin Sohn ◽  
Yun Mi Lee ◽  
...  
Keyword(s):  
Blood Glucose ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cross Linking ◽  
Kidney Weight ◽  
Zucker Diabetic Fatty Rats ◽  
Protein Cross Linking

Advanced glycation end products (AGEs) have been implicated in the development of diabetic complications, including diabetic nephropathy. KIOM-79, an 80% ethanolic extract obtained from parched Puerariae Radix, gingered Magnolia Cortex, Glycyrrhiza Radix and Euphorbia Radix, was investigated for its effects on the development of renal disease in Zucker diabetic fatty rats, an animal model of type 2 diabetes.In vitroinhibitory effect of KIOM-79 on AGEs cross-linking was examined by enzyme-linked immunosorbent assay (ELISA). KIOM-79 (50 mg/kg/day) was given to Zucker diabetic fatty rats for 13 weeks. Body and kidney weight, blood glucose, glycated hemoglobin, urinary albumin and creatinine excretions were monitored. Kidney histopathology, collagen accumulation, fibrinogen and transforming growth factor-beta 1 (TGF-β1) expression were also examined. KIOM-79 reduced blood glucose, kidney weight, histologic renal damage and albuminuria in Zucker diabetic fatty rats. KIOM-79 prevented glomerulosclerosis, tubular degeneration, collagen deposition and podocyte apoptosis. In the renal cortex, TGF-β1, fibronectin mRNA and protein were significantly reduced by KIOM-79 treatment. KIOM-79 reduces AGEs accumulationin vivo, AGE–protein cross-linking and protein oxidation. KIOM-79 could be beneficial in preventing the progression of diabetic glomerularsclerosis in type 2 diabetic rats by attenuating AGEs deposition in the glomeruli.

scholarly journals Effects of Various Levels of Luteinizing Hormone and Caprine Follicular Fluid on In Vitro Embryo Production of Shami Goat

2021 ◽  
Vol 5 (3) ◽  
pp. 575-584
Author(s):  
Omar Mardenli ◽  
Hadi Awad Hassooni ◽  
Mahdi Saleh Mohammad Al-Kerwi
Keyword(s):  
Luteinizing Hormone ◽  
Follicular Fluid ◽  
Culture Medium ◽  
Goat Breed ◽  
Embryo Production ◽  
Follicle Size ◽  
In Vitro Embryo Production ◽  
Type 3

In the current study, the hypothesis of the effects of luteinizing hormone (LH) and follicular fluid (FF) derived from follicles of varying size on in vitro embryo production of the Shami goat breed were tested. The caprine follicular fluid (cFF) was obtained from healthy female’s ovaries by aspiration method and classified into two main classes (follicles with a diameter of ≤ 2mm and ≥3mm). The resulting cFF was added to the culture medium TCM-199 through six Treatments (A, B and C with a source of follicle size of ≤ 2mm; D, E and F with a source of size of ≥3mm). LH was added only to four of the previous Treatments with the levels of 50 µg ml-1 (B and E) and100 µg ml-1 (C and F). Results of the study showed that the oocytes incubated in Treatment F achieved a clear superiority (p=0.001) in the rates of maturation (87.0%), fertilization (80.0%) and cleavage (82.3%). The oocytes incubated in the same Treatment (F) continued to outperform (p= 0.006) by achieving the best rates across cleavage stages at 2-16 cell (16%; the lower value of arrest) and blastocyst (42%). Significant differences (P=0.03) were observed among the rates of Type 1embryos (the highest rate: 45.3%; Treatment F) and Type 3 embryos (the highest rate: 45.1%; Treatment A). No significant differences were observed in the rates of morula and Type 2 embryos. It is advised to add 15% of the cFF derived from follicles with a diameter of ≥3mm and 100 µg of LH ml-1 in the maturation media to obtain higher rates of maturation and cleavage of goat oocytes.  

scholarly journals Effect of Nanostructured Scaffold on Human Adipose-Derived Stem Cells: Outcome of In Vitro Experiments

Nanomaterials ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 1822 ◽  
Author(s):  
Marina Borgese ◽  
Ludovica Barone ◽  
Federica Rossi ◽  
Mario Raspanti ◽  
Roberto Papait ◽  
...  
Keyword(s):  
Stem Cells ◽  
Culture Medium ◽  
Quantitative Polymerase Chain Reaction ◽  
Enzyme Linked Immunosorbent Assay ◽  
Adipose Derived Stem Cells ◽  
In Vitro Experiments ◽  
Fatty Acid Binding ◽  
Beneficial Effects ◽  
Angiogenesis Process

This work is addressed to provide, by in vitro experiments, results on the repercussion that a nanostructured scaffold could have on viability, differentiation and secretion of bioactive factors of human adipose-derived stem cells (hASCs) when used in association to promote angiogenesis, a crucial condition to favour tissue regeneration. To achieve this aim, we evaluated cell viability and morphology by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay and microscopy analysis, respectively. We also investigated the expression of some of those genes involved in angiogenesis and differentiation processes utilizing quantitative polymerase chain reaction (qPCR), whereas the amounts of Vascular Endothelial Growth Factor A, Interleukin 6 and Fatty Acid-Binding Protein 4 secreted in the culture medium, were quantified by enzyme-linked immunosorbent assay (ELISA). Results suggested that, in the presence of the scaffold, cell proliferation and the exocytosis of factors involved in the angiogenesis process are reduced; by contrast, the expression of those genes involved in hASC differentiation appeared enhanced. To guarantee cell survival, the construct dimensions are, generally, smaller than clinically required. Furthermore, being the paracrine event the primary mechanism exerting the beneficial effects on injured tissues, the use of conditioned culture medium instead of cells may be convenient.

scholarly journals Apolipoprotein-A1 as a Damage-Associated Molecular Patterns Protein in Osteoarthritis: Ex Vivo and In Vitro Pro-Inflammatory Properties

PLoS ONE ◽  
2015 ◽  
Vol 10 (4) ◽  
pp. e0122904 ◽  
Author(s):  
Dominique de Seny ◽  
Gaël Cobraiville ◽  
Edith Charlier ◽  
Sophie Neuville ◽  
Laurence Lutteri ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Apolipoprotein A1 ◽  
Molecular Patterns ◽  
Damage Associated Molecular Patterns

scholarly journals DAMPs Synergize with Cytokines or Fibronectin Fragment on Inducing Chondrolysis but Lose Effect When Acting Alone

2017 ◽  
Vol 2017 ◽  
pp. 1-12 ◽  
Author(s):  
Lei Ding ◽  
Joseph A. Buckwalter ◽  
James A. Martin
Keyword(s):  
Inflammatory Response ◽  
Synergistic Effect ◽  
Culture Medium ◽  
Articular Chondrocytes ◽  
Weight Bearing ◽  
Primary Cultures ◽  
Cartilage Degradation ◽  
Fibronectin Fragment ◽  
Molecular Patterns ◽  
Damage Associated Molecular Patterns

Objective and Design. To investigate whether endogenous damage-associated molecular patterns (DAMPs) or alarmins originated from mitochondria or nucleus stimulates inflammatory response in articular chondrocytes to cause chondrolysis which leads to cartilage degradation featured in posttraumatic osteoarthritis (PTOA).Materials. Primary cultures of bovine or human chondrocytes isolated from cartilage of weight-bearing joints.Treatment. Chondrocytes were subjected to mitochondrial DAMPs (MTDs) or HMGB1, a nuclear DAMP (NuD), with or without the presence of an N-terminal 29 kDa fibronectin fragment (Fn-f) or proinflammatory cytokines (IL-1βand TNF-α). Injured cartilage-conditioned culturing medium containing a mixture of DAMPs was employed as a control. After 24 hrs, the protein expression of cartilage degrading metalloproteinases and iNOS in culture medium or cell lysates was examined with Western blotting, respectively.Results. HMGB1 was synergized with IL-1βin upregulating expression of MMP-3, MMP-13, ADAMTS-5, ADAM-8, and iNOS. Moreover, a moderate synergistic effect was detected between HMGB1 and Fn-f or between MTDs and TNF-αon MMP-3 expression. However, when acting alone, MTDs or HMGB1 did not upregulate cartilage degrading enzymes or iNOS.Conclusion. MTDs or HMGB1 could only stimulate inflammatory response in chondrocytes with the presence of cytokines or Fn-f.

scholarly journals Tissue damage drives co-localization of NF-κB, Smad3, and Nrf2 to direct Rev-erb sensitive wound repair in mouse macrophages

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Dawn Z Eichenfield ◽  
Ty Dale Troutman ◽  
Verena M Link ◽  
Michael T Lam ◽  
Han Cho ◽  
...  
Keyword(s):  
Nuclear Receptors ◽  
Tissue Damage ◽  
Wound Repair ◽  
Macrophage Polarization ◽  
Expression Of Genes ◽  
Mouse Macrophages ◽  
Molecular Patterns ◽  
Damage Associated Molecular Patterns

Although macrophages can be polarized to distinct phenotypes in vitro with individual ligands, in vivo they encounter multiple signals that control their varied functions in homeostasis, immunity, and disease. Here, we identify roles of Rev-erb nuclear receptors in regulating responses of mouse macrophages to complex tissue damage signals and wound repair. Rather than reinforcing a specific program of macrophage polarization, Rev-erbs repress subsets of genes that are activated by TLR ligands, IL4, TGFβ, and damage-associated molecular patterns (DAMPS). Unexpectedly, a complex damage signal promotes co-localization of NF-κB, Smad3, and Nrf2 at Rev-erb-sensitive enhancers and drives expression of genes characteristic of multiple polarization states in the same cells. Rev-erb-sensitive enhancers thereby integrate multiple damage-activated signaling pathways to promote a wound repair phenotype.

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