scholarly journals Monocytes from circulating blood fuse in vitro with purified osteoclasts in primary culture

1984 ◽  
Vol 66 (1) ◽  
pp. 335-342
Author(s):  
A. Zambonin Zallone ◽  
A. Teti ◽  
M.V. Primavera
Keyword(s):  
Primary Culture ◽  
Phase Contrast ◽  
Laying Hens ◽  
Time Lapse ◽  
Mononuclear Phagocytes ◽  
Low Density ◽  
Medullary Bone ◽  
Membrane Contacts

The origin of osteoclasts from mononuclear phagocytes and the addition of new nuclei to already differentiated osteoclasts have already been documented by several authors, but the factors controlling these events have not yet been elucidated. With the aim of investigating this problem, monocytes, isolated from circulating blood of laying hens and cultured previously for 5 days, were added to osteoclasts isolated from the medullary bone of the same hen and cultured at low density. The cultures were either fixed and observed under phase contrast at 24-h intervals for 5 days or filmed by time-lapse cinemicrography. With both techniques the formation of extensive areas of membrane contacts, generally followed by fusion between some monocytes and osteoclasts, was observed. The absence of added resorbing factors and the possible mechanisms by which osteoclast precursors are recruited in vivo are discussed.

Effective targeted antitumor activity of the antimicrobial agent taurolidine against relapsed/refractory neuroblastoma: Cytotoxicity, target modulation and tumor xenograft studies.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e21502-e21502
Author(s):  
Lucy Swift ◽  
Chunfen Zhang ◽  
Antony Pfaffle ◽  
Paul Chew ◽  
Olga Kovalchuk ◽  
...  
Keyword(s):  
Gene Expression ◽  
Clinical Trial ◽  
Early Phase ◽  
Phase Contrast ◽  
Time Lapse ◽  
Early Phase Clinical Trial ◽  
Phase Clinical Trial ◽  
Time Lapse Video

e21502 Background: Neuroblastoma (NB) is the most common extracranial solid tumor and one of the most complex and difficult to treat diseases in pediatrics. Currently, even with highly aggressive treatment protocols, the prognosis for patients with high-risk and relapsed NB remains poor. Hence, there is a clear need to identify new agents and novel therapeutic strategies for the treatment of these children. Taurolidine (TRD) is derived from the aminosulfoacid taurine and has known anti-microbial and anti-inflammatory properties. TRD has demonstrated anti-neoplastic activity against a range of aggressive human tumors. We present mechanistic evidence and supportive preclinical data from in vitro and animal models of refractory NB for the development of an early phase clinical trial incorporating TRD. Methods: For in vitro activity studies, a panel of cell lines derived from patients with relapsed NB (n = 6) and normal control cells were treated with increasing concentrations of TRD and cell viability was measured by alamar blue assay. Phase-contrast light microscopy, western blotting, time-lapse video microscopy and analysis of global gene expression by RNA-Seq were used to evaluate target modulation and induction of cell death pathways. Bioluminescence imaging of mice bearing NB xenografts treated with TRD was used to investigate the efficacy of TRD in vivo. Results: Cell survival data showed that TRD is cytotoxic against NB cell lines in vitro (mean IC50 value 100 µM, range 65-135 µM). Phase-contrast and time-lapse video microscopy confirmed the antitumor effects of TRD. Western blot analyses identified that TRD induced target modulation and an effective apoptotic cascade, resulting in PARP cleavage. Gene expression analyses and signaling pathway activation scores indicated alterations in the Notch, MAPK and IL-10 signaling pathways. Xenograft studies further validated the in vivo activity of TRD with decreased tumor burden in treated mice and a measurable improvement in survival. Conclusions: Our study provides key pre-clinical data on the activity and mechanism of action of TRD against NB. The findings support the rationale for further evaluation of TRD for the treatment of relapsed/refractory NB patients in an early phase clinical trial.

scholarly journals THE CIRCUMFUSION SYSTEM FOR MULTIPURPOSE CULTURE CHAMBERS

1968 ◽  
Vol 39 (2) ◽  
pp. 430-450 ◽  
Author(s):  
George G. Rose ◽  
M. Kumegawa ◽  
M. Cattoni
Keyword(s):  
Phase Contrast ◽  
Cultured Cells ◽  
Morphological Characteristics ◽  
Time Lapse ◽  
Newborn Mouse ◽  
Contrast Microscopy ◽  
Free Environment ◽  
Parenchymal Cells

The circumfusion system is a complex in vitro pumping unit incorporating 12 multipurpose culture chambers through which a serum-supplemented fluid nutrient is recirculated at a rate of 4.5 ml/min per chamber. This system was used to study the differentiative responses of fetal and newborn mouse liver explants placed in the serum-free environment formed between the sheets of unperforated cellophane and cover glasses of the chambers. Hepatocytes (parenchymal cells) were discernible in 3–5 days. They retained many of their features of differentiation in the circumfusion system for more than 120 days of cultivation. The living morphological characteristics of the hepatocytes were studied by phase-contrast microscopy (direct viewing and time-lapse cinemicrography) and by special cytochemical staining. Electron micrographs were made of both fresh liver specimens and the cultured cells. Comparisons of the cultured parenchymal cells with their in vivo progenitors showed a remarkable preservation of their differentiated state.

Ultrastructure of melanocyte-keratinocyte interactions and pigment transfer in vitro

1978 ◽  
Vol 36 (2) ◽  
pp. 650-651
Author(s):  
Raul I. Garcia ◽  
Evelyn A. Flynn ◽  
George Szabo
Keyword(s):  
Direct Injection ◽  
Skin Pigmentation ◽  
Freeze Fracture ◽  
Pigment Granule ◽  
Time Lapse ◽  
Ultrastructural Level ◽  
Lanthanum Tracer ◽  
A Cell

Skin pigmentation in mammals involves the interaction of epidermal melanocytes and keratinocytes in the structural and functional unit known as the Epidermal Melanin Unit. Melanocytes(M) synthesize melanin within specialized membrane-bound organelles, the melanosome or pigment granule. These are subsequently transferred by way of M dendrites to keratinocytes(K) by a mechanism still to be clearly defined. Three different, though not necessarily mutually exclusive, mechanisms of melanosome transfer have been proposed: cytophagocytosis by K of M dendrite tips containing melanosomes, direct injection of melanosomes into the K cytoplasm through a cell-to-cell pore or communicating channel formed by localized fusion of M and K cell membranes, release of melanosomes into the extracellular space(ECS) by exocytosis followed by K uptake using conventional phagocytosis. Variability in methods of transfer has been noted both in vivo and in vitro and there is evidence in support of each transfer mechanism. We Have previously studied M-K interactions in vitro using time-lapse cinemicrography and in vivo at the ultrastructural level using lanthanum tracer and freeze-fracture.

scholarly journals Reductions of copper ion-mediated low-density lipoprotein (LDL) oxidations of trypsin inhibitors, the sweet potato root major proteins, and LDL binding capacities

2020 ◽  
Vol 61 (1) ◽  
Author(s):  
Yeh-Lin Lu ◽  
Chia-Jung Lee ◽  
Shyr-Yi Lin ◽  
Wen-Chi Hou
Keyword(s):  
Sweet Potato ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Trypsin Inhibitors ◽  
Water Soluble ◽  
Affinity Column ◽  
Low Density ◽  
Native Page

Abstract Background The root major proteins of sweet potato trypsin inhibitors (SPTIs) or named sporamin, estimated for 60 to 80% water-soluble proteins, exhibited many biological activities. The human low-density lipoprotein (LDL) showed to form in vivo complex with endogenous oxidized alpha-1-antitrypsin. Little is known concerning the interactions between SPTIs and LDL in vitro. Results The thiobarbituric-acid-reactive-substance (TBARS) assays were used to monitor 0.1 mM Cu2+-mediated low-density lipoprotein (LDL) oxidations during 24-h reactions with or without SPTIs additions. The protein stains in native PAGE gels were used to identify the bindings between native or reduced forms of SPTIs or soybean TIs and LDL, or oxidized LDL (oxLDL). It was found that the SPTIs additions showed to reduce LDL oxidations in the first 6-h and then gradually decreased the capacities of anti-LDL oxidations. The protein stains in native PAGE gels showed more intense LDL bands in the presence of SPTIs, and 0.5-h and 1-h reached the highest one. The SPTIs also bound to the oxLDL, and low pH condition (pH 2.0) might break the interactions revealed by HPLC. The LDL or oxLDL adsorbed onto self-prepared SPTIs-affinity column and some components were eluted by 0.2 M KCl (pH 2.0). The native or reduced SPTIs or soybean TIs showed different binding capacities toward LDL and oxLDL in vitro. Conclusion The SPTIs might be useful in developing functional foods as antioxidant and nutrient supplements, and the physiological roles of SPTIs-LDL and SPTIs-oxLDL complex in vivo will investigate further using animal models.

scholarly journals Monocyte tissue factor induction by lipopolysaccharide (LPS): dependence on LPS-binding protein and CD14, and inhibition by a recombinant fragment of bactericidal/permeability-increasing protein

Blood ◽  
1994 ◽  
Vol 83 (9) ◽  
pp. 2516-2525 ◽  
Author(s):  
K Meszaros ◽  
S Aberle ◽  
R Dedrick ◽  
R Machovich ◽  
A Horwitz ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Binding Protein ◽  
Mononuclear Phagocytes ◽  
Factor Vii ◽  
Peripheral Blood Mononuclear ◽  
Recombinant Fragment

Abstract Mononuclear phagocytes, stimulated by bacterial lipopolysaccharide (LPS), have been implicated in the activation of coagulation in sepsis and endotoxemia. In monocytes LPS induces the synthesis of tissue factor (TF) which, assembled with factor VII, initiates the blood coagulation cascades. In this study we investigated the mechanism of LPS recognition by monocytes, and the consequent expression of TF mRNA and TF activity. We also studied the inhibition of these effects of LPS by rBPI23, a 23-kD recombinant fragment of bactericidal/permeability increasing protein, which has been shown to antagonize LPS in vitro and in vivo. Human peripheral blood mononuclear cells, or monocytes isolated by adherence, were stimulated with Escherichia coli O113 LPS at physiologically relevant concentrations (> or = 10 pg/mL). The effect of LPS was dependent on the presence of the serum protein LBP (lipopolysaccharide-binding protein), as shown by the potentiating effect of human recombinant LBP or serum. Furthermore, recognition of low amounts of LPS by monocytes was also dependent on CD14 receptors, because monoclonal antibodies against CD14 greatly reduced the LPS sensitivity of monocytes in the presence of serum or rLBP. Induction of TF activity and mRNA expression by LPS were inhibited by rBPI23. The expression of tumor necrosis factor showed qualitatively similar changes. Considering the involvement of LPS-induced TF in the potentially lethal intravascular coagulation in sepsis, inhibition of TF induction by rBPI23 may be of therapeutic benefit.

scholarly journals Cloning of mouse ptx3, a new member of the pentraxin gene family expressed at extrahepatic sites

Blood ◽  
1996 ◽  
Vol 87 (5) ◽  
pp. 1862-1872 ◽  
Author(s):  
M Introna ◽  
VV Alles ◽  
M Castellano ◽  
G Picardi ◽  
L De Gioia ◽  
...  
Keyword(s):  
Gene Family ◽  
Tertiary Structure ◽  
Helical Structure ◽  
Distinct Pattern ◽  
Mononuclear Phagocytes ◽  
Acute Phase Reactants ◽  
Inflammatory Reactions ◽  
Reactive Protein

Abstract Pentraxins, which include C reactive protein (CRP) and serum amyloid P component (SAP), are prototypic acute phase reactants that serve as indicators of inflammatory reactions. Here we report genomic and cDNA cloning of mouse ptx3 (mptx3), a member of the pentraxin gene family and characterize its extrahepatic expression in vitro and in vivo. mptx3 is organized into three exons on chromosome 3: the first (43 aa) and second exon (175 aa) code for the signal peptide and for a protein portion with no high similarity to known sequences the third (203 aa) for a domain related to classical pentraxins, which contains the “pentraxin family signature.” Analysis of the N terminal portion predicts a predominantly alpha helical structure, while the pentraxin domain of ptx3 is accommodated comfortably in the tertiary structure fold of SAP. Normal and transformed fibroblasts, undifferentiated and differentiated myoblasts, normal endothelial cells, and mononuclear phagocytes express mptx3 mRNA and release the protein in vitro on exposure to interleukin-1beta (IL-1beta) and tumor necrosis factor (TNF)alpha. mptx3 was induced by bacterial lipopolysaccharide in vivo in a variety of organs and, most strongly, in the vascular endothelium of skeletal muscle and heart. Thus, mptx3 shows a distinct pattern of in vivo expression indicative of a significant role in cardiovascular and inflammatory pathology.

scholarly journals Cell-substratum interaction of cultured avian osteoclasts is mediated by specific adhesion structures.

1984 ◽  
Vol 99 (5) ◽  
pp. 1696-1705 ◽  
Author(s):  
P C Marchisio ◽  
D Cirillo ◽  
L Naldini ◽  
M V Primavera ◽  
A Teti ◽  
...  
Keyword(s):  
Electron Microscope ◽  
Intermediate Filaments ◽  
Immunofluorescence Microscopy ◽  
Laying Hens ◽  
Cell Types ◽  
Medullary Bone ◽  
Transformed Cell ◽  
The Core ◽  
Low Calcium Diet

The cell-substratum interaction was studied in cultures of osteoclasts isolated from the medullary bone of laying hens kept on low calcium diet. In fully spread osteoclasts, cell-substratum adhesion mostly occurred within a continuous paramarginal area that corresponded also to the location of a thick network of intermediate filaments of the vimentin type. In this area, regular rows of short protrusions contacting the substratum and often forming a cup-shaped adhesion area were observed in the electron microscope. These short protrusions showed a core of F-actin-containing material presumably organized as a network of microfilaments and surrounded by a rosette-like structure in which vinculin and alpha-actinin were found by immunofluorescence microscopy. Rosettes were superposable to dark circles in interference-reflection microscopy and thus represented circular forms of close cell-substratum contact. The core of ventral protrusions also contained, beside F-actin, fimbrin and alpha-actinin. Villin was absent. This form of cell-substratum contact occurring at the tip of a short ventral protrusion differed from other forms of cell-substratum contact and represented an osteoclast-specific adhesion device that might also be present in in vivo osteoclasts as well as in other normal and transformed cell types.

Tracings of Behavior of Myoblasts Cultured Under Couette Type of Shear Flow Between Parallel Disks

2021 ◽  
Author(s):  
Shigehiro Hashimoto ◽  
Hiroki Yonezawa
Keyword(s):  
Shear Stress ◽  
Shear Flow ◽  
Rotating Disk ◽  
Time Lapse ◽  
Mechanical Stimuli ◽  
Parallel Disks ◽  
Before And After ◽  
A Cell

Abstract A cell deforms and migrates on the scaffold under mechanical stimuli in vivo. In this study, a cell with division during shear stress stimulation has been observed in vitro. Before and after division, both migration and deformation of each cell were analyzed. To make a Couette-type shear flow, the medium was sandwiched between parallel disks (the lower stationary culture-disc and the upper rotating disk) with a constant gap. The wall shear stress (1.5 Pa < τ < 2 Pa) on the surface of the lower culture plate was controlled by the rotational speed of the upper disc. Myoblasts (C2C12: mouse myoblast cell line) were used in the test. After cultivation without flow for 24 hours for adhesion of the cells to the lower disk, constant τ was applied to the cells in the incubator for 7 days. The behavior of each cell during shear was tracked by time-lapse images observed by an inverted phase contrast microscope placed in the incubator. Experimental results show that each cell tends to divide after higher activities: deformation and migration. The tendency is remarkable at the shear stress of 1.5 Pa.

scholarly journals Two Isoforms of Npap60 (Nup50) Differentially Regulate Nuclear Protein Import

2010 ◽  
Vol 21 (4) ◽  
pp. 630-638 ◽  
Author(s):  
Yutaka Ogawa ◽  
Yoichi Miyamoto ◽  
Munehiro Asally ◽  
Masahiro Oka ◽  
Yoshinari Yasuda ◽  
...  
Keyword(s):  
Nuclear Import ◽  
Protein Import ◽  
Nuclear Protein ◽  
Time Lapse ◽  
Binding Assays ◽  
Vitro Binding ◽  
Importin Α ◽  
Nuclear Protein Import

Npap60 (Nup50) is a nucleoporin that binds directly to importin α. In humans, there are two Npap60 isoforms: the long (Npap60L) and short (Npap60S) forms. In this study, we provide both in vitro and in vivo evidence that Npap60L and Npap60S function differently in nuclear protein import. In vitro binding assays revealed that Npap60S stabilizes the binding of importin α to classical NLS-cargo, whereas Npap60L promotes the release of NLS-cargo from importin α. In vivo time-lapse experiments showed that when the Npap60 protein level is controlled, allowing CAS to efficiently promote the dissociation of the Npap60/importin α complex, Npap60S and Npap60L suppress and accelerate the nuclear import of NLS-cargo, respectively. These results demonstrate that Npap60L and Npap60S have opposing functions and suggest that Npap60L and Npap60S levels must be carefully controlled for efficient nuclear import of classical NLS-cargo in humans. This study provides novel evidence that nucleoporin expression levels regulate nuclear import efficiency.

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