scholarly journals Ultrastructural analysis of murine megakaryocyte maturation in vitro: comparison of big-cell and heterogeneous megakaryocyte colonies

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1509-1518
Author(s):  
PE Stenberg ◽  
J Levin
Keyword(s):  
Bone Marrow ◽  
Soft Agar ◽  
Small Cells ◽  
Specific Marker ◽  
Mouse Bone Marrow ◽  
Structural Level ◽  
Agar Culture ◽  
Big Cell ◽  
Mature Bone

Two morphologically distinct types of murine megakaryocyte (MK) colonies are present after three to seven days in soft agar culture: (a) “big-cell” colonies composed of ten to 30 large, mature-appearing megakaryocytes and (b) “heterogeneous” colonies consisting of approximately 100 or more cells at various stages of differentiation. Cytochemical and immunocytochemical techniques were used to study MK maturation in colonies as well as normal mouse bone marrow. Acetylcholinesterase (AChE), a specific marker for murine platelets and MK, was found in the perinuclear cisterna, endoplasmic reticulum, and occasionally, Golgi cisternae of MK in three-day big-cell colonies and immature bone marrow MK. MK in seven-day big-cell colonies and mature bone marrow MK showed additional reaction sites in the demarcation membrane system and occasional granules. In seven-day heterogeneous colonies, small cells resembled immature bone marrow MK with respect to AChE localization, whereas large cells corresponded to mature bone marrow MK. With immunogold procedures at the ultrastructural level, polyclonal antibodies against human platelet membrane glycoprotein IIIa and antimouse platelet antiserum labeled bone marrow MK and all MK from colonies grown in soft agar cultures for three to seven days. Granulocytes and macrophages in both bone marrow and soft agar cultures were negative for AChE and these immunocytochemical markers. These data indicate that the pattern of expression of AChE during maturation of MK is similar in vivo and in vitro and demonstrate, when using this marker at the fine-structural level, that a greater range of MK maturational stages is present in heterogeneous colonies than is observed in MK in big-cell colonies. Furthermore, we have confirmed that small cells in heterogeneous colonies are MK and that these colonies are composed solely of MK and their precursors.

scholarly journals Ultrastructural analysis of murine megakaryocyte maturation in vitro: comparison of big-cell and heterogeneous megakaryocyte colonies

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1509-1518 ◽  
Author(s):  
PE Stenberg ◽  
J Levin
Keyword(s):  
Bone Marrow ◽  
Soft Agar ◽  
Small Cells ◽  
Specific Marker ◽  
Mouse Bone Marrow ◽  
Structural Level ◽  
Agar Culture ◽  
Big Cell ◽  
Mature Bone

Abstract Two morphologically distinct types of murine megakaryocyte (MK) colonies are present after three to seven days in soft agar culture: (a) “big-cell” colonies composed of ten to 30 large, mature-appearing megakaryocytes and (b) “heterogeneous” colonies consisting of approximately 100 or more cells at various stages of differentiation. Cytochemical and immunocytochemical techniques were used to study MK maturation in colonies as well as normal mouse bone marrow. Acetylcholinesterase (AChE), a specific marker for murine platelets and MK, was found in the perinuclear cisterna, endoplasmic reticulum, and occasionally, Golgi cisternae of MK in three-day big-cell colonies and immature bone marrow MK. MK in seven-day big-cell colonies and mature bone marrow MK showed additional reaction sites in the demarcation membrane system and occasional granules. In seven-day heterogeneous colonies, small cells resembled immature bone marrow MK with respect to AChE localization, whereas large cells corresponded to mature bone marrow MK. With immunogold procedures at the ultrastructural level, polyclonal antibodies against human platelet membrane glycoprotein IIIa and antimouse platelet antiserum labeled bone marrow MK and all MK from colonies grown in soft agar cultures for three to seven days. Granulocytes and macrophages in both bone marrow and soft agar cultures were negative for AChE and these immunocytochemical markers. These data indicate that the pattern of expression of AChE during maturation of MK is similar in vivo and in vitro and demonstrate, when using this marker at the fine-structural level, that a greater range of MK maturational stages is present in heterogeneous colonies than is observed in MK in big-cell colonies. Furthermore, we have confirmed that small cells in heterogeneous colonies are MK and that these colonies are composed solely of MK and their precursors.

scholarly journals Effect of virulent and vaccine variants of influenza virus on the immunophenotype of dendritic cells generated from murine bone marrow

2020 ◽  
Vol 22 (3) ◽  
pp. 473-482
Author(s):  
N. K. Akhmatova ◽  
A. A. Rtishchev ◽  
S. G. Markushin ◽  
A. M. Kostinova ◽  
E. A. Akhmatova ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dendritic Cells ◽  
Influenza Virus ◽  
Morphological Characteristics ◽  
Influenza Viruses ◽  
Interleukin 4 ◽  
Specific Marker ◽  
Mouse Bone Marrow ◽  
Toll Like Receptors

The aim of this study was to generate dendritic cells from the bone marrow of mice (DC) in vitro and to assess the effect of virulent and attenuated variants of influenza virus on the maturation of DCs. Granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) were used in combination, to induce differentiation of mouse bone marrow (BM) mononucleocytes into DCs. On the 5th day, distinct variants of influenza virus were added to the cell culture, and the cells were additionally incubated for 2 days. The morphological characteristics of DCs, immunophenotype of DCs and expression of some Toll-like receptors were evaluated. On the 5th day of incubation. the DCs acquired typical morphological characteristics. DCs were large in size with an eccentrically located nucleous, often irregular in shape, with numerous processes. On the 7th day of incubation with influenza virus variants, their cytoplasm was somewhat denser. DCs acquired more processes, necessary for intercellular contacts. Expression levels of CD11c, a specific marker of BM-derived DCs, and of co-stimulatory molecules such as CD40, CD80, CD86, and MHC-II were elevated in mature DCs. Virulent versus attenuated strains of the influenza virus induced special variants of DCs differentiation, with respect to expression rates of differentiation markers, as well as expression of Toll-like receptors and costimulatory molecules. Conclusions. The in vitro cultured murine mononucleocytes derived from bone marrow can produce a large number of n-DCs, that can mature in the presence of different variants.During evolution of the DC immunophenotype treated with variant influenza viruses, we have found distinct signs of immunosuppression.The attenuated U-2 and M-26 influenza variants obtained by site-specific mutagenesis upon development of DCs immunophenotype, exhibited a decreased immunosuppressive activity and were not inferior to the cold-adapted (CA) reassortant for the most positions, but exceeded it in some instances. These studies can help to assess the criteria for evaluation the efficiency of in vitro developed influenza vaccines.

scholarly journals The effect of prostaglandins E1 and E2 on macrophage progenitor cells with high proliferative potential in mouse bone marrow in vitro

Blood ◽  
1984 ◽  
Vol 63 (6) ◽  
pp. 1348-1352
Author(s):  
AB Kriegler ◽  
TR Bradley ◽  
GS Hodgson
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Proliferative Potential ◽  
Mouse Bone Marrow ◽  
Synergistic Activity ◽  
Agar Culture ◽  
Inhibition Of Proliferation ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony

High proliferative potential macrophage progenitor cells (HPP-CFC) in 5- fluorouracil (FU) treated and normal mouse bone marrow (BM) have been shown to be less sensitive to inhibition of proliferation by prostaglandins of the E series (PGE) than low proliferative potential macrophage progenitor cells (LPP-CFC) in normal BM in agar cultures. The growth of large colonies (diameter greater than 0.5 mm) derived from HPP-CFC in FU BM, which require a combination of macrophage colony- stimulating factor (CSF-1) plus a new growth factor called synergistic activity (SA), are inhibited by 50% in the presence of 5.5 X 10(-6) M PGE1. On the other hand, LPP-CFC in normal BM, which form smaller colonies (diameter less than or equal to 0.5 mm) in the presence of CSF- 1 alone, require only 5 X 10(-8) M PGE1 for the same level of inhibition. Addition of appropriate concentrations of PGE1 to the agar culture assay should improve detection of HPP-CFC by inhibiting the proliferation of LPP-CFC. These observations suggest that the apparent negative feedback control of macrophage production by PGE operates largely on the LPP-CFC, which respond to CSF-1 alone, and is probably not involved in the regulation of the more primitive HPP-CFC.

scholarly journals Immunohistochemical localization of membrane and alpha-granule proteins in plastic-embedded mouse bone marrow megakaryocytes and murine megakaryocyte colonies

Blood ◽  
1986 ◽  
Vol 68 (3) ◽  
pp. 696-702 ◽  
Author(s):  
PE Stenberg ◽  
JH Beckstead ◽  
RP McEver ◽  
J Levin
Keyword(s):  
Bone Marrow ◽  
Human Platelet ◽  
Soft Agar ◽  
Platelet Membrane ◽  
Mouse Bone Marrow ◽  
Immunoperoxidase Technique ◽  
Membrane Glycoproteins ◽  
Agar Culture ◽  
Light Microscope Level ◽  
Murine Bone Marrow

Abstract Using an immunoperoxidase technique that permits optimal antigen localization at the light microscope level, we have detected two platelet alpha-granule constituents and three platelet membrane glycoproteins in mouse bone marrow megakaryocytes and in murine megakaryocyte colonies grown in soft agar culture for three to seven days. Using polyclonal antibodies prepared against human platelet proteins, we have demonstrated labeling for von Willebrand factor, fibrinogen, and the membrane glycoproteins IIIa and GMP-140 in both bone marrow megakaryocytes and megakaryocyte colonies after seven days of culture. Using monoclonal antibodies to membrane glycoproteins IIb and GMP-140, we have demonstrated label in mouse bone marrow megakaryocytes. Granulocyte and macrophage colonies were negative for each of these markers. Murine bone marrow megakaryocytes and megakaryocyte colonies demonstrated a similar enzyme histochemical pattern: weakly positive for alpha-naphthyl acetate esterase and negative for chloroacetate esterase. These data indicate that megakaryocytes grown in soft agar culture express many of the same glycoproteins as bone marrow megakaryocytes. Furthermore, the ability of antibodies directed against human platelet membrane glycoproteins to identify murine megakaryocyte glycoproteins indicates that these constituents have been highly conserved during evolution.

scholarly journals The effect of prostaglandins E1 and E2 on macrophage progenitor cells with high proliferative potential in mouse bone marrow in vitro

Blood ◽  
1984 ◽  
Vol 63 (6) ◽  
pp. 1348-1352 ◽  
Author(s):  
AB Kriegler ◽  
TR Bradley ◽  
GS Hodgson
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Proliferative Potential ◽  
Mouse Bone Marrow ◽  
Synergistic Activity ◽  
Agar Culture ◽  
Inhibition Of Proliferation ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony

Abstract High proliferative potential macrophage progenitor cells (HPP-CFC) in 5- fluorouracil (FU) treated and normal mouse bone marrow (BM) have been shown to be less sensitive to inhibition of proliferation by prostaglandins of the E series (PGE) than low proliferative potential macrophage progenitor cells (LPP-CFC) in normal BM in agar cultures. The growth of large colonies (diameter greater than 0.5 mm) derived from HPP-CFC in FU BM, which require a combination of macrophage colony- stimulating factor (CSF-1) plus a new growth factor called synergistic activity (SA), are inhibited by 50% in the presence of 5.5 X 10(-6) M PGE1. On the other hand, LPP-CFC in normal BM, which form smaller colonies (diameter less than or equal to 0.5 mm) in the presence of CSF- 1 alone, require only 5 X 10(-8) M PGE1 for the same level of inhibition. Addition of appropriate concentrations of PGE1 to the agar culture assay should improve detection of HPP-CFC by inhibiting the proliferation of LPP-CFC. These observations suggest that the apparent negative feedback control of macrophage production by PGE operates largely on the LPP-CFC, which respond to CSF-1 alone, and is probably not involved in the regulation of the more primitive HPP-CFC.

Immunohistochemical localization of membrane and alpha-granule proteins in plastic-embedded mouse bone marrow megakaryocytes and murine megakaryocyte colonies

Blood ◽  
1986 ◽  
Vol 68 (3) ◽  
pp. 696-702
Author(s):  
PE Stenberg ◽  
JH Beckstead ◽  
RP McEver ◽  
J Levin
Keyword(s):  
Bone Marrow ◽  
Human Platelet ◽  
Soft Agar ◽  
Platelet Membrane ◽  
Mouse Bone Marrow ◽  
Immunoperoxidase Technique ◽  
Membrane Glycoproteins ◽  
Agar Culture ◽  
Light Microscope Level ◽  
Murine Bone Marrow

Using an immunoperoxidase technique that permits optimal antigen localization at the light microscope level, we have detected two platelet alpha-granule constituents and three platelet membrane glycoproteins in mouse bone marrow megakaryocytes and in murine megakaryocyte colonies grown in soft agar culture for three to seven days. Using polyclonal antibodies prepared against human platelet proteins, we have demonstrated labeling for von Willebrand factor, fibrinogen, and the membrane glycoproteins IIIa and GMP-140 in both bone marrow megakaryocytes and megakaryocyte colonies after seven days of culture. Using monoclonal antibodies to membrane glycoproteins IIb and GMP-140, we have demonstrated label in mouse bone marrow megakaryocytes. Granulocyte and macrophage colonies were negative for each of these markers. Murine bone marrow megakaryocytes and megakaryocyte colonies demonstrated a similar enzyme histochemical pattern: weakly positive for alpha-naphthyl acetate esterase and negative for chloroacetate esterase. These data indicate that megakaryocytes grown in soft agar culture express many of the same glycoproteins as bone marrow megakaryocytes. Furthermore, the ability of antibodies directed against human platelet membrane glycoproteins to identify murine megakaryocyte glycoproteins indicates that these constituents have been highly conserved during evolution.

In Vitro, In Silico and Ex Vivo Studies of Dihydroartemisinin Derivatives as Antitubercular Agents

2019 ◽  
Vol 19 (8) ◽  
pp. 633-644 ◽  
Author(s):  
Komal Kalani ◽  
Sarfaraz Alam ◽  
Vinita Chaturvedi ◽  
Shyam Singh ◽  
Feroz Khan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
In Silico ◽  
Ex Vivo ◽  
Virulent Strain ◽  
Infection Model ◽  
Mouse Bone Marrow ◽  
Significant Activity ◽  
Macrophage Infection ◽  
In Silico Studies

Introduction: As a part of our drug discovery program for anti-tubercular agents, dihydroartemisinin (DHA-1) was screened against Mtb H37Rv, which showed moderate anti-tubercular activity (>25.0 µg/mL). These results prompted us to carry out the chemical transformation of DHA-1 into various derivatives and study their antitubercular potential. Materials and Methods: DHA-1 was semi-synthetically converted into four new acyl derivatives (DHA-1A – DHA-1D) and in-vitro evaluated for their anti-tubercular potential against Mycobacterium tuberculosis H37Rv virulent strain. The derivatives, DHA-1C (12-O-(4-nitro) benzoyl; MIC 12.5 µg/mL) and DHA-1D (12-O-chloro acetyl; MIC 3.12µg/mL) showed significant activity against the pathogen. Results: In silico studies of the most active derivative (DHA-1D) showed interaction with ARG448 inhibiting the mycobacterium enzymes. Additionally, it showed no cytotoxicity towards the Vero C1008 cells and Mouse bone marrow derived macrophages. Conclusion: DHA-1D killed 62% intracellular M. tuberculosis in Mouse bone marrow macrophage infection model. To the best of our knowledge, this is the first-ever report on the antitubercular potential of dihydroartemisinin and its derivatives. Since dihydroartemisinin is widely used as an antimalarial drug; these results may be of great help in anti-tubercular drug development from a very common, inexpensive, and non-toxic natural product.

Differentiation of Mouse Bone-Marrow Mesenchymal Stem Cells into Motor Neuron Cells in vitro

2016 ◽  
Vol 19 (2) ◽  
pp. 111-116
Author(s):  
Rafal Hussamildeen Abdullah ◽  
◽  
Shahlla Mahdi Salih ◽  
Nahi Yosef Yaseen ◽  
Ahmed Majeed Al-Shammari ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Motor Neuron ◽  
Mouse Bone Marrow ◽  
Marrow Mesenchymal Stem Cells

Expression of human adenosine deaminase in murine hematopoietic cells

1988 ◽  
Vol 8 (12) ◽  
pp. 5116-5125
Author(s):  
J W Belmont ◽  
G R MacGregor ◽  
K Wager-Smith ◽  
F A Fletcher ◽  
K A Moore ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Adenosine Deaminase ◽  
High Titer ◽  
Virus Production ◽  
Marrow Transplant ◽  
Mouse Bone Marrow ◽  
Regulation Of Expression ◽  
Murine Bone Marrow

Multiple replication-defective retrovirus vectors were tested for their ability to transfer and express human adenosine deaminase in vitro and in vivo in a mouse bone marrow transplantation model. High-titer virus production was obtained from vectors by using both a retrovirus long terminal repeat promoter and internal transcriptional units with human c-fos and herpes virus thymidine kinase promoters. After infection of primary murine bone marrow with one of these vectors, human adenosine deaminase was detected in 60 to 85% of spleen colony-forming units and in the blood of 14 of 14 syngeneic marrow transplant recipients. This system offers the opportunity to assess methods for increasing efficiency of gene transfer, for regulation of expression of foreign genes in hematopoietic progenitors, and for long-term measurement of the stability of expression in these cells.

Synergism of v-myc and v-Ha-ras in the in vitro neoplastic progression of murine lymphoid cells

1986 ◽  
Vol 6 (9) ◽  
pp. 3221-3231
Author(s):  
R C Schwartz ◽  
L W Stanton ◽  
S C Riley ◽  
K B Marcu ◽  
O N Witte
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Retroviral Vectors ◽  
Soft Agar ◽  
Lymphoid Cells ◽  
Neoplastic Progression ◽  
Direct Role ◽  
Murine Bone Marrow ◽  
Myc Oncogene

Murine bone marrow was either singly or doubly infected with retroviral vectors expressing v-myc (OK10) or v-Ha-ras. The infected bone marrow was cultured in a system that supports the long-term growth of B-lineage lymphoid cells. While the v-myc vector by itself had no apparent effect on lymphoid culture establishment and growth, infection with the v-Ha-ras vector or coinfection with both v-myc and v-Ha-ras vectors led to the appearance of growth-stimulated cell populations. Clonal pre-B-cell lines stably expressing v-Ha-ras alone or both v-myc and v-Ha-ras grew out of these cultures. In comparison with cell lines expressing v-Ha-ras alone, cell lines expressing both v-myc and v-Ha-ras grew to higher densities, had reduced dependence on a feeder layer for growth, and had a marked increase in ability to grow in soft-agar medium. The cell lines expressing both oncogenes were highly tumorigenic in syngeneic animals. These experiments show that the v-myc oncogene in synergy with v-Ha-ras can play a direct role in the in vitro transformation of murine B lymphoid cells.

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