Scanning electron microscopy of human lymphocytes during transformation and subsequent treatment with methotrexate

1977 ◽  
Vol 28 (1) ◽  
pp. 151-165
Author(s):  
C.C. Hoffmann ◽  
K.C. Moore ◽  
C.Y. Shih ◽  
R.L. Blakley
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
T Cells ◽  
T Lymphocytes ◽  
Human Peripheral Blood ◽  
Human Lymphocytes ◽  
Peripheral Blood Lymphocytes ◽  
Subsequent Treatment ◽  
Significant Area ◽  
Scanning Electron

Preparations of human peripheral blood lymphocytes containing 72–87% T-cells and 10–16% B-cells were shown by scanning electron microscopy to consist almost exclusively of cells bearing numerous microvilli, whereas thymocytes were of mixed surface morphology, with both smooth and encrusted forms numerous. T-lymphocytes purified on long nylon columns were all covered with numerous short villi. Stimulation with phytohaemagglutinin for 2 days produced T-lymphoblasts almost exclusively, and as the T-cells enlarged the microvilli lengthened, the increase in length reaching 5-fold by day 3. Addition of sufficient methotrexate on day 3 to arrest proliferation (50 nM) caused progressive loss of microvilli from the cell surface, with the eventual production of large numbers of smooth cells, the surfaces of which later became pitted, followed by the complete dissolution of the cell. T-lymphocytes were shown to form rosettes with sheep erythrocytes through direct contact of the cell membranes over a significant area, but when, as a result of methotrexate treatment, the lymphocytes had become denuded of microvilli or had reached an advanced state of dissolution, rosettes were no longer formed.

scholarly journals IDENTIFICATION OF HUMAN B AND T LYMPHOCYTES BY SCANNING ELECTRON MICROSCOPY

1973 ◽  
Vol 138 (3) ◽  
pp. 607-624 ◽  
Author(s):  
A. Polliack ◽  
N. Lampen ◽  
B. D. Clarkson ◽  
E. de Harven ◽  
Z. Bentwich ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Surface Morphology ◽  
B Lymphocytes ◽  
Cell Type ◽  
B And T Lymphocytes ◽  
Scanning Electron

In this study a variety of human lymphocytes of known B or T cell type, obtained from multiple sources, were prepared for scanning electron microscopy (SEM) by the critical point drying method. Distinction between normal B and T lymphocytes was relatively easy in most instances, on the basis of their surface architecture. Using immunological methods, between 20 and 30% of normal peripheral blood lymphocytes (PBL) were identified as B cells and from 69 to 82% as T cells. SEM results showed that 20% of the PBL had a complex villous surface and approximately 80% of cells were smaller and had a relatively smooth surface. Comparison of the above data and enrichment of B cells from PBL, by centrifugation after T cell rosettes had formed, indicated that the "villous" cells were B lymphocytes and the "relatively smooth" cells were T lymphocytes. T cells obtained from two human thymuses were also of the generally smooth cell type. Further evidence for the distinction of B and T lymphocytes, on the basis of surface morphology, was obtained from the examination of cultured lymphoid cell lines of known B or T cell derivation. Cells from cases of chronic lymphocytic leukemia also provided support for the above interpretations. Five of six untreated cases were clearly of B cell type by immunologic and SEM criteria. One unusual case showed the presence of T and B lymphocytes in almost equal numbers by SEM and a mixture of B and T cells by immunologic markers. An additional case that had received chemotherapy showed numerous atypical cells that were difficult to classify by SEM. Detailed examination of the smoother T cells showed that at least half of them had a moderate number of surface digitations and a small proportion had an intermediate surface morphology with a relatively large number of surface digitations. The latter presented difficulties in classification and may correspond to different stages of differentiation and represent subpopulations of lymphocytes. The distinction between human B and T lymphocytes on the basis of their surface architecture can be made by SEM of critical point dried samples, with relative ease in most but not all instances. The effects of stimulation, cell cycle, differentiation, intercellular contact, and density of cell population, on the surface architecture of lymphoid cells, remain to be determined.

scholarly journals SCANNING ELECTRON MICROSCOPY OF HUMAN LYMPHOCYTE-SHEEP ERYTHROCYTE ROSETTES

1974 ◽  
Vol 140 (1) ◽  
pp. 146-158 ◽  
Author(s):  
A. Polliack ◽  
Shu Man Fu ◽  
S. D. Douglas ◽  
Z. Bentwich ◽  
N. Lampen ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
T Cells ◽  
B Cells ◽  
T Lymphocytes ◽  
B Lymphocytes ◽  
Sheep Erythrocyte ◽  
Smooth Surfaces ◽  
Critical Point Drying ◽  
Scanning Electron

Human lymphocytes of known B or T derivation were examined by scanning electron microscopy (SEM) before and after rosetting with SRBC. After collection of the cells onto silver membranes the samples were prepared for SEM by the critical point drying method. Sheep RBC frequently underwent sphero-echinocyte transformation and multiple projections extended from their surfaces. This was readily noticeable after storage of SRBC in the cold and washing in Hanks, but more prominent after rosetting. These erythrocyte surface alterations were less apparent when freshly withdrawn cells were used. Spontaneous sheep erythrocyte rosettes (E-R), a marker for human T lymphocytes, were prepared with normal peripheral blood lymphocytes (PBL), thymic cells, and cultured T cells. EAC-rosettes (EAC-R), used to identify B lymphocytes with complement receptors, were prepared with normal PBL and cultured B cells. The majority of rosetting T lymphocytes had generally smooth surfaces while about 20% had an intermediate number of microvilli and 15% were more villous and indistinguishable from villous B cells. Studies of rosetting thymocytes and cultured T cells however indicated that the surface of some T cells alters on rosetting, becoming more villous and thus account for the higher numbers of villous T cells seen in E-rosettes. Point to point contact sites between SRBC and T lymphocytes were more frequent than broad zones of attachment. The majority of rosetting B lymphocytes had multiple microvilli, about 25% had a moderate number of microvilli and less than 10% had smooth surfaces similar to those of most T cells. Areas of contact between EAC and B lymphocytes were frequently broad zones of attachment. The study confirms that in many cases B and T lymphocytes can be distinguished by their surface architecture as seen under the SEM; however, about 20% of rosetting B and T cells have similar surfaces with intermediate numbers of surface microvilli and cannot be distinguished by SEM without parallel immunologic identification.

Phase Composition Influence on Melt Viscosity and Ingot Structure Formation Features in EK77 Alloy

2017 ◽  
Vol 907 ◽  
pp. 43-49
Author(s):  
Arkadiy Yu. Zhilyakov ◽  
Sergey V. Belikov ◽  
Alexander G. Gudov ◽  
Sergey P. Burmasov
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Phase Composition ◽  
Torsional Oscillation ◽  
Subsequent Treatment ◽  
Optical Metallography ◽  
Initial Structure ◽  
Melt Viscosity ◽  
Ingot Structure ◽  
Scanning Electron

Effect of the alloy initial structure before remelting on the melt viscosity, its state prior to crystallization, and on processes occurring in the alloy in solid state during its subsequent treatment was investigated using EK77 alloy. The study was performed by means of torsional oscillation of the melt with crucible, scanning electron microscopy, and optical metallography.

scholarly journals PGE2 Dependent Inhibition of Macrophage Pyroptosis By MSCs Contributes to Alleviating aGVHD

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 15-15
Author(s):  
Xueyan Sun ◽  
Yan Su ◽  
Xiao Liu ◽  
Fengqi Liu ◽  
Gaochao Zhang ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
T Cells ◽  
Nlrp3 Inflammasome ◽  
Cd4 T Cells ◽  
Inflammasome Activation ◽  
Rt Pcr ◽  
Nlrp3 Inflammasome Activation ◽  
Therapeutic Mechanism ◽  
Scanning Electron

Introduction Mesenchymal stem cells (MSCs) are being recognized as one of the treatment options for acute graft versus host disease (aGVHD), but their therapeutic mechanisms have not been fully elucidated. Pyroptosis, a novel form of inflammation related programmed cell death, often occurs in myeloid cells. Many studies have found that macrophage pyroptosis plays an important role in multiple inflammatory and autoimmune diseases (Journal of Autoimmunity, 2018). As an immune disease with involvement of various inflammatory factors, aGVHD exhibits macrophage dysfunction according to our previous study (Sci China Life Sci, 2020). However, whether macrophages undergo pyroptosis and their role in aGVHD remain unknown. MSCs have been reported to inhibit pyroptosis, and some cytokines that suppress pyroptosis can also be secreted by MSCs (Nature Immunology, 2016). Whether inhibition of macrophage pyroptosis represents a therapeutic mechanism for MSCs to alleviate aGVHD needs further exploration. Methods Twenty patients with aGVHD and 20 patients without aGVHD after hematopoietic stem cell transplantation were enrolled in our study. Macrophages were derived from CD14+ monocytes of patients and the THP-1 cell line. CD4+ T cells were isolated from peripheral blood mononuclear cells (PBMCs) of healthy volunteers. MSCs were obtained from fresh umbilical cord of healthy puerpera. Morphological analysis of macrophages was performed by scanning electron microscopy. Expression of GSDMD and NLRP3 inflammasome associated components was assessed by real-time transcription-polymerase chain reaction (RT-PCR), western blot and immunofluorescent staining. The subgroup of CD4+T cells was analyzed by flow cytometry. RT-PCR, ELISA and RNA interference were used to evaluate relevant immunomodulatory factors which were involved in the inhibitory effect of MSCs on macrophage pyroptosis. Additionally, an aGVHD mouse model was established to observe the therapeutic effect and mechanism of MSCs on macrophage pyroptosis. Results Scanning electron microscopy images showed the formation of membrane pores in macrophages of aGVHD patients. Meanwhile, expression of the pyroptosis executioner GSDMD, NLRP3 inflammasome associated components, IL-1β, IL-18, and LDH release were elevated in macrophages from aGVHD patients, indicating that macrophages in aGVHD underwent NLRP3 inflammasome activation and pyroptosis. Furthermore, NLRP3 inhibition reduced macrophages pyroptosis, suggesting that macrophages pyroptosis in aGVHD are mediated by NLRP3 inflammasome activation. Since CD4+T cells play a critical role in the pathogenesis of aGVHD, we investigated the effect of macrophage pyroptosis on CD4+T cells. In vitro, macrophage pyroptosis increased the proportion of CD69+, Th1 and Th17 cells among CD4+T cells, which was partially reversed by blocking IL-1β/IL-1R and IL-18/IL-18R signaling. We also observed that the proportion of macrophage pyroptosis was more increased in patients with III-IV aGVHD than in those with I-II aGVHD. In addition, administration of a pyroptosis inhibitor into aGVHD model mice greatly attenuated clinical and histopathological scores. Taken together, these results indicate that macrophage pyroptosis might be involved the development of aGVHD. Expression of GSDMD, NLRP3 inflammasome associated components, IL-1β, IL-18, and LDH release in aGVHD macrophages were reduced when cells were cocultured with MSCs, indicating that MSCs inhibit aGVHD macrophage pyroptosis by suppressing NLRP3 inflammasome activation. Furthermore, secretion of prostaglandin E2 (PGE2) was increased in MSCs cocultured with aGVHD macrophages, blocking which by small interfering RNA (siRNA) or inhibition of PGE2 induced CAMP-PKA signaling with antagonists both largely abrogated MSC effects. Consistently, the effect of MSCs on macrophage pyroptosis and the NLRP3 inflammasome in vivo was also dampened after transfection with prostaglandin E synthase (PTGES) siRNA, and the therapeutic effect in the aGVHD mouse model was impaired. Conclusions Our results demonstrate that macrophage pyroptosis plays a crucial role in the pathogenesis of aGVHD by promoting activation and differentiation of CD4+ T cells. MSCs suppress macrophage pyroptosis in aGVHD via PGE2/cAMP/PKA signaling, which might represent a therapeutic mechanism of MSCs for aGVHD. Disclosures No relevant conflicts of interest to declare.

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