Cell production in mouse intestinal epithelium measured by stathmokinetic flow cytometry and coulter particle counting

1983 ◽  
Vol 207 (3) ◽  
pp. 427-434 ◽  
Author(s):  
Hazel Cheng ◽  
Matthew Bjerknes
Keyword(s):  
Flow Cytometry ◽  
Intestinal Epithelium ◽  
Cell Production ◽  
Particle Counting

UV LED excited time-gated luminescence flow cytometry: evaluation for rare-event particle counting

2008 ◽  
Author(s):  
Dayong Jin ◽  
Belinda Ferrari ◽  
Robert C. Leif ◽  
Sean Yang ◽  
Lidia M. Vallarino ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Rare Event ◽  
Uv Led ◽  
Particle Counting

Pomalidomide Augments Fetal Hemoglobin Production In Primary Erythroid Cells By a Novel Mechanism Modulating BCL11A But Not KLF-1

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 314-314
Author(s):  
Abena O. Appiah-Kubi ◽  
Lionel Blanc ◽  
Sharon A. Singh ◽  
Sebastien Didier ◽  
Sehba Dsilva ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Culture System ◽  
Fetal Hemoglobin ◽  
Erythroid Differentiation ◽  
Cell Production ◽  
Expression Levels ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
F Cells

Abstract Fetal hemoglobin (HbF) is a known modifier of sickle cell disease (SCD) severity. KLF-1 is a regulator of the globin switch. It does so by increasing beta-globin production and up-regulating BCL11A, a repressor of HbF synthesis. Pomalidomide, a second generation immunomodulatory drug (IMiD), regulates HbF and F-cell production during erythropoiesis in human CD34+ cells. The mechanism by which pomalidomide enhances F-cell production is not well understood. In this study, CD34+ cells were obtained after purification of peripheral blood and positive selection and cultured using a three-phase in vitro liquid culture system which recapitulates erythropoiesis, including terminal differentiation and enucleation, in the presence of no drug, pomalidomide, hydroxyurea, or dimethyl sulfoxide (DMSO; vehicle control). Erythroid differentiation was assessed morphologically and by flow cytometry using the transferrin receptor and glycophorin A as markers of erythroid maturation. Flow cytometry was used to quantify F-cells. RT-qPCR was used to quantify mRNA expression of BCL11A, KLF-1, and gamma-globin. Western blot was used to measure the total expression levels of BCL11A. In this culture system pomalidomide increased F-cells more than hydroxyurea in both SCD and normal control erythroid cultures. There was a significant decrease in BCL11A expression levels, a repressor of HbF synthesis, with pomalidomide but not with hydroxyurea. This decrease was seen in both SCD and normal samples. KLF-1 was not affected by pomalidomide. These findings suggest a very different mechanism of action for pomalidomide versus hydroxyurea in increasing F-cell production. Pomalidomide appears to target the erythroid specific BCL11A but not the more pleiotropic transcription regulator KLF-1. Since the F-cell production was augmented in the presence of pomalidomide in controls as well as SCD erythroid cultures this study suggests a role for pomalidomide in the pharmacologic augmentation of fetal hemoglobin levels, perhaps in addition to hydroxyurea, not only in SCD but in any beta-hemoglobinopathy. Disclosures: Chan: BioTheryX Inc: Employment.

The Role of Interleukin-6 and Bone-Marrow Derived Cell Production of Hepcidin In Anemia of Inflammation

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 167-167
Author(s):  
Thomas M. Renaud ◽  
Stefano Rivella
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Knockout Mice ◽  
Interleukin 6 ◽  
Wild Type ◽  
Erythroid Progenitor ◽  
Cell Production ◽  
Time Points ◽  
Anemia Of Inflammation

Abstract Abstract 167 Anemia of inflammation is the second most common form of anemia in the general population, and its impact on patient well-being is largely underestimated. Anemia cause by inflammation is multi-factorial and includes hepcidin-induced iron restricted erythropoiesis as well as direct cytokine effects on the bone marrow, erythropoietin production and efficacy, and on the lifespan of red cells. Many murine models of anemia of inflammation are unreliable or cumbersome, but a new model introduced by Sasu et al (Blood, 2010) using a single intraperitoneal injection of heat-killed brucella abortus antigen (HKBA) has proven reproducible and robust. We have used this model to explore the role of interleukin-6 and bone marrow derived cell production of hepcidin in anemia of inflammation (AI). First, we sought to explore the effect this model of AI in wild type mice, iterleukin-6 knockout mice (IL6-KO) and hepcidin knockout mice (Hamp-KO) (n≥15 for each group). We followed these mice for 7 weeks with weekly CBC's to observe the severity and time to recovery from anemia. Wild type mice were most affected 2 weeks after injection and slowly recovered over 7 weeks (HgB at 2 week = 6.4g/dl ± 1.2). IL6-KO mice were equally affected initially, with similar hemoglobin values at 2 weeks (6.9g/dl ± 1.3) and recovered by 6 weeks. Hamp-KO mice were less affected throughout the course of anemia, with hemoglobin values of 10.8g/dl ± 0.7 at 2 weeks with resolution by week 4. IL6-KO mice began to recover more quickly than wild type mice by week three, with hemoglobin values of 10.9g/dl ± 1.5 at that time, compared to wild type mice at 3 weeks with hemoglobin values of 7.4g/dl ± 0.7 (p= 0.0001). We believe that this demonstrates that interleukin-6 and hepcidin do coordinate to contribute to anemia of inflammation, but that there may be independent effects or additional factors. To address these questions, we are currently evaluating iron-related gene expression in these groups of mice as well as evaluating iron stores at multiple time points. We also evaluated serum cytokine levels in each of these groups of mice. We found similar elevations TNF-alpha and interferon gamma in all three groups at 6 and 24 hours. We found similar elevations of IL-6 in wild type and Hamp-KO mice at 6 and 24 hours. Bone marrows and spleens form each group of mice were evaluated at 2 weeks by flow cytometry using ter119 and CD44 to evaluate specific effects on erythroid maturation. This evaluation demonstrated a a profound inhibitory effect on erythropoiesis and, in particular, on the production of erythroid progenitor cells, showing a similar profile by flow cytometry between the three groups. In vitro studies have suggested that macrophage production of hepcidin is important in the development of AI (Theurl et al 2008). We evaluated the importance of bone marrow derived cell production of hepcidin on the development of AI using bone marrow chimeras. Using 600cGy × 2 as a preparative regimen, we transplanted wild type mice with bone marrow from Hamp-KO mice. We also irradiated Hamp-KO mice and transplanted them with wild type marrow. We injected these two groups of mice as well as wild type and Hamp-KO controls, we followed them for a period of 4 weeks with weekly CBC's to evaluate the degree of anemia. Hemoglobin values of wild type mice transplanted with Hamp-KO marrow were statistically indistinguishable from those of non-transplanted wild type mice during the follow-up period (HgB values at 1 week = 6.8g ± 0.7 vs 7.29g ± 1.1; at 2 weeks = 7.3 ± 0.6 vs 6.4 ± 1.2; at 3 weeks = 8.5 ± 1.8 vs 7.4 ± 0.5; at 4 weeks = 9.1 v 1.9 vs 8.6 ± 0.5; p<0.02 for all time points). Hamp-KO mice with wild-type bone marrow were statistically indistinguishable from non-transplanted Hamp-KO mice (Hgb values at 1 week = 9.9 ± 2.4 vs 10.8 ± 0.7; at 2 weeks = 10.6 ± 1.5 vs 10.3 ± 1.0; at 3 weeks = 12.8 ± 1.2 10.8 ± 0.7; at 4 weeks 12.6 ± 1.2 vs. 13.6 ± 1.0; p<0.02 for all time points). This suggests that the production of hepcidin by bone marrow derived cells dose not play a physiologically important role in the development of anemia of inflammation. Disclosures: No relevant conflicts of interest to declare.

Further Observations on the Virus of Epizootic Diarrhea of Infant Mice. An Electron Microscopic Study

1967 ◽  
Vol 25 ◽  
pp. 110-111
Author(s):  
W. G. Banfield ◽  
G. Kasnic ◽  
J. H. Blackwell
Keyword(s):  
Electron Microscope ◽  
Infected Cell ◽  
Microscopic Study ◽  
Intestinal Epithelium ◽  
Ultrastructural Study ◽  
Osmium Tetroxide ◽  
Lead Citrate ◽  
Electron Microscopic ◽  
Virus Particles ◽  
Infected Cells

An ultrastructural study of the intestinal epithelium of mice infected with the agent of epizootic diarrhea of infant mice (EDIM virus) was first performed by Adams and Kraft. We have extended their observations and have found developmental forms of the virus and associated structures not reported by them.Three-day-old NLM strain mice were infected with EDIM virus and killed 48 to 168 hours later. Specimens of bowel were fixed in glutaraldehyde, post fixed in osmium tetroxide and embedded in epon. Sections were stained with uranyl magnesium acetate followed by lead citrate and examined in an updated RCA EMU-3F electron microscope.The cells containing virus particles (infected) are at the tips of the villi and occur throughout the intestine from duodenum through colon. All developmental forms of the virus are present from 48 to 168 hours after infection. Figure 1 is of cells without virus particles and figure 2 is of an infected cell. The nucleus and cytoplasm of the infected cells appear clearer than the cells without virus particles.

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