pkh-26 and Acute-Disease

The aim of this study was to study interactions between stromal bone marrow microenvironment and leukemic cells. We tested the hypothesis that stromal cells prevent apoptosis of AML cells by up-regulating anti-apoptotic proteins in leukemic blasts. In HL-60 and NB-4 cells, serum deprivation- and ara-C-induced apoptosis was diminished when cells were cocultured with murine MS-5 stromal cells (P < 0.02). This effect was reproduced with conditioned medium from MS-5 cells. Cocultivation with stromal cells induced Bcl-2 expression levels, both by PCR analysis and flow cytometry. In primary AML (n = 14), ara-C-induced apoptosis was significantly lower in cells cocultured with MS-5 cells than in controls (P < 0.001). This effect was partially preserved when leukemic cells were separated from stromal cells by a microporous insert (in 5/9 samples, P = 0.04). In addition, Bcl-2 levels were significantly higher in stroma-supported than in control CD34(+) AML cells (P < 0.01). Bcl-X(L) levels were higher in 5/7 samples grown on stromal layers. Of note, in AML patients resistant to induction chemotherapy (n = 6), Bcl-2 increased significantly after cultivation with stromal cells, but no such increase was noted in cells from chemotherapy-sensitive patients. In conclusion, MS-5 stromal cells prevented apoptosis in HL-60 cells and in primary AML blasts via modulation of Bcl-2 family proteins. The observed association of high Bcl-2 expression in stroma-supported AML blasts in vitro with resistance to chemotherapy in vivo suggests that the same mechanisms may be operational in vivo.

Topics: Acute Disease; Adult; Aged; Aged, 80 and over; Animals; Antimetabolites, Antineoplastic; Apoptosis; Bone Marrow Cells; Cell Adhesion; Cell Division; Culture Media, Serum-Free; Cytarabine; DNA Primers; Female; Fluorescent Dyes; HL-60 Cells; Humans; Leukemia, Myeloid; Male; Mice; Middle Aged; Organic Chemicals; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Neoplasm; Stromal Cells; Tumor Cells, Cultured; Up-Regulation

To study retinal capillary blood flow during the acute stage of induced branch retinal vein occlusion (BRVO) in the feline retina and the effect of systemic blood pressure during this event.. Known fractions of the animal blood cells were labeled by a fluorescent marker and tracked by an electro-optical device as they flowed within the retinal microcirculation. Branch retinal vein occlusion was induced by photocoagulation of a large vein at the optic disk rim. The findings were video recorded and analyzed by computerized image processing.. Reversed capillary blood cell flow from the blocked vein was noted immediately following occlusion of the vein. Two patterns of reversed capillary flow were identified: one from the blocked vein to a terminal arteriole and the other from a blocked vein to another vein via a junction with a terminal arteriole. Increased systemic blood pressure decreased the reversed flow in both flow patterns.. 1) Flow deviation to nearby retinal arterioles or veins (shunting) through the normal capillary system is an immediate hemodynamic event following acute BRVO. 2) The reversed capillary flow is affected by the systemic blood pressure.

Topics: Acute Disease; Animals; Blood Flow Velocity; Blood Pressure; Capillaries; Cats; Fluorescent Dyes; Image Processing, Computer-Assisted; Microcirculation; Organic Chemicals; Regional Blood Flow; Retinal Vein; Retinal Vein Occlusion

A new assay system using the fluorescent probe PKH 26 GL was employed to investigate the regulation of precursor B-cell acute lymphoblastic leukaemic (ALL) cell growth. PKH 26 GL is a lipophilic fluorescent probe which becomes incorporated into the plasma membrane upon the staining of cells. As the amount of probe per cell reduces at each cell division, the fluorescence can be used to measure cell proliferation. Bone marrow derived ALL cells from seven newly diagnosed cases were stained with PKH 26 GL, and cultured for 14 days in control cultures without stimulus, or in cultures with preformed human bone marrow stromal cell layers. Viable leukaemic cells from these cultures were identified on the basis of forward light scatter, 90 degrees light scatter, propidium iodide exclusion, PKH 26 GL staining and CD10 expression by flow cytometry at the beginning of the culture period and on days 2, 6, 10 and 14. The growth parameters of these leukaemic cells were determined by analysis of their pattern of PKH 26 GL fluorescence. A higher rate of proliferation and survival of cells was observed in cultures with stromal cells compared with control cultures, without stromal cells. In the presence of stromal cells, survival and proliferation continued throughout the culture period; in contrast in five of seven control cultures no viable cells could be detected after 6-10 days. Interestingly, two populations of leukaemic cells were distinguished on the basis of their different rates of proliferation, when co-cultured with stromal cells. The results indicate that this technique provides a means for studying and quantitating leukaemic cell growth within a complex stroma-dependent system.

Topics: Acute Disease; Adolescent; Bone Marrow; Bone Marrow Cells; Cell Division; Cell Survival; Child; Child, Preschool; Female; Flow Cytometry; Fluorescent Dyes; Humans; Male; Neprilysin; Organic Chemicals; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma; Stromal Cells; Tumor Cells, Cultured