Characterization of rat bone marrow lymphoid cells. I. A study of the distribution parameters of sedimentation velocity, volume and electrophoretic mobility

Various cell populations in rat bone marrow were characterized by means of a two dimensional separation using velocity sedimentation and free flow electrophoresis and by electrical sizing of the separated cells. Up to 4.5 mm/hr five different populations with discrete distributions in volume (coefficient of variation 10% to 13%) and sedimentation velocity (coefficient of variation 6% to 10%) were observed. Three of the small sized populations represented lymphocytes and small normoblasts and two of the larger sized populations represented myeloid cells. Almost all of these cells were in the G0/G1 cycle phase. In the faster sedimenting fractions which contained immature myeloid, erythroid and undefined blast cells and two S phase populations, discrete volume distributions were not evaluated. The cell populations with homogeneous volume (particularly the small lymphocytes) showed high density variations which condiserably impair the separation resolution. The cells sedimenting slower than 3.5 mm/hr were further separated by means of free flow electrophoresis into three peaks differing in electrophoretic mobility (EPM). The peaks of low and high EPM contained two populations and the peak of medium EPM contained three populations all characterized by normal volume distributions of uniform coefficient of variation between 11% and 14%. The small cells in the peaks of high and medium EPM were normolblasts and the other cells were lymphocytes. The biological significance of these results is discussed

Intestinal Epithelial Cell-Specific Deletion of PLD2 Alleviates DSS-Induced Colitis by Regulating Occludin

Ulcerative colitis is a multi-factorial disease involving a dysregulated immune response. Disruptions to the intestinal epithelial barrier and translocation of bacteria, resulting in inflammation, are common in colitis. The mechanisms underlying epithelial barrier dysfunction or regulation of tight junction proteins during disease progression of colitis have not been clearly elucidated. Increase in phospholipase D (PLD) activity is associated with disease severity in colitis animal models. However, the role of PLD2 in the maintenance of intestinal barrier integrity remains elusive. We have generated intestinal specific Pld2 knockout mice (Pld2 IEC-KO) to investigate the mechanism of intestinal epithelial PLD2 in colitis. We show that the knockout of Pld2 confers protection against dextran sodium sulphate (DSS)-induced colitis in mice. Treatment with DSS induced the expression of PLD2 and downregulated occludin in colon epithelial cells. PLD2 was shown to mediate phosphorylation of occludin and induce its proteasomal degradation in a c-Src kinase-dependent pathway. Additionally, we have shown that treatment with an inhibitor of PLD2 can rescue mice from DSS-induced colitis. To our knowledge, this is the first report showing that PLD2 is pivotal in the regulation of the integrity of epithelial tight junctions and occludin turn over, thereby implicating it in the pathogenesis of colitis

Analysis of velocity sedimentation techniques in cell separation. Influence of apparative and sample properties on separative power, resolution and sensitivity

In the present investigation the velocity sedimentation technique was analysed with respect to separation resolution, power and sensitivity. It was found that apparative modifications do not influence the resolution, which is a function of the contribution of apparative errors to the dispersion. A surprisingly small parameter of 0.15 was determined and it seems unlikely that this value can be improved. On the other hand an apparative modification is presented which improves the separation power and makes sample loading independent of the gradient filling. If cells (from rat bone marrow) were separated, a several times higher dispersion for a given cell volume was observed than was due to the apparative error. It was concluded that density variations were the major source of this dispersion. Since cell volume and density apparently show independent variations within a biological cell population the cell density cannot be disregarded if velocity sedimentation profiles are discussed in physical terms as is often done

B lymphocyte subpopulations in the mouse spleen. A study of the differentiation pathway using free flow electrophoretically separated subpopulations of direct PFC progenitor cells.

Free-flow electrophoretic separation of mouse spleen cells provides three distinct progenitor cells of direct PFC, showing high, medium and low electrophoretic mobility. All progenitor cells possess surface immunoglobulin and mouse B-lymphocyte specific antigen. The progenitor cells of high electrophoretic mobility show high cycling turnover, a spleen seeking capacity of 16%, provide PFC with a maximum 8 days after transfer and reveal an isometrical increase of the PFC dose response line as a function of the graft size. The progenitor cells of medium electrophoretic mobility are low cycling, 16% home to the spleen, a maximum of PFC is developed eight days after transfer and the PFC dose response line increases allometrically. The progenitor cells of low EPM show low cycling activity, 20% home to the spleen, a maximum of PFC is attained six days after transfer and the PFC dose response line rises isometrically. These results suggest that the electrokinetically different PFC progenitors represent biologically distinct subsets. In double transfer experiments, some evidence was obtained that progenitor cells of low electrophoretic mobility are derived from progenitors of higher electrophoretic mobility. The same observation accounts also for the formation of B lymphocytes of low EPM. Since it seemed likely that the PFC progenitor cells represent virgin cells of a single lineage, the results were discussed in the terms of differentiation pathways of B lymphocytes. A model is considered in which a progenitor of medium electrophoretic mobility provides those of high electrophoretic mobility which after passing a transient cycling stage finally produce mature resting B lymphocytes of low electrophoretic mobility