Hemopoiesis on Acetate Cellulose Membranes in the Presence of Lipopolysaccharide

The formation of hemopoietic colonies on acetate cellulose membranes in the peritoneal cavity of mice was markedly enhanced after the injection of bacterial lipopolysaccharide. In addition to granulocytic-macrophagal differentiation, the foci of erythropoiesis appeared. The stimulating effect of lipopolysaccharide was not expressed in nonirradiated mice and during the formation of hemopoietic foci on acetate cellulose membranes in the subcutaneous connective tissue.     

The characteristics of the hematopoietic cells forming colonies on cellulose acetate membranes and their distinctions from other clonogenic cells]

In order to characterize hemopoietic cells forming colonies on membranes of cellulose acetate (CFU-acm) implanted into the peritoneal cavity of mice, we studied the effect of factors stimulating and inhibiting granulocytopoiesis on these cells. Proliferation of CFU-acm can be controlled by humoral factors and this allows us to conclude that they are not identical to CFUs and probably belong to the compartment of early hemopoietic cells of the granulocyte series. We also present evidence for fractional composition, ultrastructure and bone marrow origin of cells belonging to the layer providing for hemopoietic microenvironment for the resulting foci of hemopoiesis; we also present evidence for the role of fibroblasts (fibroblast-like cells) in the maintenance of hemopoiesis. Experiments on transplantation of bone marrow from several rodent species to syn-, allo- and xenogenic recipients allowed us to study interactions of hemopoietic elements of colonies with cells of the underlying layer.     

Hemopoiesis on Stromal Sublayers Formed by Constant Lines of Fibroblasts

We studied the formation of hemopoietic colonies on an artificial sublayer in the peritoneal cavity of mice under the influence of stromal sublayers consisting of fibroblasts of six constant cell limes. All studied lines of stromal cells supported the formation of granulocytic foci and some of them supported the formation of erythroid foci as well. It was shown that hemopoiesis was preserved or, in some cases, enhanced on sublayers of fixed (metabolically inactive) cells. The treatment of fibroblasts by E. coli lipopolysaccharide did not lead, as a rule, to significant stimulation of hemopoiesis.     

Hematopoiesis on cellulose ester membranes (CEM). IV. Effect of variation in membrane composition and pore size

Artificial membranes composed of mixed cellulose nitrate and acetate esters (MCEM), polytetrafluoroethylene (Mitex), cellulose acetate (Celotate), polyvinylchloride (Polyvic), polycarbonate (Nuclepore), or linear polyethylene with pore sizes ranging from 0.45 mu to 70 mu were coated on their inner surfaces with a paste of bone marrow, diaphyseal bone, or cells proliferating in vitro on the inner surfaces of curetted femurs obtained from syngeneic mice. Squares of these membranes were folded into thirds, stapled to form an open-ended, tube-like structure, and implanted i.p. into mice. At intervals of 6-12 months, the membranes were removed and studied histologically. Control membranes not coated with any substance were also studied. Trilineal hematopoiesis, bone formation, sinusoidal structures, fat cells, and hemosiderin-laden macrophages were found with all three coatings in the tubes formed from the MCEM or Mitex membranes. The Celotate and Polyvic membranes coated with bone cells and the Polyvic membranes coated with marrow also developed trilineal hematopoiesis and new bone tissue. The remaining membranes essentially had only fibrous tissue, except for rare foci of hematopoiesis which developed on an occasional 30 mu (but not 70 mu) pore size linear polyethylene membrane. The results of these studies suggest that hematopoiesis and bone cell formation on these artificial membranes is closely linked, and that there may be some interaction between membrane constituents and the cells destined to form the hematopoietic support layer in the fostering of hematopoiesis. Whether this interaction is stimulatory or inhibitory was not defined by these studies.     

Use of the copolymer ethylenevinylacetate for studying the effect of hemopoietic cytokines

Copolymer ethylenevinylacetate is known as a polymeric carrier, which ensures a lasting release of biologically active substances. An attempt has been made to use it for testing the effects of hemopoietic cytokinins. It was shown that erythropoietin, embedded in this polymer, induces formation of erythropoietic foci on acetate-cellulose membranes in the peritoneal cavity of mice.     

Evaluation of the marrow culture on the macrophage layer covering intraperitoneally implanted membrane as an assay for hemopoietic progenitor cells

When cellulose acetate membranes are implanted into abdominal cavity of mice they turn into a foreign body overgrown with macrophages. Such macrophage layer has been shown by other authors to be able to support the growth of hemopoietic colonies formed by intraperitoneally injected hemopoietic cells. This study confirms and extends this observation by showing that both granulopoietic and erythropoietic colonies may be observed. The number of colonies grown is in linear correlation with that of injected hemopoietic cells. The frequency of erythropoietic colonies was greatly enhanced by blood letting of the host mice. Colony forming cells were most numerous in the bone marrow then in the spleen and peripheral blood and hardly in the thymus. Prior irradiation of the host mice was essential for obtaining colony growth and the optimal dose was determined to be 6.0 Gy. This technique opens the way to studies into hemopoietic progenitor cells for laboratories having no sophisticated tissue culture equipment and where necessary reagents are easily available.     

A study of hemopoiesis on a sublayer of peritoneal cells in the presence of hemopoietic cytokines

We studied the effects of erythropoietin and thrombopoietin on the clonogenic capacity and direction of differentiation of the hemopoietic cells that form colonies on acetate cellulose membrane in the peritoneal cavity of mice. An increased level of erythropoietin in the blood of recipient mice after blood letting led to the appearance of erythroid colonies upon transplantation of syngeneic hemopoietic cells but did not affect the differentiation of transplanted xenogeneic (guinea pig) hemopoietic cells. Erythropoietin transported top the stromal sublayer by a polymeric carrier also induced erythroid differentiation, while thrombopoietin transported in a similar way somewhat enhanced megakaryocytopoiesis.     

A Study of Hemopoiesis on Sublayer of Peritoneal Cells in the Presence of Hemopoietic Cytokines

We studied the effects of erythropoietin and thrombopoietin on the clonogenic capacity and direction of differentiation of the hemopoietic cells that form colonies on acetate cellulose membrane in the peritoneal cavity of mice. An increased level of erythropoietin in the blood of recipient mice after blood letting led to the appearance of erythroid colonies upon transplantation of syngeneic hemopoietic cells but did not affect the differentiation of transplanted xenogeneic (guinea pig) hemopoietic cells. Erythropoietin transported top the stromal sublayer by a polymeric carrier also induced erythroid differentiation, while thrombopoietin transported in a similar way somewhat enhanced megakaryocytopoiesis.     

Origin of hematopoietic cells in a syngenous and semisyngenous focus of heterotopic hematopoiesis]

Heterotopic hemopoiesis foci were produced by the bone marrow of C57BL/6 or (CBA X C57BL)F1 mice grafted under the renal capsule of (CBAT6T6XC57BL)F1 mice, bearing the chromosomal translocation. The cytogenetic analysis of the hemopoietic cells in the foci 20 to 120 days after the transplantation showed that in 40% of the transplants only the recipient's hemopoietic cells proliferated, whereas the rest were mosaic and contained on the average less than 20% of donor's cells both in the syngeneic and in the semisyngeneic systems. These characteristics remained stable for at least 4 months. The data obtained suggest a single inflow of not less than 10 effective hemopoietic stem cells per graft. The clone stability indicated that during the steady-state hemopoiesis the cell exchange between various regions of the hemopoietic system was not great, if any.     

Contrasting of Lowicryl K4M thin sections

Summary A method is presented for increasing the contrast of cellular structures on ultrathin sections from tissues embedded in Lowicryl K4M. The method, designated UA/MC adsorption staining, is based on the uranyl acetate/methyl cellulose staining of thawed cryosections. Ultrathin Lowicryl K4M sections were exposed to a uranyl acetate/methyl cellulose solution and the excess solution was removed with filter paper, leaving the remainder to air dry on the section. Sections on the grids were then directly observed in the electron microscope. Parameters such as methyl cellulose and uranyl acetate concentrations, duration of staining, temperature and pH were all assessed for their effect on subsequent contrast formation. Conditions were achieved which yielded intense contrast of cellular membranes, basement membranes and extracellular matrix components usually not apparent in Lowicryl K4M thin sections routinely counter-stained with uranyl acetate and lead acetate. The enhancement of the contrast of these structures does not obscure colloidal gold particles used for immunocytochemistry or lectin labeling, thus making the UA/MC adsorption staining method useful for increasing membrane contrast in routine post-embedding immuno- and lectin cytochemistry on Lowicryl K4M thin sections.     

Contrasting of Lowicryl K4M thin sections.

A method is presented for increasing the contrast of cellular structures on ultrathin sections from tissues embedded in Lowicryl K4M. The method, designated UA/MC adsorption staining, is based on the uranyl acetate/methyl cellulose staining of thawed cryosections. Ultrathin Lowicryl K4M sections were exposed to a uranyl acetate/methyl cellulose solution and the excess solution was removed with filter paper, leaving the remainder to air dry on the section. Sections on the grids were then directly observed in the electron microscope. Parameters such as methyl cellulose and uranyl acetate concentrations, duration of staining, temperature and pH were all assessed for their effect on subsequent contrast formation. Conditions were achieved which yielded intense contrast of cellular membranes, basement membranes and extracellular matrix components usually not apparent in Lowicryl K4M thin sections routinely counter-stained with uranyl acetate and lead acetate. The enhancement of the contrast of these structures does not obscure colloidal gold particles used for immunocytochemistry or lectin labeling, thus making the UA/MC adsorption staining method useful for increasing membrane contrast in routine post-embedding immuno- and lectin cytochemistry on Lowicryl K4M thin sections.     

Enzymatic protein hydrolysis in a membrane reactor related to taste properties

Potato, Vicia faba and soybean proteins were hydrolysed enzymatically in a substrate feed membrane reactor system. Alkaline proteolytic enzymes and PM-10 membranes were used for the hydrolysis of potato protein. The taste of the ultrafiltrates, which was unpleasantly bitter and potato-like, was improved by application of gelatin. Also using PM-10 membranes, Vicia faba protein isolate was hydrolysed by alkaline and acid proteolytic enzymes. The bitterness of the ultrafiltrate decreased with the formation of an isoelectric precipitate, which was probably due to association of hydrophobic peptides. The reactor equipped with a cellulose acetate membrane delivered an acceptable enzymatic hydrolysate of Promine D during the first hours of ultrafiltration. This was not the case when similar processes were performed using non-cellulosic DM-5 membranes. The usefulness of ultrafiltration for obtaining bland protein hydrolysates seems to be limited to short-term processes with cellulose acetate membranes.     

Inability of Microorganisms To Degrade Cellulose Acetate Reverse-Osmosis Membranes

Operational cellulose acetate reverse-osmosis membranes were examined for evidence of biological degradation. Numerous fungi and bacteria were isolated by direct and enrichment techniques. When tested, most of the fungi were active cellulose degraders, but none of the bacteria were. Neither fungi nor bacteria were able to degrade cellulose acetate membrane in vitro, although many fungi were able to degrade cellulose acetate membrane after it had been deacetylated. Organisms did not significantly degrade powdered cellulose acetate in pure or mixed cultures as measured by reduction in acetyl content or intrinsic viscosity or production of reducing sugars. Organisms did not affect the performance of cellulose triacetate fibers when incubated with them. The inability of the organisms to degrade cellulose acetate was attributed to the high degree of acetate substitution of the cellulose polymer. The rate of salt rejection decline was strongly correlated with chlorination of feed water and inversely with densities of microorganisms. These data suggest that microbial degradation of operational cellulose acetate reverse-osmosis membranes is unlikely.     

Cellulose acetate impregnated with polyacrylamide: a novel medium for electrophoresis

Cross-linked polyacrylamide gel containing a low proportion of methylenebis-acrylamide has been incorporated into cellulose acetate membranes. Unlike on cellulose acetate itself, electrophoresis on these modified membranes enables molecular sieving of proteins under a wide range of conditions. By modifying only part of the membrane, samples can be loaded in the normal way and sharpen as they migrate across the cellulose acetate-polyacrylamide boundary. The thin membranes retain their general ease of handling and speed of staining and destaining.     

Postnatal liver hemopoiesis in mice: generation of pre-B cells, granulocytes, and erythrocytes in discrete colonies

Morphologic analysis of hemopoietic tissue in mouse liver reveals the persistence of erythropoietic, granulopoietic, and lymphopoietic activity for approximately 2 wk after birth. Near the end of the first postnatal week, we noted a remarkable reorganization of the hemopoietic cells that was characterized by a transition from a diffuse distribution of mixed erythroid, myeloid, and lymphoid elements to a focal pattern of discrete hemopoietic colonies scattered among the cords of hepatic parenchymal cells. Each hemopoietic focus contained cells progressing along a single differentiation pathway (i.e., erythroid, myeloid, or lymphoid cells). Megakaryocytes were seen as solitary cells surrounded by hepatocytes. This pattern of colonization was observed in all strains of mice examined. In the livers of mice with known hemopoietic defects, however, differences were found in the duration of postnatal hemopoiesis. Accessory cells with macrophage-like features were consistently observed in erythropoietic foci, but were rarely seen in lymphoid foci. The latter were formed by pre-B cells identifiable by the presence of cytoplasmic mu-heavy chains and the absence of light chain expression. The occurrence of discrete colonies of erythroid, myeloid, and pre-B lymphoid cells in the postnatal liver suggests that each is derived from a single, committed precursor cell. This anatomical compartmentalization according to cell type offers a useful model system for analysis of hemopoietic differentiation and of the generation of clonal diversity among B lineage cells.     

Enhancing frequency of regeneration of somatic embryos of avocado (Persea americana Mill.) using semi-permeable cellulose acetate membranes

Regeneration of avocado via somatic embryogenesis is difficult due to poor embryo maturation, resulting in low frequencies of germination. In this study, the influence of semi-permeable cellulose acetate membranes and culture media, containing high levels of sucrose along with coconut water, on maturation and germination of somatic embryos of avocado have been evaluated. The culture of embryogenic calli on top of cellulose acetate membranes significantly increased the number of mature, white-opaque embryos that were recovered after 5 weeks of culture. These embryos showed a much more normal appearance and better quality compared with the control embryos, although the embryo size was significantly reduced. To increase the embryo size and to complete maturation, several two-step maturation treatments were tested. The culture of white-opaque somatic embryos in a modified MS medium with B5 macronutrients gelled with 10 g L^?1 agar (B5m10A medium) over a 5-week period, followed by 5 additional weeks in B5m10A with 45 g L^?1 sucrose and 20 % coconut water, yielded the best results, reducing the percentage of necrotic embryos and the number of calli formed. The beneficial effects of this maturation treatment were enhanced when using embryos that were pre-matured on cellulose acetate membranes. Following this two-step maturation treatment, the germination rate of the control somatic embryos, which were not cultured on cellulose membranes, was lower than 10 %, but it significantly improved when the embryos had been pre-matured on cellulose acetate membranes for 5 weeks, reaching a germination rate close to 40 %. The water availability was significantly reduced when somatic embryos were cultured on cellulose membranes, and after this pre-maturation treatment, the white-opaque embryos showed lower water potential and ABA content compared with the control embryos. These results suggest that culturing over cellulose membranes causes a controlled embryo desiccation that enhances the recovery of plants.     

Effect of various factors on the differentiation of hematopoietic stem cells]

The treatment in vitro of bone marrow cells in mice by phytohemagglutinin, concanavaline, or antilimphocytic globulin resulted in the suppression of exogenous hemopoietic colonies in the spleen of lethally irradiated (830r) syngenic recipients, whereas lipopolysaccharide, tuberculin, anti-theta serum or nati-gamma-globulin serum exerted no influence on the colony-forming function of hemopoietic stem cells. The morphological analysis of the ratio and cell composition of hemopoietic colonies has revealed no marked differences between the experimental and control groups. The suppression of hemopoietic stem cells by mitogens might be due both to their direct effect and indirect one, possibly, through a humoral factor.     

Experimental research on the cellular interactions during hemopoiesis

We present a review of experimental studies performed at the Laboratory of Histogenesis of the Institute of Developmental Biology, Russian Academy of Sciences, on the problem of cell interactions during hemopoiesis. Special attention has been given to original experimental models, such as production of hemopoietic foci on underlayers of fibroblasts encapsulating a foreign body in the peritoneal cavity of rodents (after intraperitoneal transplantation of hemopoietic cells) and repopulation of ectopic hemopoietic territories under the kidney capsule of mice by syngeneic or xenogeneic hemopoietic cells. We describe the competitive interactions of genetically different hemopoietic cells after the transplantation of their mixtures to irradiated mice (multicomponent radiation chimeras). Xenogeneic and multicomponent chimeras have also been obtained in long-term bone marrow culture. We have examined characteristics of hemopoiesis on stromal cell underlayers produced by cells of various origins in vitro and then transplanted into the peritoneal cavity of irradiated mice. We discuss the results obtained and possible mechanisms of these phenomena.     

Visualization of pores (export sites) correlated with cellulose production in the envelope of the gram-negative bacterium Acetobacter xylinum

The Gram-negative bacterium Acetobacter xylinum assembles a cellulse ribbon composed of a number of microfibrils in the longitudinal axis of its envelope. The zone of ribbon assembly was investigated by freeze-etch electron microscopy. Freeze-etching revealed, beneath the cellulose ribbons, a linear array of pores on the lipopolysaccharide membrane. These pores have a rim diameter of 120--150 A and a central hole or deepening of approximately 35 A. The axes of pore arrays closely coincide with linear arrays of 100 A particles on the E- and P-faces of the fractured lipopolysaccharide membranes. Pores and particles in the lipopolysaccharide membrane are probably congruent. The pores are hypothesized to be the export sites (penetration sites) for cellulose.     

Stimulating action of Brucella melitensis lipopolysaccharide on hemopoiesis in mice

The bacterial mass, brucellar protective antigen and lipopolysaccharide (LPS) obtained from B. melitensis stimulated the formation of endogenous colonies in the spleen of mice belonging to different strains, subjected to irradiation in sublethal doses. The maximum stimulating effect was observed when the antigens were introduced 24 hours prior to irradiation. LPS introduced in the optimal dose induced an increase in the number of hemopoietic stem cells (HSC) in the s-phase of the cell cycle, thus stimulating the postirradiation survival of mice irradiated in a lethal dose. 24 hours after the injection of LPS the total number of HSC in the spleen increased 1.5 times. These data indicate that LPS has a stimulating effect on hemopoiesis in mice. The effect rendered by LPS is seemingly linked with an increase in the proliferation of HSC and, to a lesser extent, depends on changes in the migration of HSC.