Biomedical subjects

N S Wolf

Publications and source records attributed to N S Wolf.

At least 19 recordsLinked to original sources

PubMed1992

An age-related reduction in the replicative capacity of two murine hematopoietic stroma cell types.

Two stromal cell types, myofibroblasts and endothelial-like cells, that were identifiable by structural and antigenic specificities, were obtained from murine bone marrow and spleen of young, middle-aged, and old mice of two strains and sexes and grown in liquid culture for 9 or 10 days. As expected, there were more total nucleated cells per organ in the old mice (with larger organs) than in the young mice. However, the concentration of stromal colony forming cells was greater in the young mice, resulting in the number of colony forming cells per organ not being significantly different in most comparisons. The in vitro replicative capacity of the two stromal cell types from both organs in all age groups was determined by clone size distribution assays. In all instances the number of cell doublings achieved was statistically significantly greater in the stromal cell clones from young mice than those from old mice. The cell doubling capacity of the middle-aged mice fell between that of the young and the old mice and in most instances that difference was also statistically significant. It was concluded that these in vitro findings constituted a biomarker of aging in these tissues and that this was significant in relation to previous in vivo and in vitro work by these authors and by others reporting the inferiority of aged bone marrow and spleen stroma to regenerate and to support hematopoiesis.

Aging↗

PubMed1990

Transplantation of hematopoietic stem cells obtained by a combined dye method fractionation of murine bone marrow.

Hematopoietic stem cells were purified from murine bone marrow cells (BMC). Their characteristic density, size, internal complexity, Hoechst 33342 dye uptake, and wheat germ agglutinin (WGA) affinity were used to distinguish them from other cells in the bone marrow. BMC suspensions were centrifuged over Ficoll Lymphocyte Separation Media (Organon Teknika, Durham, NC; density 1.077 to 1.08). The lower-density cells were drawn off, stained with Hoechst and labeled with biotinylated WGA bound to streptavidin conjugated to phycoerythrin (WGA-B*A-PE) or with WGA conjugated to Texas Red. These cells were then analyzed and sorted by an Ortho Cytofluorograph 50-H cell sorter. The cells exhibiting medium to high forward light scatter, low to medium right angle light scatter, low Hoechst intensity, and high WGA affinity were selected. Sorted BMC (SBMC) were stained with Romanowsky-type stains for morphologic assay, and were assayed in lethally irradiated (LI) mice for their ability to produce colony-forming units in the spleen (CFU-S) and for their ability to produce survival. The spleen seeding factor for day 8 CFU-S upon retransplantation of the isolated cells was 0.1. The isolated cells were found to have consistent morphology, were enriched up to 135-fold as indicated by day 8 CFU-S assay, 195-fold as indicated by day 14 CFU-S assay, and 150 sorter-selected BMC were able to produce long-term survival in LI mice with retention of donor karyotype. When recipients of this first transplantation were themselves used as BMC donors, their number of day 8 and day 12 CFU-S were found to be reduced. However, 3 X 10(5) of their BMC provided 100% survival among secondary recipients. When the previously SBMC were competed after one transplantation against fresh nonsorted BMC in a mixed donor transplant, they showed the decline in hematopoietic potency normally seen in previously transplanted BMC. We conclude that the use of combinations of vital dyes for fluorescence-activated cell sorting (FACS) selection of survival-promoting murine hematopoietic stem cells provides results comparable with those produced by antibody-selected FACS and has the advantage of a method directly transferable to human BMC.

Animals↗

PubMed1990

The effect of long-term caloric restriction on function of T-cell subsets in old mice.

The effect of caloric restriction (from weaning to old age) on CD3-stimulated CD4+ and CD8+ lymphocyte proliferation and calcium mobilization was examined. Young ad libitum (ad lib) fed, old ad lib fed, old calorically restricted, and old calorically restricted mice which were fed ad lib during the last 6 weeks of their life (restricted/refed) were compared in both BDF1 [(C57BL/6 x DBA/2)F1] and C57BL/6 mice. Proliferation of CD4+ cells was lower in old ad lib animals than in young animals; this difference was not seen in CD8+ cells. Those CD4+ cells which did proliferate in old ad lib animals underwent similar cell cycle progression as young cells. In calorically restricted and calorically restricted/refed animals, CD4+ cell proliferation was similar to the young animals, and CD8+ cells showed a higher proliferative capacity than cells from either young or old ad lib mice. Differences in proliferative capacity were not correlated with alterations in transmembrane signaling efficiency as peak [Ca2+]i was reduced in both T-cell subsets in all groups of old mice relative to young mice. Additionally, reduced [Ca2+]i was observed in the CD8+ subset for which there was no deficit in proliferation, and the enhanced proliferation seen in old restricted and old restricted/refed mice did not manifest as increased [Ca2+]i mobilization. The percentage of CD4+ cells from both mouse strains was reduced in all groups of old mice compared with young mice, while the percentage of CD8+ cells was generally similar in young and all groups of old mice. Our studies would suggest that lifelong caloric restriction of mice prevents the age-associated decrease in T-cell proliferative capacity but that the enhanced proliferation of these cells is not due to increased efficiency of transmembrane signaling.

Aging↗

PubMed1990

Transplantation studies in mice with congenital hemolytic anemia.

Sphha/sphha anemic mice have an abnormality in the erythroid membrane protein, alpha spectrin, and exhibit multiple related clinical abnormalities, including spherocytosis, shortened red cell survival, chronic hemolysis, hemosiderosis, and extramedullary hematopoiesis. In addition, these mutant mice exhibit a granulocytosis and lymphocytosis, lymph node hyperplasia, elevated serum immunoglobulins, membranoproliferative glomerulonephritis, and decreased lifespan--abnormalities that are less clearly attributable to a spectrin defect. In order to further elucidate the mechanisms of disease in these animals, we undertook a series of bone marrow transplantation experiments. Transplantation of anemic marrow into lethally irradiated congenic +/+ mice resulted in chronic spherocytosis, hemolytic anemia, peripheral leukocytosis, and extramedullary hematopoiesis. Additionally, transplant recipients of anemic marrow which had received a higher radiation dose (12 Gy) had increased numbers of peripheral blood CD4+ and CD8+ lymphocytes, a hypocellcular thymus, and a severe pneumonitis characterized by nodular areas of consolidation and edema. Mice receiving congenic +/+ marrow and irradiated with the same radiation dose exhibited minimal pulmonary abnormalities. Anemic mice transplanted with congenic +/+ marrow usually died, but the survivors exhibited reversal of some clinicopathological changes. These results would suggest that the clinical abnormalities of sphha/sphha mice are in part attributable to abnormalities of hematopoietic stem cells but may also involve defects in other cell types. The pathogenesis of the accompanying lymphoid abnormalities observed in this mutant anemic mouse and any correlation with the erythroid spectrin defect are presently unknown. The pulmonary disease that develops in the transplant recipients of anemic marrow needs to be characterized further but may represent a unique model of lung injury.

Anemia, Hemolytic, Congenital↗

PubMed1990

Dissecting the hematopoietic microenvironment. VIII. Clonal isolation and identification of cell types in murine CFU-F colonies by limiting dilution.

In this study it was found that three kinds of cells, fibroblasts, endothelial cells, and macrophages, exist in fibroblastic colony-forming unit (CFU-F) colonies, consistent with the findings of others. However, when low enough bone marrow stromal cell numbers were cultured per dish, pure fibroblast, endothelial, and macrophage colonies were observed. When the cell number cultured in each dish was increased, all colonies were of mixed cell type. This evidence strongly suggests that the macrophages and endothelial cells contained in CFU-F colonies come from locally stimulated growth resulting in contamination and not from a common progenitor with fibroblasts or each other. In some longer term experiments, adipocytes were seen to appear within the colonies of late-passage pure fibroblast cultures.

Animals↗

PubMed1990

A method to establish pure fibroblast and endothelial cell colony cultures from murine bone marrow.

Studies on the effect of the microenvironment on hematopoiesis would benefit from the availability of pure populations of nontransformed cells of each of the stromal cell types. The adherent murine bone marrow stromal cell population in this study consisted of fibroblasts, endothelial cells, and macrophages. Fibroblasts were segregated from the phagocytic endothelial cells and macrophages by allowing the phagocytic cells to ingest magnetic beads, with subsequent exposure to a magnetic field, effecting cell separation. Pure colony cultures of fibroblasts and endothelial cells were formed by varying the bead-to-cell ratio and incubation period of the cells. For complete purification of the fibroblasts, subsequent passaging was also necessary. Near confluent growth of each type was obtained with subsequent passages and sustained culture. The cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) was used to enhance endothelial cell growth. We were not able to obtain pure populations of bone marrow macrophages in near confluent culture. The three cell types were identified by cellular morphology, acid and alkaline phosphatase staining, binding with the lectins Ulex europaeus and Bandeiraea simplicifolia, and the capacity to stain for the factor VIII-related antigen (von Willebrand's Factor).

Alkaline Phosphatase↗

PubMed1990

Dissecting the hematopoietic microenvironment. VI. The effects of several growth factors on the in vitro growth of murine bone marrow CFU-F.

The effects of several growth factors on the proliferation of fibroblastic colony-forming units (CFU-F) were studied. In the present study CFU-F colonies were found to consist of fibroblasts, macrophages, and endothelial cells. Growth factors, including interleukin 3 (IL-3), interleukin 1 alpha (IL-1 alpha), epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), macrophage colony-stimulating factor (M-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and buffalo rat liver cell-conditioned medium (BRL-CM) were tested for stimulation of the proliferation of CFU-F in a standard culture in both 2% and 15% serum. Overall, the colony numbers produced in 15% serum were much higher than in 2% serum with or without growth factors. However, the influence of several growth factors on CFU-F cultured in 2% serum was relatively greater than in 15% serum when compared to controls. The stimulation of CFU-F by FGF only occurred in culture with 15% serum, and the stimulation by PDGF only occurred with 2% serum. Overall, the strongest stimulations were produced by PDGF, IL-3, and BRL-CM. Combining the other growth factors with IL-3, PDGF, or IL-1 alpha enhanced their effects only modestly. The stimulation by growth factors included increases of the cell numbers between and within colonies as well as an increase in the number of colonies. The study produced results that suggest a complex interaction mediated by growth factors between fibroblasts and other stromal cells within the CFU-F colonies and within the bone marrow itself.

Animals↗

PubMed1990

Dissecting the hematopoietic microenvironment. VII. The production of an autostimulatory factor as well as a CSF by unstimulated murine marrow fibroblasts.

Purified normal murine bone marrow-derived fibroblasts were shown to produce a factor that stimulates the in vitro growth of fibroblastic colony-forming unit (CFU-F) colonies. Conditioned medium from the purified fibroblasts (F-CM) also stimulated pure marrow fibroblasts themselves. Analysis of the F-CM detected the presence of macrophage colony-stimulating factor (M-CSF), and low levels of interleukin 1 (IL-1) and interleukin 6 (IL-6), but no detectable levels of interleukin 3 (IL-3), interleukin 5 (IL-5), granulocyte-macrophage colony-stimulating factor (GM-CSF), or granulocyte colony-stimulating factor (G-CSF). Macrophages and endothelial cells, freed from other bone marrow components, required the F-CM if no other growth factors were added. We conclude that F-CM contains an autocrine factor, which the evidence suggests is IL-1, for bone marrow fibroblasts, and a paracrine factor (CSF-1) for macrophages and/or endothelial cells.

Animals↗

PubMed1989

Cycling patterns of hemopoietic stem cell subpopulations in young and old BDF1 mice.

We have previously reported that two or more different subpopulations of bone marrow stem cells exist in mice as determined by cycling status of day-8 and day-14 CFU-S in long term bromodeoxyuridine (BrdU) infusion studies. In the present report, comparisons between cycling of stem cell subpopulations in old and young mice show that, while the general patterns persist, there are some statistically significant differences between corresponding time points of early and late CFU-S cycling patterns in young and old BDF1 mice. In both populations of CFU-S there exist cells which do not enter cycle over a five week period. The method which we have employed allowed the cycling measurements to be made in unstimulated steady-state bone marrow cell populations, since no cell death is caused in vivo.

Aging↗

PubMed1988

Life table analysis and pathologic observations in male mice of a long-lived hybrid strain (Af X C57BL/6)F1.

We have utilized a long-lived (Af X C57BL/6)F1 hybrid strain of mice for a variety of aging studies. In this report we have characterized the life expectancy and pattern of spontaneous deaths in 202 mice, malignant and nonmalignant lesions in 64 male mice dying spontaneously, and organ weights and lesions in 39 male mice killed at selected ages. The maximum age observed was 41.5 months. The principal causes of death were malignant lymphoma and alveologenic neoplasms, which were present in 56.3% and 45.3%, respectively, of the mice dying spontaneously. A variety of other neoplastic and non-neoplastic lesions that are not infrequently seen in older mice were observed in these mice. Neoplasms seen in these mice that are rare in other mice included disseminated mast cell tumors in two mice and gastric adenocarcinoma in one mouse. In comparing the diseases observed in this hybrid strain with those reported for the parent strains, there was an incidence of malignant lymphoma similar to the C57BL/6 parent, an incidence of alveologenic neoplasms intermediate between the parent strains, and a markedly reduced incidence of amyloidosis. This study provides a detailed background of baseline hematologic and morphologic data in a long-lived hybrid of two commonly used strains of mice.

Actuarial Analysis↗

PubMed1988

Evaluation of stem cell reserve using serial bone marrow transplantation and competitive repopulation in a murine model of chronic hemolytic anemia.

Serial transplantation and competitive repopulation were used to evaluate any loss of self-replicative capacity of bone marrow stem cells in a mouse model with increased and persistent hemopoietic demands. Congenic marrows from old control and from young and old mice with hereditary spherocytic anemia (sphha/sphha) were serially transplanted at 35-day intervals into normal irradiated recipients. Old anemic marrow failed or reverted to recipient karyotype at a mean of 3.5 transplants, and young anemic marrow reverted at a mean of 4.0 transplants, whereas controls did so at a mean of 5.0 transplants. In a competitive assay in which a mixture of anemic and control marrow was transplanted, the anemic marrow persisted to 10 months following transplantation; anemic marrow repopulation was greater if anemic marrow sex matched with the host. It is possible that lifelong stress of severe anemia decreases stem cell reserve in the anemic sphha/sphha mouse marrow. However, marginal differences in serial transplantation number and the maintenance of anemic marrow in a competition assay would suggest that marrow stem cells, under prolonged stress, are capable of exhibiting good repopulating and self-replicating abilities.

Aging↗

PubMed1988

Clinicopathologic features of young and old sphha/sphha mice. Mutants with congenital hemolytic anemia.

A colony of mice with congenital hemolytic anemia, sphha/sphha, were evaluated over a 3-year period. Prominent findings included decreased survivability, reticulocytosis, increased peripheral blood leukocytes, extramedullary hematopoiesis in liver and spleen, lymphoid hyperplasia and membranoproliferative glomerulonephritis. Older (12 to 21 months) anemic animals had elevated serum levels of IgG1 and IgA. There was deposition of C3, IgG, IgM, and IgA in renal glomeruli of both control and anemic mice, but deposition of IgM and IgA was more prominent and widely distributed in anemic animals and correlated with mesangial expansion and the presence of electron dense deposits in the mesangium and in glomerular capillary walls. Prominent renal tubular hemosiderosis was noted in young and old anemic mice. The relation between the hemolytic anemia and glomerular disease is unclear but these mice may be an animal model useful for exploration of changes attendant with chronic hemolysis and evaluation of renal disease that accompanies hemolytic anemia.

Age Factors↗

PubMed1986

Kinetics of early and late spleen colony development.

The kinetics of spleen colony development were studied with special emphasis upon the self-replicative capacity of CFU-S, which are responsible for the early- or late-appearing spleen colonies, and upon CFU-S migration in the days following transplantation. It was found that, while the spleen colonies had a much lower daughter CFU-S content at eight days than at 11 or 12 days, many of the 8-day colonies remained in the spleen to become 12-day colonies which had many daughter CFU-S. In addition, it was found that large numbers of CFU-S were present in the splenic space between the colonies. Evidence was developed that these "intercolonial space" CFU-S were the founders of many of the late-appearing spleen colonies and that many or all of them were transported from the bone marrow to the spleen via the blood at varying times after the transplantation. Approximately one-half of the day-12 colony founders were found to arrive in the spleen after the third posttransplantation day.

Aging↗

PubMed1985

The decline in murine splenic PHA and LPS responsiveness following serial transplantation of young and old bone marrow cells.

An analysis was made of the ability of bone marrow cells derived from young (6 months) or old (28 months) murine (A X C57BL/6) donors to differentiate to B and T lymphocytes following serial bone marrow cell transplantations into groups of young syngeneic lethally irradiated recipients. Both number and mitogenic responsiveness of splenic B and T lymphocytes were recorded for recipients of young or old bone marrow cells 9 months following each serial transplant. The data suggests that lymphopoietic stem cells senescence in a manner analogous to that predicted by the clonal succession model of ageing.

Aging↗

PubMed1985

The decline in murine splenic PHA and LPS responsiveness with age is primarily due to an intrinsic mechanism.

Changes in splenic B and T lymphocyte number and mitogenic activity with age were quantitated in (A X C57BL/6)F1 (AB6F1) hybrid mice. Although both the B and T lymphocyte proliferative reactivity to their respective mitogens, lipopolysaccharide (LPS) and phytohemagglutinin (PHA), declined significantly with age, an earlier and more marked reduction was recorded for the T cell response. The decline in B and T lymphocyte mitogenic activity with age could not be correlated with a corresponding reduction in the percentage of splenic B or T lymphocytes. The main focus of this study was to determine if the reduction in T and B lymphocyte mitogenic activity with age results primarily from a mechanism intrinsic to the lymphoid lineage itself or from adverse extracellular factors that increase with age. Bone marrow cells (BMC) derived from individual young and old donor AB6F1 mice were transplanted into the neutral environment of young, lethally irradiated syngeneic recipients. Number and mitogenic activity of splenic T and B lymphocytes were recorded for the original BMC donors as well as for the recipients of the young and old BMC lines 9 mo after the BMC transplants. A predominance of the donor (male) rather than recipient (female) karyotype within the mitogen-responding populations of recipient mice confirmed a donor BMC take. The PHA and LPS response levels exhibited by the old donors were 30% and 70% of those of the young donors, respectively. These differences in PHA and LPS reactivity recorded between young and old donors were maintained between recipients of young and old donor BMC lines. Thus, even under the influence of a young recipient environment, old BMC were incapable of giving rise to mitogen responding cells with a functional competence equivalent to that of their younger counterparts. This finding would lend further support to the theory that an intrinsic mechanism is responsible for the decline in murine mitogenic activity with age.

Aging↗

PubMed1985

A technique for the daily examination of spleen colonies in mice.

We have developed a surgical method and an insertable device for viewing the daily changes in number, shape, and development of hemopoietic spleen colonies in mice. With it we have been able to follow the spleen colony changes in individual animals over the period when macroscopic or moderately magnified counts of spleen colonies are customarily made. We have found that about half of the spleen colonies present on day 8 after transplantation do remain through day 12, while an approximately equal number disappear during this period. Further, the colonies that disappear are replaced by an equal or larger number of newly developing colonies at roughly the same temporal sequence as their disappearance. We speculate that these late-appearing colonies may be late arrivals from the recipient's previously seeded bone marrow.

Animals↗

PubMed1985

Normal cycling patterns of hematopoietic stem cell subpopulations: an assay using long-term in vivo BrdU infusion.

It was found in a long-term bromodeoxyuridine (BrdU) infusion study that two or more different subpopulations of bone marrow stem cells exist in mice. One of these subpopulations appears to be noncycling and forms approximately 10% of eight-day CFU-S. Another one, a subpopulation of slowly cycling bone marrow cells, is represented as 14-day CFU-S. The 14-day CFU-S have a regular increment in the percentage of the subpopulation entering the cycle over time, with a cell generation half-time of 21 days. The cycling status in these experiments was ascertained by in vivo continuous long-term BrdU infusion. An improved method is presented for long-term BrdU infusion with UV killing of cycled cells.

Animals↗

PubMed1984

Effect of a neutrophilia-inducing tumor on hemopoietic stem cells in mice.

The influence of neutrophilic stimulation on hemopoietic stem cells was studied in mice with tumor-induced neutrophilia. Transfusions of marrow cells from normal and neutrophilic tumor-bearing mice into lethally irradiated normal and tumor-bearing mice were performed. The number and the erythroid:granuloid (E:G) ratio of day 7 colonies in the recipient spleens and bones as well as the size of spleen colonies of recipient animals were determined. The E:G ratio of spleen and bone marrow colonies between normal and tumor-bearing mouse recipients and the number of spleen colonies did not differ significantly in either experiment. However, spleen colonies which developed in tumor-bearing irradiated mice were significantly larger than those which developed in normal recipients in both experiments. These studies indicated that while the line of differentiation taken by hemopoietic stem cells was not affected by the neutrophilic influence of the tumor, the tumor-bearing host environment appeared to enhance proliferation of transfused stem cells and/or their descendants. The stimulators of granulocytopoiesis in this model of neutrophilia appear to act on a population of progenitor cells more mature than the stem cells capable of forming 7-day colonies in the spleen and bone marrow of irradiated recipient mice.

Animals↗