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K B Udupa

Publications and source records attributed to K B Udupa.

At least 37 records · Page 2Linked to original sources
PubMed1987

Effect of donor and culture age on the function of neutrophils harvested from long-term bone marrow culture.

Neutrophils harvested from the supernatants of long-term bone marrow cultures that were initiated from young and old mice were tested for their functional activity. Superoxide generation was measured, as was the secretion of the enzymes lysozyme, myeloperoxidase, and glucuronidase, both basally and following stimulation by phorbol myristate acetate. The function of the neutrophils harvested from cultures that were initiated from young mice was identical to that of cells derived from young mouse peritoneal cavities at the time of death. A minimal decline in function in culture-derived cells was seen over the 15-18 weeks of study. For each of the above measurements examined, neutrophils obtained from cultures initiated from old mice gave values significantly lower than those in neutrophils recovered from cultures initiated from young mice. For some parameters (superoxide generation, myeloperoxidase), the rate of decline in function was more rapid for neutrophils from old rather than young mouse cultures. For other measurements the rates of decline were equal. The results indicated adequate neutrophil function in long-term bone marrow culture for extended periods of time. They also demonstrated that the age-related diminution in neutrophil function observed in vivo persisted in in vitro culture.

Aging↗
PubMed1987

Role of colony-stimulating factor in myelopoiesis in murine long-term bone marrow cultures.

Weekly medium change or midweek feeding of long-term bone marrow cultures (LTMBCs) results in a significant increase in total myeloid cell production. Proliferative myeloid cells peak 48 hours after feeding, and nonproliferative myeloid cells reach maximum levels at 72 hours. This increase in myelopoiesis is invariably preceded by a significant elevation in biologically and immunologically measurable colony-stimulating factor (CSF) in the supernatants of LTBMC. The level peaks 24 hours after medium change, then gradually returns to basal values. The decrease in CSF relates to its consumption by generating myeloid precursors because no fluctuation in the levels occur in cultures without active myelopoiesis. No significant inhibitors or promoters of CSF were detected. When highly purified L cell CSF, CSF in lung-conditioned medium, or CSF concentrated from LTBMC supernatant is added to cultures, an identical increase in myelopoiesis occurs. Anti-CSF antiserum, added to culture at the time of medium change, totally neutralizes supernatant CSF levels but does not affect myelopoiesis. These findings suggest a potential regulatory role for CSF in myelopoiesis in LTBMC. CSF appears to function within the microenvironment through a mechanism involving cell:cell interactions or by causing the production of other substances that stimulate myelopoiesis. Because exogenous CSF stimulates myelopoiesis, it is likely that it too can react either directly or through microenvironmental cells to stimulate primitive myeloid cells to divide.

Animals↗
PubMed1987

Evidence that microenvironmental factors account for the age-related decline in neutrophil function.

We measured the function of neutrophils harvested from the supernatant of long-term marrow cultures in which stromal cell cultures derived from young mice were recharged with hematopoietic cells from old mice and vice versa. The functions measured were superoxide generation and enzyme secretion (lysozyme and glucuronidase), following cell activation by either phorbol myristate acetate (PMA) or Formyl-methionyl-leucyl-phenylalanine (FMLP). In addition we measured cytosolic calcium concentration and its increase following activation by FMLP. In all culture combinations recharge resulted in the recovery of greater than 2 X 10(6) cells/flask (95% neutrophils, 98% viable). Histologic studies of cytoplasmic markers indicated that recovered neutrophils were derived from the stem cell population employed for recharge. For each neutrophil parameter measured, function was markedly improved when old hematopoietic stem cells were recharged onto a young stroma and was significantly diminished when young stem cells were recharged onto an old stroma. This applied to superoxide generation, basal and stimulated enzyme levels, and to basal cytosolic calcium concentration and its increase following activation by FMLP. These results indicate that when old hematopoietic stem cells proliferate in a young microenvironment, neutrophil function returns virtually to normal. Conversely, function diminishes when young stem cells proliferate in an old stroma. These findings demonstrate, for the first time, that neutrophil function is modulated by microenvironmental factors, hormonal, cellular, or matrix, which are decreased in the elderly. That an age-related decline in function is extrinsic to the cell and is reversible has significance for the study of neutrophil function and of cellular aging and has potential therapeutic implications.

Aging↗
PubMed1986

Influence of aging and protein deficiency on neutrophil function.

This study examined the effect of age and protein deficiency on the function of mouse neutrophils. Compared with appropriate controls aging and protein deficiency caused significant reductions in respiratory burst activity, exocytosis, and enzyme release from neutrophils. Individually, neither aging nor protein deficiency caused decreases in the ability of the neutrophil to phagocytose or kill bacteria. When aged animals were fed a protein deficient diet, however, further reductions in neutrophil function occurred that resulted in significant decreases in phagocytosis and bacterial cell kill. These findings indicate that both aging and protein deficiency compromise neutrophil function. When both variables are present the abnormalities become sufficient to affect the neutrophils' most critically important functions. The results emphasize the importance of protein deficiency in the aged and help explain the high prevalence of bacterial infection in malnourished older individuals.

Aging↗
PubMed1986

In vitro culture of proerythroblasts: characterization of proliferative response to erythropoietin and steroids.

This study characterized variables affecting the in vitro liquid culture of proerythroblasts. When bone marrow from mice depleted of haemoglobin containing cells, was cultured in vitro in the presence of human urinary erythropoietin (Ep) a significant degree of erythroid cell proliferation and maturation occurred as measured directly by 3H-thymidine (3H-TdR) incorporation into DNA (autoradiographical measurement). Proliferation increased in direct proportion to the dose of Ep added to the culture. We also demonstrated a highly significant positive correlation between proliferation measured directly by 3H-TdR incorporation into DNA or indirectly by 59Fe incorporation into haem. Ep was a potent stimulator of proerythroblast proliferation. We also examined the role of the androgenic and non-androgenic steroids on in vitro proliferation. All the hormones tested were stimulatory but only in the presence of Ep. The androgenic steroids primarily affected the more mature erythroid precursors whereas the glucocorticoids were more general growth promoters. Their addition in physiologic concentration to liquid culture reduced Ep requirements. Thus when both testosterone and hydrocortisone were added to culture the Ep concentration that produced the same degree of proliferation as a culture containing Ep alone was decreased by 90%. This finding is important as it indicates that in vitro culture conditions can be created that more closely mimic in vivo erythropoiesis where Ep requirements are far less.

Androgens↗
PubMed1986

Studies on the kinetics of the erythroid colony-forming cell.

The appearance, rate of formation, and morphologic characteristics of the erythroid colony-forming cell (CFU-E) was studied in control, exhypoxic polycythemic, and anemic mice. Polycythemia resulted in a significant reduction in CFU-E number and a lag time of approximately 8 h before colonies were visible in culture. Thereafter, the rate of formation of colonies, as assessed by the slope of the increase in their number, was identical to the control. The slower appearance of CFU-E in polycythemia was corrected by the injection of erythropoietin (Ep) 8 h prior to sacrifice, but an absolute increase in colony number only occurred when Ep was injected 24 h previously. Polycythemia also slowed the rate of colony maturation, so that at later culture times the colonies more frequently contained fewer cells and consisted of basophilic normoblasts. Anemia did not shorten the time of first appearance of colonies, but their rate of maturation was significantly more rapid than the control. These findings are best explained by a redistribution of CFU-E in various phases of the cell cycle when erythropoiesis is manipulated. Unit-gravity sedimentation studies suggested a greater fraction of CFU-E in long G1 or G0 in polycythemia. In addition, the kinetics of CFU-E development were identical to polycythemia in mice in which S and post-S-phase CFU-E were removed by hydroxyurea injection. It is likely that Ep stimulates CFU-E in all phases of the cell cycle. After stimulation by Ep, however, CFU-E in long G1 must move through the cell cycle prior to commencing cell division. This transit time of 8 h, which is unaffected by the Ep concentration in the medium, results in a lag in appearance and maturation of colonies derived from these cells. These results demonstrate that cellular variables are important in CFU-E kinetics in vitro and provide insights into factors affecting the regulation of normal erythropoiesis.

Anemia↗
PubMed1986

Evaluation of the effect of age on hematopoiesis in the C57BL/6 mouse.

The effect of age on hematopoiesis was studied in young (six-month) and old (24-month) C57BL/6 mice. In addition, studies were performed on very old (42-month) mice, housed either singly or in groups of five animals per cage. Although a reduction in hematocrit was found in the older mice, red cell mass was normal in both old and very old single-caged mice. A detailed evaluation of erythropoiesis that included plasma- and erythron-iron turnover (PIT and EIT), red cell survival, and quantitation of the marrow erythroid progenitor and differentiated cells demonstrated no age-related change in single-caged animals. Similarly, quantitation of marrow myeloid precursors was identical in these groups. These results indicate that no age-related change in basal hematopoiesis can be demonstrated even in animals approaching maximal life expectancy. When very old mice were routinely housed in groups of five per cage, however, a decrease in hematocrit was found which was accompanied by significant alterations in hematopoiesis, including reductions in total differentiated erythroid cells, erythroid burst-forming units (BFU-E), erythroid colony-forming units (CFU-E), and colony-forming-unit culture (CFU-C) levels. It is likely that in very old animals, group housing constitutes a sufficient stress to compromise hematopoiesis. These findings indicate that basal hematopoiesis is unaltered by aging, although the bone marrow's reserve capacity is markedly compromised.

Aging↗
PubMed1984

Effect of donor age on long-term culture of bone marrow in vitro.

Previous reports suggest that 33 degrees C is the optimum temperature for the long-term culture of murine bone marrow in vitro. We found that horse serum was an important variable determining our ability to culture marrow from immature (less than 10 weeks) mice at 37 degrees C. While some batches supported growth others did not. The ability of deficient horse serum to support growth could be corrected if the culture was fed at mid-week with additional medium. Marrow from aged mice (96 weeks and older) could invariably be cultured at 37 degrees C irrespective of the horse serum batch used for culture. Adult mice (20-24 weeks) gave intermediate results between immature and aged mice. All three groups of mice studied could be cultured at 33 degrees C irrespective of the batch of horse serum used or the age of the donor. These results indicate that horse serum provides a factor(s) which varies in concentration from batch to batch and is essential for normal marrow growth in vitro. The requirement for this factor is greater at higher temperature and in younger animals. Marrow from adult and aged mice was then simultaneously cultured at 33 degrees C and 37 degrees C. For adult mice the cell recovery at 33 degrees C was initially significantly higher than at 37 degrees C but at later times became less. As a result cumulative recovery at both temperatures was equal. Cumulative cell recovery and culture survival time was significantly less in the aged than the young mice cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1984

Effect of age on hematopoiesis in man.

We have shown previously that the cause of anemia in healthy elderly subjects can usually not be identified. In this study, hematopoiesis was examined in 18 healthy elderly subjects with unexplained anemia and in 15 young and 15 healthy elderly individuals without anemia. No reduction in circulating testosterone was noted, making decreased androgen levels as a cause for the anemia unlikely. The 2,3 diphospho-glycerate (2,3DPG) levels in the anemic subjects were significantly higher than their corresponding controls, suggesting that the anemia was pathologic, as no increase would be expected if the low hemoglobin was a physiologic adjustment to age. The anemia was associated with a reduction in marrow normoblast and CFU-E number, but no decrease in BFU-E levels was seen. This suggests that the mechanism of the anemia is a decrease in stem cell proliferation. This could be caused by a reduction in circulating erythropoietin or a defect in end organ response. A second possibility is that a basic cellular abnormality exists. The presence of an overall reduction in hematopoiesis in anemic elderly (decreased peripheral blood counts, reduced marrow myeloid precursors, and CFU-C levels) makes this especially likely. The abnormality may be caused by a mechanism unrelated to the aging process. The fact that nonanemic elderly also have reductions in hematopoiesis suggests that age contributes to the defect.

2,3-Diphosphoglycerate↗
PubMed1984

Erythropoiesis in the aged mouse: I. Response to stimulation in vivo.

Changes in erythropoiesis with age were studied by examining the hematocrit increase in response to hypoxia in aged mice and by assessing the change in erythropoiesis following the injection of erythropoietin in young and old polycythemic mice. The increase in hematocrit after exposure to hypoxia was more variable and generally lower in old mice than in young mice. When erythropoietin was injected into polycythemic animals, the increase in differentiated erythroid cells and 59Fe incorporation into erythroid marrow and peripheral blood cells was significantly lower in old mice than in young mice. In contrast to differentiated erythroid cells, there was less evidence of a reduced response to stimulation of the more primitive erythroid progenitor cells of aged animals. The early undifferentiated erythroid progenitor, burst-forming units, did not decrease when either young or aged mice were made polycythemic, and no change following erythropoietin injection was noted. Polycythemia suppressed the late-differentiated erythroid progenitor, erythroid colony-forming units, to a greater extent in aged animals, but when erythropoietin was injected, the percent increase over the subsequent 24 hours was identical to that in young mice. These observations indicate a reduced erythropoietic capacity with age, the abnormality being most obvious in the more mature erythroid precursors.

Aging↗
PubMed1984

Erythropoiesis in the aged mouse: II. Response to stimulation in vitro.

In this study, we present in vitro evidence that erythropoietic precursors in aged mice respond less to stimulation by erythropoietin than do precursors in young mice. The effect of age on proliferation of differentiated erythroid cells from the marrow of young and old mice was examined in liquid culture to which increasing concentrations of erythropoietin were added. Cellular proliferation was measured indirectly by 59Fe incorporation into heme and directly as tritiated thymidine incorporation into DNA. The number of normoblasts remaining in culture with and without the addition of erythropoietin was also measured. In each case, cellular proliferation was significantly lower in marrow in old than in young mice. In contrast, CFU-E colonies cultured with increasing doses of erythropoietin were similar in young and old animals. These findings indicate that aging causes a reduction in the proliferative response of differentiated erythroid cells. Failure of these cells to respond to stimulation is the likely mechanism for the reduced erythropoietic proliferative capacity found in aged animals.

Aging↗
PubMed1984

A micromethod for long-term in vitro culture of bone marrow cells.

A micromethod to study long-term culture of bone marrow cells in vitro has been developed. Using Linbro wells one-fourteenth of the cells and medium usually required for culture in a flask are plated. Thus 40-50 cultures from a single mouse can be studied at any one time. Adherent-layer formation and supernatant cell recovery were very similar when Linbro -well cultures were compared with standard large-flask cultures. In the microculture system, supernatant and adherent-layer cell numbers increased following medium change reaching a maximum 3-4 days later and decreasing by day 7. Supernatant cells were in equilibrium with those of the adherent layer as cell numbers in both compartments prior to and following medium change fluctuated identically. Following medium change, a significant increase in myeloblasts occurred at 24 h and promyelocytes and myelocytes increased 48-72 h later. In contrast no discernible pattern in daily CFU-C production was detected. These findings provide insight into the cellular kinetics of long-term marrow culture and highlight the usefulness of this method to study hematopoiesis at frequent intervals.

Animals↗
PubMed1982

Endotoxin-induced suppression of erythropoiesis: the role of erythropoietin and a heme synthesis stimulating factor.

The regulation of erythropoiesis is primarily controlled by erythropoietin (Ep). Recently, however, other factors that both stimulate and inhibit erythropoiesis have been reported. Using an in vitro liquid culture of bone marrow cells, a factor in normal mouse serum was demonstrated that markedly stimulated heme synthesis by marrow erythroid cells. In this study, the role of this heme synthesis stimulating factor (HSF) and Ep in the erythropoietic suppression caused by endotoxin administration to mice was examined. Although HSF levels did not alter appreciably after endotoxin injection, marrow erythroid cells from these animals became unresponsive to the factor. This could be reversed if Ep was added to the culture in vitro or if the hormone was injected into the mice 18 hr prior to harvesting the marrow. This marrow erythroid cell response is identical to that seen in animals in whom Ep levels are markedly reduced, such as that found in exhypoxic polycythemia, and suggest a decrease in the hormone following endotoxin administration. Additional studies demonstrated that when Ep was injected into mice 6 hr after endotoxin administration, an increase in femoral erythroid colony-forming units (CFU-E), proerythroblast number, and 59 Fe incorporation into femoral marrow cells could be demonstrated. These findings, together with the marrow erythroid cell response to the hormone, suggest that the mechanism for suppression of erythropoiesis after endotoxin injection is a reduction in the level of circulating Ep.

Animals↗
PubMed1981

The quantitation of the granulocytic/macrophage committed progenitor cell (CFUc) in man and the mouse.

A method has been developed to quantitate the morphologically unrecognizable granulocytic/macrophage committed progenitor cell (CFUc). In man this is done by relating CFUc colonies cultured in vitro to marrow normoblast number determined by ferrokinetic measurements. In the mouse total hematopoietic precursors are determined by a radioiron dilution technique and CFUc are quantitated by calculating the percentage of colonies contained in the known number of nucleated cells plated. Like other hematopoietic precursors, CFUc numbers in 14 normal men and 10 normal mice were not significantly different, the mean being 0.85 and 1.4 x 10(7) CFUc/kg body weight, respectively. The validity of these values was substantiated by demonstrating an excellent correlation in the mouse between CFUc/kg body weight and CFUc/femur. In subsequent studies CFUc were quantitated in patients with lung cancer prior to and following aggressive combination chemotherapy when hematopoietic suppression was at a maximum. Compared to a mean value of 39 CFU/10(5) cells plated from chemotherapy the value 10 days later was significantly higher, the mean being 125/10(5) cells. That this reflected a relative change in the proportion of CFUc was suggested by the finding that 10 days after chemotherapy the mean value for CFUc/kg was 1.94 x 10(7) which was not significantly greater than a mean of 1.53 x 10(7) before treatment. These studies illustrate the advantages of expressing CFUc/kg body weight and indicate the usefulness of the approach in the evaluation of CFUc growth in a wide range of physiologic and pathologic conditions. In addition to better comparisons of results obtained in groups of individuals, multiple studies in single persons in whom hematopoiesis is not in a steady state will also be possible.

Adult↗
PubMed1980

Stimulation of erythroblast formation in suspension cultures of murine marrow by a factor in normal mouse serum, and its relationship to erythropoietin.

Addition of normal mouse serum to liquid suspension cultures of murine marrow cells resulted in dose-related increases in the number of heme-synthesizing erythroid cells. Removal of late normoblasts and reticulocytes by differential immune lysis prior to culture greatly enhanced the difference in heme synthesis between cultures with or without added mouse serum, permitting detection of as little as 1 microliter/l.5 ml of medium. Cell counts on these cultures indicated that the mouse serum factor actually stimulated formation of late normoblasts through proliferation and maturation of their precursors. The effect required either the presence of Ep in the medium, or the presence in the to be cultured cell suspensions of proerythroblasts which had been induced by or had contact with Ep prior to collection of the marrow.

Animals↗
PubMed1979

In vivo erythropoietin requirements of regenerating erythroid progenitors (BFU-e, CFU-e) in bone marrow of mice.

Erythroid progenitors (B-8, B-4, CFU-e) in the femoral marrow of polycythemic mice were measured by in vitro culture assays after a single administration of BCNU or Myleran. BCNU reduced pluripotent stem cells to 40% and erythroid progenitors to less than 5% of normal. B-8, the earliest erythroid progenitors, regenerated without erythropoietin (Epo) completely within 5 days. At 14 days after BCNU, intermediate progenitors (B-4) attained 60% of their normal numbers and CFU-e attained approximately 30%. Daily injections of Epo promptly restored normal B-4 numbers and near-normal CFU-e numbers in BCNU-treated mice. After Myleran, CFU-s remained below 2% of normal for 14 days, and no regeneration of the B-8 occurred with or without daily Epo injections. The findings suggest that regneration of B-8 was dependent on cell inflow from the pluripotent stem cell compartment but was independent of the presence of Epo. Intermediate progenitors (B-4) required Epo and the presence of B-8 for complete and permanent regeneration. CFU-e were the most Epo-dependent of the three progenitors. B-4, recruited by Epo, required after their formation a second exposure to the hormone in order to progress into the CFU-e stage.

Animals↗
PubMed1978

Cell kinetics of erythroid colony-forming cells (CFU-E) studied by hydroxyurea injections and sedimentation velocity profile.

Cell kinetic characteristics of murine CFU-E were assessed by measuring femoral marrow CFU-E survival rates after injection of hydroxyurea at from 2 to 48 h before marrow collection. The results are compatible with the concept that the CFU-E represent a homogeneous non-selfsustaining population comprised of cells belonging to a single differentiation stage. Their cell cycle length was estimated to be 10 h. Marrow cells were also subjected to separation by velocity sedimentation, and the effect of preceding hydroxyurea injections on the distribution profile of CFU-E was ascertained. Variations in cell size of CFU-E were interpreted to represent CFU-E in various phases of their cell cycle. CFU-E in either G1, S or G2 at the time of their removal from the marrow were capable of forming erythroid colonies in vitro, but it appears that the CFU-E lost this capacity after completing their cell cycle within the marrow of the intact mouse.

Animals↗