Essential defects of athymic nude mice affect also the nu/+ heterozygotes.
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Biomedical subjects
E Necas
Publications and source records attributed to E Necas.
Ureter obliteration transiently depresses erythropoietin production.
BACKGROUND: The erythropoietin (EPO) gene is activated in peritubular cells of the renal cortex in response to hypoxia and EPO is secreted into the circulation. Oxygen tension in the venous blood normally determine EPO elaboration, and, therefore, oxygen consumption in the kidney appears to be an essential part of the oxygen-sensing physiological mechanism. As renal oxygen consumption is closely linked to urine production, we have compared responsiveness of the EPO gene to diminished oxygen supply in the normal kidney with that of the hydronephrotic kidney, resulting from ureter ligation. METHODS: Erythropoietin mRNA was determined in RNA extracted from murine kidneys at various times up to 61 days after ureter ligation. Mice were exposed to hypoxia for 4 h before being killed. RESULTS: Unilateral ureter ligation significantly decreased EPO mRNA levels in the affected kidney. However, the ability to increase EPO mRNA recovered between days 4 and 22, despite persisting ureter ligation. CONCLUSION: Our results demonstrate a dissociation between the excretory functions of the kidney and EPO production. Moreover, they show an early but transient suppression of EPO gene responsiveness to hypoxia after ureter ligation.
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Guinea pig serum erythropoietin (EPO) selectively stimulates guinea pig erythroid progenitors: human or mouse erythroid progenitors do not form erythroid burst-forming unit colonies in response to guinea pig serum EPO.
Erythropoietin (EPO) is the primary regulator of mammalian erythropoiesis, providing a proliferative and differentiative signal to the early EPO-responsive erythroid progenitors, burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid, as well as to later EPO-responsive erythroid progenitors. EPO is secreted by the kidney in response to hypoxia and anemia. There is an extensive biological crossreactivity between human EPO and the EPOs of other mammals. Necas et al. have reported that this crossreactivity may not include the guinea pig (Cavia porcelllus). Because the specificity of the guinea pig's erythropoietic responses may be of biological significance, we compared guinea pig hypoxic serum with mouse (m) and human (h) recombinant (r) EPOs for their ability to induce erythroid progenitor proliferation and differentiation in semisolid cultures. Guinea pig bone marrow mononuclear cells (BMMCs) formed BFU-E colonies in response to guinea pig hypoxic serum, rhEPO, or rmEPO in a dose-dependent fashion. Neither human nor mouse BMMCs responded to guinea pig hypoxic serum; however, guinea pig hypoxic serum exerted no inhibitory effect on human or mouse in vitro erythroid differentiation in the presence of rhEPO or rmEPO. The intensity of the EPO band on Western blotting analysis of guinea pig hypoxic serum was significantly greater than in nonhypoxic serum. This suggests that guinea pig erythropoiesis is mediated by EPO and stimulated by hypoxia in a fashion similar to that observed in human and mouse erythropoiesis. Furthermore, guinea pig EPO did not stimulate human or mouse erythroid differentiation in vitro, whereas guinea pig erythroid progenitors could be stimulated by human or mouse EPO, suggesting structural differences in guinea pig EPO and EPO receptor (EPOR) compared with human or mouse EPO and EPOR. These differences probably evolved after the guinea pig's ancestors diverged from myomorph rodents. Further characterization of the guinea pig EPO and EPOR should facilitate our understanding of the interaction between EPO and EPOR.
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Rescue from lethal irradiation correlates with transplantation of 10-20 CFU-S-day 12.
Repopulation by donor cells of a bone marrow ablated by irradiation is now recognized to proceed in two phases: initial repopulation that may be temporary followed by permanent engraftment of longterm repopulating cells (LTRC). While a single LTRC has been shown to be capable of restoring the entire lymph-hemopoietic system of an irradiated animal, the identity of the temporary repopulating cells has not been established unequivocally. We used the results of transplantation of subpopulations successively enriched for LTRC and containing varying numbers of CFU-S-12 (colony-forming units in the spleen at day 12 post transplantation) and progenitors to determine the likely cell type and number of cells needed for initial survival after radiation. Subpopulations from untreated and 5-fluorouracil-treated mice were discriminated on the basis of antibody reactivity, Hoechst 33342 and rhodamine 123 fluorescence intensity and light scattering properties. The minimum rescue inocula varied greatly in CFU-GEMM, BFU-E and CFU-GM content. One to two CFU-S-12 were uniformly present in all isolated suspensions that rescued 50% of lethally irradiated animals. In view of the known average seeding efficiency of CFU-S, our studies suggest that transfusion of 10-20 CFU-S day 12/13 is responsible for radioprotection. Evidence that multiple CFU-S day 12/13 are needed for initial repopulation is also supported by quantitative estimates of the number of mature cells that can be produced by CFU-S. Transfusion of a single CFU-S day 12/13 can be shown to be grossly inadequate to provide the number of peripheral blood cells needed to ameliorate the severe pancytopenia following lethal irradiation by day 12-14. Our data also indicate that 5-fluorouracil-treated marrow subpopulations appear inferior to untreated subpopulations in their ability to contribute to initial repopulation when transfused at low cell doses into lethally irradiated recipients.
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Products of red blood cell degradation inhibit responsiveness of the erythropoietin oxygen sensor.
Tissue hypoxia is less effective in increasing erythropoietin plasma levels in animals with post-transfusion polycythaemia. Since more red blood cells are decomposed under this condition, the effects of exogenous haemin and of lysed or heat-damaged red blood cells on activation of the erythropoietin gene have been studied in mice rendered hypoxic. Total RNA was extracted from the kidney and the liver and subjected to northern blot analysis with a probe containing part of the murine erythropoietin gene. Blood plasma was collected and erythropoietin levels were determined by radioimmunoassay. Erythropoietin gene activation was suppressed by haemin and increased red blood cell haemolysis. Tin (Sn) protoporphyrin, a haeme analogue which cannot bind oxygen, did not share the effect of haemin. On the other hand, when injected with haemin, Sn-protoporphyrin potentiated the suppressive effect of haemin, probably through inhibition of haemin catabolism. We conclude that the intracellular haeme concentration inhibits the kidney oxygen sensor and that this inhibition, mediated by products red blood cell degradation, is a physiological safeguard mechanism against excessive polycythaemia and its deleterious effects upon blood circulation.
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[Erythropoietin in the era of molecular biology].
Cloning of genes for erythropoietin in the mid-eighties started a new period of research and use of erythropoietin. RIA and ELISA methods of its detection were developed. It became possible to seek possibilities of its therapeutic use. The cell type which is the source of erythropoietin in the organism was assessed. The regulation of the expression of the erythropoietin gene in relation to oxygen tension was investigated. The biochemical mechanism is sought which transmits the oxygen tension to transcription signals and the stability of erythropoietin mRNA. The gene for the erythropoietin receptor was found and it is investigated in relation to the pathogenesis of primary polycythemias.
Erythropoietin↗
Hematopoietic reserve provided by spleen colony-forming units (CFU-S).
Using quantitative data available from the literature on murine hematopoiesis, the functional reserve of multipotential stem cells was calculated by comparing daily blood cell production with the potential of clonogenic spleen colony-forming cells (CFU-S) to generate blood cells. The potential of the day-8 CFU-S (CFU-S-8) population is estimated to be from 4000 to 37,000 times greater than needed in steady-state hematopoiesis. The CFU-S population may thus serve to provide a functional reserve to supply large numbers of peripheral blood cells via committed lineage-specific cells within days, whenever needed.
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[Mechanisms of oxygen transport].
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Biological Transport↗
Self-renewal of the long-term repopulating stem cell.
Self-renewal implies maintenance of all attributes of the original in the offspring and is considered characteristic of the hemopoietic stem cells. Yet, it has been questioned whether one of the most primitive hemopoietic stem cells, the long-term repopulating cell (LTRC), has that capacity. The present experiments demonstrate that single LTRCs can repopulate the lymphohemopoietic system of a lethally irradiated mouse and that the progeny of a single LTRC in a primary recipient again contains LTRCs capable of repopulating lethally irradiated secondary recipients. The transfusion of very small numbers of marrow cells (10,000-20,000 cells containing one or no LTRCs) unexpectedly provided insight into competitive marrow repopulation. At these low levels of stem cells, irradiated host stem cells or their progeny competed successfully with unirradiated donor cells. This parallels the known reemergence and marrow repopulation by host cells when the number of nonirradiated donor stem cells is reduced by serial transplantation.
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On control of the hematopoietic cell proliferation.
Hematopoietic cells, detected as spleen colony forming units (CFU-S), vary in their proliferation rate, and this can be investigated in vivo by determining the fraction of CFU-S synthesizing DNA. A large number of measurements of CFU-S number and the fraction synthesizing DNA has been obtained under standardized conditions in normal mice and after a single dose of arabinosyl cytosine (ara-C). These data are analyzed with respect to the correlation between the number of CFU-S and their DNA-synthesizing fraction since, in the past, it has been maintained that the CFU-S proliferation rate responds sensitively to changes in CFU-S numbers. The present analysis does not support this traditional view--it instead demonstrates that natural variations in CFU-S numbers occurring in the same range as those following ara-C do not trigger CFU-S proliferation. On the other hand, administration of ara-C triggers most of the surviving CFU-S into the cell cycle, while decreasing CFU-S numbers by only about 40%. Data are also presented showing changes of CFU-S numbers and their DNA-synthesizing fraction in mice given a single dose of cyclophosphamide (CY). CY reduces CFU-S numbers, but the fraction synthesizing DNA can be either increased or decreased depending on time after treatment. Both these experimental situations argue against a close relationship between CFU-S numbers and their proliferation rate and suggest a different or a more complex regulation.
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Sensitivity of CFU-E to exogenous erythropoietin in benzene-treated mice.
Normal as well as hypertransfused BDF1 mice were exposed to 300 ppm of benzene, 6 h/day, 5 days per week for 2, 4, and 6 weeks respectively. Erythroid-committed hematopoietic progenitor cells CFU-E were determined in the bone marrow, and 59Fe incorporation was measured in the peripheral blood 48 h after its injection, as an indicator of active erythroid cell production. CFU-E numbers were reduced in benzene-exposed mice at all intervals, as was 59Fe incorporation in the peripheral blood after 2 weeks of exposure. In hypertransfused mice the CFU-E suppression, caused primarily by the hypertransfusion, was aggravated by benzene. After injection of 1 IU Ep 4 days before killing the CFU-E numbers and the 59Fe incorporation increased in controls as well as in benzene-exposed animals, but the difference persisted. After nine consecutive Ep injections the difference concerning the femur disappeared after 2 and 6 weeks of benzene exposure but was still present in the peripheral blood. These results suggest that chronic benzene exposure has a negative effect on early erythroid-committed, Ep-responsive hematopoietic cells.
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Triggering of stem cell (CFU-S) proliferation after transplantation into irradiated hosts.
The paper demonstrates the beginning of increased proliferation of the bone marrow clonogenic cells, detected by the spleen colony technique, taken from normal mice and transplanted into syngeneic recipients, lethally irradiated 24 h previously. A lag period of about 10 h preceded the beginning of increased proliferation that was determined according to the sensitivity of transplanted cells to the lethal action of hydroxyurea.
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Hematopoietic stem cells in +/+, nu/+, and nu/nu C57Bl/10 mice.
Mice with the nu mutation have been shown to have a reduced incidence of spleen colony-forming units (CFU-S), with a higher variability between individual mice compared to +/+ mice. The presence of these defects also in nu/+ mice indicates that this mutation is a codominant trait and that the hematopoietic changes may not be solely caused by thymus dysgenesis. The proportion of CFU-S synthesizing DNA (approximately 40%) has not been significantly different between +/+, nu/+, and nu/nu mice.
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On the determination of hemopoietic stem cell proliferation.
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Animals↗
Haemopoietic stem cells: spleen colony-forming cells are normally actively proliferating.
The haemopoietic stem cells forming spleen colonies (CFU-S) had on average 30 to 40% of cells engaged in the DNA synthesis in normal mice continuously over 4 years. A majority of experiments aimed at the suppression of the CFU-S proliferation, which included suppression of the T-lymphocytes by means of cyclosporin A or by adult thymectomy, administration of antibacterial and antifungal agents and maintainance of mice in a sterile environment, suppression of antibody-producing cells by a successive administration of the bacterial lipopolysaccharide and cyclophosphamide and attempts to increase the total number of CFU-S in the body through massive transfusions of bone marrow cells or by grafting plugs of the bone marrow under the kidney capsulae, have not been sufficiently effective. A transient suppression of CFU-S proliferation occurred during recovery of the haemopoietic tissue from damage caused by cyclophosphamide. The results support the view that changes in CFU-S numbers and in the proportion of them in DNA synthesis may be positively correlated when CFU-S numbers fluctuate physiologically about their normal values. The failure to manipulate the CFU-S proliferation rate easily suggests that proliferation of these cells may not be under a strong 'switch on - switch off' control.
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The dysgenetic thymus in nu/nu mice is a potent source of colony stimulating factor (CSF).
During in vitro incubation, the pulmonary and thymic tissue of mice released a colony stimulating factor (CSF) supporting the development of colonies of granulocytes and macrophages from bone marrow progenitors. Cardiac, splenic, renal and bone marrow tissues were not active. The dysgenetic thymus of nude mice was a very potent source of CSF during in vitro incubation.
Aging↗
A comparison of stem cell assays using early or late spleen colonies.
The distribution of spleen colony diameters was determined 5.5, 8.0, 10.5 and 13.0 days after injection of normal bone marrow cells to lethally irradiated recipients. A relative lack of small colonies on day 8.0, as compared with days 5.5, 10.5 and 13.0, argued against a time continuum in colony appearance. The spleen colonies observed after 10 days or more probably represented a mixture of colonies which developed from the originally transplanted CFU-S and those arising from secondary CFU-S. Thus, late appearing spleen colonies may not necessarily identify a different, less mature, population of CFU-S. Administration of increasing amounts of bone marrow cells was used for comparing the linearity of the CFU-S assay for colonies observed after 8 days or after 12 to 13 days. The influence of overlapping colonies on the results was considerably augmented if large spleen colonies were observed after 12 or 13 days. Subsequently the CFU-S assay lost much of its quantitative character. We believe that some previously published data might have been misinterpreted by neglecting the important differences between 'early' and 'late' CFU-S assays.
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Individual differences in a functional organization of the hematopoietic tissue.
Random-bred ICR mice exhibited a wide range of spleen colony-forming units (CFU-S) and granulocyte-macrophage colony-forming cells (GM-CFC) between individuals. Both CFU-S and GM-CFC values were, however, relatively stable in individual mice over a period of 6 weeks, although the cellularity of the marrows changed significantly during the same period. CFU-S and GM-CFC values were only weakly correlated in individual mice. Bone marrows of mice with low CFU-S values did not have a lower cellularity than mice with high CFU-S values. A low level of CFU-S thus appeared to be compensated for by a higher clonal expansion of maturing cells. It may be concluded that there is wide variation in the organization of the stem cell compartment. Individuals may thus differ markedly in the organization of the marrow. Such differences may prevail in man and be of functional importance at times of extra demands, e.g., during cytostatic therapy.
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