Effect of age of mouse recipients on the functional activity of transplanted hematopoietic and lymphoid cells]

When transplanting the bone marrow cells from adult C57BL mice to the lethally irradiated (CBA X C57BL) F1 hybrids of different age, the decrease of the colony forming activity of the stem haemopoietic cells was observed in the spleen of the older recipients, as compared with the 3 months old ones. The joint transplantation of the bone marrow and thymus cells resulted in both the cases in the stimulation of the growth of colonies. The number of endogenous colonies of haemopoietic cells arising in the spleen of animals following the sublethal irradiation was greater in younger hybrids. After the induction of the "transplant versus host" reaction by the lymph node or spleen cells from the CBA mice, the relative weight of spleen and regional lymph node, respectively, in the older recipients exceeded those in the younger ones.     

Genetic control of allogenic inhibition of hematopoietic stem cells]

Adult mice of C57BL/6, CBA (CBA X C57BL/6) F1, (CBA X C57BL/6) F2, F1 X CBA and F1 X C57BL/6 strains were lethally irradiated and reconstituted with a constant dose of 3-10(5) C57BL/6 bone marrow cells. At the 9th day after the bone marrow transplantation the colony count was performed in spleen of irradiated recipients. In the spleen of F1, CBA and C57BL/6 mice were registered low (0--8, intermediate (6--18) and high (22-40) numbers of colonies respectively. The segregation ratios in F2 progeny were close to 2 (low): 1(intermediate): 1(high). The segregation ratios in backcross (F1 X CBA) were close to 1(low): 1(intermediate)numbers of colonies. Backcrosses (F1 X C57BL/6) were distributed to low and high numbers of colonies with the ratio 1:1. The number of spleen colonies of males and females was the same in all segregating progeny. The results of hybrid analysis suggest that a single pair of allelic genes is involved in genetic control of allogenic inhibition, and that the resistance (manifestation of inhibition) to C57BL/6 stem cells is conferred by the dominant allele.     

Abolishment of allogeneic inhibition of colony formation with the aid of various RNA classes]

The authors analysed the capacity of various temperature fractions of RNA isolated from the spleen of donors of the bone marrow cells (of mice C57BL/6I) and recipients--hybrids (CBA X C57BL/6I) F1 to abolish the depression of colony formation in the nonsyngenous organism. In the administration of bone marrow cells of mice of parental genotype C57BL/6I of the irradiated recipients F1 there is observed a sharp depression of the number of colony forming units in the spleen F1. This depression can be eliminated by preliminary incubation of the bone marrow cells of mice of parental genotype with a 63 degrees fraction of the recipient's RNA. Preliminary inculation of the bone marrow cells of mice of parental genotype with 85 degrees and cytoplasmic fractions of recipient's RNA led to a partial restoration of colony formation only. The 45 degrees and 55 degrees RNA fractions of the recipient's RNA produced no restoring action. None of the temperature RNA fractions of the RNA of donor bone marrow cells were capable of abolishment of the colony formation depression in the nonsyngenous organism. It is supposed that restoration of the colony forming capacity in the nonsyngenous organism was connected with the activity of matrix RNA of the 63 degrees fraction obtained from the recipient's spleen.     

Stimulation by normal and leukemic mouse sera of colony formation in vitro by mouse bone marrow cells

Using a modification of the agar gel method for bone marrow culture, serum from various strains of mice has been tested for colony stimulating activity. Ninety percent of sera from AKR mice with spontaneous or transplanted lymphoid leukemia and 40–50% of sera from normal or preleukemic AKR mice stimulated colony formation by C57B1 bone marrow cells. Sera from 6% of C3H and 30% of C57B1 mice stimulated similar colony formation. The incidence of sera with colony stimulating activity rose with increasing age. All colonies were initially mainly granulocytic in nature but later became pure populations of mononuclear cells. Bone marrow cells exhibited considerable variation in their responsiveness to stimulation by mouse serum. Increasing the serum dose increased the number and size of bone marrow cell colonies and with optimal serum doses, 1 in 1000 bone marrow cells formed a cell colony. Preincubation of cells with active serum did not stimulate colony formation by washed bone marrow cells. The active factor in serum was filterable, non-dialysable and heat and ether labile.     

Augmentation of erythroid burst formation by the addition of thymocytes and other myelo-lymphoid cells

Bone marrow from barrier-sustained specific pathogen-free (SPF) CBA and C57BL/6 mice gave relatively low numbers of BFU-E colonies in methylcellulose culture, as compared to conventional mice. Addition of thymocytes to the marrow cultures increased the yield of BFU-E colonies more than fourfold in SPF mice but only 1.5-fold in conventional mice. Colony size was also increased. Increased yield of BFU-E colonies was also obtained by co-culture of bone marrow with lymph node cells or with bone marrow or spleen cells from 900R whole-body-irradiated mice. The effect appeared to be cellular rather than humoral. It was not reproduced by conditioned medium from thymus or pokeweed mitogen stimulated spleen cells. The helper effect of thymus cells was eliminated or reduced by freezing and thawing, or by 48 hours of incubation after irradiation. Treatment of bone marrow cells in vitro with anti-theta serum and complement did not decrease the number of BFU-E colonies. The putative helper cells appear not to be T cells, were non-adherent to the plastic culture dish, and were cortisone resistant and radioresistant. The low BFU-E colony yield from SPF mouse marrow is presumed to be largely the result of deficiency of these non-T helper cells in SPF bone marrow, rather than of BFU-E progenitor cells.     

Influence of the maternal effect on allogenic inhibition of hematopoietic stem cells]

Bone marrow cells (0,5-10(6)) of female mice of CBA or C57BL strains were injected intravenously to lethally irradiated CBA, C57BL/6, (femaleCBA X maleC57BL/6)F1 and (femaleC57BL/6 X maleCBA)F1 mice. Spleen of recipients as assayed for colony count on the 9th day after bone marrow transplantation by the method of Till and McCullouch. Stem cells of CBA mice demonstrated failure of allogenic inhibition in (CBA X C57BL/6)F1 hybrid mice and formed the same number of colonies as in the spleen of syngenic recipients. The level of allogenic inhibition of CBA stem cells transplanted to (C57BL/6 X X CBA)F1 hybrid mice was 50%. Bone marrow cells of C57BL/6 mice formed colonies in spleen of (CBA X C57BL/6)F1 mice at least in 20 times less than in syngenic combination. In the transplantation of bone marrow from C57BL/6 mice to (C57BL/6 X CBA)F1 hybrid mice the allogenic inhibition was less pronounced (77-85%) as compared with the transfer of cells to (CBA X C57BL/6)F1 hybrid mice (95%). The sex of a recipient did not influence the number of formed colonies. The different level of allogenic inhibition of parental stem cells can not be explained by the effect of linkage with sex as the female of reciprocal hybrid mice have identical structure of sex chromosomes (X(CBA)XC57BL/6). The data obtained indicate that the maternal effect affects allogenic inhibition of stem cells in parent--F1 system. It is possible that the maternal influence may be determined by cytoplasmic factors of inheritance which affect the expressivity of recessive genes Hh, controlling the inheritance of specific haematopoietic cell antigens.     

Effect of mouse age on the ability of hematopoietic stem cells to interact with thymus cells]

The effect of the thymus cells of the C57BL/6 mice on the colony forming ability of the stem hemopoietic cells of the embryonic liver and bone marrow of young (3 months) and old (2 years) mice was studied their joint transplantation into the mice (CBAXXC57BL/6) F1. The stimulating effect of the thymus cells on the colony forming ability of the stem hemopoietic cells of different age depends both on the dose of the stem hemopoietic cells of embryonic liver and the dose of T-lymphocytes. A suggestion is put forward that the stimulating effect of the thymus cells on the colony formation is due to their interaction with the stem cells in the G2 phase of the mitotic cycle.     

Role of thymus-dependent cells in splenic colony formation]

It was shown that treatment of the bone marrow with the serum reacting with the theta-antigen, irrespective of the presence of the complement, sharply decreases the capacity of its cells to form splenic colonies. Administration of the thymus cells together with the bone marrow to the recipient largely elminates the effect of this system, significantly increasing the splenic colony formation. It is supposed that the antiserum in the bone marrow inactivates the population of cells necessary for the formation of the colonies in the spleen, but differing from the pluripotent stem cells, possibly the population of T-cells.     

Comparison of the ease of tolerance induction in thymus and bone marrow of three strains of mice

Three strains of mice which vary in their susceptibility to induction of immune tolerance with human gamma-globulin were studied to evaluate the cellular basis for their sensitivity to induction of the unresponsive state. Tolerance induction in BALB/c mice was difficult to establish, while tolerance induction was easily achieved in C57BL/6 and CBF₁ (BALB/c × C57BL/6) mice. The degree of unresponsiveness obtained with various tolerogen doses in intact C57BL/6 and CBF₁ mice was reflected in the sensitivity of their thymocytes to the production of the unresponsive state. In the BALB/c mouse strain slight immune suppression observed at low tolerogen doses was correlated with bone marrow cell unresponsiveness while significant levels of tolerance observed at a high tolerogen dose was due to suppression of thymus cells. It was apparent that CBF₁ mice had inherited both thymus cells and bone marrow cells which exhibited the sensitivity to induction of immune tolerance characteristic of those same cells of their C57BL/6 parent.     

Bone marrow colony-forming capacity in mice subjected to burn trauma]

A method of exogenous splenic colonies was applied to the study of the dynamics of the content of the colony-forming units (CFU) in the bone marrow of CBA mice to which thermal burn of the III degree of 15% of the body surface was inflicted. On the 4th and 16th days after the burn the CFU content in the bone marrow of mice decreased 1.7-2.1 times. The thymus cells of the intact mice administered simultaneously with the bone marrow of the burned mice increased, the amount of the splenic exogenous colonies formed in the recipients. The data obtained permitted to make a suggestion that not only the CFU count diminished in the bone marrow in the burned animals, but also the thymus-dependent cells necessary for normal colony formation.     

Origin of stromal bone marrow mechanocytes according to the results of typing them by isoantigens and chromosomal markers]

The bone marrow of radiochimaeras and heterotopic bone marrow transplants were used to study the origin of precursors of the fibroblasts growing in the monolayer cultures of hemopoietic tissue. In the bone marrow explants of the (C57BL/6 X CBA) F1 mice, in which the CBA bone marrow was transplanted following the lethal irradiation, the fibroblasts grown in the colonies were of recipient origin judging by isoantigens in the reaction of indirect immunofluorescence with the anti-C57BL/6-serum. At the same time in the bone marrow explants from heterotopic transplants (CBA leads to CBA X C57BL/6) the fibroblasts grown in colonies were of donor origin. The cultures of hemopoietic cells of the bone marrow of females heterotopically transplanted in the singenic male (guinea pigs Huston) contained only fibroblasts which were of donor origin judging by sex chromosomes in the metaphase plates of dividing cells. Hence, the bone marrow precursors of fibroblasts do not depend histogenetically on hemopoietic cells and are not replaced at the expense of repopulating cells of the second partner.     

"Lymph node" form of graft-versus-host reaction in mice]

Local type of the graft-versus-host reaction was induced in adult hybrid mice (CBA X C57BL) F1 by the transfer of splenic, thymus and bone marrow CBA cells subcutaneously into the right hind footpad.The weight gain of the regional knee lymph node and blast accumulation in it 7 days later were used as indices of the graft-versus-host activity. After the transfer of 5 and 20 x 10(6) splenic cells the absolute weight of the regional lymph node was 8-10 times higher than that of the contralateral control; it was also significantly greater in comparison with controls which received live syngeneic or semiallogeneic dead cells from the same source. Contrary to controls, in case of the live cells a direct dose-effect dependency could be seen. The lymph node weight gain was accompanied by a regular immunoblast accumulation. The effect of the thymus and the bone marrow cells was less pronounced than that of the splenic cells.     

Effect of thymocytes on the quantity of polypotent hematopoietic mast cells in the spleen of sublethally irradiated mice]

With the aid of Till and McCulloch's method, 5 times 10(-6) thymic cells were found to cause an increase in the number of hemopoietic endogenous spleen colonies in syngeneic donor-recipient combination. Thymic cells of C57BL mice had no effect on the number of endogenous colonies in the spleen. 40 times 10(-6) thymic cells administered 24 hr after sublethal irradiation caused an increase in the number of colony forming units in the spleen within 14 days. Possible ways of the thymus effect on hemopoiesis are discussed.     

The effect of allogeneic lymphocytes on colony formation during culturing of hematopoietic precursor cells in the peritoneal cavity

The number of colonies formed in the peritoneal cavity (on the artificial underlayer made of peritoneal cells) and in the spleen of lethally irradiated recipients, (CBA X X C57BL) F1 mice, after the intraperitoneal injection of marrow cells depends on the cell donor's genotype: syngeneic cells and cells from mice of the parent strain CBA form fewer colonies in the peritoneal cavity than in the spleen, while cells from C57BL mice produce the reverse distribution of colonies between the peritoneal cavity and the spleen. Allogenic lymphocytes, when transplanted simultaneously with hematopoietic cells, suppress colony formation in the peritoneal cavity from day 2 of cultivation and eliminate the already developed foci of hematopoiesis by day 5.     

Effect of opioid peptides on proliferation and differentiation of skeletogenic cells of rabbit bone marrow in vitro

The influence of opioid peptides DSLET and DAGO in doses 10(-5), 10(-7) or 10(-10) mg per 1 ml of the medium on colony formation in the culture of stromal bone marrow fibroblast precursors was investigated 5. 10(-6) bone marrow cells were placed in plastic containers (Costar). 12 day old cell cultures were fixed with ethanol and stained with hematoxyline-eosin. Effectiveness of fibroblast colony formation (EFFC) was detected. Grown fibroblast colonies were stained after Gomory for alkaline phosphatase. Opioid peptides DSLET and DAGO in the used doses exerted no influence on EFFC and percentage phosphatase-positive colonies, which casts doubt on a presumable direct action of opioid peptides on stromal bone marrow cell-precursors. But it does not seem unlikely that opioid peptides may affect stromal bone marrow precursors of fibroblasts through the cell environment, particularly, via macrophages.     

Kinetics of hematopoietic stem cells in the bone marrow of hepatectomized mice

Two-thirds of the liver was removed from (CBA X C57BL/6j) F1 female mice. A significant increase of the number of endogenous colonies count in the spleen of partially hepatectomized mice was observed on the 5-th day after the operation. This increase was not associated with the changes in the number of stem cells in the bone marrow as partial hepatectomy at different times after the operation exerted no effect on the number of colony-forming units (CF1) in the bone marrow.     

Murine cytomegalovirus infection can enhance hybrid resistance through modulation of host natural killer activity

Studies were undertaken to assess the effect of murine cytomegalovirus (MCMV) in two different models involving injection of parental cells into F1 hosts. In both of these systems, MCMV-induced enhancement of hybrid resistance was found. In the first model, parent-into-F1 graft-vs-host reaction, MCMV infection of (C57BL/6 x C3H)F1 (B6C3F1) hosts was found to prevent the GVHR normally induced by injection of B6 parental splenocytes into the F1 hosts. The second model involved injection of parental bone marrow into lethally irradiated B6C3F1 and (C57BL/6 x DBA/2)F1 (B6D2F1) hosts. These irradiated hosts are known to exhibit resistance to engraftment by parental C57BL/6 (B6) bone marrow. This resistance was found to be markedly enhanced by injection of the hosts with MCMV 3 days before irradiation and bone marrow injection. In contrast, engraftment into B6C3F1 hosts of syngeneic marrow, or bone marrow from the C3H parent, was not affected by MCMV infection. Engraftment of DBA/2 marrow into B6D2F1 hosts was reduced at lower doses of injected marrow, suggesting enhanced resistance against the minor Hh Ag Hh-DBA. To test whether the MCMV-induced enhancement of resistance was mediated by NK cells, splenic NK activity (YAC-1 killing) and frequency (NK1.1 staining) were assessed. Both parameters were found to be elevated at 3 days after MCMV infection but to return to normal levels by 9 days. B6 bone marrow engraftment was in fact found to be normal when the marrow was administered to F1 mice 9 days after MCMV infection. Furthermore, anti-asialoGM1 administration prevented MCMV-induced enhancement of resistance to marrow engraftment. Thus, the NK enhancement resulting from MCMV infection appears to play a major role in the enhanced HR observed in the marrow engraftment model. This effect may be of importance in clinical bone marrow transplantation, a situation in which patients are susceptible to viral infection.     

A gene-controlling response of bone marrow progenitor cells to granulocyte-macrophage colony stimulating factors

The production of granulocytes and macrophages from progenitor cells in the bone marrow is controlled, in part, by a family of humoral regulators, termed colony stimulating factors (CSF). We have examined genetic factors controlling this process using in vitro cloning techniques. The inbred mouse strain LP/J showed elevated colony formation (CFU-C) in response to one subtype of CSF (G,M-CSF) compared to other strains of mice examined including the strain C57BL/6J. This variation resulted in a shift to the left of the CFU-C dose-response curve for LP/J. No difference between LP/J and C57BL/6J was seen with another subtype of CSF (CSF-1). Maximal CFU-C response was similar in the two mouse strains with both types of CSF, and mixing experiments with both types of CSF gave the same maximal level of colony formation as the individual CSF. (C57BL/6J X LP/J)F1 progeny exhibited a CFU-C dose-response curve to CSF-2 that was intermediate between the parental types, indicating additive inheritance. Genetic analysis of backcross progeny suggested that the variation in CFU-C response is probably determined by a single primary gene, although the variability of the colony formation assay has complicated interpretation of genetic studies. These results suggest that CSF-1 and G,M-CSF act independently on a single bone marrow progenitor cell population. The properties of the genetic variation for G,M-CSF response are consistent with an alteration in cellular receptors for G,M-CSF.     

Distribution of terminal deoxynucleotidyl transferase in bovine serum albumin gradient-fractionated thymocytes and bone marrow cells of normal and leukemic mice.

The distribution of terminal deoxynucleotidyl transferase (TdT) peaks I and II, in single cell suspensions of thymuses, bone marrow, and peripheral lymphoid organs fractionated in discontinuous bovine serum albumin gradients, was examined in a variety of mouse strains and Fischer 344 rats to relate the normal patterns of thymocyte differentiation to the leukemic process. TdT peaks I and II were found in fractions A (10 to 23%), B (23 to 26%), and C (26 to 29%) of the thymus of both normal and leukemic C57BL/6 mice, whereas only peak I was found in the same fractions of AKR mice. TdT in bone marrow was found mainly in fraction A in both normal and leukemic mice. The specific activity of TdT in this fraction, which comprises only 1 to 5% of the total bone marrow cell population, was similar to that of the thymus. The cell population of fraction A of the bone marrow was found to increase (10 to 15-fold) in leukemic mice. Only low levels of TdT activity were found in either whole or fractionated bone marrow of athymic NIH Swiss (nu/nu) mice.     

Autoaggressive immunocompetent cells in the bodies of mice at remote periods following irradiation]

Lethally irradiated DBA/l mice or (C57Bl X DBA/l1 F1 hybrid mice were injected with therapeutically effective doses of isologous bone marrow cells; simultaneously syngeneic lymph node cells from either intact (control) animals or mice survived after sublethal irradiation were transplanted. In control the viability of the recipients was not affected by the presence of lymphoid cells in the mixed transplant. In contrast, the beneficial action of the bone marrow cells was abolished (killing-effect) by the lymphoid cells from mice sacrificed 6 to 12 months after the irradiation (600--700 r). The manifestation of the killing-effect depended on the number of the transplanted lymphoid cells and on the dose of the bone marrow cells in the transplant. The killing-effect was not revealed when the lymphoid cells were obtained from the donors on the 30th day after irradiation. The results suggest the autosensitization of the organism at the late postirradiation periods.