Thymodepressin protects murine bone marrow CFU-S from the hyperthermic damage

Was studied the influence of the Thymodepressin (dipeptide D-iEW--a new Russian immuno- and haemodepressant), on the hyperthermic sensitivity of haemopoietic precursors (CFU-S) and tumor model cells (EL-4 and Rauscher leukaemia). It was determined, that the injection of the Thymodepressin to donor mice, or the incubation with the preparation of the marrowy cells of normal mice provides the increasing of the CFU-S resistance to the following heating (43 degrees C). On the contrary, Thymodepressin-treated tumor cells became even more heat-sensitive. The data show that Thymodepressin can be useful for protection the haemopoietic precursors not only from radiation and chemotherapy, as it was shown earlier, but also from the hyperthermy.     

Effect of syngeneic lymphocytes, T immunodeficiency and the genotype of bone marrow donors on the differentiation of early and late splenic colonies

The formation of "early" (5-8 days) and "late" (12-14 days) colonies in spleen of lethally irradiated syngeneic or hybrid recipients after transplantation of bone marrow cells has been studied. The differentiation pattern did not depend on bone marrow cell donor's genotype and the donor-recipient combination. Erythroid to granulocyte colonies ratio (E/G) equals 2. Change of direction of bone marrow colony-forming units (CFU) differentiation has the same pattern at different stages of colony-formation. Under the influence of antigen-stimulated lymphocytes the granulopoiesis (E/G 0.3-0.5) dominanted. The thymectomy of adult animals leads to a predominant formation of erythroid colonies (E/G 3.5-5.1). When T-immunodeficiency is reversed with syngeneic lymphocytes, the differentiation of CFU is normalized at all stages of colony-formation. The process of differentiation of haemopoietic precursors, that form "early" and "late" colonies, is under T-lymphocyte control.     

Anti-immunoglobulin stimulation of murine lymphocytes. III. Enhancement of the development of responsive B cells by thymic deprivation.

The development of splenic B cells that can be induced to proliferate by soluble anti-immunoglobulin (anti-Ig) reagents requires 7 to 9 months in normal mice. We have found that this age-associated response is enhanced by thymic deprivation. Both neonatally thymectomized LAF₁ mice and thymectomized, lethally irradiated, and bone marrow-restored Balb/c mice respond earlier and more strongly to anti-Ig than their sham controls. Nevertheless, at least 3–4 months are still required after thymectomy before a response can be measured. The earlier and enhanced response to anti-Ig seen in thymectomized animals is not due simply to an increase in the total number of Ig-positive spleen cells. The age-associated response of splenic B cells to anti-Ig we have observed in normal mice may be explained by the “natural” loss of thymic influence that occurs with age.     

The role of T-system immunity in reparatory regeneration of the bone tissue in animals

It has been demonstrated in experiments on mice (CBA X C57BL) X F1, thymectomized and irradiated by 800 R, with the haemopoietic system restored by bone marrow (B-mice) that in these animals, as compared with the controls, the changes in cellular immunity (inhibition of natural killer cells and stimulation of individual functions of the phagocytizing cells) are accompanied by considerable inhibition of osteogenesis. Compensatory regeneration of broken thigh-bone in B-mice is delayed by 5--10 days in various elements of the regenerated tissue in comparison with normal mice. Anatomical formation of the provisional callus in B-mice is not completed on day 21 and approaches a 14-day regenerate of the controls. The obtained results suggest the participation of T-system immunity in the reparatory regeneration of bone tissue.     

SELF-MAINTENANCE ABILITY AND KINETICS OF HAEMOPOIETIC STROMA PRECURSORS

The use of repeated femoral curettages and repeated passages of ectopic haemopoietic foci has demonstrated the capacity of stromal precursor cells for repeated formation of haemopoietic microenvironment. During this process the stromal precursors undergo no less than ten to twelve mitoses. Precursors of bone marrow stroma proliferating slowly, if at all, in normal mice are triggered into cell cycle, as revealed by suicide methods, during formation of the ectopic haemopoietic focus.     

Presence of pluripotent haemopoietic precursors in vitro (CFU-mix) in haemopoietic tissues from mice of W/Wv genotype

Abstract. The presence or absence of haemopoietic precursors, which produce mixed colonies in vitro (CFU-mix) was examined in the bone marrow and spleen of (WB x C57BL/6) F1- W/W^v mice. Despite the failure of macroscopically evident colonyformation in the spleens of irradiated mice, haemopoietic cells of W/W^v mice did produce macroscopically-evident mixed colonies containing erythroid cells, macrophages, and often megakaryocytes, in culture medium. The size and constitution of mixed colonies derived from W/W^v mice were comparable to those of mixed colonies from congenic +/+ mice. The present results appear consistent with in vivo haemopoiesis in the W/W^v mice, which is obviously deficient, but sufficient for survival.     

Idiopathic paraproteinemia. III. Increased frequency of paraproteinemia in thymectomized aging C57BL/KaLwRij and CBA/BrARij mice

The influence of thymectomy on the appearance of idiopathic paraproteinemia (IP) during aging was investigated in mice of the C57BL/KaLwRij and the CBA/BrARij strains, which under normal conditions develop IP in high and low frequency, respectively. Compared with sham-thymectomized mice, C57BL mice thymectomized at a young adult age showed a markedly increased frequency and an earlier onset of IP during aging; this was even more pronounced in neonatally thymectomized mice. A similar effect of thymectomy was also observed in mice of the CBA strain. Restriction in the heterogeneity of the serum immunoglobulins and the appearance of transient homogeneous Ig components was another frequent finding and this often preceded the appearance of IP in mice of both strains. Thymectomy did not substantially influence either the incidence of paraproteinemias due to a B cell malignancy or the isotype distribution among the paraproteins produced. The results are compatible with the hypothesis that IP develops in three stages as a consequence of an age-related immunnodeficiency that primarily affects the T immune system.     

Effects of an antiserum for epithelial-reticular cells of the thymus on T-dependent immune response

Relations between thymic factors and Prostaglandins (PG) were studied. We investigated on the effects of different incubation times with Thymosin Fraction 5 and Indomethacin on the release by spleen cells from normal or adult thymectomized mice. Prostaglandins were measured by radioimmunoassay. Thymosin induces an increase in PGE2 release on spleen cells obtained from thymectomized mice; the same effect was not observed on spleen cells obtained from normal mice.     

Effect of T-cell mitogens and T-lymphocyte deficiency on splenic colony formation

A study was made of the influence of T-cell mitogens (Con A and PHA) on the colony formation and differentiation of hemopoietic stem cells from normal and thymectomized mice, as well as of the relationship between the colony formation and the dose of injected thymocytes. The incubation of bone marrow cells with Con A and PHA was shown to inhibit the growth of spleen colonies. This inhibition is reduced by thymocytes within the dose intervals of 0.25-2.0 X 10(7) cells/mouse. Administration of these agents serially has led to the potentiation of inhibition effect and to the inability of thymocytes to reverse it. Con A and PHA exert no effect on the differentiation of stem cells. Incubation of the bone marrow cells from thymectomized mice with Con A is much less effective in the depression of colony formation, if compared with the treatment by intact bone marrow preparations. A reversed picture was observed using antiserum to mouse brain (RAMBS). It is proposed that regulation of stem cells is governed by different subpopulations of thymocytes.     

Differentiation characteristics of circulating and bone marrow hematopoietic stem cells and the effect of thymus factors

Circulating hemopoietic stem cells (HSC) considerably differ from bone marrow HSC in active erythroid differentiation. After thymectomy of adult animals the number and differentiation of blood HSC remain unchanged, whereas during the cloning of bone marrow cells, a decrease in the number of granulocytic colonies is revealed. In in-vitro experiments, thymalin does not influence the number or differentiation of circulating HSC. On the contrary, in experiments made in vivo, it dramatically lowers erythroid specialization of blood HSC in thymectomized and sham-operated mice, which is followed by the diminution of the total number of circulating HSC. Differentiation of thymectomized mice bone marrow stem cells is completely normalized after thymalin injection. Sham-operated and thymectomized animals' HSC stimulated by thymalin injection become similar to bone marrow cells of normal mice as regards the trend of differentiation. Thymalin injection is likely to change the bone marrow HSC differentiation profile, thereby preventing the release of the cells with erythroid-oriented differentiation from the bone marrow to blood. The influence of thymalin on HSC is mediated by the environmental component which is present in the bone marrow and absent from the peripheral blood.     

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.     

Postradiation recovery of hemopoietic and stromal precursor cells in mice preinjected with prodigiozan]

Injection of prodigiozan to mice 24 h before irradiation caused, by the time of the radiation effect, a decrease in the number of haemopoietic cells-precursors (CFUs and CFU-HM) in the bone marrow and an increase in the functional activity of stromal cell-precursors--the haemopoietic microenvironment of transfer units (HMTU); in the spleen, the number of CFUs decreased, but the number of CFU-HM increased considerably. During the postirradiation period, the haemopoietic and stromal precursors were damaged to a lesser extent, and CFUs, CFU-HM and HMTU recovered more readily in prodigiozan-protected animals than in unprotected mice; the HMTU restoration preceded the increase in CFUs and CFU-HM levels.     

Lymphocyte-mediated cytotoxicity: effects of ageing, adult thymectomy and thymic factor.

Adult thymectomy, as well as ageing, depressed splenic lymphocyte-mediated cytotoxicity (LMC) in the mouse. Ageing depressed significantly LMC as early as 19 weeks of age, independently of the number of cells used for immunization. Thymectomy affected LMC only when supoptimal numbers of immunizing allogeneic cells were used. This effect peaked at 6 to 12 weeks after thymectomy. No difference between thymectomized and normal mice was observed when LMC was tested 16 to 20 weeks after thymectomy, at an age when normal control mice themselves already showed a lowered LMC due to ageing. The effect of in vivo treatment with a circulating thymic factor (TF), which was shown to disappear with ageing as well as after adult thymectomy, has been tested in adult thymectomized mice and normal young and ageing mice. TF treatment prevented LMC depression in adult thymectomized mice, whereas it depressed paradoxically splenic LMC in normal young and old mice. The possible mechanisms of the effects of adult thymectomy, ageing, and thymic factor on the different T cell subsets involved in allogeneic killer cell generation are discussed.     

Regulation of haemopoietic stem-cell proliferation in mice carrying the Slj allele

We investigated a haemopoietic stromal defect, in mice heterozygous for the Slj allele, during haemopoietic stress induced by treatment with bacterial lipopolysaccharides (LPS) or lethal total body irradiation (TBI) and bone-marrow cell (BMC) reconstitution. Both treatments resulted in a comparable haemopoietic stem cell (CFU-s) proliferation in Slj/+ and +/+ haemopoietic organs. There was no difference in committed haemopoietic progenitor cell (BFU-e and CFU-G/M) kinetics after TBI and +/+ bone-marrow transplantation in Slj/+ and +/+ mice. The Slj/+ mice were deficient in their ability to support macroscopic spleen colony formation (65% of +/+ controls) as measured at 7 and 10 days after BMC transplantation. However, the Slj/+ spleen colonies contained the same number of BFU-E and CFU-G/M as colonies from +/+ spleens, while their CFU-s content was increased. On day 10 post-transplantation, the macroscopic 'missing' colonies could be detected at the microscopic level. These small colonies contained far fewer CFU-s than the macroscopic detectable colonies. Analysis of CFU-s proliferation-inducing activities in control and post-LPS sera revealed that Slj/+ mice are normal in their ability to produce and to respond to humoral stem-cell regulators. We postulate that Slj/+ mice have a normal number of splenic stromal 'niches' for colony formation. However, 35% of these niches is defective in its proliferative support.     

Repopulation of spleens of irradiated mice after injection of spleen cell subpopulations

Haemopoietic stem cells present in the spleen of adult mice were analysed by grafting X-irradiated animals with polystyrene-nonadherent (NABS) and polystyrene-adherent (ABS) B-enriched splenocytes from syngeneic donors. The progeny of the haemopoietic stem cells present in NABS and ABS subsets were studied with respect to size, surface markers, and response to mitogens and antigens. Ninety-six per cent of the precursors of the myeloid cell lineage (CFU-S) were present in the NABS fraction (50-fold enrichment). The presence in NABS of progenitors of functional T and B lymphocytes was also demonstrated. Twelve days after grafting with NABS, more than 80% of the recipient splenocytes were large and nonadherent granulocyte-like cells. These cells had surface similarities with NABS from normal mice, since both populations reacted with peanut agglutinin and with a rabbit anti-NABS (RAN) serum.     

Repopulation of spleens of irradiated mice after injection of spleen cell subpopulations

Abstract. Haemopoietic stem cells present in the spleen of adult mice were analysed by grafting X-irradiated animals with polystyrene-nonadherent (NABS) and polystyrene-adherent (ABS) B-enriched splenocytes from syngeneic donors. The progeny of the haemopoietic stem cells present in NABS and ABS subsets were studied with respect to size, surface markers, and response to mitogens and antigens. Ninety-six per cent of the precursors of the myeloid cell lineage (CFU-S) were present in the NABS fraction (50-fold enrichment). The presence in NABS of progenitors of functional T and B lymphocytes was also demonstrated. Twelve days after grafting with NABS, more than 80% of the recipient splenocytes were large and nonadherent granulocyte-like cells. These cells had surface similarities with NABS from normal mice, since both populations reacted with peanut agglutinin and with a rabbit anti-NABS (RAN) serum.     

The effect of thymectomy on lupus-prone mice

The effect of neonatal thymectomy on the induction and/or modification of murine SLE disease was examined in several representative groups of mice with early-life SLE (MRL/Mp-lpr/lpr females, BXSB males, (NZB X W)F1 females, (NZW X BXSB)F1 males and females), late-life SLE (MRL/Mp-+/+ and BXSB females), and normal strains (BALB/c and C57BL/6 females). Our results indicated that thymectomy prevented disease only in the MRL/Mp-lpr/lpr SLE mice, and that this effect diminished as thymectomy was delayed beyond 3 wk post-natally. In the other SLE mice studied, neonatal thymectomy did not modify disease symptoms to any significant degree. Moreover, depletion of mature T cells from donor BXSB male bone marrow did not affect the expression of early-life SLE in thymectomized BXSB female recipients. Neonatal thymectomy did not induce SLE in normal mice. Of note, neonatal thymectomy did not completely deplete the Thy-1.2+ cell population, i.e., 10 to 15% remained in the spleens of the thymectomized mice. This incomplete T cell depletion, together with the previously demonstrated dependence on and hyperresponsiveness of BXSB and (NZB X W)F1 B cells to T helper cell-derived accessory signals, cast doubts on earlier conclusions that B cells from some SLE mice can autonomously proliferate and differentiate to autoantibody-secreting cells. It seems more appropriate to conclude that B cells from the various SLE mice vary in their degree of response to, and production of, T cell-derived helper signals, and thus in their expression of B cell hyperactivity and disease.     

Transfer of immunity by transfer of bone marrow cells: T-cell dependency.

Thymectomized, lethally irradiated mice reconstituted with normal bone marrow cells succumbed when challenged ip with rat Yoshida ascites sarcoma (YAS) cells 40 days after irradiation and reconstitution. In contrast, thymectomized irradiated mice reconstituted with bone marrow cells from YAS-immune donors rejected the subsequent tumor challenge. Pretreatment of the bone marrow cells from immune donors with anti-Thy 1.2 antiserum and complement completely abolished the transfer of anti-YAS resistance.Bone marrow cells from donors thymectomized 2 months before immunization enabled almost all recipients to reject YAS, but bone marrow cells from donors thymectomized 8 months before immunization protected only 50% of the recipients. Further analysis showed that mice thymectomized 8 months before immunization failed to generate anti-YAS antibody response, whereas the antibody response of mice thymectomized 2 months before immunization did not differ from that of non-thymectomized age-matched control mice. The data suggest that the immune reaction of mice against xenogeneic YAS requires long-lived T₂ lymphocytes.     

Regulation of the peritoneal macrophage functional activity by the MP-5 and MP-6 myelopeptides under stress

In mice, two-hour immobilization stress inhibited zymosan-induced production by macrophages of the oxygen radicals and cytokine IL-1β. After myelopeptides MP-5 and MP-6 were administered into mice, the stress-induced inhibition of the reactive oxygen species (ROS) and IL-1β was abrogated. MP-5 peptide stimulated spontaneous ROS production by macrophages and reduced IL-10 production under stress. Thus, under in vivo conditions and under stress, the effect of MP-5 and MP-6 myelopeptides modulates the peritoneal macrophage activity.     

DEFECT OF ERYTHROPOIESIS IN NON-LEUKAEMIC AKR MICE

The kinetics of the CFU population and of erythropoiesis were investigated in the AKR strain mouse prior to the onset of thymic leukaemias: haemopoiesis was compared in syngeneic AKR, semi-allogenic C3H and (C3H x AKR) F, mice injected with AKR stem cells. These experiments demonstrate that the reduction in the number of spleen colonies previously described by Perkins et al. (1971) in syngeneic hosts, as compared to semi-allogenic C3H hosts, is actually related to defective erythropoiesis resulting from a dysfunction of the AKR haemopoietic inductive microenvironment (HIM). Erythropoietin secretion is normal in AKR mice. the early haemopoietic events related to the stem cell: lodgement of the CFU (‘f’ factor) and doubling time, are not disturbed, but the onset of CFU proliferation is markedly delayed in the AKR strain. the main expression of the AKR HIM dysfunction is a significant reduction in the number of erythroid (E) colonies and an impaired output of red blood cells per E-colony in the syngeneic host as compared to the allogenic one. In addition, data indicate that a weakly histo-incompatible system, such as that in C3H and hybrid hosts, does not interfere with the stages of haemopoiesis except by lengthening the doubling time of the CFU. the results, on the whole, emphasize the prevalent influence of HIM.