Post-irradiation regeneration of early B-lymphocyte precursor cells in mouse bone marrow.

To examine the sequential development of early B-cell precursors in mouse bone marrow, B-lineage cells have been examined during a wave of post-irradiation regeneration. Cell phenotypes have been defined using double-immunofluorescence labelling techniques for (i) terminal deoxynucleotidyl transferase (TdT); (ii) B220 glycoprotein, detected by the binding of mAb 14.8; (iii); mu heavy chains in the cytoplasm (c mu) and at the cell surface (s mu). Three populations of mu- cells (TdT+14.8-; TdT+14.8+; TdT-14.8+) have been proposed to be early B-cell precursors which would give rise to c mu+s mu- pre-B cells and thus to s mu+ B lymphocytes. From 3 to 7 days after a sublethal dose (150 rads) of whole body gamma-irradiation, the B-lineage cells recovered rapidly to exceed normal numbers. The early B-cell precursors increased to peaks of 1.8-2.5 times normal numbers, preceding by 1 day a comparable increase and overshoot of c mu+s mu- pre-B cells, followed by recovery of s mu+ B lymphocytes. The TdT+14.8- cells peaked first at 5 days, subsiding again at 7-10 days with a shift from large- to medium-sized cells. The TdT+14.8+ cells showed a later peak (6 days), a more sustained wave in cell numbers and a delayed shift in cell size. The substantial population of 14.8+ mu- cells reached maximal observed values at 7 days and still maintained a predominantly large cell size profile at 10 days. The timing, cell-size shifts and progressive amplification of the waves of regeneration accord with a dynamic model in which the TdT+14.8-,TdT+14.8+ and TdT-14.8+ cells form three successive stages in B-cell differentiation before the expression of mu chains, presumptively including the stage of mu chain gene rearrangement. In addition, the results provide an experimental system for the enrichment of early B-cell precursors in mouse bone marrow.     

Growth of mouse ectoplacental cone cells in subcutaneous tissues. Development of placental-like cells

Ectoplacental cones of mouse embryos collected on day 8 of pregnancy were grafted into the dorsal subcutaneous tissue of host mice. The grafts were collected between days 3 and 8 after transfer and processed for light and electron microscope morphological analysis as well as for cytochemistry of nonspecific alkaline phosphatase. Fragments of normal mouse placentas collected between days 12 and 18 of pregnancy were processed similarly. About 37% of the grafts were nonhemorrhagic nodules formed by different kinds of trophoblastic cells. These cells had many morphological and cytochemical features of cells present in normal mouse placentas. Nonphagocytic giant cells, glycogen cells, as well as cells with a well-developed granular endoplasmic reticulum were similar to cells found in the placenta and were always present in the grafts. Cells showing features intermediate between the above-mentioned cells and those whose cytoplasm was poor in organelles also were found in the grafts. The latter resembled cells of layer 1 of the labyrinth of the placenta. These results suggest that trophoblastic cells of the ectoplacental cones had differentiated into placental cells following their transfer to the subcutaneous tissue.     

Specific antibody-forming B-lymphocyte colonies. I. Distribution and nature of SRBC antibody-forming B-lymphocyte colonies in mouse lyphomyeloid organs.

Wehn normal mouse spleen or lymph node cells are cultured for 7 days in agar-medium containing 2-mercaptoethanol and sheep red blood cells (SRBC), approximately one B lymphocyte colony (BLC) develops per 50-100 cells seeded. Incubation of cultures for 3 h with guinea-pig complement at day 7, demonstrated that 0.05-0.25% of all BLC form specific antibody against SRBC (SRBC-AF-BLC). The SRBC specific colonies appear centrally in lytic plaques of 2-5 mm in diameter and cells recovered from individual SRBC-AF-BLC were shown to produce antibodies of the IgM class against SRBC. Cells forming SRBC-AF-BLC are absent in the new-born and infantile spleen but appear in adult mice with a frequency of 5, 10-25 and 25-70 per 10(6) bone marrow, spleen and lymph node cells respectively. specific immunization in vivo or in vitro does not greatly affect the number of SRBC-AF-BLC-forming cells. Cytolysis of spleen cells with anti-Ig serum plus complement prior to culture reduced the number of total BLC and that of specific SRBC-AF-BLC by 93% and 94% respectively. The peak sedimentation velocity of both SRBC-AF-BLC-forming cells and total BLC-forming cells was 3.5 mm/h. Spleen cells enriched 200-300 times for cells that bind specifically to the hapten NIP were not enriched for cells forming colonies with specific antibody production against NIP. The data indicate that the cells that give rise to specific antibody-forming colonies belong to a mature virgin B cell group of small Ig-positive B lymphocytes.     

Suramin stimulates B-lymphocyte proliferation in the mouse

Suramin, a drug against trypanosomiasis and onchocerciasis, stimulates in vitro mouse B-lymphocytes to transform into blastcells, to incorporate³H-thymidine and to go into mitosis. Thymus cells (hydro-cortisone-sensitive or resistant) do not respond whereas cells from homozygous nude mice are stimulated. The large majority of the blastcells have surface immunoglobulins, and the proportion of c cells with intracellular immunoglobulin increases during the course of the cultures.     

Characterization of T-lymphocyte colony-forming cells in the mouse. II. Colony-forming cells are not confined to a selected age group of post-thymic T cells

T-cell colony-forming cells (CFC), the cells from which T-lymphocyte colonies are initiated when cells are grown in semisolid medium, have been studied. CFC have previously been shown to be more frequent in populations of rather mature thymic cells and peripheral lymphocytes than in populations of immature cortical thymocytes. We have here evaluated whether CFC in spleen and lymph nodes are confined either to newly emigrated thymocytes or to older recirculating T cells. It is shown that CFC in spleen have the same Lyt phenotype as thymic and lymph node CFC, namely Lyt-1+2+, and that the peripheral complement of CFC is rather slowly built up after birth or after total body irradiation and bone marrow reconstitution. Results obtained after fluorescence-activated cell sorting, hydrocortisone injection, or thymectomy indicate that CFC are present in a broad variety of peripheral T-cell populations in accordance with age and thus that T-cell colonies can be used to test the proliferative capacity of the 'average' peripheral T cells of the Lyt-1+2+ phenotype.     

Kinetics of colony-forming ability of mouse bone marrow cells after administration of hydrocortisone

The dynamics of the colony-forming and migration capacity of polypotent hematopoietic stem cells in the bone marrow of (CBA×C57BL) F₁ mice was studied after injection of hydrocortisone. The relative number of hematopoietic stem cells in the bone marrow was higher than in the control on the 3rd day after hydrocortisone injection. This increase was maximal on the 5th day after the injection. On the 8th day the number of hematopoietic stem cells was down to normal again.Institute of Clinical and Experimental Medicine, Siberian Branch, Academy of Medical Sciences of USSR, Novosibirsk. (Presented by Academician of the Academy of Medical Sciences of the USSR V. P. Kaznacheev.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 82, No. 9, pp. 1102–1104, September, 1976.     

The development of antigen-binding lymphocytes in foetal tissues

The time of appearance and counts of antigen-binding cells, using radioiodine-labelled flagellin and haemocyanin, was studied in the human and mouse foetus at different gestational ages: the capacity for binding radioiodine-labelled antigens was equated with acquisition of immunological funciton. Cells resembling lymphocytes from human and mouse liver and bone marrow showed antigen binding at early gestational ages, but this binding could not be prevented with species-specific antisera to immunoglobulins. Specific antigen binding to lymphocytes was detected first with thymic lymphocytes, at gestational ages of 12 weeks in humans and 14 days in mice, then with splenic lymphocytes, at 16 weeks in humans and 17 days in mice, and still later with gut lymphocytes. Relative counts of antigen-binding cells in human foetal thymus were maximal at 16–22 weeks and decreased thereafter. Lymphocyte immunocompetence, as judged by the capacity specifically to bind antigen, develops rapidly after the appearance in thymus of cells with the morphology of lymphocytes; this seemed to occur at the equivalent foetal stage in the species studied.     

Enumeration of polyclonal mitogen-responsive cells in different lymphoid tissues of the mouse.

The relative number of cells capable of responding to Con A, PHA and LPS in the spleen, blood, lymph node and Peyer's patches of CBA mice has been quantified by means of a cytological analysis technique. No difference has been found between Con A- and PHA-responsive cells in spleen and lymph node. The lymphoid tissues of T cell-deprived mice have a reduced content of PHA responsive cells, but LPS responsiveness is within normal limits. Pretreatment of peripheral lymphocyte populations with high concentrations of anti-O antiserum and complement abolishes the response of the treated cells to PHA, but not to LPS, whereas similar treatment with a cytotoxic anti-immunoglobulin serum, which has no effect on PHA-responsive cells, only partially reduces the response to LPS. The results for mitogen responsiveness are discussed with reference to other methods of quantifying T and B cells using cell-surface markers.     

In vitro frequency analysis of spleen colony-forming and marrow-repopulating hemopoietic stem cells in the mouse

Summary An assay is described for Day-12 spleen colony-forming cells (CFU-S-12) and hemopoietic stem cells with marrow-repopulating ability (MRA) in the mouse using a miniaturized stroma-dependent bone marrow culture assay in vitro. Bone marrow cells are grown in liquid culture in microtiter wells, and the resulting adherent stromal layers are depleted of all hemopoietic activity by 20 Gy gamma irradiation. Subsequently, single cell suspensions containing stem cells are overlaid in a range of concentrations, and the presence of one or more emerging phase nonrefractive cell clones (cobblestone areas) in a single well scored as positive. The frequencies of cobblestone area-forming cells (CAFC) are then calculated by employing Poisson statistics. It is shown that the CAFC Day-10 and CAFC Day-28 frequencies closely correlate with those of CFU-S-12 and MRA cells, respectively.     

Replication of influenza virus in organ cultures of human and simian urogenital tissues and human foetal tissues.

A survey of human adult tissues in organ cultures showed that influenza viruses (A/Moscow/1019/65 (h2n2) or a recombinant virus virulent for man (PR/8-A/England/939/69 Clone 7a(H3N2)) could infect uterus, bladder and conjunctiva but not oesophagus under the conditions employed; simian bladder and uterus were also susceptible. These results were similar to those already described for corresponding ferret tissues. Organ cultures of human foetal nasal mucosa, trachea, oesophagus, small and large intestine, and bladder consistently supported replication over 4 days or more with high virus yields. Lung, conjunctiva and umbilical cord were less consistently susceptible and gave lower yields. Placenta and kidney cultures allowed replication of virus in one of 8 and one of 4 experiments respectively, the yields being low and of short duration. Organ cultures of neural tissue (meninges and brain), lymphopoietic tissue (spleen, liver and thymus) and amnion did not support significant viral replication. The results are discussed in relation to possible infection of the foetus in utero with influenza virus.     

Crossreaction of antilymphocyte globulin with human granulocyte colony-forming cells.

Clinical preparations of horse antilymphocyteglobulin (ALG) were found to inhibit human bone marrow granulocyte colony growth. This effect was enhanced by complement and was dose dependent, being almost complete at ALG concentrations of 100 microgram/ml. Inhibition was a property of ALG but not of normal horse globulin. However, short incubation of ALG with bone marrow cells occasionally stimulated colony growth and normal horse globulin regularly stimulated it. Three hours' incubation of bone marrow cells with ALG was needed to produce consistent colony inhibition, which was measurable as a reduction in the expected number of colonies and as a fall in the colony: cluster ratio of surviving cell aggregates. Absorption of ALG on acute myeloid leukaemia blast cells removed the inhibiting property of the ALG while preserving its lymphocytotoxic action. Serum from two patients receiving ALG treatment inhibited colony growth for up to 48 hours after ALG administration. The results suggest the presence in ALG of antibodies specifically cytotoxic to myeloid stem cells which may relate to its myleosuppressive properties in vivo, and also indicate that it should be possible to remove antimyeloid antibodies from ALG by absorption. The use of such purified ALG would have advantages in clinical bone marrow transplantation.     

Presence of radial glia in foetal mouse cerebellum

Summary The ventricular layer (VL) of foetal mouse cerebellum at days 13–15 of gestation was studied by light and electron microscopy. In Golgi-stained material, round or ovoid cells are located in the VL. These cells have ascending processes, which extend to the pial surface. Ultrastructurally, the ascending processes are electron-lucent, contain microfilaments, some smooth endoplasmic reticulum and scant free ribosomes. They appear to be immature glial processes, oriented radially away from the ventricle. The perikarya of these glial cells lie either in the ventricular or subventricular zones.Juxtaposed along the length of these radially oriented glial processes are unidentified cells, some of which are attached to the immature glial fibres by puncta adhaerentia. These cells are elongated or ovoid with a thin rim of cytoplasm containing few organelles. These unidentified cells may represent neuroblasts (Purkinje cells, Golgi cells, cells of the deep cerebellar nuclei) or glioblasts, (precursors of astrocytes and/or oligodendrocytes) at very early stages of development.     

Abolition of allogeneic inhibition and changes in differentiation of mouse bone marrow colony-forming cells by syngeneic lymphocytes

A mixture of bone marrow cells and lymph gland lymphocytes of C57BL mice was injected intravenously into F₁ (A×C57BL) mice irradiated in a dose of 850 R. Under the influence of syngeneic lymphocytes of the C57BL mice colony-forming cells (CFUs) of the bone marrow proliferated in the F₁ mouse hybrids as if they had been in a syngeneic organism. Besides restoration of the number of CFUs capable of forming colonies in a genetically foreign organism, under the influence of lymphocytes the colonies changed their direction of differentiation, with an increase in the number of granulocyte colonies. This process was accompanied by the development of a blast transformation reaction and by hyperplasia of the lymphoid tissue of the recipients' spleens.     

Effect of poly-4-vinylpyridine on the number of colony-forming cells in mouse bone marrow and spleen

Injection of poly-4-vinylpyridine in the maximal tolerated dose into mice doubles the number of colony-forming units (CFU) in the bone marrow and reduces the number of nucleated cells by 22%. The increase in the CFU pool takes place from the second through the seventh day. The number of bone marrow cells starts to decrease 2 h after injection of the polymer and remains below the initial level for 5 days; after the second day, however, it starts to return gradually to normal, which it reaches by the seventh day. The number of CFU in the spleen increases ninefold, and this is accompanied by the development of marked splenomegaly on account of the increase in the number of cells.(Presented by Academician of the Academy of Medical Sciences of the USSR P. D. Gorizontov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 4, pp. 474–476, April, 1977.