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Sex-linked hypochromic anemia is the result of iron deficiency, apparently caused by malabsorption of iron. Progenitor cells in marrow and spleen of male anemic (genotype, sla/-) and normal (+/-) littermate mice were studied by quantitative transplantation into irradiated, but otherwise normal, recipient mice. The progeny of grafted cells were assessed by isotope uptake technics. Progenitor cells of sla/- mice were capable of extensive production of erythroid cells, which synthesized DNA and hemoglobin, and of leukocytic elements, which synthesized DNA but not hemoglobin. Proliferation of the latter cell types was assayed in recipient mice rendered polycythemic with red cell infusions to suppress erythropoiesis. The mature red cells produced by transplanted sla/- marrow and spleen were normal with respect to volume, size, and hemoglobin content. The grossly enlarged spleens of sla/- mice contained 20 times the normal number of erythropoietic progenitor units (EPU); parenteral iron therapy cured the anemia and caused the spleen to revert to normal. The ratio of EPU to leukopoietic progenitor units (LPU) was abnormally high in marrow and spleen of sla/- mice; in addition, the EPU:LPU ratio was higher in spleen than in marrow of normal and anemic mice. The high EPU:LPU ratio of sla/- bone marrow persisted after transplantation to a lesser, but significant, extent for at least 52 days. We conclude that: 1) the sla mutant gene does not affect hemopoietic cells directly, but that iron deficiency alters the ratio of EPU to LPU by committing more primitive pluripotent stem cells to erythropoiesis, and 2) EPU and LPU are separate progenitor cells with restricted potential for differentiation. Persistence of the altered EPU:LPU ratio in sla/- marrow chimeras may be explained by extensive and independent self-replication of EPU and/or LPU, especially after grafting relatively small numbers (1.6 million) of marrow cells into irradiated mice.

1. Implantation into irradiated (C57BL X 101) F1hybrids of viable liver cells from adult donor mice of the parental strains caused a wasting disease followed by death in over 90% of the recipients. 2. This “foreign liver disease” occurred after a sublethal dose of radiation was given to the recipients or after a lethal dose followed by infusion of isologous bone marrow. It did not occur in nonirradiated recipients but occurred in only 26% of sublethally irradiated F1hybrids infused with isologous bone marrow. 3. In a strain combination in which the F1hybrid was homozygous at the H-2 locus, and the parents should therefore have been antigenetically relatively compatible, the “foreign liver disease” occurred late after treatment and was enhanced by presensitization of the donor to the reciprocal parent of the hybrid. It was shown that radiation dose and liver cell dose were inter-related factors in determining the development of the disease. 4. Deaths are ascribed to an in vivoimmunologic reaction after antibody production by the grafted parental cells against F1antigens. Nevertheless, there is evidence of a possible immune response by the F1hybrid against the parent.We are grateful to Mr. W. D. Gude and Mr. T. Mack for the histological preparations.

Spleen cell suspensions of primed donor mice containing precursors of immunocytes have been transplanted into X-irradiated recipient mice 122–138 days after immunization. Following secondary stimulation with antigen (sheep erythrocytes), these precursors, called antigen-sensitive units (ASU), gave rise to progeny cells secreting specific antibody in the spleens of recipients. Single cells releasing IgM hemolysins (direct plaque-forming cells or PFC), IgG hemolysins (indirect PFC), and hemagglutinins (cluster-forming cells or CFC) were enumerated. By transplanting graded and limiting numbers of primed spleen cells, inocula were found which contained one or a few ASU reaching the recipient spleens. We estimated, thereby, the frequency of ASU detectable by our procedures in donor cell suspensions. The values obtained from direct and in-indirect plaque assays, and from cluster assays were 1 in ∼8.0 x 105, 1 in ∼4.4 x 105, and 1 in ∼5.9 x 105 nucleated spleen cells, respectively. The number of splenic ASU for direct PFC was not greater than that of unimmunized mice; however, immunization greatly increased the number of splenic ASU for indirect PFC and for CFC. By applying to each recipient spleen direct and indirect plaque tests and cluster tests, we found that positivity for each type of immunocyte was independent from that of the other two types. These results confirm the unipotent nature of splenic ASU in general, and document the commitment of ASU primed with SRBC to generate progeny cells secreting antibody of a single molecular (IgM or IgG) or functional (lysin or agglutinin) class. We concluded that splenic ASU are composed of relatively differentiated cells of the immune system of mice. With respect to specificity and class differentiation, ASU appear to be as specialized as antibody-producing cells themselves. Our results did not support the view that ASU-derived clonal populations shift from IgM to IgG antibody production.

Spleen cell suspensions of unprimed donor mice containing precursors of immunocytes have been transplanted into X-irradiated recipient mice. In the presence of antigen (sheep erythrocytes) these precursors, called antigen-sensitive units, gave rise to progeny cells secreting specific antibody. We studied quantitatively the production of cells releasing IgM hemolysins (direct plaque-forming cells), IgG hemolysins (indirect plaque-forming cells), and hemagglutinins (cluster-forming cells). We found that each of these immunocyte populations was distinct, i.e., that cells releasing agglutinins did not, as a rule, release hemolysins, and vice versa. We also found that cell populations secreting IgM hemolysins did not shift, under certain experimental conditions, to the production of IgG hemolysins during the primary immune response. By transplanting graded numbers of spleen cells, we succeeded in limiting to one or a few the number of antigen-sensitive units that reached the recipient spleen. We estimated thereby the frequency of antigen-sensitive units in donor cell suspensions and tested their potential for production of immunocytes of more than one type. Our results indicated that antigen-sensitive units were unipotent for they displayed in the spleens of unprimed donors the same restrictions of function and heterogeneity (antibody-specificity differentiation, antibody-class differentiation) found among antibody-forming cells. Furthermore, antigen-sensitive precursors for direct plaque-forming cells, indirect plaque-forming cells, and cluster-forming cells were detected in the spleens of unprimed mice in different frequencies, i.e., 1 in ∼ 106, 1 in ∼ 7 x 106, and 1 in ∼ 19 x 106 spleen cells, respectively. We concluded that relatively advanced differentiation of potentially competent cells occurs before sheep erythrocyte administration. The relevance of this finding for the broad spectrum of immunologic reactivities and for the heterogeneity of antibody responses to given antigens was discussed.

Acid phosphatase, ATP-ase and cytochrome-c oxidase activities have been studied in rat mitochondria isolated from precancerous livers and from experimental hepatomas.ATP-ase and cytochrome-c oxidase activities are increased, while acid phosphatase activity is decreased by p-DAB feeding.These enzymatic modifications seem to be secondary to morphological and structural changes of precancerous and cancerous liver mitochondria. They occur very early during experimental liver carcinogenesis, contemporaneously with the appearance of structural changes indicated by a minor swelling of precancerous and cancerous mitochondria isolated in hypotonic media, as compared to mitochondria from normal livers.No statistically significant differences have been observed between enzymatic activities of mitochondria isolated from precancerous livers and hepatomas; it is suggested that the mitochondrial damage reaches its maximum in the precancerous stages, being no furtherly modified in the established hepatomas.

Allograft reactions directed against antigens of the entire H-2ballele or of its K and D ends were compared in 3 congenic-resistant strains of mice sharing the C57BL/10 background. Lymphoma cells of B10 origin were transplanted into partially immunodepressed mice; residual host antigraft immunity was potentiated by a single administration of the antitumor agent BCNU. The immunogenicity of H-2bcomponents was distinct and additive, with strength of elicited allograft reactions decreasing in the following order: D + K>K>D.

The number of direct (γM) hemolytic plaque responses of irradiated mice, repopulated with relatively small and limiting numbers of bone marrow and thymus cells, was increased by the simultaneous immunization with two antigen complexes instead of one. Anti-sheep responses were augmented by the following antigen combinations: SRBC + HRBC, SRBC + BRBC, and SRBC + CRBC. Limiting either thymocytes or bone marrow cells indicated that the antigen mixtures acted at the level of T cells increasing severalfold the number of triggered antigen-reactive cells. It was concluded that one of the antigens could have influenced the triggering of antigen-reactive cells specific for the other by promoting synergistic or derepressive T-T cell interactions. Moreover, bone marrow precursor cells could have been activated by the thymic inducers specific for the test antigens as well as by those specific for the second of the priming antigens.

Marrow cells and 5 x 107 thymocytes of unprimed (C57BL/6 x DBA/2)F1, (C57BL/10 x WB)F1 and (C3H x C57BL)F1 donor mice were mixed in vitro and transplanted into X-irradiated syngeneic hosts. Upon injection of sheep erythrocytes, splenic plaque-forming cells (PFC) secreting IgM (direct PFC or IgG (indirect PFC) hemolytic antibody were enumerated at the time of peak responses. By grading the numbers of marrow cells, inocula were found that contained few immunocompetent cells reaching the recipient spleens, interacting with thymocytes or other accessory cells (or both), and generating PFC. The frequency of responses in BDF1 mice conformed to Poisson statistics, indicating that immunocompetent marrow cells participated in a single-hit interaction limiting PFC responses. The marrow cells assayed were not restricted for the antibody class (IgM versus IgG) to be secreted by mature PFC. Unrestricted marrow cells could have been either the precursors of PFC or accessory cells. Different results were obtained in BWF1 and C3BF1 mice. The frequency of responses in relation to the number of marrow cells grafted did not follow Poisson statistics, and the limiting cells were restricted for antibody class. Presumably, immunocompetent cells of these strains were more heterogeneous than those of BDF1 mice and participated in a multiplicity of cell-to-cell interactions. The strain differences reflected inherent properties of marrow cells and not influences of the environment in which PFC were produced. The results confirmed for bone marrow the heterogeneity of immunocompetent cells reported by others for spleen, and suggested that genetic factors such as "immune response" genes regulate cellular differentiation also for functions other than those related to antibody specificity.

Marrow cells and thymocytes of unprimed donor mice were transplanted separately into X-irradiated syngeneic hosts, with or without sheep erythrocytes (SRBC). Antigen-dependent changes in number or function of potentially immunocompetent cells were assessed by retransplantation of thymus-derived cells with fresh bone marrow cells and SRBC; of marrow-derived cells with fresh thymocytes and SRBC; and of thymus-derived with marrow-derived cells and SRBC. Plaque-forming cells (PFC) of the direct (IgM) and indirect (IgG) classes were enumerated in spleens of secondary host mice at the time of peak responses. By using this two-step design, it was shown (a) that thymus, but not bone marrow, contained antigen-reactive cells (ARC) capable of initiating the immune response to SRBC (first step), and (b) that the same antigen complex that activated thymic ARC was required for the subsequent interaction between thymus-derived and marrow cells and/or for PFC production (second step). Thymic ARC separated from marrow cells but exposed to SRBC proliferated and generated specific inducer cells. These were the cells that interacted with marrow precursors of PFC to form the elementary units for plaque responses to SRBC, i.e. the class- and specificity-restricted antigen-sensitive units. It was estimated that each ARC generated 80–800 inducer cells in 4 days by way of a minimum of 6–10 cell divisions. On the basis of the available evidence, a simple model was outlined for cellular events in the immune response to SRBC.

Marrow cells and thymocytes of unprimed donor mice were mixed in vitro and transplanted into X-irradiated syngeneic hosts. 18 hr later sheep erythrocytes were injected to induce immune responses. Splenic plaque-forming cells (PFC) secreting IgM (direct PFC) or IgG (indirect PFC) hemolytic antibody were enumerated at the time of peak responses. By transplanting graded and limiting numbers of marrow cells with 5 x 107 thymocytes, inocula were found that contained few precursors of PFC (P-PFC) reaching the recipient spleens, interacting with thymocytes, and generating PFC. However, the frequency of responses in relation to the number of grafted marrow cells did not follow Poisson statistics, presumably because the interaction of marrow cells with thymocytes was more complex than a single or a one-to-one cell event. The frequency of direct PFC responses was greater than that of indirect PFC responses in 13 of 15 groups of mice tested. This was interpreted as evidence for the existence of two classes of P-PFC, each of which was restricted to generate either direct or indirect PFC. The precursors of direct PFC were ∼ 15 times more frequent than those of indirect PFC. Since thymic antigen-reactive cells were not differentiated for antibody class, it follows that antigen-sensitive units reactive to sheep erythrocytes owe their class restriction to specialized marrow cells. Specialization of P-PFC may have arisen within marrow cell lines by differentiation, or may have been conferred upon P-PFC by interaction with other cells, including those of the irradiated host.