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Several 2'-5' oligoadenylate (2-5A) core molecules were tested for their inhibitory effect on erythroid colony-forming units (CFU-E). These compounds were introduced into the CFU-E microenvironment by vascular perfusion of isolated rat hind limbs. The marrow microvasculature did not appear to be damaged by these procedures when examined by electron microscopy. After a brief in situ exposure to 2-5A core, the marrow cells were grown in methylcellulose medium with added erythropoietin (Ep). Maximum percent inhibition of CFU-E was obtained with 2-5A trimer core. The tetramer core was less effective, and no inhibition of colony formation was seen in cultures of marrow perfused with 2-5A dimer core. From this it was concluded that the inhibitory effect of 2-5A trimer core was highly specific. Furthermore, this effect was of long duration, since when 2-5A trimer core was given just prior to Ep the CFU-E were blocked in their differentiative response to Ep for 48-72 h.

Spleen cell suspensions obtained from adult mice were separated by Ficoll/Hypaque and Percoll density gradient centrifugation. The enriched erythroblast populations were maintained in liquid culture medium for 8 hours with 10,000 units of murine interferon (IFN) alpha and beta. Exposure of these cell cultures to murine IFN alpha and beta resulted in a 48% to 70% increase in 2-5adenylate synthetase (2-5AS) activity. In parallel studies, adult mice were injected daily for 1 or 2 weeks with recombinant human IFN alpha A/D (rHuIFN alpha A/D) at a dose of 10(6) or 10(7) units/kg body weight. This treatment did not significantly affect body weight but did produce a mean 70% increase in spleen wet weight and a mean 46% increase in number of nucleated cells per spleen. This increase in number of splenic hematopoietic cells did not result in a corresponding increase in number of circulating cells. In fact, during this 1 to 2 week period the hematocrit dropped from 45% to 38% in mice injected with high dose rHuIFN alpha A/D. From these findings we propose that IFN induces an early 2-5AS activity in erythroblasts and megakaryocytes. This 2-5AS activity, which is known to inhibit protein synthesis in other cell systems, is thought to be responsible for the block or prolongation in blood cell maturation observed in the present studies.

In response to prolonged, intermittent exposure to hypoxia, the spleens of adult BALB/c mice displayed an initial increase and subsequent decrease in erythropoietic activity. The enzyme 2,5-adenylate synthetase was assayed during this period, and a direct relationship was found between the rate of red cell production and enzyme activity; that is, 2,5-adenylate synthetase activity was maximum in the spleen cell populations that contained the largest number of nucleated erythroid cells and minimum in those populations that contained the fewest nucleated erythroid cells. In contrast to this finding, synthetase activity was inversely related to the number of lymphocytes present in these spleen cell populations. On the basis of these observations, it appears that 2,5-adenylate synthetase is present in nucleated erythroid cells. If active in late erythroblasts, 2,5-adenylate synthetase may function as an inhibitor of DNA synthesis and/or hemoglobin synthesis.

Morphometric measurements of bone marrow sinus wall adventitial and endothelial cells were made in normal and leukemic samples by using a Bioquant II computer image analysis program (R & M Biometrics, Nashville TN). Electron micrographs of bone marrow from at least eight mice per group were studied in normal and day-2, day-4, and day-6 leukemic postinoculation groups. The adventitial cells abuting the sinus endothelium was found to be significantly reduced with disease progression. In addition, adventitial cell area and perimeter were found to be drastically reduced with disease progression whereas endothelial cell area and perimeter showed no significant differences. Other investigators have suggested that the marrow sinus adventitial cell cover is drastically reduced in rodent leukemias (Chen et al.; Blood, 39:99-112, 1972; Campbell; Am. J. Anat., 142:319-334, 1975). Our findings give quantitative data to support this hypothesis.

Human small intestine was obtained from fetuses at 8 to 20 weeks of gestation. The tissues were examined by electron microscopy. Lymphocytes were present as isolated cells in the mucosal mesenchyme as early as the 8th week of intrauterine life and were found, in increasing numbers, in the epithelium of specimens obtained from 11- to 20-week-old fetuses. All intraepithelial lymphocytes were located in the lateral spaces between contiguous columnar cells. Some displayed cytoplasmic pseudopods and were thought to migrate within the extracellular spaces of the epithelial layer. Surface invaginations and cytoplasmic vesicles in fetal lymphocytes suggested that these cells were capable of pinocytotic activity. The possibile origin and function of lymphocytes in fetal small intestine are discussed.

A single dose of 0.5 mCi radioiron (55Fe) was injected directly into the amniotic cavity of each of four pregnant monkeys near term. At 6 or 24 hours after 55Fe adminstration, samples of fetal jejunum, ileum, blood, spleen, liver, lung and fetal membranes were taken for radioautography and scintillation counts. Counts were also made on corresponding maternal tissues. Intestinal radioiron levels and localization indicated that fetuses swallowed and absorbed 55Fe within 6 to 24 hours after injection. Silver grains over the meconium bodies of fetal ileum suggested that intestinal epithelium retained at least some of the absorbed radioiron. However, elevated 55Fe levels in fetal blood and hematopoietic organs indicated that transport of radioiron to the circulation had occurred, presumably through fetal intestine, extraembryonic membranes and/or fetal lungs. Transfer of 55Fe across the placenta from fetus to mother occurred at a slow rate, i.e., samples of maternal organs obtained at 24 hours gave low counts.

When injected into the amniotic sacs of fetal rats in late pregnancy, 3H-leucine is rapidly absorbed and incorporated into fetal proteins as determined by liquid scintillation spectrometry. Peak values in the fetal liver and pancreas occur around four hours after injection. Approximately 21% of the radioisotope is retained in the injected fetus at 24 hours. The remainder escapes into the maternal circulation and accounts for a gradual increase in levels in noninjected fetuses from the same litter and for persistently high counts found in maternal serum as late as 24 hours after the injection. Specific activity of the radioisotope in fetal tissues is considerably higher (100- to 200-fold) when 3H-leucine is injected inth the amniotic sac rather than intravenously into the mother. The possible treatment of various fetal disorders by means of intra-amniotic injections is discussed.

The tetramer core of 2'5'-adenylate was tested in vitro for its effect on erythroid colony-forming units (CFU-E) from bone marrow of adult rats. Inhibition of CFU-E was 2'5'-adenylate dose dependent, when tested with concentrations of 0.05- 1.0 mM. Also, 2'5'-adenylate was inhibitory when incubated with 2, 6 or 10 X 10(4) cells/0.1 ml plasma clot. At 0.1 mM concentration of 2'5'-adenylate there was a 30-50% reduction of erythroid colonies for each of these three cell concentrations. There was an early decrease in 3H-thymidine incorporation when 2'5'-adenylate was added to erythropoietin-stimulated bone marrow cell suspensions prior to plating. It is suggested that in situ production of 2'5'-adenylate may represent a regulatory mechanisms in red cell maturation.

Infection of BALB/c mice with the RLV-A virus typically results in an erythropoietic dysplasia characterized by hepatosplenomegaly, erythroblastosis, erythroblastemia and severe anemia without reticulocytosis. Mice hypertransfused weekly with 75%-packed red cells for 42 days prior to RLV-A infection and viral potency controls manifested this typical RLV-A response. Mice that were hypertransfused prior to and following RLV-A infection never developed the "typical" RLV-A pathogenesis. Instead, a transplantable myeloid leukemia was established. Although the reason for altered pathogenesis remains uncertain, it seems plausible that continued hypertransfusion, presumably after establishment of an altered granulopoietic microenvironment, resulted in a completely different viral expression and development of the transplantable myeloid leukemia.

Infection of BALB/c mice with the RLV-A virus normally induces an erythropoietic dysplasia characterized by hepatosplenomegaly, erythroblastosis, erythroblastemia and severe anemia without reticulocytosis. Time to death varies between 20-30 weeks. Mice were inoculated with RLV-A after being hypertransfused with 75% packed red cells for 42 days which has been shown to eliminate erythropoiesis and modify the microenvironment to favor granulopoiesis. Following RLV-A inoculation, one group did not receive further transfusion (short-term) and another group continued with hypertransfusion weekly (long-term). The pathogenesis of RLV-A in the short-term group paralleled the characteristic RLV-A response. In the long-term group however, the characteristic RLV-A response was never observed. Instead, a granulocytic leukemia was developed. Continued hypertransfusion presumably after establishment of an altered microenvironment resulted in a completely different viral pathogenesis and the development of a transplantable myeloid leukemia.

A polycythemia-inducing strain of Friend virus was injected into adult mice. Animals were sacrificed on days 2, 5, 9, 11, and 14, and specimens of liver were fixed and embedded for light and electron microscopy. From some mice, samples of bone marrow and spleen were also processed for microscopy. Hepatic erythropoiesis was established as early as 2 to 5 days after inoculation. Often, red cell maturation occurred in typical islands. These included a central macrophage which in many instances appeared to be a Kupffer cell. The number and size of red cell foci increased through day 14. Extensive ineffective erythropoiesis involving erythroblast death occurred within these foci. This premature cell death may be related to large inclusions, thought to be autophagosomes, in the cytoplasm of erythroblasts. A few undifferentiated cells, presumed to be the reticulum cells of Friend virus disease, were observed in the liver at 9 to 14 days after infection. The morphology of these cells resembled that of primitive hemopoietic elements. These studies indicate that the liver, along with the spleen, contributes to the polycythemia in the early phase of Friend virus disease.

Fetal erythropoiesis was studied in human livers at 10 to 12 weeks of gestation. The most primitive blood cells were often observed in large indentations of the surface of hepatocytes and the plasma membranes of the two cell types were adherent at sites of attachment. Erythroid cell maturation occurred predominantly in the lumen of the sinusoids. Cell suspensions obtained from fetal livers were centrifuged, frozen at -196 degrees C, thawed and studied by electron microscopy. The primitive cells were morphologically altered by these procedures. Changes included damage to mitochondria and cell membranes and vacuole formation. Erythroblasts, by comparison, were virtually intact and even displayed some indications of reestablished functions within 10 minutes after thawing.

Horseradish peroxidase (HRP) injected into amniotic fluid is swallowed by rat fetuses and within 3-6 h reaches the gut lumen. This macromolecular protein is then absorbed by the columnar lining cells via a system of apical cytoplasmic tubules formed by invaginations of the plasma membrane. From cytoplasm subjacent to the brush border HRP is transported, within vacuoles, to the supranuclear region, where some is retained for at least 18 h, and to interepithelial spaces. Extracellular enzyme is then found throughout the epithelial basement membrane and between connective tissue cells of the mucosal and submucosal layers Finally, HRP can be detected within lumina of blood and lymphatic capillaries, strongly suggesting that it is transported from the intestine to the circulation.

Horseradish peroxidase (molecular weight, about 40,000) injected into the amniotic sacs in pregnant rats has been identified ultrastructurally, 6 to 18 hours later, within the fetal intestine in the absorptive cells and the underlying vascular endothelium. This indicates that macromolecular protein within amniotic fluid swallowed by the fetus can be absorbed and transported by fetal intestine, and may indicate that physiological compounds can be transported by this enteric route to contribute to fetal development.