Thrombocytopenia in a retrovirally-induced murine erythroleukemia.

A variant strain of Rauscher leukemia virus (RLV-A) obtained from a transplantable murine monomyelocytic leukemia causes a disease characterized by frank anemia, wasting, hepatosplenomegaly and erythroblastosis. The involvement of platelets in this disease are reported here. The RLV-A induced a severe thrombocytopenia (25 percent of control level) at the terminal stage of disease. This thrombocytopenia was not associated with disseminated intravascular coagulopathy since the prothrombin times were always within normal limits. The partial thromboplastin time was elevated in the terminal stages of disease and was found to be associated with factor deficiencies, possibly owing to the presence of anti-factor antibodies, in the intrinsic coagulation pathway, especially factor VIII. Further, splenectomy did not abolish the thrombocytopenia, since splenectomized, virally infected animals also developed severe thrombocytopenia (29 percent of control levels). The ensuing splenomegaly during progression of disease was not the cause of the thrombocytopenia. A physiological response to the severe thrombocytopenia was the production of larger size platelets. At terminal stages of the disease, platelet volume increased to 4.2 mu 3 (normal is 3.0 mu 3). An increase in platelet volume was also observed in splenectomized, virally infected animals. Electron microscopy indicated that these circulating platelets contained c-type viral particles. Viral infection was associated with decreased life span of circulating platelets, as measured by 75Se-methionine at mid and terminal stages of the disease. Our results suggest that direct viral infection of platelets and/or megakaryocytes with subsequent cell lysis is a possible cause of the observed thrombocytopenia observed in RLVA-induced disease and may also occur in other retrovirally-induced diseases.

Neonatal erythropoiesis. II. Bone marrow and splenic erythropoietic activity: data suggest erythropoietin transfer via maternal milk.

In this study we examined erythropoiesis in the bone marrow and spleen of 9 to 12-day-old neonatal rats suckled by bleeding-induced anemic mothers. Quantitative evaluations of the spleen revealed a significant decrease in total numbers of nucleated RBC/mg spleen in 11-day-old pups nursed by the anemic mother which returned to control values by day 12. A significant reduction in total numbers of nucleated RBC/mg marrow was seen in both 11 and 12-day-old pups of anemic mothers when compared to control values. These results suggest that: 1. Ep is transmitted to suckling rats via the maternal milk; 2. Ep in the neonate exerts its influence predominantly at the level of the differentiated erythroid compartment probably by causing a shortening of the mean transit time of the proliferating erythroblast compartment and/or by decrease in the maturation time of the nonproliferating orthochromatic and reticulocyte compartments; 3. the regulation of erythropoiesis in the neonate differs from that in the adult.

Detection of canine homologs of human MYC, BCL2, IGH, and TCRB genes by Southern blot analysis.

Subsets of non-Hodgkin's lymphoma in humans have been shown to involve activation of protooncogenes such as MYC and BCL2 resulting from chromosome translocation involving the IGH and TCR genes. Malignant lymphomas in the canine present histologic and clinical subsets similar to those in humans. To study the genetic nature of these lymphomas, we undertook this study to determine, by Southern blotting analysis, the extent of homology between the human and canine genes MYC, BCL2, IGH, and TCRB using human gene probes. Our results, presented here, show that the organization of these genes in the two species is very similar.

Neonatal erythropoiesis. I. Peripheral blood erythropoietic parameters: data suggest erythropoietin transfer via maternal milk.

In this study peripheral blood erythropoietic parameters in 9 to 12-day-old neonatal rats suckled by experimentally-induced anemic mothers were examined. Stimulation of erythropoiesis in these pups was judged by increases in hematocrit, hemoglobin, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration. Failure to observe increases in reticulocytes may be, in part, the result of decreased maturation time, as indicated by reticulocyte Heilmeyer maturation indices in peripheral blood. The reticulocyte maturation curve was shifted to the left in neonates nursing from anemic mothers. These results suggest that erythropoietin (Ep) is transmitted to suckling rats via maternal milk, and by escaping inactivation in their gastrointestinal tract stimulates their erythropoiesis. These findings, in agreement with our previous work, are supportive of studies by others and further indicate a maternal-neonatal erythropoietic relation in rats.

Octapeptide segments from the amino terminus of glycophorin A contain the antigenic determinants of the M and N blood groups systems.

Human red blood cells with phenotype N/N and M/M were tested in an agglutination assay with anti-N and anti-M antibodies, respectively. After incubation of the synthesized octapeptide (Leu-Ser-Thr-Thr-Glu-Val-Ala-Met) from the N-amino terminus of glycophorin A, with anti-N antibody, there was significant inhibition of the agglutination of the N-positive cells. There was also inhibition of the agglutination of the M-positive cells with anti-M antibody by the synthesized octapeptide (Ser-Ser-Thr-Thr-Gly-Val-Ala-Met) from the M-amino terminus of glycophorin A. There was no inhibition, however, of the agglutination of M-positive cells with anti-M antibodies by the N-amino terminal octapeptide. Likewise, the M-amino terminal octapeptide did not inhibit agglutination of N-positive cells with anti-N. Because the synthesized octapeptides contained no carbohydrate, the anti-N and anti-M specificity appears to be determined principally by the peptides themselves. Further studies with the use of chimeric peptides indicate that the amino terminal amino acid leucine of N-glycophorin A is a primary determinant of the N antigen, whereas the amino terminal serine of M-glycophorin A is a primary determinant for the M antigen.

Morphometric analysis of bone marrow sinus cell elements after induction of monomyelocytic leukemia in BALB/c mice.

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.

Sustained hypertransfusion and induction of a transplantable myeloid leukemia in RLV-A-infected BALB/c mice.

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.

Cytogenetic analyses of somatic chromosomes in a transplantable monomyelocytic leukemia in BALB/c mice.

In vivo cytogenetic analyses have been performed using G-, C-, and nuclear organizing region (NOR)-banding techniques, and sister chromatid exchanges (SCE) on a transplantable monomyelocytic leukemia (MML) initially induced in female BALB/c mice by the Rauscher leukemia virus (RLV). Centromeric associations have been found to be greatly increased in MML transplanted mice. Transplantability of the disease has been demonstrated at the cytogenetic level by the presence of female cells in males transplanted with MML cells. G-banding analysis has shown the existence of a marker deleted chromosome 18 in all tissues examined (bone marrow, spleen, and peripheral blood) restricted to female transplanted cells. The NOR-banding analysis has shown a slight increase in the number of Ag-NOR sites per metaphase in MML transplanted mice compared with controls and the existence of an extra chromosome having NOR in MML transplanted mice. No differences were found in C banding between controls and MML transplanted mice. In MML transplanted males, female transformed cells showed a significant reduction in SCE frequency compared with host male cells or controls.

Effect of sustained hypertransfusion on Rauscher leukemia virus-variant A (RLV-A) infection in BALB/c mice.

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.

Effects of the hormone erythropoietin in milk on erythropoiesis in neonatal rats.

Erythropoiesis was stimulated in 2- to 5-day-old normal neonatal rats nursed by phlebotomized mothers, and in 12-day-old hypertransfused neonatal suckled for 4 days by a twice-bled mother. Erythropoietic stimulation was evidenced as increases in hemoglobin levels and numbers of peripheral reticulocytes in the 2- to 5-day-olds, and by a higher 48-hour RBC-59Fe incorporation in the hypertransfused than in control 12-day-olds. The reticulocyte maturation curve was shifted to the left in 14-day-old transfused rats suckled by an anemic mother, in 14-day-old normal rats suckled by an anemic mother, and in 12-day-old transfused neonates fed cow milk to which erythropoietin (Ep) had been added. The oral administration of cow milk containing Ep to 10-day-old normal neonates induced a reticulocytosis. One-day-old rats suckled by mothers bled 72 h prior to delivery exhibited significant amounts of Ep in their plasma as compared to nondetectable levels in 1-day-old control rats of normal mothers. Decreases in relative percentages of nucleated erythrocytes were noted in spleens of hypertransfused 12-day-old neonates nursed by anemic mothers, in spleens of hypertransfused 12-day-old injected with Ep, and in spleens of 14-day-old normal rats suckled by an anemic mother. Nucleated erythrocyte numbers were reduced in both spleen and marrow of 12-day-old anemic neonates. The data suggest that: Ep is transmitted to neonatal rats via maternal milk, and in the process of gastrointestinal tract absorption, escapes inactivation, thereby stimulating erythropoiesis in these animals; Ep acts on already differentiated erythroid cells by influencing their rate of maturation.

Erythroid progenitors in Rauscher leukemia virus variant-A-induced erythropoietic dysplasia in mice.

In vitro erythropoiesis by bone marrow and spleen cells was assessed in normal mice and during progression of Rauscher leukemia virus, variant-A (RLV-A) disease in mice. As RLV-A disease progressed from early through terminal stages, there was a marked increase in the numbers of in vitro splenic CFU-E and BFU-E. Conversely, bone marrow CFU-E and BFU-E demonstrated a concomitant decrease in numbers with disease progression. At no time were erythropoietin-independent (endogenous) erythroid colonies generated. The results suggest that compartmental alterations in erythroid precursors occur during progression of RLV-A.

Viral-associated alterations in hematopoiesis in the mouse.

A variant of Rauscher leukemia virus, designated RLV-A, induces a protracted hematopoietic dysplasia characterized by hepatosplenomegaly with erythroblastosis, severe terminal anemia, thrombocytopenia, and erythroblastemia. Erythrocyte and platelet survival is reduced and the ferrokinetic data suggest that iron utilization is faulty. Stem cells (CFU-S, CFU-C, CFU-MK, CFU-E, BFU-E) are reduced in the bone marrow but increased in terminal spleens. The cause(s) of these viral-associated alterations in stem cell numbers is not known.

Erythrocyte survival studies in a rat myelogenous leukemia.

To determine the extent intrinsic erythrocyte defects and/or extrinsic factors were involved in anemia of rats bearing Shay chloroleukemia (SCL), survival of 3H-DFP labeled erythrocytes was studied in leukemic and nonleukemic hosts. Red blood cells labeled before induction of leukemia, were rapidly lost from the peripheral circulation of SCL rats in terminal stages of disease. However, labeled erythrocytes from terminal SCL animals displayed normal lifespans when transfused into nonleukemic controls. Thus the anemia of this leukemia probably resulted from extrinsic factors associated with the leukemic process. Hemorrhage appeared to be primarily responsible for the anemia of this disease.

Isolation and biochemical characterization of leukemia-associated inhibitory activity that suppresses colony and cluster formation of cells.

Leukemia-associated inhibitory activity suppresses colony and cluster formation in vitro cells derived from granulocyte-macrophage progenitor cells of normal donors. It does not inhibit these same progenitor cells from patients with leukemia and it may contribute to the proliferative advantage leukemia cells appear to possess over normal hematopoietic cells during acute leukemia. The inhibitory activity was isolated by a combination of procedures including: ultracentrifugation, Sephadex G-200, carboxymethylcellulose, SDS-polyacrylamide gel electrophoresis, thin-layer and preparative isoelectric focusing and concanavalin A-Sepharose. Leukemia-associated inhibitory activity was characterized as a glycoprotein. it was inactivated by trypsin, chymotrypsin, pronase and periodate treatment. It bound to and was eluted by alpha-methylmannose from concanavalin A-Sepharose columns and had an apparent Mr range of 450-550 000 and an isoelectric focus value between pH 4.6 and 4.9. Crude leukemia associated inhibitory activity was temperature sensitive but the more purified preparations were heat stable.

A renal inhibitor to hepatic erythropoietin [Ep] production.

Evidence is presented for the existence of a factor in renal venous blood, evoked by subtotal hepatectomy (hepx), which inhibits the production of Ep in nephrectomized (nephrx) rats exposed to hypoxia. Significant inhibitory activity is not observed in blood obtained from sites other than the renal vein. This inhibitory effect is believed to be due to the action of a specific renal inhibitory factor (RIF) which reduces the extrarenal (hepatic) Ep response to hypoxia indirectly, by decreasing the production or effectiveness of an antagonistic liver principle, the hepatic erythropoietic factor (HEF). The HEF has previously been shown to augment hepatic Ep production following hypoxia in renally-deficient animals. The RIF has no anti-Ep action and its activity is not influenced by the accumulation of metabolic wastes. A mechanism for a renal-hepatic antagonism in the Ep response to hypoxia is hypothesized.