Effects of spaceflight on rat peripheral blood leukocytes and bone marrow progenitor cells.

The white blood cell (WBC) elements and the bone marrow myeloid progenitor cell populations were analyzed to ascertain adaptation to micro-gravity and subsequent readaptation to 1 G in rats flown on the 14-day Spacelab Life Sciences-2 (SLS-2) mission. Bone marrow cells were harvested from one group of rats killed inflight (FD13) and blood was drawn from three other groups at various times. The WBC level was normal on FD14 with the exception of neutrophilia. On FD13, numbers of colony-forming units-granulocyte (CFU-G), CFU-GM, and CFU-M from flight animals were decreased compared with ground controls when incubated with recombinant rat interleukin-3 (rrIL-3) alone or in combination with recombinant human erythropoietin (rhEpo). On recovery (R + 0), flight rats had decreased numbers of total leukocytes and absolute numbers of lymphocytes and monocytes with elevated neutrophils compared with control rats. They had lower numbers of CD4, CD8, CD2, CD3, and B cells in the peripheral blood but no differences in spleen lymphocytes.

Effects of spaceflight on the number of rat peripheral blood leukocytes and lymphocyte subsets.

Experiments were carried out on peripheral blood leukocytes and spleen lymphocytes from 29 male rats that were flown during the Spacelab Life Sciences 1 (SLS-1) nine-day mission on the shuttle Columbia in June 1991 and on appropriate ground controls. On the day of landing, there was a significant decrease in the total white blood cell counts (P < 0.0001) of flight animals in comparison to controls. There was also a significant decrease in the absolute number of lymphocytes (P < 0.0001) and monocytes (P < 0.0001) in the flight animals. A slight decrease in the absolute number of eosinophils and a slight increase in the number of neutrophils were observed at landing, compared with preflight values. Immunophenotyping of the peripheral blood and spleen lymphocytes of flight and control animals indicated that, on the day of landing, there was a decrease in the absolute number of CD4 and CD8 positive cells and B lymphocytes. However, relative percentages of peripheral blood CD4+, CD8+, and B cells were not found to be depressed. No differences were discerned in the percent reactivity of spleen lymphocytes of flight animals compared with controls. The observed decrease in the number of leukocytes and lymphocytes at the immediate postflight period was transient and all values returned to the control levels by nine days postflight.

Effects of spaceflight on rat erythroid parameters.

Hematologic studies were performed on 21 ground control rats and 21 rats flown during the Spacelab Life Sciences-2 14-day mission. Group A (n = 5) was used to collect blood in flight and 9 days postflight, group B (n = 5) was injected with recombinant human erythropoietin (rhEpo), group C (n = 5) received saline as a control, and group D (n = 6) was killed in flight and tissues were collected. Results indicated no significant changes in peripheral blood erythroid elements between flight and ground control rats. The nonadherent bone marrow on flight day 13 showed a lower number of recombinant rat interleukin-3 (rrIL-3)-responsive and rrIL-3 + rhEpo-responsive blast-forming unit erythroid (BFU-e) colonies in flight rats compared with ground control rats. On landing day, a slight increase in the number of rhEpo + rrIL-3-responsive BFU-e colonies of flight animals compared with ground control rats was evident. Nine days postflight, bone marrow from flight rats stimulated with rhEpo alone or with rhEpo + rrIL-3 showed an increase in the number of colony-forming unit erythroid colonies and a decrease in BFU-e colonies compared with ground control rats. This is the first time that animals were injected with rhEpo and subsequently blood and tissues were collected during the spaceflight to study the regulation of erythropoiesis in microgravity.

Lymphatic tissue changes in rats flown on Spacelab Life Sciences-2.

Thymus, spleen, inguinal lymph node, and bone marrow specimens from rats flown on the 14-day Spacelab Life Sciences-2 mission were examined after staining of tissue sections. The primary observation was a transient retrogressive change in lymphatic tissues in the rats within a few hours after landing. There was a diffuse increase in tingible body-containing macrophages in the cortex of the thymus, thymus-dependent areas of the splenic white pulp, and inguinal lymph node. This was not observed 9 days after recovery. The in situ labeling of fragmented DNA strands catalyzed by exogenous terminal deoxynucleotidyltransferase (TdT) with ApopTag reagents (Oncor, Gaithersburg, MD) inside the tingible body-containing macrophages indicated that the process was one of apoptosis. No increase in tingible body macrophage activity was noted in thymus and spleen tissue obtained from rats in flight on flight day 13. The reaction to gravitational stress from readaptation to 1 G is the most likely explanation of the transient retrogressive change in lymphatic tissues.

Synthetic vascular grafts seeded with genetically modified endothelium in the dog: evaluation of the effect of seeding technique and retroviral vector on cell persistence in vivo.

Unique characteristics of endothelium make it an attractive target cell for gene transfer. Genetically modified endothelial cells (ECs) seeded on synthetic vascular grafts offer the potential to control neointimal hyperplasia, decrease graft thrombogenicity and improve small diameter graft patency. This study addresses the issue of synthetic vascular graft colonization with endothelial cells transduced with noninducible retroviral marker genes in the dog. Autologous endothelial cells were enzymatically harvested and transduced with either the bacterial NeoR gene or human growth hormone gene using retroviral vectors. All transduced cells were positive by polymerase chain reaction (PCR) amplification for the transduced gene sequence prior to graft seeding. Transduced ECs were seeded on Dacron grafts (n = 3) preclotted with autologous blood. These grafts exhibited complete endothelialization at times from 250 to 360 days. Recovered DNA, however, was negative for the transduced gene sequence when analyzed by PCR and Southern blotting. Expanded polytetrafluoroethylene (ePTFE) was evaluated (n = 8) using several different cell seeding protocols. Grafts were seeded at 3 densities (ranging from 6 x 10(3) to 1.5 x 10(5) cells/cm2) and 2 different adherence times. Seeding substrate was also evaluated. Grafts were either preclotted with whole blood or incubated with 20 or 120 micrograms/ml fibronectin for 60 min. Graft biopsies were evaluated from 2 to 52 wk. Limited endothelialization was present in 4 dogs as early as 2 wk, but never progressed to full luminal coverage. The remaining dogs failed to ever exhibit any luminal EC adherence. Two dogs with limited EC coverage had positive DNA by PCR for the NeoR gene sequence at 2 and 3 wk. In contrast to transduced EC's, nontransduced EC colonization of ePTFE was complete at 2 wk when seeded under conditions that transduced cells had failed to persist. Neither seeding density, adherence time, seeding substrate or retroviral vector used influenced the uniformly poor graft coverage seen with transduced cells. Results of this study indicate that despite successful gene transfer using 4 different retroviral vectors, transduced endothelial cells seeded under varying conditions appear altered in their ability to stably adhere and colonize synthetic vascular grafts in vivo.

Effects of recombinant canine granulocyte colony-stimulating factor on white blood cell production in clinically normal and neutropenic dogs.

Recombinant canine granulocyte colony-stimulating factor (rcG-CSF) was administered to clinically normal dogs, cyclic-hematopoietic dogs, and dogs undergoing autologous bone marrow transplantation, to determine whether rcG-CSF could be used to stimulate WBC production and function in normal and neutropenic dogs. To the normal dogs, rcG-CSF was administered by SC injection at rates of 1 microgram/kg of body weight, q 12 h; 2 micrograms/kg, q 12 h; or 5 micrograms/kg, q 12 h. A significant dose-dependent increase in the WBC count resulted from the stimulation of bone marrow progenitor cells. The increased WBC count was characterized by mature neutrophilia and monocytosis. Neutrophil myeloperoxidase and phagocytic activity were normal in rcG-CSF-treated normal dogs, demonstrating the production of normal functional neutrophils in response to rcG-CSF treatment. Recombinant canine G-CSF prevented neutropenia and associated clinical signs but did not completely eliminate the cycling of neutrophils in cyclic-hematopoietic dogs when it was administered at rates of 1 microgram/kg, q 12 h, and 2.5 micrograms/kg, q 12 h. The time to bone marrow reconstitution was not decreased in dogs treated with rcG-CSF at a rate of 2.5 micrograms/kg, q 12 h, for 13 days following autologous bone marrow transplantation. On the basis of our findings, we suggest that treatment with rcG-CSF is an effective way to stimulate myelopoiesis in dogs, but that the dose of rcG-CSF required to stimulate WBC production will vary depending on the cause of neutropenia. Recombinant canine G-CSF should be useful in stimulating production and maintaining function of WBC for treatment of clinical diseases seen commonly in veterinary practice.

Effects of microgravity and increased gravity on bone marrow of rats.

Astronauts have a reduction in their red cell mass when exposed to microgravity. This is probably mainly due to a physiological response to decreased energy requirements. Further studies of erythropoiesis were carried out in microgravity on rats flown on Soviet Biosatellite 2044 and in hypergravity by centrifugation at 2G. Studies included: bone marrow cell differential counts, clonal studies of RBC colony formation, and plasma erythropoietin determinations. In the bone marrow of Cosmos flight animals there was a slight increase in granulocytic cells and in centrifuged animals, a slight decrease in the percentage of erythroid cells which led to an increased M:E ratio. The bone marrow cells of flight and centrifuged rats responded to erythropoietin. Cosmos flight animals' cells formed fewer CFU-E than the controls but this was reversed in the centrifuge studies. There were no essential differences in the erythropoietin levels of test groups as compared to control groups.

Blood volume and erythropoiesis in the rat during spaceflight.

A decreased red blood cell mass (RBCM) and plasma volume (PV) have been consistently found in humans after return from spaceflight. Rats flown on the Spacelab Life Sciences-1 mission were studied to assess changes in RBCM, PV, erythropoiesis, and iron economy. The RBCM and PV increased in both ground control and flight animals as expected for growing rats. However on landing day, both the RBCM and PV, when normalized for body mass, were significantly decreased in the spaceflight animals. During an 8-d postflight observation period, iron incorporation into circulating red blood cells was diminished in the flight animals. During the first 4 d postflight, increases in reticulocyte counts were significantly smaller in the flight than the control animals. Fewer erythropoietin-responsive progenitor cells were recovered from the bone marrow of flight animals after landing than control rats. Serum erythropoietin (EPO) levels were the same in both groups. Thus, rats subjected to a 9-d spaceflight had less increase in RBCM than controls and diminished erythropoiesis during an 8-d post-spaceflight observation period. The rat, like humans, appears to require a smaller blood volume in microgravity.