Spleen-dependent turnover of CD11b peripheral blood B lymphocytes in bovine leukemia virus-infected sheep

Lymphocyte homeostasis is determined by a critical balance between cell proliferation and death, an equilibrium which is deregulated in bovine leukemia virus (BLV)-infected sheep. We have previously shown that an excess of proliferation occurs in lymphoid tissues and that the peripheral blood population is prone to increased cell death. To further understand the mechanisms involved, we evaluated the physiological role of the spleen in this accelerated turnover. To this end, B lymphocytes were labeled in vivo using a fluorescent marker (carboxyfluorescein diacetate succinimidyl ester), and the cell kinetic parameters (proliferation and death rates) of animals before and after splenectomy were compared. We show that the enhanced cell death observed in BLV-infected sheep is abrogated after splenectomy, revealing a key role of the spleen in B-lymphocyte dynamics.     

Chromium-51 labeling of sheep red blood cells.

The failure of sheep red blood cells (RBCs) labeled with Chromium-51 (Cr-51) using the ascorbic acid technique to act as a suitable intravascular marker of blood volume in a septic sheep model prompted us to investigate the technique of radiolabeling sheep erythrocytes with this isotope. Consequently, we studied thirteen sheep in which the labeling efficiency of Cr-51 as sodium chromate and hemoglobin typing was determined for each animal. Mean Cr-51 labeling efficiency of sheep RBCs was 67.5% (n = 13). Although 5 of the 13 sheep were discovered to have two types of hemoglobin (Hb) as determined by electrophoresis, overall labeling efficiency of sheep RBCs was determined to be independent of the type of hemoglobin present. However, when two types of Hb were present (Hb-A and Hb-B), Cr-51 had a higher affinity for Hb-B (80%) than Hb-A (20%) even though both Hb types are present in similar proportions (Hb-A = 53%, Hb-B = 46%). The results of this study indicate that sheep RBCs express a lower labeling efficiency for Cr-51 than do human RBCs and that Cr-51 has a higher affinity for Hb-B than for Hb-A when both hemoglobin types are present. This difference is noteworthy when interpreting Cr-51 RBC data in experimental sheep models. Furthermore, caution should be exercised when extrapolating established human protocols to animal models.     

Mouse erythrocytes as carriers for coencapsulated alcohol and aldehyde dehydrogenase obtained by electroporation in vivo survival rate in circulation, organ distribution and ethanol degradation

Alcohol dehydrogenase and aldehyde dehydrogenase (ADH and ALDH) have been coencapsulated into mouse erythrocytes by an electroporation technique. The optimal conditions were achieved as follows: 420 V, four pulses of 1 ms every 15 min. at 37 degrees C, completed by resealing: 1 h at 37 degrees C. An encapsulation yield ranging from 11-12% was obtained for ADH+ALDH-loaded erythrocytes. Carrier cell recovery was 52%. Electroporated-RBCs observed under Scanning electron microscopy exhibited a tendency toward invaginated sphero-stomatocytes. These invaginations were not found in electroporated/resealed RBCs. The intravenous administration of 51Cr-RBCs manifested a bimodal pharmacokinetic profile: an initial phase (t1/2alpha) with a rapid decrease of plasma 51Cr-RBCs followed by a slow and prolonged elimination phase (t1/2beta). The values corresponding to in vivo survival rate during the elimination phase indicated that the survival rate of 51Cr-electroporated loaded-RBCs was slightly lower (t1/2beta, 4.5 days) than 51Cr-native RBCs (t1/2beta, 5.3 days). The mean clearance values from blood of electroporated 51Cr-RBCs (unloaded and loaded) were higher (0.51 %51Cr/day and 0.54 %51Cr/day, respectively) than the obtained for native 51Cr-RBCs (0.18 %51Cr/day). The target organs for electroporated RBCs proved to be the same as for native RBCs. However, electroporated RBCs showed highest accumulation in liver, spleen and lung, since they were promptly recognized by the reticuloendothelium system. Mice induced to the state of acute ethanol intoxication and treated with ADH+ALDH-RBCs clearly showed a lower level of ethanol concentration in plasma (less than 43% ethanol) than the intoxicated mice treated with native RBCs. En consequence the clearance values of ethanol from blood in intoxicated mice treated with ADH+ALDH-RBCs (0.39 ml/min) were higher than the treated with native RBCs (0.20 ml/min). The results obtained suggest that ADH+ALDH-loaded erythrocytes could be used as a potential carrier system for in vivo removal of high levels of ethanol from blood caused by excessive alcohol consumption.     

Measurement of blood volume in fetal and neonatal sheep using red blood cells labelled with 99m technetium

Blood volume has been measured in fetal and neonatal sheep using red blood cells labelled with 99mTc. The calculated volumes were highly correlated with simultaneous measurements made using the standard 51Cr labelled red cell method, although in absolute terms the 99m Tc method provided volumes which on average exceeded by a small percentage those determined with the 51Cr method. Measurements using the 99mTc method were also made at different ages in fetal and neonatal sheep and, while no correlation could be demonstrated between blood volume and either fetal or neonatal age, neonatal blood volumes were highly correlated with body weight. The 99mTc method is considered to be a reliable technique for measuring perinatal blood volumes in sheep with the short half-life of the isotope offering additional advantages.     

Conversion of red blood cells (RBCs) from the larval to the adult type during metamorphosis in Xenopus: specific removal of mature larval-type RBCs by apoptosis

The conversion of hemoglobins (Hbs) and red blood cells (RBCs) from the larval to the adult type was monitored during normal metamorphosis in Xenopus laevis, and in artificially induced metamorphosis-arrested and precociously metamorphosed animals by means of SDS-PAGE, Hb immunohistochemistry, and double-staining with in situ DNA nick-end labeling (TUNEL) for detection of apoptosis and Hb immunostain. During normal metamorphosis, larval RBCs gradually decreased and, conversely, adult RBCs increased in number. However, in metamorphosis-arrested tadpoles, the larval-adult conversion of RBCs did not occur within 4 weeks, but did rather within 6 months after the controls metamorphosed. In order to identify possible mechanisms for the specific removal of larval RBCs from circulation in metamorphosing and metamorphosed animals, double-staining experiments with TUNEL and Hb immunostain were carried out. During metamorphic climax, many larval RBCs expressed TUNEL-positive reactions in the spleen, suggesting that the larval RBCs were specifically removed from the spleen during metamorphosis. When the larval RBCs were transferred to the circulatory system of histocompatible control adults, they survived for a long time, and no transferred RBCs showed TUNEL-positive reactions. In contrast, larval RBCs transferred to histocompatible adults that had been treated with T3 were reduced in number in the circulatory system of the recipients. Double-staining experiments demonstrated that the transferred larval RBCs underwent apoptosis in the spleen and liver of the adult recipients treated with T3, indicating that the mature larval-type RBCs were specifically removed from metamorphosing animals by apoptotic cell death under the influence of THs.     

Specific immunologic reaction in autoantibody-producing cells

Experiments were conducted on albino mongrel female mice. The capacity of the plasma obtained in 30 minutes from the animals vaccinated uith the microbial antigens of the animals to cause temporary activation of the autoimmune reaction in the recipients, in passive transfer, was revealed. The capacity of spleen cells, forming autohemolysins, to produce antibodies to sheep red blood cells after the immunization with this antigen was demonstrated by means of Cunningham and Pilarsky's method.     

The in vivo distribution of murine lymphokine activated killer cells in splenectomized host

The in vivo distribution of intravenously injected lymphokine activated killer (LAK) cells, generated in vitro with rIL-2 from normal murine splenocytes, was studied in BALB/c mice and compared with that of normal splenocytes. Both normal splenocytes and LAK cells were labeled with 51Cr, and the results were analyzed at 6, 24, and 48 hours after injection by localization index as the parameter. After injection through tail veins of mice, LAK cells were found to migrate to the spleen, lungs, liver, lymph nodes, bones and the kidneys. The apparent increased distribution pattern of LAK cells to the lung at 6 and 24 hours after injection was not detected when normal splenocytes were injected. Since almost one third of the injected LAK cells were found to localize in the spleen, it was postulated that splenectomy would affect the in vivo organ distribution of LAK cells. Accordingly, the in vivo distribution of LAK cells in splenectomized mice was further investigated. Results indicated that splenectomy enhanced the convergence of LAK cells to the lungs, liver, lymph nodes and bones. Therefore, splenectomy may augment the therapeutic effect of the adoptive transfer of LAK cells in pulmonary, hepatic, lymph node and bony metastases.     

Role of the kidneys in the mechanism of erythrodieresis in hemolytic (phenylhydrazine) and acute posthemorrhagic anemia]

The degree of hemolysis was studied comparatively in intact and nephrectomized rats after the phenylhydrazine injection. In the nephrectomized animals hemolytic (phenylhydrazine) anemia was expressed by a lesser reduction of the total erythrocyte count, of the percentage of Cr51-labeled erythrocytes, and of the intensity of the reaction for hemosiderin in the organs and tissues. A lesser degree of erythrodieresis was found in the nephrectomized rats and after an acute unsubstituted blood loss. Blood perfusion through the kidney of anemic rats led to increase of the potassium concentration in the plasma perfusate, reduction of the electrophoretic mobility of erythrocytes, their resistance, hemolysis duration, and to decrease of albumin fractions with the mol wt of from 74 500 to 27 000 in the erythrocyte stroma.     

The in vivo distribution of 51Cr-labeled Trypanosoma cruzi in mice

The optimal conditions for labeling Trypanosoma cruzi culture forms with ⁵¹CrO₄²⁻ were determined. Labeled trypanosomes or labeled human red blood cells (RBCs) were injected intravenously into normal C3H(He) female mice and the rate of clearance and organ distribution of the isotope were observed over a 30 h period. It was found that trypanosomes and xenogeneic RBCs were cleared rapidly from the peripheral blood and accumulated primarily in the liver, spleen, lungs and kidneys. A difference was noted in accumulation of trypanosomes and RBCs in these mice.     

Evidence of immunological activity in heterotropic autotransplanted splenic tissue in DBA/2 mice

After total splenectomy, a portion of the spleen was autotransplanted into subcutaneous ectopic sites of DBA/2 mice. These implanted fragments regenerated into splenic tissue that are microscopically indistinguishable from the structure of the original spleen. These implants are also immunologically functional as demonstrated by their ability to produce antibodies to sheep red blood cells (SRBC) and SRBC inhibition of splenic (implant) cell migration.     

The in vivo distribution of Cr-labeled Trypanosoma cruzi in mice

The optimal conditions for labeling Trypanosoma cruzi culture forms with ⁵¹CrO₄²⁻ were determined. Labeled trypanosomes or labeled human red blood cells (RBCs) were injected intravenously into normal C3H(He) female mice and the rate of clearance and organ distribution of the isotope were observed over a 30 h period. It was found that trypanosomes and xenogeneic RBCs were cleared rapidly from the peripheral blood and accumulated primarily in the liver, spleen, lungs and kidneys. A difference was noted in accumulation of trypanosomes and RBCs in these mice.     

Distribution of Methylene Blue after Injection into the Epidural Space of Anaesthetized Pregnant and Non-Pregnant Sheep

The aim of the study was to determine the distribution of different volumes of methylene blue solution injected into the epidural space in anaesthetized pregnant and non-pregnant sheep, to evaluate its cranial distribution and to compare between them. Fifteen pregnant and fifteen non-pregnant sheep were included in the study. Sheep were anaesthetized and received 0.05, 0.1, or 0.2 mL/kg of a lumbosacral epidural solution containing 0.12% methylene blue in 0.9% saline. Thirty minutes after the epidural injection, the ewes were euthanized. The extension of the dye within the epidural space was measured, and the correlation between the volume of the dye injected and the number of stained vertebrae was evaluated. The cranial migration of the dye between pregnant and non-pregnant sheep was also compared. The results show that the volume of methylene blue injected epidurally into pregnant and non-pregnant sheep correlated directly with its cephalic distribution into the epidural space; and a volume of 0.1 mL/kg or 0.2 mL/kg stained up to the first lumbar segment in pregnant and non-pregnant sheep, respectively. Also, the results suggest that the volume of drugs administered into the epidural space of pregnant sheep should be half the volume that would be used in non-pregnant sheep.     

Effect of radiation-induced bystander chemosignals of mice on the humoral immune response in spleen and lymph nodes of intact recipients

The ability of post-radiation (4 Gy) bystander chemosignals (the volatile components of mouse urine) to distantly modulate the humoral immune response to the sheep red blood cells in the spleen and popliteal lymph nodes of intact recipients has been investigated. It was shown that the exposure of animals to chemosignals before antigen injection resulted in the decrease and increase of the immune response in the spleen and lymph nodes, respectively. When animals were exposed to chemosignals after the antigenic stimulus, an increased immune response was observed in both spleen and lymph nodes. The contribution of radiation-induced bystander signaling in the response of socially organized animals to the effect of ionizing irradiation is discussed.     

Survival of the fittest?--survival of stored red blood cells after transfusion.

During the last 90 years many developments have taken place in the world of blood transfusion. Several anticoagulants and storage solutions have been developed. Also the blood processing has undergone many changes. At the moment, in The Netherlands, red blood cell (RBC) concentrates (prepared from a whole blood donation and leukocyte-depleted by filtration) are stored for a maximum of 35 days at 4 degrees C in saline adenine glucose mannitol (SAGM). Most relevant studies show that approximately 20% of the RBCs is lost in the first 24 hr after transfusion. Even more remarkable is that the average life span is 94 days after a storage period of 42-49 days. Such observations create the need for a parameter to measure the biological age of RBCs as a possible predictor of the fate of RBCs after transfusion. The binding of IgG to RBCs can lead to recognition and subsequent phagocytosis by macrophages. This occurs during the final stages of the RBC life span in vivo. We determined the quantity of cell-bound IgG during storage, and found considerable variation between RBCs, but no significant storage-related change in the quantity of cell-bound IgG. The significance of this finding for predicting the survival of transfused RBCs in vivo remains to be established. Hereto we developed a flow cytometric determination with a sensitivity of 0.1% for the measurement of survival in vivo based on antigenic differences. This technique has various advantages compared with the 'classical' 51Cr survival method.     

Sites and trend of erythropoiesis in anemic, normal, and splenectomized newts

Newts, Triturus cristatus carnifex (Laurenti), were anesthetized by submersion in 2% chlorbutol in tap water for 15 min, splenectomized and then rendered totally anemic two months later by treatment with acetylphenylhydrazine (APH) diluted in their tanks (25 mg/liter for 36 h, changing the solution every 12 h). In the 14 weeks following hemolysis, erythron restoration occurred with the same intermittence as it did in whole animals rendered anemic by APH treatment: Beginning the second week the red blood cell count progressively increases for about one month, followed by a period of stasis which lasts about three weeks, then by a new increase, and then by a final period of stasis. Histological examination shows that erythropoietic activity occurs partly in the circulating blood and partly in erythroblasts nestled in the crypts between the muscular trabeculae of the ventricle as well as in the atrial walls. These cells, which are not part of the freely circulating elements in the blood stream, become very abundant in both whole and splenectomized anemic newts but are also present in normal animals. Newts, thus, have three sites for erythropoiesis: the spleen, the blood stream, and the heart. The other components compensate for the elimination of the spleen without determining any lack of, or delay in, erythropoietic response.     

Pathogenicity of molecularly cloned bovine leukemia virus.

To delineate the mechanisms of bovine leukemia virus (BLV) pathogenesis, four full-length BLV clones, 1, 8, 9, and 13, derived from the transformed cell line FLK-BLV and a clone construct, pBLV913, were introduced into bovine spleen cells by microinjection. Microinjected cells exhibited cytopathic effects and produced BLV p24 and gp51 antigens and infectious virus. The construct, pBLV913, was selected for infection of two sheep by inoculation of microinjected cells. After 15 months, peripheral blood mononuclear cells from these sheep served as inocula for the transfer of infection to four additional sheep. All six infected sheep seroconverted to BLV and had detectable BLV DNA in peripheral blood mononuclear cells after amplification by polymerase chain reaction. Four of the six sheep developed altered B/T-lymphocyte ratios between 33 and 53 months postinfection. One sheep died of unrelated causes, and one remained hematologically normal. Two of the affected sheep developed B lymphocytosis comparable to that observed in animals inoculated with peripheral blood mononuclear cells from BLV-infected cattle. This expanded B-lymphocyte population was characterized by elevated expression of B-cell surface markers, spontaneous blastogenesis, virus expression in vitro, and increased, polyclonally integrated provirus. One of these two sheep developed lymphocytic leukemia-lymphoma at 57 months postinfection. Leukemic cells had the same phenotype and harbored a single, monoclonally integrated provirus but produced no virus after in vitro cultivation. The range in clinical response to in vivo infection with cloned BLV suggests an important role for host immune response in the progression of virus replication and pathogenesis.     

Regenerative capacity of the spleen in a splenectomized fish, Channa punctatus Bloch, with related investigations into changes in peripheral blood and haematopoiesis

The present investigation has provided evidence by a simultaneous study of the regenerating spleen, peripheral blood and the kidney of the haematopoietic tissue to establish the role of the spleen in the development of circulating blood cells in Channa ( Ophiocephalus ) punctatus Bloch. Regeneration of the spleen after eight to nine weeks of splenectomy was shown to be complete. Weekly observations a fortnight after splenectomy are recorded with reference to spleen morphology and weight, erythrocyte and differential leucocyte counts, haemoglobin content, haematocrit value and related red cell indices (MCV, MCH, MCHC). The results show that splenectomy leads to a macrocytic hypochromic anaemia and leucopenia, resulting in 25% reduction in haemoglobin content and a decrease in leucocyte numbers in five weeks following splenectomy. Thrombocytes and neutrophils show a relative ( P < 0·01) increase. The leucopenia is caused by large and small lymphocytes, eosinophils and basophils whose number was reduced from 50% to 67%.     

Studies on the maturation of immune responsiveness in the mouse. II. Role of the spleen.

Experiments were designed to investigate the role of the spleen in the development of the murine immune system. By using mice splenectomized within 24 hr of birth, as well as mice with a hereditary, congenital absence of the spleen, the primary immune response to sheep erythrocytes was examined. The immunocompetence of lymph node cells from spleenless or control mice was assessed in vitro, in organ and in cell suspension cultures, and in vivo, by transfer into lethally irradiated syngeneic recipients followed by antigenic stimulation. The immunologic capacities of thymus and bone marrow cells were similarly tested by injection separately or in combination into irradiated syngeneic mice. Lymph node cells from spleenless animals appeared fully competent both in vitro and in transfer experiments. Neither neonatal splenectomy nor congenital absence of the spleen significantly reduced the capacity of bone marrow or thymus cells to participate in the immune response to sheep erythrocytes.     

Tolerance-like condition developing in mice under the effect of antigen of erythrocyte lysate]

Lysate of sheep red blood cells obtained by the treatment of these cells with distilled water and purified by ultracentrifugation in cold possessed a weak immunogenicity. Its administration to mice caused the state of hyporeactivity to sheep red blood cells (a reduction of the immune response level to 10-25% of control. The capacity of the mise spleen cells to respond by immune reaction to the red blood cells following adoptive transfer was not disturbed. At the early periods after the lysate administrations the mouse spleen cells possessed a weak supressive activity in case of their transfer to the intact animals. The blood serum of mice treated with the lysate possessed a blocking activity which disappeared after the serum absorption with sheep red blood cells. A conclusion was drawn that hyporeactivity originating in mice after the lysate administration was caused by the presence in the serum of antibodies inhibiting the immune response.     

Endotoxin decreases 51CrCl3 uptake in early weaned pigs

Endotoxin, a product of bacterial infections, was used to investigate the effects acute infections on chromium (Cr) uptake in early weaned (14 d of age) pigs. At d 18, pigs were anesthetized, and a 2-mm silastic jugular catheter was inserted and passed subcutaneously to a dorsal position behind the ear. At d 21, pigs were deprived of food for 7 h and injected intraperitoneally with saline or 25 microg endotoxin (lipopolysaccharide from Escherichia coli serotype 0111:B4) per kg body wt suspended in 9 g/L saline. One hour after dosing, an oral dose of 0.7 mCi of 51CrCl3 was given by micropipet. Blood was sampled from the catheter at intervals until necropsy at 8 h after the 51CrCl3 dose. Blood and tissue samples were counted in a gamma counter. 51Cr in blood was significantly lower at 3, 4, 5, 6, and at 8 h after dosing in endotoxin-injected pigs compared to controls. Eight hours after 51CrCl3 dosing, 51Cr retention was significantly lower in the liver, heart, and kidney in endotoxin-treated pigs and tended to be lower in spleen (p<0.06) and in urine (p<0.16) with endotoxin treatment. These data suggest that during acute infection, there might be decreased Cr uptake and retention.