The effect of intravenous injection of ethyl palmitate emulsion on sequestration of thermally damaged erythrocytes in rats with experimentally induced hypersplenism, and in normal rats.

The authors attempted at experimental elimination of sequestration function of the spleen in Wistar rats using an i.v. injection of ethyl palmitate emulsion, both in "hypersplenic" animals being long-term applied i.p. methyl cellulose solution, and in control rats. In the rats clearance of 51Cr-labelled and thermally damaged erythrocytes from blood was examined and their sequestration in the spleen and liver followed. The ethyl palmitate injection resulted in both experimental groups in a significant decrease of the erythrocyte counts sequestrated in the spleen, and significant prolongation of the elimination half time for thermally damaged erythrocytes from the blood.     

Sequestration of heat damaged rat erythrocytes during experimental action of methyl palmitate on RES

The effect of an i.v. injection of methyl palmitate emulsion (MP) on the clearance of heat damaged erythrocytes from the blood and their sequestration in organs of rats was followed. Twenty-four hours following application of MP in the dose of 1.5 g/kg of body weight the survival half time of erythrocytes (T1/2(51)Cr) significantly increased, whereas the amount of sequestrated red cells in the spleen decreased. The subsequent intervals of examination, however, showed no differences as compared to the controls.     

Erythrocyte survival and haematocrit values in rats after i.v. application of ethyl palmitate and methyl palmitate emulsions

In the experiments on rats the effect of repeated application of ethyl palmitate emulsion (EP) and methyl palmitate emulsion (MP) on the survival rate of51Cr labelled erythrocytes and haematocrit values of the peripheral blood was studied. EP was applied in a total amount of 0.8 g, while MP due to its higher toxicity in a dose of 0.4 g per 100 g of body weight. The application of EP caused mild, though significant shortening of the survival period of the erythrocytes; the haematocrit values remained unchanged. Following application of MP no differences as compared to control animals could be found.     

3H-deoxythymidine incorporation in graft-versus-host disease in the Norway rat. I. Liquid scintillation studies.

Graft-versus-host-disease was produced in newborn Brown Norway (BN) rats with an intravenous (iv) injection of adult allogeneic Lewis (L) lymph node cells (experimental) and the response was compared to littermates injected with adult syngeneic BN cells (control). By 4 days the reaction in the spleen of experimental animals was such that the spleen index was 1.70 and 2.58 on day 7, and continued to increase until death. A one hour iv pulse of tritiated deoxythymidine (3HdT) administered to experimental and control animals revealed a whole organ peak incorporation of 3HdT on day 6 in experimental spleens. A second larger peak occurred on day 10 in the experimental spleen as compared to a single peak at days 6 or 7, respectively, in the experimental mesenteric and combined superficial lymph nodes. However, analysis of the incorporation of 3HdT with respect to organ weight revealed a peak incorporation in animals receiving L cells on day 4--6 with a second smaller peak on day 10 in the experimental spleen and again a single peak on day 5 or 6 in the lymph nodes. Total 3HdT incorporation within both experimental lymph node compartments became less than controls by day 15 even though experimental nodes had a larger mass. 3HdT incorporation per milligram tissue weight decreased in all tissue compartments of experimental animals by day 13--14. The contribution of donor and host cell proliferation to the various peaks observed is discussed.     

Filtrability of erythrocytes in "hypersplenic" rats.

The values of haemoglobin, reticulocytes and half-times of the filtrability of non-washed and washed erythrocytes were examined in male albino rats, Wistar strain, after i.p. injections of methylcellulose (MC) and compared with controls. In individual experiments the rats received 2 to 32 injections of MC. In injected animals, the filtrability of non-washed erythrocytes was altered. The filtrability half-times of the washed erythrocytes did not differ from the controls. Thus, the filtrability is altered for extracorpuscular reasons. "Hypersplenism" being completely developed, (after 32 MC injections), the filtrability of non-washed erythrocytes repaired when the application of MC had been discontinued, the reticulocyte values remained however increased. Problems of the mechanism of anaemia in experimental "hypersplenism" after MC injections in rats and relations between the altered filtrability of the erythrocytes and the haemolysis are discussed.     

The effect of neonatal rat graft-vs-host disease (GVHD) on Fc receptor lymphocytes.

The level of Fc receptor rosette-forming lymphocytes (Fc-RFL) was examined in spleen and lymph node cell suspension from neonatal DA and BN rats inoculated within 24 hr of birth with either allogeneic L (experimental) or syngeneic (control) lymphoid cells. In addition, these levels were compared to fetal and neonatal animals that received no injection. The indicator cells (EA) were sheep erythrocytes sensitized with one-half concentration of the highest dilution of rabbit anti-sheep erythrocyte IgG(A) which agglutinated an equal amount of 1% suspension of E. Care was taken to exclude scoring macrophages by injecting colloidal carbon at least 1 hr before killing the test animals. The spleen of 19-day DA fetal rats exhibited a level of 19.3% Fc-RFL, similar to that of animals having received adult syngeneic cells at birth (40.0%) by day 7. Thereafter the level of Fc-RFL did not vary appreciably between these two groups. However, as early as 2 days after inoculation there was a significantly greater number of Fc-RFL in the spleen of experimental DA neonates compared to controls. The lymph nodes of experimental animals did not exhibit a significantly greater number of Fc-RFL until day 6 with both tissue compartments peaking at day 10 and remaining significantly higher than controls until death. In neonatal BN animals significantly higher levels of Fc-RFL in experimental animals were not evident as early and peaked later (day 12) in both tissue compartments but again these differences remained until death. Cytotoxic alloantisera demonstrated that on days 6, 10, and 12 most, if not all, of the Fc-RFL were host in origion in both DA and BN GVHD, with a very significant host plasma cell response in such GVHD animals. One-micron tissue section revealed the presence of a great number of plasma cell especially prominent in the medulla of lymph nodes with the cortex of lymph nodes and white pulp of the spleen markedly depleted of lymphocytes indicative of cytotoxicity.     

Morphometrical studies of erythrophagocytosis in the spleen

The elimination of altered RBC was investigated morphometrically in the rat spleen by determination of the percentage of erythrophagosomes in macrophages. PHZ and diamide treated RBC led to a time dependent increase of the percentage of erythrophagosomes in comparison to normal spleens. The deformability of the target RBC was lowered as revealed by measurements with glass micropipettes. RBC-microvesicles and diamide treated RBC are primarily loaded with IgG and undergo a rapid elimination. PHZ-RBC and cells with a prolonged stay in the spleen are sequestrated by secondary mechanisms. Details of primary and secondary elimination are discussed.     

Release of erythrocytes from the spleen during exercise and splenic constriction by adrenaline infusion in the rainbow trout

Erythrocyte-supplying function of the spleen was examined in the rainbow troutSalmo gairdneri under exercise. The spleen showed remarkable reduction, about 70% in weight and about 85% in hemoglobin content, after forced exercise of 15 min. The amount of erythrocytes released from the spleen was 2.33 ml/kg body, and this amount corresponds to about 20% of the total volume of circulating erythrocytes in resting condition. No damage was observed at the spleen, splenic artery and splenic vein after the exercise. Examination of the vascular system by a corrosion casting method showed that no place other than the venous circulation exists for the erythrocytes released from the contracted spleen. The spleen was strongly constricted by infusion of adrenaline into the organ. These facts imply that the fish spleen supplies stored hemoglobin into the circulating blood in response to an increased demand of oxygen during exercise, under the control of the sympathetic nervous system.     

Determination of alpha-tocopherol in erythrocytes by gas-liquid chromatography

A method is described for the determination of submicrogram amounts of alpha-tocopherol in 0.5 ml of packed erythrocytes. The alpha-tocopherol in a lipid extract is oxidized to alpha-tocopherylquinone which is separated by thin-layer chromatography, eluted, and quantitated by gas-liquid chromatography. Calculation is based on the recovery of added alpha-tocopherol-(3)H. Erythrocytes from stock rats had an average alpha-tocopherol concentration of 344 micro g/100 ml of packed cells, while for human cells the average was 235 micro g/100 ml. The ratio of red cell to plasma alpha-tocopherol was 0.482 for rat blood, and 0.244 for human blood.     

Lymphoid tissue responses to perfluorocarbon emulsion in mice

The effects of either intraperitoneal or intravenous injection of low doses (5 or 10 ml/kg) of the proprietary emulsified perfluorocarbon-based blood substitute, Fluosol-DA 20%, on mouse lymphoid tissue and antibody production against sheep erythrocytes (SRBC) have been investigated. Mean liver weight was significantly increased and gut mesenteric lymph node (MLN) weights decreased in all animals injected with Fluosol-DA, irrespective of route of administration. In contrast, spleen weight decreased following intravenous injection of emulsion at 5 ml/kg. The mean plasma haemagglutination response to SRBC was significantly (P less than 0.01) increased in animals injected intraperitoneally with Fluosol at both doses but was similar to control in all other cases. These results show that lymphoid tissue responses to Fluosol-DA in mice are variable and that antibody production against intraperitoneally-injected SRBC is enhanced by prior injection of emulsion into the peritoneal cavity.     

IgE formation in the rat following infection with Nippostrongylus brasiliensis: I. Proliferation and differentiation of IgE-bearing cells

Sprague-Dawley rats were infected with Nippostrongylus brasiliensis larvae, and IgE formation was studied. Before infection, the serum IgE level was less than 0.4 μg/ml. The IgE level began to increase from the 10th day of infection, reached its maximum (50–100 μg/ml) at the 14th day and gradually declined. Reinfection of the rats resulted in an increase of the serum IgE level within 7 days. The IgE antibody response to N. brasiliensis antigens did not parallel the increase of IgE synthesis. In most animals, the antibody became detectable in the serum at the 21st day when the total IgE level already began to decrease. The animals showed a secondary IgE antibody response upon reinfection. Both mesenteric lymph nodes and spleen cell suspensions were examined for the presence of IgE-bearing cells (IgE-B cells) and IgE-forming cells by fluorescent antibody technique. The IgE-bearing lymphocytes became detectable in the mesenteric lymph nodes and spleen at the 8th day of infection. The proportion of the IgE-B cells in nonadherent cell population gradually increased and reached maximum at the 14th day; about 20% of immunoglobulin (Ig)-bearing cells in the mesenteric lymph nodes and 10% of Ig-bearing cells in spleen bore IgE on their surface. Evidence was obtained that these lymphocytes synthesized IgE. The IgE-forming cells were detected in both mesenteric lymph nodes and spleen of the infected animals. The number of IgE-forming cells was greater in the mesenteric lymph nodes than in spleen, indicating that the regional lymph nodes are the major source of serum IgE in the N. brasiliensis-infected animals.     

Isolation of preparative amounts of polyribosomes from normal rabbit and guinea pig spleen]

Preparative amounts of polyribosomes were isolated from normal rabbit and guinea pig spleen; up to 40 optical units of the polyribosome preparation could be obtained by centrifugation in a Spinco L-2B centrifuge with SW-27 rotor. The amount of polyribosomes isolated from spleens of immune animals was 2-3 higher than that isolated from normal animal spleens. Concentration of polyribosomal preparations by lyophylization and the storage of dried preparations do not alter the sedimentation properties of the polyribosomes. The distribution pattern of normal rabbit spleen polyribosomes in a linear sucrose gradient and the sedimentation constants of the polyribosome peaks are in good agreement with data reported by some other authors for plasmocytome polyribosomes. Using electrophoresis in agarose-polyacrylamide gel the radioactive proteins synthesized in the cell culture of normal rabbit spleen it was shown that in normal spleen the average amount of globulins makes up to 35% of total protein synthesis, as reported by some authors.     

Elastic properties of the rat respiratory system related to age

The experiments were performed on male rats of the Wistar strain under urethane anaesthesia (1.3 g/kg i.p.). Changes of oesophageal and tracheal pressures were registered in a group of 30 spontaneously breathing, supine rats, of 295 +/- 13 g average body weight during lung inflations with 1-5 ml of air. In another group of 25 rats of 70 +/- 6 g average body weight (young rats) we made the measurements during inflation with volumes 0.5-2 ml. The measurements were also performed in a group of 10 paralyzed, ventilated rats with 347 +/- 24 g average body weight and inflations 1-5 ml. Compliance of the lungs (CL), chest wall (CW) and of the respiratory system (Crs) was calculated from the linear part of the pressure-volume curve during inflation. The results indicate: 1. Cw is significantly (p less than 0.001) higher in young (134.7 ml.kPa-1.kg-1) than in adult rats (44.1 ml.kPa-1.kg-1). CL (related to body weight) is not significantly different in young and adult rats. 2. Cw is significantly (p less than 0.001) higher than CL. 3. No difference was observed in CLs Cw and Crs between paralyzed and spontaneously breathing animals.     

In vitro responses of rat lymphocytes following adult thymectomy. II. Increased inhibition by splenic adherent cells of responses to phytohemagglutinin

Adult thymectomy in rats results in a marked fall of spleen cell responsiveness to PHA over a period of days to weeks. Examination of dose-response curves showed that, with high PHA dose or ≥ 10⁶ cells/ml, there is a profound inhibition of the response with spleen cells from thymectomized animals compared with cells of matched sham-operated controls. However, when adherent cells are removed from the cell suspensions, the remaining nonadherent cells give an almost linear dose-response relationship with increasing PHA similar to that exhibited by the nonadherent cells of the controls or sometimes a slightly decreased response. Similarly, when increasing numbers of spleen cells from these animals are cultured (with admixed thymocytes to make a constant total of 2 × 10⁶ cells/ml) with PHA, the linear portion of the doseresponse curve can be extrapolated to give a similar value for the maximal potential response, which again is the same as or somewhat less than the corresponding value for sham-operated controls. A difference in inhibitory capacity is also shown in mixtures of the two spleen cell populations with LNC or with purified spleen cells. It is concluded that adult thymectomy results in increased “suppressor” activity in the spleen within a few days and may reduce slightly the number of T lymphocytes in the spleen reactive with PHA.     

Pituitary hormones regulate c-myc and DNA synthesis in lymphoid tissue

Hypophysectomy of Fischer 344 rats of both sexes led to a rapid involution of the thymus and spleen which was associated with a profound decrease in spontaneous DNA synthesis in these organs. The proportion of B lymphocytes in the spleen, of T cells and their subsets (CD4+/CD8+) in spleen and thymus, and the histological structure of the involuted organs remained normal. Treatment of hypophysectomized animals with growth hormone (GH) or prolactin (PRL) stimulated the expression of the c-myc proto-oncogene and DNA synthesis and reversed the involution in these organs. Replacement doses of adrenocorticotrophic hormone, follicle-stimulating hormone, luteinizing hormone, or thyroid-stimulating hormone had no influence on thymus or spleen size and DNA synthesis. A rapid expression of c-myc was also observed in thymuses and spleens of intact rats after the injection of GH or PRL. In vitro physiological concentrations (2.5 ng/ml) of either ovine or rat PRL or GH stimulated the incorporation of [3H]thymidine by thymus and spleen cells. These results indicate that GH and PRL regulate lymphocyte growth. This regulatory role is likely to serve as the principal mechanism of immunoregulation by these hormones.     

Interaction of diesel exhaust particles with human, rat and mouse erythrocytes in vitro

Inhaled ultrafine (nano) particles can translocate into the bloodstream and interact with circulatory cells causing systemic and cardiovascular events. To gain more insight into this potential mechanism, we studied the interaction of diesel exhaust particles (DEP) with human, rat and mouse erythrocytes in vitro. Incubation of erythrocytes with DEP (1, 10 or 100 μg/ml) for 30 min caused the highest hemolytic effect (up to 38%) in rats, compared to small but significant hemolysis in mice (up to 2.5%) and humans (up to 0.7%). Transmission electron microscopy of erythrocytes revealed the presence of variable degrees of ultrafine (nano)-sized aggregates of DEP either internalized and/or adsorbed onto the erythrocytes in the three species. A significant amount of DEP was found in rat and mouse (but not human) erythrocytes. Lipid erythrocyte susceptibility to in vitro peroxidation measured by malondialdehyde showed a significant and dose-dependent increase in erythrocytes of rats, but not humans or mice. Unlike in human erythrocytes, total antioxidant status (TAS) and superoxide dismutase (SOD) activity in rats were significantly and dose- dependently decreased. In mouse erythrocytes, DEP caused a decreased in SOD (at 10 μg/ml) and TAS (at 100 μg/ml) activities. In conclusion, DEP caused species-dependent erythrocyte hemolysis and oxidative stress, and were either taken up and/or adsorbed onto the red blood cells. Rat (and to a lesser degree mouse) erythrocytes were susceptible to DEP. Human erythrocytes showed the highest resistance to the observed effects. These species difference should be noted when using rats and mice blood as models for humans.     

Transfer of immunity to Plasmodium berghei by spleen and lymph node immune RNA.

The RNA from spleens and lymph nodes of Lewis rats immune to Plasmodium berghei protected A/J mice against a lethal challenge of the blood stages of P. berghei, NK65. The RNA was extracted by the hot phenol procedure from freshly removed spleens and lymph nodes. Protection was measured by survival and level of parasitemia as compared to controls. The levels of RNA administered were 10, 50, and 100 μg of RNA. There was observed 100% survival with 50 and 100 μg of immune spleen RNA. The maximum percentage of parasitemia was not reduced below that of the controls in the groups given immune RNA from lymph nodes, but was significantly reduced below that of the controls in the groups given immune RNA from spleens.     

Intracellular localization of anti-tumor immune RNA.

Syngeneic spleen cells from normal, non-immune Fischer 344/N rats and allogeneic spleen cells from normal Wistar-Furth rats became cytotoxic, in vitro, to chemically induced Fischer rat sarcoma (MC3-R) target cells following incubation with xenogeneic Immune RNA (I-RNA) extracted from spleens of guinea pigs immunized with MC3-R tumor cells. I-RNA extracted from intact spleen cells or from the cytoplasmic fraction of spleen cells were equally active. RNA extracted from isolated spleen cell nuclei was inactive, as were all RNA fractions from spleen cells of nonspecifically immunized guinea pigs. Syngeneic I-RNA extracted from intact spleen cells or the cytoplasmic fraction of cells from spleens of Fischer rats bearing growing MC3-R transplants mediated cytotoxic reactions against MC3-R target cells when incubated with normal Fischer rat spleen cells. RNA from the nuclei of spleen cells of rats bearing MC3-R tumors was considerably less active. All RNA fractions from spleen cells of normal non-immune Fischer rats were inactive. The immunologically active component of xenogeneic and Syngeneic I-RNA, therefore, were found to be localized in the cytoplasm of specifically sensitized lymphoid cells.     

The antimetastatic effect of a single low dose of cyclophosphamide involves modulation of galectin-1 and Bcl-2 expression

We have demonstrated that a single low dose of cyclophosphamide has an antimetastatic effect on lymphoma (L-TACB)-bearing rats by modulating the host immune response. Galectin-1, a member of the galectin family with specificity for beta-galactosides, has potent immunomodulatory properties by regulating cell-matrix interactions and T-cell apoptosis. Since galectin-1 is expressed by highly metastatic tumors, in the present study we investigated the participation of this beta-galactoside-binding protein in cyclophosphamide-induced immunomodulation. Inbred " e" rats were s.c. challenged with L-TACB. After 14 days, half of the animals received an i.p. injection of cyclophosphamide (10 mg/kg), and on day 21 tumors and spleens were excised. Cell extracts were prepared and galectin-1 expression was determined by Western blot analysis and correlated with Bcl-2 expression levels and the DNA fragmentation profile. Expression of galectin-1 was significantly decreased in tumors from cyclophosphamide-treated rats compared to non-treated rats. The same effect was observed regarding expression of Bcl-2 by tumors. In contrast, expression of Bcl-2 was significantly higher in spleens from treated animals than in non-treated rats. This effect correlated with a decreased intensity in the pattern of DNA fragmentation of spleen cells from cyclophosphamide-treated animals. Our results suggest that a single low dose of cyclophosphamide modulates the expression of galectin-1 and Bcl-2 by tumors, which could in turn influence the apoptotic threshold of spleen mononuclear cells. This mechanism could explain, at least in part, the antimetastatic effect evidenced in our tumor experimental model.     

Blood changes and fertility derangements following the injection of Candida albicans into the spleen of the experimental animals (occurrence of hypersplenism)

Summary The injection ofCandida albicans (under experimental conditions used here) into the spleens of Wistar rats and Swiss mice produces profound hematological changes which are similar in rats and mice. After injection of the organisms there was steady lowering of the white cells and of the platelets in mice and in rats. Following splenectomy there was a sharp rise of both platelets and white cells in mice and rats. Mild normocytic, normochromic anemia was observed during the experiment. Following the splenectomy the level of hemoglobin returned to normal in fourteen weeks in the rats and in thirteen weeks in the mice. An increased level of reticulocytes and siderocytes was observed during the experiment. The mean value of the spleen weights in the experimental animals was higher than in the controls. Hyperplasia was the constant histological feature of these spleens. Bone marrow was either normal or hyperplastic. plastic. These changes correspond to the picture known in human pathology as hypersplenism. The injected organisms could still be cultured from the spleen about 4–6 months after operation. Attempts to isolate the injected organisms after 6 months were unsuccessful. The experimental mice showed derangement of fertility. Of the forty females mated with normal males only seven became pregnant. The litters were normal, 7–12 babies. Thirty percent of the young died before one month. Many of them showed growth retardation.This work was supported by a Damon Runyon Memorial Fund Grant No. 720.