Effects of fish-oil constituents and plasma lipids on fibrinolysis in vitro.

An association between consumption of fish or fish oils and a reduction in coronary heart disease was established in the 1980s. The mechanisms underpinning this effect have been investigated extensively, with the focus on a reduction in platelet adhesiveness and a lowering of plasma concentrations of low-density lipoprotein (LDL)-cholesterol. Possible effects on fibrinolysis have received less attention and produced conflicting data. The present study evaluates such effects under chemically restricted conditions in vitro, in a system that, based on studies with haemostatically active drugs of known mechanisms, appears to have some relevance to the in vivo situation. Eicosapentaenoic acid (EPA)--one of the major constituents of fish oil--produced a statistically significant (P < 0.05) enhancement of fibrinolysis, when added before formation of the fibrin clot, but generally had the reverse effect when added afterwards. Docosahexaenoic acid (DHA)--the other major constituent of fish oil--had a dramatic inhibitory effect on clot formation, when added prior to clot formation, and inhibited lysis when added after the clot was formed. Maxepa (Seven Seas Ltd.)--a concentrate of EPA and DHA, and the pharmaceutical equivalent of fish oils--did not influence fibrinolysis, when added prior to clot formation. When added after the clot had formed, however, it produced significant (P < 0.05) and dose-dependent effects that varied from enhancement to inhibition. Similarly, both high-density lipoprotein (HDL)- and LDL-cholesterol, when added after clot formation, produced significant (P < 0.05) concentration-dependent effects that varied from enhancement of fibrinolysis (at the lower concentrations tested) to inhibition at higher concentrations. Our findings suggest that the effects of fish oil and lipids are more complex than simple enhancement or inhibition of fibrinolysis. Rather, the benefits may depend both on their concentration and whether they are present before or after the fibrin clots are formed.

Peripheral blood lymphocyte apoptosis: a clue to the diagnosis of acute infectious mononucleosis.

OBJECTIVE: To describe the morphology and significance of apoptotic lymphocytes in peripheral blood smears of patients with acute infectious mononucleosis. To our knowledge this has not been previously reported. DESIGN: Peripheral blood smears from 27 patients with a positive heterophile antibody test were collected and reviewed for the presence of apoptotic lymphocytes. Flow cytometry was performed on three cases to document the previously described low expression of bcl-2 in lymphocytes in infectious mononucleosis. Four control patient populations comprising 80 cases were similarly screened for the presence of apoptotic lymphocytes. SETTING: The specimens were collected over a 3-month period in two laboratories at our tertiary care hospital; all specimens were processed according to a standard protocol. PATIENTS: Young adult military recruits and their spouses, military dependent adolescents, and retired military personnel. RESULTS: Twenty-four (88.9%) of 27 peripheral blood smears of patients with acute infectious mononucleosis contained readily identifiable apoptotic lymphocytes. Three (3.75%) of 80 control peripheral blood smears were identified with rare apoptotic lymphocytes, all occurring in patients with viral upper respiratory infections. CONCLUSIONS: The finding of apoptotic lymphocytes in a peripheral blood smear is useful in the differential diagnosis of infectious mononucleosis and neoplastic hematolymphoid processes.

Epstein-Barr virus DNA is abundant and monoclonal in the Reed-Sternberg cells of Hodgkin's disease: association with mixed cellularity subtype and Hispanic American ethnicity.

One hundred twenty-five cases of Hodgkin's disease from the United States (79), Mexico City (31), and Costa Rica (15) were analyzed for the presence of Epstein-Barr virus (EBV) by in situ hybridization to EBER1 transcripts. EBV was more frequently detected in the Reed-Sternberg (RS) cells of mixed cellularity Hodgkin's disease (37 of 48 [77%]) compared with the nodular sclerosis subtype (19 of 71 [27%], P < .001). The presence of EBV was also associated with Hispanic ethnicity (P < .001). In a multivariate analysis, patient age, gender, and geographic location were less predictive of EBV positivity than were mixed cellularity histology (odds ratio = 8.3) and Hispanic ethnicity (odds ratio = 4.3). Southern blot analysis of EBV terminal repeat fragments using the Xho1a probe showed that the viral DNA was monoclonal in 17 of 17 cases having EBER1-positive RS cells. By comparison, EBV DNA was not detected by Southern analysis in 20 cases lacking EBER1 in RS cells, even when occasional background lymphocytes expressed EBER1. Because clonal viral DNA was so readily detected in EBER1-positive cases, the EBV genome is probably amplified at least 50-fold in the infected RS cells. Monoclonality of EBV DNA implies that the RS cells were infected before malignant transformation.

Complications of the Heitz-Boyer urinary diversion: case report of late development of malignancy.

We report on 3 patients with bladder exstrophy followed for more than 15 years after having undergone Heitz-Boyer urinary diversion. One patient had adenocarcinoma at the ureterocolonic anastomoses 16 years after diversion. To our knowledge this is the first reported case of a malignancy developing after a Heitz-Boyer procedure. The other 2 patients had significant problems with urinary incontinence and recurrent urinary tract infections. Initial success with this form of urinary diversion can be followed by significant long-term complications.

Hematological measurements in rats flown on Spacelab shuttle, SL-3.

Previous studies have shown that a decrease in red cell mass occurs in astronauts, and some studies indicate a leukocytosis occurs. A life science module housing young and mature rats was flown on shuttle mission Spacelab 3 (SL-3), and the results of hematology studies of flight and control rats are presented. Statistically significant increases in the hematocrit, red blood cell counts, and hemoglobin determinations, together with a mild neutrophilia and lymphopenia, were found in flight animals. No significant changes were found in bone marrow and spleen cell differentials or erythropoietin determinations. Clonal assays demonstrated an increased erythroid colony formation of flight animal bone marrow cells at erythropoietin doses of 0.02 and 1.0 U/ml but not 0.20 U/ml. These results agree with some but vary from other previously published studies. Erythropoietin assays and clonal studies were performed for the first time.

Regulation of hematopoiesis in rats exposed to antiorthostatic hypokinetic/hypodynamia: II. Mechanisms of the "anemia".

Results are presented which demonstrate a close similarity between the ability of antiorthostatic hypokinetic/hypodynamia and orthostatic hypokinetic/hypodynamia to induce anemia in laboratory rats. The "restraint anemia" (whether mediated directly by reduced activity or indirectly by possible changes in blood circulation or in altered weight-bearing capacity of the skeleton) was largely due to reduced food and/or water consumption and displayed the classical symptoms of inadequate nutrition, i.e. decreased serum erythropoietin (Ep) titers and reduced Ep sensitivity of hematopoietic tissue. Only changes in red blood cell (RBC) clearance were unique to the head-down (antiorthostatic) posture. During suspension, RBC clearance was reduced and then accelerated when suspension was terminated or the cells transfused into a normal environment. Changes in RBC clearance were due to both cell-associated and cell-independent factors and may be related to the alterations in RBC survival seen in rats during or immediately after space flight. In both suspension and weightlessness, these changes were limited to alterations in the force and/or direction of the gravity vector.

The effect of chamber restraint (with or without lower body positive pressure) on hematopoiesis (particularly erythropoiesis) in squirrel monkeys (Saimiri sciureus)

Squirrel monkeys, restrained in lower body positive pressure (LBPP) chambers in attempts to model the space flight-induced hematological consequences of a cephalad fluid shift, showed a decrease in red blood cell mass which was largely independent of actual LBPP. The only LBPP-specific hematological effect was an alteration in red blood cell (RBC) survival. This monkey model represents the second, ground-based animal system in which this phenomenon has been recorded and shown to be unique to a cephalad fluid shift. These data suggest a re-evaluation is necessary of the role of altered RBC clearance as a contributing factor to the anemia of space flight in humans.

Methods for repetitive measurements of multiple hematological parameters in individual rats.

Methods have been developed which permit frequent repetitive blood sampling of rats without perturbing physiological parameters of interest. These techniques allow a comprehensive hematological study over several weeks, in individual rats, thus permitting full documentation of selected parameters during growth and development.

Tritiated thymidine incorporation into canine cyclic hematopoietic bone marrow in vitro.

To investigate changes in the proliferative activity of bone marrow cells in canine cyclic hematopoiesis, nonadherent cells were incubated for 1 h with tritiated thymidine either immediately after the cultures were established or following an 18-h preincubation period. The data suggest that changes in thymidine incorporation show a 12- to 14-day cycle that consists of two distinct phases. During the first six days of the cycle (from peripheral neutropenia to relative neutrophilia), two peaks of incorporation were observed. During the second phase (corresponding to the neutrophilia and oncoming neutropenia), thymidine incorporation was uniformly lower than control values. The change from an apparently cyclical process to a low stable value occurred after the wave of marrow myelopoiesis and close to a time point (days 8-10 of the cycle) at which we have recently suggested significant changes in cell release and/or proliferation take place. The data can be interpreted in the context of a periphery-to-stem-cell feedback loop through an intermediate cell population, probably of myeloid precursors.

Regulation of hematopoiesis in rats exposed to antiorthostatic, hypokinetic/hypodynamia: I. Model description.

This paper provides baseline information regarding the regulation of hematopoiesis in antiorthostatic, hypokinetic/hypodynamic ("suspended") laboratory rats. The object of the study was to compare the hematological effects of suspension with those seen following space flight in man and/or rats. Observed in man after exposure to microgravity and in the suspended rats was a reduced red blood cell mass, suppressed erythropoiesis, a transient increase in hematocrit due to a reduction in plasma volume, a post-exposure hematocrit decrease, a weight loss (or failure to thrive) and a reduction in food and water consumption. A rightward shift in the oxyhemoglobin dissociation curve, observed in the rat "model", has been predicted to occur during manned space flight but has not yet been measured. Suppression of hematopoiesis is a common feature of rats during both space flight and suspension. Platelet counts showed no significant change in rats after suspension or in man during space flight. Unlike man in space but similar to space flight-exposed rats, no significant change in leukocyte number or reactivity to PHA in vitro, or in red blood cell shape distribution were observed in the suspended rats. At least in a gross sense, the rat "model" seems to reproduce many of the known hematological effects of space flight and offers promise as a 1 X g analog for understanding hematopoietic effects similar to those found in space flight.

Translation of messenger RNA from a renal tumor into a product with the biological properties of erythropoietin.

The renal tumor RCC-3-JCK, when transplanted into immunodeficient mice, caused an erythrocytic polycythemia. When grown in culture, the tumor cells secreted a substance into the culture medium that chromatographed by size-exclusion high-performance liquid chromatography similarly to purified human erythropoietin (Ep) and was positive when assayed for Ep by its ability to stimulate erythropoiesis in fetal mouse liver cells (the FMLC assay). The poly(A) + RNA was extracted from the tumor cells and injected into Xenopus oocytes, inducing the appearance of Ep(FMLC) in the oocyte culture medium. Both the tumor cells and oocyte culture media were fractionated by size-exclusion high-performance liquid chromatography, and two fractions with Ep(FMLC) activity were found in the tumor-cell culture medium. Three active fractions were found in the medium from the mRNA-injected oocytes. The largest component from both culture media had the same elution time as a human standard (Ep). The poly(A) + RNA was fractionated by sucrose density-gradient centrifugation and the 8S and 10S fractions were found to induce Ep(FMLC) synthesis when they were injected into the oocytes. We conclude that poly(A) + RNA isolated from the Ep-producing tumor RCC-3-JCK included mRNA for Ep and that the Ep was a translational product of Xenopus oocytes injected with this mRNA.

Evaluation of some serum erythropoietin concentration procedures using serum-containing and serum-free in vitro bioassays.

Studies are reported which were designed to further refine a serum-free culture method to assess the erythropoietic response of fetal mouse liver cells. The objective was to employ such a serum-free system with deproteinized serum concentrates as the test materials in a potentially highly specific assay for erythropoietin (Ep). A serum-free culture method is described which permits responses to Ep (125I-deoxyuridine incorporation) closely comparable to those observed in cultures containing optimal concentrations of sera. Mean recoveries of Ep were acceptable in each of three variations of a serum concentration procedure. However, no correlations were evident between Ep titers in whole sera and those in the concentrates (assayed in both serum-free and serum-containing cultures) with deproteinization and/or concentration procedures involving serum acidification and exposure to boiling water temperature.

Spinal cord injury and serum erythropoietin.

The origin of the temporary reduction in hematocrit and hemoglobin occurring after spinal cord injury (SCI) may be related to the neurologic dysfunction and to a change in erythropoietin (Ep) synthesis and/or secretion. Serum Ep, vital capacities, and numerous hematologic profiles were obtained in 12 cervical SCI patients during the first 12 weeks and 13th to 27th post-injury weeks, and in 8 thoracic SCI patients during the first 8 weeks and 9th to 28th weeks. Serum iron, iron binding, and saturation were obtained in 4 cervical SIC patients and blood gases in 10 patients with cervical SCI and in 6 with thoracic SCI. The results in all patients showed increased Ep values, returning to normal after 8 weeks in the thoracic SCI patients only. The few results on iron kinetics showed a tendency to low values, suggesting the necessity for further investigation. In an attempt to explain the Ep increase in SCI patients, the relative influence of the renal pelvic nerves and of the sympathetic nerves on its secretion, are examined.

Serum erythropoietin titers during prolonged bedrest; relevance to the "anaemia" of space flight.

The overall objective of these studies was to test the hypothesis that the suppression of erythropoiesis, which occurs during both spaceflight and bedrest, was mediated by reduction in circulating levels of erythropoietin. In each of two 7-day studies, groups of subjects were exposed to either horizontal or 6 degrees head-down tilt bedrest and no evidence was obtained to suggest that the erythropoietic effects were dependent on the angle of recumbency. An additional study involved six men who were exposed to horizontal bedrest for 28 days. Serum erythropoietin titers were not significantly depressed in any of the subjects but total red cell volume was decreased. Absolute increases in red cell numbers and reductions in plasma volume both elevate the haematocrit, but our data suggest that the mechanism of erythrosuppression in these two instances may be different.

Differences in some erythroid regulatory parameters between two inbred mouse strains.

Normal erythropoiesis in inbred CBA/Tr x CBA/Tr mice is shown to be associated with higher rates of red blood cell production and destruction than in age-matched, but significantly larger, C57Bl6/Tr x C57Bl6/Tr mice. As expected from these observations, serum Ep titers were significantly higher in the CBA/Tr mice than in the C57Bl6/Tr mice. These differences, which appear to result from variations in the operating point for erythropoiesis, can explain the different in vitro erythropoietin sensitivities of hematopoietic tissue from these two mouse strains.

Serum erythropoietin changes in cats with feline leukemia virus-induced erythroid aplasia.

Erythropoietin concentrations were increased significantly (p less than 0.025) in nine cats with natural feline leukemia virus infection and associated erythroid aplasia compared to six clinically normal cats. Adult cats experimentally inoculated with the Kawakami-Theilen isolate of feline leukemia virus developed a progressive simultaneous increase in erythropoietin activity and decrease in packed cell volume. These findings indicate that erythroid aplasia associated with feline leukemia virus infection is not caused by a failure in erythropoietin production.