Biomedical subjects
Publications and source records attributed to J Levin.
Mice fed azidothymidine demonstrated dramatic hyperpigmentation of their tails. Histologic examination demonstrated large quantities of melanin pigment throughout the entire epidermal layer. Control mice had scant melanin pigment localized to the basal layer. Our findings demonstrate that the mouse is a useful model for the investigation of drug-induced hyperpigmentation.
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We have enriched for bovine megakaryocytes and identified a culture system that may provide an in vitro model for platelet formation. Mature megakaryocytes with an unusually high ploidy distribution were obtained after differential centrifugation and velocity sedimentation of bone marrow cells through gradients of bovine serum albumin (BSA). The cell membranes of isolated megakaryocytes and megakaryocytes in vivo stained with antisera to human platelets and human platelet membrane GPIIIa. The microenvironment of bovine megakaryocytes in vivo was investigated using antibodies to types I and IV collagen and laminin. In an attempt to duplicate the microenvironment in vitro, bovine megakaryocytes were cultured on a reconstituted basement membrane matrix (Matrigel). The cells adhered to the gel, extended radial lamellipodia, and occasionally formed lengthy pseudopodia. Ultrastructural examination of these cells showed widening and coalescence of the megakaryocyte demarcation membranes (DMS), and inclusion of platelet granules, thin filaments, and microtubules in the processes. Very few DMS vesicles were present distally in the processes. The culture of megakaryocytes on a reconstituted basement membrane may closely model the in vivo megakaryocyte microenvironment and allow the study of thrombocytopoiesis in vitro.
Clinical and epidemiologic studies suggest that the intake of potassium chloride lowers blood pressure. To investigate whether supplemental potassium chloride (96 mmol of microcrystalline potassium chloride a day) reduced the need for antihypertensive medication in hypertensive men on a restricted-sodium diet, we conducted a randomized, placebo-controlled, double-blind clinical trial. A total of 287 men 45 to 68 years of age, 142 given potassium chloride and 145 given placebo, were followed for an average of 2.2 years after the withdrawal of their antihypertensive medication. Men in both groups received instructions on following a low-sodium diet. Overnight urinary sodium excretion fell from 63 mmol per eight hours at base line to an average of 45 mmol per eight hours during follow-up. Participants given supplemental potassium chloride had significantly higher (P less than 0.001) serum potassium levels and urinary potassium excretion (averaging 4.5 mmol per liter and 42.5 mmol per eight hours, respectively) during follow-up than participants given placebo (4.2 mmol per liter and 20.0 mmol per eight hours). Seventy-nine participants in each group required reinstitution of antihypertensive medication according to strict indications defined by the protocol. No significant differences in systolic or diastolic blood pressure were observed between the two groups. During follow-up, systolic and diastolic blood pressure averaged 130.6 and 82.5 mm Hg, respectively, for participants given supplemental potassium, and 132.5 and 83.1 mm Hg for participants given placebo. We conclude that supplemental potassium chloride does not reduce the need for antihypertensive medication in hypertensive men on a restricted-sodium diet.
Because we had observed that smoking has a pronounced effect on serum progesterone levels, we reinvestigated in healthy nonsmokers the relative progesterone levels of men and follicular-phase women. Each of eight women had multiple measurements of serum progesterone during the follicular phase of a menstrual cycle (10 days through 3 days prior to the luteinizing hormone peak of that cycle), and the average of those values was taken to represent the basal progesterone level for that woman. Seven men had blood samples drawn at 20-minute intervals between 6:00 and 9:00 AM, through an indwelling venous catheter, and the average of those values was taken. The mean follicular-phase serum progesterone level in the women was 21.4 +/- 5.4 ng/dl and the mean level in the men was 18.1 +/- 3.1 ng/dl. The difference was not statistically significant. In view of this finding, we conclude that there is essentially no ovarian secretion of progesterone during the follicular phase of the menstrual cycle.
Since its initial invention by the French worker Milian in 1901, the bleeding time has been put forward as a clinically useful test in three contexts: diagnosis (particularly of platelet disorders), prediction of clinically important bleeding, and assessment of the adequacy of various forms of therapy. Attempting a complete review of the published experience with this test, we assessed 862 articles. Original bleeding time data appeared in 664 of these articles, from which we tabulated 1083 distinct studies in humans. ROC analysis, which characterizes the sensitivity and specificity of the test, was applied in every instance in which published data were adequate (34 studies). ROCs from 27 studies of the bleeding time in association with aspirin ingestion reveal high variability in the ability of the bleeding time to detect aspirin intake, and provide evidence against claims that recently devised bleeding time methods have improved discriminatory ability based on improved reproducibility. Two ROCs from surgical studies, in which the bleeding time was used to try to predict abnormal bleeding, were statistically indistinguishable from that of a completely noninformative test. In ROCs from five studies of abnormal bleeding in uremia, the test performed approximately the same as the platelet count or hematocrit (taken singly); in one of these studies, prothrombin consumption was determined and was a better predictor of bleeding than bleeding time, hematocrit, or platelet count. In the settings of renal biopsy (one study) and massive transfusion (one study), data allowed estimation of predictive value: in no instance was there evidence that the bleeding time significantly altered a priori estimates (based on prevalence) of the risk of bleeding. Linear regression analysis was applied to data from 23 studies relating platelet count to bleeding time, to assess published claims that the bleeding time and platelet count follow a predictively useful linear relationship. In 22 of 23 instances, the inverse relationship between bleeding time and platelet count was associated with broad statistical scatter, making it impossible to predict precisely one variable given the other. The pathophysiology of an abnormal bleeding time remains poorly understood. The bleeding time is affected by a large number of diseases, drugs, physiologic factors, test conditions, and therapeutic actions, not all of them platelet-related. The test is likely to remain widely used for the diagnosis of inherited disorders of platelet function, such as von Willebrand's syndrome, despite the lack of clear criteria for its use in this context.(ABSTRACT TRUNCATED AT 400 WORDS)
To date, testing of various cytokines for the stimulation of blood cell production has not demonstrated a consistent effect on peripheral platelet levels. In this report, we provide evidence that human recombinant IL-6 increased platelet production in mice, as measured by both peripheral platelet levels and [75Se]selenomethionine (75SeM) incorporation into newly forming platelets. Peripheral white blood cell counts also were increased, but only to a modest extent, and hematocrit values were unchanged. A dose-response relationship between the amount of IL-6 administered and platelet count, 75SeM incorporation, and white blood cell count was demonstrated. Detectable megakaryocyte and granulocyte-macrophage colony-forming cells in mice that had received IL-6 also were increased in both bone marrow and spleen. These results demonstrate the ability of a purified, recombinant protein to stimulate platelet production in vivo.
It has been assumed that endogenous synthesis by the platelet precursor cell, the bone marrow megakaryocyte, is the major source of platelet alpha-granule protein. To test this hypothesis, we used mRNA phenotyping to detect in megakaryocytes the presence of mRNA transcripts specific for various proteins. Our results indicate that megakaryocytes synthesize platelet factor 4, a protein relatively specific for platelets, but do not express mRNA transcripts for the fibrinogen, albumin, or IgG found in alpha-granules. We have previously shown that megakaryocytes endocytose circulating proteins, including fibrinogen, albumin, and IgG, and incorporate them into alpha-granules. Thus, platelets appear to contain a unique type of secretory granule whose contents originate by both endogenous synthesis and endocytosis from plasma. Under basal conditions, the source of alpha-granule fibrinogen is plasma.
Detection and quantification of bacterial endotoxin in plasma by the Limulus amebocyte lysate test (or other assays for endotoxins) is hindered by the presence of inhibitors. Treatment of plasma to overcome inhibitory activities is required before plasma can be successfully assayed for endotoxin. We have conducted an investigation comparing the three most commonly used procedures (dilution-heating, trifluoroacetic acid oxidation, and chloroform extraction) for treatment of plasma before its assay for endotoxin with the chromogenic Limulus test. Initially, conditions were optimized for treatment of plasma by each of these methods. Subsequently, a direct comparison of the three plasma treatment procedures was performed with plasma spiked with known concentrations of endotoxin. The optimized dilution-heating procedure resulted in the most sensitive detection of endotoxin, with sensitivity approximately 10 times greater than the optimized trifluoroacetic acid oxidation procedure and approximately 100 times greater than treatment of plasma by chloroform extraction. Maximal detection of low concentrations of endotoxin by the chromogenic Limulus test was obtained by dilution of plasma fourfold with 0.15 mol/L NaCl followed by heating at 60 degrees C for 30 minutes. This procedure was simple, rapid, and did not involve addition of any reagents to plasma that could potentially add contaminating endotoxin.
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Murine platelet production is normally supported by high-ploidy bone marrow megakaryocytes without significant contribution from splenic megakaryocytes with predominantly low-ploidy levels. We produced sustained bone marrow ablation using radiostrontium, and examined the processes by which splenic platelet production is initiated and maintained in the absence of bone marrow function. Bone marrow hematopoiesis, measured by total nucleated cell number and viability, megakaryocyte colony-forming cells, and granulocyte-macrophage colony-forming cells, was rapidly ablated in mice by using yttrium 90-free strontium 90. Platelet count declined from normal (1224 x 10(3)/microliters) to a nadir (98 x 10(3)/microliters) 11 days after 90Sr, and then rose to a stable level (705 x 10(3)/microliters) on days 20 through 115. Peripheral leukocyte concentration decreased rapidly and remained below 25% of normal in contrast to hemoglobin levels, which were minimally lowered. Mean spleen weight rose rapidly after 90Sr to 66% above normal. Splenic megakaryocyte frequency, measured by two-color fluorescence-activated flow cytometry, rose from basal levels (0.09% +/- 0.06%) to 0.15% +/- 0.07% (p less than 0.001), total spleen nucleated cells fell to 71% of normal, and the absolute number of spleen megakaryocytes was unchanged. Total spleen megakaryocyte colony-forming cells were not significantly increased above normal whereas total spleen granulocyte-macrophage colony-forming cells increased abruptly after day 13 to 10 times normal levels. Splenectomy after hematopoietic recovery from 90Sr bone marrow ablation resulted in a rapid decline of platelet levels, followed by death. Although the spleen became the sole site of platelet production, the splenic megakaryocyte ploidy distribution was only minimally changed from normal, and the modal ploidy class remained 2N. In contrast to experimental thrombocytopenia in mice with intact bone marrow, in which megakaryocyte ploidy is increased, thrombocytopenia associated with sustained bone marrow ablation does not result in upward regulation of splenic megakaryocyte ploidy as a compensatory mechanism.
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We studied thrombopoiesis in mice after the experimental induction of sustained, immune thrombocytopenia with platelet antiserum (PAS). Utilizing light and electron microscopy and a digital image analyzer to determine platelet sectional areas, we examined platelets and megakaryocytes (MK) after 120 h of sustained, severe thrombocytopenia (120CT) and during recovery from thrombocytopenia at 48 h (48R), 72 h (72R), and 120 h (120R) after cessation of administration of PAS. Mean platelet volume (MPV), determined by electrical impedance, also was measured at each time point. Platelets at 120CT (platelet count less than 50,000/microliter), 48R (platelet count 100-200,000/microliter), and 72R (platelet count approximately 1 x 10(6)/microliter) were significantly larger in sectional area than control platelets and contained increased profiles of endoplasmic reticulum and Golgi cisternae, a lower concentration of surface-connected canalicular system, and occasional membrane complexes. The largest median platelet sectional area was detected at 48R and was the largest median value observed in response to either chronic or acute thrombocytopenia. At 120R, most platelets were normal in size and cytoplasmic appearance, although some large cells remained present in the circulation. MPV paralleled the morphometric changes in platelet sectional area. MK were increased in number at 120CT, 48R, 72R, and 120R. In addition, at least half of the MK examined at 48R contained small areas of cytoplasm, devoid of organelles, that were interspersed between larger areas of organelle-filled, undemarcated cytoplasm. The modal bone marrow megakaryocyte ploidy class, determined using two-color fluorescence-activated flow cytometry, shifted from 16N to 32N in response to sustained thrombocytopenia. In contrast, during recovery and development of rebound thrombocytosis, the relative frequency of 8N megakaryocytes was significantly increased. Because there was no consistent correlation between megakaryocyte cytoplasmic characteristics and platelet morphology, these data support the hypothesis that platelet formation is not determined by compartmentalization of MK cytoplasm into platelet areas as MK mature in the bone marrow, but involves a rearrangement of MK cytoplasm immediately prior to platelet release.
We have examined the effects of variable degrees of acute thrombocytopenia on platelet levels, mean platelet volume (MPV), and buoyant density after induction of thrombocytopenia by platelet antiserum (PAS) in mice with or without spleens. Mice were studied serially 10-16, 36, 48, 60-64, 84, 108, 144, 180, 228, 276, 348-360, 372, and 516 h after PAS treatment. MPV and platelet count (PC) x 10(6)/microliters for normal intact mice (n = 136) were 4.7 +/- 0.3 fl (SD) and 1.69 +/- 0.52 (SD), respectively. Twelve hours after PAS-induced severe thrombocytopenia (PC less than 0.05 x 10(6)/microliters), MPV increased significantly (p less than 0.01) to 6.4 fl, was maximal at 36 h (8.2 fl), remained elevated until 144 h following PAS treatment, and then returned to normal. Platelet density decreased significantly (p less than 0.05) 64 h after PAS treatment and returned to normal at 144 h. Hematocrits of repeatedly bled intact control mice decreased from 45% to 30%, accompanied by thrombocytosis (maximal PC 2.24 x 10(6)/microliters) without significant changes in either MPV or platelet density. Moderate thrombocytopenia (PC 0.1-0.2 x 10(6)/microliters) in intact mice produced significantly (p less than 0.05) increased MPV, at 5.7 fl 12 h after PAS treatment, with a peak MPV of 7.6 fl (p less than 0.001) at 36 h; MPV returned to normal at 84 h. Platelet density decreased (p less than 0.001) 12 h after PAS treatment and returned to baseline at 228 h. Control splenectomized mice (n = 185) had an MPV of 5.0 fl +/- 0.7 fl and a PC of 2.14 +/- 0.6 x 10(6)/microliters. Comparably severe and moderate thrombocytopenia in splenectomized mice produced alterations in platelet count, MPV, and density similar to those in intact mice, although maximal MPV and the degree of rebound thrombocytosis after severe thrombocytopenia were more marked in splenectomized mice. In response to reduction of the platelet mass in both intact and splenectomized mice, MPV increased in proportion to the severity of thrombocytopenia, occurred as early as 4 h after induction, and persisted during early rebound thrombocytosis. Previous observations that megakaryocyte ploidy did not shift until 48 h after onset of thrombocytopenia confirm that both initial and maximal changes in MPV in response to this stimulus are regulated by processes other than alterations of megakaryocyte DNA levels.(ABSTRACT TRUNCATED AT 400 WORDS)
Previous studies have suggested that platelet volume may be primarily regulated by the ploidy distribution of mature bone marrow megakaryocytes. However, earlier investigations from this laboratory using C57/BL mice have shown that in response to acute, severe, or moderate thrombocytopenia, platelet volume is regulated independently of megakaryocyte ploidy. Murine strains, including C57/BL, usually have a modal bone marrow megakaryocyte ploidy class of 16N. In contrast, the C3H mouse has a 32N modal bone marrow megakaryocyte ploidy class. We have examined the platelet count, platelet volume distribution, and bone marrow megakaryocyte ploidy distribution of C3H mice during steady-state thrombopoiesis and after depletion of platelets by antiplatelet serum. The platelet count and volume of normal C3H mice were not substantially different from those of C57/BL mice, but megakaryocyte frequency was marginally greater (p less than 0.05), and the ploidy distribution exhibited a a marked reduction in the proportion of 16N cells (p less than 0.001) and increased relative frequencies of 32N (p less than 0.001) and 64N (p less than 0.01) megakaryocytes. In response to acute severe thrombocytopenia, C3H mice demonstrated an increase in platelet volume equivalent to that previously reported for C57/BL mice, without a subsequent shift in the modal megakaryocyte ploidy class. The relative frequencies of 64N and 128N megakaryocytes increased significantly (p less than 0.005) compared to normal C3H mice, without a change in the frequency of 32N megakaryocytes. These studies indicate that during steady-state thrombopoiesis, a greater proportion of higher ploidy megakaryocytes (32N plus 64N) does not necessarily alter peripheral platelet count or platelet volume. Therefore, it appears that neither platelet volume nor count are primarily regulated by bone marrow megakaryocyte ploidy and that the magnitude of upward regulation of megakaryocyte ploidy is limited.
Incubation of lysates prepared from amebocytes of the horseshoe crab (Limulus polyphemus) with bacterial endotoxin results in coagulation and formation of a solid gel. Although Limulus gels remained solid indefinitely, if undisturbed, they were easily disrupted by mechanical agitation. Chemical solubility studies of gelled lysates demonstrated rapid solubilization of gels in monochloroacetic acid, a property of clots that have not been covalently stabilized; but in contrast demonstrated resistance to solubilization by urea, a property of stabilized clots. Analysis of solubilized proteins by polyacrylamide gel electrophoresis in SDS demonstrated coagulin, the designation for the activated form of coagulogen (the clottable protein) that forms a gel, only in samples derived from clotted lysate that had been previously incubated with monochloroacetic acid, but not in samples following incubation with urea, confirming the results of the chemical solubility studies. Enzymatic assays for transpeptidase (Factor XIII-like) activity in either native or gelled Limulus lysates were negative. Furthermore, analysis for covalently crosslinked peptides in gelled coagulin confirmed the absence of intermolecular gamma-glutamyl-epsilon-lysyl bonds. Therefore, the stable gels formed following coagulation of Limulus lysate by bacterial endotoxin are not covalently crosslinked.
Addition of commercial heparin to preparations of the coagulation system obtained by hypotonic lysis of Limulus amebocytes, prior to the addition of endotoxin from gram-negative bacteria, inhibited gelation. Activation of the cell-lysate proclotting enzyme, utilizing a diluted lysate preparation and synthetic substrates, was inhibited to a comparable extent with either anticoagulantly active or inactive heparin. Other glycosaminoglycans, including heparan sulfate, dermatan sulfate, chondroitin 4-sulfate, and chondroitin 6-sulfate, did not inhibit the generation of clotting enzyme activity. Experiments conducted with preparations of proactivator(s) and proclotting enzyme, obtained by fractionation of lysate with heparin-Sepharose, revealed that inhibition of endotoxin-dependent activation of amebocyte lysate by heparin is mediated by precipitation of the proactivator(s) and can be prevented by increasing the salt concentration.
The purpose of this study was to investigate the use of a radiolabeled mouse monoclonal antibody (and its F(ab')2 fragment) against alpha-fetoprotein in the scintigraphic diagnosis of hepatocellular carcinoma. Twenty-six southern African Blacks and one Caucasian with hepatocellular carcinoma and four patients with other malignant tumors of the liver were studied. Although six hepatocellular carcinomas appeared to selectively concentrate alpha-fetoprotein antibody, one of these tumors was not producing alpha-fetoprotein. Moreover, in another 18 patients with alpha-fetoprotein-producing hepatocellular carcinomas, uptake of alpha-fetoprotein antibody was at best only equal to that in nontumorous hepatic tissue, and three hepatocellular carcinomas that were not producing alpha-fetoprotein concentrated the antibody to the same extent as did the alpha-fetoprotein-producing tumors and hepatic tissue. All four tumors other than hepatocellular carcinoma concentrated alpha-fetoprotein antibody as well as did hepatic tissue. These findings suggest that the penetration of hepatocellular carcinomas by alpha-fetoprotein antibody is a passive and nonselective process. This conclusion is supported by an in vitro study in which a non-alpha-fetoprotein-producing hepatic metastasis took up as much radiolabeled alpha-fetoprotein antibody as did three of four alpha-fetoprotein-producing hepatocellular carcinomas. A likely explanation for the failure of alpha-fetoprotein monoclonal antibody to be selectively concentrated by hepatocellular carcinomas is that alpha-fetoprotein is an export protein and is not expressed on the cell membranes of malignant hepatocytes.