Relapsing hypocupraemic myelopathy requiring high-dose oral copper replacement.

Adult-onset copper deficiency with neurological manifestations is a newly recognised syndrome. Long-term oral copper replacement therapy has been the mainstay of treatment in the literature. A case of relapsing hypocupraemic myelopathy responsive to increased doses of copper replacement is reported. Standard doses of copper may not be sufficient for all patients.

Adenosine-mediated killing of cultured epithelial cancer cells.

Because micromolar concentrations of adenosine (Ado) have been documented recently in the interstitial fluid of carcinomas growing in animals, we examined the effects of low concentrations of Ado on the growth of cultured human carcinoma cells. Ado alone had little effect upon cell growth. In the presence of one of a number of Ado deaminase (ADA) inhibitors, Ado led to significant growth inhibition of all cell lines tested. Similar effects were found when ATP, ADP, or AMP was substituted for Ado. Surprisingly, the ADA inhibitor coformycin (CF) had a much greater potentiating effect than did 2'-deoxycoformycin (DCF), although DCF is a more potent ADA inhibitor. The growth inhibition of the Ado/CF combination was not abrogated by pyrimidines or caffeine, a nonspecific Ado receptor blocker. Toxicity was prevented by the addition of the Ado transport inhibitor dipyridamole or the Ado kinase inhibitor 5'-amino 5'-deoxyadenosine. S-Adenosylhomocysteine hydrolase is not involved because neither homocysteine thiolactone nor an S-adenosylhomocysteine hydrolase inhibitor (adenosine dialdehyde) potentiated toxicity of the Ado/CF combination. Unexpectedly, substitution of 2'-deoxyadenosine (the toxic moiety in congenital ADA deficiency) for Ado, did not lead to equivalent toxicity. The Ado/CF combination inhibited DNA synthesis and brought about morphological changes consistent with apoptosis. Together, these findings indicate that the Ado-mediated killing proceeds via an intracellular route that requires the action of Ado kinase. The enhanced cofactor activity of CF may be attributable to its being a more potent inhibitor of AMP deaminase than is DCF.

Clinically significant differences in the International Normalized Ratio measured with reagents of different sensitivities. SPAF Investigators. Stroke Prevention in Atrial Fibrillation.

The International Normalized Ratio (INR) system was introduced a decade ago as a way of standardizing the results of prothrombin time testing for patients taking oral anticoagulants. A strong emphasis has been placed upon using thromboplastin reagents that are very sensitive to the effects of oral anticoagulants upon the prothrombin time [i.e. reagents with low International Sensitivity Index (ISI)]. In order to assess how well the INR system functions as currently used in clinical laboratories, we compared the INRs determined using thromboplastins of differing ISIs in samples collected during a large clinical trial of oral anticoagulation for atrial fibrillation (Stroke Prevention in Atrial Fibrillation III trial). Frozen plasma was subjected to prothrombin time testing using thromboplastins with ISIs ranging from 0.97 to 2.49. INRs were calculated using machine-specific ISIs and Westgard's rules were followed to maintain quality control. An unanticipated coagulometer failure allowed a determination of the effect of machine recalibration upon the INR of control plasmas. The correlation between each pair of INRs obtained from 1181 plasmas was high (> 0.9), but the differences between reagents were statistically different from zero (P<0.001 for pairwise comparisons). Plasmas had INRs within the therapeutic range (2.0-3.0) with one reagent but not with another in an average of 20% of instances. Among the 20% discordant pairings, 43% (8.5% of the total tested) showed a difference in INR of more than 0.2 INR units above or below the target range. Low ISI thromboplastins did not perform better in this pairwise comparison than other reagents or the locally determined INR. Recalibration of a coagulometer resulted in a significant change in the INRs obtained from control plasmas (P<0.0001), which confirms and extends the observations of other authors concerning the sensitivity of the INR to coagulometer-related variables. There was a clinically significant difference in the INRs obtained with different thromboplastins, and low ISI reagents did not perform better than others. Since the risk of thrombosis rises sharply below the lower limit of the currently recommended target ranges, consideration should be given to narrowing the recommended range, or advising clinicians to aim for its mid-point. These findings illustrate the difficulties in imposing standardization upon coagulation testing after a test is in widespread use.

Assessment of coagulation system activation using spot urine measurements.

Coagulation system activation is most commonly assessed by measuring levels of one or more proteins in peripheral blood. Because faulty blood-drawing can cause activation of the coagulation system, artifactual elevations of such markers have been reported. We have therefore investigated the possibility of using randomly collected ('spot') urine samples as a non-invasive means of assessing the state of coagulation system activation. Using a commercially available enzyme-linked immunosorbent assay kit designed to measure plasma levels of fragment 1 + 2, we found immunoreactive fragment 2 in healthy control subjects, and significantly increased levels in diabetic and non-diabetic pregnant subjects, and patients with venous thromboembolism, prostate cancer, and diabetes. Measurements of excretion of immunoreactive fragment 2 are worth further study as an adjunct or alternative to plasma-based assays designed to detect or quantify coagulation system activation.

Plasma levels of factors II, VII and X and their relationship to the international normalized ratio during chronic warfarin therapy.

Monitoring of oral anticoagulant therapy is usually undertaken with the prothrombin time (PT), which is influenced by factors II, X, and VII. A number of studies have suggested that the prothrombin (factor II) level may be the most important determinant of the therapeutic efficacy of these drugs. Although some studies suggest that oral anticoagulants induce a similar residual level of plasma vitamin K-dependent proteins, others have called this into question. We therefore measured plasma levels of factors II, X, and VII in 50 patients undergoing chronic Warfarin therapy. The plasma levels of factors II, X, and VII were significantly different. Although the factor X levels of all plasmas were < 30%, levels of factors II and VII were > 30% in 14% and 50% of the samples, respectively. Multivariable analysis showed factor II levels to be the least significant of the three factors measured in determining the international normalized ratio of plasma or whole blood. Thus, plasma levels of the vitamin K-dependent coagulation factors are not equal in patients on chronic Warfarin therapy. If factor II (prothrombin) levels are indeed the major determinants of the therapeutic efficacy of Warfarin, alternative means of monitoring that more accurately reflects prothrombin levels should be evaluated.

Use of a modified activated partial thromboplastin time to detect lupus anticoagulants.

Laboratory evidence for the presence of lupus anticoagulants (LAs) is sought when patients experience thrombotic events or when coagulation assays are abnormal. Although a number of tests for LAs have been proposed, none detect all LAs, and laboratories may be confronted with the need to perform more than one test to confirm a suspected LA. Recently, a modification of the aPTT, performed by varying the initial time of incubation of the aPTT reagent with the patient's plasma, was reported to detect LAs. The difference in clotting times when plasma is subjected to a 1- or 10- minute incubation (called here the "Delta one minus ten" or DOT) using a particular micronized silica-based aPTT reagent was shown to provide good discrimination between normal and LA plasmas. Because of the low cost of this test and its relative ease of performance, we attempted to replicate the results of this test using previously characterized LA plasmas. The DOT of 23 normal plasmas was 5.1 +/- 2.1 seconds, with a range of 0.5 - 9.3 seconds. The DOT of 20 of 34 LA samples tested (59%) was > 11 seconds. The DOT was abnormal in 8 of 22 (36%) samples diagnosed with a dilute Russell's viper venom time. It was abnormal in 12 of 12 patients diagnosed by other criteria, prior to the use of the dilute Russell's viper venom time. The DOT performed with a kaolin or ellagic acid-based aPTT reagent failed to discriminate normal from LA plasma. We conclude that the DOT performed with a specific silica-based reagent is an apparently simple and moderately sensitive test for detecting the lupus anticoagulant that deserves further evaluation.

Coordinated inhibition of actin-induced platelet aggregation by plasma gelsolin and vitamin D-binding protein.

Actin is an abundant intracellular protein that is released into the blood during tissue injury and its injection into rats causes microthrombi to form in the vasculature. This report and others have shown that actin filaments are able to aggregate platelets in an adenosine diphosphate (ADP)-dependent manner. The effects on this process of two plasma actin-binding proteins, vitamin D-binding protein (DBP) and gelsolin, were examined separately and together. The addition of DBP, a monomer-binding protein, to actin filaments did not affect their ability to induce platelet aggregation. However, severing of actin filaments with gelsolin resulted in an increased degree of platelet aggregation. Preincubation of F-actin with both gelsolin and DBP resulted in a significant inhibition of aggregation. The effects of DBP and gelsolin on actin-induced aggregation paralleled their effects on exchange of actin-bound adenine nucleotides. DBP inhibited 1, N6-ethenoadenosine 5' triphosphate (epsilon-ATP) exchange with G-actin but not with F-actin. Gelsolin increased epsilon-ATP exchange with F-actin, which was largely abrogated by the addition of DBP. These results suggest that gelsolin's severing (and subsequent capping) of actin filaments not only results in an increase in the number of pointed filament ends but also in the dissociation of actin monomers containing ADP. Phalloidin, which stabilizes actin filaments while decreasing both monomer and nucleotide exchange, inhibited actin-induced aggregation, as well, indicating that depolymerization of actin filaments is not required to inhibit aggregation. Platelet activation by either G- or F-actin may thus be regulated by the local concentrations of the plasma actin-binding proteins gelsolin and DBP. Together, these proteins inhibit platelet aggregation in a manner that can be explained by their effects on actin's filament structure and the accessibility of its bound ADP. Depletion of DBP or gelsolin may allow actin released from injured tissues to stimulate purinergic receptors on platelets, and perhaps other cells, via its bound adenine nucleotides.

Actin stimulates plasmin generation by tissue and urokinase-type plasminogen activators.

The experiments reported here were carried out to define in greater detail actin's stimulation of plasmin generation by t-PA. Actin did not alter t-PA's hydrolysis of a synthetic substrate, and thus is unlikely to have a direct effect upon t-PA's proteolytic activity. When studied in a single-stage assay, actin accelerated t-PA-mediated plasmin generation from both Glu-plasminogen and Lys-plasminogen, indicating the central role of ternary complex formation. Although actin does not appear to bind two-chain urokinase (tcu-PA), it stimulates tcu-PA's cleavage of Glu-plasminogen. This finding suggests that actin alters the conformation of Glu-plasminogen to an open form. The failure of actin to increased plasmin generation by tcu-PA acting on Lys-plasminogen, which is in an open configuration, is consistent with this interpretation. Immunoglobin G, which shares with actin the property of binding to Glu-plasminogen after nicking by plasmin, did not stimulate tcu-PA's cleavage of Glu-plasminogen, indicating the uniqueness of actin's effects and suggesting interactions between actin and plasminogen at multiple binding sites. Unlike fibrin and heparin, whose stimulation of t-PA is related to polymer length actin is able to stimulate t-PA when presented in either a monomeric or polymeric form. Denaturation of actin by exposure to urea and guanidine increased its ability to stimulate plasmin generation by t-PA. Because actin's structure is maintained by a noncovalently bound adenine nucleotide (ATP or ADP), exposure to ATP/ADPases found in plasma and on cell membranes might also result in its denaturation. Actin treated with an enzyme functionally similar to such ecto-ATP/ADPases, potato apyrase, was more potent than native actin in stimulating plasmin generation by t-PA. The effects of apyrase were blocked by the addition of the plasma actin-binding proteins, gelsolin and the vitamin D-binding protein (DBP). Thus, denaturation of actin may occur in under physiologic conditions, with potential biological consequences. Actin thus appears to be unique with regard to its interactions with the fibrinolytic system and plasma actin-binding proteins may serve to protect the host from the effects of denatured actin.

Oxidative inactivation of plasmin and other serine proteases by copper and ascorbate.

Fibrin thrombi form at sites of injury, where leukocytes release a variety of oxidants. To determine whether oxidants might affect proteins of the fibrinolytic system, we examined the effects of various oxidants on plasmin. Plasmin was not inhibited by micromolar concentrations of hypochlorous acid, chloramine T, or H2O2. Neither Fe nor Cu affected plasmin alone or in the presence of H2O2. However, incubation of plasmin with 5 mumol/L Cu(I or II) in the presence of the reducing agent ascorbic acid resulted in a loss of its hydrolytic activity towards proteins as well as towards small synthetic substrates. The addition of EDTA, but not mannitol, prevented its inactivation. Inactivation was prevented by the addition of catalase and accelerated by hydrogen peroxide. Preincubation of plasmin with the competitive inhibitor alpha-N-acetyl-L-lysine methyl ester prevented inactivation by Cu(II) and ascorbate. These results together suggest site-specific oxidation of plasmin's active site. Treatment of the plasminogen activators tissue plasminogen activator and two-chain urokinase-type plasminogen activator, as well as trypsin, neutrophil elastase, and thrombin with Cu(II) and ascorbate resulted in a loss of their amidolytic and proteolytic activity, indicating the general susceptibility of serine proteases to this type of oxidation. Oxidation of the zymogens Glu-plasminogen and single-chain urokinase-type plasminogen activator by Cu(II) and ascorbate resulted in the failure of these molecules to generate active enzymes when treated with plasminogen activators or plasmin, respectively. The active site His residue may be the target of oxidative inactivation, as evidenced by the partial protection afforded plasmin by the addition of Zn(II), histidine, or the platinum derivative, platinum(II) (2,2':6',2"-terpyridine) chloride. Because platelets contain micromolar concentrations of Cu and leukocytes are rich in ascorbate, Cu-dependent site-specific oxidation might play a role in modulating proteolytic events and the life span of thrombi formed at sites of tissue injury.

Transforming growth factor beta regulation of migration in wounded rat intestinal epithelial monolayers.

BACKGROUND: In vitro studies have suggested that transforming growth factor beta 1 (TGF-beta 1) plays an important role in the regulation of proliferation of intestinal epithelial cells, effecting strong inhibition of proliferation in intestinal epithelial cell lines. Studies were undertaken to assess its role in repair after injury using an in vitro wounding model. METHODS: Wounds were created in confluent monolayers of the intestinal epithelial cell line of IEC-6. Exogenous TGF-beta 1, conditioned media from wounded IEC-6 cultures, or control media were added. Restitution was quantified as the number of cells migrating across the wound edge. Proliferation was assessed by uptake of bromodeoxyuridine and thymidine incorporation. RESULTS: Although TGF-beta was a potent inhibitor of proliferation, it promoted rapid "healing" of the monolayers through stimulation of migration of cells across the wound margin. The physiological importance of this activity was supported by the demonstration that conditioned medium from IEC-6 cells stimulated repair of the wounded monolayers. Effects of the conditioned medium could be entirely blocked by immunoneutralizing anti-TGF-beta antisera. Further, addition of protease inhibitors (aprotinin, epsilon-aminocaproic acid) that prevented the bioactivation of latent TGF-beta secreted by the IEC-6 cells also ablated the effect of the conditioned medium. In addition, expression of TGF-beta 1 messenger RNA was significantly enhanced in the wounded monolayers. CONCLUSIONS: These findings suggest that TGF-beta may play an important role in reconstitution of epithelial integrity after mucosal injury.

Histamine, actin-gelsolin binding, and polyphosphoinositides in human umbilical vein endothelial cells.

Histamine activates inositol phospholipid metabolism, increases calcium, and causes a change in shape of human umbilical vein endothelial (HUVE) cells. Changes in endothelial cell shape are determined, in part, by changes in the actin cytoskeleton. Gelsolin is an actin-binding protein with the potential to alter the actin cytoskeleton in response to changes in cell calcium and/or changes in polyphosphoinositides. Therefore, we examined the interactions of actin and gelsolin in HUVE cells in which inositol phospholipid metabolism was activated with histamine. In HUVE cells exposed to histamine we estimated actin-gelsolin binding by quantitating actin and gelsolin, immunoprecipitated with anti-gelsolin Sepharose. We estimated the relative amount of filamentous actin in the histamine-exposed HUVE cells by quantitating the amount of actin that was Triton soluble. We also measured the amount of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) in the HUVE cells before and after exposure to histamine. We found that histamine decreased the amount of actin that was immunoprecipitated with gelsolin, decreased the fraction of cell actin that was Triton soluble, and increased PIP and PIP2. These results demonstrate that histamine promotes actin filament formation in HUVE cells and that histamine-mediated changes in actin-gelsolin binding in these cells are better predicted by changes in polyphosphoinositides than by increases in cell calcium.

Effects of actin filaments on fibrin clot structure and lysis.

The muscle and cytoskeletal protein actin is released from cells as a consequence of cell death and interacts with components of the hemostatic and fibrinolytic systems, including platelets, plasmin, and fibrin. We report here that incorporation of actin filaments into fibrin clots changes their viscoelastic properties by increasing their shear modulus at low deforming stresses and by nearly eliminating their tendency to become more rigid with increasing deformation (ie, exhibit strain-hardening). The viscoelastic effects depended on the length of the actin filaments as shown by the effects of the plasma filament-severing protein, gelsolin. Binding of actin to fibrin clots also varied with actin filament length. The plasma actin-binding proteins gelsolin and vitamin D-binding protein reduced, but did not eliminate, the incorporation of actin in the clot. Fluorescence microscopy showed a direct association of rhodamine-labeled actin filaments with the fibrin network. Incubation of clots containing long actin filaments in solutions containing physiologic concentrations of gelsolin (2 mumol/L) released 60% of the actin trapped in the clot. Reduction of the actin content of a fibrin clot by incubation in a gelsolin-containing solution resulted in an increased rate of clot lysis. The ability of plasma gelsolin to shorten actin filaments may therefore be of physiologic and potentially of therapeutic importance insofar as gelsolin-mediated diffusion of actin from the clot may restore the clot's rheologic properties and render it more sensitive to the lytic action of plasmin.

Patients' preferences for learning the results of mammographic examinations.

The communication of diagnostic test results is an important aspect of the interaction between doctors and patients. Communication of mammogram results is of particular interest because the test is used to detect a common and potentially dangerous malignancy and because patients in the United States are able in some locations to obtain mammography at their own request, rather than being referred by a physician. We conducted a survey to learn about the preferences of a group of women at a traditional mammography center for learning the results of this commonly performed test. We asked women undergoing mammography to respond to questionnaires designed to learn: 1) How they felt about different methods of telling patients the results of mammograms; 2) How they were informed of the results of previous mammograms; 3) How they were told the results of the current mammograms. Patients indicated that if no abnormality is detected, they prefer to have their doctor call with the result, but if the study is 'abnormal' they wish to be told by their own physician in the office. Failing to notify the patient if the study is normal was the least preferred outcome. This group of patients did not express an interest in the most immediate form of notification (i.e. learning the result from the radiologist performing the test). Analysis of how patients felt about ways in which they were previously informed of the results of mammograms suggests that their reactions are influenced to a large extent by their clinical status. Patients undergoing mammography for diagnostic purposes, for example, were less pleased by a 'preferred' method (i.e. being told by their physician) than were those undergoing screening mammography. While patients have opinions about how they would prefer to be told their mammogram results, they are accepting a variety of methods of telling, if they are receiving good news. If abnormalities are found, patients prefer to be told in person by their own physician. Interpretations of surveys of patient satisfaction should be tempered by the finding that the clinical status of the patient alters their perceptions of satisfaction with this aspect of their physician's behavior. Patient preferences may change if increasing numbers of women are told their results by the radiologist.

Actin accelerates plasmin generation by tissue plasminogen activator.

Actin has been found to bind to plasmin's kringle regions, thereby inhibiting its enzymatic activity in a noncompetitive manner. We, therefore, examined its effect upon the conversion of plasminogen to plasmin by tissue plasminogen activator. Actin stimulated plasmin generation from both Glu- and Lys-plasminogen, lowering the Km for activation of Glu-plasminogen into the low micromolar range. Accelerated plasmin generation did not occur in the presence of epsilon-amino caproic acid or if actin was exposed to acetic anhydride, an agent known to acetylate lysine residues. Actin binds to tissue plasminogen activator (t-Pa) (Kd = 0.55 microM), at least partially via lysine-binding sites. Actin's stimulation of plasmin generation from Glu-plasminogen was inhibited by the addition of aprotinin and was restored by the substitution of plasmin-treated actin, indicating the operation of a plasmin-dependent positive feedback mechanism. Native actin binds to Lys-plasminogen, and promotes its conversion to plasmin even in the presence of aprotinin, indicating that plasmin's cleavage of either actin or plasminogen leads to further plasmin generation. Plasmin-treated actin binds Glu-plasminogen and t-PA simultaneously, thereby raising the local concentration of t-PA and plasminogen. Together, but not separately, actin and t-PA prolong the thrombin time of plasma through the generation of plasmin and fibrinogen degradation products. Actin-stimulated plasmin generation may be responsible for some of the changes found in peripheral blood following tissue injury and sepsis.

Oncologists vary in their willingness to undertake anti-cancer therapies.

Previous studies have shown that groups of cancer sub-specialists differ in their stated willingness to undergo treatment for diseases lying within their area of expertise. In order to learn whether oncologists feel similarly about other forms of cancer, medical, radiation, and surgical oncologists were asked to fill out a questionnaire indicating whether they would be willing to undergo either chemotherapy or radiation therapy for a variety of common malignancies, or recommend them to a spouse or sibling. Subjects were also asked whether they would undertake an experimental therapy (interleukin-2) for any of three malignancies, or recommend such treatment to a spouse or relative. Fifty-one oncologists (14 radiation oncologists, 14 surgical oncologists, and 23 medical oncologists) were recruited from the staff of four university teaching hospitals. Although they agreed about accepting or declining therapy for some examples, there was considerable heterogeneity in their responses. In only 37% of the 30 cases involving standard therapies did greater than or equal to 85% of the oncologists agree that they would accept or refuse therapy. Only some of the variation of the responses could be attributed to the sub-specialty orientation of the oncologists. Physicians were as willing to recommend standard therapies for themselves as a spouse or sibling. Physicians were also divided in their opinion about whether they would accept a particular experimental therapy if diagnosed with one of three neoplasms. They were significantly more likely, however, to recommend it for a spouse or sibling than to accept it for themselves. Variation in the proportion of patients who receive anti-cancer therapies may relate, in part, to differences in opinion concerning the worth of such therapies among oncologists or primary physicians. This study shows that oncologists are quite heterogeneous with regard to their personal preferences for anti-cancer treatments for a variety of malignancies. Further studies are required to learn if such attitudes (among oncologists or primary physicians) directly affect the administration of such therapies.

Actin is a noncompetitive plasmin inhibitor.

Actin, one of the most abundant cellular proteins, circulates at micromolar concentrations in peripheral blood. Because actin released from dying cells may be trapped in fibrin clots that form at sites of tissue injury, we examined the effects of actin upon lysis of fibrin clots in vitro. Incorporation of native rabbit skeletal muscle actin into fibrin clots slowed their rates of lysis for periods of up to 24 h, an effect not seen when comparable concentrations of human IgG or bovine serum albumin were added instead. Actins isolated from a variety of sources inhibited plasmin's hydrolysis of the synthetic substrate S-2251 in a noncompetitive manner, with a Ki of a 0.6-3.1 microM. Inhibition was rapid, but covalent actin-plasmin complexes were not formed. Both epsilon-aminocaproic acid and tranexamic acid prevented actin's inhibition of plasmin, suggesting that accessible lysine residues of actin interact with the kringle (lysine-binding) regions of plasmin. Neither of the high-affinity actin-binding proteins of plasma (plasma gelsolin and vitamin D-binding protein) prevented actin from inhibiting plasmin. These findings suggest that actin released into the extracellular space following cell death may modulate plasmin action, and hence a number of plasmin-dependent biological responses, at sites of inflammation and tissue injury.