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Modulation of murine macrophages and T lymphocytes by lysozyme dimer.

The effects of lysozyme dimer (2 and 20 microg/kg) administered i.p. once and four times to mice on the phagocytic and killing ability of peritoneal macrophages, interleukin-1 (IL-1) production by murine macrophages stimulated in vitro with lipopolisaccharide of E. coli and expression of thymocyte, splenocyte and mesenteric lymphonode cell CD3+, CD4+ and CD8+ markers were studied. It was found that lysozyme dimer administered once or four times at doses of 2 microg/kg and 20 microg/kg augments the phagocytic and killing activity of peritoneal macrophages. The strongest stimulating effect was noted after four injections of lysozyme dimer at a dose of 20 microg/kg. Moreover, lysozyme dimer is able to modulate the production of IL-1 by murine macrophages stimulated in vitro with LPS. Exposure to four doses of lysozyme dimer (20 microg/kg) enhances the synthesis and release of IL-1, but this drug administered once (2 microg/kg and 20 microg/kg) or four times (2 microg/kg) decreases IL-1 production by peritoneal macrophages. It was also found that administration of lysozyme dimer at a dose of 20 microg/kg, irrespective of the number of doses applied, increases the percentage of CD4+ thymocytes and splenocytes. Moreover, exposure to four doses of lysozyme dimer (2 and 20 microg/kg) increases the percentage of CD4+ and CD8+ mesenteric lymphonode cells.

Animals↗

Prospective assessment of the natural history of positive D-dimer results in persons with acute venous thromboembolism (DVT or PE).

The natural history of initially positive D-dimers for venous thromboembolism is not known. If it returns to negative in the majority of patients, it would be potentially helpful to diagnose a recurrence. In this study, we prospectively measured D-dimer levels in outpatients with a diagnosis of venous thromboembolism. There were a total of 152 patients with an average age of 57. D-dimer results were performed at baseline and repeated at one week, one month and three months. At baseline 120 of 152 (79%) had a positive D-dimer result. Of those with an initially positive result, 80% were still positive at one week and 39% were still positive at one month. Finally at three months, 13% remained positive. Seven patients had recurrent events and all had persistently elevated D-dimers at one month. This study suggests that a persistently positive D-dimer result after one month of treatment may indicate a higher risk of recurrent venous thromboembolism. D-dimer testing for the diagnosis of recurrence of venous thromboembolism deserves further study.

Adult↗

An active covalently linked dimer of human interferon-gamma. Subunit orientation in the native protein.

We have constructed and expressed a covalently linked head to tail dimer of human interferon-gamma (IFN-gamma) in which two monomers are joined head to tail via a rigid peptide hinge using genetic engineering techniques. The hinge was derived from the human immunoglobin IgA1 sequence (Hallewell, R.A., Laria, I., Tabrizi, A., Carlin, G., Getzoff, E.D., Tainer, J.A., Cousens, L.S., and Mullenbach, G.T. (1989) J. Biol. Chem. 264, 5260-5268). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the polypeptide produced by this construction migrates as a 30,000 polypeptide species. The protein elutes as a single species by molecular sieve chromatography under native conditions. The covalently linked dimer exhibits one-half the antiviral activity of native dimeric IFN-gamma; receptor binding assays show the covalently linked dimer binds to the IFN-gamma receptor with one-half the avidity of native IFN-gamma. This difference is not due to conformational differences between the two molecules, as the aromatic region of the NMR spectrum of the purified covalently linked dimer is identical with that of the wild type protein. From these data, we suggest that human IFN-gamma associates in a head to tail dimer in its active configuration. Regions of IFN-gamma are contiguous with the amino and carboxyl termini and are obscured by the hinge peptide in the covalently linked dimer. Our studies demonstrate that these regions may be important for receptor-ligand interaction.

Amino Acid Sequence↗

[D-dimer analysis in diagnosis of venous thromboembolism is reliable].

Analysis of D-dimer as an alternative to costly radiological examinations for excluding venous thromboembolism (VTE) remains controversial. In part this appears due to varying analytical performance of different D-dimer assay methods. We have evaluated a D-dimer method from BioPool, Sweden. Samples for D-dimer analysis were obtained from 86 consecutive patients admitted to the emergency unit at the Central Hospital in Kristianstad, Sweden, for symptoms consistent with venous thromboembolism. The result of the analysis was withheld for the duration of the study. In 21 patients a diagnosis of VTE was returned based upon radiological examination; 20 of these had D-dimer levels above the cut-off limit. The sensitivity for VTE using D-dimer was 95 per cent and the negative predictive value was 97 per cent. We conclude that D-dimer analysis in conjunction with a clinical investigation is helpful in excluding VTE in this category of patients.

Adult↗

[Fibrinolysis parameters: plasmin-alpha2 antiplasmin complexes (PAP) and D-dimers in acute coronary syndromes without ST segment elevation].

The aim of the paper was to determine concentrations of plasmin-alpha2 antiplasmin complexes (PAP) and D-dimers in blood of patients suffering from acute coronary syndromes without ST segment elevation. 78 patients, including 37 females and 41 males, aged 41-79 years (mean age 63.2) were included in the study. Concentrations of PAP were determined by immmunoenzymatic method using Enzygnost PAP micro kit and concentrations of D-dimers using Dade Berling Enzygnost D-Dimer micro kit. Statistically significant, higher concentrations of PAP and D-dimers, indicating increased fibrinolytic activity of blood, were observed in patients with acute coronary syndrome than in the control group. Significantly higher concentrations of D-dimers were found in the group of patients above the age of 50, when compared with younger patients. Lower D-dimers levels were observed in smokers. Gender, coexisting hypercholesterolaemia, arterial hypertension and diabetes had no impact on concentrations of PAP and D-dimers in blood of patients suffering from acute coronary syndrome without ST segment elevation.

Acute Disease↗

Elevated levels of D-dimer and fragment 1+2 upon central venous catheter insertion and factor V Leiden predict subclavian vein thrombosis.

BACKGROUND AND OBJECTIVES: Subclavian vein thrombosis is a well-recognized complication following central venous catheter insertion. We studied whether the determination of D-dimer levels, fragment 1+2 levels and factor V Leiden can identify patients at high risk of developing subclavian vein thrombosis. DESIGN AND METHODS: The presence of central venous catheter associated thrombosis was analyzed in 235 patients undergoing allogeneic bone marrow transplantation, of whom 30 (13%) developed thrombosis. A case-control study was performed with 30 patients matched for age, gender, and type of transplantation who did not develop thrombosis. Blood was sampled 3-5 days after catheter insertion. D-dimer levels were determined using a latex microparticle assay and an enzyme linked immunosorbent assay (ELISA). An ELISA was used to determine fragment 1+2 levels. The factor V genotype was determined by polymerase chain reaction. RESULTS: The levels of D-dimer and fragment 1+2 were significantly elevated in the patients who developed thrombosis. Five patients tested positive for factor V Leiden and all 5 developed subclavian vein thrombosis. Patients with high D-dimer levels (> 1300 microg/L measured by latex agglutination and >350 microg/L measured by ELISA) had a 7.0 and 6.0 times higher risk of developing subclavian vein thrombosis, respectively. A 5.5-fold increased risk of thrombosis was observed in patients with a fragment 1+2 level higher than 1.300 nmol/L. This resulted in positive predictive values of 0.78, 0.80 and 0.83 for the fragment 1+2, D-dimer and D-dimer latex agglutination assays, respectively. The accompanying negative predictive values were 0.39, 0.40 and 0.42, respectively. INTERPRETATION AND CONCLUSIONS: We conclude that the measurement of D-dimer and fragment 1+2 levels after central venous catheter insertion, as well as factor V Leiden determination, can be used to identify patients at high risk of developing symptomatic subclavian vein thrombosis.

Adult↗

[The changes and their clinical significance of D-dimer and platelet glycoprotein in patients with coronary heart disease].

OBJECTIVE: To explore the changes and their clinical significance of D-dimer and platelet glycoprotein (GP) in patients with coronary heart disease. METHODS: D-dimer and GP in 20 patients with stable angina (SA group), 48 patients with unstable angina (UA group), and 20 control cases were measured. The changes of D-dimer and GP in patients with and without coronary events were compared. The sensitivity of those changes in the diagnosis of coronary events was evaluated. RESULTS: There were significant differences of D-dimer and GP between UA group and SA group or control group (P < 0.01), while there was no significant difference between SA group and control group (P > 0.05). There were also significant differences of D-dimer and GP between patients with coronary events and patients without coronary events (P < 0.05). In the sensitivity test for detecting coronary events, D-dimer and GPIIb, GPIIIa were much more sensitive than other parameters. CONCLUSIONS: D-dimer and GPIIb, GPIIIa may be regarded as the indexes of coronary thrombosis and used for predicting the severity of coronary events.

Aged↗

Characterization of the interaction between the A2 subunit and A1/A3-C1-C2 dimer in human factor VIIIa.

Factor VIIIa is a heterotrimer of the factor VIII heavy chain-derived A1 and A2 subunits plus the factor VIII light chain-derived A3-C1-C2 subunit. While the A1 and A3-C1-C2 subunits can be isolated as a stable dimer, the A2 subunit is weakly associated with the dimer. In the human protein, the association of A2 with dimer is reversible and governed by a pH-dependent dissociation constant. Using the specific activity of factor VIIIa as an indicator of trimer concentration, the Kd (pH 6.0) was determined to be 28 nM whereas at the more physiologic pH (pH 7.4) this value was approximately 260 nM. Results from pH shift experiments confirmed the reversible binding of A2 to dimer as did the capacity for high levels of exogenous A2 subunit to inhibit the spontaneous decay of factor VIIIa activity. A2 subunit associated with the A1 subunit in the A1/A3-C1-C2 dimer based upon the capacity for free A1 subunit to inhibit the reconstitution of factor VIIIa from A2 subunit and dimer. These results indicate that the primary mechanism for the spontaneous decay of human factor VIIIa is the reversible dissociation of A2 subunit from the A1 subunit of the A1/A3-C1-C2 dimer.

Electrophoresis, Polyacrylamide Gel↗

[Negative D-dimers and exclusion of venous thromboembolism--own experience].

The assessment of D-dimer concentration has become essential step during diagnostic algorithm of venous thromboembolism (VTE). This test characterizes high sensitivity but limited specificity. Negative D-dimer with high probability excludes VTE. The aim of this study was to assess the percentage of patients treated in Department of Internal Medicine, Endocrinology and Haemostatic Disorders, Medical University of Gdańisk, who in spite of clinical signs of VTE showed normal D-dimer level. Between 2000 and 2004 in our department 57 cases with recent deep vein thrombosis (DVT) were diagnosed, in 2 cases with co-existence of pulmonary embolism (PE). The D-dimer concentration was assessed in patients' plasma with the use of immunoturbidometry. Between 57 cases with VTE, 7 patients (12%) showed normal D-dimer level (<500 microg/ml). This group consisted of 4 men and 3 women, aged from 40 to 82 years (the mean age of 58 years). In all 7 cases DVT was diagnosed, in 2 patients with concomitent PE. The final diagnosis was confirmed by compression ultrasonography and pulmonary scintigraphy. Our analysis underlines the observation that occurrence of VTE and negative d-dimer concentration is possible and may probably be related to methodological limitations. However, the lack of increase of D-dimer could also be caused by fibrinolysis alteration.

Adult↗

[Preparation and characterization of McAbs against the human crosslinked fibrin degradation product D-dimer].

D-dimer is a specific degradation product of crosslinked fibrin hydrolyzed by plasmin. Detection of D-dimer in blood is important for the diagnosis of thrombotic diseases and the surveillance of thrombolytic treatment. Therefore, D-dimer purification and preparation of anti-D-dimer McAb are necessary tasks. Using routine methods, we prepared four hybridomas which secrete anti-D-dimer McAb: HID1, HID2, HID3 and HID4. After five months in culture these hybridomas still secrete anti-D-dimer McAb. The titer of ascites McAb is 1:10(-5)-1:10(-6). HID1, HID2, and HID3 recognize the same or similar antigenic determinants and don't cross-react with coagulation factors, plasma or fibrinogen degradation products X, Y, D, and E fragments. HID4 recognizes another antigenic determinant and cross-reacts with fibrinogen degradation products X, Y and D fragments. Immunoblot results show that all of these McAbs react with a MW 190,000 band. These antibodies are being used in the development of enzyme-linked and latex immunoassay methods for the detection of D-dimer and will be useful in the diagnosis, evaluation and monitoring of thrombotic diseases and thrombolytic treatment.

Animals↗

[Study on cases of D dimer values were dissociated from FDP-E].

Determination of FDP D-dimer (D-dimer) has been recently developed for the diagnosis of thrombotic diseases with secondary fibrinolysis. We have studied the correlation between D-dimer and FDP-E concentrations in plasma from 282 patients with 630 samples. A linear correlation (r = 0.9269) was observed between the values of FDP-E and D-dimer. However, 13 out of 282 cases revealed an apparent dissociation of D-dimer concentrations from FDP-E values. Among them, 4 of these 13 cases (Group A) have shown to possess higher level of D-dimer when compared with the expected values from FDP-E, while 9 of 13 cases (Group B) revealed lower levels of D-dimer than that expected from FDP-E. All of Group A patients have been diagnosed as disseminated intravascular coagulation (DIC). On the other hand, in Group B patients, 6 of 9 were shown to have a widespread metastasis of cancer and 2 of them were under treatment with urokinase. To study whether Group B patients were under hypercoagulable or hyper-fibrinolytic state, we have examined ratios of AT III/alpha 2 PI and PIC/TAT in these cases. It has been shown that 4 of 9 patients in Group B have higher ratios of both AT III/alpha 2 PI and PIC/TAT if compared with other patients than Group B. This suggests that patients in Group B have been under hyper-fibrinolytic states.

Adult↗

[The role of D-dimer in diagnosis of ovarian cancer].

One of fibrin degradation products, D-dimer was measured in ovarian cancer (N = 28), benign ovarian tumors (N = 26), benign uterine tumors (N = 15) and normal controls (N = 66). The D-dimer value for ovarian cancers was 721 +/- 423 ng/ml, benign ovarian tumors; 299 +/- 248, benign uterine tumors; 248 +/- 141, and normal controls; 237 +/- 212, respectively. There was a significant difference between the ovarian cancer group and the other groups (p less than 0.01). When the cut off level of D-dimer was less than 400 ng/ml, the positive rate for D-dimer was 50% in stage I of ovarian cancer, 88% in stage II, 86% in stage III and 80% in stage IV. Overall, 82% of ovarian cancer patients were positive. D-dimer provides good sensitivity compared with other ovarian cancer markers such as TPA, SLX and CEA. The coefficient correlation between D-dimer and CA125, TPA, SLX or CEA was 0.476, 0.376, 0.226, -0.292, respectively. This suggest that the measurement of D-dimer is useful in diagnosing ovarian cancer.

Biomarkers, Tumor↗

Functional studies with the octameric and dimeric form of mitochondrial creatine kinase. Differential pH-dependent association of the two oligomeric forms with the inner mitochondrial membrane.

Phosphate extraction of mitochondrial creatine kinase (Mi-CK, EC 2.7.3.2) from freshly isolated intact mitochondria of chicken cardiac muscle, after short swelling in hypotonic medium, yielded more than 90% of octameric and only small amounts of dimeric Mi-CK as judged by fast protein liquid chromatography-gel permeation analysis of the supernatants immediately after extraction of the enzyme. In extraction buffer, octameric Mi-CK displayed a tendency to dissociate, albeit at a slow rate with a half-life of approximately 3-5 days, into stable dimers. Experiments with purified Mi-CK octamers or dimers, or defined mixtures thereof, incubated under identical conditions with Mi-CK-depleted mitoplasts revealed that both oligomeric forms of Mi-CK can rebind to mitoplasts. However, the association of Mi-CK was strongly pH-dependent and, in addition, octameric and dimeric Mi-CK showed different pH dependences of rebinding. Therefore, it was possible under certain pH conditions to rebind either both oligomeric forms or selectively the octamers only. Furthermore, evidence is presented that Mi-CK dimers partially form octamers upon rebinding to the inner membrane. The differential association of the two oligomeric Mi-CK forms with the inner mitochondrial membrane together with the dynamic equilibrium between octameric and dimeric Mi-CK (Schlegel, J., Zurbriggen, B., Wegmann, G., Wyss, M., Eppenberger, H.M., and Wallimann, T. (1988) J. Biol. Chem., 263, 16942-16953) suggest that both oligomeric forms are physiologically relevant. A change in the octamer to dimer ratio may influence the association behavior of Mi-CK in general and thus modulate mitochondrial energy flux as discussed in the phosphoryl creatine circuit model (Wallimann, T., Schnyder, T., Schlegel, J., Wyss, M., Wegmann, G., Rossi, A.-M., Hemmer, W., Eppenberger, H.M., and Quest, A.F.G. (1989) Prog. Clin. Biol. Res. 315, 159-176.

Animals↗

Acceleration of tetramer formation by the binding of inositol hexaphosphate to hemoglobin dimers.

The aggregation of deoxyhemoglobin dimers was studied by dropping the pH of a dilute solution of deoxyhemoglobin originally at high pH. In the presence of inositol hexaphosphate, a sharp increase in the rate of dimer association was observed. At higher concentrations of the phosphate, the rate decreased to a value close to that seen in the absence of phosphate. These observations require that inositol hexaphosphate binds to deoxyhemoglobin dimers. The dependence of the aggregation rate on phosphate concentration occurs because the reaction of a dimer containing bound phosphate with a phosphate-free dimer is 30 to 50 times faster than either the association of phosphate-free dimers or the association of dimers both containing bound phosphate.

Hemoglobins↗

Dimerization of B-type platelet-derived growth factor receptors occurs after ligand binding and is closely associated with receptor kinase activation.

Platelet-derived growth factor (PDGF) was found to induce dimerization of purified B-type PDGF receptors, as analyzed by sodium dodecyl sulfate gel electrophoresis after covalent cross-linking using disuccinimidyl suberate. PDGF-BB was 20-fold more effective than PDGF-AB; PDGF-AA was without effect. The dimerization was dose-dependent and was maximal at 0.5-2 micrograms/ml PDGF-BB; at higher concentrations dimerization was less abundant. This indicates that dimerization occurred when one PDGF-BB molecule bound two receptor molecules. The dimerization correlated to activation of the tyrosine kinase of the receptor, determined as autophosphorylation, but was not dependent on phosphorylation reactions because it occurred also in the absence of ATP. Furthermore, dimerization of the receptor correlated with the ability to phosphorylate phosphofructokinase, an exogenous substrate. The complex of ligand and receptor dimer was stable; it resisted electrophoresis under nondenaturing conditions, as well as gel chromatography. The present data indicate that intermolecular mechanisms are involved in signal transduction from the external ligand binding domain to the internal effector domains of the B-type PDGF receptor.

Animals↗

Use of the fibrin D-dimer in screening for coagulation abnormalities in preeclampsia.

Plasma from pregnant women with preeclampsia was screened for the D-dimer, a degradation product specific for crosslinked fibrin, using a monoclonal antibody (DD-3B6), latex-bead agglutination assay (DIMERTEST). Seventy-nine of 204 (38.7%) of the preeclamptic women and none of 88 healthy non-preeclamptic women were positive for the D-dimer. Presence of the D-dimer correlated consistently with elevated fibrin(ogen) degradation products, detectable fibrin monomer, and platelet count less than or equal to 100 x 10(9)/L, with a 93.0% sensitivity overall for abnormalities of the same. Among D-dimer-positive women, 66.7% had fibrin(ogen) degradation products less than 10 micrograms/mL, 60.3% had no detectable fibrin monomer, and 82.0% had platelets greater than 100 x 10(9)/L. When compared with D-dimer-negative preeclamptic women, D-dimer-positive women had significantly higher blood pressures prompting delivery, greater proteinuria, more abnormal liver function tests, and higher serum creatinine and blood urea nitrogen. In addition, they had a greater risk of cesarean section (49.4 versus 34.4%), premature delivery (58.2 versus 20.0%), low birth weight (54.5 versus 20.3%), low Apgar scores, and an abnormally high ratio of female to male offspring (1.72 versus 0.93). Testing for the fibrin D-dimer may be useful in early screening and follow-up for preeclamptic coagulopathy, and may also help to define the subset of patients with severe disease.

Adult↗

Cytostatic activity of naturally isolated isomers of secalonic acids and their chemically rearranged dimers.

Six different secalonic acids were tested for cytostatic activity against cultured mouse leukaemia L1210 cells. Secalonic acids B and E showed rather weak activity but the other four isomers showed quite strong activity, especially secalonic acids A and D which were slightly more active than adriamycin. The chemically rearranged 2,4'-dimer of secalonic acid A showed almost the same activity as the naturally isolated 2,2'-dimer, but the activity of the 4,4'-dimer was much stronger than that of both the 2,2'-dimer and adriamycin. Interestingly, in a mouse bone marrow stem cell assay, secalonic acids F and G showed almost the same toxicity as adriamycin, but secalonic acids A and D showed rather weak toxicity. Similarly, the 2,2'-dimer of secalonic acid A showed almost the same toxicity as adriamycin, but the toxicity of the 2,4'- and 4,4'-dimers was weaker than that of the 2,2'-dimer and also that of adriamycin.

Animals↗

Dissociation of nucleosomal particles by chemical modification. Equivalence of the two binding sites for H2A.H2B dimers.

Treatment of nucleosomal particles with dimethylmaleic anhydride, a reagent for protein amino groups, is accompanied by a biphasic release of histones H2A plus H2B; one H2A.H2B dimer is more easily released than the other. This behavior allows the preparation of nucleosomal particles containing only one H2A.H2B dimer, which were complemented with 125I-labeled H2A.H2B. These reconstituted particles, which contain one labeled and one unlabeled H2A.H2B dimer, were treated with the amount of reagent needed to release one of the two H2A.H2B dimers. Radioactivity was equally distributed between residual particles and released proteins, which is consistent with equivalent binding sites in the nucleosomal particle for H2A.H2B dimers, rather than with intrinsically different sites. The asymmetric release of H2A.H2B dimers would be caused by a change in the binding site of one dimer following the release of the other. This behavior might be related to the structural dynamics of nucleosomes.

Animals↗