Fibrinogen--proteolysis in acute myelogenous leukemia (AML)

Fibrinopeptides were measured as direct indices of thrombin, plasmin and elastase in plasma samples obtained from patients with AML. Peptide patterns observed were consistent with spontaneous or drug induced plasmin-specific fibrinogenolysis (AML FAB M 1/3), elastase mediated proteolysis (AML FAB M 3/4) or DIC (AML FAB 4/5). DIC was also observed in septic, agranulocytotic patients.     

TdT expression in acute myeloid leukaemia. Haemopoietic immaturity or maturational asynchrony?

A total of 412 cases of acute leukaemia were examined for the presence of nuclear terminal deoxynucleotidyl transferase (TdT) by indirect immunofluorescence. Of the 129 cases of acute myeloblastic leukaemia (AML FAB groups M1/M2) examined, 18% (n = 23) had significant proportions (greater than 10%) of TdT-positive blasts. Although most of these AML cases (n = 18) were of poorly differentiated (M1) type; 5 cases of AML showing features of granulocytic differentiation (M2) were also found to be TdT-positive. Even though TdT was generally more strongly expressed in the M1 group and associated with other markers of myeloid immaturity (Ia positive and lack of chloroacetate esterase), there was no inverse relationship with Sudan black or myeloperoxidase activity. In addition, although the proportion of AML-M1 cases with increased TdT-positive cells was slightly higher (18/95, 19%) than for the AML-M2 group (5/34, 15%) the results suggest that the presence of nuclear TdT in leukaemic myeloblasts may not only reflect cellular immaturity but may also be due to maturational asynchrony in otherwise well-differentiated blasts.     

Granulocyte proteinases as mediators of unspecific proteolysis in inflammation: a review

In severe inflammatory response, various blood and tissue cells, including polymorphonuclear granulocytes, release lysosomal proteinases, extracellularly and into the circulation. Such enzymes, as well as normally intracellular oxidizing agents produced during phagocytosis, enhance the inflammatory response by degrading connective tissue structures, membrane constituents and soluble proteins by proteolysis or oxidation. We first used polymorphonuclear elastase (E) as a marker of such release reactions. The liberated proteinase competes with susceptible substrates, including alpha 1-proteinase inhibitor (alpha 1PI) and alpha 2-macroglobulin, and is eliminated finally as inactive enzyme-inhibitor complexes by the reticulo-endothelial system. Using an enzyme-linked immunosorbent assay, we determined the plasma levels of E-alpha 1PI following major abdominal surgery, multiple trauma and pancreatogenic shock. Whereas the operative trauma was followed by up to 3-fold increase of the E-alpha 1-PI, postoperative septicemia was associated with a 10 to 20 fold increase. The increase of E-alpha 1-PI and a concomitant decrease of plasma factors, such as antithrombin III, clotting factor XIII and alpha 2-macroglobulin, were correlated. Multiple trauma causes a substantial increase of E-alpha 1-PI up to 14 hours after accident. The released elastase seems to correlate with severity of injury, but assessing the relationship to consumption of plasma factors is complicated by concomitant transfusions. In acute pancreatitis, peaks, of E-alpha 1-PI coincide with a massive consumption of antithrombin III and alpha 2-macroglobulin during shock.     

Plasma transcobalamins in haematological disorders

Plasma UBBC-B12 and transcobalamins were measured in 112 patients suffering from different haematological disorders. The data showed different patterns of changes in plasma transcobalamin profile in different haematological disorders. Plasma UBBC-B12 and transcobalamins were significantly higher than normal in untreated chronic myeloid leukaemia, acute promyelocytic leukaemia, nutritional megaloblastic anaemia and in refractory anaemias with hypercellular marrow. Normal levels of these proteins were noted in chronic lymphatic leukaemias, in primary and secondary hypereosinophilic states and in multiple myeloma. Subnormal levels of these proteins were observed in hypoplastic anaemia and acute lymphoblastic leukaemia. Chronic myeloid leukaemia patients during blast crisis and acute myeloid leukaemia patients except those suffering from acute promyelocytic leukaemia showed varying pattern of plasma transcobalamins depending on type of blast crisis or FAB subtype of AML. The significance of these changes in plasma transcobalamins have been discussed along with the experience of other workers in this field.     

Spastic paralysis of the lower extremities as the first symptom of acute myeloblastic leukemia]

A case of acute myeloblastic leukemia (AML) with spastic paralysis of the lower extremities caused by a tumor of the spinal cord as the first symptoms of the disease is presented. The tumor consisted of leukaemic cells. A diagnosis of AML type M2, according to FAB classification, was established. A complete remission was achieved after 2 courses of chemotherapy. Patient started to walk after intensive rehabilitation. After 14 months of complete remission, recurrence was observed despite an intensified therapy.     

Immunoreactive calcitonin in leukaemia.

A radioimmunoassay was used to measure concentrations of immunoreactive human calcitonin (HCT) in plasma and leucocytes from patients with various leukaemic and myeloproliferative disorders. Plasma immunoreactive HCT concentrations were increased in 32 out of 33 patients with chronic granulocytic leukaemia (CGL) and in all eight patients with acute myeloid leukamia (AML) at presentation or in relapse. Out of 11 patients with other myeloproliferative disorders, eight had increased plasma immunoreactive HCT concentrations. Buffy-coat-cell extracts and culture media from peripheral leucocytes of patients with CGL also contained increased immunoreactive HCT concentrations. In contrast, plasma from patients with chronic lymphocytic leukaemia, acute lymphoblastic leukaemia, and AML in remission had low or undetectable immunoreactive HCT concentrations. Increased plasma and cellular concentrations of immunoreactive HCT may be a consequence of abnormal proliferation of myeloid cells and might prove to be valuable in predicting relapse in patients with myeloid leukaemias.     

Is fatal pancreatitis a consequence of excessive leukocyte stimulation? The role of tumor necrosis factor alpha

This study examines the possible role of excessive leukocyte stimulation in the pathogenesis of acute pancreatitis. Levels of tumor necrosis factor alpha (TNF-alpha) and elastase-alpha 1 proteinase inhibitor (E-alpha 1PI) have been measured as markers of macrophage and neutrophil activation, respectively, in serial plasma samples from 27 patients with acute pancreatitis. Levels of TNF-alpha did not support the possibility of excessive macrophage activation, but raised levels of E-alpha 1PI in patients with either the mild or severe form of the illness indicated the activation of neutrophils. Whether this is a consequence of the illness or is a contributory factor in the pathogenesis is not clear.     

The pathomorphological diagnosis of acute leukaemias: cytology, cytochemistry and immunocytology

In 74 cases of acute leukaemia and of the blastic phase of chronic granulocytic leukaemia (CML), monoclonal antibodies of the "VI" series and a few commercially available antibodies proved to be of valuable help in establishing a definite diagnosis. In 4 out of 35 cases of AML (FAB-M 1-5), and in 2 out of 16 of the blastic phase of CML only the use of monoclonal antibodies secured the diagnosis. In the group of acute lymphoid leukaemias subtypes corresponding to various levels of differentiation, were defined. The blasts of 3 patients out of the 74 did not express any of the markers studied. Two additional cases were investigated for platelet peroxidase and studied with the antiplatelet antibodies VIPL 1, 2 and 3 by the electron microscope. These cases proved to be acute megakaryoblastic leukaemias (acute myelofibrosis).     

Immunophenotyping of acute myeloid leukaemia: relevance of analysing different lineage-associated markers

The immunophenotype of 135 previously untreated patients with FAB defined acute myeloid leukaemia (AML) was studied at diagnosis. The panel of reagents included monoclonal antibodies (MoAb) recognising myeloid-associated determinants (CD11, CD13, CD14, CD33 and others) as well as MoAb directed towards lymphoid antigens (CD7, CD10, CD19) and TdT. The results indicate that CD13 and/or CD33 are consistently expressed in AML and only rarely in ALL blasts (131/135 + ve cases, versus 4/130 in ALL). Lymphoid antigen expression was rarely detected when CD10 and CD19 were investigated in AML (0.9% and 2% + ve cases, respectively), whereas significant positivities were found for TdT and CD7 (20% and 10% respectively). Concerning FAB subtypes, two new MoAb (LAM3 and LAM7) proved very useful in the specific recognition of AML with monocytic features. The phenotype CD13+ and/or CD33+, CD9+, HLA-DR- was found to be almost exclusive for M3 AML. The response to induction chemotherapy was analysed in CD7+ and in TdT+ patients. In the latter group a statistically significant lower response rate was found with respect to TdT-ve-AML patients.     

Human leukaemia-associated antigens expressed by acute myelocytic leukaemia cells and their detection by heterologous antisera.

Antisera against human acute myelocytic leukaemias were tested in complement-dependent in-vitro cytotocity tests against leukaemia cells and normal cells as targets. After absorption with erythrocytes and spleen cells from allogeneous donors the antisera reacted with leukaemia cells, but not with leukocytes from bone marrow and the peripheral blood of children in remission, lymphocytes from healthy donors, enriched B-lymphocytes, enriched T-lymphocytes, PHA-induced blasts and cord blood lymphocytes. Extensive cross reactions were obtained in the tests against leukaemia cells. The antisera reacted not only with AML cells, but also with ALL, CLL, and CML cells. It was possible to remove the cross-reactivity with ALL cells through absorption with ALL cells or with fetal tissue, and to remove the cross reactivity with CLL cells through absorption with CLL. A complete absorption of the anti-AML sera was possible with AML and CML cells. After absorption with fetal tissue and CLL cells the antisera showed exclusively specificity for myelocytic leukaemias. Thus, AML cells contain three leukaemia-associated membrane antigen components: an antigen of fetal origin, a "CLL-specific" antigen, and an antigen that occurs on myelocytic leukaemias.     

All-trans retinoic acid induces apoptosis in acute myeloblastic leukaemia cells

The effect of all-trans retinoic acid (ATRA) on leukaemia cell differentiation, proliferation and induction of apoptosis was studied by using autonomously growing blast cells isolated from eight patients with acute myeloblastic leukaemia (AML) either at diagnosis ( n=4) or at relapse (n=4). No morphological or functional differentiation induced by ATRA was observed in any of the cases studied. In cell cultures, inhibition of leukaemia cell growth by ATRA was obvious, especially in the case of clonogenic cells, and it was both time- and concentration-dependent. Induction of apoptosis was more difficult to achieve. The cells retained over 90% viability in suspension when the ATRA exposure at any of the concentrations studied was 48 h or less. When the time of exposure to ATRA was longer than 48 h, the viability of the cells decreased in a concentration-dependent manner. Apoptosis was observed morphologically in each of the AML cases with 10-5 to 10-8 M ATRA, if the incubation time of cells in ATRA was 72 h. The percentage of apoptotic cells increased with increasing ATRA concentrations from 12± 9% of 10-8 M ATRA to 78±12% of 10-5 M ATRA. The DNA electrophoretic method was able to detect apoptosis in all the AML samples exposed to 10-7 and 10-6 ATRA for 48 h and occasionally in cases where lower concentrations and longer exposure time were used. In conclusion, the present study shows that it is possible to induce apoptotic leukaemia cell death in vitro with ATRA in AML, and this effect is dependent on both concentration and exposure time.     

Determination of 5-chloro-7-iodo-8-quinolinol (clioquinol) in plasma and tissues of hamsters by high-performance liquid chromatography and electrochemical detection

This paper describes a method of determining clioquinol levels in hamster plasma and tissue by means of HPLC and electrochemical detection. Clioquinol was separated on a Nucleosil C18 300 mm x 3.9 mm i.d. 7 microm column at 1 ml/min using a phosphate/citrate buffer 0.1M (400 ml) with 600 ml of a methanol:acetonitrile (1:1, v/v) mobile phase. The retention times of clioquinol and the IS were, respectively, 11.6 and 8.1 min; the quantitation limit (CV>8%) was 5 ng/ml in plasma and 10 ng/ml in tissues. The intra- and inter-assay accuracies of the method were more than 95%, with coefficients of variation between 3.0 and 7.7%, and plasma and tissue recovery rates of 72-77%. There was a linear response to clioquinol 5-2000 ng/ml in plasma, and 10-1000 ng/g in tissues. The method is highly sensitive and selective, makes it possible to study the pharmacokinetics of plasma clioquinol after oral administration and the distribution of clioquinol in tissues, and could be used to monitor plasma clioquinol levels in humans.     

Cell markers in the recognition of acute myeloblastic leukaemia subtypes

The diagnosis of acute myeloblastic leukaemia (AML) is based on cell morphology, cytogenetic and molecular changes, cell markers and clinical data. Our aim was to establish whether morphology and cell markers are comparable in the evaluation of AML. Bone marrow smears were analysed, and flow cytometry and monoclonal antibodies were used to determine cell type and maturity. Morphology and cell markers correlated differently in different AML subtypes.     

Activation of plasma Hageman factor and kallikrein in ongoing allergic reactions in the skin

Ten atopic subjects, sensitive to intradermal injection of less than or equal to 10 protein nitrogen units of ragweed or grass pollen antigen, underwent paired antigen and buffer skin chamber incubation over the base of denuded skin blisters. The chamber fluids were sampled over a 6-hr period for histamine and activated Hageman factor and plasma kallikrein which were complexed to C1 inhibitor. In 9 of 10 subjects significantly (p less than 0.01) increased histamine levels (74 +/- 11 ng/ml vs 1.5 +/- 0.55 ng/ml) and kallikrein-C1 inhibitor complexes (2.15 +/- 0.78 ng/ml/hr vs 0.51 +/- 0.09 ng/ml/hr, p less than 0.25) were detected at antigen sites compared with buffer sites, respectively. Increased levels of activated Hageman factor (ng/ml/hr) were detected at antigen sites (1.35 +/- 0.60) compared with buffer sites (0.11 +/- 0.05), (p less than 0.01), in 8 of 10 subjects. Whereas peak levels of histamine were obtained after 1 hr of challenge, both Hageman factor and kallikrein activation, as assessed by complex formation, tended to peak later from the 2nd to the 5th hr. This represents the first demonstration that cutaneous IgE-mediated allergic responses are associated with local activation of the intrinsic plasma coagulation-kinin pathways.     

The dual nature of the reaction between porcine elastase and human plasma alpha1 proteinase inhibitor.

Only a portion of human plasma α₁ proteinase inhibitor (α₁PI) forms a 1:1 complex with porcine elastase; the other portion is inactivated via proteolysis. High temperature (37°) and high salt (2 M) enhance complex formation. The complex is unstable, but no significant liberation of active elastase could be demonstrated. Probably the same two major products of ～50,000 and ～4,000 daltons are formed from α₁PI via proteolysis and via disintegration of the complex. Iodination of α₁PI or oxidation with chloramine-T prevents complex formation with elastase but not with trypsin. Iodinated elastase, however, forms a complex with α₁PI.     

Comparison of the cytomorphological classification with the cytochemical differentiation of acute lymphatic leukemia in children]

In 100 children with acute lymphatic leukaemia the cytomorphological subclassification of the pathological cell type was made according to Mathé and the French-American-British Co-operative group (FAB). In addition, all cases of leukaemia were differentiated according to their cytochemical type. Lymphoblasts from 10 cases of leukaemia could be subclassified immunologically. From 71 children will ALL the survival rates of those cases of leukaemia subclassified cytomorphologically and the cytochemical reactions were compiled and partially compared. Microlymphoblastic leukaemia could be found to be the most frequent type of ALL at children's age. Prolymphocytic leukaemias were characterized by a favourable survival rate and the highest percentage of ALL with the PAS type. Macrolymphoblastic and microlymphoblastic cases of leukaemia revealed no essential differences of survival rate, but significant differences of cytochemical reactions.     

Diurnal variations of plasma testosterone in men

Plasma testosterone (T) levels were assayed by a Competitive Protein Binding (CPB) technique in a group of 31 healthy males. In 22 subjects a single blood sample was taken between 8:00 and 9:00 A.M. and the mean T concentration was 6.84 ± 2.11 ng/ml. In the other 9 normal men, blood samples were taken every 4 hours. The existence of temporal variations for testosterone was confirmed by finding the highest mean plasma levels at 4:00 A.M. (9.28 ± 1.17 ng/ml) and lowest mean levels at 8:00 P.M. (2.66 ± 0.52 ng/ml).     

Elastase-type activity of human serum. Its variation in chronic obstructive lung diseases and atherosclerosis

Human serum was found to contain enzyme activities hydrolyzing succinyl trialanine paranitroanilide and 3H-kappa-elastin Sepharose substrates. Both types of activities could be partly abolished by serine active site titrants (phenylmethanesulfonylfluoride, diisopropylphosphorofluoridate) and partly by neutral chelating agents (EDTA; 1-10-phenanthroline). The combination of phenylmethanesulfonylfluoride and EDTA gave a complete inhibition of human serum elastase-type activities indicating the presence of at least two different types of elastases (serine and metalloproteases) in human serum. In nonsmokers, the average serum elastase-type activity on succinyl trialanine paranitroanilide was found equal to 78.1 ng/ml porcine pancreatic elastase equivalents and on 3H-kappa-elastin sepharose beads equal to 688.8 ng/ml. No statistically significant differences were observed in elastase levels in the sera of individuals presenting clinical symptoms of atherosclerosis. The sera of patients suffering from chronic obstructive lung diseases contained, however, higher amounts of elastase-type activities, respectively equal to 237.2 ng/ml on succinyl trialanine paranitroanilide and 1,096 ng/ml on 3H-kappa-elastin Sepharose beads and was quantitatively significant when compared with control subjects.     

Leukocyte lysozymes in various forms of leukemia]

The amount of lysozyme in the leukocytes of 47 patients with different forms of leukaemia and 6 healthy persons was investigated. The lysozyme determination was carried out in the lysate of isolated leukocytes obtained after freezing and thawing it seven times. The results expressed in microgram per 10(6) cells were compared with the simultaneously determined lysozyme concentration of serum and urine. A substantial reduction of the lysozyme amount as compared with the normal value (3.1 microgram/10(6) cells) was determined in the leukocytes of patients suffering from chronic lymphatic leukaemia, acute lymphatic leukaemia and the blastic crisis of chronic myeloid leukaemia. Different amounts of lysozyme ranging from extremely low ones to strongly elevated ones were found in leukocytes taken from patients with acute myeloblastic and chronic monocytic leukaemia. In many cases there was a lack of correlation between the lysozyme content of leukocytes on the one hand and that of serum and urine on the other hand. Possible causes underlying this lack of correlation are discussed.