The role of Willebrand factor in platelet - blood vessel interaction, including a discussion of resistance to atherosclerosis in pigs with von Willebrand's disease

Von Willebrand pigs have all the manifestations of the severe human disease. The role of Willebrand antigen (VIII R:AG) and ristocetin cofactor (VIII: RWF) was assessed in these pigs by (1) transfusion and (2) "in vitro" bleeding time assay. The skin bleeding time became normal when the level of transfused Willebrand factor (VIII R:AG/RWF) was raised in the plasma above 30 U/dl. After single or repeated transfusions, skin capillary endothelium and platelets were still distinguished from normal by VIII R:AG deficiency. When incisions in excised porcine skin ("in vitro" bleeding time) were perfused with blood and plasma fractions, haemostasis occurred when plasmatic Willebrand factor exceeded 30 U/dl whether the skin or platelets came from normal or from von Willebrand pigs. The platelet plug occluding the skin incision contained VIII R:AG by immunofluorescence. Willebrand factor appears to coat surfaces and to serve as a platelet attachment protein. These bleeder pigs are resistant to atherosclerosis. If platelets are involved in early atherosclerotic lesions, the role of Willebrand factor in platelet - blood vessel interaction may be important.     

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.     

Fibronectin, aging and related pathologies]

It could be demonstrated that plasma and tissue fibronectin (FN) increase with age. Some age dependent diseases as diabetes, osteoarthritis and Werner syndrome produce also an increase of tissue fibronectin biosynthesis. Plasma fibronectin decreases in diabetes and in breast cancer. Alternative splicing of the FN gene appears also to vary with age and in some related pathologies. Nutritional status and UV light also influence FN biosynthesis. It appears therefore that the determination of plasma FN and its isoforms as well as the study of tissue FN may be of interest for the study of chronological aging and related pathologies.     

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.     

Adriamycin in the Treatment of Acute Leukaemia

In a preliminary study a new antitumour antibiotic, adriamycin, was found to be capable of inducing complete remission in 6 out of 17 patients with acute lymphoblastic leukaemia and in one out of four with lymphoblastic lymphosarcoma despite the fact that these patients had either failed to respond or had relapsed after chemotherapy with agents recognized to be potentially successful in each condition. In five cases of acute lymphoblastic leukaemia adriamycin was used in combination with cytosine arabinoside—three achieved complete remission and two good partial remissions. This combination seems to merit further study in patients who have relapsed on the more conventional chemotherapeutic regimens in acute lymphoblastic leukaemia.In 13 patients with acute myelogenous leukaemia previously treated with daunorubicin and cytosine arabinoside no remissions were obtained with the dose range used.     

Correction of the defective extracellular matrix of Ehlers-Danlos syndrome skin fibroblasts by dexamethasone.

Skin fibroblasts derived from Ehlers-Danlos syndrome (EDS) patients lack an organized extracellular matrix (ECM) of fibronectin (FN) and often show an accumulation of cytoplasmic FN. The treatment of EDS cells of different types (I to VIII) with 10-⁷ M dexamethasone (dex), as well as cocultivation with control fibroblasts, induced in most cases the assembly of a FN-like ECM. The study of FN mRNA expression by dot-blot hybridization and of FN released into the culture media of EDS cells showed that the correction of the defective FN-ECM of EDS cells by dex treatment is associated in most cases with an increase of FN mRNA synthesis and of secreted FN.     

Vincristine and Prednisone for the Induction of Remissions in Acute Childhood Leukaemia

A total of 65 children with acute lymphoblastic leukaemia and seven with other types of acute leukaemia received treatment with a combination of vincristine and prednisone. In all 122 courses of treatment were given. Of 22 patients with acute lymphoblastic leukaemia who received this as their first treatment, all achieved complete remission. The complete remission rates were 82% for patients with acute lymphoblastic leukaemia in their first relapse, 63% in the second relapse, and much lower in subsequent relapses and in the patients with other types of acute leukaemia. Alopecia and gastrointestinal and neuromuscular toxicity occurred respectively in 51%, 29%, and 21% of instances, only the last of these side-effects of vincristine being dose-related. Most of the complete remissions were obtained with a total dose of vincristine which carried only a low risk of neurotoxicity.     

Unfavourable clinical implications for HLA-G expression in acute myeloid leukaemia

Human leukocyte antigen-G (HLA-G) molecule exerts multiple immunoregulatory functions that have been suggested to contribute to the immune evasion of tumour cells. Studies on HLA-G expression in malignant haematopoietic diseases are controversial, and the functions of HLA-G on this context are limited. In the current study, HLA-G expression was analysed in different types of patients: de novo acute myeloid leukaemia (AML, n = 54), B cell acute lymphoblastic leukaemia (B-ALL, n= 13), chronic myeloid leukaemia (CML, n= 9) and myelodysplastic syndrome (MDS, n= 11). HLA-G expression was observed in 18.5% cases of AML, 22.2% in CML and 18.2% in MDS, but not in B-ALL patients. In AML, HLA-G-positive patients had a significant higher bone marrow leukaemic blast cell percentage when compared with that of HLA-G-negative patients (P < 0.01). Total T-cell percentage was dramatically decreased in HLA-G-positive patients (P < 0.05). Cytogenetic karyotyping results showed that all HLA-G-positive AML patients (n= 5) were cytogenetically abnormal, which was markedly different from that of HLA-G-negative patients (P < 0.01). Ex vivo cytotoxicity analysis revealed that HLA-G expression in AML leukaemic cells could directly inhibit NK cell cytolysis (P < 0.01). These findings indicated that HLA-G expression in AML is of unfavourable clinical implications, and that HLA-G could be a potential target for therapy.     

Human factor VIII procoagulant activity and phospholipid interaction

The interaction between purified human factor VIII and phospholipid vesicles was investigated. The binding of factor VIII to an equimolecular mixture of phosphatidylserine (PS) and phosphatidylcholine (PC) was studied by sucrose gradient ultracentrifugation (10–40% w/v saccharose in 0.01 M Tris-HCl/0.15 M NaCl buffer (pH 7). In the absence of phospholipids all factor VIII activities (VIII : C, VIII R : WF and VIII R : AG) were found in the zone of highest sucrose density including the factor VIII related protein subunit (200 000 molecular weight). In the presence of an equimolecular mixture of PS/PC VIII R : WF activity, VIII R : AG and a factor VIII related protein still migrated to the bottom of the tube, while VIII : C activity remained at the top where phospholipids were found. Thus a dissociation phenomenon between VIII : C and the other factor VIII relateda activities was apparent in the presence of phospholipids. These results also demonstrate the binding of factor VIII : C to certain active phospholipids.     

Survival and the HL-A System in Acute Lymphoblastic Leukaemia

The relation between the HL-A antigens and survival has been studied in 58 patients with acute lymphoblastic leukaemia. Patients with the HL-A9 antigen had the best estimated median survival times, the highest ratio of survivors to non-survivors, and the lowest median age at diagnosis. The data suggest that resistance to acute lymphoblastic leukaemia may be associated with the HL-A9 antigen.     

Late marrow recurrences in childhood acute lymphoblastic leukaemia.

Thirty children with acute lymphoblastic leukemia had a recurrence in the bone marrow after treatment was stopped electively. A second haematological remission was achieved in 27 (90%), and the median duration of remission was shortest (six months) in those relapsing within six months of stopping treatment. Four of six children relapsing over one year after stopping treatment remained in second haematological remission. Leukaemic infiltration of the central nervous system developed in four patients remaining in marrow remission. It is concluded that conventional chemotherapy is unlikely to be effective in children with acute lymphoblastic leukaemia who relapse soon after stopping treatment, that "reprophylaxis" of the central nervous system probably with long-term intrathecal chemotherapy is essential, and that some patients relapsing after prolonged unmaintained remission may achieve long-term leukaemia-free survival.     

Blood coagulation factors and proteinase inhibitors in cytostatic therapy of acute leukemia

In 64 patients affected with acute leukaemia (51 patients with acute non-lymphatic leukaemia and 13 patients with acute lymphatic leukaemia) extensive investigations of blood coagulation were made during cytostatic therapy. The following conspicuous changes of haemostatasis could be observed in making the diagnosis: Lowered quick value and shortened PTT, increased fibrinogen, fibrinopeptide, A, alpha 1-antitrypsin and alpha 2-macroglobulin, diminished plasminogen and plasma fibrininectin. According to TAD (VP) protocol the induction therapy leads to hypercoagulability which can be recognized by an increase of fibrinopeptide A, coagulating factors and shortening of PTT. During the therapy with L-asparaginasis procoagulatoric as well as thromboprotective coagulating proteins are diminished. A dense laboratory control enables those disturbances of haemotasis caused by disease or therapy to be separated and contributes to preventing complications during the cytostatic induction therapy.     

Buffalo plasma fibronectin: a physico-chemical study

Plasma fibronectin (FN) of buffalo (Babulis babulis) was purified to apparent homogeneity, using gelatin-Sepharose and heparin-Sepharose affinity columns. It was found to have two subunits of molecular mass 246 kDa and 228 kDa, on SDS-gel. Its immunological cross-reactivity with anti-human plasma FN was confirmed by Western blotting. The amino acid composition was found to be similar to that of human and bovine plasma FNs. Buffalo plasma FN contained 2.23% neutral hexoses and 1.18% sialic acids. No titrable sulfhydryl group could be detected in the absence of denaturant. Reaction with DTNB indicated 3.4 sulfhydryl groups in the molecule, whereas BDC-OH titration gave a value of 3.8 -SH groups in buffalo plasma FN. Stoke's radius, intrinsic viscosity, diffusion coefficient and frictional ratio indicated that buffalo plasma FN did not have a compact globular conformation at physiological pH and ionic strength. Molecular dimensions (average length, 120 nm; molar mass to length ratio, 3950 nm(-1) and mean diameter, 2.4 nm) as revealed by rotary shadowing electron microscopy further supported the extended conformation of buffalo plasma FN. These results show that buffalo plasma FN has similar properties as that of human plasma FN.     

Repeated, 2-10 fold asparaginase treatments in children with lymphoproliferative diseases]

In 42 children being in the advanced stage of an acute lymphoblastic leukaemia as well in 7 children with lymphosarcoma a total of 83 series of treatment with L-asparaginase were carried out. During the first blastic crisis of acute leukaemia 74% of complete or partial remissions could be obtained by two treatments and 52% by the following ones. The best results were obtained by organ manifestations of acute leukaemia (80% of complete or partial remissions). Less satisfactory results were achieved in treating lymphosarcoma. All remissions were only of a short duration.     

Development of a novel polycationic adsorbent for cryogel removal.

Cryogel, prevalent in the plasma of rheumatism patients, is a plasma fibronectin (pFN)-extra domain A containing FN (EDA(+)FN)-fibrinogen (Fbg) complex formed by adding heparin (HP) at a low temperature (4 degrees C). Although EDA(+)FN does not usually exist in normal plasma, its prevalence in rheumatic patients causes cryogelation in plasma. Removal of cryogel is thus a promising and novel approach to treating rheumatism. As HP-EDA(+)FN aggregate, which is induced by the main component of cryogel, is considered to be an anion, cationic materials capable of eliminating this anionic conjugate were innovated in this study. We found that an amino group density of 100-130 micromol/g (dry weight) of adsorbents prompted selective adsorption of the EDA(+)FN-HP complex. Elimination of EDA(+)FN as high as 80% accompanied by removal of the components of total FN (pFN) (10%) and Fbg (10%) in the model patient plasma was established.     

Detection of a soluble form of the receptor for interleukin 2 in the serum of patients with hairy cell leukaemia

The sera of patients with hairy cell leukaemia (HCL) contain a factor which inhibits the binding of anti-IL-2R antibody to its target (activated T lymphocytes). The presence of this factor, which probably corresponds to the soluble form of IL-2R (sIL-2R), can be easily detected using a simple immunocytochemical inhibition assay. In a series of patients with chronic lymphoproliferative diseases the presence of sIL-2R appeared to be specific for HCL, using the sensitivity of our test, since it could not be detected in sera from normal subjects and patients with B-cell lymphocytic leukaemia or Hodgkin's disease. Thus it might be used as an additional tool for characterizing HCL patients.     

Bone marrow transplantation in childhood leukaemia--experience and strategies in the Federal Republic of Germany

BMT has gained its place in the treatment of childhood leukaemia. Nevertheless, there are still many questions open. In acute lymphoblastic leukaemia children should normally be grafted in 2nd remission (CR). Some high risk cases, however, should probably be grafted in 1st CR. It is not clear whether children with late relapses benefit more from BMT than from renewed chemotherapy. Children with a relapse during maintenance therapy, however, have a better survival rate with BMT. In acute nonlymphoblastic leukaemia certain high risk patients should be grafted in 1st CR but it has still to be shown that BMT is superior to chemotherapy in such cases. It is not clear whether children with a relapse following intensive chemotherapy (such as the BFM-protocols) will benefit from BMT at all. In chronic myelocytic leukaemia, BMT in chronic phase should be performed. Thus, for the first time cure has become possible for this disease. Waiting for acceleration or even the occurrence of a blast crisis decreases the chance of survival after BMT dramatically. Since complications of BMT such as graft-versus-host reaction or severe infections are less frequent in children, relapses remain the main problem after BMT in childhood leukaemia.     

过氧亚硝基阴离子通过JAK/STAT信号途径促进系膜细胞纤连蛋白的合成

氧化应激是糖尿病肾病的重要发病机制之一。过氧亚硝基阴离子(peroxynitrite,ONOO–)是参与氧化应激损伤的重要成员,与糖尿病及其并发症密切相关。该文观察高糖环境下ONOO–对系膜细胞合成纤连蛋白(fibronectin,FN)的影响,并探讨其作用机制。实验中,人肾小球系膜细胞分为4组:正常对照组、高糖组、高糖+尿酸组及高糖+AG490组。培养12,24,48 h后收集细胞及其上清液、并提取细胞总蛋白。采用酶联免疫吸附实验(ELISA)检测细胞上清液中FN的含量,采用免疫细胞化学和Western blot检测NT总蛋白(ONOO–生成的生物标志物)、p-JAK2及p-STAT3蛋白的表达。结果显示,与同期正常组相比,高糖组NT总蛋白、p-JAK2及p-STAT3的表达及FN含量明显增高(P<0.05),并且随着时间的延长表达逐渐增多,以48 h组最为显著;高糖+尿酸组,NT、p-JAK2、p-STAT3及FN较高糖组明显减少(P<0.05);高糖+AG490组,p-JAK2、p-STAT3及FN较高糖组明显减少(P<0.05),但NT表达与高糖组差异无统计学意义(P>0.05)。由此可见,高糖环境下系膜细胞中存在ONOO–的过量表达,ONOO–通过JAK/STAT信号途径促进系膜细胞FN的合成。     

Molecular docking of heparin oligosaccharides with Hep-II heparin-binding domain of fibronectin reveals an interplay between the different positions of sulfate groups

Fibronectin is a major component of the extracellular matrix and serves as support for cell adhesion and migration. Heparin and heparan sulfates (HS) have been reported to be high-affinity ligands for fibronectin. The strongest heparin/HS-binding site, named Hep-II, is located in the C-terminal repeat units FN12-14 of fibronectin. Mutational studies of recombinant fibronectin fragments and elucidation of the X-ray crystallographic structure of Hep-II in complex with heparin allowed localizing the main heparin/HS-binding site in FN13 to two parallel amino acid clusters: R1697, R1698, R1700 and R1714, R1716, R1745. Heparin, which is more sulfated than HS, is a better ligand for fibronectin, indicating that the sulfate density is important for the interactions. However, other studies demonstrated that the position of sulfate groups is also critical for high-affinity binding of the polysaccharides to fibronectin. In the current work, we used molecular docking of Hep-II domain of fibronectin with a series of differently sulfated dodecasaccharides of heparin to determine the implication of each sulfate position in the interaction. By using this approach, we confirmed the implication of R1697, R1698, R1700 and R1714 and we identified other amino acids possibly involved in the interaction. We also confirmed a hierarchic involvement of sulfate position as follows: 2S >> 6S > NS. Interestingly, the formation of stable complexes required a mutual adaptation between Hep-II domain and oligosaccharides, which was different according to the pattern of sulfation. Finally, we demonstrated that 3-O-sulfation of heparin stabilized even more the complex with Hep-II by creating new molecular interactions. Collectively, our models point out the complexity of the molecular interactions between heparin/HS and fibronectin.     

Combination chemotherapy for acute lymphoblastic leukaemia in adults.

Fifty-one adults with acute lymphoblastic leukaemia were entered into a trial of intense initial chemotherapy and early "prophylaxis" of the central nervous system (CNS). Initial treatment with OPAL (Oncovin (vincristine), prednisolone, adriamycin (doxorubicin), and L-asparaginase (colaspase)) followed by craniospinal or cranial irradiation and intrathecal methotrexate produced remission in 36 patients (71%). Seventeen of these patients relapsed three to 18 months after the start of remission; the remainder had been in remission for 12 to 52 months by the end of the study. The predicted median duration of complete remission was 18.5 months. None of the four patients who initially had clinical evidence of CNS disease, three of whom also had leukaemic cells identical to those found in Burkitt''s lymphoma, achieved remission. Those patients who initially had hepatomegaly or splenomegaly had a shorter remission than those without. The predicted median survival was 27 months in those who achieved complete remission, one month in those who did not, and 21 months overall. The addition of colaspase and doxorubicin to vincristine and prednisolone and the use of early CNS treatment clearly improved the remission rate among adults with acute lymphoblastic leukaemia, though the presence and length of remission was affected by the extent of disease at presentation. Burkitt-like leukaemia, which had a poor prognosis, is probably a separate disease and may benefit from a different therapeutic approach.