Antisickling properties of divanilloylquinic acids isolated from Fagara zanthoxyloides Lam. (Rutaceae).

Fagara zanthoxyloides Lam. (syn. Zanthoxylum zanthoxyloides) (Rutaceae) is the most cited Fagara species for the treatment and the prevention of sickle cell disease crisis. Sickle cell anemia (SCA) is a public health problem in many countries particularly in Africa. The present study was designed to evaluate the antisickling properties of three isomeric divanilloylquinic acids (3,4-O-divanilloylquinic acid or burkinabin A; 3,5-O-divanilloylquinic acid or burkinabin B and 4,5-O-divanilloylquinic acid or burkinabin C) identified previously by LC/MS/NMR analysis in the root bark of F. zanthoxyloides [Ouattara et al., 2004. LC/MS/NMR analysis of isomeric divanilloylquinic acids from the root bark of Fagara zanthoxyloides Lam. Phytochemistry 65, 1145-1151]. The three isomers showed interesting antisickling properties which increased from burkinabins A to C.

[Prevention of hemoglobinopathies in Brussels: a necessity?]. / Prévention des hémoglobinopathies à Bruxelles: une nécessité?

Haemoglobinopathies are the most frequent genetic diseases in the world. The estimated frequency of carriers in the world is of 200 millions while there is about 300.000 births per year of major forms of haemoglobinopathies. The neonatal screening of haemoglobinopathies performed at the Hospital Erasme on around 80% of births in Brussels since 1994 has demonstrated that the frequency of carriers of an abnormal haemoglobin is around 1.5% while more than 1/2.000 newborns has a major haemoglobinopathy. A strategy must be adopted to manage the haemoglobinopathies in Brussels.

Five years of experience with hydroxyurea in children and young adults with sickle cell disease.

The short-term beneficial effect of hydroxyurea (HU) in sickle cell disease (SCD) has been proven by randomized studies in children and adults. The Belgian registry of HU-treated SCD patients was created to evaluate its long-term efficacy and toxicity. The median follow-up of the 93 patients registered is 3.5 years; clinical and laboratory data have been obtained for 82 patients at 1 year, 61 at 2 years, 44 at 3 years, 33 at 4 years, and 22 after 5 years. On HU, the number of hospitalizations and days hospitalized dropped significantly. Analysis of the 22 patients with a minimum of 5 years of follow-up confirm a significant difference in the number of hospitalizations (P =.0002) and days in the hospital (P <.01), throughout the treatment when compared to prior to HU therapy. The probabilities of not experiencing any event or any vaso-occlusive crisis requiring hospitalization during the 5 years of treatment were, respectively, 47% and 55%. On HU, the rate per 100 patient-years of severe events was estimated to be 3.5% for acute chest syndrome, 1.2% for aplastic crisis, 0.4% for splenic sequestration; it was 0% for the 9 patients with a history of stroke or transient ischemic attack followed for an average of 4 years. No important adverse effect occurred. Long-term chronic treatment with HU for patients with SCD appears feasible, effective, and devoid of any major toxicity; in patients with a history of stroke, HU may be a valid alternative to chronic transfusion support. (Blood. 2001;97:3628-3632)

Antisickling activity of sodium cromoglicate in sickle-cell disease.

Two groups of patients with sickle-cell disease were given a single dose of sodium cromoglicate by inhalation or nasal route. The striking decrease in sickle cells after treatment by both routes lends support to the role of sodium cromoglicate in sickle-cell disease treatment.

Effects of CGRP on human osteoclast-like cell formation: a possible connection with the bone loss in neurological disorders?

Osteoclast-like cell (OCL-like) differentiation is increased in long term cultures of bone marrow taken from paralyzed areas of paraplegic patients. Among the neuropeptides recently described in bone, calcitonin gene-related peptide (CGRP) has been shown in animal studies to inhibit bone resorption in vivo and OCL-like differentiation in vitro: its deficiency could thus be a link between the neural lesion and increased OCL-like production in paraplegia and some other neurologic disorders. We therefore investigated in this study the effects of CGRP on human OCL-like formation and found that it indeed has an inhibitory effect mediated at least in part via cAMP.

In vitro antisickling activity of a rearranged limonoid isolated from Khaya senegalensis.

We previously observed that aqueous extracts of the stem bark and leaves of Khaya senegalensis exhibited a strong antisickling activity. These results prompted us to find out the constituent(s) responsible for these properties using an in vitro bio-guided fractionation. The bioassay was based on sickle cells countings, before and after deoxygenation, in blood samples taken from patients with severe sickle cell anemia and pre-incubated with the drugs to be tested. The main active constituent was identified as a rearranged limonoid whose structure was recently elucidated. In comparison with pentoxifylline used as standard, the in vitro antisickling activity of this limonoid was much higher at any concentrations and incubation conditions. In addition, it did not alter significantly the corpuscular indices.

Ferritin-associated iron induces neutrophil dysfunction in hemosiderosis.

Neutrophils (PMNs) from patients with secondary iron overload have an increased iron and ferritin content as well as a phagocytosis defect. Several serum components might be incriminated in the cellular iron accumulation. We therefore compared the effects on the PMN phagocytosis of total serum as well as the ferritin and transferrin fractions of serum derived from patients with thalassemia major and healthy control subjects. An incubation system of PMNs was developed. PMN phagocytosis was measured before and after incubation. Total serum from patients with thalassemia induced a defect that was prevented by co-incubation with deferoxamine (DFO). Gel-filtration chromatography was performed to separate the serum fraction containing transferrin and albumin from that containing ferritin. The transferrin-albumin fraction had no effect on PMN phagocytosis. On the contrary, the ferritin fraction of normal serum was deleterious to PMN phagocytosis, and the same fraction from thalassemic serum decreased PMN phagocytosis even more. Co-incubation with DFO or catalase improved this defect. Moreover, a cellular increase in the L-type subunit of ferritin was observed after the incubation of PMNs with the ferritin-containing fraction from thalassemic serum. In conclusion, serum from patients with thalassemia is toxic to PMNs, and this toxicity is due to ferritin-associated iron.

Inhibitors in German hemophilia A patients treated with a double virus inactivated factor VIII concentrate bind to the C2 domain of FVIII light chain.

To reduce the risk of transmission of hepatitis A virus, an Octapharma produced factor VIII (fVIII) concentrate treated with solvent detergent (FVIII-SD) was further pasteurized after purification. This product, Octavi SDPlus (FVIII-SDP), was marketed in Europe in 1993 to 1995. Inhibitors appeared from September to October, 1995, in 12 of 109 previously treated German hemophilia A patients. A study of similarly treated Belgian patients, who also developed inhibitors, had shown antibodies to the fVIII light chain (domains A3-C1-C2) only. In the present study, the epitope specificity of 8 German inhibitor plasmas was also found to be restricted to the light chain. In radioimmunoprecipitation assays to localize the light chain epitope(s), antibody binding to heavy chain (domains A1-A2-B) was 11-148 fold lower than to the C2 domain, and binding to recombinant A3-C1 was barely detectable. These results were supported by >95% neutralization of a high responder inhibitor titer by the C2 domain.

Increased osteoclast-like cells formation in long-term bone marrow cultures from patients with a spinal cord injury.

Patients with a spinal cord section loose a significant amount of bone. After paraplegia, bone loss occurs below the lesional level and is the more dramatic in iliac bones and in the metaphyseal area of long bones. A peak of urinary calcium and hydroxyprolinuria is observed approximately 6 weeks after their lesion. To further understand the mechanisms underlying the bone damage, we used long-term bone marrow cultures to compare osteoclast-like (OCL-like) cell formation above and below the lesional level. Seven paraplegic, one quadriparetic, one quadriplegic patients and five normal subjects were investigated. Six weeks after their spinal cord section, the number of OCL-like cells formed in iliac bone marrow cultures was significantly greater than those formed in sternal bone marrow cultures for all paraplegic patients tested. No significant differences were seen between iliac and sternal bone marrow cultures for the quadriparetic, the quadriplegic patient, or for the five normal subjects. Conditioned media (CM) from iliac marrow of paraplegic patients increased OCL-like cell formation in normal bone marrow cultures. IL-1, TNF-alpha, IL-6, and PGE2 were measured in the CM after 3 weeks of culture. IL-6 was found to be significantly higher in iliac CM compared with sternal CM in six out of seven paraplegic patients. In two patients, addition of an anti-IL-6 monoclonal antibody to the marrow cultures significantly decreased the number of OCL-like cells formed at 3 weeks. We conclude that paraplegia caused by a cord section locally induces an increase in the capacity of progenitors to form OCL-like cells in long-term bone marrow cultures. A locally increased IL-6 production in the marrow below the lesional level could be partly responsible for this observation.

Effects of non-steroidal anti-inflammatory drugs on the luminol and lucigenin amplified chemiluminescence of human neutrophils.

A panel of non-steroidal anti-inflammatory drugs commonly used for therapeutic purposes was assessed for their effects on the respiratory burst of isolated human polymorphonuclear neutrophils. Cells were stimulated with opsonised yeast and the production of reactive oxygen species was measured by amplified chemiluminescence with luminol and lucigenin which are two luminogenic agents measuring different cellular events. A special attention was devoted to the establishment of dose-effect curves and calculation of ED50. Some of the drugs tested (acemetacine, diclofenac, flufenamic acid and niflumic acid) were able to decrease both luminol and lucigenin chemiluminescence in a dose-dependent manner reflecting an inhibitory effect on the respiratory burst. The most potent derivative was flufenamic acid (ED50 8 and 78 microM, respectively, with luminol and lucigenin), followed by diclofenac (21 and 98 microM), niflumic acid (97 and 227 microM) and acemetacine (585 and 427 microM). In contrast, several other drugs (flurbiprofen, ibuprofen, ketoprofen, piroxicam) stimulated both luminol and lucigenin chemiluminescence, suggesting a pro-oxidant activity. Acetylsalicylic acid (up to 1250 microM) was a modest inhibitor (maximum 25% inhibition) showing no dose-dependent effect and tolmetin (up to 125 microM) had no significant effect in both systems. The results were in agreement using both luminogenic agents, except for indomethacin, naproxen and tenoxicam which showed different kinds of effects. The unspecific and complex nature of the measurement systems used did not allow to give a complete mechanistic interpretation of the results, but the comparison with literature data gave some pertinent explanations for both anti- and pro-oxidant effects.

Anemia in children with cancer is associated with decreased erythropoietic activity and not with inadequate erythropoietin production.

A defect in erythropoietin (EPO) production has been advocated as being the main cause of anemia presented at time of diagnosis or during treatment by adults with solid tumors. On the basis of this defect, anemic cancer patients, both adults and children, have been treated with recombinant human EPO (rHuEPO). To further elucidate the pathophysiology of anemia in children with cancer, we measured serum soluble transferrin receptor (sTfR), a quantitative marker of erythropoiesis, and serum EPO at time of diagnosis and during chemotherapy in children suffering from solid tumor or leukemia. We determined serum EPO in 111 children (55 leukemia, 56 solid tumors) at time of diagnosis. In the last 44 patients (23 leukemia and 21 solid tumors), sTfR levels were also measured. Serum EPO together with sTfR levels were also determined in 60 children receiving chemotherapy (29 leukemia, 31 solid tumors). These results were compared with those obtained from appropriate control groups. In all patients, we found a highly significant correlation between the logarithm of EPO (log[EPO]) and the hemoglobin (Hb) level. In all subsets of patients, sTfR levels were inappropriately low for the degree of anemia. Neither leukemic nor solid tumor groups showed a significant inverse relationship between log(sTfR) and the Hb level as would be expected in anemic patients with appropriate marrow response. Thus, in children with cancer, anemia is associated with a decreased total bone marrow erythropoietic activity which, in contrast to what has been reported in anemic cancer adults, is not related to defective EPO production.

In vitro antisickling activity of cromolyn sodium.

The improvement in sickle cell disease (SCD) children receiving cromolyn sodium therapy prompted us to investigate its antisickling activity in vitro. The number of sickle cells was determined in deoxygenated blood samples from 15 children with severe SCD. At the eight concentrations tested, cromolyn sodium exhibited a significantly higher activity than pentoxifylline, the standard compound. Therefore cromolyn sodium would appear to be an interesting candidate for SCD therapy and deserves further in vivo investigations.

Dose-response relationship to inhaled endotoxin in normal subjects.

Exposure to endotoxin and to its purified derivative lipopolysaccharide (LPS) is related to several occupational pulmonary diseases and to severe domestic asthma. An inhalation of a given dose of pure LPS produces both a systemic and a bronchial inflammatory response. Information on the dose-response relationship to inhaled LPS in normal subjects is a prerequisite to define the safety threshold of exposure. In the present study, the clinical and inflammatory responses to rising doses of inhaled LPS was evaluated. Nine normal volunteers were challenged weekly by inhalation with saline, 0.5, 5, and 50 microg LPS (Escherichia coli). The response determinators are the clinical symptoms, fever, FEV1, blood polymorphonuclear neutrophils (PMNs) with their level of activation (measured by luminol enhanced-chemiluminescence), and both the blood and the urine concentrations of the C-reactive protein (CRP). To assess the bronchial inflammatory response, an induced sputum was obtained 6 h after each dose of LPS, and the total and differential cell counts as well as the MPO, ECP, and TNF-alpha concentrations were measured. Compared with the saline, an inhalation of 0.5 microg LPS induces a significant decrease in the PMN luminol-enhanced chemiluminescence (p < 0.01), which could reflect a process of margination and/or extravascular sequestration of activated PMN. Inhalation of 5 microg LPS is associated with a significant rise in blood CRP (p < 0.01) and PMNs (p < 0.001) and in sputum PMNs (p < 0.05), monocytes (p < 0.05), and MPO (p < 0.05). Inhalation of 50 microg LPS was characterized by a significant increase in temperature (p < 0.01), blood PMNs (p < 0.001), blood and urine CRP (p < 0.01 and < 0.01), and sputum PMNs (p < 0.001), monocytes (p < 0.05), lymphocytes (p < 0.05), MPO (p < 0.01), TNF-alpha (p < 0.01), and ECP (p < 0.01) while five subjects develop symptoms. In normal subjects, the response to inhaled LPS is dose-related, the most sensitive markers of LPS-induced inflammation being the blood PMNs count with their level of activation, the blood CRP concentration, and the sputum PMNs count. The no-response threshold to an acute inhalation of LPS is less than 0.5 microg.

[Preventive management of patients presenting with an increased tendency to venous thrombosis]. / Attitudes préventives chez les patients présentant une tendance accrue aux thromboses veineuses.

The thrombophilias are conditions characterized by an increased tendency to thrombosis. This paper aims at presenting the actual guidelines concerning the preventive attitudes in the thrombophilias that mainly expose to venous thromboembolism. The identification of these thrombophilias resides on one hand on the patients' and their family's history of venous thrombosis, and on the other hand on the diagnosis of disorders known to be associated with an increased risk of venous thrombosis. Heparin-associated thrombocytopenia of type II is a still too often underdiagnosed syndrome in which the prevention of thrombosis requires a specific approach. The authors discuss thoroughly the preventive attitudes for patients that do not require a long-term anticoagulation, for patients in whom a long-term anticoagulation is generally recommended, and for patients in whom it is sometimes recommended. The practical use of anticoagulation is described. Lastly, a special attention is paid to situations in which the thrombotic risk is increased, such as prolonged immobilization, surgery, traumas, pregnancy and postpartum, contraception and oestrogen therapy.

[Value of in vitro study of osteoclast precursors in the case of severe infantile osteopetrosis treated by bone marrow graft]. / Intérêt de l'étude in vitro des précurseurs ostéoclastiques dans un cas d'ostéopétrose infantile sévère traité par greffe médullaire.

BACKGROUND: Pathogenesis of osteopetrosis is still debated. Testing the ability of osteoclastic progenitors to support the proliferation of functional cells may be useful in understanding pathogenesis. CASE REPORT AND METHODS: A diagnosis of osteopetrosis was made in a girl 1 month-old, born to consanguuineous parents. Bone marrow transplantation was uneffective at the age of 3 months but a second engraftment was successful at 5 months. Unfortunately, the patient died from severe thrombocytopenia at the age of 8 months. Long-term cultures of mononucleated cells from the patient's blood were performed before and after the bone marrow transplantation, with or without growth factors such as vitamin D3, IL-6 and IL-1. Similar studies were made from the patient's marrow obtained after transplantation; all results were compared with those obtained after culturing control cells from cord blood umbilical. RESULTS: Production of osteoclastic cells was mild in peripheral blood cultures; it was important in bone marrow cultures in presence of growth factors. CONCLUSION: These results suggest that osteopetrosis in our patient resulted from an intrinsic defect in progenitors of osteoclasts.

Comparative study of antiphospholipid antibody detection in eleven Belgian laboratories.

Twenty-six plasma samples have been sent to 11 different Belgian laboratories in order to detect the presence of antiphospholipid antibodies, either by immunological methods and/or by coagulation tests. A good concordance between laboratories was observed for coagulation tests. Laboratories using detection tests and performing mixing procedures and neutralisation procedures displayed the highest sensitivity as compared with laboratories which did not perform one of these two latter procedures. The concordance between laboratories for the immunological methods was much worse as compared with coagulation tests. This may be attributable either to an intrinsic problem of the immunological tests or to a selection bias due the fact that the plasmas used in this study were selected in coagulation laboratories only where the chance to find a lupus anticoagulant positive/ELISA antiphospholipid negative sample is high.

Effect of the mucoactive drug nacystelyn on the respiratory burst of human blood polymorphonuclear neutrophils.

In lung diseases such as chronic obstructive pulmonary disease (COPD) or cystic fibrosis, the activation of phagocytic cells produces high amounts of cytotoxic reactive oxygen species (ROS) that are partly implicated in the pathogenic process. In this study, the ex vivo antioxidant activity of nacystelyn (NAL), a recently developed mucoactive thiol-containing agent, was investigated using the respiratory burst of human blood polymorphonuclear neutrophils (PMNs). The ROS generation was induced by serum-opsonized zymosan and assessed with luminol- and lucigenin-enhanced chemiluminescence (ECL). The activity of NAL was compared with N-acetylcysteine (ACC) and captopril, other thiol-containing pharmacological agents having documented antioxidant properties. The three drugs significantly inhibited the ECL response of activated PMNs in the presence of luminol, a luminogenic agent which mostly reflects the production of hydroxyl and hypohalite radicals. NAL was more efficient than the other two drugs: the concentrations producing a 50% inhibition (IC50) of total luminol-ECL were 290 microM, 1580 microM and 760 microM for NAL, ACC and captopril, respectively. The inhibition of the lucigenin-ECL response of activated PMNs was less marked for all compounds suggesting a poorer reactivity with superoxide radicals. These findings demonstrate that NAL, at concentrations obtainable in vivo by inhalation, impairs the PMNs chemiluminescence response related to hydroxyl and hypohalite radicals production. As those radicals are highly cytotoxic, NAL appears as a promising agent in the prevention of oxidative lung damage caused by an active inflammatory response.

Defective polymorphonuclear leukocyte functions in children receiving chemotherapy for cancer are partially restored by recombinant human granulocyte colony-stimulating factor in vitro.

Granulocyte colony-stimulating factor (G-CSF) has important direct and priming effects on different functions of normal mature polymorphonuclear leukocytes (PMNL). Previous study has shown an alteration in respiratory burst and bactericidal activities of PMNL harvested from children with cancer treated with chemotherapy. The present study evaluates the possibility that recombinant human (rh) G-CSF could correct these defective functions in vitro. Free radical formation in defective PMNL was enhanced by rhG-CSF to a level similar to that found in normal PMNL primed by rhG-CSF. The defective bactericidal activity against Escherichia coli and Staphylococcus aureus was also corrected. This bactericidal activity was not different from that observed in normal PMNL primed by rhG-CSF. In conclusion, correction of the altered free radical-formation pathway by rhG-CSF in these cells contributed to the restoration of normal bactericidal activity against both gram-positive and gram-negative microorganisms.