Pierre Fondu

Staphylococcus aureus phagocytosis: A new cytofluorometric method using FITC and paraformaldehyde

A new method for measuring the uptake of Staphylococcus aureus by human polymorphonuclear neutrophils (PMN) using flow cytometry (FCM) is described. Bacteria were labeled with fluorescein isothiocyanate (FITC) and incubated with PMN in a suspension assay. At the end of the assay, phagocytosis was arrested by the addition of cold paraformaldehyde. Thereafter, phagocytosis was quantitated by FCM, using crystal violet to distinguish adherent versus ingested bacteria. The method was validated in multiple samples by reference to our microscopic technique; correlations of phagocytic indices were in good agreement. The FCM method was also found to be reproducible and provided a mean to detect low phagocyte values. Another feature of the approach is that FCM readings can be delayed without any appreciable alterations in the results.

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.

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.

Increased Osteoclast-Like Cells Formation in Long-Term Bone Marrow Cultures from Patients with a Spinal Cord Injury

Abstract. 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.

Dose of desferrioxamine and evolution of HIV-1 infection in thalassaemic patients.

To study the relationship between the dose of desferrioxamine (DFX) and the progression of the HIV‐1 disease in thalassaemia major patients (TMP), 64 seropositive TMP patients were studied. Cumulative incidence of CDC stage IV was calculated using a non‐parametric life‐table method. The association with the mean daily dose of DFX was tested with a Cox proportional hazards model which was also used to adjust for confounding variables. The median of the mean daily dose of DFX over the seropositive period was 40mg/kg (range 0‐65mg/kg). Age at seroconversion (P < 0.02) and splenectomy (P < 0.03) were found to be associated with the mean daily dose of DFX. 6.5 years after seroconversion, 11% of those who had been prescribed more than 40mg/kg of DFX daily had entered stage IV versus 35% of those who had been prescribed a lower dose (P < 0.01). When the dose was taken as a continuous variable it was found that the rate of progression was significantly smaller in TMP receiving a higher dose (P < 0.002). even after adjusting for age and splenectomy (P < 0.02). Although it should be noted that these results were obtained in an observational study, possibly biased by a non‐random allocation of the DFX dose, we believe that they are striking enough to support the claim that the role of DFX in the progression of HIV disease should be further evaluated.

Neutrophil dysfunctions in thalassaemia major: the role of cell iron overload

The susceptibility to infections was recorded in 13 patients with β thalassaemia major (T.P.). The following parameters were also investigated in their polymorphonuclear neutrophils (PMN): nitro blue tetrazolium (NBT) reduction, heated yeast and Escherichia coli phagocytosis, Escherichia coli killing and myeloperoxydase activity. These results were compared to those obtained in healthy controls (H.C.). The Perlss reaction was performed on PMN and graded according to a scoring system with the aim of quantifying the iron intoxication of PMN. Phagocytosis and Perlss reaction of PMN from H.C. were also studied after 20 h of incubation with thalassaemic serum. 6 T.P. out of 13 developed septicaemia during their lifetime and in all 9 septicaemic episodes were noted. Phagocytosis was greatly impaired, disclosing both cellular and serum abnormalities. The mean percentage of Perlss positive PMN was 13% in T.P., contrasting with the constant negative reaction in H.C. The incubation of PMN from H.C. with serum from T.P. induced the simultaneous appearance of a phagocytosis defect and of a positive Perls reaction. It was concluded that in β thalassaemia major the phagocytosis of PMN was altered due to a combination of serum and cellular abnormalities and that both may be related to the iron overload.

Transplanted sickle-cell disease patients with autologous bone marrow recovery after graft failure develop increased levels of fetal haemoglobin which corrects disease severity

Summary. Bone marrow transplantation (BMT) is the only curative therapy for sickle‐cell disease (SCD), but is not devoid of failure risk. Nine patients with severe SCD were grafted in our institution between 1988 and 1993. Six patients successfully engrafted, but three failed to engraft and had delayed autologous recovery.

Bone marrow transplantation for severe sickle cell anaemia

Summary. Five children with sickle cell anaemia underwent bone marrow tranplantation (BMT) for severe clinical disease. The conditioning regimen for BMT was in busulfan plus cyclophosphamide. The allograft contained more than 5.108 nucleated cells per kg recipient. Prophylaxis of GVHD consisted of methotrexate and cyclosporin A. Therapy was well tolerated. Duration of neutropenia (<0.5 × 109/1) was short (14–25 d). Platelet recovery (>50 × 109/1) occurred between day 12 and 45. The patients have been folowed up for 8–28 months. No major infections occurred and long‐term BMT‐related toxicity was limited to mild, chronic GVHD in one patient. Mean haemoglobin levels remained above 10 g/dl. Haemoglobin electrophoresis showed AS patterns in all grafted patients—all marrow donors having sickle cell trait. From our preliminary data, we conclude that BMT or sickle cell anaemia is curative, well tolerated and should be proposed for suitable patients.

Granulocyte functions in children with cancer are differentially sensitive to the toxic effect of chemotherapy.

To analyze the toxicity associated to chemotherapy upon granulocytes, different functional assays were performed, within days of drug exposure and at time of bone marrow recovery, on polymorphonuclear neutrophils (PMN) from children with cancer. There were no significant postchemotherapy changes in the expression of the different receptors studied nor in the phagocytosis ofStaphylococcus aureus 42D. By contrast, a significant decrease was observed in H2O2 production in PMN recently exposed to chemotherapy with both cytofluorometric and chemiluminescence assays. There was also a decrease in the production of O[horizontal line over dot]2 and in chemotaxis; finally, the intracellular killing of S. aureus 42D andEscherichia coli was reduced. In patients having recovered from drug-induced bone marrow aplasia, PMN functions were found to be normal except for bactericidal activity which was still defective. These observations indicate that, in patients exposed to chemotherapy, some PMN functions are transiently altered, whereas microorganism cell killing is continuously impaired.

Cardiac Surgery with Extracorporeal Circulation in Severe Factor VII Deficiency

We describe here successful open heart surgery with extracorporeal circulation and haemodilution in a child with severe congenital factor VII deficiency. Substitution therapy was realized with factor VII concentrate (SD proconvertin concentrate, Bio-Transfusion). This is the first report case of successful cardiac surgery in severe congenital factor VII deficiency.