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Biomedical subjects

J Hakim

Publications and source records attributed to J Hakim.

At least 19 recordsLinked to original sources
PubMed1992

Mechanism of lysozyme inactivation and degradation by iron.

The site-specific lysozyme damage by iron and by iron-catalysed oxygen radicals was investigated. A solution of purified lysozyme was inactivated by Fe(II) at pH 7.4 in phosphate buffer, as tested on cleavage of Micrococcus lysodeikticus cells; this inactivation was time- and iron concentration-dependent and was associated with a loss of tryptophan fluorescence. In addition, it was reversible at pH 4, as demonstrated by lysozyme reactivation and by the intensity of the 14.4-kD-band on SDS-PAGE. Desferal (1 mM) and Detapac (1 mM) added before iron, prevented lysozyme inactivation, while catalase (100 micrograms/ml), superoxide dismutase (100 micrograms/ml) and bovine serum albumin (100 micrograms/ml) gave about 30 to 40% protection by competing with lysozyme for iron binding. The denaturing effect of iron on lysozyme was studied in the presence of H2O2 (1 mM) and ascorbate (1 mM); under these conditions the enzyme underwent partly irreversible inactivation and degradation different to that produced by gamma radiolysis-generated .OH. Catalase almost fully protected lysozyme; in contrast, mannitol (10 mM), benzoate (10 mM), and formate (10 mM) provided no protection because of their inability to access the site at which damaging species are generated. In this system, radical species were formed in a site-specific manner, and they reacted essentially with lysozyme at the site of their formation, causing inactivation and degradation differently than the hydroxyl radical.

Catalase↗
PubMed1992

K562 cells produce an anti-inflammatory factor that inhibits neutrophil functions in vivo.

We have previously reported that K562, a chronic myelogenous leukemia cell line, releases a low molecular weight factor (6 to 8 Kd) that inhibits human polymorphonuclear neutrophil (PMN) adherence and adherence-related functions tested in vitro. We now report that this factor, which we have named K562 inhibitory factor (K562-IF), has potent anti-inflammatory activity in mice, associated with an inhibition of PMN functions. Its in vitro actions were less marked with mouse PMN than with human PMN. They included (1) an inhibition of both nonstimulated locomotion and locomotion induced by FMLP or serum; (2) an inhibition of the chemiluminescence induced by opsonized zymosan, but not that induced by phorbol myristate acetate or FMLP; (3) an inhibition of the degranulation stimulated by opsonized zymosan, as reflected by lactoferrin and lysozyme release; and (4) a decrease in arachidonic acid release and leukotriene B4 production by A23187-stimulated PMN. The in vivo actions of K562-IF after intraperitoneal injection included (1) an inhibition of subcutaneous PMN accumulation at the site of injection of opsonized zymosan (PMN accumulated neither outside the vessels nor intravascularly, as shown by means of histochemistry); (2) an inhibition of neutrophil accumulation in the peritoneum of mice having received sodium caseinate or opsonized zymosan intraperitoneally; and (3) lysozyme concentration in neutrophils having reached the peritoneum after opsonized zymosan treatment equal to that in blood, suggesting diminished release. PMN influx and degranulation in the peritoneum were reduced by 50% after 3 hours of treatment with 1 microgram of K562-IF (equivalent to the effect of 120 micrograms of prednisolone). Taken together, these results show that K562-IF is a potent anti-inflammatory agent that acts by inhibiting PMN functions.

Animals↗
PubMed1992

Inhibition of human neutrophil protein kinase C activity by the antimalarial drug mefloquine.

Mefloquine (alpha-(2-piperidyl)-2,8-bis(trifluoromethyl)-4-quinolinemethanol) , an antimalarial drug, has been shown to inhibit human neutrophil functions, particularly oxygen-dependent bactericidal activity. Since calcium- and phospholipid-dependent protein kinase C (PKC) has a central role in the regulation of this function, we hypothesized that its activity might be altered by mefloquine. We found that mefloquine directly inhibited PKC in a dose-dependent manner, with an IC50 of 45 microM. This inhibition appeared to be non-competitive with respect to ATP, histone and phosphatidylserine. In addition, mefloquine inhibited the binding of [3H]phorbol 12,13 dibutyrate to PKC, indicating that it interacts with the regulatory domain of PKC. By contrast, mefloquine had little or no effect on neutrophil cAMP-dependent protein kinase or its catalytic subunit. Phorbol myristate acetate-induced protein phosphorylation in intact neutrophils was also inhibited by preincubation with mefloquine at concentrations similar to those inhibiting superoxide anion production. These data suggest that inhibition of neutrophil functions by mefloquine may be due to the inhibition of cellular PKC and that mefloquine could have further biological effects in situations in which PKC is involved.

Dose-Response Relationship, Drug↗
PubMed1992

[Inhibition by pentoxifylline of procoagulant activity produced by endotoxin-active monocytes].

When appropriately stimulated, monocytes are able to initiate blood coagulation through the membrane expression of tissue factor. This procoagulant activity is thought to play a role in activating coagulation in response to inflammatory stimuli in vivo. We found that pentoxifylline, a methylxanthine derivative already reported to regulate some monocyte functions, inhibits the procoagulant activity developed by monocytes in vitro in response to endotoxin. This effect was accompanied by an early increase in intracellular levels of cyclic AMP and was mimicked by compounds that induce an increase in cyclic AMP levels. These results suggest that the suppressive effect of pentoxifylline occurs at least in part via an increase in intracellular cyclic AMP levels.

1-Methyl-3-isobutylxanthine↗
PubMed1991

Chemoattractant and opsonic activity in ascitic fluid. A study in 47 patients with cirrhosis or malignant peritonitis.

We studied prospectively the ascitic fluid of 47 patients. Thirty-five were cirrhotics (group A) and 12 had malignant peritonitis (group B). All ascitic fluid samples were initially uninfected. We measured opsonic activity by a chemiluminescent assay, and chemoattractant activity by the under agarose technique. We also measured ascitic concentrations of C3, C4, fibronectin, C-reactive protein, immunoglobulins G, A and M and total proteins. All patients were followed throughout the presence of ascites. None of the group B patients developed peritoneal infection, nor did 23 of the group A patients (group A2). Twelve group A patients (group A1) developed spontaneous bacterial peritonitis (SBP), four of them with recurrence. All indices except immunoglobulins A and M were significantly different between group A and group B patients. Comparing group A1 and group A2, only chemoattractant activity and concentrations of total proteins and C3 were significantly lower in group A1. Using a multivariate analysis with Cox's model, only C3 concentration had an independent predictive value for occurrence of SBP in cirrhotic patients.

Aged↗
PubMed1991

Gamma and pulse radiolysis study of pentoxifylline, a methylxanthine.

Pentoxifylline (Ptx) is a tri-substituted purine with anti-inflammatory properties which are thought to be due, in part, to oxygen radical scavenging. This paper reports an investigation of the reaction of Ptx with the hydroxyl radical [OH.], superoxide anion, azide radical and hydrogen peroxide generated by pulse and gamma-radiolysis, which was carried out to determine the scavenging properties of Ptx towards oxygen radicals. The superoxide anion, azide radical and hydrogen peroxide did not react with Ptx, whereas OH. reacted rapidly. In gamma-radiolysis, the action of OH. on Ptx at pH 7.4 gave rise to an end-product separated by high-performance liquid chromatography and identified by nuclear magnetic resonance and mass spectrometry as C-8-OH-Ptx (yield 0.12 x 10(-6) mol J-1). The reaction of Ptx with OH. after pulse radiolysis at pH 7-7.4 occurred with a rate constant of (7.7 +/- 1.0) x 10(9) mol-1 s-1, forming time-dependent transient radicals. The initial spectrum (2 microseconds after the pulse) showed three maxima (310, 338 and 500 nm). A decrease in the absorbance around 500 nm and an increase around 310 nm reflected a first-order reaction, suggesting a unimolecular rearrangement. It was shown by redox titration that at least two OH-adducts were formed, one with reducing and the other with oxidizing properties. These results suggest that the reducing radical may be (C-8-OH-Ptx)(.).

Cobalt Radioisotopes↗
PubMed1991

Effect of a factor released by K562 malignant cells in culture on human neutrophil bactericidal activity.

We have previously demonstrated that k562 malignant cells in culture contain and release a low-molecular-mass (8-kDa) factor that inhibits adherence-related functions of neutrophils but does not alter fMet-Leu-Phe- or phorbol ester-induced oxidative burst (M. Amar, N. Amit, T. Pham Huu, S. Chollet-Martin, M.T. Labro, M.A. Gougerot-Pocidalo, and J. Hakim, J. Immunol. 144:4749-4756, 1990). In this study, we investigated the effects of this factor, referred to as inhibitory factor 1 (IF1), on the bactericidal activity of human polymorphonuclear cells (PMNs) on Staphylococcus aureus opsonized in various ways. S. aureus was used either nonopsonized or opsonized with heat-inactivated serum or normal serum containing complement factors. The bactericidal activity of PMNs preincubated with IF1-treated or control medium was examined by counting the surviving bacteria. The ability of IF1-treated PMNs to kill bacteria was diminished when they were opsonized with normal serum. When S. aureus was not opsonized or was opsonized with heat-inactivated serum, the bactericidal activity of IF1-treated PMNs was similar to that of controls. Likewise, the phagocytosis of IF1-treated PMNs was diminished when S. aureus was opsonized with normal serum but was not altered when S. aureus was not opsonized or was opsonized with heat-inactivated serum. These results suggest that the decrease in killing might be due to defective ingestion. The chemiluminescence response of IF1-treated PMNs was inhibited when S. aureus was not opsonized or was opsonized with normal serum. No effect on chemiluminescence was observed when S. aureus was opsonized with heat-inactivated serum. These results suggest that IF1 interferes not only with S. aureus stimulation of PMNs via complement receptors but also with oxygen-dependent bactericidal activity.

Biological Factors↗
PubMed1990

Stimulation of human polymorphonuclear leukocytes potentiates the uptake of diclofenac and the inhibition of chemotaxis.

Diclofenac sodium, a non-steroidal anti-inflammatory drug, has been shown to impair the stimulation of human polymorphonuclear leukocytes (PMNs) by chemoattractants. To gain insight into the mechanism of action of this agent, we investigated the uptake of diclofenac by resting and activated PMNs and the effect of the drug on PMN locomotion. During incubation of resting PMNs at 37 degrees in the presence of 78 microM (25 micrograms/mL) diclofenac, drug uptake reached a plateau in less than 2 min. The resulting cellular to extracellular diclofenac concentration ratio (C/E) was 1.01 +/- 0.13 (mean +/- SD). Stimulation of PMNs at 37 degrees but not at 4 degrees with the chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate (PMA), induced a rise in diclofenac uptake, which was dependent on incubation time and diclofenac and stimulus concentrations. Maximal C/E was 1.83 +/- 0.18 and 4.40 +/- 0.60 (mean +/- SD) for PMNs stimulated with 10 microM fMLP and 0.16 microM PMA, respectively. The diclofenac associated with PMNs was predominantly present in the soluble fraction of disrupted cells. Interestingly, PMNs which were pretreated with diclofenac and stimulated with fMLP, exhibited impaired random and directional locomotion induced by activated serum, as compared to controls, i.e. PMNs treated with diclofenac alone or fMLP alone. Thus, stimulation of PMNs enhances diclofenac uptake and potentiates the drug impairment of chemotactic activity. These findings could explain, in part, the observed anti-inflammatory properties of this compound.

Cells, Cultured↗
PubMed1990

Production by K 562 cells of an inhibitor of adherence-related functions of human neutrophils.

Certain tumor cells generate factors that inhibit neutrophil chemotaxis. Our study was designed to explore whether such factors are produced by K 562 malignant cells and whether these have a broader effect in altering neutrophil functions. After 48 h of in vitro culture of K 562 cells, the culture medium and the cells were separated, lyophilized, and extracted with ethanol. These K 562 products, i.e., either the cell or supernatant extract, inhibited both nonstimulated locomotion and locomotion induced either by FMLP or activated serum. Furthermore, K 562 products inhibited neutrophil adherence and oxidative burst induced by opsonized zymosan, whereas oxidative burst induced by PMA or FMLP was not altered. K 562 products had an inhibitory effect on the PMN binding to iC3b-coated particles. They did not modify Mo1 expression of resting cells, did not alter the up-regulation of the receptor induced by FMLP but inhibited the FMLP-induced capping of Mo1 Ag. Con A capping was also inhibited. Actin polymerization in FMLP-stimulated PMN, as measured by flow cytometry and phalloidin binding to F-actin, was inhibited by K 562 products. The inhibitory factor present in K 562 products (cell and culture supernatant) was purified in three steps including gel filtration, ion-exchange chromatography, and IEF. The eluted active fraction corresponded to single band of about 8 kDa on SDS-PAGE. From these experiments, it is concluded that K 562 malignant cells in culture contain and release a low molecular mass factor (congruent to 8 kDa) that inhibits all adherence-related functions of neutrophils, whereas it does not alter FMLP- or PMA-induced oxidative burst. Further studies are needed to assess whether products of other tumor cells also act on the neutrophil by inhibiting adherence-related functions, Mo1 function and capping, and actin polymerization.

Actins↗
PubMed1990

Staurosporine, a protein kinase inhibitor, up-regulates the stimulation of human neutrophil respiratory burst by N-formyl peptides and platelet activating factor.

Staurosporine (STAR), a potent protein kinase C (PKC) antagonist, was found to modulate the chemoattractant-induced respiratory burst of human polymorphonuclear leukocytes (PMNs) according to drug concentration. Low STAR concentrations from 10 to 200 nM potentiated the N-formyl-methionyl-leucyl-phenylalanine (fMLP) and platelet activating factor (Paf)-induced respiratory burst, affecting both the initial rate and the total amount of superoxide anion generated. The maximal increase occurred in the presence of 100 nM STAR and optimal fMLP concentration and reached 60-100% of control values. Above 250 nM, STAR inhibited the respiratory burst with an IC50 of 360 and 320 nM for fMLP and Paf, respectively. The respiratory burst induced by PKC activators such as phorbol myristate acetate or phorbol 12, 13 dibutyrate was inhibited effectively by STAR, with a low IC50 (25 nM) for both stimuli. Thus, the use of low STAR concentrations points to two possible roles of PKC in the regulation of NADPH oxidase activity, i.e. a positive regulation in phorbol ester-treated cells and a negative regulation in chemoattractant-stimulated PMNs.

Alkaloids↗
PubMed1990

Consequences of neutrophil adhesion to physiological and pathological targets.

The ability of neutrophils to adhere in a coordinated and reversible manner to the endothelium and other tissular components is crucial to their chemoattractant-induced locomotion towards relevant targets. Opsonins play a major role in the killing effect of neutrophils by inducing close adherence between the neutrophil and the target, thus maximizing the effect of the reactive oxygen species released by the stimulated neutrophils. Reactive oxygen species are released together with degradative enzymes and other killing proteins associated with neutrophil degranulation. This targeted neutrophil activity kills invading microorganisms but, in a similar way, may be harmful to organs, cells and molecules that have been altered in some way or are involved in immune reactions. In some other pathological situations where body fluids contain proinflammatory agents, neutrophils may behave in a nontargeted and inappropriate manner. In such cases, adherence is often increased, thus slowing locomotion. Moreover, inflammatory agents often prime neutrophils for the oxidative burst induced by chemoattractants or other stimuli. The combined slow locomotion and hypersensitivity of primed neutrophils leads to a premature release of killing substances which may affect blood components, vascular cells, connective tissue or whole organs. Any disturbance of neutrophil adherence is thus potentially harmful and must be recognized and suitably treated.

Autoimmunity↗
PubMed1990

Pentoxifylline inhibition of procoagulant activity generated by activated mononuclear phagocytes.

When appropriately stimulated, monocytes are able to initiate blood coagulation through the membrane expression of tissue factor. This procoagulant activity is thought to play a role in activating coagulation in response to inflammatory stimuli in vivo. We found that pentoxifylline, a methylxanthine derivative already reported to regulate some monocyte functions, inhibits the procoagulant activity developed by U937 cells and monocytes in vitro in response to endotoxin. This effect was accompanied by an early increase in intracellular levels of cyclic AMP and was mimicked by compounds that induce an increase in the level of cyclic AMP levels. These results suggest that the suppressive effect of pentoxifylline occurs at least in part via an increase in intracellular cyclic AMP levels.

1-Methyl-3-isobutylxanthine↗
PubMed1990

Dual effect of phorbol myristate acetate on chemoattractant-induced locomotion of human neutrophils.

Phorbol myristate acetate (PMA) is a potent activator of Ca2+/phospholipid-dependent protein kinase (PKC) and was used to study the involvement of this kinase in human polymorphonuclear neutrophil (PMN) locomotion. Preincubation of PMNs with low concentrations of PMA (4 to 64 x 10(-11) M) had the following effects: (1) fMet-Leu-Phe-induced migration under agarose was increased when the chemoattractant was used at the suboptimal concentration of 10(-8)M and not at the optimal concentration of 10(-7)M; (2) no effect on spontaneous or serum- or LTB4- induced migration at either suboptimal or optimal concentrations; (3) PMA enhanced fMet-Leu-Phe-induced migration, increasing the speed of locomotion but not affecting shape changes induced by fMet-Leu-Phe; (4) the number of fMet-Leu-Phe-specific receptors expressed on the PMN membrane was not altered. Intermediate concentrations of PMA (1.6 to 4.0 x 10(-9) M) had no effect on PMN migration, whereas higher concentrations (4.0 to 16 x 10(-9) M) reduced both spontaneous and fMet-Leu-Phe-, serum-, or LTB4-induced migration in a dose-dependent manner. Effects observed with low concentrations of PMA were not associated with a translocation of cytosolic PKC even when preincubation with PMA was followed by fMet-Leu-Phe stimulation. In contrast, effects observed with higher concentrations of PMA paralleled the decrease in cytosolic PKC activity.

Cells, Cultured↗
PubMed1989

Experience with malignant tumours of the maxillary sinus in the Department of Otolaryngology Universiti Kebangsaan Malaysia, Kuala Lumpur.

Thirty one cases of malignant tumours of the maxillary sinus presenting to the Department of Otolaryngology, Universiti Kebangsaan Malaysia over a four year period from 1982 to 1986 are reviewed. 18 cases (58.1%) were squamous cell carcinoma while seven cases (22.5%) were Non-Hodgkin's lymphoma. There were four cases (12.0%) of adenoid cystic carcinoma while in two cases (6.5%) the tumours were undifferentiated. Presentation was generally late. Nasal obstruction, facial swelling and epistaxis were the main presenting symptoms. Nasal involvement was found in 61.3% of cases, while 51.6% had involvement of the palate as well. Metastasis to the cervical lymph node were uncommon (6.5%). Surgery and radiotherapy with or without chemotherapy were the main modes of treatment in the management of malignant tumours of the maxillary sinus.

Adolescent↗
PubMed1989

[Splenectomy in hematologic diseases. Indications, technics and early results. A retrospective study of 1095 cases].

Since July 1st, 1958, we have operated 1095 patients with some hematological disease. We review the chief currently accepted surgical indications for splenectomy (Hodgkin's disease no longer being one of them, or seldom so), and we communicate our results pertaining to the immediate postsurgical period. Mortality and morbidity have been 1.36% and 3.28%, respectively. In over 95% of cases, there have been no complications. The low incidence (1.36%) of infection-related complications was partly due to the important number of splenic drains placed. High quality results can only be achieved when tight collaboration exists between hematologists, radiologists and surgeons.

Drainage↗